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PubMed1999

rC5a directs the in vitro migration of human memory and naive tonsillar B lymphocytes: implications for B cell trafficking in secondary lymphoid tissues.

Human C5a is a potent chemoattractant for granulocytes, monocytes, and dendritic cells. In mice C5a has been shown to be chemotactic for germinal center (GC) B cells. To date, no information is available on the effects of C5a on human B cell locomotion. Here we demonstrate that rC5a increases polarization and migration of human tonsillar B cells. The locomotory response was due to both chemokinetic and chemotactic activities of rC5a. Moreover, memory and, at a lesser extent, naive B cell fractions from purified tonsillar populations displayed rC5a-enhanced migratory properties, whereas GC cells did not. Flow cytometry revealed C5aR (CD88) on approximately 40% memory and 10% naive cells, respectively, whereas GC cells were negative. Immunohistochemistry showed that a few CD88+ cells were of the B cell lineage and localized in tonsillar subepithelial areas, where the majority of memory B cells settle. Pretreatment of memory B cells with the CD88 mAb abolished their migratory responsiveness to rC5a. Finally, the C5 gene was found to be expressed in naive, GC, and memory B lymphocytes at both the mRNA and the protein level. This study delineates a novel role for C5a as a regulator of the trafficking of human memory and naive B lymphocytes and supports the hypothesis that the B cells themselves may serve as source of C5 in secondary lymphoid tissues.

Antigens, CD↗
PubMed1998

Novel C5a receptor antagonists regulate neutrophil functions in vitro and in vivo.

Novel recombinant human C5a receptor antagonists were discovered through modification of the C terminus of C5a. The C5a1-71T1M,C27S,Q71C monomer, (C5aRAM; CGS 27913), was a pure and potent functional antagonist. The importance of a C-terminal cysteine at position 71 to antagonist properties of C5aRAM was confirmed by studying C5a1-71 derivatives with replacements of Q71, C5a derivatives of various lengths (70-74) with C-terminal cysteines, and C5a derivatives of various lengths (71-74) with Q71C replacements. The majority of C5a1-71Q71 derivatives were agonists (C5a-like) in the human neutrophil C5a-induced intracellular calcium mobilization assay. The C5a1-71Q71C derivative was an antagonist. C5a derivatives of lengths 73 and 74 with C-terminal cysteines were agonists, while lengths 70 to 72 were antagonists. C5a derivatives of lengths 72, 73, and 74 with Q71C replacements were agonists, while, again, C5a1-71Q71C was an antagonist. C5aRAM and its adducts, including its dimer, C5aRAD (CGS 32359), were pure antagonists. Additionally, CSaRAM and CSaRAD inhibited binding of 125I-labeled recombinant human C5a to neutrophil membranes (Ki = 79 and 2 pM, respectively), C5a-stimulated neutrophil intracellular calcium mobilization (8 and 13 nM), CD11b integrin up-regulation (10 and 1 nM), superoxide generation (182 and 282 nM), lysozyme release (1 and 2 microM), and chemotaxis (11 and 7 microM). In vivo, intradermal injection of C5aRAM inhibited C5a-induced dermal edema in rabbits. Furthermore, a 5-mg/kg i.v. bolus of C5aRAD significantly inhibited C5a-induced neutropenia in micropigs when challenged with C5a 30 min after C5aRAD administration. C5aRAM and C5aRAD are novel, potent C5a receptor antagonists devoid of agonist or proinflammatory activity with demonstrated efficacy in vitro and in vivo.

Animals↗
PubMed1994

C5a stimulus-secretion coupling in rat basophilic leukaemia (RBL-2H3) cells transfected with the human C5a receptor is mediated by pertussis and cholera toxin-sensitive G proteins.

Rat basophilic leukaemia cells (RBL-2H3) were transfected with either the wild type human C5a receptor or a truncated form lacking the last 23 C-terminal residues. Transfected cells bound human C5a specifically, with affinities in the range 3-20nM, and 12-166,000 receptors per cell, similar values to those obtained on human neutrophils and monocytic cells. The stimulation of secretion by human C5a was completely inhibited by pertussis toxin and partially sensitive to cholera toxin, indicating that both wild-type and mutated receptors are coupled to G proteins. Cells transfected with the mutated receptor were equally sensitive to hC5a, suggesting that this portion of the C terminus is not an absolute requirement for signal transduction.

Animals↗
PubMed1997

Expression of an epitope tagged human C5a receptor and antibody-mediated immobilization of detergent-solubilized receptor for drug discovery screening.

In order to screen combinatorial libraries of peptides and/or small organic molecules against the human C5a receptor, we have developed a novel method for immobilizing and screening 7-transmembrane segment (7-TMS) receptors. The epitope for a high affinity monoclonal antibody (mAb179) was added to the C-terminus of the human C5a receptor, and CHO cell lines expressing the epitope tagged receptor (C5aR-KH) and the wild-type receptor (C5aR) were established. The addition of the epitope tag did not affect the affinity of C5aR-KH for C5a. The epitope tag allowed for mAb179-mediated immobilization of active receptor either in intact membranes or following detergent extraction with digitonin or Chaps. If this method is generally applicable to other 7-TMS receptors it may have broad utility for drug discovery screening as well as other applications.

Amino Acid Sequence↗
PubMed1995

IFN-gamma up-regulates the human C5a receptor (CD88) in myeloblastic U937 cells and related cell lines.

On human mature monocytes the immunomodulator IFN-gamma has been shown to down-regulate the receptor for the anaphylatoxic peptide C5a (CD88, C5aR). In this study, we show that in immature myelo-/monoblastic U937, HL60, and MonoMac6 cells, IFN-gamma induces C5aR-ligand binding activity. In U937 cells, this induction cannot be blocked by the protein kinase C inhibitor staurosporine. An increase in free cytosolic Ca2+ upon ligand binding indicates functional coupling of this receptor in U937 and HL-60 cells. G-Proteins involved in this C5a responsiveness after IFN-gamma induction are completely pertussis toxin sensitive. Our data suggest that an additional pertussis toxin-resistant pathway exists in U937 cells after induction by dibutyryl cAMP. However, this is not due to changes in the mRNA level of the pertussis toxin-insensitive G-protein subunit G alpha 16. Induction by dibutyryl cAMP, but not that by IFN-gamma, resulted in C5a-dependent release of N-acetyl-beta-D-glucosaminidase, further highlighting functional differences in the effects of the inducers. Our data show an IFN-gamma-dependent increase in C5aR expression and suggest a maturation-related change in signaling of the C5aR, presumably at the level of receptor coupling.

Acetylglucosaminidase↗
PubMed1997

Complement factors and their receptors.

In summary, recent advances in molecular cloning of anaphylatoxins and the anaphylatoxin receptors add new dimensions to our investigations and understanding of the molecular mechanisms involved in anaphylatoxin action. Combining knowledge accumulated from peptide modeling of the ligands with mutagenesis studies of these ligands and their receptors makes it possible to more accurately model interactive sites and understand the sequence of molecular interactions required for cellular activation. In addition, these new developments provide valuable tools for investigating, yet unknown, activities and cellular targets of the anaphylatoxin molecules.

Amino Acid Sequence↗
PubMed1995

Phosphorylation, dephosphorylation, and recycling of the C5a receptor in differentiated HL60 cells.

Binding of activation peptide from the fifth component of C (C5a) to its receptor triggers events leading to both stimulation of cellular responses and receptor desensitization in myeloid cells. However, although transmission of a signal to pertussis toxin-sensitive G proteins is a prerequisite to neutrophil activation, we show that the process of receptor phosphorylation is mainly independent from activation of this pathway. Treatment of cells with pertussis toxin did not modify the incorporation of phosphate mediated by a saturating concentration of C5a, indicating that agonist-occupied C5aR can be fully phosphorylated, presumably by a specific G protein-coupled receptor kinase, in the absence of activation of the Gi protein. Receptor phosphorylation was transient, with a half-life of 30 to 40 min, which suggested a role for protein phosphatases in the regulation of the state of phosphorylation of C5aR in dHL60 cells. Pretreatment of cells with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, increased the basal phosphorylation of unoccupied receptor and extended the phosphorylation mediated by C5a binding. Okadaic acid delayed, but did not suppress, the dephosphorylation process, which suggests either the involvement of additional phosphatase(s) or the degradation of nondephosphorylated receptors in the endocytic pathway. The data strongly suggest that internalized C5aR are recycled to the plasma membrane with a time course consistent with the kinetics of dephosphorylation. Dephosphorylation of C5aR might be essential to receptor recycling and resensitization during chemotaxis.

Cell Line↗
PubMed2001

Species dependence for binding of small molecule agonist and antagonists to the C5a receptor on polymorphonuclear leukocytes.

This study investigated the receptor binding affinities of a C5a agonist and cyclic antagonists for polymorphonuclear leukocytes (PMNs) isolated from human, sheep, pig, dog, rabbit, guinea pig, rat and mouse. The affinities of the two small molecule antagonists, F-[OPdChaWR] and AcF-[OPdChaWR], and the agonist, YSFKPMPLaR, revealed large differences in C5a receptor (C5aR) affinities between species. The antagonists bound to human, rat and dog PMNs with similar high affinities, but with lower affinities to PMNs from all other species. The C5a agonist also bound with varying affinities between species, but showed a different affinity profile to the antagonists. In contrast, recombinant human C5a had similar affinity for PMNs of all species investigated. The low correlation between the affinities of the antagonists and the agonist between species either suggests that different receptor residues are important for distinguishing between agonist/antagonist binding, or that the agonist and antagonist peptides bind to two distinct sites within the C5aR.

Animals↗
PubMed1998

A case of episodic angioedema associated with blood eosinophilia: upregulated C5a receptor expression on eosinophils.

A 47-year-old woman was admitted to hospital complaining of swelling and pain of the extremities, accompanied by high fever and generalized erythema. Laboratory examination showed marked blood eosinophilia with elevation of IgM, IgE, and C-reactive protein. All autoantibodies examined were negative. The heart and lungs showed no untoward findings. Biopsies of the skin and muscle revealed cellular infiltration of eosinophils around small blood vessels. Quantitation of C5a receptor (C5aR) expression by flow cytometry using anti-C5aR antibody showed upregulated expression of C5aR on blood eosinophils but downregulated expression on neutrophils. The abnormal C5aR expression on eosinophils and neutrophils became normal after spontaneous resolution of symptoms and blood eosinophilia. The possibility that C5aR expression on granulocytes is related to the pathogenesis of this syndrome may be considered.

Angioedema↗
PubMed1996

Stabilization of an isolated helical capping box in solution by hydrophobic interactions: evidence from the NMR study of bioactive peptides from the C-terminus of human C5a anaphylatoxin.

Synthetic analogues of the C-terminal portion of C5a were designed and found to be agonists of the C5a receptor [J. A. Ember et al. (1992) Jounral of Immunology, Vol. 148, p. 3165]. Nuclear magnetic resonance experiments were carried out to determine the solution conformation of the most potent analogue, the peptide C5a 65-74 (Tyr65, Phe67) (Tyr65-Ser66-Phe67-Lys68-Asp69-Met70 -Gln71- Leu72-Gly73-Arg74). Medium-range nuclear Overhauser effects (NOEs) were observed for residues 65-70 of this C5a peptide, suggesting that this region adopts a folded conformation in a significant population of the solution conformational ensemble. Quantitative analyses of (3)J(NH-alphaH) coupling constants and sequential NOE cross peaks gave an estimated helical population of 65% in the region Ser66-Met70. Additional evidence supporting the presence of a helical turn includes reduced amide-proton temperature coefficients and lowered (3)J(NH-alphaH) coupling constants in the region of Phe67-Met70. Conformational behavior of this C5a analogue peptide was studied using molecular modeling incorporating observed NOEs as constraints. The side chains of Tyr65, Phe67, and Met70 consistently form a hydrophobic cluster in all the model structures. The side chains of residues Ser66 and Asp69 can form reciprocal hydrogen bonds with the backbone NH groups of these two residues, indicating that residues Ser66-Phe67-Lys68-Asp69 (or SFKD) form a helix-stabilizing capping box (E. T. Harper and G. D. Rose (1993) Biochemistry, Vol. 32, p. 7605; H. X. Zhou et al. (1994) Proteins: Structure, Function and Genetics, Vol. 18, p. 1] even within the single turn of helical structure found in the analogue C5a peptide. A comparison of nmr results obtained for the analogue peptide and the natural decapeptide C5a 65-74 (Ile65-Ser66-His67-Lys68-Asp-69- Met70-Gln71-Leu72-Gly73-Arg74) indicated that incorporation of residues Tyr65 and Phe67 helps stabilize an isolated capping box involving residues Ser66-Asp69 in the C5a peptides through more extensive hydrophobic/aromatic interactions between residues Tyr65, Phe67, and Met70 in the analogue peptide C5a 65-74 (Tyr65, Phe67). These results constitute the first experimental demonstration of hydrophobic determinants in helical capping-box interactions, proposed recently by a statistical analysis of protein structures [J. W. Seale et al. (1994) Protein Science, Vol. 3, pp. 1741-1745]. The stabilized helical turn may also account for the greater potency of the analogue peptide C5a65-74 (Tyr65, Phe67) in receptor-binding assays.

Amino Acid Sequence↗
PubMed1991

Functional expression of a human C5a receptor clone in Xenopus oocytes requires additional RNA.

cRNA from a PCR-generated C5aR clone was prepared by in vitro transcription and microinjected into Xenopus laevis oocytes. Ligand-induced whole cell current could be detected after co-injection of cRNA for the C5aR with total RNA of the unstimulated U937 cell line, but not with either of the components injected alone. These data clearly demonstrate an absolute requirement of the C5aR for an additional human factor to become functionally expressed in Xenopus oocytes.

Animals↗
PubMed1995

Analysis of C5a receptor by monoclonal antibody.

We prepared a mouse monoclonal antibody (mAb), termed 4C8, to the human C5a receptor (C5aR, CD88) by fusing spleen cells from mice immunized with mouse Ltk- cells transfected with cDNA of human C5aR (Ltk-/C5aR) to the mouse myeloma cell line P3U1. This mAb belonging to the IgM kappa subclass, detected a 43 kDa band on cell lysates of Ltk-/C5aR by immunoblotting analysis. Flow cytometry revealed that 4C8 specifically bound to Ltk-/C5aR, suggesting that this antibody is specific for C5aR. Furthermore, 4C8 was found to partially block both intracellular Ca2+ increase in PMN stimulated by C5a and 125I-C5a binding to C5aR on PMN. When cross-linked by anti-mouse IgM, 4C8 completely inhibited the binding of C5a to C5aR on PMN and Ltk-/C5aR. Therefore, it seems likely that this mAb does not recognize the C5aR active site but sterically inhibits the binding of C5a to its receptor. Using this mAb, we detected a 50 kDa band of C5aR on cell lysates of PMN, monocytes and platelets by immunoblotting. C5aR was expressed on PMN and monocytes as determined by flow cytometry, whereas it was not demonstrated on the surface of platelets. Based on these results, this mAb should be useful for analysis of C5aR expression in various immunological conditions and inflammatory diseases.

Animals↗
PubMed1978

Chemotactic effects of the complement-derived peptides C3a, C3ai and C5a (classical anaphylatoxin) on rabbit and guinea-pig polymorphonuclear leukocytes.

The complement-derived peptides C3a, C3ai and C5a (= classical anaphylatoxin) were purified from hog serum and examined for chemotactic activity on rabbit and guinea-pig polymorphonuclear leukocytes (PMN) with the Boyden chamber technique (with filters of 3,0 micrometer pore size). When media containing albumin or serum were used all peptides induced chemotaxis of both cell species. Only C3a showed a pronounced species dependence in that it was much more active on rabbit than on guinea-pig PMN. No gross differences were found between the influence of 0.5% BSA and 10% heated (56 degree, 30 min) homologous serum added to the medium. In the absence of protein chemotaxis did not occur.

Anaphylatoxins↗
PubMed1992

Mapping of genes for the human C5a receptor (C5AR), human FMLP receptor (FPR), and two FMLP receptor homologue orphan receptors (FPRH1, FPRH2) to chromosome 19.

The genes encoding receptors for the chemotactic ligands C5a (C5AR) and FMLP (FPR) were mapped using a panel of somatic cell hybrids to chromosome 19. Because the hybridization pattern on Southern analysis suggested an intron structure or related genes in the case of FPR, genomic clones were characterized. Two structural homologues of the FMLP receptor, clones 81 (FPRH1) and 82 (FPRH2), were identified, which similarly map to chromosome 19. The structural homologues do not recognize the ligand FMLP, but are likely chemotactic receptors.

Amino Acid Sequence↗
PubMed1993

Agonist-dependent phosphorylation of N-formylpeptide and activation peptide from the fifth component of C (C5a) chemoattractant receptors in differentiated HL60 cells.

Attenuation of signaling is a key step in controlling the cytotoxic potential of leukocyte responses to chemotactic factors. Antipeptide antibodies, directed against the N-formyl chemotactic peptide receptor (FPR) and the activation peptide from the fifth component of C (C5a) anaphylatoxin receptor (C5aR) of human neutrophils, were used to analyze the ability of these receptors to be phosphorylated. Our data show that, in granulocyte-like differentiated HL-60 cells, both FPR and C5aR undergo an agonist dose-dependent phosphorylation that reaches completion in less than 2 to 3 min, consistent with the rate and the dose-dependent attenuation of signaling in phagocytes. Therefore, phosphorylation might be one of the possible mechanisms involved in the desensitization process of FPR and C5aR. Addition of either C5a or the protein kinase C activator (PMA) did not appear to induce the phosphorylation of FPR in the absence of FMLP or to modulate the phosphorylation of the latter at low concentrations of agonist. In contrast, although FMLP at a saturating concentration barely stimulated the phosphorylation of unoccupied C5aR, it markedly potentiated C5aR phosphorylation in cells exposed to low concentrations of C5a. Moreover, PMA was able to induce C5aR phosphorylation in the absence of agonist, indicating that protein kinase C or protein kinase C-activated kinase(s) could be involved in the phosphorylation of C5aR. Pretreatment of cells with staurosporine, a potent but nonspecific inhibitor of protein kinase C, resulted in the partial inhibition of both FPR and C5aR phosphorylation induced by saturating concentrations of agonist, suggesting that a kinase different from protein kinase C might be mainly responsible for the phosphorylation of these chemotactic receptors.

Amino Acid Sequence↗
PubMed1993

Probing the human receptor for C5a anaphylatoxin with site-directed antibodies. Identification of a potential ligand binding site on the NH2-terminal domain.

Molecular cloning has revealed the DNA sequence of the human receptor for the C5a anaphylatoxin (C5aR). In this study, mAb and polyclonal antibodies with specificities for deduced hydrophilic sequences of the receptor protein were employed to determine the expression and the topography of C5aR on PBL. Evidence was obtained that an antigenically homogenous receptor exists on human neutrophils, eosinophils, and monocytes that binds C5a and C5a(desArg). The assignment of epitopes to extra- or intracellular receptor domains as determined by FACS analysis of intact or permeabilized cells confirmed the general topography of the C5aR that had been predicted by hydropathy analysis. Competitive binding studies were performed to examine whether extracellular receptor domains participate in ligand binding. The occupation of the receptor by C5a inhibited only the binding of antibodies that were specific for the receptor's aminoterminal domain (C5aR-EX1). The anti-EX1 mAb S5/1 effectively antagonized C5a/C5a(desArg) biological activity. A heptameric peptide (D15DKDTLD21) was identified as the smallest receptor fragment that was recognized by the mAb S5/1 or by polyclonal rabbit anti-EX1 lg. These results imply that an amino acid sequence rich in aspartate within the receptor aminoterminus represents both an immunodominant epitope and a ligand binding site on the C5aR.

Amino Acid Sequence↗
PubMed1996

Enhanced expression of chemotactic receptors in multiple sclerosis lesions.

We have previously shown that astrocytes and microglia express the receptors for C5a, interleukin-8 (IL-8) and N-formyl-Met-Leu-Phe (FMLP) in vitro. The expression and function of chemotactic receptors in the central nervous system (CNS) is, however, largely unexplored. In this study, we examined tissue sections from normal human brain and active, chronic active and chronic silent multiple sclerosis (MS) lesions for the expression of the receptors for C5a, IL-8 and FMLP by immunohistochemistry. In normal brain tissue, the expression of all three receptors was seen at low levels on astrocytes and microglia. In contrast, expression for all three receptors was markedly elevated on foamy macrophages in the acute and chronic active MS lesions. In addition, fibrous astrocytes stained intensely for the C5a receptor in the chronic active disease. Receptor expression in the chronic silent lesion was low and similar to that seen in normal brain, with staining confined to a few hypertrophic astrocytes and foamy macrophages. These are the first studies to demonstrate expression of these receptors in the CNS and elevated receptor expression in inflammatory MS lesions. The data suggest that these chemotactic receptors may play a role in inflammatory responses in MS and possibly in other CNS diseases.

Acute Disease↗
PubMed1998

Stimulation by anaphylatoxin C5a of glycogen phosphorylase in rat hepatocytes via prostanoid release from hepatic stellate cells but not sinusoidal endothelial cells.

In the perfused rat liver, the anaphylatoxin C5a has been shown to enhance glucose output. Since hepatocytes lack C5a receptor mRNA, the metabolic effect of C5a must be elicited indirectly via C5a receptor expressing non-parenchymal liver cells. Kupffer cells were found to be able to mediate the C5a action via release of prostanoids. However, elimination of the Kupffer cells by pretreatment of the animals with gadolinium chloride reduced the metabolic effect of C5a to only about 40%. Therefore, it was investigated whether not only Kupffer cells but in addition also hepatic stellate cells or sinusoidal endothelial cells released prostanoids in response to C5a. In isolated hepatic stellate cells but not in sinusoidal endothelial cells, recombinant rat C5a induced a time- and dose-dependent release of thromboxane B2 and prostaglandins D2, E2 and F2alpha. The rate of prostanoid release was maximal within the first two minutes and then declined again. C5a-induced prostanoid release from hepatic stellate cells was smaller than that from Kupffer cells and it differed in the prostanoid ratios (PGE2/PGD2/PGF2alpha/TXB2 = 1:1:0.1:0.6 and 1:4:1:3, respectively). RrC5a activated hepatocellular glycogen phosphorylase via prostanoid release in cocultures of hepatocytes with hepatic stellate cells but not with sinusoidal endothelial cells. Thus, the part of the rrC5a-induced glucose output in the perfused rat liver, which was not abrogated by elimination of the Kupffer cells with gadolinium chloride, most likely was mediated by prostanoids released from hepatic stellate cells.

Amino Acid Sequence↗