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PubMed2000

Human complement 5a (C5a) anaphylatoxin receptor (CD88) phosphorylation sites and their specific role in receptor phosphorylation and attenuation of G protein-mediated responses. Desensitization of C5a receptor controls superoxide production but not receptor sequestration in HL-60 cells.

Upon agonist binding, the anaphylatoxin human complement 5a receptor (C5aR) has previously been found to be phosphorylated on the six serine residues of its carboxyl-terminal tail (Giannini, E., Brouchon, L., and Boulay, F. (1995) J. Biol. Chem. 270, 19166-19172). To evaluate the precise roles that specific phosphorylation sites may play in receptor signaling, a series of mutants were expressed transiently in COS-7 cells and stably in the physiologically relevant myeloid HL-60 cells. Ser(334) was found to be a key residue that controls receptor phosphorylation. Phosphorylation of either of two serine pairs, namely Ser(332) and Ser(334) or Ser(334) and Ser(338), was critical for the phosphorylation of C5aR and its subsequent desensitization. Full phosphorylation and desensitization of C5aR were obtained when these serines were replaced by aspartic acid residues. The mutation S338A had no marked effect on the agonist-mediated phosphorylation of C5aR, but it allowed a sustained C5a-evoked calcium mobilization in HL-60 cells. These findings and the ability of the S314A/S317A/S327A/S332A mutant receptor to undergo desensitization indicate that the phosphorylation of Ser(334) and Ser(338) is critical and sufficient for C5aR desensitization. The lack of phosphorylation was found to result not only in a sustained calcium mobilization and extracellular signal-regulated kinase 2 activity but also in the enhancement of the C5a-mediated respiratory burst in neutrophil-like HL-60 cells. For instance, the nonphosphorylatable S332A/S334A mutant receptor triggered a 1.8-2-fold higher production of superoxide as compared with the wild-type receptor. Interestingly, although the desensitization of this mutant was defective, it was sequestered with the same time course and the same efficiency as the wild-type receptor. Thus, in myeloid HL-60 cells, desensitization and sequestration of C5aR appear to occur through divergent molecular mechanisms.

Animals↗
PubMed1983

Detection of the complement fragment C5a in inflammatory exudates from the rabbit peritoneal cavity using radioimmunoassay.

We describe a radioimmunoassay for rabbit C5a and its use to obtain evidence of extravascular C5a generation in two inflammatory reactions in the peritoneal cavity. These observations, together with the potent activity of C5a in inducing increased microvascular permeability involving circulating PMN leukocytes, strengthen the case for considering C5a an important inflammatory mediator. These findings offer an explanation for the many different experimental inflammatory reactions where oedema formation can be suppressed either by systemic depletion of complement or by depletion of circulating PMN leukocytes.

Animals↗
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↗
PubMed1993

Inhibition of C5a-induced actin polymerization, chemotaxis, and phagocytosis of human polymorphonuclear neutrophils incubated in a glucose-based dialysis solution.

Chemotaxis and phagocytosis are important functions of phagocytic cells, which are closely related to cytoskeletal reorganization. These functions may be abnormal in phagocytes of uremic patients undergoing continuous ambulatory peritoneal dialysis (CAPD). In order to examine whether these abnormalities result from treatment, we studied actin polymerization (AP), as an index of cytoskeletal alterations, chemotaxis, and phagocytosis in polymorphonuclear neutrophils (PMNs) of healthy subjects. Polymorphonuclear neutrophils were exposed to either a hepes buffer or a glucose-based dialysis solution (GBDS) of different pH's (5.2, 7.4) and different glucose concentrations (1.36%, 2.27%, 3.86%). After incubation for 0, 5, or 20 minutes, cells were activated with 10 nmol/L C5a-complement. AP was measured as filamentous (F) actin content by NBD phallacidin staining and FACS analysis. Chemotaxis of PMNs was measured in Boyden chambers. In addition, phagocytosis of zymosan particles was assessed. Prior exposure to GBDS pH 5.2 of each glucose concentration immediately and completely inhibited AP in response to 10 nmol/L C5a-complement, reduced chemotaxis (> 95%), and completely inhibited phagocytosis. The inhibition was pH-dependent, since GBDS pH 7.4 caused less inhibition of these functions. We conclude that glucose-based dialysis solutions are cytotoxic towards neutrophils and completely inhibit their ability to display responses requiring cytoskeletal reorganization.

Actins↗
PubMed1997

Comparison of in vitro function of neutrophils from cattle deficient in plasma factor XI activity and from normal animals.

Cattle, homozygous for the genetic disorder of factor XI (FXI) deficiency, exhibit less than 2% of normal plasma FXI activity, display an increased bleeding tendency and are more prone to infectious diseases. FXI is one of the protein components of the contact activation system of coagulation that assembles on the surface of circulating neutrophils. Because of the central role of neutrophils in inflammation, the in vitro responses of neutrophils from normal and FXI deficient cattle were compared. Neutrophil degranulation was evaluated by measuring the release of myeloperoxidase and alkaline phosphatase, and the respiratory burst was evaluated by determining superoxide anion production. Neutrophils from FXI deficient animals exhibited a significant increase (P < 0.05) in the spontaneous release of granule contents compared to the cells from normal cattle. Following stimulation with C5a complement derived from normal serum, the neutrophils from the FXI deficient animals exhibited a greater increase (P < 0.05) in both alkaline phosphatase release and superoxide production. In these neutrophils, following stimulation with C3b complement from normal serum, the relative increase in myeloperoxidase release compared to the unstimulated neutrophils was lower than that observed in the neutrophils from normal animals. There was minimal superoxide production in unactivated neutrophils from either normal or FXI deficient cattle and the response to phorbol ester stimulation was similar in both groups of animals. The C5a complement from FXI deficient serum was more effective (P < 0.05) in stimulating alkaline phosphatase release and superoxide production in normal neutrophils than the equivalent fraction from FXI deficient serum while the C3b complement from the FXI deficient serum was less effective than the normal serum fraction at inducing myeloperoxidase release from normal neutrophils. The results indicate that the differences in the in vitro neutrophil function are likely related to a variation in the function of the contact activation system on the neutrophil surface between normal and FXI deficient animals.

Animals↗
PubMed1993

Levels of complement anaphylatoxin C5a in pulmonary effluent fluid of infants at risk for chronic lung disease and effects of dexamethasone treatment.

A number of studies have shown that increased numbers of neutrophils and macrophages are recruited into the airways during the development of chronic lung disease (CLD) in preterm infants. The objective of this study was to determine whether the anaphylatoxin C5a is detectable in tracheobronchial aspirate fluid of infants at risk for CLD and to evaluate the possible effects of dexamethasone (Dxm) treatment. C5a/C5a(des Arg) levels were determined by a sensitive ELISA based on a neoepitope-specific MAb. In a prospective study, 27 infants (birth weight 881 +/- 169 g, mean +/- SD) still on mechanical ventilation at d 10 postnatal age with fraction of inspired oxygen > or = 0.3 and/or peak inspiratory pressure > or = 16 cm H2O were randomly assigned to Dxm treatment at d 10 (n = 14) or d 16 (n = 13). Ten mechanically ventilated infants with no respiratory disease or who had recovered from respiratory distress syndrome did not meet these criteria on d 10 and served as a control group (birth weight 928 +/- 126 g). For the evaluation of Dxm therapy, the late treatment group was used as a control group for the early regimen. Compared with controls, C5a concentrations were higher in infants at risk for CLD on d 10 [median (25th-75th percentile): 2.40 (1.13-3.38) versus 0.82 (0.55-1.78) micrograms/L, p < 0.05].(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
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↗
PubMed1995

Altered microvascular responses to C5a in rat striated muscle during two-kidney, one clip renovascular hypertension.

OBJECTIVE: To determine how two-kidney, one clip (2-K,1C) renovascular hypertension alters microvascular responses in rat striated muscle to complement C5a, one of the most important inflammatory mediators. METHODS: 2-K,1C hypertension was induced in male Sprague-Dawley rats. Under anesthesia with pentobarbital (50 mg/kg, intraperitoneally) the cremaster muscle microcirculatory preparation with intact neurovascular connections was studied in vivo by closed-circuit videomicroscopy. Recombinant human C5a was applied topically in the tissue bath at concentrations of 10(-12), 10(-10) and 10(-8) mol/l, consecutively. Changes in the microvessel diameters in small arterioles, large arterioles and venules were measured. RESULTS: In normotensive rats complement C5a induces a significant dilation in small arterioles at low bath concentrations (10(-12) or 10(-10) mol/l), but the dilation is attenuated at a higher concentration (10(-8) mol/l). In contrast, in 2-K,1C hypertensive rats C5a constricts small arterioles at low concentrations (< 10(-10) mol/l) but dilates them at a higher concentration (10(-8) mol/l). Large arterioles and venules have minimal responses to C5a in either normotensive or 2-K,1C hypertensive rats. CONCLUSION: 2-K,1C hypertension dramatically alters C5a-induced microvascular responses in small arterioles. The alteration might be attributable to the enhanced vasoconstrictor mechanisms and impaired vasodilator mechanisms during 2-K,1C renovascular hypertension.

Animals↗
PubMed2005

Neutrophil chemotaxis in a patient with neonatal-onset multisystem inflammatory disease and Muckle-Wells syndrome.

BACKGROUND: Neonatal-onset multisystem inflammatory disease (NOMID)/chronic infantile neurologic, cutaneous, and articular syndrome is an autoinflammatory disease characterized by urticarial rash, arthropathy, and central nervous system inflammation. OBJECTIVE: To describe a 13-year-old girl with overlapping symptoms of NOMID and Muckle-Wells syndrome who has a mutation in cryopyrin (NALP3). METHODS: We examined neutrophil migration using transwell assay and time-lapse videomicroscopy. We also examined p38 mitogen-activated protein kinase (MAPK) activation in patient and control neutrophils using Western blot analysis. RESULTS: Neutrophil defects in chemotactic migration were found to a variety of chemoattractants, including interleukin 8, N-formyl-methionyl-leucyl-phenylalanine, complement C5a, and leukotriene B4. Her neutrophils exhibited elevated basal and stimulated p38 MAPK activation in response to interleukin 8, N-formyl-methionyl-leucyl-phenylalanine, complement C5a, and leukotriene B4. CONCLUSIONS: This study is the first, to our knowledge, to demonstrate defects in neutrophil chemotaxis and p38 MAPK signaling in a patient with NOMID and Muckle-Wells syndrome and a cryopyrin mutation.

Adolescent↗
PubMed2004

Antioxidative effect of propofol during cardiopulmonary bypass in adults.

AIM: To investigate the antioxidative potential of propofol (an intravenous anesthetic with a chemical structure similar to phenol-based free radical scavengers such as vitamin E) during cardiopulmonary bypass (CPB). METHODS: Thirty adult patients referred for elective cardiac procedure with CPB were included and randomly allocated to a propofol group or a control group. Patients in the propofol group received propofol (0.1 mg/kg/min) intravenously for anesthesia maintenance, whereas those allocated to the control group received fentanyl 10 microg/kg intravenously and inhaled enflurane (1 %-1.5 %). Blood samples were collected at 7 time points: before the start of CPB, at 30 and 60 min of CPB, at the conclusion of CPB, 10 min after the administration of protamine, and 12 and 24 h after the cessation of CPB. Plasma levels of free F2-isoprostanes (sensitive markers of free radicals production) and complement C5a were determined by mass-spectrometric assay and enzyme immunoassay, respectively. Neutrophil adhesion to endothelial cells was observed at 200 magnification under a light microscope. RESULTS: Levels of F2-isoprostanes, complement C5a and neutrophil adhesion rate increased significantly during and after CPB in both groups. There were significantly higher levels of F2-isoprostanes, C5a, and more neutrophils adhering to endothelial cells in the control group than those in the propofol group, respectively. CONCLUSION: Cardio-pulmonary bypass is associated with a great production of damaging free radicals. Propofol may be beneficial both as an anesthetic and as a potent free radical scavenger in patients presenting pathologies associated with free radical reactions during CPB.

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↗
PubMed1980

Granulocyte aggregometry: a sensitive technique for the detection of C5a and complement activation.

We have previously shown that complement (C) activated plasma causes granulocyte (PMN) aggregation in vitro and that C5a is responsible. The C-induced aggregation of PMNs treated with cytochalasin-B (CB) is markedly enhanced and irreversible, and the magnitude of the response is proportional to the log (concentration of activated plasma), allowing use of this technique to detect C5a and hence C-activation. To compare the sensitivity of granulocyte aggregometry to that of more standard methods of detecting C-activation, we produced graded C-activation in vitro by treating fresh serum with varying amounts of zymosan. Aggregometry was the most sensitive index of C-activation, detecting C-activation, produced by 0.02 mg zymosan/ml of serum--1/10 that required to produce C-activation detectable by C3 immunoelectrophoresis (the next most sensitive technique). Granulocyte aggregometry may also be used to detect in vivo C-activation. We have found aggregating activity in plasmas from patients with systemic lupus erythematosus, immune vasculitis, transfusion reactions, and other conditions associated with in vivo C-activation, but not in the plasmas of normal subjects.

Cell Aggregation↗
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↗