Insulin-dependent diabetes mellitus: the hypothesis of molecular mimicry between islet cell antigens and microorganisms

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MOLECIJLAR

MEDICINE

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1997

Insulin-dependent diabetes mellitus: the hypothesis of molecular mimicry between islet cell antigens and microorganisms Insulin-dependent diabetes mellitus (IDDM) in humans and the non-obese diabetic mouse is a polygenic disease, resulting from an autoimmune destruction of the insulin-secreting pancreatic p cells. At least in NOD mice, the process is mediated through aT helper l-cell-mediated cytotoxicity pathway. Although there is much circumstantial evidence to suggest that IDDM is environmentally induced, recent studies support the possibility that the inductive event involves cross-reactive immune responses to antigenic epitopes acting as molecular mimics between microbial proteins and autoantigens expressed by pancreatic insulin-secreting p cells.The following article reviews the evidence for this concept. INSULIN-DEPENDENTdiabetes mellitus (IDDM) is one of the most common and chronic endocrine disorders of children and young adults’. Once established, IDDM results in unavoidable chronic hyperglycemia, accompanied in turn by accumulating complications with increasing duration of disease.These complications include: obstructive damage to the arterioles of the kidney, heart, lower limbs and retinae; and accelerated atherosclerosis of the larger arteries, affecting the circulation to the limbs, heart and brain. These secondary problems eventually become debilitating and life threatening, exacting a terrible emotional and financial toll on the patient, his/her family and society alike. Thus there is great interest in understanding how the disease is initiated, in the hope that it could eventually be prevented.

Epidemiology Whereas IDDM (type 1 diabetes mellitus) can be thought of as an insulin deficiency disease with the majority of afflicted people showing evidence for an immunologically mediated pathogenesis, non-insulindependent diabetes mellitus (NIDDM or type 2 diabetes mellitus) represents a collection of genetically determined diseases resulting from both insulin deficiency and insulin resistance. The worldwide incidence of IDDM varies greatly, probably largely influenced by differences in the prevalence of disease susceptibility genes in different populations; however, environmental factors might also be important’. The disease is most common among people of northern European descent, with low frequencies among Black, Chinese and Japanese people.In the USA, the lifetime risk approximates 1 : 300. The incidence of disease ranges from a low of l-2 cases per 100 000 persons per year in Japanto a high of more than 40 cases per 100 000 persons per year in parts of Finland*.Thereis a modest male vs. femalepreponderance 76

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in many countries. These differences in the incidence of IDDM can largely be explained by the prevailing susceptibility-genes for IDDM, such as the human leukocyte antigen (HLA) alleles HLA-DRB 1 and DQAI/Bl, in racially distinct populations. However, poorly understood dietary and other environmental factors could also be important3. People from countries with a low incidence of IDDM (low expected genetic susceptibility), such as Japan, appear to have an increasing incidence of diabetes coincident with the westernization of their culture. Immigrants from areas of low IDDM incidence who migrate to a country with a high IDDM incidence have been thought to display an increased occurrence of the disease compared with that expected on the basis of their genes. The problem with such data is that they could merely reflect an improved diagnostic capability and better reporting of diabetes by their adoptive country. Additional evidence implying non-genetic dietary and/or environmental influences in IDDM includes (1) the rising incidence rates of IDDM in some European countries over the past three decades (as much as fourfold); (2) the higher frequency of IDDM among children who were not breast fed or who were breast fed only for a short neonatal period; (3) the falling age of disease onset; (4) the lack of disease concordance among identical twins affected by IDDM (the disease affects only one third of both members of the pair); and (5) the low numbers (10%) of patients with IDDM who do not have a close relative affected by the disease’. The rising incidence cannot be explained by the cumulation of susceptibility genes becausethe increase has beentoo fast but it could result, in part, from an artefact of improving identification. In the European studies, the latter possibility seems improbable. Thus, although such data appear compelling, the conclusion that environmental influences are ‘responsible’ would be less ambiguous if, in some geographic areas of the world, the incidence of IDDM could be shown to be falling, thus provoking a search for some ‘disappearing’ environmental factor. Unhappily we know of none. There is no genetic or immunological evidence that the disease is any different in concordant (identical) twins from that in discordant (non-identical) twins with IDDM, making their high discordance for the disease perhaps the strongest circumstantial evidence that environmental factors are involved. The incidence of disease in humans is markedly age dependent, increasing from a near absence during the early months of life to a striking peak that is coincident with pubertal development. A seasonal variability in the incidence of IDDM is also reported from all parts of the world: more people are diagnosed in the autumn/fall and winter months in both hemispheres. This suggests that common infectious viruses might be the final precipitating factor for progression to overt diabetes in a person with a contracting capacity to secrete insulin, perhaps by increasing the insulin requirements of the person or by boosting ongoing B-cell autoimmunity in some way. Epidemiology of IDDM in animal models The non-obese diabetic (NOD) mouse is a well-studied animal model of IDDM. These mice spontaneously develop severe diabetes, and most die rapidly if they are not given daily injections of insulin. In contrast to humans with IDDM, female NOD mice are affected 2-3 times more often than males. Even among female mice, the disease does not appear in a large minority of them, despite their genetic identity and apparently uniform handling and feeding. Some unexplained biological variability must therefore be responsible.

Natural history and pathogenesis of IDDM Because of its dramatic onset in people previously not suspected to be at risk for it, IDDM was once viewed as a rapidly developing

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Figure 1. Histological appearance of pancreatic specimens from patients with insulin-dependent diabetes mellitus (IDDM) of recent onset. (a) A section of pancreas stained for glucagon, somatostatin and pancreatic polypeptide [hormones of other (non-p cell) endocrine cells within the islet] with an immunochemical method involving alkaline phosphatase. All endocrine cells are stained, confirming the lack of insulin-containing p cells. (b) An islet infiltrated with chronic inflammatory cells, a condition often referred to as insulitis (Hematoxylin and Eosin).

illness that might be the result of an acute viral infection of the pancreatic islets. In fact, the disease results from a chronic autoimmune process that has been shown by retrospective studies to exist for as long as 5-10 years in a pre-clinical phase’. Indeed, the classical manifestations of IDDM - hyperglycemia, weight loss and ketosis - occur late in the natural history of the disease, and only after a majority of the pancreatic B cells have been destroyed, resulting in severe insulin deficiency. Not all B cells are simultaneously destroyed by the chronic inflammation (Fig. 1) of the pancreatic islets (insulitis), because there are disparate degrees of inflammatory involvement that vary greatly between individual islets. This is best studied in NOD mice. Whereas a complete loss of the ability to secrete insulin is usually seen within l-2 years after diagnosis in young patients, older patients often show more-slowly progressive B-cell destruction that might never become complete’. Additional characteristics that identify human IDDM as an autoimmune disease include genetic susceptibility associated with the major histocompatibility complex (MHC), especially that involving MHC class II alleles6 (see below), and the presence of autoantibodies and autoreactive T cells directed against 77

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MELILCINE

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FEHKCAKY

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Delayed-type iypersensitivity and inflammation

Microbial pathogen

Humoral immunity, allergy Nematode Figure 2. The functional relationship between T helper (Th) cell subsets in mice and humans (the situation in humans is more complex). T cells can be phenotypitally divided into polarized (Thl vs. Th2) subsets based on their cyiokine secretion profiles. Th2 cells secrete interleukin 13 (IL-i3), which can suppress IL-12-driven ThO differentiation to Thl cells, while interferon y (IFN-y) secretion by Thl cells suppresses Th2 differentiation via inhibition of the IL-Cinducing signal. ThO cells are undifferentiated precursor cells that can become Thl or Th2 cells, depending on the signals that they recieve. Thl cells are commonly involved in delayed-type hypersensitivity responses and inflammatory processes. Conversely, antigen-activated Th2 cells are driven by IL-4 itself, stimulated through mast cells, basophils and eosinophils, and are commonly associated with allergic responses and humorally mediated autoimmune diseases.

islet cellsor their antigenicconstituents’.4. An extensivenumberof humanIDDM arelessclear.Fromthese,an extensivelist of factors putativeimmunological targetsin humanIDDM havebeenidentified, that might be responsibleis derived. (1) Several target p-cell including insulin, glutamatedecarboxylase(GAD) and the protein autoantigenshave beendescribed.(2) Potentialdefectsin antigen tyrosinephosphatases IA-2 andIA-2P; but the relativeimportanceof presentationare implicated,suchas defectsin self-peptidecleavage thesep-cell antigensin the autoimmunepathogenesis of IDDM and or transportto the MHC-bindingcleft. (3) Defectsin regulationof relateddiseases remainsunclear’.In NOD mice,autoimmunityappears the immunesystem,suchasdefectiveexpression of the T-cell accesto begin againstGAD, followed by epitope spreadingto involve sorymoleculeCTLA-4, havebeenreported’.6. other antigenssuchasinsulin*.In humans,autoimmunityto insulin The aforementioned importanceof CD4’ T cellsin the diabetoand/orGAD developsearly in the courseof the disease, andtendsto genieprocesscould reflect the critical role for a subsetof thesecells to regulatethe immuneresponse. The CD4’ T cellsin mice, andto extendto IA-2 later,leadingup to the timeof diagnosis7. The immunologicalmechanisms underlying humanIDDM are someextent in humans,can be phenotypicallysubdividedon the still not fully understood.Strong evidence,both in humansand in basisof their cytokinesecretionprofiles.Thesefunctionallypolarized NOD mice, suggests that T cellsaremajorcontributorsto the patho- T-cell subsets opposeoneanotherthroughtheir reciprocaldownregugenicprocess.Indeed,in NOD micethe transferof diabetesinto irra- lation (Fig. 2)“. In both miceand humans,‘inflammatory’Thl cells diatedrecipientsinvolvesthe participationof both CD4’ MHC class arepromotedthroughthe cytokineIL-12; theypredominantlysupport II restrictedand CD8’ MHC classI restrictedT cells’. However, cell-mediatedimmuneresponses and they secreteIFN-y and IL-2. someCD4’ T-cell cloneshaveprovedcapableof transferringthe dis- Thl cellsare alsothoughtby manyto be the initial CD4’T cellsto easeto immunoincompetent irradiatedmice or severecombined stimulatep cell destructionby CD8’ cellsin NOD mice’~“.Administhe immunodeficient(SCID)-NOD mice. Studiesof this animalmodel tration of cytokinesthat promoteThl developmentexacerbates have suggestedimportant roles for several proinflamatory and development of IDDM and,conversely,administration of monoclonal immunoregulatorycytokines,includinginterferony (IFN-y), inter- antibodiesagainstcytokinesproducedby Thl cellsblocksthe develBy contrast,Th2 cellspredominantlysupport leukin2 (IL-2): IL-4, IL-10 andtumornecrosisfactor.The activity of opmentof disease7s9. antigen-presenting cells(APCs), their accessorymoleculesB7.1 and humoralimmunity andallergicresponses; they arecharacterizedby B7.2 (and their counterpartT-cell ligands),and expressionof ad- the secretionof IL-4, IL-5, IL-IO and K-13; and they generally hesionmolecules,particularly intercellular adhesionmolecule 1 appearto have a ‘protective’ role againstIDDM in NOD mice”. (ICAM-l), havebeensuggested asfactorsin the pathogenesis of the Treatmentswith recombinantIL-4 lower diabetesfrequencies13. diseasein NOD mice*.Studieson the role of cellularimmunity in However,micemadetransgenicfor IL-10 underthe insulinpromotor 78

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paradoxically develop inflammatory lesions14.Despite this, recent studies suggest that a functional imbalance between these two T-cell subsets is a key variable in determining the rate of progression to IDDM”,“. In NOD mice, an insulitis that has a marked degree of IFN=y production by either CDS’ or CD4’T cells is typical of a destructive inflammation. Conversely, those interventions that provoke an increase in content of IL-4 (Th2-like response) in the peripheral immune sites, as well as in the insulitis lesions, are associated with protection from disease. Far less is certain regarding the role of Thl vs. Th2 immunity in human IDDM. However, an inverse relationship has been reported between humoral vs. cellular autoimmunity to P-cell antigens and the risk for IDDM in prediabetic populations”. The literature suggests that islet autoantibodies are of minor pathogenic importance to IDDM, a conclusion that is consistent with the lack of diabetes seen in infants of diabetic mothers even when there is chronic transplacental passage of multiple isoforms of islet-cell IgG autoantibodies throughout the gestation period.

Molecular

mimicry

and autoimmune

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1997

disease

Theoretical basis The concept that microorganisms such as bacteria, viruses and parasites can represent environmental factors that initiate and/or sustain anti-self immune responses has stimulated research on their possible roles in certain autoimmune diseases16-19.Microorganisms provide a multitude of antigens that collectively comprise the major set of antigenic determinants (carbohydrates, lipids and proteins) that the immune system can recognize and respond to, via specific cognate receptors on inflammatory T cells (T-cell receptors) and B cells (immunoglobulins). In addition to their obvious roles in infectious diseases, microorganisms might also trigger autoimmune diseases. Microbial antigens with structural homologies to selfantigens can serve as stimuli that induce immune responses that are cross-reactive with self-tissue antigens. This might lead to a bypassing of normal mechanisms that induce tolerance, and the development of a selfperpetuating autoimmunity’“‘9. Activated T cells and B cells would thereby arise that cross-react with homologous self-antigens that are normally non-immunogenic. Thus, a protective immune response against an invading microorganism could set off an autoimmune response that might be transient and self-limited, or chronic and/or relapsing, dependingupon the genetic predisposition of the individual. Streptococcal pharyngitis provides a classical example: in most individuals it is a self-limiting condition; the immune response downregulates coincident with the disappearance of the source of the stimulatory bacterial antigen, and T-cell immunological memory is retained. However, in other individuals, dependent upon genetic factors such as their HLA-DR/DQ genotype, autoimmune responses can be induced. This autoimmunity can involve: the joints or myocardium (the arthritis and myocarditis of acute rheumatic fever); the basal ganglia of the brain (resulting in involuntary writhing

Glossary Adjuvant -A vehicle for enhancing an immune response. They often delay the absorption of an immunogenic antigen. Freund’s adjuvants are oily vehicles for enhancing the degree of the immune response following immunizations, while complete Freund’s adjuvant contains tuberculin antigens as additional immunological potentiators. Anergy- Immunological unresponsiveness. Autoantigen-A self-antigen involved in an immune response. Autoimmunedisease- Clinical syndromes where disease results from aberrant responses of the immune system to self, causing dysfunction or destruction of tissue cells. Autoimmunity-An immune response to a self-antigen. Epitope- The portion of an antigen recognized by components of the immune system such as antibodies and T cells. lmmunogen -Antigen that provokes an immune response. lnsulitis- Chronic inflammation of the pancreatic islets. Majorhistocompatibilitycomplex(MHC)-A genetic region encoding proteins that are involved in antigenic peptide presentation. Class I MHC antigens are glycoproteins expressed by most nucleated cells in humans; they present mainly intracellular peptide antigens of self or viral origin to the immune system. Class II MHC antigens are dimeric proteins that are mainly found on immunologically active cells and serve to present extracellular antigens. Molecularmimicry-The sharing of sequence and/or three-dimensional structure in regions of disparate proteins from different sources. Non-obesediabetic(NOD)mouse- An animal model for insulin-dependent diabetes mellitus (IDDM).

Polygenicdisease-A disease that results from the interaction of multiple susceptibility genes and environmental factors. Prediabetes -The pre-clinical stage of diabetes mellitus, characterized by autoantibodies to islet cell antigens, and often progressive loss of insulin secretory capacity. Severecombinedimmunodeficient @CID)mice-An inbred mouse line with a defect in antigen-receptor rearrangement (caused by recombinase deficiency), which results in the failure to develop mature B-cell or T-cell immunity. Mice with the defect cannot reject grafts and are often used for chimeric studies. SCID-NODmice- Inbred NOD mice with the SCID genetic defect. Such mice are ideal for transfer studies whereby cells from NOD mice are isolated and transplanted into the strain. These studies permit transfer of active P-cell immunity, to study the mechanisms responsible. Thl and Th2cells- T helper (Th) cells are essential to mounting an effective immune response and, in the mouse, are readily divisible into at least two functional subsets defined by the cytokines that they produce. Thl cells promote cellular immune responses and secrete the cytokines interferon p and interleukin 2. Th2 cells promote antibody responses and secrete interleukins 415 and IO. There is good evidence for these subsets in humans but the distinction is less pronounced and T cells with features of both subtypes are common.

Thymicdeletion- Normal mechanism by which potential autoreactive T cells can be destroyed in the thymus, once they express receptors to ‘self’ deteminants. Tolerance-An immunoregulatory ability to be unresponsive to an immunogen.

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Table 1. Identified homologies between #Mall autoantigens and environmentallimmunomodulatory agents associated with insulin-dependent diabetes mellitusa p-cell a4rtoantigen

Putative environmental mlmlc

GADb CA69 JunB Carboxypeptidase

Coxsackievirus P2-C protein; IAg’; HLA-DQ (IDDM-susceptible alleles); HLA-DO Bovine serum albumin. HLA-DQfi; IA-2; human herpes simplex virus types 1 and 4. HLA-DQ (IDDM-susceptible alleles); Coxsackie virus; influenza virus A.

H

(IDDM-protective

alleles); adenovirus;

hsp60.

%bb&atione: GAD, glutam~ add decarboxylase; HIA, human leukocyte antigen; hsp60, heal shot% protein 60; lCA69, islet cell autoen~ 69; IDDM, insulindependent diabetes melliis. bGAD &a has hamotogii with profnsuiin. of the Iii abwe, the authors rate the mimicry involving GAD and Coxseckii virus PZ-C protein as the most likely to be relevant to IDDM. Derived from Ref. 30 and other scwces.

movementsof the limbs and facial muscles- Sydenham’schorea); Animal modelsof autoimmunitycan supportthe concept that and the endocardium,where chronic inflammationof the valvular underappropriateexperimentalconditions,immunizationby protein can stimulatean cuspscanleadto scarringandcardiacdysfunction”. In general,if a antigenswith aminoacid similarityto self-antigens that ultimately leadsto an autoimmuneresponse, microorganism did initiate autoimmunity,the clinical featuresof the immuneresponse resultingautoimmunedisease wouldbe expectedto dependon quali- if not actual disease.Most of thesemodelsinvolve the hyperanimals tative featuresof the immuneresponse, the particularcytokinespro- immunizationof geneticallyautoimmune-disease-susceptible duced(Thl- vs. ThUike responses) and the tissuedistributionof the (for example,animalswith a particular MHC-type) with foreign, targetself-antigens. The sharingof antigenicdeterminants betweena microbial proteins that show homology to tissue-specificselfautoimmune-associated humanMHC antigens arenot microorganismand its host species,commonly termedmolecular proteins.Because to co-administer mimicry,frequentlyhappens by chance.Databasesearches of protein presentin thesemousemodels,it hasbeennecessary sequences derivedfrom mammalsandmicroorganisms revealmany additionalmicrobialproducts,particularly completeFreund’sadjupotentialCD4’ T-cell epitopes(shortpeptidesof 8-12 aminoacids) vant, to induceany appreciableanti-selfimmuneresponses, let alone that are shared,or similar enough,betweenmicroorganisms and disease.The preciserole for microbialproductsin the inductionof humansand could have relevanceto autoimmunediseasestates16. theseautoimmunemodelsis not fully characterized;however,they Thesehomologous sequences havebeenproposedto be importantin are importantin dictating whethera cellular (i.e. mostly Thl) or predominates. the pathogenesis of a number of autoimmunediseasessuch as humoral(i.e. mostlyTh2) immuneresponse rheumatoidarthritis, ankylosingspondylitisand multiple sclerosis. The molecularmimicry modeldoesnot readily explain how the However,it is difficult to predict which of thesesharedsequences autoimmunestate,onceinducedby an infectiousagent,is sustained leadingto actually yield T-cell epitopesthat are cleavedduring processingby beyondthetime that the inductiveantigenhasdisappeared, APCs and can thus be presentedto T cells in viva to generatea chronic autoimmunityanddestructionof the targetorgan.The selfspecificimmuneresponse. Also, for cell-mediatedautoimmunityto antigenbeingmimickedby the microbialproteincan serveasan imbe initiated,it is importantthat the self-peptidebe cleavedin sucha munologicaltarget,but couldit alsoserveasanelicitingantigenin its ? Oneexplanationcouldbe proway that the epitopebeing ‘mimicked’by the microorganism canbe own right to sustainits own ‘demise’ recognizedby the relevantT-cell receptor.Synthetic peptidescorre- vided by further encounterswith the inductivemicrobialimmunogen spondingto certain epitopeshave been shownexperimentallyto leadingto repeatedautoimmune attacks.This situationcouldeventuresult in cross-reactivecell-mediatedimmunity (i.e. expansionof T ally culminatein sufficient tissuedestructionand organ failure to clearly appliesto rheumaticfever, cellsthat recognizeboth the immunizingpeptideandthe mimicking resultin disease.The mechanism self-peptide)“.Furthermore,experimentsboth in humansandin lab- wheremultiple streptococcalinfectionslead to progressivecardiac oratory animalshave demonstrated immunologicalcross-reactivities damage- a situationthat canbepreventedthroughchronicantibiotic use.Alternatively,a processof intramolecular spreading of antigenicity betweenmicrobialantigensandself-tissues by serumantibodies’7. to other self-tissueantigenscouldstarta processthat would proceed Clinical evidencefor mimicry in IDDM independent of further contactwith the inciting antigen**.In the case While molecularmimicry providesa straightforwardmodel con- of IDDM, B-cell damagecould lead to exposureof otherwise antigens,which mightbe responsible for generatingsecceptually,conclusiveevidencefor it in the pathogenesis of mostauto- sequestered suchasto the cytoplasmicdomainof the immunediseases, includingIDDM, is limited.Inlkunmatoryconditions ondaryimmuneresponses, proteinsIA-2 (Ref. 23) andIA-2B (Ref. 24), asoccurs associated with autoimmuneresponses can occur following a recent transmembrane infectionwith an identifiablemicroorganism. For example,epidemic in IDDM. Autoimmunediseasemodelsbasedon molecularmimicry that autoreactiveT cells exist before the induction of Reiter’ssyndromeassociated with reactivearthritis2ican occur after presuppose infectionwith Chlamydia; ankylosingspondyhtis canfollow Yersiniaand autoimmunedisease,but are maintainedin an unresponsivestate Klebsiellainfections;andrheumatic fevercanbea consequence of group (anergy).The modeldoesnot explainwhy theseautoreactiveT cells of toleranceinduction(thymic deletion) A streptococcal infections,asmentioned above.Evenin theseseemingly escapethe majormechanism compellingclinicalinstances, thereremaingreatuncertainties aboutthe andhow they areinitially maintainedin a ‘silent’statein the peripheral immunecompartmentafter their escapefrom centraldeletionby natureof the antigensinvolvedin molecularmimicry in eachcase. 80

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the thymusZ5. An additional obstacle, which must be overcome to establish molecular mimicry as a mechanism leading to spontaneous autoimmune diseases, is the identification of the precise epitope(s) that initiate the putative cross-reactive immune responses. Difficulty arises from the aforementioned common observation of putative molecular mimics (i.e. numerous similarities between amino acid sequences of tissue antigens and microorganisms). However, the major difficulty in identifying such epitopes lies in the protracted natural history of most autoimmune diseases. Recent studies in animal models suggest this additional level of complexity because the ‘array’ of autoantigens and autoantigenic epitopes recognized during an autoimmune response expands over time (‘epitope spreading’)‘*. Indeed, intramolecular spreading (the antigenic spread from one epitope to many in the same protein) and intermolecular spreading (the antigenic spread from one protein antigen to another) of the autoimmune T-cell repertoire have been demonstrated in NOD micez6.“. Specifically, these studies demonstrate that T cells from NOD mice become activated and expand in viva against a defined group of islet cell antigens in an orderly, sequential manner. T-cell responses in the youngest NOD mice display strong reactivity to the GAD enzyme but not to other islet cell antigens, while the initial immune responsiveness to GAD is initially limited to one region of the protein only. With time, this response spreads intramolecularly to involve other regions of the protein and, eventually, after the autoimmunity to B cells in these mice has progressed to destructive islet cell inflammation (insulitis), the T-cell responses spread intermolecularly to involve other islet cell proteins (such as heat-shock protein 60, carboxypeptidase H and insulin)26’7. At least in this mouse model, the expressed autoantigenie repertoire is not fixed but rather ‘evolves’ during the course of an autoimmune disease. Therefore, exposure to environmental antigens that resemble selfa epitopes could generate a response that spreads both intramolecularly and intermolecularly to ultimately involve a much wider range of autoantigens than could have been predicted by examining the inciting antigen. This makes it difficult to predict which (if any) putative crossreactions are important in terms of disease induction vs. those that do not give rise to b autoimmune pathology, particularly in humans who are exposed to many infections.

Evidence for molecular mimicry IDDM

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IYY7

focused on only two: Coxsackie virus and rubella virus. Most of the earlier studies (those dating back to the 1950s) did not take into account the full complexity of the pathogenic nature of IDDM, and their conclusions must be interpreted in the light of our current understanding that IDDM results from a chronic autoimmune process (months to decades before clinical onset), and that susceptibility is associated with heterogeneous genetic susceptibilities (probably involving gene-dose effects). More recent models involving pathogenic roles for viruses in IDDM postulate that IDDM results from a misdirected immunological attack (i.e. molecular mimicry) upon B cells by T cells responding to an acute (the ‘hit and run’ model) or chronic viral infection’. In this model (Fig. 3), viral proteins could conceivably share amino acid sequence homology with a B-cell autoantigen and, through their molecular mimicry, lead eventually to B-cell destruction through cytotoxic T cells and IDDM. Although numerous sequence similarities between viral proteins and B-cell autoantigens are plausible, the relationship between Coxsackie virus infection and GAD autoimmunity has recently received the most attention. Another possibility that should be explored is that a viral member of the tyrosine phosphatase family could trigger an immune response to its B-cell counterpart, IA-2 or IA-2B. Alternatively, a viral protein could initiate an antibody-biased immune response. GAD Evidence for a specific molecular mimicry model involving GAD was enhanced by the finding of an 18 amino acid peptide with striking sequence homology between human GAD and the Coxsackie virus P2-C protein (Fig. 4)31.In addition, this specific region of GAD 7

_

IL-2 2 IFN-y

+ destruction

and

f

in

Environmentul agents Molecular mimicry could play a role in the pathogenesisof IDDM through sequencesimilarity of B-cell proteins with dietary and/orMHC proteins(Table 1). Both have recently been the subject of excellentreviews*&“’andwill only be briefly presentedhere. The diagnosisof IDDM following an outbreakof various viral infections has, for many years, implicatedvirusesin the pathogenesis of IDDM3’. Although many viruses have been studied, most investigatorshave

Figure 3. Hypothetical molecular mimicry model for the autoimmune basis of insulin-dependent diabetes mellitus (IDDM). (a) The process of autoimmunity might begin with an immune response by the host to a viral protein presented in the context of class II major histocompatibility (MHC) antigens (HLA-D) by an antigen-presenting cell (APC), which generates an antigen-specific Thl response. In this case, the viral protein shares a short amino acid sequence with a p-cell protein and both of these sequences are appropriately cleaved and presented with class I MHC. Therefore, a CD8+ T-cell-mediated cytolysis [assisted by growth factors such as interleukin 2 (IL-2) and augmented by interferon y (IFN-$1 is initiated, resulting in the destruction of 3 cells of the pancreatic islets and eventually IDDM. (b) The viral peptide could also be processed and could consequently stimulate the antibody-promoting Th2 pathway (IL-415 dependent), resulting in B cells producing cross-reactive antibodies that could also produce disease through a mechanism such as antibody-dependent cytolysis by macrophages. The pancreatic p cell has at least two receptor-like tyrosine phosphatases (IA-2 and IA-23) that could serve as the homologous antigen to viral antigens in IDDM if a Th2 pathway is involved.

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Evidence from human studies Additional evidence supportinga link between Coxsackie virus and IDDM arose Coxsackie virus P2-C from a recentcase-controlstudy associating IgM antibodiesto CoxsackieB virus as a Glutamatedecarboxylase marker of recent exposurein newly diag250 273 nosedIDDM patientsand age/sex-matched controls3’.In this report, humoralimmunity Figure 4. Sequence homology between Coxsackle virus and human GAD,,. The solid lines denote to CoxsackieB virus and GAD appearedto identical amino acid residues. Dashed lines denote amino acid residues with similar charge, polarity or cluster, even in people without diabetes. hydrophobicity. The numbers refer to the number of amino acid residues from the N-terminus of each However, not all publishedreports have protein. Reproduced, with permission, from Ref. 31. demonstrated a linkagebetweenimmunityto I GAD and Coxsackievirus. BecauseT-cell reactivity to antigenicepitopesis typically containsa T-cell epitopeinvolved in the GAD cellularautoimmunity different andmoreconfinedthanthat to B cells(antibodies),it is not in humanswith IDDM3*, and this region is an early target of the surprisingthat humoralimmunityto GAD in humansdoesnot appear aforementioned cellularimmunityin NOD micez6,“.GAD catalyzes to be directedat the regionof sequencesimilarity with Coxsackie the formationof the inhibitory ncurotransmitter y-aminobutyric acid viru?. One studymonitoringpolyclonalhumanT-cell responses to (GABA) from glutamine 33 , Two forms of GAD exist (GAD,, and GAD identifieda non-Coxsackie-homologous regionof GAD asthe GAD,,); GAD,, is the predominant form within the humanpancreatic predominantcellularimmuneepitope”. In NOD mice,thereappears islet cells, while GAD,, predominates in mouse islets.Within the to be no analogoussituationwherebyenteroviralinfectionsare folislet, GAD is predominantlyobservedwithin the p cells,while roles lowedby anti-GAD responses anddiabetes.On the contrary, NOD in the inhibitionof somatostatin andglucagonsecretion,aswell asin mice rearedunder gnotobioticor pathogen-freeconditionshave an regulationof proinsulinsynthesisand insulin secretion,have also enhanced, ratherthana reduced,incidenceof diabetes. beensuggested3”. Many studieshave demonstrated GAD autoantibodies in people Future directions with IDDM, aswell asthoseat risk for the disease3’x35. This autoanti- It mustbe stressed that, while thereis a linearpeptideepitopewith body response is, in part, explainedby its anti-GAD,, activity, and stronghomologiessharedbetweenGAD and Coxsackicvirus P2-C multipleconformationalepitopesarepresentwithin the molecule’1.3i. protein,this doesnot prove that an antigenicmolecularmimicry is In the NOD mouse,T-cell responses to GAD are a key eventin the responsible for IDDM. The finding does,however,providean imporinductionandpropagationof immunityto p cells,aspreviouslydis- tant focus for future investigations.The observedincreasein the cussed26X27. To identify the T-cell-reactivedeterminantsof GAD in frequencyof peripheral-blood T-cell responses to GAD in islet-cellhuman IDDM, an overlappingset of syntheticGAD,, peptideshas antihody-positivepeople destinedto develop IDDM, many years beenusedto stimulateT cellsfrom individualswith anincreased risk beforethey showclinical symptoms,in comparison with newly diagfor IDDM (autoantibody-positive,first-degreerelatives of IDDM nosedsubjectsis onepieceof datasupportingthispossibility.Thus,a patients).In newly diagnosedIDDM patientsand in those at in- pathogenicT-cell response appearsto waneat the terminationof the creasedrisk for the disease becauseof their islet cell autoantibodies, autoimmune eventsat the time of P-cell failure andclinical onsetof elevatedfrequenciesof T cellswith in vitro responsiveness to GAD diabetes.Peripheral-bloodT cells from such people, which are peptides(including amino acids 247-266 and 260-279) were ‘autoreactive’to GAD in vitro, would alsoneedto be shownto be observed32. This region(aminoacids250-273of GAD) containsthe responsiveto the CoxsackieP2-C proteinto demonstrate a stronger peptidesegment with significantsequence similarityto the P2-Cpro- link. Evenif thiswerethecase,thismolecularmimicrymightactually tein of CoxsackieB virus (Fig. 4). be an unimportant‘byproduct’of the immuneresponse,or the responsemightbe a disease-modifying or protectiveevent ratherthan Evidence from animal models an inductiveevent in the disease. These,aswell asother important Critical information to support this GAD-Coxsackie mimicry questions, canonly beanswered throughprospectivefollow-upstudies hypothesiswould involve the demonstration that peptidesfrom the in subjectsat high risk for IDDM, in which T-cell responses to Coxsackievirus sequencehomology region would stimulatean regions of GAD, and also to other antigenswith homology to immuneresponseagainstGAD and vice versa.Remarkably,recent Coxsackievirus P2-C, shouldbe examined.It would alsobe useful studieshave demonstratedthat immunizationof NOD mice with to study binding affinities of peptidesderivedfrom theseantigens eitherCoxsackievirus P2-C protein or the Coxsackievirus peptide to the classII MHC ‘binding cleft’, in an attempt to explain containingthe regionof sequence similaritywith GAD caninduceT- the well-known MHC associations in IDDM. It is unclear which cell responses in thesemicethat cross-reactwith GAD or GAD pep- homologiesbetweenmicrobial proteinsand ‘self’ proteinsoccur tides corresponding to the regionof sequencesimilarity3’.Further- solely by chanceand are irrelevant to autoimmunedisease;hommore, this ability to makea T-cell response waslimited to animals ologiesalone are insufficient to prove causalityfor any particular with an IDDM-susceptiblegenotype(classII IA”’ of NOD mice). disease process. T-cell linesfrom NOD micethat arereactiveto the regionof GAD The authors’ work cited here has been supported by grant HD with the sequencesimilarity to CoxsackieB virus can alsotransfer Acknowledgements. both insulitis and IDDM to naive recipients(D. Wegman,pers. 19469-12 from The National Institutes of Health. The American Diabetes Association and The Juvenile Diabetes Foundation International. commun.). 28

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The outstanding questions * Are environmentd agents,suchas viruses,responsible for the geographic variances of the incidence of ~~~n~epen~nt diabetes mellitus (lDDM) &at cannot be exprained on genetic grounds? l Can the casefor mole&~ mimicry providing a mechanism for autoimmune diseasessnchasIDDM be pro~n more directly? l Does the region of sequencesimilarity bet&en Coxsackie virus and giutamic acid decarboxylase provide an important epitope invoked in the induction and/or immunoregulation of IDDM, and could there be others? * If microbial agents are inducers of chronic autoimmune disease(s) such as IDDM, can IDDM be prevented through vaccin&ion? * Does the T helper cell subset (Thl vs, 7312)tbeo~ for regulation of immune responses,which explains susceptibility and resistance to diabetes in non-obese diabetic mice, extend to humans with IDDM or other autoimmune diseases?If so, is there a therapeutic opportunity based upon a switch of a pathogenic Thl effector responseto a protective Th2 response?

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