Proteolytic cleavage of single-chain, high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves behind a two-chain, high molecular weight kininogen (HKa) reported to bind to the beta2-integrin Mac-1 (CR3, CD11b/CD18, alphaMbeta2) on neutrophils and exert antiadhesive properties by binding to the urokinase receptor (uPAR) and vitronectin. We define the molecular mechanisms for the antiadhesive effects of HK related to disruption of beta2-integrin-mediated cellular interactions in vitro and in vivo. In a purified system, HK and HKa inhibited the binding of soluble fibrinogen and ICAM-1 to immobilized Mac-1, but not the binding of ICAM-1 to immobilized LFA-1 (CD11a/CD18, alphaLbeta2). 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 binding to Mac-1. Accordingly, HK, HKa, and domain 5 inhibited the adhesion of Mac-1 but not LFA-1-transfected K562 human erythroleukemic cells to ICAM-1. Moreover, adhesion of human monocytic cells to fibrinogen and to human endothelial cells was blocked by HK, HKa, and domain 5. By using peptides derived from HK domain 5, the sequences including amino acids H475-G497 (and to a lesser extent, G440-H455) were identified as responsible for the antiadhesive effect, which was independent of uPAR. Finally, administration of domain 5 into mice, followed by induction of thioglycollate-provoked peritonitis, decreased the recruitment of neutrophils by approximately 70% in this model of acute inflammation. Taken together, HKa (and particularly domain 5) specifically interacts with Mac-1 but not with LFA-1, thereby blocking Mac-1-dependent leukocyte adhesion to fibrinogen and endothelial cells in vitro and in vivo and serving as a novel endogenous regulator of leukocyte recruitment into the inflamed tissue.
Summary. Previously we demonstrated that domain 5 (D5) of high-molecular-weight kininogen (HK) inhibits neovascularization in the chicken chorioallantoic membrane (CAM) assay and further found that kallikrein cleaved HK (HKa) inhibited FGF2-and VEGF-induced neovascularization, and thus was antiangiogenic. In this study, we sought to demonstrate whether uncleaved HK stimulates neovascularization and thus is proangiogenic. The chick chorioallantoic membrane was used as an in ovo assay of angiogenesis. Low-molecular-weight kininogen stimulates angiogenesis, indicating that D5 is not involved. Bradykinin stimulates neovascularization equally to HK and LK and is likely to be responsible for the effect of HK. A murine monoclonal antibody to HK (C11C1) also recognizes a similar component in chicken plasma as detected by surface plasmon resonance. Angiogenesis induced by FGF2 and VEGF is inhibited by this monoclonal antibody and is a more potent inhibitor of neovascularization induced by VEGF than an integrin a v b 3 antibody (LM 609). Our postulate that C11C1 inhibits the stimulation of angiogenesis by HK was confirmed when either C11C1 or D5 completely inhibited angiogenesis in the CAM induced by HK. Growth of human fibrosarcoma (HT-1080) on the CAM was inhibited by GST-D5 and C11C1. These results indicate HK is proangiogenic probably by releasing bradykinin and that a monoclonal antibody directed to HK could serve as an antiangiogenic agent with a potential for inhibiting tumor angiogenesis and other angiogenesis-mediated disorders.
Objective-Plasma high-molecular-weight kininogen (HK) is cleaved in inflammatory diseases by kallikrein to HKa with release of bradykinin (BK). We postulated a direct link between HKa and cytokine/chemokine release. Methods and Results-HKa, but not BK, releases cytokines tumor necrosis factor (TNF)-␣, interleukin (IL)-1, IL-6, and chemokines IL-8 and MCP-1 from isolated human mononuclear cells. At a concentration of 600 nM, glutathione-Stransferase (GST) fusion proteins of kininogen domain 3 (D3), a fragment of domain 3, E7P (aaG255-Q292), HK domain 5 (D5), the D5 recombinant peptides HG (aa K420-D474) and HGK (aa H475-S626) stimulated secretion of IL-1 from mononuclear cells. Monoclonal antibodies (MAbs) specific for D5 or specific for D3 blocked release of IL-1 by HKa, supporting the importance of both domains. Antibodies to HK receptors on leukocytes including Mac-1, LFA-1, uPAR, and C1qR inhibited IL-1 secretion induced by tKa 98%, 89%, 85%, and 62%, respectively. Fractionation of mononuclear cells identified the responsible cell, a blood monocyte. Inhibitors of signaling pathways NFkB, JNK, and p38 but not extracellular signal-regulated kinase (ERK) decreased cytokine release from mononuclear cells. HKa increased the synthesis of IL-1 as deduced by an increase of IL-1 mRNA at 1 to 2 hours. Conclusions-HKa domains 3 and 5 may contribute to the pathogenesis of inflammatory diseases by releasing IL-1 from human monocytes using intracellular signaling pathways initiated by uPAR, 2 integrins and gC1qR. Key Words: chemokines Ⅲ cytokines Ⅲ kininogen Ⅲ monocytes Ⅲ uPAR F rom the discovery of kininogen, 1 the kallikrein-kinin system (KKS) has been intimately involved with inflammation. Plasma kallikrein cleaves HK to form BK and cleaved HK (HKa), which differs from HK because of major conformational changes. 2 BK increases capillary permeability by opening the tight junctions between endothelial cells and directly stimulates nerve endings causing pain, and is a potent vasodilator directly relaxing smooth muscles by releasing PGI 2 . The sum of these effects of BK reproduces many but not all aspects of inflammation. Emphasis in the past decade has shifted from contributions of HK to the humoral aspects of inflammation to its cellular participation, particularly interactions of HKa with leukocytes and endothelial cells. 3 Receptors on either or both of these cell types include selectins, which mediate leukocyte rolling and integrins, which mediate cell adhesion. Cellular proteases such as matrix metalloproteases degrade extracellular matrix protein in the basement membrane, facilitating neutrophil and mononuclear cell migration and emigration into tissues. The activation and participation of the KKS has been documented in inflammatory bowel disease 4 and arthritis 5 in rodents, which are models for human diseases such as rheumatoid arthritis and Crohns disease. Cytokines and chemokines released primarily but not exclusively from monocytes and tissue macrophages are known to play a central role in human inflamm...
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