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C5AR INHIBITION AMELIORATES PIROXICAM INDUCED COLITIS IN IL-10-/- MICE
U Jain1, T Woodruff2, A Stadnyk11Department of Microbiology and Immunology, Dalhousie University, Halifax, NS; 2School of Biomedical Sciences, University of Queensland, Brisbane, QLD, Australia
Aims: Piroxicam accelerates the development of colitis in IL-10-/- mice; however, the mechanisms of piroxicam induced pathogenesis are incompletely known. Piroxicam enhances mucosal exposure to bacteria and since complement is among the first processes activated to protect self from bacteria, we hypothesized that complement is locally activated and that the generation of anaphylatoxins contribute to the intestinal pathology in piroxicam fed IL-10-/- mice.
Methods: Colitis was elicited in 6 week old IL-10-/- mice (C57BL/6 background) by adding piroxicam (250 ppm) to their food for one week. Mice were gavaged daily with the selective C5aR antagonist (PMX205; 150 µg/mouse) or water (control) starting on the same day as piroxicam. On the day of euthanasia, intestinal permeability was examined using FITC-dextran, and the mice were exsanguinated and their colons excised and the length measured. Half the colon was used to obtain histological scores based on the extent of crypt damage, cell infiltration, edema, ulceration and epithelial hyperplasia. The other half was cultured and 24 h supernatants were analyzed for cytokines and anaphylatoxins. Spleen cells were stimulated with anti-CD3/CD28 antibodies and 72 h culture supernatants analysed for cytokines.
Results: Piroxicam administration to IL-10-/- but not wildtype mice resulted in increased generation of mucosal anaphylatoxins, increased intestinal permeability and severe pathology. Oral gavaging with PMX205 during the period of piroxicam ingestion significantly prevented the intestinal barrier loss and colon shortening observed in piroxicam control mice. PMX205 treated mice also showed less crypt damage, cellular infiltration and epithelial hyperplasia in colon sections. PMX205 significantly prevented the colonic production of IL-12 and IL-1beta but not of IFN-gamma compared to the control mice. Mucosal generation of anaphylatoxins, C3a and C5a was unaffected by PMX205 administration. The in vitro stimulated production of IFN-gamma but not IL-12 was significantly less in splenocytes recovered from the PMX205 treated animals.
Conclusions: Piroxicam induces increased generation of anaphylatoxins in IL-10-/- mice. C5a in turn, contributes to colon damage by increasing intestinal permeability and the local production of pro-inflammatory cytokines. Our data suggest that targeting the C5aR may therefore be a viable therapeutic option to treat inflammatory bowel disease.