To test whether there is a difference in the expression of interleukin 8 (IL8) between Crohn's disease and ulcerative colitis and to determine the main site of its synthesis this study analysed IL8 in mucosal biopsy specimens ofpatients with Crohn's disease and ulcerative colitis by enzyme linked immunosorbent assay (ELISA) and by in situ hybridisation. IL8 was measured by ELISA in 38 normal control patients, eight inflammatory control patients, 55 Crohn's disease biopsy specimens (26 patients), and 67 ulcerative colitis biopsy specimens (35 patients). IL8 mRNA was determined in samples by in situ hybridisation using a specific IL8 RNA probe. IL8 protein was significantly increased in macroscopically inflamed specimens of Crohn's disease (median 118 pg/specimen, p<0.0001), ulcerative colitis (median 140 pg/specimen, p<0001), and inflammatory controls (median 30 pg/specimen, p=0.010) compared with normal controls (median 4 pg/specimen). IL8 was also increased in uninflamed specimens of Crohn's disease (median 46 pg/specimen, p<0 001) but not of ulcerative colitis patients (median 9 pg/specimen, p=0.3). IL8 protein in the mucosa correlated significantly with macroscopic inflammation in Crohn's disease (r=0.47, p<0001) and in ulcerative colitis (r=0.60, p<0 001). IL8 mRNA was detected by in situ hybridisation in 31 of 55 biopsy specimens (56%) ofCrohn's disease patients, in 38 of 67 specimens of ulcerative colitis patients (57%), in five of eight inflammatory controls (63%) and in five of 38 normal controls (130/o). Mucosal IL8 mRNA expression correlated with mucosal IL8 protein (r=0.46, p<0.001). IL8 mRNA was only detected in inflammatory cells of the interstitium but not in mucosal epithelial cells. IL8 is produced mainly in the lamina propria of the colon in inflammatory bowel disease and correlates with mucosal inflammation. (Gut 1996; 38: 216-222)

Macrophages play an important role in the intestinal mucosal immune system. However, they are a poorly defined cell population. We therefore determined their phenotype in normal colonic mucosa. Macrophages were isolated from colonic biopsies and surgical specimens by collagenase digestion. Colonic macrophages were positively sorted by anti-CD33 magnetic beads. Flow cytometric triple fluorescence analysis was applied to study CD14, CD16, CD33, CD44, CD11b, CD11c, CD64, HLA-DR, CD80, CD86 and CD3/CD19 expression. CD33 was evaluated as a positive marker for intestinal macrophages. CD33+ cells isolated from normal colonic mucosa showed co-expression of the established intracellular macrophage marker CD68 in FACS analysis. CD33+ cells were capable of phagocytosis. Isolation of this cell population by magnetic anti-CD33 beads and culture resulted in a 4.2-40-fold increase in IL-1beta and 4.5-44-fold increase in tumour necrosis factor-alpha (TNF-alpha) secretion compared with unsorted lamina propria mononuclear cells (LPMC). Of the CD33+ cells, 90.9 +/- 6.9% (mean +/- s.d.) were CD44+. However, macrophages from colonic mucosa showed only a low expression of CD14 (10.5 +/- 3.8%), CD16 (10.1 +/- 3.9%), HLA-DR (27.3 +/- 9.2%), CD11b (17.4 +/- 6.8%), CD11c (17.8 +/- 10.4%). Furthermore, expression of CD80 (9.2 +/- 4.2%) and CD86 (15.1 +/- 7.3%) was low, suggesting a low ability of normal intestinal macrophages to activate T cells and T cell-mediated immune responses. We conclude that CD33 is useful for the isolation and flow cytometric characterization of colonic macrophages. These cells exhibit a single phenotype in normal mucosa (CD33++, CD44++, CD14-, CD16-, CD11b-, CD11c-, HLA-DRlow, CD80-, CD86-) lacking lipopolysaccharide (LPS) receptor and costimulatory molecules.

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Conclusions-IECs from normal mucosa express and secrete IL-1ra and low amounts of IL-8, but no IL-1 or IL-6. In inflamed mucosa the secretion of IL-1ra by IECs is slightly increased but may be not suYcient to antagonise the greatly increased production of proinflammatory cytokines by LPMNCs and the IECs themselves. (Gut 2000;46:350-358)

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