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PubMed1992

Wheat germ agglutinin inhibits the C5a receptor interaction: implications for receptor microheterogeneity and ligand binding site.

Wheat germ agglutinin (WGA) has been shown to inhibit the interaction of C5a with the C5a receptor on both polymorphonuclear neutrophils (PMNs) and the histiocytic cell line U937. The level of inhibition with isolated receptor preparations is 100%, and on intact cells 10 to 20% of the receptor population appear to retain their ability to bind C5a in the presence of WGA. In contrast, this lectin completely inhibits the C5a-mediated degranulation of PMN primary and secondary granules, suggesting that the population of C5a receptors responsible for mediating degranulation is also recognized by WGA. More than 50% of the receptors appear to be blocked before an effect on degranulation occurs. This inhibition by WGA does not appear to be due to down-regulation of C5a receptors from the cell surface, excessive aggregation of receptor sites, or interaction of WGA with the carbohydrate portion of the C5a molecule. The inhibition is reversed by N-acetylglucosamine but not by sialic acid. This effect appears to be specific for WGA because various other lectins do not inhibit the C5a receptor interaction. That the inhibition by WGA is due to direct binding of the lectin to N-acetylglucosamine residues on the C5a receptor is strongly supported by the ability of the cross-linked C5a-receptor complex to bind to and be specifically eluted from a WGA-Affigel affinity matrix. These observations are consistent with hypothesis that the population of C5a receptors on leukocytes exhibits microheterogeneity with respect to structure (carbohydrate content) and/or function.

Binding Sites↗
PubMed1991

Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor-Gi complex.

We have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a Kd of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the alpha and beta subunits of Gi, respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm our earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects.

Cell Membrane↗
PubMed1995

Probing the "message:address" sites for chemoattractant binding to the C5a receptor. Mutagenesis of hydrophilic and proline residues within the transmembrane segments.

The C5a anaphylatoxin ligand-receptor interaction on polymorphonuclear granulocytes stimulates chemotaxis, degranulation, and the oxidative burst. The receptor is a member of the large G-protein-coupled family. The ligand is a cationic peptide of 72 amino acids derived from the C5 component of complement and has been shown to have a number of structural requirements for interaction with the receptor. In order to probe the potential interaction sites between ligand and receptor, we constructed a series of mutated receptor molecules, targeting cysteines, prolines, and additional amino acids of interest because of combinations of charge or hydrophobicity and putative location with respect to the membrane. Transfected mutant receptors were analyzed for cell surface expression, ligand binding, and ligand-activated phospholipase C activity. The receptors created can be placed generally in four distinct classes: those which bind and signal like the natural receptor; those which bind but fail to transduce signals; those which are expressed but neither bind nor transduce signal; and those which are not expressed at the cell surface.

Amino Acid Sequence↗
PubMed1994

Stabilization of C5a receptor--G-protein interactions through ligand binding.

Binding of biotin-C5a to the C5a receptor in membrane fragments followed by detergent solubilization and purification with streptavidin-agarose affinity chromatography resulted in the isolation of a receptor complex with associated G-proteins. In contrast, when receptor was detergent-solubilized in the absence of C5a and purified by affinity chromatography with Affigel-C5a, G-proteins did not copurify. Since the results indicate that receptor ligation stabilized the receptor--G-protein interaction to allow purification of the complex, the findings emphasize the dynamic nature of the C5a receptor-effector interactions. When biotin-C5a-ligated receptor was purified from a mouse cell line overexpressing recombinant human receptor, both Gialpha2 and Gialpha3 subunits copurified, confirming that multiple transducing systems are linked to the C5a receptor. The method of stabilization of receptor-transducer complexes offers the opportunity to further elaborate the interactions of the C5a receptor with diverse transducing elements and second messenger systems.

Animals↗
PubMed1999

Chimeric receptors of the human C3a receptor and C5a receptor (CD88).

Chimeras were generated between the human anaphylatoxin C3a and C5a receptors (C3aR and C5aR, respectively) to define the structural requirements for ligand binding and discrimination. Chimeric receptors were generated by systematically exchanging between the two receptors four receptor modules (the N terminus, transmembrane regions 1 to 4, the second extracellular loop, and transmembrane region 5 to the C terminus). The mutants were transiently expressed in HEK-293 cells (with or without Galpha-16) and analyzed for cell surface expression, binding of C3a and C5a, and functional responsiveness (calcium mobilization) toward C3a, C5a, and a C3a as well as a C5a analogue peptide. The data indicate that in both anaphylatoxin receptors the transmembrane regions and the second extracellular loop act as a functional unit that is disrupted by any reciprocal exchange. N-terminal substitution confirmed the two-binding site model for the human C5aR, in which the receptor N terminus is required for high affinity binding of the native ligand but not a C5a analogue peptide. In contrast, the human C3a receptor did not require the original N terminus for high affinity binding of and activation by C3a, a result that was confirmed by N-terminal deletion mutants. This indicates a completely different binding mode of the anaphylatoxins to their corresponding receptors. The C5a analogue peptide, but not C5a, was an agonist of the C3aR. Replacement of the C3aR N terminus by the C5aR sequence, however, lead to the generation of a true hybrid C3a/C5a receptor, which bound and functionally responded to both ligands, C3a and C5a.

Anaphylatoxins↗
PubMed1998

Receptors for the anaphylatoxin C5a (CD88) on human mesangial cells.

In these studies, we determined whether there are receptors for the anaphylatoxin C5a (C5aR, CD88) on human mesangial cells (HMC). To prepare Abs to C5aR, we first synthesized an immunogenic peptide spanning residues 8-32 of the molecule, and this peptide was used to immunize rabbits. Anti-C5aR antiserum, but not preimmune serum, stained fixed and unfixed HMC in culture. By Western blotting anti-C5aR, Abs identified a 49.6-kDa protein in HMC. By reverse-transcription PCR, a cDNA product of 558 bp was amplified corresponding to the expected size of C5aR cDNA. A cDNA of the same size was amplified simultaneously from human PBL. Restriction mapping of the products amplified from HMC and from PBL gave restriction fragments of the same size. Incubation of HMC with increasing doses of C5a caused a progressive increase in the levels of the transcription factors activator protein-1 (AP-1) and cAMP response element binding protein (CREB), but C5a had no effect on the level of nuclear factor-kappaB (NF-kappaB). The effects of C5a on AP-1 were concentration and time dependent and peaked after 60 min. In contrast, the C5a metabolite C5adesArg had no significant effect on AP-1 levels. Preincubation of HMC with rabbit anti-C5aR antiserum inhibited partially the effect of C5a on AP-1. However, anti-C5aR Abs alone had no appreciable effects on AP-1. C5a caused a significant up-regulation of mRNA for the early response genes c-jun and c-fos on HMC. These results provide evidence for the presence of C5aR in adult HMC in culture and indicate that, after binding to C5aR, the anaphylatoxin C5a causes significant up-regulation of certain transcription factors and early response genes.

Adult↗
PubMed1999

Protein kinase C activation is required for cigarette smoke-enhanced C5a-mediated release of interleukin-8 in human bronchial epithelial cells.

Complement-derived anaphylatoxin C5a is a glycopolypeptide important in the regulation of inflammation. Previously, we have shown that C5a receptors (C5aR) are constitutively expressed on human bronchial epithelial cells (HBECs) grown in culture. We have also shown that the expression of C5aR is increased upon exposure of HBECs to 5% cigarette smoke extract (CSE), and that this subtoxic dose of CSE significantly enhances C5a-stimulated interleukin (IL)-8 release. To determine the intracellular signaling pathway of CSE + C5a-mediated IL-8 release, we assayed protein kinase C (PKC) activity of HBECs after exposing the cells to CSE and/or C5a. No increase in PKC activity was observed when HBECs were treated with 50 nM C5a for various times. However, PKC activity was increased by 2- to 3-fold in HBECs stimulated with 5% CSE for 1 h, as compared with cells incubated with medium only. No additional increase in PKC was observed when HBECs were treated with CSE and C5a together. When HBECs were pretreated with the PKC-specific inhibitor calphostin C (1 microM), no CSE-mediated PKC activation was observed. We then correlated PKC activation with IL-8 release in the same cells. Although HBECs required stimulation by both CSE and C5a to release maximal levels of IL-8, preincubation of CSE-stimulated HBECs with calphostin C inhibited IL-8 release by CSE + C5a. These results suggest that PKC activation by CSE alone does not result in IL-8 release, but that CSE-stimulated PKC activation is required for C5a-mediated IL-8 release from HBECs.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Gc globulin (vitamin D-binding protein) increases binding of low concentrations of C5a des Arg to human polymorphonuclear leukocytes: an explanation for its cochemotaxin activity.

The chemotactic activity of native human C5a des Arg is enhanced significantly by the normal serum and plasma protein Gc globulin (vitamin D-binding protein). Gc globulin attaches to sialic acid residues within the oligosaccharide chain of C5a des Arg to form a complex with potent chemotactic activity for human PMN. We investigated the mechanism whereby this phenomenon may occur and found that Gc globulin enhanced the binding of low concentrations of [125I]C5a des Arg to PMN, but had no effect on C5a-induced displacement of bound [125]C5a des Arg. Gc globulin bound to PMN, and probably acted as a C5a des Arg chaperon. Thus, it appears that Gc globulin, by complexing to C5a des Arg, increases the number of C5a des Arg molecules per unit of PMN membrane without affecting its affinity of binding. This phenomenon provides a plausible explanation for the enhancing effect of Gc globulin on the chemotactic activity of low concentrations of native human C5a des Arg.

Chemotaxis, Leukocyte↗
PubMed1989

1H nuclear magnetic resonance assignments and secondary structure of porcine C5ades Arg.

Sequence-specific assignments of the 1H nuclear magnetic resonance spectrum of porcine C5ades Arg are described. Assignments were facilitated by comparison of spectra obtained in H2O with partially exchanged spectra obtained in 2H2O. The sequence-specific assignments thus obtained were used to characterized the regular secondary structure in the protein, which is helical in the regions 2 to 11, 16 to 27, 35 to 41 and 45 to 64. The structure is very similar to that of human and bovine C5a.

Amino Acid Sequence↗
PubMed1994

Identification of a receptor-binding region in the core segment of the human anaphylatoxin C5a.

In order to identify regions of C5a that contribute to receptor binding and functional activity of the anaphylatoxin, a series of peptides was synthesized in which core segments have been attached to C-terminal segments via native peptidic or disulfide bonds. It has been found that residues Arg40 and Arg46 in the loop-3 region of the core induce a 1000-fold increase in the affinity of the disordered C-terminal segment of C5a. The results obtained from this work lead to the conclusion that the loop-3 region is most likely the core binding site of C5a.

Amino Acid Sequence↗
PubMed1992

Substituted 4,6-diaminoquinolines as inhibitors of C5a receptor binding.

The anaphylatoxin C5a is implicated in a number of inflammatory diseases. It is a highly cationic protein with 13 of 74 amino acids being either arginine or lysine. A search focusing on positively charged molecules, particularly amine-containing functionalities, led to the discovery of substituted 4,6-diaminoquinolines 1 [N,N'-bis(4-amino-2-methyl-6-quinolyl)urea] and 7 [6-N-(2-chlorocinnamoyl)-4,6-diamino-2-methylquinoline] as inhibitors of C5a receptor binding. These two compounds inhibited the binding of radiolabeled C5a to its receptor isolated from human neutrophils with IC50's = 3.3 and 12 micrograms/mL, respectively. Our efforts to enhance their potencies by chemical modification revealed a narrow profile of potency for effective C5a receptor binding inhibition.

Aminoquinolines↗
PubMed2001

Targeting complement in therapy.

With increasing evidence that complement activation significantly contributes to the pathogenesis of a large number of inflammatory diseases, strategies that interfere with its deleterious action have become a major focus in pharmacological research. Endogenous soluble complement inhibitors (C1 inhibitor, recombinant soluble complement receptor 1, antibodies) blocking key proteins of the cascade reaction, neutralizing the action of the complement-derived anaphylatoxin C5a, or interfering with complement receptor 3 (CR3, CD18/11b)-mediated adhesion of inflammatory cells to the vascular endothelium have successfully been tested in various animal models over the past years. Promising results consequently led to clinical trials. Furthermore, incorporation of membrane-bound complement regulators (decay-accelerating factor (CD55), membrane co-factor protein (CD46), CD59) in transgenic animals has provided a major step forward in protecting xenografts from hyperacute rejection. At the same time, the poor contribution of complement to the antitumor response, which is caused by multiple resistance mechanisms that hamper the efficacy of antibody-based tumor therapy, is increasingly recognized and requires pharmacologic intervention. First attempts have now been made to interfere with the resistance mechanisms, thereby improving complement-mediated tumor cell destruction.

Anaphylatoxins↗
PubMed2001

Distinct amplification of the C5a-receptor pathways in normodense and hypodense eosinophils of patients with atopic dermatitis.

In patients with atopic dermatitis two different types of blood eosinophils with distinct density can be isolated. The normodense cells represent the huge majority in count, whereas the hypodense eosinophils are characterized by higher effector activity. To understand the altered functional responsiveness of these two cell subtypes, the expression of C5a receptors as well as C5a-induced signal pathways and the production of reactive oxygen metabolites have been analyzed. Chemiluminescence measurements revealed significant higher production of reactive oxygen metabolites in hypodense eosinophils in comparison to normodense cells. However, no difference in the expression level of C5a receptors as well as in the C5a-induced Ca2+-transients between normodense and hypodense eosinophils were found. In contrast, hypodense eosinophils showed a significantly higher actin polymerization response and phosphatidylinositol 4,5 bisphosphate 3-kinase activation after stimulation with C5a than normodense eosinophils. Therefore, normodense and hypodense eosinophils from the blood of patients with atopic dermatitis are characterized by differential amplification of C5a-receptor signal pathways, which might explain the differences in their proinflammatory activity.

Actins↗
PubMed2002

The complement factor 5a receptor gene maps to murine chromosome 7.

Complement factor 5a (C5a) promotes local inflammation and is a potent chemoattractant for neutrophils and macrophages. We had an interest in C5a and its receptor, C5r1, because we previously identified C5a as a positional candidate gene for the quantitative trait locus Abhr2, which determines allergen-induced bronchial hyperresponsiveness in our murine model of asthma. To study the significance of C5r1 in our asthma model we first had to determine its genomic map location in mice. Genomic sequence surrounding murine C5r1 was analyzed for polymorphisms and two variable microsatellites were identified. These microsatellites were genotyped in A/J x (C3H/HeJ x A/J)F1 backcross mice (n = 355) and mapped in a panel of 164 markers spaced at approximately 10 cM intervals throughout the genome. Multipoint linkage analysis placed C5r1 on murine chromosome 7, 3.9 cM from the top of the linkage group. This map location has been previously identified as containing an additional quantitative trait locus for allergen-induced airway hyperresponsiveness, Abhr3, in this population of mice.

Animals↗
PubMed1997

Biological responses to endovascular treatment of abdominal aortic aneurysms.

PURPOSE: To review the findings of two studies investigating the apparent differences in inflammatory responses demonstrated in patients undergoing endovascular as opposed to classic surgical treatment of abdominal aortic aneurysms (AAAs). METHODS: The clinical course of seven patients treated with an endoluminal procedure (AAA-E) and seven patients undergoing conventional surgery (AAA-C) were compared (all men; ages 52 to 80 years). Blood samples were taken pre-, intra-, and postoperatively for up to 7 days. Inflammatory responses were assessed from measurement of interleukins (IL)-1 beta, IL-6, IL-8, and tumor necrosis factor (TNF-alpha); complement proteins C1q, C4, C5a, and terminal complement complexes, C5b-C9; and C-reactive proteins. Granulocyte and monocyte surface adhesion molecule expression was determined indirectly using a panel of monoclonal antibodies against CD11a, CD11b, CD11c, CD18, and L-selectin in donor white blood cells exposed to patient plasma. RESULTS: In six of the AAA-E patients, blood pressure decreases were recorded during the introduction of the device. Elevated body temperature was sustained for 2 to 5 days postoperatively in the AAA-E group. IL-6 levels were significantly higher in AAA-C patients (p < 0.0005), while TNF-alpha release was recorded in the AAA-E group only. CD11b, CD11c, and CD18 molecules on both granulocytes and monocytes were significantly upregulated 60 minutes after the endovascular procedure compared to conventional surgery. CONCLUSIONS: Endovascular aortic aneurysm repair apparently induces a significant inflammatory response, mainly involving TNF-alpha release, which differs from open AAA repair. These inflammatory responses, which may be related to the observed intraprocedural blood pressure decreases, could be caused by cell activation arising from intra-aneurysmal device manipulation.

Aged↗
PubMed2000

Induction of functional anaphylatoxin C5a receptors on hepatocytes by in vivo treatment of rats with IL-6.

In normal rat liver, anaphylatoxin C5a receptors (C5aR) are only expressed by nonparenchymal cells, mainly Kupffer cells and hepatic stellate cells, but not by parenchymal cells, i.e., hepatocytes (HC). Nevertheless, C5a stimulates glucose output by HC. This HC-specific defense reaction is induced indirectly via prostanoids secreted by the C5aR-expressing Kupffer cells and hepatic stellate cells. It is shown here that under inflammatory conditions simulated by in vivo treatment of rats with IL-6 C5aR mRNA and protein were induced in HC in a time-dependent manner. Maximal mRNA and protein expression were observed at 4-8 h and 8-10 h, respectively, after IL-6 injection. The newly expressed receptors were functional, because recombinant rat C5a significantly activated glycogen phosphorylase in HC isolated from IL-6-treated but not in HC from control rats. In perfused livers of IL-6-treated animals in contrast to control animals, recombinant rat C5a-induced glucose output was not impaired by inhibition of prostanoid synthesis and function with the cyclooxygenase inhibitor indomethacin and the thromboxane receptor antagonist daltroban. These results indicate that HC-specific defense reactions might be differently regulated under normal and inflammatory conditions as shown here for the indirect prostanoid-dependent or direct C5a-induced activation of hepatocellular glycogen phyosphorylase and glucose output in control or IL-6-treated rats, respectively.

Animals↗
PubMed1991

In vitro activation of human complement by nitrogen bubbles.

Complement activation may be responsible for some of the symptoms of decompression sickness. In the present study, complement activation was studied by exposing human sera with or without red blood cells to nitrogen bubbles. Nitrogen bubbles activated human complement as measured by generation of the fluid-phase, complement-split product C5a des Arg. In addition, we found that complement activation continued after exposure to bubbles was stopped. This continued complement activation may explain the failure of recompression treatment in some patients. Complement activation induced by nitrogen bubbles in human sera was enhanced when red cells were present. Red cells may be able to provide a stable membrane surface on which complement activation by bubbles can occur more efficiently. Complement activation by nitrogen bubbles in serum also led to the binding of activated C3 to red cells. Quantitation of bystander red-cell-bound C3d may allow the assessment of complement activation occurring by nitrogen bubbles in individuals undergoing decompression.

Complement Activation↗
PubMed1993

Regulation of eosinophil migration by adult T cell leukemia-derived factor.

Adult T cell leukemia-derived factor (ADF), originally defined as an IL-2 receptor alpha-chain (IL-2R alpha)/p55 (Tac) inducer, is a human thioredoxin homologue and has many cytokine-like activities. In this study, we examined the regulatory effect of ADF on eosinophil migration using human eosinophils and an eosinophilic subline of HL-60 human promyelocytic leukemia cells, YY-1. rADF induced migration of eosinophils from patients with hypereosinophilia, although rADF exhibited little activity on eosinophils from healthy donors. When human eosinophils were incubated with rADF (0.1-10 micrograms/ml) at 37 degrees C for 24 h, both chemotactic and chemokinetic activity of the complement anaphylatoxin peptide C5a on eosinophil migration was markedly enhanced in a dose-dependent manner. Similarly, this enhancing effect of rADF was observed in the migration assay using YY-1 cells. In contrast, rADF showed no modulation of migratory behavior of human eosinophils and YY-1 cells by IL-3, IL-5, nor granulocyte-macrophage colony-stimulating factor. Scatchard analysis of C5a receptors on YY-1 cells using 125I-C5a showed that rADF modulated neither the density nor the affinity of the cell membrane significantly. Furthermore, mutant ADF (mADF), which had no reducing activity, had no enhancing effect on C5a-induced eosinophil migration. These results indicate a possible involvement of ADF in the recruitment of eosinophils through redox regulation by a dithiol reductase activity.

Adult↗