Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 999 _____________________________ _____________________________ Pteridine Dependent Hydroxylases as Autoantigens in Autoimmune Polyendocrine Syndrome Type 1 BY OLOV EKWALL ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2001
Pteridine Dependent Hydroxylases as Autoantigens in Autoimmune Polyendocrine Syndrome Type 1
Feb 13, 2023
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nbn_se_uu_diva-596.pdf_____________________________ _____________________________
Polyendocrine Syndrome Type 1
ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2001
Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Medicine presented
at Uppsala University in 2001
ABSTRACT
Dissertations from the Faculty of Medicine 999. 81pp. Uppsala. ISBN 91-554-4941-7.
Autoimmune polyendocrine syndrome type I (APS) is a monogenous, recessively inherited disease characterised by endocrine and non-endocrine autoimmune manifestations. One fifth of
APS I patients suffer from periodic intestinal dysfunction with varying degrees of malabsorbtion,
steatorrhea and constipation. Alopecia areata is found in one third of APS I patients. By
immunoscreening human cDNA libraries derived from normal human duodenum and scalp with
APS I sera, we identified tryptophan hydroxylase (TPH) as an intestinal autoantigen and tyrosine hydroxylase (TH) as a dermal autoantigen. Forty-eight percent (38/80) of the APS I patients had
TPH antibodies (Ab) and 44% (41/94) showed TH immunoreactivity. No reactivity against TPH
or TH was seen in healthy controls. TPH-Abs showed a statistically significant correlation with
gastrointestinal dysfunction (p<0.0001) and TH-Abs were significantly correlated to alopecia
(p=0.02). TPH-Ab positive APS I sera specifically immunostained TPH containing
enterochromaffin cells in normal duodenal mucosa. In affected mucosa a depletion of the TPH containing EC cells was seen. In enzyme inhibition experiments TPH and TH activity in vitro was
reduced by adding APS I sera. TPH and TH together with phenylalanine hydroxylase (PAH)
constitute the group of pteridine dependent hydroxylases. These are highly homologous enzymes
involved in the biosynthesis of neurotransmitters. Immunoprecipitation of PAH expressed in vitro
showed that 27% (25/94) of APS I patients had antibodies reacting with PAH, but no associations with clinical manifestations was observed. An immunocompetition assay showed that the PAH
reactivity reflects a cross-reactivity with TPH.
In conclusion, we have identified TPH and TH as intestinal and dermal autoantigens in
APS I, coupled to gastrointestinal dysfunction and alopecia. We have also demonstrated
immunoreactivity against PAH in APS I patient sera reflecting a cross-reactivity with TPH.
Key words: APS I, alopecia, autoantigen, cDNA, malabsorbtion, phenylalanine hydroxylase,
pteridine, tryptophan hydroxylase, tyrosine hydroxylase.
Olov Ekwall, Department of Medical Sciences, University Hospital, SE-751 85 Uppsala, Sweden,
[email protected]
Printed in Sweden by Eklundshofs Grafiska AB, Uppsala 2001
I n m e m o r y o f B j ö r n E k w a l l
P A P E R S
This thesis is based on the following papers, which will be referred to in the text by
their roman numerals:
I. Ekwall O, Hedstrand H, Grimelius L, Haavik J, Perheentupa J, Gustafsson J,
Husebye E, Kämpe O and Rorsman F. (1998) Identification of tryptophan hydroxylase as an intestinal autoantigen. Lancet, 1998; 352(9124): 279-283.
II. Ekwall O, Sjöberg K, Mirakian R, Rorsman F and Kämpe O. (1999) Tryptophan hydroxylase autoantibodies and intestinal disease in autoimmune
polyendocrine syndrome type 1. Lancet 1999; 354(9178): 568.
III. Hedstrand H, Ekwall O, Haavik J, Landgren E, Betterle C, Perheentupa J, Gustafsson J, Husebye E, Rorsman F and Kämpe O. (2000) Identification of
Tyrosine Hydroxylase as an Autoantigen in Autoimmune Polyendocrine
Syndrome Type I. Biochem Biophys Res Commun 2000; 267(1): 456-461.
IV. Ekwall O, Hedstrand H, Haavik J, Perheentupa J, Betterle C, Gustafsson J, Husebye E, Rorsman F and Kämpe O. (2000) Pteridine dependent
hydroxylases as autoantigens in autoimmune polyendocrine syndrome type I. J Clin Endocrinol Metab 2000; 85(8): 2944-2950.
Reprints were made with the permission of the publishers.
C O N T E N T S
ABBREVIATIONS 7
B-lymphocytes 12
Antibodies 13
T-lymphocytes 14
MHC class I 19
MHC class II 21
Target organ defects 28
Mucocutaneous candidiasis 34
Pteridine dependent hydroxylases 44
TPH, PAH and TH in disease 45
CURRENT INVESTIGATION
Results 47
The identification of TPH as an autoantigen in APS I (I) 47
TPH antibodies in other autoimmune intestinal diseases (II) 49
The identification of TH as an autoantigen in APS I (III) 49
Pteridine dependent hydroxylases as autoantigens in APS I (IV) 50
Discussion 52
SUMMARY 60
ACKNOWLEDGEMENTS 63
REFERENCES 66
A B B R E V I A T I O N S
AADC Aromatic L-amino acid decarboxylase
Ab Antibody AChR Acetylcholine receptor
AIRE Autoimmune regulator (human) Aire Autoimmune regulator (mouse)
APC Antigen presenting cell APECED Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
APS I Autoimmune polyendocrine syndrome type I
BH4 Tetrahydrobiopterin
cDNA Complementary deoxyribonucleic acid CKK Cholecystokinin
CNS Central nervous system
ER Endoplasmatic reticulum GAD Glutamic acid decarboxylase
HLA Human leucocyte antigen IBD Inflammatory bowel disease
IDDM Insulin dependent diabetes mellitus
IFNγ Interferon γ
MHC Major histocompability complex PAH Phenylalanine hydroxylase
PCR Polymerase chain reaction
SCC Side chain cleavage enzyme SLE Systemic lupus erythematosus
TCR T cell receptor TH Tyrosine hydroxylase
TPH Tryptophan hydroxylase
9
I N T R O D U C T I O N
General immunology
The human immune system has evolved to ensure a dynamic defence against a wide
range of invading organisms. The first obstacle an invading pathogen must overcome
is a surface barrier e.g. keratinized skin or an enzyme coated mucosa. Pathogens able
to pass this first barrier are then met by two principally different but co-working
systems: the innate and adaptive immune systems. The innate immune system is
characterised by a similar response to re-invasion by the same type of invader,
irrespective of the number of previous encounters. On the other hand, the adaptive
immune system is characterised by its ability to strengthen the defence towards re-
invasion by the same type of invader.
The innate immune system
The components of the innate immune system are immunologically active cells,
complement, acute phase proteins and cytokines. The cells involved are phagocytic
cells including neutrophils, monocytes and macrophages; cells that release
inflammatory mediators including basophils, mast cells and eosinophils; and natural
killer cells. The innate immune response is rapid and does not require cell proliferation
before the intruder is attacked. The main limitation of the innate immune system is the
lack of specificity. The recognising receptors used are coded by germ line genes, and
the repertoire is limited to the hundreds, in contrast to 1014 – 1018 somatically
recombined receptors used in the adaptive immune system (110). The structures
recognised by the innate immune system are called “pathogen-associated molecular
patterns” and the receptors are referred to as “pattern-recognition receptors”. The
Pteridine dependent hydroxylases as autoantigens in APS I
10
general features of the pathogen-associated patterns are that they are specific for
microbial pathogens, often crucial for the survival of the pathogen, and shared by
whole classes of pathogens. Examples are lipopolysaccarides, peptidoglycans and
bacteria-specific DNA. The receptors are either secreted, endocytic or signalling.
Secreted circulating receptors bind to the surface of pathogens and mark them for
phagocytosis or destruction by the complement system. Endocytic receptors are
expressed on phagocytes and direct the recognised pathogen to lysosomes where it is
degraded. Signalling receptors induce the production of inflammatory mediators such
as inflammatory cytokines, when they encounter their counterpart. In contrast to the
adaptive immune system, the innate immune system cannot recognise self-structures.
This feature of the innate system is used as a control mechanism in the initiation of an
adaptive immune response. The initial activation of a T-cell requires two signals: the
recognition by the T-cell receptor of a peptide-MHC complex, and a co-stimulatory
signal, such as B7.1 or B7.2, controlled by the innate immune system. In this way the
system ensures that a peptide can activate a T-cell response only if it is derived from a
pathogen recognised by the innate immune system (111).
The adaptive immune system
The basis for the adaptive immune system is clonal proliferation of T-, and B-cells,
bearing receptors specific for the triggering antigens. A refined system of somatic gene
rearrangements allows the adaptive system to generate approximately 1015 different
receptors, each one specific to one unique epitope. T-cell receptors are expressed on T-
cells and recognise epitopes presented in complex with MHC. B-cells produce
antibodies when B-cell receptors are activated, and antibodies are the soluble forms of
B-cell receptors. Upon recognition of the specific epitope, the mature T-, or B-cell,
undergoes clonal proliferation leading to direct cell-mediated destruction,
Olov Ekwall
Figure 1. Overview of lymphocyte activation. T-cell receptors recognise processed
peptides presented by MHC class I or class II. A cytotoxic T lymphocyte (CTL) express
CD8, which by binding to a constant region of MHC class I restricts the CTL to
interact with MHC class I expressing cells, mainly presenting intracellular proteins of
viral, or endogenous, origin. An activated CTL kills the infected cell by inducing lysis.
A T-helper (TH) cell is in the same way restricted, by CD4 expression, to activation by
preferrably extracellular antigens presented by MHC class II. In addition to the
antigen presentation, the TH cell also requires a co-stimulatory signal, i.e. binding of
B7 to CD28, to be activated. The TH cell can be a TH1 or a TH2 cell. Activated TH1
cells produce IFNγ and IL-2, which activate CTLs or macrophages. Activated TH2
cells secrete IL-4, 5 and 6 which activate B-cells. B-cells recognise antigens directly
through membrane bound receptors and also need a second signal from an TH cell to
be activated and proliferate into antibody producing plasma cells or memory B cells
cells (From: Delves and Riott, NEJM, 2000; 343(2); 108-17).
Pteridine dependent hydroxylases as autoantigens in APS I
12
antibodies (Figure 1) (42, 43).
B-lymphocytes
B-cells are developed throughout human life from stem cells, initially in the fetal liver,
and later in the bone marrow. During differentiation the cells go through specific
developmental stages in which they, through somatic recombination, rearrange the
variable regions of the heavy and light chain of the B-cell receptor or antibody (VDJ
rearrangement). The VDJ rearrangement is mediated by recombinases coded by the
recombination-activating genes (RAG 1 and RAG 2) and is estimated to result in the
generation of about 1015 unique variable regions (2, 169). These are checkpoints that
ensure that genetic rearrangements are no longer possible after heavy and light chain
rearrangements are completed. The end products of this development are immature B-
cells leaving the bone marrow, each one expressing a unique IgM on its surface.
Immature B-cells that bind antigens to their receptors undergo clonal deletion to
prevent the occurrence of mature and activated B-cells able of producing antibodies
against self-structures (39). The immature B-cells enter the circulation and migrate to
the secondary lymphoid tissues – mainly the spleen and the lymph nodes, where they
complete their maturation. Mature cells recirculate in the periphery and the fate of
each cell is dependent upon encountering its specific antigen. When an antigen binds
to a membrane bound IgM, the binding of the antigen sends a direct signal to the
interior of the B-cell, the antigen is then internalised and presented in a complex with
MHC class II to a CD4 positive T-cell (80). The direct signal from the receptor, and
signalling from the T-cell through cytokines, are often both required to activate the B-
cell (133). The B-cell then proliferates to become antibody-producing plasma cells,
that are predominantly located in the bone marrow or on mucosal surfaces, or as long
Olov Ekwall
13
lived memory B-cells that are mainly found in the spleen and lymph nodes. In the
proliferative stage, activated B-cells further diversify the antibody specificity by
somatic hypermutation in that cells producing antibodies with the highest affinity to
the antigen are positively selected (80).
Antibodies
Antibodies are the soluble form of the B-cell receptors. An antibody consists of two
heavy chains and two light chains held together by disulphide bonds (46). Both heavy
and light chains have hypervariable regions in their amino termini and constant
regions in their carboxy termini. The hypervariable regions from one light chain and
one heavy chain together form an antigen recognition site, thus there are two antigen
recognition sites on each antibody. All antibodies or B-cell receptors produced by a
given B-cell bear the same specificity through allelic and isotypic exclusion. There are
two types of light chains, namely λ and κ. There are five different classes of heavy
chains with different heavy-chain constant (C) domains, and the constant domains
categorise the 5 major isotypes of antibodies. The C domain can be µ, δ, γ, ε or α,
giving rise to IgM, IgD, IgG, IgE and IgA, respectively. The IgGs can be further
divided into four subtypes: IgG1, IgG2, IgG3 and IgG4, and IgAs can be either IgA1
or IgA2. The isotype switching is accomplished through alternative splicing and DNA
rearrangements (158). General characteristics of the different isotypes are summarised
in table 1. IgM is mainly produced before somatic hypermutation has occurred and
therefore has a relatively low affinity. It can rapidly be secreted into the blood as a
pentamer and acts as an effective activator of the complement system. IgD has an
obscure function in the immune system. It is present on the surface of B-cells and may
have a role in the development and/or activation of the B-cell. Very low levels of IgD
can also be detected in the plasma, but the significance of this is unknown. IgG is the
Pteridine dependent hydroxylases as autoantigens in APS I
14
predominant antibody isotype found in the circulation. It is an efficient opsonisator
and activator of complement. It can also pass the placental barrier and transmit
immunological properties to the foetus before it has started its own antibody
production. IgE is primarily found beneath the skin and mucosa where it acts as an
activator of mast cells which induce a fast and powerful immune response, mainly
through the release of histamine. IgA has the ability to associate with a J chain and
form dimers that can pass through epithelial surfaces in the gut, bronchi, mammary
glands etc. The secreted IgAs can inhibit the attachment of infectious agents to the
epithelium and form a line of defence against pathogens from the outside (80).
Function IgM IgD IgG1 IgG2 IgG3 IgG4 IgA IgE
Neutralisation + - ++ ++ ++ ++ ++ -
Opsonisation - - +++ ++/- ++ + + -
Diff. into extravascular sites +/- - +++ +++ +++ +++ ++ +
Mean serum level (mg/ml) 1.5 0.04 9 3 1 0.5 2.1 3x10-5
Table 1. Characteristics of antibody isotypes and IgG subtypes.
T-lymphocytes
Immature T-cells migrate from the bone marrow to the thymus where they step-by-
step differentiate into mature cells. These mature cells are efficient in recognising and
reacting against non-self peptides, but also avoid attacking self structures. In this
Olov Ekwall
15
intriguing process, referred to as positive and negative selection of T-cells, the small
fraction of cells that survive are saved from apoptosis twice. First they are saved by
their T-cell receptor’s ability to recognise self-MHC, and then by the same receptor’s
inability to recognise self-peptides presented in a complex with MHC (Figure 2).
The T-cell receptor
Membrane bound receptors are expressed on the surface of T-cells to ensure the
specificity of the T-cell response. The T-cell receptor belongs to the immunoglobulin
superfamily of receptor molecules and shares many structural characteristics with B-
cell receptors, examples include subunits divided into variable and constant regions,
and genetic rearrangements responsible for antigenic variability (57). There are,
however, two major differences. Immunoglobulins recognise native antigens in
extracellular spaces while T-cell receptors only recognise processed peptides presented
in complex with MHC on cell surfaces. T-cell receptors only exist in a membrane
bound form while immunoglobulins can either be secreted antibodies or membrane
bound B-cell receptors.
The predominant T-cell receptor is an heterodimer consisting of two transmembrane
glycoprotein chains, α and β, linked by a disulphide bond. Although there are reports
of T-cells that, through incomplete allelic exclusion, express T-cell receptors with
more than one specificity (129), in principle all T-cell receptors on a given cell
recognise the same peptide-MHC complex. An alternative γ/δ-T-cell receptor is
expressed on a minority of T-cells found mainly in epithelial tissue in the epidermis
and small intestine. The γ/δ-receptor differs from the α/β-receptor in that it seems to
be able to recognise an antigen directly without the presence of a MHC molecule. The
physiological role of these γ/δ-T-cells is still unclear (24).
Pteridine dependent hydroxylases as autoantigens in APS I
16
In the same manner as B-cell receptors, the α-, and β-chains of the T-cell receptor are
coded by sets of genes which, during T-cell differentiation in the thymus, are
somatically recombined to form functional genes. The variable region of α-chain is
generated by ~70 Vα-segments rearranged to ~60 Jα-segments. The β-chain gene is a
result of the rearrangement of ~50 Vβ-, 2 Dβ-, and 13 Jβ-segments (42). The
variability is highest in the CDR3-region of the T-cell receptor, forming the centre of
the antigen binding groove, responsible for peptide recognition, and is lower in the
flanking MHC recognising parts (56). In contrast to immunoglobulins, T-cell receptors
do not undergo somatic hypermutation. This lowers the risk that T-cells, having passed
negative selection, mutate into self-reacting cells. This may also be functional, in the
sense that T-cell receptors must retain their ability to recognise MHC to be able to
stimulate an immune response.
The function of a T-cell is not only determined by the nature of the T-cell receptor
expressed, but also by the expression of CD4 or CD8 co-receptor molecules. A mature
T-cell is either expressing CD4 or CD8 associated with the T-cell receptor on the cell
surface. CD4 and CD8 exclusively bind to invariable parts of MHC class II and I,
respectively. The expression of CD4 or CD8 thus restricts the T-cell to interact with
peptides presented in a complex with either MHC class I or class II. Whether a T-cell
should express CD4 or CD8 is determined at the end of the T-cell differentiation in the
thymus (184).
Olov Ekwall
17
Figure 2. Positive and negative selection in the thymus. Immature, CD4+CD8+
T cells are predestined to apoptosis, and saved if they recognise MHC presented by
cortical epithelial cells. During this “positive selection”, 95% of lymphocytes are
eliminated. The surviving cells are challenged, in the medulla, by the presentation of
self peptides by dendritic cells, or macrophages. T cells that bind self peptides with too
high affinity are eliminated in this “negative selection”. The remaining fraction of
cells are exported to the periphery as CD4 or CD8 positive T cells (From: Delves and
Riott, NEJM, 2000; 343(1); 37-49).
Progenitor T-cells leave the bone marrow and enter the thymus at the edge of the
cortex as “double-negative” cells, lacking both CD 4 and CD 8 (CD 4- 8-) and a
rearranged T-cell receptor on the cell surface. The differentiation is initiated by the
rearrangement of the β-chain gene. When the β-chain is rearranged and expressed on
Pteridine dependent hydroxylases as autoantigens in APS I
18
the surface, the cells becomes “double positive” (TCRβ CD4+ CD 8+), and the
rearrangement of the α-chain is started. When the complete T-cell receptor is
expressed on “double positive” cells (TCRαβ CD4+ CD 8+), the cells are destined to
apoptosis if they are not saved in the process of positive selection, by the binding of
the T-cell receptor to MHC expressed on cortical epithelial cells (173). Ninety-five
percent of pre T-cells die in the thymus at this stage. Dendritic cells and macrophages
in the medulla then challenge the remaining cells for self-antigens bound to MHC. The
cells with T-cell receptors with high affinity to these self-antigens are directed towards
apoptosis (124). The remaining small fraction of cells then, depending on the
preference for MHC class I or II, cease to express either CD4 or CD8. The final result,
after approximately three weeks of development in the thymus, is the export to the
periphery, of single positive CD 4 or CD 8 expressing cells. These cells have a
rearranged T-cell receptor able to recognise MHC class I or II, but unable…
Polyendocrine Syndrome Type 1
ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2001
Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Medicine presented
at Uppsala University in 2001
ABSTRACT
Dissertations from the Faculty of Medicine 999. 81pp. Uppsala. ISBN 91-554-4941-7.
Autoimmune polyendocrine syndrome type I (APS) is a monogenous, recessively inherited disease characterised by endocrine and non-endocrine autoimmune manifestations. One fifth of
APS I patients suffer from periodic intestinal dysfunction with varying degrees of malabsorbtion,
steatorrhea and constipation. Alopecia areata is found in one third of APS I patients. By
immunoscreening human cDNA libraries derived from normal human duodenum and scalp with
APS I sera, we identified tryptophan hydroxylase (TPH) as an intestinal autoantigen and tyrosine hydroxylase (TH) as a dermal autoantigen. Forty-eight percent (38/80) of the APS I patients had
TPH antibodies (Ab) and 44% (41/94) showed TH immunoreactivity. No reactivity against TPH
or TH was seen in healthy controls. TPH-Abs showed a statistically significant correlation with
gastrointestinal dysfunction (p<0.0001) and TH-Abs were significantly correlated to alopecia
(p=0.02). TPH-Ab positive APS I sera specifically immunostained TPH containing
enterochromaffin cells in normal duodenal mucosa. In affected mucosa a depletion of the TPH containing EC cells was seen. In enzyme inhibition experiments TPH and TH activity in vitro was
reduced by adding APS I sera. TPH and TH together with phenylalanine hydroxylase (PAH)
constitute the group of pteridine dependent hydroxylases. These are highly homologous enzymes
involved in the biosynthesis of neurotransmitters. Immunoprecipitation of PAH expressed in vitro
showed that 27% (25/94) of APS I patients had antibodies reacting with PAH, but no associations with clinical manifestations was observed. An immunocompetition assay showed that the PAH
reactivity reflects a cross-reactivity with TPH.
In conclusion, we have identified TPH and TH as intestinal and dermal autoantigens in
APS I, coupled to gastrointestinal dysfunction and alopecia. We have also demonstrated
immunoreactivity against PAH in APS I patient sera reflecting a cross-reactivity with TPH.
Key words: APS I, alopecia, autoantigen, cDNA, malabsorbtion, phenylalanine hydroxylase,
pteridine, tryptophan hydroxylase, tyrosine hydroxylase.
Olov Ekwall, Department of Medical Sciences, University Hospital, SE-751 85 Uppsala, Sweden,
[email protected]
Printed in Sweden by Eklundshofs Grafiska AB, Uppsala 2001
I n m e m o r y o f B j ö r n E k w a l l
P A P E R S
This thesis is based on the following papers, which will be referred to in the text by
their roman numerals:
I. Ekwall O, Hedstrand H, Grimelius L, Haavik J, Perheentupa J, Gustafsson J,
Husebye E, Kämpe O and Rorsman F. (1998) Identification of tryptophan hydroxylase as an intestinal autoantigen. Lancet, 1998; 352(9124): 279-283.
II. Ekwall O, Sjöberg K, Mirakian R, Rorsman F and Kämpe O. (1999) Tryptophan hydroxylase autoantibodies and intestinal disease in autoimmune
polyendocrine syndrome type 1. Lancet 1999; 354(9178): 568.
III. Hedstrand H, Ekwall O, Haavik J, Landgren E, Betterle C, Perheentupa J, Gustafsson J, Husebye E, Rorsman F and Kämpe O. (2000) Identification of
Tyrosine Hydroxylase as an Autoantigen in Autoimmune Polyendocrine
Syndrome Type I. Biochem Biophys Res Commun 2000; 267(1): 456-461.
IV. Ekwall O, Hedstrand H, Haavik J, Perheentupa J, Betterle C, Gustafsson J, Husebye E, Rorsman F and Kämpe O. (2000) Pteridine dependent
hydroxylases as autoantigens in autoimmune polyendocrine syndrome type I. J Clin Endocrinol Metab 2000; 85(8): 2944-2950.
Reprints were made with the permission of the publishers.
C O N T E N T S
ABBREVIATIONS 7
B-lymphocytes 12
Antibodies 13
T-lymphocytes 14
MHC class I 19
MHC class II 21
Target organ defects 28
Mucocutaneous candidiasis 34
Pteridine dependent hydroxylases 44
TPH, PAH and TH in disease 45
CURRENT INVESTIGATION
Results 47
The identification of TPH as an autoantigen in APS I (I) 47
TPH antibodies in other autoimmune intestinal diseases (II) 49
The identification of TH as an autoantigen in APS I (III) 49
Pteridine dependent hydroxylases as autoantigens in APS I (IV) 50
Discussion 52
SUMMARY 60
ACKNOWLEDGEMENTS 63
REFERENCES 66
A B B R E V I A T I O N S
AADC Aromatic L-amino acid decarboxylase
Ab Antibody AChR Acetylcholine receptor
AIRE Autoimmune regulator (human) Aire Autoimmune regulator (mouse)
APC Antigen presenting cell APECED Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
APS I Autoimmune polyendocrine syndrome type I
BH4 Tetrahydrobiopterin
cDNA Complementary deoxyribonucleic acid CKK Cholecystokinin
CNS Central nervous system
ER Endoplasmatic reticulum GAD Glutamic acid decarboxylase
HLA Human leucocyte antigen IBD Inflammatory bowel disease
IDDM Insulin dependent diabetes mellitus
IFNγ Interferon γ
MHC Major histocompability complex PAH Phenylalanine hydroxylase
PCR Polymerase chain reaction
SCC Side chain cleavage enzyme SLE Systemic lupus erythematosus
TCR T cell receptor TH Tyrosine hydroxylase
TPH Tryptophan hydroxylase
9
I N T R O D U C T I O N
General immunology
The human immune system has evolved to ensure a dynamic defence against a wide
range of invading organisms. The first obstacle an invading pathogen must overcome
is a surface barrier e.g. keratinized skin or an enzyme coated mucosa. Pathogens able
to pass this first barrier are then met by two principally different but co-working
systems: the innate and adaptive immune systems. The innate immune system is
characterised by a similar response to re-invasion by the same type of invader,
irrespective of the number of previous encounters. On the other hand, the adaptive
immune system is characterised by its ability to strengthen the defence towards re-
invasion by the same type of invader.
The innate immune system
The components of the innate immune system are immunologically active cells,
complement, acute phase proteins and cytokines. The cells involved are phagocytic
cells including neutrophils, monocytes and macrophages; cells that release
inflammatory mediators including basophils, mast cells and eosinophils; and natural
killer cells. The innate immune response is rapid and does not require cell proliferation
before the intruder is attacked. The main limitation of the innate immune system is the
lack of specificity. The recognising receptors used are coded by germ line genes, and
the repertoire is limited to the hundreds, in contrast to 1014 – 1018 somatically
recombined receptors used in the adaptive immune system (110). The structures
recognised by the innate immune system are called “pathogen-associated molecular
patterns” and the receptors are referred to as “pattern-recognition receptors”. The
Pteridine dependent hydroxylases as autoantigens in APS I
10
general features of the pathogen-associated patterns are that they are specific for
microbial pathogens, often crucial for the survival of the pathogen, and shared by
whole classes of pathogens. Examples are lipopolysaccarides, peptidoglycans and
bacteria-specific DNA. The receptors are either secreted, endocytic or signalling.
Secreted circulating receptors bind to the surface of pathogens and mark them for
phagocytosis or destruction by the complement system. Endocytic receptors are
expressed on phagocytes and direct the recognised pathogen to lysosomes where it is
degraded. Signalling receptors induce the production of inflammatory mediators such
as inflammatory cytokines, when they encounter their counterpart. In contrast to the
adaptive immune system, the innate immune system cannot recognise self-structures.
This feature of the innate system is used as a control mechanism in the initiation of an
adaptive immune response. The initial activation of a T-cell requires two signals: the
recognition by the T-cell receptor of a peptide-MHC complex, and a co-stimulatory
signal, such as B7.1 or B7.2, controlled by the innate immune system. In this way the
system ensures that a peptide can activate a T-cell response only if it is derived from a
pathogen recognised by the innate immune system (111).
The adaptive immune system
The basis for the adaptive immune system is clonal proliferation of T-, and B-cells,
bearing receptors specific for the triggering antigens. A refined system of somatic gene
rearrangements allows the adaptive system to generate approximately 1015 different
receptors, each one specific to one unique epitope. T-cell receptors are expressed on T-
cells and recognise epitopes presented in complex with MHC. B-cells produce
antibodies when B-cell receptors are activated, and antibodies are the soluble forms of
B-cell receptors. Upon recognition of the specific epitope, the mature T-, or B-cell,
undergoes clonal proliferation leading to direct cell-mediated destruction,
Olov Ekwall
Figure 1. Overview of lymphocyte activation. T-cell receptors recognise processed
peptides presented by MHC class I or class II. A cytotoxic T lymphocyte (CTL) express
CD8, which by binding to a constant region of MHC class I restricts the CTL to
interact with MHC class I expressing cells, mainly presenting intracellular proteins of
viral, or endogenous, origin. An activated CTL kills the infected cell by inducing lysis.
A T-helper (TH) cell is in the same way restricted, by CD4 expression, to activation by
preferrably extracellular antigens presented by MHC class II. In addition to the
antigen presentation, the TH cell also requires a co-stimulatory signal, i.e. binding of
B7 to CD28, to be activated. The TH cell can be a TH1 or a TH2 cell. Activated TH1
cells produce IFNγ and IL-2, which activate CTLs or macrophages. Activated TH2
cells secrete IL-4, 5 and 6 which activate B-cells. B-cells recognise antigens directly
through membrane bound receptors and also need a second signal from an TH cell to
be activated and proliferate into antibody producing plasma cells or memory B cells
cells (From: Delves and Riott, NEJM, 2000; 343(2); 108-17).
Pteridine dependent hydroxylases as autoantigens in APS I
12
antibodies (Figure 1) (42, 43).
B-lymphocytes
B-cells are developed throughout human life from stem cells, initially in the fetal liver,
and later in the bone marrow. During differentiation the cells go through specific
developmental stages in which they, through somatic recombination, rearrange the
variable regions of the heavy and light chain of the B-cell receptor or antibody (VDJ
rearrangement). The VDJ rearrangement is mediated by recombinases coded by the
recombination-activating genes (RAG 1 and RAG 2) and is estimated to result in the
generation of about 1015 unique variable regions (2, 169). These are checkpoints that
ensure that genetic rearrangements are no longer possible after heavy and light chain
rearrangements are completed. The end products of this development are immature B-
cells leaving the bone marrow, each one expressing a unique IgM on its surface.
Immature B-cells that bind antigens to their receptors undergo clonal deletion to
prevent the occurrence of mature and activated B-cells able of producing antibodies
against self-structures (39). The immature B-cells enter the circulation and migrate to
the secondary lymphoid tissues – mainly the spleen and the lymph nodes, where they
complete their maturation. Mature cells recirculate in the periphery and the fate of
each cell is dependent upon encountering its specific antigen. When an antigen binds
to a membrane bound IgM, the binding of the antigen sends a direct signal to the
interior of the B-cell, the antigen is then internalised and presented in a complex with
MHC class II to a CD4 positive T-cell (80). The direct signal from the receptor, and
signalling from the T-cell through cytokines, are often both required to activate the B-
cell (133). The B-cell then proliferates to become antibody-producing plasma cells,
that are predominantly located in the bone marrow or on mucosal surfaces, or as long
Olov Ekwall
13
lived memory B-cells that are mainly found in the spleen and lymph nodes. In the
proliferative stage, activated B-cells further diversify the antibody specificity by
somatic hypermutation in that cells producing antibodies with the highest affinity to
the antigen are positively selected (80).
Antibodies
Antibodies are the soluble form of the B-cell receptors. An antibody consists of two
heavy chains and two light chains held together by disulphide bonds (46). Both heavy
and light chains have hypervariable regions in their amino termini and constant
regions in their carboxy termini. The hypervariable regions from one light chain and
one heavy chain together form an antigen recognition site, thus there are two antigen
recognition sites on each antibody. All antibodies or B-cell receptors produced by a
given B-cell bear the same specificity through allelic and isotypic exclusion. There are
two types of light chains, namely λ and κ. There are five different classes of heavy
chains with different heavy-chain constant (C) domains, and the constant domains
categorise the 5 major isotypes of antibodies. The C domain can be µ, δ, γ, ε or α,
giving rise to IgM, IgD, IgG, IgE and IgA, respectively. The IgGs can be further
divided into four subtypes: IgG1, IgG2, IgG3 and IgG4, and IgAs can be either IgA1
or IgA2. The isotype switching is accomplished through alternative splicing and DNA
rearrangements (158). General characteristics of the different isotypes are summarised
in table 1. IgM is mainly produced before somatic hypermutation has occurred and
therefore has a relatively low affinity. It can rapidly be secreted into the blood as a
pentamer and acts as an effective activator of the complement system. IgD has an
obscure function in the immune system. It is present on the surface of B-cells and may
have a role in the development and/or activation of the B-cell. Very low levels of IgD
can also be detected in the plasma, but the significance of this is unknown. IgG is the
Pteridine dependent hydroxylases as autoantigens in APS I
14
predominant antibody isotype found in the circulation. It is an efficient opsonisator
and activator of complement. It can also pass the placental barrier and transmit
immunological properties to the foetus before it has started its own antibody
production. IgE is primarily found beneath the skin and mucosa where it acts as an
activator of mast cells which induce a fast and powerful immune response, mainly
through the release of histamine. IgA has the ability to associate with a J chain and
form dimers that can pass through epithelial surfaces in the gut, bronchi, mammary
glands etc. The secreted IgAs can inhibit the attachment of infectious agents to the
epithelium and form a line of defence against pathogens from the outside (80).
Function IgM IgD IgG1 IgG2 IgG3 IgG4 IgA IgE
Neutralisation + - ++ ++ ++ ++ ++ -
Opsonisation - - +++ ++/- ++ + + -
Diff. into extravascular sites +/- - +++ +++ +++ +++ ++ +
Mean serum level (mg/ml) 1.5 0.04 9 3 1 0.5 2.1 3x10-5
Table 1. Characteristics of antibody isotypes and IgG subtypes.
T-lymphocytes
Immature T-cells migrate from the bone marrow to the thymus where they step-by-
step differentiate into mature cells. These mature cells are efficient in recognising and
reacting against non-self peptides, but also avoid attacking self structures. In this
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15
intriguing process, referred to as positive and negative selection of T-cells, the small
fraction of cells that survive are saved from apoptosis twice. First they are saved by
their T-cell receptor’s ability to recognise self-MHC, and then by the same receptor’s
inability to recognise self-peptides presented in a complex with MHC (Figure 2).
The T-cell receptor
Membrane bound receptors are expressed on the surface of T-cells to ensure the
specificity of the T-cell response. The T-cell receptor belongs to the immunoglobulin
superfamily of receptor molecules and shares many structural characteristics with B-
cell receptors, examples include subunits divided into variable and constant regions,
and genetic rearrangements responsible for antigenic variability (57). There are,
however, two major differences. Immunoglobulins recognise native antigens in
extracellular spaces while T-cell receptors only recognise processed peptides presented
in complex with MHC on cell surfaces. T-cell receptors only exist in a membrane
bound form while immunoglobulins can either be secreted antibodies or membrane
bound B-cell receptors.
The predominant T-cell receptor is an heterodimer consisting of two transmembrane
glycoprotein chains, α and β, linked by a disulphide bond. Although there are reports
of T-cells that, through incomplete allelic exclusion, express T-cell receptors with
more than one specificity (129), in principle all T-cell receptors on a given cell
recognise the same peptide-MHC complex. An alternative γ/δ-T-cell receptor is
expressed on a minority of T-cells found mainly in epithelial tissue in the epidermis
and small intestine. The γ/δ-receptor differs from the α/β-receptor in that it seems to
be able to recognise an antigen directly without the presence of a MHC molecule. The
physiological role of these γ/δ-T-cells is still unclear (24).
Pteridine dependent hydroxylases as autoantigens in APS I
16
In the same manner as B-cell receptors, the α-, and β-chains of the T-cell receptor are
coded by sets of genes which, during T-cell differentiation in the thymus, are
somatically recombined to form functional genes. The variable region of α-chain is
generated by ~70 Vα-segments rearranged to ~60 Jα-segments. The β-chain gene is a
result of the rearrangement of ~50 Vβ-, 2 Dβ-, and 13 Jβ-segments (42). The
variability is highest in the CDR3-region of the T-cell receptor, forming the centre of
the antigen binding groove, responsible for peptide recognition, and is lower in the
flanking MHC recognising parts (56). In contrast to immunoglobulins, T-cell receptors
do not undergo somatic hypermutation. This lowers the risk that T-cells, having passed
negative selection, mutate into self-reacting cells. This may also be functional, in the
sense that T-cell receptors must retain their ability to recognise MHC to be able to
stimulate an immune response.
The function of a T-cell is not only determined by the nature of the T-cell receptor
expressed, but also by the expression of CD4 or CD8 co-receptor molecules. A mature
T-cell is either expressing CD4 or CD8 associated with the T-cell receptor on the cell
surface. CD4 and CD8 exclusively bind to invariable parts of MHC class II and I,
respectively. The expression of CD4 or CD8 thus restricts the T-cell to interact with
peptides presented in a complex with either MHC class I or class II. Whether a T-cell
should express CD4 or CD8 is determined at the end of the T-cell differentiation in the
thymus (184).
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17
Figure 2. Positive and negative selection in the thymus. Immature, CD4+CD8+
T cells are predestined to apoptosis, and saved if they recognise MHC presented by
cortical epithelial cells. During this “positive selection”, 95% of lymphocytes are
eliminated. The surviving cells are challenged, in the medulla, by the presentation of
self peptides by dendritic cells, or macrophages. T cells that bind self peptides with too
high affinity are eliminated in this “negative selection”. The remaining fraction of
cells are exported to the periphery as CD4 or CD8 positive T cells (From: Delves and
Riott, NEJM, 2000; 343(1); 37-49).
Progenitor T-cells leave the bone marrow and enter the thymus at the edge of the
cortex as “double-negative” cells, lacking both CD 4 and CD 8 (CD 4- 8-) and a
rearranged T-cell receptor on the cell surface. The differentiation is initiated by the
rearrangement of the β-chain gene. When the β-chain is rearranged and expressed on
Pteridine dependent hydroxylases as autoantigens in APS I
18
the surface, the cells becomes “double positive” (TCRβ CD4+ CD 8+), and the
rearrangement of the α-chain is started. When the complete T-cell receptor is
expressed on “double positive” cells (TCRαβ CD4+ CD 8+), the cells are destined to
apoptosis if they are not saved in the process of positive selection, by the binding of
the T-cell receptor to MHC expressed on cortical epithelial cells (173). Ninety-five
percent of pre T-cells die in the thymus at this stage. Dendritic cells and macrophages
in the medulla then challenge the remaining cells for self-antigens bound to MHC. The
cells with T-cell receptors with high affinity to these self-antigens are directed towards
apoptosis (124). The remaining small fraction of cells then, depending on the
preference for MHC class I or II, cease to express either CD4 or CD8. The final result,
after approximately three weeks of development in the thymus, is the export to the
periphery, of single positive CD 4 or CD 8 expressing cells. These cells have a
rearranged T-cell receptor able to recognise MHC class I or II, but unable…
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