High-Molecular-Weight Kininogen Fragments Stimulate the Secretion of Cytokines and Chemokines Through uPAR, Mac-1, and gC1qR in Monocytes

Abstract

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...

Factor XII and uPAR upregulate neutrophil functions to influence wound healing

“…FXIa was previously described to alter the migration of human neutrophils (59), and FXI -/-mice had reduced levels of cytokines in plasma when subjected to polymicrobial sepsis (60). However, there is a growing body of evidence suggesting that components of the contact activation system may regulate inflammatory responses irrespective of their role in thrombin generation (17,(61)(62)(63)(64)(65). Here, we show that active site-inactivated FXII variants themselves promote Akt2S 474 phosphorylation independently of any proteolytic activity.…”

Urokinase and its receptors in chronic kidney disease

“…When HKa binds to uPAR on endothelial cells, it can physically disrupt the VN-uPAR signaling complex formed with co-receptors alpha(v)beta3 or alpha5 beta1 integrins, caveolin, and Src kinase resulting in anti-adhesive effect and apoptosis (73). The ligation of uPAR by HKa can stimulate cytokine and chemokine gene expression in mononuclear cells via mitogen-activated protein kinases (JNK and p38) and the nuclear factor kappa B (NFκB) signaling pathway (75). Careful studies still need to be performed to determine if VN and/or HKa participate in uPAR-dependent anti-fibrotic effects in the kidney.…”

“…In addition to effects on cell adhesion and migration, clustering between activated uPAR and one of its co-receptors may lead to trans-activation and initiation of intracellular signaling (83). Examples that may be relevant to renal disease include the JAK1/STAT1 described in the human kidney epithelial tumor cell line TCL-598 (84), the gp130/Tyk2/STAT3 reported in human mesangial cells (28), the ERK/MAPK activated in fibroblasts, endothelial cells and tumor cells (66,85,86), and the JNK/MAPK and p38/MAPK pathways in mononuclear cells (75).…”

Antithrombotic activity of kininogen is mediated by inhibitory effects of domain 3 during arterial injury in vivo

“…The remaining cleaved HK (HKa), lacking bradykinin, exhibits antiadhesive (26) and antiangiogenic (5) activity as well as enhancing cell-associated fibrinolysis (6) and releasing cytokines and chemokines to enhance inflammation (15). Although deficiency of HK results in vitro in a prolonged activated partial thromboplastin time, patients with plasma HK deficiency do not have a hemorrhagic diathesis (4).…”