Analysis of interleukin-4 and interleukin-10 and their association with the lymphocytic infiltrate in the small intestine of patients with coeliac disease.

BACKGROUND: Concentrations of pro-inflammatory cytokines are raised in the small intestine of patients with coeliac disease after ingestion of gluten but there are equivalent data on interleukin-4 (IL-4) and interleukin-10 (IL-10) producing cells. These cytokines are known to exert important regulatory effects on pro-inflammatory cytokine production from lymphocytes and macrophages. AIMS: To investigate whether there is a primary deficiency of IL-4 and IL-10 producing cells and their site of production in the small intestine of patients with coeliac disease in relation to the changes in inflammatory cell infiltrate. PATIENTS: Jejunal biopsy specimens from patients with coeliac disease (11 untreated, 10 treated) and nine disease controls were studied. METHODS: Immunohistochemical staining of sections for IL-4 and IL-10 cytokines and the cell phenotypic markers CD3 (T lymphocytes) and CD45 (total inflammatory cell infiltrate) was carried out using monoclonal antibodies. Expression of IL-4 and IL-10 messenger RNA was detected by in situ hybridisation with oligonucleotide probe cocktails for each cytokine. RESULTS: IL-4 and IL-10 mRNA and protein were detected in the lamina propria of treated and untreated coeliac patients and disease controls but not in the epithelium. A significant increase in the number of CD45 (p < 0.005) and CD3 (p < 0.05) positive cells was found in the lamina propria of patients with untreated coeliac disease compared with treated coeliac patients and disease controls but there were no differences in IL-4 or IL-10 between these groups with either method. CONCLUSIONS: There is no primary deficiency of IL-4 and IL-10 producing cells in the small intestine of patients with coeliac disease. Detectable concentrations of IL-4 and IL-10 were found in control patients which suggests that these cytokines are involved in normal mucosal immunoregulation. The increased number of T lymphocytes but not IL-4 or IL-10 producing cells in the lamina propria of patients with untreated than in those with treated disease suggests not only that the lamina propria is the major mucosal compartment for cytokine production but that newly recruited mucosal T lymphocytes are directed to a predominant Th1 and not a Th2 cytokine response in coeliac patients on a diet containing gluten.

Leucocyte typing, cytokine expression, and epithelial turnover in the ileal pouch in patients with ulcerative colitis and familial adenomatous polyposis.

BACKGROUND/AIMS: Conventional histopathology, leucocyte typing, cytokine mRNA expression, and crypt cell turnover were compared in ileal pouch biopsy specimens from patients with ulcerative colitis (UC) and familial adenomatous polyposis (FAP). METHODS: Biopsy specimens were taken from 17 patients with UC and seven with FAP at a median interval of 19 months (range 2-120) after ileostomy closure. All contained both epithelium and lamina propria. Cryostat sections were stained for lymphocyte subtypes (CD3, CD4, CD8), macrophages (CD68), common leucocyte antigen (CD45), and Ki-67, using a three stage immunoperoxidase reaction. Cytokine mRNA expression for interleukins 2 and 6, tumour necrosis factor alpha, and interferon gamma was studied using an in situ hybridisation technique. RESULTS: Lymphocyte subtype and macrophage populations in epithelium and lamina propria were similar in UC and FAP. The labelling index (Ki-67) was significantly increased in biopsy specimens from patients with UC (UC median = 43.3 (interquartile range (IQR) 38.9-48.2) v FAP 34.9 (29.9-35.2), p < 0.05). There was little or no epithelial mRNA expression for any cytokine in any of the specimens. Lamina propria mRNA expression for interleukin 2 was significantly increased in UC (UC median (IQR) 10.7 (5.4-14.2) cells per unit area v FAP 2.8 (1.5-6.6) p < 0.05) but not for tumour necrosis factor alpha, interleukin 6, and interferon gamma. CONCLUSIONS: While static morphological assessment (leucocyte type, conventional histopathological examination) was similar, tests of cell function (mRNA expression and labelling index) were different in ileal pouches in patients with UC compared with FAP. The study also showed that mRNA expression occurred almost entirely in the lamina propria.

Cytokine mRNA expression in the mucosa of treated coeliac patients after wheat peptide challenge.

This study investigated the presence of mRNA coding for interferon gamma (IFN gamma), tumour necrosis factor alpha (TNF alpha), and interleukins 2 (IL2) and 6 (IL6), in the mucosa of four coeliac patients in remission who had been challenged with either gliadin or synthetic gliadin oligopeptides. Jejunal biopsy specimens from these patients, taken before and at two, four, and six hours after challenge, were hybridised with specific 35S-labelled DNA oligonucleotide probes. The lamina propria of all the patients contained significantly increased numbers of cytokine mRNA expressing cells four hours after challenge with gliadin or an oligopeptide corresponding to amino acids 31-49 of A-gliadin (peptide A). No significant changes were seen with the peptides corresponding to aminoacids 202-220 (peptide B) or 3-21 (peptide C) of A-gliadin, with the exception of one patient who showed a significant increase in the number of TNF alpha mRNA expressing cells four hours after challenge with peptide B. In vivo studies in coeliac disease have shown that significant histological changes occur in the mucosa of treated coeliac patients four hours after challenge with either gliadin or peptide A. These findings suggest that the histological changes seen previously in the mucosa of coeliac patients after wheat peptide challenge may be caused by increased expression of cytokines within the mucosa.

Expression of tumour necrosis factor-alpha, interleukin-6, and interleukin-2 mRNA in the jejunum of patients with coeliac disease.

BACKGROUND: A T-cell-mediated immune response may be responsible for the enteropathy seen in coeliac disease (CD), but it is unclear whether this is initiated in the epithelium or the lamina propria. We studied the site and number of cells expressing mRNA encoding the cytokines interleukin-2 (IL-2), IL-6, and tumour necrosis factor-alpha in jejunal biopsy specimens from patients with untreated or treated CD and normal controls. METHODS: Tissue sections were hybridized with 35S-labelled DNA oligonucleotide probes specific for each cytokine RNA sequence. Positive cells were counted in the lamina propria and epithelial compartments. RESULTS: For each cytokine significantly greater numbers of positive cells were found in the lamina propria of untreated CD patients. Few positive cells were detected in the epithelium of all three groups. CONCLUSIONS: This study shows that the immune response to gliadin appears to occur in the lamina propria and supports cell-mediated immunity in the pathogenesis of coeliac disease.

Raised pro-inflammatory cytokines interleukin 6 and tumour necrosis factor alpha in coeliac disease mucosa detected by immunohistochemistry.

The levels of two pro-inflammatory cytokines, interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha), in coeliac disease were studied by immunohistochemistry. Jejunal biopsy specimens from patients with untreated disease, (n = 11), treated disease (n = 9), and normal controls, (n = 11) were stained to detect IL-6, TNF-alpha, CD45 (pan-leukocyte), and CD68 (macrophage surface antigen). Positive cells were identified in the epithelium (per 100 enterocytes) and in the lamina propria (per unit area). There was a significant increase in median IL-6 and TNF-alpha staining in both the lamina propria and the epithelium of untreated coeliac disease patients (lamina propria, 16.2 and 13.0 respectively; epithelium, 0.86 and 1.21, all p < 0.05) when compared with treated coeliac disease patients (lamina propria; 6.0 and 6.2, epithelium; 0.60 and 0.60) and controls (lamina propria; 6.5 and 7.5, epithelium; 0.58 and 0.60). A significant increase in the number of CD45 positive cells was found in the untreated coeliac disease lamina propria and epithelium (p < 0.05) but this was accompanied by a significant rise in CD68 positive cells in the lamina propria only (p < 0.05). Increased IL-6 and TNF-alpha in the lamina propria and epithelium of patients with untreated coeliac disease further supports their role in the immune pathogenesis of this disorder.

Detection of interferon gamma mRNA in the mucosa of patients with coeliac disease by in situ hybridisation.

In situ hybridisation has been used to study interferon gamma (IFN gamma) mRNA expression in the small intestine of patients with coeliac disease. Sections of jejunal biopsies were obtained from five patients with treated and five with untreated coeliac disease and five disease controls. These sections were hybridised with radiolabelled specific DNA oligonucleotide probes. The lamina propria of untreated coeliac disease patients contained a significantly increased number of IFN gamma producing cells compared with controls but there was no significant difference between the coeliac patients treated with a gluten free diet and controls. The results suggest that IFN gamma may play a part in the immunopathogenesis of coeliac disease.

Wheat peptide challenge in coeliac disease.

The exact nature of the cereal moiety that exacerbates coeliac disease is unknown. In-vitro studies have implicated both the N-terminal and far C-terminal domains of one of the wheat prolamins, A-gliadin. Peptides within these regions may act as epitopes that trigger immune events leading to enteropathy. We synthesized three peptides corresponding to amino-acids 3-21, 31-49, and 202-220 of A-gliadin. Four patients with coeliac disease were challenged by intraduodenal infusion of 1 g of gliadin or 200 mg of the synthetic peptides. Jejunal biopsies were taken before and at hourly intervals for 6 h after the infusion. Morphometric variables were measured and intraepithelial lymphocytes counted. Significant histological changes occurred in the small intestinal mucosa after challenge with a synthetic peptide corresponding to amino acids 31-49 of A-gliadin. The N-terminal peptide, residues 3-21 of A-gliadin, did not cause histological changes in any of the patients. In one of the four patients, minor histological changes following challenge with the peptide corresponding to residues 202-220 of A-gliadin were seen. Our results suggest that the oligopeptide corresponding to aminoacids 31-49 of A-gliadin is toxic in vivo, but there is no evidence of toxicity of the far N-terminal peptide, residues 3-21. The C-terminal peptide 202-220 may contain an epitope to which patients with coeliac disease display variable sensitivity. Since the oligopeptide corresponding to amino-acids 31-49 of A-gliadin is recognised by HLA DQ2-restricted T cells, the observed effects may be due to immune activation within the intestinal mucosa.

Rectal epithelial gamma/delta T-lymphocyte responses to local gluten challenge in coeliac disease.

The proportion of intra-epithelial lymphocytes (IEL) that utilize the gamma/delta form of the T-cell receptor (TCR) is increased in coeliac disease, but their function remains unexplained. The response of intra-epithelial lymphocytes to rectal gluten challenge in coeliac and control subjects was studied after a rectal challenge of 2 g of Frazer's fraction III. A marked rise in CD3+ IEL occurred after challenge in the coeliac patients, peaking at 6 h and returning to normal by 48 h, with no significant changes in the gamma/delta TCR+ IEL. The IEL did not significantly change after gluten challenge in the controls. Acute gluten challenge induces infiltration of the rectal mucosa by T cells in coeliac patients, which is not accompanied acutely by increased numbers of gamma/delta TCR+ IEL. This study supports the hypothesis that alpha/beta TCR+ T cells may be of importance in the early response of coeliac patients to local gluten challenge.

Gamma/delta T-cell receptor expression in the jejunal epithelium of patients with dermatitis herpetiformis and coeliac disease.

The density of jejunal intra-epithelial T cells expressing the gamma/delta form of the T-cell receptor is known to be increased in coeliac disease, the significance of which remains a mystery. The expression of the gamma/delta T-cell receptor in the jejunum of patients with dermatitis herpetiformis, coeliac disease, treated and untreated, and controls were studied. Expression of the gamma/delta T-cell receptor was significantly increased in patients with dermatitis hepetiformis (P < 0.0005) and in both untreated (P < 0.0005) and treated coeliac patients (P < 0.05) compared with controls. There were significant correlations between the indices of enteropathy, enterocyte height (P < 0.005) and villous height/crypt depth ratio (P < 0.0001), and expression of the gamma/delta T-cell receptor in the jejunum of all the patients. This argues against the hypothesis that gamma/delta T-cells have a fundamental role in the aetiology of gluten-sensitive enteropathy. It suggests that gamma/delta T cells may be involved in the effector arm of the mucosal immune response to cereal peptides in susceptible individuals.