Kusumam Joseph

Heat shock protein 90 catalyzes activation of the prekallikrein-kininogen complex in the absence of factor XII.

Bradykinin is a major mediator of swelling in C1 inhibitor deficiency as well as the angioedema seen with ACE inhibitors and may contribute to bronchial hyperreactivity in asthma. Formation of bradykinin occurs in the fluid phase and along cell surfaces requiring interaction of factor XII, prekallikrein, and high Mr kininogen (HK). Recent data suggest that activation of the kinin-forming cascade can occur on the surface of endothelial cells, even in the absence of factor XII. We sought to further define this factor XII-independent mechanism of kinin formation. Both cytosolic and membrane fractions from endothelial cells possessed the ability to catalyze prekallikrein conversion to kallikrein, and activation depended on the presence of HK and zinc ion. We fractionated the cytosol by ion exchange chromatography and affinity chromatography by using corn trypsin inhibitor as ligand. The fractions with peak activity were subjected to SDS gel electrophoresis and ligand blot with biotinylated corn trypsin inhibitor, and positive bands were sequenced. Heat shock protein 90 (Hsp90) was identified as the protein responsible for zinc-dependent prekallikrein activation in the presence of HK. Zinc-dependent activation of the prekallikrein-HK complex also depended on addition of either α and β isoforms of Hsp90 and the activation on endothelial cells was inhibited on addition of polyclonal Ab to Hsp90 in a dose-dependent manner. Although the mechanism by which Hsp90 activates the kinin-forming cascade is not understood, this protein represents the cellular contribution to the reaction and may become the dominant mechanism in pathologic circumstances in which Hsp90 is highly expressed or secreted.

Interaction of high molecular weight kininogen binding proteins on endothelial cells

Cell surface proteins reported to participate in the binding and activation of the plasma kinin-forming cascade includes gC1qR, cytokeratin 1 and u-PAR. Each of these proteins binds high molecular weight kininogen (HK) as well as Factor XII. The studies on the interaction of these proteins, using dot-blot analysis, revealed that cytokeratin 1 binds to both gC1qR and u-PAR while gC1qR and u-PAR do not bind to each other. The binding properties of these proteins were further analyzed by gel filtration. When biotinylated cytokeratin 1 was incubated with either gC1qR or u-PAR and gel filtered, a new, higher molecular weight peak containing biotin was observed indicating complex formation. The protein shift was also similar to the biotin shift. Further, immunoprecipitation of solubilized endo-thelial cell plasma membrane proteins with anti-gC1qR recovered both gC1qR and cytokeratin 1, but not u-PAR. Immunoprecipitation with anti-u-PAR recovered only u-PAR and cytokeratin 1. By competitive ELISA, gC1qR inhibits u-PAR from binding to cytokeratin 1; u-PAR inhibits gC1qR binding to a lesser extent and requires a 10-fold molar excess. Our data suggest that formation of HK (and Factor XII) binding sites along endothelial cell membranes consists of bimolecular com-plexes of gC1qR-cytokeratin 1 and u-PAR-cytokeratin 1, with gC1qR binding being favored.

Activation of the kinin-forming cascade on the surface of endothelial cells.

Abstract Activation of the plasma kallikrein-kinin forming cascade takes place upon incubation with human umbilical vein endothelial cells. The mechanism by which initiation occurs is uncertain. Zinc-dependent binding of plasma proteins to gC1qR, cytokeratin 1, and perhaps u-PAR is requisite for activation to take place. We demonstrate here that during a 2 hour incubation time plasma deficient in either factor XII or high molecular weight kininogen (HK) fails to activate, as compared to normal plasma, but with more prolonged incubation, factor XII-deficient plasma gradually activates while HK-deficient plasma does not. Our data support both factor XII-dependent (rapid) and factor XII-independent (slow) mechanisms; the latter may require a cell-derived protease to activate prekallikrein and the presence of zinc ions and HK.

High molecular weight kininogen and factor XII binding to endothelial cells and astrocytes

We have quantitated the binding of high molecular weight kininogen (HK) to human microvascular endothelial cells of lung and dermal origin as well as to astrocytes and compared the results with those reported for human umbilical vein endothelial cells (HUVEC). We also reassessed parameters of binding to HUVEC employing cells in suspension as well as cells attached to the culture plate and report similar numbers of sites varying from 6.96x10(5) to 7.71x10(5) per cell. The present study shows that HK binds with high specificity and affinity to microvascular endothelial cells (Kd = 1.86 to 4.5 nM) compared to HUVEC (Kd = 10.35 nM) but with lower affinity to astrocytes (Kd = 23.73 nM). Human cytokeratin 1, urokinase plasminogen activator receptor and gC1qR were found to be HK binding proteins present at the surface of microvascular endothelial cells and astrocytes analogous to that seen in HUVEC, as assessed by inhibition of binding with antibody to each protein. Lung microvascular endothelial cells had approximately half the number of HK binding sites as HUVEC while dermal micro vascular endothelial cells and astrocytes had only 8-10% of the sites/cell. The affinity of binding to the microvascular endothelial cells was greater than HUVEC, the affinity of binding to astrocytes was considerably less, nevertheless binding to each cell type involves gC1qR, cytokeratin 1 and u-PAR to varying degrees. We also demonstrate, for the first time, that factor XII binds to all of these cell types in a saturable and Zn(+2) dependent manner. Given that factor XII accelerates the interactions among cell surfaces and proteins of the contact activation cascade to generate bradykinin, binding of factor XII (and the prekallikrein-HK complex) may serve as a mechanism by which these proteins are concentrated locally to facilitate their interactions.

Alterations in eotaxin, MCP-4, IL-5, and IL-13 after systemic steroids for nasal polyposis

Problem: Patients with chronic hyperplastic sinusitis (CHS) with nasal polyposis (NP) have clinical improvement after the administration of oral steroids. IL-5, IL-13, eotaxin, and MCP-4 are possible mediators in this disease. Alterations in levels after steroid treatment may elucidate their relative importance. n nMethods: Nasal polyps from patients with allergic fungal sinusitis (AFS) or eosinophilic mucin rhinosinusitis (EMRS) before and after oral steroid therapy and control patient sinus mucosa were biopsied. Tissue levels of IL-5, IL-13, eotaxin, and MCP-4 were measured using ELISA. Cytokine/chemokine profiles were correlated to clinical parameters, including endoscopic grading, radiographic staging, and clinical outcomes using the Sino-Nasal Outcome Test (SNOT-20). n nResults: Twenty-one patients (control = 7, CHS/NP = 14) participated in this study. IL-5 and IL-13 levels in untreated polyps were not significantly different from the control group, but eotaxin and MCP-4 were significantly higher than control tissue (P = 0.004 and 0.003, respectively). There was a drastic reduction in the levels of IL-5, IL-13, eotaxin, and MCP-4 after treatment with systemic steroids (P < 0.03). Patients demonstrated clinical improvement after treatment with systemic steroids according to SNOT-20 scores (average presteroid 19, poststeroid 13) and mean endoscopic grading (1.75 each side presteroid, 1.13 poststeroid). n nConclusion: This study demonstrates that untreated nasal polyposis has relatively normal levels of Th2 cytokines, IL-5 and IL-13, but greatly elevated levels of the Th2 chemokines, eotaxin and MCP-4. Improved SNOT-20 scores and decreases in mean endoscopic score demonstrate clinical improvement after the administration of oral steroids. While systemic steroids significantly decreased both cytokines and chemokines, the impact on chemokines was of a much greater magnitude. n nSignificance: Treatment of CHS/NP with novel approaches to block chemokines and cytokines may lead to better control of polyps and decreased dependence on steroids in the future. n nSupport: None reported.