Streptococcal surface proteins activate the contact system and control its antibacterial activity.

Group G streptococci (GGS) are important bacterial pathogens in humans. Here, we investigated the interactions between GGS and the contact system, a procoagulant and proinflammatory proteolytic cascade that, upon activation, also generates antibacterial peptides. Two surface proteins of GGS, protein FOG and protein G (PG), were found to bind contact system proteins. Experiments utilizing contact protein-deficient human plasma and isogenic GGS mutant strains lacking FOG or PG showed that FOG and PG both activate the procoagulant branch of the contact system. In contrast, only FOG induced cleavage of high molecular weight kininogen, generating the proinflammatory bradykinin peptide and additional high molecular weight kininogen fragments containing the antimicrobial peptide NAT-26. On the other hand, PG protected the bacteria against the antibacterial effect of NAT-26. These findings underline the significance of the contact system in innate immunity and demonstrate that GGS have evolved surface proteins to exploit and modulate its effects.

Inhibition of metastasis of syngeneic murine melanoma in vivo and vasculogenesis in vitro by monoclonal antibody C11C1 targeted to domain 5 of high molecular weight kininogen.

Metastasis of malignant tumors is a major cause of morbidity and mortality. Inhibition of tumor growth in distant organs is of clinical importance. We have demonstrated that C11C1, a murine monoclonal antibody to the light chain region of high molecular weight kininogen (HK), reduces growth of murine multiple myeloma in normal mice and human colon cancer in nude mice. C11C1 inhibits angiogenesis by reducing tumor microvascular density by blocking binding of HK to endothelial cells. We now evaluate the anti-metastatic effect of C11C1 on C57BL/6 mouse lung metastatic model using B16F10 melanoma cells. The tail veins of mice were injected with 0.5 × 10(6) cells of melanoma B16F10. One group received C11C1 and the other received saline (control) intraperitoneally. When mice were killed at 28 days, 6 of 10 control mice had detectable metastatic pulmonary nodules which stained positive with an antibody against S-100 protein, a tumor antigen present in malignant melanoma cells. In the C11C1 groups, none of the mice showed metastatic foci in their lungs. We showed that C11C1 inhibits endothelial cell tube formation in a 3-D collagen fibrinogen gel model by inhibiting the rate of cleavage of HK by plasma kallikrein without changing the binding affinity for HK. These studies demonstrate that a monoclonal antibody to HK has the potential to prevent metastasis with minimal side effects.

Streptococcal inhibitor of complement-mediated lysis (SIC): an anti-inflammatory virulence determinant.

Since the late 1980s, a worldwide increase of severe Streptococcus pyogenes infections has been associated with strains of the M1 serotype, strains which all secrete the streptococcal inhibitor of complement-mediated lysis (SIC). Previous work has shown that SIC blocks complement-mediated haemolysis, inhibits the activity of antibacterial peptides and has affinity for the human plasma proteins clusterin and histidine-rich glycoprotein; the latter is a member of the cystatin protein family. The present work demonstrates that SIC binds to cystatin C, high-molecular-mass kininogen (HK) and low-molecular-mass kininogen, which are additional members of this protein family. The binding sites in HK are located in the cystatin-like domain D3 and the endothelial cell-binding domain D5. Immobilization of HK to cellular structures plays a central role in activation of the human contact system. SIC was found to inhibit the binding of HK to endothelial cells, and to reduce contact activation as measured by prolonged blood clotting time and impaired release of bradykinin. These results suggest that SIC modifies host defence systems, which may contribute to the virulence of S. pyogenes strains of the M1 serotype.

High-molecular-weight kininogen fragments stimulate the secretion of cytokines and chemokines through uPAR, Mac-1, and gC1qR in monocytes.

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)-alpha, interleukin (IL)-1beta, IL-6, and chemokines IL-8 and MCP-1 from isolated human mononuclear cells. At a concentration of 600 nM, glutathione-S-transferase (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-1beta from mononuclear cells. Monoclonal antibodies (MAbs) specific for D5 or specific for D3 blocked release of IL-1beta by HKa, supporting the importance of both domains. Antibodies to HK receptors on leukocytes including Mac-1, LFA-1, uPAR, and C1qR inhibited IL-1beta 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-1beta as deduced by an increase of IL-1beta mRNA at 1 to 2 hours. CONCLUSIONS: HKa domains 3 and 5 may contribute to the pathogenesis of inflammatory diseases by releasing IL-1beta from human monocytes using intracellular signaling pathways initiated by uPAR, beta2 integrins and gC1qR.

The role of plasma high molecular weight kininogen in experimental intestinal and systemic inflammation.

Inflammation is accompanied by activation of the plasma kallikrein-kinin system (KKS). KKS activation has been demonstrated in a variety of inflammatory human diseases. To further explore the participation of KKS in arthritis and inflammatory bowel disease, we used two experimental animal models in arthritis and enterocolitis. We found that activation of KKS is associated with arthritis induced by intraperitoneal injection of peptidoglycan-polysaccharide polymers (PG-PS) as well as the enterocolitis and systemic inflammation induced also by PG-PS when injected into the intestinal wall of genetically susceptible Lewis rats. We postulated that KKS participates in the pathogenesis of inflammatory reactions involved in cellular injury, coagulation, fibrinolysis, kinin formation, complement activation, cytokine secretion, and release of proteases. We demonstrated that therapy with a specific plasma kallikrein inhibitor modulated the experimental enterocolitis, arthritis, and systemic inflammation. The fact that deficiency of plasma high molecular weight kininogen in the genetically susceptible Lewis rat results in decreased chronic enterocolitis and systemic inflammation also supports our hypothesis. We suggest that KKS plays a similar role in idiopathic human intestinal inflammatory disease and arthritis, making kallikrein-kinin system proteins appealing targets for drug therapy in chronic inflammatory diseases such as rheumatoid arthritis and Crohn's disease.

A monoclonal antibody to high-molecular weight kininogen is therapeutic in a rodent model of reactive arthritis.

We reported that high-molecular weight kininogen is proangiogenic by releasing bradykinin and that a monoclonal antibody to high-molecular weight kininogen, C11C1, blocked its binding to endothelial cells. We now test if this antibody can prevent arthritis and systemic inflammation in a Lewis rat model. We studied 32 animals for 16 days. Group I (negative control) received saline intraperitoneally. Group II (disease-treated) received peptidoglycan-polysaccharide simultaneously with C11C1. Group III (disease-untreated) received peptidoglycan-polysaccharide simultaneously with isotype-matched mouse IgG. Group IV (disease-free-treated) and group V (disease-free isotype-treated) received saline and C11C1 or mouse IgG. Analysis of joint diameter changes showed a decrease in the C11C1 disease-treated group compared to the disease-untreated group. The hind paw inflammatory score showed a decrease in the intensity and extent of inflammation between the disease-untreated and the C11C1 disease-treated group. Prekallikrein, high-molecular weight kininogen, factor XI, and factor XII were decreased in the disease-untreated group compared to the C11C1 disease-treated group. T-kininogen was increased in the disease-untreated group when compared with the C11C1 disease-treated group. Disease-free groups IV and V did not show any sign of inflammation at any time. This study shows that monoclonal antibody C11C1 attenuates plasma kallikrein-kinin system activation, local and systemic inflammation, indicating therapeutic potential in reactive arthritis.

Inhibition of angiogenesis by antibody blocking the action of proangiogenic high-molecular-weight kininogen.

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 alphavbeta3 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.

Immunohistochemical distributions of the tissue kallikrein-kinin system in ischemic and non-ischemic mouse heart.

Kinins have been shown to play a cardioprotective role during myocardial ischemia. However, the localization of each of the components of the kallikrein-kinin system in the heart has not been determined in a cell type-specific manner. Recently, mK1 has been identified as the major tissue kallikrein with the strongest bradykinin-forming activity among the products of the mouse tissue kallikrein gene superfamily. In the study presented here, we investigated the localizations of mK1, kininogen and bradykinin B2 receptors (B2Rs) in ischemic and non-ischemic left ventricles by immunohistochemistry. Kininogen, which contains bradykinin as a surface epitope, was detected by an anti-bradykinin antibody. Changes in the amounts of mK1 and B2R were evaluated by Western blot analysis. Myocardial ischemia was induced by ligation of the left anterior descending coronary artery for 60 min followed by reperfusion for 24 h. mK1 and B2Rs were most abundantly expressed in the vascular endothelium and, to a lesser extent, in fibroblasts. No immunohistochemical signal of these molecules was detected in myocytes. Kininogen was localized in the vascular endothelium and the smooth muscle layer. Myocardial ischemia, although it had no effect on the localization of these molecules, increased the amounts of mK1 and B2R. We have obtained immunohistochemical evidence that all components of the tissue kallikrein-kinin system are present in the mouse heart. The coronary artery is the major site of kallikrein-kinin activity both in ischemic and non-ischemic hearts.

Inhibition of angiogenesis by a monoclonal antibody to kininogen as well as by kininostatin which block proangiogenic high molecular weight kininogen.

High molecular weight kininogen (HK) exhibits two activities with respect to angiogenesis after cleavage by plasma kallikrein. Cleaved HK (HKa) and its cell-binding domain 5 (D5), kininostatin, are potent antiangiogenic polypeptides. They inhibit endothelial cell migration, proliferation and tube formation. HKa and D5 inhibit angiogenesis in the chicken chorioallantoic membrane (CAM) assay. D5 stimulates apoptosis and interferes with the cell cycle. In contrast, intact HK is proangiogenic by liberating bradykinin. A monoclonal antibody to HK can inhibit angiogenesis in the CAM assay, human colon carcinoma growing as a xenograft in nude mice, and murine hybridomas growing in syngeneic hosts. Not only are the tumors decreased in volume and weight to isotype controls but the mean vascular density is decreased. Thus, both D5 and its constituent peptide and monoclonal antibody have potential for inhibiting angiogenesis and tumor growth in human therapy.

Autoantibodies to contact proteins in patients with recurrent pregnancy losses.

The kallikrein-kinin system or plasma contact system consists of three essential plasma proteins. These are coagulation factor XII, prekallikrein and high molecular weight kininogen. Deficiencies of these proteins are not associated with clinical bleeding despite marked prolongation of in vitro surface-activated coagulation time. Paradoxically, studies suggest that contact proteins have anticoagulant, profibrinolytic functions in a physiologic millieu, on endothelial cells. Recently, evidence has accumulated for the presence of the kallikrein-kinin system or plasma contact system in the fetoplacental unit. Kinins which are released within the placenta may play a role in regulating placental blood flow. This suggests that the plasma contact system may also have an important role in pregnancy. Several studies have reported the presence of autoantibodies to the contact proteins in patients with recurrent early pregnancy losses. Disruption of this system may be a risk factor for early gestational losses.

Certain autoantibodies to phosphatidylethanolamine (aPE) recognize factor XI and prekallikrein independently or in addition to the kininogens.

Recent evidence shows that many antiphospholipid antibodies (aPL) to negatively-charged phospholipid (PL) do not target anionic PL per se, but are specific for anionic PL-binding plasma proteins, for example, beta(2)-glycoprotein I (beta(2)-GPI) and prothrombin. We also reported that certain antiphosphatidylethanolamine antibodies (aPE) are not specific for phosphatidylethanolamine (PE) per se, but are directed to PE-binding plasma proteins, high molecular weight kininogen (HK), and low molecular weight kininogen (LK). Additional studies have shown that certain aPE failed to recognize purified kininogens but continued to produce aPE ELISA reactivity in the presence of semipurified HK preparations containing the HK binding proteins, factor XI (FXI) and prekallikrein (PK). We therefore investigated if certain of these aPE recognized FXI and/or PK. In this study we observed that aPE can recognize contact proteins FXI and PK independently or in combination with HK. Since contact proteins such as HK, PK and factor XII (FXII) have anti-coagulant and profibrinolytic functions, the pathophysiological role of aPE has yet to be elucidated. We propose that aPE of different specificities may initiate or promote characteristics pathological conditions in patients with thrombosis or recurrent pregnancy losses.

Evidence for the presence of a kininogen-like species in a case of total deficiency of low and high molecular weight kininogens.

Low and high molecular weight kininogens (LK and HK), containing 409 and 626 amino acids with masses of approximately 65 and 120 kDa after glycosylation, respectively, are coded by a single gene mapped to the human chromosome 3 by alternative splicing of the transcribed mRNA. The NH2-termini Glu1-Thr383 region, identical in LK and HK, contains bradykinin (BK) moieties Arg363-Arg371. LK, HK and their kinin products Lys-BK and BK are involved in several biologic processes. They are evolutionarily conserved and only 7 patients, all apparently normal, have been reported to lack them. In one of these patients (Williams' trait), a codon mutation (Arg178-->stop) has been blamed for the absence of LK and HK. However, using Western blots with 2 monoclonal anti-HK antibodies, one that recognizes the region common to LK and HK and the other that recognizes only HK, 1 detected approximately 110-kDa bands in the plasma of this LK/HK-deficient patient vs approximately 120-kDa bands in normal human and ape plasmas. With polyclonal anti-Lys-BK antibody, which strongly detects BK cleaved at its COOH-terminus in purified HK, 1 detected approximately 110-kDa bands in the normal and the deficient plasmas. Western blots with a monoclonal anti-prekallikrein (PK) antibody showed that surface activation of PK and distribution of PK activation products, both dependent on HK, were similar in these plasmas. These findings suggest that a mutant gene yielded a kininogen-like species possibly involving aberrant mRNA splicing-structurally different from normal HK, but apparently with the capacity to carry out seemingly vital HK functions.

Evidence for the presence of a kininogen-like species in a case of total deficiency of low and high molecular weight kininogens

Low and high molecular weight kininogens (LK and HK), containing 409 and 626 amino acids with masses of ~65 and 120120 kDa after glycosylation, respectively, are coded by a single gene mapped to the human chromosome 3 by alternative splicing of the transcribed mRNA. The NH2-termini Glu(1)-Thr(383) region, identical in LK and HK, contains bradykinin (BK) moieties Arg(363)-Arg(371). LK, HK and their kinin products Lys-BK and BK are involved in several biological processes. They are evolutionarily conserved and only 7 patients, all apparently normal, have been reported to lack them. In one of these patients (Williams'trait), a codon mutation (Arg(178) r stop) has been blamed for the absence of LK and HK. However, using Western blots with 2 monoclonal anti-HK antibodies, one that recognizes the region common to LK and HK and the other that recognizes only HK, I detected ~110-kDa bands in the plasma of this LK/HK-deficient patient vs ~120-kDa bands in normal human and ape plasmas. With polyclonal anti-Lys-BK antibody, which strongly detects BK eleaved at its COOH-terminus in purified HK, I detected ~110-kDa bands in the normal and the deficient plasmas. Western blots with a monoclonal anti-prekallikrein (PK) antibody showed that surface activation of PK and distribution of PK activation products, both dependent on HK, were similar in these plasmas. These findings suggest that a mutant gene yielded a kininogen-like species possibly involving aberrant mRNA splicing - structurally different from normal HK, but apparently with the capacity to carry out seemingly vital HK functions.