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Binding of von Willebrand factor to complement C1q on a cholesterol crystal surface
Abstracts / Molecular Immunology 89 (2017) 130–139
and inflammation, and can show that thrombin, under the current whole blood conditions, does not affect the degree of complement activation at the level of C3 or C5. http://dx.doi.org/10.1016/j.molimm.2017.06.075 044
137
045 Binding of von Willebrand factor to complement C1q on a cholesterol crystal surface Claudia Donat 1,∗ , Robert Kölm 1 , Sophia Thanei 1 , Marten Trendelenburg 1,2 1
Complement component C3 is highly expressed in human pancreatic islets and promotes -cell survival Klaudia Kulak 1,∗ , Ben C. King 1 , Ulrika Krus 2 , Enming Zhang 2 , Erik Renström 2 , Anna M. Blom 1 1 Section of Medical Protein Chemistry, Department of Translational Medicine, Lund University, 205-02 Malmö, Sweden 2 Department of Clinical Sciences Malmö, Lund University Diabetes Centre, Lund University, 205-02 Malmö, Sweden
Background: Pancreatic islet -cells secrete insulin, which is essential for blood glucose homeostasis. In type 2 diabetes (T2D), loss of insulin sensitivity in peripheral tissues leads to failure of cells to compensate for the increased demand for insulin, causing overt disease. Inflammation caused by the innate immune system has been shown to be a mediator of both insulin resistance and impaired insulin secretion. In particular, islet infiltration of innate immune cells and IL-1 production coincides with the course of T2D. C3 may also be involved in (patho)physiological responses in the pancreatic islet. Plasma levels of C3 have been shown to be a predictor of T2D development and are associated with incidence of T2D. In this study, we examine the regulation of the expression of C3 in human islets and investigate role of C3 in the -cell. Materials and methods: C3 expression was confirmed by RNA in situ hybridization of human pancreatic samples and western blot of isolated human islet supernatants and lysates. RNA-sequencing analysis of isolated islets from over 200 healthy and T2D donors was used to determine correlation of C3 with T2D incidence and cytokine/chemokine expression. Expression and secretion of C3 was detected by qPCR and ELISA and metabolic labeling. C3 was silenced with siRNA in INS1 832/13 cell line followed by insulin secretion and viability assays. Results and conclusions: We found that human islets express high levels of C3, in comparison to rodent islets, and expression correlates with T2D and islet inflammation. IL-1 cytokine highly upregulated expression and secretion of C3 in human islets and INS1 cells. siRNA-mediated downregulation of C3 significantly reduced INS1 viability when treated with IL-1, but ability to secrete insulin remained unaffected. These results suggest that C3 protects against cytokine-induced -cell death, but further work is required to investigate the pathway by which C3 regulates -cell survival. http://dx.doi.org/10.1016/j.molimm.2017.06.076
Laboratory of Clinical Immunology, University Hospital Basel, CH-4031 Basel, Switzerland 2 Division of Internal Medicine, University Hospital Basel, CH-4031 Basel, Switzerland Background: Atherosclerosis is a chronic inflammatory disease characterized by the formation of cholesterol crystals (CC) within atherosclerotic plaques. The versatile roles of the activated immune and hemostatic systems are still unclear. While complement C1q has been described to be protective for the progression of atherosclerosis, it has also been shown to mediate the binding of von Willebrand factor (vWF). However, the link of this interaction to CC, and potentially to atherosclerosis, remains elusive. Materials and methods: C1q was immobilized on ELISA plates and CC, before being incubated with recombinant vWF. C1q-dependent binding of vWF was measured by ELISA reader, flow cytometry, confocal microscopy, and ImageStreamX, respectively. Results and conclusions: On ELISA plates, a dose-dependent binding of vWF to immobilized C1q could be observed. With increasing concentrations, binding of vWF to C1q approaches levels comparable to the binding to its physiological binding partner collagen. Furthermore, C1q was found to bind to CC with subsequent binding of vWF. The geometric mean fluorescence intensity was significantly higher when C1q was present compared to vWF alone (p < 0.0001). This C1q-dependent binding could be confirmed using different techniques with vWF binding being virtually absent in the absence of C1q. In conclusion, C1q bound to CC was found to mediate the additional binding of vWF. This binding of vWF is likely to alter immune functions downstream of C1q, and might play a role in atherosclerosis. http://dx.doi.org/10.1016/j.molimm.2017.06.077 046 Simultaneous determination of C1-INH-containing activation complexes in human plasma Erika Kajdácsi 1,∗ , Anna Koncz 1 , Nóra Veszel 1 , László Cervenak 1 , Dominik Gulyás 1 , Péter Gál 2 , József Dobó 2 , Henriette Farkas 3 , Lilian Varga 3 1
3rd Department of Medicine, Semmelweis University, Budapest, Hungary 2 Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary 3 3rd Department of Internal Medicine, Semmelweis University, Budapest, Hungary Background: The C1-inhibitor (C1-INH) is an important regulator of complement, coagulation, fibrinolytic and contact systems. The quantity of enzyme/C1-INH complexes in the blood is possibly proportional to the level of the in vivo activation of cascadelike plasma enzyme systems. Simultaneous, parallel determination of C1-INH-containing activation complexes would be important
and inflammation, and can show that thrombin, under the current whole blood conditions, does not affect the degree of complement activation at the level of C3 or C5. http://dx.doi.org/10.1016/j.molimm.2017.06.075 044
137
045 Binding of von Willebrand factor to complement C1q on a cholesterol crystal surface Claudia Donat 1,∗ , Robert Kölm 1 , Sophia Thanei 1 , Marten Trendelenburg 1,2 1
Complement component C3 is highly expressed in human pancreatic islets and promotes -cell survival Klaudia Kulak 1,∗ , Ben C. King 1 , Ulrika Krus 2 , Enming Zhang 2 , Erik Renström 2 , Anna M. Blom 1 1 Section of Medical Protein Chemistry, Department of Translational Medicine, Lund University, 205-02 Malmö, Sweden 2 Department of Clinical Sciences Malmö, Lund University Diabetes Centre, Lund University, 205-02 Malmö, Sweden
Background: Pancreatic islet -cells secrete insulin, which is essential for blood glucose homeostasis. In type 2 diabetes (T2D), loss of insulin sensitivity in peripheral tissues leads to failure of cells to compensate for the increased demand for insulin, causing overt disease. Inflammation caused by the innate immune system has been shown to be a mediator of both insulin resistance and impaired insulin secretion. In particular, islet infiltration of innate immune cells and IL-1 production coincides with the course of T2D. C3 may also be involved in (patho)physiological responses in the pancreatic islet. Plasma levels of C3 have been shown to be a predictor of T2D development and are associated with incidence of T2D. In this study, we examine the regulation of the expression of C3 in human islets and investigate role of C3 in the -cell. Materials and methods: C3 expression was confirmed by RNA in situ hybridization of human pancreatic samples and western blot of isolated human islet supernatants and lysates. RNA-sequencing analysis of isolated islets from over 200 healthy and T2D donors was used to determine correlation of C3 with T2D incidence and cytokine/chemokine expression. Expression and secretion of C3 was detected by qPCR and ELISA and metabolic labeling. C3 was silenced with siRNA in INS1 832/13 cell line followed by insulin secretion and viability assays. Results and conclusions: We found that human islets express high levels of C3, in comparison to rodent islets, and expression correlates with T2D and islet inflammation. IL-1 cytokine highly upregulated expression and secretion of C3 in human islets and INS1 cells. siRNA-mediated downregulation of C3 significantly reduced INS1 viability when treated with IL-1, but ability to secrete insulin remained unaffected. These results suggest that C3 protects against cytokine-induced -cell death, but further work is required to investigate the pathway by which C3 regulates -cell survival. http://dx.doi.org/10.1016/j.molimm.2017.06.076
Laboratory of Clinical Immunology, University Hospital Basel, CH-4031 Basel, Switzerland 2 Division of Internal Medicine, University Hospital Basel, CH-4031 Basel, Switzerland Background: Atherosclerosis is a chronic inflammatory disease characterized by the formation of cholesterol crystals (CC) within atherosclerotic plaques. The versatile roles of the activated immune and hemostatic systems are still unclear. While complement C1q has been described to be protective for the progression of atherosclerosis, it has also been shown to mediate the binding of von Willebrand factor (vWF). However, the link of this interaction to CC, and potentially to atherosclerosis, remains elusive. Materials and methods: C1q was immobilized on ELISA plates and CC, before being incubated with recombinant vWF. C1q-dependent binding of vWF was measured by ELISA reader, flow cytometry, confocal microscopy, and ImageStreamX, respectively. Results and conclusions: On ELISA plates, a dose-dependent binding of vWF to immobilized C1q could be observed. With increasing concentrations, binding of vWF to C1q approaches levels comparable to the binding to its physiological binding partner collagen. Furthermore, C1q was found to bind to CC with subsequent binding of vWF. The geometric mean fluorescence intensity was significantly higher when C1q was present compared to vWF alone (p < 0.0001). This C1q-dependent binding could be confirmed using different techniques with vWF binding being virtually absent in the absence of C1q. In conclusion, C1q bound to CC was found to mediate the additional binding of vWF. This binding of vWF is likely to alter immune functions downstream of C1q, and might play a role in atherosclerosis. http://dx.doi.org/10.1016/j.molimm.2017.06.077 046 Simultaneous determination of C1-INH-containing activation complexes in human plasma Erika Kajdácsi 1,∗ , Anna Koncz 1 , Nóra Veszel 1 , László Cervenak 1 , Dominik Gulyás 1 , Péter Gál 2 , József Dobó 2 , Henriette Farkas 3 , Lilian Varga 3 1
3rd Department of Medicine, Semmelweis University, Budapest, Hungary 2 Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary 3 3rd Department of Internal Medicine, Semmelweis University, Budapest, Hungary Background: The C1-inhibitor (C1-INH) is an important regulator of complement, coagulation, fibrinolytic and contact systems. The quantity of enzyme/C1-INH complexes in the blood is possibly proportional to the level of the in vivo activation of cascadelike plasma enzyme systems. Simultaneous, parallel determination of C1-INH-containing activation complexes would be important