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1

Differential Expression of T cell Immunoglobulin-

and Mucin- Domain-Containing Molecule-3 (TIM-3)

According to Activity of Behçet’s disease

by

Joong Sun Lee

Major in Medicine

Department of Medical Sciences

The Graduate School, Ajou University

2

Differential Expression of T cell Immunoglobulin- and

Mucin- Domain-Containing Molecule-3 (TIM-3)

According to Activity of Behçet’s disease

by

Joong Sun Lee

A Dissertation Submitted to The Graduate School of Ajou University

In Partial Fulfillment of the Requirements for the Degree of

Ph.D. in Medicine

Supervised by

Eun-So Lee, M.D., Ph.D.

Major in Medicine

Department of Medical Sciences

The Graduate School, Ajou University

February, 2009

3

This certifies that the dissertation

of Joong Sun Lee is approved.

SUPERVISORY COMMITTEE

Sun Park

Eun-So Lee

You Chan Kim

Seonghyang Sohn

Kyung Sook Park

The Graduate School, Ajou University

December, 22th, 2008

4

Acknowledgement

Thanks to the Lord’s grace, I have been allowed health as well as patience.

In particular, I thank my thesis director, Dr. Eun-So Lee. Her attentive guidance and

dissemination of knowledge enabled this work to be accomplished. I thank Dr. Sun

Park for guiding my research work and discussing the data; Dr. Seonghyang Sohn for

her suggestion and coaching; Drs. You Chan Kim and Kyung Sook Park for

comments on the paper. I also wish to thank Mi Jin Park for her experimental

assistance and Young Bae Kim for his immunohistochemical assistance.

Above all the things, my father and mother have supported me over the years with

love and patience. I present immeasurable gratitude to my parents from the bottom of

my heart.

Finally, this paper is dedicated to my husband, Dr. Kyung Sik Seo. I want to express

my thanks to him for encouraging me, not letting me give up, loving me, and, most

of all, trusting me.

December 31th, 2008

Joong Sun Lee

i

- ABSTRACT –

Differential Expression of T Cell Immunoglobulin- and Mucin-

Domain-Containing Molecule-3 (TIM-3) According to

Activity of Behçet's Disease

Background: T cell immunoglobulin mucin-3 (TIM-3) is recently described as a TH1-

associated cell surface molecule that regulates TH1 responses and promotes tolerance in mice.

Increased TH1 immune response has been known as one possible pathogenesis of a chronic

inflammatory multisystemic disorder, Behçet's disease (BD). However, TIM-3 expression

and function in BD has not been investigated.

Purpose: In this study, its was examined that the surface expression of TIM-3, the

expression of TIM-3 protein and the TIM-3 mRNA in peripheral blood mononuclear cells

from Behçet's disease patients, and evaluated the TIM-3 expression pattern according to

clinical disease activity.

Methods: Behçet's patients (n=67), healthy control (n=13) and psoriasis patients (n=14)

were involved. Immunologic profile of Korean patients, especially in relation to T cell

immunity was examined by flow cytometry. The expression of TIM-3 as well as CD4, CD8,

CD11b and CD56 in peripheral blood mononuclear cells (PBMC) was assessed by flow

cytometry. BD group was divided according to the disease activity. TIM-3 expression was

also compared between BD in active state and BD in remission state. Western blot analysis

ii

was performed to evaluated TIM-3 protein. TIM-3 expression after simulation of PBMCs

was analyzed by flow cytometry. At the transcript level, the expression of TIM-3 was

assessed by quantitative RT-PCR. The immunohistochemical analysis was done in cutaneous

lesion with anti-TIM-3 antibody and anti-CD4 antibody.

Results: Disease activity markers, ESR and CRP, were elevated in the blood samples from

active BD compared to inactive BD. The surface expression of TIM-3 molecule was

significantly upregulated in Behçet's disease patients compared with healthy controls by flow

cytometry and the stable BD had a tendency to show higher TIM-3 than the active BD. The

immunohistochemical study in cutaneous lesion of BD showed the co-localization of TIM-3+

cells and CD4+ cells in inflammatory site. Western blot analysis also showed the upregulated

expression of TIM-3 protein in BD compared to control. Even in each patient, according to

his disease activity, the expression of TIM-3 was changed, showing a higher TIM-3

expression in remission state than in active state. In leukocytes subpopulation, there were no

significant differences in CD4, CD11b, and CD56 cells except CD8 cells decreased in BD

and psoriasis. The mean expression of TIM-3 molecule in CD8+ and CD56+ cells was

significantly increased in BD compared with controls. However the correlation between

TIM-3 frequency and TIM-3 expression in CD4 cells was highest among leukocyte

subpopulations. The stimulation of PBMC with anti-CD3, CD28 antibody in presence of IL-

2, could not up-regulate the TIM-3 expression in BD patients with simulation time and TIM-

3 expression in CD4+ and CD8+ cells were not changed after stimulation, too. The

transcription level analysis of PBMCs from BD patients revealed significantly higher mRNA

iii

expression of TIM-3 compared with controls. Moreover, in the same patient, TIM-3 mRNA

expression was significantly altered according to disease activity, increasing TIM-3 mRNA

in active state compared with remission state.

Conclusion: This study may imply the differential expression of human TIM-3 molecules by

the PBMCs of TH1-driven Behçet's disease according to disease activity and suggest that

there were altered kinetics in the expression of TIM-3 molecule and TIM-3 mRNA in

PBMCs that might modulate immunologic response in Behçet’s disease.

Key words: Behçet’s disease, Disease activity, T cell immunoglobulin and mucin (TIM)-3

iv

TABLE OF CONTENTS

ABSTRACT---------------------------------------------------------------------------------- ⅰ

TABLE OF CONTENTS----------------------------------------------------------------------------- ⅳ

LIST OF FIGURES----------------------------------------------------------------------------------- ⅵ

LIST OF TABLES------------------------------------------------------------------------------------- ⅷ

I. INTRODUCTION--------------------------------------------------------------------------- 1

II. MATERIALS AND METHODS--------------------------------------------------------- 8

A. Patients and samples------------------------------------------------------------------------- 8

B. Methods-------------------------------------------------------------------------------- 8

1. Cell preparation-------------------------------------------------------------------------- 9

2. Cell culture and stimulation of PBMCs ---------------------------------------------- 9

3. Flow cytometric analysis-------------------------------------------------------------- 10

4. Reverse transcription polymerase chain reaction (RT-PCR)--------------------- 11

5. Immunohistochemistry---------------------------------------------------------------- 12

6. Western blot analysis---------------------------------------------------------- 13

7. Statistical analysis-------------------------------------------------------------- 14

III. RESULTS--------------------------------------------------------------------------------- 15

A. Subjects characteristics-------------------------------------------------------------------- 15

B. TIM-3 expression in PBMCs and the effect of medication ------------------------- 20

C. Western blot analysis of TIM-3 protein ------------------------------------------------- 22

D. Immunohistochemical analysis of TIM-3 in erythema nodosum-like skin lesions--

v

of Behçet’s diseases ----------------------------------------------------------------------- 23

E. Correlation of TIM-3 expression with Behcet’s disease activity -------------------- 26

F. Analysis of surface markers on PBMCs and TIM-3 expression on leukocyte---------

subpopulations------------------------------------------------------------------------------ 27

G. Correlation of TIM-3+ frequency in leukocyte subpopulation with Behcet’s disease-

activity --------------------------------------------------------------------------------------- 31

H. Expression of TIM-3 in stimulated PBMCs between Behçet’s patients and healthy

controls -------------------------------------------------------------------------------------- 32

I. Expression of TIM-3 mRNA was analyzed by RT-PCR between Behçet’s disease--

- group and healthy controls -------------------------------------------------------------- 34

J. Expression of TIM-3 mRNA was analyzed by RT-PCR after stabilization of

clinical symptoms of Behçet’s disease -------------------------------------------------- 36

IV. DISCUSSION----------------------------------------------------------------------------- 38

V. CONCLUSION---------------------------------------------------------------------------- 45

REFERENCES-------------------------------------------------------------------------------- 46

국문요약 ------------------------------------------------------------------------------------- 57

vi

LIST OF FIGURES

Fig. 1. Schematic representation of human TIM protein structures------------------------------- 4

Fig. 2. Mechanisms by which Tim-3 might modulate immune function------------------------- 7

Fig. 3. The level of disease activity makers of Behçet’s disease -------------------------------- 18

Fig. 4. Cytokine profile in PBMCs from Behçet’s disease patients ex vivo --------------------19

Fig. 5. Expression of TIM-3 in PBMCs -------------------------------------------------------------20

Fig. 6. The effect of medications on the TIM-3 expression by FACS analysis --------------- 21

Fig. 7. The representative result of expression of TIM-3 protein in PBMCs from BD patients

by Western blotting--------------------------------------------------------------------------- 22

Fig. 8. TIM-3 expression in cutaneous lesion ----------------------------------------------------- 24

Fig. 9. TIM-3 expression in CD4+ cells in EN-like lesion of Behçet’s disease--------------- 25

Fig. 10. The comparison of frequcncy of TIM-3 expression between active disease and-------

stable state in the same patient ------------------------------------------------------------- 26

Fig. 11. Analysis of surface markers on PBMCs and TIM-3 expression on leukocyte----------

- subpopulations ----------------------------------------------------------------------------- 29

Fig. 12. Correlation between TIM-3 frequency and TIM-3 expression in leukocyte-------------

subpopulations ------------------------------------------------------------------------------- 30

Fig. 13. The comparison of TIM-3 expression on each cellular population of PBMCs----------

between active and stable disease state in the same patient ---------------------------- 31

Fig. 14. TIM-3 expression on stimulated PBMCs------------------------------------------------- 33

Fig. 15. Expression of TIM-3 in PBMCs assessed by RT-PCR --------------------------------- 34

vii

Fig. 16. TIM-3 mRNA was analyzed according to the disease state --------------------------- 36

viii

LIST OF TABLES

Table 1. Clinical characteristics of Behçet’s disease patients------------------------------------ 16

Table 2. Clinical characteristics and laboratory results of Behçet’s disease patients according

to disease activity----------------------------------------------------------------------------17

Table 3. Pearson correlations between the expression level of TIM-3 and TIM-3 frequency in

PBMCs subpopulation ----------------------------------------------------------------------27

1

I. INTRODUCTION

CHRACTERISTICS OF BEHÇET’S DISEASE

The clinical manifestations of what is known today Behçet’s disease (BD) were

described in as far back as the era of Hippocrates. However, in 1937, the Turkish

dermatologist Hulusi Behçet was the first to give a comprehensive description of the

symptom-complex of recurrent oral aphthous ulcers, genital ulcers, and uveitis as a disease

entity (Lee, 2000). Generally, BD was described as a systemic chronic inflammatory

disorder characterized by recurrent oral aphtha, genital ulcers, uveitis, and skin lesions

(Chajek and Fainaru, 1975; Sakane et al., 1999; Al-Mutawa and Hegab, 2004). The disease

was later recognized to affect a number of systems and organs and was associated with

arthritis (Mason and Barnes, 1969), thrombophlebitis (Cucuob et al., 2000), mucocutaneous

(Alpsoy, 2007), muscle (Afifi et al., 1980) and neurological (Farah et al., 1998) problems,

gastrointestinal (Ebert, 2008), renal (Duarte et al., 1998), cardiovascular (Atzeni et al., 2005),

and pulmonary (Erkan et al., 2001) symptoms. BD is particularly common in the Far East

and the Mediterranean basin, and is frequently noted between the 30th and 45th degree

latitudes in Asian and European populations, corresponding to the Old Silk Road, an ancient

trading route stretching between the Mediterranean, the Middle East and the Far East. In

contrast, this disorder is uncommon in the American continents, Oceania and sub-Saharan

Africa (Ohno et al, 1982; Keino and Okada, 2007). Therefore, countries that are most

affected by BD today include Turkey, Iraq, Saudi Arabia, Iran, Afghanistan, Pakistan,

northern China, Mongolia, the Koreas and Japan (Okada, 2006). A recent Korean study (Lee

2

et al, 2006) indicated that the incidence of BD had increased in recent years, and the

prevalence of male patients had increased relatively since 2001. The etiology and

pathogenesis of BD have remained unclear, however it has been assumed that a genetic

factor, infectious agent, and immune mechanism are involved in the onset of this disease

(Mizuki et al., 2000; Al-Otaibi et al., 2005). Genetic analyses have shown an association

between the HLA-B51 phenotype and BD in high prevalence countries (Sakane et al., 1999).

Furthermore, of the 27 alleles for this HLA molecule, the B*5101 allele has been shown to

be most frequently expressed in BD patients over healthy individuals (Mizuki et al., 1997).

Other related genes have been studied and may also contribute to pathogenesis. Infectious

agents, such as herpes simplex virus (Lee et al., 1996) and Streptococcus sanguis (Kaneko et

al., 1997), could act as trigger factors operating through molecular mimicry (Sakane et al.,

1997) or some other mechanism, resulting in disease perpetuation by an abnormal immune

response to an autoantigen in the absence of ongoing infection (Benoist and Mathis, 2001).

However, none of these infectious agents has proved to cause BD. The major immunological

features of BD consist of increased T- and B-cell responses to HSP, increased neutrophil

activity, and alterations in cytokine levels. Significant sequence homology exists between

mammalian and microbial HSPs. For example, they may share antigenic epitopes with

herpes simplex viruses and streptococci—micro-organisms that have been implicated in the

pathogenesis of BD (Lehner, 1997). Neutrophils from patients with BD have increased

superoxide production, enhanced chemotaxis, and excessive production of lysosomal

enzymes, indicating that the neutrophils are overactive, which leads to tissue injuries

(Ehrlich, 1997).

3

T HELPER 1 TYPE IMMUNE RESPONSE IN BEHÇET’S DISEASE

T cell deviation affects both CD4+ and CD8+ T cell clones and can be divided into two

subtypes according to the cytokine secretion profile. Type I is responsible for the production

of interleukin (IL)-2 and interferon (IFN)-γ and type II for IL-4, IL-5, and IL-10. Several

reports have documented that peripheral blood mononuclear cells (PBMCs) from BD

patients predominantly produce T helper (TH)1 cytokines, mainly IFN-γ and tumor necrosis

factor (TNF)-α, and this response is more significant in the active clinical stage, suggesting

that BD can be categorized as a TH1-mediated disorder (Raziuddin et al., 1998). Especially

the elevation of plasma IL-10 in the majority of patients and the correlation of IL-12 plasma

levels with disease activity suggest a pathogenic role of a TH1-type immune response in

active disease (Turan et al., 1997). There are many studies that present TH1 predominant

immune response in BD (Frassanito et al., 1999; Imamura et al., 2005; Nagafuchi et al.,

2005). The pathological consequences of an inappropriate TH1 response include delayed-type

hypersensitivity reactions and induction of organ-specific autoimmune diseases (Sabota et al.,

2003; Bettelli et al., 2007). Predominant induction of TH1 cells can regulate asthma, atopy

and allergies. However, if polarized TH1/ TH2 lymphocytes might play a role in the induction

and regulation of immune responses in BD is still studied. And, although TH1 and TH2 cells

can be characterized by their cytokine profiles, distinguishing these T cell subsets based on

cell surface phenotype has proven difficult.

4

T CELL IMMUNOGLOBULIN AND MUCIN DOMAIN (TIM) FAMILY

Recently, the new cell surface protein T cell immunoglobulin and mucin domain (TIM)

have been found. TIM proteins are an evolutionarily conserved family and have been

described in rodents, monkeys, and humans. Eight Tim genes (Tim-1 to Tim-8) have been

identified in the mouse genome, but only four proteins (Tim-1 to Tim-4). In humans, three

TIM genes (TIM-1, TIM-3 and TIM-4) have been identified (Kuchroo et al., 2003; Sabota et

al., 2003; Mariat et al., 2005; Meyers et al., 2005). Each of these genes is predicted to

encode a type I membrane protein with a similar structure, consisting of a signal sequence

followed by an immunoglobulin variable region (IgV)-like domain, a mucin-like domain, a

transmembrane region and an intracellular tail. All of the members characterized thus far

appear to have a role in the regulation of TH1 and TH2 effector T-cell responses.

Fig. 1. Schematic representation of human TIM protein structures. (from Nat Rev

Immunol 3: 454-462, 2003)

5

Specifically, Tim-3 acts as a regulator of TH1 cells, and Tim-2 and Tim-1 appear to regulate

TH1 cells. Tim-4, which is expressed in antigen-presenting cells (APCs), is the ligand for

Tim-1 and is thus also involved in the TH1– TH2 balance (Meyers et al., 2005; de Souza and

Kane, 2006; Kuchroo et al., 2006; Degaugue et al., 2007).

TIM-3 PROTEIN

TIM-3 is the first molecule identified to be specifically expressed on the differentiated

CD4+ TH1 and not on TH2 cells (Monney et al., 2002; Anderson and Anderson, 2006) and

found on mast cell and melanoma cells (Wiener et al., 2007). This discovery has catalyzed

investigation into how TIM-3 might regulate TH1 cells and consequently influenced the

development of organ-specific autoimmunity. Using TIM-3-Ig and an expression cloning

strategy, galectin-9 was identified as a ligand of Tim-3 (Zhu et al., 2005; van de Weyer et al.,

2006). The Tim-3-galectin-9 pathway may have evolved to ensure effective termination of

effector TH1 cells (Zhu et al., 2005; Wang et al., 2008). Numerous studies have

demonstrated that Tim-3 influences autoimmune diseases, including diabetes and multiple

sclerosis, and its role in other inflammatory diseases including allergies and cancer is

beginning to become clear. In vivo administration of Tim-3-Ig fusion protein, presumed to

block Tim-3 signaling, led to hyperproliferative responses in CD4+ T cells and increased

IFN-γ-secretion (Sabatos et al., 2003). In vivo blockade of Tim-3 using either blocking

mAbs or fusion proteins could enhance the onset and frequency of autoimmune diabetes

induced in an adoptive transfer model of disease (Sánchez-Fueyo et al., 2003). Reducing

6

Tim-3 signaling during the innate immune response to viral infection in BALB/c mice

reduces CD80 costimulatory molecule expression on mast cells and macrophages and

reduces innate CTLA-4 levels in CD4+ T cells, resulting in decreased T regulatory cell

populations and increased inflammatory heart disease (Frisancho-Kiss et al., 2006).

Galectin-9 ameliorates a mouse collagen-induced arthritis (CIA) by suppressing the

generation of TH17, promoting the induction of regulatory T cells (Seki et al., 2008). Murine

models of a wide array of human inflammatory diseases have provided compelling evidence

for a role of Tim-3 in these diseases. However, due to its recent discovery and the lack of

reagents (e.g., mAbs), there is comparatively little data on the role of TIM-3 in regulation of

human T cell biology and inflammatory disease (Anderson, 2007). In a report that first

described preferential TIM-3 expression on human TH1 cells (Khademi et al., 2004),

although levels of IFN- γ transcript among PBMCs and cerebral spinal fluid (CSF)

mononuclear cells were elevated in multiple sclerosis (MS) versus control subjects, there

was no significant increase in TIM-3 levels among PBMCs or CSF mononuclear cells among

MS patients. The first functional data demonstrating that TIM-3 negatively regulates IFN- γ

secretion from human T cells presents that TIM-3 expression is dysregulated in T cells from

MS patients (Koguchi et al., 2006). Blocking TIM-3 during T cell stimulation significantly

enhanced IFN-γ secretion in control subjects but had no effect in untreated patients with

multiple sclerosis, demonstrating a defect in TIM-3 immunoregulation (Yang et al., 2008). A

report show how a single molecule, Tim-3, by virtue of differential expression on cells of the

innate and adaptive immune systems, can both promote inflammation and terminate TH1

immunity (Anderson et al., 2007). An understanding of the pathways and molecules

7

responsible for regulation of TIM-3 expression and function will greatly facilitate the ability

to ultimately modulate the TIM-3 pathway for therapeutic benefit.

Fig. 2. Mechanisms by which Tim-3 might modulate immune function. Membrane-

bound Tim-3 on the surface of Th1 cells interacts with its ligand on CD4 T cells and

transduces an inhibitory signal into the Th1 cell, terminating the Th1 response. (from Trends

Mol Med 11:362-369, 2005)

BD is a very complex disease in clinical manifestation, etiopathogenesis, and diagnosis

as well as treatment. As mentioned above, T cell immunity, especially TH1 immune response,

plays an important role in the pathogenesis of BD. Although TIM molecules are expressed

on murine TH1 and TH2 cells, whether TIM molecules are differentially expressed on human

T cell has been reported little. Whether the expression of TIM molecules correlates with

disease activity in vivo has also little been studied. Up to now, there are still no data

concerning the expression of TIM-3 in patients with BD. Based on above reports, Tim-3 may

represent a valid therapeutic target in a wide range of peripheral and organ-specific human

inflammatory diseases. So it is reasonable that TIM-3 could have a role in the pathogenesis

8

of BD. In the present study of the postulation that alterations in the TIM-3 pathway may

underlie the TH1 shift immune response in BD, I examined the TIM-3 expression in PBMCs

of BD patients and compared it to normal control at both mRNA and protein level. This

study may provide a new insight to reveal the relationship between TIM-3 and BD and show

the pathogenesis of BD.

9

II. MATERIALS AND METHODS

A. Patients and samples

The patient population consisted of 67 patients with BD, who presented themselves for

the first time or were monitored at the Department of Dermatology, Ajou University

Hospital. BD patients met the Diagnostic criteria of the BD Research Committee of Japan.

The active group patients had at least one of the BD symptoms despite the treatment and

inactive group patients were in well-controlled states by taking anti-inflammatory

medication. The control groups consisted of 14-newly diagnosed psoriasis patients without

any other evident disease and 13 healthy volunteers as disease and healthy control groups,

respectively. The informed consent was obtained from patients prior to enrolling them into

the study. This study was approved by the Institutional Review Board (IRB no.: AJIRB-

GN3-07-098).

B. Methods

1. Cell preparation

Venous blood was sampled in sodium citrate-containing cell preparation tubes

(Vacutainer CPT; BD Biosciences, San Jose, CA. PBMC were separated by Ficoll Hypaque

density gradients (Ficoll paqueTIM plus, StemCell Technologies, Vancouver, BC, Canada).

10

Cells from the interphase were collected and washed twice with phosphate buffered saline

(PBS; Sigma, ST. Louis, MO) with 2% heat-inactivated fetal bovine serum (FBS; Gibco-

BRL, Grand Island, NY).

2. Cell culture and stimulation of PBMCs

PBMCs(1x106 cell/ml) were resuspended in culture medium (RPMI 1640 medium

supplemented with 2mM L-glutamine, 100 U/㎖ penicillin and 100 ㎍/ml streptomycin)

with 10% FBS and activated with anti-CD3 (100 ng/㎖, clone HIT3a), anti-CD28 (200 ng/㎖,

clone CD28.2) Ab and IL-2 (10 unit/㎖, eBioscience, San Diego, CA, USA). Cells were

harvested at various times, 24, 48 and 72 hr, after the incubation at 37℃ and 5% CO2 and the

TIM-3 expression on the stimulated cells were analyzed by flow cytometry. For intracellular

cytokine flow cytometric analysis, 10 ㎍/㎖ of brefeldin A (eBioscience, San Diego, CA,

USA) was added to the culture medium.

3. Flow cytometric analysis

Fluorescein isothiacyanate (FITC)-conjugated anti-CD4 (RPA-T4) anti-CD8 (HIT8a),

anti-CD11b (CBRM1/5), anti-CD56 (MEM188) antibody (eBioscience, San Diego, CA,

USA) or phycoerythrin (PE)-conjugated anti-human Tim-3 antibody (rat IgG2a, R&D

system, USA) were used for cell phenotype analysis. All mAbs were titrated to optimal

11

concentrations according to the manufacturer’s protocol. PBMCs were incubated with the

fluorescent-labeled mAbs in dark at 4℃ for 30 min. After washing with PBS, the labeled

cells were detected with Vantage flow cytometer (FACS, Becton Dickinson

Immunocytometry Systems, Mountain View, CA, USA).

4. Reverse transcription polymerase chain reaction (RT-PCR)

Total cellular RNA was extracted with the RNeasy Mini kit (Qiagen, Valencia, CA,

USA) according to the protocol provided by the manufacturer. The quantification of RNA

was determined by measuring the absorbance at 260㎚ in a spectrophotometer. First-strand

cDNA was synthesized from 2 ㎍ of total RNA. Total RNA in 13 ㎕ volume of distilled

water was mixed with oligo(dT) primer12-18, 10mM dNTP Mix (Invitrogen, Carlsbad, CA,

USA). The mixture was heated to 65℃ for 5 minutes. A final 20 ㎕ reaction mixture

consisted of 5X First-strand buffer, 0.1 M DTT, and RNaseOUTTM Recombinant RNase

Inhibitor, and SuperScriptTMIII RT (200units/㎕) (Invitrogen, Carlsbad, CA, USA) was

incubated at 50℃ for 60 min, 70℃ for 15 min and stored at -20℃. PCR reactions of 20 ㎕

consisted of 0.5 mM each primer for Tim-3, 1 ㎕ of template (i.e., equivalent of 0.1 ㎍

total RNA) and PCR SuperMix (Invitrogen, Carlsbad, CA, USA). For the co-amplification

primer, β-actin was used. RT-PCR was carried out in a MyCyclerTM Thermal Cycler System

(BIO-RAD, CA, USA). The oligonucleotide primers used were as follows: for TIM-3, sense,

12

5'-ACAGAGCGGAGG TCGGTCAGAATG-3' and antisense, 5'-

AGCCAGAGCCAGCCCAGCACAGAT-3'; and for ; β-actin, sense, 5'-

GTGGGGCGCCCCAGGCACCA-3' and antisense, 5'-CTCCTTAAT

GTCACGCACGATTTC-3'. Product sizes were 572 bp for Tim-3 and 548 bp for β-actin.

Reaction mixtures were amplified for 35 cycles for Tim-3 and 28 cycles for β-actin. Cycling

conditions were as follows: denaturing at 94℃ for 30 s, annealing at 62℃ (for Tim-3) or 60℃

(for β-actin)for 30 s and extension at 72℃ for 45 s (for Tim-3) or for 90 s (for β-actin). The

PCR products were fractionated on 2% agarose gels and visualized by ethidium bromide

staining. To quantify the transcripts, the intensities of the PCR bands were analyzed by

GelDocTM XR (BIO-RAD, CA, USA).

5. Immunohistochemistry

Formalin-fixed and paraffin-embedded tissues of erythema nodosum-like lesions in

Behcet’s disease were cut and mounted onto slides. Specimens were deparaffinated and

endogenous peroxidase activity was blocked by 3% H2O2 in methanol for 30 minutes at the

room temperature. After rinsing in phosphate-buffered saline (PBS), the specific binding

sites were blocked by blocking solution for 60 minutes at room temperature and all

specimens were incubated with antihuman TIM-3 antibody (4 mg/ml, R&D Systems,

Minneapolis, MN) and anti-CD4 antibody (1:20 dilution, Neomarker, USA) diluted in PBS

with 5% BSA, for 60 minutes at the room temperature in a humid chamber. As negative

control, a specific goat serum was used (Sigma). After washed in PBS, specimens were

13

incubated with biotinylated anti-goat antibody (Sigma) for 30 minutes at room temperature

and then washed and incubated with streptavidin/peroxidase complex reagent (Novostain

Universal Detection Kit, Novocastra Laboratories Ltd, UK) for 20 minutes. The color was

developed by the Vector NovaRED Substrate Kit (Vector Laboratories Inc., Burlingame, CA)

and the slides were counterstained with methyl-green (Dako, Denmark). All negative

controls demonstrated negligible background fluorescence.

6. Western blot

Cells were lysed in RIPA buffer (1% sodium deoxychloate, 1% Triton X-100, 50 mM

NaCl, 1% Tris–HCl (pH 7.5), and 0.1% SDS) with 10 µg/ml aprotinin, 10 µg/ml leupeptin,

and PMSF. Fifty micrograms of protein per lane were resolved by sodium dodecyl sulphate–

polyacrylamide gel electrophoresis and blotted onto nitrocellulose paper. The membranes

were blocked overnight at the room temperature with 5% non-fat dry milk in a TNET buffer

containing Tris base, EDTA, NaCl and 0.1% Tween-20. Blots were incubated with primary

antibodies followed by horseradish peroxidase-conjugated secondary antibodies. Anti-TIM-3

(R&D Systems) antibody was used. Bands were visualized with enhanced

chemiluminescence (Millipore, MA, USA). To quantify the expression, the intensities of the

bands were measured using Image-Pro Plus version 4.5 and are expressed as intensities

relative to actin.

14

7. Statistical analysis

All data were analyzed with SPSS 12.0 statistical software (SPSS Inc., Chicago, USA).

The difference between the two groups was evaluated by the Mann-Whitney U test. The

comparisons of continuous variables were performed by means of Student's t-test for

independent-samples. To investigate the relations among the variables, Pearson correlation

test was used. The results were expressed as mean ± standard deviation (SD) or standard

error mean (SEM). All p values were two tailed and p value less than 0.05 was considered to

be of statistical significance.

15

III. RESULTS

A. Subject characteristics

A total of 94 subjects entered the study (67 of BD patients, 14 of psoriasis, and 13 of healthy

controls). Baseline clinical findings of BD patients are summarized in Table 1. There were

35 men and 32 women with mean age of 37.0 yr old (range, 25 - 64). Active BD patient was

defined as those who had more than one active clinical symptom such as oral ulcer, genital

ulcer, skin lesion, ocular inflammation, GI, CNS or joint involvement at that time of

examination. Of 67 BD patients, 40 (21 males, 19 females) were in the clinically active state

with mean age of 36.8 yr (range, 25-47). The mean disease duration was 11.0 ± 7.5 years,

and 19% of patients had been taking systemic immunosuppressive agents, including

corticosteroid, during the last 6 months. In the active BD patients, 26 patients had

mucocutaneous symptoms, including oral ulcer, genital ulcer, or skin manifestation, and 14

patients other symptoms, including uveitis, gastrointestinal, central nervous system or joint

symptom. Disease activity markers, erythrocyte sedimentary ratio (ESR) and C-reactive

protein (CRP) levels, were measured in blood samples of BD patients (Table 2). In active

BD patients, both markers were elevated compared with stable BD patients (Fig.3). There

were 13 healthy controls (6 males, 7 females) with mean age of 35.6 (range, 26-48) and 14

psoriasis patients with mean age of 36.1 (range, 8-63). To evaluate cytokine profile in BD,

PBMCs were incubated for 48 hours in the presence of anti-CD3 and anti-CD28 antibody.

Brefeldin-A was added at 6 hr before intracellular cytokine analysis by flow cytometry, and

then additional 72 hr incubation with anti-CD3 antibody was done. Intracellular IFN-γ

16

frequency was higher (22.6% and 13.1% at 48 and 120 hr) than others, IL-4 (10.6% and

9.3%) and IL-17 (5.7% and 6.0%) (Fig. 4).

Table 1. Clinical characteristics of Behçet’s disease patients

Demographics of BD patients Values (mean±SD)

Age

Sex (male:female, n)

Disease duration

37.0 ± 7.7

35:32

11.0 ± 7.5

Clinical Characteristics of BD patients Case number (%)

Major symptoms

Oral ulcer

Genital ulcer

Skin lesion

Ocular lesion

Minor symptoms

Arthritis

GI involvement

Epididymitis

Cardiovascular

CNS involvement

62 (92.5)

44 (65.7)

53 (79.1)

24 (35.8)

16 (23.9)

2 (3.0)

2 (3.0)

5 (7.5)

5 (7.5)

Medication Case number (%)

Immunosuppressive agent*

Anti-inflammatory drugs**

19 (28.4)

50 (74.6)

* methylprednisolone 8-24 ㎎/d or triamcinolone 4-8 ㎎/d or cyclosporine 100-200 ㎎/d or

azathioprine 1000 ㎎/d ** colchicine 1.2 ㎎/d ± minocycline 100 ㎎/d ± pentoxiphylline 800 ㎎/d ± sulfasalazine

1000 ㎎/d

17

Table 2. Clinical characteristics and laboratory results of Behçet’s disease patients

according to disease activity.

Number M : F Age

(mean ± SD)

ESR

(mean ± SD)

CRP

(mean ± SD)

BD 67 35 : 32 37.0 ± 7.7 20.9 ± 17.7 0.90 ± 1.66

Stable 27 14 : 13 37.3 ± 6.1 12.3 ± 7.6 0.35 ± 0.69

Active1 40 21 : 19 36.8 ± 8.7 26.6 ± 20.2 1.24 ± 1.97

Mucocutaneous2 (1) 14 7 : 7 39.6 ± 9.0 30.4 ± 16.2 1.57 ± 2.04

Mucocutaneous (> 2) 12 5 : 7 34.6 ± 9.7 29.3 ± 29.2 0.99 ± 2.17

Others (1) 3 6 5 : 1 36.8 ± 9.0 18.0 ± 12.1 0.27 ± 0.40

Others (> 2) 4 8 4 : 4 35.1 ± 6.3 22.2 ± 14.6 0.90 ± 1.66

1: active BD patients as those who had more than one active clinical symptoms such as oral

ulcer, genital ulcer, skin lesion, ocular inflammation, GI, CNS or joint involvement at

that time of examination

2: mucocutaneous symptoms included oral ulcer, genital ulcer, or skin manifestations

(erythema nodosum, papulopustular eruption, Sweet’s-like)

3: Uveitis (4), GI (1), CNS (1)

4: Uveitis (4), GI (1), CNS (1), Joint (3)

CRP, C-reactive protein (normal range: 0-0.8 mg/L); ESR, erythrocyte sedimentary ratio

(normal range: female 0-20, male 0-25 mm/h)

18

Fig. 3. The level of disease activity makers of Behçet’s disease. The level of ESR and CRP

was measured in blood sample from Behçet’s disease patients. CRP, C-reactive protein

(normal range: 0-0.8 mg/L); ESR, erythrocyte sedimentary ratio (normal range: female 0-20,

male 0-25 mm/h) (**p < 0.01, * p < 0.05)

19

Fig. 4. Cytokine profile in PBMCs from Behçet’s disease patients ex vivo. Intracellular

cytokines, IFN-γ, IL-4, and IL-17, were analyzed by flow cytometry after 48 hr stimulation

of PBMCs with anti-CD3 and anti-CD28 antibody. Brefeldin-A was added at 6 hr before

flow cytometry and additional 72 hr incubation with anti-CD3 antibody was done. (thick

black bar; mean value of each subjects)

0

10

20

30

40

50

60

48h 120h 48h 120h 48h 120h

IFN-r IL-4 IL-17

%#1

#2

#3

#4

ave

20

B. TIM-3 expression in PBMCs and the effect of medication

I investigated the TIM-3 expression on PBMCs of BD patients using flow cytometry.

The percentage of TIM-3+ cells was based from its proportion to the total cells. An increased

frequency of TIM-3 expressing cells in PBMCs from BD patients compared with controls

was observed (17.90±8.09 vs. 14.70±3.88, p<0.05, Fig. 5). Then the effect of patients’

medication on the expression of TIM-3 was revealed in the BD patients. However,

medication had no effects on the TIM-3 expression both in active and total BD (Fig. 6)

Fig. 5. Expression of TIM-3 in PBMCs. Peripheral blood mononuclear cells from all

subjects were stained with PE-conjugated anti-TIM-3 antibody. Dots were the frequency of

TIM-3+ cells in PBMCs. Bars mean 95% confidence interval for mean of TIM-3. (* p < 0.05)

21

Fig. 6. The effect of medications on the TIM-3 expression. To evaluate the effects of

medications, immunosuppressive agent or anti-inflammatory drugs, on TIM-3 expression,

mean TIM-3 value was compared according to the medication state in both active disease

and total Behçet’s disease patients.

22

C. Western blot analysis of TIM-3 protein

A representative result of Western blotting is shown in Fig. 7A. The relative amount of

target protein was quantified as the density of the target protein versus GAPDH. After the

normalization of the data according to the expression of GAPDH protein, the relative density

of TIM-3 was presented percent of controls (Fig.7B). The mean value of normalized TIM-3

in BD was higher than in control. The expression of TIM-3 molecule by flow cytometry and

the amount of TIM-3 protein by Western blot were summarized in Fig.7C.

A.

B.

C.

50

60

70

80

90

100

110

120

control BD

TIM

-3 (%

of c

ontr

ol)

Western blot

TIM-3 cont1 cont2 cont3 BD1 BD2 BD3 BD4 BD5

Western blot (intensity) 4048.9 2349.1 2056.6 4336.5 3972.6 1545.3 2821.7 2982.5

Flow cytometry (%) N/A 15.53 10.72 12.64 N/A N/A 20.16 17.65

23

Fig. 7. The representative result of the expression of TIM-3 protein in PBMCs from BD

patients by Western blotting. A. Human TIM-3 protein expression was confirmed by

Western blot analysis with anti-Tim-3 polyclonal antibody. B. The mean value of TIM-3

protein in BD patients was expressed by % of controls. C. Expression of TIM-3 molecule by

flow cytometry and relative amount of TIM-3 protein by Western blot was summarized.

(N/A, not available)

D. Immunohistochemical analysis of TIM-3 in erythema nodosum-like skin

lesions of Behçet’s disease

Erythema nodosum (EN)-like lesion is one of cutaneous manifestations of BD. It is

characterized by tender, erythematous subcutaneous nodules commonly affecting the lower

extremities. Histopathologic features are the septal pannicular inflammation associated with

some peripheral lobular inflammation and a superficial and deep dermal lymphocytic

inflammatory infiltrate. As lymphocytes and macrophages are often accumulated in or

around subcutis or dermis, the TIM-3 expression was studied in tissue sections from BD

patients and classic EN patients (Fig. 8). Immunohistochemistry revealed that CD4+ cells

(Fig.9) and macrophages showed a clear positivity for TIM-3. CD4+ cells were identified in

serial sections with anti-human CD4 antibody staining. There was little difference in the

histopathologic features in both EN-liken lesion of BD and classic EN lesion.

24

A.

B.

Fig. 8. TIM-3 expression in cutaneous lesions. Immunohistochemistry with anti-Tim-3

antibody in erythema nodosum-like lesion of BD (A) and classic erythma multiforme (B).

Two representatives out of ten different patients. The background staining of the control

slides was negligible (x 400).

25

A.

B.

Fig. 9. TIM-3 expression on CD4+ cells in EN-like lesion of Behçet’s disease.

Immunohistochemistry with anti-TIM-3 antibody in erythema nodosum-like lesion (x200)

(A). Inflammatory cells are stained with anti-CD4 antibody in serial sections (x200) (B).

Outside box is magnification from an inside box (x400) )

26

E. Correlation of TIM-3 expression with Behcet’s disease activity

To know whether TIM-3 expression increases with the improvement of clinical

manifestations in a BD patients, the TIM-3 expression in five paired blood samples from BD

patients with shift of their clinical status was evaluated. Among the active BD patients at the

first study examination, five patients (patient’s number 2, 15, 24, 26 and 29) have turned out

to be in the clinically stable state. The mean duration to become in the stable state was 9.2

months and their rechecked laboratory tests, ESR and CRP, were also within normal range.

All five patients showed the significantly elevated TIM-3 expression (7.51 ± 1.96, p<0.05)

compared to before their active disease state (19.03 ± 9.80) (Fig. 10).

Fig. 10. The comparison of frequency of TIM-3 expression between active disease and

stable state in the same patient. When the Behçet’s patients who have had active disease

became in the stable state, flow cytometric anyalsis was done again. (* P < .05)

0

5

10

15

20

25

30

35

40

#2 #15 #24 #26 #29 average

TIM

-3 (

%)

active

stable

*

27

F. Analysis of surface markers on PBMCs and TIM-3 expression on leukocyte

subpopulations

In the previous studies, the distribution of Tim-3 is not restrited to the T cell

comparetment, and Tim-3 expression has been reported on the mouse dendritiec cells, the

mouse monocytes and macrophages, as well as the human natural killer and NK-T cells. So

population of PBMCs from BD patients was divided to CD4+, CD8+, CD11b+, CD25+,

CD56+, CTLA-4+, and foxp3+ cells and TIM-3 expression on each subpopulation was

anlayzed. Table 3 showed that there was no correlation between the expression level of TIM-

3 and TIM-3 expressing CD25+, CTLA-4+, and foxp3+ cells, and then CD4, CD8, CD11b,

and CD56 cells were objects of further analysis (Pearson correlation coefficient; 0.739,

0.567, 0.532, and 0.543, p < 0.01).

Table 3. Pearson correlations between the expression level of TIM-3 and TIM-3

frequency in PBMCs subpopulation.

TIM3+ TIM3+ CD4+

TIM3+ CD8+

TIM3+ CD11b+

TIM3+ CD25+

TIM3+ CD56+

TIM3+ CTLA4+

TIM3+ foxp3+

TIM3+ Pearson correlation 1.000 .739** .567** .532** -.062 .543** .242 -.036

Sig. (2-tailed) . .000 .001 .002 .745 .002 .255 .858

**Correlation is significatn at the 0.01 level.

28

The expression of various cell surface markers, CD4, CD8, CD11b and CD56 was

determined by flow cytometry immediately after blood sampling. As shown in figure 11A,

CD8 expression was significantly decreased in BD group (mean ± SEM, 20.80 ± 1.27) and

psoriasis (23.54 ± 2.53) compared to control group (30.36 ± 1.59) (p<0.05). To examine the

expression of TIM-3 in leukocyte subpopulations, PBMCs were double-stained with PE-

conjugated anti-TIM-3 antibody versus FITC-conjugated anti-CD4, CD8, CD11b, or CD56

antibody. Mean value of TIM-3 expression in CD8+ and CD56+ cells were significantly

increased in BD (mean ± SEM, 29.69 ± 1.8 and 43.31 ± 2.5) compared to control (17.27 ±

4.4 and 23.84 ± 2.5), not in CD4+ and CD11b+ cells (Fig. 11B). However, a significant

correlation exists between TIM-3 frequency and TIM-3 expression in all subpopulations

(TIM-3 to TIM-3 in CD4 cells, R=0.715; in CD8 cells, R=0.466; in CD11b cells, R=0.428;

in CD56 cells, R=0.543, p < 0.01) (Fig. 12).

29

A.

B.

Fig. 11. Analysis of surface markers on PBMCs and TIM-3 expression on leukocyte

subpopulations. A. The frequency of CD4+, CD8+, CD11b+ and CD56+ cells in PBMC was

expressed percent of total PBMCs. B. The percentage of TIM-3 expression in the CD4+,

CD8+, CD11b+ and CD56+ cells. Data show the mean ± SEM. (* p < 0.05)

0

5

10

15

20

25

30

35

40

45

CD4 CD8 CD11b CD56

freq

uen

cy in P

BM

Cs(

%)

**

0

10

20

30

40

50

60

70

80

CD4 CD8 CD11b CD56

TIM

-3+

freq

uen

cy (%

)

control

psoriasis

BD

*

**

30

A. B.

C. D.

Fig. 12. Correlation between TIM-3 frequency and TIM-3 expression in leukocyte

subpopulations. Linear regresssion analysis showed the correlation between TIM-3

frequency and TIM-3 expression in each subpopulations. Independent variables were

expression of TIM-3 in CD4+ cells (A), in CD8+ cells (B), CD11b+ cells (C), and CD56+ cells

(D). R; Pearson correlation coefficient

R2 Linear = R2 Linear =

R2 Linear = R2 Linear =

31

G. Correlation of TIM-3+ frequency in leukocyte subpopulation with Behcet’s

disease activity

In the analysis of cellular population, there were no changes in CD4, CD8, CD11b and

CD56+ cells after disease stabilization, however the percentage of TIM-3 expression on

CD4+ cells (22.70 ± 9.77) and CD56+ cells (57.07 ± 19.25) was significantly increased after

disease stabilization compared to active state (7.23 ± 4.46, 20.17 ± 9.47) (Fig. 13).

A.

B.

0

10

20

30

40

50

#2

#15

#24

#26

#29

aver

age

#2

#15

#24

#26

#29

aver

age

#2

#15

#24

#26

#29 #2

#15

#24

#26

#29

aver

age

CD4 CD8 CD11B CD56

freq

uen

cy

in P

BM

C (

%)

active

stable

0

10

20

30

40

50

60

70

80

90

#2

#15

#24

#26

#29

aver

age

#2

#15

#24

#26

#29

aver

age

#2

#15

#24

#26

#29

aver

age

#15

#24

#26

#29

aver

age

CD4+ CD8+ CD11b+ CD56+

TIM

-3+

fre

quen

cy (

%)

active

stable

*

*

32

Fig. 13. The comparison of TIM-3 expression on each cellular population of PBMCs

between active and stable disease state in the same patient. A. the frequency of CD4+ cell

in PBMCs, the frequency of coexpressing Tim-3+ and CD4+ cells, and the expression level of

TIM-3+ cells on CD4+ cells, B. the ame as A about CD8+ cells, C. the same as A about

CD11b+ cells, D. the same as A about CD56+ cells (* p <0.05)

H. Expression of TIM-3 in stimulated PBMCs from Behçet’s patients and

healthy controls

It is known that TIM-3 expression is predominantly detected in TH1-differentiated

CD4+ cells (Sabota et al., 2003), so after stimulation of PBMCs with anti-CD3 and –CD28

antibody in presence with IL-2, TIM-3 expression was analyzed by flow cytometry at each

time points (at 24, 48 and 72 hours after stimulation). There was differential expression of

TIM-3 between BD and control. TIM-3 expression was up-regulated in control as

stimulation time, but not in BD group (Fig. 14A). The frequency of CD4 and CD8 cells was

increased in control as stimulation time, but not in BD group (Fig. 14B). However, the

frequency of TIM-3 expression in CD4+ and CD8+ cells showed similar pattern between tow

groups (Fig. 14C).

33

A.

B. C.

Fig. 14. TIM-3 expression on stimulated PBMCs. Flow cytometric analysis was done in

healthy control group and Behçet’s disease. At each time points, the mean value was

compared between two groups. A. expression of TIM-3 on stimulated PBMCs, In figure B,

TIM-3 expression level on each cellular population (left; analysis of CD4+ cells, right;

analysis of CD8+ cells) (* p <0.05)

05

101520253035

0h 24h 48h 72h

TIM

-3 (%

)

control

BD

0

5

10

15

20

25

30

35

0h 24h 48h 72h 0h 24h 48h 72h

CD4 CD8

% in

PB

MC

0

10

20

30

40

50

60

0h 24h 48h 72h 0h 24h 48h 72h

in CD4 cells in CD8 cells

TIM

-3 fr

eque

ncy

(%)

34

I. Expression of TIM-3 mRNA was analyzed by RT-PCR between Behçet’s

disease group and healthy controls

Next, to examine TIM-3 expression on mRNA level, quantitative RT-PCR was used in

healthy controls, active BD patients and non-active BD patients (Fig.15A). The relative

expression of TIM-3 mRNAwas significantly increased in active BD patients compared with

those of controls (Fig. 15B).

A.

B.

0

50

100

150

200

250

control Total Active Stable

normal Behcet's disease

Rel

atev

e m

RN

A e

xpre

ssio

n

(% o

f co

ntr

ol)

*

0 1 2 3 4 Control

0 1 2 3 4 0 1 2 3 4 Active Stable

Behçet’s disease

35

Fig. 15. Expression of TIM-3 in PBMCs assessed by RT-PCR. RT-PCR for Tim-3 was

conducted and the volume of bands were expressed as percent of control measured by

intensity*band area (㎜) relative to β–actin expression measured by GelDocTM XR program.

A. left; PBMC from normal healthy controls, center; PBMCs from active Behçet’s disease

patients, right; PBMCs from non-active Behçet’s disease patients (lane 1; 100bp DNA

ladder, lane 2-9; non-active BD), B. relative comparison of mean PCR bands between active

Behçet’s disease and control. (* p <0.05)

36

J. Expression of TIM-3 mRNA was analyzed by RT-PCR after stabilization of

clinical symptoms of Behçet’s disease

Among patients showing stabilization of their disease, the PBMCs from five patients

were estimated using RT-PCR to evaluate the TIM-3 expression between two clinical states.

The signal of TIM-3 bands after disease stabilization was weaker than in active state (Fig.

16A) and relative expression level analysis showed also significantly decreased (p<0.01) in

stable state of BD patients (Fig. 16B).

A.

B.

Fig. 16. TIM-3 mRNA was analyzed according to the disease state. RT-PCR for Tim-3

was conducted and the volume of bands were expressed as intensity*band area (㎜) relative

to β–actin expression. In the same patient, PBMCs were taken at the active disease state and

0

50

100

150

200

active stable

Rel

ativ

e m

RN

A e

xpre

ssio

n *

0 #1 #2 #3 #4 0 #1 #2 #3 #4 Active state Stable state

37

the stable state. A. left; TIM-3 mRNA expression on the active state of Behçet’s disease,

right; after stable state of Behçet’s disease patient in prior the active state, B. the relative

comparison of the mean PCR bands value between two states. (* p <0.01)

38

IV. DISCUSSION

Behçet’s disease (BD) is a systemic inflammatory disorder characterized basically by

recurrent oral ulcers, genital ulcers, and uveitis. At times, BD may evolve to a widespread

disorder involving the skin, joints, and the pulmonary, vascular, and central nervous systems.

Recent findings have supported the concept that immunologic abnormalities, which are

possibly induced in genetically susceptible individuals, are important in the pathogenesis of

BD (Lehner, 1999). Immunological investigations have demonstrated the presence of

immune dysregulation among the patients with BD. The disease is characterized by

infiltration of lymphocytes and neutrophils into the affected organs. Because cytokines are

involved in the regulation of functions of lymphocytes and phagocytes, they play an

important role in the pathogenesis of BD (Gul, 2001). As for most other autoimmune

disorders and vasculitis, the TH1 type cytokine profile is predominant in BD (Direskeneli,

2001). Increased expression of IL-8, monocyte chemoattractant protein 1, IFN-γ, and IL-12

mRNA in BD lesions compared with normal skin. Except for IL-10, TH2 cytokines (i.e., IL-4

and IL-13) were absent. These suggest a direct role of TH1 lymphocytes (Ben Ahmed et al.,

2004). In the recent report, TH1 lymphocyte dominance in peripheral circulating blood

measured by flow cytometry may play a role in the pathogenesis of BD uveitis (Ilhan et al.,

2008). In the immunoperoxidase staining of oral lesions, TH1 cytokines and the TH1-

associated chemokine receptors were increased in both patient groups as compared to normal

controls, indicating the involvement of a TH1 immune response in the immunopathology of

both BD and aphthous stomatitis (Dalghous et al., 2006).

39

TIM molecules constitute a recently described family of molecules expressed on T

cells. Tim-3 has been recently implicated in murine TH1 cell responses and in experimental

autoimmune encephalomyelitis (EAE), a TH1-related murine autoimmune disease of the

CNS (Money et al., 2002). Engagement of Tim-3 down-regulated TH1 responses (Zhu et al.,

2005), demonstrating a role in the inhibition of effector TH1 cells during normal immune

responses and in the induction of peripheral tolerance (Sabota et al., 2003; Sánchez-Fueyo et

al., 2003). From recent studies, TIM-3 molecule is regarded as one of surface markers of TH1

subset. There were a few human diseases studied about TIM-3 untill now (e.g., multiple

sclerosis, systemic lupus erythematosus, allergic disease, some cancers) (Graves et al., 2005;

Wiener et al., 2007; Wang et al., 2008; Yang et al., 2008). However, there was no report

that it is studied whether the TIM-3 is related in immune response in BD or not. For the first

time, this study shows the TIM-3 expression in PBMC of BD patient and evaluates its

expression according to the disease activity.

In the clinical characteristics, the active BD patients were defined as those who had

more than one active clinical symptom such as oral ulcer, genital ulcer, skin lesion, ocular

inflammation, GI, CNS or joint involvement at that time of examination and active BD

patient showed higher clinical disease activity marker (serum ESR and CRP level) than

stable BD patient (p < 0.01 and p < 0.05). Among active symptoms, mucocutaneous

symptoms had more increased ESR and CRP level (Table. 2). There were several studies

about cytokine production profile in BD, which result in different patterns. However, it is

generally said that BD has the TH1 cytokine dominant pattern. Recently it has been reported

that TIM-3 is expressed on TH17 cells (Nakae et al., 2007). Ex vivo flow cytometric

40

intracellular cytokine analysis showed higher expression of IFN-γ than IL-4 and IL-17 after

anti-CD3 and anti-CD28 antibody treatment (Fig. 4). These data may suggest study the the

characteristics of the study-subjects are consistent with general BD ones.

For the first time, the surface expression of TIM-3 in PBMC from BD patietns was

analyzed by flow cytometry. TIM-3 expression was significantly increased in Behçet’s

disease patients compared with controls (p<0.05) (Fig. 5). Behçet’s disease is known as TH1

response dominant disease through the study of cytokine production, and less study about

surface marker analysis. Ilhan et al. showed that TH1 lymphocyte dominance in peripheral

circulating blood may play a role in the pathogenesis of BD uveitis (Ilhan et al. 2008). TIM-

3 is mainly expressed on TH1 cells and this result may be consistent with the former concept.

Though there was no significant difference in TIM-3 expression between active and stable

BD, active BD had a tendency to be the lower expression level of TIM-3 than stable BD. To

rule out the possibility of medication effects in BD patients, BD group was divided,

medicated or not. Medications had no effects on TIM-3 expression in BD patient, active state

or not (Fig. 6). Total protein of TIM-3 analysis by Western blotting also revealed similar

pattern in flow cytometry, increased TIM-3 protein in BD compared with controls (Fig. 7).

TIM-3 was also expressed in cutaneous lesions by imunohistochemistry. Erythema nodosum

(EN)-like lesion is one of characteristic cutaneous manifestation of BD. Punch biopsies from

BD patients with EN-like lesion revealed TIM-3+ cells among the infiltrated cells around

subcutaneous tissue and around superficial perivascular lesions similar to classic EN lesion

(Fig. 8). In serial cut tissue, CD4+ cells were found to be co-localized by TIM-3+ cells in

EN-like lesion (Fig. 9). This might suggest that TIM-3 is involved in disease manifestation

41

not only in peripheral blood inflammatory cells but also in disease organ.

Then, I investigated the TIM-3 expression pattern according to the clinical disease

activity in PBMCs from the same patient. The mean duration turning active to inactive state

was 9.2 months. All five patients in the inactive state showed the increased TIM-3

expression (mean ± SD; 7.51 ± 1.96, p<0.05) than the ones in the active state (19.03 ± 9.80)

(Fig. 10). It was the same pattern in the comparison between the active BD group and the

stable BD group, but with statistical significance in individual change. I hypothesized that

the decreased expression of TIM-3 in the active state plays a role in the disease manifestation

of BD since TIM-3 is known as those down-regulate TH1 responses. And this is consistent

with the former reports that anti-Tim-3 treatment, blockade of Tim-3, during the

development of EAE (Monney et al., 2002) and diabetes in non-obese diabetic mice

(Sánchez-Fueyo et al., 2003) showed clinically exacerbated disease, which indicated that

Tim-3 negatively regulates the development of TH1-mediated disease. From this result, it

might be conceived that altered TIM-3 expression may influence the clinical activity of

Behçet’s disease.

In addition to its expression in T cells, TIM-3 mRNA is present in macrophage and

other non-T cells, although this functional relevance is not known (Anderson et al. 2007). To

determine the source of TIM-3 expressing cells, the surface markers of PBMCs were stained

with anti-CD4, CD8, CD11b, CD56, CTLA-4, CD25, and foxp3 antibody. TIM-3 expression

in CD25+, CTLA-4+, and foxp3+ cells were very low (data not shown) and there were no

correlation between TIM-3 frequency and TIM-3 expression level in CD25+, CTLA-4+, and

foxp3+ cells (Table 3). And then CD4, CD8, CD11b, and CD56 cells were included

42

following study. Among cell populations, only CD8+ cells were down-regulated in Behçet’s

disease (Fig. 11A). However, the frequency of TIM-3 expression in CD8 and CD56 cells

was increased in BD compared with in controls (Fig. 11B). CD11b+ cells showed the highest

expression of TIM-3, it may be explained in a context that TIM-3 is constitutively expressed

CD11b macrophage (Monney et al. 2002). The correlation analysis between TIM-3

expression and TIM-3 frequency in cell population reveled that there were significant

correlations in all subpopulations, especially in CD4+ cells (R=0.715, p<0.01) (Fig. 12).

These data might suggest that there are complex patterns of TIM-3 expression in

inflammatory cells; the immunologic characteristics in Behçet’s disease, the rate of TIM-3

expressing cells, and the correlation with total TIM-3 frequency. According to the clinical

activity in the same patient, the percentage of each subpopulation was not changed. However,

the frequency of TIM-3 expression in CD4+ and CD56+ cells was lower in the active state

than in the instable state (Fig. 13). These data were the consistent tendency that in active BD

group, the TIM-3 was expressed lower than in the stable BD group.

It is known that TIM-3 expression is predominantly detected in TH1-differentiated

CD4+ cells (Sabota et al., 2003), so after the stimulation of PBMCs with anti-CD3 and –

CD28 antibody in presence with IL-2, TIM-3 expression was analyzed by flow cytometry.

Contrast to expectation, there were no differences in the TIM-3 expression on stimulated

PBMCs with time (Fig. 14A). TIM-3 had a tendency to increase in control but not in BD

without significance, and the difference between two groups was significant with time.

These data may suggest that there is an impaired response of TIM-3 expression in BD

patients and may contribute to the abnormal immune response in BD pathogenesis.

43

Next, TIM-3 expression at mRNA level was estimated by quantitative RT-PCR. It was

found that PBMCs from BD patients expressed higher TIM-3 mRNA than those from

healthy controls and there were no significant differences between active and stable groups

(Fig. 15). In the same patient, TIM-3 mRNA level in the active disease was significantly

higher than in the stable state as well (Fig. 16). In this result, TIM-3 expression at transcript

level was paradoxically contrary to the cell surface expression level. Previous study found

that monocytes, microglia from active border regions of multiple sclerosis (MS) lesions

showed higher level of TIM-3 mRNA than did those from the quiescent center of MS lesions,

from adjacent normal appearing white matter, or from noninflamed white matter tissue

(Anderson et al. 2007), consistent result with this study in Fig. 15. On the contrary, other

report revealed that MS cerebrospical fluid clones expressed lower levels of TIM-3 mRNA

than control suggesting failure to up-regulate T cell expression of TIM-3 in inflammatory

sites may represent a novel, intrinsic defect that may contribute to the pathogenesis of MS

and other human autoimmune disease (Koguchi et al. 2006). Even in one disease, multiple

sclerosis, different results have been reported. Further experiments will be needed to study

TIM-3 expression in various human diseases.

To sum up, the present study provides preliminary data of the expression of TIM-3 and

its relation to immunopathogenesis in BD patients. It revealed a significant increased cell

surface TIM-3 in BD patients compared with controls and the same tendency was shown in

the expression in TIM-3 protein. The TIM-3 mRNA level was also increased in BD patients

compared with controls. However, in the same patient according to the disease activity the

reverse expression of TIM-3 was found between flow cytometry and RT-PCR. In addition,

44

TIM-3 expression in stimulated PBMCs was altered in BD patients, which had no change in

the expression with stimulation time. The up-regulated surface expression of TIM-3, TIM-3

protein, and TIM-3 mRNA in PBMCs from BD as well as presence of TIM-3 in cutaneous

lesion was first described in this study. The differential expression of TIM-3 according to the

disease activity and in various cellular population might contribute to the modulate immune

response related to pathogenesis of Behçet’s disease. Further studies on the functional effect

of TIM-3 in immunologic abnormalities and on TIM-3 role in signal pathway are needed to

elucidate the exact role of TIM-3 in BD.

45

V. CONCLUSION

1. Expression of cell surface TIM-3, TIM-3 protein, and TIM-3 mRNA in PBMCs from

Behçet’s disease patient as well as TIM-3 expression in cutaneous lesion was firstly

described in this study.

2. TIM-3 expression in BD patients was significantly up-regulated compared with those in

healthy controls presented by flow cytometry, Western blot, and RT-PCR.

3. According to the disease activity, there was differential expression of TIM-3; patients

showed that the surface TIM-3 expression was significantly decreased in the active

disease state compared to in the stable state, however, TIM-3 mRNA was decreased in

the active compared to in the stable.

4. There were different expressions of TIM-3 in CD4+, CD8+, CD11b+, and CD56+ cells

and correlation between TIM-3 frequency and their TIM-3 expression.

5. PBMCs from Behçet’s disease patients have altered responses of TIM-3 expression in

the stimulation.

This study may imply the differential expression of human TIM-3 molecules by PBMCs of

TH1-driven Behçet's disease according to the disease activity and suggest that there were

altered kinetics in the expression of TIM-3 molecule and TIM-3 mRNA in PBMCs that might

modulate immunologic response in Behçet’s disease.

46

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57

- 국문요약 -

베체트병 활성도에 따른 TIM-3 의 발현 양상

아주대학교 대학원의학과

이 중 선

(지도교수: 이 은 소)

연구배경: T cell immunoglobulin mucin-3 (TIM-3)는 최근 1 형 T 세포(TH1)

특이 분자로 밝혀졌고 TH1 반응이 우세한 질병의 발병기전 및 면역 반응을

조절하는 역할을 한다고 알려졌다. 증가된 TH1 면역반응은 만성 전신정 염증성

질환인 베체트병의 주요한 발병요인 중의 하나이다. 하지만 아직까지

베체트병에서 TIM-3 의 역할에 대한 연구는 되어있지 않다.

연구목적: 이 연구는 베체트병에서 TIM-3 분자의 세포표면 발현, TIM-

3 단백질의 발현 및 mRNA 의 발현을 말초혈액단핵구에서 확인하고 또한

질병활성도에 따른 발현 양상을 알아보고자 했다.

재료 및 방법: 베체트병 환자 (67 명), 정상 대조군 (13 명), 건선 환자

(14 명)를 대상으로 하였다. 베체트병 환자는 질병의 활성도에 따라 활동성과

비활동성으로 구분하였다. 유세포분석기를 통해 한국인 베체트병의 면역학적

양상을 알아보고, 세포표면의 TIM-3, CD4, CD8, CD11b 및 CD56 의 발현을

58

알아보았다. 단백질 수준의 TIM-3 발현 분석은 웨스턴블롯팅을 이용하였다.

TIM-3 발현은 환자군과 대조군에서 그리고 같은 환자에서 질병이 활동성일

때와 비활동성일 때를 비교하였다. 중합효소연쇄반응을 통하여 TIM-3

전사수준의 발현을 분석하였다. 베체트병 환자의 피부 병변에서는

면역조직화학적으로 TIM-3 의 발현을 살펴보았다.

결과: 베체트병 활성지표인 적혈구침강속도와 C 반응성단백은 활동성 베체트병

환자에서 비활동성 보다 높았다. 세포 표면 TIM-3 발현은 정상에 비하여

베체트병 환자에서 현저하게 높았고 비활동성 환자에서 활동성 환자보다 높은

경향을 보였다. 환자의 피부병변의 침윤된 염증세포에서 TIM-3 가 발현됨을

확인하였고 연속된 조직 슬라이드에서 CD4+세포가 같은 위치에서 확인되었다.

웨스턴블랏팅에서도 단백질 수준의 TIM-3 가 정상보다 환자에서 더 많이

발현되었다. 또한 같은 환자에서도 질병활성도에 따라 TIM-3 발현에 차이를

보였는데, 비활동성 질환 상태일 때 활동성 상태보다 높은 발현을 보였다.

말초혈액단핵구 중에서 CD8 양성 세포만 베체트병 환자에서 정상보다 낮았으며,

CD8, CD56 세포에서 TIM-3 의 발현은 베체트병 환자에서 정상보다 높았다.

하지만 TIM-3 발현 수준은 CD4 세포에서의 TIM-3 발현 정도와 가장 높은

상관관계를 보였다. 말초혈액 단핵구를 자극 후 시간에 따른 TIM-3 발현에는

변화가 없었고, CD4, CD8 세포의 TIM-3 발현능에도 자극 시간에 따른 변화는

보이지 않았다. 또한 TIM-3 mRNA 수준의 발현도 활동성 환자에서 정상에

비해 높게 나타났고, 같은 환자에서 질환이 비할동성일 때 보다 활동성 일 때

TIM-3 mRNA 가 높게 발현함을 확인하였다.

59

결론: 이 연구는 베체트병에서 TIM-3 의 발현을 세포표면, 단백질 수준 및

유전자 수준에서 확인하였고 질병 활성도에 따라 TIM-3 발현에 차이가 있음을

확인하였으며 이는 TIM-3 가 면역학적 반응을 조절하여 베체트병의 발병에

관계가 있을 가능성을 제시한다.

핵심어: 베체트병, 질병활성도, T cell immunoglobulin and mucin(TIM)-3