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The Journal of Immunology, 1998, 160: 5646-5652.
Copyright © 1998 by The American Association of Immunologists

Receptors for the Anaphylatoxin C5a (CD88) on Human Mesangial Cells1

W. A. Wilmer*, P. T. Kaumaya{dagger}, J. A. Ember{ddagger} and F. G. Cosio2,*

* Department of Internal Medicine, Division of Nephrology, and {dagger} Department of Microbiology and Medical Biochemistry, The Ohio State University, Columbus, OH 43210; and {ddagger} The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-{kappa}B (NF-{kappa}B). The effects of C5a on AP-1 were concentration and time dependent and peaked after 60 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
C5a is a soluble by-product of the activation of the complement component C5 (reviewed in Refs. 1 and 2). The proinflammatory actions of C5a were among the first recognized effects of complement activation and include chemotaxis for leukocytes and monocytes; induction of leukocyte degranulation, oxygen radical production, and cytokine release (3, 4); induction of the expression of adhesion molecules on endothelial cells (5, 6); and others (reviewed in 1 . These effects of C5a are mediated by binding to a specific G protein-linked membrane receptor (C5aR)3 (7). The presence of this receptor was initially shown, by functional assays, in circulating peripheral blood leukocytes and in mast cells (1, 8). However, more recent studies utilizing Abs to the C5aR have suggested that this receptor is more widely expressed than previously suspected (5, 9, 10, 11, 12).

To our knowledge, no previous study has examined the presence of C5aR in human mesangial cells (HMC). The presence of C5aR on these cells was suggested by results of previous experiments in which we demonstrated that incubation of HMC with C5a causes an up-regulation of mRNA for the complement-regulatory protein decay-accelerating factor (DAF) (13, 14). The presence of C5aR in HMC may be of pathogenic significance because complement activation occurs frequently in human glomerulonephritis (15), and the presence of complement receptors on glomerular cells suggests the possibility that activated complement products may interact directly and specifically with those cells. Previous studies have argued that the sublytic effects of activated complement products on organ cells may play important roles in the pathogenesis of complement-mediated tissue damage. These cellular effects of activated complement components may be receptor dependent or independent. For example, terminal complement components (C5b-9) can deposit on cell membranes, triggering signal-transduction pathways and causing phenotypic changes (16, 17, 18), yet no receptors for C5b-9 have been recognized. We and others have been unable to demonstrate receptors for degradation products of C3 (19, 20) on adult HMC. Of interest, recent studies demonstrated receptors for C1q in fetal HMC (21).

The recent description of the human C5aR gene sequence (7) has considerably simplified the study of C5aR in tissue cells. Although the functional effects of C5a/C5aR interactions on circulating cells are clear, the role of C5aR on tissue cells is far less clear. For example, previous studies showed that C5a causes contraction of smooth muscle 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 cause an up-regulation of selectin expression on human endothelial cells (5), thus allowing leukocyte adhesion to the vessel wall in vivo (6). In these studies, we assessed whether C5aR are present in adult HMC in culture. To determine the functional roles of these receptors, we also assessed whether the interaction of C5a with HMC causes activation of transcription factors or changes in mRNA levels for early response genes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of polyclonal anti-C5aR (anti-C5aR (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) peptide) antiserum

For this procedure, we followed methods similar to those described in previous publications to prepare Abs against the 9–29 peptide of the C5aR (23). In brief, the immunogenicity map of the C5aR molecule was determined using previously described methods (24). This analysis identified, among others, the sequence of amino acids from 8 to 32 as having a high immunogenic potential. This sequence is unique for C5aR (11, 23) and is located near the amino-terminal portion of the molecule, in proximity to the binding site for C5a (25). The C5aR peptide 8–32 was synthesized, linked to a promiscuous T cell epitope to enhance its immunogenicity (26, 27), and injected s.c. into two rabbits. Both animals produced high titer Abs against the immunizing peptide, as detected by an ELISA. For this assay, the immunizing peptide or a control peptide of the same length was used to coat the bottom of microtiter plates. Wells were incubated with increasing dilutions of serum obtained from rabbits before immunization or at several time points after the immunization. Binding of rabbit Abs to the plate was detected by incubation with peroxidase-labeled goat anti-rabbit IgG (Zymed Laboratories, Grand Island, NY), followed by peroxidase substrate.

Preparations of HMC in culture

The methods used in our laboratory for the isolation and characterization of HMC in culture have been described previously (13). The cells used in these experiments were isolated from four different kidneys obtained from potential cadaveric transplant donors, but deemed unsuitable for transplantation. HMC were cultured in 75-mm flasks in Media 199 (Life Technologies, Grand Island, NY) containing 10% heat-inactivated FCS.

Identification of C5aR on HMC

C5aR on cultured HMC were identified by immunocytochemistry, flow cytometry, and Western blot analysis. Rabbit anti-C5aR antiserum or preimmune serum control was used to stain HMC, as follows: HMC cultured in chamber slides (Nunc) were fixed with 1% paraformaldehyde and air dried overnight. These cells were first incubated with preimmune serum, rabbit anti-C5aR antiserum. In some experiments, HMC were also incubated with anti-C5aR Abs in the presence of 2 mg/ml of the immunizing C5aR peptide. In most experiments, HMC were incubated with Ab for 60 min at room temperature with a final dilution of antiserum of 1/500. After washing the unbound Abs with PBS containing 1% BSA (Sigma, St. Louis, MO), HMC were incubated with peroxidase-labeled (or FITC-labeled) goat anti-rabbit IgG Abs. The peroxidase substrate was developed by incubation with 3-amino-9-ethylcarbazole (Sigma). Cells were visualized directly by light or immunofluorescence microscopy. For FACS, cells were first detached from the culture surface by incubation with cold 10 mM EDTA and vigorous pipetting. Before fixation, cells were stained with anti-C5aR antiserum or control antiserum (both diluted 1/500) at 4°C. The secondary Abs used for these experiments were FITC-labeled goat anti-rabbit Abs (Zymed), and these cells were analyzed by FACS, as described previously (28). In some experiments, the results obtained with the rabbit anti-C5aR antiserum against the 8–32 peptide prepared for these studies were compared with results obtained using a different rabbit anti-C5aR antiserum raised against the 9–29 peptide (23) (kindly provided to us by Dr. Hugli, The Scripps Clinic and Research Institute, La Jolla, CA).

To identify the size of the cell membrane Ag recognized by anti-C5aR antiserum, we performed Western blot analyses. In these experiments, HMC membranes were harvested in a lysis buffer that contained 20 mM HEPES, pH 7.9, and protease inhibitors. After cell lysis, the particulate fraction was pelleted by centrifugation at 4°C and resuspended in lysis buffer containing 0.1% Triton X-100 (Sigma). The lysate was clarified by centrifugation. Following quantitation of proteins by the BCA method (Pierce, Rockford, IL), using BSA as standard, 25 µg of membrane proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes via semidry technique (29). Thereafter, the blots were quenched in PBS/0.1% Tween, 2% BSA for 120 min and incubated with rabbit anti-C5aR antiserum or preimmune rabbit serum for 60 min. After washing, the blots were incubated with biotinylated goat anti-rabbit Ab for 45 min, washed, and incubated with horseradish peroxidase-streptavidin complexes (Zymed). Following a final wash, bands of interest were demonstrated using enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL).

Identification of C5aR mRNA in HMC

For these experiments, we used RT-PCR following a protocol similar to 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 transcription using the SuperScript Preamplification Kit (Life Technologies). 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 764–783 of the published C5aR sequence (7, 30) were prepared (Oligos, Etc, Wilsonville, OR). A mixture of 1 µM concentration of each primer together with buffer and Taq polymerase was heated at 95°C for 5 min, then held at 80°C, while 5 µl of the solution containing cDNA were added to the tube. cDNA was amplified for 40 cycles, and the amplified products were separated in a 2% agarose gel. The predicted size of the C5aR product is 558 bp (7, 30).

To further characterize the cDNA product amplified by RT-PCR, we used restriction enzyme mapping. In these experiments, we reamplified the cDNA product obtained after the first amplification cycle before exposing this product to restriction enzymes. Purified cDNA was treated with 10 U of the restriction enzymes PstI (Boehringer Manneheim, Indianapolis, IN) or ScaI (Life Technologies) for 60 min at 37°C. The digests were run on a 2% agarose gel.

Stimulation of HMC with C5a: effects on transcription factors and on mRNA levels for early response genes

In these experiments, HMC were incubated with 2 µg/ml of C5a or its metabolite C5adesArg at 37°C for variable periods of time in the presence of media containing 10% FBS. rC5a was purchased from Sigma. C5adesArg was prepared by incubating 10 µM C5a in 1% BSA with 5 U of carboxypeptidase B (Sigma) at 37°C for 30 min (5). Cleavage of C5a by this enzyme was stopped by incubating the mixture at 56°C for an additional 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 and then 56°C for 30 min.

To assess the effects of C5a on the activity of nuclear transcription factors, we performed electrophoretic mobility shift assays (EMSA). Briefly, conditioned HMC were chilled for 5 min before harvest. Media were then removed and the cells were washed twice in cold PBS, scraped off the culture surface, and centrifuged at 4°C. Nuclear proteins were obtained using the protocol of Dignam (32) with modifications by Osborn (33) with the addition of the protease inhibitor leupeptin (5 µg/ml) (Sigma) to each buffer. Protein concentrations were measured by the BCA method (Pierce) using BSA as a standard. Ten micrograms of nuclear protein were added to an incubation buffer containing 10 mM Tris, 100 mM NaCl, 1 nM EDTA, 4% glycerol, and 2 µg of poly(dI-dC) (Pharmacia, Piscataway, NJ). Double-stranded oligonucleotides containing two consensus 12-O-tetradecanolyphorbol 13-acetate (TPA)-responsive elements, or two consensus cAMP-responsive elements, or two consensus NF-{kappa}B motifs (Life Technologies) were end labeled with [{gamma}-32P]ATP by T4 kinase. Unbound 32P was separated from the oligonucleotide mixture with DNA-spin columns (Worthington Biomedical, Freehold, NJ). A total of 0.2 ng of the labeled oligonucleotide probe (TPA-responsive element, cAMP-responsive element, or NF-{kappa}B) was added to the incubation buffer for 20 min at room temperature, and the protein-DNA complexes were resolved at 4°C on 5% nondenaturing polyacrylamide gels in 0.25x TBE buffer at 175 V. The gels were dried and exposed to autoradiography film at -70°C. DNA-protein complexes were demonstrated as labeled bands with retarded migration through the gel. Specificity of the bands as complexes containing AP-1, cAMP response element binding protein (CREB), or NF-{kappa}B transcription factors was confirmed by incubating the nuclear protein and labeled probe with 50x unlabeled specific probe or an irrelevant probe at the same concentration (Life Technologies).

To determine the specificity of the effects of C5a on transcription factors, in some experiments cells were incubated with C5a or its metabolite C5desArg at equal concentrations. To determine the participation of C5aR on the response of HMC to C5a, cells were preincubated at 37°C for 30 min with polyclonal anti-C5aR antiserum (1/4000) or equal concentrations of preimmune serum before the incubation with C5a.

In additional experiments, we assessed, by Northern blot analyses, the effects of C5a on mRNA levels for the early response genes c-fos and c-jun. These methods have been previously described (34). In brief, RNA was isolated from HMC as described above. Fifteen micrograms of total RNA were loaded on a 1% agarose gel and electrophoresed. RNA was transferred to nylon membranes and hybridized with 32P-labeled cDNA for c-jun or c-fos (American Type Culture Collection, Rockville, MD). Labeled bands were identified by autoradiography and quantitated by densitometry. 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 gel was equal.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of C5aR on HMC

Figure 1Go displays HMC stained with rabbit preimmune serum (Fig. 1GoA), rabbit anti-C5aR (8–32 peptide) antiserum (Fig. 1GoB), or anti-C5aR antiserum in the presence of an excess of immunizing peptide (Fig. 1GoC). 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 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 1GoD displays the results of a representative experiment (n = 4) analyzing the presence of C5aR on the surface 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. 1GoD), most likely because the background staining of cells in solution is higher than that obtained when cells were stained adherent to a culture surface.



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  		FIGURE 1. A–C, FITC staining of HMC with preimmune rabbit serum (A), rabbit anti-C5aR antiserum (B), and anti-C5aR antiserum in the presence of the C5aR peptide used for immunization (C). D, Flow-cytometry analysis of HMC stained with preimmune rabbit serum (···) or rabbit anti-C5aR antiserum (——). The figure displays the results of one experiment that is representative of four separate experiments. FITC intensity is expressed in a logarithmic scale.

 
Figure 2Go 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 calculated 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).



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  		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 labeled rabbit IgG). Results of one experiment that is representative of four separate experiments.

 
Identification of C5aR mRNA in HMC

Figure 3GoA 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 visually 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 fragments of the same size from both cell types (Fig. 3GoB).



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  		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 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.

 
Effects of C5a on mRNA levels for early response genes and on transcription factor activity

Figure 4Go demonstrates the effects of C5a incubation on the mRNA levels of the early response gene c-fos and c-jun. As can be seen in this representative experiment (one of three experiments), C5a resulted in significant up-regulation of c-jun mRNA in HMC and had a lesser effect on the levels of c-fos mRNA.



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  		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. Ctrl refers to cells incubated in the absence of C5a. The same Northern gel was stained first for c-jun, and then for c-fos. Bottom part of the figure displays 18S RNA, stained with ethidium bromide, in each of the lanes.

 
Figure 5Go demonstrates the effects of C5a on the DNA binding of the transcription factors AP-1 (Fig. 5GoA) and CREB (Fig. 5GoB). Increasing concentrations of C5a caused a time-dependent up-regulation in the binding of AP-1 and CREB to labeled oligonucleotides. DNA binding of both AP-1 and CREB appears to peak after 60 min of incubation with the anaphylatoxin; thereafter, the levels of transcription factor binding fall, although they remain higher than in unstimulated cells. After a 60-min incubation with 2 µg/ml of C5a, quantitation of AP-1 bands by densitometry revealed a significant up-regulation of AP-1 levels by C5a (control, 2.4 ± 1.9; C5a, 4.11 ± 1.9, n = 5, p = 0.01 by paired t test). In separate experiments (Fig. 6GoA), C5adesArg (2 µg/ml) had no significant effect on the levels of AP-1 in HMC (control, 2.4 ± 1.7; C5adesArg, 3.9 ± 1.4, n = 3, NS). To assess the participation of C5aR on the effects of C5a on transcription factor levels, HMC were incubated with anti-C5aR antiserum or preimmune serum (both diluted 1/1000) before and during exposure to C5a. As can be seen (Fig. 6GoB), rabbit anti-C5aR Abs partially blocked the effects of C5a on AP-1 binding, but preimmune rabbit serum had no significant effect. The quantitative data for three separate experiments include: controls, 2 ± 1; C5a, 5.3 ± 3.3; C5a + anti-C5a Abs, 3.7 ± 2.3; and C5a + preimmune, 5.2 ± 3.1, all values expressed in arbitrary densitometric units. In additional experiments, HMC were incubated with increasing concentrations (1/4000 to 1/500) of anti-C5aR or preimmune antiserum. Neither sera alone had significant effects on AP-1 levels on HMC (data not shown). In contrast to the effects of C5a on AP-1 and CREB levels, the binding of the transcription factor NF-{kappa}B did not change significantly after incubation of HMC with C5a at the same concentrations and times of incubation used in the above experiments (data not shown).



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  		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 50x unlabeled oligonucleotide. Lane labeled "ns comp" includes nuclear proteins incubated with labeled oligonucleotide in the presence of 50x 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.

 


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  		FIGURE 6. Specificity of the effects of C5a on AP-1 binding. A, AP-1 EMSA of HMC incubated with 2 µg/ml C5a or 2 µg/ml C5desArg for 60 min. Results of one experiment that is representative of three experiments. B, AP-1 EMSA of HMC preincubated with C5a (2 µg/ml C5a for 60 min) alone; C5a at the same concentration in the presence of anti-C5aR Abs (diluted 1/1000); and C5a in the presence of control preimmune Abs (diluted 1/1000). The results of this experiment are representative of a total of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study provide convincing evidence for the expression of receptors for C5a on adult HMC. The estimated molecular mass of these receptors in these cells is similar to that shown in other cells, although it should be noted that there appears to be variations in the size of C5aR in different cells (7, 10, 23, 35, 36). The C5aR in polymorphonuclear leukocytes (PMN) has been shown to be significantly smaller than 49.6 kDa (23), which is the size of C5aR in HMC. However, the putative C5aR cDNA product amplified from HMC and leukocytes, using primers derived from the structure of the leukocyte C5aR (7), is of the same size, suggesting that variations in the molecular mass of C5aR in different cells may be due to posttranslational modification of the molecule.

The rabbit anti-C5aR antiserum prepared in these studies is of high affinity and recognizes a portion of the C5aR molecule that appears to be in close proximity to the C5a binding site (23, 25). It is likely that this proximity explains the partial inhibition of C5a effects caused by anti-C5aR Abs. In addition, these data lend credence to the postulate that the effects of C5a on HMC are mediated through binding to specific membrane receptors. The functional effects of C5a detected in these studies required concentrations of C5a that are significantly higher than those required to stimulate PMNs (1), but similar to the doses of C5a used in other studies that assessed the effects of C5a on endothelial cells (5). It is perhaps reasonable to expect that circulating cells would be responsive to low concentration of circulating C5a, while in tissues, cells may be exposed to higher concentrations of the anaphylatoxin generated locally. In fact, the chemotactic effects of C5a will predict that the concentration of this peptide should be higher in tissues than in the circulation, thus directing the migration of circulating cells toward inflammatory sites.

In these studies, we have assessed basic functional effects of C5a on HMC, including effects on the levels of certain transcription factors and mRNA levels for the early response genes. These results support two basic postulates: 1) HMC in culture have functional C5aR that, after interacting with its ligand, leads to activation of intracellular signal transduction and regulation of mRNA levels of certain genes; 2) the observed changes in mRNA levels for early response gene suggest that binding of C5a to HMC may lead to additional phenotypic changes on these cells. Regarding the latter, previous studies from our laboratory and others (14, 37, 38, 39, 40) suggest that the deleterious effects of complement activation on glomerular cells are limited by the fact that activated complement products themselves stimulate the synthesis of complement-regulatory proteins that inhibit complement activation on the cell surface (41). For example, terminal complement components (C5b-9) stimulate the synthesis of DAF (14) and CD59 (38) by HMC, and these proteins protect the cell from complement-mediated lysis (14, 41). C5a may also contribute to this protective feedback loop, because C5a also causes an increase in DAF and CD59 mRNA levels in HMC (13, 14). Finally, we have provided evidence that these mechanisms are likely to be operational in vivo, because although the normal glomerulus contains little DAF, in complement-mediated glomerulonephritis, DAF expression is markedly up-regulated on mesangial cells (37).

Our knowledge about the function of C5aR in tissue cells is limited, because it is only recently that these receptors have been recognized in cells other than those of myeloid lineage. Thus, we can only speculate about other possible effects of C5a on HMC based on the effects of C5a on other cells. For example, because HMC are contractile cells (42, 43) and C5a stimulates smooth muscle cell contraction (22), it is attractive to postulate that C5a may cause HMC contraction directly binding to C5aR, and that this effect may cause alterations in glomerular blood flow (42, 44). Perhaps related to this postulated effect, injection of C5a directly into the renal artery and local generation of C5a during complement-mediated glomerulonephritis cause a reduction of renal blood flow (45, 46). We also postulate that C5a/C5aR may participate in the changes that occur in the kidney during exposure to endotoxin. This postulate is based on the following observations: C5a binding to C5aR leads to up-regulation of adhesion molecules on endothelial cells (5) and margination of inflammatory cells on pulmonary vessels during complement activation in vivo or after injection of LPS (6). Circulating leukocytes also marginate in the glomerulus after injection of LPS (47) and, of interest, the injection of endotoxin (LPS) into experimental animals causes an up-regulation of C5aR mRNA levels in the kidney and other organs (11). Finally, previous studies have shown that HMC have the capacity to produce a variety of cytokines (reviewed in 42 , and because C5a can stimulate cytokine release from inflammatory cells (3, 48), it may be postulated that this anaphylatoxin may have similar effects on HMC. These postulates, although speculative at this point, suggest several pathogenic pathways through which C5a and HMC C5aR may participate in glomerular inflammation.


   Acknowledgments
 
We thank Dr. Hugli from The Scripps Research Institute for his support, Ms. Susan Martin for expert secretarial assistance, and Mr. Alan Bakaletz for technical assistance.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant 1PO1 AI-HL 40150. Back

2 Address correspondence and reprint requests to Dr. Fernando G. Cosio, The Ohio State University, Division of Nephrology, N210 Means Hall, 1654 Upham Drive, Columbus, OH 43210-1250. Back

3 Abbreviations used in this paper: C5aR, receptors for the anaphylatoxin C5a; AP-1, activator protein-1; CREB, cAMP response element binding protein; DAF, decay-accelerating factor; EMSA, electrophoretic mobility shift assay; HMC, human mesangial cells; NF-{kappa}B, nuclear factor-{kappa}B; PMN, polymorphonuclear leukocyte. Back

Received for publication May 6, 1997. Accepted for publication February 5, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hugli, T. E.. 1984. Structure and function of the anaphylatoxins. Springer Semin. Immunopathol. 7:193.[Medline]
  2. Hebert, L. A., F. G. Cosio, D. J. Birmingham. 1996. Complement and complement regulatory proteins in renal diseases. E. G. Nielson, and W. G. Couser, eds. Immunologic Renal Diseases 377. Lippincott-Raven, Philadelphia.
  3. Okusawa, S., K. B. Yancey, J. W. M. van der Meer, S. Endres, G. Lonnemann, K. Hefter, M. M. Frank, J. F. Burke, C. A. Dinarello, J. A. Gelfand. 1988. C5a stimulates secretion of tumor necrosis factor from human mononuclear cells in vitro: comparison with secretion of interleukin 1ß and interleukin 1{alpha}. J. Exp. Med. 168:443.[Abstract/Free Full Text]
  4. Morgan, E. L., S. Sanderson, W. Scholz, D. J. Noonan, W. O. Weigle, T. E. Hugli. 1992. Identification and characterization of the effector region within human C5a responsible for stimulation of IL-6 synthesis. J. Immunol. 148:3937.[Abstract]
  5. Foreman, K. E., A. A. Vaporciyan, B. K. Bonish, M. L. Jones, K. J. Johnson, M. M. Glovsky, S. M. Eddy, P. A. Ward. 1994. C5a-induced expression of P-selectin in endothelial cells. J. Clin. Invest. 94:1147.
  6. Mulligan, M. S., E. Schmid, G. O. Till, T. E. Hugli, H. P. Friedl, R. A. Roth, P. A. Ward. 1997. C5a-dependent up-regulation in vivo of lung vascular P-selectin. J. Immunol. 158:1857.[Abstract]
  7. Gerard, N. P., C. Gerard. 1991. The chemotactic receptor for human C5a anaphylatoxin. Nature 349:614.[Medline]
  8. Chenoweth, D. E., T. E. Hugli. 1978. Demonstration of specific C5a receptor on intact human polymorphonuclear leukocytes. Proc. Natl. Acad. Sci. USA 75:3943.[Abstract/Free Full Text]
  9. Burg, M., U. Martin, C. Rheinheimer, J. Kohl, W. Bautsch, E. C. Bottger, A. Klos. 1995. IFN-{gamma} up-regulates the human C5a receptor (CD88) in myeloblastic U937 cells and related cell lines. J. Immunol. 155:4419.[Abstract]
  10. Gasque, P., P. Chan, M. Fontaine, A. Ischenko, M. Lamacz, O. Gotze, B. P. Morgan. 1995. Identification and characterization of the complement C5a anaphylatoxin receptor on human astrocytes. J. Immunol. 155:4882.[Abstract]
  11. Haviland, D. L., R. L. McCoy, W. T. Whitehead, H. Akama, E. P. Molmenti, A. Brown, J. C. Haviland, W. C. Parks, D. H. Perlmutter. 1995. Cellular expression of the C5a anaphylatoxin receptor (C5aR): demonstration of C5aR on nonmyeloid cells of the liver and lung. J. Immunol. 154:1861.[Abstract]
  12. Buchner, R. R., T. E. Hugli, J. A. Ember, E. L. Morgan. 1995. Expression of functional receptors for human C5a anaphylatoxin (CD88) on the human hepatocellular carcinoma cell line HepG2. J. Immunol. 155:308.[Abstract]
  13. Shibata, T., F. G. Cosio, D. J. Birmingham. 1991. Complement activation induces the expression of decay accelerating factor on human mesangial cells. J. Immunol. 147:3901.[Abstract]
  14. Cosio, F. G., T. Shibata, B. H. Rovin, D. J. Birmingham. 1994. Effects of complement activation products on the synthesis of decay accelerating factor and membrane cofactor protein by human mesangial cells. Kidney Int. 46:986.[Medline]
  15. Hebert, L. A., F. G. Cosio. 1992. The role of the complement system in renal injury. Semin. Nephrol. 12:408.[Medline]
  16. Carney, D. F., T. J. Lang, M. L. Shin. 1990. Multiple signal messengers generated by terminal complement complexes and their role in terminal complement complex elimination. J. Immunol. 145:623.[Abstract]
  17. Lovett, D. H., G. M. Hansch, M. Goppelt, K. Resch, D. Gemsa. 1987. Activation of glomerular mesangial cells by the terminal membrane attack complex of complement. J. Immunol. 138:2473.[Abstract]
  18. Cybulsky, A. V.. 1991. Release of arachidonic acid by complement C5b-9 complex in glomerular epithelial cells. Am. J. Physiol. 261:F427.[Abstract/Free Full Text]
  19. Ross, G. D., M. E. Medof. 1985. Membrane complement receptors specific for bound fragments of C3. Adv. Immunol. 37:217.[Medline]
  20. Fisher, E., M. D. Appay, M. D. Kazatchkine. 1984. The human glomerular C3 receptor. Mol. Immunol. 21:1223.[Medline]
  21. Van den Dobbelsteen, M. E. A., F. J. van der Woude, W. E. M. Schroeijers, N. Klar-Mohamad, L. A. van Es, M. R. Daha. 1996. Both IgG- and C1q-receptors play a role in the enhanced binding of IgG complexes to human mesangial cells. J. Am. Soc. Nephrol. 7:573.[Abstract]
  22. Marceau, F., D. deBlois, C. Laplante, E. Petitclerc, G. Pelletier, J. H. Grose, T. E. Hugli. 1990. Contractile effect of the chemotactic factors f-met-Leu-Phe and C5a on the human isolated umbilical artery: role of cyclooxygenase products and tissue macrophages. Circ. Res. 67:1059.[Abstract/Free Full Text]
  23. Morgan, E. L., J. A. Ember, S. D. Sanderson, W. Scholz, R. Buchner, R. D. Ye, T. E. Hugli. 1993. Anti-C5a receptor antibodies: characterization of neutralizing antibodies specific for a peptide, C5aR(9–29), derived from the predicted amino-terminal sequence of the human C5a receptor. J. Immunol. 151:377.[Abstract]
  24. Kaumaya, P. T. P., A. M. VanBuskirk, E. Goldberg, S. K. Pierce. 1992. Design and immunological properties of topographic immunogenic determinants of a protein antigen (LDH-C4) as vaccines. J. Biol. Chem. 267:6338.[Abstract/Free Full Text]
  25. Oppermann, M., U. Raedt, T. Hebell, B. Schmidt, B. Zimmermann, O. Gotze. 1993. Probing the human receptor for C5a anaphylatoxin with site-directed antibodies: identification of a potential ligand binding site on the NH2-terminal domain. J. Immunol. 151:3785.[Abstract]
  26. Kaumaya, P. T. P., S. Kobs-Conrad, A. M. DiGeorge, V. C. Stevens. 1994. "De novo" engineering of peptide immunogenic and antigenic determinants as potential vaccines. C. Basava, and G. M. Anantharamaiah, eds. Peptides: Design, Synthesis, and Biological Activity 133. Birkhauser, Boston.
  27. Kaumaya, P. T. P., S. Kobs-Conrad, Y. Hoon Seo, H. Lee, A. M. VanBuskirk, N. Feng, J. F. Sheridan, V. Stevens. 1993. Peptide vaccines incorporating a "promiscuous" T-cell epitope bypass certain haplotype restricted immune responses and provide broad spectrum immunogenicity. J. Mol. Recognit. 6:81.[Medline]
  28. Cosio, F. G., X. Shen, D. J. Birmingham, M. E. van Aman, L. A. Hebert. 1990. Evaluation of the mechanisms responsible for the reduction in erythrocyte complement receptors when immune complexes form in vivo in primates. J. Immunol. 145:4198.[Abstract]
  29. Okubo, S., F. Niimura, H. Nishimura, F. Tkemoto, A. Fogo, T. Matsusaka, I. Ichikawa. 1997. Angiotensin-independent mechanism of aldosterone synthesis during chronic extracellular fluid volume depletion. J. Clin. Invest. 99:855.[Medline]
  30. Richard, S., C. A. Farrell, A. S. Shaw, H. J. Showell, P. A. Connelly. 1994. C5a as a model for chemotactic factor-stimulated tyrosine phosphorylation in the human neutrophil. J. Immunol. 152:2479.[Abstract]
  31. Chomczysnski, P., N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156.[Medline]
  32. Dignam, J. D., R. M. Lebovitz, R. G. Roeder. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11:1475.[Abstract/Free Full Text]
  33. Osborn, L., S. Kunkel, G. Nabel. 1989. Tumor necrosis factor alpha and interleukin-1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa B. Proc. Natl. Acad. Sci. USA 86:2336.[Abstract/Free Full Text]
  34. Cosio, F. G., W. A. Wilmer. 1995. Effects of high glucose concentration on c-fos: role of protein kinase C. Kidney Int. 51:S-3.
  35. Boulay, F., L. Mery, M. Tardif, L. Brouchon, P. Vignais. 1991. Expression cloning of a receptor for C5a anaphylatoxin on differentiated HL-60 cells. Biochemistry 30:2993.[Medline]
  36. Watanabe, H., M. Kuraya, R. Kasukawa, H. Yanagisawa, M. Yanagisawa, T. Fujita. 1995. Analysis of C5a receptor by monoclonal antibody. J. Immunol. Methods 185:19.[Medline]
  37. Cosio, F. G., D. D. Sedmak, J. D. Mahan, Jr N. S. Nahman. 1989. Localization of decay-accelerating factor in normal and diseased kidneys. Kidney Int. 36:100.[Medline]
  38. Shibata, T., T. Kohsaka. 1995. Effects of complement activation on the expression of CD59 by human mesangial cells. J. Immunol. 155:403.[Abstract]
  39. Quigg, R. J., B. P. Morgan, V. M. Hollers, S. Adler, A. E. I. Sneed, C. F. Lo. 1995. Complement regulation in the rat glomerulus: Crry and CD59 regulate complement in glomerular mesangial and endothelial cells. Kidney Int. 48:412.[Medline]
  40. Nangaku, M., R. L. Meek, J. Pippin, K. L. Gordon, B. P. Morgan, R. J. Johnson, W. G. Couser. 1996. Transfected CD59 protects mesangial cells from injury induced by antibody and complement. Kidney Int. 50:257.[Medline]
  41. Holers, V. M., J. L. Cole, D. M. Lublin, T. Seya, J. P. Atkinson. 1989. Human C3b and C4b regulatory proteins: a new multi-gene family. Immunol. Today 6:188.
  42. Mene, P., M. S. Simonson, M. J. Dunn. 1989. Physiology of the mesangial cell. Physiol. Rev. 69:1347.[Free Full Text]
  43. Kreisberg, J. I., M. Venkatachalam, D. Troyer. 1985. Contractile properties of cultured glomerular mesangial cells. Am. J. Physiol. 249:F457.
  44. Schlondorff, D. O.. 1987. The glomerular mesangial cell: an expanding role for a specialized pericyte. FASEB J. 1:272.[Abstract]
  45. Pelayo, J. C., D. E. Chenoweth, T. E. Hugli. 1986. Effects of the anaphylatoxin C5a on renal and glomerular hemodynamics in the rat. Kidney Int. 30:62.[Medline]
  46. Sekse, I., B. M. Iversen, M. R. Daha, J. Ofstad. 1992. Acute effect of passive Heymann nephritis on renal blood flow and glomerular filtration rate in the rat: role of the anaphylatoxin C5a and the alpha-adrenergic nervous system. Nephron 60:453.[Medline]
  47. Cosio, F. G., J. T. Innes, Jr N. S. Nahman, J. D. Mahan, R. M. Ferguson. 1987. Cyclosporine potentiates the nephrotoxic effects of endotoxin. Transplantation 44:425.[Medline]
  48. Goodman, M. G., D. E. Chenoweth, W. O. Weigle. 1982. Induction of interleukin 1 secretion and enhancement of humoral immunity by binding of human C5a to macrophage surface receptors. J. Exp. Med. 1156:912.



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