δ-Opioid Receptor- and [Met]-enkephalin-like Immunoreactivity in Colonic Mucosa from Patients with Crohn’s Disease and Ulcerative Colitis

Inflammatory bowel disease (IBD) is a condition of unknown aetiology that seriously affects gut function. It has been proposed that changes in opioid and other peptides contribute to the pathophysiology of IBD (Shanahan, 1998). In this investigation we have used immunohistochemistry to determine the distribution of the δ-opioid receptor (δOR) and its endogenous ligand, [Met]enkephalin, in the colonic mucosa of healthy gut and patients with two forms of IBD (Crohn’s disease and ulcerative colitis (UC)). Immunofluorescence and ABC immunohistochemistry using anti- δOR (Insight Biotechnology, UK) and anti-[Met]enkephalin (Affiniti, UK) antibodies were carried out on paraffin embedded specimens of healthy, Crohn’s Disease and UC human colon. Anti-chromogranin A antibody (Abcam, UK) was used to identify neuroendocrine cell populations. The number of immunoreactive (IR) crypts and neuroendocrine cells per field of view were counted at x10 and x20 magnifications, respectively. Statistical analysis was carried out using a Kruskall-Wallis t-test with Dunn’s post test where p<0.001 was considered significant. Colocalisation experiments with anti-chromogranin A antibody showed that [Met]enkephalin-like IR was present in a subpopulation of neuroendocrine cells. A significant difference was seen in the number of [Met]enkephalin-like IR neuroendocrine cells per field of view in Crohn’s disease compared to healthy and UC colon. [Met]enkephalin-like IR was also observed apical to the nuclei in enterocytes lining the mucosal crypts. In healthy gut, the apical IR was seen in very few crypts and only occurred at their base. However, in Crohn’s disease the apical staining was more widespread and seen throughout the entire crypt. 0.9±0.2 [Met]enkephalin-like IR crypts were seen per field of view in healthy specimens (n=12) which was significantly increased to 7.9±0.4 in Crohn’s disease (n=8). [Met]enkephalin-like IR in UC was also localised to the apical side of enterocytes but the distribution was similar to that seen in healthy specimens with 2.0±0.4 IR crypts per field of view (n=13). δOR-like IR was also seen in a subpopulation of neuroendocrine cells but not in enterocytes. No significant difference was noted in the number of δOR-like IR neuroendocrine cells in IBD (n=7) compared to health (n=7). The mucosal differences observed in [Met]enkephalin-like IR in enterocytes between the two forms of IBD could aid in the differential diagnosis of these distressing conditions. Experiments are ongoing to determine whether [Met]enkephalin-like IR is localised in the same neuroendocrine cells as δOR-like IR