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-γ (IFN-γ), tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) 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°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-1β 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-1 β levels (P< 0.05). An increase in IFN-γ levels was also seen when coeliac biopsies were cultured with FFIII and L-NMMA (P < 0.05). This pattern was not seen with TNF-α. 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-1β. 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.