Gluten-induced nitric oxide and pro-inflammatory cytokine release by cultured coeliac small intestinal biopsies

OBJECTIVES: To determine whether there was increased nitric oxide (NO) production from coeliac small intestinal biopsies cultured in vitro with gluten and whether the inhibition of NO production could prevent gluten-induced enterotoxicity. The relationship between NO production with the pro-inflammatory cytokines interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) was evaluated. DESIGN: Small intestinal biopsies from ten patients with treated coeliac disease and six controls were studied. METHODS: Small intestinal biopsies were taken from each patient and set up in culture with Frazer's fraction III (FFIII), a peptic/tryptic digest of gluten, FFIII plus L-NMMA and L-NMMA alone, culture medium alone and ovalbumin which served as a control protein. The biopsies were cultured for 20 h at 37 degrees C. The supernatants were then collected and analysed for nitrite using the Greiss reaction; cytokine levels were determined using ELISA kits. Enterocyte height was determined by microscopy using a calibrated eyepiece graticule and cryostat sections of the cultured biopsies. RESULTS: Coeliac biopsies cultured with FFIII produced significantly greater nitrite compared to culture medium alone (P< 0.05) and this could be blocked with L-NMMA (P< 0.01). A reduction in enterocyte height was seen in coeliac biopsies cultured with FFIII compared to culture medium alone (P < 0.01) and this was ameliorated but not completely blocked when FFIII was cultured with L-NMMA. These changes were not seen in the controls. There was a significant reduction in IL-1beta levels in the supernatant of coeliac biopsies cultured with FFIII compared to culture medium alone (P< 0.05), but when cultured with FFIII and L-NMMA there was a significant increase in IL-1beta levels (P< 0.05). An increase in IFN-gamma levels was also seen when coeliac biopsies were cultured with FFIII and L-NMMA (P< 0.05). This pattern was not seen with TNF-alpha. CONCLUSIONS: Increased levels of NO can be found when coeliac biopsies are cultured with gluten in an in vitro small intestinal culture system, and NO may play a role in the observed enterotoxicity as the inhibition of NO production ameliorates the enterocyte damage. The results suggest that NO is involved in the regulation of pro-inflammatory cytokines, particularly IL-1beta. This is likely to be one of many pathways leading to the observed mucosal pathology in coeliac disease and demonstrates the close interactions between them.