Properdin binding to the surface of leptospira promotes complement activation and controls proliferation of non-pathogenic bacteria in vitro

Abstracts / Immunobiology 217 (2012) 1129–1222

complement-sufficient serum (CSS) to mimic oxidative stress in the presence of complement proteins. We have previously shown that oxidative stress down-regulates the level of complement inhibition at the cell surface of RPE cells, effectively reducing the cells ability to fights off complement attack. Here we examine the immediate and transient effects of complement activation. ARPE-19 were grown as monolayers on transwell plates or as subconfluent cells on coverslips. Effects on RPE tight junctions were determined by transepithelial electrical resistance (TER) measurements. Effects of sublytic MAC on intracellular signaling was analyzed by Western blotting; on VEGF secretion by ELISA; and on membrane potential and ion channel activity by patch-clamp recordings and Ca2+ imaging (Fura-2). Current clamp and Ca2+ imaging of individual ARPE-19 cells revealed that treatment with CSS led to a rapid and sustained depolarization of the RPE cells, with a concomitant Ca2+ signal. The sustained phase of the Ca2+ signal is dependent upon the activity of the voltage-dependent calcium channel (VDCC). Co-treatment with H2 O2 + CSS but not with either H2 O2 or CSS shifted the voltagedependence of the VDCC to more positive values. Treatment of ARPE-19 cell monolayers with H2 O2 + CSS results in the rapid phosphorylation of the VDCC, secretion of VEGF followed by a loss TER. Both loss of TER and VEGF secretion can be blunted by VDCC inhibition. Depolarization of RPE cells in response to CSS together with the parallel shift of L-type channel voltage-dependence in response to oxidative stress leads to a substantial increase in VDCC activity (Ca2+ influx). Phosphorylation of the VDCC, which is known to prevent rundown of the channel, ensures prolonged Ca2+ influx and membrane depolarization required for VEGF secretion. Taken together, identifying how sublytic MAC induces VEGF expression and secretion might offer opportunities to selectively inhibit pathological VEGF release only. http://dx.doi.org/10.1016/j.imbio.2012.08.188 187 Properdin and complement amplification loop in the pathogenesis of experimental autoimmune myasthenia gravis (EAMG) Jindrich Soltys 1 , Cordula Stover 2 , Xiaobo Wu 3 1

Department of Neurology & Psychiatry, Saint Louis University School of Medicine, Saint Louis, MO, USA 2 Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, UK 3 Department of Medicine; Division of Rheumatology, Washington University School of Medicine, St. Louis, MO, USA An increased concentration of serum complement fragment Bb in Musk Ab + myasthenia gravis patients suggests that complement alternative pathway (AP) and its regulators might be involved in disease pathogenesis. In this report, we explore the roles of feedback amplification loop and positive regulator properdin (P) on EAMG pathology by comparing the clinical, pathological, and immunological aspects of disease. We hypothesized that a lack of AP amplification or properdin can prevent the destruction of the neuromuscular junction (NMJ). Active EAMG was induced by subcutaneous injections of acetylcholine receptor (AChR) to WT, factor B−/− (fB−/− ) and P−/− mice. Disease severity was assessed by behavioral assessments. The level of C3, C5b-9 (MAC) and the AChR specific antibodies was determined by ELISA. Serum was analyzed for the presence of IL-2, IL-4, IL-6, TNF-a, IFN-g, IL-17A and IL-10 cytokines. Deposition of C3 and C5b-9 (MAC) at the NMJ was analyzed by immunofluorescence staining. Production of AChR specific IgG, IgG1 and IgG2b antibodies was comparable among fB−/− , P−/− and WT control mice. Compared to WT EAMG, clinical scores though were significantly reduced in P−/− EAMG and a trend in the

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same direction for less disease phenotype was also noted in the fB−/− EAMG. Significantly smaller amounts of C5b-9 (MAC) were also detected in fB−/− EAMG and P−/− EAMG mice. This result correlated with reduced complement mediated destruction of the NMJ in fB−/− and P−/− EAMG. Surprisingly, cytokines, including IFN-g and IL-17, were increased in the serum of fB−/− EAMG and P−/− EAMG. Data indicate that complement AP activation is involved in the pathogenesis of EAMG. Our observations suggest that modulation of AP via properdin remains a potential approach to alter the outcome of EAMG. In contrast to our prediction, cytokine levels were higher in serum of fB−/− and P−/− mice. http://dx.doi.org/10.1016/j.imbio.2012.08.189 188 Properdin binding to the surface of leptospira promotes complement activation and controls proliferation of nonpathogenic bacteria in vitro Adriana G.P. Martínez 1 , Viviana P. Ferreira 2 , Tatiana R. Fraga 1 , Silvio de Arruda Vasconcellos 3 , Mohamed R. Daha 4 , Angela S. Barbosa 5 , Lourdes Isaac 1 1

Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, SP, Brazil 2 Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Health Sciences, OH, USA 3 Faculty of Veterinary and Zootechnology, University of São Paulo, SP, Brazil 4 Department of Nephrology, University of Leiden, The Netherlands 5 Laboratory of Bacteriology, Institute Butantan, São Paulo, SP, Brazil Leptospirosis is one of the most important worldwide zoonosis and is caused by pathogenic Leptospira. Activation of the alternative pathway (AP) of complement is crucial for killing non-pathogenic L. biflexa and properdin (P) acts effectively since this bacterium proliferates in P-depleted human serum. The aim of this work was to investigate if P binds equally to both non-pathogenic and pathogenic Leptospira and if this binding requires previous deposition of C3b. Several non-pathogenic and pathogenic serovar of Leptospira were incubated with different sources of properdin [normal human serum (NHS), unfractionated purified P, physiological dimers, trimers, tetramers of P (P2, P3, P4), or non-physiological aggregates (Pn)]. The binding was evaluated by ELISA and Western blot. All strains interacted with unfractionated purified P as well as P present in total NHS, but no significant binding differences were observed. However, non-pathogenic Leptospira bound significantly more to physiological forms P2 and P3 (0.79 ± 0.2 and 0.64 ± 0.2, respectively) than the pathogenic serovar (0.51 ± 0.01 and 0.45 ± 0.03, respectively); p < 0.05. Also, the data show that the interaction of Leptospira and P occurs independently of previous deposition of C3b fragments on the bacteria surface. In addition, pathogenic, but not non-pathogenic, secreted leptospiral proteases were able to degrade purified properdin in 30 kDa fragment. Among several recombinant leptospiral membrane proteins tested, lipoprotein LIC11087, present only in pathogenic Leptospira, was the major ligand for P. In conclusion, the physiological forms of P bind most efficiently to L. biflexa and we propose that this may play a role in limiting non-pathogenic Leptospira survival by potentially triggering activation of the AP. On the other hand, pathogenic Leptospira can degrade properdin, which may contribute to complement evasion and pathogen survival. http://dx.doi.org/10.1016/j.imbio.2012.08.190