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

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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-␬B (NF-␬B). 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-␬B, nuclear factor-␬B; PMN, polymorphonuclear leukocyte. previous publications to prepare Abs against the 9–29 peptide of the C5aR

(23). In brief, the immunogenicity map of the C5aR molecule was deter- Etc, Wilsonville, OR). A mixture of 1 ␮M 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 ␮l 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. and on mRNA levels for early response genes Preparations of HMC in culture In these experiments, HMC were incubated with 2 ␮g/ml of C5a or its The methods used in our laboratory for the isolation and characterization of metabolite C5adesArg at 37°C for variable periods of time in the presence HMC in culture have been described previously (13). The cells used in of media containing 10% FBS. rC5a was purchased from Sigma. these experiments were isolated from four different kidneys obtained from C5adesArg was prepared by incubating 10 ␮M C5a in 1% BSA with 5 U potential cadaveric transplant donors, but deemed unsuitable for transplan- of carboxypeptidase B (Sigma) at 37°C for 30 min (5). Cleavage of C5a by Downloaded from tation. HMC were cultured in 75-mm flasks in Media 199 (Life Technol- this enzyme was stopped by incubating the mixture at 56°C for an addi- ogies, Grand Island, NY) containing 10% heat-inactivated FCS. tional 30 min. To control for possible effects of temperature, the C5a preparation used in these experiments was also incubated for 30 min at 37°C Identification of C5aR on HMC and then 56°C for 30 min. To assess the effects of C5a on the activity of nuclear transcription C5aR on cultured HMC were identified by immunocytochemistry, flow factors, we performed electrophoretic mobility shift assays (EMSA). cytometry, and Western blot analysis. Rabbit anti-C5aR antiserum or pre- Briefly, conditioned HMC were chilled for 5 min before harvest. Media immune serum control was used to stain HMC, as follows: HMC cultured http://www.jimmunol.org/ were then removed and the cells were washed twice in cold PBS, scraped in chamber slides (Nunc) were fixed with 1% paraformaldehyde and air off the culture surface, and centrifuged at 4°C. Nuclear proteins were ob- dried overnight. These cells were first incubated with preimmune serum, tained using the protocol of Dignam (32) with modifications by Osborn rabbit anti-C5aR antiserum. In some experiments, HMC were also incu- (33) with the addition of the protease inhibitor leupeptin (5 ␮g/ml) (Sigma) bated with anti-C5aR Abs in the presence of 2 mg/ml of the immunizing to each buffer. Protein concentrations were measured by the BCA method C5aR peptide. In most experiments, HMC were incubated with Ab for 60 (Pierce) using BSA as a standard. Ten micrograms of nuclear protein were min at room temperature with a final dilution of antiserum of 1/500. After added to an incubation buffer containing 10 mM Tris, 100 mM NaCl, 1 nM washing the unbound Abs with PBS containing 1% BSA (Sigma, St. Louis, EDTA, 4% glycerol, and 2 ␮g of poly(dI-dC) (Pharmacia, Piscataway, NJ). MO), HMC were incubated with peroxidase-labeled (or FITC-labeled) goat Double-stranded oligonucleotides containing two consensus 12-O-tetrade- anti-rabbit IgG Abs. The peroxidase substrate was developed by incubation canolyphorbol 13-acetate (TPA)-responsive elements, or two consensus with 3-amino-9-ethylcarbazole (Sigma). Cells were visualized directly by ␬

cAMP-responsive elements, or two consensus NF- B motifs (Life Tech- by guest on September 29, 2021 light or immunofluorescence microscopy. For FACS, cells were first de- nologies) were end labeled with [␥-32P]ATP by T4 kinase. Unbound 32P tached from the culture surface by incubation with cold 10 mM EDTA and was separated from the oligonucleotide mixture with DNA-spin columns vigorous pipetting. Before fixation, cells were stained with anti-C5aR an- (Worthington Biomedical, Freehold, NJ). A total of 0.2 ng of the labeled tiserum or control antiserum (both diluted 1/500) at 4°C. The secondary oligonucleotide probe (TPA-responsive element, cAMP-responsive ele- Abs used for these experiments were FITC-labeled goat anti-rabbit Abs ment, or NF-␬B) was added to the incubation buffer for 20 min at room (Zymed), and these cells were analyzed by FACS, as described previously temperature, and the protein-DNA complexes were resolved at 4°C on 5% (28). In some experiments, the results obtained with the rabbit anti-C5aR nondenaturing polyacrylamide gels in 0.25ϫ TBE buffer at 175 V. The gels antiserum against the 8–32 peptide prepared for these studies were com- were dried and exposed to autoradiography film at Ϫ70°C. DNA-protein pared with results obtained using a different rabbit anti-C5aR antiserum complexes were demonstrated as labeled bands with retarded migration raised against the 9–29 peptide (23) (kindly provided to us by Dr. Hugli, through the gel. Specificity of the bands as complexes containing AP-1, The Scripps Clinic and Research Institute, La Jolla, CA). cAMP response element binding protein (CREB), or NF-␬B transcription To identify the size of the cell membrane Ag recognized by anti-C5aR factors was confirmed by incubating the nuclear protein and labeled probe antiserum, we performed Western blot analyses. In these experiments, with 50ϫ unlabeled specific probe or an irrelevant probe at the same con- HMC membranes were harvested in a lysis buffer that contained 20 mM centration (Life Technologies). HEPES, pH 7.9, and protease inhibitors. After cell lysis, the particulate To determine the specificity of the effects of C5a on transcription fac- fraction was pelleted by centrifugation at 4°C and resuspended in lysis tors, in some experiments cells were incubated with C5a or its metabolite buffer containing 0.1% Triton X-100 (Sigma). The lysate was clarified by C5desArg at equal concentrations. To determine the participation of C5aR centrifugation. Following quantitation of proteins by the BCA method on the response of HMC to C5a, cells were preincubated at 37°C for 30 min (Pierce, Rockford, IL), using BSA as standard, 25 ␮g of membrane pro- with polyclonal anti-C5aR antiserum (1/4000) or equal concentrations of teins were separated by 10% SDS-PAGE and transferred to nitrocellulose preimmune serum before the incubation with C5a. membranes via semidry technique (29). Thereafter, the blots were In additional experiments, we assessed, by Northern blot analyses, the quenched in PBS/0.1% Tween, 2% BSA for 120 min and incubated with effects of C5a on mRNA levels for the early response genes c-fos and c-jun. rabbit anti-C5aR antiserum or preimmune rabbit serum for 60 min. After These methods have been previously described (34). In brief, RNA was washing, the blots were incubated with biotinylated goat anti-rabbit Ab for isolated from HMC as described above. Fifteen micrograms of total RNA 45 min, washed, and incubated with horseradish peroxidase-streptavidin were loaded on a 1% agarose gel and electrophoresed. RNA was trans- complexes (Zymed). Following a final wash, bands of interest were dem- ferred to nylon membranes and hybridized with 32P-labeled cDNA for onstrated using enhanced chemiluminescence (ECL; Amersham, Arlington c-jun or c-fos (American Type Culture Collection, Rockville, MD). La- Heights, IL). beled bands were identified by autoradiography and quantitated by densi- Identification of C5aR mRNA in HMC tometry. Densitometry of 18S ribosomal RNA stained with ethidium bromide was used to confirm that the loading of RNA in all of the lanes of the For these experiments, we used RT-PCR following a protocol similar to gel was equal. that described previously (30). In brief, total RNA was isolated from HMC and from peripheral blood cells by the RNAzol B method (Tel-Test, Friendswood, TX) (31). cDNA was prepared from the RNA by reverse Results transcription using the SuperScript Preamplification Kit (Life Technolo- Identification of C5aR on HMC gies). RNA was then removed from the solution by treatment with RNase H for 20 min at 37°C. Primers corresponding to nucleotides 226–245 and Figure 1 displays HMC stained with rabbit preimmune serum (Fig. 764–783 of the published C5aR sequence (7, 30) were prepared (Oligos, 1A), rabbit anti-C5aR (8–32 peptide) antiserum (Fig. 1B), or anti- 5648 C5a RECEPTORS ON MESANGIAL CELLS

FIGURE 2. Western blot analysis of HMC membrane proteins stained with rabbit anti-C5aR antiserum. A unique protein band of 49.6 kDa (arrow) was identiﬁed in immunoblots using an anti-C5aR antiserum (lane labeled aC5aR Ab), but not blots stained with preimmune rabbit IgG (lane Downloaded from labeled rabbit IgG). Results of one experiment that is representative of four separate experiments.

C5aR antiserum in the presence of an excess of immunizing peptide (Fig. 1C). As can be seen, HMC stain positive with anti-C5aR serum, and the stain is not present when cells are incubated with the antiserum in the presence of the immunizing peptide. Identical http://www.jimmunol.org/ staining patterns were obtained using other rabbit anti-C5aR (9–29 peptide) antiserum previously described in the literature (23) (data not shown). It should be noted that at low concentrations of Abs, there was significant variability in the intensity of C5aR staining among cells. However, at higher concentrations, all cells were noted to be positive compared with HMC stained with preimmune rabbit serum. Figure 1D displays the results of a representative experiment (n ϭ 4) analyzing the presence of C5aR on the surface by guest on September 29, 2021 of HMC by flow cytometry. It should be noted that, compared with direct visual counting of adherent HMC, the estimate of C5aR- positive HMC analyzed by flow cytometry is low (for example, 30% positive cells in the experiment shown in Fig. 1D), most likely because the background staining of cells in solution is higher than that obtained when cells were stained adherent to a culture surface. Figure 2 displays the results of Western blot analysis of cell membrane proteins stained with anti-C5aR (8–32 peptide) Abs. As can be seen, these Abs identify a unique protein band with a cal- culated molecular mass of 49.6 kDa. This experiment was repeated using anti-C5aR (9–29 peptide) Abs previously described (12, 23), and those Abs identified a protein band of similar molecular mass as the band identified using Abs produced in our laboratory (data not shown).

Identification of C5aR mRNA in HMC Figure 3A displays the size of the DNA product amplified by RT- PCR from HMC and from total RNA of peripheral blood cells. The size of the amplified product is 558 bp in both cell lines, which is the expected size for C5aR. It should be noted that in these and all other experiments, the amount of amplified C5aR cDNA was vi- sually less in HMC than in peripheral blood leukocytes. Restriction enzyme treatment of the C5aR cDNA amplified from HMC and from peripheral blood cells resulted in the generation of DNA FIGURE 1. A–C, FITC staining of HMC with preimmune rabbit serum fragments of the same size from both cell types (Fig. 3B). (A), rabbit anti-C5aR antiserum (B), and anti-C5aR antiserum in the presence of the C5aR peptide used for immunization (C). D, Flow-cytometry Effects of C5a on mRNA levels for early response genes and on analysis of HMC stained with preimmune rabbit serum (⅐⅐⅐) or rabbit anti- transcription factor activity C5aR antiserum (——). The figure displays the results of one experiment that is representative of four separate experiments. FITC intensity is ex- Figure 4 demonstrates the effects of C5a incubation on the mRNA pressed in a logarithmic scale. levels of the early response gene c-fos and c-jun. As can be seen The Journal of Immunology 5649 Downloaded from

FIGURE 3. Top, Size of the cDNA product amplified by RT-PCR from peripheral blood leukocytes (lane 1) and HMC (lane 2) utilizing C5aR primers. The product of interest is identified by an arrow on the left side of http://www.jimmunol.org/ the figure and corresponds to a 558-bp size. Right line displays DNA size markers. Bottom, Restriction analysis of cDNA product amplified from PMN (lanes 1, 2, and 3) and HMC (lanes 4, 5, and 6) utilizing C5aR primers. Lanes 1 and 4, Intact cDNA product; lanes 2 and 5, cDNA treated with PstI; lanes 3 and 6, cDNA treated with ScaI. Reference DNA size lanes are shown on the first lane of the gel.

FIGURE 4. Effects of C5a on c-jun and c-fos mRNA levels in HMC.

The concentration of C5a used in each lane is identified above each lane. by guest on September 29, 2021 in this representative experiment (one of three experiments), C5a Ctrl refers to cells incubated in the absence of C5a. The same Northern gel resulted in significant up-regulation of c-jun mRNA in HMC and was stained first for c-jun, and then for c-fos. Bottom part of the figure had a lesser effect on the levels of c-fos mRNA. displays 18S RNA, stained with ethidium bromide, in each of the lanes. Figure 5 demonstrates the effects of C5a on the DNA binding of the transcription factors AP-1 (Fig. 5A) and CREB (Fig. 5B). In- creasing concentrations of C5a caused a time-dependent up-regulation in the binding of AP-1 and CREB to labeled oligonucleo- nificantly after incubation of HMC with C5a at the same concen- tides. DNA binding of both AP-1 and CREB appears to peak after trations and times of incubation used in the above experiments 60 min of incubation with the anaphylatoxin; thereafter, the levels (data not shown). of transcription factor binding fall, although they remain higher than in unstimulated cells. After a 60-min incubation with 2 ␮g/ml Discussion of C5a, quantitation of AP-1 bands by densitometry revealed a The results of this study provide convincing evidence for the ex- significant up-regulation of AP-1 levels by C5a (control, 2.4 Ϯ 1.9; pression of receptors for C5a on adult HMC. The estimated mo- C5a, 4.11 Ϯ 1.9, n ϭ 5, p ϭ 0.01 by paired t test). In separate lecular mass of these receptors in these cells is similar to that experiments (Fig. 6A), C5adesArg (2 ␮g/ml) had no significant shown in other cells, although it should be noted that there appears effect on the levels of AP-1 in HMC (control, 2.4 Ϯ 1.7; to be variations in the size of C5aR in different cells (7, 10, 23, 35, C5adesArg, 3.9 Ϯ 1.4, n ϭ 3, NS). To assess the participation of 36). The C5aR in polymorphonuclear leukocytes (PMN) has been C5aR on the effects of C5a on transcription factor levels, HMC shown to be significantly smaller than 49.6 kDa (23), which is the were incubated with anti-C5aR antiserum or preimmune serum size of C5aR in HMC. However, the putative C5aR cDNA product (both diluted 1/1000) before and during exposure to C5a. As can amplified from HMC and leukocytes, using primers derived from be seen (Fig. 6B), rabbit anti-C5aR Abs partially blocked the ef- the structure of the leukocyte C5aR (7), is of the same size, sug- fects of C5a on AP-1 binding, but preimmune rabbit serum had no gesting that variations in the molecular mass of C5aR in different significant effect. The quantitative data for three separate experi- cells may be due to posttranslational modification of the molecule. ments include: controls, 2 Ϯ 1; C5a, 5.3 Ϯ 3.3; C5a ϩ anti-C5a The rabbit anti-C5aR antiserum prepared in these studies is of Abs, 3.7 Ϯ 2.3; and C5a ϩ preimmune, 5.2 Ϯ 3.1, all values high affinity and recognizes a portion of the C5aR molecule that expressed in arbitrary densitometric units. In additional experi- appears to be in close proximity to the C5a binding site (23, 25). ments, HMC were incubated with increasing concentrations (1/ It is likely that this proximity explains the partial inhibition of C5a 4000 to 1/500) of anti-C5aR or preimmune antiserum. Neither sera effects caused by anti-C5aR Abs. In addition, these data lend cre- alone had significant effects on AP-1 levels on HMC (data not dence to the postulate that the effects of C5a on HMC are mediated shown). In contrast to the effects of C5a on AP-1 and CREB levels, through binding to specific membrane receptors. The functional the binding of the transcription factor NF-␬B did not change sig- effects of C5a detected in these studies required concentrations of 5650 C5a RECEPTORS ON MESANGIAL CELLS Downloaded from http://www.jimmunol.org/ FIGURE 5. Effects of C5a on the binding of the transcription factors AP-1 (A) and CREB (B). In both figures, values above each lane describe the concentration of C5a and/or the time of incubation with the anaphylatoxin. Negative control (“comp” lane) includes nuclear proteins incubated with labeled probe in the presence of 50ϫ unlabeled oligonucleotide. Lane labeled “ns comp” includes nuclear proteins incubated with labeled oligonucleotide in the presence of 50ϫ unlabeled nonspecific oligonucleotide. The arrow on the side of the figure identifies the band of interest. Each of these experiments is representative of three additional experiments.

C5a that are significantly higher than those required to stimulate ment-mediated glomerulonephritis, DAF expression is markedly by guest on September 29, 2021 PMNs (1), but similar to the doses of C5a used in other studies that up-regulated on mesangial cells (37). assessed the effects of C5a on endothelial cells (5). It is perhaps Our knowledge about the function of C5aR in tissue cells is reasonable to expect that circulating cells would be responsive to limited, because it is only recently that these receptors have been low concentration of circulating C5a, while in tissues, cells may be recognized in cells other than those of myeloid lineage. Thus, we exposed to higher concentrations of the anaphylatoxin generated can only speculate about other possible effects of C5a on HMC locally. In fact, the chemotactic effects of C5a will predict that the based on the effects of C5a on other cells. For example, because concentration of this peptide should be higher in tissues than in the HMC are contractile cells (42, 43) and C5a stimulates smooth circulation, thus directing the migration of circulating cells toward muscle cell contraction (22), it is attractive to postulate that C5a inflammatory sites. may cause HMC contraction directly binding to C5aR, and that In these studies, we have assessed basic functional effects of this effect may cause alterations in glomerular blood flow (42, 44). C5a on HMC, including effects on the levels of certain transcrip- Perhaps related to this postulated effect, injection of C5a directly tion factors and mRNA levels for the early response genes. These into the renal artery and local generation of C5a during comple- results support two basic postulates: 1) HMC in culture have func- ment-mediated glomerulonephritis cause a reduction of renal tional C5aR that, after interacting with its ligand, leads to activa- blood flow (45, 46). We also postulate that C5a/C5aR may par- tion of intracellular signal transduction and regulation of mRNA ticipate in the changes that occur in the kidney during exposure to levels of certain genes; 2) the observed changes in mRNA levels endotoxin. This postulate is based on the following observations: for early response gene suggest that binding of C5a to HMC may C5a binding to C5aR leads to up-regulation of adhesion molecules lead to additional phenotypic changes on these cells. Regarding the latter, previous studies from our laboratory and others (14, 37–40) on endothelial cells (5) and margination of inflammatory cells on suggest that the deleterious effects of complement activation on pulmonary vessels during complement activation in vivo or after glomerular cells are limited by the fact that activated complement injection of LPS (6). Circulating leukocytes also marginate in the products themselves stimulate the synthesis of complement-regu- glomerulus after injection of LPS (47) and, of interest, the injec- latory proteins that inhibit complement activation on the cell sur- tion of endotoxin (LPS) into experimental animals causes an up- face (41). For example, terminal complement components (C5b-9) regulation of C5aR mRNA levels in the kidney and other organs stimulate the synthesis of DAF (14) and CD59 (38) by HMC, and (11). Finally, previous studies have shown that HMC have the these proteins protect the cell from complement-mediated lysis capacity to produce a variety of cytokines (reviewed in Ref. 42), (14, 41). C5a may also contribute to this protective feedback loop, and because C5a can stimulate cytokine release from inflammatory because C5a also causes an increase in DAF and CD59 mRNA cells (3, 48), it may be postulated that this anaphylatoxin may have levels in HMC (13, 14). Finally, we have provided evidence that similar effects on HMC. These postulates, although speculative at these mechanisms are likely to be operational in vivo, because this point, suggest several pathogenic pathways through which C5a although the normal glomerulus contains little DAF, in comple- and HMC C5aR may participate in glomerular inflammation. The Journal of Immunology 5651

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