pteridine dependent hydroxylases as autoantigens in autoimmune polyendocrine syndrome type 1

Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 999

_____________________________ _____________________________

Pteridine Dependent Hydroxylases asAutoantigens in Autoimmune

Polyendocrine Syndrome Type 1

BY

OLOV EKWALL

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2001

Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Medicine presented

at Uppsala University in 2001

ABSTRACT

Ekwall, O. 2001. Pteridine dependent hydroxylases as autoantigens in autoimmune polyendocrinesyndrome type I. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala

Dissertations from the Faculty of Medicine 999. 81pp. Uppsala. ISBN 91-554-4941-7.

Autoimmune polyendocrine syndrome type I (APS) is a monogenous, recessively inheriteddisease 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 tyrosinehydroxylase (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 TPHcontaining 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 associationswith 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]

Olov Ekwall 2001

ISSN 0282-7476

ISBN 91-554-4941-7

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 tryptophanhydroxylase 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

INTRODUCTION

General immunology 9

The innate immune system 9

The adaptive immune system 10

B-lymphocytes 12

Antibodies 13

T-lymphocytes 14

The T-cell receptor 15

The major histocompability complex 18

MHC class I 19

MHC class II 21

Tolerance 22

The “Danger” hypothesis 23

Autoimmunity 24

Genetic factors 25

Molecular mimicry 26

Aberrant expression of antigen 27

Defect immunoregulation through cytokines 28

Target organ defects 28

Superantigens 28

The TH1/TH2 paradigm 29

Apoptosis 29

Autoantigens, autoantibodies and autoreactive T-cells 30

Autoimmune polyendocrine syndrome type I 32

Mucocutaneous candidiasis 34

Hypoparathyroidism 35

Addison’s disease 35

Hypogonadism 36

Alopecia 36

Intestinal dysfunction 37

Vitiligo 37

Autoimmune hepatitis 38

Pernicious anaemia 38

Insulin-dependent diabetes mellitus 39

Minor components 39

Genetics 40

Autoimmune polyendocrine syndrome type II 42

Pteridine dependent hydroxylases 44

Structure 44

Function and tissue distribution 45

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

FUTURE PERSPECTIVES 61

ACKNOWLEDGEMENTS 63

REFERENCES 66

A B B R E V I A T I O N S

AADC Aromatic L-amino acid decarboxylase

Ab AntibodyAChR Acetylcholine receptor

AIRE Autoimmune regulator (human)Aire Autoimmune regulator (mouse)

APC Antigen presenting cellAPECED Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy

APS I Autoimmune polyendocrine syndrome type I

BH4 Tetrahydrobiopterin

CAH Chronic active hepatitisCD Cluster of differentiation

cDNA Complementary deoxyribonucleic acidCKK Cholecystokinin

CNS Central nervous system

EAE Experimental autoimmune encephalomyelitisEC Enterochromaffin

ER Endoplasmatic reticulumGAD Glutamic acid decarboxylase

HLA Human leucocyte antigenIBD Inflammatory bowel disease

IDDM Insulin dependent diabetes mellitus

IFNγ Interferon γ

Ig Immunoglobulin

ITT In vitro transcription and translationMG Myasthenia gravis

MHC Major histocompability complexPAH Phenylalanine hydroxylase

PCR Polymerase chain reaction

PKU PhenylketonureaRT-PCR Reverse transcriptase - polymerase chain reaction

SCC Side chain cleavage enzymeSLE Systemic lupus erythematosus

TCR T cell receptorTH Tyrosine hydroxylase

TPH Tryptophan hydroxylase

Olov Ekwall

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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,

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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).


12

complement mediated lysis, neutralisation or phagocytosis through opsonisation by

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

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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


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 - - +++ ++/- ++ + + -

Activation of NK cells - - ++ - ++ - - -

Activation of mast cells - - + - + - - +++

Complement activation +++ - ++ + +++ - + -

Distribution

Transport across epithelium + - - - - - +++ -

Transport across placenta - - +++ + ++ +/- - -

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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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).


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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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


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 to interact

with self peptides in complex with MHC (Figure 2).

The major histocompability complex

All nucleated cells in the body express the major histocompability complex (MHC)

antigens on their surface. MHC antigens present processed pathogen-derived, or

endogenous, peptides for T-cells (87, 88). There are two classes of MHC: class I and

class II. MHC class I present peptides for CD 8 positive T-cells and MHC class II

present peptides for CD 4 positive T-cells. MHC class I consists of two subunits, one

membrane bound α-chain with three domains, α1, α2 and α3, and β2-microglobulin.

MHC class II molecules have one α, and one β chain, each with two domains called

α1/α2 and β1/β2, respectively. Human MHC antigens are also called human leukocyte

antigens (HLA). All human MHC, with the exception of β2-microglobulin, are coded

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by genes clustered on chromosome 6. A number of other proteins engaged in

processing and presentation of peptides are also encoded by genes in the MHC region.

HLA is polygenic and polymorhpic in that there are multiple genes for each HLA

locus, and there are up to as many as 200 possible alleles for each locus. Three genes

encode MHC class I (A, B and C), and four sets of genes encode MHC class II (DP,

DQ, DR1 and DR2). In addition, most individuals are heterozygous at HLA loci,

because the alleles on both chromosomes are seldom identical. This results in a total of

six different class I molecules and eight different class II molecules expressed in one

individual. The diversity is even higher if class II α and β chains from different

chromosomes are taken into account. The combination of HLA alleles expressed is

called the HLA haplotype. The HLA haplotype determines the range of peptides that

can be presented in complex with MHC and this is probably the basis for the

association between HLA haplotype and increased risk for different autoimmune

diseases (142).

MHC class I

MHC class I is expressed on all nucleated cells, and presents peptides generated within

the cell (Figure 3) (87). Endogenous or viral proteins are degraded in proteasomes and

are loaded into MHC class I in the endoplasmatic reticulum by two proteins:

“Transporters associated with Antigen Processing” -1 and –2 (TAP-1 and TAP-2). The

MHC class I-peptide complex is then transported through the Golgi apparatus to the

cell surface and presented for CD 8 positive T-cells (60). The α1 and α2 domain of the

α-chain form a peptide-binding groove where the peptide is presented. The groove is

closed at the ends restricting the length of the presented peptide to 8 – 10 amino acids

(106). Different allelic variants of MHC class I have different preferences to the

peptide bound in the cleft, and two or three residues in the peptide sequence are crucial


20

Figure 3. Antigen processing. The upper part of the figure illustrates the processing of

antigens presented by MHC class I. Viral or endogenous proteins are degraded in

proteasomes into peptides. Peptides are transported to ER where they are loaded into

MHC class I, and the complexes are transported through the Golgi apparatus to the

surface of the cell. The lower part shows the processing of antigens presented by MHC

class II. Extracellular proteins enter the cell through endocytosis, or phagocytosis,

into early endosomes (a). MHC class II are synthesised in ER and transported through

Golgi into primary lysosomes (b), which fuse with endosomes and MHC class II

compartments (c) are formed. In MHC class II compartments the proteins are

degraded, peptides are loaded into MHC class II, and the complexes are exported to

the surface (From: Klein and Sato, NEJM, 2000; 343(10); 702-709).

for the binding. These so called anchor residues are similar in all peptides bound to a

given MHC class I molecule. The MHC class I-peptide complex is finally presented to

CD 8 positive cytotoxic T-cells resulting in cell death through the release of cytotoxic

effector proteins e.g. pore forming perforin and apoptosis inducing gramzymes from

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the cytotoxic T-cell. The mechanism is primarily designed to serve as a defence

against viruses and intracellular bacteria.

MHC class II

MHC class II is expressed on a subgroup of immune cells including B-cells, activated

T-cells, macrophages, dendritic cells and thymic epithelial cells. Other cells have also

been found to present MHC class II in the presence of interferon-γ (87). While class I

molecules present peptides derived from cytosolic proteins, MHC class II present

peptides from extracellular pathogens, degraded in endocytic vesicles (Figure 3).

MHC class II chains are synthesised separately in the endoplasmatic reticulum where

they are brought together and stabilised by the invariant chain protein, which binds the

peptide binding groove and thereby blocks premature peptide binding. The MHC

class II-invariant chain complex is transported in vesicles that eventually fuse with

endocytic vesicles, containing degraded extracellular proteins, where the invariant

chain is released and a peptide is loaded into the groove (26). The peptide binding cleft

of MHC class II shares many features with the corresponding cleft on MHC class I,

but one important difference is that the cleft is open in its ends, allowing longer

peptides to be presented. In addition, the MHC class II groove has pockets where

anchor residues determine whether a peptide is allowed to be presented by a specific

allelic variant of MHC class II (28). The result of the presentation of the MHC class I-

peptide complex for CD 4 positive T-cells is dependent on whether the T-cell is an

TH1 or TH2 cell (117). Mainly through the secretion of interferon-γ, IL-2, TNF-α and

TNF-β, TH 1 cells activate macrophages to destroy intracellular micro-organisms,

initiate T-cell mediated cytotoxicity, and activate B-cells to produce opsonizing

antibodies. The TH1 response is classically demonstrated by the delayed type


22

hypersensitivity reaction. Activated TH2 produce IL-4, IL-5, IL-10 and IL-13, which

in turn, activate B-cells to produce immunoglobulins of different isotypes (145). TH2

cells also activate mast cells and eosinophils. TH1 and TH2 cells are thought to arise

from a common precursor. The course of differentiation is determined by the genetic

background, the physical form of the immunogen, the type of adjuvant, the dose of

antigen and the co-stimulatory factors present at the time of antigen presentation (154).

Tolerance

The combination of a potent immune system with an ingenious capability to generate

an enormous range of B- and T-cell reactivity along with thousands of self peptides

displayed requires the immune system to have a number of mechanisms to prevent

reaction with self structures. Central tolerance develops in the fetus and is based on the

negative selection of developing T-, or B-cells in the thymus or bone marrow, where

cells reacting with self antigens undergo apoptosis (113). There is a need for a second

line of defence against self reactivity. This is called the peripheral tolerance and it is

needed as cells can escape central tolerance mechanisms. These cells are reactive to

self antigens that are of low affinity, or expressed at low concentration, or not at all in

the thymus or bone marrow. Peripheral tolerance is developed postnatally and the

general principles are the need for dual signals to activate T- and B-cells, and low or

absense of exposure to the antigens (172). B-cells require co-stimulation by T-helper

cells to become activated and thus rely on T-cell tolerance, or a “hole” in T-cell

reactivity, for their own tolerance. An autoreactive B-cell that does not receive a co-

stimulatory signal from a TH-cell becomes anergic. There are also Fas ligand

expressing T-cells with specificity for self peptides that induce apoptosis in self

reactive T-, or B-cells (172). Ignorance of self antigens by autoreactive T-cells can

occur if:

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1. The antigen is anatomically sequestered behind an endothelial barrier e.g. the

blood-brain barrier.

2. The antigen is presented in amounts too low to be detected.

3. The antigen is presented on cells with few or no MHC molecules.

T-cells can also become anergic if the antigen is presented by a non-professional APC

lacking the ability to produce a co-stimulatory signal e.g. B7.1 or B7.2. The central

and peripheral mechanisms of tolerance are believed to be our main protection against

autoimmune disease (103).

The “Danger” hypothesis

An alternative model of viewing immune regulation and initiation of immune

responses is called the danger hypothesis (7, 108). This model recognises the deletion

of self reactive lymphocytes in the thymus, but does not see the self/non-self

discrimination as the on/off-button. Instead it stresses the importance of APCs in the

periphery as gatekeepers in the immune system. Autoreactive T-cells need a second

signal from an activated APC to be activated. The APC, according to this model,

becomes activated by endogenous alarm signals if the tissue is in danger. These alarm

signals can be of two principal types: “pre-packaged” as the extracellular exposition of

intracellular cell components e.g. DNA, RNA, mitochondria, or inducible as e.g. heat

shock proteins and IFNα. The type of alarm signal determines which immunological

effector response a given stimuli will provoke.


24

Autoimmunity

In a normal physiological state a number of autoreactive T-, and B-cells circulate in

the body without causing disease. Transient autoimmune reactions are also common

especially in connection with infectious disease or trauma. Autoimmune diseases arise

when the immune system turns itself against self structures in a sustained and

uncontrolled way, resulting in tissue damage or direct pathogenic action by

autoantibodies. The classical definition of an autoimmune disease, formulated in 1957

(181), includes four criteria:

1. The existence of an autoantibody or cell mediated immunity.

2. The identification of the corresponding antigen.

3. The induction of disease in an experimental animal by immunisation with the

antigen.

4. The transfer of disease to a healthy individual by transfer of T-cells, B-cells or

autoantibodies.

Revised criteria have been formulated, but in principle these original criteria are still

valid (149). Autoimmune diseases are common, affecting around 3 percent of the

population, women are more frequently affected (3:1 sex ratio) (79). The autoimmune

diseases can be divided into three classes: systemic disease e.g. systemic lupus

erythematosus (SLE), organ specific destructive disease e.g. insulin-dependent

diabetes mellitus (IDDM) and organ specific non-destructive disease e.g. myasthenia

gravis. Systemic autoimmune diseases are characterised by autoreactivity against cell

constituents present in most cells in the body, e.g. antinuclear antibodies, and a

heterogeneous clinical presentation often mediated through immune complexes. Organ

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25

specific destructive diseases often display tissue specific intracellular enzymes as

autoantigens, e.g. glutamic acid decarboxylase (GAD) or 21-hydroxylase. The disease

leads to a destruction of target cells which is probably cell mediated. Organ specific

non-destructive diseases show autoantibodies against tissue specific cell surface

receptors, e.g. acetylcholine receptor antibodies, directly driving the

pathophysiological process, and the target tissue is not destroyed. The disease

discussed in this thesis, APS I, is a typical organ specific destructive disease, with a

number of target organs. The mechanisms underlying autoimmune diseases are largely

unknown, but some possible mechanisms and risk factors will be discussed below.

Genetic factors

Many autoimmune diseases, especially organ specific diseases show a familial

clustering. Any given individual affected by one autoimmune disease has an increased

risk of being affected by a second autoimmune disease. Different HLA alleles can be

protective or increase the susceptibility to different autoimmune diseases. For instance

the DRB1*0401 allele increases the risk of getting rheumatoid arthritis sevenfold (96),

and the DQB1*0302 allele increases the risk of getting IDDM. With the exception of

the strong association between HLA-B27 and ankylosing spondylitis, most disease

associations have been found with different class II loci (162). It is difficult to

determine exactly which gene is responsible for a particular disease association as

many genes in the MHC region are tightly linked and combinations of alleles are

needed to increase susceptibility (121). The HLA associations are, in principle,

understandable as different HLA alleles have varying preferences when it comes to

“choosing” what peptides to present for T-cells, both in the development of central

tolerance in the thymus and in the presentation for effector T-cells in the periphery.

Substitution of one single amino acid in the peptide binding groove has been shown to


26

have major influence on peptide binding (95). Other genes encoded in the MHC

complex that have been shown to increase the risk of autoimmunity are genes

encoding complement components, especially C1, C2 and C4, in SLE (51, 120). Genes

outside the MHC complex are also thought to contribute to the genetic component in

autoimmune disease. Association studies in human IDDM have resulted in a number

of candidate genes, among them the gene encoding insulin (82). The genetic

components in most autoimmune diseases seem, however, to be very complex and

gene polymorphisms coupled to disease are to be seen as permissive rather than

causing the disease. One exception is APS I, which is a monogenous autosomal

recessive disease with total penetrance and as such, can be of value to study

mechanisms underlying autoimmune disease in general.

Molecular mimicry

The basis for molecular mimicry in autoimmune disease is that a foreign infectious

particle, or food constituent, has a linear or conformational epitope that is homologous

with a self antigen. The foreign particle must be structurally similar enough to share

determinants, but dissimilar enough to provoke an immune response. Several

mechanisms for the autoimmune reaction are possible (6, 103):

1. A viral antigen is presented in complex with MHC class I to a cytotoxic T-cell.

The cytotoxic T-cell is then activated, clonally expanded and can attack other

cells presenting a self-peptide that is homologous to the viral epitope.

2. An ignorant or anergic TH cell with self specificity is activated by a cross reactive

foreign peptide presented in complex with class II and co-stimulatory factors by a

professional APC.

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27

3. The foreign antigen activates a B-cell which proliferates and cross reacts with a

homologous self antigen.

4 . The foreign antigen activates a B-cell which proliferates, undergoes somatic

hypermutation leading to epitope spreading and cross reacts with a homologous

self antigen through epitope spreading.

Once the tolerance is broken by molecular mimicry, the autoimmune process can

continue without the presence of the foreign particle, as tissue damage leads to a non-

physiological exposition of self peptides. A number of homologies between viral or

bacterial epitopes and human autoantigens have been described (126). For example:

the homology between the AChR α subunit (the predominant autoantigen in

myasthenia gravis) and herpes simplex virus glycoprotein D (152), and the shared

determinants of GAD and the P2-C protein of coxackie B virus (11).

Aberrant expression of antigen

Intracellular self-antigens, or antigens normally presented in immunologically

privileged compartments e.g. brain or testis, can under special circumstances be

accessible to the immune system. Infections, trauma or inflammation can break down

vascular or cellular barriers. In sympathetic ophthalmia, trauma against one eye

releases intraocular proteins and an autoimmune reaction against both eyes follows

(159). Another example is that mice infected with coxsackie virus with a tropism for

pancreatic islets develop a chronic autoimmune inflammation against islet cells which

is not related to molecular mimicry (72). Aberrant MHC class II expression in target

cells normally only expressing MHC class I, e.g. due to stimulation by inflammatory

cytokines, may lead to the presentation of intracellular self peptides for CD 4 positive

TH cells.


28

Defect immunoregulation through cytokines

The peripheral tolerance relies on a number immunoregulatory mechanisms leading to

ignorance of self epitopes. Potentially self reactive TH-cells are kept ignorant by TS-

cells, IL-10, TGF-β and prostaglandins. Cytokine imbalance has also been shown to

promote autoimmunity. An experimentally deleted or mutated IFNγ gene in pancreatic

β-cells decrease the autoimmune destruction of the β-cells (175), and in IDDM

patients an increased expression of IFNγ has been demonstrated (73). SLE patients, as

well as their healthy relatives, have a defect IL-10 regulation of TS-cells (64).

Target organ defects

Pre-morbid abnormal target organ function or aberrant MHC expression are

considered to be related to an increased risk of developing an autoimmune disorder.

Animal strains with high susceptibility for autoimmune disease, e.g. EAE receptive

animals, have been shown to upregulate MHC class II expression in target organs

(156). Obese strain chickens, prone to autoimmune thyroiditis, have a defective iodine

uptake in their thyroid gland. This has been experimentally shown to be a permissive

trait to develop thyroiditis (160).

Superantigens

Superantigens are proteins produced by pathogens that have the ability to activate

CD 4 positive T-cells by binding both the outer faces of MHC class II and the Vβ

domain of the TCR. A superantigen has a relative specificity for certain Vβ chains,

and is capable of polyclonally activating 2 – 20 % of all T-cells. It has been proposed

that bacterial or viral superantigens may have a role in the development of rheumatoid

arthritis by activating anergic self reactive T-cells (130).

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29

The TH1/TH2 paradigm

Studies, mainly those involving different animal models, have shown that organ

specific destructive autoimmune diseases have a predominant TH1 cytokine profile,

while systemic diseases show a bias towards TH2 (146). Manipulation of TH

differentiation has resulted in a changed cause of the disease, e.g. in EAE where

addition of an anti-B7.1 antibody reduced the incidence, while anti-B7.2 increased the

severity of the disease (94). It has been proposed that an imbalance in the TH1/TH2

response may be a prerequisite for autoimmune diseases to evolve. This can open the

way for new therapeutic strategies by modulating the TH1/TH2 balance (146).

Apoptosis

Apoptosis, or programmed cell death, is a mechanism for controlled cell destruction

which, in contrast to necrosis, occurs without inflammatory responses. Apoptosis is

mediated via Fas and its’ ligand, Fas L, in the immune system and has an important

role in the modulation of the immune response. Defect regulation of apoptosis could

have a possible role in autoimmunity by several mechanisms (33), (30):

1. Defect elimination of autoreactive lymphocytes.

2. Aberrant cleavage of intracellular autoantigens can result in the presentation of

new, not tolerated, epitopes.

3. In contrast to apoptosis, defective apoptosis can lead to a bias towards perforin

mediated cell destruction which promotes autoimmunity through an uncontrolled

exposition of intracellular material.


30

The existence of Fas mutations in patients with autoimmune lymphoproliferative

syndrome (52) supports the role of apoptosis in autoimmune disease. Experimental

support also exists, e.g. the very low incidence of diabetes in perforin-negative NOD

mice (83).

Autoantigens, autoantibodies and autoreactive T-cells

Self structures identified through reactivity with autoantibodies or self reactive T-cells,

associated with autoimmune diseases, are called autoantigens. A large number of B-

cell autoantigens have been identified through immunohistochemistry,

immunoblotting and screening of cDNA libraries. T-cell autoantigens are harder to

identify and it is difficult to verify the association with a disorder in a large patient

sample, mainly due to the MHC restriction of T-cell reactivity. The effector

mechanisms in autoimmune diseases are mediated both through autoantibodies and

through T-cells. They can be classified in analogy with type II, III and IV

hypersensitivity reactions (38). Type II reactions are antibody mediated with cell

surface antigens resulting in phagocytosis or complement mediated lysis, as in

autoimmune haemolytic anaemia (45), receptor mediated stimulation as in Graves’

disease (14) or receptor blockade as in myasthenia gravis (101). Type III reactions are

immune complex mediated with extracellular antigens, matrix derived or soluble, and

can be exemplified by SLE (92). Type IV reactions are T-cell mediated, organ specific

destructive diseases and the antigens proposed are often intracellular as GAD in

IDDM (12) and 21-OH in Addison’s disease (180).

The role of autoantigens in the aetiology of autoimmune diseases, and the value of

their identification, is not obvious. B-cell activation and subsequent autoantibody

production requires activated TH-cells. The specificity of these TH-cells and the

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specificity of the autoantibody do not necessarily have to be the same, even if spatial

and time factors suggest a shared antigen. In myasthenia gravis, both autoantibody

specificity and T-cell epitopes are predominantly found within the same region of the

α subunit of AChR (140). Insulin and GAD65 are both reported as B-, and T-cell

autoantigens in IDDM (13, 119, 131, 132).

The value of a number of antibodies as disease-specific diagnostic markers is

undisputed. The occurrence of autoantibodies often precedes the clinical onset of the

disease (23), and can thus be used to screen persons at risk of developing disease. The

titres of some autoantibodies, e.g. anti-dsDNA antibodies in SLE (167), are correlated

to disease activity, while others are of less value for monitoring the disease.


32

Autoimmune polyendocrine syndrome type I

Autoimmune polyendocrine syndrome type I (APS I; OMIM acc no. 240300), also

known as autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy

(APECED), is an organ-specific autoimmune disease. The disease affects multiple

organs (Table 2) and is characterised by circulating tissue-specific autoantibodies

(Table 3).

Neufeld

1980

Brun

1982

Ahonen

1990

Zlotogora

1992

Betterle

1998

Cases 106* 166* 68 23 41

Origin Mixed Mixed Finnish Iranian Jewish Italian

Sex ratio f/m 1.4 1.7 0.8 1.1 2.4

Chronic candidiasis 78% 75% 100% 17% 83%

Hypoparathyroidism 82% 89% 79% 96% 93%

Adrenal insufficiency 67% 60% 72% 22% 73%

Hypogonadism 12% 45% 36% 36% 43%

Alopecia 26% 20% 29% 13% 37%

Intestinal dysfunction 24% 25% 18% NR 15%

Vitiligo 9% 4% 13% NR 15%

Autoimmune hepatitis 11% 9% 12% NR 20%

Pernicious anaemia 15% 16% 13% 9% 15%

Thyroid disease 10% 12% 4% 4% 10%

IDDM 2% 1% 12% 4% 2%

*a majority from a literature search; NR: not reported

Table 2. Clinical characteristics of different populations of APS I patients.

The disease is rare and can be traced back in the literature as sporadic case reports for

almost 100 years (27, 37, 40, 41, 98, 136, 166). The disease was recognised as a

familial syndrome in the mid-forties (161), and reported under different names during

the following decades (44, 75, 116, 163, 177, 182). A more precise definition of the

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syndrome, based on a large clinical review, was made in 1980 (122). APS I was then

defined as the occurrence of two out of three cardinal symptoms: chronic

mucocutaneous candidiasis, hypoparathyroidism and autoimmune Addison’s disease,

or the occurrence of one of these three symptoms in siblings to APS I patients. APS I

was separated from other polyglandular syndromes such as APS II and APS III. The

wide clinical variation in APS I referring to the number of, type of and age at onset for

different components, was thoroughly described in a large Finnish study of 68 patients

in 1990 (5).

As will be discussed later, APS I is enriched in some genetic isolates e.g. Finland,

Sardinia and among Iranian Jews. The clinical components vary in frequency between

different populations (5, 17, 185). Although the clinical course of the disease is highly

variable, the three main components usually develop in a specific order with

candidiasis occurring before the age of 5, hypoparathyroidism before the age of 10 and

finally Addison’s disease usually before the age of 15 (5, 17). Note that all patients do

not develop all three main components, and that only two out of three are required for

the diagnosis. In general, the earlier the age at onset of the first symptom indicates a

predisposition to developing a higher number of components, and conversely, patients

who have a late first manifestation of the disease are more likely to have fewer

components (5).

In addition to the three main components, APS I patients develop other endocrine and

non-endocrine symptoms in varying frequencies (Table 2). The different components

of APS I will be discussed in detail below.


34

Related autoantigens Reference

Chronic candidiasis -

Hypoparathyroidism Calcium sensing receptor 100, 64

Adrenal insufficiency 21-OH (17-OH, SCC) 180 (93, 179)

Hypogonadism SCC 178

Alopecia TH 67

Intestinal dysfunction TPH 49

Vitiligo SOX9, SOX10 68

Autoimmune hepatitis CYP1A2, CYP2A6, AADC 35, 36, 58

Pernicious anaemia H+,K+-ATPase 84

Thyroid disease TPO, TG 17, 165

IDDM GAD65, ICA, insulin, IA-2 65, 89, 170

Table 3. Manifestations in APS I and related autoantigens

Mucocutaneous candidiasis

Chronic mucocutaneous candidiasis is often the first clinical manifestation of APS I

and it can appear very early in childhood. The diagnosis is based on microscopic

examination and culturing for Candida albicans. The candidiasis affects the nails, the

dermis and the oral, vaginal and oesophageal membranes. In most patients the

candidiasis responds to therapy and is restricted to a limited part of the body surface.

In rare cases the candidiasis can cause serious clinical problems. General candidiasis

after immunosuppressive treatment has been reported to be the cause of death in one

APS I patient (17). The occurrence of candidiasis in APS I has been seen as an

evidence for a T-cell deficiency (34, 104) and experimental studies have shown

defective T-cell suppressor functions (10). Other studies have shown that APS I

patients lack “anti-candidial factors” in their serum (102), but that they are capable of

producing anti-candidial antibodies indicating a correct B-cell response (138).

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35

Hypoparathyroidism

Hypoparathyroidism is often the first endocrine component of APS I and usually

appears before the age of 10 (mean 7.5 yr.) (5, 17). Patients with subnormal plasma

calcium levels, supranormal plasma phosphate levels and normal renal function are

diagnosed as having hypoparathyroidism (5). Parathyroid autoantibodies by

immunofluorescence (22), and autoantibodies reacting with the extracellular domain

of the calcium sensing receptor (100) have been reported. These results have not been

confirmed in other studies. On the contrary, a recent study indicates that we are still

lacking a good serologic test for hypoparathyroidism (164).

Addison’s disease

There are a number of causes to Addison’s disease, or adrenal insufficiency.

Historically, and world-wide, tuberculosis is probably the major cause of Addison’s

disease. Other causes are metastatic malignancies, haemorrhage, sarcoidosis or rare

hereditary diseases such as adrenoleucodysdrophy. In the western world, adrenal

insufficiency is most often caused by an autoimmune destruction of the suprarenal

cortex. The term “Addison’s disease” refers to the autoimmune variant of the disease

in this paper. Addison’s disease most often occurs as an isolated disease, but can also

be one component of APS I, or other syndromes. The laboratory diagnosis of

Addison’s disease is defined as: low plasma cortisol, and/or low urinary cortisol,

and/or high plasma ACTH concentrations, and/or defect response to acute and/or

prolonged ACTH stimulation. Autoantibodies against the adrenal cortex in idiopathic

Addison’s disease have been described since the 1950s (8, 21). 21-OH has been

identified as the major autoantigen in Addison’s disease (180). 17α-OH and side-chain

cleavage enzyme (SCC) are presented as an adrenal autoantigen in APS I patients with

Addison’s disease (93) (179). A recent multivariate analysis of the diagnostic values of


36

different autoantigens in APS I points out 21-OH and SCC as associated with

Addison’s disease in APS I (164). In APS I, the disease has an earlier age at onset

(peak 10-12 yr.) compared to isolated Addison’s disease or Addison’s disease in APS

II (peak 20-30 yr.) (122, 123). Historically, Addison’s disease has been a major cause

of death in APS I, but with modern substitution therapy the clinical management is

satisfactory. As in isolated Addison’s disease, future management will probably

include substitution with androgens to women with Addison’s disease, in addition to

the usual treatment with glucocorticoid and mineralocorticoid supplementation (9, 59).

Hypogonadism

Hypogonadism in APS I shows a female predominance and is mainly diagnosed at

puberty, but can also be detected as secondary amenorrhea before the age of 40. The

diagnosis of ovarian failure is based on amenorrhea, high levels of FSH or LH, and

slow, absent or regressive pubertal development. Testicular failure is diagnosed

through high base-line, or post-GRH stimulatory, levels of FSH or LH, and low

testosterone levels (5). Both 17α-OH and SCC have been proposed as autoantigens

associated with gonadal failure (18, 171, 178). A recent multivariate analysis of ten

different autoantibodies in a large sample of APS I patients shows that SCC alone is

statistically associated with hypogonadism (164).

Alopecia

Alopecia is present in APS I both as alopecia areata and as the more severe form

alopecia totalis. Alopecia areata is defined as a transient patchy hair loss on the scalp.

When the hair loss covers the whole scalp and becomes permanent the condition has

developed into an alopecia totalis. Alopecia totalis is twice as common as alopecia

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37

areata in APS I (69). Autoantibodies against hair follicles have been shown, by

indirect immunofluorescence, to be associated with alopecia totalis in APS I (69).

Intestinal dysfunction

Different descriptions and names such as malabsorbtion (5, 17), celiac syndrome (37,

44), constipation (29), steatorrhea and diarrhoea (41, 78, 116) have been used in the

literature to characterise the intestinal dysfunction seen in APS I. This illustrates the

great variations of the clinical manifestations of the gastrointestinal dysfunction seen

in APS I. We define the intestinal dysfunction as periodic steatorrhea, diarrhoea or

severe constipation. The dysfunction is often therapy resistant and results in marked

weight loss and/or growth failure. APS I patients with intestinal dysfunction as their

first manifestation of the disease generally have a high number of disease components

(5). Although the malabsorbtion often is severe no specific shortages are seen. Faecal

fat is often elevated. Exocrine pancreas insufficiency (176) and low levels of

cholecystokinin (71, 77) have been reported. Some reports have described a

covariation between intestinal symptoms and hypocalcemia (5, 71, 135). The intestinal

symptoms have been explained in the literature as being secondary to hypocalcemia

(71), candidial overgrowth (153), exocrine pancreas defect, or intestinal

lymphangiectasia (16). Substitution with pancreatic enzymes, normalisation of serum

calcium (71) and immunosuppressive treatment with cyclosporine (176) or

methylprednisolone (128), has been proposed for treatment.

Vitiligo

Vitiligo is characterised by loss of pigment formation by melanocytes in the skin.

Different patterns of distribution are acro-facial, focal, segmental and generalised

vitiligo. In APS I vitiligo often occurs as symmetrical depigmented patches on the


38

face, neck, extensor surfaces of hands, wrists and legs and in the axillae. Complement-

fixing autoantibodies have been described as associated with vitiligo in APS I (137).

Recently, the transcription factors SOX 9 and SOX 10, expressed in melanocytes, have

been identified as autoantigens associated with vitiligo in APS I (68).

Autoimmune hepatitis

Autoimmune chronic active hepatitis is probably the most feared component of APS I.

The clinical course is highly variable, ranging from asymptomatic changes of liver

enzymes to fulminant hepatitis with a fatal outcome (5, 112). The histopathologic

picture resembles that of idiopathic autoimmune chronic active hepatitis and markers

of viral hepatitis are not present. Autoantibodies against cytochromes P4501A2,

P4502A6 and aromatic L-amino acid decarboxylase (AADC) have been associated

with autoimmune hepatitis in APS I (35, 36, 58). Treatment is based upon

immunosuppressive therapy in combination with corticosteroids (112). The

determination of liver enzymes should be done with regularity to promote early

detection and intervention.

Pernicious anaemia

Pernicious anaemia is secondary to vitamin B12 malabsorbtion due to a shortage of

intrinsic factor caused by a disturbed parietal cell function, coupled to autoimmune

gastritis. Clinical features are megaloblastic anaemia and achlorhydria. Lymphocytic

infiltrates and autoantibodies directed towards parietal cells have been found (116,

122). H+K+-ATPase and intrinsic factor have been described as autoantigens

associated with autoimmune gastritis (84, 150).

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39

Insulin-dependent diabetes mellitus

Insulin-dependent diabetes mellitus (IDDM) shows a great variation in occurrence

between different populations of APS I patients, with frequencies ranging from 1 to 12

percent (5, 17, 122, 143). Autoantibodies against glutamic acid decarboxylase

(GAD65), tyrosine phosphatase IA-2 (IA-2), insulin and/or islet cell antibodies (ICA)

are present in APS I patients with IDDM (65, 89, 170). In a multivariate analysis of

the clinical associations of a panel of autoantigens in APS I, only IA-2 was shown to

have a predictive value (164). AADC which has been cloned from a β-cell cDNA

library as an autoantigen in APS I does not seem to correlate to IDDM (76, 147), but

surprisingly, it is statistically associated with autoimmune hepatitis (58).

Minor components

Ectodermal dystrophy with hypoplastic enamel of the teeth, pitted nail dystrophy and

tympanic membrane dystrophy affects 77, 52 and 33 percent of APS I patients,

respectively (5). Keratoconjunctivitis is seen in 8 - 41% of APS I patients as corneal

opacities, bulbar conjunctival injection and corneal neovascularisation (5, 17, 109).

Autoimmune thyroid disease, mainly Hashimoto’s thyroiditis, has been reported in

2 – 13% of APS I patients (5, 17, 85). Thyroid autoantibodies are present in a high

proportion of APS I patients with and without thyroid disease (17, 165). The

prevalence of acquired asplenia, due to a progressive destruction of the spleen, has not

been systematically examined, but has been described in a high proportion in small

sample of APS I patients (55, 134). Cholelithiasis is also reported as over-represented

in APS I patients (55).


40

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41

identified by positional cloning and named autoimmune regulator (AIRE) (118, 168).

The AIRE gene encodes a 545 aa, 57.5 kDa protein with a conserved nuclear

localisation signal, two plant homeodomain (PHD)-type zinc fingers, four LXXLL

motifs, one SAND and one HSR domain (Figure 4). These features are all markers for

nuclear, DNA-binding proteins suggesting a role for AIRE in transcriptional control.

AIRE has also been shown in vitro to interact with transcriptional coactivators and

activate transcription (19, 139).

AIRE is expressed mainly in thymic epithelial cells and to a lesser degree also in the

spleen, lymph nodes, fetal liver and mononuclear leukocytes (20, 118, 186). Recent

studies in mice also demonstrate Aire expression in a variety of tissues outside the

immune system e.g. kidney, testis, adrenal glands, liver and ovary (66). The sub-

cellular localisation of wild type AIRE has been described mainly as restricted to the

nucleus, but a cytoplasmatic expression has also been described (20, 70, 144). In the

nucleus AIRE is found in dots resembling, but not co-localising with, the nuclear

bodies formed by e.g. promyelotic leukaemia protein (PML), Sp 100 or Sp140.

The function of AIRE is not yet understood. The expression pattern in man and

experimental studies using murine Aire, show that AIRE is likely to be involved in the

thymic selection process and so indicates a role for AIRE in the induction of self-

tolerance (186).

To date, 29 different disease causing mutations of AIRE have been described which

are clustered in four mutational hotspots. Although 5 – 10 % of patients who fulfil the

clinical criteria for APS I lack mutations in AIRE exons, or only have one allele

mutated in the exonic parts, the general agreement is that both AIRE alleles must be


42

mutated to cause the disease (19). A possible explanation for these APS I patients with

no detectable mutations is probably that mutations do exist in promoter regions or

intronic parts of the AIRE gene which have not been examined by sequencing. In

Finland, Sardinia and among Iranian Jews marked founder effects are seen. 89% of

Finnish APS I chromosomes have the major Finnish mutation, R257X, 92% of

Sardinian APS I chromosomes have the common Sardinian mutation, R139X, and

100% of APS I patients among Iranian Jews are homozygous for a single amino acid

change, Y85C (19, 148). Most mutations are, mainly through frame shifts, resulting in

truncated forms of AIRE, lacking DNA-binding domains or nuclear receptor binding

domains.

In experimental studies of sub-cellular localisation of mutated AIRE variants, an

altered intracellular distribution pattern of the protein was found (19, 144). Almost all

mutations examined, except the common Iranian missense mutation, resulted in a

cytoplasmatic location of the mutated protein. In transcription activation assays

truncated mutated AIRE constructs showed no, or markedly lowered, activation of

transcription. The transcriptional activation ability of the AIRE construct with the

common Iranian mutation was the same as wild type AIRE. The fact that the

experimental behaviour of the Iranian mutation does not differ much from wild type

AIRE may explain the milder clinical phenotype that can be noticed among Iranian

Jewish APS I patients.

Autoimmune polyendocrine syndrome type II

The diagnosis of autoimmune polyendocrine syndrome type II (APS II) requires the

presence of Addison’s disease plus autoimmune thyroid disease and/or insulin-

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43

dependent diabetes mellitus (IDDM) (122). Other endocrinopathies and associated

autoimmune diseases are also present, at low frequencies. APS II shows a female

predominance (123, 165), is inherited in a dominant fashion (47), and has been shown

to be associated with HLA-DR3 and -DR4 (105, 125). The age at onset for Addison’s

disease is higher in APS II (peak at age 20 – 30) compared to APS I (peak at age 10 –

12) (123).


44

Pteridine dependent hydroxylases

Tryptophan hydroxylase (TPH; EC 1.14.16.4), tyrosine hydroxylase (TH; EC

1.14.16.2) and phenylalanine hydroxylase (PAH; 1.14.16.4) together constitute the

enzyme family of tetrahydrobiopterin dependent hydroxylases. The three enzymes are

closely related, share functional and structional characteristics and are thought to have

evolved from a common progenitor gene (63).

Structure

The functional structures of the enzymes are homologous with an N-terminal

regulatory domain, a central catalytic domain and a C-terminal tetramerization

domain. The regulatory domain is specific for each enzyme with a 20% linear

homology between the three enzymes. It determines substrate specificity and thus

reflects the unique properties of each enzyme. The central catalytic domain, containing

the active site, is highly conserved and has an overall homology of around 65%. TPH

is a 230 kDa tetramer consisting of identical subunits, each with a molecular mass of

58 kDa. TH consists of four 55-59 kDa subunits, as four isoforms (TH 1 - 4) due to

alternative splicing, forming tetramers with molecular masses of 204-217 kDa. PAH is

found in an equilibrium between tetrameric and a dimeric forms composed of 50 kDa

subunits (74). The crystal structures for the catalytic domain of TH and complete PAH

have been determined (50, 91). The structures are essentially identical, and the TPH

structure can be predicted, based on the structures of TH and PAH (174). The crystal

structure determinations are not only useful in understanding the regulation of

enzymatic activity, but may also be of great value in determining the possible

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45

conformational epitopes involved in autoimmunity directed against these enzymes

(62).

Function and tissue distribution

TPH, TH and PAH are monooxygenases, incorporating one atom of oxygen into the

substrate and reducing the other atom to water, and thereby catalyze the hydroxylation

of different amino acids. Tetrahydrobiopterin (BH4) supplies the two electrons

required. Molecular oxygen and ferrous iron are also needed in the reaction (Figure 7).

The regulatory properties differ in detail between the enzymes, but in general they are

regulated by their substrates, BH4 and phosphorylation of serines (53). TPH and TH

have central roles in the biosynthesis of the neurotransmitters serotonin and dopamine

(Figure 7). TPH catalyzes the hydroxylation of tryptophan into 5-OH tryptophan and is

the rate-limiting enzyme in the synthesis of serotonin. It is expressed in serotonergic

cells in the central nervous system and the intestine (183). TH is the rate limiting

enzyme in the biosynthesis of catecholamines where it converts tyrosine into L-Dopa,

and is mainly found in the adrenal medulla and catecholaminergic neurons throughout

the body. PAH is primarily produced in the liver where it catalyses the conversion of

phenylalanine to tyrosine. This serves two purposes: it provides an endogenous supply

of tyrosine, making tyrosine a non-essential dietary component, and it is also rate

limiting in the catabolism of phenylalanine (74).

TPH, PAH and TH in disease

Mutations in PAH are the most common cause of phenylketonurea (PKU). In PKU,

serum levels of phenylalanine are elevated to toxic levels causing brain damage

leading to mental retardation. A second result of PAH dysfunction is that tyrosine

becomes an essential dietary amino acid. The treatment is based on a diet low in


46

phenylalanine and supplemented with tyrosine (48). Defects in the production, or

recycling, of BH4 also leads to hyperphenylalaninaemia in combination with TPH and

TH deficiencies. These conditions have a more severe clinical presentation than

isolated PAH dysfunction. TH defects and polymorphisms have been coupled to

bipolar affective disorders, schizophrenia and parkinsonism (61). Hitherto, no disease

couplings have been made with TPH dysfunction, although theoretically TPH defects

could very well be involved in affective disorders, schizophrenia, migraine, drug abuse

and intestinal movement disorders (115).

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C U R R E N T I N V E S T I G A T I O N

Results

The identification of TPH as an autoantigen in APS I (paper I)

Periodic gastrointestinal dysfunction represents a significant management problem in

APS I, affecting 25-30% of the patients. Before this study, the pathogenesis of the

gastrointestinal dysfunction was largely unknown, but explained in the literature as

being secondary to hypocalcemia (71), candidial overgrowth (153), exocrine pancreas

defect or intestinal lymphangiectasia (16). We undertook this study with a belief that

the intestinal dysfunction, as well as the majority of the other manifestations of APS I,

was of autoimmune origin. The aim of the study was to identify a potential intestinal

autoantigen associated with APS I and possibly other autoimmune intestinal diseases.

A commercially available, human duodenal cDNA library was immunoscreened with

sera from seven APS I patients. A positive clone was identified as tryptophan

hydroxylase (TPH, EC 1.14.16.4) and was used for in vitro transcription/translation

(ITT) followed by immunoprecipitation with sera from 80 APS I patients. Forty-eight

percent (38/80) of the APS I patients had TPH antibodies (Ab), whereas in a large

number of sera from patients with other autoimmune diseases (n=372) and healthy

blood donors (n=70) no reactivity against TPH was detected. A correlation with

clinical symptoms showed that 89% (17/19) of APS I patients with gastrointestinal

dysfunction were TPH-AB positive, compared to 34% (21/61) without (p < 0.0001).

Immunostainings of normal human small intestine with patient sera and specific

antibodies were performed. These showed that TPH-Ab positive APS I sera


48

specifically immunostain TPH containing enterochromaffin cells in normal duodenal

mucosa. We also observed that 3/8 APS I patient sera stained goblet cells and that 3/8

patient sera reacted with Paneth cells (Figure 5). When immunostaining duodenal

biopsies from APS I patients with gastrointestinal dysfunction and TPH-Abs, a total

absence of enterochromaffin cells was seen. Finally, enzyme inhibition assays

demonstrated that TPH-Ab positive APS I sera almost completely inhibited TPH

activity at a dilution of 1:100.

Figure 5. Immunostainings of normal human duodenum with APS I showing

immunoreactivity with goblet cells (left), EC cells and Paneth cells (right).

In this study we identified TPH as an endogenous intestinal autoantigen in APS I and

established a statistical association between TPH-Ab and gastrointestinal dysfunction.

We also confirmed the reactivity against TPH with enzyme inhibition assays and

immunohistochemical stainings of normal and affected tissues.

Olov Ekwall

49

TPH antibodies in other autoimmune intestinal diseases (paper II)

We reported the identification of TPH as an intestinal autoantigen in APS I in paper I.

The gastrointestinal symptoms in APS I are in some respects related to both the

clinical presentation and the suggested autoimmune background of more common

conditions such as inflammatory bowel disease, irritable bowel syndrome and celiac

disease. Autoimmune enteropathy in children and paraneoplastic pseudoobstruction of

the gut are two rare disaeses with clinical or pathogenic features related to the

intestinal disease in APS I. In paper II, we wanted to find out if TPH-antibodies are

present in these other intestinal diseases with a possible autoimmune pathogenesis.

Sera from 22 patients with ulcerative colitis, 36 patients with Crohn’s disease, 47

patients with irritable bowel syndrome, 25 patients with celiac disease, 3 children with

autoimmune enteropathy and two patients with ANNA-1 seropositive paraneoplastic

pseudoobstruction of the gut were screened for the presence of TPH-antibodies using

ITT and immunoprecipitation of TPH (97, 114, 155). Immunoreactivity against TPH

could not be detected in sera from these patient groups. This illustrates the high

specificity of TPH-antibodies as a marker for gastrointestinal disease in APS I.

The identification of TH as an autoantigen in APS I (paper III)

Ectodermal manifestations in APS I such as alopecia, vitiligo, nail and enamel

dystrophy are frequent. Alopecia areata, characterized by sudden patchy hair-loss, has

been reported in frequencies varying from 13 to 37% in different populations of APS I

patients (5, 17, 185). In an earlier study we showed that APS I patients with alopecia

have autoantibodies directed against hair follicles (69). In this investigation, we set out

to identify the antigen responsible for this reactivity.


50

A λ-ZAP EXPRESS cDNA library was constructed from normal human scalp and

immunoscreened with serum from an APS I patient in a 1:3000 dilution. Ten positive

clones were identified, and one of them coded for TH isoform 2. The TH clone was

used for ITT followed by immunoprecipitation with sera from 94 APS I patients from

Finland, Sweden, Norway and Italy. Forty-four percent (41/94) of the APS I patients

showed TH reactivity, whereas in a large number of sera from patients with IDDM

(n=224), Addison’s disease (n=20), and healthy blood donors (n=65) no reactivity

against TH was detected. A correlation with clinical symptoms showed that 62%

(18/29) of APS I patients with alopecia were TH-Ab positive, compared to 35%

(23/65) without (p = 0.02). No correlation with any of the other APS I components

was found. Western blot with all four TH isoforms (TH1-4) showed equal reactivity

with all isoforms. In analogy with the findings regarding TPH antibodies, although

less pronounced, TH reactive APS I sera inhibited TH enzyme activity in vitro in an

enzyme activity assay. Although TH expression in keratinocytes and hair follicles has

been described using RT-PCR (31, 32), we were not able to stain keratinocytes or hair

follicles with APS I sera or specific TH antibodies.

In conclusion, TH was identified as a dermal autoantigen in APS I, significantly

associated with alopecia. We also demonstrated that TH-Ab positive sera from APS I

patients inhibit TH activity.

Pteridine dependent hydroxylases as autoantigens in APS I (paper IV)

In papers I and III we identified TPH and TH as autoantigens in APS I associated with

intestinal dysfunction and alopecia, respectively. These two enzymes, together with

phenylalanine hydroxylase (PAH), constitute the group of biopterin dependent

Olov Ekwall

51

hydroxylases, which all are involved in the biosynthesis of neurotransmitters. Since

TPH, TH and PAH display a high linear and conformational homology (74, 174), we

wanted to investigate if APS I patients also have antibodies against PAH. We were

also interested in to what extent cross-reactivity occurred between the three enzymes.

A clone encoding PAH was used for ITT followed by immunoprecipitation with sera

from 94 APS I patients and 70 healthy controls. Twenty-seven percent of the APS I

patients had PAH antibodies. No reactivity was detected in the controls. No

statistically significant associations with the main clinical components of APS I were

found with PAH antibodies. A weak correlation with CAH (p=0.065) was observed.

Altogether 59 sera from the 94 APS I patients reacted with at least one of TPH, TH or

PAH, while 35 showed no reactivity. Nineteen of the sera contained antibodies

towards all enzymes, 12 to TPH only and 12 to TH only. No sera showed antibodies

that reacted solely to PAH. An immunocompetition assay was designed in which

[35S]-methionine labeled and non-labeled TPH, TH and PAH were expressed by ITT.

A fixed amount of labeled protein was mixed with increasing proportions of non-

labeled proteins, and an immunoprecipitation was performed. In this way the ability of

the non-labeled protein to compete with the labeled protein for antibody binding could

be determined. We demonstrated that the reactivity against PAH represents a cross

reactivity with TPH while antibodies against TPH and TH are directed towards

epitopes unique for the two enzymes.


52

Discussion

The identification of TPH as an intestinal autoantigen in paper I and the subsequent

finding of the absence of TPH containing EC cells in patient biopsies strongly suggests

that the gastrointestinal dysfunction seen in APS I is not secondary to other disease

components. On the contrary, the deletion of TPH containing EC cells in an otherwise

intact mucosa suggests a very specific immune attack against these cells. The

possibility of a congenital EC cell aplasia, or specific EC cell apoptosis, leading to a

loss of tolerance against TPH cannot be completely excluded. However, this is in

conflict with preliminary observations in an APS I patient with severe gastrointestinal

dysfunction and loss of TPH containing EC cells. In this patient Cyclosporin A

treatment resulted in clinical remission as well as EC cell reappearance (176) (Cheri L

Deal, Montreal, personal communication).

In the gut, serotonin is classically believed to regulate motility and serotonin depletion

in mice has been shown to result in diarrhoea and decreased glucose uptake (90).

Serotonin receptors have also been found in the pancreas, and an enteropancreatic

communication mediated by serotonin has been proposed (86). One study has shown a

role for EC cell derived serotonin in mediating cholecystokinin (CKK) independent

stimulation of pancreatic secretion in response to luminal stimuli (99). The clinical

presentation of the gastrointestinal dysfunction in APS I is heterogeneous and

characterised by weight loss, fat malabsorbtion, diarrhoea, constipation, abdominal

cramps, growth failure and exocrine pancreas insufficiency in varying frequencies.

The loss of serotonin producing EC cells in APS I may explain both a dysfunction of

the exocrine pancreas and a dysregulation of intestinal motility. You can also speculate

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53

that the EC cells and/or serotonin have an as yet unknown, regulatory function for

energy absorption as some patients only suffer from marked weight loss, without

specific shortages. An interesting clinical observation is that an APS I patient with

pronounced malabsorbtion, frequent diarrhoea and marked weight loss regained

weight and normalised her defecation habits when treated with citalopram, a serotonin

reuptake inhibitor (O Kämpe, personal communication).

A recent case report describes an APS I patient with severe malabsorbtion in whom a

deficient CKK secretion, due to a transient loss of CKK producing EC cells, was

observed (77). Analysis of TPH antibodies or specific immunostaining for serotonin

producing cells was not performed. CKK deficiency has been reported before (71),

and treatment with CKK substitution has given varying clinical results (71, 176). One

can not exclude the possibility that CKK and serotonin are produced by the same EC

cells and that TPH autoreactivity can induce a loss of both serotonin and CKK

production.

A large group of autoantigens are the intracellular enzymes, e.g. GAD65 and 21-OH.

It is a common feature of the corresponding autoantibodies to inhibit the activity of the

autoantigen. The ability of APS I patient sera to decrease the enzymatic activity of

both TPH and TH in vitro is striking (Papers I and III) and it is tempting to believe that

the autoantibodies execute their action in vivo by directly affecting enzyme activity.

However, only anecdotal reports exist of autoantibodies having intracellular activity

(141) and the in vitro results can not be extrapolated to the in vivo situation without

further experimental evidence (107). Still, the observed enzyme inhibition patterns

strengthens the role for TPH and TH as autoantigens of importance in APS I. It is also


54

noteworthy that PAH-Ab positive patient sera, possibly reflecting a cross-reaction with

TPH, does not affect PAH activity (Paper IV).

In immunostainings, three out of eight APS I patient sera showed immunoreactivity

against intestinal goblet cells (Figure 5). We do not know the significance of this

finding, but it is also interesting that sera from patients with ulcerative colitis and

Crohn’s disease are reported to have antibodies against goblet cells (54). The identity

and pathogenic role of the goblet cell antigen in inflammatory bowel disease (IBD) has

not been determined. If postulating that APS I patients and IBD patients recognise the

same goblet cell structure, APS I sera may be used to identify the antigen also of

interest for IBD. APS I sera, with high titre, high affinity, autoantibodies have been

proved suitable for immunoscreening cDNA libraries (49, 67, 147). Another finding

with unknown significance is that Paneth cells were also recognised by three out of

eight APS I patient sera (Figure 5). Paneth cells secrete small antimicrobial peptides

called cryptdins into the lumen and thus participate in the innate immune system

(127). An impaired Paneth cell function could, hypothetically, lead to a disturbed

microbial environment in the lumen.

We have used a strategy based on immunoscreening of cDNA libraries in the

identification of TPH and TH as autoantigens. Both these antigens are expressed in a

small fraction of cells in duodenum and scalp, respectively. Initial immunoblotting

experiments using whole tissues failed to show reactivity, probably because the

expression levels of these infrequently expressed proteins were below the detection

level. This illustrates the suitability of the immunoscreening methodology for

identifying rare proteins.

Olov Ekwall

55

Approximately 10 percent of APS I patients present with gastrointestinal dysfunction

as their first manifestation of the syndrome and they usually have a more severe form

of APS I (5). Since TPH antibodies are not found in other diseases with a similar

clinical presentation (Paper II), measurement of TPH-antibodies can be a valuable

diagnostic tool to identify APS I patients among young children presenting with

atypical gastrointestinal disease. An early diagnosis is desirable as it enables an early

treatment of other components of the syndrome as soon as they appear.

In paper III, TH is identified as an APS I autoantigen associated with alopecia. TH

exists in four alternatively spliced isoforms, TH1-4, which are structurally almost

identical with only minor differences in the N-terminal regulatory domain. APS I sera

do not discriminate between the four isoforms. TH is mainly expressed in the central

nervous system (CNS), the sympathetic nervous system and the adrenal medulla where

it converts tyrosine into L-dopa. Interestingly, TH expression has also been found, by

RT-PCR, in human hair follicles and keratinocytes (31, 32), and a role for TH in

keratinocyte differentiation has been suggested (151). We were unable to immunostain

hair follicles or keratinocytes by use of specific TH antibodies or APS I sera, but nerve

endings adjacent to hair follicles were clearly TH positive. The number and density of

TH containing nerve fibres has been reported to fluctuate in a hair cycle dependent

manner (25) and an autoimmune attack against TH containing cells may affect hair

growth.

Twenty-seven percent of APS I patients (25/94) had antibodies reacting with PAH

(Paper IV). All these patients also reacted with TPH, and some of them reacted with

TH as well. In immunocompetition experiments PAH reactivity could always be

inhibited by adding TPH, while adding TH did not affect PAH reactivity. Nor did


56

PAH or TPH inhibit TH reactivity. No inhibition of PAH activity in vitro was seen

when adding PAH-Ab positive APS I sera. TPH and TH reactivities acted

independently of each other, both in the general reactivity pattern as well as in

immunocompetition experiments. These observations together strongly suggest that

the PAH reactivity seen is reflecting a cross-reactivity with TPH and that TPH and TH

antibodies are specific for each enzyme. The autoantibodies in APS I are not

monoclonal, but possibly oligoclonal. The partial inhibition of TPH reactivity seen

when adding PAH possibly reflects that some epitopes are shared and some are unique

for TPH. In a theoretical model, visualised in figure 6, three subsets of antibodies

exist. One only reacting with TH, one only reacting with TPH, and one primarily

reacting with TPH but also cross reacting with PAH through shared epitopes. These

shared epitopes are presumably located in the catalytic domain as the most widespread

homologies are found there and because cross reactivity with the regulatory domain

probably would have resulted in an inhibiting action of the cross-reacting antibodies.

It is tempting to interpret the weak, not statistically significant, correlation between

PAH-Abs and chronic active hepatitis (CAH) seen in paper IV as a possible

pathogenic mechanism considering that PAH is mainly expressed in the liver. The

possibility that these cross-reacting antibodies may have an effect in vivo, if PAH is

displayed on the cell surface, cannot be ruled out. When screening a large number of

patient sera from patients with both autoimmune and infectious hepatitis we were not

been able to find PAH antibodies (unpublished data).

The possibility of both linear and conformational epitopes makes the determination of

epitopes responsible for initiation of autoimmune responses difficult. There are still

only limited numbers of proteins for which we know the three dimensional structure.

Olov Ekwall

57

The crystal structures for TH and PAH have been determined and the TPH structure is

soon to be revealed (61, 91). The combination of this knowledge and the intriguing

patterns of reactivity against TPH, TH and PAH in APS I patients, should make it

possible to perform epitope mappings, taking both linear and conformational epitopes

in account. Chimeric proteins with regions of interest substituted between the

enzymes, could be constructed and expressed in vitro to search for structural as well as

linear epitopes.

Figure 6. Cross reactivity hypothesis. Immunisation with TPH can either result in the

generation of TPH specific antibodies only (I), or antibodies reacting with both TPH

and PAH through shared epitopes (II). TH immunisation only seems to induce TH

specific antibodies (III).


58

Why does the immune system target intracellular enzymes? It is apparent that enzymes

of vital importance for the function of the target organ, or target cell, are chosen as

autoantigens in APS I and in other tissue specific autoimmune diseases (15, 157).

Figure 7. The figure outlining the biosynthesis of serotonin, dopamine and GABA

illustrates that key enzymes in the synthesis of neurotransmitters are autoantigens in

APS I. Cofactors required for the reactions mediated by TPH, TH and PAH are

marked.

In this case, a whole family of enzymes, all involved in the generation of

neurotransmitters, are targeted by the immune system (Figure 7). Speculations can be

made about explanations for this preference by the immune system:

1. Key enzymes are evolutionary conserved and often very homologous between

species. This increases the risks of autoimmunity induced by cross-reactivity with

infectious agents.

2. The central tolerance for enzymes is based on the presentation of the enzyme by

itself in the thymus or bone marrow. In the periphery, the enzymes are displayed

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59

bound to their substrates, and may then have a changed conformation leading to

the exposition of new, not tolerated, epitopes for the immune system.

3. The tolerance of intracellular enzymes that are normally expressed at low levels in

subpopulations of cells is primarily based on ignorance. This means that there are

potentially self reactive T-cells under normal conditions, but they are ignorant

since the number of self epitopes presented at a given time do not reach the

threshold of activating these cells. If, for instance, due to trauma, infection or

target organ stress, the amount of enzyme presented for the self reactive T-cells

increases over the threshold, T-cells are activated and the autoimmune process

starts.

In some autoimmune diseases, e.g. IDDM and myasthenia gravis, it has been shown

that autoantibodies and T-cells are directed against the same autoantigen (119, 131,

140). In APS I, no such T-cell studies have been reported, so it is still difficult to

conclude if B-cell autoantigens such as TPH, TH and PAH are just representing an

epiphenomenon, or if they represent structures that initially trigger the immune

response. The role of the autoantibodies as diagnostic markers in APS I is undisputed.

By mutational analysis of the AIRE gene approximately 90% of APS I patients are

identified (19) and by analysing a battery of APS I specific autoantibodies the

sensitivity is also around 90% (164). Since a genotype–phenotype correlation has not

yet been described in APS I, autoantibody analysis is still, by giving important clinical

information on the risk of developing different components of APS I, a valuable

complement to mutational analysis.


60

S U M M A R Y

In this thesis we present the original identification of TPH as an endogenous intestinal

autoantigen in APS I associated with gastrointestinal dysfunction (Paper I). We have

shown that TPH autoantibodies are specific for APS I, and thereby a good diagnostic

tool to identify APS I patients among young children presenting with atypical

gastrointestinal disease (Paper II). TH is originally identified as a dermal autoantigen

in APS I associated with alopecia (Paper III). Autoantibodies reacting with PAH are

demonstrated in sera from APS I patients, probably reflecting a cross-reactivity with

TPH (Paper IV).

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61

F U T U R E P E R S P E C T I V E S

The physiological role of serotonin, and serotonin producing EC cells, in the gut

requires further study. APS I patients, and hopefully also Aire deficient mice, with a

total loss of serotonin producing EC cells in an otherwise intact mucosa represent

suitable models. Does serotonin, or serotonin producing EC cells through other

mediators affect absorption in the gut?

The epitope mapping of TPH, TH and PAH, as outlined in the discussion, should be

reasonably easy to carry out. This might give us information that makes it easier to

understand the preferences of the immune system.

TPH and TH are both rate limiting in the synthesis of neurotransmitters thought to be

involved in the pathogenesis of different diseases e.g. affective disorders,

schizophrenia and Parkinson’s disease. They ought to be suitable as targets for

pharmaceutical intervention aiming at an increased or decreased production of

serotonin or dopamine. APS I sera have been shown to markedly inhibit TPH and TH

activity, and in other studies both inhibitory and stimulatory antibodies have been

identified (81). If an epitope mapping study identifies binding sites responsible for

inhibition and possibly stimulation of TPH or TH, then a suitable drug could be

engineered to mimic this binding.

The same strategy as we have used in identifying TPH and TH as autoantigens could

be applied to other conditions. In IBD, we have already seen a clear staining pattern of

Goblet cells, similar to that given by a subgroup of APS I patients. The identification


62

of the antigen responsible for this Goblet cell staining could supply important

information regarding the pathogenesis of IBD.

Hopefully the ongoing studies of Aire knock-out mice will provide us with new

insights into the pathogenesis of APS I, and autoimmune disease in general. The report

of Aire being expressed in thymic epithelial cells involved in negative selection

suggest that Aire is participating in the generation of central tolerance, and that

mutations may lead to loss of self tolerance and subsequent autoimmune

manifestations (186). We will have the perfect animal model to investigate organ

specific autoimmune disease if the Aire depleted mice develop autoimmune diseases.

Target organs, and infiltrating T-cells, can be studied at the time of the initial attack,

and not as in humans when the organ destruction has already taken place.

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63

A C K N O W L E D G E M E N T S

The work was performed at the Department of Medical Sciences, Uppsala University,Sweden. It has been made possible through the generous financial support from the

Swedish Medical Research Council, the Torsten and Ragnar Söderberg Foundation,the Swedish Society of Medicine, the Claes Groschinsky Memorial Foundation, the

Förenade Liv Mutual Group Life Insurance Company, the Lennander Foundation, theAgnes and Mac Rudberg Foundation, the Swedish Diabetes Association, the Torsten

Nilsson Foundation, the Åke Wiberg Foundation, the Ernfors Family Foundation,

AstraZeneca and the Swedish Society for Medical Research, which is hereby fullyacknowledged.

Scientific research is never a “one-mans work”, and many people have contributed tothis thesis. I would like to take this opportunity to thank them all, and especially:

Sverker Ljunghall, the former head of the department, who not only talked aboutbringing molecular biology into the clinical wards, but also took practical measures by

accepting a number of us “clinical molecular biologists” as PhD students.

Kjell Öberg, the present head of the department, for supplying good workingconditions and for signing all kinds of papers - just before the DHL-guy comes.

Olle Kämpe, my supervisor, for creating an inspiring atmosphere in the group byalways sharing scientific visions and knowledge. For understanding that skiing, good

food and laughter also brings science forward and, of course, for being the only one in

the group who knows how the pH-meter really works.

Fredrik Rorsman, my second supervisor, for sharing a room (with a poor view), for

your knowledge in molecular biology and for always, even in times of hard clinicalwork, having a couple of minutes to discuss experimental data. Thank you also for

letting me inherit your apartment, your childrens clothes, skates, skis and………

Eystein Husebye, “our man in Norway”, for not only supplying valuable patient

material, but also for valuable moments in front of arcade games around the world,

and for always, in an almost magical way, being first in line at check-in desks, cashiersetc.

Håkan Hedstrand, for being a good friend, for travel companionship, for amazinginventions, for thinking differently and for always contributing with new aspects and

different approaches to scientific problems.

Gennet Gebre-Medhin, for great friendship, for always being generously helpful, for

sharing a desk and my taste in “smågodis” and for always having time for a chat.


64

Annika Söderbergh, who has left us for Arboga, for friendship and for always being

honest. Even though sometimes…….

Filip Sköldberg, for friendship, for critical thinking and for always having the latest

lab protocol – directly from some obscure web site.

Thomas Nilsson, for never hesitating to offer help and for late nights under the elk,listening to old 78 rpm records.

Eva Landgren, for your interest in GI-autoimmunity, for strengthening ourcompetence in basic science and for trying to get the lab a little bit more organised.

Jakob Skov, our “one-man brat pack”, for allowing the group to have futureperspectives.

Åsa Hallgren and Katrin Österlund, for being genuinely nice and friendly and for

expert technical help whenever needed.

Ola Winqvist, for always having an answer, and for tacos and Jacuzzi in La Jolla.

Gunnel Nordmark, for a positive attitude, and for adding a rheumatological, clinicalperspective into discussions.

Sophie Bensing, Lars Rönnblom, Anders Rönnblom, Roger Nilsson, Per Maritsand Patrik Larsson for contributing with knowledge and good spirits at our weekly

Monday seminars.

My co-authors Jan Gustafsson, for supplying patient sera, and for showingcontinuous interest, Jan Haavik, for knowing everything worth knowing about “our

enzymes”, Lars Grimelius, for invaluable help with immunohistochemistry, JaakkoPerheentupa, for generously sharing your patient sera and clinical knowledge of

APS I and Vanda A. Lennon, Klas Sjöberg and Corrado Betterle for patient sera.

Ismo Ulmanen, Petra Eskelin, Maria Halonen and Meelis Kolmer, “The Helsinki

connection”, and Cheri Deal, Leanne Ward and Patricia Crock, “The Canadian-

Australian connection”, for fruitful collaborations in the past, present and future.

Kerstin Westermark, Therese Dahlman and Majstin Wik for first introducing me

to the group, and for guiding me in my first fumbling efforts to unravel the mysteriesof thyroid cancer.

Peter Stålberg for being a good friend, for Hurricanes in New Orleans, pike-fishing,and my daily dose of irony.

Olov Ekwall

65

Håkan Melhus, Andreas Kindmark, Maria Branting, Fredrik Stiger, SaraJohansson and other members of the Osteoporosis group for laughters and lateralthinking in the coffee corner.

Anna Schölin, Mikael Kullin, Jan Melin, Britt Skogseid, Ann-Christin Syvänenand all other friends and members of various research groups at the Centre forClinical Research for small talk, sharing instruments, advice, etc.

Cindy Wong, Maud Marsden and Charlotte Sweeney for expert linguistic revision,and Cindy also for always being so nice and caring.

Elisabet Dew, Anna Carlbom-Härd, Elizabeth Dehlin and Lena Hultström forhelp with practical matters at the department throughout the years.

The pike-fishers for giving me something to look forward to each autumn.

My mother Barbro for continuous love and support. My late father Björn, who left usmuch too early, for having been the best of fathers. He will always continue to be an

inspiration for me in my research.

Axel and Erland, my two bold boys, for giving me an excuse to play with LEGO and

RC-cars.

Anna-Karin, my princess, for being the love of my life as well as an inspiring

colleague in research and in clinical work – and everything in between.


66

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8. Anderson JR, Goudie RB, Gray KG, Timbury GC. Auto-antibodies in

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9. Arlt W, Callies F, van Vlijmen JC, Koehler I, Reincke M, Bidlingmaier M,Huebler D, Oettel M, Ernst M, Schulte HM, Allolio B. Dehydroepiandrosterone

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