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PubMed1993

Relationship of C5a receptor modulation to the functional responsiveness of human polymorphonuclear leukocytes to C5a.

The relationship of C5a receptor expression on human polymorphonuclear leukocytes (PMN) to the functional response of these cells to C5a was studied using flow cytometry. C5a receptor expression was determined with a fluorescein conjugate of C5a and oxidative burst activity was monitored by conversion of dichlorofluorescein to dichlorofluorescein (DCF) as a measure of H2O2 production. These studies showed that after incubation of PMN with increasing concentrations of C5a, and allowing for internalization of bound ligand, more than 40% of the cell surface C5a receptors were internalized before the DCF response to optimal concentrations of C5a was decreased below the levels for untreated control cells. Although C5a responsiveness was lost after preincubation with 10(-8) M C5a, cells remained responsive to formyl peptide. In other studies, cells were preincubated with unlabeled C5a under conditions that provided for internalization of nearly all C5a receptors. PMN were then cultured for up to 90 min and monitored for C5a receptor reexpression and return of cell function. In these studies, the DCF response of PMN to C5a returned to 100% much earlier than the cells regained full expression of C5a receptors. The DCF response to formyl peptide remained intact throughout the period of C5a receptor reexpression. These studies showed that once > 40% of the original population of C5a receptors are reexpressed on the PMN, that these cells regain 100% of their functional responsiveness to C5a in the DCF assay. Evaluation of the affinity and number of C5a receptors using 125I-labeled C5a after receptor reexpression showed that maximal receptor reexpression was approximately 73% of that obtained with control cells and the Kd of reexpressed receptors was 0.60 vs 0.94 nM for control cells. These studies demonstrate that only a portion of the total C5a receptors expressed on PMN are essential to stimulate a 100% functional response in PMN and that the reexpressed receptors are capable of transducing a signal that activates the oxidative burst in these cells.

Complement C5a↗
PubMed1994

Mutation of aspartate 82 of the human C5a receptor abolishes the secretory response to human C5a in transfected rat basophilic leukemia cells.

C5a is a potent chemoattractant for monocytes, neutrophils and other leukocytes. The receptor for human C5a is a member of the rhodopsin superfamily of G protein-coupled receptors and contains an aspartate residue (Asp82) within the putative second transmembrane domain conserved in all other G protein-linked receptors. We investigated the role of this residue and also the carboxy-terminal 23 residues of the C5a receptor in ligand binding and signal transduction by expressing wild-type and mutant receptors in the rat basophilic leukemia cell line RBL-2H3. Wild-type and truncated receptors coupled efficiently to effector systems, resulting in the C5a-dependent discharge of granule contents. In contrast RBL cells transfected with receptors in which Asp82 had been mutated to asparagine did not respond to human C5a by secretion despite binding human C5a with high affinity. We conclude therefore that Asp82 is not involved in the interaction with ligand but is essential for the proper transduction of the ligand binding signal.

Animals↗
PubMed1997

C5a receptors are detectable on mast cells in normal human skin and in psoriatic plaques but not in weal and flare reactions or in uticaria pigmentosa by immunohistochemistry.

The expression of the receptor for the anaphylatoxin C5a on mast cells was studied with three monoclonal antibodies directed to the N-terminal domain of the C5a receptor. Human skin was investigated by immunohistology applied to sequential 2 micron sections of acrylate-embedded tissues. All anti-C5a receptor antibodies stained c-kit+ or tryptase+ cells which were metachromatic after toluidine blue staining in normal human skin. The binding of anti-C5a receptor antibodies was inhibitable by a peptide representing the first 31 amino acids of the C5a receptor. A similar expression of C5a receptors was found on mast cells in chronic psoriatic plaques. However, C5a receptors were not detectable on mast cells in weal and flare reactions or in lesional skin of uticaria pigmentosa. These findings suggest that (1) anti-C5a receptor antibodies directed to the N-terminal domain of the receptor are suitable tools for the identification of mast cells in acrylate-embedded sections of human skin, (2) mast cell activation in weal and flare reactions results in C5a receptor downregulation or receptor blockade and (3) mast cells in urticaria pigmentosa lack a typical marker of normal human skin mast cells.

Antibodies, Monoclonal↗
PubMed1994

The amino terminus of the human C5a receptor is required for high affinity C5a binding and for receptor activation by C5a but not C5a analogs.

The binding domain of the human C5a receptor consists of two distinct and physically separable subsites. One of these sites binds the C-terminal 8 amino acids of C5a and is as yet undefined, while the second site lies in the N terminus of the receptor and interacts with the core of C5a. Two deletion mutants were prepared to probe the importance of this second site. Removal of residues 2-22 decreased the binding affinity for C5a by 600-fold, while extending the deletion through residue 30 caused a further 75-fold decrease. Thus, the N terminus is responsible for at least 45% of the total energy for the binding of C5a. The five aspartic acids present in the deleted segments appear to be critical residues, as their conversion to alanines accounts for most of the affinity lost in the two truncations. Despite its importance for binding, the N terminus is not necessary for signal transduction, as a C-terminal peptide analog of C5a was able to stimulate G protein activation and to generate a Ca2+ flux through a receptor lacking residues 2-22. However, intact C5a was a very poor activator of this truncated receptor. These results imply that interaction between the N terminus of the receptor and C5a produces a conformational change in C5a that allows it's C terminus to properly interact with and activate the receptor.

Amino Acid Sequence↗
PubMed1991

Characterization of the C5a receptor on guinea pig platelets.

Guinea pig (gp) platelets react to nanomolar doses of the complement-derived anaphylatoxin C5a with a shape change, aggregation and release of biogenic amines and nucleotides from their granules. We have investigated the specific receptor for C5a on gp platelets which mediates these biological effects. Competitive binding studies with 125I-labeled guinea pig C5a (125I-gpC5a) revealed approx. 4000 binding sites/cell with Kd = 6 x 10(-9) M. The more than 60-fold higher biological activity (ATP-release from gp platelets) of gpC5a versus recombinant human C5a (rhuC5a) and the different binding behavior of gpC5a and rhuC5a point to a species restriction in the gp platelet system. Cross-linking of 125I-gpC5a to gp platelets (250 microM DSS) and analysis by SDS-PAGE under reducing conditions resulted in labeling of a single band with a molecular mass of 32 kDa (ligand-receptor complex). Because of these characteristics, the C5a receptor on gp platelets clearly differs from all previously described C5a receptors.

Adenosine Triphosphate↗
PubMed1999

Cloning and preliminary pharmacological characterization of the anaphylatoxin C5a receptor in the rabbit.

1 The rabbit receptor for C5a was cloned from a genomic library and found to be 79.5% identical to the human homologue, the highest degree of similarity found so far in nonprimate laboratory animals. 2 The rabbit C5a receptor stably expressed in RBL cells binds human 125I-C5a (2 nM). Unlabelled C5a and the C-terminal analogue N-acetyl-Tyr-Ser-Phe-Lys-Pro-Met-Pro-Leu-D-Ala-Arg (Ac-YSFKPMPLaR) were found to be competitors of that binding, the peptide analogue retaining approximately 0.1% of the affinity of human C5a. 3 The order of potency human C5a>Ac-YSFKPMPLaR was conserved in bioassays based on rabbits (relaxation of the isolated portal vein and pulmonary artery; acute in vivo neutropenia), but with a decreasing potency gap between the two compounds, a likely consequence of the resistance to peptidases of the analogue. 4 The molecular definition of the rabbit C5a receptor evidenced a high preservation degree of sequence and pharmacologic properties relative to the human ortholog receptor, thus defining a set of molecular tools for the investigation of the role of C5a in physiologic and pathologic models based on the rabbit (e.g. atherosclerosis, inflammation).

Amino Acid Sequence↗
PubMed1994

C5A anaphylatoxin and its seven transmembrane-segment receptor.

The molecular cloning of the C5a receptor places this molecule in the superfamily of G-protein coupled receptors. This superfamily is characterized by the presence of signature motifs including seven hydrophobic domains which span the cell membrane and impart a predicted serpentine topology to the receptor proteins. The identification of other members of this family, including receptors for the chemokines IL-8 and Mip-1/Rantes, thrombin, formyl peptide, and platelet activating factor, provide new tools for understanding structure-function relationships relevant to the inflammatory process. This review focuses on the recent biological studies concerning the ligand C5a and its cellular receptor, the structure/activity relationships so far discerned, signal transduction mechanisms, progress toward identification of receptor antagonists, and some likely directions for future studies. Where appropriate, relevant work on related seven transmembrane segment receptors is discussed.

Amino Acid Sequence↗
PubMed2002

Expression and function of the C5a receptor in rat alveolar epithelial cells.

Although alveolar epithelial cells (AEC) form an important barrier for host defenses in the lung, there is limited information about ways in which AEC can directly participate in the lung inflammatory response. In the current studies, primary cultures of rat AEC (RAEC) have been shown to specifically bind recombinant rat C5a at high affinity and in a saturable manner. This binding was enhanced in a time-dependent manner by pre-exposure of RAEC to LPS, IL-6, or TNF-alpha, the increased binding of C5a being associated with increased levels of mRNA for the C5a receptor (C5aR). Exposure of RAEC to C5a also caused increased expression of mRNA for C5aR. As compared with exposure of RAEC to LPS or to C5a alone, exposure to the combination caused enhanced production of TNF-alpha, macrophage inflammatory protein-2, and cytokine-induced neutrophil chemoattractant-1, as well as increased intracellular levels of IL-1beta. These data indicate that RAEC, when activated, have enhanced binding of C5a in association with increased mRNA for C5aR. The functional outcome is enhanced release of proinflammatory mediators. These data underscore the phlogistic potential of RAEC and the ability of C5a to enhance the phlogistic responses of RAEC.

Animals↗
PubMed1994

C5a as a model for chemotactic factor-stimulated tyrosine phosphorylation in the human neutrophil.

Human neutrophils were exposed to the chemotactic factors C5a, FMLP, IL-8, leukotriene B4, and PAF, for 30 s, and subsequently analyzed for protein tyrosine phosphorylation by immunoblotting whole cell lysates with a polyclonal antiphosphotyrosine Ab. All chemotactic factors caused the rapid de novo tyrosine phosphorylation of a broad band of approximately 120 kDa and increased the phosphotyrosine content of several other proteins, including two with molecular masses of 60 and 56 kDa that were present in the unstimulated neutrophil. Tyrosine phosphorylation was evident as early as 10 s after stimulation and was maintained for 1 to 3 min before dephosphorylation occurred. The extent of tyrosine phosphorylation was dependent on the concentration of chemotactic factor, with stimulation observed at concentrations as low as 10 to 100 nM. To investigate the pathway used by chemotactic factors to transduce this signal, neutrophils were treated with PMA. PMA also stimulated tyrosine phosphorylation in the neutrophil but with a slower response time and a different pattern of affected proteins. Additional experiments suggested that tyrosine phosphorylation is involved in the regulation of the neutrophil respiratory burst because the tyrosine kinase inhibitor, herbimycin A, inhibited C5a-induced protein tyrosine phosphorylation and also prevented C5a- and FMLP-induced superoxide anion production. Herbimycin A also inhibited PMA-induced tyrosine phosphorylation and superoxide anion production. To confirm that the ability to stimulate tyrosine phosphorylation was intrinsic to the C5a receptor, tyrosine phosphorylation was examined in both undifferentiated U937 cells (C5a receptor negative) and cAMP differentiated U937 cells (C5a receptor positive). C5a induced tyrosine phosphorylation only in differentiated U937 cells. Analysis of the C5a receptor mRNA using the PCR confirmed its presence in differentiated and its absence in undifferentiated U937 cells. Therefore, C5a stimulates tyrosine phosphorylation via a receptor-mediated mechanism and U937 cells provide a system in which G-coupled receptor-mediated tyrosine phosphorylation can be investigated.

Benzoquinones↗
PubMed1996

Expression and modulation of C5a receptor (CD88) on skin dendritic cells. Chemotactic effect of C5a on skin migratory dendritic cells.

Although it is known that dendritic cells (DC) migrate in response to inflammatory stimuli. There is little information about the expression of receptors for chemotactic factors on DC. The present study has demonstrated by double immunostaining and flow cytometry of Langerhan's cell (LC)-enriched epidermal cell suspensions that a small subpopulation (5-6%) of epidermal resident DC (rLC) expresses receptors for C5a (C5aR). Epidermal rLC positive for C5aR show a round-shape morphology, were located next to the basement membrane and express HLA-DR molecules higher than C5aR negative rLC. These observations suggest that rLC would express C5aR as part of their process of maturation during tissue trafficking. To investigate whether epidermal LC up-regulate C5aR along their differentiation pathway. LC were differentiated in vitro after culture in epidermal cell suspensions supplemented with granulocyte macrophage colony-stimulating factor (GM-CSF). As a result, in vitro differentiated LC increased the expression of C5aR up to 69% of the DC population. In accordance with this observation, interdigitating DC of secondary lymphoid organs (lymph node and tonsil) also expressed (5aR. Migratory CD1a positive DC that spontaneously migrated out of dermal or split-skin organ explants were also positive for C5aR and were used for chemotaxis and chemokinesis assays in response to human recombinant C5a (rC5a). Optimum migration to rC5a was observed at 10(-8)M with a sigmoidal dose response curve. Checkboard analysis demonstrated that locomotion in response to rC5a was chemotaxis and not chemokinesis.

Antigens, CD↗
PubMed1997

C5aR ligand peptide 3D QSAR study performed with an applied linear conformation.

A 3D quantitative structure-activity relationship study (QSAR) of binding and activation of the human C5a receptor by peptide analogs of the C-terminal binding domain of C5a anaphylatoxin is reported. Using published C5a analog affinity and activity data, this paper seeks to elucidate the pharmacophore for the high affinity C-terminal binding domain of the C5a peptide with the molecular modeling technique of comparative molecular field analysis (CoMFA). In order to model peptides for which there was incomplete conformational data, an arbitrary linear conformation was imposed upon the highly flexible C5a analogs. The resulting models yield a crossvalidated q2 of 0.889 and 0.787, for receptor-ligand affinity and EC50 calcium release activity, respectively, suggesting these models have good predictive ability for other test peptides.

Amino Acid Sequence↗
PubMed1998

A neuronal C5a receptor and an associated apoptotic signal transduction pathway.

1. We report the first experimental evidence of a neuronal C5a receptor (nC5aR) in human cells of neuronal origin. Expression of nC5aR mRNA was demonstrated by the reverse transcriptase-polymerase chain reaction (RT-PCR) in TGW human neuroblastoma cells. 2. Expression of a functional C5aR was supported by the finding that C5a evoked a transient increase in the intracellular calcium level as measured by flow cytometry (FACS). 3. To analyse the function of the nC5aR, an antisense peptide fragment of the C5aR was used. Previous data showed that a C5aR fragment (a peptide termed PR226) has C5aR agonist and antagonist effects in U-937 cells depending on the concentration of the peptide. We found that a multiple antigenic peptide (MAP) form of the same peptide (termed PR226-MAP) induced rapid elevation of nuclear c-fos immunoreactivity and resulted in DNA fragmentation, a characteristic sign of apoptosis, in TGW cells. 4. Early electrophysiological events characteristic of apoptosis were also detected: intermittent calcium current pulses were recorded within 1-2 min of peptide administration. C5a pretreatment delayed the onset of this calcium influx. 5. We also demonstrated that the apoptotic pathway is linked to nC5aR via pertussis toxin-sensitive G-proteins. 6. Although the function of C5a and its receptor on neurons is unknown, these results suggest that an abnormal activation of this signal transduction pathway can result in apoptosis and, subsequently, in neurodegeneration.

Animals↗
PubMed1976

Chemotactic responses of normal human basophils to C5a and to lymphocyte-derived chemotactic factor.

Peripheral blood basophils from normal human subjects responded chemotactically to the complement component C5a and also to the supernatant fluid from cultured human lymphocytes stimulated by mitogens or specific antigen. Basophil chemotactic factor (BCF) was detected in stimulated lymphocyte cultures within 24 hr and increased for the first 3 days. BCF was found after stimulation of lymphocytes by B or T cell mitogens as well as by specific antigen. The amount of BCF in culture fluids was independent of cell proliferation. Monocyte chemotactic factor (MCF) and BCF could not be separated from one another by Sephadex or DEAE chromatography. Both activities were eluted from Sephadex G-100 columns in a m.w. region of about 15,000 daltons and were found after ion exchange chromatography on DEAE in a peak eluted by 0.1 M NaCl. These findings suggest that mitogen or antigen-stimulated lymphocytes secrete a molecule that is chemotactic for both monocytes and basophils. Thus, in the absence of a basophil-specific chemotactic molecule, the preferential accumulation of basophils in certain types of delayed hypersensitivity skin reactions must be due to additional factors such as basophil chemotaxis augmentation factor.

Basophils↗
PubMed1993

Human chemotaxis receptor genes cluster at 19q13.3-13.4. Characterization of the human C5a receptor gene.

The human C5a anaphylatoxin and formyl peptide receptor genes, as well as two genes with high sequence identity to the formyl peptide receptor, FPRH1 and FPRH2, have been mapped to chromosome 19 (Lu et al., 1992). Further analysis reveals that these genes are present in the 19q13.3 band adjacent to the 13.3-13.4 interface. MRNAs for the C5a and formyl peptide receptors, as well as for FPRH1, are expressed in cAMP differentiated U937 cells and human eosinophils, while all four transcripts are expressed in human lung. This observation opens the possibility for coordinate regulation of these genes. In order to initiate the mapping of fine structure at this locus, genomic clones have been analyzed. All four of the genes have a similar structure, with the receptor protein encoded in a single exon. Detailed characterization of the C5a receptor gene reveals a two exon structure, with the 5' untranslated sequence and initiating methionine located in the first exon. An intron of approximately 9 kb separates exon 1 from the receptor-encoding exon 2. The region of genomic DNA flanking the 5' untranslated sequence possesses promoter activity when transfected into the myeloid-derived rat basophilic leukemia RBL-1 cells, but the same region is inactive when transfected into nonmyeloid cells. Deletional analyses indicate that C5a receptor 5' flanking region contains both cell-specific suppressor and promoter regions.

Animals↗
PubMed1995

Identification of the major phosphorylation sites in human C5a anaphylatoxin receptor in vivo.

Interaction of human C5a anaphylatoxin with cell surface receptors mediates cell activation and receptor desensitization. Treatment of differentiated HL60 cells or transiently transfected COS-7 cells with C5a or phorbol 12-myristate 12-acetate (PMA) results in rapid hyperphosphorylation of the C5aR. In an attempt to gain more insight into the function of phosphorylation in the desensitization of C5aR, we have initiated experiments to identify phosphoacceptor sites at the amino acid level after stimulation of cells with either C5a or PMA. In this report we show that C5aR is phosphorylated exclusively on serine residues in both differentiated HL60 and transfected COS-7 cells irrespective of the stimulus used. Peptide mapping after cyanogen bromide cleavage of phosphorylated C5aR indicates that despite the presence of a protein kinase C consensus motif the third cytoplasmic loop is not phosphorylated when cells are challenged with either C5a or PMA. Thus, whether the cells are stimulated with C5a or PMA, the phosphorylation sites appear to be restricted to serine residues in the carboxyl tail. Phosphoamino acid analysis of a series of mutants in which an individual serine residue was replaced by a threonine residue indicates that the C5aR undergoes C5a-dependent phosphorylation to the maximal stoichiometry of 6 mol of PO4/mol of receptor at Ser314, Ser317, Ser327, Ser332, Ser334, and Ser338. Simultaneous substitution of serine residues by alanine at positions 332, 334, and 338 affected neither the binding of C5a nor the cell surface expression of the mutant, but resulted in a dramatic reduction (more than 80%) of both C5a- and PMA-mediated phosphorylation as compared to the wild type receptor. This result suggests that phosphorylation on the segment extending from Ser332 to Ser338 is required for the subsequent phosphorylation of the carboxyl-terminal tail of C5aR.

Amino Acid Sequence↗
PubMed1996

Amplification of the inflammatory response: adhesion molecules associated with platelet/white cell responses.

Cardiopulmonary bypass (CPB) causes leukocyte and platelet activation, resulting in upregulation of the adhesion receptor CD11b/CD18 on leukocytes and upregulation of P-selectin, the adhesion receptor that binds the activated platelet to polymorphonuclear neutrophils (PMNs) and monocytes. Our laboratory has studied the expression of activation-dependent adhesion receptors during in vivo CPB. Both PMN and monocyte CD11b were upregulated during CPB but with differing time courses. Peak PMN CD11b levels occurred at the end of the hypothermic phase of bypass, whereas monocyte CD11b levels increased steadily throughout the course of CPB, peaked at 2-4 h after CPB, and remained significantly elevated as late as 18-24 h post CPB. The percentage of P-selectin-positive platelets increased significantly during bypass, peaking around the end of bypass and remaining elevated in the early post-bypass period. The level then returned to normal by 18 h post-bypass. Monocyte-platelet binding paralleled the increase in P-selectin-positive platelets during bypass and similarly remained elevated in the post-bypass period. PMN-platelet binding also increased but peaked early during CPB. Upregulation of these adhesive receptors and formation of platelet-leukocyte conjugates may influence the prothrombotic activity of monocytes and the proinflammatory activity of PMNs in the post-CPB period. Our laboratory has developed an in vitro model of extracorporeal circulation, and recirculation of blood on this circuit results in significant activation of PMNs and monocyte CD11b expression, increasing progressively over time. Likewise, the percentage of P-selectin-positive platelets increased and was paralleled by the formation of leukocyte-platelet conjugates comparable to the pattern found in vivo. Generation of the complement fragments C5a and the C5b-9 membrane-attack complex may contribute to platelet P-selectin expression and formation of leukocyte-platelet conjugates during CPB. The in vitro model has been used to test the cellular effects of complement inhibition employing a monoclonal antibody that blocks cleavage of C5 into C5a and C5b to determine the role of early vs. late complement components in the cellular activation induced by CPB. Preliminary results demonstrate that blockage of the formation of C5a and the C5b-9 membrane-attack complex during simulated extracorporeal circulation effectively inhibits platelet and PMN activation and the formation of leukocyte-platelet conjugates.

Blood Platelets↗
PubMed1999

Human T cells express the C5a receptor and are chemoattracted to C5a.

The anaphylatoxin C5a is a potent mediator of inflammation that exerts a broad range of activity on cells of the myeloid lineage. In this study, we present the first evidence that human T cells express the C5a receptor (C5aR) and are chemotactic to C5a. Using FACS analysis, we found that the C5aR was expressed at a low basal level on unstimulated T cells and was strikingly up-regulated upon PHA stimulation in a time- and dose-dependent manner. CD3+ sorted T cells as well as Jurkat T cells were shown to express C5aR mRNA as assessed by RT-PCR. Moreover, semiquantitative RT-PCR analysis demonstrated that C5aR mRNA was down-regulated in purified T cells upon long-term PHA stimulation. To demonstrate that C5a was biologically active on T cells, we investigated the chemotactic activity of C5a and observed that purified CD3+ T cells are chemotactic to C5a at nanomolar concentrations. Finally, using a combination of in situ hybridization and immunohistochemistry, we showed that the T cells infiltrating the central nervous system during experimental allergic encephalomyelitis express the C5aR mRNA. In summary, these results suggest that C5a exerts direct effects on T cells and could be involved in the trafficking of T cells under physiological and pathological conditions, including inflammatory diseases of the central nervous system.

Animals↗