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PubMed1992

Complement, neutrophil, and macrophage activation in women with severe preeclampsia and the syndrome of hemolysis, elevated liver enzymes, and low platelet count.

Activation of complement, neutrophils, and macrophages was studied in 14 women with severe preeclampsia, 11 of whom had the syndrome of hemolysis, elevated liver enzymes, and low platelet count; in 14 women with normal pregnancies; in seven normal pregnant women undergoing cesarean deliveries; and in 15 healthy nonpregnant women. Activation of complement, neutrophils, and macrophages was measured by plasma determinations of complement split products, polymorphonuclear (PMN) elastase, and neopterin, respectively. Women with severe preeclampsia had increased levels of C5a, terminal complement complex, PMN elastase, and neopterin at delivery and 1 day postpartum as compared with the normal pregnant group. One week postpartum, neopterin remained higher in preeclamptic women, whereas the complement components and PMN elastase had returned to normal. Cesarean delivery after normal pregnancy did not increase the levels of complement split products, PMN elastase (except for one value), or neopterin. The nonpregnant women had normal PMN elastase and neopterin levels. Accordingly, complement, neutrophils, and macrophages are activated in women with severe preeclampsia at delivery. The plasma levels of PMN elastase correlated positively to the formed terminal complement complexes in vivo. An in vitro study was performed to elucidate further the connection between complement and leukocyte activation. Recombinant C5a incubated in whole blood and in a neutrophil cell suspension gave a dose-dependent release of PMN elastase. Both the clinical and the in vitro results indicate that activation of the complement system may affect the function of neutrophils. This study supports the theory that the pathologic manifestations of severe preeclampsia may be explained by complement-induced release of biologically active substances from activated leukocytes.

Biopterins↗
PubMed1999

Role of C3 cleavage in monocyte activation during extracorporeal circulation.

BACKGROUND: We previously demonstrated that inhibiting formation of terminal complement components (C5a and C5b-9) prevents platelet and neutrophil (PMN) but not monocyte activation during simulated extracorporeal circulation (SECC). This study examined whether earlier complement inhibition during SECC, blocking C3a formation, would additionally prevent monocyte activation. METHODS AND RESULTS: SECC was established by recirculating heparinized whole blood from human volunteers on a membrane oxygenator. CAB-2, a chimeric protein constructed from genes encoding the complement regulatory proteins CD46 and CD55, inactivates the C3/C5 convertases and blocks in vitro generation of C3a, C5a, and C5b-9. CAB-2 was used in 4 experiments at a final concentration of 300 micrograms/mL and 4 experiments at 30 micrograms/mL; 4 control runs used vehicle alone. Samples were assayed for C3a and C5b-9, monocyte activation (CD11b upregulation), PMN activation (CD11b upregulation and elastase release), and platelet activation (P-selectin expression and monocyte-platelet conjugate formation). CAB-2 at both doses significantly inhibited formation of C3a and C5b-9 during SECC. High-dose CAB-2 significantly blocked monocyte and PMN CD11b upregulation and PMN elastase release. CAB-2 also inhibited formation of platelet activation-dependent monocyte-platelet conjugates. CONCLUSIONS: Blockade of complement activation early in the common pathway inhibited monocyte CD11b upregulation during SECC, suggesting that early complement components contribute most to monocyte activation during SECC. As expected, PMN and platelet activation were blocked by terminal complement inhibition. This investigation further elucidates the relation between complement and blood cell activation during simulated cardiopulmonary bypass.

Blood Platelets↗
PubMed1996

1,25(OH)2D3 and cAMP synergistically induce complement 5a receptor messenger RNA.

Complement 5a receptor (C5aR) mediates both acute and chronic participation of monocytes in the immune response. In the human U937 monoblast, C5aR is maximally expressed 4 days after treatment with 1,25(OH)2D3 (or cycloheximide) and prostaglandin E2 combined. The authors asked whether these agents altered expression of C5aR messenger RNA (mRNA). Unstimulated U937 cells expressed neither C5aR mRNA nor C5a binding. Complement 5aR mRNA rose 3 hours after prostaglandin E2 application and fell to basal levels by 12 hours. This early rise in C5aR mRNA did not cause an acute rise in C5a binding, which gradually increased between 1 and 4 days. Neither 1,25(OH)2D3 nor cycloheximide induced expression of C5aR mRNA in the absence of prostaglandin E2 but did enhance prostaglandin E2-stimulated C5aR mRNA expression and C5a binding. The authors observed a late increase in C5aR mRNA at day 3 in treated cells. Inhibition of this late rise in mRNA with 5,6-dichlorobenzimidazole riboside attenuated C5a binding by 65%, indicating its importance in the generation of C5a binding sites. The expression of functional C5aR is, therefore, a complex process involving regulation at transcriptional and posttranscriptional levels.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Analysis of membrane processes for blood purification.

Physical phenomena play an important role in membrane processes for blood purification. They largely determine the separation performance of these devices and they interact with chemical and biological phenomena to determine their biocompatibility, or lack thereof, in the clinical setting. In the first part of this paper, analyses of physical phenomena which determine the separation and purification characteristics are reviewed for several processes, including hemodialysis, hemofiltration, combined hemodialysis and ultrafiltration, and membrane plasmapheresis with cross-flow microfiltration. Special attention is given to transport of high-molecular weight solutes in hemodialysis, for use in subsequent analyses, and to the factors which determine filtrate flux in membrane plasmapheresis, because recent findings in this area provide an understanding of filtration processes in general. The second part concerns the problem of biocompatibility, especially as manifested in renal prostheses. After reviewing some of the pathways to bioincompatibility, exploratory analyses are presented using relatively simple models. The objective of these analyses is to provide an initial quantitative framework for examining the likelihood of monocyte secretion of interleukin-1 being stimulated by various routes. Issues examined, for which illustrative calculations are presented, include (1) transport of endotoxin fragments across regenerated cellulose and other membranes, (2) anaphylatoxin C5a concentrations in conventional hemodialysis and (3) the effects of equilibrium and reaction phenomena, ultrafiltration, diffusive membrane permeation and membrane adsorption on the disposition of C5a which is generated at the membrane surface.

Animals↗
PubMed2003

Structure-function relationships of human C5a and C5aR.

Using peptides that represent linear regions of the powerful complement activation product, C5a, or loops that connect the four alpha helices of C5a, we have defined the ability of these peptides to reduce binding of (125)I-C5a to human neutrophils, inhibit chemotactic responses of neutrophils to C5a, and reduce H(2)O(2) production in neutrophils stimulated with PMA. The data have defined likely sites of interaction of C5a with C5aR. The peptides had no functional activity per se on neutrophils and did not interfere with neutrophil responses to the unrelated chemotactic peptide, N-formyl-Met-Leu-Phe. Although previous data have suggested that there are two separate sites on C5a reactive with C5aR, the current data suggest that C5a interacts with C5aR in a manner that engages three discontinuous regions of C5a.

Amino Acid Sequence↗
PubMed1993

Site-specific mutations in the N-terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor.

C5a is an inflammatory mediator that evokes a variety of immune effector functions including chemotaxis, cell activation, spasmogenesis, and immune modulation. It is well established that the effector site in C5a is located in the C-terminal region, although other regions in C5a also contribute to receptor interaction. We have examined the N-terminal region (NTR) of human C5a by replacing selected residues in the NTR with glycine via site-directed mutagenesis. Mutants of rC5a were expressed as fusion proteins, and rC5a was isolated after factor Xa cleavage. The potency of the mutants was evaluated by measuring both neutrophil chemotaxis and degranulation (beta-glucuronidase release). Mutants that contained the single residue substitutions Ile-6-->Gly or Tyr-13-->Gly were reduced in potency to 4-30% compared with wild-type rC5a. Other single-site glycine substitutions at positions Leu-2, Ala-10, Lys-4, Lys-5, Glu-7, Glu-8, and Lys-14 showed little effect on C5a potency. The double mutant, Ile-6-->Gly/Tyr-13-->Gly, was reduced in potency to < 0.2%, which correlated with a correspondingly low binding affinity for neutrophil C5a receptors. Circular dichroism studies revealed a 40% reduction in alpha-helical content for the double mutant, suggesting that the NTR contributes stabilizing interactions that maintain local secondary or tertiary structure of C5a important for receptor interaction. We conclude that the N-terminal region in C5a is involved in receptor binding either through direct interaction with the receptor or by stabilizing a binding site elsewhere in the intact C5a molecule.

Amino Acid Sequence↗
PubMed1986

Airways hyperreactivity and inflammation produced by aerosolization of human C5A des arg.

The premise of this study was that complement fragments would lead to granulocyte infiltration and to associated changes in airways reactivity. Inflammation was induced in large airways (greater than 1.0 mm) by aerosolization of the complement chemotactic factor, C5a des arg, which was isolated from activated human serum. Pulmonary function (resistance, compliance, and lung volume) and histamine reactivity were assessed at 4 and at 48 h after C5a des arg or a saline sham aerosol. Four hours after exposure to C5a des arg, the animals exhibited evidence of significant bronchoconstriction and hyperinflation. In addition, an increased responsiveness to histamine was demonstrated, which was not correlated with the degree of bronchospasm. By 48 h, these alterations were partially or completely resolved. Histologic examination and quantitation demonstrated significant accumulation of neutrophils, which was limited to airways 0.5 mm in or larger. Saline-treated animals also demonstrated a neutrophil infiltration, but significantly less than those treated with C5a des arg. However, these animals did not demonstrate hyperreactivity to histamine, and there was little change in baseline mechanical function. The mild inflammation associated in the saline control animals was demonstrated to be a result of intubation of the airways. Granulocyte dependence of the effects of C5a des arg was partially established by abrogation of these effects when the animals were rendered granulocytopenic with nitrogen mustard. We conclude that the physiologic responses of the airways to C5a des arg were largely granulocyte dependent. However, since granulocyte accumulation could occur without airways dysfunction, it is suggested that cell activation is an important step in producing neutrophil-associated airways hyperresponsiveness.

Aerosols↗
PubMed1987

Identification of an antigenic epitope and receptor binding domain of human C5a.

Nine different murine anti-human C5a monoclonal antibodies have been produced and characterized. They exhibit Kas for the 125I-labeled ligand that range from 0.4 to 48 X 10(8) M-1, and they display limited cross-reactivity with C5a from other species. Each of these antibodies has been found to compete with the granulocyte C5a receptor for binding site(s) on the C5a polypeptide. Exploration of the antigenic topography of C5a revealed that the immunodominant portion of this glycopolypeptide resides between residues Lys20 and Arg37, with the area surrounding Cys27 being particularly important. In addition, a specific C5a derived tryptic peptide containing these amino acid residues competes with 125I-C5a for binding to the receptor. These observations are consistent with previously published data and suggest that this area of the C5a molecule is an important part of the receptor "recognition domain", and thus plays a critical role in the C5a receptor interaction.

Antibodies, Monoclonal↗
PubMed1998

Reduced expression of C5a receptors on neutrophils from cord blood.

AIM: To describe further functional deficiencies of neonatal neutrophils by measuring the expression of C5a receptors. METHODS: C5a uptake was measured using flow cytometry with fluorescein isothiocynate labelled recombinant C5a. The response of neutrophils to stimulation with C5a and fMLP was tested by measuring migration and exocytosis of myeloperoxidase and lactoferrin. RESULTS: C5a mean fluorescence on neutrophils from neonates was significantly lower (22.4 (SD 3.5)) than in adult controls (31.5 (3.1)). Neutrophils from neonates migrated poorly towards both C5a and fMLP compared with those from adult controls. Exocytosis of myeloperoxidase, but not lactoferrin from neonatal neutrophils stimulated with C5a, was significantly lower than in adult controls. fMLP stimulation, on the other hand, resulted in significantly higher exocytosis in neonates. CONCLUSION: The lower expression of C5a receptors on neutrophils from neonates could be related to reduced C5a mediated exocytosis of myeloperoxidase.

Antigens, CD↗
PubMed1993

N-linked glycosylation of the C5a receptor.

The recent cloning of the cDNA encoding the human C5a receptor reveals a single potential site for N-linked glycosylation. Previous studies have suggested the presence of at least one carbohydrate moiety in the C5a receptor. Enzymatic digestion with Endoglycosidase F confirmed this presence and a point mutation at the predicted site of glycosylation was sufficient to lower the apparent molecular weight of the receptor as determined by SDS-PAGE. Removal of the carbohydrate moiety failed to abolish expression of the receptor and brought about only a slight reduction in the dissociation constant of the C5a receptor suggesting that the role of glycosylation in the binding of C5a by its receptor is limited.

Animals↗
PubMed1992

Anaphylatoxin generation and distribution during in vitro LDL apheresis.

The extent of anti-apo-B IgG-Sepharose-induced complement activation in serum and plasma (heparin 2 U/ml and ACD-B 1:20) was investigated using an in vitro model of LDL apheresis. The total volume of serum or plasma loaded to the chromatography column was collected in defined aliquots. The washing, desorption and regeneration fluids were processed in the same way. From the obtained values of generated complement split products C3a (desarg), C4a (desarg), C5a (desarg) and complement proteins C3, C4, C5, the conversion rates of the precursor were calculated. In the experiments with serum, 19% of C3, 8% of C4 and 2.3% of C5 were converted by the immunoadsorbent, whereas with plasma 7, 6, and 0.6%, respectively, were found. Furthermore, only 60-74% of total anaphylatoxins were found in the effluent during the loading process. The residual 26-40% was removed from the column with the subsequent washing fluids. Therefore, in the clinical routine, only a reduced part of generated anaphylatoxins will be retransfused to the patient. The fact that C5 is converted to the most limited extent to its biologically active fragment additionally contributes to the understanding of the good clinical tolerability of the LDL apheresis.

Anaphylatoxins↗
PubMed1985

Characterization of a C5a receptor on human polymorphonuclear leukocytes (PMN).

Elucidation of the interactions between C5a and granulocytes is central to understanding the role of C5a in inflammation. In this study, interactions between C5a and PMN have been studied at two levels. Binding of human C5a to intact human cells has been characterized by using the radiolabeled ligand 125I-C5a. Binding is shown to be reversible, saturable, and to reach equilibrium in 60 to 90 min at 0 degrees C. Results show high affinity C5a binding sites with Kd = 2 X 10(-9) M and a range of 50,000 to 113,000 binding sites per PMN. These values for C5a receptors are comparable with the number of fMLP and LTB4 receptors on PMN. Binding of C5a to PMN fails to reach equilibrium at 37 degrees C because there is an irreversible loss of available surface receptors caused by an active internalization of the ligand-receptor complex. Interactions between C5a and human PMN were characterized further by cross-linking experiments, with the use of ethylene glycol bis succinimidylsuccinate (EGS). Cross-linking of 125I-C5a to intact PMN followed by subcellular fractionation revealed a single radioactive band present only in the plasma membrane fraction and visualized by autoradiography. Similar experiments resulted in a covalent linkage between 125I-C5a and a component in the isolated plasma membrane of PMN. The covalent complex containing C5a and a putative receptor has been visualized by autoradiography as a single 60,000 Mr complex on SDS-PAGE. The complex is not present when experiments are performed in the presence of excess unlabeled C5a or in the absence of EGS. Therefore, the putative receptor for C5a on human neutrophils is estimated to be approximately 48,000 Mr, assuming contribution of 12,000 to 13,000 daltons by the ligand 125I-C5a.

Cell Membrane↗
PubMed2002

Contractile effect of anaphylatoxin C5a and of a mimetic peptide on the human umbilical artery: further evidence for leukocyte-dependent vasomotion.

Reproducible and concentration-dependent contractile effects were recorded when the human umbilical artery was exposed to recombinant C5a (>or=1 nM). The synthetic mimetic peptide Ac-YSFKPMPLaR was about 100-fold less potent in this respect, and its effect was largely prevented by treatment with the TP prostanoid receptor antagonist SQ 29548. The selective cyclooxygenase-1 inhibitor flurbiprofen, but not by the cyclooxygenase-2 inhibitor L-745337, prevented the contractile effect of both C5a and Ac-YSFKPMPLaR. Cells positive for C5a receptor (CD 88) immunoreactivity were scattered in the umbilical artery structure and were apparently more abundant in the periphery. The macrophage marker CD 68 was also expressed by dispersed cells, with a distribution similar to CD 88; both markers were co-expressed in some cells represented in consecutive sections. Immunoblot for C5a receptors has been applied to tissue or cells extracts: a specific approximately 42-kd major band observed in peripheral blood leukocytes was also expressed by the fresh umbilical artery, but not by cultured smooth muscle cells derived from the umbilical artery. Macrophages dispersed in the connective structure of the vessel wall may, in response to C5a receptor agonists, release prostanoids formed by cyclooxygenase-1 that indirectly contract smooth muscle cells. Leukocyte-dependent vasospasm may be relevant in situations in which complement is activated and may trigger thromboembolic complications via the secretion of thromboxane-like eicosanoids also active on circulatory platelets.

Antigens, CD↗
PubMed1999

Characterization of a polymorphism in the coding region of the human C5a anaphylatoxin receptor.

A polymorphism was identified in the coding region of the human C5a anaphylatoxin receptor gene leading to C to T transition at nucleotide position 450 (a silent substitution in the Ala150 codon, GCC to GCT). Its distribution was studied in a population of healthy volunteers from the Québec city region (prevalence of 2.8%) and among patients with end-stage renal failure who had previously undergone renal graft (prevalence 1.4%, not significantly different from that of the control group). This new marker provides a valuable tool to assess the risk for putative C5a-associated disorders with genetic determinism.

Alleles↗
PubMed1990

Interaction between the C5a receptor and Gi in both the membrane-bound and detergent-solubilized states.

C5a elicits a variety of responses from the polymorphonuclear leukocyte all of which utilize G proteins as transducing elements. In the present study, we report the consequences of the interaction between the C5a receptor and the G proteins and describe a system which may allow identification of the transducing proteins. C5a binding to polymorphonuclear leukocyte membranes is inhibited by pertussis, but not cholera, toxin and by a variety of guanine nucleotides. In the absence of nucleotide, we observed a single class of sites with a Kd of 17 pM. The presence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) did not alter this affinity but did result in a concentration-dependent decrease in the number of binding sites. Surprisingly, we did not observe the concomitant appearance of a low affinity state implying that, if such a state exists, its affinity is below our limit of detection (5 nM). The receptor and G protein retained their functional interaction following solubilization of the membrane in digitonin. In the absence of nucleotide, we observed a single class of sites with a Kd of 28 pM. Addition of GTP gamma S suppressed binding, and, as was found in membranes, this inhibition is due almost entirely to a decrease in the number of sites. Again we failed to detect the appearance of a lower affinity state. Gel filtration studies of the detergent-solubilized receptor and receptor-C5a complexes indicate that the receptor is precoupled to G protein in the absence of ligand (C5a).

Binding Sites↗
PubMed1985

Identification of the polymorphonuclear leukocyte C5a receptor.

The peptide C5a is thought to play an important role in the inflammatory response primarily through its action on the polymorphonuclear leukocyte (PMN). The receptor for C5a on human PMN has now been identified by affinity labeling. Cross-linking 125I-C5a to intact PMN with disuccinimidyl suberate produced a species that had a molecular mass on sodium dodecyl sulfate gels of 5.2 X 10(4) daltons. We believe this species represents a complex between C5a and its receptor for the following reasons. The band is eliminated if the cross-linking experiment is performed in the presence of a large excess of unlabeled C5a, but is unaffected by the presence of nonspecific protein or the chemotactic factors N-formyl-Met-Leu-Phe and leukotriene B4. The 5.2 X 10(4)-dalton species is not observed if the cross-linker is omitted. Finally, the dose-response curves for the inhibition of binding of 125I-C5a by unlabeled C5a and the inhibition of cross-linking are similar. Subtraction of the molecular mass of C5a from that of the complex gives a molecular mass for the binding moiety of the C5a receptor of 4.0 X 10(4) daltons.

Binding, Competitive↗