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PubMed1994

Degranulation from human eosinophils stimulated with C3a and C5a.

It is unclear what actually induces eosinophil degranulation in vivo. We examined eosinophil cationic protein (ECP) release from normal human eosinophils (Eos) in response to C3a and C5a. C3a and C5a induced remarkable ECP release when Eos were preincubated with cytochalasin B. ECP release induced by C3a or C5a was greater than that induced by PAF at a concentration of 10(-7) M. The ED50 for ECP release was 3 x 10(-8) M and 3 x 10(-9) M for C3a and C5a, respectively. C3a or C5a elicited a rapid and transient rise in [Ca2+]i. These results suggest that C3a and C5a may contribute to hypersensitivity diseases by inducing eosinophil degranulation.

Blood Proteins↗
PubMed1985

Pharmacological characterization of the slow component of deactivation of guinea-pig isolated ileum to the spasmogenic action of C5adesArg.

The slow component of deactivation of guinea-pig isolated ileum to C5adesArg was studied to analyse the mechanism of loss and subsequent recovery of sensitivity. Neither cycloheximide (10(-3) M) nor colchicine (5 X 10(-5) M), vinblastine, lumicolchicine, or cytochalasin B (each 2 X 10(-5) M) affected significantly the spasmogenic effect of C5adesArg or the course of deactivation produced by repeated applications; chloroquine (2 X 10(-4) M) inhibited the spasmogenic effect unspecifically without interfering with deactivation. Recovery from slow deactivation was totally blocked by chloroquine and considerably diminished by colchicine and vinblastine, but was not affected by the other agents. It is proposed that recovery involves lysosomal processing of C5a receptors (occupied by the peptide) but does not require biosynthesis of new receptors.

Animals↗
PubMed1986

Characterization of the plasma membrane bound GTPase from rabbit neutrophils. I. Evidence for an Ni-like protein coupled to the formyl peptide, C5a, and leukotriene B4 chemotaxis receptors.

We have characterized the GTPase activity of the Ni-like guanine-nucleotide-binding regulatory protein in rabbit neutrophil plasma membranes. The low Km (3.64 +/- 0.87 X 10(-7) M) GTPase copurified with the formyl peptide receptor in the plasma membrane fraction obtained by discontinuous sucrose density gradient centrifugation. The Vmax (23.9 +/- 2.91 pmol/mg/min) and Km of the unstimulated enzyme were similar to those reported for Ni in other cell types. The activity of the unstimulated enzyme was both magnesium and sodium dependent and linear over the first 4 min of the assay. The chemoattractants, formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, and leukotriene B4 (LTB4) stimulated the GTPase in purified neutrophil plasma membrane preparations, whereas other secretagogues, such as A23187 and PMA, were without effect. Lineweaver-Burk analysis showed an fMLP-induced increase in Vmax (31.94 +/- 4.80 pmol/mg/min) (33.1 +/- 9.5%) but not in Km. The dose-response curve for fMLP stimulation showed an ED50 of 4.1 +/- 1.0 X 10(-8) M and an overall 22.2 +/- 3.1% maximal stimulation. C5a (30 micrograms/ml) increased the activity of the GTPase 21.3 +/- 5.7% and 10(-7) M LTB4 produced a 32.2 +/- 5.4% increase. Activated pertussis toxin treatment of neutrophil plasma membranes inhibited by 72.5 +/- 14.3% the stimulation of GTPase activity induced by fMLP; however, activated cholera toxin had no effect on the inhibition of fMLP stimulation, suggesting a direct role for an Ni-like protein in the coupling process. In contrast to the lack of inhibition of fMLP stimulation by activated cholera toxin treatment of plasma membranes, both pertussis toxin and to a lesser extent cholera toxin treatment reduced fMLP, C5a, and LTB4 stimulation of the GTPase in sonicates prepared from pretreated whole cells. Pertussis toxin inhibited fMLP stimulation of the GTPase by 75 +/- 7%, C5a stimulation was inhibited by 83 +/- 13%, and LTB4 stimulation was inhibited completely. Sonicates prepared from neutrophils treated similarly with cholera toxin showed a smaller inhibition of GTPase activity (50 +/- 4% and 14 +/- 9% for fMLP and LTB4, respectively) with the exception of C5a, where CT inhibition (81 +/- 32%) equaled pertussis toxin inhibition. Similarly, pertussis toxin completely inhibited the release of the granule enzyme N-acetyl-glucosaminidase by all three chemoattractants, whereas cholera toxin, except with C5a stimulation, had little or no effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1999

Selective blockade of membrane attack complex formation during simulated extracorporeal circulation inhibits platelet but not leukocyte activation.

OBJECTIVE: Complement activation is induced by cardiopulmonary bypass, and previous work found that late complement components (C5a, C5b-9) contribute to neutrophil and platelet activation during bypass. In the present study, we blocked C5b-9 formation during extracorporeal recirculation of whole blood to assess whether the membrane attack complex was responsible for both platelet and leukocyte activation. METHODS: In a simulated extracorporeal model that activates complement (C3a and sC5b-9), platelets (CD62P expression, leukocyte-platelet conjugate formation), and leukocytes (increased CD11b expression and neutrophil elastase), we examined an anti-human C8 monoclonal antibody that inhibits C5b-9 generation for its effects on cellular activation. RESULTS: Anti-C8 significantly inhibited sC5b-9 formation but did not block C3a generation. Anti-C8 also significantly inhibited the increase in platelet CD62P and monocyte-platelet conjugate formation seen with control circulation. Moreover, compared with control circulation, in which the number of circulating platelets fell by 45%, addition of anti-C8 completely preserved platelet counts. In contrast to blockade of both C5a and sC5b-9 during simulated extracorporeal circulation, neutrophil activation was not inhibited by anti-C8. However, circulating neutrophil and monocyte counts were preserved by addition of anti-C8 to the extracorporeal circuit. CONCLUSIONS: The membrane attack complex, C5b-9, is the major complement determinant of platelet activation during extracorporeal circulation, whereas C5b-9 blockade has little effect on neutrophil activation. These data also suggest a role for platelet activation or C5b-9 (or both) in the loss of monocytes and neutrophils to the extracorporeal circuit.

Antibodies, Monoclonal↗
PubMed1989

Chemotactic factor generation and cell accumulation in acute lung injury induced by endotracheal acid instillation.

We studied the time course of chemotactic factor generation and inflammatory cell accumulation in the rabbit aspiration pneumonia model. Two major potent chemotactic factors, leukotriene B4 (LTB4) and C5a, in bronchoalveolar lavage fluid (BALF) were measured by radioimmunoassay, and cell analysis was also done. The level of LTB4 increased only in the early phase (2-6 h) after endotracheal acid instillation. The level of C5a increased gradually almost in parallel with the total protein level in BALF, and reached a maximum at 24 h. Neutrophil accumulation occurred early and reached a maximum at 24 h. In contrast, the number of alveolar macrophages increased from days 1 to 7. These findings suggest that the increases in LTB4 and C5a are responsible for accumulation of neutrophils and that C5a may be an important chemotactic factor for alveolar macrophage.

Animals↗
PubMed1997

Nucleotide and corrected amino acid sequence of the functional recombinant rat anaphylatoxin C5a.

For bacterial expression of rat anaphylatoxin C5a, the cDNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using rat liver RNA and degenerate primers designed according to the published amino acid sequence [1]. Surprisingly, the amino acid sequence deduced from cDNA differed at positions 55 (N for K), 63 (K for H), 67 (E for N), 68 (S for E) and 69 (H for S) from the published sequence. The overall amino acid composition, however, was unchanged because these 5 amino acids were located at different positions compared to the published sequence. As a consequence, the proposed N-glycosylation site was absent, suggesting O-glycosylation of the mature molecule. Recombinant rat C5a with a 6 histidine tag at the N-terminus was expressed in bacteria, purified and renatured. The peptide was as potent as recombinant human C5a in eliciting lysosomal enzyme release from human granulocytes.

Amino Acid Sequence↗
PubMed1993

Induction of inflammatory cytokines in the pleural effusion of cancer patients after the administration of an immunomodulator, OK-432: role of IL-8 for neutrophil infiltration.

When OK-432, a well-known streptococcal preparation for an anti-tumour drug, was administered into the pleural cavity of patients with malignant pleurisy, a rapid and prominent leukocytosis, predominantly consisting of neutrophils, was observed in the cavity. Neutrophil infiltration usually peaked 6-9 h after OK-432 administration, and levelled down after 24 h. Prior to the neutrophil accumulation, transient but marked elevation of various inflammatory cytokine levels including IL-1 beta, TNF-alpha, IL-8 and G-CSF was observed. In particular, IL-8 levels increased more than 10-fold, while GM-CSF did not change significantly. A good correlation between IL-8 levels and neutrophil chemotactic response was observed particularly during 0-3 h. Specific neutralization or removal of IL-8 by antibody column abrogated half of the neutrophil chemotaxis, while neutralization of C5a removed around 40%. Sequential removal of IL-8 and C5a abrogated totally 80% of chemotaxis, confirming that these two factors are mostly responsible for the neutrophil chemotaxis in the pleural fluids. These results have suggested that rapid neutrophil infiltration induced by OK-432 in vivo is ascribable largely to IL-8 and in part to C5a.

Adult↗
PubMed1986

Evaluation of low dose anaphylatoxic peptides in the pathogenesis of the adult respiratory distress syndrome (ARDS). Monitoring of early C5a effects in a guinea-pig in vivo model after i.v. application.

A guinea-pig in vivo model is presented that allows the infusion of purified C5a via a central vein catheter and the monitoring of its effects on granulocytes and platelets, the most important cells in the pathogenesis of several lung disorders, e.g. shock lung. After the infusion of C5a, which was adjusted to a quantity that caused slight and transient alterations of lung physiology, granulocytes disappeared from circulation within 1 min. Simultaneously the granulocyte content of the lung increased about three-fold as judged by histological evaluations. Morphologic destructions were not observed. After the drop a rebound of circulating Polymorpho-nuclear leucocytes (PMN) occurred, which was significantly higher than control values and the appearance of banded cells indicated a mobilization from bone marrow stores. Studies with 51-chromium labelled PMNs revealed that most, but not all, of the granulocytes returned to circulation after transient sequestration. The number of platelets also decreased after C5a infusion, but the rebound was delayed compared with the PMNs and did not exceed control values. The changes in circulating cells, lung histology, and lung physiology are comparable to those occurring during the onset of shock lung and thus strengthen the supposed importance of C5a concerning the pathogenesis of that syndrome.

Animals↗
PubMed1990

Functional loss of chemotactic factor inactivator in the adult respiratory distress syndrome.

The adult respiratory distress syndrome (ARDS) is often characterized by a neutrophilic alveolitis, which may be mediated in part by the neutrophil chemoattractant, C5a. Chemotactic factor inactivator (CFI) can decrease C5a-directed neutrophil chemotaxis. Thus, a loss of CFI activity in the ARDS lung could lead to an increased ability of C5a to attract neutrophils. Lung CFI levels were measured antigenically and functionally in bronchoalveolar lavage (BAL) fluid obtained from 29 patients with ARDS and in 14 normal control subjects. Antigenic levels of CFI were found to be markedly elevated in ARDS BAL fluid (1,855 +/- 437 ng/ml) compared with that in normal BAL (29 +/- 10 ng/ml, p less than 0.005), but, in contrast, CFI functional activity was markedly decreased in ARDS BAL fluid compared with that in normal BAL fluid (31 +/- 7% inhibition versus 76 +/- 5% inhibition, p less than 0.01). Furthermore, although purified CFI readily inhibited the ability of C5a to attract neutrophils (92% inhibition), this activity was decreased when BAL fluid from patients with ARDS was incubated with CFI (47 +/- 10%, p less than 0.01). These findings suggest that patients with ARDS are functionally deficient in CFI, leading to an increased ability of C5a to attract neutrophils.

Aminopeptidases↗
PubMed2003

Elevation of basal intracellular calcium as a central element in the activation of brain macrophages (microglia): suppression of receptor-evoked calcium signaling and control of release function.

Microglia-brain macrophages are immune-competent cells of the CNS and respond to pathologic events. Using bacterial lipopolysaccharide (LPS) as a tool to activate cultured mouse microglia, we studied alterations in the intracellular calcium concentration ([Ca 2+]i) and in the receptor-evoked generation of transient calcium signals. LPS treatment led to a chronic elevation of basal [Ca 2+]i along with a suppression of evoked calcium signaling, as indicated by reduced [Ca 2+]i transients during stimulation with UTP and complement factor 5a. Presence of the calcium chelator BAPTA prevented the activation-associated changes in [Ca 2+]i and restored much of the signaling efficacy. We also evaluated downstream consequences of a basal [Ca 2+]i lifting during microglial activation and found BAPTA to strongly attenuate the LPS-induced release of nitric oxide (NO) and certain cytokines and chemokines. Furthermore, microglial treatment with ionomycin, an ionophore elevating basal [Ca 2+]i, mimicked the activation-induced calcium signal suppression but failed to induce release activity on its own. Our findings suggest that chronic elevation of basal [Ca 2+]i attenuates receptor-triggered calcium signaling. Moreover, increased [Ca 2+]i is required, but by itself is not sufficient, for release of NO and certain cytokines and chemokines. Elevation of basal [Ca 2+]i could thus prove a central element in the regulation of executive functions in activated microglia.

Animals↗
PubMed1992

Kinetics of C5a release in cardiac lymph of dogs experiencing coronary artery ischemia-reperfusion injury.

Previous studies of myocardial ischemia suggest that complement activation may play a central role in the inflammatory response during reperfusion. Our previous work has demonstrated neutrophil chemotactic activity to be present in reperfusion canine cardiac lymph after myocardial ischemia and infarction. To evaluate the contribution of the complement-dependent anaphylatoxin C5a to this neutrophil chemotactic activity, rabbit antiserum to canine C5a was prepared. At dilutions > 1:500 but < 1:2,000, the antiserum abolished the ability of zymosan-activated dog serum to induce a ruffled, bipolar morphology in isolated neutrophils used as a bioassay of chemotactic stimulation. This antiserum did not affect similar morphological changes in neutrophils exposed to platelet activating factor (10(-7)-10(-6) M) or recombinant human interleukin-8 (10(-9)-10(-8) M); thus, we deemed it functionally specific for canine C5a. In a pattern similar to what we previously reported, cardiac lymph collected before a 1-hour ligation of the left circumflex coronary artery had little ability to alter the morphology of canine neutrophils (shape change index, 11.3 +/- 4.6, mean +/- SEM; n = 7), but by 1 hour of reperfusion, lymph activated neutrophils significantly in five of seven dogs (mean shape change index, 72.6 +/- 17.7; p < 0.01). At 2 hours of reperfusion, neutrophil activation by lymph occurred in six of seven dogs (mean shape change index, 103.1 +/- 22.2). At 3 hours of reperfusion, cardiac lymph of only three of six dogs caused neutrophil activation, and at 4 hours of reperfusion, this activity was evident in lymph from only two of five dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Induction of interleukin-8 synthesis from monocytes by human C5a anaphylatoxin.

Interleukin-8 (IL-8) is an important mediator of inflammation and has been shown to be a potent chemotactic/cell activator for polymorphonuclear neutrophils (PMNs). T lymphocytes, and basophils. Cellular sources of IL-8 include monocytes, PMNs, endothelial cells, epithelial cells, and keratinocytes when stimulated by factors such as lipopolysaccharide, IL-1, and tumor necrosis factor-alpha. This report demonstrates that C5a, in addition to being a direct mediator of inflammation, can induce both IL-8 synthesis and high levels of release from monocytes. Natural human C5a and a synthetic C-terminal analogue peptide of C5a each induced IL-8 synthesis and release from CD14+ human peripheral blood mononuclear cells. Antigenic reactivity based on enzyme-linked immunosorbent assay gave evidence that IL-8 was present in the culture supernatants of stimulated peripheral blood mononuclear cells. Proof that supernatant levels of IL-8 attain biologically significant quantities was provided by human PMN chemotaxis assays. The quantity of IL-8 recovered from C5a-activated monocytes in peripheral blood mononuclear cells is up to 1,000-fold greater than that released from comparable numbers of PMNs under similar conditions. Therefore, IL-8 released from C5a-activated monocytes may play a significant role in expanding and prolonging cellular infiltration and activation at sites of infection, inflammation, or tissue injury. This observation suggests an important humoral amplification loop for inflammatory events involving both complement activation and cytokine release.

Antigens, CD↗
PubMed1993

Evaluation of the C-terminal C5a effector site with short synthetic C5a analog peptides.

Biological activities have been determined for a series of 18 peptides based on the C-terminal sequence of human or rat C5a. Lysosomal enzyme release was tested in two cell types, the promyelotic leukemia cell line U937 and polymorphonuclear leukocytes. In addition, an ATP-release assay with guinea pig platelets was performed. It was demonstrated that the C-terminal octapeptide 67-74 of human C5a represents the minimal sequence required to induce a measurable biological signal in all assays. Extending this peptide to a length of 21 amino acids produced at best only a slight enhancement of potency. Amino acid replacements with either tryptophanyl or phenylalanyl residues in positions between 65-69 either increased potency (at position 67), or abrogated potency (at position 66) in the two lysosomal enzyme assays. N-terminal acylation with the fluorenylmethoxy-carbonyl-aminohexanoyl group slightly enhanced C5a potency. In desensitization experiments with guinea pig platelets all peptides with a C5a activity were able to desensitize not only the C5a but also the C3a responses.

Acetylglucosaminidase↗
PubMed1994

Reversibility of tachyphylaxis to C5A in guinea pig tissues, perfused human placental lobule, and umbilical artery.

The spasmogenic effect of C5a is mediated by histamine and/or eicosanoids. Tachyphylaxis to this effect of C5a occurs rapidly, but the spasmogenic effects of C5a on a guinea pig lung parenchymal strips, field-stimulated ventricular papillary muscle, and human umbilical artery were completely restored by a 1-h period of drug-free rest, whereas that of guinea pig ileum was not. Perfusion of the isolated human placental lobule with C5a caused a transient pressor response that was largely abolished by indomethacin (5 microM), indicating mediation by cyclooxygenase metabolites. This pressor response to C5a was also completely restored following a 1-h rest period. The results show that tissue rest reverses tachyphylaxis to the spasmogenic effects of C5a in tissues where the response is mediated by cyclooxygenase metabolites. Where the response is mediated by histamine released by mast cells, restoration does not occur, presumably because of the catastrophic nature of mast cell degranulation. Histamine released in guinea pig papillary muscle by C5a may be from non-mast-cell sources.

Animals↗
PubMed1987

Inhibition of C5a-induced basophil degranulation by disodium cromoglycate.

Injection of purified porcine C5a into 24-hr basophil-rich cutaneous basophil hypersensitivity sites in the dose range 10(-12)-10(-10) moles/site produced cutaneous basophil anaphylaxis (CBA). The H1 antihistamine antagonist mepyramine, given orally (3.0-30 mg/kg), inhibited the vasopermeability, but not the basophil degranulation, characteristic of CBA. The antiallergy agent disodium cromoglycate (DSCG), administered intravenously (3.0-30 mg/kg), inhibited vasopermeability and basophil degranulation. DSCG inhibition of mast cell degranulation was not important in the inhibition of CBA, since intact mast cells were found to be depleted at basophil-rich sites and absent at C5a-induced CBA sites from animals treated with DSCG. C5a at 10(-11) moles/site also induced vasopermeability and mast cell degranulation in normal guinea pig skin. Vasopermeability, but not mast cell degranulation, was inhibited by mepyramine at 30 mg/kg p.o. However, DSCG at 10 mg/kg i.v. failed to inhibit either the vasopermeability or the mast cell degranulation of this reaction. These results indicate that C5a induces the degranulation of both basophils and mast cells in the guinea pig, and that C5a-induced degranulation of basophils, but not mast cells, is inhibited by DSCG.

Anaphylaxis↗
PubMed1989

Comparison of LTB4- and C5a-stimulated chemotaxis of isolated human neutrophils: difference revealed by cell migration in thick filters using the multiwell cap procedure.

Under comparable conditions (90 min incubation in 2% albumin buffer) using 3 micron pore cellulose nitrate filters and the multiwell cap procedure (Evans et al., Bioscience Reports 6:1041, 1986), C5a was more potent than LTB4 as a chemoattractant (EC'50 s = 0.5 and 4 nM) and caused 5 times as many cells to completely traverse the filter. During the 90 min incubation, no cells traversed the filter in the absence of chemoattractant. The cap assay was modified to study migration of cells within the filter. During the 30 min incubation, EC50 values for C5a and LTB4 were comparable (1.2 and 1.6 nM) in causing cells to enter the filters but C5a was superior in causing the cells to move as measured by the mean distance of migration (EC'50 s = 0.25 and 1.8 nM). Our studies support the view that LTB4 acts primarily to cause cell adhesion (and penetration) of the endothelium and that C5a plays a major role in cell migration (McMillan and Foster, Agents and Actions 24: 114, 1988).

Chemotaxis, Leukocyte↗
PubMed1999

In vitro and in vivo dependency of chemokine generation on C5a and TNF-alpha.

Under a variety of conditions, alveolar macrophages can generate early response cytokines (TNF-alpha, IL-1), complement components, and chemotactic cytokines (chemokines). In the current studies, we determined the requirements for TNF-alpha and the complement activation product C5a in chemokine production in vitro and in vivo. Two rat CXC chemokines (macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (CINC)) as well as three rat CC chemokines (MIP-1alpha, MIP-1beta, and monocyte chemoattractant protein (MCP)-1) were investigated. Chemokine generation in vitro was studied in rat alveolar macrophages stimulated with IgG immune complexes in the absence or presence of Abs to TNF-alpha or C5a. The rat lung injury model induced by IgG immune complex deposition was employed for in vivo studies. Abs to TNF-alpha or C5a were administered intratracheally or i.v., and effects on chemokine levels in bronchoalveolar lavage fluids were quantitated by ELISA. Both in vitro and in vivo studies demonstrated the requirements for TNF-alpha and C5a for full generation of CXC and CC chemokines. In vitro and in vivo blockade of TNF-alpha or C5a resulted in significantly reduced production of chemokines. Supernatant fluids from in vitro-stimulated macrophages revealed by Western blot analysis the presence of C5a/C5adesArg, indicating intrinsic generation of C5a/C5adesArg by alveolar macrophages and explaining the higher efficiency of intratracheal vs i.v. blockade of C5a in reducing chemokine production. These results underscore the central role of both TNF-alpha and C5a, which appear to function as autocrine activators to promote CXC and CC chemokine generation by alveolar macrophages.

Animals↗