Binding of human C3a and the synthetic nonapeptide C3a (tyr-70-77) to rat peritoneal mast cells.
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Animals↗
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Ex vivo biocompatibility evaluation of a new modified cellulose membrane.
To evaluate membrane biocompatibility, an open loop ex vivo model was designed simulating the hemodialysis procedure. Blood was withdrawn continuously from healthy nonuremic donors, heparinized, and pumped through a module containing the membrane to be studied. C3a generation in the module was determined at various time points comparing the cuprammonium cellulose (CC) membrane and four types of modified cellulose (MC) membrane, each with a different degree of hydroxyl (OH-) group substitution. In other studies, C3a generation in the ex vivo mode was compared with that during in vivo dialysis. In the ex vivo model, C3a generation with MC membranes was reduced by 70% compared with CC. However, within the MC group, the degree of C3a generation did not correlate with the degree of OH-group substitution. In vivo studies confirmed the reduced degree of C3a generation with the MC membrane compared with CC. Additionally, validation studies using the CC membrane showed excellent agreement between C3a generation during ex vivo perfusion and in vivo dialysis. The results suggest that a group of new MC membranes causes substantially less complement activation than the CC membrane but that the degree of complement activation with various subtypes of MC membranes is not related to the degree of OH-group substitution.
Anaphylaxis↗
Studies on C3ahu binding to human eosinophils: characterization of binding.
C3a purified to chemical homogeneity from human serum binds preferentially to human eosinophils greater than neutrophils. Little or no binding is found with human platelets. Maximum binding to eosinophils at 37 degrees C occurs within 15 min. Dilution of 125I-C3a by either cold C3a or washing away unbound 125I-C3a and reincubating at 37 degrees C reveals a T1/2 of approximately 30 min. C3adesArg neither binds to eosinophils nor inhibits the binding of 125I-C3a. The binding of C3a to human eosinophils may reflect a physiologic role of C3a in eosinophil motility or function.
Blood Platelets↗
Effects on complement activation of a new continuous autotransfusion system.
Allogeneic blood transfusions may subject patients to risks of infection and allergic reactions. Various techniques for transfusion of shed blood have been developed. The aim of this study was to evaluate a new continuous autotransfusion system (Fresenius CATS) as regards its impact on the complement system, and on erythrocytes and leucocytes. Eighteen consecutive patients undergoing hip replacement surgery were studied. Complement variables (C4d, factor Bb, C3a and terminal complement complex, SC5b-9) and free haemoglobin, haemoglobin, leucocytes, platelets, albumin and protein were determined in the patient's blood preoperatively, 1 min before the start of transfusion, 15 and 60 min after transfusion; and in the reservoir, in the waste bag and in the retransfusion blood. Increased concentrations of C3a and SC5b-9 were found in the collected reservoir blood (P < 0.05). The washing and centrifugation procedure reduced these concentrations (< 0.001). High levels of free haemoglobin were found in the collected blood as well as in the processed product. The median haemoglobin level in the processed blood was 260gL-1 (range 104-289gL-1). Inflammatory mediators from the complement cascade are removed by continuous autotransfusion technique. The processed blood contains high levels of free haemoglobin.
Adult↗
Anaphylatoxin C3a induces rapid protein phosphorylation in guinea pig platelets.
We investigated C3a signal transduction using guinea pig platelets. Anaphylatoxin C3a induced cytosolic calcium influx and turnover of inositol phospholipids and caused protein phosphorylation via specific C3a receptors on guinea pig platelets. Phosphorylation of 40 kDa and 20 kDa proteins was detected within 30 s after C3a stimulation. From phosphoamino acid analysis of both proteins, it was found that about 80% was serine and 20% was threonine. Phosphorylation was decreased by pretreatment of platelets with calcium blockers, diltiazem and TMB-8. A protein kinase C inhibitor, staurosporine, effectively blocked phosphorylation of both proteins, but H-7 did not. The effects of these inhibitors on platelet aggregation were correlated with their effects on protein phosphorylation. These results suggest that protein-serine/threonine phosphorylation is important for C3a signal transduction in guinea pig platelets and that the isoenzyme of protein kinase C or another kinase probably participates in such protein phosphorylation. C3a also caused protein-tyrosine phosphorylation of 56 kDa and 33-36 kDa proteins within 30 s. Staurosporine effectively blocked phosphorylation of these bands. It is suggested that tyrosine kinase also may be involved in C3a signalling in guinea pig platelets.
Animals↗
Plasma levels of main granulocyte components in patients dialyzed with polycarbonate and cuprophan membranes.
Plasma levels of granulocyte lactoferrin, granulocyte myeloperoxidase and granulocyte elastase in complex with alpha 1-proteinase inhibitor (E-alpha 1PI) were investigated in regular hemodialysis patients dialyzed with hollow-fiber dialyzers made from polycarbonate (FD 100) or cuprophan (GFS 120 H). Plasma levels of all these main granulocyte components increased significantly during hemodialysis. E-alpha 1PI levels were significantly higher in patients dialyzed with the polycarbonate compared with the cuprophan membrane, whereas the increases of myeloperoxidase and lactoferrin were not different for the two dialyzers. On the other hand, plasma C3a levels were higher in patients dialyzed with the cuprophan compared with the polycarbonate dialyzer. Therefore, granulocyte activation during hemodialysis does not necessarily need complement activation.
Blood Proteins↗
Quantitation of C3a by ELISA using a monoclonal antibody to a neoantigenic C3a determinant.
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Anaphylatoxins↗
Effects of complement activation on platelet-activating factor and eicosanoid synthesis in rat mesangial cells.
The effects of in vitro complement activation and of isolated complement components (C3a, C5a, C3b) on the activity of the microsomal enzyme acetyl coenzyme A: 1-0-alkyl-glycero-3-phosphocholine acetyl transferase (AcTr) in cultured mesangial cells and on the synthesis of prostaglandin E2 (PGE2) were assessed. In vitro complement activation induced by the introduction of purified cobra venom factor (CVF) in culture media containing human serum enhanced mesangial cell PGE2 synthesis and had no effect on microsomal AcTr activity. When media containing C6-deficient serum were used, the stimulatory effect of CVF on PGE2 synthesis was abolished. Introduction of CVF in these media enhanced mesangial cell AcTr activity. These effects were partially reversed when the C6-deficient serum was supplemented with C7-deficient serum to allow formation of the C5b-9 complex. Isolated C3a, C5a, and C3b had opposite effects on PGE2 synthesis and on AcTr activity. Specifically, all components enhanced mesangial cell PGE2 synthesis. In contrast, AcTr activity was enhanced by C3a alone; C5a had no effect, whereas C3b had an inhibitory effect. The observations indicate that in response to complement activation and specific anaphylatoxin agonists, mesangial cell eicosanoid synthesis is not coupled with changes in the activity of AcTr and PAF synthesis.
Acetyltransferases↗
Recurrent hematuria: a novel clinical presentation of hereditary complete complement C4 deficiency.
A 10-year-old boy suffered from recurrent attacks of fever, vomiting, and hematuria. During disease flares, circulating immune complexes were detected in the serum. Elevated levels of Bb, Ba, and C3a indicated complement activation through the alternative pathway. Complement C4 was undetectable. C4 phenotyping by agarose gel electrophoresis showed complete C4 deficiency. Restriction fragment length polymorphism (RFLP) studies showed a homozygous deletion of the C4B and 21-hydroxylase A genes. A mild mesangioproliferative glomerulonephritis with mesangial deposits of immunoglobulin (1g) G, IgM, IgA, Clq, C3, properdin, and terminal complement complex was probably caused by immune complex deposition and alternative complement pathway activation. Treatment with low-dose prednisolone substantially reduced the frequency of further episodes.
Biopsy↗
[Reduction of neutrophil activation by prostaglandin E1 in open heart surgery].
This study was designed to demonstrate the effect of prostaglandin E1 (PGE1) on neutrophil activation in open heart surgery. Twenty adult patients undergoing cardiopulmonary bypass (CPB) for various cardiac operations were divided into 2 groups. PGE1 group consisted of 10 patients (7 males and 3 females) and the control group consisted of 10 patients (6 males and 4 females). In PGE1 group patients, 20-50 ng/kg/min of PGE1 was administered intravenously from the induction of anesthesia to the completion of CPB. Blood samples were taken before, during, after CPB, and in the morning of the first postoperative day. Differential counts of white blood cells, plasma neutrophil elastase (PNEL) activity, serum complements activity (C3a, CH50) and superoxide production of neutrophils were measured. Superoxide production by isolated neutrophils was evaluated utilizing luminol dependent chemiluminescence. After the initiation of CPB complements were activated markedly, and PNEL activity increased significantly in both groups. Although after CPB PNEL activity turned to decrease, it was still significantly higher on the first postoperative day than the preoperative value. There were no significant differences between two groups as for complements activation and PNEL activity. The total number of white blood cells unchanged during CPB and neutrophilia appeared after CPB, but no significant difference between two groups. Superoxide production of neutrophils relatively decreased during CPB and significantly increased after CPB in the control group. However, in PGE1 group superoxide production was reduced after CPB, especially on the first postoperative day. These results showed that PGE1 reduced neutrophil-mediated superoxide production in open heart surgery. In conclusions, PGE1 is useful agent to reduce the hazardous effects of neutrophils after CPB.
Adult↗
Cloning and characterization of rat C3a receptor: differential expression of rat C3a and C5a receptors by LPS stimulation.
The anaphylatoxin C3a, generated during complement activation, is a factor known to mediate various inflammatory reactions. The human C3a receptor (C3aR) was recently cloned and identified to be a member of the G-protein-coupled receptor family. C3aR is characterized by seven transmembrane domains including a large second extracellular loop that appears to be a unique feature of this receptor. Here we report the isolation of the rat C3aR clone and confirm that the isolated cDNA coded for rat C3aR based on C3a binding analysis to stably transfected cells. Northern blot analysis of rat C3aR revealed expression in various tissues, similar to that of human C3aR but dissimilar to rat C5aR. We found that expression of rat C3aR in various tissues did not increase significantly after LPS injection, whereas rat C5aR expression is greatly increased. These results suggest that expression of C3aR and C5aR is independently regulated in rat cells and tissues.
Amino Acid Sequence↗
Biocompatibility of a silicone-coated polypropylene hollow fiber oxygenator in an in vitro model.
BACKGROUND: A silicone-coated microporous hollow-fiber membrane oxygenator has been developed to prevent plasma leakage during long-term use. The objective of this study was to evaluate the biocompatibility of the oxygenator. METHODS: A silicone-coated oxygenator was compared with an uncoated oxygenator in an in vitro model of cardiopulmonary bypass. Simulated circulation was maintained for 6 h at 37 degrees C. RESULTS: Platelet counts decreased significantly (p < 0.05) and leukocyte counts tended to decline; however, the differences between groups were not significant. Concentrations of C3a increased significantly in both groups (p < 0.05), but levels were significantly less in the silicone-coated oxygenator (p = 0.008). In contrast, concentrations of C4a, beta-thromboglobulin, and granulocyte elastase increased significantly (p < 0.05), but the differences between groups were not significant. CONCLUSIONS: Silicone coating over a microporous hollow-fiber membrane may improve biocompatibility by reducing C3a activation.
Coated Materials, Biocompatible↗
Increase in prostanoid formation in rat liver macrophages (Kupffer cells) by human anaphylatoxin C3a.
Human anaphylatoxin C3a increases glycogenolysis in perfused rat liver. This action is inhibited by prostanoid synthesis inhibitors and prostanoid antagonists. Because prostanoids but not anaphylatoxin C3a can increase glycogenolysis in hepatocytes, it has been proposed that prostanoid formation in nonparenchymal cells represents an important step in the C3a-dependent increase in hepatic glycogenolysis. This study shows that (a) human anaphylatoxin C3a (0.1 to 10 micrograms/ml) dose-dependently increased prostaglandin D2, thromboxane B2 and prostaglandin F2 alpha formation in rat liver macrophages (Kupffer cells); (b) the C3a-mediated increase in prostanoid formation was maximal after 2 min and showed tachyphylaxis; and (c) the C3a-elicited prostanoid formation could be inhibited specifically by preincubation of C3a with carboxypeptidase B to remove the essential C-terminal arginine or by preincubation of C3a with Fab fragments of a neutralizing monoclonal antibody. These data support the hypothesis that the C3a-dependent activation of hepatic glycogenolysis is mediated by way of a C3a-induced prostanoid production in Kupffer cells.
Animals↗
Demonstration of a specific C3a receptor on guinea pig platelets.
Guinea pig platelets reportedly contain receptors specific for the anaphylatoxin C3a based on both ligand-binding studies and functional responses. A portion of the human 125I-C3a that binds to guinea pig platelets is competitively displaced by excess unlabeled C3a; however, the majority of ligand uptake was nonspecific. Uptake of 125I-C3a by guinea pig platelets is maximal in 1 min, and stimulation of guinea pig platelets by thrombin, ADP, or the Ca2+ ionophore A23187 showed little influence on binding of the ligand. Scatchard analysis indicated that approximately 1200 binding sites for C3a exist per cell with an estimated Kd of 8 x 10(-10) M. Human C3a des Arg also binds to guinea pig platelets, but Scatchard analysis indicated that no specific binding occurred. Because the ligand-binding studies were complicated by high levels of nonspecific uptake, we attempted to chemically cross-link the C3a molecule to a specific component on the platelet surface. Cross-linkage of 125I-C3a to guinea pig platelets with bis(sulfosuccinimidyl)suberate revealed radioactive complexes at 105,000 and 115,000 m.w. on SDS-PAGE gels by autoradiographic analysis. In the presence of excess unlabeled C3a, complex formation was inhibited. No cross-linkage could be demonstrated between the inactive 125I-C3a des Arg and the putative C3a-R on guinea pig platelets. Human C3a, but not C3a des Arg induces serotonin release and aggregation of the guinea pig platelets. Human C3a was unable to induce either serotonin release or promote aggregation of human platelets. Uptake of human 125I-C3a by human platelets was not saturable, and Scatchard analysis was inconclusive. Attempts to cross-link 125I-C3a to components on the surface of human platelets also failed to reveal a ligand-receptor complex. Therefore, we conclude that guinea pig platelets have specific surface receptors to C3a and that human platelets appear devoid of receptors to the anaphylatoxin.
Anaphylatoxins↗
The neuropharmacology of immune complex activity in the rat hypothalamus.
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Anaphylaxis↗
Murine macrophage behavior on peptide-grafted polyethyleneglycol-containing networks.
Polyethyleneglycol-based networks were employed as substrates to graft bioactive peptides to study macrophage interactions with materials. Our overall objective was to utilize biologically active factors to stimulate certain macrophage function on materials suitable for implantation in connective tissues. In this study, we sought to explore the bioactivity of several peptides derived from extracellular matrix adhesion proteins and macrophage-active proteins that are normally soluble. The candidate peptides examined corresponded to residues 63 to 77 of complement component C3a (C3a(63-77)), residues 178 to 207 of interleukin-1 beta (IL1beta(178-207)), residues 1615 to 1624 of fibronectin (FN(1615-1624)), endothelial-macrophage activating polypeptide II, complement component C5a inhibitory sequence, macrophage inhibitory peptide, and YRGDG; materials lacking peptides were used as negative controls. An established murine cell-line IC-21 was employed as a macrophage model, and human dermal fibroblasts were used for comparison. Our results showed that the substrates without grafted peptides were free from artifactual cell adhesion associated with the adsorption of serum or cellularly secreted proteins for long duration of culture. Of all grafted samples, IL1beta(178-207)- and C3a(63-77)-grafted surfaces supported higher adherent macrophage densities. C3a(63-77)- and FN(1615-1624)-grafted surfaces supported higher adherent fibroblast densities. From competitive inhibition studies, cell adhesion was determined to occur in a receptor-peptide specific manner. The presence of grafted YRGDG in addition to IL1beta(178-207), C3a(63-77), or FN(1615-1624) synergistically increased macrophage and fibroblast adhesion. Materials grafted with IL1beta(178-207) or C3a(63-77) co-grafted with or without YRGDG did not support the formation of multinucleated giant cells from the fusion of adherent macrophages in vitro.
Amino Acid Sequence↗
Solution conformation of carboxy-terminal fragments of the third component of human complement C3: proton nuclear magnetic resonance study of C3a, des-Arg-C3a, and C3a Arg69.
A proton nuclear magnetic resonance (NMR) study is reported of the solution conformation of human C3a, that is released on activation of C3, the third component of complement. The intact C3a was used along with des-Arg-C3a, which is formed on cleavage of Arg-77 at the C terminal of C3a, and C3a Arg69, which is a 69-residue fragment produced on tryptic digestion of C3. A method of carboxypeptidase digestion/difference spectroscopy (Endo & Arata (1985) Biochemistry 24, 1561-1568) was extensively used for the spectral assignments of Ile-43, Ile-60, Leu-63, Tyr-15, and Tyr-59. On the basis of the results of nuclear Overhauser effect (NOE) measurements, we discuss the solution conformation of the C3a molecule. It has been concluded that removal of Arg-77, which is essential for expression of the biological activity of C3a, does not induce any significant change in the solution conformation of the C3a molecule. The C3a molecule is known to consist of a core region that comprises segment Tyr-15-Tyr-59. We conclude that in solution the C terminal segment sticks out of the core and takes on a helix-like conformation. Possible roles of the core region and the N terminal segment in maintaining the conformation of the C terminal segment are briefly discussed.
Amino Acid Sequence↗
Synthetically modified cellulose: an alternative to synthetic membranes for use in haemodialysis?
Renal replacement therapy relies predominantly on the use of cellulose-based membranes. Such membranes have a biocompatibility profile which is inferior to membranes manufactured from synthetic polymers. Synthetically modified cellulose (SMC) is a new, low-flux haemodialysis membrane in which hydroxyl groups have been replaced with benzyl groups. The biocompatibility profile characterized by changes in white cell and platelet counts and the activation of complement components (C3a, C5a and C5b-9) have been studied in vivo and compared with those of cellulose acetate, unmodified cellulose (Cuprophan ) and low-flux polysulphone (Fresenius Polysulfone) in the same group of patients. For SMC, the white cell count at 15 min declined to 65.6% of pretreatment level, compared with 63.8% for the cellulose acetate, 79.6% for low-flux polysulphone and 28.1% for Cuprophan, thereafter returning to pretreatment levels. Both modified cellulose membranes were superior to unmodified cellulose (P = 0.001); the differences between the modified cellulose membranes were not significant statistically. The changes induced by all three cellulose-based membranes exceeded those for low-flux polysulphone (P = 0.001). Associated with the neutropenia was a reduction in platelet count, but this was independent of membrane type. The mean time-averaged concentrations of C3a(des Arg) over 150 min were 1168 ng ml(-1) (SMC), 1030 ng ml(-1) (cellulose acetate), 1297 ng ml(-1) (Cuprophan) and 790 ng ml(-1) (low-flux polysulphone). Equivalent values for C5a(des Arg) were 6.12 (SMC), 2.98 (cellulose acetate), 11.03 (Cuprophan) and 1.33 ng ml(-1) (low-flux polysulphone). C5b-9 values were 385 (SMC), 386 (cellulose acetate), 177 (Cuprophan) and 185 ng ml(-1) (low-flux polysulphone). For each of the complement components the differences between the membranes were significant [P = 0.0009 (C3a(des Arg)), P = 0.0001 (c5a(des Arg) and C5b-9)]. The levels of C5b-9 generated during dialysis also showed a significant positive correlation compared to C5a for all membranes considered as a single group (Pearson's correlation coefficient = 0.870, P = 0.0001). It is concluded that the modification of the cellobiosic unit is a promising approach to improve the biocompatibility profile of cellulose-based membranes. The two different methods of modification lead to similar improvements in biocompatibility compared with unmodified cellulose, but as yet do not match that of low-flux polysulphone.
Biocompatible Materials↗