СЗа Receptor

C3a Receptor Julia A. Ember1 and Tony E. Hugli2,* 1

Pharmingen, 10975 Torreyana Road, San Diego, CA 92121, USA The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA

2

* corresponding author tel: 858-784-8158, fax: 858-784-8307, e-mail: [email protected] DOI: 10.1006/rwcy.2000.23002.

SUMMARY C3a receptor (C3aR) is a G protein-coupled receptor of the rhodopsin superfamily. The receptor contains the characteristic seven transmembrane domains connected by intra- and extracellular loops, with the Nterminus having an extracellular orientation and the C-terminus being intracellular and the region to which the G proteins bind. C3aR has a major distinguishing feature, which is an extraordinarily large extracellular loop between the fourth and fifth transmembrane helices. This loop in human C3aR contains 175 residues: in most G protein-coupled receptors the corresponding extracellular loop region is 30±40 residues in length. The C3aR is widely distributed in both myeloid cells and nonmyeloid tissue cells. The C3aR on eosinophils and basophils is thought to be particularly important biologically since these cells are chemotactically activated by the ligand C3a. Neutrophils also express C3aR, but the ligand can only induce calcium mobilization in these cells and does not cause granular release, oxygen radical generation, or chemotaxis. C3aR expression has been shown to be upregulated on astrocytes and other brain cells during inflammation. Only one form of C3aR has been detected in human, rat, and mouse, but two isoforms of C3aR have been described in the guinea pig (C3aR-S and C3aR-L for small and large forms).

BACKGROUND

1996) has occurred in the last 10 years. This has permitted comparisons between the receptors to these related ligands and a significant structural difference was observed. For example, we learned that the C3aR has novel and unique structural characteristics compared with most other G protein-coupled receptors, including the C5aR, which is an unusually large extracellular loop. The C3aR has now been cloned from several animal species, including mouse (Hsu et al., 1997; Tornetta et al., 1997), guinea pig (Fukuoka et al., 1998a), and rat (Fukuoka et al., 1998b) and the sequences of these receptor molecules are compared in Figure 1. The patterns of identity between C3aR obtained from different species show relatively high levels in the N-terminal extracellular region and for the transmembrane segments. The second intracellular loop is also highly conserved, perhaps because the two cysteinyl residues participate in critical disulfide bonds. The large second extracellular loop has only modest homology, while the C-terminal intracellular region, which contains the G protein-binding site, is highly conserved. The major unique feature in C3aR compared to other rhodopsin family receptors is the unusually large second extracellular loop; this region may be particularly important for binding the C3a molecule (Figure 2). The only species of C3aR for which isoforms have been detected is the guinea pig (Fukuoka et al., 1998a). The full-length form of gpC3aR is designated C3aR-L and the isoform with a 34 residue deletion in the second extracellular loop is designated C3aR-S.

Discovery

Alternative names

Cloning of both the C5a receptor (C5aR) and C3aR (Ames et al., 1996; Crass et al., 1996; Roglic et al.,

C3aR or the C3 anaphylatoxin receptor has no other alternative names; however it was first reported as an

Figure 1 The complete protein sequences for the C3a receptor from human (Hu), guinea pig (Gp), rat (Rt), and mouse (Mo) are presented. The alignments were optimized for maximal identity and the seven transmembrane regions have been identified by a line and roman numerals. The residue positions that have been conserved in all species are denoted by asterisks. These C3aRs are similar to each other in size, but are considerably larger than the C5aR. The distinguishing feature between the C3aR and C5aR is the large second extracellular loop region, comprised of approximately 170 residues. The expressed C3aR on guinea pig cells has been estimated to be much larger than the 54 kDa nude protein reported here (Fukuoka and Hugli, 1988). This difference may be accounted for by glycosylation, since multiple oligosaccharide groups could be attached at several potential sites in the C3aR.

C3a Receptor 2175 Figure 2 A model is proposed illustrating the interactions between C3a and its respective receptor. The design for this model was adapted from the C5a/C5aR model proposed by Siciliano et al. (1994). C3aR is a G protein-coupled transmembrane receptor of the rhodopsin superfamily. The C3a molecule has at least two major binding sites on C3aR. A noneffector binding site (site 1) exists on the C-terminal helical region of C3a which either makes contact with the large extracellular loop (as shown here) or with other exposed regions of the receptor, including the extracellular Nterminal region. Site 2 contains the C-terminal effector region of C3a, including the sequence LGLAR, which is shown penetrating into the `pore' formed by the seven transmembrane domains of C3aR. This model for C3a/C3aR interaction corresponds to a model originally proposed for multisite binding of C5a with its receptor (Chenoweth and Hugli, 1980).

Exterior

Mutation studies have shown that a number of charged residues at either end of this loop (D159, D325±D327), and in the adjacent membrane region (R161, E162, and R340), participate in ligand binding and are important for C3a-mediated cell activation via the C3aR molecule (Sun et al., 1999). However, major portions of this large loop have been deleted without functional consequences (Chao et al., 1999; Fukuoka et al., 1999). A three-dimensional model of the human C3aR molecule, based on the known structure of the rhodopsin receptor, has been proposed (Sun et al., 1999).

Main activities and pathophysiological roles The C3a receptor is expressed on leukocytes, monocytes, platelets, and mast cells (Ember et al., 1998). Brain cells such as astrocytes (Ischenko et al., 1998) express C3aR which is upregulated during inflammation (Nataf et al., 1999). C3a is a potent chemotactic factor for eosinophils (and basophils) but not neutrophils (Daffern et al., 1995), suggesting that C3aR plays a specific role in asthma and/or allergic disorders. Although there are C3aR on neutrophils, C3a stimulation results in no granular release or oxygen burst, but does induce calcium mobilization (Norgauer et al., 1993; Takafuji et al., 1994). Eosinophil Activation and Migration

Cytoplasm

orphan cDNA clone (AZ3B) by Roglic et al. (1996). No CD code designation has yet been assigned to this receptor molecule.

Structure C3aR is a member of the rhodopsin superfamily and contains the seven transmembrane helical motif, with an N-terminus exposed to the extracellular surface of the cell and the C-terminus having an intracellular orientation. One of the extracellular interhelical loops (loop 2 between helix 4 and 5) is unusually large, being 175 residues in length in human C3aR. C3aR molecules from all species known to date have this extraordinarily large second extracellular loop.

Until recently, the effects of C3a on granulocytic cell types have remained controversial. Earlier studies have reported that C3a could induce degranulation, aggregation, and chemotaxis of human neutrophils (Damerau et al., 1980; Showell et al., 1982a,b; Nagata et al., 1987), suggesting that C3a could exert a direct effect on these cells via the C3aR. However, when neutrophils were purified free of eosinophils (i.e. > 98% pure) the neutrophil cells no longer polarize, undergo chemotaxis, or release granular enzymes in the presence of C3a (Daffern et al., 1995). On the other hand, purified eosinophils were activated by C3a, and adding C3a-stimulated eosinophils to purified neutrophils activated the neutrophils as well. Consequently, it is the eosinophils stimulated by C3a that in turn release factors capable of stimulating the neutrophils. Thus, an indirect stimulation of neutrophils by C3a was presumably mediated by contaminating eosinophils in the earlier cell preparations. The C3a effect is specific since a bioactive synthetic analog of C3a (C3a 57±77 peptide) was also active, while the nonreceptor-binding des Arg

2176 Julia A. Ember and Tony E. Hugli form of C3a failed to stimulate these cells. The ED50 for chemotaxis of eosinophils to C3a was recorded at 100 nM, which is much higher than that for C5a on these same cells; however, it must be remembered that the levels of C3a that can be generated in blood is approximately 6 mM or 20-fold greater than that of C5a. Although neutrophils are not activated by C3a to migrate, release enzymes, or exhibit an oxidative burst, they do express the C3aR and C3a does induce intracellular calcium mobilization (Norgauer et al., 1993; Takafuji et al., 1994). C3a effects on the eosinophil include chemotaxis, granule release, oxidation burst, as well as upregulation of 2-integrins and shedding of L-selectins (Jagels et al., 2000). Monocyte Activation Evidence for C3aR on monocytic cells is less wellestablished in functional or molecular terms. It has been claimed that C3aR is expressed on leukocytes and monocytes but not on B or T cells (Martin et al., 1997). Human monocytes exhibit increased intracellular calcium levels, much like neutrophils, when stimulated by C3a, but not by C3a(des Arg) (Zwirner et al., 1997). However, both C3a and C3a(des Arg) reportedly exert equal effects on cytokine synthesis in human monocytes (Haeffner-Cavaillon et al., 1987; Takabayashi et al., 1996, 1998). C3a and C3a(des Arg) suppress cytokine synthesis and polyclonal immune responses in human B lymphocytes (Fischer and Hugli, 1997). Since C3a(des Arg) does not bind to the C3aR expressed on RBL-2H3 cells, it was concluded that the effects that can be mediated by both intact C3a and the des Arg derivative in monocytes and B lymphocytes must be C3aRindependent effects (Wilken et al., 1999). Therefore, the true actions of C3a mediated through the C3aR on monocytes and lymphocytes must yet be defined and separated from the nonspecific (i.e., non-C3aR) effects on these cell types. These insights suggest that much of the earlier work using PBMCs to evaluate the effects of C3a in mixed-cell systems will need to be carefully re-evaluated in terms of both direct and indirect cell activation, as well as specific versus nonspecific activation processes. Platelets and Mast Cells Little attention has been given to C3aR on platelets since early evidence indicated that human platelets were devoid of a C3a receptor based on chemical crosslinking experiments and on a lack of functional responses (Fukuoka and Hugli, 1988). These studies

identified a curiosity, namely that guinea pig, but not human, platelets express a high molecular weight protein (95±105 kDa) that binds C3a with an estimated Kd of 8  10ÿ10 M. The size of this molecule is nearly twice the size of the cloned guinea pig C3a receptor. This difference might be explained by the fact that guinea pig C3aR has five N-glycosylation sites compared to only two sites in human C3aR. The guinea pig platelets responded to C3a, but not to C3a(des Arg), indicating specificity of the interaction. Serotonin was released and aggregation was induced well below micromolar levels of C3a in guinea pig platelets. Since earlier reports claimed that human platelets were activated by C3a and C3a(des Arg) (Polly and Nachman, 1983), we now conclude that both specific and nonspecific effects of C3a on contaminating cells led to the release of mediators such as PAF, causing the human platelets to respond in an indirect manner, much as human neutrophils respond to C3a in the presence of eosinophils. Based on current evidence, one must conclude that C3aR is not expressed on the human platelet. Direct evidence for C3aR on human or animal (rat) mast cells does not exist. In both the rat and human mast cells, the activation process, including histamine release, is induced by both C3a and C3a(des Arg), suggesting that stimulation of these cells is not through the C3aR. A crosslinking study was used to identify the molecule on the rat mast cell to which the C3a (and C3a des Arg) binds (Fukuoka and Hugli, 1990). The conclusion was that the primary binding site for C3a on the mast cell was the enzyme chymase. In vivo studies supported this hypothesis, since activating levels of C3a (and C3a des Arg) were degraded by chymase when injected in the peritoneal cavity of the rat (Kajita and Hugli, 1991). The mechanism of mast cell activation by C3a, like that of the human neutrophils, platelets, and B lymphocytes (Wilken et al., 1999) appears to be C3aR-independent. Tissue Responses C3a and synthetic C3a analogs are known to induce a visible wheal-and-flare reaction when injected into human and animal skin (Hugli and Erickson, 1977). This response is not species-specific, is dose-dependent, and has a short duration of 15±30 minutes with little sensation (i.e. slight itching) (Hugli, 1981). Lung strips and ileal strips from guinea pigs undergo reversible and tachyphylactic contraction when exposed to C3a (Stimler et al., 1983). Pathology C3a is not known to cause a significant physiologic response when injected into research animals. This is

C3a Receptor 2177 probably due to the rapid removal of the essential Cterminal arginine by carboxypeptidases M and N. However, there is evidence that C3a can produce pulmonary injury (Stimler et al., 1980) and that intrabronchial instillation of milligram quantities of C3a in the guinea pig did cause severe bronchial constriction and death in some instances (Huey et al., 1983; Regal and Klos, 2000).

GENE Recent molecular studies resulted in cloning of human C3aR, first as an orphan G protein-coupled receptor (Roglic et al., 1996), having an extra large extracellular loop, which was later identified as C3aR by Crass et al. (1996) and Ames et al. (1996). The C3aR cDNA clone was isolated from human neutrophils and from differentiated leukocytic cell lines HL60 and U937. The cDNA clones had an open reading frame of 1446 base pairs coding for 482 amino acid residues with a calculated Mr of 54 kDa. Human C3aR has three potential N-glycosylation sites and has been estimated by western blot analysis to be 60 kDa when expressed on a human astrocyte cell

line. These results suggest that human C3a may contain N-linked oligosaccharides contributing up to 10% of the total weight. Estimates of guinea pig platelet C3aR based on crosslinking experiments were 105±115 kDa, suggesting a larger contribution from oligosaccharides attached at the five potential Nglycosylation sites in guinea pig C3aR. The genes encoding C3aR have been mapped to band position p13.2-3 of chromosome 12 in humans and 6F1 in mouse (Hollmann et al., 1998; Paral et al., 1998).

Accession numbers Human: Q16581, Z73157, U62027, NM004054 Mouse: O09047, U77461, U77460, AF053757, U97537 Rat: U86379 Guinea pig: U86378, AJ006402

Sequence See Figure 3.

Figure 3 Nucleotide sequence for human C3aR. Sequence: 1449 bp mRNA BASE COUNT 331a 380c 310g ORIGIN: Homo sapiens 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441

ATGGCGTCTT CCCCCAGTAA AATGGGCTGG TTCCTCCACC CACTTGGCTC ATCATTGTCC TGTCTTGTGG TCTATCTGTG CGGGAAATCT TCATTAGATT GTTCAGCCGC CATCCTTGGA TCACTCCCTA CCTGCTGATG AGCCCACTGG TCTAGCAATT GGCCAATTCA CTAGTGGTGG TTCCGAATGC GTGGTGGTGG TTGCTTACTG ATTGCTCTAG GATTTTAGGA GAGCTCACAC ACTGTGTGA

TCTCTGCTGA TTCTCTCCAT TGCTGTGGGT TCACCTTGGC TCCAGGGACA TCAACATGTT TATTCAAGCC GATGTATCTG TCACTACAGA ATCCAGACTT CTGGAGAAAT CAGTCCCCAC GGGGTTCTGC TGGTCTCACC ATAACTCTGA CCTTCTACGA CAGATGACGA GTTTCCTGCT AAAGGGGCCG CTGTCTTTCT ACCCAGAAAC CATCTGCCAA AGAAAGCAAG GTTCCACCCA

428t

GACCAATTCA GGTCATTCTC GGCTGGCCTG GGACCTCCTC GTGGCCCTAC TGCCAGTGTC AATCTGGTGT GGTGGTGGCT CAACCATAAT TTATGGAGAT GAATGATAGG TGTCTTCCAA TAGGTTAACA TAAAATCCCC TGCTTTTCTC GTCTGAGCTA TCAAGTGCCA GCCCTCTGTT CTTCGCCAAG TGTCTGCTGG TCCCTTGGGG TAGTTGCTTT GCAGTCCATT CTGTCCCTCA

ACTGACCTAC AGCCTTACTT AAGATGCAGC TGCTGCCTCT GGCAGGTTCC TTCCTGCTTA CAGAATCATC TTTGTGATGT AGATGTGGCT CCACTAGAAA TTAGATCCTT CCTCAAACAT AGTCAAAATC AGTGGGTTTC TCTACTCATT CCACAAGGTT ACACCCCTCG ATCATGATAG TCTCAGAGCA ACTCCATACC AAAACTCTGA AATCCCTTCC CAGGGAATTC AACAATGTCA

TCTCACAGCC TTTTACTGGG GGACAGTGAA CCTTGCCCTT TATGCAAGCT CTGCCATTAG GCAATGTAGG GCATTCCTGT ACAAATTTGG ACAGGTCTCT CCTCTTTCCA TTCAAAGACC TGTATTCTAA CTATTGAAGA TAAAGCTGTT TCCAGGATTA TGGCAATAAC CCTGTTACAG AAACCTTTCG ACATTTTTGG TGTCCTGGGA TTTATGCCCT TGGAGGCAGC TTTCAGAAAG

ATGGAATGAG ATTGCCAGGC CACAATTTGG CTCGCTGGCT CATCCCCTCC CCTGGATCGC GATGGCCTGC GTTCGTGTAC TCTCTCCAGC TGAAAACATT AACAAATGAT TTCTGCAGAT TGTATTTAAA TCACGAAACC CCCTAGCGCT TTACAATTTA GATCACTAGG CTTCATTGTC AGTGGCCGTG AGTCCTGTCA TCATGTATGC CTTGGGGAAA CTTCAGTGAG AAATAGTACA

2178 Julia A. Ember and Tony E. Hugli

PROTEIN

Sequence See Figure 4.

Description of protein C3aR is an integral membrane protein, consisting of 482 amino acids, forming a single polypeptide chain. It belongs to the rhodopsin-like receptor superfamily, characterized by seven hydrophobic transmembrane regions connected with three extra- and three intracellular loops. The orientation of C3aR was determined by immunohistochemical methods, and it was determined that the N-terminal end is located extracellularly, while the C-terminal end is intracellular.

Relevant homologies and species differences A 50±60% homology exists between the protein sequences of C3aR from various species. A consensus sequence of XKSXXKX occurs in the intracellular loop 2 domain between transmembrane helices 5 and 6 of C3aR from all species. This motif represents a phosphorylation site for kinase C (Fukuoka et al., 1998a). A total of six Thr/Ser residues have been conserved at the C-terminal end of C3aR, representing potential phosphorylation sites that can become modified as a result of C3a binding. The large extracellular loop between transmembrane helices 4 and 5 is a region with the lowest level of sequence identity. An N-glycosylation site at Asn9 in human C3aR has been conserved, but other N-glycosylation sites vary between C3aR from different species (Figure 1).

Affinity for ligand(s) The C3a/C5a hybrid mutagenesis studies were aimed at investigating the C3aR-binding sites (Bautsch et al.,

1992). The conclusion from these studies confirmed that a secondary or noneffector-binding site is required for optimal affinity of C3a binding to its receptor. Studies to determine the architecture and to localize binding sites on the C3a receptor has been done. As described earlier, the recently cloned human C3aR contains an unusually large extracellular loop (between transmembrane helices 4 and 5), a feature which is unique among G protein-coupled receptors (Ames et al., 1996; Crass et al., 1996; Roglic et al., 1996). Since it has been postulated that the large extracellular loop might contain some or all the structural determinants for C3a binding, this region has been a focus of recent studies. Generation of loop deletion and point mutations of the receptor (Chao et al., 1999; Sun et al., 1999) indicates a multisite cooperative C3a/C3aR-binding interaction (Figure 2).

Cell types and tissues expressing the receptor The C3a receptor has been demonstrated on guinea pig platelets (Fukuoka and Hugli, 1988), human alveolar macrophages, neutrophils, basophils (Glovsky et al., 1979), and eosinophils (Daffern et al., 1995) by either functional assays or using chemical crosslinking techniques. Flow cytometry was used to identify C3aR on peripheral monocytes and umbilical vein endothelial cells, as well as the Raji cell line and differentiated HL-60 and U937 monocytic cell lines (Roglic et al., 1996). Northern blot analysis showed high levels of mRNA for C3aR in human lung and spleen with lower levels in heart, placenta, kidney, thymus, testis, ovaries, small intestine, colon, and several regions of the brain (Ames et al., 1996). This wide distribution of message for the C3aR is surprising, since C3a was believed to have a more limited functional and physiologic role than C5a. Observations of C3a as a chemotactic factor for eosinophils and persumably basophils, but not neutrophils, suggests a specialized role for C3a in inflammatory responses involving these cell types (Daffern et al., 1995). There is much new biology to

Figure 4 Amino acid sequence for human C3aR. Sequence c3aR protein: human, Homo sapiens 1-MASFSAETNS HLALQGQWPY REIFTTDNHN SLPRGSARLT GQFTDDDQVP LLTDPETPLG TV-482

TDLLSQPWNE GRFLCKLIPS RCGYKFGLSS SQNLYSNVFK TPLVAITITR KTLMSWDHVC

PPVILSMVIL IIVLNMFASV SLDYPDFYGD PADVVSPKIP LVVGFLLPSV IALASANSCF

SLTFLLGLPG FLLTAISLDR PLENRSLENI SGFPIEDHET IMIACYSFIV NPFLYALLGK

NGLVLWVAGL CLVVFKPIWC VQPPGEMNDR SPLDNSDAFL FRMQRGRFAK DFRKKARQSI

KMQRTVNTIW QNHRNVGMAC LDPSSFQTND STHLKLFPSA SQSKTFRVAV QGILEAAFSE

FLHLTLADLL SICGCIWVVA HPWTVPTVFQ SSNSFYESEL VVVAVFLVCW ELTRSTHCPS

CCLSLPFSLA FVMCIPVFVY PQTFQRPSAD PQGFQDYYNL TPYHIFGVLS NNVISERNST

C3a Receptor 2179 be learned from the recent discoveries that C3a receptors are as widely distributed as are receptors for cytokines and chemokines. Recent reports suggest that C3a may induce differing responses from mixed PBMCs exposed to LPS, depending on the adhesive state of these cells. It was reported that C3a suppressed cytokine production when these cells were cultured in polypropylene tubes, which prevents cell±matrix adhesion. However, C3a enhanced LPS-induced cytokine production of the cultures when they were carried out in standard polystyrene tissue culture plates (Takabayashi et al., 1996). Recent studies in our laboratory, using purified monocytes, have demonstrated a clear suppressive effect of C3a on LPS-induced production of inflammatory cytokines (Fischer et al., 1999). In contrast, C3a enhances LPS-induced production of the immunosuppressive cytokine IL-10. One interesting observation common to all three studies is that C3a(des Arg) retains biological activity in each of these assays. Evidence that the actions of C3a(des Arg) occur through specific receptor interactions is still not convincing. Most functional responses of myeloid cells, including essentially all eosinophil responses, and the immunosuppressive effects of C3a on humoral immune responses in mixed PBMC populations, are not elicited by C3a(des Arg) at submicromolar concentrations. On the other hand, in the systems just mentioned above, as well as in mast cell activation, the potency of C3a(des Arg) ranges from being equipotent with C3a, to having approximately 10% of the activity of the parent molecule. The residual activity of C3a(des Arg), although physiologically relevant, has been attributed to nonspecific poly cation effects, as originally defined by Mousli et al. (1992). Biochemically, C3a and C3a(des Arg) are highly charged cationic proteins at physiologic pH. It was found that in rat peritoneal mast cells, C3a and C3a(des Arg) were nearly equipotent in inducing histamine release (Johnson et al., 1975). It was further found that a number of otherwise unrelated cationic molecules also induced histamine release, and that their potency correlated with their charge-to-mass ratio. Based primarily on these data, it was concluded that rat mast cells do not bear specific C3a receptors, and that the activating effects of C3a were C3aRindependent and related to the cationic nature of the molecule. The potential effects of C3a on nonmyeloid cells has not been explored in great detail, partly due to a lack of information concerning the receptor. Since C3aR has recently been identified and cloned, and antibodies generated to the molecule, it is only a matter of time until the biologic manifestations

associated with this receptor have been identified. Although northern blot analysis has suggested a wide distribution of C3aR, little evidence for direct C3a stimulation on nonmyeloid cells has been reported. Again, the presence of mast cells and myeloid cells in these tissues prevents conclusive assignment of receptor expression to other particular cell types. It is anticipated that the molecular tools now available will accelerate characterizations of the cellular distribution of C3aR. Many tissue effects of C3a are virtually identical to those of C5a and this supports the notion that macrophages and mast cells are prominent players at the tissue level. Intradermal injection of C3a leads to a classical wheal-and-flare response, but without a significant leukocyte infiltration. These results suggest that the chemotactic properties of C3a for eosinophils may be insufficient at these levels to induce eosinophil recruitment (Fernandez et al., 1978). Alternatively, C3a may be converted in vivo to the chemotactically inactive des Arg form before eosinophil recruitment can occur. The lack of neutrophil or monocyte recruitment suggests that local mast cell activation per se is insufficient to generate chemotactic signals for either of these circulating cell types.

Regulation of receptor expression The C3aR is downregulated upon ligand binding and is cross-desensitized by C5a (Settmacher et al., 1999). C3aR upregulation has been reported on astrocytes during inflammation (Nataf et al., 1999).

DOWNSTREAM GENE ACTIVATION

Genes induced IL6 mRNA expression was increased in human astrocytes by C3a; however, the protein IL-6 was not generated.

THERAPEUTIC UTILITY

Effects of inhibitors (antibodies) to receptors No neutralizing antibody yet exists to C3aR. Several anti-C3aR peptide antibodies have been generated but no neutralizing antibodies have been reported.

2180 Julia A. Ember and Tony E. Hugli

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Stimler, N. P., Bloor, C. M., and Hugli, T. E. (1983). C3a-induced contraction of guinea pig lung parenchyma: role of cyclooxygenase metabolites. Immunopharmacology 5, 251±257. Sun, J., Ember, J. A., Chao, T.-H., Fukuoka, Y., Ye, R. D., and Hugli, T. E. (1999). Identification of ligand effector binding sites in transmembrane regions of the human G protein-coupled C3a receptor. Protein Sci. 8, 1±8. Takabayashi, T., Vannier, E., Clark, B. D., Margolis, N. H., Dinarello, C. A., Burke, J. F., and Gelfand, J. A. (1996). A new biologic role for C3a and C3a desArg. J. Immunol. 156, 3455±3460. Takabayashi, T., Vannier, E., Burke, J. F., Tompkins, R. G., Gelfand, J. A., and Clark, B. D. (1998). Both C3a and C3a(desArg) regulate interleukin-6 synthesis in human peripheral blood mononuclear cells. J. Infect. Dis. 177, 1622±1628. Takafuji, S., Tadokoro, K., Ito, K., and Dahinden, C. A. (1994). Degranulation from human eosinophils stimulated with C3a and C5a. Int. Arch. Allergy Immunol. 104, 27±29. Tornetta, M. A., Foley, J. J., Sarau, H. M., and Ames, R. S. (1997). The mouse anaphylatoxin C3a receptor: molecular cloning, genomic organization and functional expression. J. Immunol. 158, 5277±5282. Wilken, H.-C., GoÈtze, O., Werfel, T., and Zwirner, J. (1999). C3a (desArg) does not bind to and signal through the human C3a receptor. Immunol. Lett. 67, 141±145. Zwirner, J., Gotze, O., Moser, A., Sieber, A., Begemann, G., Kapp, A., Elsner, J., and Werfel, T. (1997). Blood- and skinderived monocytes/macrophages respond to C3a but not to C3a(desArg) with a transient release of calcium via a pertussis toxin-sensitive signal transduction pathway. Eur. J. Immunol. 27, 2317±2322.