Characterization of a C3a receptor in rainbow trout and Xenopus: the first identification of C3a receptors in nonmammalian species.
ABSTRACT Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs. Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immunofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2). In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR. However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor, this event occurred before the emergence of teleost fish.
- SourceAvailable from: Ivan Mulero[show abstract] [hide abstract]
ABSTRACT: Mast cells are important as initiators and effectors of innate immunity and regulate the adaptive immune responses. They have been described in all classes of vertebrates and seem to be morphologically and functionally similar. However, early studies had shown that fish and amphibian mast cells were devoid of histamine. In this study, we take a fresh look at the evolution of histamine and find that the mast cells of fish belonging to the Perciformes order, the largest and most evolutionarily advanced order of teleosts, are armed with histamine. More importantly, histamine is biologically active in these fish where it is able to regulate the inflammatory response by acting on professional phagocytes. In addition, the actions of histamine in these immune cells seem to be mediated through the engagement of H(1) and H(2) receptors, which, together with the H(3) receptor, are well conserved in bony fish. We propose that the storage of histamine in vertebrate mast cells and its use as an inflammatory messenger was established in primitive reptiles (Lepidosauria) approximately 276 million years ago. This same feature seems to have developed independently in Perciform fish much more recently in the Lower Eocene, between 55 and 45 million years ago, a short period during which the great majority of Percomorph families appeared.Proceedings of the National Academy of Sciences 01/2008; 104(49):19434-9. · 9.74 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: In mammals, interaction of CD28 with CD80 or CD86 molecules provides costimulatory signals for T cell activation that leads to increased IL-2 gene and protein expression by activated T cells. Thus far, CD80 and CD86 have been cloned and functionally characterized only in mammals and birds. To shed light into the evolution of CD80 and CD86, we have cloned and functionally characterized a rainbow trout (rt) molecule (rtCD80/86) that shows the highest degree of sequence conservation and phylogenetic relationship with CD80 and CD86 molecules. Moreover, its genomic organization was almost identical to that of human CD86. Rainbow trout possess one membrane-bound and two soluble CD80/86 transcripts, all of which are derived from the same rtCD80/86 gene. The membrane-bound form exhibited its highest degree of expression in lymphoid tissues, particularly on B cells. Incubation of trout leukocytes with LPS and bacteria leads to up-regulation of rtCD80/86 gene expression. Importantly, we show that trout and other teleost fish contain a single CD80/86 gene, thus suggesting that this gene may represent the ancestor from which CD80 and CD86 arose by gene duplication in more evolved species. To gain further insights into the function of rtCD80/86, we have identified and cloned trout IL-2 and have shown that recombinantly produced trout CD80/86 up-regulates the expression of IL-2 in trout blood leukocytes. Significantly, this finding indicates that the capacity to modulate IL-2 expression is a primordial function that has been conserved both in fish and mammalian CD80/CD86 molecules throughout 350 million years of evolution.The Journal of Immunology 08/2009; 183(1):83-96. · 5.52 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: As physical barriers that separate teleost fish from the external environment, mucosae are also active immunological sites that protect them against exposure to microbes and stressors. In mammals, the sites where antigens are sampled from mucosal surfaces and where stimulation of naïve T and B lymphocytes occurs are known as inductive sites and are constituted by mucosa-associated lymphoid tissue (MALT). According to anatomical location, the MALT in teleost fish is subdivided into gut-associated lymphoid tissue (GALT), skin-associated lymphoid tissue (SALT), and gill-associated lymphoid tissue (GIALT). All MALT contain a variety of leukocytes, including, but not limited to, T cells, B cells, plasma cells, macrophages and granulocytes. Secretory immunoglobulins are produced mainly by plasmablasts and plasma cells, and play key roles in the maintenance of mucosal homeostasis. Until recently, teleost fish B cells were thought to express only two classes of immunoglobulins, IgM and IgD, in which IgM was thought to be the only one responding to pathogens both in systemic and mucosal compartments. However, a third teleost immunoglobulin class, IgT/IgZ, was discovered in 2005, and it has recently been shown to behave as the prevalent immunoglobulin in gut mucosal immune responses. The purpose of this review is to summarise the current knowledge of mucosal immunoglobulins and B cells of fish MALT. Moreover, we attempt to integrate the existing knowledge on both basic and applied research findings on fish mucosal immune responses, with the goal to provide new directions that may facilitate the development of novel vaccination strategies that stimulate not only systemic, but also mucosal immunity.Developmental and comparative immunology 11/2011; 35(12):1346-65. · 3.29 Impact Factor
Characterization of a C3a Receptor in Rainbow Trout and
Xenopus: The First Identification of C3a Receptors in
Hani Boshra,2* Tiehui Wang,2†Leif Hove-Madsen,2‡John Hansen,2§Jun Li,2*
Anjun Matlapudi,* Christopher J. Secombes,†Lluis Tort,¶and J. Oriol Sunyer3*
Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because
C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish
implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa
molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs.
Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immu-
nofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser
extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout
leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2).
In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR.
However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra
piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR
characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor,
this event occurred before the emergence of teleost fish. The Journal of Immunology, 2005, 175: 2427–2437.
endogenous danger signals that induce the development of an in-
flammatory response and trigger the activation of several key in-
nate immune processes. All three anaphylatoxins share a high de-
gree of homology and have been found to possess overlapping
functions. The C5a anaphylatoxin is considerably more potent than
C3a and C4a in inducing biologically relevant responses (2, 3).
The role of C4a in inflammation is speculative to date. Common
functions of C3a and C5a in mammals include their ability to in-
duce chemotaxis, respiratory burst, and the expression of several
proinflammatory cytokines in a variety of leukocytes (4–7). The
major known tasks attributed solely to C3a involve the induction
ctivation of the complement system results in the gen-
eration of anaphylatoxin molecules C3a, C4a, and C5a
(1). In mammals these molecules are considered to be
of chemotaxis specifically in eosinophils and mast cells as well as
the inhibition of the polyclonal Ab response (8–12). In the last
year, several reports have demonstrated additional important roles
of C3a in innate and adaptive immunities. In this regard, a new
study demonstrates that C3a has previously unforeseen antibacte-
rial properties (13). Another report has shown that human mono-
cyte-derived dendritic cells can be chemoattracted to C3a after
up-regulation of the C3aR with IFNs (14). Recent studies have
demonstrated a novel role of the C3aR in the retention of hemo-
poietic stem/progenitor cells in bone marrow (15). The role of C3a
in adaptive immunity has been demonstrated recently in a study
showing that C3a down-regulates the Th2 response to epicutane-
ously introduced Ag (16). Thus, it is becoming apparent that
the functions of C3a in immunity are greater than previously
C3a and C5a elicit their biological activities through binding to
C3aR and C5aR, respectively. These two receptors are members of
the rhodopsin family of G protein-coupled receptors, and they have
been characterized in a variety of mammalian species, including
humans (17, 18), rat (19, 20), dog (21), mouse (22, 23), and guinea
pig (19, 24). A C5a-like receptor (C5L2) recently characterized in
mice and humans has been shown to bind C5a and its desarginated
derivative (C5adesArg). In contrast to C5aR, C5L2 appears to be
uncoupled from heterotrimeric G proteins. Because C5L2 seems to
lack the capacity to transduce signals, it has been suggested that it
acts as a decoy receptor, thereby modulating the concentration of
both C5a and C5adesArg(25, 26). C3aR and C5aR have a wide
cellular distribution and have been shown to be expressed in cells
of myeloid and nonmyeloid origin (27–35).
In mammals, the C3aR is the only member of the rhodopsin
family of seven-transmembrane, G protein-coupled receptors with
an unusually large second extracellular loop (ECL2) between the
fourth and fifth transmembrane regions (TM4 and TM5) (3). It is
*Department of Pathobiology, University of Pennsylvania School of Veterinary Med-
icine, Philadelphia, PA 19104;†Scottish Fish Immunology Research Center, Univer-
sity of Aberdeen, Aberdeen, Scotland;‡Cardiology Department, Hospital de Sant Pau,
Barcelona, Spain;§Western Fisheries Research Center, Seattle, WA 98115; and¶Unit
of Animal Physiology, Department of Cell Biology, Physiology, and Immunology,
Universitat Autonoma de Barcelona, Bellaterra, Spain
Received for publication January 20, 2005. Accepted for publication April 15, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Science Foundation Grant MCB-0417078, the
National Research Initiative of the U.S. Department of Agriculture Cooperative State
Research, Education and Extension Service, Grant 2004-01599; a contract from the
European Community (Q5RS-2001-002211); and Grant AGL2000-0349 from the
Ministerio de Ciencia y Tecnologı ´a.
2H.B., W. T., HM. L., H.J., and L.J. contributed equally to this study.
3Address 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,
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
worth noting that there appears to be very little sequence homol-
ogy of this loop among species. Interestingly, studies of guinea pig
C3aR have identified two alternatively spliced receptors, lacking
34 residues of the large ECL2. No functional differences could be
found in the expressed guinea pig spliced C3aR products (36).
Little is known about the evolution of C3aR and C5aR. How-
ever, anaphylatoxin activity has been demonstrated in primitive
invertebrate species, where C3a-like peptides have been shown to
induce hemocyte chemotaxis in tunicates, thereby implying the
presence of a C3a-like receptor in these animals (37, 38). A recent
report in trout showed for the first time in teleosts the presence of
three C3a molecules generated from three trout C3 isoforms (C3-1,
C3-3, and C3-4). Each of the three C3a isoforms stimulated to a
significant degree the respiratory burst of trout head kidney leu-
kocytes, suggesting that teleost fish contain a C3aR (39). Recently,
recombinant trout C5a has been produced and shown to play a
prominent role in inducing leukocyte chemotaxis (40, 41) and re-
spiratory burst (40). Thereafter, a C5aR was identified in rainbow
trout, representing the first cloned (42, 43) and functionally char-
acterized anaphylatoxin receptor in a nonmammalian species (42).
It is well known that C3aR and C5aR share a high degree of ho-
mology, to the extent that it has been hypothesized that both re-
ceptors represent the duplication products of a single ancestral re-
ceptor. Thus, the presence of a bona fide C5aR in teleosts is
relevant because it may be indicative of the existence of C3aR in
these animal species. Thus, this study was initiated to explore the
above-mentioned hypothesis with the idea of finding a homologue
of C3aR in rainbow trout. In this study we report the character-
ization of a trout molecule whose primary structure appears to be
highly similar to that of mammalian C3aRs (MC3aRs).4However
this trout C3a-like receptor (TC3aR) was found to lack a signifi-
cant portion of the large extracellular loop between TM4 and TM5
characteristic of MC3aRs, which is substituted instead by a much
smaller loop. We also demonstrate the ability of this trout receptor
to interact with trout C3a.
Materials and Methods
Rainbow trout (100–200 g) were obtained from Limestone Springs Fish
Farm. Fish were maintained in aquarium tanks using a water recirculation
system involving extensive biofiltration, UV sterilization units, and ther-
mostatic temperature control. Water temperature was maintained continu-
ously at 12–14°C.
cDNA cloning of TC3aR and sequence phylogenetic analysis
Trout cDNA was generated from trout liver as previously described (42)
using an Oligotex Direct mRNA kit (Qiagen), according to the manufac-
turer’s recommendations. mRNA (2.0 ?g) was reverse transcribed to neg-
ative strand cDNA with oligo(dT) (0.05 ?g/?l) and 40 U of SuperScript
reverse transcriptase II (Invitrogen Life Technologies) for 1 h at 42°C. A
primer set was designed on the basis of a 729-bp established sequence tag
(EST; GenBank accession no. CA373689) from rainbow trout (Oncorhyn-
chus mykiss) that was found to be homologous in sequence to mammalian
C3aRs. The forward primer (5?-TCAAAGATGGGGGACAACA-3?) and
the reverse primer (5?-CAGACAGCGATGACCAGTA-3?) were used to
generate a 713-bp DNA fragment using the proofreading enzyme Pfu
Turbo (Stratagene) according to the manufacturer’s recommendations and
with 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.
This fragment was also used as a probe for Northern and Southern blotting.
PCR products were cloned into a TOPO zero blunt vector (Invitrogen Life
Technologies) and sequenced with a 3100 DNA analyzer (Applied Bio-
systems). Consensus sequences were generated from comparisons of re-
peated amplifications from trout liver mRNA using SeqMan and MegAlign
(DNA Star) software. The full-length cDNA of TC3aR was obtained by
performing 5? and 3? RACE using SMART cDNA prepared from liver
tissues, as described by Wang et al. (44). 3? RACE using a forward primer
(5?-GGGACAACATGGATTTCTCAG-3?) based on our initial TC3aR
EST yielded a 1463-bp fragment. When sequenced, the 3? end was found
to possess a stop codon, a 3?-untranslated region, and a poly(A) tail. 5?
RACE was performed using a primer corresponding to the 3? untranslated
region (5?-CCAACAGCTTTACACAAAACGCCATC-3?) and yielded an
additional 13 bp located 5? to the original EST. Sequence alignments and
the phylogenetic tree were generated using the Clustal X software package
(45). TM regions in TC3aR were predicted using TMpred software (46).
TRIzol reagent (Invitrogen Life Technologies) was used to obtain the total
RNA from various tissues and leukocytes of rainbow trout. RNA was quan-
tified, and 10 ?g/lane was size-fractionated on agarose-formaldehyde gels
and transferred to nylon-supported nitrocellulose membranes (Bio-Rad) by
capillary blotting. The blot was thereafter exposed to UV cross-linking to
fix the RNA to the membrane. The 713-bp cDNA corresponding to our
initial TC3aR EST was gel purified and radiolabeled with [32P]dCTP using
the Ready-to-Go labeling system (Amersham Biosciences) and was puri-
fied using ProbQuant G-50 microcolumns (Amersham Biosciences). The
blot was prehybridized in warm Express hybridization solution (BD Clon-
tech) at 68°C for 30 min in a hybridization oven (Problot 6; Labnet). The
blot was hybridized at 68°C for 60 min in Express hybridization solution
with 1–2 ? 106cpm of labeled probe/ml. After hybridization, the blot was
rinsed three times in 2? SSC with 0.1% SDS for 30 min at room temper-
ature, then washed in 0.1? 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) in an autoradiography cassette (Fisher Sci-
entific). The expression of TC3aR RNA was normalized for equal loading
and transfer to 28S RNA.
Genomic DNA (10 ?g) isolation and blotting procedures have been pre-
viously described (42). For Southern blotting, the 713-bp cDNA fragment
corresponding to our initial EST was generated by PCR. The gel-purified
PCR product was then randomly labeled (Amersham Biosciences) with
[32P]dCTP and used as a probe (65°C) under stringent conditions (0.25?
SSC/0.25% SDS, 64°C final wash). The blot was exposed to film for
Generation of Abs against TC3aR
A 20-aa peptide corresponding to the N-terminal region of TC3aR (EHYG-
NFSENYVTESYGEFDC) was synthesized by Biosynthesis. Matrix-as-
sisted laser desorption mass spectrometry was used to determine the purity
of the peptide. The synthesized peptide was coupled to keyhole limpet
hemocyanin by the glutaraldehyde method and was used to raise Abs in
rabbits (Biosynthesis). Ab titers were determined by ELISA. The Ig frac-
tion of the antiserum was first purified using a HiTrap protein G column
according to the instructions of the manufacturer (Amersham Biosciences).
Thereafter, specific Abs against the TC3aR peptide were purified by af-
finity chromatography using the synthetic peptide coupled to cyanogen
bromide-activated Sepharose (Amersham Biosciences).
Isolation of PBLs and head kidney leukocytes (HKLs)
PBLs were isolated as previously described (42). Briefly, blood was col-
lected from trout through the caudal vessel using a heparinized syringe and
a 21-gauge needle. After extraction, the blood was immediately diluted
(1/5) with EMEM (American Type Culture Collection) supplemented with
100 U/ml penicillin, 100 ?g/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) density gradient and cen-
trifuged at 400 ? 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 sup-
plemented with 100 U/ml penicillin and 100 ?g/ml streptomycin. HKLs
were isolated as previously reported (39).
Flow cytometric analysis
Flow cytometric analysis of PBLs and HKLs was performed using condi-
tions previously described (42). Briefly, 1 ? 106cells were suspended in
1? PBS/2% FCS and incubated for 30 min at room temperature with
affinity-purified rabbit anti-TC3aR, alone or in combination with anti-trout
4Abbreviations used in this paper: MC3aR, mammalian C3aR molecule; ECL2, sec-
ond extracellular loop; [Ca2?]i, intracellular calcium; EST, established sequence tag;
HKL, head kidney leukocyte; ICD, intracellular domain; IF, immunofluorescence;
PKC, protein kinase C; TC3aR, trout C3aR; XC3aR, Xenopus C3aR; TM, transmem-
2428CLONING AND CHARACTERIZATION OF FIRST NONMAMMALIAN C3aR
IgM (mAb 1.14 provided by Dr. G. Warr, Medical University of South
Carolina, Charlestown, SC) or anti-trout thrombocyte (mAb28.D7) mAbs.
As a negative control, cells were incubated with Abs purified from preim-
munized rabbit serum or control mouse IgG. After two washes in PBS/2%
FCS, cells were stained with either FITC-conjugated anti-rabbit IgG or
allophycocyanin-conjugated anti-mouse IgG. Cells were washed two
more times, and cell cytometric analysis was performed using a standard
FACScan (BD Biosciences). For each sample, 20,000 individual cells were
analyzed, and the resulting data were analyzed using the program
CellQuest (BD Biosciences).
Indirect immunofluorescence microscopy
Indirect immunofluorescence microscopy was performed using cells
stained with anti-TC3aR in a similar manner as described for cell cyto-
metric assays, with an additional step of incubation with Hoechst stain
are indicated by upward and downward arch symbols, respectively. Residues important for C3aR function are shown in bold and include the following:
Tyr200(trout numbering) and the PKC recognition domain in the trout sequence (FKSQRA). The serine/threonine C-terminal phosphorylation sites in trout
and Xenopus are also in bold (see Results and Discussion). Predicted N-glycosylation sites are circled. Asterisks denote identities in all sequences, and 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 TM1 and the C-terminal valine of the trout sequence, respectively. The peptide sequence used to generate polyclonal Abs (at the N
terminus of the trout sequence) is boxed.
Amino acid comparisons of TC3aR and XC3aR with MC3aR sequences. Putative TMs are overlined. Extracellular and intracellular domains
2429 The Journal of Immunology
(according to the manufacturer’s recommendations) to visualize the cell
Generation of TC3a and C3adesArg
TC3a was generated as previously described (39) from C3-1, the most
active and abundant TC3 isoform (47). Briefly, trout C3-1, Bf/C2, and
factor D molecules were purified from trout serum as described previously
(47, 48). To produce the C3a anaphylatoxin, purified TC3-1 (5 mg), trout
factor B/C2 (250 ?g), and trout factor D (30 ?g) were incubated for 30 min
at room temperature in the presence of 5 mM Mg2?in PBS. To purify the
C3a fragment, the total reaction mixture (0.8 ml) was passed through a
Superdex 200 gel filtration column (Amersham Biosciences) equilibrated
with PBS, pH 7.4. The purity of the C3a was determined by SDS-PAGE
and N-terminal sequencing. In mammals, the C-terminal Arg of C3a is
critical for several of its functions, including its ability to induce intracel-
lular calcium mobilization in leukocytes and other cell types. To remove
the C-terminal Arg from the TC3a, this anaphylatoxin was treated with
carboxypeptidase B (20%, w/w) for 1 h at room temperature, then purified
on a Superdex 75 gel filtration column (Amersham Biosciences) equili-
brated with PBS, pH 7.4 (39).
Measurement of intracellular calcium [Ca2?]imobilization by
Trout leukocytes (HKLs) were incubated with the acetoxymethyl ester of
the fluorescent calcium indicator fluo-3 (5 ?M) for 15 min. Cells were then
washed twice with Tyrode’s solution and left on coverslips for cell attach-
ment and de-esterification of fluo-3 for at least 30 min before experiments
were begun. Fluorescence was measured using a confocal microscope
(Leica SP2; AOBS) with the excitation beam at 488 nm attenuated to 2.5%.
Fluorescence emission was collected between 500 and 650 nm. Experi-
ments were begun by flushing the cells with control Tyrode’s solution to
remove debris and nonsticking cells. Baseline fluorescence was recorded in
the control solution for at least 10 min before C3a (5 nM) was added alone
or in combination with anti-TC3aR IgG or the control preimmune IgG. To
determine the time course of the action of C3a with or without Ab, fluo-
rescence was measured in 10–15 randomly selected cells every 30 s over
a 15-min period. The fluorescence intensity was expressed as a percentage
of the baseline fluorescence (control) before addition of C3a with or with-
out Ab, and the peak fluorescence was measured after addition of C3a
alone, C3a (5 nM) with the control preimmune IgG (40 nM), or C3a with
anti-TC3aR IgG (40 nM). For statistical evaluation of the results, values
from each animal were averaged, giving one value for each condition per
animal. Statistical analysis was performed on the average values from each
animal, with n representing the number of animals. Student’s t test for
unpaired samples was used, and differences were considered statistically
significant at p ? 0.05.
Isolation and sequence analysis of TC3aR cDNA
We originally identified a 729-bp EST from rainbow trout that
showed a high degree of homology to mammalian C3aR. Using 5?
and 3? RACE, we obtained a 1476-bp product (TC3aR) that in-
cluded an initiation site, an open reading frame encoding for 364
aa, and a 3? polyadenylation signal. When aligned with C3aR from
human, mouse, rat, and guinea pig, a significant degree of homol-
ogy was observed throughout the molecule (Fig. 1), with one ex-
ception: a region spanning ?137 aa in MC3aRs was noticeably
absent in TC3aR. Upon additional analysis, it was shown that this
portion of sequence corresponded to a large segment of the ECL2
of mammalian C3aR (represented in Fig. 2). C3aR in mammals is
the only rhodopsin receptor known to have such a large ECL2 (49,
50). In the alignment of Fig. 1, we also included a sequence from
Xenopus tropicalis (a diploid frog) highly homologous to TC3aR
and MC3aR. Like TC3aR, the Xenopus molecule (XC3aR) lacked
a large portion of the ECL2 (113 residues), although this region
contained 24 more residues than the ECL2 of TC3aR. The XC3aR
sequence was found after a multiple alignment analysis of TC3aR
and MC3aRs sequences with available Xenopus genomic scaffolds
located at the Ensembl Genome Browser web site of the Wellcome
Trust Sanger Institute (?www.ensembl.org/?). More excitingly, in
silico analysis of the Xenopus scaffold containing the XC3aR gene
showed that XC3aR and MC3aR genes were located in syntenic
regions, further supporting designation of the trout and Xenopus
molecules as C3aR (Fig. 3). We were unable to find the corre-
sponding syntenic region in the genome of zebrafish, although we
did find a molecule highly homologous to TC3aR and XC3aR in a
different region of the zebrafish genome (data not shown). This
lack of synteny in regions of the genome between the zebrafish and
Xenopus (or mammals) has been observed for other genes (i.e.,
genes within the MHC region (51)). It should be stressed that
exhaustive in silico analysis of the genomes of Xenopus or ze-
brafish did not yield a C3aR-like molecule with an extra large
ECL2 similar in size to that of mammals. Similarly, analysis of the
trout or salmon ESTs (?244,837 ESTs) deposited at the National
Center for Biotechnology Information or at the Institute for
Genomic Research did not yield either a C3aR-like molecule with
an extra large ECL2. All the above results combined suggest that
C3aR in fish and amphibians lacks the additional extra piece of
sequence that is uniquely present in the ECL2 from all MC3aR
Hydropathy analysis confirmed that TC3aR and XC3aR did pos-
sess seven-transmembrane domains normally associated with
C3aR (and all rhodopsin receptors) in higher vertebrates (Fig. 4).
As stated above and shown in Figs. 1 and 2, there was a noticeable
difference in the amount of residues of ECL2 between TM4 and
TM5 of TC3aR and XC3aR compared with the same region of
MC3aR molecules. In humans, the entire ECL2 contains 175 aa
(50), whereas in TC3aR and XC3aR, the area spanning the ECL2
is significantly shorter, comprising 36 and 62 aa, respectively (Fig.
2). It is worth noting that MC3aR is fully functional even after
deletion of 65% of the residues of ECL2 (52). In fact, mutagenesis
studies have shown that the crucial residue (Tyr174in humans)
required for the interaction of C3aR with C3a is located at the
beginning of the ECL2 (53). Significantly, this critical residue is
conserved in the ECL2 of TC3aR and XC3aR. When aligned with
other MC3aRs, this tyrosine is located two amino acids down-
stream from Cys172(human numbering). In TC3aR and XC3aR, the
the ECL2 in trout, Xenopus, and human C3aR. The ECL2 of C3aR is
shown between TM4 and TM5. Numbers in parentheses indicate the
amount of residues within the ECL2.
Schematic structure of C3aR showing the different sizes of
2430 CLONING AND CHARACTERIZATION OF FIRST NONMAMMALIAN C3aR
corresponding tyrosine is also two residues from Cys198(trout num-
bering). It should be noted that mammalian and trout C5aR also pos-
sess a homologous tyrosine, but in both cases this residue is located
four amino acids downstream from their respective cysteines.
To find meaningful percentages of amino acid identities be-
tween TC3aR and the mammalian and Xenopus C3aR sequences,
the sequence alignments were performed excluding the extra ?137
residues of the ECL2 from MC3aRs or excluding the extra 26
residues of the ECL2 from XC3aR lacking in TC3aR. The analysis
showed that TC3aR presented a significantly greater degree of ho-
mology to C3aR than to C5aR molecules or other members of the
rhodopsin gene family. Thus, TC3aR showed 40% identity to
XC3aR, 38.3% identity to murine C3aR, 34.0% identity to human
orphan receptor ChemR23, 32% identity to human formyl peptide
receptor 1, 29.8% identity to TC5aR, and 25.3% identity to
Further analysis of the structure of TC3aR and XC3aR indicated
that similar to MC3aRs and MC5aRs, these sequences possessed a
serine/threonine-rich C terminus in which these residues may rep-
resent phosphorylation sites that become modified as a result of
ligand stimulation (36). Conservation of post-translational modi-
fications, including N-linked glycosylation, was also observed. To
date, all characterized MC3aRs have been found to possess three
to eight glycosylation sites, in contrast with the one or two con-
tained in C5aR molecules. It is interesting that one to four (de-
pending on the species) of these glycosylation sites are localized in
the ECL2 (36). TC3aR and XC3aR were both found to possess
four potential glycosylation sites (Fig. 1). Despite their consider-
ably shorter ECL2, both TC3aR and XC3aR still contained two
N-linked glycosylation sites within that region. This observation is
significant, because it indicates a higher degree of conservation of
TC3aR to C3aR rather than to MC5aR, in which the ECL2 is
devoid of glycosylation.
In our previous characterization of TC5aR, we found that TMs
among C5aR molecules were more highly conserved than their
intra/extracellular regions (42). This does not appear to be the case
with TC3aR. Although TM2 and TM3 showed the highest degree
of conservation among all C3aR TMs (61.5 and 68.2%, respec-
tively), there also existed a high degree of conservation of all three
C3aR intracellular domains (ICD), with sequence identity values
ranging from 46.7% (ICD3) to 85.7% (ICD1). The extracellular
domains, especially the N terminus and ECL2 regions, remained
the least conserved of the receptor, with ?6% sequence identity.
It has been hypothesized that C3aR serves as a putative substrate
for protein kinase C; in all MC3aRs, this motif has been found to
be conserved as XKSXXKX (36). Although TC3aR does not pos-
sess this exact motif, it contains a sequence signature that is con-
sistent with protein kinase C (PKC) recognition (FKSQRA) (54),
which is also located in IC3. PKC recognition domains analogous
to those mentioned above in mammalian and trout C3aRs were
absent in the IC3 of all cloned C5aR, including TC5aR.
In Fig. 5, a phylogenetic tree was constructed using TC3aR,
XC3aR, MC3aR, C5aR, TC5aR, along with other mammalian rho-
dopsin receptors. Both trout and Xenopus C3aR molecules clus-
tered with the MC3aR molecules (Fig. 5). The tree composite also
suggests that TC3aR is the most ancestral of all C3aR molecules.
Northern blot analysis of TC3aR
Total RNA was obtained from a variety of trout tissues and leu-
kocytes and was separated by formaldehyde agarose gel electro-
phoresis. After transfer to nylon membranes, TC3aR RNA expres-
sion was detected using a 713-bp P32labeled probe, corresponding
Xenopus (X). The scaffold was found at the Ensembl Genome Browser web site of the Wellcome Trust Sanger Institute (?www.ensembl.org/?). The scaffold
spans a genomic fragment of 500 kb. XC3aR is contained within bases 120,206–122,817. The entire scaffold was found to be syntenic with the region from
human chromosome 12 that contains the human C3aR gene (H; lower part of the figure). This region spans from 6.8 to 8.2 Mb of human chromosome
12. The lines connecting the human and Xenopus genomic regions link the gene orthologs between the two species. Below the gene name, is indicated either
the GeneScan number (upper part; Xenopus scaffold) or the Ensembl gene identification number (lower part; human chromosome 12 region). The graphic
indicates the order in which the genes are positioned in the genomic regions of Xenopus and human; however, the distances from gene to gene are not
The genomic regions containing Xenopus and human C3aR are syntenic. The upper part of the figure represents genomic scaffold 811 from
2431The Journal of Immunology
to our original TC3aR EST. In all samples, no more than one band
was observed, which was estimated to be ?2.4 kb (Fig. 6). In
mammals, C3aR mRNA has been detected at sizes ranging from
2.1 kb (human and mouse) (17, 22) to 3 kb (guinea pig) (36).
Normalization of the TC3aR signal with 28S rRNA indicated that
expression was strongest in blood leukocytes. A significant level of
TC3aR message was found in the rest of the samples tested within
a relatively short period of exposure (1 day). It is difficult to state
whether this expression was due to unavoidable blood contamina-
tion of the sampled organs or was the true expression of TC3aR in
these tissues. In this regard, the expression of TC3aR in gills (an
organ rich in blood leukocytes) was found to be comparable to that
of PBLs (data not shown).
Southern blot analysis
As was the case for Northern blot analysis, the 713-bp probe was
labeled with32P and used as a probe for Southern blotting. This
probe was used because analysis of genomic DNA by PCR indi-
cated that no introns were present within this fragment. Each of the
three restriction enzymes yielded two or three different digestion
products (Fig. 7). Fish 2 and 3 showed additional bands in the blot,
providing evidence of allelic variation in these animals. Taking
into account that no restriction sites exist within the probe for the
enzymes used in the digestion, the Southern blot data appear to
suggest that two TC3aR genes exist in the trout genome. The later
was almost expected due to the quasi-tetraploid nature of rainbow
trout. It should be pointed out, however, that screening of trout
liver and head kidney libraries by RT-PCR or colony blotting us-
ing the 713-bp EST fragment failed to yield other variants or iso-
forms of TC3aR. Moreover, the Northern blot analysis detected
only a single band. Significantly, multiple sequence alignment
analysis of our TC3aR sequence with all ESTs comprised at the
Institute for Genomic Research and National Center for Biotech-
nology Information Unigene EST indexes (?155,000 trout ESTs)
did not yield any EST significantly similar in primary and second-
ary structures to TC3aR. Although not definitive, these facts sug-
gest that only one of the two TC3aR genes is expressed.
Binding of anti-TC3aR to PBLs
Polyclonal Abs were generated against a 20-aa peptide corre-
sponding to a portion of the putative N-terminal extracellular
region of TC3aR (boxed residues in Fig. 1). Anti-peptide
specific Abs were affinity purified using a column to which the
peptide had been coupled. When used for flow cytometric
analysis, ?83% of all PBLs were stained with the Ab, as
indicated in the shift of fluorescence shown in the histogram in
Fig. 8A. Incubation of the Ab preparation with a molar excess
of the TC3aR peptide inhibited ?90% of the Ab staining of
PBLs, providing additional evidence of the specificity of the Ab
(data not shown). As shown in Fig. 8, staining was localized to
two distinct cell populations, designated R1 and R2. The cells
in R1 (?51% of the PBLs), displayed low forward and side
scatters and showed the strongest staining. The R2 population
(?25% of the PBLs) exhibited the highest forward and side
scatters (composed of granulocytes in trout), although they
stained to a lesser degree compared with the R1 population.
Granulocytes in mammals have also been shown to express
C3aR (35, 55). The R3 population (?17% of the PBLs) repre-
sented the negative cells, because these cells displayed the same
fluorescence intensity as those stained with the preimmune
polyclonal rabbit IgG (Fig. 8D). Costaining analysis using the
anti-TC3aR and an anti-trout thrombocyte mAb showed that
?95% of the TC3aR-negative cells (shown in R3, Fig. 8D)
were, in fact, thrombocytes (data not shown). As expected, the
anti-TC3aR did not stain trout erythrocytes (data not shown).
The fact that the anti-TC3aR did not stain thrombocytes and
erythrocytes supports the specificity of the anti-TC3aR and is in
agreement with the lack of staining of thrombocytes and RBCs
in humans when using anti-human C3aR (55).
Costaining analysis using the anti-TC3aR in combination
with a mAb specific for trout IgM (mAb 1.14) that stains B
lymphocytes in trout (56, 57) showed the presence of TC3aR in
all B cells (Fig. 8F). This pattern was displayed in all fish
analyzed (n ? 8). The double-positive cells (representing
?36% of the PBLs) displayed the same low forward and side
scatter properties of the R1 population in Fig. 8 (data not
shown). This was expected, because B cells are small agranular
cells. It should be noted that this double-positive population
showed some variability among different individuals, ranging
from ?29 to 55% of the PBLs analyzed.
Trout HKLs displayed a very similar staining pattern, in which
B cells showed the highest binding to anti-C3aR, and ?95% of
granulocytes stained, although once again, to a lesser extent (data
C3aR. TM plotting of C3aR amino acid sequences from trout (upper
panel), Xenopus (middle panel), and human (lower panel) were obtained
using the TMpred program, based on the method of Hofman and Stoffel
(46). TM domains 1–7 and ECL2 are also indicated.
Prediction of TM regions of TC3aR, XC3aR, and human
2432 CLONING AND CHARACTERIZATION OF FIRST NONMAMMALIAN C3aR
Immunofluorescence (IF) microscopy
IF microscopy using the affinity-purified Abs indicated that TC3aR
is expressed on the cell surface of trout granulocytes and lympho-
cyte-like cells of PBLs (Fig. 9) and HKLs (data not shown), and
that the staining pattern was punctuated and patchy (Fig. 9). In
agreement with the flow cytometric results, it could be observed
that in lymphocytes the patchy areas were generally more abun-
dant than in granulocytes (Fig. 9). A similar scattered pattern of
C3aR staining has been shown on human PMN (55) and astrocytes
Inhibition of C3a-mediated intracellular calcium [Ca2?]i
mobilization by anti-TC3aR Abs
In mammals, it is well known that C3a induces increases in
[Ca2?]iin a variety of cells (59–62). We determined first
whether TC3a could have a similar effect on trout leukocytes,
then we investigated the ability of the anti-TC3aR to inhibit
potential C3a-mediated increases in [Ca2?]i. To evaluate the
[Ca2?]i-mobilizing capacity of TC3a, its effect on fluo-3 fluo-
rescence was measured, as shown in Fig. 10. Because analysis
of [Ca2?]iusing confocal microscopy have never been per-
formed with trout leukocytes, we first optimized the experimen-
tal conditions to obtain stable baseline fluorescence measure-
ments over time. This led to measurements in which the average
baseline fluorescence in control cells increased only by 1.5 ?
2.1% over a 10-min period (85 cells from six trout). The middle
panel of Fig. 10A shows one example of fluorescence from
control cells after 10-min exposure to control Tyrode’s buffer.
Most of the cells showed no or very little fluorescence. In the
right panel of Fig. 10A, the increase in cell fluorescence
induced by exposure to TC3a (5 nM) is clearly shown. As
observed in that figure, a majority of the cells were stimulated
(turned to green) by C3a, representing 68 ? 4% of the cells
examined (59 cells from a total of three fish). The left panel in
and other related members of the rhodopsin
gene family. Amino acid alignments were per-
formed using Clustal X, which was used to
generate an unrooted neighbor-joining tree.
Numbers on the branches indicate total recov-
ery from 1000 bootstrap replications. The phy-
logenetic tree was created using alignments
performed with the entire mammalian C3aR
sequences. Accession numbers used to con-
struct the tree are as follows: TC5aR,
AY438032; 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; TC3aR, AJ616902;
human C3aR, AAH20742; guinea pig C3aR,
O88680; rat C3aR, O55197; mouse C3aR,
AAH03728; mouse Dez, NP_032179; human
ChemR23, CAA75112; human formyl peptide
receptor 1 (FPR1), P21462; human FPR1,
NM_002029; mouse FPR1, NP_038549; and
mouse FPRL1, NP_032068.
Phylogenetic tree of TC3aR
micrograms of total RNA from the indicated tissues of rainbow trout was
electrophoresed, blotted onto a nitrocellulose membrane, and hybridized
with a DNA probe corresponding to a 713-bp fragment of TC3aR.
Ethidium bromide-stained 28S is shown as a loading control.
Tissue-specific expression of TC3aR by Northern blot. Ten
rainbow trout was digested with EcoRI, EcoRV, and HindIII. The blot was
then hybridized with a DNA probe corresponding to a 713-bp fragment of
TC3aR. Values on the right of the blot indicate kilobases.
Southern blot analysis. Genomic DNA from four individual
2433 The Journal of Immunology
Fig. 10A shows a transmission (brightfield) image of the mi-
croscope field with the cells selected for fluorescence analysis.
Fig. 10B shows the time course of the increase in fluo-3
fluorescence after addition of 5 nM C3a. To verify that the
effect of C3a was specific, the action of the desarginated form
of C3a (C3adesArg) on [Ca2?]imobilization was also analyzed.
In contrast to C3a, C3adesArgdid not increase [Ca2?]iin trout
leukocytes, suggesting that the stimulatory effect was a direct
effect of C3a (Fig. 10C). This result is in agreement with the
situation in mammals, in which C3adesArgdoes not have an
influence on [Ca2?]imobilization (3, 63).
To verify that the cloned trout receptor (TC3aR) has the capac-
ity to interact with the C3a ligand, we investigated whether the
anti-TC3aR could block the C3a-mediated increase in [Ca2?]ithat
is shown in Fig. 10C. To this end, cells were exposed to 5 nM C3a
in the presence or the absence of an 8-fold excess of the anti-
TC3aR IgG. Preimmune IgG was used as a control. Fig. 10C
shows that the anti-TC3aR almost completely abolished the stim-
ulatory effect of C3a in inducing increases in [Ca2?]i, whereas
preimmune IgG had no effect. Thus, the inhibitory action of the
anti-TC3aR in C3a-mediated increases in [Ca2?]isupports the idea
that TC3aR is a bona fide C3aR.
Our current knowledge of the structure and function of C3aR mol-
ecules comes from the study of MC3aRs, with nothing being
known about the evolutionary origins of this important proinflam-
matory molecule. Thus, up to this point, no C3aRs have been iden-
tified in nonmammalian species. The present study was therefore
undertaken to identify a homologous receptor in an evolutionarily
old vertebrate species, with the goal of better understanding the
important structural elements and functions that have been con-
served throughout the evolution of this receptor.
Recent studies by us have shown that teleost fish contain C3a
and C5a anaphylatoxins that play important roles in chemotaxis
and respiratory burst processes, implying the presence of anaphy-
latoxin receptors in these species. We (42) and others (43) have
recently reported the characterization of a bona fide TC5aR in
rainbow trout. These findings suggested that the duplication event
giving rise to C5aR and C3aR from a common ancestor might have
occurred before the emergence of teleost fish.
In this study we have characterized a 364-residue molecule in
rainbow trout that is highly homologous to all known MC3aRs.
Several lines of evidence indicate that the trout molecule reported
in this study represents a true C3aR: 1) the overall primary and
secondary structures of TC3aR show a significantly higher degree
of homology to C3aR than to C5aR or other members of the large
rhodopsin family of seven-transmembrane, G protein-coupled re-
ceptors; 2) the phylogenetic tree composite illustrates that TC3aR
clustered with XC3aR and all known mammalian C3aR molecules;
3) the fact that, similar to TC3aR, the XC3aR sequence lacked a
large piece (113 residues) of the ECL2 along with evidence that
the XC3aR gene was found to reside in a genomic region syntenic
to the region containing C3aR in mammals; and 4) functional ev-
idence showing that anti-TC3aR Abs inhibited C3a-mediated
[Ca2?]imobilization in trout leukocytes.
stained with affinity-purified rabbit anti-TC3aR (3 ?g/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–E, Forward (FSC-H) and side (SSC-H) scatter analyses of positively (R1
and R2) and negatively (R3) stained cells after incubation with anti-
TC3aR. F, Double staining of trout PBLs with anti-TC3aR and anti-trout
surface IgM. Cells were costained with affinity-purified rabbit anti-TC3aR
(3 ?g/ml) and anti-trout IgM (3 ?g/ml). One experiment of eight per-
formed is shown.
Binding of anti-TC3aR to trout PBLs. A, Histogram of cells
were stained with 3 ?g/ml affinity-purified rabbit anti-TC3aR (green flu-
orescence) and with Hoechst staining to visualize the nucleus (blue fluo-
rescence). Asterisks denote control cells incubated with preimmune rabbit
IgG (3 ?g/ml).
Indirect IF analysis of trout PBLs with anti-TC3aR. Cells
2434CLONING AND CHARACTERIZATION OF FIRST NONMAMMALIAN C3aR
A significant feature of TC3aR was the lack of 137 residues of
the mammalian ECL2 region, which is unusually large in MC3aR
molecules. MC3aR is the only member of the rhodopsin family of
seven-transmembrane, G protein-coupled receptors with an unusu-
ally large ECL2. It is striking, however, that the only residue in the
ECL2 that appears to be pivotal for the interaction of C3a with
C3aR (Tyr174) in humans (53) is positioned at the beginning of the
loop, and it is conserved in the trout and Xenopus C3aR sequences.
Because the extra piece (137 residues) of the loop present in mam-
malian ECL2 does not seem to play a role in C3a binding, it has
been proposed that it might bind to additional ligands and/or it
might associate with surrounding cell surface proteins (53). Com-
bined with our results, these findings suggest that the ancestral
molecular architecture of C3aR did not include this extra piece of
sequence, which was probably acquired later in evolution, after the
appearance of amphibians, but before the emergence of mammals.
Northern blot analysis showed that PBLs were the most plentiful
source of TC3aR message. In mammals, C3aR is expressed in a
wide variety of organs, although tissue distribution varies consid-
erably between species. In guinea pigs, C3aR has been found to be
expressed primarily in macrophages and spleen, with residual ex-
pression in liver, brain, and lung (36). However, in mice, C3aR is
expressed mainly in heart and lung tissue, with no significant ex-
pression in spleen (22). This contrasts with human C3aR, which is
found to be primarily expressed in placental, heart, and lung tis-
sues, with no appreciable levels found in brain (17). The high
expression of C3aR in PBLs was also confirmed at the protein
level, using Abs against TC3aR. Significantly, flow cytometric
analysis showed a high degree of TC3aR expression in trout B
cells, which suggests an important role for this receptor in fish
immunity. The presence of C3aR in mammalian B cells is incon-
clusive at this time. Although two studies using anti-human C3aR
Abs showed no C3aR staining in circulating B cells (35, 61), an-
other study demonstrated the presence of C3aR at the cDNA and
protein levels in human activated-tonsil derived B cells (9). A sim-
ilar situation was shown with regard to C3aR expression in human
T cells. Although unchallenged circulating T cells were shown to
be devoid of C3aR (35, 55), activated human T cells were dem-
onstrated to express a functional C3aR (64). Thus, it is possible
that the expression of C3aR in mammals depends on the activation
state of lymphocytes. Our data, however, seem to suggest that all
circulating lymphocytes in trout express TC3aR. The staining re-
sults obtained for TC3aR are very similar to those previously re-
ported for TC5aR (42). Like the anti-TC3aR, anti-TC5aR Abs
were shown to stain all B cells as well as the granulocyte popu-
lation of PBLs. In addition, anti-TC5aR, similar to anti-TC3aR,
did not stain the thrombocyte population, a finding in agreement
with the lack of staining of thrombocytes in humans when using
anti-human C3aR (55). It is worth noting that although several
studies have convincingly demonstrated that human platelets do
not express C3aR (65, 66), C3a has been reported to activate
guinea pig platelets (67), implying the presence of such receptors
in the platelets of these animals. It is therefore possible that in
mammals, the expression of C3aR in platelets is species specific.
In conclusion, our findings represent the first structural and
functional characterization of a C3aR in a nonmammalian species.
The data presented in this study support the hypothesis that if
C3aR and C5aR diverged from a common ancestor, then this event
occurred before the emergence of teleost fish. Given the new array
of roles recently demonstrated for C3a and C3aR in mammals
(13–16), one anticipates that the study of these molecules in fish
may identify unexpected functions of these molecules in higher
We thank Xinyu Zhao (Biomedical Imaging Core, School of Medicine,
University of Pennsylvania, Philadelphia, PA) for her excellent technical
assistance with the IF analysis for Fig. 9.
The authors have no financial conflict of interest.
mobilization in trout leukocytes. A. Trout C3a stimulates [Ca2?]imobili-
zation in leukocytes. Confocal images of Fluo-3 fluorescence in trout leu-
kocytes before (control) and 10 min after exposure of the cells to 5 nM
C3a. The left panel in A shows the brightfield image of the cells. Rectan-
gles enclose the selected cells where changes in fluorescence were fol-
lowed. B, Time course of the change in fluorescence after addition of TC3a.
The panel shows the average changes in fluorescence of the total number
of cells analyzed (10–15 cells) in a representative experiment (n ? 3). C,
An 8-fold excess of anti-TC3aR inhibits TC3a-mediated [Ca2?]imobili-
zation, although the preimmune IgG is not inhibitory. For comparison, the
effect of TC3a alone is shown. To show that the effect of TC3a was spe-
cific, the action of the desarginated form of C3a (desArg TC3a) on [Ca2?]i
mobilization was also analyzed. The values represent the average peak
fluorescence of all analyzed cells (10–15 cells/experiment; n ? 3) after
10-min incubation with TC3a in the presence or the absence of preimmune
IgG or anti-TC3aR IgG. The asterisk indicates a significant difference (p ?
0.01) between TC3a- and desArg TC3a-treated cells as well as between
preimmune IgG- and anti-TC3aR IgG-treated cells.
Anti-TC3aR inhibits TC3a-mediated intracellular calcium
2435The Journal of Immunology
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2437The Journal of Immunology