B cells produce immunoregulatory molecules in both HLA-B27 transgenic rats with colitis and non-transgenic littermates

Abstract

Proteinase-Activated Receptor-4 (PAR-4) is 7-transmembrane domain G protein-coupled receptor equally activated by thmmbin attd trypsin. Since enzymatic activities of potential PAR-4 agonists such as thrombin and trypsin are largely increased into the luminal content of inflamnmtory' bowel disease patients. We hypothesized that local PAR-4 activation caused inflammation and is imphcated in the generation of inflammation in a model of Dextran Sodium Sulfate (DSS)-induced colitis. Methods: PAR-4 expression in mouse and human colon was stud~ed by RT-PCR Groups of mice received into their distal colon either a selective PAR-4 agonist (the PAR-4-activating peptide AYPGKF, 200p.g) or a control peptide (inacuve on PAR-4). Pepducin, a novel cell penetrating palmitoylated peptide, based on the bunmn PAR-4 3rd intracellular loop, acts as a PAR-4 antagonist. We have used this PAR-4 antagonist to prove that the effects of intracolnnic injection of PAR-4 agonists were due to PAR-4 activation, by treating mice that have received AYPGKF, with the PAR-4 antagonist or its vehicle. Then, to ins.estigate the role of PAR-4 in the pathogenesis of colitis, 2.5 % DSS was added to the nmuse drinking water to induce colitis, and these mice were treated twice daily, (50p~g/day; 100p~g/day), with the PAR-4 antagonist or its vehicle. At different times after PAR-4-AP intracolic injection or 7 days after the induction of DSS colitis, mice were sacrificed and inflammatory parameters (myeloperoxidase activity, macroscopic damage score, wall thickness) were measured in the mouse colon, Results: PAR-4 signal was detected both in mouse and human colon, Int racolonic administration of AYPGKF, but not the control peptide, caused an inflammatory reaction characterized by increased granulocyte infiltration and edema. Inflammation which reached a maxinmm 10 h after the intmcolonic injection and was back to normal 48h later, was completely inhibited by' PAR-4 antagonist treatment. Seven days afier the reduction of DSS colitis, increased wall thickness and damage score were significantly reduced, in a dose-dependent manner, in mice treated with the PAR-4 antagomst compared to vehicle-treated mice, but MPO acti~aty was not significantly different in all DSS-treated groups. Conclusion: PAR-4 activation triggers inflammation in mouse colon and plays an active role in the generation of inflammation in the DSS-induced colitis model, However, the pro-inflammatory role of PAR-4 did not seem to be associated with granulocyte recruitment.