C5a Receptor

C5a Receptor Tony E. Hugli1 and Julia A. Ember2,* 1

Department of Immunology, M/S IMM18 The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037-1092, USA 2 Pharmingen, 10975 Torreyana Road, La Jolla, CA 92121, USA * corresponding author tel: 858-784-8158, fax: 858-784-8307, e-mail: [email protected] DOI: 10.1006/rwcy.2000.23001.

SUMMARY Although the biological effects and functional properties of the potent chemotactic factor C5a have been known since the 1960s, the C5a receptor (C5aR) was not demonstrated on human leukocytes or characterized until the late 1970s. Only in 1991 was the gene structure and derived protein sequence for human C5aR first reported. The C5aR, also known as CD88, is a member of the rhodopsin superfamily of receptors with wide cellular and tissue distribution in both humans and animals. As the receptor for a major humoral chemotactic factor and potent immunomodulatory factor, it is a key component of inflammatory and immunoregulatory processes. The nature and variety of cellular functions associated with the engagement of C5aR by its natural ligand, and the physiologic consequences of the mediated cellular responses, place C5aR in the same category functionally as chemokine and cytokine receptors.

BACKGROUND Early functional and chemical crosslinking studies using purified C5a indicated that C5a receptors (CD88) exist on neutrophils (Chenoweth and Hugli, 1978), monocytes (macrophages) (Chenoweth et al., 1982, 1984), basophils (Schulman et al., 1988; MacGlashan and Hubbard, 1993), and eosinophils (Gerard et al., 1989), as well as platelets (Fukuoka et al., 1988; Kretzschmar et al., 1991) and mast cells (Johnson et al., 1975; Fukuoka and Hugli, 1990) from human and nonhuman species. C5aR is expressed on myeloid cell lines, such as U937 and HL-60, but only after these cells differentiate to a mature stage of development. Recently, evidence has been presented

that C5aR is expressed on liver parenchymal cells, lung vascular smooth muscle, lung and umbilical vascular endothelial cells, bronchial and alveolar epithelial cells, as well as astrocytes and microglial cells (Haviland et al., 1995; Lacy et al., 1995; Gasque et al., 1997). The distribution of C5aR is predominant in human heart, lung, spleen, spinal cord, and in many regions of the brain. The size of C5aR was estimated to be approximately 40±48 kDa based on a variety of chemical crosslinking techniques that chemically attached [125I]C5a to C5aR on neutrophils (Heideman and Hugli, 1984; Huey and Hugli, 1985; Rollins and Springer, 1985). The ligand, human C5a, is a glycoprotein of 11± 12 kDa, and is frequently mentioned under the collective name anaphylatoxin. Anaphylatoxins are small, bioactive fragments released from C3, C4, and C5 during complement (C) activation. The term anaphylatoxin was coined by Friedberger (1910). It is a descriptive label for an activity found in complementactivated serum that produced rapid anaphylactoidlike death when C-activated serum was injected into laboratory animals. Anaphylatoxin has remained the generic name for molecules now chemically identified as C3a, C4a, and C5a. As a group, the anaphylatoxins are humoral mediators mainly recognized for their proinflammatory and immunoregulatory functions that serve as bioactive sentinels in host defense.

Discovery Early characterization of C5aR (CD88) on human neutrophils clearly established three facts that later played a prominent role in the elucidation of the C5a±C5aR molecular interactions and in isolation of the receptor gene (Chenoweth and Hugli, 1978;

2162 Tony E. Hugli and Julia A. Ember Huey and Hugli, 1985). It was determined that (1) C5a (125I-labeled) binds to a receptor on human neutrophils with nanomolar affinity; (2) there are approximately 100,000 copies of the receptor per cell; and (3) certain degradation products of C5a (i.e. C5a desArg and C5a(1±69)) compete with the intact factor for binding C5aR. The observation that a C5a fragment devoid of the C-terminal effector site (i.e. C5a(1±69)) binds to the receptor without activating the cell led to the hypothesis that both a primary effector site and secondary binding sites exist on the C5a ligand and that each is important for optimal engagement of the ligand with the receptor (Chenoweth, 1978; Gerard et al., 1979; Chenoweth and Hugli, 1980). The C5a receptors are much more widely distributed than was previously believed in terms of both cells and tissues. It is now clear that the C5a receptors are present not just on myeloid inflammatory cells but on a variety of tissues as well. C5aR exists on both mast cells and macrophages in the skin, digestive, vascular, and pulmonary tissues, along with numerous other receptor-bearing tissue cells, suggesting that particular tissues and organs may be more highly responsive to C5a stimulation than others.

Figure 1 Proposed model illustrating the interaction between C5a and the C5a receptor. The design for this model was adapted from the C5a/C5aR model proposed by Siciliano et al. (1994). C5aR is a G protein-coupled transmembrane receptor of the rhodopsin family. The model for C5a interaction with C5aR indicates that the noneffector site (site 1) on C5a binds to the N-terminal region of the C5a receptor while the C-terminal effector site (site 2) of C5a penetrates the `pore' formed by the circular arrangement of the seven transmembrane helices.

Alternative names

Cytoplasm

Exterior

C5aR, CD88, anaphylatoxin receptor, complement receptor (Rother et al., 1992).

Structure Human C5aR is an integral membrane glycoprotein, consisting of 350 amino acids forming a single polypeptide chain. It belongs to the rhodopsin-like receptor superfamily, characterized by seven hydrophobic, transmembrane helical regions connected by three extra- and three intracellular loops. Orientation of C5aR in the membrane was determined by immunohistochemical techniques. It was determined that the N-terminal end of C5aR is exposed on the extracellular surface, while the C-terminal end extends into the cell from the intracellular surface of the membrane. A model of the C5aR molecule has been proposed (Gerard and Gerard, 1994b) (Figure 1).

Main activities and pathophysiological roles It was known by the late 1960s that anaphylatoxins possessed potent spasmogenic activity (i.e. the ability to contract smooth muscle tissues) and could both

enhance vascular permeability and recruit white blood cells when injected into the skin, either of animals (Dias da Silva and Lepow, 1967; Cochrane and MuÈller-Eberhard, 1968) or of humans (Lepow et al., 1970; Wuepper et al., 1972; Vallota and MuÈllerEberhard, 1973). Many other actions have since been attributed to C5a, including a host of cellular effects that imply important immunomodulatory functions (Morgan et al., 1993), as well as a number of tissuespecific effects (Ember et al., 1998). Cellular Activities Cellular responses mediated by the ligand-specific activation of C5aR reflect the prominent proinflammatory character of the C5a molecule. Perhaps the biologic property most closely identified with C5a± C5aR interactions is the potent chemotactic activity for granulocytes, particularly neutrophils and eosinophils. C5a induces chemotactic migration of neutrophils and eosinophils in vitro at an EC50 between 0.5 and 2.0 nM (Fernandez et al., 1978; Morita et al.,

C5a Receptor 2163 1989; Daffern et al., 1995). Potent activators of inflammation are released by C5a from all granulocytes that possess C5aR. Elastase, peroxidase, glucuronidase, and lactoferrin are released from neutrophils. C5a released peroxidase, major basic protein (MBP), eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP) from eosinophils (Henson, 1971; Goetzl and Austen, 1974; Takafuji et al., 1994) and arachidonate and vasoamines from basophils (Siraganian and Hook, 1976; Hartman and Glovsky, 1981; MacGlashan and Hubbard, 1993). C5a also activates the NADPHoxidase pathway in granulocytes, leading to an oxidative burst (Goetzl and Austen, 1974; Elsner et al., 1994). Cellular responses to C5a have been well characterized in vitro and described in detail in numerous articles and reviews (Chenoweth and Goodman, 1983; Hugli, 1984; Ember et al., 1998). Treatment of granulocytes with cytochalasin B, or an equivalent microfilament-disrupting agent, is required to elicit optimal responses to C5a in vitro, with the exception of chemotaxis. This same requirement is shared by other granulocyte-activating factors, including f-MLF (fMLP) and IL-8. Experimental evidence suggests that adhesive interactions with other cell types or with extracellular matrix replaces the effect of cytochalasin B during in vivo cellular responses to C5a (Becker et al., 1974; Henson et al., 1978; Laurent et al., 1991). Consistent with this hypothesis, C5a has been shown to act as a proadhesive stimulus for granulocytes. C5a±C5aR interaction in both neutrophils and eosinophils leads to an increased expression of 2 integrins and concurrent shedding of L-selectin (Kishimoto et al., 1989; Lundahl et al., 1993; Neeley et al.,1993). Tissue-Specific Activities Recent evidence shows that C5a/C5aR plays an important role in immune injury in the lung (Mulligan et al., 1996, 1997; Schmid et al., 1997) and in postischemic vascular and tissue injury (Ito and Del Balzo, 1994; Amsterdam et al., 1995; Ivey et al., 1995). This supports the contention that regulation of selected complement activation products such as C5a may be of therapeutic value. Discovery that both C3a and C5aR may exist on numerous cell types other than circulating white cells, such as hepatocytes, lung, epithelial cells (Haviland et al., 1995), endothelial cells (Foreman et al., 1994), and the astrocytes and microglial cells in brain tissue (Gasque et al., 1995b, 1997), implicates these anaphylatoxins in vascular diseases, pulmonary diseases, and degenerative neurologic diseases.

GENE Recent molecular studies resulted in cloning of the C5aR (Boulay et al., 1991; Gerard and Gerard, 1991). The gene structure for the human C5aR was reported by two separate groups in 1991. One group used a library obtained from dibutyryl-cAMP-induced human myeloid U937 cells (Gerard and Gerard, 1991) and the other group used a dibutyryl-cAMPinduced human myeloid HL-60 cell library (Boulay et al., 1991). Both groups obtained cDNA clones with open reading frames of 1050 base pairs coding for an identical protein of 350 amino acid residues with a calculated Mr of 39,320. A single glycosylation site was located at Asn5 of the first extracellular domain of C5aR. The presence of an N-linked oligosaccharide group presumably explains the difference in size between the nude protein of 39 kDa and the 40± 48 kDa estimate for C5aR expressed on human leukocytes. The size of the native C5aR was estimated using a variety of chemical crosslinking techniques to attach [125I]C5a to C5aR on human neutrophils (Heideman and Hugli, 1984; Huey and Hugli, 1985; Rollins and Springer, 1985). The effect of glycosylation of C5aR on C5a binding was explored by replacing Asn5 with an Ala residue (Pease and Barker, 1993). When both the Ala5-C5aR mutant molecule and wild-type C5aR were expressed on Chinese hamster ovary cells and compared, the dissociation constants were 20 nM and 13 nM, respectively. These results suggest that glycosylation of the C5aR has little influence on ligand binding and hence on C5a-induced cellular functions.

Accession numbers Human (Homo sapiens): X57250, X58674, M62505, M76672 Chimpanzee (Pan troglodytes): X97730 Orang-utan (Pongo pygmeus): X97732 Rhesus monkey (Macaca mulatta): X97731 Gorilla (Gorilla gorilla): X97733 Mouse (Mus musculus): S46665, L05630, S50577 Rat (Rattus norwegicus): X95990 Dog (Canis familiaris): X65860 Rabbit (Oryctolagus cuniculus): AF068680

Sequence Base count C5aR cDNA: 189 A, 348 C, 293 G, 262 T. See Figure 2.

2164 Tony E. Hugli and Julia A. Ember Figure 2 Nucleotide sequence for human C5a receptor. Sequence Base Count

189 a

348 c

293 g

262 t

C5aR

cDNA

Origin: Homo sapiens 1 61 121 181 241 301 361 421 481 541 601 661 721 761 841 901 961 1021 1081

ATGAACTCCT GACCTCAACA TTGGTCATCT GTGACGGCAT GCCGACTTCC CACTGGCCCT TACGCCAGCA CCCATCTGGT TGGGGTTTAG TACTTTCCAC GCCGTGGCCA TGTTACACTT CTCAAGGTGG ACGGGGATAA CTGGACTCCC GTGGTGGCCG AACGTGTTGA GACACTATGG TGTCCCTTCC

TCAATTATAC CCCCTGTGGA TTGCAGTCGT TCGAGGCCAA TCTCCTGCCT TTGGCGGGGC TCCTGCTCCT GCCAGAACTT CCCTGCTGCT CAAAGGTGTT TCGTCCGGCT TCATCCTGCT TGGTGGCAGT TGATGTCCTT TGTGTGTCTC GCCAGGGCTT CTGAAGAGTC CCCAGAAGAC TT

CACCCCTGAT TAAAACTTCT CTTCCTGGTG GCGGACCATC GGCGCTGCCC CGCCTGCAGC GGCCACCATC CCGAGGGGCC GACCATACCC GTGTGGCGTG GGTCCTGGGC CCGGACGTGG GGTGGCCAGT CCTGGAGCCA CTTTGCCTAC CCAGGGCCGA CGTGGTTAGG CCAGGCAGTG

Chromosome location and linkages The genes encoding C5aR, along with two structurally related formyl peptide receptors (FPRH1 and FPRH2), have been mapped to band position q13.2 in human chromosome 19 (Bao et al., 1992).

PROTEIN

Accession numbers

TATGGGCACT AACACGCTGC GGAGTGETGG AATGCCATCT ATCTTGTTCA ATCCTGCCCT AGCGCCGACC GGCTTGGCCT TCCTTCCTGT GAATACAGCC TTCCTGTGGC AGCCGCAGGG TTCRTTATCT TCGTCACCCA ATCAACTGCT CTGCGGAAAT GAGAGCAAGT TAGGCGACAC

ATGATGACAA GTGTTCCAGA GCAATGCCCT GGTTCCTCAA CGTCCATTGT CCCTCATCCT GCTTTCTGCT GGATCGCCTG ACCGGGTGGT ACGACAAACG CTCTACTCAC CCACGCGGTC TCTGGTTGCC CCTTCCTGCT GCATCAACCC CCCTCCCCAG CATTCACGCG GTCATGGGCC

GGATACCCTG CATCCTGGCC GGTGGTCTGG CTTGGCGGTA ACAGCATCAC GCTCAACATG GGTGTTTAAA TGCCGTGGCT GCGGGAGGAG GCGGGAGCGA GCTCACGATT CACCAAGACA CTACAAGGTG GCTGAATAAG CATCATCTAC CCTCCTCCGG CTTCACAGTG ACTGTGGCGA

Figure 3 Amino acid sequence for human C5a receptor. Sequence C5aR protein, human, Homo sapiens MNSFNYTTPDYGHYDDKDTLDLNTPVDKTSNTLRVPDILALVIFAVVFLV GVLGNALVVWVTAFEAKRTINAIWFLNLAVADFLSCLALPILFTSIVQHH HWPFGGAACSILPSLILLNMYASILLLATISADRFLLVFKPIWCQNFRGA GLAWIACAVAWGLALLLTIPSFLYRVVREEYFPPKVLCGVDYSHDKRRER AVAIVRLVLGFLWPLLTLTICYTFILLRTWSRRATRSTKTLKVVVAVVAS FFIFWLPYQVTGIMMSFLEPSSPTFLLLNKLDSLCVSFAYINCCINPIIY VVAGQGFQGRLRKSLPSLLRNVLTEESVVRESKSFTRSTVDTMAQKTQAV

Human (Homo sapiens): P21730 Chimpanzee (Pan troglodytes): P79240 Orang-utan (Pongo pygmeus): P79234 Rhesus monkey (Macaca mulatta): P79188 Gorilla (Gorilla gorilla): P79175 Mouse (Mus musculus): P30993 Rat (Rattus norwegicus): P97520 Dog (Canis familiaris): P30992

the rhodopsin superfamily of receptors, otherwise known as GTP-binding, protein-coupled receptors or seven transmembrane-spanning receptors. C5aR is an integral membrane protein.

Sequence

Relevant homologies and species differences

See Figure 3.

Description of protein The C5aR protein deduced from the cDNA clones identified the characteristic structure of a member of

Sequence comparisons between C5aR and other members of the rhodopsin superfamily indicated that a relatively close homology exists only with human neurokinin A (substance K) and formyl peptide receptors (Gerard and Gerard, 1991). Even receptors having the highest degree of sequence

C5a Receptor 2165 identity exhibit only 25% (substance K) and 35% (formyl peptide) structural identity to C5aR. More recently, the cDNA sequences for mouse C5aR (Gerard et al., 1992), guinea pig (Fukuoka et al., 1998), rat (Akatsu et al., 1997), canine, and a partial

sequence for bovine C5aR have been determined (Perret et al., 1992) (Figure 4). It is interesting to note that the extracellular N-terminal region, which is believed to be a ligand-binding site, is poorly conserved between species while the transmembrane

Figure 4 The complete protein sequences for the C5a receptor from human (Hu), dog (Dg), guinea pig (Gp), rat (Rt), and mouse (Mo). The alignments were optimized to obtain 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 an asterisk. The C5a receptors from various species are similar in size and each molecule has a potential glycosylation site near the N-terminus.

2166 Tony E. Hugli and Julia A. Ember helical regions and the intracellular C-terminal region, which binds the G proteins, are highly conserved.

spinal cord, and in various regions of the brain (Haviland et al., 1995; Nataf et al., 1999).

Affinity for ligand(s)

Regulation of receptor expression

Characterization of the binding affinity and numbers of receptors on neutrophils, estimated to average 80,000 copies with an affinity of approximately 2 nM (Huey and Hugli, 1985; Shapira et al., 1995), provided critical information that indicated differentiated leukocytic cell lines would be appropriate sources from which to isolate and clone the C5aR gene. The cloned C5aR was expressed in COS-7 cells and high-affinity C5a binding was demonstrated. The Boulay group (Boulay et al., 1991) concluded that there were both high-affinity (Kd=1.7 nM) and lowaffinity (Kd=20±25 nM) C5aRs expressed on these COS cells, while the Gerard group (Gerard and Gerard, 1991) described only the high-affinity receptors (Kd=1.4 nM) and demonstrated G protein-dependent phosphorylation of phosphatidylinositol in response to C5a.

Challenge of C5aR-bearing cells by C5a causes a rapid phosphorylation of serine residues in the Cterminal region of C5aR. Agonist-binding to C5aR causes a rapid internalization of the complex into endosomes that cluster near the nucleus within 10 minutes. Under continuous exposure to C5a, and in the absence of protein synthesis, C5aR is maintained in a highly phosphorylated state but is not degraded. Confocal microscopy and ligand-binding studies indicated that internalized receptors were recycled to the plasma membrane. During this process receptors are dephosphorylated. Therefore phosphorylation plays a key role in the intracellular trafficking of the C5aR. Phosphorylated C5aRs may be recognized by adapter proteins that interact with the endocytic machinery. Truncation of the intracellular C-terminal end of C5aR by deletion of residues 314±350 disrupted expression, while deletion of only the last 26 residues (i.e. 326±350) did not affect expression or ligandbinding of the C5aR (Klos et al., 1994). IL-3 has been shown to regulate C5aR expression in neurons and in astrocyte-targeted IL-3 transgenic mice (Paradisis et al., 1998).

Cell types and tissues expressing the receptor Early functional and chemical crosslinking studies using purified factors indicated that C5aR (CD88) exists on neutrophils (Chenoweth and Hugli, 1978), monocytes (macrophages; Chenoweth et al., 1982, 1984), basophils (Schulman et al., 1988; MacGlashan and Hubbard, 1993), and eosinophils (Gerard et al., 1989), as well as platelets (Fukuoka et al., 1988; Kretzschmar et al., 1991) and mast cells (Johnson et al., 1975; Fukuoka and Hugli, 1990). C5aR is expressed on differentiated myeloid cells, such as U937 and HL-60. C5aR is expressed on liver parenchymal cells, lung vascular smooth muscle, lung and umbilical vascular endothelial cells, bronchial and alveolar epithelial cells, HepG2 cells, a hepatoma cell line (Buchner et al., 1995; McCoy et al., 1995), mesangial cells (Wilmer et al., 1998) as well as astrocytes and microglial cells (Haviland et al., 1995; Lacy et al., 1995; Gasque et al., 1997), on cultured human fetal astrocytes and astrocyte cell lines (Gasque et al., 1995a; Lacy et al., 1995). C5a induces smooth muscle contraction in virtually all tissue types tested, including ileal, uterine, bronchial, and vascular smooth muscle (Hugli, 1981; Hugli et al., 1987). Tissue distribution of C5aR was most predominant in human heart, lung, spleen,

Release of soluble receptors The C5aR is not released as a soluble receptor and does not circulate.

SIGNAL TRANSDUCTION

C5a/C5aR-Binding Sites Cloning of the C5a receptor (Boulay et al., 1991; Gerard and Gerard, 1991) provided new opportunities for elucidating the requirements for ligand± receptor interactions between C5a and its receptor. Antibodies were generated to C5aR peptides that mimic portions of the N-terminal extracellular region of the molecule. These antibodies are not only excellent markers of cells or tissues expressing the receptor (Buchner et al., 1995; Gasque et al., 1995b, 1997; Haviland et al., 1995), but they block binding and cellular activation by intact C5a (Morgan et al., 1993; Oppermann et al., 1993). Truncation of

C5a Receptor 2167 N-terminal residues 1±22 in C5aR (DeMartino et al., 1994; Siciliano et al., 1994) abrogated binding of intact C5a, but had no effect on binding C-terminal peptide analogs of C5a (DeMartino et al., 1994; Siciliano et al., 1994). Point mutations converting five aspartic acid residues in the N-terminal region of C5aR to alanines (i.e. Asp10, 15, 16, 21, 27 ! Ala) resulted in significant loss in binding affinity for C5a. These data indicated a critical role for the identified aspartic acid residues in C5a±C5aR interactions (DeMartino et al., 1994; Mery and Boulay, 1994). Studies focused on locating the C5a effector-binding site on C5aR have also used point mutation techniques. Replacement of Glu199 and Arg206 in C5aR by alanines produced major depression in C5a binding, suggesting that these residues contribute to the effector-binding site in C5aR. It has been concluded that specific residues in the second extracellular loop and residues on the fifth intramembrane helix help form the primary effector binding site on C5aR. Based on these mutational results, a model was designed proposing a noneffector interaction site involving the aspartic acid side-chains in the Nterminal portion of C5aR and the cationic side-chain of Arg40 (and possibly Arg37 and Lys12) in human C5a. An effector interaction site occurs between anionic residues in the second extracellular loop and on the fifth intramembrane helix of C5aR, and basic residues Arg62, His66, Lys67, and Arg74 of the human C5a molecule. Based on this model, contact between C5a and the N-terminal portion of C5aR defines the noneffector site that promotes a cooperative interaction with the effector-binding site, resulting in cellular activation (Siciliano et al., 1994).

Associated or intrinsic kinases There are several neutrophil signal transduction pathways regulated by Gi-coupled C5aR: one involves the activation of phospholipase C (PLC). The ligand-bound receptor interacts with pertussis toxin (PTX)-sensitive G proteins, such as the Gi proteins, and releases the subunits, which then stimulate PLC and phosphatidylinositol 3-kinase (PI-3 kinase) activities, followed by activation of phospholipase A2 (PLA2) and phospholipase D (Jiang et al., 1996). Postreceptor activation of the PLC/PKC pathway modulates intracellular calcium fluxes which are presumed to be important for neutrophil degranulation. Another pathway involves activation of PI-3 kinase and the generation of phosphatidylinositol triphosphate (PIP3) (Chang et al., 1990; Kammerer et al., 1990; Wingrove et al., 1992), which may be critical for cytoskeletal reorganization and the chemotactic

response. There are reports showing PTX-insensitive G16 subunits coupled to C5aR; G16 is known to activate PLC. However, the PTX-sensitive pathways appear to be predominant in mature leukocytes, since neutrophil responses to C5a were largely PTXsensitive (Buhl et al., 1994, 1995).

Cytoplasmic signaling cascades A number of biologic responses are initiated in leukocytes when C5a binds to C5aR, a seven membrane-spanning receptor coupled to regulatory heterodimeric guaninine nucleotide-binding proteins (Gi proteins; Gerard and Gerard, 1991, 1994a; Rollins et al., 1991). C5aR and other seven transmembrane receptors coupled to Gi are capable of activating the Ras/Raf/MAP kinase pathway (Buhl et al., 1994; Gerard and Gerard, 1994a). The subunits of Gi activate the mitogen-activated protein kinase (MAP) pathway in a Ras-dependent manner. Raf-1 binds to Ras-GTP, which activates Raf-1 kinase activity. Activated Raf-1 phosphorylates and activates mitogen-activated protein kinase/Erk kinase (MEK-1), which in turn phosphorylates and activates MAP kinase. The MAP kinase cascade may contribute to the functional assembly of the NADPH oxidase responsible for C5aR-mediated oxygen radical generation in neutrophils. Despite the persistence of the activating ligand C5a, the cellular responses mediated through C5aR are transient and cells rapidly become refractory to further stimulation, a phenomenon termed homologous desensitization. Receptor phosphorylation appears to be the key mechanism by which many G proteincoupled receptors are regulated. It has been shown that C5aR is phosphorylated exclusively at serine residues localized at the carboxyl end, by a member of the G protein-coupled receptor kinase family (Giannini et al., 1995). Despite the fact that a putative PKC consensus motif is present in the third cytoplasmic loop of C5aR, C5a-dependent phosphorylation is mainly resistant to PKC inhibitors. Therefore, PKC is not a major enzyme involved in agonist-dependent phosphorylation of C5aR.

DOWNSTREAM GENE ACTIVATION

Transcription factors activated NFB based on PTX-sensitivity. Others: undetermined.

2168 Tony E. Hugli and Julia A. Ember

Genes induced C5aR engagement by C5a induces IL-1, IL-6, IL-8, and TNF from human monocytes (Okusawa et al., 1987, 1988; Scholz et al., 1990; Ember et al., 1994), IL-6 mRNA in human astrocytes (Sayah et al., 1999), -integrin in human neutrophils and eosinophils (Jagels et al., 1999), and L-selectin shedding (Jagels et al., 2000).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors Although the proinflammatory effects of anaphylatoxins are indisputably beneficial in the context of localized infections or injuries, there are a number of noninfectious diseases and syndromes in which anaphylatoxins appear to play a deleterious role. Perhaps the most direct link between complement activation and a pathologic response results from extracorporeal circulation of the blood, either during hemodialysis or in coronary bypass surgery (Craddock et al., 1977; Kirklin et al., 1983; Howard et al., 1988). This postpump syndrome is characterized by mild respiratory distress, pulmonary hypertension and occasional vascular leakage. The etiology of these physiologic changes appears to be identical to those responses observed in experimental animal studies. The responses to intravenous administration of C3a or C5a (i.e. increased capillary permeability and edema, bronchoconstriction, pulmonary vasoconstriction, leukocyte aggregation in the lung vasculature, and possibly peripheral vasodilation), mimic this syndrome in humans. This syndrome appears to be an entirely complement-driven process, subsequent to complement activation through contact with nonbiocompatible materials composing the blood contact surfaces of dialysis and perfusion apparatus. Although the short-term consequences of these effects appear to result in minimal morbidity, there is concern that repeated intravascular complement activation, as occurs in chronic dialysis patients, may lead to longterm pathology of the lung (Craddock et al., 1977). Furthermore, in the setting of cardiopulmonary bypass, the leukocyte aggregation in the lung and impaired pulmonary blood flow may detrimentally

affect perfusion of the heart and other organs following surgery, and may contribute to an ARDSlike syndrome which develops in a small percentage of bypass patients (Kirklin et al., 1983; Howard et al., 1988). The length of time a patient requires ventillatory support following surgery has been correlated with C3a levels in the blood following reperfusion (Moore et al., 1988). Acute respiratory distress syndrome (ARDS) and multiple system organ failure (MSOF) are two related syndromes which develop most frequently as a consequence of severe polytrauma or septicemia (Faist et al., 1983; Murray et al., 1988; Parsons et al., 1989). The progressions of ARDS and MSOF are similar and characterized in the early stages by increased vascular permeability, impaired organ perfusion and, in the case of ARDS, respiratory insufficiency. Later stages are characterized by a continuation of the early malfunctions, with progressive damage to endothelium, necrosis, leukocyte infiltration and tissue necrosis and remodeling (Shoemaker et al., 1980; Herndon and Traber, 1990). In ARDS the damage is localized primarily to the lung, whereas in MSOF damage is disseminated not only to the lung but also to the liver, kidneys, and digestive tract.

Phenotypes of receptor knockouts and receptor overexpression mice Targeted disruption of mouse C5aR expression resulted in no developmental or biological defects in myeloid cell lineages, as well as hepatocytes and epithelial cells. C5aR-deficient mice bred normally and displayed no gross defects when maintained under barrier conditions. On the other hand, deficient mice were unable to clear intrapulmonary-instilled Pseudomonas aeruginosa, despite a marked increase of neutrophil influx, and succumbed to pneumonia. C5aR-deficient mice challenged with sublethal inocula of Pseudomonas become superinfected with secondary bacterial strains. It is concluded that C5aR has a nonredundant function and is required for mucosal host defense in the lung (Hopken et al., 1996).

THERAPEUTIC UTILITY Recent evidence that C5a plays an important role in immune injury in the lung (Mulligan et al., 1996, 1997; Schmid et al., 1997) in postischemic vascular and tissue injury (Ito and Del Balzo, 1994; Amsterdam

C5a Receptor 2169 et al., 1995; Ivey et al., 1995) supports the contention that regulation of selected complement activation products may be of therapeutic value. The discovery that both C3a and C5a receptors may exist on numerous cell types other than circulating white cells, such as hepatocytes, lung epithelial cells (Haviland et al., 1995), endothelial cells (Foreman et al., 1994), or the astrocytes and microglial cells in brain tissue (Gasque et al., 1995b, 1997), has serious implications for anaphylatoxins playing a role in vascular diseases, pulmonary diseases, and degenerative neurologic diseases.

Effect of treatment with soluble receptor domain Soluble N-terminal C5aR peptides failed to act as antagonist of C5a in receptor-binding studies, possibly because of low affinity for the ligand.

Effects of inhibitors (antibodies) to receptors Antibodies generated against peptides based on the extracellular loop sequence of C5aR were used to confirm receptor expression and to investigate ligand binding to the C5aR. Antibodies generated to peptides that mimic portions of the N-terminal extracellular region of C5aR are not only excellent markers of cells and tissues expressing the receptor (Buchner et al., 1995; Gasque et al., 1995b, 1997; Haviland et al., 1995), but also block C5a binding and cellular activation by the intact ligand (Morgan et al., 1993; Oppermann et al., 1993). Antibodies generated against the N-terminal region of C5aR effectively block ligand binding and could be used as therapeutic agents. No clinical uses of C5aR inhibitors have been recorded.

References Akatsu, H., Miwa, T., Sakurada, C., Fukuoka, Y., Ember, J. A., Yamamoto, T., Hugli, T. E., and Okada, H. (1997). cDNA cloning and characterization of rat C5a anaphylatoxin receptor. Microbiol. Immunol. 41, 575±580. Amsterdam, E. A., Stahl, G. L., Pan, H. L., Rendig, S. V., Fletcher, M. P., and Longhurst, J. C. (1995). Limitation of reperfusion injury by a monoclonal antibody to C5a during myocardial infarction in pigs. Am. J. Physiol. 268, H448±H457. Bao, L., Gerard, N. P., Eddy, R.L. Jr., Shows, T. B., and Gerard, C. (1992). Mapping of genes for the human C5aR (C5AR), human FMLP receptor (FPR), and two FMLP

receptor homologue orphan receptors (FPRH1, FPRH2) to chromosome 19. Genomics 13, 437±440. Becker, E. L., Showell, H. J., Henson, P. M., and Hsu, L. S. (1974). The ability of chemotactic factors to induce lysosomal enzyme release. 1. The characteristics of the release, the importance of surfaces, and the relation of enzyme release to chemotactic responsiveness. J. Immunol. 112, 2047±2054. Boulay, F., Mery, L., Tardif, M., Brouchon, L., and Vignais, P. (1991). Expression cloning of a receptor for C5a anaphylatoxin on differentiated HL-60 cells. Biochemistry 30, 2993±2999. Buchner, R. R., Hugli, T. E., Ember, J. A., and Morgan, E. L. (1995). Expression of functional receptors for human C5a anaphylatoxin (CD88) on the human hepatocellular carcinoma cell line HepG2. Stimulation of acute-phase protein-specific mRNA and protein synthesis by human C5a anaphylatoxin. J. Immunol. 155, 308±315. Buhl, A. M., Avdi, N., Worthen, G. S., and Johnson, G. L. (1994). Mapping of the C5a receptor signal transduction network in human neutrophils. Proc. Natl Acad. Sci. USA 91, 9190±9194. Buhl, A. M., Osawa, S., and Johnson, G. L. (1995). Mitogenactivated protein kinase activation requires two signal inputs from the human anaphylatoxin C5a receptor. J. Biol. Chem. 270, 19828±19832. Chang, Y. H., Jagels, M. A., and Abraham, E. (1990). Haemorrhage produces depressions in alloantigen-specific immune responses in the mouse through activation of suppressor T cells. Clin. Exp. Immunol. 80, 478±483. Chenoweth, D. E., and Goodman, M. G. (1983). The C5a receptor of neutrophils and macrophages. Agents Actions 12, (Suppl.)252±273. Chenoweth, D. E., and Hugli, T. E. (1978). Demonstration of specific C5a receptor on intact human polymorphonuclear leukocytes. Proc. Natl Acad. Sci. USA 75, 3943±3947. Chenoweth, D. E., and Hugli, T. E. (1980). Human C5a and C5a analogs as probes of the neutrophil C5a receptor. Mol. Immunol. 17, 151±161. Chenoweth, D. E., Goodman, M. G., and Weigle, W. O. (1982). Demonstration of a specific receptor for human C5a anaphylatoxins on murine macrophages. J. Exp. Med. 156, 67±78. Chenoweth, D. E., Soderberg, C. S., and von Wedel, R. (1984). Dibutyryl cAMP induced expression of C5a receptors on U937 cells. J. Leukoc. Biol. 1984, 36, 241(abstract). Cochrane, C. G., and MuÈller-Eberhard, H. J. (1968). The derivation of two distinct anaphylatoxin activities from the third and fifth components of human complement. J. Exp. Med. 127, 371±386. Craddock, P. R., Fehr, J., Brigham, K. L., Kronenberg, R. S., and Jacob, H. S. (1977). Complement and leukocyte-mediated pulmonary dysfunction in hemodialysis. N. Engl. J. Med. 296, 769±774. Daffern, P. J., Pfeifer, P. H., Ember, J. A., and Hugli, T. E. (1995). C3a is a chemotaxin for human eosinophils but not for neutrophils. I. C3a stimulation of neutrophils is secondary to eosinophil activation. J. Exp. Med. 181, 2119±2127. DeMartino, J. A., Van Riper, G., Siciliano, S. J., Molineaux, C. J., Konteastis, Z. D., Rosen, H., and Springer, M. S. (1994). The amino terminus of the human C5a receptor is required for high affinity C5a binding and for receptor activation by C5a but not C5a analogs. J. Biol. Chem. 269, 14446±14450. Dias da Silva, W., and Lepow, I. H. (1967). Complement as a mediator of inflammation. II. Biological properties of anaphylatoxin prepared with purified components of human complement. J. Exp. Med. 125, 921±946. Elsner, J., Opermann, M., Czech, W., Dobos, G., Schopf, E., Norgauer, J., and Kapp, A. (1994). C3a activates reactive

2170 Tony E. Hugli and Julia A. Ember oxygen radical species production and intracellular calcium transients in human eosinophils. Eur. J. Immunol. 24, 518±522. Ember, J. A., Sanderson, S. D., Hugli, T. E., and Morgan, E. L. (1994). Induction of interleukin-8 synthesis from monocytes by human C5a anaphylatoxin. Am. J. Pathol. 144, 393±403. Ember, J. A., Jagels, M. A., and Hugli, T. E. (1998). In ``The Human Complement System in Health and Disease'' (ed J. Volanakis and M. Frank), Characterization of complement anaphylatoxins and their biological responses pp. 241±284. Marcel Dekker, New York. Faist, E., Baue, A. E., Dittmer, H., and Heberer, G. (1983). Multiple organ failure in polytrauma patients. J. Trauma 23, 775±787. Fernandez, H. N., Henson, P. M., Otani, A., and Hugli, T. E. (1978). Chemotactic response to human C3a and C5a anaphylatoxins. I. Evaluation of C3a and C5a leukotaxis in vitro and under stimulated in vivo conditions. J. Immunol. 120, 109±115. Foreman, K. E., Vaporciyan, A. A., Bonish, B. K., Jones, M. L., Glovsky, M. M., Eddy, S. M., and Ward, P. A. (1994). C5ainduced expression of P-selectin in endothelial cells. J. Clin. Invest. 94, 1147±1155. Friedberger, E. (1910). Weitere Untersuchungen uber Eisissanaphylaxie: IV. Mitteilung. Immunitaetaforsch. Exp Ther. 4, 636±690. Fukuoka, Y., and Hugli, T. E. (1988). C5a receptors on guinea pig platelets. Fed. Proc. 2, A1007 (abstract). Fukuoka, Y., and Hugli, T. E. (1990). Anaphylatoxin binding and degradation by rat peritoneal mast cells. Mechanisms of degranulation and control. J. Immunol. 145, 1851±1858. Fukuoka, Y., Ember, J. A., Yasui, A., and Hugli, T. E. (1998). Cloning and characterization of the guinea pig C5a anaphylatoxin receptor: Interspecies diversity among the C5a receptors. Int. Immunol. 10, 275±283. Gasque, P., Fontaine, M., and Morgan, B. P. (1995a). Complement expression in human brain. Biosynthesis of terminal pathway components and regulators in human glial cells and cell lines. J. Immunol. 154, 4726±4733. Gasque, P., Chan, P., Fontaine, M., Ischenko, A., Lamacz, M., GoÈtze, O., and Morgan, B. P. (1995b). Identification and characterization of the complement C5a anaphylatoxin receptor on human astrocytes. Relevance to inflammation in the brain. J. Immunol. 155, 4882±4889. Gasque, P., Singhrao, S. K., Neal, J. W., GoÈtze, O., and Morgan, B. P. (1997). Expression of the receptor for C5a (CD88) is upregulated on reactive astrocytes, microglia and endothelial cells in inflammed human CNS. Am. J. Pathol. 150, 31±41. Gerard, N. P., and Gerard, C. (1991). The chemotactic receptor for human C5a anaphylatoxin. Nature 349, 614±617. Gerard, C., and Gerard, N. P. (1994a). The pro-inflammatory seven-transmembrane segment receptors of the leukocyte. Curr. Opin. Immunol. 6, 140±145. Gerard, C., and Gerard, N. P. (1994b). C5a anahylatoxin and its seven transmembrane-segment receptor. Annu. Rev. Immunol. 12, 775. Gerard, C., Chenoweth, D. E., and Hugli, T. E. (1979). Molecular aspects of the serum chemotactic factors. J. Reticuloendothel. Soc. 26, 711±718. Gerard, N. P., Hodges, M. K., Drazen, J. M., Weller, P. F., and Gerard, C. (1989). Characterization of a receptor for C5a anaphylatoxin on human eosinophils. J. Biol. Chem. 264, 1760± 1766. Gerard, C., Bao, L., Orozco, O., Pearson, M., Kunz, D., and Gerard, N. P. (1992). Structural diversity in the extracellular faces of peptidergic G-protein-coupled receptors. J. Immunol. 149, 2600±2606.

Giannini, E., Brouchon, L., and Boulay, F. (1995). Identification of the major phosphorylation sites in human C5a anaphylatoxin receptor in vivo. J. Biol. Chem. 270, 19166±19172. Goetzl, E. J., and Austen, K. F. (1974). Stimulation of human neutrophil leukocyte aerobic glucose metabolism by purified chemotactic factors. J. Clin. Invest. 53, 591±599. Hartman, C.T. Jr., and Glovsky, M. M. (1981). Complement activation requirements for histamine release from human leukocytes: influence of purified C3a hu and C5a hu on histamine release. Int. Arch. Allergy Appl. Immunol. 66, 274±281. Haviland, D. L., McCoy, R. L., Whitehead, W. T., Akama, H., Molmenti, E. P., Brown, A., Haviland, J. C., Parks, W. C., Perlmutter, D. H., and Wetsel, R. A. (1995). Cellular expression of the C5a anaphylatoxin receptor (C5aR): demonstration of C5aR on nonmyeloid cells of the liver and lung. J. Immunol. 154, 1861±1869. Heideman, M., and Hugli, T. E. (1984). Anaphylatoxin generation in multisystem organ failure. J. Trauma 24, 1038±1043. Henson, P. M. (1971). The immunologic release of constituents from neutrophil leukocytes. I. The role of antibody and complement on nonphagocytosable surfaces or phagocytosable particles. J. Immunol. 107, 1535±1546. Henson, P. M., Zanolari, B., Schwartzman, N. A., and Hong, S. R. (1978). Intracellular control of human neutrophil secretion. I. C5a-induced stimulus-specific desensitization and the effects of cytochalasin B. J. Immunol. 121, 851±855. Herndon, D. N., and Traber, D. L. (1990). In ``Pathophysiology and Basic Concepts of Therapy'' (ed E. A. Deitch), Pulmonary failure and acute respiratory distress syndrome. Multiple organ failure pp. 192±214. Thieme, New York. Hopken, U. E., Lu, B., Gerard, N. P., and Gerard, C. (1996). The C5a chemoattractant receptor mediates mucosal defence to infection. Nature 383, 86±89. Howard, R. J., Crain, C., Franzini, D. A., Hood, C. I., and Hugli, T. E. (1988). Effects of cardiopulmonary bypass on pulmonary leukostasis and complement activation. Arch. Surg. 123, 1496±1501. Huey, R., and Hugli, T. E. (1985). Characterization of a C5a receptor on human polymorphonuclear leukocytes (PMN). J. Immunol. 135, 2063±2068. Hugli, T. E. (1981). The structural basis for anaphylatoxin and chemotactic functions of C3a, C4a, and C5a. Crit. Rev. Immunol. 1, 321±366. Hugli, T. E. (1984). Structure and function of the anaphylatoxins. Springer Seminar Immunopathol. 7, 193±219. Hugli, T. E., Marceau, F., and Lundberg, C. (1987). Effects of complement fragments on pulmonary and vascular smooth muscle. Am. Rev. Respir. Dis. 135, S9±S13. Ito, B. R., and Del Balzo, U. (1994). Effect of platelet depletion and inhibition of platelet cyclooxygenase on C5a-mediated myocardial ischemia. Am. J. Physiol. 267, H1288±H1294. Ivey, C. L., Williams, F. M., Collins, P. D., Jose, P. J., and Williams, T. J. (1995). Neutrophil chemoattractaants generated in two phases during reperfusion of ischemic myocardium in the rabbit. Evidence for a role for C5a and interleukin-8 (comment). J. Clin. Invest. 95, 2720±2728. Jagels, M. A., Daffern, P. J., Zuraw, B. L., and Hugli, T. E. (1999). Mechanisms and regulation of PMN and eosinophil adherence to human airway epithelial cells. Am. J. Respir. Cell Mol. Biol. 21, 418±427. Jagels, M. A., Daffern, P. J., and Hugli, T. E. (2000). C3a and C5a enhance granulocyte adhesion to endothelial and epithelial cell monolayers: epithelial and endothelial priming is required for C3a-induced eosinophil adhesion. Immunopharmacology 46, 209±222.

C5a Receptor 2171 Jiang, H., Kuang, Y., Wu, Y., Smrcka, A., Simon, M. I., and Wu, D. (1996). Pertussis toxin-sensitive activation of phospholipase C by the C5a and fMet-Leu-Phe receptors. J. Biol. Chem. 271, 13430±13434. Johnson, A. R., Hugli, T. E., and MuÈller-Eberhard, H. J. (1975). Release of histamine from rat mast cells by the complement peptides C3a and C5a. Immunology 28, 1067. Kammerer, R. C., Merdink, J. L., Jagels, M. A., Catlin, D. H., and Hui, K. K. (1990). Testing for fluoxymesterone (Halotestin) administration to man: identification of urinary metabolites by gas chromatography-mass spectrometry. J. Steroid Biochem. 36, 659±666. Kirklin, J. K., Westaby, S., Blackstone, E. H., Kirklin, J. W., Chenoweth, D. E., and Pacifico, A. D. (1983). Complement and the damaging effects of cardiopulmonary by pass. J. Cardiovasc. Surg. 86, 845±857. Kishimoto, T. K., Jutila, M. A., Berg, E. L., and Butcher, E. C. (1989). Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors. Science 245, 1238± 1241. Klos, A., Matje, C., Rheinheimer, C., Bautsch, W., KoÈhl, J., Martin, U., and Burg, M. (1994). Amino acids 327-350 of the human C5a-receptor are not essential for [125I]C5a binding in COS cells and signal transduction in Xenopus oocytes. FEBS Lett. 344, 79±82. Kretzschmar, T., Kahl, K., Rech, K., Bautsch, W., KoÈhl, J., and Bitter-Suermann, D. (1991). Characterization of the C5a receptor on guinea pig platelets. Immunobiology 183, 418±432. Lacy, M., Jones, J., Whittemore, S. R., Haviland, D. L., Wetsel, R. A., and Barnum, S. R. (1995). Expression of the receptors for the C5a anaphylatoxin, interleukin-8 and FMLP by human astrocytes and microglia. J. Neuroimmunol. 61, 71±78. Laurent, F., Benoliel, A. M., Capo, C., and Bongrand, P. (1991). Oxidative metabolism of polymorphonuclear leukocytes: modulation by adhesive stimuli. J. Leukoc. Biol. 49, 217±226. Lepow, I. H., Willms-Kretschmer, R. A., Patrick, R. A., and Rosen, F. S. (1970). Gross and ultrastructural observations of lesions produced by intradermal injection of human C3a in man. Am. J. Pathol. 61, 13±20. Lundahl, J., Hallden, G., and Hed, J. (1993). Differences in intracellular pool and receptor-dependent mobilization of the adhesion-promoting glycoprotein Mac-1 between eosinophils and neutrophils. J. Leukoc. Biol. 53, 336±341. MacGlashan, D.W. Jr., and Hubbard, W. C. (1993). IL-3 alters free arachidonic acid generation in C5a-stimulated human basophils. J. Immunol. 151, 6358±6369. McCoy, R., Haviland, D. L., Molmenti, E. P., Ziambaras, T., Wetsel, R. A., and Perlmutter, D. H. (1995). N-formylpeptide and complement C5a receptors are expressed in liver cells and mediate hepatic acute phase gene regulation. J. Exp. Med. 182, 207±217. Mery, L., and Boulay, F. (1994). The NH2-terminal region of C5aR but not that of FPR is critical for both protein transport and ligand binding. J. Biol. Chem. 269, 3457±3463. Moore, F.D. Jr., Warner, K. G., Assousa, S., Valeri, C. R., and Khuri, S. F. (1988). The effects of complement activation during cardiopulmonary bypass. Ann. Surg. 208, 95±103. Morgan, E. L., Ember, J. A., Sanderson, S. D., Scholz, W., Buchner, R., Ye, R. D., and Hugli, T. E. (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±388. Morita, E., SchroÈder, J.-M., and Christophers, E. (1989). Differential sensitivities of purified human eosinophils and

neutrophils to defined chemotaxins. Scand. J. Immunol. 29, 709±716. Mulligan, M. S., Schmid, E., Till, G. O., Friedl, H. P., Hugli, T. E., Johnson, K. J., and Ward, P. A. (1996). Requirement and role of C5a in acute lung inflammatory injury in rats. J. Clin. Invest. 98, 503±512. Mulligan, M. S., Schmid, E., Till, G. O., Hugli, T. E., Friedl, H. P., Roth, R. A., and Ward, P. A. (1997). C5a-dependent upregulation in vivo of lung vascular P-selectin. J. Immunol. 158, 1857± 1861. Murray, J. F., Matthay, M. A., Luce, J., and Flick, M. R. (1988). An expanded definition of the adult respiratory distress syndrome. Am. Rev. Respir. Dis. 138, 720±723. Nataf, S., Stahel, P. F., Davoust, N., and Barnum, S. R. (1999). Complement anaphylatoxin receptors on neurons: new tricks for old receptors? Trends Neurosci. 22, 397±402. Neeley, S. P., Hamann, K. J., White, S. R., Baranowski, S. L., Burch, R. A., and Leff, A. R. (1993). Selective regulation of expression of surface adhesion molecules Mac-1, L-selectin, and VLA-4 on human eosinophils and neutrophils. Am. J. Respir. Cell Mol. Biol. 8, 633±639. Okusawa, S., Dinarello, C. A., Yancy, K. B., Endres, S., Lawley, T. J., Frank, M. M., Burke, J. F., and Gelfand, J. A. (1987). C5a induction of human interleukin 1. Synergistic-effect with endotoxin or interferon- . J. Immunol. 139, 2635±2639. Okusawa, S., Yancey, K. B., Van Der Meer, J. W. M., Endres, S., Lonneman, G., Hefter, K., Frank, M. M., Burke, J. F., Dinarello, C. A., and Gelfand, J. A. (1988). C5a stimulates secretion of tumor necrosis factor from human mononuclear cells in vitro: comparison with secretion of interleukin 1 and interleukin 1. J. Exp. Med. 168, 443±448. Oppermann, M., Raedt, U., Hebell, T., Schmidt, B., Zimmermann, B., and GoÈtze, O. (1993). Probing the human receptor for C5a anaphylatoxin with site-directed antibodies: identification of a potential ligand binding site on the NH-2terminal domain. J. Immunol. 151, 3785±3794. Paradisis, P. M., Campbell, I. L., and Barnum, S. R. (1998). Elevated complement C5a receptor expression on neurons and glia in astrocyte-targeted interleukin-3 transgenic mice. GLIA 24, 338±345. Parsons, P. E., Worthen, G. S., Moore, E. E., Tate, R. M., and Henson, P. M. (1989). The association of circulating endotoxin with the development of the adult respiratory distress syndrome. Am. Rev. Respir. Dis. 140, 294±301. Pease, J. E., and Barker, M. D. (1993). N-linked glycosylation of the C5a receptor. Biochemistry 31, 719±726. Perret, J. J., Raspe, E., Vassart, G., and Parmentier, M. (1992). Cloning and functional expression of the canine anaphylatoxin C5a receptor. Biochemistry 288, 911±917. Rollins, T. E., and Springer, M. S. (1985). Identification of the polymorphonuclear leukocyte C5a receptor. J. Biol. Chem. 260, 7157±7160. Rollins, T. E., Siciliano, S., Kobayashi, S., Cianciarulo, D. N., Bonilla Argudo, V., Collier, K., and Springer, M. S. (1991). Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor-Gi complex. Proc. Natl Acad. Sci. USA 88, 971±975. Rother, K., Worner, I., Prior, B., and Hansch, G. M. (1992). Leukocyte mobilization from bone marrow by complement receptor 3 (CR3) interaction. FASEB J. 6, A1893 (abstract). Sayah, S., Ischenko, A. M., Zhakhov, A., Bonnard, A. S., and Fontaine, M. (1999). Expression of cytokines by human astrocytomas following stimulation by C3a and C5a anaphylatoxins: specific increase in interleukin-6 mRNA expression. J. Neurochem. 72, 2426±2436.

2172 Tony E. Hugli and Julia A. Ember Schmid, E., Warner, R. L., Crouch, L. D., Friedl, H. P., Till, G. O., Hugli, T. E., and Ward, P. A. (1997). Neutrophil chemotactic activity and C5a following systemic activation of complement in rats. Inflammation 21, 325±333. Scholz, W., McClurg, M. R., Cardenas, G. J., Smith, M., Noonan, D. J., Hugli, T. E., and Morgan, E. L. (1990). C5amediated release of interleukin 6 by human monocytes. Clin. Immunol. Immunopathol. 57, 297±307. Schulman, E. S., Post, T. J., Henson, P. M., and Giclas, P. C. (1988). Differential effects of the complement peptides, C5a and C5a desArg on human basophils and lung mast cell histamine release. J. Clin. Invest. 81, 918±923. Shapira, L., Champagne, C., Gordon, B., Amar, S., and Van Dyke, T. E. (1995). Lipopolysaccharide priming of superoxide release by human neutrophils. Inflammation 19, 289±295. Shoemaker, W. C., Appel, P. L., Czer, S. C., Bland, R., Schwartz, S., and Hopkins, J. A. (1980). Pathogenesis of respiratory failure (ARDS) after hemorrhage and trauma. Crit. Care Med. 8, 504±512. Siciliano, S. J., Rollins, T. E., DeMartino, J., Konteatis, Z., Malkowitz, L., Van Riper, G., Bondy, S., Rosen, H., and Springer, M. S. (1994). Two-site binding of C5a by its receptor: an alternative binding paradigm for G protein-coupled receptors. Proc. Natl Acad. Sci. USA 91, 1214±1218. Siraganian, R. P., and Hook, W. A. (1976). Complement induced histamine release from human basophils. II. Mechanism of the histamine reaction. J. Immunol. 116, 639±646. 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. Vallota, E. H., and MuÈller-Eberhard, H. J. (1973). Formation of C3a and C5a anaphylatoxins in whole human serum after

inhibition of the anaphylatoxin inactivator. J. Exp. Med. 137, 1109±1123. Wilmer, W. A., Kaumaya, P. T., Ember, J. A., and Cosio, F. G. (1998). Receptors for the anaphylatoxin C5a (CD88) on human mesangial cells. J. Immunol. 160, 5646±5652. Wingrove, J. A., Discipio, R. G., Chen, Z., Potempa, J., Travis, J., and Hugli, T. E. (1992). Activation of complement components C3 and C5 by a cysteine proteinase (gingipain-1) from Porphyromonas (Bacteroides) gingivalis. J. Biol. Chem. 267, 18902±18907. Wuepper, K. D., Bokisch, V. A., MuÈller-Eberhard, H. J., and Stoughton, R. B. (1972). Cutaneous responses to human C3 anaphylatoxin in man. Clin. Exp. Immunol. 11, 13±20.

LICENSED PRODUCTS Serotec sells a rabbit anti-human CD88 polyclonal (Catalog no. AHP353) and mouse anti-human CD88 monoclonals (Catalog no. MCA1283 and 1284) for research. Both interact with linear sequences of the Nterminal region of CD88 (C5aR). A patent exists for a neutralizing polyclonal antibody to CD88 (C5aR). US Patent No. 5,480,974 issued 2 January 1996 (USSN 08/079,051 filed 21 April 1995); Edward L. Morgan, Julia A. Ember and Tony E. Hugli, co-inventors, Antibodies to Human C5a Receptor.