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At least 19 recordsLinked to original sources
PubMed1991

Complement C5a receptor assay for high throughput screening.

The complement C5a receptor on U937 cells, a human histiocytic lymphoma cell line, stimulated with dibutyryl-cAMP have been stabilized for at least 3 months at a dilute, ready to use concentration. [125I]-Bolton Hunter labeled C5a, (recombinant, human) has been prepared by reverse phase HPLC to 2200 Ci/mmol. Using a filtration binding assay the Kd from receptor saturation analysis is 10-40 pM and there are 50,000-100,000 receptor sites per cell. These reagents have permitted the development of a reliable, reproducible and convenient drug screening assay, in kit format, for compounds acting at the C5a receptor.

Complement C5a↗
PubMed1990

Thromboxane A2 and peptidoleukotrienes contribute to the myocardial ischemia and contractile dysfunction in response to intracoronary infusion of complement C5a in pigs.

Intracoronary infusions of activated complement C5a result in myocardial ischemia, contractile dysfunction, and leukocyte accumulation. The hypothesis was tested that the generation of the coronary vasoconstrictors, thromboxane A2 and the 5-lipoxygenase leukotrienes (LTC4 and LTD4), contributes to the C5a-induced decrease in coronary blood flow and contractile function. The left anterior descending coronary artery in anesthetized swine was cannulated and servo pump-perfused with arterial blood at constant pressure and measured flow. Regional subendocardial contractile function was assessed with sonomicrometry. The interventricular vein was cannulated for sampling of coronary venous blood for leukocyte count. The responses in left anterior descending coronary artery blood flow and percent segment shortening to intracoronary infusions of LTC4 (1 microgram), LTD4 (1 microgram), thromboxane agonist U46619 (7.5 micrograms), and C5a (500 ng) were assessed before and after 1) LTD4/LTE4 receptor blockade with leukotriene receptor blocker LY171883 (10 mg/kg i.v.) (n = 5), 2) thromboxane A2/prostaglandin H2 receptor blockade with thromboxane receptor blocker BM13505 (2 mg/kg i.v.) (n = 5), and 3) combined thromboxane and leukotriene receptor blockade (n = 5). In the absence of receptor blockade, intracoronary C5a decreased coronary flow (50-60%) and regional segment function (60-70%) compared with the preinfusion levels. This was accompanied by a fall in coronary venous blood leukocyte levels by 5-6 x 10(6) cells/ml in the absence of alterations in arterial blood leukocyte count. Intracoronary injections of LTD4, LTC4, or U46619 also resulted in prompt decreases in coronary blood flow (50-60%) and segment function (70-80%) from preinfusion levels. Leukotriene receptor blockade with LY171883 abolished these responses to LTD4 and LTC4. Administration of LY171883 also attenuated (p less than 0.05) the myocardial response to C5a; coronary flow and segment function decreased by approximately 28% from preinfusion levels. Thromboxane receptor blockade with BM13505 eliminated the response in coronary flow and segment function to intracoronary U46619. Similar to LY171883, administration of BM13505 blunted (p less than 0.05) the C5a-induced decreases in coronary flow and contractile function, which fell by approximately 20-25% from the preinfusion level. After the combined LTD4/LTE4 receptor and thromboxane A2/prostaglandin H2 receptor blockade, intracoronary C5a resulted in little change in both coronary blood flow and segment shortening. In contrast to the flow and function effects, the C5a-induced myocardial leukocyte extraction was not decreased by leukotriene and/or thromboxane receptor blockade.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Activation of leukocytes with complement C5a is associated with prostanoid-dependent constriction of large arteries in atherosclerotic monkeys in vivo.

Activated leukocytes release a variety of substances which have been shown in vitro to modulate vascular tone. The chemotactic peptide complement C5a is a physiological activator of leukocytes. We injected human recombinant complement C5a (10 and 100 micrograms) into the blood-perfused hind limb of normal and atherosclerotic cynomolgus monkeys and examined vascular responses. In both normal and atherosclerotic monkeys, the high dose of C5a produced about 65% decrease in leukocyte cell count in venous blood drainage from the hind limb. Injection of C5a produced a pronounced increase in resistance of large arteries (segment from iliac artery to dorsal pedal artery) in atherosclerotic, but not in normal monkeys. The constrictor effect of C5a in atherosclerotic monkeys was abolished by the thromboxane A2 receptor antagonist SQ 29,548 (2 mg/kg i.v.). The platelet-activating factor antagonist WEB 2086 (5 mg/kg, i.v.) did not alter vascular responses to C5a. We conclude that activation of leukocytes produces constriction of large arteries in atherosclerotic, but not normal, monkeys in vivo. This response may be mediated in part by release of thromboxane A2.

Animals↗
PubMed1987

Effect of low dose methotrexate on neutrophil chemotaxis induced by leukotriene B4 and complement C5a.

When mediators of inflammation such as complement component C5a or leukotriene B4 are introduced into an air pouch created in mice, these mediators induce the migration of neutrophils into the air pouch. Pretreatment of mice with low doses of methotrexate inhibits leukotriene B4 or C5a induced neutrophil migration into the air pouch. Inhibition of neutrophil chemotaxis by methotrexate may, at least in part, account for the rapid onset of antiinflammatory activity that was observed in clinical trials with methotrexate in rheumatoid arthritis.

Animals↗
PubMed1989

Thromboxane is produced in response to intracoronary infusions of complement C5a in pigs. Cyclooxygenase blockade does not reduce the myocardial ischemia and leukocyte accumulation.

Activated polymorphonuclear leukocytes (PMNs) contribute to myocardial injury during ischemia and reperfusion. There is evidence that activation of the complement pathway may be one of the mechanisms of PMN activation during ischemia. Intracoronary infusion of complement C5a during normal perfusion pressure is associated with decreased coronary flow, contractile dysfunction, and PMN accumulation. The mechanisms responsible for these changes have not been identified. Thromboxane A2 (TXA2) is a potential mediator of this myocardial ischemic response. Activated PMNs produce TXA2, a known coronary vasoconstrictor, and TXA2 was shown to be a mediator of the pulmonary hypertensive response to activated complement. The goal of the present study was to determine if an enhanced TXA2 production is associated with the myocardial response to C5a and whether cyclooxygenase blockade would reduce the myocardial ischemia. In open-chest pigs, intracoronary C5a (500 ng) caused reversible reductions in blood flow (50.0% of control), regional contractile function (25.8% of control), leukocyte trapping (1.0 x 10(6) cells/g myocardium or a peak artery-coronary venous difference of 5.3 x 10(3) cells/microliters blood), and increased coronary venous TXB2 (the TXA2 breakdown product) from 1.6 pmol/ml to a peak of 6.9 pmol/ml. Cyclooxygenase blockade with aspirin or indomethacin, which prevented TXB2 production, did not alter the response in flow, function, or PMN trapping. Ibuprofen, a known direct inhibitor of PMNs in addition to its cyclooxygenase blockade effect, reduced the response slightly. The pig coronary vascular bed was responsive to the TXA2 agonist U46619, which reduced flow and function without PMN trapping. Mechanical reductions in coronary flow to levels equivalent to those during the C5a infusions did not increase coronary venous TXB2 nor cause PMN trapping but did cause equivalent contractile dysfunction. Incubation of whole blood with C5a at concentrations equivalent to those achieved in vivo did not cause TXB2 production. We conclude that 1) TXA2 is produced in response to intracoronary C5a and 2) cyclooxygenase blockade does not prevent the C5a-induced myocardial ischemia, contractile dysfunction, and PMN trapping. The TXA2 production likely involves a vascular site or a blood cell-vascular interaction. This model system indicates the potential for persistently activated PMNs to cause continued ischemia during myocardial reperfusion.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1988

Complement C5a (desArg) generation in serum exposed to damaged aortic endothelium.

Complement activation by injured endothelial cells was investigated using ex vivo rabbit thoracic aortas as a source of endothelium and a neutrophil aggregation (NA) bioassay to detect the complement cleavage product C5a (desArg). Endothelium, oxygen-starved by incubating aortas 30 min with Tyrode's buffer, activated complement as demonstrated by a 57% increase in the NA response induced by serum from buffer-treated aortas as compared to serum from untreated control aortas. Incubation of MgEGTA serum in injured aortas resulted in a 27% (P less than 0.025) weaker NA response than normal serum, indicating participation by both classical and alternative pathways of complement activation. Serum from aortas incubated 30 min with 100 micrograms/ml cholestane-3 beta, 5 alpha, 6 beta-triol, a cytotoxic cholesterol oxidation derivative, induced a NA response comparable to that from serum from aortas treated 30 min with Tyrode's buffer. Heat inactivation of serum prior to aortic incubation abolished NA activity and serum incubated in deendothelialized aortas lacked NA activity. Fractionation of serum samples from these experiments on Sephadex G-100 revealed a single peak of NA activity corresponding to the molecular weight of C5a (desArg). Endothelial cell injury was demonstrated by the inability to exclude Trypan blue dye and by scanning electron microscopy. These data demonstrate that damaged arterial endothelium can effectively activate the complement system, resulting in the production of an anaphylatoxic inflammatory mediator.

Animals↗
PubMed1988

Complement C5a receptor antagonism by protamine and poly-L-Arg on human leukocytes.

It is shown that protamine selectively and dose-dependently inhibits complement C5a-induced leukocyte responses such as histamine release from basophils, chemiluminescence and beta-glucuronidase release from neutrophils. Protamine produces parallel rightward displacements of the C5a dose-response curves. The inhibitory capacity of the polypeptide is reversible and disappears following repeated washing of exposed cells. In neutrophils poly-L-Arg similarly and specifically antagonizes C5a-induced chemiluminescence and enzyme release. This polymer alone, however, degranulates basophils and neutrophils, leading to histamine and enzyme release, respectively. It is concluded that on human neutrophils the arginine-rich polycations protamine and poly-L-Arg exhibit a competitive C5a receptor antagonism. In addition, protamine inhibits the C5a receptors on basophils. It is hypothesized that molecular conformations of the arginine-rich polycations might bind reversibly to, and block negatively charged groups at the C5a-receptor sites.

Basophils↗
PubMed1990

Complement C5a activation of phospholipase D in human neutrophils. A major route to the production of phosphatidates and diglycerides.

The contribution of phospholipase D (PLD) to the production of phosphatidic acid (PA) and diglyceride (DG) by C5a-stimulated human neutrophils has been studied. Membrane-associated 1-O-alkyl-phosphatidylcholine (alkyl-PC) was double labeled with 3H and 32P by incubating neutrophils with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC. Upon stimulation with recombinant C5a, these labeled neutrophils produce 1-O-alkyl-phosphatidic acid (alkyl-PA) and, in the presence of ethanol, 1-O-alkyl-phosphatidyl-ethanol (alkyl-PEt), containing both 3H and 32P. Formation of radiolabeled alkyl-PEt parallels that of radiolabeled alkyl-PA and requires both extracellular Ca2+ and cytochalasin B. Furthermore, the 3H/32P ratios of alkyl-PA and alkyl-PEt formed during stimulation are very similar to that of th substrate alkyl-PC. These results demonstrate that, in C5a-stimulated neutrophils, alkyl-PA and alkyl-PEt are formed from alkyl-PC almost exclusively by PLD-catalyzed hydrolysis and transphosphatidylation, respectively. Upon C5a stimulation, neutrophils labeled with 3H and 32P also produce 1-O-[3H]alkyl-diglyceride [( 3H]alkyl-DG) and [32P]orthophosphate [( 32P]PO4), but not [32P]phosphocholine. [3H]Alkyl-DG and [32P]PO4 are formed in parallel, although temporally lagging behind alkyl-PA. Propranolol, a PA phosphohydrolase (PPH) inhibitor, decreases the formation of both [3H]alkyl-DG and [32P]PO4, although increasing alkyl-PA accumulation. These data support the conclusion that alkyl-DG is formed from alkyl-PC by the combined activities of PLD and PPH and not by phospholipase C (PLC). Furthermore, by using [3H]acyl-PC-labeled neutrophils, it is demonstrated that, like alkyl-PC, 1-acyl-PC is also degraded sequentially by PLD and PPH to 1-acyl-DG. Propranolol does not inhibit phosphoinositide-specific PLC and yet it causes almost complete inhibition of the total DG mass accumulation in C5a-stimulated neutrophils. We conclude that, in cytochalasin B-treated neutrophils stimulated with C5a, PLD-catalyzed hydrolysis of PC determines the levels of both PA and DG with potentially important ramifications for neutrophil-mediated defense functions.

Alkylation↗
PubMed1986

Complement mediated inhibition of immune precipitation and solubilization generate different concentrations of complement anaphylatoxins (C4a, C3a, C5a).

Complement prevents the formation of insoluble immune complexes (inhibition of immune precipitation (IIP], and solubilizes preformed immune aggregates (solubilization (SOL]. Since the mechanism of complement activation differs in these two reactions, it is possible that they differ also in the amount of complement fragments released, in particular the anaphylatoxins C3a, C5a and C4a. We measured C4 and C3 consumption, and the formation of complement anaphylatoxins during IIP and SOL using two different immune complex models (BSA, rabbit anti-BSA; tetanus toxoid (TT), human anti-TT). At equal immune complex concentrations in both models, SOL was more efficient than IIP at cleaving C3, and more C3a and C5a was released. Comparing the two reactions, C3a formation was followed by more C5 cleavage (C5a) during SOL. Similarly C4a formation (classical pathway activation) was followed by more C3 cleavage (C3a: classical and alternative pathway activations), during SOL. It is suggested that in vivo SOL of insoluble complexes is rapidly accompanied by a damaging phlogistic reaction, whereas IIP produces less inflammation.

Antigen-Antibody Complex↗
PubMed1989

The release of C5a in complement-activated serum does not require C6.

The influence of terminal complement components on the generation and release of the complement C5a fragment was investigated by comparing the levels of C5a in complement-activated serum with the levels of C5a produced in serum depleted of complement C6. In order to investigate the release of C5a, a modified C5a assay was developed that utilizes an anti-C5b monoclonal antibody to remove C5, C5b, and C5b-C5a complexes from samples prior to C5a assay. The modified assay was developed because the standard methodology, which includes an acid-precipitation step designed to dissociate C5a and C5b, cannot distinguish free C5a from the C5a that is bound to C5b. Therefore, the standard methodology is not capable of monitoring the influence of terminal components on C5a/C5b dissociation. Levels of C5a were measured in complement-activated whole human serum, in serum depleted of C6, and in serum containing inhibitory levels of anti-C6 Fab using both the modified C5a assay and the standard methodology. Sera were complement-activated with either zymosan to activate the alternative complement pathway or with antibody-coated sheep erythrocytes to activate the classical pathway. The levels of free C5a in C6-depleted sera after activation were equivalent to the C5a levels in activated whole serum, indicating that C6 is not required for the release of C5a from C5b. In addition, the quantity of C5a detected in zymosan-activated sera using the standard acid-precipitation methodology was greater than C5a levels when assayed using the modified immunoadsorption technique, confirming that acid-treatment enhances the C5a dissociation and promotes C5a recovery. Since the other terminal components, C7, C8, and C9, bind to C5b only after C5b only after C6 is bound, these results indicate that none of the terminal components are required for the release of C5a. Although the terminal components could influence the rate of C5a release, the quantity of C5a released in serum was entirely independent of terminal components.

Animals↗
PubMed1992

Induction of two K+ currents by complement component C5a in mouse macrophages.

Puff application of complement component C5a (5 x 10(-8) M) onto peritoneal macrophages from thioglycollate-stimulated mice induced two kinds of outward current at a holding potential of -68 mV, a slowly-rising sustained outward current and a spike-like transient outward current. Quinidine (2 x 10(-4) M) and tetraethylammonium (10(-2) M) partially suppressed both types of outward current. Charybdotoxin (2 x 10(-6) M) markedly suppressed the spike-like outward current. Reversal potentials in bath solutions of different external K+ concentrations were dependent only on K+ concentrations. The transient current was not suppressed in Ca(2+)-free EGTA-containing solution, but was completely abolished in BAPTA-containing solution. One kind of single channel responding to C5a, which has a single-channel conductance of 29 pS, was recorded from cell-attached patches. These results suggest that C5a activates a Ca(2+)-dependent and another type of K+ current.

Animals↗
PubMed1979

Enhancement of hexose uptake in human polymorphonuclear leukocytes by activated complement component C5a.

The polymorphonuclear leukocyte (PMNL) depends on glucose as a source of energy for motility, chemotaxis, phagocytosis, and bactericidal activity. Activated complement (C5a) at low concentrations stimulates carrier-mediated carbohydrate transport in PMNLs as measured by the uptake of 2-deoxy-D-[(3)H]glucose. Human PMNLs were preincubated at 37 degrees C for 15 min with zymosan-activated human serum or various purified preparations of human C5a. A concentration-dependent increase in deoxyglucose transport (>700% of control) into PMNLs occurred with all test substances. Reaction was linear for 30 min, and uptake of deoxyglucose followed saturation kinetics. C5a caused a decrease in the K(m) for deoxyglucose, from 0.53 to 0.11 mM, without altering the V(max) (44 nmol/30 min per 5 x 10(6) PMNLs in control and 46.6 with C5a). The optimal concentration of C5a for enhanced carrier-mediated transport of deoxyglucose was similar to that which promoted optimal chemotaxis. Activated serum from C5-deficient mice had little or no effect on deoxyglucose transport whereas that from normal syngeneic mice enhanced deoxyglucose transport. C5a did not enhance deoxyglucose transport into isolated erythrocytes, platelets, or lymphocytes. The deoxyglucose within the cell was primarily in the phosphorylated form, and hexokinase activity was not increased in PMNLs stimulated with C5a, indicating that hexokinase was not rate limiting and that enhanced transport was the mechanism of the C5a activity. Insulin at physiologic concentration (10 ng/ml) had no effect on deoxyglucose transport in PMNL and did not act as a competitive inhibitor of C5a. This insulin-like bioactivity could be detected with the amount of C5a that would be present after activation of 0.1-0.5% of the C5 in 1 ml of serum. This suggests that uptake of [(3)H]deoxyglucose by PMNLs might serve as a highly sensitive test for activation of the fifth component of complement.

Biological Transport↗
PubMed1988

Differential effects of the complement peptides, C5a and C5a des Arg on human basophil and lung mast cell histamine release.

The ability of purified anaphylatoxins to induce human lung mast cell mediator release was investigated. In eight anti-IgE responsive (histamine release = 22 +/- 5%, mean +/- SEM) mast cell preparations of 1-96% purity, C5a and C5a des Arg (0.55 pg/ml to 55 micrograms/ml), failed to elicit or potentiate histamine release; lung fragments were similarly unresponsive. The related peptide C3a was also inactive. All anaphylatoxins failed to induce mast cell leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) release. LTC4 release was also negligible from basophils where C5a was a potent histamine release stimulus. Supernatants from C5a-challenged mast cells remained fully active on basophils, excluding carboxypeptidase inactivation of C5a as an explanation for the lung mast cell results. In contrast to lung, skin mast cells were C5a-responsive (histamine release = 8 +/- 1%, at 55 micrograms/ml, n = 2). We conclude that C5a, though devoid of activity on the human lung mast cell, is a human basophil and skin mast cell secretagogue. These findings demonstrate significant organ-specific heterogeneity in mast cell responsiveness.

Basophils↗
PubMed1991

Modulation of the chemotactic properties of complement fragments C5a and C3 by the anti-inflammatory agent, SC-41930.

Cleavage of the fifth component of complement yields C5a, a potent neutrophil (PMN) and eosinophil chemoattractant, and modulator of microvascular permeability. Similarly, but to a lesser degree, C3 increases vascular permeability and histamine release. SC-41930 (7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8- propyl- 2H-1-benzopyran-2-carboxylic acid), an orally-active antiinflammatory agent was tested in an in vivo model of dermal PMN chemotaxis induced by r-hu-C5a and hu-C3. Intradermal injection of C5a in the guinea pig resulted in a significant dose-dependent influx of PMNs at 4 hours as assessed by the dermal levels of myeloperoxidase (MPO). SC-41930 (20 mg/kg) given orally to guinea pigs with intradermal injections of 1 microgram C5a significantly (p less than 0.001) reduced dermal MPO content. SC-41930 was less potent against C3, requiring 40 mg/kg to significantly reduce dermal MPO levels. Agents such as SC-41930, which nullify complement's proinflammatory properties, may well have therapeutic potential.

Animals↗
PubMed1988

Recruitment of actin to the cytoskeletons of human monocyte-like cells activated by complement fragment C5a. Is protein kinase C involved?

U-937 cells differentiated by exposure to dibutyryl cyclic AMP respond to complement fragment C5a with a marked increase in cytoskeletal F-actin, which can be detected by fluorescence-activated cell sorting (f.a.c.s.) analysis of their rhodamine phalloidin-stained cytoskeletons. The C5a-induced increase in F-actin content can be prevented by prior exposure of the cells to cytochalasin B and pertussis toxin. It is insensitive to removal of extra cellular Ca2+, to cholera toxin or to neomycin. Phorbol myristate acetate (PMA), an activator of protein kinase C, does not induce actin polymerization in the differentiated cells. Both C5a and PMA stimulate superoxide production. The action of C5a on superoxide formation is also inhibited by neomycin, a phospholipase inhibitor. These results suggest that the cytoskeletal response to C5a requires activation of a G protein, but probably does not involve phospholipase C and protein kinase C, and is not highly dependent on the availability of Ca2+. Phospholipase C and kinase C may, however, be components of the pathway leading from C5a binding to superoxide production.

Actins↗
PubMed1989

Tertiary structure of human complement component C5a in solution from nuclear magnetic resonance data.

The tertiary structure for the region 1-63 of the 74 amino acid human complement protein C5a in solution was calculated from a large number of distance constraints derived from nuclear Overhauser effects with an angular distance geometry algorithm. The protein consists of four helices juxtaposed in an approximately antiparallel topology connected by peptide loops located at the surface of the molecule. The structures obtained for the helices are compatible with alpha-helical hydrogen-bonding patterns, which provides an explanation for the observed slow solvent exchange kinetics of the amide protons in these peptide regions. In contrast to the peptide region 1-63, no defined structure could be assigned to the C-terminal region 64-74, which increasingly acquires dynamic random coil characteristics as the end of the peptide chain is approached. An average root-mean-square deviation of 1.6 A was obtained for the alpha-carbons of the first 63 residues in the calculated ensemble of C5a structures, while the alpha-helices were determined with an average root-mean-square deviation of 0.8 A for the alpha-carbons. A comparison between the solution structure of C5a and the crystal structure of the functionally related C3a protein, as well as inferences for the interaction of C5a with its receptor on polymorphonuclear leukocytes, is discussed.

Amino Acid Sequence↗
PubMed1992

[The effect of erythropoietin therapy on biocompatibility in hemodialysis].

In order to evaluate the treatment with erythropoietin (EPO) on selected indicators of biocompatibility the authors examined 8 patients dialyzed for prolonged periods before treatment (HTK = 0.23, median), during EPO treatment (Recormon, administered by the s.c. route, HTK = 0.28) in the course of 4-hour haemodialysis on dialyzers with a Cuprophan membrane. The examination before and during treatment was made under equal conditions. Heparinization was also equal despite the fact that during EPO in four patients the residual blood volume in the dialyzer was increased. Comparison of the results before treatment and during EPO treatment did not reveal at any of the collection times (before dialysis, during the 15th, 10th, 60th and 235th minute of the procedure significant differences in the number of leucocytes, plasma concentrations of the C5a complement component, number of thrombocytes and activated coagulation times. Plasma concentrations of the thrombin-antithrombin III complex were in EPO during the 60th minute of haemodialysis significantly lower (p < 0.05) than before EPO. The authors conclude that EPO treatment does not have a significant effect on changes in the number of leucocytes in blood during haemodialysis nor on the activation of complement by an alternative way. EPO does not lead to a greater activation of the coagulation system during haemodialysis; the lower concentration of the thrombin-antithrombin III complex suggests the opposite. Explanation of this finding, similarly as detection of the cause of the increased residual blood volume in some patients, calls for further investigation.

Adult↗