Effects of Epithelial IL-13Rα2 Expression in Inflammatory Bowel Disease

Background: Mucosal IL-13 Receptor alpha 2 (IL13RA2) mRNA expression is one of the best predictive markers for primary non-responsiveness to infliximab therapy in patients with inflammatory bowel disease (IBD). The objective of this study was to understand how IL-13Rα2, a negative regulator of IL-13 signaling, can contribute to IBD pathology.Methods:IL13RA2 knockout (KO) and wild type (WT) mice were exposed to dextran sodium sulfate (DSS) in drinking water to induce colitis. Furthermore, mucosal biopsies and resection specimen of healthy individuals and IBD patients before the start of anti-tumor necrosis factor (anti-TNF) therapy were obtained for immunohistochemistry and gene expression analysis.Results: After induction of DSS colitis, IL13RA2 KO mice had similar disease severity, but recovered more rapidly than WT animals. Goblet cell numbers and mucosal architecture were also more rapidly restored in IL13RA2 KO mice. In mucosal biopsies of active IBD patients, immunohistochemistry revealed that IL-13Rα2 protein was highly expressed in epithelial cells, while expression was restricted to goblet cells in healthy controls. Mucosal IL13RA2 mRNA negatively correlated with mRNA of several goblet cell-specific and barrier genes, and with goblet cell numbers.Conclusions: The data suggest that IL-13Rα2 on epithelial cells contributes to IBD pathology by negatively influencing goblet cell recovery, goblet cell function and epithelial restoration after injury. Therefore, blocking IL-13Rα2 could be a promising target for restoration of the epithelial barrier in IBD

Flow cytometric measurement of cytoplasmic free calcium in human peripheral blood T lymphocytes with fluo-3, a new fluorescent calcium indicator.

Cytoplasmic free calcium [( Ca2+]i) is a key intracellular messenger in many cell types. We have used fluo-3, a recently developed calcium probe, to study [Ca2+]i in resting and stimulated human peripheral blood T lymphocytes. The spectral properties of fluo-3 permit analysis of [Ca2+]i in flow cytometers with a 488 nm argon laser excitation source and fluorescein filter settings. The data obtained with fluo-3 are both qualitatively and quantitatively in good agreement with the data in the literature. After stimulation of T lymphocytes with the mitogens phytohaemagglutinin, concanavalin A and with OKT3, and anti-CD3 monoclonal antibody, a biphasic [Ca2+]i response was observed, with an early EGTA-insensitive [Ca2+]i rise, followed by an EGTA-sensitive sustained [Ca2+]i plateau. Non-mitogenic monoclonal antibodies directed against the CD5, CD28 and CD7 T cell surface antigens elicited [Ca2+]i rises only when crosslinked on the cell surface with goat anti-mouse IgG. Conversion of fluorescence data into absolute [Ca2+]i values by means of a non-disruptive calibration procedure, yielded a [Ca2+]i of 107 +/- 18 nM (mean +/- SD, n = 13) in resting T lymphocytes. Time-dependent loss of cellular dye content limits the precision of the calibration procedure in experiments of longer duration. We conclude that fluo-3 promisingly extends the potential application field of flow cytometers with 488 nm argon lasers to [Ca2+]i studies in T lymphocytes