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Functional recruitment of C4b-binding protein to Salmonella Rck
2236
Abstracts / Molecular Immunology 47 (2010) 2198–2294
the augmentation of adaptive alloimmunity by C5aR, which has relevance for therapeutic strategies. doi:10.1016/j.molimm.2010.05.117 195 Elevated cytokine concentrations in serum compared to plasma samples from healthy humans is not explained by in vitro complement activation J.K. Ludviksen a , L.T. Hennø b , O.L. Brekke a,c , D. Christiansen a , H. Fure a , E.W. Nielsen d , T.E. Mollnes a,c,e a
Research Laboratory and Department of Laboratory Medicine, Nordland Hospital, Bodø, Norway b Medical Faculty, University of Tromsø, Norway c Institute of Medical Biology, University of Tromsø, Norway d Department of Anesthesiology, Nordland Hospital, Bodø, Norway e Institute of Immunology, Oslo University Hospital Rikshospitalet and University of Oslo, Norway Background: Cytokines are potential biomarkers for sepsis and other inflammatory conditions. Pre-analytical sample conditions are crucial for many biomarkers and efforts needs to be made to obtain reliable results. Thus, the role of anticoagulants, storage temperature and post-sample in vitro complement activation on the concentrations of 27 different cytokines were examined. Methods: Venous blood was obtained from ten donors using Vacuette tubes containing EDTA, heparin or citrate. Plasma samples were put on ice and centrifuged immediately at +4 ◦ C or stored at +4 ◦ C or 25 ◦ C for 1 and 4 h before centrifugation. Serum was prepared with or without gel. Cytokines were analyzed using the Bio-Plex Human Cytokine assay. Complement activation was measured as the terminal complement complex (TCC) by ELISA. Results: The concentrations of TNF-a, IL-6, MIP-1a, G-CSF, FGF basic and IFN-g were lower in plasma compared to serum. IL-2, IL4, IL-7, IL-8, IL-10, IL-12, IL-17 and PDGF-bb were not detectable in immediately centrifuged plasma, but detectable in serum. In comparison, the MCP-1 concentration was highest in heparin plasma. Storage at 25 ◦ C for 4 h significantly increased IL-2, IL-6, IL-8, IFN-g and GM-CSF concentrations in EDTA plasma. In contrast, IL-10 and IP-10 were unaffected by storage at both temperatures. TCC was significantly elevated only in the serum from tube with gel, indicating that complement activation could not explain the increased cytokine concentrations in serum compared with plasma. Conclusions: The concentration of most cytokines in plasma samples centrifuged immediately at +4 ◦ C were significantly lower than in serum, indicating that cytokines in humans should be analyzed in EDTA or citrate plasma. The higher cytokine levels in serum compared to plasma could not be explained by in vitro complement activation. doi:10.1016/j.molimm.2010.05.118 196 Functional recruitment of C4b-binding protein to Salmonella Rck Derek K. Ho a , Hanna Jarva a , Anna M. Blom b , Seppo Meri a
a
Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland b Department of Laboratory Medicine, Medical Protein Chemistry, Malmö University Hospital, Lund University, Malmö, Sweden Complement is a key component of innate immune defence against microbial pathogens. Accordingly, resistance to comple-
ment mediated killing, or serum resistance, is a common trait of pathogenic bacteria. Rck is a 19-kDa outer membrane protein encoded on the virulence plasmid of Salmonella enterica serovars Typhimurium and Enteritidis. When expressed in either E. coli or S. enterica Typhimurium, Rck confers LPS-independent serum resistance as well as the ability to bind to and invade cultured mammalian cells. Recently, functional binding of the complement regulatory protein C4b-binding protein (C4bp) has been demonstrated in a homologous protein from Yersinia enterocolitica, Ail. We hypothesized that Rck-mediated serum resistance is due to C4bp binding. We observed that E. coli expressing Rck bound C4bp using both the purified protein and heat-inactivated serum. No binding was detected in the absence of Rck expression. Reduced C4b deposition (C4c epitope) was observed in the presence of Rck despite no loss in IgG binding. Cofactor assays revealed that C4bp bound to Rck is functional for factor I-mediated cleavage of C4b. In competition assays, radiolabeled C4bp binding to Rck was reduced by increasing concentrations of unlabeled protein. No effect was observed by increasing heparin or salt concentrations, suggesting mainly nonionic interactions. We generated a double point mutation in the second surface exposed loop of Rck, K89F/A90K, which led to deficient C4bp binding. Accordingly, we observed restored C4b and MAC deposition compared to WT Rck, as well as loss of survival in serum bactericidal assays. These data suggest that C4bp binding to Rck may play an important role in resistance to complementmediated killing of Salmonella. doi:10.1016/j.molimm.2010.05.119 197 CRASP2 of Candida albicans binds C4b-binding protein and mediates fungal contact with human endothelial cells Shanshan Luo a , Anna M. Blom b , Steffen Rupp c , Bernhard Hube d , Uta-Christina Hipler e , Christine Skerka a , Peter F. Zipfel a,f,∗ a
Department of Infection Biology, Leibniz-Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute, Jena, Germany b Department of Laboratory Medicine, Section of Medical Protein Chemistry, University of Lund, Malmö, Sweden c Fraunhofer-Institute for Grenzflächen- und Bioverfahrenstechnik IGB, Stuttgart, Germany d Department of Microbial Pathogenicity Mechanisms, LeibnizInstitute for Natural Product Research and Infection Biology, Hans-Knöll-Institut, Jena, Germany e Friedrich-Schiller-University, Clinic of Dermatology and Allergology, Jena, Germany f Friedrich-Schiller-University, Jena, Germany Candida albicans binds and utilizes several human complement inhibitors for immune evasion, such as C4b-binding protein (C4BP), Factor H and Factor H like protein 1 (FHL-1). Here, we identified Candida CRASP2 as the first fungal C4BP binding protein. Recombinant CRASP2 binds C4BP and this interaction is of ionic nature. The CRASP2 binding domains within C4BP were localized to the complement control protein module (CCP) 4, CCP7 and CCP8. C4BP bound to CRASP2 maintains complement inhibitory activity. CRASP2 binding was increased about 2 fold in a CRASP2 overexpressing Candida strain and was decreased by 20% in a knock out strain. In addition CRASP2 surface expression was up-regulated upon co-culture of C. albicans with human non-phagocytic epithelial, endothelial cells and phagocytic monocytes as shown by flow cytometry. The C. albicans surface CRASP2 bound to human CR3 deficient endothelial cells and mediated fungal adhesion and invasion to cell barrier, as shown by flow cytometry. In addition, CRASP2 expression and sequence variation were tested in thirteen clinical
Abstracts / Molecular Immunology 47 (2010) 2198–2294
the augmentation of adaptive alloimmunity by C5aR, which has relevance for therapeutic strategies. doi:10.1016/j.molimm.2010.05.117 195 Elevated cytokine concentrations in serum compared to plasma samples from healthy humans is not explained by in vitro complement activation J.K. Ludviksen a , L.T. Hennø b , O.L. Brekke a,c , D. Christiansen a , H. Fure a , E.W. Nielsen d , T.E. Mollnes a,c,e a
Research Laboratory and Department of Laboratory Medicine, Nordland Hospital, Bodø, Norway b Medical Faculty, University of Tromsø, Norway c Institute of Medical Biology, University of Tromsø, Norway d Department of Anesthesiology, Nordland Hospital, Bodø, Norway e Institute of Immunology, Oslo University Hospital Rikshospitalet and University of Oslo, Norway Background: Cytokines are potential biomarkers for sepsis and other inflammatory conditions. Pre-analytical sample conditions are crucial for many biomarkers and efforts needs to be made to obtain reliable results. Thus, the role of anticoagulants, storage temperature and post-sample in vitro complement activation on the concentrations of 27 different cytokines were examined. Methods: Venous blood was obtained from ten donors using Vacuette tubes containing EDTA, heparin or citrate. Plasma samples were put on ice and centrifuged immediately at +4 ◦ C or stored at +4 ◦ C or 25 ◦ C for 1 and 4 h before centrifugation. Serum was prepared with or without gel. Cytokines were analyzed using the Bio-Plex Human Cytokine assay. Complement activation was measured as the terminal complement complex (TCC) by ELISA. Results: The concentrations of TNF-a, IL-6, MIP-1a, G-CSF, FGF basic and IFN-g were lower in plasma compared to serum. IL-2, IL4, IL-7, IL-8, IL-10, IL-12, IL-17 and PDGF-bb were not detectable in immediately centrifuged plasma, but detectable in serum. In comparison, the MCP-1 concentration was highest in heparin plasma. Storage at 25 ◦ C for 4 h significantly increased IL-2, IL-6, IL-8, IFN-g and GM-CSF concentrations in EDTA plasma. In contrast, IL-10 and IP-10 were unaffected by storage at both temperatures. TCC was significantly elevated only in the serum from tube with gel, indicating that complement activation could not explain the increased cytokine concentrations in serum compared with plasma. Conclusions: The concentration of most cytokines in plasma samples centrifuged immediately at +4 ◦ C were significantly lower than in serum, indicating that cytokines in humans should be analyzed in EDTA or citrate plasma. The higher cytokine levels in serum compared to plasma could not be explained by in vitro complement activation. doi:10.1016/j.molimm.2010.05.118 196 Functional recruitment of C4b-binding protein to Salmonella Rck Derek K. Ho a , Hanna Jarva a , Anna M. Blom b , Seppo Meri a
a
Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland b Department of Laboratory Medicine, Medical Protein Chemistry, Malmö University Hospital, Lund University, Malmö, Sweden Complement is a key component of innate immune defence against microbial pathogens. Accordingly, resistance to comple-
ment mediated killing, or serum resistance, is a common trait of pathogenic bacteria. Rck is a 19-kDa outer membrane protein encoded on the virulence plasmid of Salmonella enterica serovars Typhimurium and Enteritidis. When expressed in either E. coli or S. enterica Typhimurium, Rck confers LPS-independent serum resistance as well as the ability to bind to and invade cultured mammalian cells. Recently, functional binding of the complement regulatory protein C4b-binding protein (C4bp) has been demonstrated in a homologous protein from Yersinia enterocolitica, Ail. We hypothesized that Rck-mediated serum resistance is due to C4bp binding. We observed that E. coli expressing Rck bound C4bp using both the purified protein and heat-inactivated serum. No binding was detected in the absence of Rck expression. Reduced C4b deposition (C4c epitope) was observed in the presence of Rck despite no loss in IgG binding. Cofactor assays revealed that C4bp bound to Rck is functional for factor I-mediated cleavage of C4b. In competition assays, radiolabeled C4bp binding to Rck was reduced by increasing concentrations of unlabeled protein. No effect was observed by increasing heparin or salt concentrations, suggesting mainly nonionic interactions. We generated a double point mutation in the second surface exposed loop of Rck, K89F/A90K, which led to deficient C4bp binding. Accordingly, we observed restored C4b and MAC deposition compared to WT Rck, as well as loss of survival in serum bactericidal assays. These data suggest that C4bp binding to Rck may play an important role in resistance to complementmediated killing of Salmonella. doi:10.1016/j.molimm.2010.05.119 197 CRASP2 of Candida albicans binds C4b-binding protein and mediates fungal contact with human endothelial cells Shanshan Luo a , Anna M. Blom b , Steffen Rupp c , Bernhard Hube d , Uta-Christina Hipler e , Christine Skerka a , Peter F. Zipfel a,f,∗ a
Department of Infection Biology, Leibniz-Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute, Jena, Germany b Department of Laboratory Medicine, Section of Medical Protein Chemistry, University of Lund, Malmö, Sweden c Fraunhofer-Institute for Grenzflächen- und Bioverfahrenstechnik IGB, Stuttgart, Germany d Department of Microbial Pathogenicity Mechanisms, LeibnizInstitute for Natural Product Research and Infection Biology, Hans-Knöll-Institut, Jena, Germany e Friedrich-Schiller-University, Clinic of Dermatology and Allergology, Jena, Germany f Friedrich-Schiller-University, Jena, Germany Candida albicans binds and utilizes several human complement inhibitors for immune evasion, such as C4b-binding protein (C4BP), Factor H and Factor H like protein 1 (FHL-1). Here, we identified Candida CRASP2 as the first fungal C4BP binding protein. Recombinant CRASP2 binds C4BP and this interaction is of ionic nature. The CRASP2 binding domains within C4BP were localized to the complement control protein module (CCP) 4, CCP7 and CCP8. C4BP bound to CRASP2 maintains complement inhibitory activity. CRASP2 binding was increased about 2 fold in a CRASP2 overexpressing Candida strain and was decreased by 20% in a knock out strain. In addition CRASP2 surface expression was up-regulated upon co-culture of C. albicans with human non-phagocytic epithelial, endothelial cells and phagocytic monocytes as shown by flow cytometry. The C. albicans surface CRASP2 bound to human CR3 deficient endothelial cells and mediated fungal adhesion and invasion to cell barrier, as shown by flow cytometry. In addition, CRASP2 expression and sequence variation were tested in thirteen clinical