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) produ ction from coeliac small intestinal biopsies cultured in vitro with gluten and whether the inhibition of NO production could prevent gluten-induced en terotoxicity. The relationship between NO production with the pro-inflammat ory cytokines interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (T NF-alpha) and interleukin-1 beta (IL-1 beta) was evaluated. Design Small intestinal biopsies from ten patients with treated coeliac dis ease and six controls were studied. Methods Small intestinal biopsies were taken from each patient and set up i n culture with Frazer's fraction III (FFIII), a peptic/tryptic digest of gl uten, FFIII plus L-NMMA and L-NMMA alone, culture medium alone and ovalbumi n 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 nitrit e using the Greiss reaction; cytokine levels were determined using ELISA ki ts. Enterocyte height was determined by microscopy using a calibrated eyepi ece 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 block ed with L-NMMA (P< 0.01). A reduction in enterocyte height was seen in coel iac biopsies cultured with FFIII compared to culture medium alone (P< 0.01) and this was ameliorated but not completely blocked when FFIII was culture d with L-NMMA. These changes were not seen in the controls. There was a sig nificant reduction in IL-1 beta levels in the supernatant of coeliac biopsi es 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-1 be ta levels (P< 0.05). An increase in IFN-gamma levels was also seen when coe liac biopsies were cultured with FFIII and L-NMMA (P< 0.05). This pattern w as not seen with TNF-alpha. Conclusions Increased levels of NO can be found when coeliac biopsies are c ultured 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 prod uction ameliorates the enterocyte damage. The results suggest that NO is in volved in the regulation of pro-inflammatory cytokines, particularly IL-1 b eta. This is likely to be one of many pathways leading to the observed muco sal pathology in coeliac disease and demonstrates the close interactions be tween them, fur J Gastroenterol Hepatol 11:529-535 (C) 1999 Lippincott Will iams & Wilkins.