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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↗
PubMed1990

Measurement of the chemotactic complement fragment C5a in rheumatoid synovial fluids by radioimmunoassay: role of C5a in the acute inflammatory phase.

Evidence suggests that complement is activated in rheumatoid joints. A sensitive radioimmunoassay for the activation fragment of C5, C5a, which is a potent chemoattractant for neutrophils, was therefore developed. A mean C5a concentration in 22 rheumatoid joint fluids of about 2.5 x 10(-9) mol/l was found. This concentration of C5a is sufficient to induce two of the characteristic features of the acute inflammatory phases of rheumatoid arthritis: neutrophil accumulation and microvascular plasma protein leakage. In animal models it has been shown that C5a is a potent inducer of inflammatory oedema by a neutrophil dependent mechanism. A striking feature of the acute inflammatory phases of rheumatoid arthritis is the appearance of high numbers of neutrophils in the synovial fluid. It is suggested that C5a might have a role in mediating neutrophil accumulation and, as a consequence, may be important in acute joint swelling and pain.

Adult↗
PubMed1985

Chemical synthesis of a gene encoding the human complement fragment C5a and its expression in Escherichia coli.

A gene coding for the C5a fragment of the fifth component of human complement has been chemically synthesized, cloned, and expressed in Escherichia coli. The 253-base-pair gene fragment was built through a two-step enzymic assembly of 16 oligonucleotides, the average length of each being 32 residues. The oligonucleotides were synthesized by using the phosphoramidite method. The gene was cloned in a pBR322-derivative plasmid downstream from the lac up-promoter mutant, UV5-D. The expression of C5a was detected and measured by immunoassay and a radioligand binding assay. C5a from E. coli was comparable to C5a purified from human serum in inhibiting binding of human 125I-labeled C5a to its putative receptor on polymorphonuclear leukocytes. Studies of smooth muscle contraction in isolated guinea pig ileum showed that the recombinant C5a was biologically active and produced cross-tachyphylaxis with human serum-derived C5a. The results demonstrate the feasibility of expressing C5a anaphylatoxin in bacteria and provide a system for mutagenesis of the C5a protein.

Animals↗
PubMed1985

Subnormal sensitivity of neutrophils to complement split product C5a in rheumatoid arthritis: relation to complement catabolism and disease extent.

The capacity of circulating neutrophils for activation by complement was studied in outpatients with classical or definite rheumatoid arthritis during treatment with dextropropoxyphene only. Analysis of dose-response in the Boyden chamber assay of chemotaxis showed that sensitivity to the potent, complement derived anaphylatoxin, C5a, was markedly decreased, especially in those patients with few joints involved. In contrast, peak response to C5a was within the normal range. Increased complement 3c split products in plasma of the patients suggested involvement of complement cascade reactions. Subnormal sensitivity of neutrophils to phlogistic mediators released by complement may tend to limit their recruitment and potentially tissue destroying secretion locally in rheumatoid arthritis.

Adult↗
PubMed1991

The role of protein kinase C activation and inositol phosphate production in the regulation of cell-surface expression of Mac-1 by complement fragment C5a.

The adhesion of leukocytes to endothelial and other cell types is an essential part of the acute inflammatory response. One means by which adherence can be increased is by activation of the CD11/CD18 family of leukocyte glycoproteins. Chemotactic peptides, lipid mediators, phorbol esters and tumour necrosis factor are all able to increase the cell surface expression of one member of this family, CD11b/CD18 or Mac-1, by an unknown signal transduction mechanism. In this report, regulation of Mac-1 expression by C5a is shown to be independent of protein kinase C (PK-C) activation. The inhibitor of PK-C, H-7, has no effect on the action of C5a and only a slight effect on phorbol ester-induced up- and down-regulation of Mac-1, at a concentration that inhibits superoxide production in response to both factors by 40%. Inositol phospholipid hydrolysis, an important pathway leading to PK-C activation, and the transient increases in cytosolic Ca2+ associated with inositol phosphate production, are also shown to be not essential processes in C5a-stimulation of Mac-1 expression.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Group B streptococci inactivate complement component C5a by enzymic cleavage at the C-terminus.

Incubation of recombinant human C5a (rC5a) with the 7360 strain of group B streptococci (GBS) destroyed the ability of rC5a to stimulate chemotaxis or adherence of purified human polymorphonuclear leucocytes (PMNs). Treatment of 125I-labelled rC5a with GBS 7360 correspondingly decreased rC5a binding to human PMNs. This also resulted in an approx. 600 Da decrease in the molecular mass of rC5a as determined by SDS/PAGE. Incubation of rC5a with the GBS strain GW, which only minimally altered the ability of rC5a to activate human PMNs, did not affect rC5a binding to PMNs and did not alter the molecular mass of rC5a on SDS/PAGE. Plasma-desorption m.s. of rC5a inactivated by GBS 7360 showed that the GBS cleaved the rC5a between histidine-67 and lysine-68 near the C-terminus of rC5a. This mechanism of inactivation of C5a by proteolytic cleavage at the C-terminus of C5a is consistent with the known critical role of the C-terminus in C5a activation of human PMNs. This C5a-cleaving proteinase activity may contribute to the pathophysiology of GBS infections.

Adult↗
PubMed1986

High-level expression of a gene encoding the human complement factor C5a in Escherichia coli.

The synthetic C5a gene was initially found to be expressed poorly in Escherichia coli. We undertook studies to determine the reasons for poor expression and to increase expression. The work was focused on the role of the mRNA structure in C5a expression and stability of its product in E. coli. We present data on the effects of varying the sequence at the 5' end of mRNA as well as different ribosome-binding sites on expression. Evaluation of the stability of C5a showed rapid degradation of C5a in wild-type E. coli (half-life 3-5 min). Screening of several protease-deficient strains of E. coli showed that C5a was much more stable in an htpR strain carrying a mutation in the sigma subunit of RNA polymerase that is specific for heat shock promoters. The mutation is associated with a proteolytic deficiency. The half-life of C5a was increased to 20 min. By manipulating the expression vector, the regulatory region for the C5a gene, the host strain, growth conditions and methods for recovering the protein, C5a levels were increased 300-fold over previously reported amounts to about 3% of total cellular protein.

Base Sequence↗
PubMed1988

Solid-phase radioimmunoassay of human complement fragment C5a.

In this competitive RIA for determining concentrations of human C5a in biological fluids and in buffers, labeled C5a and sample are allowed to compete for binding to a limited amount of goat antibody to human C5a in solution. Free and bound tracer are then separated by a second antibody (rabbit anti-goat IgG) immobilized on paramagnetic particles. Total incubation time for this assay is 70 min. Sensitivity, precision, and analytical recovery of this assay compare well with those of a reference method.

Binding, Competitive↗
PubMed1988

Leukotriene production in human neutrophils primed by recombinant human granulocyte/macrophage colony-stimulating factor and stimulated with the complement component C5A and FMLP as second signals.

Neutrophils (PMN) preincubated with recombinant human granulocyte/macrophage colony-stimulating factor (rhGM-CSF) for 2 h and then stimulated with the chemotactic factors, C5a or FMLP, produce substantial amounts of the lipoxygenase products 5-Hete, LTB4, and omega-oxidised LTB4 metabolites (4.36 +/- 0.95 (SEM) pM (n = 21) LTB4 and LTB4 metabolites/10(6) PMN). No lipoxygenase metabolites are detected by HPLC and RIA if purified PMN are stimulated by either GM-CSF or chemotactic factors in the absence of exogenous arachidonate. The priming effect of GM-CSF upon chemotactic factor induced generation of lipid mediators is a relatively slow process, clearly evident after 1 h and optimal after 2 h. Leukotriene generation is measurable with 0.8 U GM-CSF/10(6) PMN and is maximal with 80 U (10(-11)-10(-9) M). Upon activation of primed PMN with chemotactic factors, leukotriene synthesis is induced very rapidly. Already 2.5 min after activation the major lipoxygenase metabolites present are 20-OH LTB4 and 20-COOH LTB4. Our study shows that the synthesis of lipoxygenase metabolites from endogeneous AA can be initiated in PMN through receptor mediated processes by the appropriately timed combination of biological soluble inflammatory mediator peptides. Furthermore, these results indicate that GM-CSF not only enhances effector cell functions but can qualitatively change the mediator profile formed after activation with a second triggering signal. Such a mechanism might be important in amplifying inflammatory responses. Alternatively, lipid mediators formed might also have an intracellular or autocoid role and be responsible for the enhancement of other PMN functions like oxygen radical release.

Adult↗
PubMed1989

Identification of receptor-binding residues in the inflammatory complement protein C5a by site-directed mutagenesis.

C5a is an inflammatory mediator potentially involved in a number of diseases. To help define which of its 74 residues are important for receptor binding and response triggering, changes in the amino acid sequence of C5a were introduced by site-directed mutagenesis. Synthetic C5a-encoding genes incorporating point mutations were expressed in Escherichia coli, and the mutant proteins were purified to homogeneity. Modifications of the C5a molecule causing parallel reductions in binding to polymorphonuclear leukocyte membranes and in stimulation of polymorphonuclear leukocyte locomotion (chemokinesis) suggest that carboxyl-terminal residues Lys-68, Leu-72, and Arg-74 interact with the receptor. Substitutions in the disulfide-linked core of C5a revealed involvement of Arg-40 or nearby residues, because potency losses were associated with only localized conformational changes as detected by NMR. Surprisingly, a substitution at core residue Ala-26, which did not alter C5a core structure, appeared from NMR results to reduce potency by causing a long-distance conformational change centered on residue His-15. Thus, at least three discontinuous regions of the C5a molecule appear to act in concert to achieve full potency.

Amino Acid Sequence↗
PubMed1991

Attenuation of spontaneous pseudopod formation in human neutrophils by pentoxifylline.

The effects of pentoxifylline (PTX) on spontaneous pseudopod formation in neutrophils in response to the tripeptide formyl-Met-Leu-Phe (fMLP), endotoxin, human complement C5a, and leukotriene B4 (LTB4) were examined in autologous plasma. Unseparated supernatant leukocyte suspensions from fresh heparinized venous human blood were incubated with PTX (0-5 mM) for 25 min and then stimulated for 5-25 min within a range of concentrations of fMLP, endotoxin, complement C5a, and LTB4. The cell suspensions were fixed with glutaraldehyde and stained with crystal violet in acetic acid; the percentage of neutrophils with pseudopods was determined under high-resolution light microscope. The results show that PTX significantly decreases formation of pseudopods in the presence of all four stimulators. The mechanism of pseudopod suppression appears to be independent of the adenosine receptor. PTX and its analogues, HWA 138 and HWA 448, decreased pseudopod formation by similar amounts when stimulated with 10(-8)M fMLP. These results suggest that PTX may improve microvascular perfusion and attenuate neutrophil-mediated injury by reducing the degree of neutrophil pseudopod formation in free suspension and microvascular entrapment.

Actins↗
PubMed1979

Induction of lysosomal enzyme secretion by alveolar macrophages in response to the purified complement fragments C5a and C5a des-arg.

Purified C5a and its "inactive" form, C5a des-arg, were shown to induce secretion of acid hydrolases from rabbit alveolar macrophages (AM) in a concentration-dependent manner. Secretion increased with time to 5 times above controls by 72 hr. Concentrations of these enzymes in the cell lysates did not decrease during the incubation, suggesting that synthesis of new enzyme was occurring. The lysosomal enzyme secretion was accompanied by increased pinocytosis and release of proteolytic enzymes from the macrophages. At no time was significant lactic dehydrogenase liberated, indicating that secretion was selective and not due to cell death. Data presented also suggest that C5a des-arg induced secretion from the macrophages of a chemotactic factor for neutrophils. It was concluded that C5a and C5a des-arg may play a role in lung injury by interactions with AM, inducing the secretion of acid hydrolases and proteolytic enzymes that can cause tissue damage, and by regulating the influx of other inflammatory cells into the interstitium and air spaces.

Anaphylatoxins↗
PubMed1991

Human neutrophil migration into skin chambers is associated with production of NAP-1/IL8 and C5a.

Respiratory burst activity initiated by the chemoattractants fMLP, rC5a and rNAP-1/IL8 was investigated in human exudated and peripheral blood neutrophils. Exudated cells were isolated after migration into a skin chamber and the respiratory burst activity was measured as chemiluminescence amplified by luminol and horseradish peroxidase. The response to fMLP (5 x 10(-8) mol/l) was significantly enhanced (p less than 0.01) in the exudated cells but was significantly decreased after stimulation (5 x 10(-8) mol/l) with rC5a and rNAP-1/IL8 (p less than 0.05 and p less than 0.01, respectively). Analysis revealed that, in the chamber fluid, the activated complement C5a was generated during exudation (p less than 0.01). Determinations of NAP-1/IL8 showed that this substance was also produced and released into the chamber fluid (p less than 0.01). No correlation was found between the number of exudated cells and the amount of C5a or NAP-1/IL8 in the exudation fluid, thus indicating that, in vivo, the exudation process is controlled by multiple factors and not by the quantity of a single chemoattractant. The present study shows that NAP-1/IL8 and C5a are produced in humans during an aseptic inflammation, and that this occurs in parallel to the migration of neutrophils into the skin chambers. The significant desensitization of the exudated cells to NAP-1/IL8 and C5a reflects a previous exposure to these attractants. These results suggest that the novel tissue-derived cytokine NAP-1/IL8 plays a role in human neutrophil exudation in vivo.

Cell Movement↗
PubMed1991

Regulation of complement 5a receptor expression in U937 cells by phorbol ester.

Receptors for the anaphylactic portion of complement, C5a, are not initially expressed in the monoblastic U937 cell line, but appear as the cell is induced to differentiate by the synergistic actions of 1,25(OH)2D and cyclic adenosine monophosphate (cAMP). Phorbol myristate acetate (PMA), which activates the protein kinase C pathway (PKC), does not cause C5a receptor (C5aR) expression when used as a single agent. The induction of C5aR by the synergistic actions of 1,25(OH)2D and cAMP, however, can be augmented as much as 180% by the addition of PMA. C5aR arising in cells exposed to 1,25(OH)2D and 8,4-chlorophenylthio-cAMP have an affinity constant of about 0.4 nM as assessed by cold competition analysis. We show here that when phorbol augmentation of receptor number occurs, the affinity constant is increased by 3.6-fold. In an effort to ascertain whether the change in C5aR Kd involved a PKC-dependent event we examined whether 5-60 min exposure of C5aR-positive cells to PMA would change C5aR Kd. Acutely, PMA caused a downregulation of receptor binding with decreases in apparent receptor number out of proportion to changes in Kd. One hundred nanomolar PMA, which effects nearly complete translocation of PKC to the membrane, consistently caused a 70-90% decrease in C5a surface binding. This downregulation was proportional to PMA dose and exposure time. Micromolar concentrations of the microtubule depolymerizing agents colchicine and vinblastine caused a less drastic downregulation, about 50% of the maximal phorbol effect. Our data suggest that activation of the PKC system might acutely limit the macrophage's ability to respond to C5a; chronically, phorbols upregulate receptor expression, most likely through positive effects on C5aR gene expression.

Calcitriol↗
PubMed1989

[Effects of nicotine and cigarette smoke extracts on plasma level of complement C3a and C5a, thromboxane B2 and 6-keto PGF1 alpha in rabbits].

In the present investigation we measured plasma levels of complement C3a and C5a, thromboxane B2 (TxB2) and 6-keto PGF1 alpha in anesthetized rabbits by radioimmunoassay after the intravenous injection of nicotine and cigarette smoke extracts. Plasma levels of complement C3a in rabbits showed a significant increased and reached a maximum value 60 minutes after the intravenous injection of nicotine and cigarette smoke extract. Plasma levels of complement C5a in rabbits showed a significant increase and reached a maximum value 30 minutes after the intravenous injection of nicotine. Plasma levels of complement C5a in rabbits showed a significant increase and reached a maximum value 60 minutes after the intravenous injection of cigarette smoke extract. Plasma levels of TxB2 in rabbits showed a significant increase after the intravenous injection of nicotine and cigarette smoke extract. Plasma levels of 6-keto PGF1 alpha in rabbits did not show any change after the intravenous injection of nicotine and cigarette smoke extracts. The above results may suggest that the intravenous injection of nicotine and cigarette smoke extracts induces an increase of plasma levels of complement C3a and C5a and TxB2 in anesthetized rabbits.

6-Ketoprostaglandin F1 alpha↗
PubMed1985

Model structure for the inflammatory protein C5a.

The complement cleavage product C5a is a potent stimulant of inflammatory processes; thus, inhibition of C5a activity is of therapeutic interest. The three-dimensional structure of the major portion of C5a was modeled from the homologous C3a crystal structure by comparative modeling techniques. The model shows that core residues of C5a are completely conserved, while external residues differ from C3a. Even though the amino-terminal 12 residues of C3a are disordered in the crystal, this sequence in C5a may form an amphipathic helix. The distribution of species sequence differences in the complete C5a structure suggests a possible receptor binding site.

Amino Acid Sequence↗