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PubMed1987

Guillain-Barré syndrome: activated complement components C3a and C5a in CSF.

Plasma and spinal fluid levels of complement activation products C3a and C5a were quantitated by radioimmunoassay in a group of 16 patients suffering from acute monophasic Guillain-Barré syndrome (GBS). Median CSF levels of C3a (118 ng/ml) and of C5a (9.6 ng/ml) were significantly elevated when compared with samples from a control group of patients with noninflammatory neurologic diseases. Plasma concentrations of these anaphylotoxic peptides were not significantly different between the two populations. Our findings indicate that the complement system is activated in the CSF of patients with acute GBS. Complement activation products may contribute to the inflammatory changes observed in this disorder.

Complement C3↗
PubMed1986

C5a fragment of bovine complement. Purification, bioassays, amino-acid sequence and other structural studies.

C5a and des-Arg-C5a have been purified from bovine serum in milligram amounts. The progress of the purification was followed by measuring the chemotactic activity of the complement fragments. The two polypeptides induce activation of neutrophil-oriented locomotion and secretion with very similar dose/response effects. After preparing a rabbit antiserum to bovine C5a/des-Arg-C5a, a competitive enzyme-linked immunosorbent assay (ELISA) was set up for the detection of C5a from 5 ng/mol to 1 microgram/ml. The complete primary structure of bovine C5a, which consists of 74 amino acids, has been determined by sequence analysis of the tryptic peptides, aligned by peptides derived from a chymotryptic digest, and by partially sequencing the intact molecule. Bovine C5a has a sequence homology of 78% and 70% with porcine and human C5a, respectively, reacts with an antiserum to porcine C5a and is recognized by cell surface receptors on human neutrophils. Finally, the secondary structure of bovine C5a was investigated by circular dicroic spectroscopy and predicted from the amino acid sequence. A comparison of the content and distribution of alpha-helical and/or hydropathic regions, suggests that the three-dimensional structure of C5a might be modeled from the known crystal structure of the homologous C3a molecule.

Amino Acid Sequence↗
PubMed1979

Chemotactic factor for tumor cells derived from the C5a fragment of complement component C5.

Previously, we have stablished that the fifth component of complement (C5) serves as an important source of mediators that have locomotory (chemotactic) activity for leukocytes and tumor cells. C5a, a fragment (Mr 11,200) derived from the NH2-terminal portion of the alpha chain of C5, is the major chemotactic peptide for leukocytes. The present studies demonstrate that cleavage of C5a with trypsin generates a derivative peptide that is chemotactic for tumor cells (Walker carcinosarcoma). This fragment has an estimated Mr of 6000 as assessed by gel filtration and does not require the COOH-terminal arginine of C5a, because equivalent amounts of chemotactic activity for tumor cells can be generated from des-Arg-C5a by digestion with trypsin. The C5a-derived chemotactic peptide for tumor cells demonstrates peak activity at approximately 1 pM. These studies emphasize the key role of the C5a region of the C5 molecule in the generation of peptides that affect locomotory responses of cells.

Animals↗
PubMed1986

Leukocyte aggregation response to quantitative plasma levels of C3a and C5a.

Granulocyte aggregation (GA) response has previously been described as a sensitive assay for complement activation in sepsis. Complement component C5a has been implicated as the plasma factor responsible for GA. The quantitative interaction of complement components C3a and C5a with GA, however, is not clearly defined. This study evaluates the relationship of GA responses to plasma levels of C5a and C3a in zymosan-activated plasma (ZAP). The C3a and C5a levels were measured by radioimmunoassay in serial dilutions of ZAP. Granulocyte aggregation responses of normal human leukocytes were determined for each ZAP dilution. The C5a levels in a 1:16 dilution of ZAP were higher than in normal plasma (22 +/- 7 vs 9 +/- 3 ng/mL), as were GA responses (24 +/- 1 vs 11 +/- 2 percentage of maximum light transmission). The C3a levels in a 1:8 dilution of ZAP are elevated above those of normal plasma (656 +/- 167 vs 411 +/- 29 ng/mL). Correlation coefficients were .9809 for C3a vs GA, .9788 for C5a vs G, and .9860 for C3a vs C5a. Complement components C3a and C5a are involved in GA in vitro. Granulocyte aggregometry can detect low levels of activated complement in ZAP but may not be specific for C5a. The relative contribution of C3a and C5a to observed GA is not clear from the data.

Cell Aggregation↗
PubMed1994

Effect of platelet depletion and inhibition of platelet cyclooxygenase on C5a-mediated myocardial ischemia.

Activation of the complement cascade is involved in the myocardial injury resulting from transient ischemia and reperfusion. We previously showed that the complement anaphylatoxin C5a causes myocardial ischemia in vivo, mediated in part via thromboxane (Tx) A2. In the present study, we assess the role of platelets in the C5a-induced myocardial ischemia and Tx release. The left anterior descending coronary artery of anesthetized pigs was perfused with arterial blood at constant pressure and measured flow (coronary blood flow). Segment function (percent segment shortening) was measured with sonomicrometry, and regional coronary venous blood was sampled and assayed for TxB2 (by radioimmunoassay). We found that the C5a-induced decrease in coronary blood flow and percent segment shortening and the release of Tx were indistinguishable whether the left anterior descending coronary artery bed was perfused with normal arterial blood, with arterial blood obtained from animals depleted of platelets (cyclophosphamide, n = 6), or with arterial blood from aspirin-treated animals (n = 9) in which the platelets were unable to produce Tx. These data demonstrate that platelet-derived Tx does not contribute to the C5a-induced myocardial ischemia and Tx release in this model and that these cells do not play an integral role in this phenomenon.

Animals↗
PubMed1988

Role of arachidonate metabolites in C5a-induced bronchoconstriction.

The complement cleavage product, C5a, causes a bronchoconstriction in the guinea pig as evidenced by a decrease in dynamic lung compliance and an increase in pulmonary resistance. Previous studies had demonstrated that the antihistamine pyrilamine and the cyclooxygenase inhibitor indomethacin inhibited the C5a-induced bronchoconstriction but the leukotriene (LT)D4 antagonist L-649,923 did not. As an extension of those studies, the purpose of the present study was to determine the contribution of specific cyclooxygenase products and/or LTB4 in mediating C5a-induced bronchoconstriction. To assess the role of the various potential mediators, plasma levels of thromboxane (TX)B2, prostaglandin (PG)D2 and PGF2 alpha were monitored. In addition, guinea pigs were treated either with the TX synthetase inhibitor U-63557A, treated with the TX receptor antagonist SQ 29,548 or made tachyphylactic to the bronchoconstrictor actions of LTB4. C5a challenge caused an increase in plasma concentrations of TXB2, which peaked before the maximum of the bronchoconstriction. However, no significant increase in plasma concentrations of PGD2 or PGF2 alpha was seen. Both U-63557A at 80 mg/kg and SQ 29,548 significantly inhibited the C5a-induced bronchoconstriction, whereas 10 mg/kg of U-63557A did not. The inability of 10 mg/kg of U-63557A to inhibit the response could be explained by both incomplete inhibition of TX synthesis as well as possibly by the increased plasma concentrations of the potent bronchoconstrictor PGD2, which occurred with C5a challenge in the presence of U-63557A. In animals tachyphylactic to LTB4, the maximum of the C5a-induced bronchoconstriction was no different from control.(ABSTRACT TRUNCATED AT 250 WORDS)

Airway Resistance↗
PubMed1994

Complement peptides C3a- and C5a-induced mediator release from dissociated human skin mast cells.

The complement peptides C3a and C5a have been shown previously to release histamine from human basophils but not human lung mast cells. As skin mast cells differ from those of the lung in both immunocytochemical and functional properties, we examined the ability of these anaphylatoxins to release preformed and newly generated mediators from human dispersed skin mast cells. In concentration-response studies, both C3a and C5a released histamine in a concentration related manner with C5a being 40-50 times more potent. However, the extent of histamine, 15-20%, was considerably less than that released from basophils. This was not due to catabolism of the peptides by mast cell proteases, mast cell supernatants that contained C5a being effective in releasing basophil histamine. Removal of the C-terminal arginine from C3a and C5a abolished their activity on skin mast cells. In time-course studies, histamine release induced by C3a and C5a was complete within 15 seconds. Complement-induced histamine release is a non-cytotoxic process as evidenced by 2-deoxy-D-glucose and antimycin A, inhibitors of glycolysis and oxidative phosphorylation, respectively. In contrast to IgE-dependent stimulation, anaphylatoxin-induced histamine release from human skin mast cells is independent of extracellular calcium. Both C3a and C5a at concentrations that induced 10-16% net histamine release caused a negligible release of the newly generated mediator, PGD2. The results suggest that C3a and C5a stimulate human skin mast cells in a manner similar to substance P and related basic secretagogues. However, the activation site for C3a and C5a appears to be different to that for substance P as the substance P antagonist (D-Pro4, D-Trp7,9,10) SP4-11 inhibited histamine release stimulated by substance P but not that induced by C3a and C5a.

Anaphylatoxins↗
PubMed1988

Bacterial lipopolysaccharide enhances chemoattractant-induced elastase secretion by human neutrophils.

Bacterial lipopolysaccharide (LPS) has previously been shown to enhance a number of chemoattractant-induced responses by human neutrophils. The possible role of elastase, a neutral protease with broad substrate specificity, in neutrophil-mediated vascular injury of a variety of diseases prompted us to examine a) whether or not LPS enhances the direct chemoattractant-induced secretion of elastase, b) the quantitative requirements of LPS and chemotactic factors, and c) some structural requirements of LPS for this effect. Our results show that LPS at 10 ng/ml and above, enhanced formyl-methionyl-leucyl-phenylalanine-induced neutrophil secretion of elastase, as well as secretion of myeloperoxidase and vitamin B12-binding protein. This effect was independent of cytochalasins or surface stimulation, and thus may occur during chemotactic factor stimulation in vivo. LPS also enhanced neutrophil secretory responses to the complement fragments C5a, C5a des arg, and, to a lesser degree, to leukotriene B4 and platelet-activating factor. This enhancement effect appeared to require the presence of the lipid A moiety and/or parts of the core polysaccharide but not the O-antigen portion of the LPS molecule. Our findings identify a possible LPS-dependent mechanism of neutrophil elastase-mediated tissue injury in Gram-negative infections.

Antigens, Bacterial↗
PubMed1986

Effects of anti-C5a antibodies on the adult respiratory distress syndrome in septic primates.

In vitro and in vivo studies have suggested that human complement component C5a plays a key role in neutrophil injury in the adult respiratory distress syndrome (ARDS). First, using leukocyte aggregometry, we demonstrated that the addition of a recently developed rabbit anti-human polyclonal antibody to C5a des arg to endotoxin-activated plasma prevented leukocyte aggregation in vitro. We then administered the anti-C5a des arg antibody to septic primates (Macaca fascicularis). Three groups of primates, control, septic, and anti-C5a antibody treated septic, were studied (n = 4 in each group). A 30-min infusion of Escherichia coli (1 X 10(10)/kg) resulted in severe sepsis and ARDS. Primates were killed 4 h after completion of the E. coli infusion. Septic animals not treated with anti-C5a antibody had 75% mortality (3/4), decreased oxygenation, severe pulmonary edema, and profound hypotension. Septic primates treated with anti-C5a antibodies did not die and did not develop decreased oxygenation (P less than 0.05) or increased extravascular lung water (P less than 0.05). They also had a marked recovery in their mean arterial blood pressure (P less than 0.05). This study demonstrates that treatment with rabbit anti-human C5a des arg antibodies attenuates ARDS and some of the systemic manifestations of sepsis in nonhuman primates.

Animals↗
PubMed1995

Limitation of reperfusion injury by a monoclonal antibody to C5a during myocardial infarction in pigs.

The complement system has been implicated in reperfusion injury during acute myocardial infarction. We therefore attempted to reduce reperfusion injury with a monoclonal antibody (MAb) to the complement component, C5a. In 13 control pigs and 9 pigs pretreated with this MAb, ischemia was induced by a 50-min occlusion of the left anterior descending coronary artery, followed by 3 h of reperfusion. Infarct area (as percent of risk area) was reduced from 58 +/- 5% in controls to 38 +/- 7% (P < 0.05) in MAb-treated animals. Heart rate-systolic blood pressure product, left ventricular (LV) first derivative of pressure, LV end-diastolic pressure, and coronary blood flow were similar (P > 0.05) in the two groups. At 15 min of reperfusion, immunoreactive factor Bb began to increase significantly (P < 0.05) in regional coronary venous plasma, consistent with activation of the alternative complement pathway. The anti-C5a MAb did not attenuate formation of the membrane attack complex (C5b-9) as assessed by a hemolytic complement assay. Myocardial myeloperoxidase activity, a marker of tissue neutrophil concentration, was similar in the risk regions of the two groups, suggesting that neutrophil infiltration was unaltered by the MAb. However, in vitro the MAb (15 and 30 micrograms/ml) reduced C5a-stimulated neutrophil aggregation (67.4 and 70.9%), chemotaxis (52.5 and 81.4%), degranulation (66.7 and 75.8%), and superoxide generation (26.7 and 100%). In conclusion, myocardial infarction-reperfusion is associated with activation of the alternative complement pathway. Furthermore, a MAb to C5a that inhibits neutrophil cytotoxic activity, but neither the membrane attack complex nor myocardial neutrophil accumulation, decreases infarct size in pigs. These data suggest an important role of the alternative complement pathway and C5a in the propagation of ischemia cardiac damage during reperfusion.

Animals↗
PubMed1995

Binding of Gc globulin (vitamin D binding protein) to C5a or C5a des Arg is not necessary for co-chemotactic activity.

Gc globulin (vitamin D binding protein) has been shown to augment significantly the leukocyte chemotactic activity of the activated complement peptides C5a and C5a des Arg. However, the mechanism of chemotaxis enhancement is not known. Natural C5-derived peptides contain a carbohydrate side chain that comprises approximately 25% of the mass of the 11-kDa peptides. Previous studies have demonstrated that Gc globulin binds to C5-derived peptides via sialic acid residues on this carbohydrate side chain. The necessity of this carbohydrate side chain for chemotaxis enhancement by Gc globulin was investigated by using both natural (glycosylated) and recombinant (carbohydrate-free) peptides. The dose-response curves of neutrophil chemotaxis to recombinant C5a or C5a des Arg plus Gc globulin were identical to those observed with the naturally derived peptides, despite the fact that natural C5a bound to Gc globulin while the recombinant C5a failed to bind this protein. Moreover, neutrophils pretreated with Gc globulin then washed before addition to the chemotaxis assay displayed significantly enhanced movement to C5a alone. These results indicate that the binding of C5a/C5a des Arg to Gc globulin is not necessary for their chemotactic activity to be augmented. Furthermore, these results demonstrate that the co-chemotactic activity of Gc globulin is generated on the cell surface, independent of C5a binding to its receptor.

Chemotaxis, Leukocyte↗
PubMed1988

Human C5a and C5a des Arg exhibit chemotactic activity for fibroblasts.

C5a and C5a des Arg are chemotactic factors for inflammatory cells but it is not known whether these agents are chemoattractants for fibroblasts. Accordingly, C5a, purified from zymosan-activated human, and C5a des Arg, prepared by incubating C5a with immobilized porcine carboxypeptidase B, were studied for fibroblast chemotactic activity. We observed that both C5a and C5a des Arg stimulated human skin fibroblasts and fetal bovine ligament fibroblasts to migrate in a concentration-dependent fashion, and that the migratory responses were similar in magnitude to the responses achieved with optimal concentrations of two known fibroblast chemoattractants, platelet-derived growth factor and human fibrinopeptide B. The peak responses to C5a and C5a des Arg occurred at approximately 10(-9)M. With ligament fibroblasts, there was a greater response to C5a des Arg than to C5a, but with human fibroblasts there was no difference. Cochemotaxin, which enhances the chemotactic activity of C5a des Arg for neutrophils, had no effect on C5a des Arg fibroblast chemotactic activity but appeared to increase the fibroblast chemotactic activity of C5a. These results indicate that the effects of C5a and C5a des Arg in vivo may extend to the recruitment of mesenchymal cells. Moreover, the findings represent another example of an activity retained by C5a after removal of its carboxyl terminal arginine.

Antibodies↗
PubMed1987

Rapid and simple measurement of human C5a-des-Arg level in plasma or serum using monoclonal antibodies.

A new sandwich immunoassay method for measuring human C5a-des-Arg was developed using monoclonal antibody specifically reactive with C5a-des-Arg. Monoclonal antibodies were obtained from a panel of hybridomas produced by fusion of mouse myeloma cells, P3 X 63-AG8,653, with spleen cells from a CBF1(C57BL/6 X BALB/c) mouse immunized with purified C5a. The reactivities of these monoclonal antibodies against C5a, C5a-des-Arg and C5 were tested by solid-phase radioimmunoassay. One of the antibodies reacted with C5a-des-Arg, but not with C5a and C5. By use of this antibody for capturing antibody in sandwich immunoassay, a rapid and simple method was developed for measuring C5a-des-Arg without previous removal of C5. The sensitivity of this assay system was approximately 1 ng/ml for C5a-des-Arg.

Animals↗
PubMed1976

Induction of platelet aggregation by the complement-derived peptides C3a and C5a.

The effect of two complement-derived peptides, hog serum C3a and C5a, on platelet aggregation in platelet-rich plasma and suspensions in Tyrode solution was investigated. 1. Guinea-pig platelets were aggregated by both C3a and C5a; the spasmogenically inactive product of C3a, C3ai, also induced aggregation. Threshold concentrations were in the range of 10(-6)--10(-9) M depending on the peptide and platelet preparation. 2. Cat platelets were aggregated by C5a (threshold concentrations 10(-7)--10(-8) M) but not by C3a. 3. Platelets from pig, rabbit and man were not aggregated by either of the two peptides in concentrations of up to 5 X 10(-6) M. 4. When C5a was administered repeatedly in subthreshold doses guinea-pig platelets became tachyphylactic to C5a but were still aggregable by C3a or ADP. Conversely, platelets desensitized to C3a still reacted to C5a or ADP. Tachyphylaxis towards C5a developed also when platelets were incubated with C5a in the absence of free Ca2+ under which condition they do not react. The tachyphylaxis in this case became evident after recalcification of the medium. The lack of cross-desensitization indicates that C3a and C5a react via different receptors. 5. C3a and C5a were injected i.v. into guinea pigs. Histological examination of the lungs revealed that some of the smaller vessels (20-30 mu in diameter) were occluded by platelet aggregates. In addition signs of severe acute emphysema were seen in animals treated with C5a, but only slight emphysema in C3a-treated animals. Intravenous injections of C3a into guinea pigs caused but weak respiratory distress and drowsiness and never killed an animal (at doses of up to 20 mg per kg body weight), whereas C5a caused the well-known severe respiratory failure and death already at doses of 0.03 mg/kg body weight.

Anaphylatoxins↗