Jian Cheng Qi

Naturally occurring free thiols within β2-glycoprotein I in vivo: nitrosylation, redox modification by endothelial cells, and regulation of oxidative stress-induced cell injury

β2-Glycoprotein I (β2GPI) is an evolutionary conserved, abundant circulating protein. Although its function remains uncertain, accumulated evidence points toward interactions with endothelial cells and components of the coagulation system, suggesting a regulatory role in vascular biology. Our group has shown that thioredoxin 1 (TRX-1) generates free thiols in β2GPI, a process that may have a regulatory role in platelet adhesion. This report extends these studies and shows for the first time evidence of β2GPI with free thiols in vivo in both multiple human and murine serum samples. To explore how the vascular surface may modulate the redox status of β2GPI, unstimulated human endothelial cells and EAhy926 cells are shown to be capable of amplifying the effect of free thiol generation within β2GPI. Multiple oxidoreductase enzymes, such as endoplasmic reticulum protein 46 (ERp 46) and TRX-1 reductase, in addition to protein disulfide isomerase are secreted on the surface of endothelial cells. Furthermore, one or more of these generated free thiols within β2GPI are also shown to be nitrosylated. Finally, the functional significance of these findings is explored, by showing that free thiol-containing β2GPI has a powerful effect in protecting endothelial cells and EAhy926 cells from oxidative stress-induced cell death.

Novel Assays of Thrombogenic Pathogenicity in the Antiphospholipid Syndrome Based on the Detection of Molecular Oxidative Modification of the Major Autoantigen β2-Glycoprotein I

Objective Beta-2-glycoprotein I (β2GPI) constitutes the major autoantigen in the antiphospholipid syndrome (APS), a common acquired cause of arterial and venous thrombosis. We recently described the novel observation that β2GPI may exist in healthy individuals in a free thiol (biochemically reduced) form. The present study was undertaken to quantify the levels of total, reduced, and posttranslationally modified oxidized β2GPI in APS patients compared to various control groups. Methods In a retrospective multicenter analysis, the proportion of β2GPI with free thiols in serum from healthy volunteers was quantified. Assays for measurement of reduced as well as total circulating β2GPI were developed and tested in the following groups: APS (with thrombosis) (n = 139), autoimmune disease with or without persistent antiphospholipid antibodies (aPL) but without APS (n = 188), vascular thrombosis without APS or aPL (n = 38), and healthy volunteers (n = 91). Results Total β2GPI was significantly elevated in patients with APS (median 216.2 μg/ml [interquartile range 173.3–263.8]) as compared to healthy subjects (median 178.4 μg/ml [interquartile range 149.4–227.5] [P < 0.0002]) or control patients with autoimmune disease or vascular thrombosis (both P < 0.0001). The proportion of total β2GPI in an oxidized form (i.e., lacking free thiols) was significantly greater in the APS group than in each of the 3 control groups (all P < 0.0001). Conclusion This large retrospective multicenter study shows that posttranslational modification of β2GPI via thiol-exchange reactions is a highly specific phenomenon in the setting of APS thrombosis. Quantification of posttranslational modifications of β2GPI in conjunction with standard laboratory tests for APS may offer the potential to more accurately predict the risk of occurrence of a thrombotic event in the setting of APS.

Redox control of β2-glycoprotein I-von Willebrand factor interaction by thioredoxin-1.

Summary.  Background: β2‐Glycoprotein I (β2GPI) is an abundant plasma protein that is closely linked to blood clotting, as it interacts with various protein and cellular components of the coagulation system. However, the role of β2GPI in thrombus formation is unknown. We have recently shown that β2GPI is susceptible to reduction by the thiol oxidoreductases thioredoxin‐1 and protein disulfide isomerase, and that reduction of β2GPI can take place on the platelet surface. Methods: β2GPI, reduced by thioredoxin‐1, was labeled with the selective sulfhydryl probe Na‐(3‐maleimidylpropionyl)biocytin and subjected to mass spectrometry to identify the specific cysteines involved in the thiol exchange reaction. Binding assays were used to examine the affinity of reduced β2GPI for von Willebrand factor (VWF) and the effect of reduced β2GPI on glycoprotein (GP)Ibα binding to VWF. Platelet adhesion to ristocetin‐activated VWF was studied in the presence of reduced β2GPI. Results:  We demonstrate that the Cys288–Cys326 disulfide in domain V of β2GPI is the predominant disulfide reduced by thioredoxin‐1. Reduced β2GPI in vitro displays increased binding to VWF that is dependent on disulfide bond formation. β2GPI reduced by thioredoxin‐1, in comparison with non‐reduced β2GPI, leads to increased binding of GPIbα to VWF and increased platelet adhesion to activated VWF. Conclusions:  Given the importance of thiol oxidoreductases in thrombus formation, we provide preliminary evidence that the thiol‐dependent interaction of β2GPI with VWF may contribute to the redox regulation of platelet adhesion.

IL-16 Regulation of Human Mast Cells/Basophils and Their Susceptibility to HIV-1

AIDS patients often contain HIV-1-infected mast cells (MCs)/basophils in their peripheral blood, and in vivo-differentiated MCs/basophils have been isolated from the blood of asthma patients that are HIV-1 susceptible ex vivo due to their surface expression of CD4 and varied chemokine receptors. Because IL-16 is a ligand for CD4 and/or an undefined CD4-associated protein, the ability of this multifunctional cytokine to regulate the development of human MCs/basophils from nongranulated progenitors residing in cord or peripheral blood was evaluated. After 3 wk of culture in the presence of c-kit ligand, IL-16 induced the progenitors residing in the blood of normal individuals to increase their expression of chymase and tryptase about 20-fold. As assessed immunohistochemically, >80% of these tryptase+ and/or chymase+ cells expressed CD4. The resulting cells responded to IL-16 in an in vitro chemotaxis assay, and this biologic response could be blocked by anti-IL-16 and anti-CD4 Abs as well as by a competitive peptide inhibitor corresponding to a sequence in the C-terminal domain of IL-16. The additional finding that IL-16 induces calcium mobilization in the HMC-1 cell line indicates that IL-16 acts directly on MCs and their committed progenitors. IL-16-treated MCs/basophils also are less susceptible to infection by an M/R5-tropic strain of HIV-1. Thus, IL-16 regulates MCs/basophils at a number of levels, including their vulnerability to retroviral infection.

Beta2-glycoprotein I protects thrombin from inhibition by heparin cofactor II: Potentiation of this effect in the presence of anti–β2-glycoprotein I autoantibodies

OBJECTIVE Beta2-glycoprotein I (beta2GPI) is an important autoantigen in the antiphospholipid syndrome (APS). In vitro studies suggest that it may have multifaceted physiologic functions, since it displays both anticoagulant and procoagulant properties. We have previously reported that beta2GPI can directly bind thrombin, a key serine protease in the coagulation pathway. The present study was undertaken to examine the influence of beta2GPI on thrombin inactivation by the serpin heparin cofactor II (HCII). The effect of anti-beta2GPI antibodies was also examined. METHODS HCII inactivation of thrombin was assessed using chromogenic and various platelet functional assays. The influence of intact and proteolytically cleaved beta2GPI and anti-beta2GPI antibodies was determined in these systems. RESULTS beta2GPI protected thrombin against inactivation by HCII/heparin. Cleavage of beta2GPI at Lys317-Thr318 abrogated its protective effect. Patient polyclonal IgG and murine monoclonal anti-beta2GPI antibodies potentiated the procoagulant influence of beta2GPI in this system. CONCLUSION These novel findings suggest that beta2GPI may regulate thrombin inactivation by HCII/heparin. The observation that anti-beta2GPI antibodies potentiate the protective effect of beta2GPI on thrombin in this system, thereby promoting a procoagulant response, may potentially delineate one of the pathophysiologic mechanisms contributing to the prothrombotic tendency in patients with APS.

The diacylglycerol-dependent translocation of Ras guanine nucleotide-releasing protein 4 inside a human mast cell line results in substantial phenotypic changes, including expression of interleukin 13 receptor α2

Ras guanine nucleotide-releasing protein 4 (RasGRP4) is a mast cell (MC)-restricted guanine nucleotide exchange factor and diacylglycerol (DAG)/phorbol ester receptor. An RasGRP4-defective variant of the human MC line HMC-1 was used to create stable clones expressing green fluorescent protein-labeled RasGRP4 for monitoring the movement of this protein inside MCs after exposure to phorbol 12-myristate 13-acetate (PMA), and for evaluating the proteins ability to control gene expression. RasGRP4 resided primarily in the cytosol. After exposure to PMA, RasGRP4 quickly translocated to the inner leaflet of the cells plasma membrane. 15-30 min later, this signaling protein translocated from the plasma membrane to other intracellular sites. The translocation of RasGRP4 from the cytosol to its varied membrane compartments was found to be highly dependent on Phe548 in the proteins C1 DAG/PMA-binding domain. Extracellular signal-regulated kinases 1 and 2 were activated during this translocation process, and c-kit/CD117 was lost from the cells surface. Transcript-profiling approaches revealed that RasGRP4 profoundly regulated the expression of hundreds of genes in HMC-1 cells. For example, the expression of the transcript that encodes the interleukin (IL) 13 receptor IL-13Rα2 increased 61- to 860-fold in RasGRP4-expressing HMC-1 cells. A marked increase in IL-13Rα2 protein levels also was found. The accumulated data suggest RasGRP4 translocates to varied intracellular compartments via its DAG/PMA-binding domain to regulate signaling pathways that control gene and protein expression in MCs, including the cells ability to respond to IL-13.

Beta-lapachone inhibits proliferation and induces apoptosis in retinoblastoma cell lines.

AimsTo investigate the cytotoxicity of beta-lapachone, a potent agent that may selectively target tumour cells, in retinoblastoma (RB) cell lines.MethodsGrowth inhibitory effects of beta-lapachone were evaluated in Y79, WERI-RB1, and RBM human retinoblastoma cell lines. Pro-apoptotic effects of beta-lapachone were evaluated in Y79 cells by detection of caspase 3/7 activity, by enzyme-linked immunosorbent assay for nucleosome fragments, and by cellular morphological analysis.ResultsBeta-lapachone induced significant dose-dependent growth inhibitory effects in all three retinoblastoma cell lines. The 50% growth inhibitory concentration (IC50) of this agent was 1.9 μM in Y79 cells, 1.3 μM in WERI-RB1 cells, and 0.9 μM in RBM cells. Beta-lapachone also induced proapoptotic effects in RB cells. Treatment of Y79 cells with 1.9 μM beta-lapachone (IC50) resulted in a peak, fourfold induction of caspase 3/7 activity at 72 h post-treatment; a peak, 5.6-fold increase in nucleosome fragments at 96 h post-treatment; and a peak, 1.7-fold increase in the frequency of apoptotic cells at 48 h post-treatment, relative to vehicle-treated controls.ConclusionBeta-lapachone induced potent cytotoxic effects in RB cell lines at low micromolar concentrations, suggesting this agent could be useful in the clinical management of RB.

Deletion of the Antiphospholipid Syndrome Autoantigen β2‐Glycoprotein I Potentiates the Lupus Autoimmune Phenotype in a Toll‐like Receptor 7–Mediated Murine Model

The BXSB.Yaa mouse strain is a model of systemic lupus erythematosus that is dependent on duplication of the Toll‐like receptor 7 gene. The objective of this study was to systematically describe the amplified autoimmune phenotype observed when the soluble plasma protein β2‐glycoprotein I (β2GPI) gene was deleted in male BXSB.Yaa mice.

Hemopoietic cells with features of the mast cell and basophil lineages and their potential role in allergy.

Human mast cells (MCs) and basophils play a key role in the pathogenesis of allergic disorders, not only by producing inflammatory and fibrogenic mediators, but also by directly and indirectly secreting various cytokines and chemokines. Although mast cells and basophils have differences in many properties, recent evidence suggests that human MCs and basophils may be derived from a common progenitor, and their contents and phenotypes may be reversibly altered in a variety of allergic disorders. The study of FcetaRI signalling of mast cell and basophils offers new opportunities for therapeutic interventions based on the specific inhibition of the earliest events in allergic diseases. This article reviews the origin, differentiation, morphology and phenotypic properties of MCs and basophils, focussing particularly on the possible pathogenic role of MCs and basophils in allergy and biochemical targets for therapeutic interventions in allergic diseases.

An Antibody Raised Against In Vitro-derived Human Mast Cells Identifies Mature Mast Cells and a Population of Cells that are Fc∊RI+, Tryptase−, and Chymase− in a Variety of Human Tissues:

Selective markers for human mast cells are of paramount importance for understanding their role in physiological and pathological processes. A mouse monoclonal antibody (MAb) designated 2C7, raised against in vitro-derived human mast cells, was used in immunoenzymatic analysis of sections from a variety of human organs. Double immuno-labeling with 2C7 and tryptase, chymase, Fc∊RIα, and c-kit was performed on cryostat tissue sections from skin, colon, uterus, breast, stomach, bladder, and lung. MAb 2C7 stained greater than 93% of the tryptase+ or chymase+ mast cells in all tissues examined. In addition, the majority of cells stained with the tryptase or chymase also stained for Fc∊RIα. However, there were a significant number of Fc∊RIα+ cells in all tissues studied that were tryptase− and/or chymase−. In contrast, MAb 2C7 in double immunoenzymatic staining co-localized with 93–96% of the Fc∊RIα+ cells in all tissues. Analysis for c-kit expression on the different tissues revealed that the majority of tryptase+ or chymase+ cells in skin, uterus, bladder, and lung stained with c-kit. However, only approximately 70-78% of tryptase+ cells in colon and stomach were c-kit+. These data suggest that MAb 2C7 appears to identify mature mast cells and a population of Fc∊RIα+, chymase−, and tryptase− cells in a variety of human tissues.