gluten immunochemistry gluten immunochemistry the immunochemistry of triticeae glutens is important in several inflammatory diseases. it can be subdivided into innate responses (direct stimulation of immune system), class ii mediated presentation (hla dq), class i mediated stimulation of killer cells, and antibody recognition. the responses to gluten proteins and polypeptide regions differs according to the type of gluten sensitivity. the response is also dependent on the genetic makeup of the human leukocyte antigen genes. in gluten sensitive enteropathy, there are four types of recognition, innate immunity (a form of cellular immunity priming), hla-dq, and antibody recognition of gliadin and transglutaminase. with idiopathic gluten sensitivity only antibody recognition to gliadin has been resolved. in wheat allergy, the response pathways are mediated through ige against other wheat proteins and other forms of gliadin. illustration of two alpha gliadins showing two proteolytically resistant sites. top shows 6 t-cells sites in 33mer, and bottom shows innate immune peptide and two cxcr3 binding sites. innate immunity underlying conditions the normal intestine illustration of the brush border membrane of small intestinal villiwheat proteins interact with the immune system by means of dq2-mediated programmed cell death (apoptosis) of the gut in sensitive individuals. new research is finding that the coeliac gut may be predisposed to sensitivity in the absence of hla genetic factors. how diet proteins reach the blood the fate of digestible protein in the small intestine in the normal gut, proteins are digested to peptides by pepsin (stomach), trypsin and chymotrypsin (derived from the pancreas and activated in the gut). peptides are further digested when they approach the villi, where brush border peptidases break proteins into amino acids. over much of the small intestine only small solutes, like water, can cross the tight junctions, however some regions of the intestine peptides as large as 500 daltons (4 amino-acids residues in length) can cross. the gluten sensitive gut the fate of gluten in coeliac disease or eia there is a growing body of evidence that the gluten-sensitive intestine differs from the normal gut, several gluten peptides can enter behind the brush border membrane cells. for example, a "33mer" of α-2 gliadin is a magnitude larger than the size exclusion of the tight junction, ω-5 gliadin peptides have been found in the blood stream of people with exercise-induced anaphylaxis, aided by salicylates. and the innate 25 is capable of reaching mononuclear cells in coeliac gut, but in normal gut is broken down by brush border peptidases. it may be this lower peptidase activity that explains the presence of these peptides behind the brush border membrane. recently, it was found that an α-9 gliadin peptide was capable of binding the "cxcr3" receptor, increasing zonulin production and weakening tight junctions, this may explain how, generally, larger peptides can enter the gluten-sensitive gut. innate immunity to gluten refers to an immune response that works independently of t-cell receptor or antibody recognition of the 'innate' peptide. this peptide acts directly on cells, such as monocytes, stimulating their growth and differentiation. innate immunity to gluten is complicated by an apparent role gluten has in bypassing normal host defense and peptide exclusion mechanisms in the gut. while not truly innate, these activities allow gliadin to enter into areas where many lymphocytes patron. in bypassing these filters gliadin alters the normal behavior of both digestive cells, called enterocytes or epithelial cells, and lymphocytes. this increases the potential of causing sensitivity (see underlying conditions). one potential explanation of why certain people become sensitive is that these individuals may not produce adequate peptidases in some areas of the gut, allowing these peptides to survive. other explanation for some may be that food chemicals or drugs are weakening the defenses. this can be the case with ω5-gliadin allergy with salicylate sensitivity. there is no clear reasoning, either from genetics or from long-term studies of susceptible individuals why these gut peptide restrictions would change. illustration of the innate peptide and cxcr3 sites on alpha-9 gliadin once inside, α-9 gliadin 31-55 shows the ability to activate undifferentiated immune cells that then proliferate and also produce inflammatory cytokines, notably interleukin 15 (il-15). this produces a number of downstream responses that are pro-inflammatory. the other peptide that may have innate behavior is the "cxcr3" receptor binding peptides, the receptor exists on enterocytes, the brush border membrane cells. the peptide displaces an immune factor and signals the disruption of the membrane seal, the tight junctions, between cells. alpha gliadin 31-43 gluten bears an innate response peptide (irp) found on α-9 gliadin, at positions 31-43 and on α-3, 4, 5, 8, and 11 gliadins. the irp lies within a 25 amino-acid long region that is resistant to pancreatic proteases. the 25mer is also resistant to brush border membrane peptidases of the small intestine in coeliacs. irp induced the rapid expression of interleukin 15 (il15) and other factors. thus irp activates the immune system. studies show that, while in normal individuals the peptide is trimmed over time to produce inactive peptide, in coeliacs a 19mer may lose a residue from one end or the other, after prolonged incubation that 50% remains intact. intraepithileal lymphocytes and il15 the release of il15 is a major factor in coeliac disease as il15 has been found to attract intraepithelial lymphocytes (iel) that characterize marsh grade 1 and 2 coeliac disease. lymphocytes attracted by il-15 are composed of markers enriched on natural killer cells versus normal helper t-cells. one hypothesis is that il-15 induces the highly inflammatory th1 response that activates t-helper cells (dq2 restricted gliadin specific) that then orchestrate the destructive response, but the reason why inflammatory cells develop prior to gliadin specific helper cells is not known. the irp response differs from typical responses that stimulate il15 release, such as viral infection. in addition, other cytokines such as il12 and il2, which are typically associated with t-helper cell stimulation, are not involved. in these two ways the innate peptide activation of t-cells in coeliac disease is strange. il-15 appears to induce increases in mica and nkg2d that may increase brush-border cell killing. in addition, innate immunity to irp peptide is involved in coeliac disease, dermatitis herpetiformis and possibly juvenile diabetes. irp targets monocytes and increases the production of il-15 by an hla-dq independent pathway, a subsequent study showed that both this region and the "33mer" could create the same response, in cells from both treated coeliacs and non-coeliacs. however, unlike the non-coeliacs, the treated coeliac cells produce the disease marker nitrite. this indicates that another abnormality in people with coeliac disease that allows stimulation to proceed past the normal healthy state. after extensive study, there is no known genetic association for this that appears to stand out at present, and implicates other environmental factors in the defect. infiltrating peptides some alpha gliadin have other direct-acting properties. other gliadin peptides, one in a glutamine rich region and another peptide, "qvlqqstyqllqelccqhlw", bind a chemoattractant receptor, cxcr3. gliadin binds to, blocks and displaces a factor, i-tac, that binds this receptor. in the process it recruits more cxcr3 receptor, increases myd88 and zonulin expression. the factor it displaces, i-tac, is a t-cell attractant. this peptide may also be involved in increased risk for type 1 diabetes as zonulin production is also a factor. this triggering of zonulin ultimately results in the degradation of tight junctions allowing large solutes, such as proteolytic resistant gliadin fragments to enter behind the brush border membrane cells. one study examined the effect of ω-5 gliadin, the primary cause of wd-eia, and found increased permeability of intestinal cells. other studies show that ige reactivity to ω-5 gliadin increases greatly when deamidated or crosslinked to transglutaminase. hla class i restrictions to gliadin hla class i restrictions to gliadin are not well characterized. hla-a2 presentation has been investigated. the hla-a antigens can mediate apoptosis in autoimmune disease and hla a*0201 in with the hla-dq8 haplotypes has been documented. the class i sites were found on the carboxyl end of gliadin at positions 123–131, 144–152, and 172–180. the involvement of class i responses may be minor, since antibodies to transglutaminase correlate with pathogenesis and recognition of extracellular matrix and cell surface transglutaminase can explain the destruction within coeliac disease. this process involves antibody-dependent cellular cytotoxicity. with regard to a receptor called fos, euphemistically called the "death receptor", enterocytes appear to overexpress the receptor in coeliac lesions, there is speculation that class i presentation of gliadin, ttg or other peptides that invokes signalling. the role of class i receptor in cell-mediated programmed cell (enterocyte) death is not known. mic these proteins are called mhc class i polypeptide-related sequence a and b. discovered by sequence homology analysis these proteins are found on the surface of enterocytes of the small intestine, are believed to play a role in disease. studies to date have revealed no mutation that would increase risk for mica. hla-dq recognition of gluten illustration of hla-dq with peptide in the binding pocket hla dq receptor with bound peptide and tcr understanding dq haplotypes and dq isoforms dq haplotypes illustration of dq antigen genetics, click image for details each individual has 22 pairs of autosomes. the hla complex in humans is a large region, ~3 million nucleotides, on chromosome 6, within this region there are a large number of genes. the dq represent 2 genetic loci that lie next to each other. one gene is called dqa1 and the other is called dqb1. there are many alleles at each genetic locus. further information: hla-dq, hla-dqa1, and hla-dqb1 dq antigen, a cell surface receptor, is composed of two polypeptide subunits. there are dozens of alleles at each locus and many create unique subunit isoforms. there are a large number of possible combinations. evolution of humans has limited the most common isoforms. these are more common isoforms encoded by haplotypes, almost all of the time, one passed without change from a person's mother and father from conception. each allele at each locus has an official name. for the alpha subunit the names are given by the gene-alleles. for example, the major dq1 alleles are given as dqa1*0101, *0102, *0103, *0104. dq1 refers to the dq α1 groups of isoforms (historically by serotype) which is the 01 portion of the allele number, the last two digits identify a specific allele in that group. all other dq serotypes refer to beta chain groups – dq2, dq3-(dq7, dq8, dq9), dq4, dq5, dq6. a common way to write a phenotype (both alleles a person has) as dqa1*0101/*0102. this is not enough information to identify a person's isoforms. we also need information about beta chain, the best way to do this is to refer to common haplotypes. hla-dq haplotypes are commonly written in a style: hla-dqa1*0101:dqb1*0501. when considering a person's haplo-phenotype the form dqa1*0102:dqb1*0602/dqa1*0501:dqb1*0201 is the same as dqa1*0102/*0501 dqb1*0602/*0201. when drawn out the form can be used to identify all potential isoforms. (see image below) relationship of haplotypes to antigens there are many potential dq isoforms as a result of cis- and trans-haplotype pairing (see image on left). of course the cis-haplotypes are more common. typically most individuals can produce 4 isoforms, but the 2 isoforms tend to be the most abundant. there are instances where this may not be true, such as when the two betas or two alphas are very similar in structure. most important with regard to isoforms - different subunit isoform pairings can result in the binding of different foreign or self antigens. from a disease defense perspective the more different kinds of peptides that can be presented, the more likely the immune system will detect pathogens and remove them quickly. as a consequence the hla genes are kept extremely variable in most mammalian populations relative to other genes. dq2.5 and gluten sensitivity isoform pairings in dq2.5 homozygotes result in one isoform for coeliac disease however there appears to be one isoform that has a higher role. this isoform is dq α5-β2 (dq2.5). because the beta chain is β2, historically it has been called dq2. not all dq2 isoforms are pathogenic, but at least 2 appear to be more associated with disease. dq2.5 isoform is not rare, 25% of americans caucasians carry the isoform, whereas >90% of people with coeliac disease carry the isoform. dq2 is also increased in gluten-sensitive idiopathic neuropathy. the dqa1*0501:dqb1*0201 haplotype is the most frequent source of