Oats induced villous atrophy in coeliac disease.

The current trend is to allow coeliac disease (CD) patients to introduce oats to their gluten free diet. We sought further data from the clinical setting with regards to oats consumption by coeliac patients. Several oat products were tested for wheat contamination using a commercial enzyme linked immunoassay (ELISA) kit, and six samples were examined by an ELISA using a cocktail of monoclonal antibodies, mass spectrometry, and western blot analysis. Nineteen adult CD patients on a gluten free diet were challenged with 50 g of oats per day for 12 weeks. Serological testing and gastroduodenoscopy was performed before and after the challenge. Biopsies were scored histologically and levels of mRNA specific for interferon gamma were determined by reverse transcription-polymerase chain reaction analysis. Oats were well tolerated by most patients but several reported initial abdominal discomfort and bloating. One of the patients developed partial villous atrophy and a rash during the first oats challenge. She subsequently improved on an oats free diet but developed subtotal villous atrophy and dramatic dermatitis during a second challenge. Five of the patients showed positive levels of interferon gamma mRNA after challenge. Some concerns therefore remain with respect to the safety of oats for coeliacs.

Keratinocyte growth factor and coeliac disease.

BACKGROUND: Coeliac disease is characterised by increased epithelial renewal associated with a mucosal T cell response to gliadin. Keratinocyte growth factor (KGF) is produced by cytokine activated gut stromal cells and may be a link between mucosal T cell activation in untreated coeliac disease and epithelial hyperplasia. AIMS: To characterise expression of KGF in coeliac disease. METHODS: KGF transcripts in coeliac disease were measured by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) and localised using in situ hybridisation. KGF production by gluten reactive CD4+ T cell clones was examined. In addition, KGF transcripts were measured following ex vivo challenge of coeliac biopsies with a peptic-tryptic digest of gliadin. RESULTS: KGF transcripts were elevated in coeliac biopsies compared with normal controls but were not different from non-coeliac disease controls. By in situ hybridisation, KGF mRNA containing cells were present in the upper half of the lamina propria, most abundantly just under the epithelium. There was no signal from cells within the epithelium. Gluten reactive T cell clones did not make KGF. In vitro challenge of coeliac biopsies generated a strong interferon gamma response but a specific KGF response could not be detected because of an extremely high number of KGF transcripts in all cultured biopsies. CONCLUSIONS: KGF is overexpressed in coeliac biopsies and in tissues with non-coeliac enteropathy. No evidence was found for KGF production by intraepithelial lymphocytes or lamina propria T cells.

Measurements of cytokine mRNA expression by quantitative polymerase chain reaction in studies of celiac disease.

Cytokines are known to play a key regulatory role in immune responses. The onset or progression of immunopathology in various diseases is often associated with aberrant production of one or more cytokines. It is therefore of considerable interest to characterize cytokine "profiles" associated with disease processes. Many methods employed for identification and quantification of cytokines produced by different cell types rely on the responsiveness of indicator cell lines. Such bioassays are technically restrictive owing to the time required for performance and because of sensitivity and specificity problems. Enzyme-linked immunosorbent assays (ELISAs), on the other hand, detect both biologically active and inactive cytokines without discrimination. These assays are easy to use, but the commercial kits are usually expensive. Both bioassays and ELISAs are unable to identify actual cytokine production and do not account for cytokines consumed by cells. In addition, the minute amounts of cytokine protein often produced in autocrine or paracrine microenvironments may not be easily detectable in a sample, especially when tissue or cells are available in only small quantities (1). Furthermore, although cells producing cytokine protein may be detected by immunocyto/histochemistry, only a limited number of antibodies with good performance are available (2), and the possibility of confusing synthesis with cellular uptake of cytokines exists.

Regional specialization in the mucosal immune system: what happens in the microcompartments?

Mucosal immunity is an important arm of the immune system because it operates in tissues involved in everyday infectious defence as well as in tolerance against innocuous environmental and dietary antigens. Here, Per Brandtzaeg and colleagues discuss compartmentalized regulation of mucosal B cells and mechanisms that might explain the strikingly regionalized effector disparity of the human mucosal immune system.

Different regulatory pathways employed in cytokine-enhanced expression of secretory component and epithelial HLA class I genes.

The transmembrane secretory component (SC, or pIg receptor) plays a crucial role in mucosal immunity by translocating dimeric IgA and pentameric IgM through exocrine epithelia. This receptor is up-regulated by cytokines in parallel with increased epithelial HLA expression. By use of the human epithelial cell line HT-29m3, we show that IFN-gamma, TNF-alpha and IL-4 activate transcription of the SC gene. This activation was slow, suggesting mediation via newly synthesized protein factors. IFN-gamma and TNF-alpha, but not IL-4, also up-regulated expression of HLA class I genes. However, this gene induction was rapid and did not depend on new protein synthesis. Nuclear run-on experiments showed that the transcription rate of HLA class I genes nearly peaked after only 30 min of IFN-gamma or TNF-alpha stimulation, whereas the SC transcription rate did not peak until after 20-36 h of IFN-gamma, TNF-alpha or IL-4 stimulation. Gel electrophoresis mobility shift assays demonstrated binding of nuclear proteins from cytokine-stimulated HT-29 cells to consensus elements in the promoter of the SC gene, involving the binding site for the nuclear factor-kappaB p50 subunit after TNF-alpha stimulation, and IFN-stimulated response element after IFN-gamma stimulation (and weakly after TNF-alpha. Our observations in vitro likely parallel events in vivo by which activated mucosal T cells and macrophages enhance pIg receptor-mediated external transport of secretory IgA and IgM and up-regulate epithelial HLA expression.

HLA restriction patterns of gliadin- and astrovirus-specific CD4+ T cells isolated in parallel from the small intestine of celiac disease patients.

Celiac disease is a common HLA-DQ2-associated enteropathy caused by an abnormal T-cell-mediated immune response to ingested wheat gliadin proteins. We have previously isolated in situ activated mucosal T cells from celiac disease patients and demonstrated that these T cells were gliadin specific and predominantly DQ2 restricted. In contrast to this, gliadin-specific T cells isolated from peripheral blood display a variable HLA restriction pattern, thereby indicating that the skewed DQ restriction of T cells resident in the celiac lesions could be dictated by a preference for DQ-mediated antigen presentation in the mucosa of CD patients. To address this, we analyzed the HLA restriction of T cells recognizing astrovirus, a common gastroentetitis virus, isolated from intestinal mucosa of six celiac disease patients. As an internal control, gliadin-specific T cells were isolated and analyzed in parallel. The gliadin-specific mucosal T cells were marked in their DQ2 restriction, whereas the parallel astrovirus-specific T cells were predominantly restricted by DR molecules. Our data indicate that the repertoire of T cells present in celiac lesions is determined by the priming antigen(s) and not by a skewing in the expression of functional HLA class II isotypes in the disease affected small intestinal mucosa.

Gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease.

BACKGROUND & AIMS: Celiac disease appears to be a T cell-mediated enteropathy induced by gluten in genetically predisposed individuals. Duodenal biopsy specimens from patients with celiac disease and histologically normal controls were investigated to see if cytokine expression is related to disease activity. METHODS: Cytokine messenger RNA (mRNA) expression was determined by quantitative reverse-transcription polymerase chain reaction and in situ expression by immunohistochemistry. RESULTS: In normal controls, mRNA levels were usually below the quantitative limit, even after in vitro gluten stimulation. By contrast, interferon (IFN)-gamma mRNA was increased more than 1000-fold in untreated disease. In vitro gluten stimulation of specimens from treated patients (gluten-free diet) increased IFN-gamma mRNA to the levels of untreated patients. In addition, increased mRNA levels for interleukin (IL)-2, IL-4, IL-6, and tumor necrosis factor alpha were found after such stimulation, whereas mRNA for IL-5, IL-10, and IL-12p40 was usually below the quantitative level. Biopsy specimens from untreated patients contained on average 10-fold more lamina propria cells positive for IFN-gamma than normal controls, whereas cells containing IL-4 were rare in both subject groups. CONCLUSIONS: The results show that mucosal gluten exposure in patients with celiac disease rapidly elicits high levels of IFN-gamma expression and lower levels of IL-2, IL-4, IL-6, and tumor necrosis factor alpha even in the virtual absence of IL-12.

Cytokine profiles of cultured microvascular endothelial cells from the human intestine.

BACKGROUND AND AIMS: Cytokine production by endothelial cells, has, for practical reasons, been chiefly studied in human umbilical vein endothelial cells (HUVEC) but, because tissue-specific differences apparently exist, the role of human intestinal microvascular endothelial cells (HIMEC) as a source of mucosal cytokines was also assessed. METHODS: The expression of cytokine transcripts in HIMEC was screened by means of reverse transcription polymerase chain reaction (RT-PCR) and compared with cytokine profiles of HUVEC. Production of cytokines was investigated by bioassay and enzyme linked immunosorbent assay (ELISA). RESULTS: In the basal unstimulated state, HIMEC and HUVEC cultures contained detectable mRNA for interleukin (IL)-3, IL-7, IL-8, IL-11, IL-14, IL-15, tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and granulocytemacrophage colony stimulating factor (GM-CSF). However, message was undetectable for IL-2, IL-4, IL-5, IL-9, IL-10, IL-12p40, IL-13, and interferon (IFN)-gamma in the resting as well as the stimulated state. Stimulation of HIMEC and HUVEC with recombinant human (rh) IL-1 beta or rhTNF-alpha induced cell associated bioactive IL-1 alpha but not IL-1 beta, as well as enhanced secretion of both IL-6 and IL-8. Furthermore, transcript levels for GM-CSF and TNF-alpha were enhanced by rhIL-1 beta or rhTNF-alpha in both cell types. Supernatants from Th1-like or Th0-like gluten reactive intestinal T cell clones derived from patients with coeliac disease elicited cytokine profiles in both HIMEC and HUVEC similar to those revealed after rhIL-1 beta or rhTNF-alpha stimulation. CONCLUSIONS: These data demonstrate that the intestinal microvascular endothelium may contribute to the cytokine network of the intestinal mucosa with the ability to respond to locally generated cytokines and to produce potent inflammatory mediators.

CD4+ T cells with specific reactivity against astrovirus isolated from normal human small intestine.

BACKGROUND & AIMS: The gut is the largest immunologic organ in the human body, but little is known about the antigen specificity of mucosal T cells. This study sought to determine whether T cells resident in the duodenal mucosa could recognize astrovirus, a common and clinically important gastroenteritis virus. Serum antibodies against astrovirus are prevalent, indicating frequent viral exposure and postinfectious induction of systemic immune responses. Mucosal immune responses may conceivably mediate protection on astroviral reinfections. METHODS: Small intestinal biopsy specimens with normal histology were obtained from 8 adults and challenged in an organ culture system with inactivated human astrovirus. T cells activated by the viral challenge were isolated either by immunomagnetic positive selection of mucosal resident cells or by collecting cells emigrating into the culture supernatant. RESULTS: Astrovirus-specific, mucosal T-cell lines were isolated from all 8 subjects. Analysis of 29 CD4+ T-cell clones from 3 subjects showed predominant HLA-DR restriction of astrovirus responses. Most of the T-cell clones showed a Th1-like cytokine profile when restimulated with astrovirus. CONCLUSIONS: Helper T cells residing in normal, duodenal mucosa of adult subjects recognize a common enteropathogenic virus. These mucosal CD4+ T cells are presumably important in mucosal defense against recurrent astroviral infections.

Cytokine profiles differ in newly recruited and resident subsets of mucosal macrophages from inflammatory bowel disease.

BACKGROUND & AIMS: Most macrophages in the normal intestinal mucosa have a mature phenotype. In inflammatory bowel disease (IBD), a monocyte-like subset (CD14+ L1+) accumulates. The aim of this study was to characterize its potential with regard to cytokines. METHODS: Lamina propria mononuclear cells were adherence-separated, with or without depletion of CD14+ cells, and production of cytokines was investigated by bioassay, enzyme-linked immunosorbent assay, or immunocytochemistry. RESULTS: Tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), and IL-1 receptor antagonist were found mainly in cells positive for myelomonocytic L1. In undepleted IBD cultures, TNF-alpha, IL-1alpha and beta, and IL-10 were markedly up-regulated by pokeweed mitogen stimulation; IL-1alpha and beta and IL-10 were also up-regulated by stimulation of interferon gamma and lipopolysaccharide in combination. The latter stimulation had no effect on normal control or CD14-depleted IBD cultures. Indomethacin caused a marked increase of TNF-alpha, particularly in undepleted IBD cultures, whereas IL-10 and IL-4 decreased TNF-alpha and IL-1beta in both CD14+ and CD14 macrophages. CONCLUSIONS: In IBD mucosa, macrophages with a monocyte-like phenotype are primed for production of TNF-alpha and IL-1alpha/beta and may therefore be of significant pathogenic importance [corrected]. However, this CD14+ subset, as well as the mucosal resident macrophages, have preserved responsiveness to several down-regulatory factors such as the macrophage deactivators IL-10 and IL-4.

Mucosal immunity--a major adaptive defence mechanism.

The epithelial glycoprotein called secretory component (SC) is quantitatively the most important receptor of the immune system because it is responsible for external transport of locally produced polymeric IgA (pIgA) to generate remarkably large amounts of secretory IgA. Antibodies of this type constitute the major mediators of specific humoral immunity. Transmembrane SC belongs to the Ig supergene family and functions as a common pIg receptor, also translocating pentameric IgM externally to form secretory IgM. The B cells responsible for mucosal pIg production are initially stimulated in organized mucosa-associated lymphoepithelial structures, particularly the Peyer's patches in the distal small intestine; from these inductive site they migrate as memory cells to exocrine tissues all over the body. Mucous membranes are thus furnished with secretory antibodies in an integrated way, ensuring a variety of specificities at every secretory effector site. There is currently great interest in exploiting this integrated or "common" mucosal immune system for oral vaccination against pathogenic infectious agents and also to induce tolerance in T cell-mediated autoimmune diseases. However, much remains to be learned about mechanisms for antigen uptake and processing necessary to elicit stimulatory or suppressive mucosal immune responses. Moreover, evidence is emerging for the existence of considerable regionalization with regard to functional links between inductive sites and effecter sites of mucosal immunity.

Gluten activation of peripheral blood T cells induces a Th0-like cytokine pattern in both coeliac patients and controls.

Coeliac disease is apparently a T cell-mediated disease, precipitated in the proximal small intestine of susceptible individuals by gluten. Preferential presentation of gluten peptides most probably takes place in coeliac mucosa by the disease-associated HLA-DQ2 and -DQ8 molecules. In peripheral blood, however, both HLA-DR, -DQ and -DP-restricted T cell responses to gluten have been observed. We examined gluten-specific T cell clones (TCC) derived from peripheral blood for cytokine production to see if their profiles were related to the HLA restriction or the disease state of the donors. As previously found for mucosal TCC, the main product was interferon-gamma (IFN-gamma), often with additional IL-4, IL-5, IL-6, IL-10, tumour necrosis factor, and transforming growth factor-beta. Regardless of restriction element or disease state, gluten-reactive TCC from peripheral blood therefore seem to secrete cytokines compatible with a Th0 profile.

Gluten specific, HLA-DQ restricted T cells from coeliac mucosa produce cytokines with Th1 or Th0 profile dominated by interferon gamma.

Coeliac disease is precipitated in susceptible subjects by ingestion of wheat gluten or gluten related prolamins from some other cereals. The disease is strongly associated with certain HLA-DQ heterodimers, for example, DQ2 (DQ alpha 1*0501, beta 1*0201) in most patients and apparently DQ8 (DQ alpha 1*0301, beta 1*0302) in a small subset. Gluten specific T cell clones (TCC) from coeliac intestinal lesions were recently established and found to be mainly restricted by HLA-DQ2 or HLA-DQ8. Antigen induced production of cytokines was studied in 15 TCC from three patients, 10 being DQ2 and five DQ8 restricted. Cell culture supernatants were prepared by stimulation with gluten peptides in the presence of DQ2+ or DQ8+ Epstein-Barr virus transformed B cells as antigen presenting cells (APC). Supernatants were analysed for cytokines by bioassays, ELISA, and CELISA. Cellular cytokine mRNA was analysed semi-quantitatively by slot blotting and polymerase chain reaction (PCR). All TCC were found to secrete interferon (IFN) gamma, often at high concentrations (> 2000 U/ml); some secreted in addition interleukin (IL) 4, IL 5, IL 6, IL 10, tumour necrosis factor (TNF), and transforming growth factor (TGF) beta. The last TCC thus displayed a Th0-like cytokine pattern. However, other TCC produced IFN gamma and TNF but no IL 4, or IL 5, compatible with a Th1-like pattern. In conclusion, most DQ8 restricted TCC seemed to fit with a Th0 profile whereas the DQ2 restricted TCC secreted cytokines more compatible with a Th1 pattern. The TCC supernatants induced upregulation of HLA-DR and secretory component (poly-Ig receptor) in the colonic adenocarcinoma cell line HT-29.E10, most probably reflecting mainly the high IFN gamma concentrations. This cytokine, particularly in combination with TNF alpha, might be involved in several pathological features of the coeliac lesion. The characterised cytokine profiles thus support the notion that mucosal T cells activated in situ by gluten in a DQ restricted fashion play a central part in the pathogenesis of coeliac disease.