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The Journal of Immunology, 2004, 172: 4381-4390.
Copyright © 2004 by The American Association of Immunologists

Cloning, Expression, Cellular Distribution, and Role in Chemotaxis of a C5a Receptor in Rainbow Trout: The First Identification of a C5a Receptor in a Nonmammalian Species1

Hani Boshra2,*, Jun Li2,*, Rodney Peters2,*, John Hansen{dagger}, Anjan Matlapudi* and J. Oriol Sunyer3,*

* Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104; and {dagger} Center of Marine Biotechnology, University of Maryland Marine Biotechnology Institute, Baltimore, MD 21202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
C3a, C4a, and C5a anaphylatoxins generated during complement activation play a key role in inflammation. C5a is the most potent of the three anaphylatoxins in eliciting biological responses. The effects of C5a are mediated by its binding to C5a receptor (C5aR, CD88). To date, C5aR has only been identified and cloned in mammalian species, and its evolutionary history remains ill-defined. To gain insights into the evolution, conserved structural domains, and functions of C5aR, we have cloned and characterized a C5aR in rainbow trout, a teleost fish. The isolated cDNA encoded a 350-aa protein that showed the highest sequence similarity to C5aR from other species. Genomic analysis revealed the presence of one continuous exon encoding the entire open reading frame. Northern blot analysis showed significant expression of the trout C5a receptor (TC5aR) message in PBLs and kidney. Flow cytometric analysis showed that two Abs generated against two different areas of the extracellular N-terminal region of TC5aR positively stained the same leukocyte populations from PBLs. B lymphocytes and granulocytes comprised the majority of cells recognized by the anti-TC5aR. More importantly, these Abs inhibited chemotaxis of PBLs toward a chemoattractant fraction purified from complement-activated trout serum. Our data suggest that the split between C5aR and C3aR from a common ancestral molecule occurred before the emergence of teleost fish. Moreover, we demonstrate that the overall structure of C5aR as well as its role in chemotaxis have remained conserved for >300 million years.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Activation of the complement system results in the activation of complement C3, C4, and C5 components, leading to the generation of C3a, C4a, and C5a anaphylatoxins, respectively (1). Anaphylatoxins play a key role in inflammatory responses. Because of their common evolutionary ancestry and structural similarities, they elicit overlapping functions. For example, some of the common roles of C3a, C4a, and C5a in mammals include their ability to induce smooth muscle contraction, chemotaxis, respiratory burst, and production of PGs and eicosanoids (2). It is well known, however, that C5a is by far the most potent of all three anaphylatoxins, whereas C4a is the weakest in inducing biologically relevant responses (3). C5a plays an important role in promoting: 1) chemotaxis to a variety of leukocytes, including neutrophils and macrophages (2, 4, 5); 2) activation of the respiratory burst in phagocytes (6); 3) expression of TNF-{alpha} and IL-6 in monocyte-derived macrophages (7) as well as expression of IL-1{beta}, RANTES, and IL-8 in umbilical vein endothelial cells (8); 4) production of PGs and eicosanoids (9); 5) release of histamine by mast cells and basophils (10, 11); and 6) up-regulation of CR3 (12, 13) and P-selectin (14). In addition, C5a has been shown to play an important role in adaptive immunity. In this regard, it can act as an adjuvant by enhancing the Ab response (15) as well as by promoting Ag-specific cytotoxic (CTL) responses (16).

C5a exerts its effects through the C5a receptor (C5aR or CD88), a member of the rhodopsin family of seven-transmembrane(seven-TM) G protein-coupled receptors (2). C5aR appears to have awide cellular distribution and has been shown to be expressed in cells of myeloid (17, 18, 19) and nonmyeloid origin (20, 21). To date, C5aRs have been cloned only in mammalian species. A relatively low homology level has been observed across the mammalian C5aR sequences; for example, human C5aR is only 65, 67, 68, and 70.8% identical with C5aR from mouse, guinea pig, dog, and rat, respectively (22, 23, 24, 25, 26, 27). All these C5aR molecules possess an extracellular N-terminal region, seven helical hydrophobic TM regions, and an intracellular C-terminal domain. It is of interest that the coding region of the C5aR genes analyzed to date is intron-less.

Not long ago it was thought that C5aR was the only receptor for C5a. However, a second C5a receptor, C5L2, has recently been cloned and characterized in humans and mice (28, 29). C5L2 shows the highest sequence identity to C5aR. Unlike C5aR, C5L2 appears to be uncoupled from heterotrimeric G proteins. Also in contrast to C5aR, C5L2 does not induce significant activation of mitogen-activated protein kinases, mediate calcium fluxes, or stimulate chemotaxis (29). C5L2 has a high affinity for both C5a and its des-arginated form (C5adesArg)4 (29, 30). Evidence for the binding of C5L2 to C3a, C4a, and their des-arginated forms, however, is controversial (29, 31). As C5L2 appears to lack the capacity to transduce signals, it has been proposed that it acts as a decoy receptor, thereby modulating the concentrations of both C5a and C5adesArg (29). Because of the high sequence similarity between C5L2 and C5aR as well as their linkage in the same region of chromosome 19 (28), it seems probable that the two genes originated by gene duplication from a common ancestor.

The complement system is an ancient mechanism of defense that predates the appearance of vertebrate species, being present even in invertebrate deuterostome species (32, 33, 34, 35). Teleost fish have a complement system that includes all three pathways of complement activation (36). In addition, C3, C4, and C5 molecules have been identified, cloned, and purified in these animal species (37). Moreover, teleost fish are the only group of animals containing multiple forms of functionally active C3 proteins that are the products of different genes (38, 39, 40, 41, 42, 43). In rainbow trout, our animal model, three C3 isoforms (C3-1, C3-3, and C3-4) have been cloned, and all three are present as functional C3 proteins in plasma (38, 44). An additional C3 protein (C3-2) has been reported in rainbow trout that apparently lacks hemolytic activity (45). Trout C4 has recently been cloned, purified, and functionally characterized in our laboratory (46). In addition, a C5 trout molecule has been purified (47) and partially cloned (48).

The roles of anaphylatoxins in inflammation and other immunoregulatory processes have only been studied in mammalian species to date. Very little is known about the structure and function of such molecules in lower vertebrate species. In teleost fish, it has been shown that trout serum has the ability to chemoattract trout leukocytes (49). However, specific anaphylatoxins and their receptors remain to be characterized in such lower vertebrates. Accordingly, the evolutionary aspects related to the structure and function of anaphylatoxin receptors have not been elucidated to date.

It is likely that C3a, C5a, and formyl peptide receptors arose by gene duplication from the same common ancestor. In addition, genomic data and phylogenetic analysis seem to support the idea that C5aR and C5L2 were duplicated from a common ancestor. Although deuterostome invertebrate species contain bona fide C3 molecules, they appear to be devoid of C5, suggesting that the fifth component of the complement system emerged for the first time in vertebrates (35). In this regard, no C5 homologues have been found in agnathan fish, the most primitive living vertebrate species. Cartilaginous fish, however, appear to have a lytic pathway and a functional C5 homologue, although sequence data are lacking to confirm the identity of this component (50). Thus, teleost fish are the most primitive species in which a bona fide C5 molecule has been identified (48, 51). We hypothesize that as C5 molecules first emerged in cartilaginous or teleost fish, the possibility exists that the C5aR also originated in these species.

To shed light on the evolution of the primary structure and functional roles of the C5a receptor, we have now identified and cloned a C5aR molecule from a teleost fish. In this study we show for the first time in a nonmammalian species the presence of a C5aR and describe its cloning, expression analysis, genomic organization, and evolutionary relationship with mammalian C5a receptors. In addition, we report that Abs raised against the receptor positively stain distinct leukocyte populations. We also demonstrate a functional role for trout C5a receptor (TC5aR) in chemotaxis, as shown by inhibition studies using Abs generated against the N-terminal portion of the receptor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Fish

Rainbow trout (100–200 g) were obtained from Limestone Springs Fish Farm (Richland, PA). Trout plasma and serum were provided by Dr. S. Lapatra (Clear Springs Foods, Buhl, ID). Fish were maintained in aquarium tanks using a water recirculation system involving extensive biofiltration, UV sterilization units, and thermostatic temperature control. Water temperature was maintained continuously at 12–14°C.

Complementary DNA cloning of TC5aR and sequence and phylogenetic analysis

TC5aR cDNA was generated from trout liver mRNA that was isolated with an Oligotex Direct mRNA kit (Qiagen, Valencia, CA) according to the manufacturer’s recommendations. mRNA (2.0 µg) was reverse transcribed to negative strand cDNA with oligo(dT) (0.05 µg/µl) and 40 U of Superscript reverse transcriptase II (Invitrogen, Carlsbad, CA) for 1 h at 42°C. Two primer sets designed on the basis of rainbow trout (Onchorhyncus mykiss) and Atlantic salmon (Salmo salar) expressed sequence tags (ESTs) similar to the 5' and 3' ends of C5aR (GenBank accession no. CA364569 and CB517717, respectively) were used to generate overlapping C5aR sequences encompassing the entire putative ORF of TC5aR. The forward primer (5'-GAAATCTAAGTCACCTCCAATCA-3') and the reverse primer (5'-GGACACACAGAACTTCACATA-3') generated a 1067-bp fragment using the proofreading enzyme Pfu Turbo (Stratagene, La Jolla, CA) according to the manufacturer’s recommendations and using the following thermocycling conditions: 95°C for 2 min; 40 cycles of 95°C for 30 s, 55°C for 1 min, and 72°C for 1 min; and 72°C for 10 min. PCR products were cloned into a TOPO zero blunt vector (Invitrogen) and sequenced with a 3100 DNA analyzer (PE Applied Biosystems, Foster City, CA). Consensus sequences were generated from comparisons of repeated amplifications from trout liver mRNA using SeqMan and MegAlign (DNA Star, Madison, WI) software. Sequence alignments and the phylogenetic tree were conducted using the Clustal X software package (52). TM regions in TC5aR were predicted using TMpred software (53).

Northern blotting

Total RNA was prepared from various tissues and leukocytes of rainbow trout using TRIzol reagent (Invitrogen). RNA was quantified, and 10 µg/lane was size-fractionated on agarose-formaldehyde gels and transferred to nylon-supported nitrocellulose membranes (Bio-Rad, Hercules, CA) by capillary blotting. The blot was thereafter exposed to UV cross-linking to fix the RNA to the membrane. A 541-bp probe was generated using 5'-GGCTCCATCCCCCAGTTTGTCT-3' as forward primer and 5'-CCTGGGTTCTTTCTGATGTG-3' as reverse primer. The probe was radiolabeled with [32P]dCTP using the Ready-to-Go labeling system (Amersham Pharmacia Biotech, Arlington Heights, IL) and was purified using ProbQuant G-50 microcolumns (Amersham Pharmacia Biotech). The blot was prehybridized in warm Express hybridization solution (BD Clontech, Palo Alto, CA) at 68°C for 30 min in a hybridization oven (Problot 6; Labnet, Edison, NJ). The blot was hybridized at 68°C for 60 min in Express hybridization solution with 1–2 x 106 cpm of labeled probe/ml. After hybridization, the blot was rinsed three times in 2x SSC with 0.1% SDS for 30 min at room temperature and then washed in 0.1x SSC with 0.1% SDS with continuous agitation at 50°C for 40 min.

After washing, the blot was exposed to x-ray film (Kodak XB; Eastman Kodak, Rochester, NY) in an autoradiography cassette (Fisher Scientific, Pittsburg, PA). Expression of C5aR RNA was normalized for equal loading and transfer to 28S RNA.

Southern blotting

Genomic DNA (10 µg) isolation and blotting procedures have been previously described (54). For Southern blotting, a 1038-bp cDNA fragment obtained by RT-PCR was generated using the forward primer (5'-GAAATCTAAGTCACCTCCAATCA-3') and the reverse primer (5'-CCTGGGTTCTTTCTGATGTG-3'). The gel-purified PCR product was then randomly labeled (Amersham Pharmacia Biotech) with [32P]dCTP and used as a probe (65°C) under stringent conditions (0.25x SSC/0.25% SDS, 64°C final wash). The blot was exposed to film for 6 days.

Abs against TC5aR

Two peptides corresponding to the N-terminal region of TC5aR (C5aR6–20 (SILTEEELSLYNITD) and C5aR22–36 (EFVKPGGLGPVLGPR)) were synthesized by Biosynthesis (Lewisville, TX). Matrix-assisted laser desorption mass spectrometry was used to determine the purity of the peptides. The synthesized peptides were coupled to keyhole limpet hemocyanin by the glutaraldehyde method and used to raise Abs in rabbits (Biosynthesis). Ab titers were determined by ELISA. The Ig fraction of the antiserum was first purified using a HiTrap protein G column, according to the instructions of the manufacturer (Amersham Pharmacia Biotech). Thereafter, specific Abs against the C5aR peptides were purified by affinity chromatography using the synthetic peptides coupled to cyanogen bromide-activated Sepharose (Amersham Pharmacia Biotech).

Isolation of PBLs

Blood was obtained from the caudal vessel with a heparinized syringe. After extraction, the blood was immediately diluted (1/5) with EMEM (American Type Culture Collection, Manassas, VA) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 U/ml heparin, then placed on ice. The blood cell suspension was thereafter layered onto a 51/34% discontinuous Percoll (Sigma-Aldrich, St. Louis, MO) density gradient and centrifuged at 400 x g for 30 min. The band of cells lying at the interface was collected, and the cells were washed with HBSS or kept in EMEM supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin.

Flow cytometric analysis

For each experiment, 1 x 106 PBLs in PBS containing 2% FCS were incubated at room temperature for 30 min with affinity-purified rabbit anti-TC5aR6–20 or anti-TC5aR22–36 alone or in combination with either anti-trout IgM (mAb1.14) or anti-trout thrombocytes (mAb28.D7). As a control, cells were incubated with the same concentration of preimmune polyclonal rabbit IgG or control mouse IgG. After two washes with PBS, cells were stained for 30 min with FITC-conjugated anti-rabbit IgG and/or PE-conjugated anti-mouse IgG. Thereafter, cells were washed twice in PBS, and flow cytometric analysis was conducted using a standard FACScan (BD Biosciences, Mountain View, CA). For each sample, 20,000 individual cells were analyzed. Data were analyzed with the program CellQuest (BD Biosciences).

Chemotaxis and inhibition of cell migration by anti-TC5aR Abs

In mammals, zymosan-activated serum is routinely used as source of C5a to perform C5a-mediated chemotaxis (55, 56). Preliminary experiments showed that samples of trout serum activated with either zymosan or rabbit erythrocytes (RaRBC; Cocalico Biologicals, Reamstown, PA) were both potent chemoattractants for trout PBLs (data not shown). However, at lower dilutions of the activated serum (1/80 to 1/160), the RaRBC-activated serum was more potent than the zymosan-activated serum in inducing chemotaxis; therefore, RaRBC-activated serum was used thereafter as the source of C5a. A fraction containing ~8-kDa peptides (expected size of anaphylatoxins) showing strong chemoattractant properties was then purified from RaRBC-activated trout serum by gel filtration chromatography. To this end, trout serum (1 ml) was incubated with 40 µl (108 cells/ml) of RaRBC for 30 min at room temperature. The reaction mixture was then centrifuged at 3000 rpm for 10 min at 4°C. The supernatant (activated serum) was removed carefully and brought to 20 mM in EDTA. Activated serum (0.5 ml) was diluted with 0.5 ml of PBS (pH 7.5), loaded onto a Superdex 200 column (Amersham Pharmacia Biotech), and eluted at a flow rate of 0.5 ml/min with PBS. Fractions containing proteins with molecular masses of ~8 kDa were identified via 8–16% SDS-PAGE, pooled, and then passed through an additional step of gel filtration chromatography (Superdex 200 column; Amersham Pharmacia Biotech). The resulting fraction (named as chemoattractant fraction) was shown to be almost as potent as activated trout serum in inducing chemotaxis of PBLs (see Fig. 8A). Checkerboard analysis showed that the migration of trout leukocytes toward the C5a-containing fraction was due to a chemotactic (directional), rather than a chemokinetic (random), response (data not shown).



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  		FIGURE 8. Anti-TC5aR inhibits chemotaxis of PBLs. Chemotactic responses of trout leukocytes were assayed in a 96-well chemotaxis chamber. The chemoattractant preparations (RaRBC-activated trout serum or the gel filtration-purified chemoattractant fraction) were placed in the lower wells of the chamber, and the trout cells (4 x 105) were added to the upper wells. The upper and lower wells were separated by a 3-µm pore size polycarbonate filter. The chamber was incubated for 60 min at 20°C, and the cells migrating to the bottom wells were counted. For the inhibition studies in B, the leukocytes had previously been incubated for 10 min with various amounts of anti-TC5aR (0–75 µg/ml) or equal amounts of preimmune polyclonal rabbit IgG (control). A, The chemoattractant preparations used are as follows: a 1/40 dilution of trout serum previously activated with RaRBC (activated serum), the chemoattractant fraction at a final concentration of 2 µg/ml, the chemoattractant fraction at a final concentration of 0.5 µg/ml, and PBS as a negative control. B, Chemotaxis toward the chemoattractant fraction (2 µg/ml) was inhibited by preincubating the PBLs with increasing amounts of affinity-purified anti-TC5aR. Results in B are presented as a percentage of the migrated control cells (i.e., cells that had been preincubated with equal amounts of rabbit preimmune IgG). Results are expressed as the mean ± SEM of five experiments (involving five different fish) performed in triplicate.

 
Chemotactic migration was assessed using a 96-well chemotaxis chamber (MBC96; NeuroProbe, Gaithersburg, MD). For our chemotaxis experiments, 29 µl of the chemoattractant fraction (in PBS-5 mM Ca2+Mg2+) were placed in the bottom wells of the chamber to give a final concentration of 2 µg/ml. Trout PBLs (4 x 105) were placed in the upper wells of the chamber. The PBLs had previously been incubated for 10 min with various amounts of anti-TC5aR or preimmune polyclonal rabbit IgG (0–75 µg/ml), or with PBS. The upper and lower wells were separated by a 3-µm pore size polycarbonate filter. The chamber was then incubated for 60 min at 20°C, and the cells that had migrated to the bottom wells were counted. Cell migration was calculated as the average number of five randomly selected high power fields (x400) per well. For the inhibition experiments, data are presented as the percent migration of PBLs that had previously been incubated with the control preimmune polyclonal rabbit IgG.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Isolation and sequence analysis of TC5aR cDNA

Using primers based on Atlantic salmon and rainbow trout ESTs that showed a high degree of similarity to human C5aR, we first generated a cDNA fragment spanning ~1.1 kb. The sequenced DNA had a 1050-bp open reading frame (ORF) that encoded a 350-aa protein (Fig. 1) and included a stop codon. Using the same primers, we obtained a PCR fragment of identical size using trout genomic DNA, indicating the presence of one continuous exon encoding the entire ORF (data not shown). The deduced amino acid sequence of the obtained TC5aR cDNA showed the highest degree of homology to C5aR molecules from other vertebrate species (see Fig. 1). TC5aR showed 35.3% identity to human C5aR, 31.7% identity to human C5L2, 28.3% identity to human formyl peptide receptor 1 (FPR), and 24.4% identity to human C3aR.



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  		FIGURE 1. Amino acid comparison of TC5aR with mammalian C5aR and C5L2 sequences. Putative TM domains are overlined. Extracellular and intracellular domains are indicated by downward and upward arch symbols, respectively. Residues required for C5aR function and interaction with C5a are shown in bold and include the following: N-terminal glutamic/aspartic acid residues, as well as N-terminal prolines; the C5aR-specific DRF motif located after TM3; Cys109 and Cys187; C-terminal phosphorylation sites (Ser319, Ser331, Ser336, Ser340, and Ser342) (putative roles of these residues are described in Results and Discussion). Predicted N-glycosylation sites are circled. The numbers in parentheses indicate the amino acid identity as a percentage of TC5aR. Asterisks denote identities in all sequences, whereas gaps are denoted by dashes. Colons indicate strongly similar amino acids, whereas single dots infer weakly similar residues. The N and C termini are indicated by lines linked to the TM1 and the C-terminal glutamine, respectively.

 
Hydropathic analysis of the deduced amino acid sequence of TC5aR was nearly identical with that for rat C5aR (Fig. 2) or other C5aRs (data not shown), indicating the presence of seven TM domains in TC5aR. In general, the TM domains were more conserved than the intracellular and extracellular regions (Fig. 1), as has also been observed when making comparisons among mammalian C5aR molecules. Thus, the TM portions of TC5aR varied in the extent of their homology to those found in humans, with TM1 and TM2 showing the highest level of identity (61 and 47%, respectively); TM3–7 demonstrated the strongest variability, with levels of homology of 26–45% to its human counterpart. Similar results were obtained when the C5aR TM domains were compared across mammalian species (data not shown). Extra/intracellular domains also had varying degrees of homology. The N-terminal extracellular domain of TC5aR retained almost no overall homology to any mammalian C5aR (Fig. 1). This is also consistent with previous studies in mammals indicating that the region of greatest variation lies in the N terminus of C5aR. Only one extracellular (between TM2 and TM3) and one intracellular (between TM3 and TM4) region showed a high degree of homology (61 and 75% identity, respectively). The other extra- and intracellular regions showed very low levels of identity (5–33%).



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  		FIGURE 2. Prediction of TM regions of trout and rat C5aR. TM plotting of C5aR amino acid sequences from trout (upper panel) and rat (lower panel) were obtained using the TMpred program, based on the method of Hofman and Stoffel (53 ). TM domains 1–7 are indicated.

 
As is true for C5aR in higher vertebrates, TC5aR possesses an N-linked glycosylation site in its N-terminal domain (Asn17; trout numbering; Fig. 1). Interestingly, however, it has a second potential glycosylation site at the C terminus (Asn317) that has no counterpart in human C5aR. Another potential N-linked glycosylation site is present at Asn199 (Asn-Thr-Thr), although its probability was calculated to be <50% using the NetNGlyc 1.0 program. The latter two glycosylation sites are absent from all known mammalian C5aRs.

Conservation of cysteines in trout and mammalian C5aRs also indicates a high degree of structural homology between TC5aR and those of higher vertebrates. It has previously been suggested that Cys109 and Cys186 form an important disulfide bridge between extracellular domains that is necessary for G protein-coupled signaling (25). These two cysteines are present in all known C5aRs of higher vertebrates. As shown in Fig. 1, these cysteines are also present in TC5aR. Another characteristic of C5aR is its high proline content at the N terminus (25). In higher vertebrates, this content varies from 8–17%. TC5aR possesses three prolyl residues in its first 35 aa, falling within this range (8.5%). The acidity of the N terminus is another important structural feature associated with mammalian C5aR. An indicator of such acidity lies in its aspartic and glutamic acid contents, which vary from 5 in dogs to 8 in rats. TC5aR has seven such residues (shown in bold in Fig. 1). It has been shown that phosphorylation of C5aR in higher vertebrates occurs at serine residues in the C terminus; in humans, such phosphorylation occurs at Ser314, Ser327, Ser332, Ser334, and Ser338 (22, 25). Putative homologous TC5aR phosphorylation sites are located at Ser319, Ser331, Ser336, Ser340, and Ser342.

In humans, interactions between the C5a ligand and its receptor involve at least two distinct sites (57, 58). One site, the binding site, involves the acidic residues at the N terminus (17, 59), whereas the other site involves a less-defined interaction with the region comprising residues Arg206 and Glu282 (human numbering) (60). It is worth noting that whereas several similarities exist between the N terminus of TC5aR and other C5aRs, there are also some differences. For example, in humans, Tyr14, Thr19, and Asp27 are hypothesized to contribute to the C5a binding site in C5aR. This hypothesis is supported by the same conserved residues in mice, rats, dogs, humans, and guinea pigs (25); yet, whereas trout contains Tyr16 (trout numbering) and Thr19, it lacks Asp27. Other residues shown to be important in the second region of C5aR interacting with C5a are Arg206 and Asp282 (human numbering) (58). Although the Arg is present in TC5aR (Arg203), the aspartic acid residue is changed to histidine. The availability of a C5aR sequence from trout, a primitive vertebrate species, will now be instrumental in further identifying and defining conserved residues involved in the second site of C5aR that is suspected to interact with C5a.

A second C5a-binding receptor, C5L2, has recently been identified in humans and mice (28, 29). Although highly homologous to C5aR, this receptor does not seem to be involved in the G protein-coupled signaling usually associated with C5aR, because it lacks an essential Arg in the aspartic acid-arginine-phenylalanine (Asp-Arg-Phe) (DRF) motif following the third transmembrane domain (29). In mouse and human C5L2, this Arg is substituted by Leu (28, 29). It is more likely that our cloned TC5aR is a homologue of C5aR in higher vertebrates, based on several observations: 1) a greater degree of sequence identity of TC5aR with human C5aR than with C5L2 (35.3 vs 31.7%, respectively; Fig. 1), 2) the conservation of the arginine of the C5aR-specific DRF motif (Fig. 1), and most importantly, 3) the functional evidence obtained in this study that Abs specific for TC5aR inhibit chemotaxis of trout PBLs (Fig. 8).

Although TC5aR possesses many of the structural properties associated with C5aR from mammals, it should be noted that commonalities also exist between TC5aR and C5L2, mainly involving cysteine content and positions. In this regard, all C5aR of mammals possess 9–10 cysteines, whereas human or mouse C5L2 and TC5aR have 15 cysteines residues each, eight of which are in conserved positions. In addition, only the TC5aR and C5L2s molecules possess two consecutive cysteines in their respective TM2 region, and only TC5aR and C5L2 molecules have two cysteines in their N-terminal extracellular domain. These similarities indicate that TC5aR contains structural features of both C5aR and C5L2. Thus, it is tempting to speculate that TC5aR represents the stage before the duplication of C5aR and C5L2 from a common ancestral molecule. This hypothesis is supported to some extent by the phylogenetic tree composite. The phylogenetic tree of the C5aR, C5L2, C3aR, and formyl peptide receptor sequences was drawn using the neighbor-joining method. TC5aR branched as an outgroup to the mammalian C5aR and C5L2 cluster (Fig. 3). According to this hypothesis, the divergence of C5aR and C5L2 from a common ancestral molecule may have happened after the emergence of teleost fish, in a more evolutionary modern animal species.



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  		FIGURE 3. Phylogenetic tree of TC5aR and other related members of the rhodopsin gene family. An amino acid alignment (Clustal X) of the mature proteins was used to generate the unrooted neighbor-joining tree. Numbers on the branches indicate the total recovery from 1000 bootstrap replications. Accession numbers of the molecules used to construct the tree are as follows: TC5aR, AY438032; TC3aR, CA373689; human C5aR, NP_001727; guinea pig C5aR, O70129; rabbit C5aR, Q9TUE1; rat C5aR, NP_446071; mouse C5aR, AAB97774; dog, C5aR, P30992; human C5L2, BAA95414; mouse C5L2, BAC35303; human C3aR, AAH20742; guinea pig C3aR, O88680; rat C3aR, O55197; mouse C3aR, AAH03728; mouse Dez, NP_032179; human ChemR23, CAA75112; human FPR, P21462; human FPR1, NM_002029; mouse FPR1, NP_038549; and mouse FPRL1, NP_032068.

 
We have recently found an EST in the database encoding for a partial product (no stop codon was found) of 243 aa that showed the highest sequence homology to mammalian C3aR. In the phylogenetic tree, this molecule clustered with all known C3aR molecules (Fig. 3). The presence of this receptor in teleost would give further support to our hypothesis that the split between C5aR and C3aR from a common ancestral molecule occurred before the emergence of teleost fish. The characterization of this putative trout C3aR is underway in our laboratory. It should be noted that TC5aR did branch separately from all C3aR molecules, including the putative trout C3aR EST (Fig. 3). However, we designated the trout clone C5aR, not C3aR, based not only on the phylogenetic tree, but also on the conserved primary and secondary structures and key residues commonly associated with C5aR in higher vertebrates. Moreover, TC5aR, similarly to mammalian C5aRs, lacked the 144 extra aa characteristic of the second extracellular domain of C3aR molecules (2).

Northern blot analysis of TC5aR

Expression of rainbow TC5aR message was examined by Northern blot analysis. Total RNA was isolated from a variety of trout tissues and leukocytes, separated by formaldehyde agarose gel electrophoresis, and transferred to nylon membranes. The blot was thereafter hybridized with the 541 bp of 32P-labeled TC5aR probe. The size of the TC5aR message (2.37 kb) was very similar to that observed for human, guinea pig, and mouse C5aRs, which range from 2.3–2.5 kb (Fig. 4). Normalization of the C5aR signal with the 28S band indicated that the strongest signal was detected in the head kidney, followed by blood leukocytes, caudal kidney, liver, and spleen, respectively. Similarly, PBLs and bone marrow are the most plentiful sources of message in both mouse and human C5aR. It should be noted that teleost fish do not have bona fide bone marrow; instead, the head kidney is considered to be the functional and structural homologue of mammalian bone marrow. It should be stressed that weak signals of the same size were detected in brain and heart after longer exposure of the film (2 days) This pattern is also in agreement with the weak expression of the C5aR message in the same organs in mouse, human, and guinea pig (25, 29).



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  		FIGURE 4. Tissue-specific expression of TC5aR by Northern blot. Ten micrograms of total RNA from indicated tissues of rainbow trout was electrophoresed, blotted to a nitrocellulose membrane, and hybridized with a TC5aR probe. Expression of TC5aR message was found in head kidney, caudal kidney, blood leukocytes, liver, and spleen. Weaker signals were observed in brain and heart after longer exposure of the film. Ethidium bromide-stained 28S is shown as a loading control.

 
Southern blot analysis of TC5aR

The entire coding region of the TC5aR gene was amplified and used as a probe for Southern blotting (Fig. 5). Two bands could be observed for each individual and digest, with the exception of fish 1, which showed signs of allelic variation (three bands). As salmonid fish possess a tetraploid past, one would expect the presence of two independent loci. Taking into account that no introns exist within the coding region of TC5aR and that no restriction sites exist within the probe for the enzymes (EcoRV or HindIII) used in the digestion, the Southern blot data suggest that two C5aR genes exist in the trout genome. However, expression analysis showed that one single band was detected in the Northern blots. In addition, multiple sequence alignment analysis of our TC5aR sequence with all rainbow trout ESTs comprised in the The Institute for Genomic Research and National Center for Biotechnology Information Unigene EST indexes (>102,000 ESTs) resulted in the same sequence. Thus, taking into account our expression analysis and the alignment analysis with all available trout ESTs, it is reasonable to suggest that only one of the two C5aR genes is expressed. Supporting the latter, only one sequence could be amplified from trout cDNA when using the primers described to amplify TC5aR.



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  		FIGURE 5. Southern blot analysis. Rainbow trout genomic DNA from four different individuals was digested with EcoRV and HindIII. The blot was then hybridized with a cDNA probe corresponding to the entire coding region of TC5aR. Values on the left of the blot represent kilobases.

 
Binding of anti-TC5aR to trout PBLs

Abs generated against peptides based on the extracellular N-terminal domain sequence of several of the mammalian C5aRs have proven to be very effective in recognizing the cells expressing the receptor (17, 18, 61, 62, 63). Two anti-peptide Abs recognizing two different areas of the N-terminal extracellular region of TC5aR (see Fig. 1) were produced. Specific Abs against the peptides were affinity-purified using columns to which each individual peptide was coupled. Immunoprecipitation analysis with a 1:1 mixture of both anti-C5aR6–20 and anti-C5aR22–36 affinity-purified Abs showed that a protein of 43 kDa was recovered from leukocyte lysates of peripheral blood (data not shown). The molecular mass obtained for the immunoprecipitated protein is very close that predicted for TC5aR (39.2 kDa). Binding of the anti-TC5aR to PBLs was assessed by flow cytometry. Approximately 86% of the trout PBLs were positively stained with both Abs, as indicated by the shifts in the fluorescence intensity shown in the histograms of Fig. 6A (anti-TC5aR6–20) and 6F (anti-TC5aR22–36). The staining patterns seen for both Abs were practically identical, strongly suggesting their reactivity against the same molecule. Thus, anti-TC5aR6–20 and anti-TC5aR22–36 positively stained two different regions (R1 and R2) in an equal manner. R2 was comprised of the cell population with the highest forward and side scatter (large cells (L)) and a cell population with the lowest forward an side scatter (small cells-1 (S1); Fig. 6, C and H). R1 contained the population of cells displaying the strongest staining (36% of PBLs). The cells of R1 (S2) had similar forward and side scatter values as the S1 cell population of R2 (Fig. 6, B and G). For both Abs (anti-TC5aR6–20 and anti-TC5aR22–36), R3 contained the negative cells (16% of PBLs) or the cells that showed the same fluorescence intensity as those stained with the preimmune polyclonal rabbit IgG (Fig. 6, D and I). This region (R3) was comprised of a well-defined population of small cells (S3) showing a forward scatter slightly larger than that of the positively stained S1 population. It should be noted that the region containing the negative cells (R3) showed some variability among different individuals, ranging from ~10–20% of the total cell number analyzed.



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  		FIGURE 6. Binding of anti-TC5aR6–20 and anti-TC5aR22–36 to trout PBLs. A and F, Histogram of cells stained with affinity-purified rabbit anti-TC5aR6–20 and anti-TC5aR22–36, respectively (2.5 µg/ml), followed by FITC-conjugated goat anti-rabbit IgG (line histogram; green). As a control, cells were incubated with preimmune rabbit IgG (filled histogram; blue). B–J, Forward (FSC-H) and side (SSC-H) scatter analyses of positively (R1 and R2) and negatively (R3) stained cells after incubation of PBLs with the anti-TC5aR6–20 (B–E) and anti-TC5aR22–36 (G–J) Abs. One experiment of five is shown.

 
Costaining analysis using the anti-TC5aR6–20 and a monoclonal anti-trout IgM Ab (mAb 1.14) that recognizes surface IgM in trout B lymphocytes (64, 65) showed that all IgM-positive cells were also C5aR positive (R1 in Fig. 7A), indicating the presence of the C5aR in all trout B cells. As expected, B lymphocytes (all contained in R1; Fig. 7A) displayed low forward and side scatters (Fig. 7C). In addition, they comprised ~71% of the cells contained in the small population of PBLs shown in Fig. 7B. However, ~17% of the small cells that were TC5aR positive were also IgM negative (shown as SIg- in Fig. 7D). As shown previously in Fig. 6, C and H, the TC5aR-positive cells also included the entire large population of PBLs (shown as L in Fig. 7, B and D).



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  		FIGURE 7. Double staining of PBLs with anti-TC5aR and anti-trout surface IgM. A, Cells were costained with affinity-purified rabbit anti-TC5aR6–20 (2.5 µg/ml) and anti-trout IgM (2.5 µg/ml). B, Forward (FSC-H) and side (SSC-H) scatter analyses of the entire PBL sample. C–E, Forward (FSC-H) and side (SSC-H) scatter analyses of positively costained cells (C; R1 of A), cells stained only with the anti-TC5aR6–20 (D; R2 of A) and negatively stained cells (E; R3 of A). One experiment of three performed is shown.

 
Costaining analysis using a monoclonal anti-trout thrombocyte Ab showed that these cells were both C5aR and Ig negative (data not shown). Thrombocytes encompassed ~10–15% of PBLs, as previously shown by others (65, 66). In addition, thrombocytes comprised >95% of the PBLs that were negative for C5aR. Thus, the majority of cells within R3 of histograms D and I from Fig. 6 are thrombocytes. The fact that the anti-C5aR Abs did not stain thrombocytes further substantiates the specificity of the anti-C5aR and is in agreement with the lack of C5aR expression on the surface of mammalian thrombocytes (67).

In conclusion, ~86% of the trout PBLs were positively stained with the anti-TC5aR Abs. Approximately 83% of the cells stained by the anti-C5aR Ab were comprised of B lymphocytes and granulocytes (Fig. 7). The rest of the TC5aR-positive cells (~17%) formed part of a well-defined population with low side and forward scatter that could not be identified because of the current lack of specific markers for rainbow trout T lymphocytes, NK cells, and other cell types. The majority of the PBLs that were not immunoreactive with the anti-TC5aR (R3 in histograms from Fig. 6, A and F) appeared to be thrombocytes. In mammals, studies have shown that similar Abs against areas of the extracellular N-terminal domain of C5aR stain macrophages, neutrophils, monocytes, T cells, and B cells from PBLs or from other tissues (17, 18, 62, 67). Although our results show that B lymphocytes appear to express C5aR, contradictory results, have been reported on the presence of C5aR in human and murine B cells (68, 69). In this regard, it has been shown that C5aR is expressed in human naive, germinal center, and memory B lymphocytes at both the mRNA and protein levels (68). However, a recent report described the characterization of a novel mAb that recognizes murine C5aR. The authors showed that B cells were not stained by the Ab (69). The contrasting results from the studies depicted above might be explained by the fact that the presence of C5aR was observed in human B cells, whereas the opposite result was seen in murine B cells. Therefore, the possibility exists that the expression pattern of C5aR in B cells is variable among mammalian species. Clearly more studies need to address the presence of C5aR in mammalian B lymphocytes to resolve this controversy.

Inhibition of chemotaxis by anti-TC5aR Abs

One of the most compelling pieces of evidence in this study indicating the presence of a bona fide C5aR in trout is the inhibition of chemotaxis of PBLs elicited by the anti-TC5aR Abs (Fig. 8B). In mammals, C5aR plays a major role in chemotaxis, and Abs against the N-terminal extracellular domain of C5aR have been shown to inhibit chemotaxis (62). To investigate whether this function is conserved in TC5aR, we evaluated the ability of our anti-C5aR Abs to inhibit chemotaxis of PBLs. We first showed that RaRBC-activated trout serum is a powerful chemoattractant for PBLs. We obtained by gel filtration a fraction containing ~8-kDa peptides (expected mass of anaphylatoxins) purified from RaRBC-activated trout serum that showed chemoattractant properties similar to those displayed by the activated trout serum (Fig. 8A). This fraction was then used for the inhibition studies with the anti-TC5aR Abs. Anti-TC5aR6–20 and anti-TC5aR22–36 were combined (1:1) and used for the inhibition experiments. It should be noted that at all concentrations tested, the preimmune polyclonal rabbit IgG control had no inhibitory effect. In contrast, preincubation of the anti-TC5aR Abs with the PBLs almost completely inhibited their migration to the lower well of the chemotaxis chamber containing the trout chemoattractant fraction (Fig. 8B). The anti-TC5aR Abs inhibited chemotaxis in a dose-dependent manner, and maximal inhibition (~80%) was achieved with 75 µg/ml of the Ab. In addition, both the anti-TC5aR6–20 and anti-TC5aR22–36 Abs, when used individually, were equally effective in inhibiting chemotaxis (data not shown). The fact that both anti-TC5aR Abs inhibited chemotaxis suggests that both regions (TC5aR6–20 and TC5aR22–36) are important for interaction with the ligand. These data also indicate that the C5aR is the most important, if not the only, receptor mediating complement-dependent chemotactic responses in trout. Although in mammals C5a is the most potent chemoattractant anaphylatoxin, very little is known about the role of fish or other nonmammalian vertebrate anaphylatoxins in chemotaxis. Recent studies in our laboratory have shown that none of the three purified C3a anaphylatoxin molecules derived from the three trout C3 isoforms appears to play a role in the chemotaxis of trout leukocytes (70). However, we have shown that complement-activated trout serum and the gel filtration-purified chemoattractant fraction both elicit strong chemotactic responses (Fig. 8A). Combined with the inhibition of chemotaxis by anti-TC5aR Abs, these findings suggest that C5a is an important chemotactic anaphylatoxin in fish.

Concluding remarks

To our knowledge, the present study represents the first structural and functional characterization of a C5aR in a nonmammalian species. Our sequence and phylogenetic analyses suggest that the divergence of C5aR and C3aR from a common ancestral molecule occurred before the emergence of teleost fish. The functional data also represent the first direct evidence of involvement of C5aR from a primitive vertebrate species in chemotaxis. In conclusion, we have shown that the overall structure of C5aR as well as its role in chemotaxis have remained conserved for >300 million years.


   Acknowledgments
 
We thank Dr. D. McClellan for editorial assistance.


   Footnotes
 
1 This work was supported by National Science Foundation Grant MCB-0118387. The nucleotide sequence data reported in this paper have been submitted to GenBank (accession no. AY438032). Back

2 H.B., J.L., and R.P. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. J. Oriol Sunyer, Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, 413 Rosenthal, 3800 Spruce Street, Philadelphia, PA 19104. E-mail address: sunyer{at}vet.upenn.edu Back

4 Abbreviations used in this paper: C5adesArg, des-arginated form; EST, expressed sequence tag; FPR, formyl peptide receptor; ORF, open reading frame; R, region; RaRBC, rabbit erythrocyte; S, small cell; TC5aR, trout C5a receptor; TM, transmembrane. Back

Received for publication October 22, 2003. Accepted for publication January 22, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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