Regulation of leukocyte recruitment by polypeptides derived from high molecular weight kininogen

Proteolytic cleavage of single-chain, high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves be hind a two-chain, high molecular weight kininogen (HKa) reported to bind to the beta2-integrin Mac-1 (CR3, CD11b/CD18, alphaM beta2) on neutrophils an d exert antiadhesive properties by binding to the urokinase receptor (uPAR) and vitronectin. We define the molecular mechanisms for the antiadhesive e ffects of HK related to disruption of beta2-integrin-mediated cellular inte ractions in vitro and in vivo. In a purified system, HK and HKa inhibited t he binding of soluble fibrinogen and ICAM-1 to immobilized Mac-1, but not t he binding of ICAM-1 to immobilized LFA-1 (CD11a/CD18, alphaL beta2). This inhibitory effect could be attributed to HK domain 5 and to a lesser degree to HK domain 3, consistent with the requirement of both domains for bindin g to Mac-1. Accordingly, HK, HKa, and domain 5 inhibited the adhesion of Ma c-1 but not LFA-1-transfected K562 human erythroleukemic cells to ICAM-1. M oreover, adhesion of human monocytic cells to fibrinogen and to human endot helial cells was blocked by HK, HKa, and domain 5. By using peptides derive d from HK domain 5, the sequences including amino acids H475-G497 (and to a lesser extent, G440-H455) were identified as responsible for the antiadhes ive effect, which was independent of uPAR. Finally, administration of domai n 5 into mice, followed by induction of thioglycollate-provoked peritonitis , decreased the recruitment of neutrophils by A similar to 70% in this mode l of acute inflammation. Taken together, HKa (and particularly domain 5) sp ecifically interacts with Mac-1 but not with LFA-1, thereby blocking Mac-1- dependent leukocyte adhesion to fibrinogen and endothelial cells in vitro a nd in vivo and serving as a novel endogenous regulator of leukocyte recruit ment into the inflamed tissue.