The detection and localization of inducible nitric oxide synthase production in the small intestine of patients with coeliac disease

Objectives To investigate whether there are increased numbers of inducible nitric oxide synthase (iNOS) containing cells in the small intestine of patients with coeliac disease and the localization of nitric oxide synthase production. Design Small intestinal biopsy specimens from patients with coeliac disease (11 untreated, 10 treated) and nine disease controls were studied. Methods Histochemical staining of sections for NADPH-diaphorase activity was performed, which gives an indication of NOS activity. iNOS protein was detected with immunohistochemistry and iNOS mRNA expression was detected using in situ hybridization with an oligonucleotide probe cocktail for iNOS. Cell phenotype was detected using monoclonal antibodies to CD3 (T-lymphocytes) and CD45 (total inflammatory cell infiltrate). Results There was significantly greater NADPH diaphorase staining in the lamina propria of patients with untreated coeliac disease (P<0.005). The same pattern was found for immunohistochemical and in situ hybridization methods of staining for iNOS in each of the patient groups (P< 0.005) but no epithelial staining was seen with any method. The pattern of iNOS staining in the lamina propria appeared in a similar distribution to that of the inflammatory cell infiltrate. At least 80% of the significantly increased total inflammatory cell infiltrate (CD45) in the lamina propria of patients with untreated coeliac disease was lymphocytic (CD3) whilst the iNOS staining cells made up less than 15% of the total inflammatory cell infiltrate. Conclusions There is a significant increase in the number of NOS staining cells of the inducible isoform in the lamina propria of patients with untreated coeliac disease. The lamina propria and not the epithelium is the site of iNOS production in coeliac disease. It appears that inflammatory cells other than T-lymphocytes are likely to be the cellular sources of iNOS production within the lamina propria. This is the first study to demonstrate increased numbers of iNOS producing cells in the small intestine of patients with untreated coeliac disease and suggests a role for nitric oxide in the pathogenesis of the histological changes seen in coeliac disease although it may be a non-specific inflammatory response to immune activation by gluten in susceptible individuals.