Pathogenesis of CD

In genetically susceptible individuals, ingestion of cereal prolamins from wheat, barley, rye, and possibly oats initiates an inflammatory disorder during which the small intestinal mucosa is damaged. This process is accompanied by malabsorption, activation of the intestinal immune system, and

Cereal peptides

Gluten-free diet

Innate immune response


Adaptive immune response, CD4 mediated, Th1 skewed

Inflammation of intestinal mucosa

Antibody production

Damage of intestinal mucosa

Gliadin antibodies Autoantibodies

Histologic analysis

Serologic testing

Fig. 1. Brief survey of pathogenesis and diagnosis of CD. Food peptides from wheat and related cereals are partially digested and absorbed. Remaining peptides can be modified by tTG. Toxic peptides bind to MHC class II molecules (antigen presentation). For binding, specific HLA alleles (DQ2 or DQ8) are required. Peptide presentation to CD4 T cells initiates mechanisms of adaptive immune response. As a result, the intestinal mucosa is damaged, which can be analyzed in intestinal biopsy material (gold standard for final CD diagnosis). Further, antibodies against gliadin and against tTG as autoantigen are produced, from which autoantibodies are regarded as the main help in diagnosis. Due to their lower diagnostic validity, gliadin antibodies have lost importance. However, according to new findings, this species of antibodies may gain new weight if for detection deamidated gliadin peptides are applied. The therapy of CD consists in elimination of the offending cereal proteins from food (so-called gluten-free diet). Furthermore, cereal peptides are also able to activate innate immune responses.

production of antibodies against the corresponding cereal food proteins and of autoantibodies against tTG (Fig. 1). The intolerance against prola-mins is generally assumed to be lifelong [25, 26]; however, some observations question this claim [27, 28]. The main therapy consists in exclusion of the disease-triggering cereal proteins from the diet (so-called gluten-free diet).

Before gluten proteins from food are able to interact with cells of the intestinal immune system, they have to pass through the gastrointestinal tract.

Most food macromolecules are nearly completely digested to monomers and short oligomers by the high hydrolytic potential of the gastrointestinal luminal fluid and plethora of enzymes residing in the microvillus membranes of intestinal enterocytes. Due to their high proline content, gliadins are poorly digested and larger peptides remain. In the past, it was believed that a peptidase could be missing in CD patients resulting in the accumulation of toxic peptides in the intestinal lumen [29]. Several results suggested a defective mucosal digestion of gliadin peptides [30-33]; however, a peptidase specifically missing in CD patients was never identified. It was shown that a peptide comprising 33 amino acids from the N-terminal part of a2-gliadin, a representative of a-gliadin, remains remarkably stable after digestion with peptic and pancreatic enzymes and even after incubation with rat or human brush border membrane preparations [34]. Shorter peptides from this 33-mer also resisted intestinal digestion. Dipeptidyl peptidase IV and dipeptidyl carboxypeptidase I were identified as rate-limiting enzymes in digestive breakdown [35]. Another peptide (26-mer) from 7-gliadin (rich in proline and glutamine) also resisted proteolysis [36]. Supplementation of digestion with a bacterial enzyme, prolyl endopeptidase, led to a more efficient hydrolysis of gliadin peptides [37]. Prolyl endopeptidase decreased the amount of intact gliadin peptides crossing the intestinal biopsy specimens of CD patients, but could not completely prevent the intestinal passage of toxic or immunostimulatory metabolites [38]. However, pretreatment of food gluten with prolyl endopeptidase reduced gluten-induced malabsorption in CD [39] and destroyed epitopes important for subsequent activation of T cells [40]. Enzyme therapy based on animal digestive extracts also was successful in reducing symptoms in CD patients [41]. Collectively, these data demonstrate that even if there is no specific peptidase deficiency demonstrable in CD patients, large gluten peptides likely reach and interact with the intestinal epithelium.

The mechanism by which gluten-derived peptides are absorbed through the intestinal epithelium in order to interact with immune cells is still largely unknown. Binding of gliadin peptides was demonstrated to rat micro-villus membrane proteins [42-46], to isolated glycoproteins of rat brush border membranes [47], to cells of the intestinal epithelial cell line HT-29 [48,49], to isolated rat enterocytes and human enterocytes from CD patients in remission as well as from controls [50], and to crypt cells of human biopsies from patients with active CD but not from disease controls or patients in remission [51]. In spite of these intensive investigations, the mechanism of binding still remains unknown.

After binding, gliadin is endocytosed [52] and translocated to HLA-DR antigen-containing endocytic vesicles [53] and Golgi complexes [54] of small intestinal enterocytes in active CD. Alternatively, gliadin may be transported paracellularly across the epithelium. It was found that zonulin, a protein involved in regulation of permeability of tight junctions is increased in intestinal tissues during acute CD [55]. In vitro, gliadin-induced zonulin release in intestinal epithelial cells was found to open tight junctions and increase intestinal permeability [56], thus favoring passage of environmental antigens possibly involved in the pathogenesis of CD.

Following absorption from the intestinal mucosa, gluten peptides stimulate intestinal T lymphocytes to promote antibody production and release of inflammatory mediators. Stimulation of lymphocytes by gluten is seen as main step in the chain of pathogenetic events in CD and is, therefore, of central interest. Other effects of gluten peptides on intestinal epithelial cells [57, 58] and other cells [59-62] which do not seem to be directly related with activity intestinal immune system ("direct toxicity'' of gluten peptides) and induction of innate immune responses (for review see [63, 64]) will not be discussed here.

The intestinal mucosa harbors distinct populations of T lymphocytes. For example, CD4+ T lymphocytes are predominant within the lamina propria. Other T-lymphocyte populations reside in the epithelium (intraepithelial lymphocytes). The majority of these are CD8+ with a minority CD4— and CD8—. In CD, the number of T cells in the lamina propria and epithelium is increased. Although intraepithelial T cells have potent cytolytic and immu-noregulatory capacity, their role in the pathogenesis of CD is not well understood. It has been assumed that intraepithelial lymphocytes, by upre-gulation of natural killer receptors, kill enterocytes. Gluten can induce natural killer receptors as well as expression of their ligands on enterocytes by stimulating the expression of interleukin-15 [65]. However, CD4+ T cells are considered the key component of the anti-gluten response [66]. The CD4+ T cells of the lamina propria are restricted by MHC class II molecules. In CD patients, these lamina propria CD4+ lymphocytes can be stimulated by gluten peptides bound to MHC class II molecules on the surface of antigen-presenting cells. This interaction results in release of proinflammatory cyto-kines such as interferon-7 [67,68] and the ultimate destruction of the intestinal mucosa. Thus, the CD4-mediated proinflammatory immune response in CD is Th1 skewed. Another effect of stimulation of CD4 T cells of lamina propria is the induction antibodies to gliadin and the autoantigen tTG.

The central event in stimulation of lamina propria T cells of CD patients is presentation of gluten-derived peptides. There are two prerequisites for stimulation: (1) presence of the DQ2 or DQ8 haplotype of MHC molecules and (2) modification of gluten peptides for optimal fit into the binding pocket of DQ molecules.

In a large population-based twin study [69], it was shown that in Italy 75% of monozygotic twin pairs were concordant for CD compared with only 11% of dizygotic twin pairs. From these results, it was concluded that environmental factors apart from gluten have only little or even no effect on the pathogenesis of CD. From 15 concordant monocygotic twin pairs, 12 had the DQ2 haplo-type, 1 had the DQ8 haplotype, and 2 had a DRB1*7 allele. The DRB1*7 allele was shown to be linked with the DRB4*01 allele so that these two individuals were positive for Dw53, which is also strongly implicated in the pathogenesis of CD [70]. However, only a small percentage of the 30% of Europeans who inherit HLA-DQ2 and/or DQ8 actually develop CD. Thus, other genes than HLA should be implicated in CD. It was calculated that HLA genes confer only up to 40% of the genetic risk for CD and that the rest is attributable to non-HLA genes [71]. However, large genome-screening studies failed until now to identify other genes that exert a major effect. Rather, a wealth of genetic characteristics that individually exerts little effect might collectively contribute to gluten intolerance together with HLA.

The role of HLA molecules in the pathogenesis of CD is seen in their function of antigen presentation. After binding gluten peptides, MHC molecules associate with T-cell receptors of intestinal CD4+ T cells, resulting in a stimulation of T cells. Peptides derived from native gliadin, however, showed low affinity for DQ2 [72]. Studies of peptide-binding motifs of DQ2 and DQ8 revealed the importance of negatively charged residues in several positions [73-76] not typical for native gliadin peptides.

In 1997, tTG was identified as autoantigen of CD and gliadin was described to be a preferred substrate for this enzyme [1]. Subsequently, gliadin was found to be cross-linked to tTG in tissue sections of monkey esophagus [77]. Hypotheses were then proposed to explain how gliadin cross-linked to tTG could cause the synthesis of autoantibodies directed against tTG (formation of a neoantigen, epitope spreading, intramolecular help) [77, 78]. However, tTG was not only shown to be able to cross-link gliadin to amine donors but also to deamidate glutamine residues within gliadin, thus giving rise to glutamic acid residues. Selective deamidation of gliadin by tTG [79-83] was shown to strongly enhance gliadin-specific T-cell reactivity. However, deami-dation of gluten peptides by tTG is not always a prerequisite for binding to MHC molecules and initiation of immune responses [16, 84, 85].

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