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CUTTING EDGE |
*
Institute of Medical Microbiology, Hannover Medical School, Hannover, Germany; and Departments of
Pulmonary Pharmacology and
Molecular Biology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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60% identical to
the huC3aR was isolated from a genomic DNA library and found to be
expressed in guinea pig heart, lung, and spleen. HEK-293 cells
cotransfected with this clone, and a cDNA encoding G
-16 specifically
bound (Kd = 1.6 ± 0.7 nM) and responded
functionally to C3a with an intracellular calcium mobilization
(ED50 = 0.18 ± 0.02 nM). Human rC4a weakly bound to
both the hu- and gpC3aR (IC50 > 1 µM). However, only
HEK-293 cells expressing the gpC3aR responded functionally to rC4a
(ED50 = 8.7 ± 0.52 nM), while cells expressing the
huC3aR did not (c 
1 µM). Thus, through an
interaction with the C3aR, huC4a may elicit anaphylatoxic effects in
guinea pigs but not in man. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Human C4a, first described in 1979, is regarded as the third anaphylatoxin (3) due to its structural similarity to C3a and C5a, its dependence on a carboxyl-terminal arginine residue for biologic activity, and its proinflammatory properties in guinea pigs. Although less active than C3a and C5a, in guinea pigs, human C4a induces smooth muscle contraction, increases vascular permeability (3), and induces granule secretion from platelets and O2- generation in macrophages (4, 5).
These biologic effects of C4a are subject to low dose desensitization (tachyphylaxis). Preincubation with substimulatory concentrations of C4a abrogates the functional response toward a subsequent 100% stimulus. In addition, cross-desensitization in guinea pig ileal contraction assays was observed between C4a and C3a, but not between C4a and C5a (3, 4). Based on these observations, C3a and C4a are thought to act on a common receptor. This view, however, has recently been questioned by Murakami and coworkers, who failed to detect desensitization of C3a-induced responses after pretreatment of guinea pig macrophages with human C4a (5). Furthermore, C4a did not inhibit binding of 125I-labeled C3a to guinea pig macrophages (5). These findings would indicate distinct and separate receptors for C3a and C4a in guinea pigs.
In man, convincing evidence for anaphylatoxic effects of human C4a is still missing. C4a has been reported to inhibit C3a-induced chemotaxis of macrophages (6), although at extremely low concentrations (10-16 M!). Also, C4a and C4a analogue synthetic peptides lead to a dose-dependent wheal and flare generation when injected into human skin (3, 7), although no negative controls were included in these experiments. Evidence for C4a effects in species other than man or guinea pig has not been reported.
Recently, the C3aR from man (huC3aR)4 (8, 9, 10) and mouse (11, 12) have been cloned. Stably transfected cell lines expressing these receptors and human neutrophils have been tested for functional response towards serum-purified human C4a and found to be completely unresponsive (11, 13). This would suggest the existence of a separate C4a receptor in man and mouse, on the premise that C4a is an anaphylatoxin in these species as well. However, no positive control demonstrating biologic activity of the C4a preparation used in these experiments was presented.
Highly purified human C4a is difficult to prepare and, due to the high C4a concentrations required in most test systems, even trace contamination with other biologically active molecules, especially C3a and C5a, may jeopardize the experiments, as has been reported in one of the above-mentioned investigations for serum-purified C4a (13). This possible contamination may well be the cause for some of the discrepant results presented above. Using human rC4a and cells functionally expressing a cloned guinea pig (gp) or huC3aR, we provide evidence that C4a functions as an agonist of the gpC3aR but not the huC3aR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Human C3a was obtained from Advanced Research Technologies (San Diego, CA), 125I-labeled human C3a from NEN-DuPont (Boston, MA), and rC5a from Sigma (St. Louis, MO). The C3a carboxyl-terminal analogue synthetic peptide (WWGKKYRASKLGLAR, (W63,W64)C3a(63-77) (14)) was obtained from Bachem (King of Prussia, PA), and the C3a synthetic peptide P117 and control peptide P251 were prepared as described (9). Oligonucleotides were obtained from MWG Biotech (Ebersberg, Germany). N-terminal hexahistidine-tagged rC4a or rC4a with an N-terminal methionine residue was expressed in Escherichia coli and purified to homogeneity. The binding and functional activities of these two preparations of rC4a were equivalent.
Guinea pig platelet release assay
Functional characterization of rC4a was performed in a guinea pig ATP release assay, as described (15). Guinea pigs from strains C2BB/R+ (C3aR positive) and C2BB/R- (C3aR-negative) from our own breeding colonies were used as platelet donors (16). Desensitization was measured by preincubation of the platelets with the deactivating stimulus (a concentration leading to <10% ATP release, determined empirically at the beginning of each experiment) for 5 min at 37°C and subsequent addition of a 100% stimulus of either C3a (c = 10 nM), rC4a (c = 4 µM), or rC5a (c = 250 nM).
Cloning of the guinea pig C3aR
A partial DNA sequence of the gpC3aR was obtained by PCR
amplification of genomic DNA using oligonucleotide primers derived from
regions conserved between the human and mouse C3aR sequences
(8, 9, 10, 11, 12). This fragment was used to screen a guinea pig
genomic DNA library in 
FIXII (Stratagene, La Jolla, CA). A clone
containing a genomic insert, which encoded the gpC3aR, was identified.
The open reading frame of this clone plus an extra 48 bp of genomic DNA
sequence at the 3' end was subcloned into pcDNA3 (Invitrogen, San
Diego, CA) and designated pSL94.
Receptor characterization
Competitive binding assays were performed essentially as
described (9). A microtiter plate-based calcium
mobilization assay, utilizing a fluorometric imaging plate reader
(FLIPR; Molecular Devices, Sunnyvale, CA), was used for the functional
characterization of HEK-293 cells transiently expressing either the
gpC3aR or huC3aR and G-16 (17). Briefly, cells were
transfected using Lipofectamine Plus reagent (Life Technologies,
Gaithersburg, MD), and the following day they were plated in
poly-D-lysine-coated 96-well black/clear plates (Becton
Dickinson, Bedford, MA). After 18 to 24 h, the medium was
aspirated from each well, and the cells were loaded with Fluo-3-AM
(Molecular Probes, Eugene, OR). At initiation of the assay,
fluorescence is read once every second for 60 s and then every
3 s for the following 60 s. Agonist was added at 10 s,
and the maximal fluorescence count above background after addition was
used to define maximal activity for that concentration of agonist.
FLIPR software normalizes fluorescence readings to give equivalent
readings for all wells at zero time.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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To avoid the problem of contamination in serum-purified human C4a
preparations, we expressed rC4a. As shown in Figure 1
A, human rC4a was
approximately three orders of magnitude less potent in a guinea pig ATP
release assay than C3a, but induced ATP release at high nanomolar
concentrations (ED50 
400 nM), comparable with the
ED50 of 600 nM determined previously for serum-purified C4a
in the serotonin release assay (4). Furthermore, in this
assay, complete cross-tachyphylaxis between human C3a and rC4a, but not
C5a, was observed (Fig. 1B). In addition, platelets of the
C3aR-negative guinea pig strain C2BB/R-, an inbred strain
of our own breeding colonies with a functional C3aR deficiency
(16), did not respond to either C3a or rC4a (data not
shown). These data demonstrate the functional activity of the rC4a and
confirm previous data on guinea pig cells showing cross-tachyphylaxis
between C3a and C4a (3, 4). The simplest explanation of
these data would be that C3a and C4a act through the same receptor.
However, postreceptor mechanisms may account for the observed
cross-tachphylaxis, as in the case of the FMLP-R and C5aR
(18). We, therefore, set out to clone a C4a-binding C3aR
from the guinea pig.
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The coding regions of many G protein-coupled receptors, including
the human C3aR (19), are not interrupted by introns.
Therefore, we cloned the gpC3aR directly from a genomic DNA library.
Using primer combinations conserved in the C3aR sequences of man
(8, 9, 10) and mouse (11, 12), a genomic DNA
fragment was amplified with high homology to the huC3aR. Using this
fragment as a probe to screen a guinea pig library, a genomic clone
was isolated with an open reading frame of 1428 bp, which encoded a
protein of 475 residues with a calculated molecular mass of 53,570 Da
and four potential N-glycosylation sites (Fig. 2). The sequence encodes a G
protein-coupled receptor with seven hydrophobic transmembrane domains,
a large second extracellular loop of 165 amino acid residues, and a
high homology to the C3aR sequences of man, mouse, and rat
(8, 9, 10, 11, 12, 20). However, only half of the residues are
conserved in all four known C3aR sequences (240/475 = 50.5%), and
only 37 of the 165 residues in the second extracellular loop (22.4%)
in the gpC3aR are found at the same position in the other C3aR
sequences. The peptide sequence of this gpC3aR was disclosed in a
recently published book (2); however, the nucleotide
sequence has not been published nor does it appear in GenBank, and no
functional or binding data have been presented. It is unlikely that
this receptor is a pseudogene, since by RT-PCR, expression of this
transcript was detected in guinea pig heart, lung, and spleen RNA (data
not shown).
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,
competitive displacement studies revealed the presence of a high
affinity receptor for C3a with an apparent Kd of
2 nM. As previously shown with the huC3aR, this binding was specific
for C3a, because 125I-labeled C3a could be displaced in
a dose-dependent manner by the synthetic C3a analogue peptide P117, but
not by the unrelated peptide P251 (9). In addition,
supramicromolar concentrations of rC4a (IC50 > 1 µM)
were able to competitively displace radiolabeled C3a from both the
guinea pig and huC3aR. Although human C4a is able to bind weakly to
both the gpC3aR and huC3aR, the affinity of this interaction is three
orders of magnitude lower than the affinity of C3a binding to the same
receptors.
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To compare the functional activity of the gpC3aR and
huC3aR, we cotransfected the cDNA for each receptor, together with a
cDNA clone encoding G-16, into HEK-293 cells and tested for
intracellular calcium mobilization in response to rC4a, rC5a, C3a, or a
C3a analogue peptide. Previously, we reported that cotransfection of
the huC3aR sequence with G-16 was necessary to render transfected
HEK-293 cells responsive to C3a (9). As shown in Figure 4A, cells expressing the
gpC3aR responded in a dose-dependent manner not only to C3a
(ED50 = 0.18 ± 0.02 nM) and the C3a peptide
(ED50 = 0.15 ± 0.01 nM), but also to rC4a
(ED50 = 8.7 ± 0.52 nM). Cells transfected with the
cDNA for the gpC3aR or G-16 alone did not respond to any of the
peptides tested (data not shown).
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B). Previously,
comparable results were obtained with stable cell lines expressing the
cloned huC3aR (13). The gpC3aR and huC3aR responded
differently to human rC4a as well as to the C3a synthetic peptide,
which was virtually equipotent with C3a vs the gpC3aR but approximately
an order of magnitude less potent than C3a vs the huC3aR. This may not
be an unexpected result, as the biological assay originally used to
characterize this "superagonist" C3a analogue peptide were all
performed on guinea pig cells (14). Minimal activity was
noted with rC5a on cells expressing either the gpC3aR or huC3aR, but
only at supramicromolar concentrations (ED50 > 10
µM). These data confirm that the receptor naturally expressed on guinea pig platelets is promiscuous with respect to human C3a and rC4a, and they extend these observations to demonstrate that both anaphylatoxins are also potent agonists of the cloned gpC3aR. In contrast, rC4a is not active on the huC3aR or mouse C3aR (this study and 13 . The C3aR is a single-copy gene in man (19) and guinea pig (data not shown). Thus, human C4a cannot have a biologic effect in man that is mediated via the C3aR, whereas such a pathway apparently exists in guinea pigs. However, it remains to be determined whether gpC4a will also stimulate the gpC3aR.
Our data are difficult to reconcile with the findings of Murakami and coworkers who describe separate receptors for C3a and C4a on guinea pig macrophages (5), although these differences may result from the different reagents and assays used. While there are no additional reports describing a C4aR that is distinct from the C3aR, our data do not exclude such a possibility. Through an interaction with the C3aR, human C4a may function as an anaphylatoxin in guinea pigs, but not in mouse or man.
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Acknowledgments |
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Footnotes |
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2 Part of this work was presented at the 28th Annual
Meeting of the Deutsche Gesellschaft für Immunologie,
Würzburg, Germany, 1997.
3 Address correspondence and reprint requests to Dr. Wilfried Bautsch, Institute of Medical Microbiology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30623 Hannover, Germany. E-mail address:
4 Abbreviations used in this paper: huC3aR, human C3a receptor; gp, guinea pig (i.e., gpC3aR); FLIPR, fluorometric imaging plate reader; c, concentration.
Received for publication May 22, 1998. Accepted for publication June 30, 1998.
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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3).