Does dysregulation of immune responses in the tonsils play an important role in the pathology of psoriasis? Sigrún Laufey Sigurðardóttir Thesis for the degree of Philosophiae Doctor University of Iceland Faculty of Medicine School of Health Sciences 2014
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Does dysregulation of immune responses in the tonsils play an important role in the pathology of
psoriasis?
Sigrún Laufey Sigurðardóttir
Thesis for the degree of Philosophiae Doctor University of Iceland Faculty of Medicine
School of Health Sciences 2014
Does dysregulation of immune responses in the tonsils play an important role in the pathogenesis of
psoriasis?
Sigrún Laufey Sigurðardóttir
Thesis for the degree of Philosophiae Doctor
Supervisors: Helgi Valdimarsson professor emeritus and Andrew Johnston PhD Doctoral committee: Johann Eli Guðjónsson MD PhD,
Björn Rúnar Lúðvíksson MD PhD University of Iceland
School of Health Sciences Faculty of Medicine
2014
Er truflun í stjórnun ónæmissvara í kverkeitlum mikilvægur orsakaþáttur psoriasis?
Sigrún Laufey Sigurðardóttir
Ritgerð til doktorsgráðu
Umsjónarkennarar: Helgi Valdimarsson professor emerítus, Andrew Johnston PhD. Doktorsnefnd: Jóhann Elí Guðjónsson PhD, Björn Rúnar Lúðvíkson PhD.
Háskóli Íslands Heilbrigðisvísindasvið
Læknadeild 2014
“If we knew what it was we were doing, it would not be called research, would it?”
ʊ Albert Einstein
Thesis for a doctoral degree at the University of Iceland. All right reserved. No part of this publication may be reproduced in any form without the prior permission of the
copyright holder.
© Sigrún Laufey Sigurðardóttir ISBN (apply at ISBN 978-9935-9186-3-5) Printing by Háskólaprent Reykjavík, Iceland 2014
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Ágrip
Bakgrunnur
Sóri (psoriasis) er krónískur bólgusjúkdómur í húð og er tíðni hans um 2% meðal hvítra
manna. Sjúkdómurinn kemur snemma fram og einkennist af rauðum, þykkum,
hreisturkenndum skellum sem algengast er að finna á olnbogum, hnjám og í hársverði. Þessar
skellur valda óþægindum og hafa neikvæð áhrif á lífsgæði sjúklinganna.
Íferð sjúkdómsvaldandi T frumna í húð er talið vera upphafsskrefið í þróun sjúkdómsins.
Nýlegar rannsóknir hafa sýnt að þessar T frumur hafa Th1/Th17 svipgerð og leiða til
bólgumyndunar og offjölgunar keratínfrumna sem loks myndar húðskellurnar. Í dag er ekki
vitað hver uppruni þessara sjúkdómsvaldandi frumna er en þekkt er að hálsbólgur af völdum
streptokokka geta leitt til eða orsakað versnun í sóraútbrotum. Þetta bendir til þess að
kverkeitlar geti haft áhrif á myndun sóra hjá ákveðnum sjúklingum.
Kverkeitlar eru staðsettir innarlega í hálsi þar sem þeir starfa sem varnarstöðvar fyrir bæði
öndunar- og meltingarveg. Þeir eru hluti af svo kölluðum Waldeyer’s hring er tilheyrir
eitlakerfi slímhúðar (MALT). Kverkeitlar innihalda mikinn fjölda ónæmisfrumna og bregðast
skjótt og örugglega við sýklum. Þeirra helsta einkenni eru kímmiðjur sem taka upp stærsta
hluta vefjarins.
Markmið
Aðalmarkmið verkefnisins var að reyna að skýra hvers vegna endurteknar streptokokka
hálsbólgur geti leitt til myndunar á sóra eða versnunar í sjúkdómseinkennum. Ennfremur var
markmiðið að reyna að greina hvaða þættir væru mikilvægir fyrir þessi tengsl kverkeitla og
sóra.
Efni og aðferðir
Tuttugu og fimm kverkeitlar úr sórasjúklingum voru bornir saman við fjörutíu og einn
kverkeitil úr einstaklingum með endurteknar sýkingar og átta kverkeitla með ofvöxt. Þessir
kverkeitlar fengust úr reglubundnum kverkeitlatökum á Landspítalanum Háskólasjúkrahúsi.
Strok var tekið af öllum kverkeitlunum og ræktað upp til að greina örveruflóruna.
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Kverkeitlar voru litaðir með venjulegri vefjalitun (H&E), ónæmisfræðilegri vefjalitun eða
litun með flúorljómandi mótefnum til að meta tjáningu á örverudrepandi peptíðum og
efnatogum. Fjöldi og staðsetning ónæmisfrumna var jafnframt metinn með sömu aðferð.
Svipgerð T frumna úr kverkeitlum og blóði var greind með flæðifrumusjá. T frumur og
angafrumur voru ennfremur einangraðar og litaðar. Hnattkjarna hvítfrumur úr kverkeitlum
sem og einangraðar kverkeitla T frumur voru örvaðar með örverudrepandi peptíðinu LL-37
ásamt eða án DNA. Hjá sórasjúklingum voru frumur örvaðar með samsvarandi peptíðum úr
M protein streptokokka og keratínum úr húð og svipgerð T frumna ákvörðuð. Þetta var
framkvæmt bæði við upphaf rannsóknarinnar og tveimur mánuðum eftir kverkeitlatöku
Niðurstöður
Sýkingar af völdum streptokokka var algengari meðal sórasjúklinga. Átti það sérstaklega við
um sýkingar af völdum streptokokka af flokki C. Enginn marktækur munur var á tjáningu á
örverudrepandi peptíðinu LL-37 en hins vegar hafði peptíðið margvísleg ónæmifræðileg áhrif.
Tjáning þess var einskorðuð við ífarandi hvítfrumur og angafrumur í kímmiðjum. Þá tjáðu T
frumur úr kverkeitlum psoríasissjúklinga marktækt meira af húðrötunarsameindinni CLA
(cutaneous lymphocyte associated antigen) og var fylgni á milli fjölda þeirra í kverkeitlum og
blóði. Ennfremur tjáðu þessar frumur ýmsar sameindir er tengjast Th1/Th17 ónæmissvari. Við
örvun með hinum samsvarandi peptíðum brugðust T frumurnar við á Th1/Th17 miðaðan hátt.
Þá var vefjafræðilegur munur á milli hinna mismunandi kverkeitlahópa.
Niðurstaða
Út frá fengnum niðurstöðum er hægt að álykta að kverkeitlar séu mikilvægir fyrir meingerð
psoriasis hjá ákveðnum einstaklingum og að hinar meinvaldandi T frumur í blóði og húð
sórasjúklinga gætu átt uppruna sinn í kverkeitlum.
Lykilorð: Psoriasis, kverkeitlar, kímmiðjur, CLA, T frumur.
9
Abstract
Background
Psoriasis is a chronic inflammatory skin disease affecting around 2% of the Caucasian
population. It occurs early in life and is characterized by thick, erythematous scaly plaques
that are most commonly located on elbows, knees and scalp. They cause discomfort and have
negative effect on the life quality of the patients.
The infiltration of pathogenic T cells into skin is thought to be the initiating step in the
development of psoriasis. Recent studies have shown that these T cells are of the Th1/Th17
phenotype and drive the hyperproliferation of keratinocytes, inflammation and ultimately
plaque development. The origin of these T cells is currently unknown, but the association of
streptococcal throat infection and the initiation and exacerbation of the disease suggests the
involvement of the palatine tonsils in the pathogenesis of psoriasis for a subset of patients.
The palatine tonsils are located on the inside of the throat where they act as guardians of
the respiratory and the digestive tract. They form a part of the Waldeyer’s ring and belong to
the mucosa-associated lymphoid system (MALT). Tonsils are packed with various immune
cells and initiate a strong immune response against invading pathogens. One of their most
distinctive features are the germinal centres that occupy a great part of the lymphoid tissue.
Aim
The overall aim was to clarify why recurrent streptococcal infections can lead to the
initiation or exacerbation of psoriasis. Moreover to identify the distinguishing factor
underlying their pathological importance in psoriasis.
Materials and methods
Twenty-five psoriasis tonsils were compared to forty-one recurrently infected tonsils and
eight hypertrophic tonsils obtained through routine tonsillectomy at the National University
Hospital, Reykjavik, Iceland. Tonsil swabs were analysed for bacterial infection in all tonsils.
Histological comparison of tonsil sections was performed using H&E staining,
immunohistological staining or immunofluorescent staining for antimicrobial peptides,
chemokines and immune cells. T cell phenotypes in tonsils and blood were determined using
flow cytometry (FACS) analysis. T cells and DC were also isolated and stained. Tonsil
mononuclear cells (TMC) and isolated T cells were stimulated with the antimicrobial peptide
10
LL-37 with or without exogenous human DNA. At study entry and two month post
tonsillectomy, peripheral blood mononuclear cells (PBMC) and TMC from psoriasis patients
were stimulated with homologous peptides from streptococcal M6 protein and epidermal
keratin 17 and the T cell phenotype determined.
Results
Streptococcal infections were more frequent among psoriasis patients, in particular
infections by group C streptococci. No difference was observed in the expression of
antimicrobial peptide LL-37. However, we observed that LL-37 had a number of
immunomodulatory effects and that its expression within the tonsils was restricted to
infiltrated leukocytes and GC-DCs. Tonsil T cells from psoriasis tonsils had a higher
expression of the skin-homing molecule cutaneous lymphocyte-associated antigen (CLA) and
their frequency in tonsil correlated with their levels in blood. Furthermore, these cells
expressed various Th1/Th17-associated molecules. These skin-homing T cells responded to
the keratin/streptococcal peptides in a Th1/Th17 manner. Finally, a clear histological
difference was observed between the tonsils of psoriatic and non-psoriatic individuals.
Conclusion
It can be concluded that palatine tonsils are important for the pathogenesis of psoriasis in a
subset of patients and that the pathogenic psoriasis T cells in the blood and skin of these
patients may have originated from within the tonsils.
Keywords: Psoriasis, tonsil, germinal centre, CLA, T cells.
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Acknowledgements
I would like to thank Helgi Valdimarsson, Professor emeritus and former Head of the
Department of Immunology, Landspitali - the National University Hospital of Iceland for his
guidance and support as well as the opportunity to do this project.
I particularly wish to thank Andrew Johnston for encouraging me to enter the mysterious
world of the PhD study. Your endless support, enthusiasm for Immunology, friendship and
advice has been invaluable throughout the years.
I would like to thank Inga Skaftadóttir for her assistance and good advice and my former
colleagues at the Department of Immunology for their companionship and support.
I thank Björn Rúnar Lúðvíksson Professor and current Head of the Department of
Immunology, for providing facilities for the work and the doctoral committee for their
contribution.
I thank the staff of the Ear, Nose and Throat Department, Landspitali-Fossvogur, Reykjavik
and Hannes Hjartarson MD at the Medical Clinic, Glæsibæ, Reykjavík for their assistance.
I sincerely thank my friends and family, in particular my sister, Svanborg Þórdís and my
parents; Elfa Ólafsdóttir and Sigurður G. Sigurðsson for all their help, encouragement and
support.
Most of all I thank my son, Víðir Davíð, for his love and understanding. I love you to the
moon and back.
The work described in this thesis was performed at the Department of Immunology,
Landspitali – the National University Hospital, Reykjavik, Iceland.
The work was funded by The Icelandic Research Fund (grant number 080448021-23), The
Icelandic Research Fund for Graduate Students, The Research Fund of The University of
Iceland for Doctoral Studies and the Landspitali University Hospital Research Fund.
This work is dedicated to the loving memory of my mother, Elfa Ólafsdóttir
12
Contents
Ágrip .......................................................................................................................................... 7�Abstract ..................................................................................................................................... 9�Acknowledgements ................................................................................................................. 11�Contents ................................................................................................................................... 12�List of abbreviations ............................................................................................................... 15�List of figures .......................................................................................................................... 18�List of tables ............................................................................................................................ 18�List of papers .......................................................................................................................... 19�Declaration of contribution ................................................................................................... 20�1� Introduction ....................................................................................................................... 21�
1.1� The immune system ............................................................................................................... 21�1.2� The innate immune system .................................................................................................... 21�1.3� The adaptive immune system ................................................................................................. 24�
1.3.1� T cell phenotypes .............................................................................................................. 25�1.3.2� T memory cells ................................................................................................................. 29�
1.4� The skin immune system ....................................................................................................... 29�1.5� Psoriasis ................................................................................................................................. 31�
1.5.1� The clinical manifestations of psoriasis ............................................................................ 31�1.5.2� The psoriatic skin .............................................................................................................. 31�1.5.3� Psoriasis triggers ............................................................................................................... 34�
1.6� The palatine tonsils ................................................................................................................ 34�1.6.1� Histological features ......................................................................................................... 35�1.6.2� Secondary lymphoid follicles ........................................................................................... 36�1.6.3� The germinal center reaction ............................................................................................ 37�1.6.4� Infections and the crypt epithelium .................................................................................. 39�1.6.5� Expression of PRRs in tonsils ........................................................................................... 40�1.6.6� Hypertrophic and recurrently infected tonsils................................................................... 41�
1.7� Microbial diversity and the microbiome ................................................................................ 41�1.7.1� The microbiome of the skin .............................................................................................. 42�1.7.2� The microbiome of the palatine tonsils ............................................................................. 43�
1.8� -Haemolytic Streptococcus .................................................................................................... 44�1.9� The association of streptococcal throat infection and psoriasis ............................................. 45�
1.9.1� Streptococcal M protein and psoriasis .............................................................................. 46�1.9.3� Peptidoglycan and psoriasis .............................................................................................. 48�
1.10�Antimicrobial peptides ........................................................................................................... 48�1.10.1�Defensins .......................................................................................................................... 49�1.10.2�The anti-microbial peptide hCAP18/LL-37 ...................................................................... 52�
1.12�Concluding remarks ............................................................................................................... 57�2� Aims .................................................................................................................................... 59�
2.1� The role of LL-37 in palatine tonsils (paper I) ....................................................................... 59�2.2� Histological and microbiological characteristics of psoriasis tonsils (paper II) .................... 59�2.3� The effect of tonsillectomy (paper III) .................................................................................. 59�
13
3� Materials and methods ...................................................................................................... 61�3.1� Study approval ....................................................................................................................... 61�3.2� Tonsil material and participants in the study ......................................................................... 61�3.3� Preparation of tonsil tissue ..................................................................................................... 62�3.4� Swabs and bacterial typing .................................................................................................... 62�3.5� Tonsil cell isolation procedures ............................................................................................. 62�
3.5.1� Tonsil mononuclear cell (TMC) isolation and culture ...................................................... 62�3.5.2� Peripheral blood mononuclear cell (PBMC) isolation and culture ................................... 62�3.5.3� Isolation of CD4+ T cells ................................................................................................. 62�3.5.4� Isolation of myeloid dendritic cells .................................................................................. 63�3.5.5� Isolation of plasmacytoid dendritic cells .......................................................................... 63�3.5.6� Isolation of CD68+ macrophages ..................................................................................... 63�
3.6� Cell stimulation with hCAP-18/LL-3 peptide ........................................................................ 64�3.7� Staining procedures ................................................................................................................ 64�
3.7.1� Haemotoxylin and eosin staining ...................................................................................... 64�3.7.2� Immunohistostaining (IHC) .............................................................................................. 64�3.7.3� Fluorescence immunohistochemistry (FIH) ..................................................................... 66�
3.8� Histological evaluation .......................................................................................................... 68�3.9� Cytokine detection and quantification in cell supernatant ..................................................... 68�3.10�Expression of cell surface receptors ...................................................................................... 69�3.11�Real-time RT-PCR ................................................................................................................. 69�3.12�Peptide stimulation ................................................................................................................ 70�
3.12.1�Homologous keratin and streptococcal peptide antigens .................................................. 70�3.12.2�Determination of peptide-reactive T cells......................................................................... 71�
3.13�HLA-Cw*0602 typing ........................................................................................................... 71�3.14�Expression of the data and statistics ...................................................................................... 72�
4� Results ................................................................................................................................. 73�4.1� hCAP-18/LL-37 expression and function in palatine tonsils (paper I) .................................. 73�
4.1.1� hCAP18/LL-37 is expressed by leukocytes in the squamous epithelium ......................... 73�4.1.2� hCAP18/LL-37 is expressed by leukocytes within the tonsil crypts ................................ 75�4.1.3� hCAP18/LL-37 is expressed in the lymphoid follicles ..................................................... 75�4.1.4� LL-37 modulates chemokine and cytokine expression of tonsil mononuclear cells ........ 76�4.1.5� LL-37 influences the expression of tonsil mononuclear cell surface receptors. ............... 78�4.1.6� Tonsil CCL5 and CXCL9 expression is concomitant with LL-37 ................................... 79�4.1.7� DNA-LL-37 complexes instigate a Th1 response in the TMC cultures ........................... 80�
4.2� Psoriasis tonsils are histologically and microbiologically distinctive with skin homing T
cells (Paper II) ........................................................................................................................ 81�4.2.1� -haemolytic streptococcus is more common in psoriasis tonsils ...................................... 81�4.2.2� Psoriasis tonsils are histologically different ..................................................................... 82�4.2.3� Hypertrophic tonsils have enlarged lymphoid follicles .................................................... 83�4.2.4� The number of CD68+ macrophages correlates with follicle size .................................... 83�4.2.5� Psoriasis tonsils have a higher frequency of skin-homing (CLA+) T cells ...................... 84�4.2.6� Skin homing T cells in psoriasis tonsils express IL-23R. ................................................. 84�4.2.7� Other histological features ................................................................................................ 86�
4.3� Tonsillectomy improves psoriasis by decreasing the frequency of peptide-reactive T cells in
blood (paper III). .................................................................................................................... 87�4.3.1� Tonsillectomy improves psoriasis, evaluated by PASI score ........................................... 87�
14
4.3.2� IL-8 decreases in blood after tonsillectomy ...................................................................... 88�4.3.3� Peptide–specific CD8+ T cells in blood decrease following tonsillectomy ..................... 88�4.3.4� Peptide-specific CD8+ T cells in tonsils correlate with levels in blood ........................... 88�4.3.5� Improvement in PASI score is correlated to the frequency of circulating peptide-specific
CD8+ T cells . ................................................................................................................... 88�5� Discussion ........................................................................................................................... 89�
5.1� Tonsils and psoriasis .............................................................................................................. 96�5.2� Hypothetical scenarios ........................................................................................................... 98�5.3� The distinguishing factors of psoriasis patients ................................................................... 100�
6� Conclusions ...................................................................................................................... 103�7� Final conclusion ............................................................................................................... 105�8� References ........................................................................................................................ 107�9� Original Publications ....................................................................................................... 129�
15
List of abbreviations
AMP Anti-microbial peptides
ANOVA Analysis of variance
AP-1 Activation protein 1
APC Antigen presenting cell
APRF Acute phase response factor
B cell Lymphocyte that forms and matures in the bone marrow
Bcl-6 B cell lymphoma protein 6
BCR B cell receptor
Blimp-1 B lymphocyte induced maturation protein 1
BM Bone marrow
BSA Bovine serum albumin
CAMP Cathelicidin antimicrobial peptide
CARD caspase recruitment domain
CD Cluster of differentiation
CLA Cutaneous lymphocyte-associated antigen
CCL C-C chemokine ligand
CCR Cysteine-Cysteine chemokine receptor
CCL Cysteine-Cysteine-ligand
CXCL Cysteine-X-Cysteine chemokine ligand, X being a variable amino acid
CXCR Cysteine-X-Cysteine chemokine receptor, X being a variable amino acid
DAB 3,3’-diaminobenzidine
DC Dendritic cell
DEFB4 Defensin beta 4 gene
DNA Deoxyribonucleic acid
dsRNA Double stranded RNA
DZ Dark zone
EDTA Ethylenediaminetetraacetic acid
EGFR Epithelial growth factor receptor
ELISA Enzyme- linked immunosorbent assay
FACS Fluorescence activated cell sorting
FDC Follicular dendritic cell
FHC Fluorescent immunohistochemistry
Foxp3 Forkhead box 3
FPR2 G-protein-coupled formyl peptide receptor 2
GAS Group A ȕ-hemolytic streptococcus
GC Germinal centre
GC-DC Germinal centre dendritic cells
GI Gastrointestinal tract
GM-CSF Granulocyte macrophage colony stimulating factor
GP Gram positive
GPCR G protein coupled receptor
GWAS Genome wide association studies
hCAP-18 Human cathelicidin antimicrobial protein 18
H&E Haemotoxylin and eosin staining
HBD Human ȕ defensin
HD Human defensin
HLA Human leukocyte antigen
HNP Human neutrophil peptide
HT Hypertrophic tonsils
ICAM Intercellular adhesion molecule
ICOS Inducible costimulator
IDC Interdigitating dendritic cell
16
iDC Immature dendritic cell
iE-DAP J-D-glutamyl-meso-diaminopimetic acid
IFN Interferon
Ig Immunoglobulin
IHC Immunohistostaining
IL Interleukin (a class of cytokine)
iNOS Inducible nitric oxide synthase
IRF Interferon regulatory factor
IRAK IL-1R-associated kinase
iTreg Induced T regulatory cells
JAK-STAT Janus kinase - signal transducers and activators of transcription
K16 Keratin 16
KC Keratinocytes
L Litre
L Ligand (as part of abbreviations for chemokines)
LC Langerhans cell
LES-DC Lympho-epithelial symbiosis dendritic cell
LN Lymph node
LL-37 Leucine leucine-37, a cathelicidin
LRR leucine-rich repeat
LPS Lipopolysaccharide
LT Lymphotoxin
LTA Lipoteichoic acid
LTB4 Leukotriene B4
LZ Light zone
MAL MyD88-adapter-like protein
MALT Mucosal-associated lymphoid system
MAPK mitogen-activated protein kinase
M-CSF macrophage colony stimulating factor
MD-2 Myeloid differentiation factor 2
mDC Myeloid dendritic cell
MDP Muramyl dipeptide
MHC Major histocompatibility complex
ȝm micrometer
Min minutes
mm millimetre
MyD88 Myeloid differentiation factor 88
MZ Marginal zone
NALP3 NACHT-LRR-PYD containing protein 3
NET Neutrophil extracellular traps
NF Nuclear factor
NF-țB Nuclear factor țB
NLR Nod-like receptor
NO Nitric oxide
NOD nucleotide oligomerization domains
NK Natural killer cells
nTreg Natural T regulatory cells
p probability
PASI Psoriasis area severity index
PBMCs Peripheral blood mononuclear cell
PBS Phosphate-buffered saline
PD-1 Programmed death -1
pDC Plasmacytoid dendritic cell
PGE2 Prostaglandin E2
PGN Peptidoglycan
17
PGRP Peptidoglycan recognition protein
PRR Pattern recognition receptor
PSOR Psoriasis susceptibility locus
PST Psoriasis tonsil
PT Palatine tonsil
PYD Pyrin domain
R Receptor
RNA Ribonucleic acid
RNS Reactive nitrogen species
ROR Retinoic acid-related orphan receptor
ROS Reactive oxygen species
rRNA Ribosomal RNA
RT Recurrently infected tonsils
RTHT Recurrently infected tonsils with hypertrophy
S1-P Sphingosine-1 phosphate
SCID Severe combined immunodeficiency
SD Standard deviation
sIgA Secretory IgA
SpeC Streptococcal pyogenic exotoxin C
SLE Systemic lupus erythematosus
SLO Streptolysin O
STAT Signal transducer and activator of transcription
Tbet T box transcription factor
T cell lymphocyte that forms in the bone marrow but matures in the thymus
TBM Tingible body macrophage
Tc T cytotoxic cell, CD8+ T cell
TCM T central memory cell
TCR T cell receptor
TEM T effector memory cell
TFH T follicular helper cells
TFH-GC T follicular helper cells- Germinal center
TFH-EF T follicular helper cells- Extrafollicular
TGF-ß Transforming growth factor beta
Th Helper T cell
Th1 Type 1 T helper cell
Th2 Type 2 T helper cell
Th17 Type 17 helper T cell
Th22 Type 22 helper T cell
TIR Toll/IL-1 receptor
TJ Tight junction
TLR Toll-like receptor
TMC Tonsil mononuclear cell
TNF-D Tumor necrosis factor alpha
TRIF TIR-domain-containing adapter-inducing interferon-Eҏ TRAM TRIF-related adaptor molecule
TRM T resident memory cell
Treg T regulatory cells
TX group Tonsillectomized group
VBDRE Vitamin D responsive element
18
List of figures
Figure 1. The interactions between psoriasis and streptococcal throat infections.............. 47
Figure 2. The expression of neutrophil elastase in tonsil. .................................................. 74
Figure 3. LL-37 positive cells are observed migrate through some follicles. .................... 74
Figure 4. TMC stimulated with LL-37 for 1 hour influences the production of CCL5. .... 77
Figure 5. TMCs stimulated with LL-37 for 17 hours influences the
chemokine and cytokine production. .................................................................. 77
Figure 6. LL-37 stimulation for 1 hour has limited effect on the expression
of T cell surface receptors. .................................................................................. 78
Figure 7. Stimulation with LL-37 for 17 hours influenced T cell expression
of surface receptors. ............................................................................................ 79
Figure 8. Infected palatine tonsils are more common among psoriasis smokers
than non- smokers. .............................................................................................. 82
Figure 9. Various subpopulations of CD4+ T cells differ between RT and PST tonsils .. 85
Figure 10. CD8+ T cell expression of CXCR6 and CD69 differs between
RT and PST tonsils. .......................................................................................... 85
Figure 11. The tonsil squamous epithelium form structures similar to rete ridges. ........... 86
List of tables
Table 1. Five different dendritic cell phenotypes along with Langerhans cells
can be found in the palatine tonsil. ..................................................................... 36
Table 2. List of bacteria isolated from skin and tonsils. .................................................... 44
Table 3. Antibodies used for immunohistostaining (IHC) and
fluorescent immunohistochemistry (FHC). ......................................................... 66
Table 4. List of antibodies used for evaluation of cell surface receptor
expression and intracellular cytokine production, using FACS analysis.. .......... 70
Table 5. There is a positive association between streptococcal infections in
tonsils and throat swabs prior to tonsillectomy. .................................................. 82
19
List of papers
This thesis is based on the following original publications, which are referred to in the text by
their Roman numerals (I-V):
I. Sigurdardottir SL, Thorleifsdottir RH, Guzman AM, Gudmundsson GH, Valdimarsson
H, Johnston A. The anti-microbial peptide LL-37 modulates immune responses in the
palatine tonsils where it is exclusively expressed by neutrophils and a subset of
dendritic cells. Clinical Immunology, 142 (2): 139-149, 2012.
II. Sigurdardottir SL, Thorleifsdottir RH, Guzman AM, Valdimarsson H, Johnston A. The
association of sore throat and psoriasis may be explained by histologically distinct
tonsils and increased expression of skin homing molecules by tonsil T cells. Clinical
and experimental immunology, 174(1):139-51, 2013.
III. Thorleifsdottir RH, Sigurdardottir SL, Sigurgeirsson B, Olafsson JH, Sigurdsson MI,
Petersen H, Arnadottir S, Gudjonsson JE, Johnston A, Valdimarsson H. Improvement
of psoriasis after tonsillectomy is associated with a decrease in the frequency of
circulating T cells that recognize streptococcal determinants and homologous skin
determinants. Journal of Immunology, 188 (10):5160-5, 2012.
IV. Sigurdardottir SL, Thorleifsdottir RH, Valdimarsson H, Johnston A. The Role of the
Palatine Tonsils in the Pathogenesis and Treatment of Psoriasis. British Journal of
Dermatology, 168(2):237-42, 2013.
V. Johnston A, Sigurdardottir SL, Ryon JJ: Isolation of mononuclear cells from tonsillar
tissue. In: Current Protocols in Immunology. Chapter 7: Unit 7.8, 2009.
VI. Valdimarsson H, Thorleifsdottir RH, Sigurdardottir SL, Gudjonnson JE, Johnston A.
Psoriasis—as an autoimmune disease caused by molecular mimicry. Trends in
Immunology, 30(10): 494-501, 2009.
20
Declaration of contribution
All experiments done and published in papers I and II were planned and evaluated by Sigrún
Laufey Sigurðardóttir (SLS), Andrew Johnston (AJ) and Helgi Valdimarsson (HV) while
paper III was planned mainly by Ragna Hlín Þorleifsdóttir (RHT), AJ and HV with the
participation of SLS.
Paper I. All tonsils were processed, sliced and prepared by SLS. All immunohistology,
fluorescent staining, isolation of tonsil mononuclear cells, T cells and dendritic cells, in vitro
cell culturing and stimulation, FACS staining and analysis, ELISA measurements and data
analysis were done by SLS. AJ and AMG did all the RT-PCR analysis. The manuscript was
written by SLS and AJ with the final contribution of all authors.
Paper II. All tonsils were processed, sliced and prepared by SLS. All immunohistology,
histological measurements, FACS staining and FACS analysis of tonsil T cells were done by
SLS. Throat swabs of RT tonsils were done by SLS but throat swabs of PST tonsils were
equally done by RHT and SLS. FACS staining of blood T cells was equally done by RHT and
SLS. The manuscript was written by SLS and AJ with the final contribution of all authors.
Paper III. All tonsils were equally processed and prepared by SLS and RHT. The effects
of tonsillectomy on PASI score were evaluated by RHT. IL-8 levels in blood were measured
by AJ. The frequency of peptide specific T cells in blood and tonsils was done by RHT with
the assistance of SLS. The manuscript was written by RHT, AJ and HV with the contribution
of all authors to the final version of the paper.
Paper IV. The manuscript was written by SLS and AJ with the contribution of all authors
to the final version of the paper.
Paper V. The book chapter was written by SLS and AJ.
Paper VI. The manuscript was written by HV with the contribution of all authors to the
final version of the paper.
21
1 Introduction
1.1 The immune system The first physical barriers against infection are the epithelia, which need to be breached for a
successful infection to take place. Pathogens may gain access to the body through a number
of different routes such as the skin, lung, gastro-intestinal tract and eyes. The immune system
is comprised of a widespread network of specific cells in the blood, peripheral tissues and
lymphoid organs and maintains health by keeping infections at bay. It is commonly divided
into the innate immune system that is present in both animals and plants and the adaptive
immune system that is exclusively present in jawed vertebrates.
Infections and tissue damage stimulate cells to send out an alarm signals by releasing
alarmins and cytokines, which activate resident immune cells and recruit circulating immune
cells from the blood. Cytokines are small signaling molecules that are important for cellular
interactions. They are proteins, peptides or glycoproteins with inhibitory or stimulatory
effects on antibody production or inflammation. Interleukins (IL) are a common form of
cytokines that are released by neutrophils, macrophages, dendritic cells, T cells and B cells.
Chemokines are small chemotactic cytokines, ranging 8-11kDa in size that are critical in
directing cells to the appropriate compartments in tissue, such as luring peripheral leukocytes
to inflamed or injured areas (reviewed by1).
1.2 The innate immune system The innate immune system is independent of an earlier encounter of the pathogen and its fast
reaction stems from a circulating army of immune cells expressing germ line encoded pattern-
recognition receptors (PRRs) such as Toll-like receptors (TLR), nucleotide oligomerization
domains (NOD)-like receptors (NLR) or scavenger and complement receptors. They
recognize conserved microbial molecular patterns that distinguish the microbial from host
cells and importantly each receptor binds to a specific class of bacterial molecule thus
significantly increasing the capacity of pathogen recognition. Binding to PRRs initiates an
alarm-signalling pathway, leading to inflammatory responses through the release of pro-
inflammatory cytokines and phagocytosis of the microbe and thereby its killing and digestion.
Activation of TLRs is important for the regulation of the immune response as it induces
inflammatory responses and activates the adaptive immune response (reviewed by2).
The structural characteristics of TLR are uniform with extracellular leucin-rich repeat
(LRR) motifs, a transmembrane helix and cytoplasmic Toll/IL-1 Receptor (TIR) domain3.
22
Activation of the TLR leads to their dimerization and subsequent recruitment and binding of
the cytoplasmic adaptor protein myeloid differentiation factor 88 (MyD88) to its cytoplasmic
domain. Thereafter the IL-1R-associated kinase 4 (IRAK-4) and IRAK-1 as well as the
MyD88-adapter-like protein (MAL), that is necessary for TLR 2 and TLR4 signaling, are
recruited and bind to the adaptor molecule. This activates signaling cascades further
downstream resulting in the activation of transcription factors such as nuclear factor (NF)-ț%
and the activator protein (AP)-1 that mediate production of proinflammatory cytokines,
chemokines and antimicrobial peptides (AMP). TLR 7 and TLR9 can also activate the
transcription factor IFN-regulatory factor (IRF)-7 resulting in the production of type 1
interferon (IFN-Į and ȕ) (reviewed by2).
The signaling pathway of TLR3 is independent of MyD88 but dependent on the TIR-
domain-containing adapter-inducing interferon-E�(TRIF) and the TRIF-related adaptor
molecule (TRAM). They activate other downstream signaling cascades resulting in the
activation of the transcription factor IRF3 and type 1 IFN production or (NF)-ț% and
inflammatory cytokines production. Interestingly, TLR4 is able to use both signaling
pathways (reviewed by2).
The ten TLRs identified in humans are either surface bound (TLR1, 2, 4, 5 and 6) or
intracellular in endosomes (TLR3, 7, 8, 9 and 10). TLR2 forms a heterodimer with TLR1 or
TLR6 and recognizes bacterial lipoprotein. TLR4 forms a heterodimer with myeloid
differentiation factor 2 (MD-2) that recognises lipopolysaccharide (LPS), a structural
component of the outer membrane protein of Gram-negative bacteria4. TLR3, 7 and 9 are
activated by motifs in microbial nucleic acids and TLR5 recognizes conserved patterns in
bacterial flagellin (reviewed by2). TLR10 forms a heterodimers with TLR2 in the phagosomes
where it recognises tri-acylated lipopeptides but the function of TLR10 is thought to be
limited to a sensory role5.
Nod-like receptors (NLR), are found within the cytosol6. They have a ligand sensing LLR
domain, a NOD containing domain (NACHT) and a signal-mediating domain that is
composed of caspase recruitment and activation domain (CARD) or pyrin domains (PYD)
(reviewed by6). Among the best-studied NOD proteins are the NOD1 and NOD2 that detect
the peptidoglycan components J-D-glutamyl-meso-diaminopimelic acid (iE-DAP)7 and
muramyl dipeptide (MDP) respectively8. Binding to NLR results in stimulation of signalling
cascades resulting in the activation of NF-kB or AP-1 and the production of pro-inflammatory
cytokines and AMPs. A mutation in the gene coding for NOD2 (CARD15), causes a loss-of-
23
function mutation in NOD2 that is associated with Crohn’s disease9, 10
and results in
disruption of intestinal defences11
.
NLRs can form large cytoplasmic protein complexes with the cysteine protease caspase-1.
They are linked together with the adaptor protein apoptosis associated speck-like protein
containing a CARD (ASC). The whole complex is called inflammasome and is important for
the processing of the inactive pro-forms of the inflammatory cytokines IL-1E and IL-18 into
their highly inflammatory mediating form. The best-studied inflammasome is the NALP3
(NACHT-LRR-PYD containing protein 3) that is for example activated by a number of
danger signals, including Listeria monocytogeneses12 and Staphylococcus aureus infections13
.
Neutrophils are the first cells to infiltrate a damaged or infected tissue14
where they sense
pathogens using all TLRs except TLR315
. They are short-lived and particularly efficient at
destroying invading pathogens using their respiratory burst and degranulation where the
intracellular granules, containing damaging enzymes, AMP, harmful reactive oxygen species
(ROS) and reactive nitrogen species (RNS), fuse with the phagosome containing the engulfed
pathogen. At death, neutrophils release extracellular traps (NETs) containing DNA, histone
proteins and antimicrobial enzymes that have been shown to efficiently kill the bacteria,
including Staphylococcus aureus16. Neutrophils can also synthesise the pro-inflammatory
prostanoids, prostaglandin E2 (PGE2) and leukotriene B4 (LTB4), and are highly efficient in
recruiting other immune cells from the blood, including monocytes14
.
Once in the peripheral tissues monocytes can differentiate into macrophages, which are
very proficient phagocytes, clearing debris and dead cells and engulfing bacteria using PRRs
and scavenger receptors. They also release ROS and RNS and are a major source of pro-
inflammatory cytokines such as interleukin (IL)-1E and tumour necrosis factor (TNF)-D as
well as chemokines, prostanoids and AMP. Macrophages are also potent antigen presenting
cells (APC), and as such may present fragments of ingested material obtained from the
affected site to the cells of the adaptive immune system in the lymph nodes (reviewed by17
).
They influence the immune response by stimulating Th1 responses against intracellular
infections and tumors (M1 macrophages) or Th2 responses against extracellular microbes and
mediating immune regulation (M2 macrophages)(reviewed by18
). The interactions between
tissue cells and resident and infiltrating immune cells are co-ordinated using cytokines and
chemokines for cell-cell communication, activation and recruitment. Other innate immune
cells are the dendritic cells (DC), mast cells, natural killer cells, natural killer T cells and JGT
cells, most of which are out of the scope of the current discussion. If the innate immune
system does not manage to overcome the infection or the pathogen is of an earlier encounter,
the adaptive immune system becomes recruited and activated.
24
1.3 The adaptive immune system Bone marrow (BM) derived monocytes differentiate into immature DCs (iDC) that reside in
the peripheral tissue. They have high endocytic activity that enables them to constantly
sample their microenvironment, searching for pathogens with their PRRs. The endocytosed
material is broken down and its peptide antigens displayed on the cell surface in the context of
major histocompatibility complex (MHC) molecules, which in humans are known as human
leukocyte antigens (HLA). After pathogen encounter the DC matures and shuts off its
endocytic activity while massively upregulating the expression of its HLA-peptide complexes
and a host of surface molecules such as the co-stimulatory molecules CD80, CD86-binding
CD28 on T cells and CD40-binding CD40L on T cells that are necessary for T and B cell
priming. It also upregulates CCR7 that facilitates its homing through the lymphatic vessels to
the tissue-draining lymph node (LN) where CCR7 binds to the chemokines CCL19 or CCL21
found in the lymphatics and the LN. The DC localizes within the T cell areas and presents its
antigens to naïve T cells. In this respect, DC link together the innate and the adaptive immune
system (reviewed by19
).
T cell precursors originate in the BM and migrate to the thymus where maturation and
rearrangement of their DE or JG T cell receptor genes (TCR) occurs. TCR is a disulfide-linked
heterodimer with a variable domain that binds the antigen presented by APCs and a constant
region inserted to the cell membrane. Those T cells that either fail to bind or bind too strongly
are negatively selected (die)20
while cells with intermediate affinity are positively selected and
differentiate into either CD4+ or CD8+ T cells that recognize peptide antigens in MHC class I
and MHC class II respectively. This selective process aids in the recognition and elimination
of potentially harmful self-reactive T cells (reviewed by21
). T cells are thought to be cross-
reactive, which means that they can react to more than one particular peptide-MHC ligand
(reviewed by22
).
Mature naïve T cells express the L-selectin (CD62L) and both CXCR4 and CCR7, which
enables them to leave thymus and migrate to the T cell zones within the secondary lymphoid
tissues such as LNs or spleen23
following a CXCL12 gradient, the ligand for CXCR4
(reviewed by24
). They circulate until they find an APC displaying the peptide fragment for
which they were selected, in context of an appropriate HLA molecule. Full activation of the T
cells takes 4-5 days and through TCR binding (signal 1), activation by co-stimulatory
molecules (signal 2) and CD40L-CD40 binding or PRR binding or cytokine stimulation
(signal 3); they differentiate into short lived T effector cells that either migrate to an inflamed
25
area or become TFH assisting in the germinal center (GC) reaction (reviewed by25
). Anergy or
cell death can result from stimulation without the co-stimulatory molecules.
The chemokines and cytokines present during the priming of the naïve T cell influence
both its phenotype and the homing receptors it expresses after differentiating into T effector
cell. Competition between CCR7 ligands and sphingosine-1 phosphate (S1P) inhibits further
migration of the cell and in order to leave the LN and migrate to tissue, desensitization of
CCR7 to its ligands and re-sensitization to S1P must occur. The S1P then binds its receptor
S1P1 on the T cell surface and mediates its egress (reviewed by24
). The T cells also increase
the expression of various homing molecules such as CCR9 or CLA26
. For successful
infiltration and diapedesis of the cell, complex interactions occur between adhesion
molecules, selectins, integrins, and the homing receptors. The selectins mediate reversible
binding that causes a rolling motion while activated integrin secure firm binding and the
homing receptor binds to the chemokines forming a chemokine gradient to the site of interest
(reviewed by24
). The T cells undergo clonal expansion and complex cellular interaction occurs
with the modulation of the immune response and clearance of the infection or stimulation of a
wound healing response. After antigen elimination, T cells are eliminated although a few T
cells become memory T cells.
Chemokine receptors can also license a cell to localize in particular tissue area. In this
respect, CCR7 and CXCR4 allow T cells to accumulate within the T cell zones in the tonsil
and lymph nodes27, 28
and CXCR5 draws B cells to the GC29
. Different homing receptors have
been used to characterise the various T cell phenotypes. In this respect, Th1 cells typically
express the homing receptors CCR5 and CXCR3, Th2 cells express CCR4 and CCR8, Th17
cells express CCR6 and T follicular helper cells CXCR5. Expression of homing receptors on
T regulatory cells depends on the cytokine milieu at differentiation (reviewed by24
).
1.3.1 T cell phenotypes Depending on the type of pathogen and the tissue where the encounter took place, the DC
secretes particular cytokines to assist the T cell and B cell responses. The DC-T cell
interaction can result in Th1, Th2, Th9, Th17, Th22 effector CD4+ T cells or types of T
regulatory cells. Each cell type has its unique role and feature and the decision is dependent
on the combination of chemokines and cytokines in its microenvironment30
.
Naïve T cells acquire a Th1 phenotype in the presence of IFN-Ȗ and IL-12 derived from
DC activated by PRRs (reviewed by31
). They are characterized by a massive IFN-Ȗ production
that activates macrophages as well as the immune response against intracellular microbes32
.
26
They also secrete lymphotoxin Į (LTĮ), IL-10 and IL-2 (reviewed by30
), which is important
for the development of both CD4+33
and CD8+ memory T cells34
. Th1 cells utilize the T box
transcription factor (T-bet), which is critical along with STAT4 (signal transducer and
activator of transcription 4) for IFN-Ȗ production and maintenance of the Th1 phenotype
while suppressing Th2 responses35
. Th1 cells are part of the cell-mediated immunity and can
participate in autoimmunity.
The Th2 phenotype is induced in the presence of IL-4 and IL-2 with activation of the IL-4
induced transcription factor STAT6 and GATA336
or the IL-2 induced STAT537
. They secrete
IL-4, IL-5, IL-10, IL-13, IL-25 (IL-17E) and amphiregulin and are active against extracellular
parasites and important in asthma and allergic diseases (reviewed by30
). Th2 cells are
important for the humoral immunity by stimulating antibody production, as IL-4 is an
important feedback cytokine for Th2 class differentiation and IgE class switching in B cells38
.
IL-5 recruits eosinophils39
while IL-10 suppresses Th1 proliferation and DC function. IL-13 is
active against extra-and intra cellular parasites and contributes to asthmatic responses40
. IL-25
is produced by lung epithelial cells in response to allergens41
and acts as amplification factor
for Th2 responses, mucus secretion and recruitment of eosinophils while amphiregulin
stimulates epithelial cell proliferation (reviewed by30
). Until recently, IL-9 secretion was
thought to be entirely from Th2 cells but recently, a Th9 phenotype has been characterized.
The Th9 phenotype is characterised by IL-9, IL-21 and IL-10 production, activation of
the transcription factor PU.142
and expression of the IL-25 receptor chain IL-17RB in greater
abundance than the other T cell phenotypes43
. IL-9 induces hematopoiesis, B cell and mast
cell development and function and T cell maturation, in particular CD4+ T cells (reviewed
by44
). Th9 cells are more common in peripheral blood of allergic than healthy individuals.
They stimulate mucin secretion41
and activate and recruit mast cells in the lungs45
while
protecting against intestinal parasites46
. They are present in both normal and inflamed skin
where they appear to promote inflammation47
48
but may be important in stage IV melanoma
where they are infrequent compared to healthy skin47
. Stimulation with TGF�E can induce IL-
9 production by Th1749
and Th2 cells and with IL-4 drive the differentiation of Th9 cells50
.
Furthermore, Th17 cells can co-express IL-17A and IL-9 after long term in vitro culture49
while the Th17 related cytokine IL-23 can inhibit the IL-9 secretion51
. Th17 derived IL-9
contributes to the inflammatory response but the effects on T regulatory cell development are
conflicting52
.
27
Th17 phenotype is induced in the presence of IL-1E53 54
, TGFE54, IL-6, IL-21 and IL-23
55
with Il-6 and IL-1E being the key cytokines54
. IL-6 inhibits activation of T regulatory cells
and TGFȕ suppresses Th1 response while stimulating IL-17/IL-22 response56
. IL-23 is
necessary to sustain the Th17 phenotype 57
by affecting their differentiation58
and survival59
.
Activated Th17 cells, characteristically express IL-17A, IL-17F, IL-2260
, IL-2660
, CCL2061
and the IL-23 receptor (IL-23R). They utilize the transcription factors retinoic acid related
orphan receptor (ROR)Jt, RORD, STAT3 and interferon regulatory factor (IRF) 4 (reviewed
by30
). They are active against extracellular bacteria and fungi, stimulate epithelial AMP
production61, 62
and are associated with various autoimmune diseases63
. IL-17A and IL-17F
induce the production of pro-inflammatory cytokines and chemokines64-66
and increase the
recruitment of neutrophils67, 68
. IL17A induces production of PGE265
, NO69
and matrix
metalloproteinases64
. IL-21 increases differentiation of Th17 cells57
, promotes B cell
differentiation70
and IgG class switching71
. All IL-17+ peripheral blood T cells express
CCR672
, which along with CCR4 is important for their migration to epithelia54
.
Th22 cells were originally identified as skin-homing memory T cells that produced IL-22
independently from IFNJ, IL-4 and IL-1773, 74
. These cells express the chemokine receptors
CCR4, CCR6 and CCR1074
but exhibit a low expression of the transcription factors RORJt
and T-bet73
. The Th22 phenotype can be induced by IL-6 and TGF-E secreting plasmacytoid
DC73
and their proliferation is dependent on IL-2362
. IL-22 is a member of the IL-10 family of
cytokines and the IL-22 receptor is composed of IL-22R1 and IL-10R2 heterodimer75
and
confined to non-hematopoietic cells such as epithelial cells76
. IL-22 is important for epithelial
defenses as it induces AMP production62
, inhibits keratinocyte differentiation and increases
cellular mobility through matrix metalloproteinases77
. IL-22 induces acanthosis (epidermal
hyperplasia) by stimulating keratinocyte proliferation and inflammation in dermis by STAT3
activation78
.
T regulatory cells are of two main types: thymus derived CD4+CD25+ Treg (nTreg) and
peripheral induced Treg (iTreg: Tr1 and Th3) that stem from primed naïve CD4+CD25- cells.
The Treg express high levels of CD25, the IL-2 receptor. They are important in maintaining
peripheral tolerance, preventing autoimmune disease and limiting chronic inflammatory
disease but they can also have harmful effects by limiting anti-tumor responses. The
transcription factor forkhead box P3 (FOXP3) is required for their maintenance, function and
development79, 80
and overexpression in conventional T cells induces Treg phenotype49
. Treg
phenotype is induced in the presence of TGF-E and IL-2, where IL-2 activation of STAT5 is
28
important for Foxp3 induction81
. Furthermore, a continued TGF-E expression is necessary to
maintain the phenotype82
. The suppressive effects of Treg on T effector cells are mediated
through TGF-E, IL-10 and IL-35. Another subgroup of Treg is located within the gut and at
inflamed sites and is identified by CD103 (DE integrin) expression, which is inducible in
iTreg cells83
.
TFH or T follicular helper cells have a high expression of CD40L, ICOS (inducible
costimulator) and PD-1 (programmed death 1) that are important for interaction with B cells.
They produce the cytokines IL-21, CXCL13 and IL-4. The transcription factor Bcl-6 (B cell
lymphoma protein 6) is the main inducer of the TFH phenotype84
. It is stimulated by IL-12 that
leads to the upregulation of CXCR5 and ICOS85
and downregulation of the T zone homing
molecule CCR786
87, 88
. Tfh differentiation is inhibited by the transcription factor Blimp-1
(Blimp-1) 89
. Tfh can be divided into two groups90
: TFH -GC and TFH -EF.
TFH-GC are located in the germinal centers. They are CD4+CXCR5
+CD57
+
CD45RO+CCR7
-CD69
+ and interact with centrocytes but not naïve B cells
90. They
are
influenced by cytokines as IL-10 induces their activity while IFNJ�and TGF-B1 reduce it.
Interestingly, TFH-GC secrete CXCL13 and may thereby influence migration into the germinal
centers91
.
The other group is the TFH-EF that is extrafollicular90
. They are
CXCR5low
BCL6low
IL7Rlow
CD4+ with low levels of ICOS90
. They provide help to naïve and
memory B cells and participate in the initial phase of the germinal center reaction.
Interestingly, TFH-EF produce higher levels of IL-10 and IL-21 than TFH-GC but are unable to
interact with GC-B cells in this state as they induce apoptosis through Fas-FasL interactions
90. Next they are thought to migrate into the LZ and participate in the differentiation of the
centrocytes, as they induce the ICOS and PD-1 levels in concordance to TFH-GC after
stimulation with naïve B cells.90
.
A potential third group is the peripheral blood CXCR5+ TCM with high ICOS levels. They
express high levels of CXCL13 and migrate to the follicles and assist in the GC reaction
where they induce immunoglobulin secretion and plasma cell differentiation92
. They are
thought to be important for secondary humoral immune responses.
29
1.3.2 T memory cells Naïve T cells express protein tyrosine phosphatase CD45RA, which is altered during
differentiation so that T effector or memory cells express the CD45RO isoform that facilitate
recognition of antigen by binding to the TCR93
. T memory cells can be divided into three
groups: Central memory T cells (TCM), effector memory T cells (TEM) and tissue resident
memory cells (TRM). TCM have a high expression of CCR7 and CD62L that allow them to
migrate between lymphoid tissues. They have low effector function but differentiate into
effector T cells upon antigen stimulation94
. They are important for long lasting memory as
they have high proliferation activity and are more independent from stimulation by co-
stimulatory molecules. TEM have a low expression of CCR7 and CD62L while expression of
chemokine receptors that bind to inflammatory chemokines, such as CCR5 and CXCR3, is
induced. Some TEM have increased CLA expression94
. TEM are important for immediate
immune response as they retain their effector function and are faster acting than the TCM,
which first need to differentiate and proliferate. Finally TRM are in fact TEM that are resident in
the affected area as they do not exit the non-lymphoid tissue (reviewed by95
). They express
CD103 and CD69, which are important for their resident nature as CD69 prevents
sphingosine-1 phosphate (S1P)-mediated egress from tissue96
and CD103/E7 integrin enforces
maintenance in epithelia via E-cadherin binding. TRM are much more efficient than TCM in
providing long-term protection against re-infection97
.
1.4 The skin immune system The skin is a barrier against the external environment, protects against harmful pathogens and
toxins and participates in the regulation of the body’s temperature and immune response98
. It
is heterogeneous in regard to its thickness, hair and gland density, humidity, acidity and
temperature99
. In this regard, the forearms are cool, desiccated areas while armpits are warm
and humid. These physiological factors influence the composition of the commensal flora that
occupies the skin after birth and protect the host from colonization of harmful bacteria100
.
The skin consists of three main layers, the four-layered epidermis, the dermis and the
subcutaneous fat. The outermost layer of the epidermis, the stratum corneum contains
terminally differentiated keratinocyte, the corneocytes that are enlarged, flattened, cornified
cells without nuclei and organelles that form a watertight crosslinked barrier by connecting
through the tight junction proteins, e.g. claudin. It is continually shredded and replaced by
new keratinocytes that originate from the stratum basal as the basal cells divide and the new
cells migrate and differentiate on the way to the stratum corneum98
.
30
Epidermal keratinocytes recognize microbiota through TLR101
102
, NLR103
and mannose
receptors104
on their surface that provides a fast response with secretion of AMP, such as
human beta defensin-2 (HBD-2). They also express chemokine receptors and release various
chemokines, such as CCL20 and CXCL9, CXCL10 and CXCL11 that attract effector T cells
or CXCL1 and CXCL8 that attract neutrophils102
. They also secrete cytokines such as the
pro-inflammatory cytokine IL-1E and IL-17 that induces HBD-262
. Langerhans cells (LC) are
APC located within the lower epidermis where they form part of the immune surveillance
team105
. LC penetrate keratinocyte tight junctions by lengthening their dendrites and thereby
scan the upper layer of dermis as they form dens networks leading to the uptake of antigens in
co-operation with keratinocytes105
. Epidermis contains melanocytes that produce melanin,
iDC and rarely CD8+ T cells while the dermis has both iDC and DC of myeloid origin
(CD11c+ DC, mDC), macrophages, pDC, NKT cells, mast cells, fibroblasts, JG T cells
(reviewed by106
) and CD103+ DCs107
.
CD4+ helper T cells are resident in skin108
. They are mostly TEM mediating Th1 response.
They express high levels of the skin homing molecules cutaneous lymphocyte associated
antigen (CLA), which binds E-selectin on the surface of endothelial cells, and CCR4 and
CCR6. Most of the cells co-express all three receptors108
. Around 80% of the T cells are TRM
cells and around 90% of CLA+ T cells are resident cells108
. CCR4 homes to its ligands
CCL17 or CCL22 that allows the cell to adhere and migrate to the skin through the high
endothelial venules while CCR6 homes to the skin through the high endothelial venules in
response to CCL20109. CCR8 has recently been identified as a skin homing molecule
110 and
may regulate localization of TRM surveillance cells in peripheral tissue. CCL1 is the ligand for
CCR8, which is most commonly produced by LC111
. Epidermal keratinocytes induce CCR8
expression by naïve T cells111
that correlates positively with CLA but negatively with CCR4
production and expression is inhibited by IL-12 111
. Some T cells express CXCR6 with
CCR8108
and a few are TRM Treg108
. Throughout the dermis are both nerves and blood and
lymphatic vessels where cells enter or leave the skin106
. Some of the TCM migrate from skin to
draining LN, re-enter the circulation and migrate to distal LNs or cutaneous sites of
nonspecific inflammation112
. These newly identified cells have a new phenotype,
CCR7+CD62L+CD69-CD103+/-CD62E+, with upregulation of CD40L and IL-2 secretion
after stimulation112
.
31
1.5 Psoriasis Psoriasis is a chronic inflammatory skin disease. The most common form, psoriasis vulgaris,
affects around 2% of individuals of both sexes 113
, occurs early in life (age 15-30) with 10-
40% of the individuals developing psoriatic arthritis that becomes severe and deforming for
about 5% of the patients114
with significant comorbidity 115
. Psoriasis has very negative
impact on quality of life116
due to the obvious cutaneous symptoms and discomfort.
1.5.1 The clinical manifestations of psoriasis The clinical manifestations are very distinctive with thick, erythematous, scaly plaques that
are commonly located on the knees, elbows and scalp although they can occur throughout the
body114
. The keratin composition of involved psoriatic epidermis is abnormal with keratins 6,
16, and 17 increased in their expression, compared with normal skin where keratins 5 and 14
are expressed by basal keratinocytes and keratins 1 and 10 expressed subra-basally117
. The
thickening of the epidermis is caused by hyperproliferaton of keratinocytes that is thought to
be driven by infiltrating T cells118, 119
. The keratinocytes present in the outermost layer of the
skin, the stratum corneum, show abnormal maturation with parakeratosis (the retention of the
cell nuclei) and form a disrupted outer barrier117
. The shedding of the outermost layer is
obvious with large visual flakes that negatively influence the quality of life of the patient. A
distinctive histological feature are the rete ridges present in the thickened epidermis that form
elongated projections into the dermis. The plaques are erythematous due to angiogenesis and
widening of the vessel diameter. Furthermore, the psoriatic plaques are heavily infiltrated
with various inflammatory cells.
1.5.2 The psoriatic skin Psoriasis skin has a markedly different cell composition compared to healthy skin. Increased
numbers of T cells are seen with CD8+
T cells infiltrating the epidermis and dermis while
CD4+ T cells dominate in the dermis
120, 121. CD11c
+ DCs are found in the lower epidermis,
neutrophils accumulate in the stratum corneum and LC’s are clustered in the spinous layer of
the thickened epidermis. Although initially attributed to keratinocyte (KC) dysregulation,
psoriasis is now considered a T cell mediated skin disease122-124
caused by interactions
between KC, APC and T cells. In this respect, blocking T cell entry into epidermis prevents
hyperplasia in a mouse model of psoriasis125
and treatments that target T cells are effective for
patients123, 126
. Cells of the innate immunity are also important and neutrophils and mast cells
that release IL-17 and other inflammatory mediators, are the most common IL-17+ cells in the
dermis127
and DC and macrophages appear to be the dominant IL-22 producing cell type128
.
32
Myeloid DC can also stimulate Th1/Tc1 (T1)- and Th17/Tc17 (T17)-mediated responses and
the differentiation of IFNJ+IL-17
+ T cells
129. In fact, psoriasis skin lesions contains at least
two types of mDC; cells similar to mDC in healthy skin and cells that are immature and more
inflammatory and numerous than in healthy skin129
. They are along with macrophages the
major source of TNFD and inducible nitric oxide synthase (iNOS) in psoriasis130
The infiltrated T cells in the psoriasis lesions are CD3+CD45RO+ memory cells131
expressing the activation markers CD25 and CD69121
. They have undergone clonal expansion
as observed by the rearrangement of their TCR132-134
and reflect a high CD4:CD8 ratio131
.
Both CD4+ and CD8+ T cells in skin are characterized by the expression of the skin homing
molecules CLA135, 136
, CCR4, CCR10, CCR6 (reviewed in109
) and CCR8110
, and CLA is
considered important as it is detectable very early in the plaque formation137
.
Psoriasis appears to be a mixed T1/T17 disease, as recent studies indicate that both IFN-Ȗ
and IL-17/IL-22-producing CD8+ T cells play a key role in the formation of psoriasis
plaques120
(paper III). They form the greater proportion in the epidermis of psoriasis lesions138
139
138 while Th17 cells are primarily located in the dermis and peripheral blood
140. The
psoriasis lesions contain cells that are typically around 20% IFN-J-producing T1131
and 40%
IL-17A-producing T17 cells along with cells expressing and co-expressing other signature
cytokines such as TNF-D�and IL-22108
128
where the IL-17 production138
and TCR activity128
was dependent on TNF-D. T1/T17 cells are often found co-localized in the plaques140
120
and
CD4+ and CD8+T cells co-expressing IFN-Ȗ and IL-17 have also been identified141
.
CD4+IL17+Foxp3+ T cells have been observed in severe psoriasis skin lesions142
and
interestingly, T reg from patients with severe psoriasis can differentiate into IL-17A secreting
cells in the presence of IL-23142
.
T22 and Tc17 cells are more common in psoriasis than in healthy skin128
. Furthermore,
double positive cells are found140
with around 17% being CD4+IL-22+IL-17
+ T cells, 10% of
CD8+IL-22
+IL-17
+ phenotype
141 and 21% being CD8+ IL17+IFN-Ȗ + cells. Tc17 cells are
resistant to Treg suppression138
and a negative correlation exists between Treg: Tc17 cells and
PASI score, thus reflecting the importance of these cells141
. T17 cells that co-express either
CCR6 and CXCR672
or CXCR354
have been identified in skin and CD8+CXCR6+ T cells are
increased in the peripheral blood and skin of psoriasis patients. They appear to have skin
homing potential as shown by skin graft experiments in severe combined immunodeficiency
(SCID) mice143
. Its sole ligand CXCL16 is upregulated by keratinocytes, DC and monocytes
in the psoriasis skin143
.
33
The T1/T17 cytokines have various functions. IFN-J upregulates a collection of genes
involved in the immune response. IL-17A upregulates keratinocyte genes involved in
epidermal barrier function and the activation and migration of neutrophils to an inflamed area
while upregulating various cytokines, chemokines and metalloproteinases144
. Therapy with an
IL-17 antibody (ixekizumab) dose-dependently improves psoriasis lesions145
. IL-22 causes
massive tissue immune response, as expression of IL-22R is limited to epithelial cells61
. This
stimulates both keratinocyte hyperproliferation146
and AMP production62
. IL-23 is necessary
for proliferation and survival of T17 cells144
and the gene coding for its receptor is associated
with increased risk of psoriasis147
. However, none of these cytokines are present or function
alone and they form strong synergistic interactions as part of a complex cytokine network that
drives the tissue changes seen in psoriasis lesions.148
. In this respect, T1 and T17 immune
responses appear to interact as IL-17 and IFN-J�synergistically upregulate keratinocyte
production of pro-inflammatory cytokines148
and the antimicrobial peptide HBD-2120
.
Furthermore, IFN-Ȗ produced by T1 cells stimulates mDC to secrete IL-1ȕ and IL-23 that
induce differentiation of T17 cells. IFN-Ȗ and IL-17 stimulate APC120
and keratinocytes149
respectively, to produce CCL20, which induces T17 cell migration to the area due to their
CCR6 expression. In fact, all peripheral blood T17 cells are CCR6+72
and the expression is
particularly high for CLA+CD4+ memory T cells in the peripheral blood of psoriasis
patients150
. Furthermore, correlation has been observed between CLA+CD8+ and
CD8+CD25+ circulating T cells and disease severity (psoriasis area severity index, PASI)135
.
Interestingly, CCR6, CCL20150
, IL-17R151
, IL-23R152
and IL-23153
are all overexpressed in
psoriasis plaques making them particularly susceptible to the T17 skewed cytokines.
Furthermore CCL20 produced by IL-17+ T cells60
can sustain inflammatory conditions by
promoting migration and proliferation.
TNFD�is produced by the Th1 cells154
and together with IL-17 synergistically upregulates
various important genes for psoriasis pathology155, 156
. Interestingly, together these cytokines
activate components of the innate immune system by stimulating keratinocyte production of
HBD-3 and 4 and the chemokine CXCL8 and CXCL1, which attract innate immune cells to
the area155
156
. They also stimulate growth and pigment production by melanocytes, possibly
reflecting the observed changes in pigmentation in psoriasis plaques 156
.
34
1.5.3 Psoriasis triggers Psoriasis is a complex genetic disease
157 with a high heritability of 60-90%
158. Population
studies indicate that first degree relatives are more likely to get the disease114
. Furthermore the
concordance is higher among monozygotic than dizygotic twins with an observed difference
in frequency in northern European individuals (60-72% and 15-30% respectively)159, 160
than
individuals in Australians (35% versus 12% respectively)161
. Despite recent advances in
technology, it has been postulated that our current knowledge only covers around 25% of the
disease heritability162
. Psoriasis susceptibility loci identified in GWAS (genome-wide
association study), now number more than 40 and have been linked to genes coding elements
of both the innate163-167
and the adaptive immune system147, 163, 167
as well as to proteins
important for skin barrier function (reviewed by168
). The identified genes have various
important roles on a cellular and physiological level with some influencing the inflammatory
response and mediating the interactions between cells165, 168
.
Environmental factors such as trauma114
, stress114
, smoking169
and rapid withdrawal from
immunosuppressive drugs (such as steroids or TNF-Į inhibitors170
171
) play a role in the
elicitation of psoriasis. Obesity can increase the severity of the disease172
. Throat infections
by ȕ-haemolytic streptococci is the most convincing trigger and has been associated with both
the initiation and exacerbation of psoriasis (paper IV). This interaction is not well
characterized but psoriasis patients appear more vulnerable to throat infections than their
aged-matched household controls173
.
1.6 The palatine tonsils The palatine tonsils are secondary lymphoid organs located on the inside of the throat where
they are frequently exposed to invading pathogens and foreign antigens. They can be found
among all mammals, except rodents, and their number, morphology and location differ
between species174, 175
. The paired palatine tonsils in humans are important for the immune
defence of the mucosal area of the mouth and throat as they form a part of Waldeyer´s ring,
named after Heinrich Wilhelm Gottfried von Waldeyer-Hartz (b.1836- d.1921), the anatomist
who first described it 176
. Also included are the nasopharyngeal tonsil (adenoid), the paired
tubal tonsils and the lingual tonsil. Waldeyer´s ring belongs to the mucosa associated lymph
system (MALT) that protects the opening of both the respiratory and the digestive tracts176
.
The palatine tonsils are most active between the age of three and ten in humans177
after which
involution begins with proliferation of fibrous tissue178
.
35
1.6.1 Histological features The palatine tonsils are only partially capsulated, unlike lymph nodes or spleen. They are
coated with a thick stratified non-keratinized avascular squamous epithelium that forms a
barrier against the outer surface area 176
. No afferent lymphatics are present in the tonsil thus
the blood vessels serve as the entry site for lymphocytes176
. The palatine tonsils are packed
with cells and can contain up to 1x109 lymphoid cells of which B cells are the most prominent
followed by CD3+ T cells that are largely CD4+ T helper cells179
. Age appears to influence
the cellular composition within the tonsil as the percentage of CD4+ T helper cells increases
with age until the individual reaches the middle age (35 years) while CD19+ B cells and
CD8+ T cells become less frequent 179
. Interestingly, lymphoid infiltration in tonsils begins
as early as the 14-16th
week of development 175
The surface epithelium invaginates into tunnels (crypts) which branch throughout the
tissue where some interconnect deep within it. The crypts are lined by a discontinuous
reticulated epithelium that is only one cell thick in parts, the lymphoepithelium 176
. The
crypts increase the surface area of the tonsils significantly as each human tonsil can contain
from ten up to thirty crypts176
. Tonsils of other mammals differ in having fewer crypts where
for example cows have two crypts and rabbits only one174 175
. The reticulated crypt
epithelium is unique with epithelial cells, intra-epithelial lymphocytes, stroma cells,
macrophages, neutrophils and DCs clustered together. This optimizes antigen sampling
where APC transports the antigen to the extrafollicular T cell areas and later to the B cell
follicles180
where it induces an adaptive immune response. The crypts are the main entry side
for pathogens and other orally transmitted antigens and get gradually filled with degenerated
cells and debris which increase during each infection leading to an ideal habitat for anaerobic
bacteria181
.
The existence of specialized antigen and microbe endocytotic cells, the M cells, within the
crypt epithelium has been debated182, 183
particularly as the reticulated epithelium functions in
a similar way. In the sub-epithelial tissue there are numerous primary follicles which are
present as early as the 16th
week of gestation180
and secondary lymphoid follicles184
with
germinal centres (GC) which appear shortly after birth180
.
36
1.6.2 Secondary lymphoid follicles Tonsils contain numerous secondary lymphoid follicles with germinal centres (GC) that form
from primary follicles around six days after antigen stimulation. The follicle is typically
divided into three areas by their appearance after H&E staining: the dark zone (DZ) and the
light zone (LZ) that constitute the GC and then the mantle zone (MZ), each with their own
cell composition. These areas are functionally different but all contribute to the development
of memory B-cells and plasma cells. Various cell types such as naïve B-cells which are
precursor cells of the DZ centroblasts, mature B-cells, T-cells and dendritic cells can be found
in the MZ surrounding the GC of the follicle. The extra-follicular area is also packed with
different T-cell types with a very high CD4:CD8 T cell ratio185
, macrophages, iDC and high
endothelial venules through which B and T cells migrate from blood into the tissue. In
addition to the LC located in the squamous epithelia, six different types of DC have recently
been identified based on different methods and markers186-191
. These cells are of different
phenotype and are spatially arranged accordingly (table 1).
Table 1. Five different dendritic cell phenotypes along with Langerhans cells can be found in the palatine tonsil.
Type Phenotype Location
GC-DC CD11c
+lin
-CD3
-CD4
+CD1a
-CD13
+
CCL18+ CD54
highfascin
+CD40
low
GC dark zone
fDC CD11c
+CD3
-CD4
+CD13
-CD21
+
CD35+CD54
+ CNA.42
+fascin
+
GC light zone
IDC
CD11c+lin
-CD3
-CD4
+CD2
+
CD54low
CD40high
CD1a-fascin
+
S100ȕ+ (IDC-1) S100ȕ-
(IDC-2)
Extrafollicular area near T-cell
areas
mature cells
pDC CD11c-lin
-CD4
+CD123
+CD54
+ BDCA-2
+
Around HEV
immature cells
LES-DC CD11c
-CD123
-CD1a
+ CD35
-
fascin- S100ȕ+
FcȖRr-
Cryptreticulated endothelium
LC CD1a+fascin
- S100ȕ+
Squamous epithelium
crypts
Abbreviations: GC-DC (germinal centre DC), fDC (follicular DC), IDC (interdigitating DC), pDC
(plasmacytoid DC), LES-DC (lympho-epithelial symbiosis DC), LC (Langerhans cells)186, 187 188, 189 182, 183 191,
192.
Two of these have a functional role in the GC whereas the other three are located in
different parts of the extra-follicular area, which also contains CD4+
T-cells, CD8+ T-cells,
macrophages and high endothelial venules. CD68+ tingible body macrophages (TBM) are
located throughout the GC. Their role is to remove all cells that do not survive the selection
37
phase and have subsequently entered apoptosis193
. B cells are the most prominent cell types
within the GC but T follicular helper cells, GC-DC, mDC, fDC (follicular DC) and naïve B
cells are also present within the LZ193
.
The follicle is a very dynamic structure and the cellular composition, as well as its
histology, is dependent on its developmental stage. Under normal conditions, the GC have a
limited lifespan, where 3-4 weeks after the initial antigen exposure a dissolution process takes
place as the GC gradually loses its structure and decreases in size194
. It is therefore likely that
the GCs in the tonsils are at different stages of development or dissolution, which involves
expression of various molecules such as cytokines, chemokines and possibly anti-microbial
peptides.
1.6.3 The germinal center reaction After an antigen enters the crypts it is taken up through the reticulated epithelium and
transported to the extrafollicular area by APC that present it to naïve CD4+ T cells in the T
cell zone 195
. The interactions with the APC stimulate the differentiation of the T cells into a
not quite fully developed follicular T helper cell phenotype, TFH-EF. This occurs by binding of
ICOS to ICOSL and CD40L to CD40 on the APC88
and by subsequent induction of Bcl-684
that enhances CXCR5 expression86
87
, thus stimulating migration88
. The activated T cells
migrate to the T zone/follicle border where they encounter activated CXCR5+B cells that
present the antigen in their B cell receptor, thus initiating the GC reaction. The T cell
mediated helps is through CD40L, ICOS and IL-21 secretion. The B cells can become either
very short-lived plasma cells or enter the GC to undergo differentiation into either long-lived
plasma cells or memory T cells.
Next, some of the T cells localize to the LZ in response to the high concentration of the
CXCR5 ligand, CXCL13 released by follicular DC (fDC)86
where they become fully
developed TFH-GC after interactions with centrocytes. The number of B cells in the follicles
correlates with the number of TFH cells196
88
. The TFH-GC are CD57+91
and secrete IL-21, which
is needed for normal GC development86, 91
. They are very effective in stimulating centrocytes
to produce all variants of immunoglobulin (IgA, IgG, IgM, IgE) and mainly induce class
switching to IgG1-3 and IgA1, which is dependent on CD40L-CD40 interactions91
.
The activated B cell induces the expression of CXCR4 and migrates to the DZ where the
CXCR4 ligand, CXCL12, is highly expressed197
. Within the DZ, CXCR4high
CD83low
CD86low
B cells (centroblasts) undergo somatic hypermutation of the immunoglobulin variable gene
(IgV) and clonal expansion with the aid of GC-DC198
. This process leads to the differentiation
38
of centroblasts into various clones of centrocytes that migrate to the LZ (centrocytes) towards
the CXCL13 gradient. There they encounter follicular DCs (fDC) that are clustered in the
middle of the follicle. The fDC have captured the antigen and present it unprocessed to the
centrocytes, often by immune complexes29
. In fact, fDC can retain the antigen for some time,
thus functioning as an antigen storage199
. The centrocytes undergo positive selection by
having their antigen specificity tested200
first by follicular DC and then by T follicular helper
cells. The terms centroblasts and centrocytes are used for clarity although research indicate
that tthey do not differ in size200
.
The centrocytes are of the CXCR5+CXCR4
lowCD83
highCD86
high phenotype and CXCR5 is
thought to distinguish between the DZ and LZ zones201
197
. The GC B cells die by apoptosis
unless a high expression of anti-apoptotic molecules is maintained or binding of CD40-
CD40L to a TFH occurs. This has great relevance for negative selection of self-reactive B cells
(reviewed by 193
). FDC upregulate B cell CD86 expression, which promotes the APC
potential of the cells202
. Within the GC, antibody producing plasma cells (IgG, IgA, IgM,
IgE)180
and memory B cells develop with the assistance of the TFH and DC subsets. The
transcription factor Bcl-6 is influenced by IL-21 released by TFH to affect the differentiation
of B cells and is necessary for the production of high affinity antibodies and plasma cell
formation70
. IL-21 appears to stimulate IgG1 production
Only cells with the highest affinity B-cell receptor for that particular antigen survive and
differentiate into plasmablasts and later plasma cells. Cells of intermediate affinity are thought
to become memory B cells and those with low affinity go through apoptosis and are removed
by the TBM. Until recently, it was thought that the selection occurred by competition for the
antigen presented by the fDC. However, recent evidence indicates that it is the access to TFH
that is the limiting factor as the number of T cells is limited and only B cells expressing high
levels of MHC receive help from the TFH cells. The TFH also undergo proliferation and
apoptosis in the LZ through antigen receptor signaling and co-stimulatory interactions. The
role of mDC within the follicle is not clear (reviewed by 193
).
It is not well known what determines the fate of the centrocytes. But it has been shown
that centrocytes expressing Blimp-1 and BCR with high affinity are likely to become
plasmablast that differentiate further outside the GC into plasma cells203
. This is dependent on
other factors such as NF-kB activation that upregulates Blimp-1 and IL-21 secretion from TFH
203. B memory cell differentiation is less well known. In fact it is thought that surviving
apoptosis during the LZ selection process might be enough as it has been shown that they
39
undergo shorter GC reaction203
. It has also been suggested that cells with intermediate affinity
of BCR become memory B cells or even re-cycle to the DZ (reviewed by 193
). Negative
selection to inhibit autoreactive B cells is defective in patients producing autoantibodies
(reviewed by 193
).
Other T cells than TFH, have been identified in the GC. In this respect Th17 cells have been
shown to induce centrocyte proliferation, the formation of GC and mediate isotype class
switching to IgG1, IgG2a, IgG2b, and IgG3204
. Furthermore, CD8+ early TEM cells that are
CXCR5+ICOS+CD69+ have been identified. They respond to CXCL13 and home to the GC
where they are likely to participate in the GC reaction as they express the co-stimulatory
molecules CD28, OX40 (CD134) and CD70 that are important for interactions with B cells205
.
They appear to be able to leave the follicle and return to the periphery due to their expression
of the inflammation homing receptor CCR5 that reacts to CCL5205
. They produce IFNJ and
contain granzyme A but lack perforin202
.
Natural Treg in follicles share features of Treg and TFH. They are CD4+CD25+CD69-
CXCR5+PD-1high
but do not express CD40L206
. They activate and are dependent on Bcl-6
and are able to directly suppress B cell antibody secretion and probably limit the GC reaction
by suppression of B cells and TFH function, most likely by their high IL-10 production207
. In
mice, CD8+ CXCR5+ICOS+ CD44+ Tregs have been identified in the GC that appear to be
important for prevention of autoimmune disease by mediating self-tolerance208
.
1.6.4 Infections and the crypt epithelium The palatine tonsils are situated within the throat so that they can efficiently fight off invading
microbes and initiate adaptive immune response within the GC. In this respect, the tonsil
crypts increase the available area for antigen sampling but at the same time expose the tonsil
more readily to bacterial interference. Therefore a good balance has to be present between
those important roles for the tonsils to have an optimal activity.
The first barrier that invading bacteria have to pass is the mucosal layer covering the tonsil
which traps and neutralizes the microbe using digestive enzymes and IgA from saliva as well
as IgG209
and neutrophils arriving through the tonsil crypts210, 211
. IgG coats the bacteria and
marks it for phagocytosis whereas IgA prevents attachment of the bacteria. However, the IgA
producing cells are less numerous than IgG secreting cells212
. Furthermore, the squamous
epithelium appears to lack the components needed to transfer secretory Ig (sIgA) through the
membrane and to the surface213
. It is therefore likely that the sIgA travelling through the
crypts is negligible in comparison with the sIgA from the salivary glands which distributes
40
throughout the oral area209
. Bacterial swabs from tonsils show that around 90% of the
bacteria are coated with IgG antibodies while IgA coating increased with a longer duration of
tonsillitis indicating that if insufficient, IgA might contribute to initiation of infection due to
less effective phagocytosis of the bacteria209
. Interestingly, bacteria from healthy individuals
are coated with lysozyme, IgG and sIgA antibodies whereas during acute tonsillitis by
S.pyrogenes, the lysozyme coating was increased while sIgA was decreased214
.
AMPs are also present in the saliva 215
from where they cover the mucosal layer of the
tonsil and possibly the tonsil crypts. Studies indicate that bacterial infections are restricted to
the crypts216
as bacteria can only adhere to the squamous epithelium217
218
or its shed
endothelial cells219
. This might be due to the different cytokeratin and glycoconjugate
expression220
of the membranes that influence both adherence and opportunity of infiltration.
Furthermore, the crypt epithelium contains the integral membrane protein, claudin-4 that
forms part of a channel through the tight junction (TJ) barrier221
that might allow access by
loosening the intercellular junction222
. Bacteria might also be able to infiltrate via DC as
bacterial–epithelial TJ “crosstalk” and DC also express various TJ which the bacteria might
interact with 222
. The existence of biofilms223
has been verified in chronically diseased tonsils,
particularly in crypts224
. It appears to offer protection to the bacteria by hindering access of
the immune system and antibiotics and is therefore thought to contribute to chronic and
recurrent infections224
.
1.6.5 Expression of PRRs in tonsils The low likelihood of bacterial infection through the squamous epithelium is reflected in their
lack of TLR2 and TLR4 expression while maintaining a high level of the intracellular TLR3
that is important against viral defences225
as well as NOD1 and NOD2228.
Furthermore, crypt
epithelium expresses TLR2 and TLR1/2 is observed in the local macrophages and DC 226
.
Tonsil T cells express all except TLR6 and TLR8227
and during recurrent infection, CD4+
T cells downregulate TLR9 whereas TLR2, 3 and 5 is upregulated in CD8+ T cells227
. Tonsil
B cells express TLR1, 2, 7, 9 and 10, regardless of infection or maturation stage228
. T cells
express a variety of NLR, such as NOD1, NOD2 and NALP3, particularly in the T cell zones
among CD4+ T cells229, 230
. Furthermore, this expression is very strong within the GC.
Interestingly, tonsillar infection by GAS (Group A ȕ-hemolytic streptococcus) or H.influenza,
does not influence mRNA expression of NOD1, NOD2 or NALP-3228
. Furthermore, NOD2
and NOD1 act synergistically with ĮCD3 and ĮCD28 to induce proliferation of T cells but are
ineffective by themselves but have stimulatory effects on monocytes227
.
41
1.6.6 Hypertrophic and recurrently infected tonsils The most common clinical tonsil diseases are recurrent tonsillitis (RT), characterized by
frequent infections with sore throat symptoms, and abnormal enlargement of the tonsil
(hypertrophic tonsils, HT) causing discomfort for the patients. The enlargement may be due to
lack of apoptosis within GC or repeated irritations due to viral infections231
that lead to a
greater overall follicular tissue area compared to the RT tonsils71
as they contain the same
total number of follicles54, 71
. The numbers of TBM generally correlate with GC size, as they
are necessary for its homeostasis232
. Furthermore, APCs are functionally similar in RT and
HT tonsils233
. However, the cellular composition of the tonsils is markedly different with RT
tonsils having fewer B cell, helper T cells and Treg234
but more numerous CD68+
macrophages particularly in crypts232
.
What causes recurrent tonsillitis is unknown but RT tonsils have fewer and partially
disrupted TJ within the crypt epithelium which influences its cellular interactions222
. This
could establish an ideal infection site for microbes and in fact inflammation within the crypts
(cryptitis) amplifies with each inflammation possibly due to increased infiltration of
macrophages, neutrophils and DC235
. The immune response gets stronger and more damaging
as noticed by the presence of fibrosis in RT tonsils236
. An intermediate condition is a RT
tonsil with hypertrophy (RTHT) that has both larger and more numerous follicles compared to
RT tonsils237
.
1.7 Microbial diversity and the microbiome “No man is an island, entire of self” (John Donne b.d.1572-d.d. 1631). Instead, humans are
occupied by millions of microbes, assembled from birth. Their combination is dependent on
opportunistic encounters through lifestyle, environment and genetic factors and is therefore
different between individuals but relatively stable throughout life of the individual238
.
Commensal bacteria occupy areas with a high bacteria exposure, such as the skin, the
Waldayer’s ring and the Gastro-intestinal tract (GI). Certain bacterial hierarchy exists where
some are residents while others become temporary “hitchhikers” with the local bacterial
flora239
. The microbial-host relationship is symbiotic and has developed through co-evolution
of humans and microbes239
. Commensal bacteria protect the host forming stable niches that
inhibit colonization of pathogens. They can stimulate angiogenesis240
and assist in host
antimicrobial activity241
242
and tissue healing by stimulating AMP secretion through TLR243
.
Interestingly, follicles in spleen and lymph nodes244
do not develop normally245
without the
42
bacteria and epithelial cell homeostasis in GI is dependent on TLR-mediated recognition by
the host 243
.
How do the commensal bacteria manage to stay under the radar of the immune system?
Various hypotheses have been put forward such as non-stimulatory interactions with the
TLRs, dampening of the innate immunity by Treg-mediated tolerance and lack of danger
signalling (reviewed by246
). In this respect, intestinal epithelial cells regulate
communications with commensal bacteria by negative regulation of TLR and IL-1R247
and
by minimizing TLR expression at sites of frequent encounters of the commensal bacteria,
during steady state (reviewed by248
). Finally, commensal bacteria can prevent activation of
the NF-kB pathway by inhibiting degradation of its masking molecule IkB249
and by
butyrate production through fermentation that induces IL-10 production resulting in anti-
inflammatory responses in the GI250
.
Recently, identification of the variable regions of the conserved 16S ribosomal RNA
(rRNA) gene has been used to identify different species of the microbiome. This has greatly
expanded the current knowledge of the commensal bacteria of humans. However, this
method does not distinguish between live, dying or dead microbes nor does it accurately
predict all habituations, as it does not distinguish between free and phagocytosed cells.
Phylogenetically related microbes are more likely to co-occur symbiotically than
microbes of distant relation as they compete for the niche251
. Analyzing the microbiome is
important for understanding the role of microbes in the development and sustainment of
human disease as changes in the composition of the microbiota due to external factors such
as infections, can lead to the development of diseases e.g. diabetes252
, ulcer253
, crohn’s254
and autoimmune arthritis255
.
1.7.1 The microbiome of the skin The skin microbiome
100, 256 differs between physiologically diverse body sites and individual
factors such as age, environment, transmission opportunity, previous exposures, genotype,
lifestyle and underlying diseases, influence the microbiome (reviewed by257
). The microbiome
reaches into the subepidermal compartments256
where free ranging bacteria have been
detected in the dermis and adipose tissue256
. Although the overall bacterial diversity differed,
the commensal bacteria Staphylococcus epidermis and Pseudomonas spp. were frequently
identified256
. Their presence near the basal layer indicates direct interaction with host cells,
indicating effects on immune responses256
. The cutaneous viral biome reflects high variability
43
with preferential habituation of multiple Papillomaviruses, circovirus and
polyomaviruses258.
Today, two studies have been published comparing psoriasis lesions to healthy or
uninvolved skin but due to different sampling methods the results are not unanimous259
260
.
In general, the most common phylum of bacteria were Firmicutes along with Actinobacteria
and Proteobacteria259 260
. Firmicutes were the most commonly isolated bacteria in psoriasis
skin and comparison to healthy skin showed either significant difference 259
or not260
.
Proteobacteria were more common in the psoriasis trunk skin260
while Actinobacteria were
more common in healthy skin259
260
The genus Streptococci were the most common bacteria
isolated from psoriasis skin260
unlike staphylococci260. Furthermore, Propionibacteria was
more common in healthy skin than lesions while uninvolved skin had intermediate bacteria
levels259
260
. These studies indicate that the bacterial flora in the psoriasis skin is altered
which might be due to the disease itself or as a causal factor of the abnormal immune
responses present in the lesions.
Interestingly, studies based on 16S rRNA anlyzis of peripheral blood monocytes from
psoriasis patients showed that they had phagocytized more bacteria compared to monocytes
from healthy blood261
. However, no correlation was observed to PASI.
1.7.2 The microbiome of the palatine tonsils A recent 16S rRNA study of surface swabs from various oral cavity components shows a
similar microbiome, indicating comparible niches262
. Phylum Firmicutes was generally
identified (~45%) while Bacteroidetes, Fusobacteria, Proteobacteria and Actinobacteria
were less frequent 262
. Streptococcus (~23%), Veillonella, Prevotella and Fusobacterium,
were the most common genera with each ~10%263-265
. In healthy adult tonsils, Bacteriodetes
was the dominant phylum unlike RT tonsils where Firmicutes are most common phylum in
adults and Proteobacteria in children262
.
Traditional bacterial culture methods of tonsil surface and core swabs show different
colonization of bacteria species. RT tonsils are characterized by Staphylococcus aureus
infections in adults (30-53,5%)263
and children (65,9%)265
followed by Haemophilus
influenza and S. pyogeneses (~15%)265
. Furthermore, S.pyogenes is the most commonly
found bacterium in peritonsillar abscesses266
. HT tonsils are associated with H.influenza
(~31%), S.pyogenes (~23%), S. aureus (~24%) and S.pneumonia (~13%)263
. Finally,
Actinomyces present in crypts of both HT (~62%) and RT tonsils (~27%), are associated
with higher tonsil volume indicating a role in the hypertrophy216
.
44
Table 2. List of bacteria isolated from skin and tonsils.
Phylum Genus Species Actinobacteria Actinomyces Actinomyces spp. Propionibacterium
Bacteriodetes Prevotella P. melaninogenica P.histolitica
Firmicutes Streptococcus S.pyogenes S.intermedius S.pneumonia
Staphylococcus S.aureus
Veillonella
Fusobacterium Fusobacterium F.necrophorum
Proteobacteria Haemophilus Pseudomonas
H. influenza
Pseudomonas spp.
Spirochaetes Treponema
Tonsils are a very heterogeneous tissue as reflected by a recent study comparing isolated
16S rRNA from crypts of healthy adults, RT adults, HT and RT children. It identified the
presence of a cryptal core microbiome that was independent of age and tonsil pathology,
constituting 8 phylum of bacteria that change in composition between groups and
individuals267
. Phyllum Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria,
Actinobacteria, and Spirochaetes constituted around 70% of all bacteria isolated with values
>1%. Genus streptococcus and prevotella were present in all crypts. The main results were a
greater difference in bacterial species between children and adults than between
pathology267
. Surprisingly, no difference was observed for S.pyogenes and H. influenza
values did not differ between HT and RT tonsils. In fact, bacterial species such as
S.pyogenes and S.aureus that are usually associated with tonsillitis were found in very low
proportions while F.necrophorum, S. intermedius and P.histicola or P. melaniogenica were
significantly related to adult RT. These differences may be due to the growth conditions
within the tonsils as well as the methodology used. Interestingly it has been shown that
tonsillectomy decreases the level of anaerobic bacteria in the oropharyngeal area of
individuals with recurrent tonsillitis268
1.8 E-Haemolytic Streptococcus Streptococcus is a Gram-positive (GP) E-haemolytic bacteria belonging to the Lactobacilli
family. They are classified further according to Lancefield serotyping into groups A - F. They
are spherical in shape and can cause invasive infections. Group A Streptococcus, in particular
Streptococcus pyogenes is one of the most common causes for sore throat and associated with
both acute and recurrent infections269, 270
. It can survive intracellularly in macrophages261
and
up to 7 days in epithelial cells without symptoms271, 272
.
45
Streptococcus anginosus are a group of heterogenous streptococci bacteria important in
infection of certain body fluid and internal organs among other places (reviewed by)273
.
Group A, C and G streptococci express the virulence factor, M protein, on their surface. It has
two polypeptide chains forming a D-helical coiled-coil structure and interrupts the host
phagocytosis response274
. Streptococcal superantigens can stimulate T cells unspecifically by
binding to both TCR and the MHC class II molecule simultaneously, leading to polyclonal
activation of T cells. The TCR binding is most commonly to the VE region of the variable
chain275
. Activation by superantigen has been linked to guttate psoriasis276
. GAS is localized
to the tonsillar reticulated crypts277
and biofilm.
1.9 The association of streptococcal throat infection and psoriasis Streptococcus is the most commonly identified bacteria in the psoriasis skin
260 and an
association exists between initiation and acute exacerbation of psoriasis and throat infection
by the M protein expressing streptococci (reviewed in paper IV). Interestingly a similar
relationship is present between D-streptococci and pustulosis palmaris et plantaris, which is a
psoriasis-like disease restricted to the palms and soles278
. It is not known why psoriasis
patients are much more vulnerable to symptomatic throat infections than their household
controls173
. Studies indicate that antibiotic use is not effective 279
280, 281
however,
tonsillectomies can have beneficial effects for psoriasis patients with repeated throat
infections282, 283
(paper III-IV).
Skin homing of T cells depends on the expression of CLA284
, which is inducible by
inflammatory mediators, such as superantigens and IL-12, that are released during throat
infection by streptococcus285-287
. S.pyogenes enhances the maturation and activation of pDC
and mDC that induce the expression of co-stimulatory molecules and the production of pro-
inflammatory cytokines and chemokines, resulting in a Th1 polarization of naïve T cells288
.
The extracellular cysteine proteinase, streptococcal pyogenic exotoxin (Spe) B has the ability
to degrade a wide range of chemokines released in response to inflammation by epithelia
although the chemotactic abilities of CXCL8 and CCL5 remain unaffected and CXCL9
preserves its anti-microbial activity289
. S.pyogenes direct the immune response in a Th17
orientation by inducing TGF-E290, which also enhances integrin expression (D5E1) and
invasion into epithelial cells291
. It releases streptolysin O (SLO) that perforates neutrophils
and leads to exaggerated host response292
.
Two different hypotheses have been put forward to explain possible roles of streptococcus
in the development of psoriasis. Both depend on the idea of molecular mimicry, which occurs
46
when amino acid sequences between a foreign peptide and a self-peptide are similar enough
to cause cross-reaction between the shared sequences and potentially autoreactive T cells.
These cells have a weak affinity for the self-antigen, escaped the negative selection in the
thymus and become activated and stimulated in the periphery by cross-reaction. This causes
loss of tolerance for the self-antigen, leading to inflammation and potential tissue damage or
autoimmunity (reviewed by22
,paper VI).
1.9.1 Streptococcal M protein and psoriasis The first theory focuses on the importance of M protein in psoriasis. Homologous peptides,
shared by streptococcal M protein and epidermal keratins stimulate peripheral CLA+ T cells,
possibly through molecular mimicry293
. This is further reflected by the strong positive
correlation that is present between the frequency of these cells in blood and the severity of the
psoriasis plaques (paper III) and the full or partial disease remission after tonsillectomy (paper
III). Furthermore, T cells isolated from psoriasis tonsils and corresponding psoriasis plaques
are oligoclonal by sharing the same TCRVB gene rearrangements, indicating a mutual
origin294
. The pathological T cells in the psoriasis skin might therefore originate in tonsils
from where they migrate with blood to the skin. Skin homing T cells are expanded in the
psoriasis blood135
and may have Th1, Th22 or Th17 phenotypes as they are more numerous in
their blood295
and skin120, 140, 149
than healthy individuals. These T cells are thought to be
mainly CD8+ cells that are able to recognize the auto-epitope presented in the HLA class I
molecule on the surface of the APC. This conclusion is based on the accumulation of these
cells in the dermis and genetic research indicating their involvement.
47
Figure 1. The interactions between psoriasis and streptococcal throat infections.
(1) Throat infection leads to the migration of streptococci into the tonsil crypts and stroma. The bacteria release
superantigens and IL-12 that upregulate CLA expression by the T cell. (2) An antigen presenting cell engulfs the
bacteria and presents its fragments on its surface with the aid of MHC complexes. This allows it to introduce a T
cell to bacterial components such as peptidoglycan (PG) and M protein peptides. The T cell becomes an effector
cell, ready to respond upon bacteria encounter. (3) CD8+ T cells can also recognize and bind PG through TLR2,
which they express on their surface unlike CD4+ T cells. PGN within APC may also bind intracellular innate
immune receptors and stimulate the response. Internalized streptococcus can also lead to a similar process when
the environmental conditions improve and it becomes externalized and infectious again. The T effector cell
expressing the skin homing molecule CLA is now able to migrate from the tonsil to the skin where it may react
to skin keratins that are homologous to M protein peptides. Image from paper VI.
In this respect, Psoriasis susceptibility locus 1 (PSORS1) has most convincingly been
associated with psoriasis (reviewed by paper VI). It is situated on chromosome 6p21 where
the major histocompatibility complex (MHC) is located296
. Within that region is the
susceptibility gene HLA-C that encodes for the MHC class I allele Cw*0602297
. The protein
product that HLA-Cw*0602 codes binds and presents short peptides that have been
implicated in the pathogenesis of psoriasis293, 298-300
. Furthermore, studies indicate that
64.2% of patients with chronic plaque psoriasis are positive for HLA-Cw*0602 while 6.8%
are homozygous for the allele301
. Interestingly, smoking can induce the expression of psoriasis
risk genes such as HLA-Cw6 (reviewed by169
). However, the psoriasis lesions develop
through interactions of various immune cells as earlier discussed.
48
1.9.3 Peptidoglycan and psoriasis The second theory is based on the involvement of bacterial peptidoglycans in psoriasis.
Peptidoglycans (PGN) form part of the streptococcal cell wall and can bind to a TLR and
thereby initiate an immune response. It has been suggested that PGN originating from tonsils
are important for triggering the Th1 mediated response characterizing the affected psoriasis
skin302
. This is based on the finding that PGN containing cells, mostly CD68+ macrophages,
are more frequent in psoriasis skin than normal or non-affected skin where they are found in
close proximity to the CD4+ T cells303
. Furthermore, the CD4+ T cells respond in an HLA-
restricted way to the PGN by producing IFNJ303. Lastly, polymorphisms in peptidoglycan
recognition proteins (PGRP) 3 and 4 are associated with psoriasis and are also found within
tonsil GC164
. PGRP 1-4 are PRRs localized in the PSORS4 gene locus that recognize
muramyl pentapeptide or tetrapeptide part of the PGN but can also bind LPS and LTA
(reviewed by 304
). They can e.g. monitor normal gut microbiota and thereby protect against
tissue damage, inflammation and colitis305
. It is therefore possible that PGN are important for
the development of psoriasis.
1.10 Antimicrobial peptides Antimicrobial peptides (AMP) are key elements of mucosal and skin defenses as effectors of
the innate immune system and a first line of defence against infection. They are amphipathic,
positively charged, small, 12-50 amino acid long proteins or peptides306
that are fast acting
and highly efficient against both Gram positive and Gram negative bacteria, fungi, protozoa
and even some viruses306
. AMPs are either produced by local cells, such as epithelial cells, at
the site in question or released during degranulation of intra vesicles within infiltrating cells
such as neutrophils.
All AMPs need to be post-transcriptionally cleaved by proteases to become efficient and
active peptides307
. They are structurally preserved and categorized accordingly into looped or
extended structure, ß-sheet or D-helix307
. The amphipathic characteristic is the main reason for
the direct and efficient antimicrobial activity, as their cationic half interacts with the
negatively charged phospholipids within the microbial membrane through electrostatics. The
hydrophobic part causes insertion of the peptide into the bacterial membrane, which initiates
membrane disruption and eventual lysis of the microbe (reviewed by308
). AMPs are also
potent immunomodulators by attracting various immune cells to the site of inflammation and
by influencing the direction of the immune response.
The focus of this discussion will be on human defensins and cathelicidin as they are
present in both the palatine tonsils and skin and are important for the pathology of psoriasis.
49
1.10.1 Defensins Defensins have ß-sheet structures and are classified into D, ß or T-defensin
307 depending on
the intramolecular disulphide pair binding of their six-cysteine residues, which contain
different amino acids307
. At least 8 genes encoding D- and ß-defensin are located in a cluster
on chromosome 8p23 where the human ß-defensin genes are found clustered together 309
.
Both human D- and ß defensin have been shown to be important for the innate immune
response but stop codons present in all the six genes identified for T-defensins, interfere with
the gene translation, thus rendering them inactive310
.
1.10.1.1 Human D-defensins
In leukocytes, D-defensins are encoded from three exons present in one locus and the third
exon encodes for the active AMP311
. Genes for other defensins have only two exons311
. Six
D-defensins have been identified in humans; the human neutrophil peptides (HNP) 1-4 and
the human defensins (HD) 5 and 6312
. HNP1-4 are produced and stored in neutrophil
azurophilic granules from where they are released by degranulation312
. HNP1-3 have also
been identified in human natural killer cells (NK), Ȗį T cells and B cells313
. They have been
identified in psoriasis plaques314
and palatine tonsils315
. HNP1-2 have chemotactic effects on
monocytes316
and, along with HNP-3, resting naïve CD4+T cells, CD8+ T cells, and iDCs317
.
They can influence the immune response by inducing pro-inflammatory cytokine secretion318
,
interacting synergistically with other AMPs319
320
and chemokines315
, inducing anti-
inflammatory responses during neutrophil necrosis or apoptosis321
, stimulating woundhealing
responses in fibroblasts322
and acting effectively against intracellular infections323
.
The enteric HD5 and 6 are produced by Paneth cells in the intestine and by cells within the
female urogenital tract312
and their deficiency has been associated with Crohn’s disease324
.
HD5 is also produced and induced during infection in the kidney and urinary tract where it is
relevant for maintaining sterile urine325
. It is effective against both Gram positive and Gram
negative bacteria326
. HD6 protects against enteric bacteria by specific methods as it surrounds
and traps the bacteria with fibrils and nano-nets that form by self-assembly, thus inhibiting
their translocation across the epithelium327
.
50
Human E-defensins
Today, 28 different isoforms of human E-defensins (HBD) have been identified328
most from
epithelia, particularly gingival tissue, keratinocytes, testis and epididymis but also by various
cells of the immune system (reviewed by 309
). GWAS have reported copy number variations
in the defensin gene cluster166, 329
, most likely due to gene duplications330
. The defensins
share a common two exon gene structure, the intronic region is either small (2 kb) in the case
of the inducible defensins (DEFB4 (HBD-2), DEFB103 (HBD-3) and DEFB104 (HBD-4)) or
large (10kb), in the case of the constitutively expressed DEFB1 gene (HBD-1)331
.
HBD-1 is constitutively expressed by various epithelia such as in palatine tonsil332
and
suprabasal layers of skin 333
. It is inducible during inflammation334
and effective against Gram
negative bacteria with chemotactic effects on memory T cells and iDC in blood335
. Reduction
of its disulphide bonds alters its structure and enhances its antimicrobial activity336
. A genetic
variation in its DEFB1 gene is a potential risk factor for Crohn’s disease and may determine
disease phenotype and the colonic localization of the disease337
.
HBD-2 expression at steady state is low but inducible and significantly enhanced in
inflammatory disorders 338-340
such as psoriasis lesions341
, from where it was originally
isolated314
. It is widely expressed by the gastrointestinal and respiratory tracts339, 340, 342
,
platelets343
, monocytes and mDC344
. It is chemotactic for peripheral memory T cells and
iDC335
and binds to the CCR6 receptor335
. Its binding to CCR2345
attracts macrophages, mast
cell346
, activated neutrophils347
and monocytes to the scene.
A high copy number of its gene, DEFB4, is a risk factor for Crohn’s disease348
and
psoriasis27, 195
. In this respect, although disputed349
, HBD-2 serum levels correlate with
disease severity350
but are inversely correlated with IL-17351
. HBD-2, HBD-3 and LL-37 are
highly expressed in both psoriasis and atopic dermatitis352
353
. HBD-2 is important for wound
healing322
and a correlation has been observed between HBD-2, the disruption of the skin
barrier and the seriousness of the disease354
.
HBD-2 expression is induced by IL-1ȕ and TNF-Į334 in epidermis, LPS through TLR2
355
and TLR4356
in endothelial cells, IL-17A and IL-22 in airway epithelium76, 357
and skin62
and
IFNJ, TNFD, IL-6 and IL-1E in keratinocytes351
. However keratinocyte expression is
suppressed by IL-13, IL-10 and IL-4351
. Interestingly IL-17 and IFNJ have synergistic effects
on keratinocyte production of HBD-2 and HBD-3358
and the keratinocytes show enhanced
bactericidal activity against Staphylococcus aureus358. HBD-2 induces IL-22 production by
51
CD3/CD28 stimulated T cells while suppressing IL-17A production and enhancing IL-10 and
IFNJ production351
.
Interestingly, HBD-2 mediates different effects on commensal and pathogenic oral
bacteria. In this respect, the common indigenous bacteriums Streptococcus mitis is
unsusceptible to the peptide while known cariogenic species such as S. mutans and S.
sobrinus were targeted359, 360
.
HBD-3, originally isolated from psoriasis scales, is produced by neutrophils,
keratinocytes361
, the various epithelia361, 362
and non-epithelial tissue such as the heart, skeletal
muscle 153
, tonsil and esophagus362
. It is effective against fungal, Gram negative and Gram
positive infections, particularly by aerobic bacteria, and is more effective against anaerobic
bacteria than HBD-2363
. Its antimicrobial effects are by inhibiting biosynthesis of the bacterial
cell wall of staphylococci364
. Keratinocyte killing of Staphylococcus aureus is dependent on
HBD-3360
.
HBD-3 attracts monocytes153,
macrophages362
335, 365
, iDC, mast cells366
and memory T
cells335
. Its chemotactic effect on leukocytes is through the CCR2 and CCR6 receptors345
367
.
In fact, HBD-3 interactions with CCR6 can suppress neutrophil apoptosis368
.
HBD-3 induces proinflammatory cytokine production and enhances expression of co-
stimulatory molecules such as CD86369
by monocytes. It influences macrophage370
and LC
stimulation by inducing CCR7 expression and Th1 polarization of T cells371
. It also stimulates
the maturation of mDC and monocytes and activates APC through TLR1 and 2 dependent
pathways369
.
HBD-3 expression is induced by IFN-Ȗ, IL-1ȕ, TNF-Į 362
, TLR agonists361
and IL-2262
in
keratinocytes and CXCL8 and CCL2372
in blood mononuclear cells. It is suppressed by the
Th2 cytokines IL-13 and IL-4 in keratinocyte373
. HBD-3 and prostaglandin D2, released from
mast cells by degranulation form a positive feeback loop aided by histamine366
374
375
that
together influences skin vascular permeability366
. Skin wounding transactivates epidermal
growth factor receptor, which induces HBD-3 expression 359
.
HBD-4 is highly expressed in the testis and is active against both Gram negative and Gram
positive bacteria365
. It is expressed at low levels in various tissues such as lungs376
but
appears to be absent from skin87
. Although it is present in neutrophils365
, it does not appear to
influence neutrophils or eosinophil but is chemotactic for monocytes365
and macrophages335,
365. It is also inducible in human primary keratinocytes upon exposure to bacteria and
52
inflammatory cytokines, such as IL-1E, IFNJ or TNFD{Harder, 2004 #2096;365
. It influences
mast cells and vascular permeability in a similar manner to HBD-3366
Keratinocyte activation by HBD-2, 3 and 4 stimulate them to secrete IL-18, IL-20 and IL-
8, all of which are important in the pathology of psoriasis377
and together these defensins have
synergic effects on keratinocyte antimicrobial activity378
and IL-18 secretion 377
.
The palatine tonsils can express a number of AMPs such as HBD-1332, 379
, HBD-2379, 380
,
HBD-3379, 381
and HBD-4382
where they are expressed by the epithelium of both the surface
and crypts as well as within the follicles 382
. All these E-defensins are also found upregulated
within the psoriasis plaques where they are chemotactic and attract immune cells to the
area341
.
1.10.2 The anti-microbial peptide hCAP18/LL-37 The human cationic antimicrobial peptide-18 (hCAP-18) is the only identified member of the
cathelicidin family in humans383
. It has been detected in genital, airway, intestinal and ocular
epithelia 384, 385
386
387, 388
, skin, sweat glands389
, saliva and salivary glands390, 391
,
sweat glands392, 393
and colon mucosa393
It is predominantly expressed by neutrophils and
stored within their granules394, 395
. It has also been reported in mast cells313
, monocytes313
(paper I) and macrophages (paper 1) , DC313
, B-cells, NK cells and Ȗį-T-cells 313
but not in
Įȕ-T-cells 396
.
1.10.2.1 Regulation and processing of hCAP18/LL-37
hCAP-18 is translated from a 4-exon gene, CAMP (cathelicidin antimicrobial peptide), into a
precursor molecule that consists of a cathelin pro-domain and a C-terminal antimicrobial
peptide397
. The CAMP gene has a promoter region containing binding sites for NF-țB 398
, a
vitamin D responsive element (VDRE) 396
as well as two IL-6 response elements: nuclear
factor for IL-6 (NF-IL6) and acute-phase response factor (APRF, also known as signal
transducer and activator of transcription-3 [STAT-3])385, 399, 400
. A free functional peptide is
released by an enzymatic cleavage401
by proteinase 3 from neutrophils402
or kallikrein from
keratinocytes403
. Interestingly, further processing of LL-37 to smaller active peptides may
occur after secretion404
. Until recently, the precursor protein was thought inactive and
inhibitive to the free peptide but recently it was shown to be antimicrobial405
. Furthermore,
the previously described cathepsin L inhibitor-function of the cathelin pro-domain406
, which
limits tissue damage during infection and potentially inhibits viral entry into cell404
, is more
likely to be a function of the active LL-37 peptide itself407
.
53
1.10.2.2 hCAP-18/LL-37 has anti-microbial effects
The active LL-37 peptide forms an amphipathic Į-helix with a broad-spectrum antimicrobial
activity toward Gram negative and Gram positive bacteria403, 408
and anti-fungal409 and anti-
viral effects (e.g. HIV121
and influenza410-412
). The structural design of the peptide is crucial
for the anti-microbial function of the peptide as it can become inserted into the bacterial
membrane, leading to its disruption and ultimately bacterial lysis413
. LL-37 prevents the
development of biofilm, which is essential to limit the colonization by bacteria320
, and
stimulates D-defensin release by neutrophils 414
.
Patients with the chronic periodontal disease, Morbus Kostmann, have neutropenia with
HNP1-3 and LL-37 deficiency causing severe infections in the mouth mucosa414
. This
emphasizes the importance of the peptide in mucosal innate defences. LL-37 has anti-bacterial
effect on the intracellular Gram negative bacteria Brucella suis415 and is even more efficient
than antibiotics in killing the intracellular Gram positive bacteria Staphylococcus aureus416.
LL-37 expression by endothelial cells in the urinary tract is induced during bacterial
infections417
while its production is more regulated in other places such as skin and colon. In
this way, butyrate induces its production during infection, reduces inflammation418
and
attenuates the inhibition that the bacterium Shigella has on the production of the peptide by
colon epithelia 419
.
LL-37 can bind to LPS and cause lysis of the bacteria420
. It can also have anti-sepsis
effects421
by binding and inactivating free LPS421
422
and thereby diminishing the production
of LPS-induced TNF-Į423 as well as IL-1E, IL-6 and IL-10 production from monocytes
424,
TNF-Į production from macrophagesand CXCL8 and MCP-1425
. This occurs with the
peptide in low doses but not in high426
. During septic-shock, LL-37 levels are low whereas
TNFĮ concentrations are high426
. Following recovery, the LL-37 levels become gradually
higher again426
, which might indicate that LL-37 is down-regulated during septic-shock to
inhibit a further stimulation of the immune response427
. Stimulation of macrophages with the
Gram positive bacterial cell wall component lipoteichoic acid (LTA), up-regulates production
of LL-37427
and attenuates TNF-D and IL-6 production313, 428
.
54
1.10.2.4 Other functions of the active LL-37 peptide
LL-37 is a strong chemoattractant 429
for neutrophils313
, mast cells 430
, monocytes and CD4+,
but not CD8+, T-cells 313, 429
. The LL-37 induced chemotaxis of monocytes, neutrophils and
eosinophils has been shown to depend on the G-protein-coupled formyl peptide receptor
(FPR) 2431
,429
which is also expressed by T-cells{ macrophages, airway epithelium and
endothelial cells432 433 434
. This has recently been disputed 432-434
and the importance of other
receptors such as the purinergic P2X7 receptor and epithelial growth factor receptor (EGFR)
has been highlighted434
.
Binding of LL-37 to the P2X7 receptor in monocytes, neutrophils435
and human gingival
fibroblasts 436
436
leads to the release of CXCL8437
and IL-1ß434
. LL-37 also inhibits neutrophil
apoptosis 438
using the FPR2 and P2X7432
receptors. In addition, LL-37 appears to signal by
transactivation of the EGFR433
, which induces keratinocyte migration and thus promotes
wound healing439
. Furthermore, LL-37 has a role in angiogenesis440
. Various other receptors
have been identified although their function has not been fully clarified. With neutrophils, LL-
37 binds to the G protein coupled receptor (GPCR), CXCR2, a receptor for CXCL8441
, which
stimulates CXCL8 secretion. Other LL-37 binding GPCR are MrgX2 in mast cells442
and
P2Y11 in glial cells398
.
The active form of vitamin D, 1,25-dihydroxyvitamin D (3) (1,25(OH)2D3), binds to the
vitamin D receptor, which induces cathelicidin expression in keratinocytes, monocytes and
neutrophils443
. In skin it therefore contributes to its natural defences444
. In macrophages,
ligand binding to TLR can upregulate the expression of the vitamin D receptor and thereby
induce the expression of LL-37445
. Binding to TLR induces LL-37 secretion423, 446
and LL-37
can moreover modulate the immune response to a TLR ligand binding435
.
1.10.2.5 Immunomodulatory effects of LL-37
Responses to cellular stimulation with the LL-37 peptide differs between cell types, the
surrounding area and the concentration of the peptide. In this respect, LL-37 stimulates IL-1ȕ
secretion from monocytes447
while suppressing it by neutrophils378
. Similarly, LL-37 can
attenuate or magnify the immune response. LL-37 has anti-bacterial effects in low doses378
but the sensitivity and magnitude of the response is even greater in combination with other
AMPs such as HBD2344, 448
produced by various cells in the crypts, such as epithelial cells,
macrophages, monocytes and pDC449
.
55
Monocyte differentiation with LL-37 and macrophage colony stimulating factor (M-CSF)
or granulocyte M-CSF (GM-CSF) results in pro-inflammatory macrophages that secrete
higher levels of IL-23p40 following LPS stimulation447, 450
than when cultured with the
chemokines alone. Most studies indicate that LL-37 has little effect on lymphocytes by
itself424
but after CD3/CD28 stimulation of T cells it enhances production of IFN-Ȗ, IL-4, IL-
13 and TNFĮ while suppressing IL-17, IL-22, IL-1b, IL-6 and IL-10 production 378, 423, 450
.
When peripheral blood mononuclear cells are stimulated with the peptide, it interacts
synergistically with components of the innate immune system (IL-1ȕ, GM-CSF451
,
CXCL8450
) while suppressing the influence of components important for the stimulation of T
cells (IL-4, IL-12, and IFN-Ȗ451). Similar results have been found for epidermal keratinocyte
where stimulation with LL-37 in high concentration (25ug/mL) together with IFNJ or TNFD
decrease their production of CXCL10 and CCL5 although CXCL8 production was
enhanced451
. Interestingly, high doses of the peptide inhibited CXCL10 production whereas
low doses stimulated both CXCL10 and CCL5 production424
.
LL-37 production by keratinocytes and neutrophils is influenced by the surrounding
cytokines. The pro-inflammatory cytokines TNF-D, IL-1ȕ and IL-6 along with IFN-Ȗ and the
Th17 cytokines IL-17 and IL-22 enhanced its production424
. On the contrary, IL-10, IL-4 and
IL-13 as well as cyclosporine A suppressed its production424
. Furthermore, LL-37 induces the
release of IL-6, TNF-Į, GM-CSF and IL-1ȕ from keratinocytes 452
453
. The anti-inflammatory
cytokine IL-10 and the Th2 cytokine IL-13 suppress LL-37 production313
while IL-6
synergizes with IFN-Ȗ to give a fast down-regulation424
. Furthermore, serum level of LL-37 is
inversely correlated with serum levels of interleukin-17, TNFĮ, IL-6, IL-1ȕ and IL-22451
.
LL-37 reduces the stimulatory effect of the TLR3 ligand, polyinosinic:polycytidylic acid
(polyI:C) a synthetic double stranded RNA (dsRNA), as measured by lower CCL5, CXCL10
and CXCL8 production454
while having synergic effect on flagellin stimulation in
keratinocytes leading to enhanced CXCL8 production451
. High concentration of LL-37 in
combination with the Th17 cytokines IL-17 or IL-22 induces the expression of IL-6 and
CXCL8455
. LL-37 stimulates Th1 polarization of monocyte-derived DC446
and attenuate their
activation by TLR ligands456
. Furthermore, pDC may take up LL-37 complexed to self-DNA
from psoriasis skin, which activates TLR9, leading to the secretion of IFN-D that activates
immune responses dependent on T cells457
. In the presence of TLR9 ligands, LL-37 enhances
keratinocyte secretion of IFN-ȕ458. LL-37 can also form a complex with self-RNA released
56
from necrotic cells that activate pDC and mDC through TLR7 and TLR8 respectively458
.
These self-RNA and LL-37 intertwined complexes have been located in psoriasis plaques459
.
Cytosolic DNA present within psoriasis keratinocytes, but absent in healthy cells, activate
inflammasomes to induce the post-transcriptional activation of IL-1E. However, LL-37 can
bind to the DNA and thus interfere with the process and lessen the inflammatory response459
.
1.10.2.6 LL-37 in skin
LL-37 is important for wound healing460
461
and recently it has been shown to be important for
sufficient stiffness of endothelial cells and maintenance of fluid homeostasis by affecting the
permeability of the skin barrier387
. Its expression in skin is dependent on the inflammatory
conditions, as it is upregulated in keratinocytes during inflammatory disorders462
and strongly
expressed in psoriatic plaques unlike atopic dermatitis352
LL-37 has also been shown to induce a pathological condition in the skin. In this respect, it
induces an inflammatory response in rosacea463
and is increased in various autoimmune
diseases464
. It is found in high levels in the skin and serum of SLE patients464
with positive
correlation to the level of pDC and IFN-D341, 352, 465. LL-37 is induced in psoriasis skin
compared to atopic dermatitis341
although one study did not observe any difference in mRNA
expression124
. The cytokines TNF-Į and IFN-Ȗ are understood to play a major role in the
pathogenesis of psoriasis352
and are also potent inducers of AMPs in the skin 457
. LL-37
expression is co-localized with a higher expression of TLR9 among keratinocytes in psoriasis
plaques where it could induce the secretion of type I interferons and contribute to the
pathology of the disease424
. Serum levels of LL-37 are higher among psoriasis patients than
healthy individuals424
. Interestingly, cyclosporine A treatment which inhibits T cell activation
and has been shown to abrogate T cell influx in skin, reduces the LL-37 serum
concentrations424
. Serum LL-37 levels correlate with IFN-Ȗ and IL-10 in psoriasis patients
while it was inversely linked to IL-17424
. This suggests that a positive feedback loop might be
present between LL-37 and IFNJ�while IL-17 linked loop might be negatively regulated262
.
57
1.12 Concluding remarks Psoriasis is a complex incurable skin disease that greatly influences the quality of life of the
patient. It is considered to be a T cell mediated skin disease but the causal factor has not been
identified. Its immunology has been thoroughly studied, showing the importance of both the
innate and the adaptive immune system.
The strong association between throat infections by E-haemolytic streptococci and
psoriasis initiation or exacerbation, indicates that the pathogenic T cells might originate in
tonsils. Studying the effects of tonsillectomy on the level of circulating skin homing T cells
and by comparing the immunology of recurrently infected tonsils from individuals with or
without psoriasis, might contribute greatly to our understanding of the disease.
59
2 Aims
The overall aim of the study was to investigate the role of palatine tonsils in the pathogenesis
of psoriasis in patients with known association of initiation or exacerbation following
recurrent throat infections.
2.1 The role of LL-37 in palatine tonsils (paper I) i. To evaluate the LL-37 expression in the palatine tonsils and establish which cell
types express the peptide.
ii. To compare the LL-37 expression of RT and PST tonsils.
iii. To evaluate if LL-37 influences the immune response of tonsil mononuclear cells
differently in PST and RT tonsils.
iv. To assess the influence of LL-37 on DNA uptake by tonsil DC and thereby its
potential importance in GC immune response.
2.2 Histological and microbiological characteristics of psoriasis tonsils (paper II) i. To compare the bacteriology of recurrently infected tonsils of individuals with or
without psoriasis, particularly in regard to E-haemolytic streptococci.
ii. To compare the histological characteristics of psoriasis and recurrently infected
tonsils, particularly in regard to the number and size of GC.
iii. To compare the surface receptor expression of T cells in PST and RT tonsils, in
particular the expression of skin homing molecules.
iv. To evaluate if there is a correlation between the expressions of surface molecules
on T cells in blood and tonsil.
2.3 The effect of tonsillectomy (paper III) i. To evaluate the effect of tonsillectomy on PASI score.
ii. To evaluate post-tonsillectomy levels of IL-8.
iii. To evaluate the frequency of tonsil T cells specific for the homologous M
protein/keratin peptides and compare their levels in blood before and after
tonsillectomy.
iv. To evaluate if there exists a correlation between blood and tonsil levels of peptide
specific T cells.
61
3 Materials and methods
3.1 Study approval The Ethics Committee of the Landspítali University Hospital, Iceland and The Data
Protection Authority approved all studies. Furthermore, The Icelandic Bioethics Committee
(VSNb2006090015/03-15) approved of the collection of personal data from psoriasis patients.
All study participants gave informed consent and the study was conducted in compliance with
good clinical practice and according to the Declaration of the Helsinki Principles.
3.2 Tonsil material and participants in the study Recurrently infected tonsils (n=41) and hypertrophic tonsils (n=8) were obtained through
routine tonsillectomies at The National University Hospital, Iceland. Information about the
participants’ sex, age and frequency of tonsillitis was gathered while withholding any
personal information. Tonsils were also obtained from a group of psoriasis patients (n=25).
Those individuals were 18 years of age or older with a history of recurrent throat infections
associated with exacerbation of their skin disease.
All patients had a confirmed diagnosis of psoriasis by their dermatologist prior to
participation. Furthermore, all patients had a known association between streptococcal throat
infection and worsening in their psoriasis plaques. All participants with psoriasis were off
treatment for a minimum of 4 weeks before entering the study and 8 weeks afterwards and
antibiotics were not permitted during the study. However, after these 12 weeks the patients
received treatment as needed as evaluated by their doctor.
The psoriasis patients were monitored for at least 2 years and personal information
gathered at each visit, first at study entry, then two months post-tonsillectomy and finally
every six months. Patients were evaluated using the standard method of Psoriasis Area and
Severity Index (PASI) scoring by a treatment-blinded observer at study entry and after 2, 6,
12, 18 and 24 months. Heparinized venous blood was collected at study entry and at 2, 12
and 24 months evaluation. An ear-nose and throat specialist examined the patients after
completion of the study to confirm the absence of tonsil remnants.
62
3.3 Preparation of tonsil tissue Immediately after excision, the tonsils were stored in cold sterile saline until processed.
Tissue from each tonsil was snap frozen in Tissue-Tek OCT compound (Sakura Finetek,
Zoeterwoude, NL) and stored at -70°C until prepared for immunohistochemistry. The tonsil
tissue was also prepared for ex-vivo cell culture studies.
3.4 Swabs and bacterial typing Swabs were taken from the surface epithelia and within crypts using a specimen collection
swab (Therapak Pharma Services Ltd., Middlesex, UK). Bacterial typing from throat swabs
was carried out by culture on sheep blood agar and Streptococcus subspecies were identified
using a Streptex kit (Thermo Fisher Scientific, Remel products, Lenexa, KS, USA).
3.5 Tonsil cell isolation procedures
3.5.1 Tonsil mononuclear cell (TMC) isolation and culture Tonsil mononuclear cells were isolated as previously described
466. Briefly, tonsils were
minced into 3 mm pieces, passed through a tea-sieve and washed with Hanks' balanced salt
solution (Gibco, Invitrogen Ltd, Paisley, UK). TMC were collected at the interphase fraction
formed by Ficoll density gradient centrifugation (Sigma-Aldrich, St.Louis, MO, USA) and re-
suspended in RPMI-1640 medium (Gibco) supplemented with 10% heat-inactivated fetal calf
serum (Gibco), 100 U mL-1
penicillin (Sigma), and 100 µg mL-1
streptomycin (Sigma) for cell
culture, or re-suspended in PBS for FACS (fluorescence activated cell sorting) analysis.
3.5.2 Peripheral blood mononuclear cell (PBMC) isolation and culture Heparinized venous blood was drawn from the psoriasis patients prior to the tonsillectomy.
PBMCs were collected at the interphase fraction formed by Ficoll and the same isolation
procedure followed as described for the TMCs. In the final step cells were resuspended in
supplemented RPMI-1640 for cell culturing or PBS for FACS analysis.
3.5.3 Isolation of CD4+ T cells TMC were suspended in cold MACS buffer (PBS containing 0.5% BSA (Sigma) and 2mM
EDTA) at a density of 107
cells mL-1
and the cells labeled with anti-CD4-PE (clone 3.9,
Miltenyi Biotec, San Diego, CA, USA) for 15 minutes at 4ºC in the dark, washed twice with
cold MACS buffer, re-suspended and incubated with anti-PE microbeads (Miltenyi Biotec)
for 15 minutes at 4ºC in the dark, after which the cells were washed twice with MACS buffer.
CD4+ T cells were magnetically isolated on a MACS MS column (Miltenyi), collected,
centrifuged and counted. The cells were resuspended in RPMI-1640 medium (Gibco)
63
supplemented with 10% heat inactivated fetal calf serum (Gibco), 100 U mL-1
penicillin
(Sigma), and 100 µg mL-1
streptomycin (Sigma). In some cases the isolated CD4+ T cells
were collected in an eppendorf tube and filled with 400 µL RIPA buffer (Cell Signaling
Technology, Danvers, MA, USA) and kept at -20°C for a later RT-PCR evaluation.
3.5.4 Isolation of myeloid dendritic cells Myeloid dendritic cells (mDCs) were negatively selected from bulk TMC cultures using a
myeloid dendritic cell isolation kit and LD column as instructed by manufacturer (Miltenyi).
The mDCs were then labeled with anti-CD11c-PE-cy5 (clone 3.9, Biolegend) on ice for 30
minutes in the dark and washed twice with cold MACS buffer. Cells were re-suspended and
incubated with anti-PE microbeads (Miltenyi) for 15 minutes at 4ºC in the dark, after which
the cells were washed twice with MACS buffer. CD11c+ cells were isolated on a MACS MS
column (Miltenyi), collected, centrifuged and counted and thereafter stained and isolated
using anti CD13-PE (cloneL138, Becton-Dickinson, San Jose, CA, USA (BD)) and anti-PE
beads (Miltenyi) with MS column (Miltenyi) for RT-PCR or anti-CD13 APC (clone WM15,
BD) and anti-APC beads (Miltenyi) for cell staining. An aliquot of the isolated cells was
analyzed after each isolation step using a FACScalibur cytometer and CellQuest software
(BD).
3.5.5 Isolation of plasmacytoid dendritic cells Plasmacytoid DCs (pDC) were negatively isolated from bulk TMC using plasmacytoid
dendritic cell magnetic isolation kit II and LD column as instructed by the manufacturer
(Myltenyi Biotec). The pDCs were then labeled with and anti-CD123-PE-cy5 (6H6,
Biolegend) on ice for 30 minutes in the dark and washed twice with cold MACS buffer. Cells
were re-suspended and incubated with anti-PE microbeads (Miltenyi Biotec) for 15 minutes at
4ºC in the dark, after which the cells were washed twice with MACS buffer. CD123+ cells
were isolated on a MACS MS column (Miltenyi Biotec), counted and phenotyped.
3.5.6 Isolation of CD68+ macrophages CD68+ macrophages were positively selected from bulk TMC cultures using magnetic
isolation. Cells were stained using the same basic method as when isolating CD4+ T cells.
TMC were stained with anti-CD68-PE (clone Y1/82A, Biolegend) on ice for 30 minutes in
the dark and washed twice with cold MACS buffer. Cells were re-suspended and incubated
with anti-PE microbeads (Miltenyi Biotec) for 15 minutes at 4ºC in the dark, after which the
cells were washed twice with MACS buffer. CD68+ cells were isolated on a MACS MS
column (Miltenyi Biotec), counted and phenotyped.
64
3.6 Cell stimulation with hCAP-18/LL-3 peptide Isolated TMCs were seeded at a final density of 2x10
6 cells mL
-1 and isolated CD4+ T cells at
106mL
-1. All TMCs were treated with 0, 1, 5 or 10µg mL
-1 of LL-37 peptide (Innovagen,
Sweden) for either 1 hour, 3 hours or 17 hours at 37°C in a humidified atmosphere of 5% CO2
and 95% air. Stimulation of TMC with LL-37 with or without human DNA 1:5 proportion,
with DNA concentrations of 0-2 µg mL-1
was carried out at a cell density of 1x106 cells mL
-1
for 17 hours. All the CD4+ isolated T cells were stimulated for 17 hours. Cells were
harvested and stained for FACS analysis or collected and kept in RIPA buffer (Cell Signaling
Technology) or RNA later (Sigma) at -30°C. Supernatant was stored at -70°C until required.
3.7 Staining procedures
3.7.1 Haemotoxylin and eosin staining Frozen tonsil tissue was sliced at -20°C into 5 µm sections using Leica CM 3050-S Cryostat
(Leica Biosystems, Wetzler, Germany). Sections were mounted onto a glass slide, air-dried
and fixed in cold aceton for 10 minutes and then washed in PBS. Sections were stained with
haemotoxylin (Thermo Shandon, Pittsburgh, PA, USA) for 20 seconds, washed with PBS and
then dipped into 37 mM ammonia solution after which sections were incubated in PBS for 10
minutes. Thereafter, the sections were stained with eosin (Sigma) for 60 seconds and
dehydrated in alcohol gradient of 70%-80% and 100% and fixed in Accustain (Sigma-
Aldrich, St. Louis, MO, USA) and mounted with Mountex (Histolab products AB, Göteborg,
Sweden). Each section was evaluated by a blinded observer.
3.7.2 Immunohistostaining (IHC) Fresh frozen 5 µm sections of tonsil were mounted onto a glass slide, air-dried and fixed in
2% paraformaldehyde for 15 minutes or acetone for 10 minutes, blocked with 1% hydrogen
peroxide in PBS containing 3% of the appropriate host serum, washed in PBS and blocked
with 1.5% of the appropriate serum dependent on the host of the secondary antibody used.
Sections were incubated with polyclonal or monoclonal antibodies (table 3) at an optimal
concentration determined by previous titration, for 30 minutes and thereafter stained
according to the Vectastain Elite ABC IgG or IgM kit protocol. The staining was visualized
using 3,3’-diaminobenzidine (DAB,� BD), counterstained with haematoxylin, dehydrated,
fixed and mounted as previously described. As a negative control, the primary antibody was
omitted or the appropriate isotype control antibodies used at the same concentration as the
primary antibody.
65
3.7.2.1 Specificity of the LL-37 staining
The antigen-specificity of the LL-37 staining was verified by incubating overnight at 4 °C the
synthetic LL-37 peptide (Innovagen) at a 10-fold molar excess with the LL-37 antibody. This
mixture was used for immunostaining the next day according to protocol. To exclude staining
due to contamination, the LL-37 antibody (Innovagen) was incubated at a 10-fold molar
excess with lipopolysaccharide (Sigma) overnight at 4°C and the mixture used for
immunostaining the following day.
Previously published data on LL-37 staining in the tonsils293
revealed a strong and general
staining within the squamous epithelium in contrast to our data. A generous gift of Dr. Ole
Sorensen (Lund, Sweden) of rabbit LL-37 anti-serum used in those experiments was obtained.
After titration of the anti-serum a comparison was done to the purchased LL-37 antibody
(Innovagen) used in our experiments using DAB substrate for visualization.
3.7.2.2 Double staining with LL-37
Some sections were double stained where the staining procedure of the primary antibody (LL-
37) was completed by visualizing with DAB (BD) and then the staining procedure was
repeated for the second antibodies: CCL5 (R&D Systems), CXCL9 (R&D Systems), CCR4
(Abcam) and CCR6 (R&D Systems). Those sections were mounted directly with Mounting
Gel (Sigma-Aldrich). One blocking step was added to the staining protocol, where
Avidin/Biotin blocking kit (Vector) was used after blocking with the appropriate serum.
Vectastain Elite goat or mouse IgG kit protocol (Vector) was used and the second antibody
was visualized using either AEC (BD systems) or New Fuchsin substrate kit (BD systems)
giving a red colour in contrast to the brown colour of the DAB. These sections were mounted
with Gelmount aqueous mounting medium (Sigma).
3.7.2.3 Staining of isolated tonsil dendritic cells
After isolation, cells were incubated on a glass slide at 37°C for two hours allowing them to
regain their dendritic morphology. The slides were air-dried for 30 minutes and fixed with
100% ethanol for 10 minutes. The following day slides were stained with an antibody against
LL-37 as previously described.
66
Fluorescence immunohistochemistry (FIH) Fresh frozen tissue sections were air-dried, fixed in 2% w/v paraformaldehyde in PBS and
blocked first with 50 mM ammonium chloride and then with 10% of the appropriate serum.
Thereafter tissue sections were incubated overnight in a humidified chamber at 4°C with the
primary antibody (table 3). The next day, after washing in PBS, the following fluorescently
labeled secondary antibodies were added at room temperature in the dark for one hour:
TexasRed goat anti-rabbit IgG (Invitrogen, Eugene, Oregon, USA), AlexaFluor488 goat anti-
mouse IgG (Invitrogen) or AlexaFluor350 goat anti-mouse IgM (Invitrogen). The samples
were mounted with Gelmount aqueous mounting medium (Sigma). Either the primary
antibodies were omitted for negative controls, or replaced with the appropriate isotype control
antibodies (Dako).
3.7.2.4 Double and triple fluorescent staining
For double and triple immunostaining I utilised primary antibodies from different host species
or different Ig isotypes, which allowed us to follow the same procedure as used for single
immunostaining. After completion of the blocking steps, all primary antibodies were
incubated at once overnight in a humidified chamber at 4°C. In this way, the LL-37 antibody
was diluted to the appropriate concentration and the second and third primary antibody added
to the mixture in the right concentration. These primary antibodies and the optimal
concentrations are listed in table 3.
The following day, after washing with PBS, the secondary antibodies were added to the
sections using the same method as with the primary antibodies. In this way, the appropriate
secondary antibodies were mixed together according to the right concentration and added to
the sections for one hour after which they were washed with PBS and mounted with
Gelmount aqueous mounting medium (Sigma). The secondary antibodies were the same as for
the primary staining protocol except for CCR6 and CCR4 which were pre-labelled using a
Zenon Alexa fluor 488 Goat IgG labelling kit (Invitrogen) as instructed by manufacturer and
using biotinylated anti-goat IgG secondary antibody (Vector) and Streptavidin AlexaFluor
488 conjugate (Invitrogen) for their visualization.
67
Table 3. Antibodies used for immunohistostaining (IHC) and fluorescent immunohistochemistry (FHC). 1IHC, 2FHC. Antibody Dilution Clone Type Manufacturer LL-37 1/1640
1, 2 n/a Polyclonal rabbit IgG Innovagen, Lund,
Sweden
CD68 1/20001
1/10002
KP1 Monoclonal
mouse anti-human IgG
Santa Cruz Biotechnology,
California, USA
Neutrophil elastase
1/500 1, 2
NP57 Monoclonal
mouse anti-human IgG
DAKO, Glostrup,
Denmark
CD15 1/500 1, 2
C3D-1 Monoclonal
mouse anti-human IgM
DAKO
CD13 1/32 VS5E Monoclonal
mouse anti-human IgG
Novocastra, Newcastle
upon Tyne, UK
CD11c 1/52 5D11 Monoclonal
mouse anti-human IgG
Novocastra
CD123 1/1002 IL-23 RD Monoclonal
mouse anti-human
BD
CCL5 1/201 n/a Polyclonal
goat anti-human IgG
R&D systems
CXCL9 1/201 n/a Polyclonal
goat anti-human IgG
R&D systems
CCL18 1/102 n/a Polyclonal R&D systems
CCR4 1/301 n/a Polyclonal
goat anti-human IgG
Abcam, Cambridge, UK
CCR6 1/301 53103 Monoclonal
mouse anti-human IgG
R&D systems
Cytokeratin8 1/10002 M20 Monoclonal
anti-cytokeratin
peptide 8
Sigma-Aldrich
fDC 1/1001 CNA.42
Monoclonal
mouse anti-human IgG
DAKO
Ki67 1/4001 D3B5 Monoclonal
rabbit anti-human IgG
Cell Signaling Technology
(Beverly, MA)
Caspase-3 1/2001 D3E9 Monoclonal
rabbit anti-human IgG
Cell Signaling Technology
MCA8746 1/50001,2
MAC387 Mouse anti-human IgG Serotec
Mature mØ MCA1122
1/201, 2
RFD7 Mouse anti-human IgG Serotec
68
3.8 Histological evaluation Histological characteristics of PST, RT and HT tonsils were compared using H&E stained
tonsil tissue. The circumference of the follicles, germinal centres and mantle zones were
evaluated using Axiovision 4.6.3 software (Carl Zeiss microscopy, Jena, Germany) at 25 or
100x magnification in two to four locations within the same tissue. Measurements were
gathered in mm2. Follicles were counted in the visual field at 25x magnification from a
minimum of four locations within each tonsil tissue. All tonsil sections were coded
beforehand and evaluated by a blinded observer. The number of macrophages within
germinal centres and follicles were counted at randomly selected locations in 3-4 follicles at
100x magnification for one tissue section for each tonsil. The number of crypts was counted
at 25x magnification to distinguish between long, short and intertwined crypts while the
thickness of the squamous epithelium was measured at 100x magnification.
Immunohistologically stained cells were counted in the 400x visual field from three locations
within the tonsil tissue. Evaluation of cell staining and condition of tissue was done by a
blinded observer using a three level grading system comparing RT, HT and PST tonsils.
3.9 Cytokine detection and quantification in cell supernatant Enzyme linked immunosorbent assay (ELISA) is a sensitive method for measuring soluble
analytes such as cytokines and chemokines. Its basic principle is an antibody binding
specifically to an antigen of unknown concentration that is detected using an appropriate
secondary antibody coupled to an enzyme that changes the colour of a specific substrate.
Either the colour absorbance (colorimetric) or fluorescent signal (fluorimetric) of the substrate
is measured and concentration of the samples evaluated from a set of standards of known
concentration. Non-specific binding is typically avoided by selecting specific antibodies that
perform well under these conditions. Bovine serum albumin is often added to reagents and
solutions to help reduce non-specific binding in the assay.
Cell culture supernatants were collected and stored at –70°C until used. CCL5, CXCL1
and CXCL9 were measured using Duo-Set ELISA assays as instructed by manufacturer
(R&D Systems). 100 PL of supernatants and standards in the appropriate dilutions were
measured in duplicates. The optimal density of each well was determined using a microplate
reader set at 450nm (Titertek Multiscan ® Plus MK II; ICN Flow Laboratories, Irvine, UK)
and each sample evaluated using a 7 point standard curve.
Serum CXCL8 was measured using the Quantikine Elisa kit (R&D systems) as instructed
by manufacturer. Cytokine concentration was measured at study entry and after 24 months.
69
50 PL of standard or serum was measured in duplicates. The optical density was determined
at 450 nm and CXCL8 concentrations calculated from the standard curve.
CCL5, CXCL1 and CXCL9 were measured using DuoSet ELISA assays as instructed by
manufacturer (R&D Systems). The assays had the following detection limits: CCL5, 15.6 -
1000 pg/ml, CXCL1, 31.2 – 2000 pg/ml and CXCL9, 15.6 - 1000 pg/ml. (Cytokine
concentrations were calculated from a 7-point standard curve.
3.10 Expression of cell surface receptors Flow cytometry was used to evaluate the expression of various cell surface receptors on the
surface of isolated TMCs. Isolated tonsil mononuclear cells were stained for 30 minutes on
ice with various antibodies (table 4) or the appropriate isotype controls. Cells were washed
twice with PBS and fixed in 0.5% PFA. Stained cells were analysed on a FACSCalibur (BD)
flow cytometer with CellQuest (BD) software.
3.11 Real-time RT-PCR Total RNA was isolated from cells (RNeasy Mini kit, Qiagen) and 200 ng of RNA template
was reverse transcribed using a high capacity cDNA reverse transcription kit and random
primers (ABI, Foster City, CA, USA). RT-qPCR was carried out on a 7900HT Fast real time
PCR system (ABI). Primers for IFN-J (Hs00989291_m1), MX-1 (Hs00182073_m1), CCL5,
CXCL9, CCR4, CCR6, TBX21 and the housekeeping gene RPLP0 (36B4, Hs99999902_m1)
were obtained from ABI. Results were normalized to the expression of RPLP0.
70
Table 4. List of antibodies used for evaluation of cell surface receptor expression and intracellular cytokine production, using FACS analysis. AF is an abbreviation for Alexa Fluor.
Antibody Label Clone Manufacturer
CD4 PE Clone 3.9 Biolegend, San Diego, CA, USA
CD4 Percp-cy5.5 RPA-T4 Biolegend, San Diego, CA, USA
CD8 Percp-cy5.5 RPA-T8 Biolegend San Diego, CA, USA
CD13 PE L138 Becton Dickinson (BD), NJ, USA
CD11c PE-cy5.5 Clone 3.9 Biolegend, San Diego, CA, USA
CD62L FITC DREG-56 Biolegend, San Diego, CA, USA
CD54 PE HA58 Becton Dickinson (BD), NJ, USA
CLA FITC HECA-452 Biolegend, San Diego, CA, USA
CD69 PE L78 Becton Dickinson (BD), NJ, USA
CD25 APC BC96 Biolegend, San Diego, CA, USA
CCR4 PE 205410 R&D Systems, Minneapolis, USA
CCR5 APC HEK/1/85a Biolegend, San Diego, CA, USA
CCR6 APC 53103 R&D Systems, Minneapolis, USA
CCR7 APC 150503 R&D Systems, Minneapolis, USA
CCR8 APC 191704 R&D Systems, Minneapolis, USA
CCR10 APC 314305 R&D Systems, Minneapolis, USA
CXCR4 PE 12G5 Biolegend, San Diego, CA, USA
CXCR5 FITC 51505 R&D Systems, Minneapolis, USA
CXCR5 FITC 51505 R&D Systems, Minneapolis, USA
CXCR6 PE 56811 R&D Systems, Minneapolis, USA
IL23R PE 218213 R&D Systems, Minneapolis, USA
IL-17A AF 647 eBio64CAP17 eBioscience, San Diego, USA
IFN- Ȗ PE 4S.B3 Biolegend, San Diego, CA, USA A
3.12 Peptide stimulation
3.12.1 Homologous keratin and streptococcal peptide antigens
As previously described293
streptococcal M6 protein and keratin 17 (K17) share amino acid
sequences homology. The significance of this finding for psoriasis was evaluated by
stimulating with overlapping residue peptides of M6 protein and K17. This was thought to be
an efficient method to identify the peptides of most significance. K17 was divided into a
complete set of overlapping residue peptides that were compared to the M6 protein using the
FASTA algorithm which is a DNA and protein sequence alignment software package used to
identify similar protein or nucleic acid sequences. The results showed that the homologous
71
peptides were restricted to the conserved C-terminal end of the M6 protein. The homologous
K17 peptides were found in two regions located in the coil-forming part of the keratin.
Furthermore, four to six amino acids were shared by each homologous M and K17 peptide.
From those, only peptides predicted to bind to HLA-Cw*0602 were selected leading to 64
short, overlapping peptides that were mostly 9-12 amino acids long. 16 peptide pools were
established by blending together 8 peptides (paper III, supplement table 1).
3.12.2 Determination of peptide-reactive T cells
Isolated peripheral blood mononuclear cells (PBMCs) or TMCs were stimulated and cultured
in cell culture tubes (Nunc, Thermo Fisher Scientific, Roskilde, Denmark) for 16 hours at a
final density of 1x106
cells mL-1
. The cells were stimulated with the 16 peptide pools (2 ȝg/ml
total peptide each) and anti-CD28 and anti-CD49d needed as costimulation (1 ȝg/ml each;
Serotec). Brefeldin A solution (3 ȝg/ml; eBioscience, San Diego, CA, USA) was added to the
cell cultures after two hours of stimulation to inhibit the secretion of cytokines into the cell
culture and establish an intracellular build up which can be evaluated with FACS staining. A
final cell culturing for 14 hours was completed at 5° slant at 37°C in a humidified 5% CO2
atmosphere. Positive controls for cytokine stimulation were used; anti-CD3 antibodies (1
ȝg/ml; Serotec Scandinavia) and streptokinase (200 U/ml; Hoechst Marion Roussel,
Stockholm, Sweden). As most of the responding T cells express the skin homing molecule,
CLA466, 467
it was used to identify the cells of interest along with anti-CD4, anti-CD8
antibodies (table 1). After staining on ice for 20 minutes in the dark, cells were washed first
with twice with PBS and then fixed with 500 ȝl cold (4°C) 2% paraformaldehyde for 10
minutes and then with PBS. Finally, cells were prepared for intracellular cytokine staining by
treating them with a permeabilizing buffer (0.5% BSA, 0.1% saponin, 0.1% sodium azide;
Sigma-Aldrich) for 10 min at room temperature. Cells were washed again and resuspended.
Cells were stained with anti–IFN-Ȗ-PE and anti–IL-17A-AF647 (Table 3) for 20 min at 4°C in
the dark. Finally cells were washed in 1.5 mL permeabilizing buffer, centrifuged at 500 x g,
resuspended in a fixing buffer consisting of permeabilizing buffer with 1% paraformaldehyde.
Lymphocytes were analyzed using a FACSCalibur flow cytometer capturing a minimum of
200,000 events guided by appropriate isotype control Abs.
3.13 HLA-Cw*0602 typing
Genotyping of blood mononuclear cells for HLA-Cw*0602 alleles was performed as
previously described297
.
72
3.14 Expression of the data and statistics
Initially, data were tested for normality after which a parametric or non-parametric test was
selected as appropriate. P values of less than or equal to 0.05 were considered significant.
The effects of LL-37 were evaluated using one-way ANOVA for paired samples with
Dunnett's multiple comparison test as a post-test. In general, comparison of two groups was
done using a 2-tailed t-test or Mann Whitney test as appropriate. Comparison of the bacterial
infections between groups was analysed using Fisher’s exact t-test. Correlation data were
evaluated using Spearman´s test or linear regression as appropriate.
Intracellular flow cytometry data were interpreted as positive when more than 0.05%
events were judged positive compared with unstimulated control samples or fluorescence
minus one controls. Evaluation of the intracellular peptide stimulation data was done by
determining the percentage of CLA+ T cells in all the 16 peptides pools after which the
results were normalized using square root normalization and averaged for each patient. After
testing for normality, comparison between time points and different groups was evaluated
using ANOVA test for repeated measurements. A specific statistical program, R version 2.1,
was used evaluate the correlation between peptide responses (The R Foundation, Vienna,
Austria).
73
4 Results
4.1 hCAP-18/LL-37 expression and function in palatine tonsils (paper I) The expression of hCAP-18/LL-37 in RT, HT and PST tonsils was evaluated using
immunohistochemistry of fresh frozen 5 Pm sections stained with 1PgmL-1
of polyclonal
rabbit IgG hCAP-18/LL-37 antibody and visualized using DAB chromogen. LL-37 staining
was observed within the crypt reticulated epithelium and in infiltrated areas of the squamous
epithelium (Figure 1b-d, paper I). LL-37 positive cells were found scattered around the tissue
and in some tonsils LL-37 positive follicles were identified (Figure 1a-c, paper I).
4.1.1 hCAP18/LL-37 is expressed by leukocytes in the squamous epithelium Contrary to previous publications
466 467
480
, hCAP18/LL-37 expression within the squamous
epithelium was only identified in leukocyte infiltrated areas (n=40). This is contrary to
previous publications where LL-37 has been reported to be strongly expressed by tonsil
squamous epithelium, in a similar manner to inflamed skin466
480
. To ascertain if the observed
difference was due to variation in methodology or reagents, I obtained the rabbit anti-serum
used in previous study466
for comparison (generously provided by Dr. Sørensen, Lund
University, Sweden). Staining with the original concentration of the anti-serum466
resulted in
a strong epithelial staining with a high non-specific background throughout the tissue (Figure
1e, paper I). Application of the anti-serum in the same concentration as used in our study
reduced the background staining without reducing the leukocyte staining within the squamous
epithelium (Figure 1f, paper I). This strongly suggested that the squamous epithelium did not
express the peptide.
To determine whether epithelial cells or leukocytes were expressing hCAP18/LL-37, a
double immunofluorescent staining was performed using antibodies against LL-37 and
leukocytes. In this respect, CD15 (Figure 1g-I, paper I) and neutrophil elastase was used to
identify neutrophils (Figure 2) and CD68 for tissue macrophages (Figure 1j-m, paper I).
Fluorescently labelled secondary antibodies were used for visualization giving a red signal for
LL-37 and green colour for the leukocytes. A complete co-localization, resulting in yellow
signal, was observed between LL-37 and CD15 (Figure 1i, paper I) and elastase (Figure 2d-f).
However, LL-37 seldom co-localized with CD68 in the squamous epithelium (Figure 1j-m,
paper I).
74
Figure 2. The expression of neutrophil elastase in tonsil.
Fresh frozen tonsils were cut into 5 ȝm sections and prepared for immunohistochemical detection. Single
staining with neutrophil elastase was visualized using the Fuchsin colour (a-c). Neutrophils were clearly visible
in infiltrated areas of the squamous epithelium from and within the stroma (a, 5x and b, 40x magnification). In
infected tonsils, neutrophils were observed migrating through the tissue (c, 10x magnification). Double immune-
fluorescence microscopy revealed that the LL-37 positive cells in the squamous epithelium (e) were neutrophils
(d) with the majority co-localizing (f, 10x magnification).
Figure 3. LL-37 positive cells are observed migrate through some follicles.
In some tonsils, LL-37 expressing cells can be found in both the MZ and the GC (a, see arrows). These cells
appear to be migrating through the GC and are most likely neutrophils on their way to the surface as shown with
neutrophil elastase staining (1b) (40x magnification).
75
4.1.2 hCAP18/LL-37 is expressed by leukocytes within the tonsil crypts Previous studies have shown the crypt reticulated epithelium to have a strong expression of
LL-37220
and here I confirm those findings (Figure 1c, paper I). Furthermore, the LL-37
staining within the tonsil tissue, was strongest within the cryptal area. Double and triple
immunofluorescent stainings was performed to ascertain whether the crypt epithelial cells or
if other cell types associated with the epithelium or crypts were the source of the peptide
expression (Figure 2, paper I). To specifically identify the crypt reticulated epithelial cells, I
used an antibody against cytokeratin 8456
which has been shown to be a specific marker for
these cells. The leukocytes were distinguished as before using antibodies against CD15,
neutrophil elastase and CD68. All sections were also stained for LL-37 (Figure 2, paper I).
The crypt reticulated epithelial cells did not express LL-37 as no co-localization was
detected with cytokeratin 8 (Figure 2a-d, paper I). On the other hand a strong co-localization
was observed between the neutrophil markers and LL-37 (Fig 2e-h) as within the squamous
epithelium. To verify that neutrophils and not macrophages were the main LL-37 expressing
cell type, a double fluorescent staining was performed. Various macrophage markers were
selected to identify macrophages in different maturation states. In this respect, LL-37 was
double stained with CD68 (Figure 3a-c, paper I) identifying a 110 kD intracellular
glycoprotein belonging to the scavenger receptor family that is commonly used to identify
macrophages, RFD7 (Figure 3d-f, paper I) that identifies mature tissue macrophages or
MCA8746 (Figure 3g-i, paper I). Macrophages were rarely co-localized with LL-37 thus
confirming that neutrophils are the dominant LL-37 expressing cell type within the crypts.
In some tonsils, a few infiltrating LL-37 positive cells, identified as neutrophils, were
observed close to the follicles and in rare instances within them (Figure 3).
4.1.3 hCAP18/LL-37 is expressed in the lymphoid follicles Immunohistochemistry revealed the presence of LL-37 within the follicles of a subset of
tonsils. Further analysis showed LL-37 expression in 14 out of 40 tonsils (36%) examined,
regardless of pathology (Figure 1c and 4a, paper I). Given the function of the follicles in
driving humoral responses and the expression of Fc receptors on many follicular cells, the
specificity of the LL-37 staining was first verified by incubating the LL-37 antibody with a
ten-fold molar excess of the LL-37 peptide overnight at 4°C before staining the sections. This
completely abolished all LL-37 staining in the tissue (Figure 4a, paper I) demonstrating that
the signal was most likely from the antibody binding LL-37 in the tissue, rather than
antibody-Fc receptor interactions.
76
To determine the identity of the LL-37 expressing follicular cells, a double- labelled
fluorescent immunohistochemistry was administered using CD68 for GC macrophages and a
number of specific DC markers: PNA.42 for follicular DC, CD11c and CD13 for GC-DC and
CCL18 for CCL18+ GC-DC. As before, CD68+ macrophages (Fig. 4d, paper I) did not
express LL-37 while follicular DC (Fig. 4c, paper I) and CCL18+ DC were weakly positive
for the peptide (Fig. 4b, paper I). Interestingly, CD11c+ DCs expressed LL-37 to a certain
extent and CD13+ DCs were most clearly co-localized with the peptide staining (Figure 4f,
paper I). As CD13+ DCs also express CD11c, it seems that CD11c+CD13+ DC cells are the
main cell type expressing the peptide within the tonsil follicles. Staining of the tonsil tissue
with CD13 revealed that it is strongly expressed in general by cells in the extrafollicular area
(Figure 4g, paper I). However, the co-localization detected within the follicles was absent in
the extrafollicular area (Figure 4g, paper I).
4.1.3.1 The CD11c+CD13+ follicular dendritic cells express LL-37
The CD11c+CD13+ phenotype of follicular cells was verified by magnetic cell isolation and
flow cytometry. CD11c+ cells were obtained by negative selection from bulk TMC cultures
and then positively selected for CD11c and thereafter CD13 using LD and MS magnetic
columns respectively. Lineage (CD3, CD14, CD16, CD19, CD20
and CD56) negative
CD11c+CD13+HLA+DR+ cells were identified by flow cytometry (figure 5 a-d, paper I). An
aliquot was added to a glass slide, incubated for an hour at 37°C to allow cells to regain their
cell morphology and then stained for the presence of LL-37 (Figure 5 e, paper I). Dendritic
cells of the lin-HLA-DR+CD11c+ phenotype had DC morphology and expressed LL-37
(Figure 5e, paper I).
4.1.4 LL-37 modulates chemokine and cytokine expression of tonsil mononuclear cells
In order to study the significance of the observed LL-37 expression by the GC-DC, isolated
TMCs were cultured in vitro with the LL-37 peptide at 0, 1, 5 or 10 PgmL-1
concentrations
and CCL5 and CXCL9 chemokine production evaluated. These chemokines are of particular
interest as both chemokines can amplify antimicrobial defences468
469
. Furthermore, CCL5 has
been shown to stimulate both early perivascular recruitment315
of T cells and follicular
migration of T cells470
and CXCL9 has immunomodulatory effects471
.
77
Figure 4. TMC stimulated with LL-37 for 1 hour influences the production of CCL5.
TMCs were stimulated in complete medium with LL-37 and the chemokine production evaluated using ELISA.
LL-37 significantly decreased the CCL5 production of TMCs when stimulated with 1 or 5PgmL-1
LL-37
(p=0.008, n=10).
Figure 5. TMCs stimulated with LL-37 for 17 hours influences the chemokine and cytokine production.
Tonsil mononuclear cells were isolated and stimulated with LL-37 for 17 hours and the chemokine and cytokine
production measured using ELISA method. LL-37 dose dependently increased the production of CCL27 (a,
p=0.0044, n=14) while decreasing CXCL1 production (b, p=0.0198, n=13). LL-37 induced the production of
IL-17 (c, p=0.0373, n=6, non-parametric Friedman test), CXCL9 (d, p=0.022, n=6) and at 1 PgmL-1
concentration of the peptide CXCL10 (d, p=0.0096, n=6). Although LL-37 did not influence the production of
IFNJ, it stimulated production of CXCL10 in low doses and CXCL9 in high doses (see also figure 5c in thesis
and figure 6b in paper 1) indicating a role in enhancing the effect of of IFNJ.
78
Stimulation for 1 or 3 hours had limited effects on CCL5 production by TCM (Figure 4).
However, a dose- dependent response was observed for the production of CCL5 and CXCL9
(p=0.022 and 0.0003 respectively, Figure 6a and b, paper I) and CCL27 after 17 hours
stimulation (Figure 5a, p=0.0044). Interestingly, the production of CXCL1 was slightly but
significantly decreased (Figure 5b, p=0.0198). Stimulation with 1 PgmL-1
of LL-37
significantly increased the production of IL-17 (Figure 5c, p= 0.0373) and CXCL10 (Figure
5d, p= 0.0096). LL-37 stimulation did not influence the production of IFND, IL12-p40,
CCL3, CCL4, CCL2, VEGF, TNFD or IFNJ. Although LL-37 had limited effect on IFNJ it
induced the production of the IFNJ-induced chemokines CXCL9 and CXCL10 indicating a
synergistic effect on IFNJ.
4.1.5 LL-37 influences the expression of tonsil mononuclear cell surface receptors.
Isolated TMC were stimulated with LL-37 for 1 or 17 hours as previously described. Cells
were stained with fluorescently labelled antibodies for 30 minutes on ice, washed and
analysed using flow cytometry. Stimulation with 5 PgmL-1
LL-37 for 1 hour decreased CD8+
T cell expression of the GC marker CD57 (figure 6, p= 0.0443). The adhesion molecule CD54
was significantly decreased after stimulation with 10 PgmL-1
LL-37, for both CD8+ and
CD4+ T cells (figure 6, p= 0.0412 and 0.0394 respectively).
Figure 6. LL-37 stimulation for 1 hour has limited effect on the expression of T cell surface receptors.
LL-37 stimulation influenced CD8+ T cell expression of the GC related molecule, CD57 (a, p=0.0443, n=6) in
intermediate doses and expression of the adhesion molecule CD54 in high doses in both CD8+ T cells (b,
p=0.0412, n= 6) and CD4+ T cells (c, p= 0.0394, n=11). Statistical significance determined with ANOVA and
indicated with * P<0.05.
79
Longer stimulation of TMC for 17 hours with the LL-37 peptide has greater influence on
the immune response. In this respect, LL-37 significantly decreased the frequency of tonsil
CD4+ T cells expressing the homing molecules CCR6 (p=0.0001, n=14, Fig. 6d, paper I) and
CCR4 (p=0.0292, n=14, Fig. 6c, paper I), CD54 (p=0.0017), CLA (p= 0.0372) and CXCR6
(p=0.037) while enhancing CXCR3 expression at 5ug/mL concentration (p=0.0131) (figure
7b). LL-37 had some effect on CD8+ T cells by inducing CD57 expression (p= 0.0196) and
reducing the frequency of CCR4+ T cells (p= 0.0131) (figure 7a).
Figure 7. Stimulation with LL-37 for 17 hours influenced T cell expression of surface receptors.
Stimulation with LL-37 in high doses induced CD8+ T cell expression of CD57 (p=0.0196, n=5) while reducing
expression of CCR4 (p= 0.0131, n=10). Expression of the adhesion molecule CD54 was decreased with higher
concentration of LL-37 (p=0.0017, n=9). The homing receptor CLA was lower when stimulated with 1 or 5
Pg/L (p=0.0372, n=10). The chemokine receptors CXCR3 (p=0.0131, n= 9) and CXCR6 (p= 0.037, n= 9) were
influenced by LL-37 at 5 Pg/mL and 10 Pg/mL respectively.
4.1.6 Tonsil CCL5 and CXCL9 expression is concomitant with LL-37 The perceived interactions between LL-37, CCL5 and CXCL9 gave reason to examine if an
association was present between expression of the chemokines and the peptide within the
tissue. Tonsil tissue sections were cut into 5Pm thick sections, dried, fixed in acetone and
double-stained with LL-37 and CCL5 or CXCL9 using DAB (brown) for LL-37 and Fuchsin
red for the chemokines. CXCL9 was strongly expressed in the area closest to the GC’s
(Figure 7a and b, paper I) as well as the extrafollicular area and the crypt reticulated
epithelium. Various infiltrating cells in the tissue stroma, cryptal area and vessels were also
shown to expressed CXCL9. Most of these areas appeared to express LL-37 as well.
Interestingly, some follicular CXCL9 expression was present that co-localized to some extent
with LL-37 (Figure 7a, paper I). Furthermore, histological comparison of the GC expression
revealed that LL-37 expressing follicles had a stronger CXCL9 expression (Figure 7a and b,
80
paper I) although no difference was observed in CXCL9 expression elsewhere within the
tissue. CCL5 expression in the tonsils showed a similar pattern as it was strongly expressed
by the crypt reticulated epithelium, by infiltrating cells and the extrafollicular area,
particularly in close proximity to the follicles (Figure 7c and d, paper I). Follicular CCL5
expression co-localized somewhat with LL-37 (Figure 7c, paper I). Tonsils with positive LL-
37 follicles had an overall stronger expression of CCL5 (Figure 7c and d, paper I).
4.1.7 DNA-LL-37 complexes instigate a Th1 response in the TMC cultures Plasmacytoid dendritic cells (pDC) do not respond to self DNA alone but when it is bound to
LL-37 the resulting complex has been shown to bind TLR9458
and activate pDC. Furthermore,
both myeloid (mDC) and pDC have been shown to be activated by self-RNA bound to LL-37
by TLR8 and TLR7 respectively456
. The palatine tonsils contain a mixture of various cells,
including mDC and pDC that constitute less than 1% of the total TMC population (figure 5b,
paper I). To investigate the influence of LL-37 on tonsil dendritic cells and T cells, I
stimulated TMC culture for 3 hours with 0, 1, 5 or 10 PgmL-1
of LL-37 peptide or in complex
with human DNA in 1:5 proportion. After stimulation the cells were collected and preserved
in RIPA buffer for RT-PCR evaluation or stained for flow cytometry and the expression of
surface receptors evaluated. A significant increase in IFN-Ȗ and MX-1 mRNA transcript
expression was measured after stimulation with the complex whereas the peptide or the DNA
alone did not have any influence (Figure 6e and 6f, p<0.05 for both, paper I).
Isolated CD4+ T cells did not appear to be influenced by the LL-37 as neither CCR4 nor
CCR6 levels changed and the secretion of the chemokine CXCL9 remained unaffected, even
in the presence of DNA (data not shown).
81
4.2 Psoriasis tonsils are histologically and microbiologically distinctive with skin homing T cells (Paper II)
4.2.1 E-haemolytic streptococcus is more common in psoriasis tonsils Throat infections by ȕ-haemolytic streptococci have been shown to be associated with the
severity of skin plaques (paper III). In order to investigate if the bacterial flora in recurrently
infected tonsils differed between psoriasis patients and individuals without the disease, tonsil
swabs were analysed and compared. All the psoriasis patients had a history of worsening in
skin symptoms ensuing a throat infection176 while the RT tonsils were obtained during routine
tonsillectomy, providing a group gathered by chance. All participants in the study had on
average four symptomatic infections annually. However, E-haemolytic Streptococci in general
(group A, B, C and G) and Streptococcus C were more frequently isolated from psoriasis
tonsils (Figure 1, p=0.021 and p=0.036 respectively, paper II). Furthermore, infections by
other bacterial species (Figure 1, 6/25 versus 2/41, p=0.0455, paper II) and co-cultured
bacteria (6/25 versus 2/41, p=0.0455) were also more common. There was no difference in
the number of un-infected tonsils while the bacterial species isolated were more numerous in
the psoriasis tonsils (24 cases of bacteria in 25 PST tonsils versus 21 case of bacteria in 41 RT
tonsils (Table 1, p=0.0001, paper II).
The squamous epithelium acts as a barrier against infections which generally occur through
the reticulated crypt epithelium due to its thin and disrupted membrane472
. A comparison of
the bacterial swabs from the crypts and surface showed no difference in the number of
isolated bacterial species within the same tonsil (data not shown). A clear correlation was
observed between a positive streptococcal throat swab some time prior to tonsillectomy and a
positive bacterial culture from the isolated psoriasis tonsils (table 5, p= 0.0001). In contrast,
tonsils that were negative for streptococcal infections (n=11) were mostly negative in the
throat swab (n=10).
Tobacco smoking appears to increase the vulnerability to bacterial infections among the
psoriasis patients (Figure 9, p=0.052). However, it is not clear how smoking affects the RT
tonsils as no personal data were gathered from that group. Furthermore, smoking did not
appear to influence the histological structure of the PST tonsils although some studies have
shown various negative effects473
.
82
Figure 8. Infected palatine tonsils are more common among psoriasis smokers than non- smokers.
PST tonsils from smokers were more often infected than the non-smokers (p=0.052). Further analysis of the data
revealed no difference in the total number of psoriasis tonsils infected by streptococcus. Y- axis represents the
number of psoriasis tonsils for each column. Fisher´s exact test.
Table 5. There is a positive association between streptococcal infections in tonsils and throat swabs prior to tonsillectomy.
There is a significant correlation between streptococcal infections prior to tonsillectomy and culture of the
bacteria from the tonsils post tonsillectomy, p=0.0001. Fisher’s exact t-test.
Tonsils
Throat swab Streptococcus Negative
Streptococcus (n=18) 17 1
Negative (n=11) 1 10
4.2.2 Psoriasis tonsils are histologically different The histological characteristics of the tonsils were investigated to determine if any underlying
differences were present in recurrently infected tonsils from individuals with or without
psoriasis. Tonsils were cut into 5 mm sections, air-dried, fixed and stained with haemotoxylin
and eosin. The number of lymphoid follicles was obtained and the circumference of the
follicles measured to include both the GC and the MZ. Psoriasis tonsils had on average the
smallest follicles (Figure 2c and 2d, paper II) while the hypertrophic tonsils had the largest
(Figure 2a and 2d, paper II). The difference was both due to larger mantle zones (Figure 2f,
paper II) and larger GC (Figure 2e, paper II) within the HT tonsils. The RT follicles were
mostly larger than the PST tonsils (Figure 2b and 2d, paper II) while the MZ area was
comparable in size (Figure 2f, paper II). Interestingly, some follicles had very small GC
(Figure 2b and 2c) while others had negligible MZ (Figure 2a and 2b, paper II).
83
4.2.3 Hypertrophic tonsils have enlarged lymphoid follicles Calculation of the relative proportion of the GC and the MZ area showed that hypertrophic
tonsils had follicles dominated by GC compared with RT and PST tonsils (Figure 3a-3d,
p=0.005 and p= 0.02 respectively, paper II). Conversely, follicles in psoriasis tonsils were
predominantly made up of the MZ, which was proportionally a larger part of the follicle than
in the HT (Figure 3e, p=0.012, paper II) and the RT tonsils (Figure 3e, p=0.01, paper II).
Despite HT tonsils having proportionally larger tissue area covered by follicles (Figure 3f,
paper II), no difference was observed between the numbers of follicles (Figure 2g, paper II).
The tonsil stroma contains mostly follicles and extrafollicular space. Evaluation of the size of
the follicles as a percentage of the total tissue area might reveal both histological and
functional differences between the RT, HT and the PST tonsils. Follicles covered the largest
part of the total tissue area within the HT tonsils compared with PST and RT tonsils (Figure
3f, p=0.0004 and p=0.014 respectively, paper II) whereas no difference was observed between
the RT and PST tonsils. The difference seemed to be both due to the larger size of the GC in
the HT tonsils compared to the RT (Figure 3g, p=0.017, paper II) and the PST tonsils (Figure
3g, p<0.0001, paper II) as well as the MZ area (Figure 3h: RT, p=0.008; PST, p= 0.018, paper
II). However, the PST and RT tonsils differed only in respect to the GC as a proportion of
total tissue (Figure 3g, p=0.02, paper II).
4.2.4 The number of CD68+ macrophages correlates with follicle size Tingible body macrophages (TBM) are located within the GC (Figure 4a, paper II) where they
remove apoptotic cells. Tissue sections were cut, air-dried, fixed and stained for the TBM
marker CD68 and visualized using DAB chromogen. The GC TBMs were counted and linear
correlation between the number of TBM and the size of the follicle or GC calculated for all
tonsils. The total number of TBM within the GC correlated positively with both the size of
the GC (Figure 4e-f, paper II) and the size of the whole follicle (Figure 4b-d, r2=0.71, paper
II) regardless of pathology. Interestingly, when the data were analysed separately for the two
tonsil groups, the positive correlation remained strong (Figure 4c, PST tonsils, r2=0.74; Figure
4d, RT tonsils, r2=0.72).
Interestingly, PST tonsils contained fewer TBM per mm2 of GC or follicular area than the
RT tonsils (Figure 4g, p=0.025; 4h, p=0.06, paper II).
84
4.2.5 Psoriasis tonsils have a higher frequency of skin-homing (CLA+) T cells The known association of sore throat and psoriasis gave reason to evaluate the skin homing
potential of tonsil T cells. Isolated TMCs were stained with antibodies against CD4, CD8 and
the skin homing marker CLA. Cells were collected and analysed using flow cytometry.
Psoriasis tonsils had a significantly higher frequency of CLA+ T cells compared with RT
tonsils (Figure 5a-b, paper II). This was evident for both the CD4+ (p=0.024) and CD8+ T
cell populations (p=0.01). Furthermore, a relatively strong correlation was observed between
the frequency of CLA+ T cells in blood and tonsil (Figure 5c and d, paper II) of psoriasis
patients for both CD8+ T cells (r=0.65, p=0.01) and CD4+ (r=0.62, p=0.024) T cells.
4.2.6 Skin homing T cells in psoriasis tonsils express IL-23R.
Tonsil mononuclear cells were isolated and stained for the IL-23 receptor (IL-23R) for
evaluation of Th17 cells. Psoriasis tonsils had an increased frequency of both IL-23R+CD4+
T cells (p=0.002) and IL23R+CD8+ T cells (p=0.004) in comparison with RT tonsils (Figure
5e and 5f respectively, paper II). There was a slight difference in the number of CD4+ T cells
co-expressing IL-23R and CLA that were more common in the PST tonsils (Figure 6g,
p=0.05, paper II) whereas the results for the CD8+ T cells were comparable.
PST and RT tonsils differed in the expression of various other markers. In this respect,
PST tonsils had a higher frequency of CD4+ T cells expressing markers for the central
memory phenotype, CCR7 and CD62L (Figure 6h, p<0.0001 for both, paper II).
Furthermore, various subpopulations of CCR7+ CD4+ T cells were more frequently present
in the PST tonsils (Figure 9). In this respect, co-expression with CD54 (p=0.0179) or CCR6
(p=0.0188) alone or combined (p= 0.0361) was significantly higher. Additionally, the PST
tonsils had a higher frequency of CD54+CCR6+ CD4+ T cells (p=0.0001, figure 9).
85
Figure 9. Various subpopulations of CD4+ T cells differ between RT and PST tonsils
Psoriasis tonsils had a higher frequency of CD4+ T cells co-expressing the adhesion molecule CD54 and the
Th17 marker CCR6 (p=0.0001, n=8 vs. 14). They also had a higher frequency of CCR7+ CD4+ T cells
expressing CD54 alone (p=0.0179, n=10 vs. 14), CCR6 alone (p=0.0188, n=8 vs. 10) or co-expressed with
CCR6 (p=0.0361, n=8 vs. 10).
In contrast, CD4+ T cells from the RT tonsils expressed the activation markers CD25 or
CD69 (Figure 6h, p=0.0002 and Fig 5i; p=0.0005, paper II) alone or concomitantly (Figure 6i,
p= 0.0005, paper II) significantly more frequently. Furthermore, the CD8+CD69+ T cells
from RT tonsils were also more frequent while PST tonsils had a higher frequency of
CD8+CXCR6+ cells (Figure 10, p=0.0012 and p= 0.0319 respectively).
Figure 10. CD8+ T cell expression of CXCR6 and CD69 differs between RT and PST tonsils. CD8+ T cells in PST tonsils have a higher expression of CXCR6 (p=0.0012, n=9 vs. 7) while RT tonsils have a
higher expression CD69 (p=0.319, n=11 vs. 19).
86
Interestingly, CD4+ T cells from RT tonsils had a higher expression of CCR5 (Figure 6i,
p= 0.009, paper II) and a higher co-expression of the skin homing molecules of CLA and
CCR10 as well as CCR4+CCR10+ (Figure 6i, p=0.047 and p=0.006 respectively, paper II).
However, PST and RT tonsils had a similar expression of the chemokine receptors CCR4 and
CCR10 (data not shown). Furthermore, psoriasis tonsils had a higher frequency of CD4+ T
cells co-expressing CLA and CCR6 (Figure 6i, p= 0.047, paper II).
4.2.7 Other histological features
Figure 11. The tonsil squamous epithelium form structures similar to rete ridges. The tonsil squamous epithelium from an individual with severe psoriasis has histological features that resemble the rete
ridges present within the skin. The “rete ridges” formation in the tonsil squamous epithelium at a) 50 x magnification b) 100x
magnification and c) 400 x magnification.
An interesting observation was made when the palatine tonsils of an individual with a high
PASI score of 36 was evaluated. A feature similar to the rete ridges within the psoriasis skin
was found in the squamous epithelium. This histological feature was not as distinctive in the
other tonsils and completely absent in most. Unfortunately it was not possible to evaluate if
there was a correlation between a high PASI score and the presence of these histological
changes as most of the psoriasis patients had a low to average PASI score.
87
4.3 Tonsillectomy improves psoriasis by decreasing the frequency of peptide-reactive T cells in blood (paper III).
4.3.1 Tonsillectomy improves psoriasis, evaluated by PASI score Out of twenty-nine participants in the study, fifteen patients with chronic plaque psoriasis
were randomly selected to undergo tonsillectomy (TX group) while fourteen individuals
served as a control group. Disease characterization and personal information were comparable
between the groups (Table 1, paper III). The severity of the psoriasis was evaluated using
PASI score (psoriasis area severity index) that considers the plaques in regard to redness
(erythema), thickness (induration) and scaling (desquamation) along with the percentage of
affected body area. The PASI score was evaluated at the study entry and at fixed time points
throughout the study to register any changes following tonsillectomy. In order for the study to
remain unbiased, a double numerical code was used where the identity of the participants was
not revealed until the end of the study. This ensured that the individual evaluating the PASI
score had no knowledge of which participants underwent tonsillectomy.
The TX group had a significantly lower PASI score throughout the study period compared
to at study entry and the control group (Figure 1A, p=0.0001, paper III). This was apparent
from the first evaluation two months after tonsillectomy (Figure 1A and 1C, paper III). In
fact, improvement in the PASI score ranged from 30-90% in the TX group with around 60%
of the TX patients obtaining 50% reduction in the severity of their skin lesions while 15-30%
of the patients showed some improvement at different time points (Figure 1B, paper III).
Furthermore, two patients did not show any improvement (patient 4 and 6) in PASI score nor
in the frequency in peptide-reactive skin homing T cells (Figure 4A, paper III), which adds
further relevance to the results from this study. A similar pattern of improvement was not
observed among the individuals in the control group during the same time period (Figure 1D,
paper III).
Use of topical treatment was significantly more common among the individuals in the
control group (86%) than among the TX individuals (27%) whereas a few patients had
phototherapy in both groups (Table II, paper IIII). Additionally, one tonsillectomized patient
needed systemic therapy with methotrexate due to severe arthritis (Table II, paper III). The
great majority of the participants were HLA-Cw*0602 positive (Table 1, TX group 11/15;
control group 13/14, paper III) but due to the small number of participants it is difficult to
verify the finding that improvement was not associated with the carriage of the HLA-
Cw*0602 allele (data not shown).
88
4.3.2 IL-8 decreases in blood after tonsillectomy Measurement of the chemokine IL-8 in serum is a good indicator of the inflammatory activity
in the blood of the patients and possibly a more objective way to evaluate the effect of the
tonsillectomy or systemic inflammation. Peripheral blood was collected at study entry and
after 24 months. The IL-8 concentration in serum was measured using by ELISA. IL-8
decreased significantly following tonsillectomy (Figure 2, p= 0.034, paper III) and no such
change was observed in the control group even though they received more topical treatment
(Figure 2A and 2B, paper III).
4.3.3 Peptide–specific CD8+ T cells in blood decrease following tonsillectomy To evaluate the relevance of peptide specific T cells in psoriasis, PBMCs were isolated and
stimulated for 16 hours with homologous M protein and keratin peptides. Cells were stained
for flow cytometry with antibodies against CLA and CD8 as well as antibodies against the
intracellularly expressed IFN-J (Th1 cells) or IL-17 (Th17 cells). Interestingly, tonsillectomy
significantly reduced the amount of peptide reactive CLA+CD8+ T cells in the TX group as
evaluated two months after the operation for each of the sixteen peptide pools tested (Figure
3A, paper III). Similar results were not observed for the control group (Figure 3B, paper III).
4.3.4 Peptide-specific CD8+ T cells in tonsils correlate with levels in blood Evaluation of the frequency of peptide-reactive skin homing CD8+ T cells from TX patients
revealed that there was a strong positive correlation between their frequency in blood and
tonsil at study entry for both IFN-J producing (Figure 4A, r=0.788, p< 0.0001, paper III) and
IL-17 producing cells (Figure 4B, r=0.644, p<=0.015, paper III).
4.3.5 Improvement in PASI score is correlated to the frequency of circulating peptide-specific CD8+ T cells .
Comparison of the circulating peptide-specific IFN-J producing skin homing CD8+ T cells in
blood of the TX patients and the control group, revealed a significant time-dependent decline
in the TX group only (p= 0.003, Figure 5A, paper III). A good positive correlation was
observed between PASI and the blood levels of the peptide reactive skin homing IFN-J (r=
0.594, p<0.001, Figure 5B, paper III) and IL-17 producing CD8+ T cells (Figure 1 in
supplements, r=0.560, p<0.001, paper III). As previously noted, no correlations were
observed in the control group. Importantly, the frequency of circulating T cells responding to
anti-CD3 antibody or streptokinase did not change after tonsillectomy indicating the
specificity of the response.
89
5 Discussion
Psoriasis is a complex autoimmune skin disease thought to be mediated by the infiltration of
clonally expanded T cells 132-134
, predominantly of the CD4 phenotype131
to skin. The origin
of these pathogenic cells is unknown but various lines of evidence suggest the involvement of
the palatine tonsils.
In this respect streptococcal throat infections have been associated with the initiation and
exacerbation of psoriasis (paper IV). Furthermore, despite the high prevalence of
streptococcal throat infections among the general public263-265
, their occurrence is even greater
among psoriasis patients173
. This is particularly interesting, as proteins from E-haemolytic
streptococci have been shown to induce T cell expression of the skin homing molecule
cutaneous lymphocyte-associated antigen (CLA)285-287
, a T cell phenotype more frequent in
the peripheral blood of psoriasis patients than healthy individuals135
. Additionally, T cell
clones carrying similar TCRVB gene rearrangements (which confers the specificity of the T
cell receptor) 294
have been isolated from the tonsils and skin of the same patients, suggesting
that these T cells, despite being isolated from distant tissues are responding to the same
antigens. In addition, tonsillectomies have been shown to decrease skin disease severity in
some psoriasis patients with a history of recurrent throat infections282, 283
(paper III-IV).
Based on these findings, it was hypothesised that the palatine tonsils were important for the
pathogenesis of psoriasis among individuals that experienced either episodes of guttate
psoriasis or exacerbation of chronic plaque psoriasis following streptococcal infection.
In this thesis I investigated the characteristics of psoriasis tonsils. The overall aim was to
clarify why only some individuals develop psoriasis, or experience exacerbation, after
recurrent streptococcal throat infections. Moreover to identify the distinguishing factor
underlying their pathological importance in psoriasis.
I hypothesised that the tonsils of psoriasis patients might be either hypersensitive to
infection, or their innate immune responses were dysregulated resulting in more frequent
infections, inflammation and symptoms of sore throat, as experienced by the psoriasis
patients. This would be reflected in different functionality, histology and/or the phenotypic
characteristics of the psoriasis immune cells.
Here I show that although streptococcal infections are one of the most common causes of
sore throat in the general population, they are even more frequent among psoriasis patients
(paper II). This was particularly evident for infections by group C streptococci (paper II). As
90
a means of assessing whether the innate immune responses were dysregulated, I examined
antimicrobial peptide expression but found no difference in the tonsil expression of LL-37
despite its overexpression within psoriasis plaques and its association with psoriasis
pathogenesis (paper I). However, I show that LL-37 has a number of immunomodulatory
effects and that its expression within the tonsils is restricted to infiltrating leukocytes and GC-
DCs (paper I). Interestingly, the phenotypes of T cells within psoriasis tonsils and recurrently
infected tonsils differed with greater expression of skin homing molecules and molecules
associated with Th1/Th17 polarization (paper II). Additionally I show that skin homing T
cells from psoriasis tonsils respond to keratin peptides in a Th1/Th17 dependent manner
(paper III) and that certain histological characteristics of tonsils are dependent on their
pathological state (paper II).
The aim of the first study (paper I) was to investigate whether the expression of the
cathelicidin antimicrobial peptide (AMP) LL-37 was dysregulated in psoriasis tonsils. LL-37
was selected as a representative of the innate immune system due to its association with the
pathogenesis of psoriasis 351, 463-465
and its upregulated expression within the lesional skin341,
462 and serum
424.
No difference was observed in the tonsil expression of LL-37 between the tonsil groups.
Contrary to previous reports474 467
LL-37 was only detected in leukocyte-infiltrated areas of
tissue and 36% of the tonsils contained lymphoid follicles that were positive for the peptide.
Further analysis revealed that neutrophils and some macrophages were the main LL-37
expressing cell types within the tonsil epithelium while CD11c+CD13+ DCs expressed LL-37
within the germinal centre. I confirmed that these LL-37 positive follicular GC were active
and stimulation with the peptide showed that LL-37 promoted a Th1 mediated immune
response in a DC dependent manner. Furthermore, the presence of DNA altered its
modulatory effect possibly by acting as a co-factor or a damage-associated molecular pattern.
Finally, LL-37 was found to influence the T cell expression of a number of homing
molecules.
From these results I conclude that in tonsils, LL-37 likely has immunomodulatory effects
on both the innate and the adaptive immune system and may contribute to the overall
germinal centre reaction in follicles.
The lack of LL-37 expression by the tonsil squamous epithelium is interesting as it is
expressed by a number of other squamous epithelia in the oral cavity such as the lining of the
mouth, tongue and oesophagus391
as well as within the saliva and salivary glands385
.
91
Furthermore, it has been described in the lung epithelia393
and colon epithelium474
, the areas
guarded by the tonsils. Other epithelia shown to express LL-37, albeit in more distant tissues
are the squamous epithelia of the cervix and vagina220
.
The absence of LL-37 expression from the tonsil squamous epithelium suggests that the
surface is already well-defended by other factors such as the thickness of the epithelium,
secretion of saliva and mucus in the oral area and expression of other anti-microbial peptides
and proteins, such as lysozyme, released by infiltrated or localized cells224, 379
. Within the
crypts, LL-37 appears to have an important role in anti-microbial defences by preventing
microbial biofilm development 413
320
even low concentration of the peptide are sufficient to
effectively work against the development of Streptococcus pyogenesis biofilms 475
.
Secreted LL-37 can modulate the tonsil immune response by attracting more leukocytes to
the area, influencing pDC and T cells and by stimulating the release of other AMPs378
, some
of which act in synergy with LL-37, e.g. human E-defensins 344, 448
.
The decreased expression of CXCL1 following LL-37 stimulation is interesting as LL-37
may also signal via CXCR2, the receptor for CXCL1440
. CXCL1 is mainly expressed by the
surface epithelium, high endothelial venules (HEV) and in crypts476
, the main contact sites to
the external environment. One may speculate that competitive binding to the receptor may
influence the migration of the neutrophils into the tonsil. In this respect, under certain
conditions LL-37 could act as a negative feedback regulator and thereby participate in the
regulation of inflammation by reducing the influx of neutrophils, thus dampening the immune
response.
The production of IFN-Ȗ transcripts by tonsil mononuclear cells following stimulation with
the LL-37-DNA complex is noteworthy, as it has been shown that LL-37 modulates the
immune response to IFN-Ȗ by isolated B cells, macrophages, monocytes and DC477
. Our
results suggest that LL-37 and IFN-Ȗ can work synergistically as LL-37 induced the
production of the IFN-Ȗ-induced chemokines CXCL9 478
, CXCL10 and CCL5187
without
significantly inducing the production of IFN-Ȗ itself. Both CD11c+ DC and macrophages
have been shown to produce CXCL9 and CXCL10 in tonsil GC although the expression is
highest within the T cell zone479
. Therefore, cellular interactions and cytokine release are
likely to play an important role in the activity of LL-37.
I have shown that some tonsil follicles have relatively pronounced LL-37 expression
levels, and one might speculate that such follicles induce a greater production of CCL5,
CXCL9 and CXCL10, possibly through IFN-Ȗ that would increase the homing of naïve T
92
cells into the follicles471
, thus strengthening the GC reaction. In adddition, IFN-Ȗ might
influence B cell IgG class switching in the follicle447, 450
. The stronger LL-37 expression by
some tonsil follicles might reflect follicles in a higher state of reactivity, due to the stage of
follicular development or disintegration, frequency of inflammation (tonsillitis), the types of
microbes colonizing the tonsil, or the time since last symptomatic infection.
The potential interactions between IFN-Ȗ and LL-37 could also be mediated through
CCL18, which is expressed by CD4+CD11c+ DC within the GC and the T cell zones187
. This
chemokine promotes the interaction of naïve T cells and iDC within an IL-10 dominated
environment leading to the production of T regulatory cells in the tissue. CCL18 also attracts
naïve T cells and spleen marginal zone B cells (CD38-) into GC187
. Its expression varies
between follicles and might depend on the activation and progression of the GC480
. IFN-Ȗ
inhibits the production of CCL18458
and the induced IFN-Ȗ production brought about by the
LL-37-DNA complex might therefore disrupt the suppression of the specific immune
responses within the tonsils and thereby increase the likelihood of an autoimmune response.
The effect of LL-37 might depend on the extent of dying cells releasing RNA or DNA447, 450
into their surroundings, the LL-37 concentration in the microenvironment, the different cell
types in the surrounding area and the availability of LL-37 released by e.g. neutrophils.
Additionally, LL-37 might directly induce the GC reaction by inducing apoptosis among
CD4+CD25+foxp3+ T regulatory cells481
.
In the same manner, the LL-37 expressed by the follicular DC might drive their inhibition
of B cell apoptosis within the GC, and their capacity of limiting the accumulation of
autoantibodies (reviewed by482
) or the antigen presentation through immune complexes29
. In
this respect, LL-37 could be vital for the monitoring of the immune response by fortifying
specific immune responses dependent on the microenvironment.
The role of CCL27 in tonsils is not as clear. In tonsils, pDC that co-express CCR6 and
CCR10 are found in close proximity to CCL27 that is produced by basal epithelial cells492
.
CCL27 is upregulated in inflamed skin such as atopic dermatitis483
and its interaction with its
receptor, CCR10, is thought to contribute to the migration of CLA-expressing memory T cells
to skin493
. However, a recent study showed that CCL27, CCR4 and CCR10 are
downregulated in lesional skin compared to non-lesional skin of psoriasis patients483
.
The decreased expression of CLA and CCR4 by LL-37 stimulated tonsil T cells, might
indicate a regulation of the response due to lack of danger signal with increased expression of
CD54, leading to reduced efflux of T cells from the tonsils.
93
LL-37 may also have a role in influencing the local Th17/Th1 T cell polarization. LL-37
could decrease the Th17 response within the tonsil424
by promoting an IFN-Ȗ Th1 response.
This is in concordance with the fact that serum concentrations of LL-37 and IL-17 are
inversely correlated in psoriasis patients but not healthy individuals 424
. Interestingly HBD-2
shows a similar pattern351
and so far is the only anti-microbial peptide shown to correlate with
the disease severity (PASI score)349
351
424
.
I conclude that although no difference was observed in the LL-37 expression between the
different tonsil groups, it does not exclude the possibility that the immune response differs
significantly during acute inflammation, a scenario not examined in this study. LL-37 appears
to have immunomodulatory effects in the tonsils where it may influence the germinal centre
reaction and participate in orchestrating the immune response.
Comparison of the bacteriology between recurrently infected tonsils from individuals with
or without psoriasis revealed that infections were generally more common among the
psoriasis tonsils (p=0.0001), particularly infections by streptococci expressing M protein
(Lancefield groups A, B, C and G, p=0.021). In fact, group C streptococcus was the most
frequent infection (Paper II, Table X, p=0.036) and tobacco smoking tended to increase the
vulnerability to infections among the patients (p=0.052). These results confirm that psoriasis
patients are predisposed to streptococcal throat infections; however I have yet to determine
whether this is due to a vulnerability to initial infection or a dysregulation in the immune
response resulting in a less efficient clearance of the bacteria and a higher carrier rate.
The persistence of streptococcal infections272, 273
lies in the ability of the bacteria to remain
dormant and undetected within the host cells 283
279, 484-486
until external conditions become
more permissive for growth. Streptococcal superantigens have been shown to induce T cells
to express the skin homing molecule CLA 135, 285-287, 293, 487
, which is significantly more
frequently expressed by T cells in the blood of psoriasis patients compared to healthy
individuals135
. Indeed, it has been shown that peripheral CD8+ T cells (particularly CLA+
cells) secrete IFN-Jҏ in response to stimulation by homologous peptides from human epidermal
keratins and streptococcal M protein, conceivably by a cross-reaction293
. The findings that
psoriasis tonsils have a higher frequency of potentially skin homing T cells (paper II), there is
a correlation between their frequency in tonsils and blood of psoriasis patients (paper II) and
that the frequency of CLA+CD8+ T cells in the blood correlates with disease severity488
gives
a basis to hypothesise that the plaque forming T cells originate from tonsils.
94
To test this hypothesis, psoriasis patients with a history of skin disease exacerbation during
bouts of streptococcal sore throat were recruited and half of the patients underwent
tonsillectomy and the other half served as a control group. Blood was drawn at study entry
and two months post-tonsillectomy and peripheral blood T cells stimulated with a number of
peptide pools containing the homologous peptides designed from the sequences of keratin 17
and streptococcal M6 proteins (paper III).
The frequency of the peptide-reactive CLA+CD8+IFN-J+ T cells decreased significantly
post-tonsillectomy. Furthermore, a correlation was observed between the levels of IFN-Ȗ
producing CLA+CD8+ peptide-specific T cells in blood and tonsils (r=0.788, p<0.001).
Additionally, a significant correlation was observed between the frequencies of peptide
specific CLA+CD8+ T cells producing IL-17 in tonsils and blood (r=0.644, p=0.015). The
frequency of these specific cells decreased following tonsillectomy and correlated with the
measured disease severity (PASI) score. The control group showed very little variation in all
measured parameters during the study.
Post-operative improvement was not found to be associated with the carriage of the HLA-
Cw*0602 allele which has previously been shown to have a strong association with
psoriasis301
489
297
. This is interesting as both peptides from keratins and M proteins have been
shown to mediate a stronger response among skin homing CD8+ T cells in individuals
expressing the allele293
. However, the results presented in this thesis are consistent with other
studies highlighting the importance of CD8+ T cells in the pathogenesis of psoriasis141
128, 135,
488 (reviewed in paper VI). In this way, it has been shown that CD8+T cells are prerequisite
for the development of a psoriasis plaque125
. Furthermore, the pathogenic T cells are clonally
expanded as observed by their rearranged TCR 132-134
and their activated memory
phenotype131
. Additionally, CD8+ T cells are more numerous in psoriasis tonsils.
Furthermore, CD4+ and CD8+ T cells have a different preference for location within the
skin where CD4+ T cells home to the dermis140
while CD8+ T cells progress into the
epidermis120, 121
138
139
. It is possible that CD8+ T cells play a greater role in the initiation of
the plaque formation but once the plaque is formed, a complex interplay between various cell
types within the skin ignites the process and maintains the plaque with the assistance of the
CD4+ T cells.
Today, psoriasis is considered to be a mixed Th1/Th17 disease within a predominant IL-
17, IL-22 and IFN-J cytokine milieu140
141
128
. Analysis of tonsil T cells revealed a similar
pattern as CD4+ and CD8+ T cells expressing IL-23R or co-expressing CLA and IL-23R or
95
CCR6 were more common in the psoriasis tonsils, indicating a potential Th17 bias. This is in
concordance with the fact that CD8+ T cells in psoriasis skin had been shown to express IL-
17 and IL-22128
. Furthermore, the existence of double-positive IL-17/IFN-Ȗ+ cells has also
been described140
and the Tc17 cells have been shown to be resistant to suppression by T
regulatory cells138
. The innate immune system can also contribute to the Th17 skewing as IL-
17 may also be produced by neutrophils and mast cells, and macrophages have been shown to
produce IL-22128
, leading to hyperproliferation of keratinocytes. Furthermore, in vitro
monocyte-derived dendritic cells can stimulate Th17/Tc17 (T17) and Th1/Tc1 (T1) responses
and differentiation of IFNJ+IL-17
+ T cells
129.
T cells from psoriasis tonsils differed in the expression of a number of phenotypic surface
markers when compared with T cell from recurrently infected tonsils. In this respect,
psoriasis tonsils had a higher frequency of T cells expressing CD62L and CCR7, which are
important for homing to lymph nodes and T cell areas within secondary lymphoid organs27, 28
.
Furthermore, T cells from psoriasis tonsils appear to be less activated with a lower expression
of the activation markers CD69 and CD25.
The observed difference in T cell phenotypes gave reason to investigate if a histological
difference existed between the different tonsil groups. As previously described184, 490
, the
hypertrophic tonsils had large and clearly defined follicles that occupied a large part of the
tissue. In the psoriasis tonsils, the follicles were less distinct and smaller and occupied a
smaller part of the total tissue area. Also, the extrafollicular area appeared to be not very
dense. On the other hand, the recurrently infected tonsils showed intermediate histological
features with the most numerous crypts. The GC:MZ ratio was the lowest in the psoriasis
tonsils, indicating a larger T cell zone, which is in accordance with the higher CD62L and
CCR7 expression among the T cells. Interestingly no difference was observed in CXCR5
expression between the groups, the molecule necessary for homing to the GC. Its expression
is negatively correlated with CCR7 so that when upregulated, CCR7 is downregulated. This
may indicate that the more numerous CCR7+ T cells are due to increased proliferation in the
T cell zone or an increased influx of T cells to the tonsil tissue without any influence on T
follicular helper cells.
Follicles within the same tissue were of different sizes that can partly be explained by the
plane of sectioning, where some follicles are shown at the “tip” of the follicle whereas others
are at their widest point. This could also be due to different developmental stage of the follicle
as on rare occasions the MZ or the GC were hardly noticeable.
96
The GCs are important for the adaptive immune response, as it is the home of B cell
proliferation, selection, differentiation and immunoglobulin class switching. The large size of
the follicles in the hypertrophic tonsils indicates a highly active GC whose size reflects
repeated stimulation possibly due to viral infection231
. Furthermore lack of apoptosis or
induced proliferation are considered contributing factors231
and are possibly mediated by the
Th2 cytokine IL-4231
. These tonsils have been shown to contain a higher number of basophils
in comparison with RT tonsils, indicating a potential allergic response 231
.
The histological characteristics of the psoriasis tonsils were quite different to RT tonsils
and suggest a dysfunctional regulation of the GC immune response leading to a weaker GC
reaction. This is supported by the significantly smaller number of tingible body macrophages
(CD68+) per mm2 of both GC and follicle area within the psoriasis tonsils. These
macrophages ingest cellular debris and are thought to downregulate the GC reaction491
. It is
conceivable that this could lead to the accumulation of apoptotic cells with increased release
of DNA into the surrounding area. This might interrupt its function and increase the risk of
the GC acting as a source of autoantigens.
5.1 Tonsils and psoriasis As the only encapsulated secondary lymphoid tissue within the human body, the palatine
tonsil is in many respects extraordinary. The multilayered squamous epithelium resembles the
skin epidermis while the mesenchymal cells in the underlying tissue have been shown to have
the ability to function in a similar manner to dermal fibroblasts492
.
As reviewed earlier, there is evidence that the pathogenic T cells found in psoriatic skin
may have originated from the tonsils, however, the mechanism by which this comes about is
not well-established. One interesting observation that highlights the extraordinary nature of
the tonsils is that lymphoid progenitor cells have been observed within tonsils, typically in
the periphery of the T cell zone close to the fibrous septae493
. Moreover, recently it was
shown that extrathymic T cell development occurs in the fibrous scaffolds of the tonsils494
. In
this interesting study by McClory and coworkers, five different T cell phenotypes of different
developmental stages, similar to those in the thymus, were observed. These T cells displayed
similar gene expression as their thymic counterparts and were able to differentiate into active
T cells in vitro494.
97
Tonsil T cells have been shown to display great plasticity in phenotype. In this respect,
tonsils have been shown to contain CD8+CD25+Foxp3+ T cells that express high levels of
IFN-Ȗ, TNF-D and IL-17495
. This interesting plasticity is dependent on IL-6 that is commonly
expressed within inflamed tissue. It can enhance the co-expression of IFN-Ȗ and IL-17 or
lead to a dominant Th17 phenotype. As IL-6 can also stimulate LL-37 production from
keratinocytes and neutrophils 424
one might speculate that in the presence of DNA, a LL-37-
DNA complex could form. This complex might then induce IFNJ and thereby reduce the
presence of the T regulatory phenotype. Foxp3+ T regulatory cells are 10 times higher in
tonsil than blood and more suppressive 496
. Their depletion in tonsils has been shown to
induce allergen-induced proliferation of tonsil T cells thus disrupting the normal non-
responsiveness to food and air born allergens496
. This underlines their importance for a
balanced tonsil immune response. The observed difference in this thesis in the surface marker
expression between the different tonsil groups may reflect the underlying difference in the
microenvironment of the T cells. It is also possible that tonsil T cells may exhibit greater
plasticity than other T cells. It is possible that a portion of the larger Th17 population
observed within the psoriasis tonsils stem from the Foxp3 T regulatory cells.
The tonsil DCs have been shown to be able to cross-present soluble antigens497
and the
results presented in this thesis support the hypothesis that T cell dependent molecular mimicry
is important for the pathogenesis of psoriasis. Streptococcal infections were more common in
psoriasis tonsils, which contained a higher percentage of skin-homing (CLA+) T cells and
these levels correlated with levels in blood. A proportion of these T cells responded
specifically to homologous peptides from streptococci and skin keratins. This is in line with
the finding that T cell clones isolated from skin and tonsil carry similar TCRVB
rearrangements294
, suggesting responses to the same antigens, consolidating a link between
tonsils and psoriasis plaques.
98
5.2 Hypothetical scenarios Two hypothetical scenarios may be put forward to explain the development of the pathogenic
T cells observed in psoriasis:
I. In the first scenario naïve T cells, originating from thymus, migrate to the tonsil where
they become concentrated within the T cell zones. Recurrent streptococcal throat infections
promote the presentation of streptococcal peptide antigens by APC in the tonsils and
recognition by tonsil T cells as part of the immune response. This scenario also increases the
potential for presentation of streptococcal peptides that have similar peptide sequences to that
of proteins upregulated in psoriasis skin, such as peptides from keratin 17 and streptococcal
M6 protein. This process may be aided and abetted by the broad-spectrum activation of T
cells and APC induced by superantigens released by the bacteria285, 286, 498
. Moreover, the
ability of some bacteria to become internalized in epithelia and lie dormant for some time 486,
499 also contributes to the high exposure to the bacteria and recurrence of bacterial-driven
tonsil inflammation.
The streptococcus bacterium stimulates the T cells to express CLA, thus allowing them to
home to the skin. The T cells undergo clonal expansion that significantly increases their
frequency. Some of the streptococcal components enter the bloodstream where they become
internalized and presented by APCs. Once in the skin, the CLA+CD4+ T cells localize in the
dermis where they are presented to the streptococcal component through MHC class II
molecules on the surface of APCs. The CLA+CD8+ T cells inhabit the epidermis where they
encounter the homologous keratin peptides presented by the local APC through HLA class I.
There they induce the proliferation of keratinocytes, which is one of the representative
features of psoriasis.
II. In the second scenario, extrathymic T cell development occurring in the fibrous scaffold
provides T cells that are more vulnerable to developmental error as their surrounding is a
highly active tissue exposed to various foreign antigens. Furthermore, the developmental
process may be under less controlled situations than provided by the thymus. These tonsil T
cells may migrate from the scaffold area to the T cell zone or the crypts where they become
activated, lose the tolerance for self-antigen and become inflammatory T cells. These cells
then undergo a similar activation process as the thymus-derived T cells. However, it is
interesting to speculate if the keratin composition of the tonsils themselves is a contributing
factor to the molecular mimicry as keratin 16 can be observed scattered around the tissue. In
99
this way, instead of becoming activated within the skin, some T cells may directly respond to
keratin 16 within the tonsil itself.
The cryptal area is highly active as it is the main port of entry for the invading microbes
but also the main defence site as it is occupied with a wide variety of immune cells.
Neutrophils are particularly numerous, releasing LL-37 along with other factors into their
surrounding. In the constant battle between the immune system and the spread of the bacteria,
apoptosis rates are high within the crypts. They are also filled with various other debris
leading to an optimal environment for the bacteria. Plasmacytoid DC and myeloid DC located
within the crypts become activated when engulfing LL-37 bound to self-DNA from the
apoptotic cells that accumulate in the crypts. The repeated streptococcal infection enhances
the likelihood of cross-reaction between the peptide keratins and streptococcal M-protein
peptides presented by the pDC or mDC. This leads to abnormal responses of T cells that result
in the differentiation of autoreactive T cells. Both LL-37 and streptococci induce a Th1 or
Th17 polarization of naïve T cells288
. Furthermore, the Th17 response stimulates the
incorporation of the bacteria into epithelial cells291
.
Although the results represented in this thesis support the important role of streptococcal
M protein in psoriasis, it does not exclude the participation of peptidoglycans in the initiation
of the disease. Peptidoglycans have been shown to stimulate CD4+ T cells into producing
IFNJ303. Furthermore, it has been proposed that CD8+ T cells in epidermis are responding to
similar peptides sequences whereas CD4+ T cells in dermis recognize peptidoglycans (paper
VI).
100
5.3 The distinguishing factors of psoriasis patients What factors distinguish psoriasis patients from other individuals that are frequently exposed
to streptococcal throat infections? What contributes to the relationship observed between
skin and tonsils?
MacFadden and colleagues have postulated if the high carrier rate of streptococci observed
among psoriasis patients may be a survival trait against the once deadly scarlet fever epidemic
caused by invasive streptococci500
. The potential of engulfing streptococci and incubating it
intracellularly reduced the mortality risk of the affected individual. The cost of this trait was
the polarization of the immune response in a Th1/Th17 direction and the development of
psoriasis. In this respect, peripheral blood mononuclear cells from psoriasis patients have a
higher proliferative response upon stimulation with streptococcal antigen compared to healthy
individuals288
. Furthermore, CLA expression is also induced by the release of bacterial
superantigens and IL-12285, 286, 498
.Streptococcus can also mediate a Th1 response by
influencing DC cells288 and Th17 response through TGF-E290
induction.
Another factor is the genetic predisposition of psoriasis patients. Psoriasis has a
complicated genetic interaction with genes linked to the various function of both the epithelial
barrier and the general immune function of the body. An individual with a particular
genotype, yet to be identified, might be more vulnerable to throat infections from an early age
with some triggering effect possibly linked to various hormonal changes following
adolescence when the disease commonly becomes exposed. In this way it has been shown that
individuals with polymorphism in the TLR4 gene have up to 3-fold increase in risk of GAS
infections501
and psoriasis patients have increased occurrence of both streptococcal infections
and malignancy than the general population indicating a general abnormal reaction 502
.
A third factor may be the development of the palatine tonsils themselves. Recurrent
exposure to extrogenous antigens and underlying genetics may lead to a disruption in the
normal developmental process of the palatine tonsils. In this way, a dysregulation of the tonsil
innate immune system may increase the vulnerability to infection. Studies on germ-free
piglets have shown that the microbial environment during maturation influences the
immunological architecture of the tonsil503
. This along with the fact that there exists a
histological difference between the hypertrophic, recurrently infected and psoriasis tonsils,
indicate that this is a possible scenario. A long history of repeated infections, potentially from
as early as the first year of life might disturb the regular immune response, leading to
impaired defences with insufficient reaction against the invading microbe. Other underlying
101
illnesses or serious infections might shed the same effect. The smaller GC observed within the
psoriasis tonsils were less defined with small marginal zones indicating a disrupted GC
development and reaction. It is possible that the GC are short lived with a high disintegration
rate or that the new GC do not develop in a normal way.
Many of the infectious bacteria such as streptococci, can form intracellular reservoirs
within macrophages and endothelial cells that are particularly difficult to clear. Individuals
with lessened defences or a genetic predisposition are more likely to end up with the dormant
microbe that re-emerges when conditions are favourable. The bacteria multiply, releasing
various bacterial substances that induce an immune response, leading to the reappearance of
symptomatic sore throat. In this respect, the carriage rate of streptococci (A, C and G) in the
present study was very high, or 37%, as all the participants had a known association of
worsening in skin symptoms during throat infection. It is more likely that the general carriage
rate is around 9% among psoriasis patients as previously shown173
which is still very high as
it is 10 times more common than among their household controls.
One might also postulate that psoriasis patients or individuals predisposed to the disease
have a longer duration of throat infections than other individuals with recurrent infections. As
of yet, no study is available that has focused on the infectious history of the patient.
103
6 Conclusions
This study is to our knowledge, the first prospective, randomized and observer-blinded study
on the effect of tonsillectomy in psoriasis. It is also the first detailed comparative study of the
tonsil of psoriasis patients versus other recurrently infected or hypertrophic tonsils and as such
an important contribution to the understanding of the association between tonsillitis and
psoriasis.
In this thesis it was shown that psoriasis tonsils are histologically different and confirmed
that they are more frequently colonized by ß-haemolytic streptococci than non-psoriasis
tonsils. The psoriasis tonsils contained T cells with a significantly altered phenotype and a
clear Th1/Th17 trend, both in the expression of surface molecules and in the response to the
homologous keratin K17 and streptococcal M6 peptides. Furthermore, a clear correlation was
observed between the frequency of the skin-homing and keratin-responsive T cells in blood
and tonsil. Investigation of the innate immune component LL-37 revealed no difference in its
expression between the tonsil groups, indicating that decreased LL-37 expression was not the
underlying reason for the increased streptococcal colonization. However, LL-37 did appear to
have immunomodulatory effects in tonsils and may contribute to the overall germinal centre
reaction in the lymphoid follicles.
These findings are in concordance with the hypothesis that the palatine tonsils of psoriasis
patients have a dysregulated innate immune response or a hypersensitivity to infection. A
more thorough study is needed to determine which factors in the tonsils predispose to the
streptococcal colonization and give rise to the increase in symptomatic sore throats, as well as
the link to the psoriatic skin disease.
One of the major strengths of this study is the clearly defined psoriasis patient group; the
disease severity was on average mild (PASI 11, range 4.5- 36.6) with 50% of the patients with
PASI � 10 but all patients had reported an association between streptococcal throat infections
and worsening of their disease. The detailed comparison of the psoriasis tonsils with other
recurrently infected tonsils and hypertrophic tonsils was carried out to determine whether the
observed histological difference was in relation to infection or other factors. The outcome of
this comparison contributed significantly to the study.
As a possible future avenue of research, a larger, randomized and observer-blinded trial for
tonsillectomy would be useful to confirm the findings of the current study and help refine
what factors predict whether a patient will have a significant and prolonged clinical
improvement in their skin disease following tonsillectomy. In such a study it is critically
104
important to obtain a control cohort matched for age, gender, disease history, and disease
severity to follow up in comparison to the tonsillectomized group, as psoriasis may
spontaneously go into remission. Our findings indicate that emphasis should be placed on a
detailed comparison between the psoriasis tonsils and recurrently infected tonsils as the
hypertrophic tonsils were histologically very different. A detailed history of previous
infections and other illnesses should be gathered from both study groups to evaluate if our
hypothesis on the pathological process in tonsils is correct. One interesting aspect is a
comparison of the tonsil and skin microbiomes, which, using new DNA sequencing
technology, can give a much more detailed survey of the microbial flora in these two distinct
body sites; whether tonsil abnormalities (or genetic background) give rise to altered bacterial
colonization or whether the histological differences occur as a result of the different bacteria
in the tonsil remains unclear, however it is tempting to speculate that these events give rise to
the activation of an altered T cell repertoire. Although analysis of the tonsil T cells of the non-
tonsillectomized group is not possible, using throat swabs, I could analyse the microbiome
and analyse specificities of skin-homing T cells from the blood of the control group to try to
shed light on the tonsil-blood-skin immune axis.
As the psoriasis tonsil T cells express more skin-homing molecules the next step would be
to determine if these cells migrate to skin and actively participate in the pathogenesis. In this
respect, tonsil, blood and skin samples should be gathered and compared for various factors
such as skin-homing markers and TCR rearrangements that can be determined using next-
generation DNA and RNA sequencing.
It is currently not fully understood why skin-homing T cells target discrete patches of skin
leading to the formation of the lesions. Damage to the skin or wear and tear at sites of high
wear (elbows and knees) may release inflammatory mediators to help trigger lymphocyte
infiltration of skin, or re-activation of resident memory T cells. The antigens driving the
expansion and infiltration of these cells are currently the topic of speculation. In this respect
it has been shown that psoriasis T cells from skin respond to peptide sequences from
streptococcal M protein and here I show that the same applies for tonsil T cells. This does not
exclude the possibility that the pathogenic T cells respond to infections by other bacteria or
bacterial components. In fact, the T cell population that responded to the homologous M6 and
K17 peptides was proportionally very small while the observed difference in T cell phenotype
was general. This indicates that the microenvironment is different and that the psoriasis tonsil
T cells are responding to other factors as well. It is also possible that the peptide-specific T
cells in tonsils can modify the local microenvironment, leading to alterations of the T cell
105
phenotypes. In this respect it would be interesting to evaluate if the extrathymic T cells in
tonsils are a contributing factor to the disease.
7 Final conclusion
These results indicate that palatine tonsils are an important contributing factor to the disease.
The new finding of extrathymic T cell development within tonsils renders further support to
the hypothesis that the pathogenic T cells may originate in tonsils.
107
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9 Original Publications
131
Papers I - V
133
Paper I
The anti-microbial peptide LL-37 modulates immuneresponses in the palatine tonsils where it is exclusivelyexpressed by neutrophils and a subset of dendritic cellsSigrun L. Sigurdardottir a, b, Ragna H. Thorleifsdottir a, b,Andrew M. Guzman d, Gudmundur H. Gudmundsson c,Helgi Valdimarsson a, b, Andrew Johnston d,⁎
a Department of Immunology, Landspitali-University Hospital, Reykjavik, Icelandb Department of Medicine, University of Iceland, Reykjavik, Icelandc Institute of Biology, University of Iceland, Reykjavik, Icelandd Department of Dermatology, University of Michigan, Ann Arbor, MI, USA
Received 15 September 2010; accepted with revision 29 September 2011Available online 7 October 2011
KEYWORDSPalatine tonsils;Anti-microbial peptides;LL-37;Leukocytes;Psoriasis
Abstract Antimicrobial peptides are essential elements of epithelial defense against invadingmicro-organisms. The palatine tonsils are positioned at the entry of the airway and the gut andas such are ideally situated to act as immune sentinels in the pharynx protecting against micro-bial invasion. Tonsils express a number of antimicrobial peptides including hCAP18/LL-37. Herewe clearly define the expression of hCAP18/LL-37 in the tonsils showing unequivocally thathCAP18/LL-37 is mainly expressed by infiltrating neutrophils and follicular CD11c+CD13+HLA-DR+ dendritic cells, rarely by macrophages, and never by the epithelium itself. To explore pos-sible functions for follicle-derived LL-37, we stimulated tonsil mononuclear cells with LL-37 invitro and observed the secretion of the proinflammatory cytokines CCL5 and CXCL9, expressionof IFN-γ and MX-1 and down-regulation of chemokine receptors CCR4 and CCR6 which are in-volved in tissue-selective T cell trafficking. Taken together, these data illustrate new potentialimmunoregulatory functions for hCAP18/LL-37 in the tonsils.© 2011 Elsevier Inc. All rights reserved.
Abbreviations hCAP-18, Cathelicidin human cationic antimicrobial protein-18; DC, Dendritic cells; TMC, Tonsil mononuclear cells; RT-qPCR,Real-time, reverse transcription quantitative PCR.⁎ Corresponding author at: Department of Dermatology, University of Michigan, 6427 Medical Science Building 1, 1301 East Catherine Street,
Ann Arbor, MI 48109 USA. Fax: +1 734 615-7277.E-mail address: [email protected] (A. Johnston).
1521-6616/$ - see front matter © 2011 Elsevier Inc. All rights reserved.doi:10.1016/j.clim.2011.09.013
ava i l ab l e a t www.sc i enced i r ec t . com
C l i n i ca l Immuno logy
www.e l sev i e r . com / l o ca t e / y c l im
Clinical Immunology (2012) 142, 139–149
1. Introduction
Mucosal defense is crucial for maintaining a barrier betweeninternal body milieu and potentially invasive organisms fromour environment. The paired palatine tonsils (PT) belong tothe mucosa-associated lymphoid tissues of the human phar-ynx, forming Waldeyer's ring, which are instrumental in pro-tecting the entrance to the gut and airway. PT promoteeffective immune surveillance and efficient antigen sam-pling as their surface is lined by stratified squamous epithe-lium that extends into deep and branched crypts lined byreticulated epithelium, populated by lymphocytes, dendriticcells (DC), neutrophils and macrophages [1]. PT are alsopacked with B cell-rich lymphoid follicles, specialized forpriming humoral responses.
The most common clinical tonsil diseases are recurrentinfections typically by Streptococcus pyogenes[2,3] orStaphylococcus aureus[3] and abnormal enlargement of thetonsil often associated with Haemophilus influenzae infec-tions [3,4]. As a first line of defense, PT express various an-timicrobial peptides including the human β-defensins [5]and the cathelicidin human cationic antimicrobial protein-18 (hCAP-18) [6,7]. hCAP-18 is expressed as an inactivepro-protein requiring enzymatic processing to release activepeptide, LL-37 which forms an amphipathic α-helix withanti-microbial activity against Gram-negative and -positivebacteria [8–12].
hCAP-18/LL-37 is expressed by a number of cell types butpredominantly by the specific granules of neutrophils [13].LL-37 has been detected in squamous epithelia [14], colonmucosa [15–17] and in highly differentiated epithelia suchas lungs [12,18]. LL-37 has been detected in inflamed skin[14], particularly psoriasis plaques and rosacea [19] andbeen shown to be important for wound healing and mucosaldefenses [20,21]. LL-37 has a number of immunological func-tions such as influencing DC differentiation [22] and actingas a chemoattractant [13,23] particularly for neutrophils[24,25].
LL-37 expression in tonsils has received some recent at-tention [6,7], yet, its tissue localization, cellular sources,and role in tonsil immune responses are poorly character-ized. Here we show that neutrophils are the main source ofLL-37 in the tonsils and importantly we show that tonsil epi-thelial cells do not express this peptide. Moreover, we dem-onstrate for the first time that a population of follicularCD11c+CD13+ DC express LL-37. On a functional level, treat-ment with exogenous LL-37 increased tonsil mononuclearcell production of RANTES (CCL5) and MIG (CXCL9) while sup-pressing a number of cell surface receptors involved in lym-phocyte homing. Taken together these results indicate thatin addition to its direct antimicrobial actions, LL-37 has a po-tential immunomodulatory role in mucosal defenses.
2. Materials and methods
2.1. Tissue preparation
Palatine tonsils were obtained from 40 individuals undergoingroutine tonsillectomies due to hypertrophy or recurrent tonsil-litis at The National University Hospital, Reykjavik, Iceland.The study was approved by the National Bioethics Committee
of Iceland. This study was conducted in compliance with goodclinical practice and according to the Declaration of HelsinkiPrinciples. Immediately after excision, the tonsils were storedin cold sterile saline until processed. Tissue from each tonsilwas snap frozen in Tissue-Tek OCT compound (Sakura Finetek,Zoeterwoude, NL) and prepared for immunohistochemistry,and also prepared for ex-vivo cell culture studies.
2.2. Immunohistochemistry
Fresh frozen 5 μm sections of tonsil were air-dried and fixed in2% paraformaldehyde for 15 min or cold acetone for 10 min,blocked with 1% hydrogen peroxide in phosphate buffered sa-line (PBS) containing 3% goat serum, washed in PBS andblocked for 20 min with 1.5% goat serum (Vector laboratories,Burlingame, CA, USA). Sections were incubated with polyclon-al anti-rabbit LL-37 antibody (1.07 μg mL!1, Innovagen, Lund,Sweden) for 30 min and thereafter stained according to theVectastain Elite rabbit IgG kit protocol (Vector). The stainingwas visualized using 3,3′-diaminobenzidine ((DAB), Becton-Dickinson, San Jose, CA, USA (BD)) and counterstained withhematoxylin (Thermo Shandon, Pittsburgh, PA, USA) followedby a dip into 37 mM ammonia solution. Finally the sectionswere dehydrated in alcohol with a concentration gradient of70%–90%–100%, fixed in Accustain (Sigma-Aldrich, St. Louis,MO, USA) and mounted with Mountex (Histolab products AB,Göteborg, Sweden). As a negative control, the primary anti-body was omitted. To demonstrate antigen-specific stainingand exclude staining due to contamination, synthetic LL-37peptide (Innovagen) was incubated at a 10-fold molar excesswith the LL-37 antibody overnight at 4 °C and the mixtureused for immunostaining. Double staining was performed bystaining for LL-37 first and then repeating the staining proce-dure for the second primary antibodies CCL5 (R&D Systems)and CXCL9 (R&D Systems), using an Avidin/Biotin blocking kit(Vector), Vectastain Elite goat IgG kit protocol (Vector) and vi-sualizing using New Fuchsin substrate kit (BD). These sectionswere mounted with Gelmount aqueous mounting medium(Sigma). Comparison to previously published data on LL-37staining (Ball et al., 2007) [7] was done by titration of the rab-bit LL-37 anti-serum (a generous gift of Dr. Ole Sorensen,Lund, Sweden) using DAB substrate for visualization.
2.3. Fluorescence immunohistochemistry
The fresh frozen sections were air-dried, fixed in 2% w/v para-formaldehyde in PBS and blocked first with 50 mM ammoniumchloride and then with 10% goat serum. Thereafter tissue sec-tions were incubated overnight in a humidified chamber at4 °C with the following antibodies: monoclonal mouse anti-human follicular dendritic cell (clone CNA.42, Dako, Glostrup,Denmark),monoclonal mouse anti-human CD15 (C3D-1, Dako),monoclonal mouse anti-human neutrophil elastase (NP57,Dako), mouse anti-human CD68 (c514H12, Serotec, Oxford,UK), mouse anti-human macrophage marker MCA8746(Serotec, clone MAC387) for tissue macrophages, mouse anti-human macrophage mature marker (Serotec, clone RFD7),polyclonal rabbit anti-human LL-37 (1.75 μg mL!1, Innovagen),CD11c (5D11, Novocastra, Newcastle upon Tyne, UK), CD13(VS5E, Novocastra), and anti-cytokeratin 8 (M20, Sigma). Thenext day, after washing in PBS, the following fluorescently
140 S.L. Sigurdardottir et al.
labeled secondary antibodies were added at room tempera-ture in the dark for 1 h: TexasRed goat anti-rabbit IgG (Invitro-gen, Eugene, Oregon, USA), AlexaFluor488 goat anti-mouseIgG (Invitrogen) or AlexaFluor350 goat anti-mouse IgM (Invitro-gen). The samples were mounted with Gelmount aqueousmountingmedium (Sigma). The primary antibodieswere eitheromitted for negative controls or replaced with the appropriateisotype control antibodies (Dako).
2.4. Tonsil mononuclear cell culture
Tonsil mononuclear cells (TMC) were isolated as previouslydescribed [26]. Briefly, tonsils were minced into 3 mmpieces and passed through a tea-sieve, and washed withHanks' balanced salt solution (Gibco, Invitrogen Ltd, Paisley,UK). TMC were collected at the interphase fraction formed byficoll density gradient centrifugation (Sigma) and re-suspendedin RPMI-1640 medium (Gibco) supplemented with 10% heatinactivated fetal calf serum (Gibco), 100 U mL!1 penicillin(Sigma), and 100 μg mL!1 streptomycin (Sigma). TMC wereseeded at a final density of 2!106 cells mL!1 and treated withthe LL-37 peptide ( 0, 1, 5 or 10 μg mL!1) (Innovagen) for 3 or17 h at 37 °C in a humidified atmosphere of 5% CO2 and 95%air. Stimulation with LL-37 with or without human DNA in 1:5proportion (0, 0.2, 1 or 2 μg mL!1), was carried out at a celldensity of 1!106 cells mL!1 for 17 h.
2.5. Cytokine detection
Cell culture supernatants were collected and stored at!70 °C until used. CCL5, CXCL1 and CXCL9 were measuredusing ELISA assays as instructed by manufacturer (R&DSystems).
2.6. Expression of cell surface receptors
Isolated TMC were stained for 30 min on ice with antibodiesagainst CD4-PerCP-Cy5.5 (clone RPA-T4, Biolegend, SanDiego, CA, USA), CD8-PerCP-Cy5.5 (RPA-T8, Biolegend),CCR4-PE (205410, R&D), CCR6-APC (53103, R&D), or the ap-propriate isotype controls. Cells were washed twice with PBSand fixed in 0.5% paraformaldehyde in PBS. Stained cellswere analyzed using a FACScalibur flow cytometer with Cell-Quest (BD) software.
2.7. Isolation of tonsil DC
To verify the presence of CD11c+CD13+ DC in tonsils, mye-loid dendritic cells (mDCs) were negatively selected frombulk TMC cultures using a myeloid dendritic cell isolationkit (Miltenyi Biotec, Auburn, CA) and LD columns (Miltenyi)as instructed by the manufacturer. The mDCs were then la-beled with CD11c-PE-cy5 antibody (clone 3.9, Biolegend)on ice for 30 min in the dark and washed twice with coldMACS buffer (PBS containing 0.5% BSA and 2 mM EDTA).CD11c+ cells were positively isolated using a MACS MS col-umn (Miltenyi) and thereafter stained and isolated usingCD13-PE antibody (L138, BD) and anti-PE beads (Miltenyi).An aliquot of the isolated cells was analyzed by FACS; the re-mainder was incubated on a glass slide at 37 °C for 2 h
allowing the dendritic cells to restore morphology. Theslides were air-dried for 30 min and fixed with 100% ethanolfor 10 min. The following day slides were stained with an an-tibody against LL-37 as previously described. The non-myeloid cells were also stained for LL-37.
2.8. Isolation of tonsil CD4+ T cells
Tonsil CD4 T cells were isolated from bulk TMC cultures usinganti-CD4-PE antibody (clone 3.9, Biolegend) and anti-PEmicrobeads (Miltenyi) and resuspended in RPMI-1640 medi-um (Gibco) supplemented with 10% heat inactivated fetalcalf serum (Gibco), 100 UmL!1 penicillin (Sigma), and100 μg mL!1 streptomycin (Sigma). Cells were seeded at aconcentration of 106 mL!1 and stimulated for 17 h with LL-37 (0, 1, 5 or 10 μg mL!1) with or without human DNA (0,0.2, 1 or 2 μg mL!1) in 1:5 proportion.
2.9. Real-time RT-PCR
Total RNA was isolated from cells (RNeasy Mini kit, Qiagen)and 200 ng of RNA template was reverse transcribed usinga high capacity cDNA reverse transcription kit and randomprimers (ABI, Foster City, CA, USA). RT-qPCR was carriedout on a 7900HT Fast real time PCR system (ABI). Primersfor IFN-γ (Hs00989291_m1), MX-1 (Hs00182073_m1 and thehousekeeping gene RPLP0 (36B4, Hs99999902_m1) wereobtained from ABI. Results were normalized to the expres-sion of RPLP0.
2.10. Statistical analyses
Data were tested for normality and one-way ANOVA forpaired samples with Dunnett's multiple comparison test aspost-test or 2-tailed t-tests were used as appropriate. Pvalues less than or equal to 0.05 were considered significant.
3. Results
3.1. Neutrophils are themain source of hCAP18/LL-37expression in the tonsil tissue
Previous studies have indicated that tonsil epithelia expressLL-37 [6,7]. However, we show that LL-37 expression in thetonsil squamous epithelium is limited to leukocyte-infiltratedareas containing mostly neutrophils (n=40, Figs. 1a–d). Thisis in contrast to these two previous reports [6,7], thereforeby way of comparison, we also stained tonsil sections withthe antiserum used in [6] which resulted in a high level ofnon-specific background staining at the dilutions used previ-ously (Figs. 1e and f).
To analyze whether the observed LL-37 expression was byepithelial cells or the leukocyte infiltrate, we performeddouble immunofluorescent staining using antibodies againstLL-37 and CD15 (Figs. 1g–i) or LL-37 and neutrophil elastase(not shown) to identify neutrophils, or CD68 to identify tis-sue macrophages (Figs. 1j–m). There was a complete co-localization of LL-37 and CD15 (Fig. 1i) or neutrophil elastase(not shown). LL-37 very rarely colocalized with CD68(Figs. 1j–m).
141The anti-microbial peptide LL-37 modulates immune responses in the palatine tonsils
The crypt reticulated epithelium has previously been shownto heavily express LL-37 [6,7] and we confirm that the tonsilcrypts are the dominant sources of LL-37 (Fig. 1c). Yet, a vari-ety of cell types are closely associatedwith the crypt reticulat-ed epithelium, thus to delineate which cells were the source ofthe peptide expression, we performed double and triple immu-nofluorescent microscopy on tonsil sections (Fig. 2). We la-beled the crypt reticulated epithelial cells with antibodiesagainst the epithelial marker cytokeratin 8 [27], in combina-tion with antibodies against CD15, neutrophil elastase, CD68
and LL-37 (Fig. 2). LL-37 did not co-localize with cytokeratin8 (Figs. 2a–d). However, strong co-localization was observedwith LL-37 and the neutrophil markers (Figs. 2e–h). Macro-phages, identified by either CD68 (Figs. 3a–c), RFD7(Figs. 3d–f) or MCA8746 (Figs. 3g–i) rarely co-localized withLL-37, confirming that neutrophils are the main cell typeexpressing LL-37 in and bordering the tonsil crypts. Interesting-ly, LL-37 was found in rare instances to co-localize with twoother macrophage markers (clones RFD7 and MAC387)(Figs. 3d–i).
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Figure 1 Neutrophils are the predominant source of LL‐37 in the palatine tonsils. 5 μm fresh frozen sections of palatine tonsil wereprepared for immunohistochemical detection of hCAP18/LL‐37. LL‐37 was found expressed in the infiltrated areas of the squamousepithelium (a,b), by leukocytes in the stroma (a), the crypts (c) and some follicles (negative in a, positive in c, see arrows). Notably,LL‐37 expression is only present in areas of the squamous epithelium infiltrated by leukocytes (b) and LL‐37 is strongly expressed inthe crypts (c). Comparison of the affinity purified polyclonal IgG antibody from Innovagen (d) and anti‐serum used in previous publi-cations at dilutions of 1:500 (e) and 1:5000 (f) showing significant levels of non‐specific staining. Double immunofluorescence micros-copy of tonsil showing neutrophils (g, CD15, green) and LL‐37 (h, red) with the majority of the signals co‐localizing (i, yellow). CD68(j, green) and LL‐37 (k, red) did not co‐localize (l, m) indicating that CD68+ macrophages are not sources of LL‐37 in the tonsilepithelium.
142 S.L. Sigurdardottir et al.
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Figure 2 Neutrophils rather than reticulated epithelial cells are responsible for LL‐37 expression in the tonsil crypts. Triple immunoflu-orescence microscopy of 5 μm fresh frozen tonsil sections revealed that crypt epithelium cells (a, cytokeratin 8, green) did not express LL‐37 (b, red) while CD15+ neutrophils (c, blue) completely co‐localized with the peptide (d, purple). Neutrophils labeled using both neutro-phil elastase (e, green) and CD15 (g, blue) are the main source of LL‐37 (f, red) as demonstrated by the co‐localization of all 3 signals (h).High magnification image (inset) showing LL‐37 positive cells contain a multilobular nucleus which is characteristic of neutrophils. Mainscale bar 210 μm, inset 12 μm.
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Figure 3 Macrophages are not a significant source of LL‐37 expression in the tonsils. Double immunofluorescence microscopy of 5 μmfresh frozen tonsil sections demonstrating that CD68+ macrophages (a, green) very rarely colocalized with LL‐37 (b, red) in or near thetonsil crypt (c, close up) nor anywhere else in the tonsil. Mature macrophages (RFD7, d, green) co‐localized rarely (f, yellow) with LL‐37 (e, red). Tissue macrophages (MCA8746, g, green) were observed close to LL‐37 positive areas (h, red) but seldom co‐localized.Scale bar 0.21 mm.
143The anti-microbial peptide LL-37 modulates immune responses in the palatine tonsils
3.2. CD11c+CD13+DCwithin lymphoid follicles expressLL-37
LL-37 expression was observed within the follicles of 14 outof 40 (36%) of the tonsils examined. In some instances afew infiltrating cells that were LL-37 positive were foundclose to the follicle or within it. This staining was specificas it could be abolished by pre-incubating the antibodywith a 10-fold molar excess of LL-37 peptide overnight be-fore staining the tissue (Fig. 4a). To determine which cellsin the follicles expressed LL-37, we used double-labeled im-munohistochemistry with the following markers: CD68 forgerminal center macrophages, CCL18 for CCL18+ DC, CD11cand CD13 for germinal center dendritic cells and PNA.42for follicular DC. CCL18+ DC and follicular DC were weaklypositive for LL-37 (Fig. 4b) whereas no co-localization wasobserved for PNA.42 fDC (Fig. 4c) CD68+ macrophages(Fig. 4d). However, LL-37 co-localized somewhat withCD11c (Fig. 4e) and strongly associated with CD13+ cells inthe follicles (Fig. 4f). Given that the CD13+ cells alsoexpressed CD11c, these follicular CD11c+CD13+ cellsappeared to express the peptide. Interestingly CD13 is alsostrongly expressed by cells in the extrafollicular areas ofthe tonsils but this wasn't associated with LL-37 expression(Fig. 4g). The existence of the CD11c+CD13+ tonsil cell phe-notype was verified by flow cytometry, staining for lineage(CD3, CD14, CD16, CD19, CD20, and CD56) negative, HLA-
DR+, CD11c+, and CD13+ cells (Figs. 5a–d) and isolation ofCD11c+CD13+myeloid DCwhich displayed a dendritic morphol-ogy and generally stained strongly positive for LL-37 (Fig. 5e).Taken together, these data indicate that CD11c+CD13+ DC inthe tonsil follicles express LL-37.
3.3. LL-37 modulates chemokine and chemokinereceptor expression by cultured tonsil mononuclearcells
To investigate the functional consequences of tonsil DC ex-pression of LL-37, we cultured TMC in vitro with various con-centrations of synthetic LL-37 peptide. We found that LL-37dose-dependently enhanced the production of CCL5(p=0.0003) and CXCL9 (p=0.022) after 17 h of stimulation(Figs. 6a and b) whereas CXCL1 production tended to decrease(p=0.032), not shown. Not only could exogenous LL-37 alterTMC chemokine expression, we observed that LL-37 couldalter the expression of chemokine receptors by tonsil T cells.Using flow cytometry we found that stimulation of TMC withLL-37 peptide significantly decreased the frequency of tonsilCD4+ T cells expressing the homing molecules CCR4(p=0.0292, n=14, Fig. 6c) and CCR6 (p=0.0001, n=14,Fig. 6d) on their surface. Stimulation of isolated CD4+ tonsilT cells with LL-37 did not have any effect on their chemokineproduction (not shown).
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Figure 4 CD11c+CD13+ dendritic cells within lymphoid follicles express LL-37. LL-37 was specifically detected in the tonsil follicles (a,middle image), the staining was abolished by pre-incubating the antibody with a 10-fold molar excess of LL-37 peptide overnight beforestaining the tissue (a, left image) and staining was absent using an isotype control antibody (a, right image). Double immunofluorescencedetection on 5 mm fresh frozen tonsil sections revealed that CCL18+ DC (green, b), follicular DC (PNA.42 marker, green, c) and CD68+macrophages (green, panel d) co-localize (yellow, b–d) weakly with LL-37 (red, all panels) within the germinal centers. However,CD11c+ DC (panel e, green) partially co-localized (yellow) whereas CD13+ DC (panel f, green) substantially co-localized (yellow) withLL-37 (red, e–g). Interestingly CD13 expression is much more strongly expressed by cells in the extrafollicular areas of the tonsils thanwithin the follicles (panel g) but this was not associated with LL-37 expression. Figs. 4g and a left and middle image scale bar0.21 mm, Fig. 4a right image scale bar 0.2 mm; Figs. 4b–f left and middle image scale bar 0.05 mm, Figs. 4b–f right image scale bar0.025 mm.
144 S.L. Sigurdardottir et al.
3.4. Tonsil CXCL9 and CCL5 expression is associatedwith LL-37
Given that LL-37 induced the production of CCL5 and CXCL9by TMC cultures, we investigated whether increased tissueLL-37 expression was associated with enhanced CCL5 orCXCL9 expression in tonsils. CXCL9 was strongly expressedaround the germinal centers (Figs. 7a and b), particularlyin those close to crypts, by the crypt reticulated epitheliumand by infiltrating cells throughout the tissue stroma, vesselsand cryptal area most of which appeared to be also positivefor LL-37 (not shown). The extrafollicular area surroundingthe follicle was strongly CXCL9 positive and interestingly,some expression was observed within the follicles that co-localized somewhat with LL-37 (Fig. 7a). Comparison of ton-sil sections with and without germinal center LL-37 expres-sion showed a tendency towards an overall stronger CXCL9expression in the LL-37 positive tonsils (Figs. 7a and b). Nodifference was observed in the CXCL9 staining of infiltratingcells, crypt epithelium or around vessels. CCL5 was stronglyexpressed by infiltrating cells, the crypt reticulated epithe-lium (not shown) and patches of strong expression wereseen in the extrafollicular area surrounding the follicles(Figs. 7c and d). CCL5 expression was observed within thefollicles where it co-localized partially with LL-37 (Fig. 7c)
and the overall expression appeared stronger in tonsilswith follicular LL-37 expression (Figs. 7c and d).
3.5. DNA-LL-37 complexes induce a Th1 signature inTMC cultures
It has recently been shown that plasmacytoid DC take up selfDNA coupled to LL-37 leading to activation of TLR9 [28] andboth plasmacytoid DC and myeloid DC are activated by selfRNA-LL-37 complexes via TLR7 and TLR8 respectively [22].As palatine tonsils contain both plasmacytoid and myeloidDC within the TMC population (Sigurdardottir SL, unpub-lished observation), we stimulated TMC with LL-37 with orwithout human DNA for 3 h to investigate the effects of LL-37 on tonsil DC and T cells. We found significantly increasedIFN-γ and MX-1 mRNA transcript expression after 3 h stimula-tion with the complex (pb0.05, both, Figs. 6e and f). This in-crease was not observed when cells were stimulated by LL-37 or DNA alone.
4. Discussion
The palatine tonsils play an important role as an active bar-rier against bacteria and viruses. The squamous epithelium is
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Figure 5 Lineage negative CD11c+CD13+HLA‐DR+ from palatine tonsils. Tonsil mononuclear cell suspensions were prepared as de-scribed and were found to contain a population of lineage negative CD11c+CD13+HLA‐DR+ cells as identified by flow cytometry (a–d).When isolated on glass slides, lin‐HLA‐DR+CD11c+ cells had a dendritic cell morphology (e) and expressed hCAP18/LL‐37 (DAB immu-nocytochemistry, scale bar 20 μm).
145The anti-microbial peptide LL-37 modulates immune responses in the palatine tonsils
thick and avascular and although bacteria are found in themucosal layer covering the epithelium, studies disagree ontheir ability to penetrate the epithelium [29–32]. Here weshow that tonsil squamous epithelial cells do not expressLL-37 but delegate this function to neutrophils (Fig. 1).This is in contrast to the results of Song et al. [6] which islikely due to a high level of non-specific background stainingas the authors used a 10–20! higher concentration of the an-tibody than we concluded appropriate by serial dilution,which we replicated here for comparison (Figs. 1e and f).Our data also contrast with those of Ball et al. [7] which islikely due to a difference in the antibody used and the meth-odology selected. We concluded that the antibody usedherein is specific for LL-37, especially as this LL-37 stainingwas entirely blocked by overnight pre-incubation of the anti-body with LL-37 peptide (Fig. 4g). Interestingly, neithergroup reported follicular LL-37 expression.
It is interesting that unlike skin [14,19] and colon mucosa[15–17], tonsil epithelia do not themselves express LL-37and perhaps the thickness of the tonsil epithelium alongwith its expression of human β-defensins [5] and the abilityto call on neutrophils are sufficient for its defense. Antimi-crobial defense of the tonsil crypts is crucial due to bacterialbiofilm formation [33,34], accumulation of debris anddegenerated cells in the crypts [35] and the intrinsic physicalweakness of the crypt wall, as this is only 1 cell thick in
places [36]. In addition, some leukocytes throughout thetonsil stroma were found to express LL-37 whereas the con-nective tissue did not (Fig. 1a), and these neutrophils arelikely to be on route to the epithelial compartments. Theconsiderable LL-37 production by the neutrophils located inthe crypts (Figs. 1c, 2d, and 2h) strengthens crypt defensesby contributing anti-microbial activity, diminishing biofilmdevelopment [37] and acting as a chemoattractant forother leukocytes. Furthermore, LL-37 acts synergisticallywith human β-defensins [38] which are produced by thecrypt epithelium, crypt monocytes, macrophages and plas-macytoid DC [39,40]. It also stimulates α-defensin releaseby neutrophils which further contribute to the crypt immunedefense [41].
The most common tonsillar diseases are recurrent tonsilli-tis (RT) due to repeated infections and tonsillar hypertrophy(HT) or enlargement of the tonsil. The hypertrophic tonsilsare often without detectable infections and differ in their un-derlying histology from the recurrently infected tonsils ([42]and S.L. Sigurdardottir, unpublished observation). We foundfollicular LL-37 expression varied between donors with 36%tonsils examined being positive for LL-37 regardless of under-lying pathology (Figs. 1a, c and 4a). Furthermore, in a giventonsil, follicles were either all positive, to a different extent,or strikingly all negative. Staining with various DC markersrevealed that CD11c+CD13+ DC, which have previously been
DNA DNA+LL-37 LL-370.000
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-1 m
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CX
CL9
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(b)
(c)
(d)
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Figure 6 LL‐37 induced chemokine secretion and altered chemokine receptor expression by tonsil mononuclear cells. Mononuclearcells were prepared from fresh palatine tonsil (n=6) as described and stimulated for 17 h with synthetic LL‐37 peptide (1–10 μg/ml).Cell culture supernatants were assayed for the presence of CCL5 and CXCL9 by ELISA and cells analyzed for chemokine receptorexpression by flow cytometry. Both CCL5 (a, p=0.0003) and CXCL9 (b, p=0.022) were significantly induced by LL‐37. The fractionof CD4+ tonsil T cells expressing CCR4 (c, p=0.0292, n=14) or CCR6 (d, p=0.0001, n=14) was diminished after treatment withLL‐37. LL‐37 had no effect on CCR4 or CCR6 expression nor CXCL9 secretion of isolated CD4+ T cells. LL‐37 (10 μg/ml), in combinationwith human DNA (2 μg/ml), induced IFN‐γ (e) and MX‐1 (f) mRNA expression in mononuclear cell cultures (mean+SEM, n=7). Statis-tical significance determined by 1‐way ANOVA or 2‐tailed t‐test as appropriate and indicated as *pb0.05.
146 S.L. Sigurdardottir et al.
described in the thymus [43] and tonsils [44,45] appeared tobe responsible for follicular LL-37 expression. Isolation ofthese cells showed thatmost stained strongly for LL-37 where-as a few were negative or weakly positive for the peptide(Fig. 5e). This might reflect their activation status, histologi-cal position or even differences in LL-37 uptake from theirmicroenvironment. LL-37might thus have an important immu-nomodulatory role in the functional activities of the follicles.The follicular LL-37 expression co-localized somewhat withCCL5 and CXCL9 (Fig. 7) and appeared overall stronger in ton-sils with LL-37 positive follicles. As it has been shown thatCCL5 stimulates follicular migration of T cells [48] whileCXCL9 has immunomodulatory properties [49] and togetheramplify antimicrobial defenses [50–52], our results suggestthat tonsils with follicular LL-37 expression are more reactivein general possibly due to differences in the frequency of ton-sillitis, colonizing microbes, time elapsed since the lastinfection or different stages of follicle development or disinte-gration. To further illustrate this point, we showed that LL-37peptide complexed to human DNA induced IFN-γ and MX-1mRNA expression by cultured TMC after 3 h stimulation(Fig. 6e) which are characteristic of a Th1 immune response,and given the localization, this Th1 environment may contrib-ute to altered IgG class switching in the LL-37+ follicles [46].Interestingly, we did not detect any type I interferon, IL-6 orTNF-α mRNA in contrast to previously described work usinghighly enriched blood-derived pDC cultures [28]. The TMC cul-tures used here consist mostly of B- and T cells with plasmacy-toid and myeloid DC constituting only 0.90% and 0.93% of thecell population respectively (Sigurdardottir SL, unpublishedobservation). This may give a more physiological insight intothe true response of the pDC to activation with DNA-LL-37complexes in situ, as T cell-antigen presenting cell interac-tions are critical for the polarization of the immune response.Furthermore, the TMC are isolated from a highly stimulatedenvironment (tonsils with a history of inflammation or infec-tion) which might influence their baseline stimulatory condi-tion in culture. Studies have shown that LL-37 by itself haslittle effect on lymphocyte chemokine/cytokine production[57,58] but synergistically enhances stimulation by innatecomponents (IL-1β, GM-CSF) [57,59] and suppresses stimula-tion by T cell associated components (IL-4, IL-12, and IFN-γ)[57]. To investigate this further we stimulated isolated CD4+T cells and TMC with the LL-37 peptide and evaluated expres-sion of CCR4 and CCR6 as well as the production of CCL5 andCXCL9. LL-37 had no effect on the isolated CD4+ T cells (notshown) whereas it suppressed CCR4 and CCR6 expression byCD4+ T cells within the TMC population (Fig. 6e). Interestingly,the vast majority of resting tonsil CD4+ T cells expressedCCR4, which is associated with homing of lymphocytes to theskin [54], indicating a potential role for CCR4 in interactionsbetween the tonsils and skin, which is particularly interestinggiven the role of the tonsils in the T-cell mediated skin diseasepsoriasis [55,56]. The observed decreased CCR6 expressionmight indicate that LL-37 influences the inflammatory processof the TMC culture by decreasing Th17 responses [60] and pro-moting a Th1 environment which is supported by the observedincreases in IFN-γ and MX-1 mRNA (Fig. 6f). LL-37 stimulatedthe production of CCL5 and the IFN-γ-induced chemokineCXCL9 (Figs. 6a and b). These results are supported by the ob-served chemokine staining of the follicles (Fig. 7). These twowidely-acting chemoattractants are likely to be produced as a
first response to stimulation especially as CCL5 stimulates Tcell perivascular recruitment [47]. CXCL1 expression has beenreported to be expressed in the crypts [53] and our results indi-cate that LL-37 can attenuate CXCL1 levels (not shown) whichmay influence neutrophil chemoattraction.
LL-37 appears to have a complex role in reinforcing spe-cific immune responses dependent on other stimuli and thesurrounding environment. In this respect it enhances syner-gistically stimulation by components of the innate immunesystem [38,57,59] while suppressing stimulation by T cellcomponents [57]. Furthermore, LL-37 enhances IL-1β re-lease by LPS-stimulated monocytes [61] while decreasing itfrom LPS-stimulated neutrophils [58]. LL-37 concentrationappears to be important with inhibition at low doses andstimulation at high doses [23,62]. It is mainly expressed byneutrophils and stored in their granules [8]. When the neu-trophils are stimulated, they degranulate and secrete largeamounts of LL-37 into the surrounding area where it canact as a co-stimulator for the enhancement of danger signalsespecially in the presence of LPS and IL-1 [57,59].
LL-37 could therefore act as an extra level of immuneregulation by amplifying danger signals when present athigh concentrations and suppressing inappropriate reactionsat low concentrations. LL-37 is robustly expressed in variousinflammatory diseases [19], including lesional psoriatic skin[14], where its properties as a leukocyte chemoattractantand TLR agonist may contribute to the immunopathogenesisof the disease [22,28,63]. LL-37 has been convincingly shownto be essential during bacterial infections [20] and its ab-sence from neutrophils in Morbus Kostmann patients leavesthe body susceptible to massive bacterial infections [21].During these situations, LL-37 is released in the presence ofa danger signal and is necessary as an antimicrobial peptideand to co-stimulate and help direct the immune response.
(b)
(c) (d)
LL-37 (+) LL-37 (-)
CC
L5C
XC
L9
(a)
Figure 7 LL‐37 positive germinal centers have a strong CXCL9and CCL5 expression. In germinal centers positive for LL‐37 (a,c) a stronger CXCL9 (a) and CCL5 (c) expression was observedthan within LL‐37 negative follicles (b,d) positive only forCXCL9 (b) and CCL5 (d). A partial co‐localization was observedfor LL‐37 and CXCL9 (a) as well as LL‐37 and CCL5 (c). Interest-ingly, a few cells positive for both chemokines were observed atthe edge of the follicle as well as within it. Images 400! magni-fication. Scale bar 0.05 mm.
147The anti-microbial peptide LL-37 modulates immune responses in the palatine tonsils
5. Conclusion
In tonsils, LL-37 is expressed by infiltrating neutrophils andDC located within the tonsil follicles. In addition to its anti-microbial properties, LL-37 is a strong chemoattractant andwe now show that in the absence of other stimuli LL-37 canalter the immune response via alteration of chemokine se-cretion and chemokine receptor expression involved in tis-sue-selective T cell trafficking.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors would like to acknowledge support from The Ice-landic Research Fund (grant 080448021-23), LandspitaliUniversity Hospital Research Fund and The Research Fund ofThe University of Iceland for Doctoral Studies. The authorsthank the staff of the Ear, Nose and Throat department,Landspitali-Fossvogur, Reykjavik for their assistance. AndrewJohnston is supported by the Babcock Endowment Fund.
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149The anti-microbial peptide LL-37 modulates immune responses in the palatine tonsils
135
Paper II
The association of sore throat and psoriasis might be explained byhistologically distinctive tonsils and increased expression ofskin-homing molecules by tonsil T cells
S. L. Sigurdardottir,*†
R. H. Thorleifsdottir,*†
H. Valdimarsson* and A. Johnston‡
*Department of Immunology,
Landspitali-University Hospital, Reykjavik,
Iceland and †Department of Medicine, University
of Iceland, Reykjavik, Iceland, and ‡Department
of Dermatology, University of Michigan, Ann
Arbor, MI, USA
Summary
Recent studies have highlighted the involvement of the palatine tonsils in thepathogenesis of psoriasis, particularly among patients with recurrent throatinfections. However, the underlying immunological mechanism is not wellunderstood. In this study we confirm that psoriasis tonsils are infected morefrequently by !-haemolytic Streptococci, in particular Group C Streptococcus,compared with recurrently infected tonsils from patients without skindisease. Moreover, we show that tonsils from psoriasis patients containedsmaller lymphoid follicles that occupied a smaller tissue area, had a lowergerminal centre to marginal zone area ratio and contained fewer tingiblebody macrophages per unit area compared with recurrently infected tonsilsfrom individuals without skin disease. Psoriasis patients’ tonsils had a higherfrequency of skin-homing [cutaneous lymphocyte-associated antigen(CLA+)] CD4+ and CD8+ T cells, and this correlated significantly with theirfrequency of blood CLA+ T cells. The psoriasis patients also had a higher fre-quency of tonsil T cells expressing the interleukin (IL)-23 receptor that wasexpressed preferentially by the CLA+ T cell population. In contrast, recur-rently infected tonsils of individuals without skin disease had a higher fre-quency of tonsil T cells expressing the activation marker CD69 and a numberof chemokine receptors with unknown relevance to psoriasis. These findingssuggest that immune responses in the palatine tonsils of psoriasis patientsare dysregulated. The elevated expression of CLA and IL-23 receptor bytonsil T cells may promote the egression of effector T cells from tonsils to theepidermis, suggesting that there may be functional changes within thetonsils, which promote triggering or exacerbation of psoriasis.
Keywords: CLA, follicle, palatine tonsils, psoriasis, streptococcus
Accepted for publication 3 June 2013
Correspondence: A. Johnston, Department of
Dermatology, University of Michigan, Medical
Science Building I, Room 6427, 1301 Catherine
Street, Ann Arbor, MI 48109-5675, USA.
E-mail: [email protected]
Introduction
Psoriasis vulgaris is a chronic inflammatory skin diseasecharacterized by highly noticeable erythematous, thickened,scaly plaques [1]. The plaques reflect a massive keratinocytehyperproliferation driven by an inflammatory infiltrate richin CD4+, CD8+ and γδ-T cells [2–4]. Psoriasis affectsapproximately 2% of people of both sexes [5], with dimin-ished quality of life [6] and significant co-morbidities [7].While psoriasis has been established to be a complex geneticdisease [8], environmental factors such as trauma and stresscan play a role in its elicitation [1]. Throat infections byβ-haemolytic Streptococci have been associated with its ini-tiation and acute exacerbation [9–15]. This interaction is
not well characterized, but psoriasis patients are more vul-nerable to throat infections than their aged-matched house-hold controls [11]. The palatine tonsils are important formucosal defences due to their location at the opening of therespiratory and digestive tracts as part of the efficient lym-phoid defence system, termed Waldeyer’s ring [16]. Tonsilsare coated with a thick squamous epithelium that extendsinto branched crypts lined by reticulated epithelium [16].Tonsils contain numerous secondary lymphoid follicles thatform after antigen stimulation. These have a distinguishedmantle zone that surrounds a highly organized anddynamic structure, the germinal centre (GC), which isdivided into dark and light zones. Within the dark zone, Bcells undergo somatic hypermutation and clonal expansion
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Clinical and Experimental Immunology ORIGINAL ARTICLE doi:10.1111/cei.12153
139© 2013 British Society for Immunology, Clinical and Experimental Immunology, 174: 139–151
while the light zone is the den of antigen selection and cellproliferation. B cells that do not survive are removed bytingible body macrophages located throughout the GC [17].The mantle zone contains various cell types, including naiveB cells, mature B cells, T cells and dendritic cells. The extra-follicular area is also packed with T cells, mainly of theCD4+ phenotype.
The association between streptococcal throat infectionand the onset or exacerbation of psoriasis has beenobserved in many studies [15], and this observation gaverise to the hypothesis that the T cells that drive psoriaticskin lesions might originate in tonsils, from where theymigrate to the skin and stimulate plaque formation [18].This is supported by the fact that skin T cells tend to beoligoclonal [18] and T cells isolated from the skin and tonsilof the same individual have been shown to carry the sameTCRVB gene rearrangements, indicating a common origin[14].
Tonsillectomy appears to be beneficial for some psoriasispatients who have a history of skin disease exacerbationtriggered by sore throat [15,19]. There is a strong correla-tion between disease improvement and a decline in thefrequency of skin-homing T cells in the blood that recog-nize homologous peptides, present in both the streptococ-cal M protein and keratins in psoriatic skin [19]. Thisskin-homing characteristic is associated with the expres-sion of cutaneous lymphocyte-associated antigen (CLA),which has been shown to be induced by inflammatorystimuli such as interleukin (IL)-12 and streptococcalsuperantigens [20–22] released during streptococcal throatinfections. CLA+ tonsil T cells can be T helper type 17(Th17), Th22 or Th1 polarized, as these phenotypes havebeen shown to be increased in psoriatic skin [23–25] andblood [26].
It is currently not understood why, following streptococ-cal infection, only some people experience psoriatic out-breaks. One possible explanation is that the tonsil micro-environment of these individuals promotes the generationof inflammatory T cells that drive the skin disease. Weinvestigated this possibility by comparing the histology ofrecurrently infected tonsils from psoriasis patients withthose of individuals without skin disease, finding thattonsils from psoriasis patients contained smaller lymphoidfollicles covering less tissue area, the proportion of germi-nal centre to marginal zone area was smaller and there werefewer tingible body macrophages per unit area. In addition,we examined the expression of a number of phenotypic Tcell markers on CD4+ and CD8+ tonsil T cells using flowcytometry, and found that psoriasis patients’ tonsils had ahigher frequency of CLA+, CD4+ and CD8+ T cells and therewas a significant correlation between tonsil and bloodCLA+ T cell frequency. The psoriasis patients also had ahigher frequency of tonsil T cells expressing IL-23 receptor,which was also expressed preferentially by the CLA+ T cellpopulation.
Our results show that the tonsils of psoriasis patients aredistinct histologically from non-psoriasis tonsils, both withregard to follicular morphology and the number of tingiblebody macrophages present within the GC. Furthermore,psoriasis tonsils had a higher frequency of T cells expressingCLA and IL-23 receptor. These findings might, to someextent, explain why tonsillectomy can have a beneficialeffect in psoriasis.
Materials and methods
Study cohort and tissues
Palatine tonsils were obtained from eight patients withhypertrophic tonsils (HT), 66 patients with recurrent infec-tions, 25 of whom were psoriasis patients (PST), and 41were without skin disease (RT). Tonsils were obtainedthrough routine tonsillectomies at the National UniversityHospital, Reykjavik, Iceland or as part of a clinical trial fortonsillectomy as a treatment for psoriasis [19]. A completemedical history was gathered from the psoriasis patients.For non-psoriatic donors, the age, sex and frequency oftonsil infections was obtained. All participants signedinformed consent. The study was approved by the NationalBioethics Committee of Iceland and conducted in compli-ance with good clinical practice and according to the Decla-ration of Helsinki Principles.
Tissue preparation and mononuclear cell isolation
Tonsils were stored immediately in cold sterile saline afterexcision until processed. For bacterial analyses, swabs weretaken from both the crypt and the surface epithelium of theRT and the PST tonsils. For histological evaluation, tonsiltissue was snap-frozen in Tissue-Tek optimum cutting tem-perature (OCT) compound (Sakura Finetek, Zoeterwoude,NL) and kept at −70°C until processed. Tonsil mononuclearcells were isolated as described previously [27]. Briefly,tonsils were minced into 3-mm pieces, passed through atea-sieve and washed with Hanks’s balanced salt solution(HBSS; Gibco, Invitrogen, Paisley, UK). Peripheral bloodmonuclear cells (PBMC) were isolated from the heparinizedvenous blood of psoriatic individuals prior to tonsillectomy.All mononuclear cells were collected at the interphase frac-tion formed by density gradient centrifugation on Ficoll(Sigma-Aldrich, St Louis, MO, USA). Cells were thenwashed twice in phosphate-buffered saline (PBS) andresuspended in PBS for fluorescence activated cell sorter(FACS) staining.
Evaluation of T cell surface receptor expression
Isolated mononuclear cells were stained for 30 min on icewith antibodies against CD4 (clone RPA-T4; Biolegend, SanDiego, CA, USA), CD8 (RPA-T8; Biolegend), CLA (HECA-
S. L. Sigurdardottir et al.
140 © 2013 British Society for Immunology, Clinical and Experimental Immunology, 174: 139–151
452; Biolegend), CCR4 (205410; R&D Systems, Minneapo-lis, MN, USA), CCR5 (HEK/1/85a; Biolegend), CCR6(53103; R&D Systems), CCR7 (150503; R&D Systems),CCR8 (191704; R&D Systems), CCR10 (314305; R&DSystems), CXCR4 (12G5; Biolegend), CXCR5 (51505; R&DSystems), CXCR6 (56811; R&D Systems), IL-23R (218213;R&D Systems) CD62L [DREG-56; Biolegend), intercellularadhesion molecule 1 (ICAM1) (HA58; Becton-Dickinson,San Jose, CA, USA (BD)], CD69 (L78; Becton-Dickinson),CD25 (BC96; Biolegend) or the appropriate isotype con-trols. Cells were washed twice with PBS and fixed in 0·5%paraformaldehyde (PFA). Stained cells were analysed on aFACSCalibur (Becton-Dickinson) flow cytometer withCellQuest (Becton-Dickinson) software.
Immunohistochemistry. Fresh frozen 5-μm sections oftonsil were air-dried and fixed in cold acetone for 10 minand stained with haematoxylin (Thermo Shandon, Pitts-burgh, PA, USA), followed by a dip into 37 mM ammoniasolution. Sections were dehydrated in alcohol with a con-centration gradient of 70–90–100%, fixed in Accustain(Sigma-Aldrich) and mounted with Mountex (HistolabProducts AB, Göteborg, Sweden). After fixing, some sec-tions were blocked with 1% hydrogen peroxide in PBS con-taining 3% mouse serum, washed in PBS and blocked for20 min with 1·5% mouse serum (Vector Laboratories,Burlingame, CA, USA), then incubated with monoclonalanti-CD68 (KP1; Santa Cruz Biotechnology, Santa Cruz,CA, USA) for 30 min and stained according to theVectastain Elite rabbit immunoglobulin (Ig)G kit protocol(Vector Laboratories). The staining was visualized using3,3′-diaminobenzidine (DAB; Becton-Dickinson) andcounterstained with haematoxylin, as described previously.As a negative control, the primary antibody was omitted.
Bacterial culture and typing. Bacterial typing from throatswabs was carried out by culture on sheep blood agar andStreptococcus subspecies were identified using a Streptex kit(Thermo Fisher Scientific, Remel, Lenexa, KS, USA).
Histological measurements
Tonsil tissue was evaluated by measuring the size of thetissue area and the circumference of follicles, germinalcentres and mantle zones. Measurements were collected in
mm2 at 25× or ×100 magnification in two to four locationswithin the same tissue using Axiovision version 4·6·3 soft-ware (Carl Zeiss, Jena, Germany). Follicles were alsocounted in the visual field of ×25 magnification from atleast four locations within the same tonsil tissue. All tonsilsections were coded and evaluated by a blinded observer.Macrophages were counted within the germinal centre at×100 magnification. The number of crypts was counted at×25 magnification. On average, three to four follicles wereselected randomly per tissue section of each tonsil. Onetissue section was utilized from every tonsil.
Statistics
Statistical significance was determined using the Mann–Whitney U-test, Student’s t-test, Fisher’s exact test, Spear-man’s test or linear regression where appropriate.
Results
!-haemolytic Streptococcus is isolated more commonlyfrom psoriasis tonsils
Given the previously reported associations between tonsilinfections with β-haemolytic Streptococci and psoriasis[9–15], we first analysed the bacterial colonization of recur-rently infected tonsils from individuals with and withoutpsoriasis. All the donors of the recurrently infected tonsils(RT) reported that they had, on average, four symptomaticinfections annually, and the group of psoriasis patients(PST) had been selected for a clinical trial on the basis thatthey had a history of worsening of their skin disease follow-ing throat infection [19]. Thus, we found that bacteriacould be cultured from the tonsils of both groups with 68%of PST tonsils and 44% of RT tonsils positive for one ormore species (Table 1, P = 0·0001, Fisher’s exact test two-tailed P-value). We found that Group C Streptococcus wasthe dominant isolate from psoriatic tonsils and this was sig-nificantly more frequent in psoriatic than non-psoriatictonsils (Table 1 and Fig. 1, 40 versus 14·6%, P = 0·021). Inaddition, all groups of β-haemolytic Streptococci (Groups A,B, C and G) were found to be over-represented significantlyin psoriasis tonsils compared with recurrently infectedtonsils (Table 1 and Fig. 1, P = 0·036 all groups combined).Interestingly, co-cultured bacteria were found more
Table 1. Bacterial cultures from psoriasis and recurrently infected tonsils.
Tonsil
Bacteria cultured
Streptococcus of group:
Staphylococcus aureus Escherichia coli NoneA B C G Anginosus
PST (25) 2 (8%) 1 (4%) 10 (40%) 3 (12%) 2 (8%) 4 (16%) 2 (8%) 8 (32%)RT (41) 2 (4·9%) 1 (2·4%) 6 (14·6%) 4 (9·8%) 6 (14·6%) 2 (4·9%) 0 (0%) 18 (56%)
PST: psoriasis tonsils; RT: recurrently infected tonsils.
The association of psoriasis and sore throat
141© 2013 British Society for Immunology, Clinical and Experimental Immunology, 174: 139–151
commonly in psoriatic tonsils (six of 25 versus two of 41,P = 0·0455), as were infections by bacterial species otherthan streptococcus (Table 1, six of 25 versus two of 41,P = 0·0455). No significant difference was observed betweenswabs taken from the crypts and the surface epithelium ofthe same tonsil. Interestingly, tobacco smoking appeared toinfluence the likelihood of bacterial infections among thepsoriasis patients (P = 0·052, Fisher’s exact test), but thisinformation was not gathered from the RT group. Smokingdid not appear to affect histological features of the tonsils.
Tonsils from psoriasis patients contain small folliclesdominated by mantle zone
To investigate whether there are underlying histological dif-ferences in recurrently infected tonsils from individualswith and without psoriasis, we measured the number andsize of lymphoid follicles in haematoxylin-stained tonsilcryosections. Whole follicles were measured so that theyincluded both the germinal centre (GC) and the mantlezone (MZ). Hypertrophic tonsils had, on average, the largestfollicles (Fig. 2a,d), while the smallest were present in thepsoriasis tonsils (Fig. 2c,d). This difference was due both tolarger GC (Fig. 2e) and larger MZ within the HT tonsils(Fig. 2f). The RT follicles were most often larger than the
psoriasis tonsils (Fig. 2b,d), but did not differ with regard tothe size of the MZ area (Fig. 2f). Interestingly, some follicleshad very small GC (Fig. 2b,c), while others had negligibleMZ (Fig. 2a,b).
Hypertrophic tonsils are dominated by enlargedlymphoid follicles
The relative proportions of the GC and the MZ areasrevealed that follicles were dominated typically by a GC inthe hypertrophic tonsils compared with RT and PST tonsils(Fig. 3a–d, P = 0·005 and P = 0·02, respectively). However,follicles in PST tonsils were dominated by their MZ, whichwas a significantly larger proportion of the follicle structurecompared with that of HT (Fig. 3e, P = 0·012) or RT tonsilfollicles (Fig. 3e, P = 0·01). No difference was observed inthe number of follicles per unit area of tissue (Fig. 3f),despite the hypertrophic tonsils having a proportionallylarger tissue area containing follicles (Fig. 3g). Tonsil stromaconsists mainly of follicles and extrafollicular space. Evalua-tion of the follicle size, as a percentage of tissue that is filledby follicles, could give further indications of histologicaland functional differences between the tonsils. In hyper-trophic tonsils, the follicles account for the largest part ofthe total tissue area compared with PST and RT tonsils
Psoriasis tonsils Recurrently infected tonsils
S·anginosus
4%
4%4%
4%4%
8%
4%
4%
S· aureus
Group CStreptococcus
24%
Group BStreptococcus
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4%E· coli
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4%
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7·4%
2·4%
4·9%
12·2%
2·4%
2·4%
2·4%
2·5%
S·aureus
Group CStreptococcus
7·4%
Group BStreptococcus
Group GStreptococcus
Group AStreptococcus
No bacteria cultured 56%
Fig. 1. Analysis of bacteria isolated from recurrently infected tonsils of psoriasis patients and controls revealed that tonsils from psoriasis patientswere infected more frequently by β-haemolytic Streptococci. Psoriasis tonsils (PST, left) were infected more frequently by β-haemolytic Streptococcithan were the recurrently infected tonsils not associated with skin disease (RT, right, P = 0·021), with a strong bias towards Streptococcus C infection(P = 0·036). Two distinct bacterial species could be cultured from some of the tonsils, and this occurred significantly more frequently with PSTtonsils (P = 0·001). In terms of co-infections, the co-culture of Streptococcus G and C was the only common factor between the two tonsil groups.No difference was observed in the overall number of uninfected tonsils [32% in PST versus 56% in recurrently infected tonsils (RT)]. Infections bybacterial species other than streptococcus were more frequent among the psoriasis tonsils (six of 25 versus two of 41, P = 0·0455). Fisher’s exact test,two-tailed P-values.
S. L. Sigurdardottir et al.
142 © 2013 British Society for Immunology, Clinical and Experimental Immunology, 174: 139–151
(Fig. 3f, P = 0·0004 and P = 0·014, respectively), while nodifference was observed between the PST and RT tonsils.The difference appeared to be due to both the larger size ofthe GC in the hypertrophic tonsils compared with PST(Fig. 3g, P < 0·0001) and RT tonsils (Fig. 3g, P = 0·017) andthe MZ area (Fig. 3h: RT, P = 0·008; PST, P = 0·018). Thedifference between PST and RT tonsils was observed only inthe evaluation of the GC as a proportion of total tissue(Fig. 3g, P = 0·02).
The number of CD68+ macrophages correlates withfollicle size and is lower in the PST tonsils
The tingible body macrophages express CD68 on theirsurface and are located within the GC (Fig. 4a). Theirnumber correlated with both the size of the follicle(Fig. 4b–d) and the GC in all tonsils (Fig. 4e–f). Interest-ingly, evaluation of the number of macrophages per mm2 ofGC or follicle size revealed that they were fewer in the PSTtonsils than the RT tonsils (Fig. 4g, P = 0·025; 4h, P = 0·06).
The frequency of skin-homing (CLA+) T cells isincreased in the tonsils of psoriasis patients
Given the association of sore throat and the onset or exacer-bation of psoriasis, the skin-homing potential of tonsil T
cells was evaluated by analysing the frequency of CLA+ Tcells using flow cytometry. Our gating strategy is illustratedin Fig. 5. The frequency of CLA+ T cells was significantlyhigher in the PST tonsils compared with RT tonsils(Fig. 6a–b); this applied to both the CD4+ (P = 0·024) andthe CD8+ T cell populations (P = 0·01). Furthermore, therewas a fairly strong correlation between the frequencies ofCLA+ T cells in the tonsils and blood (Fig. 6c,d) of psoriasispatients for both CD4+ (r = 0·62, P = 0·024) and CD8+ Tcells (r = 0·65, P = 0·01).
IL-23R is expressed preferentially by skin-homing Tcells in psoriasis tonsils
Analysis of the Th17-associated IL-23 receptor (IL-23R)revealed an increased frequency of both IL-23R+CD4+ Tcells (P = 0·002) and IL-23R+CD8+ T cells (P = 0·004) in thepsoriatic tonsils compared with RT tonsils (Fig. 6e,f). CD4+
T cells co-expressing CLA and IL-23R were marginally morecommon in the PST tonsils (Fig. 6g, P = 0·04), with no dif-ference observed for the CD8+ T cells (not shown). Psoriatictonsils also had a higher frequency of CD4+ T cells express-ing the CD62L and CCR7+ (Fig. 6h, P < 0·0001 for both),suggestive of a central memory phenotype. Interestingly,CD4+ T cells expressing the activation markers CD69 orCD25 (Fig. 6h,i; P = 0·0002 and Fig. 6i; P = 0·0005) alone or
(b)(a)
Average size of follicle Average size of germinal center
P = 0·0014P = 0·008P = 0·01 P = 0·02P = 0·0009P < 0·0001P < 0·0001
Average size of mantle zone
Tonsils
Man
tle z
one
(mm
2 )
Ger
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HT RT PST HT RT PSTHT RT PSTTonsilsTonsils
0·350·300·250·200·150·100·050·00
Fig. 2. The follicles of psoriasis tonsils are smaller and have smaller germinal centres than hypertrophic and recurrently infected tonsils.Hypertrophic tonsils (HT, a) are tightly packed with enlarged follicles with noticeably large germinal centres. The recurrently infected tonsils (RT, b)and the psoriasis tonsils (PST, c) contain follicles of various sizes. Measurements of the follicular circumference revealed that PST follicles weresmaller than the HT (d, P < 0·0001) and RT follicles (d, P = 0·008). The germinal centres were also significantly smaller in comparison to the othergroups (e, HT, P < 0·001; RT, P = 0·0014), while the mantle zone of both RT (f, P = 0·02) and PST (f, P = 0·009) tonsils was smaller than in the HTgroup. All measurements were made in mm2 at ×25 magnification in two to four visual fields per tonsil. Statistical significance calculated withStudent’s t-test or Mann–Whitney U-test, as appropriate; n.s.: not significant.
The association of psoriasis and sore throat
143© 2013 British Society for Immunology, Clinical and Experimental Immunology, 174: 139–151
concomitantly (Fig 5i, P = 0·0005) were significantly morefrequent in the RT tonsils. This did not apply to CD8+ Tcells. T cells from RT tonsils also had greater expressionof CCR5 (Fig. 6i, P = 0·009). Furthermore, a higherco-expression of CLA with the skin-homing-associatedmolecules CCR10 (Fig. 6i, P = 0·047) and CCR4+CCR10+
(P = 0·006) was observed in the RT tonsils. Interestingly,CLA expression accompanying CCR6 (Fig. 6i, P = 0·047)
was more common among the CD4+ T cells in the PSTtonsils. No difference was observed between the PST and RTtonsils for CCR4 or CCR10 alone (data not shown).
Discussion
The palatine tonsils form an important part of the mucosaldefence system of the upper respiratory tract and the
25
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P = 0·01
Germinal centre area of total tissue Mantle zone area of total tissue (%)Follicular area of total tissue (%)
P = 0·014P < 0·0001
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Fig. 3. Psoriasis tonsils have smaller germinal centres and larger mantle zones. The germinal centre occupies a smaller part of the follicle in thepsoriasis tonsils (PST, c) compared with the hypertrophic (HT, a, P = 0·005) and the recurrently infected tonsils (RT, b, P = 0·02) (a–d). Conversely,the mantle zone is the larger portion of the follicle in the PST tonsils compared with the HT (e, P = 0·019) and the RT tonsils (e, P = 0·012). The RTand HT tonsils appeared to be proportionally similar (d,e). Although the tonsils differed in the size of their follicles, there was no difference infollicle number (f). Evaluation of the total tissue area covered by follicles revealed that the HT tonsils had a significantly larger follicle area than boththe PST and the RT tonsils (g, P = 0·0004 and P = 0·014, respectively). Germinal centres (GC) and mantle zones (MZ) were analysed further todistinguish which was the influencing factor. HT tonsils had both a larger GC and MZ than both the PST (h, P < 0·0001; i, P = 0·018) and the RTtonsils (h, P = 0·017; i, P = 0·08). However, the difference between RT and PST tonsils was present only for the GC (h) that was significantly smallerin the PST tonsils (P = 0·02). Data points represent average measurements of all follicles in each tonsil in mm2 at ×25 magnification in two to fourvisual fields for each tonsil. Statistical significance was determined using Student’s t-test or Mann–Whitney U-tests as appropriate and indicated oneach panel. Follicles shown at ×100 magnification.
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144 © 2013 British Society for Immunology, Clinical and Experimental Immunology, 174: 139–151
opening of the digestive tract. Due to their location, infec-tions by bacteria, virus and fungi are common, althoughonly few cause symptomatic sore throat. In some instancesinfections can become persistent, leading to the eventualremoval of the tonsils by tonsillectomy. Comparison of thebacteriology of recurrently infected tonsils from psoriasispatients and individuals without skin disease confirmedthat bacterial infections were more common among thepsoriasis tonsils (Table 1, P = 0·0001) and that tobaccosmoking appeared to increase their vulnerability to infec-tion (P = 0·0529).
Psoriasis has a complex genetic background, andnumerous linkage studies, followed by genome-wide asso-ciation studies (GWAS), have resulted in the identificationof 41 genetic susceptibility loci [28], 36 of which are asso-ciated with genes that have a known immunologicalfunction [28] and might be important to the immuneresponses of the tonsils. These include genes involved in Tcell and innate cell function such as ERAP1, IL12B, IL23R,IL23A, NOS, TYK2 and STAT3, as well as TNIP1,TRAF3IP2, TNFAIP3, CARD14 and REL, important fornuclear factor (NF)-κB signal transduction. Thus, the dif-ferences in bacterial colonization and tonsil histologyreported here may be partly the consequence of particularallele carriage at these loci influencing tonsil innate andadaptive immune responses.
Of note is the ability of some bacteria, including strepto-cocci, to penetrate and survive in host cells as facultativeintracellular bacteria [29,30], which renders antibiotic treat-ment challenging [31–35] and probably influences theoutcome of bacterial swab tests, which typically only cultureextracellular organisms. Moreover, tonsils are typically notexcised during infectious episodes, probably adding to theunderestimation of bacteria species. The recent introduc-tion of high-throughput DNA/RNA sequencing for studiesof the microbiome, which does not rely on the ability ofbacteria to grow on selection media, are now revealing thetrue diversity of tonsil flora [36,37].
The bacterial infections noted here were mainly byβ-haemolytic Streptococci (serotypes A, B, C and G,P = 0·021), in particular Group C Streptococci (Table 1,P = 0·036), which have been associated with psoriasis[9–12]. Streptococcal infections have been shown to inducethe expression of the skin-homing molecule CLA on thesurface of T cells [20–22,38–40]. We have reported previ-ously that psoriasis patients have an increased frequency ofCLA+ T cells in the blood [40], and in this study we showthe same pattern in the psoriasis tonsils (Fig. 6a,b). Peptidessharing homologous sequences from streptococcal Mprotein and human epidermal keratins have been shown tostimulate CLA+ T cells from the blood of psoriasis patients[39] and lesional T cell clones have been shown to respondin a HLA-restricted manner to streptococcal peptidoglycan[41]. We have postulated that these T cells play an impor-tant role in psoriasis [18], as improvement after tonsillec-
tomy correlates closely with their reduction in blood [19]and T cell clones with similar TCRVB gene usage have beenisolated from the tonsils and skin lesions of psoriasispatients [14]. The finding that psoriasis tonsils are infectedmore commonly by streptococcus and have a higher T cellCLA expression in tonsils that correlate with levels in blood(Fig. 6c,d) is consistent with the notion that streptococcalantigen-specific T cells are involved in the pathogenesis ofpsoriasis, and that effector T cells generated in the tonsilscould migrate through the circulation to the skin.
The epidermal proliferation characteristic of psoriasis isnow thought to be driven, at least in part, by IL-17- andIL-22-secreting T cells [23,42]. T cells releasing IL-17, IL-22and related cytokines commonly express IL-23R and CCR6and these cells were more frequent in the psoriasis tonsils(Fig. 6), suggesting a potential Th17 cytokine bias. Anumber of studies have shown increased expression ofcytokines by T cells from RT compared with HT tonsils,with a predominance of Th1 over Th2 cytokines [43–45].When cells from HT and RT tonsils were stimulated withintact, heat-inactivated Haemophilus influenzae and GroupA Streptococcus, H. influenza induced IL-1α, IL-1β, tumournecrosis factor (TNF)-α, IL-6, IL-8, IL-2, interferon(IFN)-γ, TNF-β and IL-10 production in both tonsilgroups, but Group A Streptococcus induced significantlyhigher frequencies of IFN-γ-positive cells in the RT group[46]. Similar findings emerged when T cells from HT andRT tonsils were stimulated with isolated streptococcalM-protein, with S. pyogenes-positive RT tonsil T cells secret-ing more IFN-γ [45]. These reports all indicate thatcytokine production by tonsil lymphocytes is elevated in RTcompared with HT, and suggest that the pattern of cytokineexpression probably differs accordingly to match the patho-genic challenge. Since the emergence of Th17, Th22 andTh9 phenotypes, further studies analysing these T cellphenotypes in terms of cytokine and adhesion/homingmolecule expression and localization in tonsils are nowwarranted.
Although a number of case reports [15] and our recentprospective study [19] suggest that tonsillectomy had apositive outcome on the disease activity of some psoriasispatients, this might be restricted to individuals with agenetic predisposition [e.g. human leucocyte antigen(HLA)-Cw6 carriers] and a history of skin disease exacerba-tion with sore throat. Follow-up periods vary in thesereports, and although several years of remission have beenreported it is uncertain whether or not this is permanent,given the dynamic nature of the immune response and thepossibility of tonsil regrowth.
Psoriasis tonsils differed from recurrently infected tonsilsby containing more CD4+ T cells with lymphoid migratingabilities, expressing more CD62L+ and CCR7+, both ofwhich are important for homing to lymph nodes and T cellareas [47,48]. These findings, along with the differentialexpression of the activation markers CD69 and CD25,
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145© 2013 British Society for Immunology, Clinical and Experimental Immunology, 174: 139–151
P = 0·025
MZ
MØ
1·00
0·75
0·50
0·25
0·00
1·00
0·75
0·50
0·25
0·00
0·4
0·3
0·2
0·1
0·0
0·03
0·02
0·01
0·00
1·00
0·75
0·50
0·25
0·00
1·50
1·25
1·00
0·75
0·50
0·25
0·00
Siz
e of
folli
cle
(mm
2 )S
ize
of fo
llicl
e (m
m2 )
0 50 100
(b)
(d)
CD68+ macrophages
CD68+ macrophages and follicle size
CD68+ macrophages and follicle size (RT)
150
P < 0·0001
200
0 50 100CD68+ macrophages
150 200
r 2 = 0·71
Siz
e of
folli
cle
(mm
2 )S
ize
of g
erm
inal
cen
tre (
mm
2 )
0 5025 10075
(c)
(e) (f)
(g) (h)
(a)
CD68+ macrophages
CD68+ macrophages and follicle size (PST)
CD68+ macrophages in germinal centre (PST)
CD
68+ m
acro
phag
es p
er m
m2 fo
llicl
e
125
0 5025 10075
CD68+ macrophages
125
P < 0·0001r
2 = 0·74
P < 0·0001r
2 = 0·85
P < 0·0001r
2 = 0·72
Siz
e of
ger
min
al c
entre
(m
m2 )
CD68+ macrophages in germinal centre (RT)
0 5025 10075CD68+ macrophages
150125 175
P < 0·0001
P = 0·006
r 2 = 0·55
0·025
0·020
0·015
0·010
0·005
0·000
Number of macrophages/mm2 germinal centreNumber of macrophages/mm2 follicle area
CD
68 m
acro
phag
es p
er m
m2 L
Z
RTPSTTonsils
RTPSTTonsils
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146 © 2013 British Society for Immunology, Clinical and Experimental Immunology, 174: 139–151
implicate a dysregulation of innate immune mechanisms inthe PST tonsils that influences the differentiation of T cells.This was evaluated further by comparing the histologicalcharacteristics of the tonsil. The histology of hypertrophicand recurrently infected tonsils has been studied thoroughly[49,50] whereas, hitherto, psoriasis tonsils have been histo-logically undefined. In this study we show that psoriasistonsils have unique histological characteristics that distin-guish them from other tonsils. Tonsil follicles can differwith respect to their reactivity regardless of pathology [51].Hypertrophic tonsils have large noticeable and clearly
defined follicles with an extrafollicular area that is densewith multiple cells (Fig. 2a). The psoriasis tonsils, however,have follicles that are often less defined, and theextrafollicular area appears less densely packed (Fig. 2c); theRT tonsils appear to have an intermediate characteristic(Fig. 2b), with more crypts than HT tonsils (P = 0·018).PST follicles are smaller (Fig. 2) and cover less tissue area(Fig. 3). The composition of the follicles was also different,as they have a lower GC : MZ ratio than the other tonsils.Not all follicles were similar in size and structure in thethree groups, as some had proportionally smaller GC and
Fig. 4. The number of CD68+ macrophages correlate with the size of the follicle. CD68+ tingible body macrophages were counted within thegerminal centre (GC) (a, see arrow). The circumference of the follicle and the GC were measured and macrophages per square mm of either follicleor GC evaluated. A clear correlation is present between the number of CD68+ macrophages in the germinal centre and its size as well as the size ofthe whole follicle, regardless of pathology (b, r2 = 0·71). A positive correlation is also observed when analysed separately for psoriasis tonsils (PST) (c,r2 = 0·74) and recurrently infected tonsils (RT) (d, r2 = 0·72). A similar correlation is observed when the number of macrophages is analysed inregard to the size of the germinal GC alone, although the correlation is stronger for the psoriasis tonsils (e, r2 = 0·74 versus f, r2 = 0·74). The numberof CD68 macrophages per mm2 area of GC was evaluated for both the GC (g) and the whole follicle (h). Macrophages were more numerous permm2 of both areas in the recurrently infected tonsils. A total of five PST tonsils and 15 RT tonsils were analysed with, on average, four follicles beingmeasured per tonsil. Statistical significance was determined using linear regression, t-test or Mann–Whitney U-test with P < 0·05 consideredsignificant.◀
IgG
2b is
otyp
e co
ntro
l
99·03%0·00%
0·97%0·00%
Sid
e-sc
atte
r
0
256
512
768
1024
CD4+
CLA
exp
ress
ion
0·00%
0·00%
17·36%
82·64%
CLA+
CD4
IgM
isot
ype
cont
rol
0·00%
0·00%
0·84%
99·16%
IL-2
3R e
xpre
ssio
n
92·55%0·00%
7·45%0·00%
Forward-scatter
Sid
e-sc
atte
r
10240
256
512
768
1024
Lymphocytes
(b) (d) (f)
(e)(c)(a)
0 256 512 768 104
104
103
103
102
102
101
101
100
CD4104103102101100
CD4104103102101100
CD4104103102101100
CD4104103102101100
100
104
103
102
101
100
104
103
102
101
100
104
103
102
101
100
Lymphocytes
CD4+
CLA+
Fig. 5. Fluorescence activated cell sorter (FACS) gating strategy on tonsil mononuclear cells and examples of surface phenotype analysis. Followingtonsil mononuclear cell isolation on Ficoll, cells were immunophenotyped using fluorescently labelled antibodies and flow cytometry. In thisexample we acquired 10 000 events then determined interleukin (IL)-23R expression on CD4+cutaneous lymphocyte-associated antigen (CLA)+
lymphocytes by first gating on lymphocytes (a), CD4-positive cells (b), then using an immunoglobulin (Ig)M isotype control (c) to determineCLA-positive cells (d). Gating on CD4+CLA+ lymphocytes, we determined IL-23R expression (e,f).
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147© 2013 British Society for Immunology, Clinical and Experimental Immunology, 174: 139–151
CLA+CD4+ T cell correlationin blood and tonsil
CLA+CD4+ T cells in tonsil (%)
CLA+CD4+ T cells in tonsil
IL23R+CD8+ T cells in tonsil
IL23
R+ C
D8+ T
cel
ls (
%)
CLA+CD8+ T cell correlationin blood and tonsil
CLA+CD8+ T cells in tonsil (%)
CLA
+ CD
8+ T c
ells
in b
lood
(%
)
CD
4+ IL23
R+ T
cel
ls (
%)
IL23R+CD4+ T cells in tonsil
CLA+IL23R+CD4+ T cells in tonsil
Fre
quen
cy o
f CD
4+ T c
ells
(%
)
Fre
quen
cy o
f CD
4+ T c
ells
(%
)C
LA+ IL
23R
+ CD
4+ T c
ells
(%
)C
LA+ C
D4+ T
cel
l in
bloo
d (%
)C
LA+ C
D4+ T
cel
ls (
%)
RTPSTTonsils
RTPSTTonsils
RTPSTTonsils
CCR5+ CD25+ CD69+
CD25+CLA+
CCR6+CLA+
CCR10+CLA+
CCR4+
CCR10+
(i)
(a)
(c)
(e)
(g)
******
*
*
**
**
**
***
***
(h)
(f)
P = 0·004P = 0·002
P = 0·04
P = 0·024
CLA+CD8+ T cells in tonsil
CLA
+ CD
8+ T c
ells
(%
)
(b)P = 0·01
r = 0·62, P = 0·01
(d)
r = 0·65, P = 0·01
10
20
30
0
5
10
15
0
CD62L CCR7 CD69
20
10
40
30
0
15
10
25
20
0
5
2010
405060708090
100
30
0
5
10
15
0
10
20
30
0
0
5
5
10
10
15
15
20
20
25
25
30
30
35
00
5
5
10
10
15
15
20
20
25
25
30
30
35
0
RTPSTTonsils
RTPSTTonsils
Fig. 6. The frequency of skin-homing[cutaneous lymphocyte-associated antigen(CLA)+] T cells is higher in the psoriasis tonsils(PST) patients, and these cells expressinterleukin (IL)-23R preferentially. Thefrequency of CD4+ (P = 0·024) and CD8+
(P = 0·01) T cells expressing CLA was higher inthe PST than the control tonsils (a,b). Therewas a correlation between the frequency ofCLA+ T cells in the blood and tonsils ofpsoriasis patients CD4+ r = 0·62, P = 0·01, CD8+
r = 0·61, P = 0·02 (c,d). PSTs had a higherfrequency of CD4+ (P = 0·002) and CD8+ (f,P = 0·004) T cells expressing IL-23R (e,f).Furthermore, the IL-23R was expressedpreferentially by CLA+ CD4+ T cells, and suchco-expression more frequent in the PST tonsils(g, P = 0·04). The frequency of CD4+ T cellsexpressing CD69 or CD25 alone (h, P = 0·0002and i, P = 0·0005) or together (i, P = 0·0005)was higher in the recurrently infected tonsils(RT) tonsils. CCR5 expression was also higherin the RT tonsils (i, P = 0·009). Interestingly,CD69+ CD8+ T cells were more frequent in theRT tonsils (P = 0·03, not shown). Furthermore,CD4+ T cells co-expressing CLA and CCR10 (i,P = 0·047) or CCR4 and CCR10 (i, P = 0·006)were more frequent in the RT tonsils. CD4+ Tcells expressing CCR7 (h, P < 0·0001), CD62L(h, P < 0·0001) or CLA and CCR6 (i, P = 0·047)were more frequent in PST than RT tonsils.Dotted lines indicate 95% confidence intervalswith Spearman’s correlation in (c) and (d). Forgrouped data, statistical significance wasdetermined using Student’s t-test orMann–Whitney U-test as appropriate. Box-plotsshow median and 95% confidence intervals forPST (n = 11, filled boxes) and RT (n = 22, greyboxes) tonsils.
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148 © 2013 British Society for Immunology, Clinical and Experimental Immunology, 174: 139–151
larger MZ. On rare occasions, follicles with limited MZ orhardly noticeable GC were observed. Germinal centres areimportant for the humoral immune response, as B cellproliferation, differentiation and immunoglobulin class-switching occurs in the GC. The hypertrophic tonsils in thisstudy had the largest follicles and GC, which also coveredthe greatest tissue area indicating a highly active GC, whichis in accordance with earlier studies [49,50]. Why the folli-cles become enlarged is not understood, but it has been sug-gested that viral infections causing repeated irritation orlack of cell apoptosis within the GC might be the causalfactor [52]; future microbiome studies of the tonsils mightshed light on the viral hypothesis. It is possible that thesmaller size of the psoriasis follicles is due to a dysfunc-tional regulation of the GC immune response. Analysis ofthe tingible body macrophages within the GC can give indi-cations of its function, as their role is to ingest cellulardebris within the germinal centre [53] and possibly todown-regulate the GC reaction [54]. Interestingly, thenumber of CD68 macrophages correlated strongly withboth the size of the follicle and the GC. However, psoriasisfollicles had fewer macrophages per mm2 of both GC andfollicle area than the RT tonsils. This could cause an accu-mulation of apoptotic cells within the GC that might inter-rupt its function and increase the risk of autoantigens.Interestingly, with smaller follicles the T cell area, theextrafollicular area, becomes proportionally enlarged. Itcould be hypothesized that the combination of increasedincidences of streptococcal infections and smaller follicleswith possible dysfunction in the regulation of the immuneresponse might lead to insufficient clearance of the bacteria,thus causing enhanced intracellular reservoirs of the bacte-rium within macrophages and epithelial cells. These couldreinfect later, when conditions are favourable. In thisrespect, it has been shown that approximately 9% of psoria-sis patients are symptomless carriers of streptococcus,which is 20% more common than in their household con-trols [11]. However, in the present study the psoriasispatients were selected on the basis of a known association ofsore throat and exacerbation of their disease resulting in acarrier rate of 44% for Streptococcus Groups A, C and Gcombined.
These results suggest that the immune response in thetonsils of psoriasis patients is abnormal. These data are con-sistent with the idea that the infiltrating T cells which drivepsoriatic skin disease might originate in tonsils where strep-tococcal infection induces a skin-homing phenotype.CD68+ macrophages containing peptidoglycan, thought tohave originated in the tonsils, have been found in increasednumbers in psoriatic skin lesions [52], and lesional CD4+ Tcell clones have been shown to respond in an HLA-restricted manner to this streptococcal peptidoglycan [52].Thus, the recurrent tonsil infections could lead to the matu-ration of skin-homing T cells that recognize streptococcalmembrane and cell wall moieties [55] which, after migra-
tion to the skin, could react with streptococcal epitopes [52]or alternatively skin-specific epitopes via molecularmimicry [18,56], leading to the development of psoriasisplaques.
Acknowledgements
The authors would like to acknowledge support from theIcelandic Research Fund (grant number 080448021-23), theIcelandic Research Fund for Graduate Students, LandspitaliUniversity Hospital Research Fund and the Research Fundof the University of Iceland for Doctoral Studies. Theauthors thank the staff of the ear, nose and throat depart-ment, Landspitali-Fossvogur, Reykjavik for their assistance.Andrew Johnston is supported by the Babcock EndowmentFund and the American Skin Association.
Disclosures
The authors declare that there are no conflicts of interest.
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55 Baker BS, Brown DW, Fischetti VA et al. Skin T cell proliferativeresponse to M protein and other cell wall and membrane proteinsof group A streptococci in chronic plaque psoriasis. Clin ExpImmunol 2001; 124:516–21.
56 Besgen P, Trommler P, Vollmer S, Prinz JC. Ezrin, maspin,peroxiredoxin 2, and heat shock protein 27: potential targets of astreptococcal-induced autoimmune response in psoriasis. JImmunol 2010; 184:5392–402.
The association of psoriasis and sore throat
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Paper III
The Journal of Immunology
Improvement of Psoriasis after Tonsillectomy Is Associatedwith a Decrease in the Frequency of Circulating T Cells ThatRecognize Streptococcal Determinants and Homologous SkinDeterminants
Ragna Hlin Thorleifsdottir,*,†,‡ Sigrun Laufey Sigurdardottir,*,‡ Bardur Sigurgeirsson,†
Jon Hjaltalin Olafsson,†,‡ Martin Ingi Sigurdsson,‡,x Hannes Petersen,‡,{
Sigurlaug Arnadottir,*,‡ Johann Eli Gudjonsson,‖ Andrew Johnston,‖ andHelgi Valdimarsson*,‡
Exacerbation of chronic psoriasis can be associated with streptococcal throat infections, and T cells that respond to peptide sequen-ces common to streptococcal M proteins and skin keratins have been detected in patients’ blood. To our knowledge, we haveconducted the first blinded, prospective study to assess the impact of tonsillectomy on psoriasis. Twenty-nine patients with chronicpsoriasis and history of exacerbation after sore throat were randomly assigned to tonsillectomy (n = 15) or control (n = 14) groupsand monitored for 2 y clinically and by enumeration of circulating skin homing T cells that respond to short homologous Mprotein or keratin peptides. Thirteen patients (86%) showed sustained improvement after tonsillectomy ranging from 30 to 90%reduction in disease severity. Furthermore, there was a close correlation between the degree of clinical improvement in individualpatients and reduction in the frequency of peptide-reactive skin-homing T cells in their circulation. No corresponding clinical orimmunologic changes were observed among the controls. These findings indicate that tonsillectomy may have a beneficial effect onchronic psoriasis because the palatine tonsils generate effector T cells that recognize keratin determinants in the skin. TheJournal of Immunology, 2012, 188: 5160–5165.
P soriasis is a common inflammatory skin disease that can beassociated with arthritis and tends to have a fluctuatingcourse. Several distinct but overlapping clinical variants
have been identified, but chronic plaque-type lesions are mostcommon. Psoriatic plaques are characterized by a marked kera-tinocyte hyperproliferation, altered differentiation, and keratin ex-pression, and they are associated with dermal and epidermal in-filtration of leukocytes. It is now generally accepted that thesechanges are triggered and maintained by oligoclonal T lympho-cytes, indicating that the psoriatic process is driven by conventionalAgs (1). The pathological epidermal hyperplasia coincides withepidermal influx of a1b1 integrin-positive CD8
+ T cells and can beprevented by specific blocking of this integrin (2). Recent studiesindicate that, in addition to Th1 cells, Th17 cells also have an
important role in psoriasis (3), as well as IL-17–producing CD8+
T cells (4), and that the keratinocyte hyperproliferation might bedriven by the Th17 cytokine IL-22, either directly or indirectly (5,6). IFN-g is also a powerful inducer of the chemokine CCL20,a ligand for CCR6, which is expressed by T cells that can produceIL-17. In this way IFN-g may play a major role in enhancing theIL-17 response (4). Psoriasis has a strong genetic component, witha 40–70% concordance in identical twins (7, 8). Several suscep-tibility alleles have now been identified and confirmed (9), andHLA-Cw*0602 has recently been implicated as the strongestsusceptibility allele in psoriasis (10). On the basis of this andvarious other pathological features of psoriasis, we have arguedthat CD8+ T cells are likely to be the ultimate effector cells thatrecognize autoepitopes presented in the context of HLA-Cw6 orother HLA class I molecules on the surface of APCs and kerati-nocytes (11). The pathogenic activity of these CD8+ T cells is,however, likely to require a local interaction with CD4+ T cells,involving cross-presenting dendritic cells (11).A strong association between streptococcal throat infection and
the acute guttate variant of psoriasis (an early onset form) has beendemonstrated in many studies. Furthermore, it has been demon-strated in a prospective study that chronic plaque psoriasis may alsoexacerbate after such infections and, furthermore, the psoriasispatients had an !10-fold higher frequency of streptococcal throatinfections than age-matched household controls (12). Moreover,worsening was exclusively associated with throat infection by thethree groups of b-hemolytic streptococci (A, C, and G) that ex-press M protein on their surface (12), a major virulence factorcomposed of two polypeptide chains with a helical coiled-coilconfiguration. Interestingly, mAbs raised against group A strep-tococci cross-reacted with keratin defined as the a helical coiled-
*Department of Immunology, Landspitali University Hospital, 101 Reykjavik, Ice-land; †Department of Dermatology, Landspitali University Hospital, 101 Reykjavik,Iceland; ‡Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland; xDepart-ment of Medicine, Landspitali University Hospital, 101 Reykjavik, Iceland; {Depart-ment of Ear, Nose, and Throat, Landspitali University Hospital, 101 Reykjavik, Iceland;and ‖Department of Dermatology, University of Michigan, Ann Arbor, MI 48109
Received for publication October 4, 2011. Accepted for publication March 12, 2012.
This work was supported by the Icelandic Research Fund, the Icelandic ResearchFund for Graduate Students, the University of Iceland Research Fund, and the Sci-ence Fund of the National University Hospital in Iceland.
Address correspondence and reprint requests to Prof. Helgi Valdimarsson, Depart-ment of Immunology, Landspitali University Hospital, Hringbraut, 101 Reykjavik,Iceland. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: CLA, cutaneous lymphocyte-associated Ag; PASI,Psoriasis Area and Severity Index; TX, tonsillectomy.
Copyright! 2012 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/12/$16.00
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coil autoantigen in human skin (13, 14). Although psoriasis ismediated by T cells and not by Abs, these findings potentiallylinked keratin to psoriasis.An extensive homology between streptococcal M protein and
keratin was first reported !20 y ago (15). Of !4200 mammalianproteins that were compared, human type 1 keratins that are up-regulated in psoriasis (16) showed the strongest homology withthe streptococcal M protein. On the basis of this and other find-ings, it was proposed in 1995 that psoriasis can be initiated bystreptococcal superantigens and maintained by T cells that rec-ognize streptococcal M protein determinants in the palatine tonsilsand homologous keratin determinants in the skin (17). Subse-quently, a markedly increased frequency of T cells that recognizesuch determinants was detected in the blood of patients withchronic psoriasis compared with allergic dermatitis patients andHLA-Cw*0602–positive healthy controls (18, 19). Notably, thegreat majority (.90%) of the circulating T cells that responded tothe homologous K and M peptides expressed the skin-homingentity cutaneous lymphocyte-associated Ag (CLA) (19).There are several reports of partial or complete remission of
psoriasis following tonsillectomy (20, 21), including three patientswho were shown to have identical T cell clones in their palatinetonsils and skin lesions (20). However, to our knowledge, no con-trolled prospective trial has been reported. As most psoriasis pa-tients have a fluctuating disease activity and spontaneous remissionsare not uncommon, matched patient controls are essential to de-termine whether tonsillectomy has any beneficial effect.To our knowledge, we have conducted the first blinded, pro-
spective study to assess the clinical and immunologic impact oftonsillectomy on chronic psoriasis. We argued that if T cells,primed against streptococcal M protein determinants in the pala-tine tonsils, play a pathogenic role in psoriasis, then the numbersof these cells should decrease in the circulation after tonsillectomyand this should be associated with reduced disease activity.
Materials and MethodsStudy population and clinical follow-up
Twenty-nine patients with chronic plaque psoriasis were recruited and ran-domly allocated into tonsillectomy (TX) and control groups. The study wasapproved by the Icelandic National Bioethics Committee (VSNb2006090015/03-15). Written informed consent was obtained from each patient. Patientswere eligible for the study if they were$18 y age, had been diagnosed by adermatologist with chronic plaque psoriasis, and had a history of psoriasisexacerbation during or shortly after throat infections. Patients with heartand lung diseases were excluded. The patients were off treatment, in-cluding antibiotics, for at least 4 wk before they entered the study and for2 mo thereafter. Subsequently, the participants were allowed to havetreatment according to what they and their dermatologists thought indi-cated. Their disease course was followed for at least 2 y and their diseaseseverity assessed by the Psoriasis Area and Severity Index (PASI) (22),which is the standard method for evaluating changes in the extent andactivity of this disease. The clinical evaluation was observer-blinded withregard to tonsillectomy. The participants were evaluated clinically at studyentry and after 2, 6, 12, 18, and 24 mo and blood samples were obtained atstudy entry and after 2, 12, and 24 mo. The patients’ need for anti-psoriasistreatment during the follow-up was also monitored. The patients were allexamined for tonsillar remnants at the end of the study.
Homologous peptide Ags
The amino acid sequence of keratin 17 was split into a complete set ofoverlapping residue peptides that were then used as a library to comparewith the sequence of the M6 protein using the FASTA algorithm (23). Thehomologous M peptides were restricted to the conserved C-terminal half ofthe protein whereas the homologous keratin peptides were present in twocoil-forming regions of keratin 17. Each homologous M peptide sharedfour to six amino acids with the corresponding K peptide, and they werefurther selected on the basis of predicted binding to HLA-Cw*0602 aspreviously described (19). Thus, 64 short, mostly 9- to 12-aa overlapping
peptides derived from human cytokeratin 17 or streptococcal M6 protein(homologous K/M peptides) were selected on the basis of sequence ho-mology and predicted binding to HLA-Cw*0602 as previously described(19). The peptides were blended into 16 peptide pools, each containing 8peptides. Supplemental Table I shows the size and location of the over-lapping homologous K/M peptides, and supplemental Table II shows theirsequences and lists some relevant references.
Enumeration of peptide-reactive T cells
The frequency of T cells that respond to amino acid sequences common tokeratin and M protein was determined as previously described (19). Briefly,PBMCs or tonsillar mononuclear cells were isolated from heparinizedvenous blood of psoriatic individuals or tonsillar tissue (24) by Ficoll(Sigma-Aldrich, St. Louis, MO) density gradient sedimentation. Single-cell suspensions of tonsillar mononuclear cells were prepared as described(24). The PBMCs or tonsillar mononuclear cells were cultured at a densityof 1 3 106 cells/ml in complete RPMI 1640 in cell culture tubes (Nunc,Thermo Fisher Scientific, Roskilde, Denmark) and stimulated for 16 h withthe 16 peptide pools (see Supplemental Table I) at a final concentration of2 mg/ml, in the presence of the costimulatory Abs to CD28 and CD49d (1mg/ml each; Serotec Scandinavia, Oslo, Norway). After the first 2 h thesecretion inhibitor brefeldin A was added (10 mg/ml; Sigma-Aldrich) andthe cultures incubated for a further 14 h at a 5˚ slant at 37˚C in a humid-ified 5% CO2 atmosphere. Anti-CD3 (1 mg/ml; Serotec Scandinavia) andstreptokinase (200 U/ml; Hoechst Marion Roussel, Stockholm, Sweden)were used as positive controls. The great majority (.90%) of the T cellsresponding to the homologous K and M peptides express the skin-homingentity CLA (19). After the incubation, the mononuclear cells were there-fore stained with anti–CLA-FITC (BioLegend, San Diego, CA) and anti-CD4 or CD8-PerCP mAbs (BioLegend) on ice, in the dark for 20 min.After two washes in PBS, the cells were fixed in 500 ml cold (4˚C) 2%paraformaldehyde for 10 min at room temperature and after a further washthey were treated with permeabilizing buffer (0.5% BSA, 0.1% saponin,0.1% sodium azide; Sigma-Aldrich) for 10 min at room temperature,followed by a wash and resuspension in the same buffer. The cells werethen stained with anti–IFN-g-PE (BioLegend) and anti–IL-17A-AF647 for20 min at 4˚C in the dark, washed in 1.5 ml permeabilizing buffer,centrifuged for 5 min at 500 3 g, and resuspended in 250 ml per-meabilizing buffer supplemented with 1% paraformaldehyde. Lympho-cytes were analyzed using a FACSCalibur flow cytometer (BD Biosciences)gating on light scatter and CD4, CD8, and CLA expression, capturinga minimum of 200,000 events guided by appropriate isotype control Abs.The source and characteristics of the various Ab conjugates used for theflow cytometric analyses are shown in Supplemental Table III.
HLA-Cw*0602 typing
Genotyping of blood mononuclear cells for HLA-Cw*0602 alleles wasperformed as previously described (10).
Serum IL-8 ELISA
Serum IL-8 was used as a serological inflammatory marker and measured atstudy entry and after 24 mo by Quantikine ELISA (R&D Systems, Min-neapolis, MN) as directed by the manufacturer.
Table I. Baseline demographic information and disease characteristicsof the patients
TX Group(n = 15)
Control Group(n = 14)
Males/females 3/12 6/8Age, y (6SD) 35.3 6 9.9 35.9 6 9.8Body mass index 6 SD 25.2 6 5.3 25.4 6 3.6Duration of psoriasis, y (6SD) 19.9 6 9.5 20.5 6 11.7Age at onset, y (range) 15 (4–35) 15 (2–28)Family history (n) 12 12Psoriatic arthritis (n) 4 1Psoriatic nails (n) 10 9HLA-Cw*060-positive 11 13PASI score 6 SD 11.0 6 5.7 9.3 6 3.7Prior treatments (n)Topical 8 8UVB 5 4Systemic 1 0
Values are means unless specified otherwise.
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Sigrún Laufey Sigurðardóttir
Expression of the data and statistics
Flow cytometric responses were rated positive when .0.05% of the CLA+
CD4 or CD8+ T cell populations expressed IFN-g or IL-17 brighter thanthe corresponding unstimulated culture or fluorescence minus one control.
The frequency of T cells that responded to each of the 16 peptide poolswas determined as a percentage of CLA+ cells in each of the T cell sub-populations and then expressed as an average for each patient after squareroot normalization.
Data were tested for normality using the Kolmogorov–Smimov test. PASIscores and peptide responses were compared between the groups and dif-ferent time points using an ANOVA test for repeated measurements. Forpeptide response measurements, the square root of the peptide responses wasused to better approximate normality. Correlation between peptides responseswas performed using R, version 2.10 (The R Foundation, Vienna, Austria).
ResultsClinical findings
Twenty-nine patients with chronic plaque psoriasis were recruitedand randomly allocated into TX and control patient groups. De-mographic information about the participants at study entry andtheir disease characteristics are presented in Table I.As depicted in Fig. 1A the mean PASI score decreased signif-
icantly in the TX group, both with time after tonsillectomy andcompared with the controls (p , 0.001). Thus, 13 of 15 tonsil-lectomized patients showed an improvement ranging from 30 to90% reduction of the PASI score (Fig. 1C), and up to 60% (9 of15) reached 50% reduction in skin lesions at some stage during thestudy (Fig. 1B). The improvement was in most cases observed
within 2 mo and was generally maintained throughout the 2-yfollow-up (Fig. 1C). No consistent corresponding clinical changeswere observed among the control patients (Fig. 1D). Furthermore,12 (86%) of the controls used topical treatment at some time pointduring the study compared with only 4 (27%) in the TX group(Table II). However, three patients in each group had been givenphototherapy and one patient in the TX group was started onmethotrexate after 12 mo because of arthritis. There was no clearassociation between the degree of improvement and carriage ofthe HLA-Cw*0602 allele, but more patients need to be studied inthis context.
The effect of tonsillectomy on serum concentration of IL-8
To objectively assess changes in inflammatory activity, serumlevels of IL-8 were measured at study entry and after 24 mo, and, asshown in Fig. 2, a slight but significant decrease was observed inthe tonsillectomized patients but not in the control patients despitetheir more frequent topical treatment (Table II).
FIGURE 1. Clinical changes during the 2-y follow-up. (A) A significant decrease in the mean PASI score was observed in the tonsillectomized patientsboth with time and when compared with the controls (p , 0.001). (B) Percentage of tonsillectomized patients that reached 50% improvement (PASI50),75% improvement (PASI75), and 90% improvement (PASI90) at each time point. (C) Percent changes in the PASI scores of individual participantsthroughout the 2-y of study, with the scores at study entry for each patient being set as 0. Thirteen of 15 participants in the TX group improved clinically(range, 30–90%) after tonsillectomy. Patients 4 and 6 (arrows) did not show significant changes in blood frequencies of peptide-reactive skin-homingT cells. No corresponding clinical improvement was observed in the controls (D).
Table II. Psoriasis treatments during the 2-y follow-up
Treatment 6–24 mo TX Group (n = 15) Control Group (n = 14)
Topical, n (%)a 4 (27) 12 (86)Phototherapy, n (%) 3 (20) 3 (21)Systemic, n (%) 1 (7)aCorticosteroid creams and vitamin D analog creams.
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The effect of tonsillectomy on the frequency of the circulatingpeptide-reactive T cells
The frequency of T cells responding to each of 16 pools of ho-mologous M protein and keratin peptides (see Supplemental TableI) was determined at study entry and after 2, 12, and 24 mo by flowcytometry as a percentage of skin homing (CLA+) cells in eachT cell subpopulation. Fig. 3A shows the marked general declineusually observed in the frequency of circulating CD8+ T cellsresponding to each of the 16 peptide pools in one representativepatient before and 2 mo after tonsillectomy. Similar declines wereobserved in the tonsillectomized patients after 12 and 24 mo,whereas no consistent corresponding changes were detected in thecontrol patients (Fig. 3B). The average responses of each patientto all 16 peptide pools were then calculated at each time pointafter square root normalization (Figs. 4, 5, Supplemental Fig. 1).
As shown in Fig. 4, there was a very strong positive correlationbetween the frequency of peptide-reactive skin homing CD8+
T cells in tonsils and blood at the time of the tonsillectomy. Thisapplied to both IFN-g– and IL-17–producing CD8+ T cells (r =0.788, p , 0.001 and r = 0.644, p = 0.015, respectively). A sig-nificant correlation was also observed between the frequency ofIFN-g–producing skin-homing CD4+ T cells in tonsils and blood(r = 0.679, p = 0.001), but a corresponding correlation was notobserved for IL-17–producing skin-homing CD4+ T cells (r =0.315, p = 0.273) (data not shown). After tonsillectomy, the fre-quency of circulating peptide-reactive IFN-g–producing skinhoming CD8+ T cells decreased significantly compared with thecontrols (p = 0.003) (Fig. 5A). Furthermore, there was a highlysignificant correlation between the extent of clinical improvement(decreases in PASI scores) of individual patients and the degree ofdecline in the frequency of peptide-reactive skin homing IFN-g+
CD8+ T cells in their blood (r = 0.594, p , 0.001). As depictedin Supplemental Fig. 1, similar associations were observed forpeptide-reactive skin-homing IL-17–producing CD8+ T cells inthe tonsillectomized patients (r = 0.560, p , 0.001). A weakerassociation was found for skin-homing IL-17+CD4+ T cells(p = 0.003), but the association was not significant for IFN-g+
CD4+ T cells (p = 0.137). No associations between changes inPASI scores and frequency of circulating peptide-reactive CD8+ orCD4+ T cells were found in the control patients (Fig. 5C, Sup-plemental Fig. 1C). No decreases were observed after tonsillec-tomy in the frequencies of circulating T cells that responded toanti-CD3 Ab stimulation or to the control Ag streptokinase (datanot shown).
DiscussionIt has previously been reported that psoriasis patients have in theircirculation T cells that recognize determinants that streptococcal Mprotein share with some human keratins (18, 19) and the greatmajority of these T cells are CLA+ (19). We now report thatpatients with chronic psoriasis and a history of disease exacer-bation in association with sore throat generally improve aftertonsillectomy, and concurrently the numbers of circulating T cellsthat recognize these shared determinants show a marked decline.These findings indicate that effector T cells originating from thepalatine tonsils may be involved in the pathogenesis of psoriasis.First, there is a very close correlation between the frequency ofsuch T cells in the tonsils and peripheral blood (Fig. 4), suggestingthat the tonsillar T cells are recirculating. Second, the extent of thedecline in the numbers of these T cells in the circulation correlatesfairly closely with the degree of clinical improvement of indi-vidual patients (Fig. 5B).Note that this study was designed to detect maximal numbers of
T cells that are specific for determinants that streptococcal Mproteins share with human type 1 keratins. Thus, the study does notdistinguish between T cells that recognize primary, dominant
FIGURE 2. Serum concentrations of IL-8. Serumlevels of IL-8 were measured at study entry and after24 mo, and (A) a slight but significant decrease wasobserved in the tonsillectomized patients (p = 0.034)but not in the control patients (B) (p = 0.275) despitemore topical treatment (Table II) (Wilcoxon matchedpairs signed rank test).
FIGURE 3. Tonsillectomy was associated with a striking reduction inthe frequency (percentage) of circulating T cells that recognized the 16homologous peptide pools. Frequency (percentage) of circulating CLA+
CD8+ T cells producing IFN-g after stimulation with the homologouskeratin and M protein peptide pools at study entry and after 2 mo: (A)representative tonsillectomized psoriasis patient; (B) representative controlpsoriasis patient. The responses of each patient to all 16 peptide pools werethen calculated for each time point and expressed, after square root nor-malization, as an average for each patient (see Fig. 5, Supplemental Fig. 1).
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autoepitopes and determinants that reflect epitope spreading orT cells that may respond exclusively to either keratin or M proteindeterminants.Furthermore, our observations do not exclude the possibility that
other Ags may be involved in the pathogenesis of psoriasis, in-cluding other streptococcal (25) or peptidoglycan components(26). Note in this context that we selected M protein and keratinpeptides that were predicted to bind relatively strongly with HLA-Cw*0602, which is carried by ,50% of patients with chronicpsoriasis (27), although this may vary between populations (28,29). Second, we selected for this study patients who reported
aggravation of their disease in association with sore throat,which only applies to !40% of patients with chronic psoriasis inIceland (R.H. Thorleifsdottir, J.H. Eysteinsdottir, J.H. Olafsson,B. Sigurgeirsson, M.I. Sigurdsson, and H. Valdimarsson, manu-script in preparation). It remains to be investigated whetherpatients who have not noticed worsening in association with sorethroat also improve after tonsillectomy. Furthermore, two of thepatients in our study did not improve after tonsillectomy (seearrows in Fig. 1C), although no tonsillar remnants could bedetected in these patients after the operation. Thus, indications fortonsillectomy of patients with chronic psoriasis remain to be pre-
FIGURE 4. There was a signifi-cant correlation between the fre-quency of peptide-reactive skin-homing CD8+ T cells in the bloodand tonsils at the time of the tonsil-lectomy. (A) IFN-g–producing pep-tide-specific CD8+ T cells (r = 0.788,p , 0.001). (B) IL-17–producingpeptide-specific CD8+ T cells (r =0.644, p = 0.015). Data expressed asa square root normalized average ofpeptide-reactive skin-homing T cells(Spearman rank correlation test).
FIGURE 5. Changes in the bloodfrequencies of IFN-g–producingpeptide-specific CD8+ T cells in thetonsillectomized and control patients.(A) Box plot shows a significantdecrease in the average frequencyof peptide-reactive IFN-g–producingskin-homing (CLA+) CD8+ T cells inthe tonsillectomized compared withthe controls (p = 0.003, ANOVA). (B)Close correlation throughout the 2-ystudy period (three data points perpatient) between clinical improve-ment (percentage reduction of PASI)and percentage reduction in the bloodfrequency of skin-homing IFN-g pro-ducing peptide-reactive CD8+ T cells(r = 0.594, p , 0.001). No suchcorrelation was observed in the con-trols (r = 20.023, p = 0.896). The Tcell frequency data are expressed asa square root normalized average ofpeptide-reactive skin-homing T cells.PASI and T cell frequency at studyentry were set as 0 for each partici-pant. In (B) and (C), the vertical axisshows percentage changes in the PASIscores and the horizontal axis per-centage changes in the frequency ofpeptide-reactive CLA+CD8+ T cellsduring the study period (Spearmanrank correlation test).
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cisely defined. It is therefore not possible at this stage to estimatehow large a proportion of patients with chronic psoriasis mightbenefit from tonsillectomy, and information is also lacking abouthow long such improvement may last beyond the 2-y follow-up inthe present study. Note that the tonsillectomized patients not onlybenefited in terms of reduction of skin lesions but also required lesssymptomatic treatment than the control patients, and longer termfollow-up studies should therefore also focus on that issue.Recent studies have indicated that CD8+ T cells may play
a more direct role than CD4+ T cells in the pathogenesis of pso-riasis (2, 4, 11). Thus, the great majority of T cells in lesionalepidermis are CD8+ and, furthermore, psoriasis lesions do notdevelop when CD8+ T cells are prevented to migrate from dermisinto epidermis (2). Our data support this, as there is a strongercorrelation between clinical improvement and reduction in thefrequency of cross-reactive CD8+ than CD4+ T cells.Our findings may help to identify some of the autoepitopes that
are recognized by T cells in psoriatic lesions. It has been reportedby many groups that these lesions are infiltrated by oligoclonalT cells (1), and although most of these clones are transient (1) andprobably reflect autoepitope spreading (30), others are dominantand persist or even reappear in lesional skin after treatment-induced remission (31, 32).Only symptomatic treatments are currently available for psori-
asis, and symptoms usually relapse when treatment is discontinued.It is still not clear if and to what extent Ag-specific immuno-therapeutic measures, which are curative in some animal models ofautoimmunity, are directly applicable to human autoimmune dis-eases. This is probably partly because epitope spreading makes itdifficult to identify primary and dominant autoepitopes in humans(30, 33). However, identification of circulating T cells that respondto homologous M protein and keratin determinants in patientswith treatment-induced remission (32) may help to identify pri-mary autoepitopes that might be targeted for highly specific im-munotherapy for psoriasis.
AcknowledgmentsWe appreciate the excellent technical assistance of Thor Fridriksson,Andrew M. Guzman, Cynthia S. Chen, Dr. Trilokraj Tejasvi, and Phillip E.Stuart.
DisclosuresThe authors have no financial conflicts of interest.
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11. Gudjonsson, J. E., A. Johnston, H. Sigmundsdottir, and H. Valdimarsson. 2004.Immunopathogenic mechanisms in psoriasis. Clin. Exp. Immunol. 135: 1–8.
12. Gudjonsson, J. E., A. M. Thorarinsson, B. Sigurgeirsson, K. G. Kristinsson, andH. Valdimarsson. 2003. Streptococcal throat infections and exacerbation ofchronic plaque psoriasis: a prospective study. Br. J. Dermatol. 149: 530–534.
13. Shikhman, A. R., and M. W. Cunningham. 1994. Immunological mimicry be-tween N-acetyl-beta-D-glucosamine and cytokeratin peptides: evidence for amicrobially driven anti-keratin antibody response. J. Immunol. 152: 4375–4387.
14. Swerlick, R. A., M. W. Cunningham, and N. K. Hall. 1986. Monoclonal anti-bodies cross-reactive with group A streptococci and normal and psoriatic humanskin. J. Invest. Dermatol. 87: 367–371.
15. McFadden, J., H. Valdimarsson, and L. Fry. 1991. Cross-reactivity betweenstreptococcal M surface antigen and human skin. Br. J. Dermatol. 125: 443–447.
16. Leigh, I. M., H. Navsaria, P. E. Purkis, I. A. McKay, P. E. Bowden, andP. N. Riddle. 1995. Keratins (K16 and K17) as markers of keratinocyte hyper-proliferation in psoriasis in vivo and in vitro. Br. J. Dermatol. 133: 501–511.
17. Valdimarsson, H., B. S. Baker, I. Jonsdottir, A. Powles, and L. Fry. 1995. Pso-riasis: a T-cell-mediated autoimmune disease induced by streptococcal super-antigens? Immunol. Today 16: 145–149.
18. Gudmundsdottir, A. S., H. Sigmundsdottir, B. Sigurgeirsson, M. F. Good,H. Valdimarsson, and I. Jonsdottir. 1999. Is an epitope on keratin 17 a majortarget for autoreactive T lymphocytes in psoriasis? Clin. Exp. Immunol. 117:580–586.
19. Johnston, A., J. E. Gudjonsson, H. Sigmundsdottir, T. J. Love, andH. Valdimarsson. 2004. Peripheral blood T cell responses to keratin peptides thatshare sequences with streptococcal M proteins are largely restricted to skin-homing CD8+ T cells. Clin. Exp. Immunol. 138: 83–93.
20. Diluvio, L., S. Vollmer, P. Besgen, J. W. Ellwart, S. Chimenti, and J. C. Prinz.2006. Identical TCR b-chain rearrangements in streptococcal angina and skinlesions of patients with psoriasis vulgaris. J. Immunol. 176: 7104–7111.
21. Nyfors, A., P. A. Rasmussen, K. Lemholt, and B. Eriksen. 1975. Improvement ofrefractory psoriasis vulgaris after tonsillectomy. Dermatologica 151: 216–222.
22. Fredriksson, T., and U. Pettersson. 1978. Severe psoriasis: oral therapy witha new retinoid. Dermatologica 157: 238–244.
23. Pearson, W. R., and D. J. Lipman. 1988. Improved tools for biological sequencecomparison. Proc. Natl. Acad. Sci. USA 85: 2444–2448.
24. Johnston, A., S. Sigurdardottir, and J. Ryon. 2009. Isolation of mononuclear cellsfrom tonsillar tissue. Curr. Protoc. Immunol. Chapter 7: Unit 7.8.
25. Besgen, P., P. Trommler, S. Vollmer, and J. C. Prinz. 2010. Ezrin, maspin,peroxiredoxin 2, and heat shock protein 27: potential targets of a streptococcal-induced autoimmune response in psoriasis. J. Immunol. 184: 5392–5402.
26. Baker, B. S., A. Powles, and L. Fry. 2006. Peptidoglycan: a major aetiologicalfactor for psoriasis? Trends Immunol. 27: 545–551.
27. Mallbris, L., K. Wolk, F. Sanchez, and M. Stahle. 2009. HLA-Cw*0602 asso-ciates with a twofold higher prevalence of positive streptococcal throat swab atthe onset of psoriasis: a case control study. BMC Dermatol. 9: 5.
28. Fry, L., A. V. Powles, S. Corcoran, S. Rogers, J. Ward, and D. J. Unsworth. 2006.HLA Cw*06 is not essential for streptococcal-induced psoriasis. Br. J. Dermatol.154: 850–853.
29. Gudjonsson, J. E., A. Karason, E. H. Runarsdottir, A. A. Antonsdottir,V. B. Hauksson, H. H. Jonsson, J. Gulcher, K. Stefansson, and H. Valdimarsson.2006. Distinct clinical differences between HLA-Cw*0602 positive and negativepsoriasis patients: an analysis of 1019 HLA-C- and HLA-B-typed patients. J.Invest. Dermatol. 126: 740–745.
30. Vanderlugt, C. L., and S. D. Miller. 2002. Epitope spreading in immune-mediated diseases: implications for immunotherapy. Nat. Rev. Immunol. 2: 85–95.
31. Curran, S. A., O. M. FitzGerald, P. J. Costello, J. M. Selby, D. J. Kane,B. Bresnihan, and R. Winchester. 2004. Nucleotide sequencing of psoriatic ar-thritis tissue before and during methotrexate administration reveals a complexinflammatory T cell infiltrate with very few clones exhibiting features thatsuggest they drive the inflammatory process by recognizing autoantigens. J.Immunol. 172: 1935–1944.
32. Vollmer, S., A. Menssen, and J. C. Prinz. 2001. Dominant lesional T cell receptorrearrangements persist in relapsing psoriasis but are absent from nonlesionalskin: evidence for a stable antigen-specific pathogenic T cell response in pso-riasis vulgaris. J. Invest. Dermatol. 117: 1296–1301.
33. Steinman, L. 2004. Immune therapy for autoimmune diseases. Science 305: 212–216.
The Journal of Immunology 5165
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SUPPLEMENTARY FIGURE 1. Changes in the blood frequencies of IL-17 producing peptide-reactive CD8+ T cells the tonsillectomized and control patients. Panel a) shows by a box plot the average frequency of peptide-reactive IL-17 producing skin homing CD8+ T cells in the tonsillectomized and control patients (p = 0.009, ANOVA). Panel b) shows a close correlation between clinical improvement (percent reduction of PASI) and percent reduction in the blood frequency of skin homing IL-17 producing peptide-reactive CD8+ T cells (r = 0.594, p < 0.001). No such correlation was observed in the controls (panel c, r = 0.274, p = 0.130). Data expressed as a square root normalized average peptide-reactive skin homing T cells. PASI and T cell frequency at study entry was set as zero for each participant. Spearman's rank correlation test.
pept
ide-
reac
tive
IL-1
7 pr
oduc
ing
CLA
+CD
8+ T
cel
ls
0.0
0.2
0.
4
0
.6
0.8
1.0
TX Controls, p = 0.009, ANOVA
a)
0 2 12 24 Months
PASI
D
ecre
ase
(%) !
Incr
ease
(%)
b) TX
Decreased (%) ! Increased (%) frequency of peptide-reactive IL-17
producing CLA+CD8+ T cells
Decreased (%) ! Increased (%) frequency of peptide-reactive IL-17
producing CLA+CD8+ T cells
PASI
D
ecre
ase
(%) !
Incr
ease
(%)
c) Controls
r = 0.274, p = 0.130 r = 0.560, p < 0.001
Supplemental Table I. Composition of peptide pools1
1 2 3 4 5 6 7 8
9 31-K17-12
130-K17-9
206-K17-9
236K17-12
372-K17-9 261-M6-9 262-M6-
12 355-M6-9
10 101-K17-12
134K17-12
212K17-12
238-K17-9
379-K17-12 282-M6-9 285-M6-
12 368-M6-
16
11 125-K17-9
135-K17-9
217-K17-9
239K17-12
379-K17-9
299-M6-12 146M49 382-M6-
12
12 127-K17-9
139-K17-9
220-K17-9
309-K17-9
396-K17-9 299-M6-9 M146 384-M6-9
13 128-K17-9
162-K17-12
220-K17-12
334-K17-12
404-K17-9 306-M6-9 338M6-
12 464-M6-
12
14 128-K17-10
165-K17-12 145K10 338-K17-
12 407-K17-
9 319-M6-
12 344-M6-
12 M159
15 146K17 168-K17-9 145K17 351-K17-
12 415-K17-
12 324-M6-9 354-M6-12 GAGE
16 146K9 179-K17-9
231-K17-9
366-K17-12
255-M6-12 327-M6-9 345-M6-9 BMRF-1
1 White boxes are human keratin peptides, light gray boxes are streptococcal peptides and dark grey boxes are controls.
Supplemental Table II. Peptide sequences derived from keratin and M6 protein.
Peptide name 1 Sequence 2 Peptide name Sequence
31-K17-12 ISSVLAGASCPA 464-M6-12 ALTVMATAGVAA3
101-K17-12 GGGFAGGDGLLV4
125-K17-9 RLASYLDKV 306-M6-9 NLTAELDKV
127-K17-9 ASYLDKVRA
128-K17-10 SYLDKVRALE
146K17 SYLDKVRALEEANADLEVKI5 M146 AKKQVEKALEEANSKLAALE5,6
146K9 SYLDKVQALEEANNDLENKI5 146M49 AKKKVEADLAEANSKLQALE5
128-K17-9 SYLDKVRAL
130-K17-9 LDKVRALEE 344-M6-12 KALEEANSKLAA
134-K17-12 RALEEANADLEV 354-M6-12 AALEKLNKELEE
135-K17-9 ALEEANADL 345-M6-9 ALEEANSKL
309-K17-9 KTEELNKEV 355-M6-9 ALEKLNKEL
139-K17-9 ANADLEVKI
162-K17-12 YSPYFKTIEDLR4
165-K17-12 YFKTIEDLRNKI4
168-K17-9 TIEDLRNKI
179-K17-9 ATIENAHAL
206-K17-9 ARTGLRQTV 324-M6-9 SRQGLRRDL
217-K17-9 DVNGLRRVL 282-M6-9 SRKGLRRDL
372-K17-9 QIQGLIGSV
212-K17-12 QTVEADVNGLRR 319-M6-12 QISDASRQGLRR
220-K17-12 GLRRVLDELTLA 285-M6-12 GLRRDLDASREA
220-K17-9 GLRRVLDEL 327-M6-9 GLRRDLDAS
145K17 LRRVLDELTLARTDLEMQIE5
145K10 LRRVLDELTLTKADLEMQIE5 382-M6-12 EAEAKALKEQLA
231-K17-9 ARTDLEMQI 261-M6-9 DIGALKQEL
236-K17-12 EMQIEGLKEELA 384-M6-9 EAKALKEQL
238-K17-9 QIEGLKEEL 262-M6-12 IGALKQELAKKD
239-K17-12 IEGLKEELAYLR 368-M6-16 KLTEKEKAELQAKLEA
334-K17-12 LRRVLQGLEIIL7 M149 KLTEKEKAELQAKLEAEAKA5,6
338-K17-12 LQGLEIILQSQL7 255-M6-12 EQKSKQDIGALK4
351-K17-12 MKASLENSLEET 338-M6-12 AKKQVEKALEEA6
366-K17-12 YCMQLSQIQGLI4,7
379-K17-12 SVEEQLAQLRCE 299-M6-12 QVEKDLANLTAE
379-K17-9 SVEEQLAQL 299-M6-9 QVEKDLANL
396-K17-9 QEYQILLDV
404-K17-9 VKTRLEQEI
407-K17-9 RLEQEIATY GAGE YRPRPRRY8
415-K17-12 YRRLLEGEDAHL4 BMRF1 YRSGIIAVV9
1 Peptide name indicates the start position in the protein sequence, protein name and sequence length. 2 Sequences used in Johnston A. et al. Clin Exp Immunol 2004; 138:83-93, except where indicated. 3 Examples of sequence homologies shared by K and M peptides in bold face. 4 Sequence or partial sequence used in Dionne S.O. et al. Immunogenetics 2004; 56:391-8. 5 Sequence used in Gudmundsdottir A.S. et al. Clin Exp Immunol 1999; 117:580-6. 6 Sequence used in Sigmundsdottir H. et al. Scand J Immunol 1997; 45:688-97. 7 Sequence used in Shen Z. et al. J Dermatol Sci 2005; 38:25-39. 8 A peptide used as a negative control for Cw6 binding (van den Eynde B. et al. J. Exp. Med. 1995 182 689-698). 9 A peptide from Epstein-Barr Virus Protein BMRF1 as a positive control (Steven N.M. et al., J. Exp. Med. 1997 185 1605-1617).
Supplemental Table III. Antibodies used for flow cytometric analysis.
Target Conjugate Source Clone IsotypeCD4 PerCP-Cy5.5 BD Biosci SK3 G1CD8 PerCP-Cy5.5 BD Biosci SK1 G1IgG1 control PerCP-Cy5.5 BD Biosci X40 CLA FITC BioLegend HECA-452 Rat IgMRat IgM control FITC BioLegend IFN-! PE BioLegend 4S.B3 G1IL-17A A647 eBioscience eBio64CAP17 G1
139
Paper IV
REVIEW ARTICLEBJD
British Journal of Dermatology
The role of the palatine tonsils in the pathogenesis andtreatment of psoriasisS.L. Sigurdardottir,1 R.H. Thorleifsdottir,1 H. Valdimarsson1 and A. Johnston2
1Department of Immunology, Landspitali-University Hospital, Reykjavik, Iceland2Department of Dermatology, University of Michigan, Ann Arbor, MI, U.S.A.
CorrespondenceAndrew Johnston.E-mail: [email protected]
Accepted for publication3 August 2012
Funding sourcesThis work was funded in part by grants from TheBabcock Endowment, National PsoriasisFoundation U.S.A. and The American SkinAssociation to A.J., and from The IcelandicResearch Fund, Landspitali Hospital Research Fundand The Research Fund of the University ofIceland for Doctoral Studies to S.L.S.
Conflicts of interestNone declared.
DOI 10.1111/j.1365-2133.2012.11215.x
Summary
Psoriasis is a common chronic skin disease with strong genetic associations andenvironmental triggers. Patients with psoriasis develop sore throats much morefrequently than nonpsoriatic individuals and it is well documented that strepto-coccal throat infections can trigger the onset of psoriasis, and such infectionscause exacerbation of chronic psoriasis. It is now generally accepted that psoriaticlesions are caused by abnormal reactivity of specific T lymphocytes in the skin.However, it has been shown in recent years that activation of specific immunityis always preceded by activation of nonspecific innate immune mechanisms, andthat abnormalities in the innate immune system can cause dysregulation in spe-cific immune responses. Here we explore the possible immune mechanisms thatare involved in the link between infection of the tonsils and this inflammatoryskin disease. Moreover, we survey the literature and discuss the suitability oftonsillectomy as a treatment for psoriasis.
Psoriasis, a T-cell-mediated autoimmunedisease
Psoriasis is a common chronic inflammatory skin disease, mostoften characterized by thickened erythematous scaly plaques,and appears in a variety of forms with distinct characteristics.The most common form, psoriasis vulgaris, affects 1–3% ofthe Caucasian population,1 usually persists – with 40% devel-oping seronegative arthritis – and has a very negative impacton quality of life.2 Psoriasis is generally regarded as a T-cell-mediated autoimmune disease,3–5 with treatments targeting Tcells being very effective;6,7 indeed, blocking of T-cell entryinto the epidermis prevents development of hyperplasia in amouse model of psoriasis.8 There is considerable literature insupport of psoriasis being driven by T cells recognizing pep-tide antigens (reviewed in Valdimarsson et al.9). T cells frompatients with psoriasis show increased responses to homolo-gous peptides from streptococcal M proteins10,11 and humanepidermal keratins;10,12,13 thus, it is possible that psoriasismay occur as a consequence of T cells cross-reacting with epi-topes from streptococcal M proteins and human keratins.3,5
The palatine tonsils might play a major role in psoriasis asthey are a common site for streptococcal infections and, as
immune sentries loaded with antigen-presenting cells and Tcells, are likely a major site for T-cell priming against suchantigens. Here we detail how the immune function of the ton-sils could lead to the initiation and exacerbation of psoriasisand we survey potential interventions.
The tonsils, immunological sentries of thepharynx
The mucosal surfaces of the mouth, throat and upper respira-tory tract are the first major barrier protecting the host frompotentially invading microorganisms, and integral to thisdefence are the tonsils, which provide a protective immunolog-ical ring (Waldeyer’s ring) at the openings of both the digestiveand respiratory tracts.14 The palatine tonsils have a surface linedby a stratified squamous epithelium that extends into deep andbranched crypts, increasing their total surface area. The surfaceof the crypts is lined with a reticulated epithelium, whichin parts may be only one cell thick. The reticulated tonsilepithelium has a unique cellular composition where epithelialcells, stromal cells, intraepithelial lymphocytes, dendritic cells(DCs), neutrophils and macrophages are all situated in closeproximity, which is ideal for productive antigen sampling. In
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BJD ! 2012 British Association of Dermatologists 2013 168, pp237–242 237
the subepithelial space are B-cell-rich lymphoid follicles thatfoster the development of memory B cells and antibody pro-ducing plasma cells, a process that requires the interaction of anumber of B-cell, T-cell and DC subsets.15 Six phenotypes ofDC have been identified in the tonsils,16,17 each with a differentlocation and function. DCs are particularly adept at antigensampling and processing and presentation of peptide antigens,and influence the T helper 1 (Th1) ⁄Th2 ⁄Th17 balance ofT-cell-mediated responses.18
The involvement of streptococci in psoriasis
The most frequent tonsil diseases are recurrent infections andabnormal enlargement of the tonsils (hypertrophy), whichmay result in the need for tonsillectomy. Streptococcus pyogenes in-fections are associated with acute and recurrent throat infec-tions,19,20 while Haemophilus influenzae infections are thought tostimulate hypertrophic responses.21 Throat infection withb-haemolytic streptococci is the only well-defined externaltrigger that has been convincingly associated with initiationand acute exacerbation of psoriasis.22–26 This association wascorroborated in a prospective study that concluded thatpatients with psoriasis reported a sore throat 10 times moreoften than household controls, and that streptococcal throatinfections can cause exacerbation of chronic plaque psoriasis.24
Furthermore, a-streptococci are likely to have a role in pustu-losis palmaris et plantaris (PPP), a chronic, recurrent, inflam-matory skin disease restricted to the palms and soles.27 Thesetwo inflammatory skin diseases appear to benefit from tonsil-lectomy,28–30 suggesting the importance of the palatine tonsilsin inflammatory diseases outside the throat. Tonsils from indi-viduals with PPP differ from recurrently infected tonsils withregard to histology and the expression of skin homing mole-cules by T cells. Furthermore, the size of the T-cell area intonsils has been associated with skin improvement after tonsil-lectomy.30 In this respect, we have recently demonstrated that,compared with nonpsoriatic individuals, tonsils from patientswith psoriasis contain an increased frequency of T cells withskin homing potential (Sigurdardottir, manuscript in prepara-tion). Moreover, superantigens from b-haemolytic streptococcihave been demonstrated to enhance T-cell expression of cuta-neous lymphocyte-associated antigen (CLA),31,32 a carbo-hydrate moiety expressed on 80% of T cells in the skin,33 andthe frequency of CLA+CD8+ T cells in the blood of patientswith psoriasis correlates with their disease severity.34 Theimportance of specific effector ⁄memory T cells is now gener-ally recognized in psoriasis,4 but it remains to be elucidatedwhy and where the effector T cells are activated. In thisregard, the innate immune response of the tonsils may play acrucial role in the instigation and subsequent regulation of theT-cell-mediated adaptive immune response. Thus, aberranttonsil responses to streptococcal infection could lead to alteredT-cell expansion and trafficking to the skin where they inducean inflammatory response that initiates the psoriasis plaque.This is further supported by the fact that similar oligoclonal Tcells have been isolated from tonsils and psoriatic plaques of
the same individual,35 with disease remission following tonsil-lectomy.35
The potential of tonsillectomy for thetreatment of psoriasis
Almost a century ago, Winfield36 presented a series of six casereports that first drew attention to the occurrence of psoriasisfollowing inflammation of the tonsils. b-Haemolytic strepto-coccus was later identified as the infectious agent,20,23,37 lead-ing to the onset of guttate psoriasis, or exacerbation ofchronic plaque psoriasis.
Streptococci are not fully eradicated with antibiotics such aspenicillin or erythromycin that do not penetrate host cells,38
as they are facultative intracellular bacteria39,40 unaffected byconventional antibiotics. Rifampicin and clindamycin are alsoactive intracellularly38 and eliminate both extra- and intra-cellular streptococci;41–44 however, even treatment with anti-biotics is likely to have only a temporary benefit, as about20% of healthy, asymptomatic individuals carry b-haemolyticstreptococci in their throat,24 making it likely that the bacte-rium recolonizes the throat of the patients after cessation oftreatment. With respect to the treatment of psoriasis with anti-biotics, there is a lack of adequately controlled clinical trials(see Owen et al.45).
A search of the PubMed database using the search terms‘psoriasis’, ‘tonsils’ and ‘tonsillectomy’ revealed 71 articlesfrom 1960 to 2012. After limiting our search to those studieson ‘chronic plaque psoriasis’ or ‘guttate psoriasis’ or ‘psoriasisvulgaris’, excluding ‘palmoplantar pustulosis’ or ‘pustulosispalmaris et plantaris’, and following up citations within thesepublications, we noted that there exist 12 original case reportsor studies on the effect of tonsillectomy on psoriasis(Table 1). In the first report, Whyte and Baughman46
described the improvement of psoriasis and decrease in anti-streptolysin O titres following tonsillectomy of three patientswith guttate psoriasis. This was followed by the first con-trolled trial, a 1967 report from Germany47 that described 92patients with psoriasis with chronic tonsillitis, of whom 56underwent tonsillectomy whereupon 34 (61%) improved and22 (39%) appeared unchanged. This compares with the con-trol group of 36 patients with psoriasis who did not have atonsillectomy, of whom 8 (22%) improved and 28 (78%)were unchanged. The authors did not give details of whetherthe control group was matched for age and sex, nor did theyuse a quantitative scoring system for disease severity.
Later, Nyfors et al.48 conducted an uncontrolled retrospect-ive study, which examined the effect of tonsillectomy on 74young (mean age 14Æ2 years) patients with psoriasis vulgaristhat proved to be refractory to topical treatments. During thefollow-up period (mean 4Æ5 years), 32% of patients remainedclear of psoriasis, 39% had a considerable improvement, 22%were unchanged and 7% worsened. Another case report49
describes the effect of tonsillectomy on two young sisters withguttate psoriasis that developed 1 week after catching a coldand severe sore throat. Interestingly, both sisters carried the
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BJD ! 2012 British Association of Dermatologists 2013 168, pp237–242
238 The role of the palatine tonsils in psoriasis, S.L. Sigurdardottir et al.
human leucocyte antigen-B37 allele, which is part of one ofthe psoriasis risk haplotypes.50 In both cases the skin lesionscleared almost completely within 2 months of tonsillectomy;however, the treatment was complicated by the postoperativeuse of antibiotics and topical corticosteroid cream.
Over 10 years later, Hone et al.51 reported an uncontrolledprospective study on 13 patients (mean age 17 years) withrecalcitrant psoriasis. The group consisted of six patients withguttate psoriasis and seven with chronic plaque psoriasis.From the group as a whole, seven patients showed completeclearance and three showed significantly improved psoriasisfollowing tonsillectomy, with noticeable improvement withina month. The data for the patients with guttate psoriasis wasmore impressive, as the disease cleared in five out of sixpatients, with the remaining patient having a significantimprovement. The authors claim that the patients served as
their own controls, yet they omitted to acknowledge thatpatients with guttate psoriasis might have spontaneous remis-sions. In the same year, a Japanese study including 35 patientswith psoriasis vulgaris52 reported that 49% of the patientsshowed a clear improvement in their clinical symptoms fol-lowing tonsillectomy, which was more effective for women(84% having some degree of improvement) than men (31%).It was also more effective in younger patients: 83% of thoseunder the age of 20 years showed some degree of improve-ment, which fell to 68% in the under-30s category.
In 1998, Rosenberg et al.53 reported clearance of psoriasisin nine of 14 patients following tonsillectomy, and a signifi-cant improvement in the remaining five. All 14 patients hadevidence of streptococcal colonization that could not beresolved by antibiotics. A year later, McMillin et al.54 gavedetails of two case reports. The first was a 5-year-old girl
Table 1 A summary of all case reports and studies to date detailing the effects of tonsillectomy on psoriasis
Authors Country, yearNumberin study Disease
Age range(mean) Outcome
Follow-upperiod
Whyte and Baughman46 U.S.A., 1964 3 Guttate psoriasis 15–23 (20) ‘Occasional plaques’ aftertonsillectomy
1 year
Cepicka and Tielsch47 Germany, 1967 92 Psoriasis associatedwith infection
NR Symptom free in 61% aftertonsillectomy, vs. 8% innontonsillectomized group
2–5 years
Nyfors et al.48 Denmark, 1976 74 Psoriasis vulgaris 4–33 (14Æ2) All lesions cleared in 32%,‘considerable improvement’in 39%, unchanged in 22%,worsened in 7%
4Æ5 years(7–204months)
Saita et al.49 Japan, 1979 2 Guttate psoriasis 7, 11 Both ‘cleared almostcompletely’ within 2 monthsof tonsillectomy
NR
Hone et al.51 Ireland, 1996 13 6 Guttate psoriasis7 Chronic plaquepsoriasis
6–28 (17) Guttate: 5 ⁄6 cleared, 1 ⁄6improved
Plaque: 2 ⁄7 cleared, 2 ⁄7improved, 3 ⁄7 unchanged
6–52 (26)months
Kataura and Tsubota52 Japan, 1996 35 Psoriasis vulgaris NR ‘Remarkably effective’ 29%,‘effective’ 20%, ‘partiallyeffective’ 11%, unchanged31%, worsened 9%
3 months
Rosenberg et al.53 U.S.A., 1998 14 NR NR All lesions cleared in 9 ⁄14cases, improvement in 5 ⁄14
NR
McMillin et al.54 U.S.A., 1999 2 1 Guttate psoriasis1 Severe psoriasis
115
All lesions cleared 16 months
Ozawa et al.55 Japan, 1999 385 Generalized pustularpsoriasis
NR ‘Effective’ in 16Æ7% of cases NR
Takahara et al.56 Japan, 2001 7 Psoriasis (?) 9–46 (23) All lesions cleared in 3 ⁄7,80% of lesions cleared in2 ⁄7, no change in 2 ⁄7
2–9 years
Diluvio et al.35 Germany, 2006 3 Recalcitrant nonpustularchronic plaque psoriasiswith guttate flares
21, 29, 33 Complete remission (PsoriasisArea and SeverityIndex score = 0)
> 3 years
Thorleifsdottir et al.29 Iceland, 2012 29 Chronic psoriasiswith historyof exacerbation aftersore throat
19–54 (35Æ5) 13 ⁄15 (86%) with 30–90%clinical improvement. Noconsistent clinical changesin control group
2 years
NR, not reported.
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BJD ! 2012 British Association of Dermatologists 2013 168, pp237–242
The role of the palatine tonsils in psoriasis, S.L. Sigurdardottir et al. 239
who had severe early-onset psoriasis, a history of enlargedtonsils and one episode of streptococcal pharyngitis. The sec-ond was an 11-year-old boy with early-onset guttate psoriasisand recurrent streptococcal pharyngitis. As with the earlierreport from Hone et al.,51 the children showed a markedimprovement in their psoriasis as early as 1 month after ade-notonsillectomy and both were clear of psoriasis at a 16-month follow-up examination. In the same year, Ozawaet al.55 conducted a multicentre retrospective study based onphysician questionnaires, which included 385 cases of gener-alized pustular psoriasis. They reported that tonsillectomy was‘effective’ for 16Æ7% of the patients. A more recent reportcame from Japan in 2001, where seven patients with psoria-sis underwent tonsillectomy and were followed up for2–9 years after surgery.56 All lesions had cleared in threepatients and 80% of lesions cleared in two patients, with nochanges in the disease of the remaining two. Of the fivepatients showing improvement, four had a history of tonsilli-tis making their skin lesions worse. The group of Prinzfollowed three patients with recalcitrant chronic plaque psori-asis with guttate flares for 3 years after tonsillectomy andreported complete remission.35 Moreover, analysis of T-cellreceptor beta-chain variable region gene rearrangementssuggested that T cells from the same T-cell clones weresimultaneously present within the skin and tonsils of thesepatients.35
These data are compelling but case reports carry limitationsinherent in their study design (or lack thereof), given that thepatients are managed in a noncontrolled environment, theobservation is based on an individual or small group, whichmay not reflect a larger population, and some of the effectsnoted may have simply arisen from the natural history ofthe disease, rather than the treatment. Thus, we recentlycompleted a prospective, controlled, observer-blinded trial29
involving 29 patients with chronic psoriasis and a history ofexacerbation after sore throat, who were randomly assignedto tonsillectomy (n = 15) or control (n = 14) groups andwere followed for 2 years. Of this group, 13 patients (86%)showed sustained improvement after tonsillectomy rangingfrom 30% to 90% reduction in disease severity. In addition toclinical follow-up, we assessed blood T cell responses to poolsof short peptides derived from human epidermal keratins andstreptococcal M proteins, and found a close correlationbetween the degree of clinical improvement in individualpatients and reduction in the frequency of peptide-reactiveskin-homing T cells in the blood.29 No corresponding clinicalor immunological changes were observed among the controlgroup.
Outlook
All of the studies and case reports reviewed here conclude thattonsillectomy may be of considerable benefit to selectedpatients. Psoriasis is influenced by multiple genetic polymor-phisms and environmental factors,57 resulting in a range ofdisease severity and course among patients, which is likely
reflected in the response to treatment.58,59 Some patients withrecalcitrant guttate or chronic plaque psoriasis, particularly thosewith early-onset psoriasis that is exacerbated by streptococcaltonsillitis, appear to have long-term remissions following tonsil-lectomy. In our recent study,29 tonsillectomized patients notonly benefited in terms of reduction of skin lesions but they alsorequired less symptomatic treatment than the control patients,indicating that longer-term follow-up studies should also focuson that issue. Thus, in some cases, tonsillectomy could becomean adjunct to existing therapy, to reduce the dosage of drugs orallow the use of a less potent drug to control the disease. Thisbecomes an interesting consideration in our era of expensivebiological treatments. However, it remains to be seen howeffective tonsillectomy is in the long term and exactly whichpatent groups will gain the most benefit.
What’s already known about this topic?
• There is a well-established association between strepto-coccal tonsillitis and psoriasis, yet studies on the efficacyof tonsillectomy for psoriasis have never been affordedmainstream attention.
What does this study add?
• We have reviewed tonsil immunology and how tonsilinfection could lead to skin disease.
• We have also reviewed all available reports of the out-come of tonsillectomy on psoriasis, including a recentobserver-blinded prospective study, and identifiedpatients who may potentially benefit from this procedure.
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11 Sigmundsdottir H, Sigurgeirsson B, Troye-Blomberg M et al. Circu-lating T cells of patients with active psoriasis respond to strepto-coccal M-peptides sharing sequences with human epidermalkeratins. Scand J Immunol 1997; 45:688–97.
12 Gudmundsdottir AS, Sigmundsdottir H, Sigurgeirsson B et al. Is anepitope on keratin 17 a major target for autoreactive T lympho-cytes in psoriasis? Clin Exp Immunol 1999; 117:580–6.
13 Shen Z, Wang G, Fan J-Y et al. HLA DR B1*04, *07-restricted epi-topes on keratin 17 for autoreactive T cells in psoriasis. J DermatolSci 2005; 38:25–39.
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15 MacLennan IC, Gulbranson-Judge A, Toellner KM et al. The chang-ing preference of T and B cells for partners as T-dependent anti-body responses develop. Immunol Rev 1997; 156:53–66.
16 Stent G, Reece JC, Baylis DC et al. Heterogeneity of freshly isolatedhuman tonsil dendritic cells demonstrated by intracellular markers,phagocytosis, and membrane dye transfer. Cytometry 2002; 48:167–76.
17 Summers KL, Hock BD, McKenzie JL et al. Phenotypic characteriza-tion of five dendritic cell subsets in human tonsils. Am J Pathol2001; 159:285–95.
18 Steinman RM, Hemmi H. Dendritic cells: translating innate toadaptive immunity. Curr Top Microbiol Immunol 2006; 311:17–58.
19 Koch RJ, Brodsky L. Qualitative and quantitative immunoglobulinproduction by specific bacteria in chronic tonsillar disease. Laryngo-scope 1995; 105:42–8.
20 Hope-Simpson RE. Streptococcus pyogenes in the throat: a studyin a small population, 1962–1975. J Hyg (Lond) 1981; 87:109–29.
21 Brodsky L, Moore L, Stanievich JF, Ogra PL. The immunology oftonsils in children: the effect of bacterial load on the presence ofB- and T-cell subsets. Laryngoscope 1988; 98:93–8.
22 Fry L. Psoriasis. Br J Dermatol 1988; 119:445–61.23 Telfer NR, Chalmers RJ, Whale K, Colman G. The role of strepto-
coccal infection in the initiation of guttate psoriasis. Arch Dermatol1992; 128:39–42.
24 Gudjonsson JE, Thorarinsson AM, Sigurgeirsson B et al. Streptococ-cal throat infections and exacerbation of chronic plaque psoriasis: aprospective study. Br J Dermatol 2003; 149:530–4.
25 Wardrop P, Weller R, Marais J, Kavanagh G. Tonsillitis and chronicpsoriasis. Clin Otolaryngol Allied Sci 1998; 23:67–8.
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27 Mrowietz U. Pustular eruptions of palms and soles. In: Fitzpatrick’sDermatology in General Medicine (Fitzpatrick TB, Wolff K, eds), 7th edn,Vol. 1. New York: McGraw-Hill Medical, 2008; 215–18.
28 Yokoyama M, Hashigucci K, Yamasaki Y. Effect of tonsillectomy inpatients with pustulosis palmaris et plantaris. Acta Otolaryngol 2004;124:1109–10.
29 Thorleifsdottir RH, Sigurdardottir SL, Sigurgeirsson B et al.Improvement of psoriasis after tonsillectomy is associated with adecrease in the frequency of circulating T cells that recognizestreptococcal determinants and homologous skin determinants.J Immunol 2012; 188:5160–5.
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31 Leung DY, Gately M, Trumble A et al. Bacterial superantigensinduce T cell expression of the skin-selective homing receptor, thecutaneous lymphocyte-associated antigen, via stimulation of inter-leukin 12 production. J Exp Med 1995; 181:747–53.
32 Sigmundsdottir H, Gudjonsson JE, Valdimarsson H. Interleukin-12alone can not enhance the expression of the cutaneous lymphocyteassociated antigen (CLA) by superantigen-stimulated T lympho-cytes. Clin Exp Immunol 2003; 132:430–5.
33 Picker LJ, Treer JR, Ferguson-Darnell B et al. Control of lymphocyterecirculation in man. II. Differential regulation of the cutaneouslymphocyte-associated antigen, a tissue-selective homing receptorfor skin-homing T cells. J Immunol 1993; 150:1122–36.
34 Sigmundsdottir H, Gudjonsson JE, Jonsdottir I et al. The frequencyof CLA+ CD8+ T cells in the blood of psoriasis patients correlatesclosely with the severity of their disease. Clin Exp Immunol 2001;126:365–9.
35 Diluvio L, Vollmer S, Besgen P et al. Identical TCR beta-chain rear-rangements in streptococcal angina and skin lesions of patientswith psoriasis vulgaris. J Immunol 2006; 176:7104–11.
36 Winfield JM. Psoriasis as a sequel to acute inflammations of thetonsils: a clinical note. J Cutan Dis 1916; 34:441–3.
37 Norrlind R. Psoriasis following infections with hemolytic strepto-cocci. Acta Derm Venereol 1950; 30:64–72.
38 Tulkens PM. Intracellular distribution and activity of antibiotics. EurJ Clin Microbiol Infect Dis 1991; 10:100–6.
39 LaPenta D, Rubens C, Chi E et al. Group A streptococci efficientlyinvade human respiratory epithelial cells. Proc Natl Acad Sci USA1994; 91:12115–19.
40 Osterlund A, Popa R, Nikkila T et al. Intracellular reservoir of Strepto-coccus pyogenes in vivo: a possible explanation for recurrent pharyngo-tonsillitis. Laryngoscope 1997; 107:640–7.
41 Foote PA Jr, Brook I. Penicillin and clindamycin therapy in recur-rent tonsillitis. Effect of microbial flora. Arch Otolaryngol Head Neck Surg1989; 115:856–9.
42 Brook I, Leyva F. The treatment of the carrier state of group Abeta-hemolytic streptococci with clindamycin. Chemotherapy 1981;27:360–7.
43 Orrling A, Stjernquist-Desatnik A, Schalen C. Clindamycin in recur-rent group A streptococcal pharyngotonsillitis – an alternative totonsillectomy? Acta Otolaryngol 1997; 117:618–22.
44 Brook I. Failure of penicillin to eradicate group A beta-hemolyticstreptococci tonsillitis: causes and management. J Otolaryngol 2001;30:324–9.
45 Owen CM, Chalmers RJ, O’Sullivan T et al. A systematic review ofantistreptococcal interventions for guttate and chronic plaque pso-riasis. Br J Dermatol 2001; 145:886–90.
46 Whyte HJ, Baughman RD. Acute guttate psoriasis and streptococcalinfection. Arch Dermatol 1964; 89:350–6.
47 Cepicka W, Tielsch R. [Focal infections and psoriasis vulgaris]. Der-matol Wochenschr 1967; 153:193–9.
48 Nyfors A, Rasmussen PA, Lemholt K, Eriksen B. Improvement ofrecalcitrant psoriasis vulgaris after tonsillectomy. J Laryngol Otol1976; 90:789–94.
49 Saita B, Ishii Y, Ogata K et al. Two sisters with guttate psoriasis re-sponsive to tonsillectomy: case reports with HLA studies. J Dermatol1979; 6:185–9.
50 Jenisch S, Henseler T, Nair RP et al. Linkage analysis of human leu-kocyte antigen (HLA) markers in familial psoriasis: strong disequi-librium effects provide evidence for a major determinant in theHLA-B ⁄-C region. Am J Hum Genet 1998; 63:191–9.
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51 Hone SW, Donnelly MJ, Powell F, Blayney AW. Clearance of recal-citrant psoriasis after tonsillectomy. Clin Otolaryngol Allied Sci 1996;21:546–7.
52 Kataura A, Tsubota H. Clinical analyses of focus tonsiland related diseases in Japan. Acta Otolaryngol Suppl 1996;523:161–4.
53 Rosenberg EW, Skinner RB, Noah PW. Anti-infectious therapy inpsoriasis. in: Psoriasis (Roenigk HH, Maibach HI, eds), 3rd edn.New York: Marcel Dekker, 1998; 373–9.
54 McMillin BD, Maddern BR, Graham WR. A role for tonsillectomyin the treatment of psoriasis? Ear Nose Throat J 1999; 78:155–8.
55 Ozawa A, Ohkido M, Haruki Y et al. Treatments of generalized pus-tular psoriasis: a multicenter study in Japan. J Dermatol 1999;26:141–9.
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57 Elder JT, Bruce AT, Gudjonsson JE et al. Molecular dissection ofpsoriasis: integrating genetics and biology. J Invest Dermatol 2010;130:1213–26.
58 Papp KA, Langley RG, Lebwohl M et al. Efficacy and safety of us-tekinumab, a human interleukin-12 ⁄23 monoclonal antibody, inpatients with psoriasis: 52-week results from a randomised, dou-ble-blind, placebo-controlled trial (PHOENIX 2). Lancet 2008;371:1675–84.
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Paper V
UNIT 7.8Isolation of Mononuclear Cells fromTonsillar Tissue
Andrew Johnston,1 Sigrun L. Sigurdardottir,2 and Judith J. Ryon3
1Department of Dermatology, University of Michigan, Ann Arbor, Michigan2Department of Immunology, Landspitali-University Hospital, Reykjavik, Iceland3National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
ABSTRACT
Located on the inside of the throat, the paired palatine tonsils form part of the firstmajor barrier protecting the digestive and respiratory tracts from potentially invadingmicroorganisms. The tonsils have a surface of stratified squamous epithelium that extendsinto deep and branched crypts lined by reticulated epithelium, which in parts may onlybe one cell thick. Organized in the sub-epithelial space are B cell rich lymphoid follicles.T cells are mostly located in the extra-follicular spaces with a very high CD4:CD8 T cellratio. In addition to the T and B cell subsets, six phenotypes of dendritic cells (DC) havebeen identified in the tonsils: Langerhans cells in the squamous epithelium, germinalcenter DC, and follicular DC in the germinal center, and another three DC phenotypesthat are located in the extra-follicular area (interdigitating DC, plasmacytoid DC, andlympho-epithelial symbiosis-DC). Here, we describe the isolation of tonsil mononuclearcells from fresh human tonsil. Curr. Protoc. Immunol. 86:7.8.1-7.8.4. C! 2009 by JohnWiley & Sons, Inc.
Keywords: tonsils ! T cells ! B cells ! lymphocytes
BASICPROTOCOL
This unit describes a protocol for the isolation of lymphoid cell populations from tonsillartissue. The procedure can also be applied with little or no modification to spleen andlymph node tissue.
Human tonsils are the most readily available lymphoid organs and are often used as asource of large numbers of cells characteristic of local lymphoid tissue. Mononuclearcells are isolated by mechanical disruption of tonsil tissue followed by Ficoll densitygradient centrifugation. Cells may then be separated into subpopulations according togeneral protocols used for peripheral blood lymphocytes (UNITS 7.1–7.7).
CAUTION: When working with human blood, cells, or infectious agents, biosafetypractices must be followed (see Chapter 7 introduction).
NOTE: All solutions and equipment coming into contact with cells must be sterile, andproper sterile technique must be used accordingly.
Materials
Fresh human palatine tonsilsIsotonic saline or Hanks balanced salt solution (HBSS) with antibiotics (see
recipe), 4"CComplete RPMI medium supplemented with 10% (v/v) human AB serum
(APPENDIX 2A) with 5 µg/ml gentamicin and 0.5 µg/ml amphotericin B
Forceps, sterile150 # 25–mm petri dishes
Current Protocols in Immunology 7.8.1-7.8.4, August 2009Published online August 2009 in Wiley Interscience (www.interscience.wiley.com).DOI: 10.1002/0471142735.im0708s86Copyright C! 2009 John Wiley & Sons, Inc.
ImmunologicStudies inHuman
7.8.1
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Isolation ofMononuclear
Cells fromTonsillar Tissue
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Disposable sterile scalpels (Swann-Morton, cat. no. 0511 or equivalent)Straight iris scissors (Fisher, cat. no. S17338 or equivalent)Dissecting forceps (VWR, cat. no. 382027-384 or equivalent)3-in. stainless steel sieve with 250-µm mesh (Thomas Scientific, cat. no. 8323-N48
or equivalent)5-ml plastic syringe plunger50-ml polycarbonate centrifuge tubes3-ml transfer pipet (single-use Pasteur pipets; Sarstedt, cat. no. 861171)40-µm plastic cell strainer (Fisher, cat. no. 22363547)Low-speed centrifuge with fixed-angle or swinging-bucket rotor (e.g., Sorvall
RT-6000B with H-1000B rotor)
Additional reagents and equipment for Ficoll-Hypaque cell separation (UNIT 7.1) andcryopreservation of cells (APPENDIX 3G)
1. Obtain fresh tonsils extracted from individuals undergoing routine tonsillectomy.Keep tissue in cold isotonic saline in a plastic container on ice until processing.
Since tissue viability decreases with time, process the tissue within 1 to 3 hr.
2. Using sterile forceps, place specimen on a large plastic petri dish and keep moistenedwith 2 ml HBSS with antibiotics.
3. Cut tissue into 3- to 10-mm fragments with scissors or scalpels.
4. Using sterile forceps, place tissue pieces into a stainless steel sieve that has been setinside a large plastic petri dish containing 2 ml HBSS.
5. Push the lymphoid cells present in the tissue through the sieve using the flat end of a5-ml plastic syringe plunger. Rinse remaining tissue two to three times with HBSSuntil clear.
6. Transfer equal volumes of cell suspension from the petri dish to 50-ml centrifugetubes using a disposable sterile transfer pipet.
7. Add more cold HBSS and process remaining tissue fragments by repeating steps 4to 6 twice more to free as many lymphocytes as possible from stroma. Discard anymaterial remaining in the sieve.
8. After transferring all cells to centrifuge tubes, break up any cell clumps, suspend thecells by repeated pipetting, and pass through a sterile cell strainer. The final volumeof cell suspension should be 35 ml/tube.
Six to twelve tubes are usually required.
9. Overlay the cell suspension on 10 ml Ficoll-Hypaque (UNIT 7.1). Centrifuge 20 min ina Sorvall H-1000B rotor at 1800 rpm (1000 # g), room temperature. Accelerate anddecelerate without braking.
10. Collect mononuclear cells from interface and discard cell pellet containing fibroblastsand other cell debris (see UNIT 7.1).
11. Wash cells three times, each time with HBSS and resuspend in complete RPMI-10medium for immediate use, or cryopreserve and store in liquid nitrogen freezer forfuture use.
ImmunologicStudies inHuman
7.8.3
Current Protocols in Immunology Supplement 86
REAGENTS AND SOLUTIONSUse deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2A; for suppliers, see APPENDIX 5.
Hanks balanced salt solution with antibiotics
HBSS (APPENDIX 2A) containing:100 U/ml penicillin100 µg/ml streptomycin5 µg/ml gentamicin0.5 µg/ml amphotericin B
COMMENTARY
Background InformationInvestigators in the early 1970s first sep-
arated tonsillar lymphocytes from stroma bygentle teasing with small-gauge needles, andshowed that these cells could be stimulatedin vitro to produce immunoglobulin if the ra-tios of T and B cells were adjusted appro-priately (Hoffmann et al., 1973; Sloyer et al.,1973). Preparation of single-cell suspensionswas facilitated by the passage of cells througha fine-mesh sieve (Watanabe et al., 1974). To-day, tonsils are most often used as a source oflarge numbers of human B cells, which are of-ten further separated by size using countercur-rent centrifugation (Muraguchi et al., 1983),or into fractions of differing density on Percollgradients (Nakagawa et al., 1988). These tech-niques are described in relation to monocyte/macrophage isolation in UNIT 7.6. Because ofthe large number of lymphocytes that can beextracted from a pair of tonsils, they are alsoa useful source of dendritic cells (Summerset al., 2001) despite comprising only 1% of thetonsil lymphoid population. In addition, theyare also useful as a source of tissue NK cells,which, along with dendritic cells, participatein tonsillar innate immune responses (Strowiget al., 2008)
Critical ParametersStorage in ice-cold isotonic saline solu-
tion or HBSS and rapid processing of cellsas soon as possible after surgical removal arecritical to maintaining viability. Routine useof antibiotics—including amphotericin B—isnecessary to prevent contamination, althoughovergrowth can occur despite these precau-tions.
Anticipated ResultsTonsil specimens typically weigh from
2 to 10 g. In general, larger tonsils fromyounger patients will contain large germinalcenters and yield the greatest number of vi-able mononuclear cells. Total mononuclear
cell yields will range from 5 # 108 to 5 #109 cells per pair of tonsils. B cells com-prise 60% to 70% of the cell population afterFicoll-Hypaque centrifugation, while 30% to40% are T cells and 1% to 8% are monocytes(Watanabe et al., 1974). Dendritic cells com-prise <1% of the cell population (Hart andMcKenzie, 1988).
Time ConsiderationsThe total time required for this proce-
dure depends on tonsil size. Average process-ing time is $2 hr. After separation on Fi-coll, mononuclear cells may be cryopreserved(APPENDIX 3G). Further separation into subsetscan be performed after thawing.
Literature CitedHart, D. and McKenzie, J. 1988. Isolation and
characterization of human tonsil dendritic cells.J. Exp. Med. 168:157-170.
Hoffmann, M., Schmidt, D., and Oettgen, H. 1973.Production of antibody to sheep red blood cellsby human tonsil cells in vitro. Nature 243:408-410.
Muraguchi, A., Butler, J., Kehrl, J., and Fauci, A.S.1983. Differential sensitivity of human B cellsubsets to activation signals delivered by anti-µantibody and proliferative signals delivered bya monoclonal B cell growth factor. J. Exp. Med.157:530-546.
Nakagawa, T., Nakagawa, N., Ambrus, J.L., andFauci, A.S. 1988. Differential effects of Inter-leukin 2 vs. B cell growth factor on humanB cells. J. Immunol. 140:465-469.
Sloyer, J., Veltri, R., and Sprinkle, P. 1973. In vitroIgM antibody synthesis by human tonsil-derivedlymphocytes. J. Immunol. 111:183-188.
Summers, K.L., Hock, B.D., McKenzie, J.L. andHart, D.N. 2001. Phenotypic characterization offive dendritic cell subsets in human tonsils. Am.J. Pathol. 159:285-295.
Strowig, T., Brilot, F., Array, F., Bougras, G.,Thomas, D., Muller, W.A., and Munz, C. 2008.Tonsillar NK cells restrict B cell transformationby the Epstein-Barr virus via IFN-gamma. PloSPathog. 4:e27.
Isolation ofMononuclear
Cells fromTonsillar Tissue
7.8.4
Supplement 86 Current Protocols in Immunology
Watanabe, T., Yoshizaki, K., Yagura, T., andYamamura, Y. 1974. In vitro antibody forma-tion by human tonsil lymphocytes. J. Immunol.113:608-616.
Key ReferenceWatanabe et al., 1974. See above.Describes protocol and expected yields, as well asthe determination of optimal conditions for in vitrosynthesis of immunoglobulin by tonsil lymphocytes.
143
Paper VI
Psoriasis – as an autoimmune diseasecaused by molecular mimicryHelgi Valdimarsson1, Ragna H. Thorleifsdottir1, Sigrun L. Sigurdardottir1,Johann E. Gudjonsson2 and Andrew Johnston2
1 Department of Immunology, Landspitali University Hospital, Reykjavik, Iceland2 Department of Dermatology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
Psoriasis is strongly associated with streptococcalthroat infection, and patients have increased occurrenceof such infections. Psoriatic lesional T cells are oligoclo-nal, and T cells recognizing determinants common tostreptococcal M-protein and keratin have been detectedin patients’ blood. We propose that CD8+ T cells inpsoriatic epidermis respond mainly to such determi-nants, whereas CD4+ T cells in the dermis preferentiallyrecognize determinants on the streptococcal peptido-glycan that might itself act as an adjuvant. The strepto-coccal association might reflect the concurrence ofsuperantigen production promoting skin-homing of ton-sil T cells, M-protein mimicking keratin determinants,and adjuvant effects of the peptidoglycan. Accordingly,improvement of psoriasis after tonsillectomy should beassociated with fewer T cells that recognize keratin andstreptococcal determinants.
IntroductionPsoriasis is a common inflammatory skin disease that canbe associated with arthritis. It tends to have a fluctuatingcourse that can sometimes be linked to environmentalfactors. The disease has a strong genetic component, witha 40–70 % concordance in identical twins [1,2]. Severaldistinct but overlapping clinical phenotypes have beenidentified, which might be genetically determined to alarge extent [1,3]. Psoriatic skin lesions are characterizedby a marked keratinocyte hyperproliferation and altereddifferentiation associated with dermal and epidermal infil-tration of leukocytes, and it is now generally accepted thatthese pathological changes are triggered and maintainedby T lymphocytes [4,5]. As shown in Figure 1, the majorityof the T cells infiltrating the dermis are of the CD4 phe-notype, whereas CD8+ T cells predominate in the lesionalepidermis [5,6]. Furthermore, the pathological epidermalhyperplasia coincides with epidermal influx of a1b1 integ-rin positive T cells and can be prevented by specific block-ing [5]. In normal human skin basal keratinocytes expresskeratins K5 and K14, and suprabasal keratinocytesexpress mostly K1 and K10, whereas K6 and K16 areexpressed during wound healing and in psoriatic lesions,and these keratins together with K17 predominate inpsoriatic skin [7]. The homology between M-protein andkeratins is mostly restricted to K14, K16 and K17. Inter-estingly, expression of these psoriasis-associated keratins
can be induced in vitro by addition of the pro-inflammatorycytokines IL-1b [8] or IFN-g [9]. Recent studies indicatethat, in addition to Th1 cells, Th17 cells have an importantrole in psoriasis [10], including IL-17-producing CD8+ Tcells [6], and that the keratinocyte hyperproliferationmight be driven by the Th17 cytokine IL-22 either directlyor indirectly [11]. IFN-g is a powerful inducer of thechemokine CCL20, a ligand for C-C chemokine receptor-6 (CCR6), which is expressed by T cells, including CD8+ Tcells that can produce IL-17 [6]. Thus, IFN-g might have atriple role in psoriasis: first, by inducing andmaintaining aputative K17-dependent autoimmune loop [9]; second, bystimulatingmyeloid dendritic cells (DC) to induce T cells toproduce IL-17; and third, at the same time, stimulateepidermal production of CCL20, which attracts the IL-17-producing cells to the epidermis [6].
Recent reviews have mostly addressed the clinical,genetic and cytokine aspects of psoriasis, and only one[12] of the approximately 500 review articles that havebeen published on psoriasis in the past three years hasfocused on the fundamental question of whether psoriasisis an autoimmune disease.
Others and we have recently reviewed the genetic andpathogenic aspects of psoriasis [13–15]. Notably, over 60%of psoriasis patients carry one or two class I HLA-Cw*0602alleles compared with a population frequency of 10–15%,and this HLA allele was recently strongly implicated as animportant susceptibility allele in psoriasis [16]. On thebasis of this and other pathogenic features of the disease,we argued that CD8+ T cells are likely to be the ultimateeffector cells that recognize auto-epitopes presented by thebinding pockets of HLA-Cw6 or other HLA class I mol-ecules on the keratinocyte surface. This pathogenic activityis, of course, generated through a complex interplay be-tween CD4+ and CD8+ T cells as well as antigen presentingcells (APCs), including cross-presenting DCs [15]. Here, wefocus mainly on reports that have addressed the issue of Tcell specificity and the nature of the auto-antigen(s) inpsoriasis.
Psoriasis, streptococcal infections, and the palatinetonsilsAn association between streptococcal throat infection andthe acute guttate form of psoriasis (an early onset form) hasbeen demonstrated in many studies, and the reportedincidence of streptococcal infections preceding this typeof psoriasis ranges between 56% and 97% [reviewed in Ref.
Review
Corresponding author: Valdimarsson, H. ([email protected]).
494 1471-4906/$ – see front matter ! 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.it.2009.07.008
Sigrun Laufey Sigrurdardottir
Sigrún Laufey Sigurðardóttir
17]. Furthermore, we demonstrated in a prospective studythat chronic plaque psoriasis is also exacerbated after suchinfections and that the psoriasis patients had about a ten-fold higher frequency of streptococcal throat infectionsthan age-matched household controls [18]. As has beenestablished for acute guttate psoriasis, worsening ofchronic psoriasis was associated only with throat infectionby the three groups of b-hemolytic streptococci (A, C andG)that express M-protein on their surface [18]. M-protein, amajor virulence factor composed of two polypeptide chainsin a complexwith an alpha-helical coiled-coil configuration,is anchored in the bacterial cell membrane. In contrast to avery variable N-terminal part of the protein, the C-term-inal membrane-anchored half is highly conserved in differ-ent bacterial strains and serotypes. No association hasbeen found between psoriasis and particular M serotypes[19].
Non-symptomatic carriage of streptococci in the tonsilcrypts is common and these bacteria are efficiently inter-nalized by crypt macrophages and epithelial cells, wherethey are protected from most commonly used antibiotics.Streptococcal carriage might be associated with increasedincidence of symptomatic streptococcal throat infections[20]. There are several reports of partial or completeremission of psoriasis following tonsillectomy, but no con-trolled prospective trial has been reported. As most psor-iasis patients have a fluctuating disease activity andspontaneous remissions are not uncommon, matchedpatient controls are essential to determine whether ton-sillectomy has any beneficial effect.
Reports on oligoclonality of lesional T cells in psoriasisSince 1994, at least 14 studies reported by nine indepen-dent groups have indicated that chronic psoriasis lesionsare infiltrated by oligoclonal T cells, and failure to identifyoligoclonal lesional T cells has been reported by only one
group that studied patients with the acute guttate form butnot chronic plaque psoriasis [21]. These studies, which aresummarized briefly in Table 1, involve over 70 patients,including 27 with psoriatic arthritis, and collectively theymake a strong case for the notion that the T cells, whichmediate chronic psoriasis, are driven by conventional anti-gen(s). Furthermore, follow-up studies have shown thatwhile minor clones tend to be transient, dominant clonescan persist for a long time and even re-appear in lesionalskin after drug-induced remission [23,24,29,34]. Moreover,identical T cell clones have been detected in both skin andsynovial lesions in patients with psoriatic arthritis (PsA)[31,32]. Notably, in the four studies where CD4+ and CD8+
T cells in the bloodwere analyzed separately, oligoclonalitywas detected among the CD8+ but not the CD4+ T cells[25,26,33,34].
A study reported by researchers who had previouslyidentified a number of CD4+ and CD8+ T cell clones insynovial fluid from patients is of special interest [33]. Theyargued that, as psoriasis usually relapses soon after treat-ment with methotrexate is discontinued, T cell clones withspecificities that are relevant in the very early stages of theinflammatory process should persist during the treatment[34]. Interestingly, most of the expanded clones did notpersist and the T cell clones that could be detected duringthe treatment (about 12% of the clones that were detectedin active tissue before the treatment with methotrexate)were of the CD8+ T cell lineage, and were found in bothjoint fluid and blood [34]. These findings highlight the well-known phenomenon that the greatmajority of lymphocytesthat infiltrate active inflammatory sites are polyclonal, andnot specific for the antigen(s) that initiated the inflamma-tory changes [reviewed in Ref. 35]. In autoimmune reac-tions, some of these polyclonal T cells might, however,recognize and proliferate clonally in response to newauto-antigens exposed or activated by the inflammatory
Figure 1. Sections of psoriatic skin lesions counterstained with haematoxylin/eosin for CD4+ and CD8+ T cells (brown). Note that the dermis is infiltrated mostly by CD4+ Tcells, whereas most of the CD8+ T cells are located in or around the epidermal ridges. The broken line indicates the border between the epidermis and dermis.
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process, a phenomenon called epitope or determinantspreading [reviewed in Ref. 36]. It should be noted in thiscontext that minor transient T cell clones [30] are likely torepresent epitope spreading whereas dominant clones,especially those that persisted during methotrexate treat-ment [34], are more likely to be directed against the auto-epitope(s) that initiated the autoimmune process. Thus,targeting clones that are secondary to the inflammatoryprocess might explain, to some extent, why it has proveddifficult to treat autoimmune diseases in humans withmodified peptides, an approach that has consistently beensuccessful in animalmodels of experimental autoimmunityinduced with defined epitopes [reviewed in Ref. 37].
The most recent report on T cell oligoclonality in psor-iasis indicates that T cell clones, which expand in thepalatine tonsils, might be involved in maintaining psor-iatic lesions [38]. Thus, three patients with a history ofmoderate to severe plaque psoriasis for 1.5, 6, and 10 yearsall experienced at least 3 years remission after tonsillect-omy. They all showed highly restricted T cell receptor(TCR) Vb spectratypes of lesional T cells. Furthermore,sequencing of the CDR3 segment of the TCR Vb-chain alsoidentified a number of identical clonal rearrangementswithin the lesional skin of all three patients and, moreover,several of these lesional clonotypes were detected amongthe cutaneous lymphocyte antigen (CLA) positive T cellswithin their tonsils. This and the remission of the patients
after the tonsillectomy strongly suggest an egress of patho-genic T cells from the tonsils to the skin.
The consistent finding of oligoclonal T cells in psoriaticlesions strongly indicates that chronic psoriasis is drivenby conventional antigen(s) but does not provide any infor-mation about the identity or nature of the antigen(s).However, it is now possible to identify T cell clones inthe blood with very sensitive functional assays that allowthe identification of individual T cells that respond to shortpeptides by cytokine production (e.g. bymeans of ELISPOTor flow cytometric detection of cytokine production) [39,40].In this way we, and others, have tested a number ofpsoriasis patients and non-psoriatic controls for T cellactivation in the presence of short (9–20 amino acid) pep-tides with determinants that are shared by streptococcalM-protein and human keratins.
T cells cross-reacting with human keratin andstreptococci can be detected in the blood of psoriasispatientsAn extensive homology between streptococcal M-proteinand keratin was first reported about 17 years ago, and thisapplied to segments of identical amino acid sequences andoverall homology [41]. Of about 4200 mammalian proteinsthat were compared, human type 1 keratins showed thestrongest homology with the streptococcal M6 protein. Onthe basis of this and other findings, we proposed in 1995
Table 1. Reports on oligoclonal T cells in psoriatic skin and joint lesions.
Ref. No.patients
Tissuesstudied
TCR-Vßbias
Methods Oligoclonality Comments
CDR3sequencing
Spectratyping
22 7 E Vß 3, 13.1 Yes No Yes (7/7) Study confined to lesional epidermalT cells, oligoclonality of CD8+T cells detected in blood
23 4 E Vß 3, 13.1 Yes No Yes (4/4) Same patients as in Ref. 22, clonalpersistence > 15 months
24 11 E & D Vß 2, 6 Yes No Yes (3/3) Clonal persistence for at least18 months
25 5 E & D Vß 5.1, 12,16 No Yes Yes (4/4) Oligoclonality among CD8+T cells in blood
26 10 E & D Inconsistent Yes Yes Yes (4/6) Early or expanding lesion,T cell oligoclonality also in blood
27 6 E & D Vß 9, 20 Yes No Yes (6/6) Concordant identical twinswith identical lesional clones
28 10 E & D Inconsistent No Yes Yes (9/10) Identical clones identified insymmetrical skin lesions
29 2 E & D Vß 2, 3, 6,13, 17
Yes No Yes (2/2) Dominant clones persist,non-dominant clones transient
30 5 E Inconsistent Yes Yes Yes (5/5) Oligoclonality restrictedto dominant clones
31 9 E, D, S Inconsistent Yes No Yes (9/9) Homologous clones in skinand joint lesions
32 3 E, D, S Inconsistent Yes No Yes (3/3) Homologous clones in skinlesions and synovial membrane
33 6 SF NR Yes Yes Yes (6/6) Oligoclonality more pronouncedin CD8+ T cells and identical CD8+T cell clones identified in blood
34 9 S & SF NR Yes Yes Yes (9/9) Dominant CD8+ T cell clones observedin synovial tissue and blood afterMTX-induced remission
38 3 E, D, T NR Yes Yes Yes (3/3) Homologous T cell clones intonsils and skin lesions
D, dermis; E, epidermis, MTX, methotrexate; NR, not recorded; S, synovial tissue; SF, synovial fluid; T, tonsillar tissue.
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that psoriasis is initiated by streptococcal superantigensand maintained by T cells that recognize streptococcal M-protein determinants in the palatine tonsils and homolo-gous keratin determinants in the skin [42]. Subsequently,the frequency of T cells in the blood that were activated byshort homologous M and K peptides to produce IFN-g wasdetermined by the ELISPOT technique in two sets ofexperiments involving a total of 31 patients with chronicpsoriasis, 6 patients with allergic dermatitis and 29healthy controls [43,44]. The responses were strikinglyhigher in the psoriasis patients than in the controls, whileno difference was observed in the responses to a controlantigen (streptokinase/streptodornase (sk/sd)). Notably, Mand K peptides that share an ALEEAN amino acidsequence induced stronger and more consistent T cellresponses than any of the other peptides. Minimalresponses were detected when the patients’ T cells wereexposed to control K peptides that did not share sequenceswith M-protein. Moreover, the responses to the homolo-gous peptides could not be detected after only two weeks oftreatment with UVB irradiation, which drives epidermal Tcells into apoptosis, whereas the responses to the sk/sdcontrol antigen were unaffected by this treatment [43,44].
There is increasing evidence that CD8+ T cells have akey role in the pathogenesis of some autoimmune diseases[45,46], including psoriasis [5,6]. With this in mind and thestrong association of psoriasis with the HLA-Cw*0602allele, we next split the amino acid sequence of K17 intoa complete set of overlapping nine residue peptides thatwere then used as a library to compare with sequences ofthe M-protein [47]. Sets of peptides from K17 and M-protein that showed sequence similarities as well as motifspredicted to bind to HLA-Cw6, were selected together withfour K17 and M peptides that did not share sequences orhad a poor predicted binding for HLA-Cw6. These peptideswere tested against blood lymphocytes from 12 HLA-Cw6positive and 11 HLA-Cw6 negative patients with chronicpsoriasis and 11 healthy HLA-Cw6 positive individualswith a family history of psoriasis. T cells from all thepsoriatic patients responded by production of IFN-g toone or more (up to 10) of the K peptides, whereas thecontrols either did not respond to any or showed onlyborderline responses to 4 or fewer of these peptides. TheHLA-Cw6 negative psoriatic patients showed T cellresponses that were intermediate between those of theHLA-Cw6 positive patients and the controls. Interestingly,the responses were mostly confined to the CD8+ T cellpopulation. Moreover, the great majority (>90%) of theresponding cells expressed the skin homing CLA determi-nant. The frequency of T cells responding to theM-peptideswas significantly higher in both groups of patients,whereas the controls responded equally well to the controlantigens sk/sd andCandida albicans cellular proteins [47].In another study of the potential contribution of CD4+ Tcells, the whole K17 molecule was scanned for sequencespredicted to be HLA DRB1*04, *07 restricted T cell epi-topes [48]. These HLA class 2 alleles are significantlyassociated with psoriasis, although not as strongly asHLA-Cw6. T cells isolated from the blood of 52 HLADRB1*04 and/or *07 positive psoriasis patients and 48matched controls were testedwith a number of overlapping
K17 peptides with motifs that were predicted to associatewith either or both of these HLA class 2 alleles. Two of thepeptides induced relatively strong proliferation and IFN-gproduction by T cells from the patients, whereas T cellsfrom the non-psoriatic controls showed negligibleresponses. Interestingly, one of these two peptides con-tains the ALEEAN sequence that had been reported toactivate T cells from psoriasis patients [43,44,47]. Theother peptide had an amino acid sequence that had notbeen reported to be recognized by psoriatic T cells.
More recently, it was reported that it was possible toabolish the T cell proliferation and IFN-g production,induced by these peptides, by single alanine residue sub-stitutions at a critical TCR contact position [49]. Moreover,these altered peptide ligands induced production of IL-4,and the anti-inflammatory cytokines IL-10 and TGF-b and,furthermore, supernatants from the T cell cultures inhib-ited keratinocyte proliferation in vitro [49].
Does the streptococcal peptidoglycan have a role inpsoriasis?It has been reported that T cell lines (TCLs) grown fromlesional dermis, and therefore consisting predominantly ofCD4+ T cells, do not respond by proliferation to recombi-nant M-protein [50]. Moreover, cell wall components fromstreptococci that did not express M-protein induced T cellresponses, in terms of IFN-g production, similar to cell wallcomponents that included M-protein, indicating that thedermal CD4+ T cells showed little response to M-proteindeterminants [50]. The response of the dermal TCLs todifferent streptococcal cell wall components, including thepeptidoglycan (PG) was investigated further, and it wasestimated that at least half of the streptococcal cell wall-specific T cells responded to PG. Interestingly, streptococ-cal PG was detected within lesional dermal macrophagesthat were within clusters of dermal T cells or in the dermalpapillae [51]. PG is known to interact with a number ofreceptors of innate immunity [52,53], including Toll-likereceptor 2 (TLR2), nucleotide-binding oligomerizationdomains 1 and 2 (NOD 1 and NOD 2) and the recentlyidentified family of PG recognition proteins 1–4 (PGRP 1–4). Furthermore, the genes for TLR2, NOD1, NOD2 andPGRP 2, 3, and 4 are all located in regions that have beenreported to show linkage with psoriasis [14], althoughsome of these loci remain to be confirmed. On the basisof these findings and the location of the genes for the PGbinding proteins, it was proposed that PGmight be amajoretiological agent in psoriasis, possibly because psoriasispatients have an altered innate immune response to PG[12]. This interesting proposal is consistent with, andcomplements, our molecular mimicry model.
Psoriasis and three concurrent features of streptococcalthroat infectionsA crucial issue in this context is that the TCLs werecultured exclusively from lesional dermis but not the epi-dermis [50,51], which was used for other experiments notinvolving T cell responses to M-protein or PG [54]. There-fore, the TCLs consisted mostly of CD4+ T cells [50,51] thatpredominate in the lesional dermis. In contrast, thelesional epidermis is infiltrated mostly by CD8+ T cells,
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which are required for the development of psoriatic lesions[5]. As shown in Figure 2 and elaborated in Box 1, wepropose that APCs in the tonsils and draining lymph nodespresent various streptococcal determinants to T cells. Atthe same time, CLA expression on the resultant effector Tcells might be enhanced by streptococcal superantigen [21]
(Figure 2, steps 1 and 3)) which facilitates their egress intothe skin. As most CD4+ T cells in psoriatic lesions localizeto the dermis, they associate with APCs that might bepresenting streptococcal components, including PG [51],and some might therefore expand clonally in response toantigen determinants on PG (Figure 3, steps 5 and 7).
Figure 2. Psoriasis and three concurrent features of streptococcal throat infections. Step 1: Infection or colonization of tonsils by superantigen-producing microbes such asStreptococcus spp. increases expression of skin-homing cutaneous lymphocyte-associated antigen (CLA) by tonsillar T cells. Steps 2 and 3: Streptococcal throat infectionsor their persistence promotes maturation of both effector CD4+ (blue) and CD8+ (red) T cells that recognize streptococcal components, including PG (red) and M-proteindeterminants (black). CD8+ T cells (but not CD4+ T cells) also express Toll-like receptor 2 on their surface (TLR2) (purple), which can bind PG. Macrophages and antigen-presenting cells (APC) with internalized streptococcal components. Internalized PG might also provide adjuvant activity by binding to intracellular innate immune receptorswithin APCs.
Box 1. Proposed etiological and pathogenic sequence in psoriasis
1) Genetically determined abnormal innate immune response asso-ciated with deviated T cell responses (e.g. IFN-g or IL-17 bias?)against microbes containing PG (proposed in Ref. [12] and here)
2) Increased frequency of streptococcal throat infections in psoriasispatients [18]
3) Infection and colonization of tonsils by superantigen-producingmicrobes increases expression of the skin-homing cutaneouslymphocyte antigen (CLA) by tonsil T cells [21] (Figure 2, step 1)
4) Streptococcal throat infections or their persistence promotematuration of effector T cells that recognize streptococcalcomponents including PG and M-protein determinants [12] (andproposed here, Figure 2, steps 2 and 3).
5) Association between streptococcal throat infections and psoriasisis strong due to concurrence of superantigen production, PG thatbinds to innate immune receptors (e.g. TLR2) and M-protein thatmimics type 1 keratins in the epidermis (proposed here, Figure 2steps 1–3).
6) Some streptococcal components, including PG and M-protein,enter the bloodstream where they are taken up by monocytes/macrophages (proposed in Ref. 12 and here, Figure 3, step 1) NBMacrophages are not found in efferent lymph nor have they beenobserved to migrate from (lymphoid) tissues into the circulation.
7) Increased frequency in the blood of psoriasis patients of bothCD4+ and CD8+ T cells that respond to antigen determinants that
are common to streptococcal M-protein and type 1 keratins[43,44,47–49] (Figure 3, step 2).
8) Re-circulating CLA+ effector CD4+ T cells migrate mainly intolesional dermis but CLA+ effector CD8+ T cells migrate mostlythrough the dermis and into the epidermis [5,6] (Figure 3, steps 3and 4).
9) Entry of CD8+ T cells is required for the characteristic keratinocyteproliferation in psoriasis [5] (Figure 3 step 4).
10) Streptococcal components, including PG, are presented mainly inthe dermis and hence primarily to CD4+ T cells (Figure 3 step 5). Kpeptides that share determinants with streptococcal M-proteinare predominantly presented in the epidermis and thereforemainly to CD8+ T cells (proposed here, Figure 3, step 6).
11) Antigen presenting cells at the dermal–epidermal junctionpresent PG determinants to CD4+ T via HLA class II (Figure 3,step 7), and present keratin determinants to CD8+ T cells via HLAclass I molecules (Figure 3, step 8). At the same time PG insideantigen-presenting cells might mediate an adjuvant effect forboth CD4+ and CD8+ T cells via intracellular innate immunereceptors (proposed here, Figure 3, steps 7 and 8).
12) Recruitment of naıve T cells to the tonsils and draining lymph nodesis probably required for maintaining persistent psoriatic plaques.There is a close correlation between the frequency of CLA+ T cells inthe blood of psoriasis patients and their disease severity [59].
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Conversely, most CD8+ T cells migrate into the lesionalepidermis [5] where APCs would present predominantly Kpeptides, some of which will share sequences with strep-tococcal M-protein (Figure 3, step 6). It is therefore pos-tulated that most of the oligoclonal T cells observed inlesional epidermis [22] are specific for such cross-reactivedeterminants that are presented by HLA class I moleculeson the surface of keratinocytes, and on DCs located in theepidermis and at the dermis–epidermis junction (Figure 3,step 8). A similar interaction might take place, to someextent, in draining lymph nodes between both naıve andeffector CD8+ T cells and DCs that have migrated to lymphnodes from lesional epidermis. At the same time, PG insideAPCs in psoriatic lesions (Figure 3, steps 7 and 8), thetonsils and lymph nodes draining lesional skin might havean adjuvant effect via intracellular innate immune recep-tors, and this effect might be different or exaggerated inpsoriasis patients [12]. Furthermore, it has been reportedrecently that CD8+ T cells express TLR2 (a receptor forPG), ligation of which lowers their TCR activationthreshold [55] (Figure 3, step 8). Thus, the key to thestrong association between psoriasis and streptococcalthroat infections might be the concurrence in the pathogenof (a) components with adjuvant activity, (b) amino acid
sequences that mimic human keratin, and (c) the release ofstreptococcal superantigens that have been reported topromote the expression of the skin-homing CLA epitope[21].
Recirculation of pathogenic T cells in psoriasisA recent proposal has suggested that psoriasis lesionsmight be initiated and maintained by T cells that residein the skin without the need for recruitment from thecirculating T cell pool [56]. This hypothesis is based mainlyon the observation that transplants of uninvolved (i.e. non-psoriatic) skin from psoriasis patients onto SCID mice,which are also deficient in type I and type II interferonreceptors (and therefore also with greatly impaired NK cellfunction, AGR129mice) became lesional after 4 to 5 weeks,and a marked proliferation of the human T cells in thegraft, especially the epidermal CD8+ T cells, was observedduring this latent period [57]. This is a challenging prop-osition but thus far it has not been reported how long thepsoriatic lesions last in this animal model. It is welldocumented that DCs have a very short half-life at inflam-matory sites and, furthermore, that a substantial pro-portion of effector T cells also become apoptotic duringan ongoing immunological response [reviewed in Ref. 58],
Figure 3. Recruitment of T cells, macrophages, and/or antigen-presenting cells (APCs) into psoriatic lesions. Step 1: Some streptococcal components, including PG (red)and M-protein (black), enter the bloodstream and are taken up by monocytes/macrophages that migrate as APCs into developing psoriatic lesions. Step 2: There is anincreased frequency in the blood of psoriasis patients of both CD4+ and CD8+ T cells, which respond to antigen determinants that are common to both streptococcal M-protein and type 1 Ks. Step 3: Re-circulating CLA+ effector CD4+ T cells migrate mainly into the lesional dermis. Step 4: CLA+ effector CD8+ T cells migrate mostly through thedermis and into the epidermis. Entry of CD8+ T cells into the epidermis is required for the characteristic keratinocyte proliferation of psoriasis. Step 5: Streptococcalcomponents, including PG, are presented mainly in the dermis and hence mostly to CD4+ T cells. Step 6: K peptides that cross-react with streptococcal M-proteindeterminants are presented predominantly in the epidermis and therefore to CD8+ T cells. Step 7: APCs at the dermal–epidermal junction can present PG to CD4+ T cells viaHLA class II molecules. Step 8: The same junctional APCs present keratin determinants to CD8+ T cells via HLA class I. PG might also lower the activation threshold of CD8+ Tcells expressing TLR2 [55].
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making it likely that recruitment of central memory andnaıve T cells is required for maintaining persistent T cell-dependent inflammation. It is therefore unlikely that psor-iatic plaqueswould persist for years without recruitment ofmemory and naıve T cells from the re-circulating pool.Furthermore, a close correlation between the frequencyof CLA+ T cells (i.e. those homing to the skin) in the bloodand disease severity has been reported in psoriasispatients [59]. It should be noted that the AGR129 micedescribed above have no lymphoid cells (including func-tional NK cells), and therefore it is possible that theimmunocytes in the transplanted human graft could havea greatly extended survival in this context.
Concluding remarksStudies published in recent years have been uniformlyconsistent with the conceptual framework, outlined in1995, that chronic psoriasis is an autoimmune diseasemediated by effector T cells that are primed against con-ventional streptococcal antigens in the palatine tonsils andsubsequently cross-react to keratin determinants in theskin [42]. Thus, T cells in the blood of psoriasis patientsrecognize determinants that are shared by streptococcalM-protein and human keratin, and these T cells disappearduring effective treatment of the disease. However, otherstreptococcal components, such as PG, might be importantin the pathogenesis of psoriasis both as an adjuvant and anantigen recognized directly by T cells in the tonsils, lesionalskin, and draining lymph nodes. Because CD8+ T cellsegress preferentially to the epidermis, they have the oppor-tunity to recognize keratin determinants that are sharedwith streptococcal M-protein and presented by epidermalAPCs. The great majority of the infiltrating CD4+ T cellsreside in the dermis, where they can recognize streptococ-cal components, including PG, that are preferentiallypresented there. In either case, an improvement of psor-iasis after tonsillectomy should be associated with reducedfrequency of circulating T cells that recognize streptococcaldeterminants because the palatine tonsils are the majorfocus for streptococcal infections and carriage. We arecarrying out a controlled, prospective and observerblind study on the clinical and immunological impact oftonsillectomy in patients with chronic psoriasis (Box 2).Removal of the tonsils has generally been associated with a
substantial clinical improvement coinciding with amarkeddecrease in the frequency of cross-reactive keratin andM-protein-specific CD8+ T cells in the blood, whereasreduction in the frequency of corresponding cross-reactiveCD4+ T cells has not been consistent [60]. We conclude thatidentification of antigen determinants that are recognizedby dominant T cell clones in psoriasis are of crucial import-ance because they might be the appropriate prime targetsfor highly specific immunotherapy.
AcknowledgementsThe authors have been funded by the European Union Training andMobility of Researchers Programme, The Icelandic Research Fund, TheUniversity of Iceland Research Fund and The Science Fund ofLandspitali University Hospital. The help of the Icelandic Associationof Psoriasis and Eczema Patients (SPOEX) in recruiting volunteers isgreatly appreciated.
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Box 2. Some testable predictions
1) Abnormal responses of tonsil T cells from psoriasis patients tostreptococcal components including PG and M-protein (underinvestigation).
2) Raised serum levels of peptidoglycan and/or increased frequencyin the blood of psoriasis patients of monocytes/macrophagesthat contain PG (under investigation).
3) Increased frequency of CLA+ T cells that respond to streptococcalPG in the blood of psoriasis patients (as has been reported forCLA+ T cells) that respond to homologous M-protein and keratinpeptides [47] (under investigation).
4) CD8+ T cells isolated from lesional epidermis should preferen-tially respond to homologous M-protein and K peptides (underinvestigation).
5) Psoriasis should improve after tonsillectomy and the improve-ment should coincide with reduction in the blood of CLA+ T cellsthat respond to streptococcal components and K determinants(strongly suggestive data are available [60].
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