P049 A possible role of mucosal cytokine profile for predicting response to infliximab therapy in ulcerative colitis

Abstract

leads to altered cytokine production and the development of Crohn’s like ileitis, which in fact presents one of the few mouse models in which intestinal inflammation develops spontaneously. SHIP1 is expressed exclusively in hematopoietic lineage and bone marrow parenchymal cells, and colitis can be transferred to wild type mice by SHIP1 / splenocytes. These data strongly suggest a role for SHIP1 in immune cells in the pathogenesis of CD. We therefore investigated SHIP1 activity and expression in peripheral blood mononuclear cells (PBMCs) from CD patients. Methods: We developed a SHIP1 activity assay by precipitating endogenous SHIP1 from PBMC lysates and incubating precipitates with PtdIns(3,4,5)P3. The accumulation of free phosphate as a result of SHIP1 activity was measured by Malachite green assay. The specificity and quantitative range of this assay were confirmed by using SHIP1 deficient cells, and testing different concentrations of protein. Total SHIP1, SHIP2 and PTEN levels were determined by quantitative Western blot analysis (Odyssey). Results: We performed SHIP1 activity assays on PBMCs of 34 CD patients. In every individual measurement, SHIP1 activity in PBMCs from healthy donors was taken as control (n = 25). We identified 5 patients with SHIP1 expression below detection limit and hence low SHIP1 activity. The remaining patients showed a significant upregulation of SHIP1 protein expression levels, but reduced SHIP1 activity after correction for SHIP1 expression. Differences in SHIP1 activity and expression patterns were not caused by altered PBMC composition as no significant differences in B-cell, T-cell or monocyte percentages were observed between CD patients (n = 15) and healthy controls (n = 9). SHIP2 and PTEN protein levels were normal in all CD patients. Conclusions: Our results demonstrate for the first time that immune cells from 15% of CD patients have decreased SHIP1 expression and activity levels, which may affect immune cell function through interference with phospholipid levels. The remaining 85% of CD patients show a decreased activity of SHIP1, although this may be compensated for by an increased SHIP1 expression. Together, these data suggest a role for impaired SHIP1 activity in the development of disease in a subset of CD patients.