Pathomechanisms in celiac disease

Best Practice & Research Clinical GastroenterologyVol. 19, No. 3, pp. 373–387, 2005

6

Pathomechanisms in celiac disease

Frits Koning* PhD

Associate Professor

Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre,

P.O. Box 9600, 2300 RC Leiden, The Netherlands

Detlef Schuppan MD, PhD

Professor and Lecturer in Medicine

Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School,

330 Brookline Ave, Boston, MA, USA

Nadine Cerf-BensussanInserm EMI-0212, Faculte Necker-Universite Rene Descartes-Paris V, Paris, France

Ludvig M. Sollid MD, PhD

Professor

Institute of Immunology, University of Oslo, Rikshospitalet University Hospital, N-0027 Oslo, Norway

Celiac disease is an inflammatory disorder of the small intestine caused by an immune response toingested wheat gluten and similar proteins of rye and barley. It affects at least 1 in 200 individuals,corresponding to roughly three million patients in Western Europe and Northern America alone.Data accumulated since the discovery of gluten specific T cells in the intestine of celiac diseasepatients the early 1990s have allowed the deciphering of the interplay between the triggeringenvironmental factor, gluten, the main genetic risk factor, the HLA-DQ2/8 haplotypes and theautoantigen; the enzyme tissue transglutaminase (tTG). This established a key role of adaptiveimmunity orchestrated by lamina propria T cells responding to a set of gluten derived peptides.More recent work points to an important contribution of innate immunity triggered by a distinctgluten peptide and driven by the proinflammatory cytokine Interleukine-5 (IL-15). Together, theseobservations provide a unique explanation for the disease inducing capacity of gluten.

Key words: gluten; tissue transglutaminase; HLA-DQ; IL-15; inflammation.

doi:10.1016/j.bpg.2005.02.003available online at http://www.sciencedirect.com

1521-6918/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: C31 71 526 6673; Fax: C31 71 521 6751.

E-mail address: [email protected] (F. Koning).

http://www.sciencedirect.com

374 F. Koning et al

BACKGROUND

Celiac disease (CD) is a permanent intolerance to gluten for which the only known cureis a lifelong gluten free diet.1 Wheat gluten is a complex mixture of at least 100 relatedproteins. The major components of gluten are the gliadins and glutenins which can bothbe subdivided into distinct protein families.2 As wheat is used in many food products,exposure to relatively large amounts of gluten starts very early in life. Usually gluten isintroduced into the diet at the age of 6 months and a child of 12 months age eatsbetween 6 and 9 g gluten daily.

Virtually all CD patients share certain HLA-DQ molecules.3–5 The function of HLA-molecules is to bind peptides and to ‘present’ these to T cells. When such peptides arepathogen-derived, the T cells can mount a response which will ultimately result in theeradication of the pathogen. In the case of CD, however, the immune system ismisguided by responding to HLA-DQ-bound gluten peptides,6–13 causing inflammationand tissue damage in the small intestine which leads to flattening of the intestinal villi.The result can be a strongly reduced absorptive capacity in the intestine whichmanifests itself as chronic diarrhoea, fatigue, and a failure to thrive.1 Despite the factthat the primary lesion is localised in the gut, CD predominantly presents with varietyof extraintestinal symptoms.

GENETIC ASPECTS

A high prevalence (10%) among first-degree relatives of CD patients indicates thatsusceptibility to develop celiac disease is strongly influenced by inherited factors.14

Familial clustering is stronger in CD than in most other chronic inflammatory diseaseswith a multifactorial etiology.15 The strong genetic influence in CD is furthersupported by a high concordance rate (w75%) in monozygotic twins.16 Both HLAand non-HLA genes contribute to the genetic predisposition.15,17 The presence ofcertain HLA genes appears to be necessary but sufficient for CD development. Thecharacteristic of the HLA association suggest that the HLA genes are involved in aprocess that controls CD development.

HLA GENES

Most CD patients carry the DR3-DQ2 haplotype (the DRB1*0301-DQA1*0501-DQB1*0201 haplotype), or are DR5-DQ7/DR7-DQ2 heterozygotes (i.e. they carry theDRB1*11/12-DQA1*0505-DQB1*0301/DRB1*07-DQA1*0201-DQB1*0202 haplo-types).3,18 CD patients with these DR-DQ-haplotype combinations thus share thesame functional DQ molecule on the cell surface, encoded by genes carried in cis (e.g.DQA1*05 and DQB1*02 carried on the same haplotype) or trans position (e.g. DQA1*05carried on a different haplotype to DQB1*02).3,19 The CD patients who are DQA1*05and DQB1*02 negative frequently carry the DRB1*04-DQA1*03-DQB1*0302 haplotype(i.e. DR4-DQ8 haplotype). Available genetic and functional data favor DQ8 as theprimary disease susceptibility determinant in these patients.5 The very few remainingCD patients who are neither DQ2 (DQA1*05/DQB1*02) nor DQ8 (DQA1*03/DQB1*0302) carry either the a-chain or the b-chain of the DQ2 heterodimer(i.e. DQA1*05 or DQB1*02).19

Pathomechanisms in celiac disease 375

NON-HLA GENES

Much less is known about non-HLA genes in this disorder. There are several reportsthat imply involvement of the gene for the negative costimulatory molecule CTLA4, ora neighboring gene (such as those encoding CD28 or ICOS). However, the overalleffect of this gene is small.20–23 Several genome searches for risk factors have beenperformed in CD.24–31 With the exception of HLA genes, there is relatively littleconsensus between the results, which most likely indicates that each of the non-HLAgenes have a relatively modest effect. Gene–gene interactions and diseaseheterogeneity in the out-bred human population could also produce such a picture.The region that has most consistently been linked to CD is on the long arm ofchromosome 5 (5q31-33).28,31 In meta-analysis of data from several Europeanpopulations this region reached genome wide significance leaving little doubt thatthere exist a susceptibility gene in this region. There is also accumulating evidence for asusceptibility factor on chromosome 11q32 and on chromosome 19p13.29

DIAGNOSIS OF CD

A representative small intestinal histology, revealing significant villous atrophy (lesionsof Marsh stage II and especially III) remains the gold standard for the diagnosis of CD.Serological screening for CD followed by histological confirmation has revealedunexpectedly high prevalences of the condition, between 1:100 and 1:220, in manygeographical regions, such as Europe, the USA, India, North Africa, the near and middleEast, mostly clinically asymptomatic or oligosymptomatic. Diagnosis of these CD casesby screening could be important, since early institution of a gluten-free diet may preventlong-term sequelae, such as osteoporosis, secondary autoimmunity, or evenmalignancy.

In screening of normal populations IgA and IgG anti-gliadin (AGA) antibodies displaysensitivities of 82 and 89%, and specificities of 90 and 66%, resp.,33 both unacceptablylow. Moreover, IgA AGA will identify ten times as many positives as there are biopsy-proven celiacs34 and only long-term positivity of IgA AGA indicates the presence of CDwith higher specificity.35 IgA autoantibodies to endomysium (EMA) or reticulin(fibronectin and collagen containing extracellular fibrils) are a sensitive and,importantly, the most specific serological tool to screen for CD. Mean sensitivityreported from reference laboratories in Europe is 93 and 87%, and specificity is 99 and99.7%, respectively.33 However, lower sensitivities have been observed in the USA36

which appears to be a problem of standardization, and in a CD reference center in theNetherlands37 for less severe intestinal lesions (II and IIIa). With the identification oftissue transglutaminase (tTG) as the target CD autoantigen recognised by EMA38 thedevelopment of easy to perform and observer-independent ELISA tests has becomepossible. For population screening the IgA autoantibodies to tTG serve as an excellenttool to predict celiac disease with a sensitivity and specificity of 94 and 97% onceoptimised assays based on human recombinant tTG are used. It should be noted thatthe quality of the commercially available human recombinant tTG-assays is variable,making selection of a good supplier necessary.39 The original assay versions based onguinea pig tTG should not be used anymore, since despite similar sensitivity, specificityis lower (93%).

376 F. Koning et al

TISSUE TRANSGLUTAMINASE, THE CD AUTOANTIGEN

The enzyme tissue transglutaminase, tTG or transglutaminase 2 is expressed by almostall cell types and is usually retained intracellularly in an enzymatically inactive form. tTGcan be released to the extracellular space to become associated with the extracellularmatrix.40,41 This release is increased when cells are under mechanical or inflammatorystress. tTG belongs to a family of at least eight calcium-dependent transamidatingenzymes that catalyze the covalent and irreversible cross-linking of a protein with aglutamine residue (glutamine donor) to a second protein with a lysine residue(glutamine acceptor), resulting in the formation of an 3-(g-glutamyl)-lysine isopeptidebond40–42 (Figure 1). tTG displays a high specificity for only certain protein-boundglutamine residues as glutamine donor substrates (see below), whereas the lysine-containing glutamine acceptor substrates are numerous. tTG is only active in thepresence of high calcium concentrations, as are found in the extracellular space, whereit contributes to the stabilization of the extracellular matrix.43 However, intracellularactivation and subsequent crosslinking can also occur when cellular integrity is lost andextracellular calcium floods the cell, as found in apoptosis. The latter function of tTGcan prevent leakage of potentially harmful molecules from virus-infected or dying cells.In this line, tTG and tTG-derived crosslinking plays a role in a variety ofneurodegenerative disorders, such as Alzheimer’s and Huntington’s disease.44,45

Under certain conditions, i.e. when no primary lysines are available as glutamineacceptors or at low pH, as can prevail in intestinal inflammation, tTG merely

NH2

HC- (CH2)2- C

O

HN

O = C

C = O

R - CH

NH

R - CH

HC- (CH2)2- C

O

N - (CH2)4- CH

HHN

C = O

O = C

NH

NH

O = C

CH - R

C = O

NH

+

CH - R

C = O

H2N-(CH2)4- CH

NH

O = C

NH

tTG/Ca++

NH3

NH2

HC- (CH2)2- C

O

HN

O = C

C = O

R - CH

NH

R - CH

HC- (CH2)2 -C

HN

C = O

O = C

NH

+ H2OtTG/Ca++

NH3

O

O

Figure 1. Protein crosslinking and deamidation by tTG. Tissue transglutaminase catalyzes either the

deamidation (top) or cross-linking of proteins (tottom). Bottom: tTG will crosslink a glutamine residue in one

protein to a lysine residue in another protein at relatively high pH (O7). This results in the formation of an

irreversible isopeptidyl bond. Top: in the absence of a suitable lysine residue and at relatively low pH (!7) the

glutamine residue in the target protein is converted into a glutamic acid residue. In both cases the reaction is

calcium-dependent. Deamidation results in the generation of gluten peptides with the negatively charged

residue glutamic acid which facilitates gluten peptide binding to HLA-DQ2 and HLA-DQ8.


deamidates a target glutamine in the substrate protein, transforming the neutralglutamine to a negatively charged glutamic acid residue.46

tTG is upregulated in wound healing, angiogenesis and apoptosis. Transforminggrowth factor-b (TGFb), tumor necrosis factor-a, interleukin-6, retinoids, andcorticosteroids stimulate, and bone morphogenetic proteins-2 and-4, and histaminedownregulate tTG expression.40–42 On the other hand, tTG tethers the inactiveproform of the pluripotent cytokine transforming growth factor beta (TGFb, latentTGFb) to the cellular surface which allows its proteolytic activation to mature TGFb bythe enzyme plasmin.47 Inhibition of this surface activation by the patients’ autoantibodiescould be relevant for CD pathophysiology, since TGFb can induce the differentiation ofhuman intestinal epithelial cells, which are less differentiated in active CD.48

THE GLUTEN SPECIFIC T CELL RESPONSE

The role of the disease associated HLA-DQ molecules became clear in 1993 whenHLA-DQ restricted, gluten-specific T cells were indeed isolated from intestinal biopsiespatients and found to be absent in healthy controls.6 The disease associated DQ2 andDQ8 molecules predispose to CD by preferential presentation of gluten peptides toCD4C T cells in the lamina propria. This finding, in light of the nature of the HLAassociation, also implicate that the gluten reactive DQ restricted T cells somehowcontrols the inflammatory response leading to the celiac lesion. In 1998 the identity ofthe first gluten peptides that were recognised by such T cells was reported8,9 (Table 1).These results also solved a mystery that had surrounded T cell recognition of glutenpeptides for several years: HLA-DQ2 and HLA-DQ8 preferentially bind peptidesthat contain amino acids with a negative charge, whereas such peptides are not found inthe gluten molecules. It was found that the enzyme tissue transglutaminase (tTG), thetarget of the autoantibodies in CD, can modify gluten peptides, which introduces thenegative charges required for binding to HLA-DQ-molecules49,50 (Figure 1). Thus,ingested gluten molecules are degraded to peptides by gastrointestinal enzymes,modified by tTG, bind to HLA-DQ2 or HLA-DQ8, and trigger an inflammatory T cellresponse. Further, work has revealed the identity of a series of gluten peptides that cantrigger such T cell responses, indicating that T cells can also recognise particular glutenpeptides in their native form.

The now known source proteins for these T cell stimulatory peptides are the a-gliadins, g-gliadins and the low and high molecular weight glutenins6–13,51–54 (Table 1).Particulary the latter came as a surprise as the glutenins were thought not to beinvolved in CD pathogenesis. The situation is further complicated by the fact that someof these peptides are in fact found repetitively in gluten molecules and that there alsoexist many homologous peptides in gluten molecules. Also, recent work has indicatedthat due to the high proline content, gluten fragments can be particulary resistent to

Table 1. Amino acid sequence of typical immune stimulatory gluten peptides.

a-gliadin PQPQLPYPQ and PFPQPQLPY

g-gliadin FPQQPQQPF and PQQSFPQQQ

LMW-glutenin FSQQQQSPF

HMW-glutenin QGYYPTSPQ

378 F. Koning et al

enzymatic degradation in the gastrointestinal tract, which further enhances theirimmunogenic potential.54 Thus, a large number of immunogenic gluten peptides arepresent in a variety of gluten proteins.

THE ROLE OF TISSUE TRANSGLUTAMINASE IN GLUTENMODIFICATION

Considering the abundance of glutamine residues in gluten (w30–40%) tTG has manypotential target sites in gluten. Mass spectral analysis of the deamidated T cellstimulatory gluten peptides, however, showed a highly restricted pattern ofdeamidation that often coincides with the positions where negative charges arepreferred in the HLA binding motif, thus favouring the binding of gluten peptides toHLA-DQ2 and/or -DQ8. The specificity of gluten deamidation by tTG, therefore, is acrucial factor in the generation of toxic gluten peptides. The specificity of tTG hastherefore been investigated and was found to be very straightforward in the case ofgluten: in the sequences QP and QXXP the Q is not a target for tTG while in thesequences QXP, QXXF(Y, W, M, L, I, or V) and QXPF(Y, W, M, L, I, or V) the Q is atarget for deamidation by tTG.52,55 As glutamine and proline are the two mostabundant amino acids in gluten, averaging 36 and 15%, respectively, the abovementioned sequences are very frequently found in gluten peptides and a highly accurateprediction of tTG target glutamines can be made. We have shown that this informationcan be used to design algorithms that predict gluten peptides that have T cellstimulatory properties.52 Strikingly, such algorithms also identify many peptides with Tcell stimulatory properties in other cereals that are known to cause disease in CDpatients, in particular barley and rye.52 Selective modification of glutamine by tTG thusresults in the generation of a large repertoire of peptides derived from various cerealsthat all can bind to HLA-DQ2 or HLA-DQ8 and stimulate T cells in the intestine ofpatients (Figure 2).

The strength of the T cell response to these gluten peptides, however, is influencedby the level of HLA-DQ2 expression: gluten presentation by antigen presenting cellshomozygous for HLA-DQ2 results in at least fivefold higher T cell responses comparedto gluten presentation by antigen presenting cells heterozygous for HLA-DQ2.56

Strikingly, this correlates with the risk of CD development: HLA-homozygousindividuals have an increased risk of disease development compared to heterozygousindividuals.18 The strength of the gluten specific T cell response thus appears todetermine the likelihood of disease development.

IL-15 ORCHESTRATES INTRAEPITHELIAL LYMPHOCYTE CHANGESIN CD

The scheme described above links the toxicity of cereal-derived peptides to their specificrecognition by T cells, and thereby provides a strong rationale for the interplay betweenenvironmental and genetic factors. Recent data suggest, however, that certain parts ofgluten may stimulate the innate part of the immune system and that interleukin-15 (IL-15)is a central player in this part of the gluten induced immune response (Figure 3).

The massive increase in IEL is considered a diagnostic criterion of CD, and is not ausual feature of inflammatory conditions in the small intestine. While high counts of IEL

Figure 2. The celiac small intestinal lesion. Depiction of the intestinal mucosa with emphasis on the factors taking part in the development and control of celiac disease.

(a) The parts of gluten which are resistant to processing by luminal and brush border enzymes will survive digestion, and can be transported across the epithelial barrier as

polypeptides. Gluten peptides are deamidated by tissue transglutaminase (tTG or TG2), which, in the intestinal mucosa, is mainly located extracellularly in the subepithelial

region, but is also found in the brush border. CD4C T cells in the lamina propria recognise predominantly deamidated gluten peptides, presented by HLA-DQ2 or -DQ8

molecules on the cell surface of antigen presenting cells (APC). (b) Immunofluorescence staining of TG2 (red), HLA-DQ (green) and T cells (CD3; blue) in the small

intestinal mucosa of an untreated celiac disease patient. (Immunofluorescent image courtesy of H. Scott, Rikshospitalet). Artwork by Nature Publishing Group.

Path

om

echanism

sin

celiacdisease

379

NKG2D

IEL

MICA

IL15IL15

IL15IL15TcR?

?

CytotoxicityCytotoxicityIFN-γIFN-γ IL-15IL-15

gliadinpeptide 31-49gliadinpeptide 31-49

IL-15IL-15

Figure 3. Mechanisms leading to the activation of IEL by IL-15 in celiac disease. Peptide 31-49, common to the

N-termini of a-gliadins, induces the production of the cytokine IL-15 in epithelial cells and macrophages via as

yet unknown relays. In turn, IL-15 arms IEL by stimulating their cytotoxic properties and their expression of

the innate immune receptor NKG2D. Furthermore, Il-15 induces the expression of MICA, the epithelial

ligand of NKG2D. Binding of NKG2D to MIC can then trigger the cytotoxicity of IEL against epithelial cells. In

IL-15 stimulated IEL, as it is the case in active celiac disease and in refractory celiac sprue, MIC-NKG2D

mediated cytotoxicity of IEL can thus occur independently of a specific signal given via the T cell receptor

(TcR).66,67 The contribution of additional signals provided either via the T cell receptor and/or other innate

receptors is not excluded.

380 F. Koning et al

are observed in rare conditions such as tropical sprue, and some cases of commonvariable immunodeficiencies, the numbers of IEL are normal or at most moderatelyincreased in Crohn’s disease and in autoimmune enteropathy. In uncomplicated CD,changes affect the two main subsets of IEL. CD8C IEL bearing an ab T cell receptor,which form a large subset of effector cells connected to the adaptive immune system,increases in active CD only. The second subset of IEL characterised by a gd T cellreceptor and effector functions linked to innate immunity and response to epithelialstress, expands in latent CD before the appearance of villous atrophy57 and remainsincreased several years after gluten free diet.58 Moreover, it is now admitted thatenteropathy-associated T cell lymphomas and clonal refractory sprue (RCS), rare butcharacteristic complications of CD represent two stages of a malignant process arisingfrom the IEL compartment.59,60 These observations point to a severe impairment ofintraepithelial homeostasis in CD which cannot be the mere consequence of laminapropria T cell activation. One in vitro study has suggested that, not only CD4Cintestinal T cells, but also some CD8C Tab cells, might be engaged into a specificresponse against gliadin.61 Specific recognition of gliadin cannot however account for allintraepithelial lymphocyte changes and particularly for the early and persistent increasein Tgd IEL. An alternative although non-exclusive hypothesis is an alteration of theinnate immune mechanisms which control lymphocyte-epithelial interactions. IL-15emerged as a likely candidate, on the basis of several observations. This cytokine plays akey role in the homeostasis of murine Tab and Tgd IEL and promotes proliferation andactivation of human IEL in vitro. Engineered IL-15 over-expression in the intestine leadsto a massive expansion of CD8 intestinal T cells accompanied by a CD-likeenteropathy.62 Moreover transgenic mice overexpressing IL-15 develop leukemias of


either T CD8C/K or NK phenotype.63 Indirect evidence of the presence of IL-15 wasfirst provided by Jabri et al who showed that IEL in active CD strongly expressed CD94,an NK receptor rapidly and specifically induced by IL-15 on control IEL.64 Consistentwith a distinctive role of IL-15 in CD, Mention et al demonstrated a massive increase inthe expression of IL-15 in the intestine of patients with active CD but not with Crohn’sdisease.65 Complementary observations from three groups have now delineatedseveral contributions of IL-15 to the pathogenesis of CD.

A first series of results suggests that IL-15 promotes the abnormal expansion of IELand might thereby favour the emergence of lymphoma. This hypothesis is based on exvivo studies in refractory celiac sprue, where epithelium-derived IL-15 appearedmandatory for the survival and growth of the abnormal clonal lymphocytes whichinfiltrate the epithelium.66 A second series of results indicates that IL-15 stimulates theeffector functions of IEL, their secretion of interferon gamma as well as theircytotoxicity and thereby promotes epithelial damage. Strikingly, IL-15 not onlyenhances specific cytotoxic signals mediated via the T cell receptor in IEL, but also armsinnate cytotoxic mechanisms which enable normal CD8 Tab IEL in uncomplicated CD,and clonal IEL from refractory sprue to kill enterocytes independently of the T cellreceptor. Two recent studies have thus delineated how IL-15 might trigger epitheliallysis in CD by activating the NKG2D-MIC pathway66,67 (Figure 3). Meresse et alobserved that IL-15 enhances expression the NK receptor NKG2D on IEL, while Hueet al showed that addition of IL-15 to intestinal organ cultures induced expression of itsligand, the non-classical MHC class I molecule MIC on epithelial cells, which thenbecome the target of NKG2DC IEL.67 Meresse et al also demonstrated that, in CD8abT cells which have recently been activated via their T cell receptor, signals provided byIL-15 can synergize with the activation pathway of NKG2D and trigger lymphocytecytotoxicity independently of any further engagement of the T cell receptor.66 Since,CD8ab T IEL enter the epithelium subsequently to their recent activation in intestine-associated lymphoid organs, NKG2D can be activated in the presence of IL-15 even inthe absence of a cognitive antigen. That this pathway is operating in vivo in CD issuggested by the massive up-regulation of MIC in the epithelium of patients with activeCD and RCS, and by the demonstration that IEL isolated from biopsies of these patientsspontaneously kill MIC-positive targets.65–67 The contribution of additional innatereceptors is however likely since the T cell receptor independent-cytotoxicity of IL-15-activated IEL against enterocyte cell lines was only partially dependent on NKG2D.66,67

Altogether these data indicate that IL-15 can orchestrate the rapid mobilization ofIEL even in the absence of an intraepithelial specific signal. This pathway uncovered bythe study of CD likely plays a key role as a front line defence against intestinalintracellular pathogens, many of which may trigger the production of IL-15 by epithelialcells (Figure 3). Why this pathway is abnormally activated in celiac patients is not yetfully elucidated, but recent data point to the role of the toxic gliadin peptide 31-43/49.

GLIADIN PEPTIDE 31-43 ACTS AS A TRIGGER OF AN INNATERESPONSE ORCHESTRATED BY IL-15 IN CD

Incubation of intestinal biopsies from patients on a GFD with peptide 31-43 for 4 hoursleads to activation of lamina propria dendritic cells including increased expression ofIL-15.68 Furthermore, preincubation with peptide 31-43 boost the expression of theCD25 activation marker on CD4C lamina propria T cells in response to gliadin-derived

382 F. Koning et al

T cell epitopes. This observation, consistent with the known enhancing effect of IL-15secreting dendritic cells on T cell activation, led Maiuri et al to suggest that peptide 31-43 induces in CD an innate response necessary to initiate the T cell adaptiveresponse.68 Hue et al then showed that the innate response orchestrated by IL-15 inthe epithelium might also be initiated by this peptide. Using the longer peptide 31-49,they observed that this peptide, like the gliadin Frazer’s fraction, but unlike a 33 merpeptide including three distinct gliadin T cell epitopes, induced within 12 hours theexpression of MIC on epithelial cells. This inducing effect was blocked by an anti-IL-15antibody and on the contrary reproduced in the presence of soluble IL-15, indicatingthat IL-15 acted as a relay of the 31-43/49 peptide.67

Taken together, these observations suggest that gluten contain not only peptidesable to bind the pocket of HLA-DQ2 molecules to elicit a specific T cell response(Figure 2), but also peptides activating the innate immune system, that cooperate toinduce intestinal inflammation in genetically predisposed individuals (Figure 3). Removalof gluten from the diet, which interrupts both the innate and adaptive immuneresponse, alleviates inflammation. Yet, while it now clear that adaptive immunity canonly be triggered by gliadin peptides in HLA-DQ2/8 individuals, how peptide 31-43/9works and why it is only active in celiac patients, remains unknown. The intracellularrelay(s) that lead(s) to the induction of IL-15 need(s) also to be delineated. Noticeably,Mention et al have observed that in patients on a gluten-free-diet, epithelial expressionof IL-15 decreased but remained above controls.65 This observation, which may explainthe prolonged increase in Tgd IEL after gluten exclusion, advocates a possible defectin the regulation of IL-15 in celiacs, a hypothesis comforted by the study of RCS, wherethe massive expression of IL-15 becomes autonomous of gluten and self-sustainslymphoid expansion and epithelial damage. Uncovering such a defect will howeverrequire first to decipher the complex mechanisms which control IL-15 translation andexpression.66

CONCLUSION AND PERSPECTIVES

CD is currently the autoimmune related disorder where the relationships betweenenvironment and genetics are best understood. Gluten possesses unique structuralfeatures that allow triggering of both the adaptive and innate arms of the immuneresponse. Precise structural data have enlightened the links between this triggeringenvironmental agent, the main genetic risk factor and the autoantigen, and thesequences of events that lead to the activation of lamina propria CD4C T cells by alarge set of gluten-derived peptides in HLA-DQ2/8 individuals only are now welldelineated. More recent evidence introducing the proinflammatory cytokine IL-15 intothe game has provided a clue to the changes in the intraepithelial compartment, ahallmark of the disease. IL-15, directly or indirectly via the activation of innate immunereceptors, can drive the expansion and activation of IEL. IL-15 can also participate tothe triggering of the gluten-specific CD4C response in the lamina propria and promotethe emergence of lymphoid malignancies. The fact that an a-gliadin peptide, whichappears not to be recognised by intestinal CD4C T cells, can up-regulate IL-15production in the intestine of CD patients suggests that the innate response in the IELcompartment may be initiated autonomously, and perhaps explain why the expansion ofIEL can be observed very early on in latent CD before the onset of villous atrophy.These findings however open new questions. How this peptide can trigger IL-15


production and why only in CD patients? How is the production of IL-15 regulated inCD and what are the exact contributions and relays of this cytokine in the intestinalinflammatory process. More generally, many questions remain on the mechanisms thatcontrol the development of the disease only in a very small fraction of HLA-DQ2/8individuals, with a very wide spectrum of severity and at different windows of age. Howthe additional but as yet unknown genes suggested by familial and linkage studies impacton the sensitivity to gluten? What is the contribution of other environmental factors?That alpha-interferon, a cytokine produced in response to viral infections, can triggerCD in at risk individuals, comfort clinical observations of overt CD revealed by aninfectious episode. Alpha-interferon increases expression of HLA genes, stimulatesdendritic cell maturation and might thereby promote the specific T cell response togliadin. Intestinal infections with intracellular agents can also stimulate the localproduction of IL-15 and might synergize with peptide 31-43/9 to promote the innateresponse.

One burning unsolved question is how we can utilize these findings for the benefit ofthe patients. A strict gluten-free diet is a safe and efficient treatment but imposes on thepatients a life-long social burden, and there is a strong demand for alternativetherapeutics. The discovery of multiple redundant T cell epitopes in gliadins andglutenins together with the complexity of wheat genetics strongly jeopardize thefeasibility of wheat detoxification. Identification of these peptides should allowhowever, the development of immunological tests more appropriate to measure thesafety of food products for CD patients than current techniques. The demonstration ofdominant T cell epitopes offers the opportunity to produce analogues, which might beused to alleviate the specific T cell response to gluten, but the efficiency and safety ofthis approach has still to be proven. That immunogenicity of gliadin peptides largelydepends on their deamidation by tTG leads to consider the possibility to block thisenzyme. Yet, it is striking that inactivation of tTG in mice leads to the development ofautoimmunity, ascribed to a defect in the activation of the anti-inflammatory cytokineTGF-b and to a diminished clearance of apoptotic cells.69 Another suggestion by Shanet al is to increase the intraluminal digestion of gluten-derived proteins to decrease theamount of potentially harmful peptides reaching the mucosa, using an exogenous prolyl-endopeptidase. The ability of this enzyme to detoxify gluten in the intestinal lumen ofCD patients is currently under screening. Finally, very recent findings stress thepossibility to manipulate the innate response. Identification of a putative endogenousreceptor for peptide 31-43 and of the signalling pathway elicited by this peptide mayprovide a target for intervention. Another possibility might be to block IL-15 or signalsactivated by IL-15. Blocking MIC-NKG2D interactions might help to prevent IL-15induced epithelial damage but may not be sufficient since IL-15 activated IEL appear touse additional receptors to kill epithelial cells. Neutralising directly IL-15 should havemore impact on the innate intraepithelial lymphocyte response and might also alleviatethe adaptive response in lamina propria. This approach deserves more particularly to beconsidered in the small subset of patients who become refractory to the gluten-free-diet and develop a clonal lymphoproliferative disease of very poor prognosis.Accessibility of clinically validated tools to block IL-15 remains however one majorbarrier.

In conclusion, the past ten years of research on CD have provided one of the moststriking demonstrations that mechanisms and concepts uncovered by fundamentalimmunology can be readily transferred to the understanding of a human disease. CDreciprocally appears as a model disease to decipher the control of the intestinal immune

384 F. Koning et al

response, the mechanisms of autoimmunity and lymphomagenesis. One majorchallenge ahead is now to transfer this knowledge into practical benefit for the patients.

Practice points

† celiac disease is more commen as previously thought: 0.5–1% of the generalpopulation is a patient. Many patients are thus unrecognized

† tissue transglutaminase specific antibodies are highly specific indicators ofdisease

† the large majority of CD patients are HLA-DQ2 positive (95%). The remainderare mostly HLA-DQ8 positive. HLA-typing can be useful for excluding thediagnosis of CD, particularly among family members of CD patients

Research agenda

† identification of non-HLA genes that predispose to CD† identification of the molecular pathways through which these genes predispose

to CD† elucidation of the mechanism through which the 31-43 gluten peptide induces

IL-15 production† development of novel therapy for CD† development of improved diagnostic tools for CD† development of safer foods for CD patients

ACKNOWLEDGEMENTS

FK was supported by the Celiac Disease Consortium, an Innovative Cluster approvedby the Netherlands Genomics Initiative and partially funded by the Dutch Government(BSIK03009) and grants from the European Community (BHM4-CT98-3087 andQLK1-CT-2000-00657). DS was funded by the German Research Council (DFG Schu646/11-3), project QLK1-CT-1999-00037 of the European Community, and theGerman Celiac Association. NCB is supported by the Institut National de la Sante et dela Recherche Medicale (INSERM), Assistance Publique-Hopitaux de Paris(AP-HP) andFondation Princesse Grace. LMS was supported by the Research Council of Norway,the European Community (BHM4-CT98-3087, QLK1-CT-1999-00037 and QLK1-CT-2000-00657), the Norwegian Cancer Society and the NIH (DK65965; together with C.Khosla).

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