Molecular Immunology 36 (1999) 877±884
Expression of the anaphylatoxin C5a receptor in non-myeloid cells J. Zwirner a,*, A. Fayyazi b, O. GoÈtze a a
Department of Immunology, University of GoÈttingen, D-37075, GoÈttingen, Germany b Department of Pathology, University of GoÈttingen, D-37075, GoÈttingen, Germany
Abstract C5a, a 74 amino acid peptide cleaved from the complement protein C5, represents the most potent anaphylatoxin and possesses in¯ammatory as well as immunomodulatory acitivities. In the past, expression of the receptor for the anaphylatoxin C5a (C5aR) has been thought to be restricted to cells of myeloid origin. However, recent evidence suggests that the C5aR is constitutively expressed in non-myeloid cells including epithelial, endothelial and smooth muscle cells in the human liver and lung. These ®ndings are contrasted by results from our laboratory which demonstrated that in the normal human liver and lung C5aR expression is detectable exclusively in macrophages and macrophage-derived cells (Kuper cells). Interestingly, we found evidence that C5aR expression may be inducible in epithelial cells as C5aR mRNA was observed in vivo in human keratinocytes of the in¯amed but not of the normal skin. Herein we review the work of our laboratory and others on the expression of the C5aR in various human non-myeloid cells types. A better understanding of the expression patterns of this important anaphylatoxin receptor may provide new insights in the pathophysiological role of C5a in vivo. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Anaphylatoxin; C5a receptor; Monoclonal antibody; Immunohistochemistry; Complement
1. Introduction The complement-derived anaphylatoxin C5a is a potent mediator of in¯ammation and possesses immunomodulatory activities (Goldstein, 1992; KoÈhl and Bitter-Suermann, 1993). C5a induces the chemoattraction and degranulation of leukocytes. It also induces smooth muscle contraction, histamine release from mast cells, vasodilation and increased vascular permeability. In addition, C5a possesses immunoregulatory activities through the induction (tumor necrosis factor (TNF)-a, interleukin (IL)-1, IL-6 and IL-8) and suppression (IL-12) of cytokines in monocytes (Cavaillon et al., 1990; Schindler et al., 1990; Montz et al., 1991; Ember et al., 1994; Wittmann et al., 1999). * Corresponding author. Tel.: +49-551-395811; fax: +49-511395843. E-mail address: [email protected]
C5a mediates its eects by binding to a speci®c high-anity receptor, C5aR/CD88, a member of the rhodopsin subfamily of G protein coupled receptors with seven transmembrane segments (Boulay et al., 1991; Gerard and Gerard, 1991; Siciliano et al., 1994). Traditionally, expression of the C5aR was thought to be restricted to cells of myeloid origin. Speci®c binding sites for C5a have been demonstrated on neutrophils (Chenoweth and Hugli, 1978), eosinophils (Gerard et al., 1989), basophils (Kurimoto et al., 1989) and monocytes (Werfel et al., 1992). With the cloning of the human C5aR cDNA (Gerard and Gerard, 1991; Boulay et al., 1991), speci®c reagents for the detection of receptor expression could be generated such as antibodies which recognize the N-terminal region of the C5aR (Oppermann et al., 1993; Morgan et al., 1993; Haviland et al., 1995; Werfel et al., 1996). Haviland et al. (1995) provided evidence for the constitutive expression of the C5aR in epithelial, endo-
0161-5890/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 1 6 1 - 5 8 9 0 ( 9 9 ) 0 0 1 0 9 - 1
J. Zwirner et al. / Molecular Immunology 36 (1999) 877±884
Table 1 Published data on the expression of the C5aR in non-myeloid cellsa Cell type
Hepatic stellate cells (Ito cells)
Proximal tubular cells Bronchial epithelial cells
Alveolar epithelial cells
Smooth muscle cells
Lung Brain HUVEC HUVEC HUVEC Lung, intestine, kidney Lung, liver, kidney, intestine Liver/HepG2 HepG2 Liver Liver (cultured cells) Liver (tissue/cultured cells) Liver (cultured cells) Liver (cultured cells) Liver (tissue/cultured cells) Kidney (cultured cells) Kidney (cultured cells) Kidney Lung Lung (cultured cells) Lung Lung Lung Lung Lung Lung Lung, intestine, skin Lung, intestine, liver, kidney Skin (cultured cells/Hacat) Skin
IC IC LBS, FS RT-PCR, FS FS ISH IC, ISH IC, ISH, NB IC, NB IC, ISH RT-PCR IC, FS RT-PCR FS IC, FS IC, RT-PCR, IC, RT-PCR, IC, ISH IC IC, RT-PCR, ISH IC IC ISH IC IC ISH IC IC, RT-PCR, ISH
Y Yd N Y Y Ne Nf Y Y Nf N N Y Y Yf Y Y Y Y Y Ne Nf Y Ne Nf Y Ne Nf N Yd
Haviland et al. (1995) Gasque et al. (1997) Lundberg et al. (1996) Foreman et al. (1994) Ikeda et al. (1997) Fayyazi et al. (1999a) Fayyazi et al. (1999b) Haviland et al. (1995) McCoy et al. (1995) Fayyazi et al. (1999b) Schieferdecker et al. (1997) Schlaf et al. (1999) Schieferdecker et al. (1997) Schieferdecker et al. (1998) Schlaf et al. (1999) Braun and Davis III (1998) Wilmer et al. (1998) Fayyazi et al. (1999b) Haviland et al. (1995) Floreani et al. (1998) Fayyazi et al. (1999) Fayyazi et al. (1999b) Haviland et al. (1995) Fayyazi et al. (1999a) Fayyazi et al. (1999b) Haviland et al. (1995) Fayyazi et al. (1999a) Fayyazi et al. (1999b) Werfel et al. (1996) Fayyazi et al. (1999)
FS WB, FS WB, FS
Data on lymphocytes and on cells of the central nervous system are not included. IC, Immunochemistry; RT-PCR, reverse transcriptase-polymerase chain reaction; FS, functional study; LBS, ligand binding study; ISH, in situ hybridization; NB, Northern Blotting; WB, Western blotting. c C5aR expression detected: Y=yes; N=no. d De novo expression in in¯amed tissue. e In normal and in¯amed tissue. f In normal tissue. b
thelial and smooth muscle cells of the human liver and lung and also in parenchymal cells of other solid organs. These data suggest that C5a confers previously unexpected functions on a variety of dierent tissue cells. Also in 1995, two groups demonstrated that in addition to microglia (the resident macrophages of the central nervous system), human astrocytes, a non-myeloid cell type, express the C5aR (Gasque et al., 1995; Lacy et al., 1995). The list of non-myeloid cells which may express the C5aR has been increasing since then. It now also includes hepatic stellate cells (Ito cells), renal mesangial cells and neuronal cells (Schieferdecker et al., 1997; 1998; Wilmer et al., 1998; Braun and Davis III, 1998; Farkas et al., 1998; Stahel et al., 1997). Most recently, evidence for the expression of the C5aR in human T and B lymphocytes has been presented (Nataf et al., 1999; Ottonello et al., 1999) although these cell types have been regarded in the past to be devoid of binding sites for C5a (Werfel et
al., 1992; Zwirner et al., 1999). In many of the dierent cell types, however, the functions of the expressed C5aR have not been fully elucidated. Herein we review experimental evidence on the expression of the C5aR in various human non-myeloid cell types (see also Table 1). Emphasis is placed on the most recent work of our laboratory (Fayyazi et al., 1999a,b). We also discuss methodological problems encountered with reagents used for the demonstration of C5aR expression. 2. C5aR expression in the liver Haviland et al. (1995) reported that several non-myeloid tissue cells including liver parenchymal cells (hepatocytes) express the C5aR constitutively. However, in recent reports on liver cells from the normal rat, in contrast to the human liver, C5aR mRNA and protein
J. Zwirner et al. / Molecular Immunology 36 (1999) 877±884
Fig. 1. Expression of the C5aR in the normal human liver. C5aR protein was detected in Kuper cells (®lled arrows) but not in hepatocytes (®lled arrow heads). C5aR expression was detected by immunohistochemistry as described (Fayyazi et al., 1999b). In brief, morphologically normal, adult human tissue was obtained from unaected areas of liver (n = 2) following surgical tumor excision. Anti-C5aR mAb P12/1 was incubated on acetone-®xed cryostat sections, followed by biotin-conjugated polyclonal goat anti-mouse Ig and peroxidase-conjugated streptavidin. The sections were stained with 3-amino-9-ethylcarbazole. Counterstaining was performed in hemalaun. Original magni®cation, 400.
were not detectable in hepatocytes and Kuper cells represented the predominant C5aR expressing cell population (Schieferdecker et al., 1997; Schlaf et al., 1999). The con¯icting data on the expression of C5aR in hepatocytes of rat and human origin prompted us to reinvestigate the pattern of C5aR expression in normal human tissue samples of the liver (Fayyazi et al., 1999b). The immunohistochemical examination of normal human liver tissue revealed a prominent C5aR expression in mononuclear cells lining hepatic sinusoids (Kuper cells) (Fig. 1) whereas expression of C5aR protein could not be noted in hepatocytes (Fig. 1), bile duct epithelial cells, vascular smooth muscle or endothelial cells. These data were con®rmed by in situ hybridization showing that only Kuper cells but not hepatocytes express C5aR mRNA (Fayyazi et al., 1999b). A prominent C5aR expression in Kuper cells is not surprising since these cells are part of the mononuclear phagocyte system as are blood monocytes which
express high numbers of C5aR molecules (Oppermann et al., 1993; Zahn et al., 1997). However, our results do not agree with previously published data which indicated an abundant expression of the C5aR in nonmyeloid cells of the human liver. Since Haviland et al. (1995) used in their studies a polyclonal C5aR antiserum, it may be speculated that the staining of non-myeloid liver cells may not speci®cally indicate C5aR expression but may be caused by a cross-reactive epitope of unknown origin. Such a phenomenon has been described for anti-C5aR monoclonal antibodies (mAbs). In addition to their binding to C5aR expressing cells, the mAbs P12/1 and D12/1 have been shown to display a weak reactivity against a desmosomal antigen in keratinocytes unrelated to the C5aR (Werfel et al., 1996). In the normal liver, however, we did not detect any reactivity against epithelial antigens making the mAbs P12/1 and D12/1 appropriate tools for the detection of C5aR expressing cells in acetone-®xed hepatic tissue sections. It is also noteworthy, that the mAbs P12/1 and D12/1 along with polyclonal anti-C5aR antisera have been applied in immunohistochemical studies on the characterization of C5aR expression in human brain (Gasque et al., 1997). In this study, the mAbs detected a minimal constitutive expression of the C5aR in astrocytes and microglia of the normal brain. In in¯amed tissue, however, receptor expression was upregulated in these as well as in endothelial cells. Since Haviland et al. (1995) did not disclose the source of the tissues used in their study, it may be speculated that C5aR expression in non-myeloid cells of the liver may also be induced by in¯ammation. This notion is supported by our own observation that keratinocytes in in¯amed but not in normal human skin revealed detectable levels of C5aR transcripts (Fayyazi et al., 1999a). The concomitant expression of C5aR and IL6 mRNA in keratinocytes aected from lichen planus may be indicative of a role of IL-6 in the induction of C5aR expression in the in¯amed skin (Fayyazi et al., 1999a). The expression of C5aR in the hepatoma-derived cell line HepG2 (Haviland et al., 1995; McCoy et al., 1995) may not necessarily re¯ect receptor expression in wildtype hepatocytes. Instead, it may be interpreted as the consequence of the cells' malignant transformation. 3. C5aR expression in the lung In addition to hepatocytes, it was reported that endothelial, smooth muscle as well as alveolar and bronchiolar epithelial cells in the human lung express the C5aR constitutively (Haviland et al., 1995). The cellular localization of C5aR production within the lung was demonstrated exclusively by immunohisto-
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Fig. 2. Expression of the C5aR in the normal human lung. C5aR protein was detected in interstitial (a; ®lled arrow) and alveolar macrophages (b; ®lled arrow) but not in bronchial epithelial cells (a; open arrow), in alveolar epithelial cells (b; ®lled arrow head), in smooth muscle cells of the bronchial wall (a; open arrow head) and in endothelial cells of vessels. V denotes a vessel within the bronchial wall (a). C5aR expression was detected by immunohistochemistry as described in the legend of Fig. 1. Adult human lung tissue was obtained from unaected areas of lung (n = 3) following surgical tumor excision. Original magni®cation, 400.
chemistry using a polyclonal antiserum against the C5aR. We investigated C5aR expression in the normal human lung using the mAbs P12/1 and D12/1 (Fayyazi et al., 1999b). These antibodies react with the N-terminal region of the human C5aR. The immunohistochemical examination revealed a prominent C5aR expression in alveolar and interstitial macrophages (Fig. 2). However, C5aR expression could not be observed in bronchial and alveolar epithelial cells, in vascular smooth muscle and in endothelial cells (Fig. 2). These results are supported by our recent ®ndings that in the normal and in¯amed human lung only macrophages express C5aR mRNA as detected by in situ hybridization on paran-embedded sections (Fayyazi et al., 1999a). It has been suggested that the C5aR-speci®c mRNA detected by Northern blot analysis of total RNA preparations from lung tissue may be derived primarily from epithelial, endothelial and smooth muscle cells and not from in®ltrating neutrophils, monocytes or macrophages (Haviland et al., 1995). But the considerable numbers of C5aR expressing macrophages in the lung, as detected in our stu-
dies might suce to yield distinct C5aR mRNA signals in Northern blot analyses. The immunohistochemical ®ndings reported might possibly be a consequence of antibody binding to antigens other than CD88 as suggested above. Indeed, it has been reported that a monoclonal antibody may bind to two unrelated proteins (CD74 and CD80) although the primary amino acid sequences of the two proteins had only a low homology (Freeman et al., 1998). It is also noteworthy that a polyclonal antiserum against the human C3aR has been shown to be reactive against the human B cell line Raji and certain cell types depending on the antibody concentration used although Raji cells did not express detectable amounts of C3aR mRNA (Martin et al., 1997). These examples highlight the need for stringent speci®city controls of polyclonal antisera to exclude the possibility of false positive staining results. Antibody titers used should be speci®ed. It was reported that the binding of polyclonal Ig against the C5aR to structures in paran-embedded tissue sections could be inhibited by their pretreatment with C5a ligand. Unfortunately, experimental details were not given (Haviland et al., 1995). In contrast to
J. Zwirner et al. / Molecular Immunology 36 (1999) 877±884
Fig. 3. Expression of the C5aR in the normal human kidney. C5aR protein was detected in epithelial cells of proximal tubuli (P), and in few leukocytes localized in glomeruli (G) and within the interstitium. C5aR expression was detected by immunohistochemistry as described in the legend of Fig. 1. Adult human kidney tissue was obtained from unaected areas of kidney (n = 2) following surgical tumor excision. Original magni®cation, 400.
these ®ndings, we were unable to inhibit the binding of anti-C5aR mAbs P12/1 and D12/1 to cryosections from normal human lung which had been ®xed with acetone (10 min at room temperature) or paraformaldehyde (4%, 10 min at room temperature) and treated with an excess of C5a. Blocking of the antibody binding to the C5aR requires binding of C5a to the N-terminus of the receptor and to a second site in its transmembrane region (Siciliano et al., 1994; DeMartino et al., 1994; 1995). In our own experiments both paraformaldehyde and acetone treatment of human tissue led to a loss of C5a binding to the receptor leaving thus the speci®c antibody binding to the receptor unaected. Taken together, our investigations have failed to provide evidence for the expression of C5aR in epithelial, endothelial and smooth muscle cells of the normal human lung. 4. C5aR expression in the intestine and kidney It has been suggested that the signi®cant levels of C5aR mRNA detected by Northern blot analyses in the spleen, heart, kidney and intestine may be derived from cell types other than tissue macrophages, presum-
ably parenchymal cells (Haviland et al., 1995). Therefore, we studied C5aR expression in the normal human intestine and kidney using the mAbs P12/1 and D12/1 (Fayyazi et al., 1999b). The immunohistochemical examination of normal human intestine revealed a prominent C5aR expression in tissue macrophages whereas C5aR expression could not be observed in epithelial cells, in vascular smooth muscle and in endothelial cells. These results are supported by the ®ndings that in the normal and in¯amed human intestine only macrophages express C5aR mRNA as detected by in situ hybridization (Fayyazi et al., 1999a). These results suggest that macrophages and not parenchymal cells are the most likely source of C5aR mRNA identi®ed in Northern blot analyses of normal intestinal tissue. In the normal human kidney, the mAbs P12/1 and D12/1 bound not only to interstitial macrophages but also to cells of the proximal tubuli (Fig. 3). In situ hybridization indicated the presence of C5aR mRNA in these cells (Fayyazi et al., 1999b). In contrast, no expression of C5aR mRNA or protein could be observed in the epithelial cells of other tubuli, vascular smooth muscle cells or glomerular/vascular endothelial cells. Visualization of C5aR expression by the anti-C5aR mAbs in a highly specialized epithelial cell type in the
J. Zwirner et al. / Molecular Immunology 36 (1999) 877±884
kidney may be taken as evidence for the speci®city and sensitivity of the immunohistochemical procedures. The epithelium of proximal tubuli in the kidney has been demonstrated in the past to express various cytokines and complement components, either constitutively or upon stimulation (Prodjosudjadi et al., 1995; Gerritsma et al., 1996; 1998). Since C5a has been shown to possess immunoregulatory activities through the induction of cytokines (TNFa, IL-1, IL-6, and IL8) in human monocytes (Cavaillon et al., 1990; Schindler et al., 1990; Montz et al., 1991; Ember et al., 1994) it is tempting to speculate that C5aR-dependent pathways may also be involved in the cytokine production within tubular epithelial cells and thereby in the complement-mediated pathophysiology of a wide range of nephritides (McLean, 1993; Johnson, 1997).
5. C5aR expression in cultured primary cells An early study by Lundberg et al. (1996) failed to identify speci®c C5a binding sites on human umbilical vein endothelial cells (HUVEC) and the release of prostacylcin upon C5a stimulation. Subsequently, however, C5a has been shown to induce the expression of P-selectin and the secretion of von Willebrand factor in HUVEC although binding studies with 125I-C5a again failed to provide unequivocal evidence for C5aR expression (Foreman et al., 1994). These results may indicate a low level of the C5aR expression in these cells. In a more recent publication, Ikeda et al. (1997) demonstrated that C5a induces tissue factor activity in HUVEC. However, a strong increase in tissue factor activity induced by 10 mM C5a as compared to 1 mM was measured. As the anity constant for the binding of C5a to its receptor is in the range of 1 nM (Burg et al., 1995; Rothermel et al., 1997) receptor saturation should be accomplished by C5a concentrations far below 10 mM. The discrepancy between the increasing eectiveness of exceedingly high C5a concentrations and receptor saturation at much lower ligand concentrations is yet unresolved and makes the interpretation of the results quite dicult. Evidence for the expression of the C5aR has also been demonstrated in human cultured bronchial epithelial and renal mesangial cells (Floreani et al., 1998; Braun and Davies III, 1998; Wilmer et al., 1998). These ®ndings may suggest that cultured primary cells reach a state of activation which induces their expression of C5aR while the receptor is not detectable under resting conditions. This notion is supported by the ®nding that C5aR expression in mesangial cells could only be detected in cultured cells whereas no receptor expression was detectable in vivo (Braun and Davies III, 1998).
6. Conclusions The results of our experiments con®rm previous ®ndings that the C5aR may also be expressed in nonmyeloid cells. In contrast to studies by others, however, only proximal tubular cells of the normal human kidney could be shown to express the C5aR constitutively, whereas receptor expression in other non-myeloid cells (such as keratinocytes and hepatocytes) may be inducible under in¯ammatory conditions in vivo or under tissue culture conditions in vitro. However, the mechanisms that lead to the induction of C5aR expression are not understood. They will be the focus of future studies. The data presented herein also demonstrate that monoclonal antibodies are well suited for the investigation of C5aR expression. When using polyclonal antisera for the identi®cation of receptor expression, however, a careful characterization of the speci®city of the staining reaction should be performed. Taken together, our ®ndings point to an as yet unknown role of C5a in human renal physiology and in the pathogenesis of human skin disorders beyond its well-de®ned function as a chemoattractant and activator of leukocytes.
Acknowledgements This work was supported by grants from the Stiftung der UniversitaÈt GoÈttingen and the Deutsche Forschungsgemeinschaft, project Go 410/7-2.
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