Immunopathogenesis of celiac disease

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Samuel Gee in Britain who first described the disease as we currently know it and who ...... León F, Camarero C, Pena R, Eiras P, Sanchez L, Baragaño M, et al.
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Revisión Inmunología Vol. 24 / Núm 3/ Julio-Septiembre 2005: 313-325

Immunopathogenesis of celiac disease F. León1, L. Sánchez2, C. Camarero3, C. Camarero4, G. Roy5 Clinical Discovery-Immunology, Bristol-Myers Squibb, Princeton, USA. 4Pathology Department, Hospital de Cruces, Barakaldo, Spain; Departments of 2Hematology, 3Pediatrics, and 5Immunology, Hospital Ramón y Cajal, Madrid, Spain.

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INMUNOPATOGENIA DE LA ENFERMEDAD CELIACA

Recibido: 2 Septiembre 2005 Aceptado: 15 Septiembre 2005

RESUMEN La Enfermedad Celiaca (EC) o Enteropatía Sensible al Gluten, es una alteración del intestino delgado proximal producida por una intolerancia inmunológica permanente a las prolaminas del gluten. La EC provoca una intolerancia a la ingesta de cereales habituales en la dieta occidental: el trigo, el centeno, la cebada y, posiblemente, la avena. La EC es la enfermedad crónica gastrointestinal más frecuente, afectando al 1% de la raza caucásica. El contacto con el gluten desencadena una inflamación crónica intestinal de base autoinmune, cuya lesión histológica característica es una atrofia vellositaria reversible. Requiere para su manifestación un contexto genético conferido por la presencia de determinados alelos del HLA. El diagnóstico se basa en la demostración de una relación causal entre la ingesta de gluten y la presencia de una enteropatía característica. El único tratamiento es la difícil exclusión del gluten de la dieta. La hipótesis patogénica más aceptada consiste en el desarrollo de una respuesta inmune aberrante al gluten y el desencadenamiento de una enteropatía mediada por linfocitos T activados localmente. Aunque los mecanismos que desencadenan la EC no se conocen, se ha descrito una reacción inmune de los linfocitos CD4+ de la lámina propia frente a péptidos del gluten deamidados por la enzima transglutaminasa tisular (tTG) y presentados por HLA-DQ2 o DQ8, así como una reacción en el epitelio intestinal mediada por linfocitos intraepiteliales (LIE) CD8+ citotóxicos. El estudio de los mecanismos inmunológicos que subyacen en la EC podría permitir el avance en el conocimento de la patogenia de otras enfermedades inflamatorias y autoinmunes, además de abrir nuevas vías terapéuticas para la EC.

ABSTRACT Celiac Disease (CeD), or Gluten Sensitive Enteropathy (GSE), is an immunologically-mediated intolerance to dietary prolamins. A cellular immune response against these cereal-derived proteins impairs the absorption of nutrients by the small intestine and leads to a loss of the normal mucosal architecture and subsequent clinical and metabolic complications. Prolamins are a component of gluten, present in wheat, rye, barley and oats, common ingredients of Western diets, and CeD is the commonest chronic gastrointestinal disease in Caucasians (1% prevalence). Ingestion of gluten provokes a chronic inflammatory response that induces a flattening of intestinal villi in genetically-conditioned subjects. This auto-aggression is reversible when gluten is withdrawn from the diet, and a radical avoidance of those cereals is the only available management of CeD. The temporal association between gluten intake and histological changes constitutes the basis for the diagnosis. The most accepted pathogenic mechanism of CeD is the presentation of gluten peptides by HLA-DQ2 and -DQ8-bearing antigen presenting cells (APC) to T lymphocytes in the intestinal mucosa. Those peptides would be modified by the enzyme tissue transglutaminase (tTG) and become high-avidity binders for those HLA molecules. The ensuing immune reaction would involve CD4+ lymphocytes in the lamina propria and CD8+ cytotoxic intraepithelial lymphocytes (IEL) in the epithelium. Further research is needed to define the mechanisms that lead to the absence of immunologic tolerance to gluten and to develop potential strategies to restore it. KEY WORDS: KEY WORDS: Celiac/ Transglutaminase/ IEL/ Gluten/ Autoimmunity.

PALABRAS CLAVE: Celíaca/ Transglutaminasa/ LIE/ Gluten/ Autoinmunidad.

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HISTORICAL PERSPECTIVE As early as in II BC, Areteus of Cappadocia already wrote on a clinical entity very similar to CeD, but it was Samuel Gee in Britain who first described the disease as we currently know it and who first advocated for diet as the management, in 1887(1). In the 50’s, Dicke observed the relationship between gluten intake and CeD after noticing a reduction in the numbers of CeD patients in Holland during the food shortages provoked by World War II(2), as well as their increase when conditions subsequently improved. Historically, the diagnosis of CeD relied on the clinical symptoms and signs exclusively, and the characteristic pathologic changes of the gut mucosa observed during autopsies were initially considered just a postmortem artifact. When endoscopy was introduced in the 50’s and jejunal biopsies were studied(3,4), those changes became a hallmark of CeD: villous atrophy, cryptal hyperplasia and increased lymphoid density(5). Nowadays, the causal relationship between gluten intake and histological changes remains at the core of the diagnosis of CeD. From the 70’s, a number of associated antibodies (Abs) and auto-antibodies were discovered: anti-reticulin, -gliadin, -jejunal and -endomysial antibodies(6). These Abs allowed for a highly specific and sensitive screening of CeD and its implementation in at-risk populations demonstrated that CeD is very prevalent, often asymptomatic and notably under-diagnosed (the «celiac iceberg»)(7). In addition, two markers have been found to be present in virtually all CeD patients: the major histocompatibility antigen (HLA) HLA-DQ2(8) and an increase in intraepithelial lymphocytes (IEL) bearing the γδ Tc-receptor (TcR)(9). These findings prompted a pathogenic hypothesis for CeD in which gluten peptides would be presented to mucosal CD4+ Tc by HLA-DQ2, in a mode that would break tolerance. This abnormal or exacerbated presentation hypothesis has gained momentum since the discovery of the biochemical modification of gluten peptides by the enzyme tissue transglutaminase (tTG)(10), which happens to be the antigen of anti-endomysial autoantibodies(11). Supporting this theory, gluten-specific HLA-DQ2-restricted Tc clones have been found in the gut mucosa of CeD patients(12,13). Immuno-dominant epitopes had been long known in the gluten sequence(14-17) but it was not until recently that the mechanism by which those peptides could gain access to the jejunal epithelium was elucidated. In vitro digestion of recombinant α2-gliadin with gastric and pancreatic enzymes generates a 33-aminoacid peptide rich in proline and glutamine, very stable and resistant to gastric enzymatic digestion, which contains multiple copies of the 3 epitopes most frequently

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recognized by mucosal Tc of CeD patients(18). In addition, this 33-mer peptide is very susceptible to deamidation by tTG, rendering a highly immuno-stimulatory peptide amenable to DQ2 presentation to α-gliadin-specific Tc(19). Finally, homologues of this peptide have been found in all the cereals that are toxic for CeD patients, but not in non-toxic cereals. The pieces of the puzzle seem falling into place, and CeD may be the first auto-aggressive disease in which the specific antigen and HLA restriction is known.

OVERVIEW OF CELIAC DISEASE Celiac Disease (CeD, CIE10: Q90.1; MIM: 212750)(1,20-23) or Gluten-Sensitive Enteropathy (GSE) is an immunemediated chronic small bowel disease caused by intolerance to prolamins, the proteic and alcohol-soluble fraction of gluten, a major component of some of the commonest cereals in the western diet: wheat, rye, barley(2,24,25). The effects of gluten in celiacs can manifest weeks or months after its ingestion. There is considerable clinical heterogeneity in the presentations of CeD. The classic presentation is more frequent in children, with gastrointestinal (GI) symptoms and signs (distended abdomen, diarrhea, failure to thrive, muscle mass loss) and potential malabsorption (26). Also in children, but particularly in adults(27), CeD can manifest itself with a wide variety of presentations, including metabolic defects (vitamin deficiencies, iron-deficiency anemia), dermatological signs (dermatitis herpetiformis), reproductive or neuro-psychiatric abnormalities, etc... Dermatitis herpetiformis (DH) is, rather than a sign of CeD, a gluten-sensitive entity in itself, with sub-epidermal IgA deposits and blister formation, in which 10% of the patients have no evidence of GI disease and 90% are asymptomatic except for the skin lesions. CeD generally starts in childhood, with a first peak of incidence at 5 years of age. The second peak is in the 4th decade of life, but the onset of CeD can take place at any age. CeD is more frequent in females, with a 2:1 bias with respect to males(21). The prevalence of CeD in the general population ranges from 1/90 (Italy) to 1/300 (England(28)) in Europe(29-31), and 1/250 in North-America(32,33). These figures include the atypical forms of CeD which will be described in detail later, namely silent (asymptomatic) and latent (no atrophy) CeD(34-36). CeD is rare in Asia, unsurprisingly given the importance of the diet and the genetic background in this disease; accordingly, its frequency is increased in Asian immigrants to Western countries(37). The diagnosis is increasingly done in adults, given the improvement of screening techniques and the high rate of

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Figure 1. Normal jejunal mucosa. Left image, haematoxylin-eosin (HE), original magnification 200 x. Right image, scanning electron microscopy (SEM), original magnification 300 x.

Figure 2. Normal jejunal epithelium. Left image, apical or lumen side of a jejunal villus, HE, original magnification 2000 x. Right image, enterocite brush border, electron microscopy (EM), original magnification 22000 x.

asymptomatic disease(38,39): in New Zealand, the prevalence in adults is estimated to be 1.2%, of which 90% are undiagnosed silent CeD cases(40). Only 25% of new diagnoses in adults are done in patients with symptoms from childhood. Globally, the incidence and prevalence of CeD have increased steadily(7,41). Large screening studies performed in healthy blood donors and school-age children(39,42-44) have shown that the prevalence of sub-clinical forms out-weights clinical CeD 3 to 1, what has been termed the «CeD iceberg»(7,30). Populations at risk for CeD include type I Diabetes Mellitus (DM-I)(45), Down Syndrome(46,47), IgA Deficiency(48) and firstdegree relatives of CeD patients(49). The screening of CeD is performed by means of the detection of associated serum antibodies and autoantibodies, such as anti-reticulin(50), anti-gliadin (AGA)(51), anti-endomysial (EMA)(6) and, most recently, anti-transglutaminase. The

titer of these antibodies decreases after treatment, thus constituting useful tools to monitor dietary compliance and complications(52-56). The confirmation of the diagnosis is based upon the demonstration of a causal relationship between gluten intake and the characteristic enteropathy of CeD (cryptal hyperplasia, increased lympho-plasmocytoid cellularity in the lamina propria(5) and increased IEL(57,58)). If GFD is not established, the histological lesion progresses towards epithelial atrophy and loss of epithelial villi (Figs. 1-3) and, at the cellular level, loss of enterocyte microvilli (Figs. 4-5). For this reason, a small bowel biopsy is mandatory, and is obtained by capsule(4) or oral endoscopy(3,59). These alterations are not pathognomonic of CeD, and can also be observed in other diseases(60) such as in gastroenteritis and post-enteritis syndrome, in food allergy –particularly to soy and cow milk(61)-, in parasite

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Figure 3. Epithelial atrophy in Celiac Disease, scanning EM. Scanning electron microscopy images of normal apical jejunal mucosa (left, original magnification 960 x) and active Celiac Disease jejunal mucosa (right, original magnification 450 x). Note cryptal cavities and remainders of villi in this Total Atrophy stage

Figure 4. Epithelial atrophy in Celiac Disease, EM. Electron microscopy images of normal microvilli on the apical surface of a normal enterocyte (left, original magnification 22000 x) and severe brush border lesion in Celiac Disease (right, original magnification 22000 x).

Figure 5. Schematic and histological representation of the stages of Celiac Disease enteropathy. From normality (1) to total atrophy (5). HE, original magnification 200 x.

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infestations (Giardia, Cryptosporidium and nematodes), etc., in children. In adults, these histological changes can be observed in tropical sprue, immuno-deficiencies, lymphomas, Crohn’s disease, and other diseases. This lack of specificity of the intestinal biopsy makes it necessary to repeat the study 2 or 3 times, depending of age at onset and the clinical picture, in order to firmly demonstrate a causal relationship between gluten intake and the enteropathy. A first biopsy is performed as initial diagnosis, a second one after gluten withdrawal and improvement of the clinical signs, and a third one during a gluten challenge(62). In 1990, the criteria of the European Society of Pediatric Gastroenterology and Nutrition (ESPGAN)(63) were revised in order to require a single biopsy in children over 2 years of age, and two biopsies in those asymptomatic patients in which improvement with diet could not be assessed clinically. There are still three situations in which a third biopsy is mandated during

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challenge: when the onset takes place before 2 years of age, when the first study is ambiguous (because serology or histology are negative), or if the patient demands it(63). Treatment of CeD is based on lifelong withdrawal of gluten from the diet, what facilitates the repair of the mucosal architecture, the normalization of total IEL numbers (except for γδ IEL, which remain elevated) and a clinical and functional recovery. Gluten-free diet (GFD) is expensive and difficult to follow for many patients. The safety threshold is only 50 mg of gluten per day and the average consumption is 13 gr/day(64), what illustrates the difficulty of carrying out this restrictive diet. Occasionally, after a period of adequate response to therapy, the patient fails to respond any longer, situation that is termed «Refractory Celiac Disease» and is often accompanied by signs of lymphomagenesis that can evolve into a full-blown lymphoma(65,66). Complications arise in the absence of complete GFD and include osteoporosis, chronic anemia, increased susceptibility for autoimmune disease(67), reproductive abnormalities(68), obstetric complications(69), jejunal ulcers, etc. Neoplasias appear in 10% of untreated patients, mainly digestive cancer (mouth, pharynx, esophagus(70)) and intestinal Tc lymphomas(71). In childhood, due to the loss of absorption through the villi (Figs. 3-4) complications are mainly nutritional, metabolic and growth abnormalities, but also a growing number of malignant complications(72). Importantly, a correct GFD eliminates the increased risk for complications, stressing the need for early diagnosis and for screening of subjects at risk. The etiology of CeD is unknown(73-75). Hypothesis include toxic, enzymatic, infectious(76,77) and immune origins(78), being the immune hypothesis the most accepted since the discovery of the involvement of the gut-associated lymphoid tissue (GALT) in the mucosal effects of gluten in CeD(79).

GENETICS OF CELIAC DISEASE The genetic component of CeD is clear(80) and is based on the significant association of CeD with certain HLA class II alleles. Up to 20% of first-degree relatives of CeD patients are affected by the disease. Monozygotic twins have a 80% concordance rate (81,82), of which the HLA complex explains 40%(74). In 91% of patients, CeD is linked to the HLA heterodimer HLA-DQA1*0501, DQB1*0201, serologically defined as HLA-DQ2, and encoded in cis in haplotype DR3DQ2 and in trans in haplotype DR5/DR7-DQ2(8,83-86). Most DQ2-negative patients possess the heterodimer HLADQA1*0301, DQB1*0302 (in haplotype DR4-DQ8)(87). However, given that HLA-DQ2 is present in 25-30% of healthy Caucasians and that the prevalence of CeD is 1% in this population, it

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seems clear that factors other than the HLA must play a role in the development of the disease. However, little is known about the role of non-HLA genes in CeD. Recent studies have suggested an association with polymorphisms in the negative regulator molecule CTLA-4(88-90), what is consistent with its function and with findings in other autoimmune diseases. The other genetic region associated so far to CeD is the long arm of chromosome 5 (5q 31-33)(91-93) that harbors several genes of importance for the immune system.

ROLE OF GLUTEN IN CELIAC DISEASE The pathogenic role for gluten present in wheat is known since the 50s(94). Gluten proteins can be classified according to their solubility into glutenins (alcohol insoluble) and gliadins (α, β, γ and w; alcohol soluble(99)). Not only gliadins in gluten, but also secalins in rye and hordeins in barley, as well as possibly avidins in oats, are toxic for celiac patients(95). Some studies suggest glutenins can also damage the intestinal mucosa(96-98). The prolamins of gluten are characterized for their high content in the aminoacids glutamine and proline, while the prolamins of rice and corn have a lower content. Oat prolamins (avenins) have an intermediate content of those aminoacids, and a high consumption may be also toxic in CeD(25). The toxicity of repetitive glutamine/proline- rich sequences has been demonstrated in vitro in mucosal explant cultures(100,101) and in vivo in proximal and distal intestine(102). Gluten seems to have a direct effect on the epithelium, both on enterocytes and on IEL, what could play a role in the initial bias of the mucosal immune response. The exposure of intestinal biopsies from celiacs to gluten in vitro induces an increase of HLA-DR expression on enterocytes and lamina propria macrophages in less than an hour(103,104). It is not clear whether the effect on enterocytes is due to a toxic mechanism(74) or whether an aberrant processing pathway for gluten exists in those cells in CeD patients(105). Gliadins provoke a reorganization of the actin cytoskeleton in healthy mucosa, and anti-actin antibodies are found in CeD(106). Regarding IEL, γδ IEL are able to directly recognize native unprocessed gluten(107). Finally, gluten activates the alternative complement pathway(108). Regardless of these findings, the primary triggering event(s) in CeD remain(s) unknown.

AUTOIMMUNITY IN CELIAC DISEASE Active CeD is characterized by the presence of serum antiendomysial IgA antibodies (EMA). These antibodies provide the screening diagnosis of CeD with a sensitivity and specificity

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close to 100%(109). And CeD has a high rate of association with other autoimmune disorders such as Type I Diabetes Mellitus, Dermatitis Herpetiformis, autoimmune thyroiditis, vasculitis, autoimmune alopecia and hepatitis, etc., which are also unusually frequent in relatives of CeD patients(110). All in all, the risk of autoimmunity for untreated celiacs is >30% after 20 years, for only 5% in celiacs treated by 2 years of age(111). This fact suggests that active CeD confers susceptibility, by unknown mechanisms, to organ-specific autoimmunity, stressing the relevance of an early diagnosis and GFD.

TISSUE TRANSGLUTAMINASE AND ANTI-TTG AUTOANTIBODIES Tissue transglutaminase (tTG) is an ubiquitous enzyme that catalyzes covalent bonds between the g-carboxyl group of glutamine and the amino group of lysine(10,112). tTG is normally intracellular, being more abundant in fibroblasts, mononuclear cells and endothelium. It has a double physiologic function, being involved in apoptosis and in tissue repair. On one hand, it prevents the extra-cellular release of cytoplasmic material during apoptosis by means of stabilizing it through covalent bonds(113). On the other hand, if tTG is released from the cell, it contributes to the assembly of extra-cellular matrix during tissue repair. In non-physiologic situations in which there is an excess of glutamyl-donor molecules with respect to receptor molecules, tTG can catalyze the deamidation of glutamine into glutamic acid. And, interestingly, gliadins, with over 30% content in glutamine, are important sources of glutamyl groups(10,96). The connection between tTG and CeD may be inferred from the fact that deamidation of gluten peptides in vitro renders them more immunogenic(114-116) and amenable of enhanced presentation at the HLA-DQ2/8 binding pocket (117-119) , though deamidation is not an absolute requirement(14,17). The determination of endomysial IgA antibodies (EMA) by immuno-fluorescence became the gold standard for the screening of celiac disease, but is a time-consuming and subjective technique(56). For these reasons, the identification of tTG as the main(11) (though not only(120)) auto-antigen of EMA antibodies represented a break-through, since it allowed for the development of objective and standardized solid phase assays such as enzyme-linked immuno-sorbent assay (ELISA)(121,122). The current model for the role of tTG is as follows (Fig. 6)(124,125): tTG would deamidate gluten peptides and generate neo-epitopes (and gluten-tTG complexes) that would be presented by HLA-DQ2 or DQ8 to lamina propria

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lymphocytes (LPL)(118). The production of autoantibodies(126) may be due to the recognition of the neo-epitopes by intestinal Bc, that can also act as APC by means of their HLA-DQ2 expression(123). LPL would become activated after recognition of gluten peptides in DQ2 and could provide help to B cells, allowing for the expansion of the auto-reactive clones. There are evidences to support these hypothesis, such as an increase in expression(127) and activity(10) of tTG in CeD mucosa (also during GFD), or the isolation of HLA-DQ-2-restricted LPL Tc clones with specificity for deamidated gluten(12), though the model is not formally proven to date. The role of anti-tTG antibodies in the pathogenesis of CeD is not clear either, though they are generally considered an epiphenomenon rather than pathogenic(128,129). AntitTG antibodies are able to block the activity of tTG in vitro(130,131), but the net effect of this blockade in vivo is unknown: it might be beneficial by decreasing the generation of gluten neo-epitopes (118,132), but it could also be detrimental by inhibiting the synthesis of TGF-β, an important mediator of mucosal healing(133) in whose biosynthesis tTG plays a key role(130,134).

INTRAEPITHELIAL LYMPHOCYTES IN CELIAC DISEASE The immune response to gliadins takes place at the lamina propria and epithelial levels. The role of lamina propria lymphocytes, LPL, seems clearer than that of intraepithelial lymphocytes (IEL), despite the fact that the variations in IEL subsets and their global increase are a constant feature of CeD. Indeed, the first detectable abnormality in CeD is an increase in αβ and γδ IEL(75,135), what was also the first histopathologic feature described(101). The increased IEL % in the epithelium is due both to their increased proliferation and to a reduction in epithelial cells during the flat mucosa stage of CeD(136,137). The increase in T or CD3+ IEL is also the first detectable event during the gluten challenge test. A second abnormality observed in CeD, which turned out to be nearly pathognomonic, is the increase in TcR-γδ+ IEL(9,138), determined initially by immuno-histochemistry(139141) and subsequently quantified by flow-cytometry (137). Both αβ and γδ IEL proliferate in situ in CeD(142). However, while the increase in αβ IEL correlates with disease activity(137) and is corrected by the GFD, the increase in γδ IEL is constant in relative terms: while these cells average 4% of all IEL in healthy controls, they represent a 25% in celiacs(137), in all phases of disease. The γδ IEL increase is not totally specific of CeD, since it has been occasionally found in other situations, such as cow’s milk intolerance, food allergy (140),

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Figure 6. Pathogenic model of Celiac Disease. Gliadin peptides get access to the mucosal immune system of the small bowel and undergo deamidation by tissue transglutaminase (tTG), extracellularly or in lamina propria APC. The peptides are then presented by APC in HLA-DQ2 or DQ8 to CD4 + Tc, which produce Th1 cytokines such as INF-γ and TNFα. These pro-inflammatory cytokines induce an increased production of matrix metalloproteases and contribute to epithelial apoptosis. Epithelial cell death is likely predominantly induced by NK-receptor-bearing intra-epithelial lymphocytes (IEL) that exert cytolytic actions via recognition of non classic MHC on enterocytes that produce the stimulating cytokine IL-15, closing in the pathogenic circle. Lamina propria CD4+ Tc can also stimulate Bc production of antibodies such as anti-tTG or anti-gliadin Abs.

Figure 7. IEL changes along the course of Celiac Disease. The first abnormality detected in the mucosa of CD patients, even prior to histologic or antibody findings, is an increase in TcR-γδ IEL, practically pathognomonic of CD. When exposure to gluten stimulates the immune system of the gut, TcR ab IEL increase in numbers and activation markers, and CD3(NK and NK-like) IEL literally vanish. These two parameters, unlike the increase in γδ IEL, tend to normalize during Gluten Free Diet (GFD). Subsequently, lamina propria CD4+ Tc increase and get activated and contribute to induce the rest of the pathogenic pathway leading to mucosal atrophy. The changes in IEL subsets are very characteristic of CD and the IEL «lymphogram» has a high sensitivity and specificity in the diagnosis of CD, even of atypical forms (potential, latent).

cryptosporidiasis(140), giardiasis(140,144), Sjögren Syndrome(145) and IgA deficiency(146). However, the increase in γδ IEL in a minority of patients with these conditions tends to be mild and transient(143) or in some cases may correspond to latent CeD. The γδ IEL increase is not observed in other common intestinal disorders(147), and it can be stated that CeD is the only disease in which γδ IEL are increased systematically, permanently and intensely(139-141). Besides, the increase in γδ IEL is observed in all phases of the disease (even in GD) (Fig. 7), what makes this parameter a helpful diagnostic tool in latent and potential CeD(137).

The third and last abnormality described in IEL subsets in CeD is the profound decrease in CD3- CD103+ IEL(138), also termed «NK-like» IEL. The combined determination of gd and CD3- IEL provides specificity to the pathology study in CeD(137). The NK-like or CD3- IEL subset is best studied by flow-cytometry(148,149), given the need for multi-color staining since these cells possess no specific markers. CD3- IEL are numerically the second subset in the healthy small bowel: 50.25±2.57% in children < 3 years, 28.28±2.23% in >3 yearold subjects (mean±SD) and they become virtually undetectable in active CeD (Fig. 7). There are several sub-populations

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within CD3- IEL: Tc precursors (more abundant in very young children); the most prevalent true NK cells(149) (with decreasing numbers with age); and, occasionally, oligoclonal premalignant CD3- IEL in refractory CeD (in elderly individuals mainly)(177). The physiologic role of CD3- IEL is unknown, though for NK IEL is likely related to their cytotoxic capacity(149). The reasons for their disappearance from the epithelium at all phases of active CeD (including latent and potential, though they re-appear on GFD)(136) are also unclear. The analysis by flow-cytometry of the 3 parameters (αβ, γδ and CD3- IEL), termed «IEL lymphogram» (Fig. 7) has a high specificity and sensitivity in the clinics in the diagnosis of CeD, and is particularly helpful in atypical presentations(178).

CURRENT PATHOGENIC MODEL OF CELIAC DISEASE The current most accepted view of the pathogenesis of CeD is as follows(Fig. 6): Gluten would gain access to the Peyer’s patches physiologically via M cells, or supraphysiologically during periods of increased epithelial permeability(150). Gluten would be processed by Peyer’s patches dendritic cells(179) and presented to CD4+ Tc(151). In the absence of deamidation, the presentation of gluten peptides on HLA-DQ2 or DQ8 would induce a Th2 response(152). However, in the presence of tTG activity and deamidation of gluten peptides in the antigen-presenting cells, with enhanced avidity for DQ2/DQ8, the response would be biased towards Th1. IEL, which produce Th1 cytokines(180), could also predispose the initial response to gluten under certain conditions, such as infections. Presentation of gluten to Tc could be carried out not only by dendritic cells(153-155), but also by macrophages, Bc and even enterocytes, that express HLA class II(156). Enterocytes can present antigens to LPL via evaginations through the basement membrane(157), and can express costimulatory molecules under inflammatory conditions(158-160). The primed CD4+ Tc would then recirculate to the lamina propria(161,162), and subsequent contact with gluten would induce their activation(135) and proliferation(75), with production of pro-inflammatory cytokines such as TNF-α(163,164). This would result in synthesis and release of metalloproteases MMP-1 y MMP-3(165-167) and of Keratinocyte Growth Factor by stromal cells(168), what would induce cryptal hyperplasia(101,169) (Fig. 6). The next stage, villous atrophy (Figs. 3-5), would be (at least partially) due to enterocyte death induced by IEL(129,170,171). CD8+ IEL from CeD patients have been shown to respond to gluten peptides presented by HLA-A2(172). Additionally, there is an over-expression of membranebound IL-15 on enterocytes in active CeD and refractory sprue(173), what induces the expression of the NK receptors

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CD94(174) and NKG2D by CD3+ IEL. The ligand for NKG2D, MIC-A (a non-classical HLA class I molecule) is overexpressed on enterocytes in active CeD, what supports the involvement of the MIC-A / NKG2D pathway in the epithelial atrophy of CeD(175, 176) (Fig. 6).

FRONTIERS IN CELIAC DISEASE RESEARCH The basic knowledge of the pathogenesis of CeD should be translated into therapeutic alternatives to the gluten-free diet. One of those under research is the possibility of eliminating the toxic gluten 33-mer peptidic sequence, what has been achieved in vitro and in vivo by exposure to bacterial prolyl endopeptidases, that have been proposed as an oral therapy(18). Another approach is the inhibition of tTG, but it is unclear how that would interfere with important physiologic processes. Yet another alternative is the genetic modification of gluten to eliminate the toxic sequences in the gliadins, but such sequences exist also in glutenins, which are essential to the dietary properties of cereals. Finally, a number of basic questions remain to be answered: What factors other than HLA are needed in order to initiate the disease? Genetic factors or of other nature (infection, gluten over-exposure, age of its dietary introduction)?. What is the role of IEL subsets in CeD?. In summary, CeD is an autoimmune or auto-aggressive disease in which an autoantigen and an HLA-restriction have been found, but it remains orphan of therapeutic approaches. Further research is needed to define the mechanisms that lead to absence of immunologic tolerance to gluten, and to develop potential strategies to restore it.

CORRESPONDENCE TO: Francisco León Clinical Discovery-Immunology Pharmaceutical Research Institute Bristol-Myers Squibb Rt. 206 and Province Line Rd. Princeton, NJ 08543 Phone: 609-252-6254. Fax: 609-252-6816 E-mail: [email protected]

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