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PubMed1986

Anaphylatoxin C5a fails to promote prostacyclin release in cultured endothelial cells from human umbilical veins.

Subcultured endothelial cells from human umbilical veins respond to histamine and melittin with increased prostacyclin production, measured as 6-keto-prostaglandin F1 alpha by radioimmunoassay. However, no response to leukotriene C4 was observed. Primary cultured cells, on the other hand, respond to leukotriene C4 and the histamine response was 7 times more potent for these cells than for subcultured cells. In contrast, neither primary cultures nor subcultures of endothelial cells released prostacyclin following application of either human anaphylatoxin C5a (100 nM) or C3a (1 microM). In addition, these endothelial cells appear to have no specific binding sites for 125I-C5a. However, endothelial cells released prostacyclin in the presence of human polymorphonuclear leukocytes that were activated with C5a. We conclude that involvement of endothelial cells in the haemodynamic response to anaphylatoxin is an indirect function, i.e. C5a activates circulating or tissue cells which in turn stimulate the endothelial cell to produce prostacyclin.

6-Ketoprostaglandin F1 alpha↗
PubMed1991

The priming effects of the products of stimulated mononuclear cells on the response of neutrophils to C5a des arg.

Certain recombinant human cytokines have been shown to enhance polymorphonuclear leucocyte (PMN) responses to subsequent stimulation. Mononuclear cells (MNC) from normal healthy individuals were stimulated for 5 h with 1 micrograms/ml bacterial lipopolysaccharide (LPS) in order to induce production and secretion of inflammatory cytokines into the surrounding medium. These mononuclear cell conditioned media (MNCM) were then used to prime PMN isolated from healthy volunteers. Preincubating the PMN with MNCM for 15 min at 4 degrees C followed by washing and warming to 37 degrees C caused a 344% increase (n = 26) in the rate of superoxide anion production in response to zymosan-activated serum (ZAS), a source of C5a des arg. This effect could not be reproduced with recombinant human forms of interleukin 1 beta (Il-1 beta) or granulocyte-macrophage-colony stimulating factor (GM-CSF), although, with the latter, there was some effect when the preincubation stage was carried out for 60 min at 37 degrees C. Only recombinant human tumour necrosis factor-alpha (rh-TNF-alpha) gave a similar PMN priming effect to that seen with MNCM. This effect could not be reversed by washing away either the MNCM or rh-TNF-alpha. The priming effect could be markedly reduced (74.8%, n = 6) by employing the use of polyclonal antibody to TNF-alpha in the preincubation step; assaying for TNF-alpha in these MNCMs showed that the degree of priming corresponded to the amount of TNF-alpha present. Rh-TNF-alpha alone appeared to have very little direct stimulatory effect on respiratory burst activity. The results show that TNF-alpha produced by LPS stimulated MNC after 5 h binds to a PMN surface receptor in the cold and warming of the cells to 37 degrees C allows for an immediate and dramatic response to ZAS stimulation. This suggests that TNF-alpha is the important cytokine upregulating PMN responses to other physiological mediators, including C5a des arg during the early phases of an inflammatory reaction.

Cells, Cultured↗
PubMed2005

A critical role for sphingosine kinase in anaphylatoxin-induced neutropenia, peritonitis, and cytokine production in vivo.

The aim of our study was to investigate the roles played by sphingosine kinase (SPHK) in the anaphylatoxin C5a-triggered responses in vivo. Our data show that i.v. administration of C5a triggers a rapid neutropenic response, but pretreating mice with the SPHK inhibitor, N,N-dimethylsphingosine (DMS), 10 min before the C5a i.v. administration substantially inhibited the C5a-triggered neutropenia. Similarly the i.v. administration of C5a caused a rapid increase in the serum levels of TNF-alpha and IL-6, and this increase in cytokine levels was blocked by DMS. We then induced acute peritonitis with C5a. The C5a i.p. injection triggered a fast recruitment of neutrophils, later followed by monocytes, into the peritoneal cavity. Vascular permeability was also observed: when we i.v. injected Evans blue before C5a i.p. injection, we could observe a continued influx of the dye into the peritoneum. In mice pretreated with DMS, there was a significant reduction on the C5a-triggered neutrophil and monocyte infiltration, as well as a marked reduction on the Evans blue influx. Our data also show that the i.p. administration of C5a caused a rapid increase in TNF-alpha and IL-6 levels in the peritoneal cavity, and this increase in cytokine levels was substantially inhibited in mice pretreated with the SPHK inhibitor. Taken together, these observations suggest a potential role for SPHK in the C5a-triggered inflammatory responses in vivo.

Animals↗
PubMedDate not supplied

Effect of LTB4 and its isomers on human leucocyte migration into skin chambers.

The in vivo chemotactic effect of LTB4 and of its isomers, 6-trans-LTB4, 12 epi-6-trans-LTB4 and 5S, 12S-DHETE, was tested with a skin chamber technique in healthy volunteers and in parallel in vitro with an under-agarose technique. LTB4 had an in vivo chemotactic effect at 10(-7) mol/l in 24-hour experiments, while its isomers had no in vivo chemotactic effect at this concentration. LTB4 was also in vitro a more effective attractant than its isomers. In addition, C5ades Arg was tested using zymosan-activated serum, and was found to have an in vivo chemotactic effect at 1.5 X 10(-10) mol/l. However, when LTB4 and C5ades Arg were studied in 6-hour experiments in skin chambers there was an alteration in relative potency, LTB4 being relatively more potent at shorter test durations. This is most likely due to metabolisation of LTB4 in the presence of PMN:s and precludes a strict comparison of the in vivo chemotactic effects of LTB4 and C5ades Arg. When zymosan-activated serum or LTB4 was replaced by PBS after six hours in skin chamber experiments more leukocytes accumulated in the chambers at 24 hours than in chambers containing PBS for the whole 24 hour period. The reason for the increased migration even after the removal of the chemo-attractants as well as the relevance of LTB4 and C5a as chemo-attractants in the inflammatory process is discussed.

Chemotaxis, Leukocyte↗
PubMed1991

Evidence for the involvement of multiple signalling pathways in C5a-induced actin polymerization and nucleation in human monocyte-like cells.

The signal transduction mechanisms involved in complement fragment C5a-induced recruitment of actin to the cytoskeleton have been investigated using U-937 cells differentiated by exposure to dibutyryl cyclic AMP. Two parameters of cytoskeletal activation were compared: F-actin formation and nucleation of polymerization of pyrenyl-actin in whole cell lysates. The dose dependency of these responses to C5a was clearly different to that observed for [3H]inositol phosphate formation and also markedly different from that observed for the production of reactive oxygen intermediates (ROI). Further evidence to dissociate inositol lipid hydrolysis from these cytoskeletal responses was obtained by treating cells with neomycin, phorbol myristate acetate and pertussis toxin and by modulating the levels of intracellular Ca2+ using quin 2. Inhibition of [3H]inositol phosphate and ROI production was not correlated with effects on actin recruitment or nucleation. In addition, these agents had differing effects on F-actin formation and nucleation activity. The results show that the production of inositol phosphates is not required for stimulating either F-actin formation or nucleation activity and also that ligand-induced polymerization of actin depends primarily upon an increase in the availability of G-actin rather than nucleation sites. These cytoskeletal responses are apparently controlled by different signalling pathways which diverge at an early stage.

Actins↗
PubMed2004

The possibilities and pitfalls for anti-complement therapies in inflammatory diseases.

The complement system is a key component of innate immunity, acting to protect the host from micro-organisms such as bacteria and other "foreign" threats, including tumor cells. However, excessive activation of complement can injure the host and can even be life threatening. These toxic effects are caused primarily by the excessive production of the anaphylatoxins C3a and C5a during complement activation and excessive formation of membrane attack complex on the host cell membrane. Many inflammatory diseases, including rheumatoid arthritis and glomerulonephritis, are thought to involve excessive activation of complement, both for their development and perpetuation. Uncontrolled complement activation is also implicated in post-ischemic inflammation and tissue damage and in sepsis. Therefore, it is important to regulate the complement system to treat disease. There are still no broadly applicable agents for the therapeutic regulation of excessive complement activation. However, there are now some agents in the development that might provide useful anti-complement therapies in the near future. Current strategies include the use of neutralizing antibodies, small synthetic antagonists, soluble recombinant forms of the natural complement regulators, and gene therapies to control excessive complement activation. Here we describe these new agents, their strengths and weaknesses and progress in testing the agents in relevant animal models.

Animals↗
PubMed1985

Modulation of neutrophil-reduced pyridine nucleotide content following stimulation with phorbol myristate acetate and chemotactic factors.

Modulation of NAD(P)H in human neutrophils (PMN) following stimulation with phorbol myristate acetate (PMA) or chemotactic factors was determined by flow cytometry. Stimulation of PMN with 1 microgram/ml of PMA results in a time-dependent decrease in fluorescence, attributable to the oxidation of NAD(P)H. The decrease in fluorescence did not occur with PMN from a patient with chronic granulomatous disease (CGD) and was observed in only half of PMN from the mother of the patient. Loss of fluorescence in normal PMN was maximal following 7-15 min of stimulation with PMA. Simultaneous measurement of PMA-stimulated NAD(P)H oxidation and H2O2 production showed that NAD(P)H oxidation occurred as an all-or-none response while H2O2 production showed a graded response. These data suggest that with PMA stimulation, a threshold exists beyond which constitutive NAD(P)+ reduction is suppressed and complete oxidation of NAD(P)H occurs, while H2O2 production is proportional to the concentration of PMA. PMA-stimulated oxidation of NAD(P)H was reversible, and fluorescence returned to the initial level or higher after 40-60 min. Oxidation of NAD(P)H also occurred when cytochalasin B-treated PMN were stimulated with 25 nM C5a or 100 nM formyl-methionyl-leucyl-phenylalanine (f-MLP), but occurred more rapidly, peaking at 1 to 3 min. Fluorescence also returned by 5-6 min. This response to C5a and f-MLP was graded and proportional to the concentration of chemotactic factor used. Comparative studies showed that the cytochalasin-B treatment was essential for measurement of NAD(P)H oxidation, in response to C5a and F-MLP.

Complement C5↗
PubMed1991

Formation of C5a during cardiopulmonary bypass: inhibition by precoating with heparin.

A novel enzyme immunoassay based on direct detection of C5a by a monoclonal antibody (C17/5) specific for a neoepitope exposed in C5a/C5adesArg was used to measure in vivo and in vitro C5a formation during cardiopulmonary bypass. In vivo, we observed a significant threefold to fourfold increase in patient plasma C5a/C5adesArg levels from baseline values (5.6; 1.6 to 12.9 ng/mL) (median and range) up to 42 hours postoperatively (17.5; 6.5 to 46.0 ng/mL) when two different uncoated cardiopulmonary bypass circuits were used. Coating of the extracorporeal circuit with end-point-attached heparin completely abolished C5a formation in vitro during circulation of blood through the circuit for 120 minutes. The C5a concentration (median and range) was 3.2 (2.6 to 15.9) ng/mL at the start and 3.1 (2.7 to 15.0) ng/mL at the end of the experiment. In the uncoated setups the corresponding C5a concentrations were 10.1 (6.2 to 17.5) and 19.7 (13.1 to 24.3) ng/mL. Finally, heparin-coated cardiopulmonary bypass circuits were examined in vivo. C5a levels did not increase significantly during the cardiopulmonary bypass period in the heparin-coated group in contrast to the uncoated group, but the postoperative increase in C5a levels was similar in the two groups. We conclude that heparin coating improves biocompatibility by completely abolishing C5a formation in vitro. The discrepancy between the in vitro and the in vivo findings is probably related to the complicated biological turnover of C5a.

Antibodies, Monoclonal↗
PubMed1990

Regulation of the human autologous T cell proliferation by endogenously generated C5a.

The immunomodulating role of endogenously synthesized C5 and subsequently generated C5a was studied in a serum-free human autologous mixed leukocyte reaction (AMLR) using either separated T and non-T cell populations or unfractionated mononuclear leukocytes of human peripheral blood. Monoclonal mouse IgG or Fab fragments against human C5/C5a were used as probes for the evaluation of the biological effects of C5a. The reduction of DNA synthesis after the addition of nanogram amounts of anti-C5/C5a mAb was dose-dependent, reaching maximum levels of 30-50%. Of special importance was the availability of a mAb that recognizes a neoepitope present on C5a and not on serum-derived C5. The demonstration of the specificity of its inhibitory effect suggests that C5 is synthesized under the in vitro conditions employed and that the subsequently generated C5a exerts biological effects on T cell proliferation.

Antibodies, Monoclonal↗
PubMed1991

Inflammatory properties of neutrophil-activating protein-1/interleukin 8 (NAP-1/IL-8) in human skin: a light- and electronmicroscopic study.

Neutrophil-activating protein-1/interleukin 8 (NAP-1/IL-8), purified to homogeneity from lipopolysaccharide-stimulated human peripheral blood monocytes, was injected intracutaneously into human skin. Sequential biopsy specimens were taken in order to investigate the sequence of ultrastructural changes induced by the cytokine. Whereas intracutaneous injection of 100 ng of NAP-1/IL-8 per site caused no macroscopic changes, by histology infiltration with polymorphonuclear leukocytes (PMN) and monocytes was present within 1 h and increased at 3 and 5 h. No lymphocyte infiltration was noted. The first ultrastructural changes (30 min) consisted of the presence of cytoplasmic 7-nm microfilament bundles, as well as numerous protrusions of the luminal plasma membrane of endothelial cells (EC). As a striking feature, multiple 100- to 160-nm electron lucent vesicles could be observed in the EC cytoplasm. These structures differed from plasmalemmal vesicles and suggest secretory activity. When PMN and monocytes appeared in the vascular lumen (1 h and later), the number of 100-160-nm electron-lucent vesicles had decreased significantly. In contrast to C5a-injected skin sites, mast cell degranulation was absent.

Complement C5a↗
PubMed1988

C5a-induced bronchoconstriction: absence of a role of circulating white blood cells and platelets.

C5a causes an intense bronchoconstriction when injected intravenously into the guinea pig. The present study was designed to determine if circulating white blood cells or platelets were important target cells for the induction of bronchoconstriction on intravenous injection of C5a. To accomplish this, we examined the effect of C5a on numbers of circulating cells as well as the effect of depletion of various circulating cell populations on C5a-induced bronchoconstriction. Intravenous injection of C5a caused a precipitous drop in circulating granulocytes which preceded the maximum bronchoconstrictor response. The levels of circulating platelets and mononuclear cells were unchanged by intravenous injection of C5a. Depletion of either white blood cells or platelets did not significantly alter either the maximum bronchoconstrictor response to C5a or the time course of the response. Thus, these studies suggest that C5a-induced bronchoconstriction in the guinea pig is not dependent on an interaction of C5a with circulating white blood cells or platelets.

Airway Resistance↗
PubMed1992

Effect of adenylate cyclase activators on C5a-induced human neutrophil aggregation, enzyme release and superoxide production.

The effect of adenylate cyclase activators on C5a- and f-Met-Leu-Phe-induced human neutrophil aggregation, enzyme release and superoxide production was investigated. C5a-stimulated superoxide production was markedly inhibited by adenylate cyclase activators, and the order of potency was PGE1 greater than isoproterenol greater than epinephrine greater than PGF2 alpha, which correlated with intracellular cAMP levels. However, neutrophil aggregation was inhibited by PGE1, PGE2, isoproterenol and epinephrine only at concentrations greater than 10(-6) M. Lysozyme release was inhibited only via PGEs in the presence of the phosphodiesterase inhibitor, methylisobutylxanthine. These results suggest that in the human neutrophil: (1) C5a-induced superoxide production is more sensitive to regulation by cAMP than neutrophil aggregation or enzyme release, and (2) the type of receptor occupied as well as the threshold level of cAMP are important in the regulation of neutrophil aggregation and enzyme release stimulated by C5a.

1-Methyl-3-isobutylxanthine↗
PubMed2005

CD44 and annexin A2 mediate the C5a chemotactic cofactor function of the vitamin D binding protein.

The vitamin D binding protein (DBP) is a plasma protein that significantly enhances the chemotactic activity of C5a and C5a(desArg) (cochemotactic activity). The objective of this study was to investigate how DBP mediates this process using neutrophils and U937 cells transfected with the C5a receptor (U937-C5aR cells) and comparing chemotaxis to C-activated serum (DBP dependent) vs purified C5a (DBP independent). Binding to the cell surface is essential for this protein to function as a chemotactic cofactor, and DBP binds to a chondroitin sulfate proteoglycan (CSPG) on neutrophil plasma membrane preparations. To determine whether a CSPG also functions to mediate cochemotactic activity, U937-C5aR cells were grown in chlorate to inhibit CSPG sulfation or treated with chondroitinase AC. Either treatment significantly inhibited chemotaxis only to C-activated serum. CD44 is a major cell surface CSPG on leukocytes, and functions to facilitate chemotaxis. Treatment of cells with anti-CD44 blocks chemotaxis of neutrophils and U937-C5aR cells to C-activated serum but not purified C5a. DBP binds to CD44 on the cell surface as evidenced by coimmunoprecipitation, confocal microscopy, and cell binding studies. Annexin A2 associates with CD44 in lipid rafts; therefore, its potential role in mediating cochemotactic activity was investigated. Results demonstrate that anti-A2 inhibits neutrophil and U937-C5aR chemotaxis specifically to C-activated serum, blocks DBP binding to cells, and colocalizes with anti-DBP on the cell surface. These results provide clear evidence that CD44 and annexin A2 mediate the C5a chemotactic cofactor function of DBP.

Annexin A2↗
PubMed1996

Thrombin primes responsiveness of selective chemoattractant receptors at a site distal to G protein activation.

To define the molecular basis of human chemoattractant receptor regulation, rat basophilic leukemia RBL-2H3 cells, which are thrombin-responsive, were transfected to stably express epitope-tagged receptors for C5a, interleukin-8 (IL-8), formylpeptides (e.g. N-formyl-methionyl-leucyl-phenylalanine (fMLP)), and platelet-activating factor (PAF). Here we demonstrate that both thrombin and a synthetic peptide ligand for the thrombin receptor (sequence SFLLRN) caused phosphorylation and heterologous desensitization of the receptors for C5a, IL-8, and PAF but not that for formylpeptides as measured by agonist-stimulated [35S]guanosine 5'-3-O-(thio)triphosphate binding to membranes. Consistent with the PAF receptor phosphorylation, both thrombin and thrombin receptor peptide inhibited phosphoinositide hydrolysis, Ca2+ mobilization, and degranulation stimulated by PAF. Unexpectedly, despite heterologous desensitization at the level of receptor/G protein activation, there was enhancement ("priming") by thrombin of subsequent activities stimulated by C5a and IL-8 as well as fMLP. The priming effect of thrombin was blocked by its inhibitor, hirudin. However, two other activators of the thrombin receptor, the peptide SFLLRN and trypsin, stimulated Ca2+ mobilization in RBL-2H3 cells but did not cause priming. In addition, SFLLRN and the thrombin receptor antagonist peptide FLLRN both inhibited thrombin-induced Ca2+ mobilization but not priming. Furthermore, the proteolytically active gamma-thrombin, which does not stimulate the tethered ligand thrombin receptor and caused little or no Ca2+ mobilization in RBL-2H3 cells, effectively primed the response to fMLP. These data demonstrate that heterologous receptor phosphorylation and attenuation of G protein activation are not, by themselves, sufficient for the inhibition of biological responses mediated by C5a and IL-8. Moreover, thrombin appears to utilize mechanism(s) independent of its tethered ligand receptor to selectively prime phospholipase C-mediated biological responses of the C5a, IL-8, and formylpeptide receptors but not PAF. Because C5a, IL-8, and formylpeptide activate phospholipase Cbeta2, whereas PAF stimulates a different phospholipase C, the striking selectivity of thrombin's priming may be mediated via its ability to enhance receptor-mediated activation of phospholipase Cbeta2.

Amino Acid Sequence↗
PubMed2000

Analysis of receptor/ligand interactions using whole-molecule randomly-mutated ligand libraries.

We report a novel method for the analysis of protein ligands using a whole molecule mutagenesis/phage display system. The cDNA for the inflammatory polypeptide C5a was used as template in a PCR reaction doped with mutagenic nucleoside triphosphates (dP and 8-oxo-2'deoxyguanosine (8-oxodG)) to allow introduction of mutations in a highly controlled manner throughout the cDNA. The resultant library of mutants was displayed on the surface of phage and functional polypeptides were selected by several rounds of selection against the cells bearing the receptor for C5a. Following selection only a limited number of residues in C5a were found to be mutated, suggesting that mutations in key residues involved in the maintenance of structure and in receptor binding had been eliminated. The selected C5a sequences had a higher affinity for receptor than wild type phage-C5a conjugates. As this method for analysing the functional characteristics of proteins does not rely on knowledge a priori of structure, it may be useful for affinity maturation or analysis in a wide range of protein ligand/receptor systems.

Amino Acid Sequence↗
PubMed1993

Chronic myelogenous leukemia-derived basophilic granulocytes express a functional active receptor for the anaphylatoxin C3a.

The receptor for the inflammatory peptide C3a has scarcely been examined on human cells. This work demonstrates that human tumor-derived basophilic granulocytes express C3a receptors, and presents parts of the hitherto unknown C3a-signal transduction. When incubated with IL-3, these cells specifically liberated histamine on C3a stimulation. Independent from IL-3, 240,000 +/- 100,000 receptors per cell with a Kd of 5.6 +/- 0.9 nM were determined. [Ca2+]i increased from 120 +/- 35 nM to 300 +/- 80 nM after a C3a challenge, as measured by digital imaging fluorescence microscopy, and rested at its basal level in the presence of C3a-desArg, the immediate catabolic product of C3a in vivo. This [Ca2+]i increase could be completely desensitized homologously by C3a as well as inhibited by up to 75% by pertussis toxin. Thus, tumor-derived basophils are suitable for cloning of the human C3a receptor.

Anaphylatoxins↗