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PubMed1996

Expression cloning of the human C3a anaphylatoxin receptor (C3aR) from differentiated U-937 cells.

A cDNA clone encoding the human C3a anaphylatoxin receptor (C3aR) was isolated from a pcDNAI/Amp expression library prepared from U-937 cells which had been differentiated with dibutyryl cAMP to a macrophage-like phenotype. The cDNA clone contained an insert of 4.3 kbp and was able to confer to transfected human HEK-293 cells the capacity to bind specifically iodinated human C3a. Chinese hamster ovary cells co-transfected with this cDNA clone and a G-protein alpha subunit (G alpha-16) became functionally responsive to C3a and a C3a analog synthetic peptide, as measured by increased phosphoinositide hydrolysis. As inferred from the cDNA sequence, the clone encodes a 482-residue polypeptide with seven hydrophobic membrane-spanning helices and a high homology to the human C5a and formyl-Met-Leu-Phe receptors. Uniquely among the family of G-protein coupled receptors, the C3aR contains an exceptionally large second extracellular loop of approximately 175 residues. Northern hybridizations revealed an approximately 2.3-kb transcript as the major and an additional approximately 3.9 kb-transcript as a minor transcription product of the C3aR. The C3aR appears to be widely expressed in different lymphoid tissues, as shown by Northern hybridizations, providing evidence for a central role of the C3a anaphylatoxin in inflammatory processes.

Amino Acid Sequence↗

PubMed1999

Inhibition of IgE-mediated triggering of mast cells by complement-derived peptides interacting with the Fc epsilon RI.

Mucosal type mast cells, in contrast to the serosal type ones, do not respond to cationic agents, or to the complement-derived peptides C3a and C5a. Earlier we have found that while C3a does not activate the rat mucosal type mast cells (line RBL-2H3), it strongly inhibits the IgE-mediated triggering of these cells, by interfering with the Fc epsilon RI-initiated signaling pathway. In the present study we further investigated the mechanism of this process. It is shown, that C3a interacts with the beta-chain of the Fc epsilon RI complex. Binding of the complement peptide to the cells apparently causes a decrease in the proximity of the IgE-binding Fc epsilon RI. Investigating certain sequences of C3a we found that the inhibition is caused by the C-terminal sequences of the complement-peptide, ranging from positions 56 to 77 and also by a shorter sequence, ranging from positions 56 to 64. The inhibitory effect of these peptides was observed both in the case of RBL-2H3 cells and mouse bone marrow derived mast cells.

Amino Acid Sequence↗

PubMed1985

Fast deactivation of guinea-pig isolated ileum to C5adesArg: a possible cyclic AMP-dependent mechanism.

The fast component of deactivation of guinea-pig isolated ileum to the spasmogenic action of the complement peptide C5adesArg was further differentiated from the slow component which had been previously analysed (Damerau et al., 1985a, b). Fast deactivation differs from the slow component in the following characteristics: (a) it is unspecific in that it is also induced by C3a, another complement peptide, (b) it depends on the spasmogenic effect of the peptides, and (c) it does not occur at 16 degrees C. In contrast to the slow component, in which the deactivation is thought to be caused by blockade of C5a receptors, fast deactivation seems to be due to a transient increase of intracellular cyclic AMP evoked by C5adesArg and C3a; it is prevented by GDP beta S (5 X 10(-4) M) which blocks activation of adenylate cyclase, and prolonged by agents which sustain cyclic AMP elevations, namely 5 X 10(-4) M theophylline and 5 X 10(-4) M) GTP gamma S.

Animals↗

PubMed2000

Activated human T lymphocytes express a functional C3a receptor.

The C3a molecule is an anaphylatoxin of the C system with a wide spectrum of proinflammatory effects predominantly on cells of myeloid origin. In this study we investigated the expression of the high affinity receptor for C3a (C3aR) in human T lymphocytes using receptor-specific mAb. C3aR expression was detected in CD4(+) and CD8(+) blood- or skin-derived T cell clones (TCC) from birch pollen-sensitized patients with atopic dermatitis. No significant difference in C3aR expression in CD4(+) or CD8(+) TCCs could be observed. In contrast to C3a(desArg), C3a led to a transient calcium flux in TCCs expressing the C3aR, whereas C3aR-negative TCCs were unreactive. Circulating T cells from patients suffering from severe inflammatory skin diseases expressed the C3aR, whereas no expression of C3aR could be found in unstimulated T lymphocytes from patients with mild inflammatory skin diseases or from healthy individuals. Type I IFNs, which are potent stimulators of cellular immunity, were identified as up-regulators of C3aR expression in vitro in freshly isolated or cloned T lymphocytes. Moreover, C3aR(+) T cells were found at the sites of injection in IFN-beta-treated patients with multiple sclerosis. These data provide direct evidence for the expression of C3aR on activated human T lymphocytes; this may point to a biological function of C3a in T cell-dependent diseases.

Antibodies, Monoclonal↗

PubMed1993

Estimation of local vital reactions in severely burned tissues of guinea pig skin using C3a or C3a desArg as a marker.

We observed changes in levels of anaphylatoxin C3a and/or its desArg (C3a/C3a desArg) peptides in the local site of severely burned skin tissues of guinea pigs using immuno-Western blotting methods. The C3a/C3a desArg peptides, which were probably generated during complement activation, were detected at significant levels from 30 min to 72 h following burn injury in an area limited to 3 mm from the wound edge. Levels of these peptides showed a tendency to be highest in that area 24 h after burn infliction. In postmortem injuries, C3a/C3a desArg peptides could not be detected. These peptides were detected in antemortem wounds up to at least 2 days at 22 degrees C and up to 3 days at 4 degrees C after death, although decreases in levels were found. Lower concentrations of these peptides were also found in postmortem burns in which postmortem hypostasis appeared strongly. These results suggest that, except for injuries of the area with obvious postmortem hypostasis, detection of C3a/C3a desArg can be useful for estimation of vital reactions in many kinds of wounds during inflammation.

Animals↗

PubMed1997

Complement activation of electrogenic ion transport in isolated rat colon.

The complement cascade is an important component in many immune and inflammatory reactions and may contribute to both the diarrhoea and inflammation associated with inflammatory bowel disease. Isolated rat colonic mucosae were voltage clamped in Ussing chambers. Basolateral addition of zymosan-activated whole human serum (ZAS) induced a rapid onset, transient inward short circuit current (SCC). This response was concentration dependent and was significantly attenuated by pre-heating ZAS at 60 degrees C for 30 min. Depletion of complement from normal human serum with cobra venom factor (CVF) significantly lowered SCC responses. Chloride was the primary charge carrying ion as responses to ZAS were abolished in the presence of the loop diuretic bumetanide. The complement component C3a stimulated ion transport but not to the same extent as whole serum. Exogenous C5 was without effect. The cyclooxygenase inhibitor piroxicam significantly attenuated the response to ZAS. These findings support the possibility that complement activation may contribute to the pathophysiology of secretory diarrhoea since activation of electrogenic chloride secretion converts intestinal epithelia to a state of net fluid secretion.

Animals↗

PubMed1998

Anaphylatoxin C3a but not C3a(desArg) is a chemotaxin for the mouse macrophage cell line J774.

Varying results have been published regarding the functional reactivity of different cell types, including human monocytes, to the anaphylatoxin C3a and its degradation product C3a(desArg). To further delineate the functions of C3a and C3a(desArg) on this cell type we used the murine macrophage (Mø) cell line J774A.1 which is known to respond to the anaphylatoxin C5a. J774 cells specifically bound fluorescein isothiocyanate-labeled recombinant human C3a (rC3a). The cells migrated along rC3a concentration gradients in a dose-dependent manner with an optimal concentration of about 3 nM (rC5a:7 nM) and a half-maximal effective concentration (EC50) of about 1.2 nM (rC5a: 2 nM). The degradation product rC3a(desArg) was devoid of chemotactic activity. mRNA for the recently cloned G protein-coupled mouse high-affinity C3a receptor (C3aR) was detected in J774 cells, suggesting that this receptor represents the binding site for C3a on J774 Mø. In support of the specific nature of C3a-stimulated cellular mobility, RBL-2H3 transfectants expressing the human C3aR were also shown to migrate along gradients of rC3a (optimal concentration about 8 nM; EC50 about 3.5 nM) whereas rC3a(desArg) was again inactive. In summary, our findings demonstrate for the first time a specific, receptor-mediated chemoattraction of cells of the monocytic lineage to the anaphylatoxin C3a which may contribute to the accumulation of Mø at sites of inflammation.

Animals↗

PubMed1997

Complement activation during storage of whole blood, red cells, plasma, and buffy coat.

BACKGROUND: The process of separating whole blood into components and the storage of blood components may cause the release of toxic metabolites from the complement cascade. The aim of this study was to determine whether the storage of blood components leads to the activation of the complement cascade and the release of anaphylatoxins. STUDY DESIGN AND METHODS: Blood from 12 healthy volunteers was collected and stored either as whole blood or as components: red cells in saline-adenine-glucose-mannitol solution, plasma, and buffy coat. The concentrations of anaphylatoxins and other complement proteins in the various blood components were intermittently analyzed during a 5-week storage period. RESULTS: Increasing levels of anaphylatoxins were demonstrated during the storage of whole blood and plasma. Elevated concentrations of the anaphylatoxins C3a and C5a were observed during the storage of whole blood. Increased C5a levels were observed after 7 days of storage. High concentrations of C3a were found in plasma after 14 days of storage. Low or non-detectable levels of C3a; C5a, and other complement components were found in red cells stores in saline-adenine-glucose-mannitol solution. CONCLUSION: The study demonstrated activation of complement during the storage of whole blood and plasma but not in red cells in storage solution. The transfusion of larger volumes of stored whole blood or plasma may contribute to the risk of development of organ dysfunction. Therefore, it is advisable to use red cells in storage solution.

Anaphylatoxins↗

PubMed1995

Role of protein adsorption on haemodialysis-induced complement activation and neutrophil defects.

The present clinical study investigated the role of protein adsorption on complement activation and neutrophil functions during in vivo haemodialysis. The parameters were measured simultaneously at the arterial and venous sites of a cuprophan (CU) dialyser with or without pretreatment with human albumin, human immunoglobulins or human total plasma proteins (PLP). Leukocyte count, complement activation (C3a des arg), oxygen radical production and chemotaxis were measured at time zero and 15 min at the arterial and venous sites of the dialyser. Leukopenia observed at both sites was prevented only with PLP treatment. Complement activation was maximal at the venous site, but was not prevented by any of the treatments. Neutrophil oxygen radical production and chemotaxis were significantly decreased only at the venous site and restored to normal with any of the three treatments. Complement activation was maximal at the venous site, but was not prevented by any of the treatments. Protein adsorption on the dialyser membrane seems to modulate the bioincompatibility parameters in a different way. Depending on the functions tested, the protein fractions have different protecting effects, indicating the multifactorial mechanism implicated in the CU haemodialysis-induced leukopenia, complement activation and neutrophil defect.

Adsorption↗

PubMed1983

Generation of C3a anaphylatoxin from human C3 by human mast cell tryptase.

Tryptase, the dominant neutral protease of human pulmonary mast cell secretory granules, has the capacity in vitro to generate C3a anaphylatoxin from purified human C3. Only the alpha-chain of C3 is cleaved, and major fragments with apparent m.w. of 105,000, 39,500, 34,000, 29,000, and 9000 are detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under reducing conditions. Fragments of 34,000 and 9000 m.w. are detected without reduction. A portion of the 9000 m.w. protein corresponds to C3a by virtue of its co-migration in SDS polyacrylamide gels with purified C3a and with trypsin-generated C3a, by its detection in a radioimmunoassay for C3a, and by its contractile activity on the guinea pig ileum bioassay. In the presence of heparin, another component of the mast cell secretory granule, the rate of appearance and the distribution of C3 cleavage fragments as assessed in SDS polyacrylamide gels are not appreciably changed with the exception that no C3a material can be detected in the SDS polyacrylamide gels or by radioimmunoassay and bioassay of the unresolved reaction mixture. Enhanced catabolism of authentic C3a by tryptase occurs in the presence of heparin and by analogy when C3a is generated from C3 by tryptase in the presence of heparin. Whereas tryptase secreted by activated human mast cells may generate C3a, a potentially important additional mediator of immediate hypersensitivity events, the concomitant release of heparin may serve to down-regulate C3a irrespective of its mechanism of generation.

Anaphylatoxins↗

PubMed1988

Regionally variable pulmonary artery responses to C3a.

Products of the complement (C) cascade may have direct effects on pulmonary vascular tissue and contribute to pulmonary vasoconstriction in states of C activation. We studied the effects of C3a, a C-derived vasoactive peptide, on isolated rabbit hilar (HPA) and main pulmonary arteries (MPA). C3a elicited concentration-dependent constriction of HPA (10(7) M to 5 x 10(7) M) but minimal response in MPA at all concentrations tested. The difference between HPA and MPA responses was significant (P less than 0.05, paired t test). To evaluate HPA desensitization to C3a, the peptide was reapplied at 60 min in some tissues and at 120 min in others. All tissues consistently exhibited less constriction at 60 min than observed with previous exposures. Histamine contribution to the HPA response to C3a was determined by exposing the tissues for 30 min before C3a application to pyrilamine (1 x 10(-5) M), an histamine H1-receptor antagonist. Pyrilamine reduced the HPA response to C3a by 70-85%. We conclude that 1) isolated rabbit PA responses exhibit regional variability to C3a over a range of concentrations; 2) C3a desensitizes HPA for at least 60 min, but the tissue demonstrates variable recovery within 120 min; and 3) HPA responses to C3a are reduced by pyrilamine, an H1-receptor antagonist.

Animals↗

PubMed1999

Identification of ligand effector binding sites in transmembrane regions of the human G protein-coupled C3a receptor.

The human C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor (GPCR) composed of seven transmembrane alpha-helices connected by hydrophilic loops. Previous studies of chimeric C3aR/C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site-directed mutagenesis, and mutant receptors were stably expressed in the RBL-2H3 cell line. Ligand binding studies demonstrated that R161A (helix IV), R340A (helix V), and D417A (helix VII) showed no binding activity, although full expression of these receptors was established by flow cytometric analysis. C3a induced very weak intracellular calcium flux in cells expressing these three mutant receptors. H81A (helix II) and K96A (helix III) showed decreased ligand binding activity. The calcium flux induced by C3a in H81A and K96A cells was also consistently reduced. These findings suggest that the charged transmembrane residues Arg161, Arg340, and Asp417 in C3aR are essential for ligand effector binding and/or signal coupling, and that residues His81 and Lys96 may contribute less directly to the overall free energy of ligand binding. These transmembrane residues in C3aR identify specific molecular contacts for ligand interactions that account for C3a-induced receptor activation.

Amino Acid Sequence↗

PubMed1991

Effect of extracorporeal membrane oxygenation on leukocyte function in neonates.

The impact of extracorporeal membrane oxygenation (ECMO) on neonatal leukocyte content and function was examined in six patients. Patients were treated with ECMO for a mean of 134 h (range 44-246 h). Absolute neutrophil counts decreased from 14679 +/- 2291/mm3 to 7791 +/- 1672/mm3 after 2 h of ECMO. However, neutrophil phagocytosis and oxidative burst remained unchanged during the first 48 h of bypass. Monocyte counts also decreased during bypass, and at times were undetectable in 50% of patients. Monocyte HLA-DR content was decreased compared to normal cord blood prior to initiation of ECMO, and remained low throughout ECMO. However, the content increased significantly after termination of bypass. Plasma C3a levels increased transiently, paralleled by an increase in neutrophil CR3 expression. While moribund infants had some impairment of host defenses prior to ECMO, there was no further impact of ECMO per se on the parameters measured, other than transient complement activation and decreased monocyte counts.

Complement Activation↗

PubMed1996

Differential regulation of the C3a and C5a receptors (CD88) by IFN-gamma and PMA in U937 cells and related myeloblastic cell lines.

We have analyzed the induction of the receptor for the anaphylatoxic peptide C3a (C3aR) by the immunomodulator IFN-gamma, the phorbol ester PMA, and dibutyryl cAMP (Bt2cAMP) in comparison with the C5a receptor (C5aR, CD88). For U937 cells, IFN-gamma and Bt2cAMP up-regulated the C3aR to the same extent, whereas Bt2cAMP was 20-fold more effective in C5aR induction. PMA increased the expression of the C5aR, and acted synergistically with IFN-gamma. In contrast, PMA did not increase specific 125I-hC3a binding, and actually antagonized C3aR induction by IFN-gamma. Two related human cell lines of the myeloblastic/monocytic lineage, HL-60 and Mono Mac 6, showed inducibility of the C3aR similar to U937 cells. The two receptors showed subtle differences in signal transduction. Despite comparable numbers of both receptors, IFN-gamma potentiated activation of the C5aR but not the C3aR, as measured by an increase in free cytosolic Ca2+ upon ligand activation. Interestingly, Bt2cAMP-treatment led to a functional response to C3a in U937 cells. Such differences in receptor regulation and signaling might underlie the partly differing physiologic effects of C3a and C5a on, for example, chemotaxis, induction of oxidative burst, or immunoregulatory functions.

Acetylglucosaminidase↗

PubMed1990

Complement activation and the prognostic value of C3a in patients at risk of adult respiratory distress syndrome.

In vivo and in vitro studies have shown that complement activation plays an important role in the pathogenesis of the adult respiratory distress syndrome (ARDS). In a prospective study of polytrauma patients at risk of ARDS (n = 38) complement parameters were determined over a period of 14 days in serial plasma samples (obtained every 6 h during the first 48 h). Polytrauma induced a rapid and remarkable complement activation. Low levels of the complement proteins C3, C4, C1 inhibitor (C1 INH) factor I and factor H during the first 48 h indicated complement consumption in all patients. Elevated C3a levels in the first few hours after injury were associated with the later development of ARDS. A more sensitive indicator than C3a alone was the calculated C3a:C3 ratio discriminating ARDS and non-ARDS patients. A second rise of C3a levels and C3a:C3 ratio from day 4 on paralleled the course of extravascular lung water. To assess the mode of complement activation, the activation-specific protein complexes C1rC1s-C1 INH and C3b(Bb)P were measured in some of the patients. We demonstrate that in the first 48 h complement activation occurred via the alternative pathway only and was later followed by an additional activation via the classical pathway. Our observations suggest that monitoring of C3a and C3 in plasma can identify polytrauma patients at high risk for ARDS at an early stage of the disease.

Adolescent↗

PubMed1996

Effects of inhibition of complement activation using recombinant soluble complement receptor 1 on neutrophil CD11b/CD18 and L-selectin expression and release of interleukin-8 and elastase in simulated cardiopulmonary bypass.

The inflammatory response to cardiopulmonary bypass includes activation of complement and induction of several neutrophil activation pathways. A recombinant soluble form of complement receptor 1 was used as a specific inhibitor of complement activation in simulated cardiopulmonary bypass circuits. Substantial complement activation was observed in these circuits with progressive accumulation of both plasma C3a and terminal complement complex. Soluble complement receptor 1 resulted in a significant reduction in C3a levels (p < 0.01) but did not inhibit terminal complement complex generation. A marked rise in neutrophil CD11b/CD18 expression, simultaneous loss of L-selectin expression, and a progressive accumulation of plasma elastase-alpha 1-antitrypsin occurred and were not affected by soluble complement receptor. However, generation of interleukin-8 in the circuits was inhibited (p < 0.05) by pretreatment with soluble complement receptor. These data suggest that changes in neutrophil activation seen during cardiopulmonary bypass may not be induced directly by anaphylatoxin generation.

CD11 Antigens↗

PubMed1997

Alternative pathway activation of the complement system in preterm infants with early onset infection.

The increased incidence of infection in preterm neonates has been related in part to their relative deficiency of most complement components, because complement is known to participate in the defense against bacterial and viral infections. In a prospective study, complement activation products were determined in 52 preterm infants. Twenty preterm infants suffered from proven early onset infection, 11 infants were presumed to suffer from infection, which could not be confirmed. Twenty-one preterm infants without infection or perinatal asphyxia formed the control group. EDTA plasma was obtained within the first 6 h after birth, and follow-up examinations were done in 15 patients with proven infection during the next 24 h. The complement activation products C3a-desArg, C3bBbP, and sC5b-9 were measured with enzyme immunoassay systems. In preterm neonates with early onset infection, a significant elevation of C3a-desArg was found in the very early course of the disease. C3a-desArg generation resulted from alternative pathway activation as shown by a concurrent increase of C3bBbP concentration. In addition, significantly higher concentrations of sC5b-9 predicted infection in the first few hours after birth. Thus, despite very low levels of native complement proteins, preterm babies are able to generate remarkable amounts of activation products of the complement cascade. The elevation of these activation products preceded by hours significant changes of routine laboratory markers of infection, such as leukocyte count, differential blood count, and C-reactive protein. Thus they might help to identify preterm neonates with severe systemic infection earlier than other laboratory parameters.

Age of Onset↗

PubMed1992

Functional activities of synthetic anaphylatoxic peptides in widely used biological assays.

A comparison study was carried out between the modern ATP release assay (ARA) with guinea-pig platelets and the traditional guinea-pig ileum contraction assay (ICA). The biological activities of the anaphylatoxin C3a and synthetic C3a analogue peptides were determined in both assays. In dose-response curves with C3a, a human C3a peptide with the last 21 amino acids of the C terminus (C3a 56-77) and a peptide with 13 amino acids which was acylated N-terminal with the aromatic fluorenylmethoxycarbonyl group and an aminohexanoyl group (Fmoc-Ahx YRRGRAAALGLAR) were tested. The ARA turned out to be 100 times more sensitive than the ICA. In contrast to previous reports the 21 amino acid long C3a analogue peptide did not exhibit full C3a activity but only 7% (ARA) or 12% (ICA). The potentiation of biological activity in the ARA by coupling non-peptide acyl-residues N terminal to peptide C3a analogues could be confirmed with Fmoc-Ahx-YRRGRAAALGLAR in the ICA. In addition, the tri-peptide Fmoc-Ahx-LAR displayed C3a specific activity in the ICA demonstrated by desensitization experiments.

Adenosine Triphosphate↗