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Human Mesangial Cells Receptors for the Anaphylatoxin

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Human Mesangial Cells Receptors for the Anaphylatoxin Receptors for the Anaphylatoxin C5a (CD88) on Human Mesangial Cells W. A. Wilmer, P. T. Kaumaya, J. A. Ember and F. G. Cosio This information is current as J Immunol 1998; 160:5646-5652; ; of September 29, 2021. http://www.jimmunol.org/content/160/11/5646 References This article cites 43 articles, 20 of which you can access for free at: Downloaded from http://www.jimmunol.org/content/160/11/5646.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: by guest on September 29, 2021 http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Receptors for the Anaphylatoxin C5a (CD88) on Human Mesangial Cells1 W. A. Wilmer,* P. T. Kaumaya,† J. A. Ember,‡ and F. G. Cosio2* In these studies, we determined whether there are receptors for the anaphylatoxin C5a (C5aR, CD88) on human mesangial cells (HMC). To prepare Abs to C5aR, we first synthesized an immunogenic peptide spanning residues 8–32 of the molecule, and this peptide was used to immunize rabbits. Anti-C5aR antiserum, but not preimmune serum, stained fixed and unfixed HMC in culture. By Western blotting anti-C5aR, Abs identified a 49.6-kDa protein in HMC. By reverse-transcription PCR, a cDNA product of 558 bp was amplified corresponding to the expected size of C5aR cDNA. A cDNA of the same size was amplified simultaneously from human PBL. Restriction mapping of the products amplified from HMC and from PBL gave restriction fragments of the same size. Incubation of HMC with increasing doses of C5a caused a progressive increase in the levels of the transcription factors activator protein-1 (AP-1) and cAMP response element binding protein (CREB), but C5a had no effect on the level of nuclear factor-kB (NF-kB). The effects of C5a on AP-1 were concentration and time dependent and peaked after 60 Downloaded from min. In contrast, the C5a metabolite C5adesArg had no significant effect on AP-1 levels. Preincubation of HMC with rabbit anti-C5aR antiserum inhibited partially the effect of C5a on AP-1. However, anti-C5aR Abs alone had no appreciable effects on AP-1. C5a caused a significant up-regulation of mRNA for the early response genes c-jun and c-fos on HMC. These results provide evidence for the presence of C5aR in adult HMC in culture and indicate that, after binding to C5aR, the anaphylatoxin C5a causes significant up-regulation of certain transcription factors and early response genes. The Journal of Immunology, 1998, 160: 5646–5652. http://www.jimmunol.org/ 5a is a soluble by-product of the activation of the com- tivation occurs frequently in human glomerulonephritis (15), and plement component C5 (reviewed in Refs. 1 and 2). The the presence of complement receptors on glomerular cells suggests C proinflammatory actions of C5a were among the first rec- the possibility that activated complement products may interact ognized effects of complement activation and include chemotaxis directly and specifically with those cells. Previous studies have for leukocytes and monocytes; induction of leukocyte degranula- argued that the sublytic effects of activated complement products tion, oxygen radical production, and cytokine release (3, 4); in- on organ cells may play important roles in the pathogenesis of duction of the expression of adhesion molecules on endothelial complement-mediated tissue damage. These cellular effects of ac- by guest on September 29, 2021 cells (5, 6); and others (reviewed in Ref. 1). These effects of C5a tivated complement components may be receptor dependent or in- are mediated by binding to a specific G protein-linked membrane dependent. For example, terminal complement components receptor (C5aR)3 (7). The presence of this receptor was initially (C5b-9) can deposit on cell membranes, triggering signal-trans- shown, by functional assays, in circulating peripheral blood leu- duction pathways and causing phenotypic changes (16–18), yet no kocytes and in mast cells (1, 8). However, more recent studies receptors for C5b-9 have been recognized. We and others have utilizing Abs to the C5aR have suggested that this receptor is more been unable to demonstrate receptors for degradation products of widely expressed than previously suspected (5, 9–12). C3 (19, 20) on adult HMC. Of interest, recent studies demonstrated To our knowledge, no previous study has examined the presence receptors for C1q in fetal HMC (21). of C5aR in human mesangial cells (HMC). The presence of C5aR The recent description of the human C5aR gene sequence (7) has on these cells was suggested by results of previous experiments in considerably simplified the study of C5aR in tissue cells. Although the which we demonstrated that incubation of HMC with C5a causes functional effects of C5a/C5aR interactions on circulating cells are an up-regulation of mRNA for the complement-regulatory protein clear, the role of C5aR on tissue cells is far less clear. For example, decay-accelerating factor (DAF) (13, 14). The presence of C5aR in previous studies showed that C5a causes contraction of smooth mus- HMC may be of pathogenic significance because complement ac- cle cells, although this effect may be due to the interaction of the anaphylatoxin with mast cells or leukocytes present in the vessel wall (1, 22). In addition, recent studies showed that C5a/C5aR interactions *Department of Internal Medicine, Division of Nephrology, and †Department of Mi- crobiology and Medical Biochemistry, The Ohio State University, Columbus, OH cause an up-regulation of selectin expression on human endothelial 43210; and ‡The Scripps Research Institute, La Jolla, CA 92037 cells (5), thus allowing leukocyte adhesion to the vessel wall in vivo Received for publication May 6, 1997. Accepted for publication February 5, 1998. (6). In these studies, we assessed whether C5aR are present in adult The costs of publication of this article were defrayed in part by the payment of page HMC in culture. To determine the functional roles of these receptors, charges. This article must therefore be hereby marked advertisement in accordance we also assessed whether the interaction of C5a with HMC causes with 18 U.S.C. Section 1734 solely to indicate this fact. activation of transcription factors or changes in mRNA levels for early 1 This work was supported in part by National Institutes of Health Grant 1PO1 AI-HL 40150. response genes. 2 Address correspondence and reprint requests to Dr. Fernando G. Cosio, The Ohio State University, Division of Nephrology, N210 Means Hall, 1654 Upham Drive, Materials and Methods Columbus, OH 43210-1250. Preparation of polyclonal anti-C5aR (anti-C5aR (8–32) peptide) 3 Abbreviations used in this paper: C5aR, receptors for the anaphylatoxin C5a; AP-1, antiserum activator protein-1; CREB, cAMP response element binding protein; DAF, decay- accelerating factor; EMSA, electrophoretic mobility shift assay; HMC, human mes- For this procedure, we followed methods similar to those described in angial cells; NF-kB, nuclear factor-kB; PMN, polymorphonuclear leukocyte. previous publications to prepare Abs against the 9–29 peptide of the C5aR Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 5647 (23). In brief, the immunogenicity map of the C5aR molecule was deter- Etc, Wilsonville, OR). A mixture of 1 mM concentration of each primer mined using previously described methods (24). This analysis identified, together with buffer and Taq polymerase was heated at 95°C for 5 min, among others, the sequence of amino acids from 8 to 32 as having a high then held at 80°C, while 5 ml of the solution containing cDNA were added immunogenic potential. This sequence is unique for C5aR (11, 23) and is to the tube. cDNA was amplified for 40 cycles, and the amplified products located near the amino-terminal portion of the molecule, in proximity to the were separated in a 2% agarose gel. The predicted size of the C5aR product binding site for C5a (25). The C5aR peptide 8–32 was synthesized, linked is 558 bp (7, 30). to a promiscuous T cell epitope to enhance its immunogenicity (26, 27), To further characterize the cDNA product amplified by RT-PCR, we and injected s.c. into two rabbits. Both animals produced high titer Abs used restriction enzyme mapping. In these experiments, we reamplified the against the immunizing peptide, as detected by an ELISA. For this assay, cDNA product obtained after the first amplification cycle before exposing the immunizing peptide or a control peptide of the same length was used this product to restriction enzymes. Purified cDNA was treated with 10 U to coat the bottom of microtiter plates. Wells were incubated with increas- of the restriction enzymes PstI (Boehringer Manneheim, Indianapolis, IN) ing dilutions of serum obtained from rabbits before immunization or at or ScaI (Life Technologies) for 60 min at 37°C. The digests were run on several time points after the immunization. Binding of rabbit Abs to the a 2% agarose gel. plate was detected by incubation with peroxidase-labeled goat anti-rabbit IgG (Zymed Laboratories, Grand Island, NY), followed by peroxidase Stimulation of HMC with C5a: effects on transcription factors substrate.
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