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At least 19 recordsLinked to original sources
PubMed1992

Complement activation and polymorphonuclear neutrophil leukocyte elastase in sepsis. Correlation with severity of disease.

Complement activation is necessary for an adequate immune and inflammatory response to infections. Activation releases anaphylatoxins that cause vasodilation, increase vascular permeability, and trigger release of polymorphonuclear neutrophil leukocyte (PMN) lysosomal enzyme and oxygen radicals. Under normal circumstances, an orderly progression of such events has a beneficial antimicrobial effect. The same mechanism, however, when uncontrolled, may damage host tissues. To provide information about the clinical importance of such events in sepsis, different complement parameters (C3, C4, and the desarginated forms of C3a [C3a(des)-Arg] and C5a [C5a(des)-Arg]), PMN elastase, and malondialdehyde (a by-product of membrane peroxidation by oxygen radicals) were measured daily in 26 septic patients and correlated with two objectively assessed and previously validated severity scores (acute physiology and chronic health evaluation [APACHE II] and Sepsis Severity Score [SSS]). Nonsurvivors (n = 12) had significantly greater and longer lasting complement activation than that in survivors, as reflected by higher levels of catabolic peptides (C3a(des)-Arg) and lower levels of native proteins (C3 and C4). C3a(des)-Arg, C3, C4, and the C3a(des)-Arg-C3 ratio were correlated with Sepsis Severity Scores. Polymorphonuclear neutrophil leukocyte elastase levels were higher in nonsurvivors and were correlated with C3a(des)-Arg and the C3a(des)-Arg-C3 ratio. Malondialdehyde levels were significantly higher in all patients than in controls, without, however, any relationship to severity of disease or clinical outcome. Since the higher and more persistent the complement activation and polymorphonuclear neutrophil leukocyte stimulation, the worse the patient's prognosis, we conclude that these mechanisms may be important in the clinical development of sepsis.

Adult↗
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↗
PubMed1992

Vascular occlusion of the retina: an experimental model. I. Leukocyte aggregates.

A new technique was developed to study the pathogenesis of retinal arteriolar occlusion in the pig. Using a catheter with its tip in front of the exit of the ophthalmic artery, autologous microparticles could be injected directly into the retinal arterial system. These microparticles were C5a-des-Arg stimulated leukocytes, which were aggregated to pellets of different sizes ranging from 0.26 to 1.0 mm. For better adhesion of the aggregates to the endothelium, endothelial damage was induced by the injection of additional substances, including methomidate and endothelial antibodies. In a further series of experiments systemic hypoxemic conditions were created over several hours prior to injection of the leukocyte aggregates. This resulted in cotton-wool spots and arterial branch occlusions with retinal edema. Furthermore, retinal hemorrhages occurred. This experimental model seems to be appropriate for mimicking retinal arteriolar occlusion syndromes.

Animals↗
PubMed1988

Anaphylatoxin-induced release of histamine from in vitro perfused guinea pig kidney.

The effect of porcine C5a des Arg and C3a, given as a bolus injection, in the isolated constant flow pump-perfused guinea-pig kidney was investigated. Only C5a des Arg showed activity which was manifested by a dose-dependent increase in perfusion pressure (PP, due to vasoconstriction) and histamine release. Although histamine release was substantial, it alone could not account for the increase in PP. The two more likely causes are a direct vasoconstrictor effect and the release of other mediators.

Anaphylatoxins↗
PubMed1992

Production of monoclonal antibodies against the human anaphylatoxin C5a des Arg and their application in the neoepitope-specific sandwich-ELISA for the quantification of C5a des Arg in plasma.

A panel of ten murine monoclonal antibodies (MAbs) was raised against the human anaphylatoxin C5a des Arg. The MAbs were shown to abrogate or significantly inhibit the chemotactic activity in zymosan activated serum. MAb 4A2E10E2 and MAb 3G3C4 were used as capture and detecting antibody, respectively, in a sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of C5a des Arg. This ELISA was shown to be very sensitive (detection limit 20 pg/ml) and could be applied directly to plasma/serum samples. The lack of interference by plasma components, in particular C5, suggested specificity for an epitope on C5a (des Arg) which is concealed in native C5 and exposed on the activation fragment only, i.e., a 'neoepitope'. The mean C5a des Arg level in EDTA-plasma from 25 healthy individuals assessed at a 1/20 dilution was 11.2 ng/ml (SD 3.4; range 6.4-16.8 ng/ml). The applicability of the assay was investigated in patients treated with haemodialysis using different membranes. Markedly elevated plasma levels of C5a des Arg were found in blood returning from the dialyzer following contact with cuprophane membranes.

Animals↗
PubMedDate not supplied

C5a/C5ades-Arg-induced increase in blood pressure in the guinea pig: role of thromboxane.

Cleavage of the complement protein C5 by activation of the complement system yields a low molecular weight fragment C5a. Knowledge of the alterations in blood pressure induced by C5a as well as the mediators responsible for the blood pressure changes may provide information concerning the potential role of C5a in the adverse hemodynamic responses associated with complement activation. The purpose of this study was to characterize changes in mean arterial pressure in the guinea pig after intravenous challenge with a combination of guinea pig C5a plus C5a(des-Arg) (C5a/C5a(des-Arg)) and determine the mediators responsible for the transient increase in blood pressure which was observed. Mean arterial pressure was monitored in mechanically ventilated pentobarbital-anesthetized guinea pigs. Intravenous injection of C5a/C5a(des-Arg) consistently caused a marked but transient rise in blood pressure. A transient hypotensive response was also seen with injection of markedly higher doses of guinea pig C5a/C5a(des-Arg). Various pharmacological antagonists were used to determine the mediators responsible for the increase in blood pressure induced by guinea pig C5a/C5a(des-Arg). We found that the LTD4 antagonist L-649,923 did not inhibit the transient rise in blood pressure. However, the cyclooxygenase inhibitor indomethacin inhibited the C5a/C5a(des-Arg)-induced pressor response as did the thromboxane synthetase inhibitor U-63557A and the thromboxane receptor antagonist SQ 29,548. In addition, the C5a/C5a(des-Arg)-induced pressor response was not inhibited by the H1 antagonist pyrilamine, but was inhibited in part by the alpha-adrenergic antagonist phentolamine. Also, the response was reduced in animals depleted of circulating platelets or white blood cells. Thus, the results of our studies suggest that intravenously injected guinea pig C5a/C5a(des-Arg) causes release of the vasoconstrictor thromboxane, most likely from circulating white blood cells or platelets, resulting in a transient rise in blood pressure in the guinea pig. In addition, release of catecholamines may contribute to the pressor response observed.

Adrenergic alpha-Antagonists↗
PubMed1990

Effects of proinflammatory mediators on canine neutrophil chemotaxis and aggregation.

The present study investigated in vitro the qualitative and quantitative neutrophil function-activating properties of the most important non-cytokine participants in the acute inflammatory cell response. Apart from the results obtained with leukotriene (LT) D4, similar qualitative relationships were found for the several mediators tested regarding effects on neutrophil migration and aggregation. Thus, LTB4, PAF-acether and zymosan-activated serum all activated both functions, while f-Met-Leu-Phe had no effect. In all cases, the half-maximal eliciting concentration (EC50) for induction of chemotaxis was much lower than for aggregation, indicating that high and low receptor affinity responses were being studied, respectively. LTD4 induced modest aggregation, but was virtually without effect on migration. Using PAF-acether as stimulus, the mechanism of aggregation was studied in more detail. The results indicate that LTB4, PAF-acether, the complement-split products C5a/C5a desArg, and perhaps LTD4, may play a role as stimulants of neutrophil functions in inflammatory processes in vivo.

Analysis of Variance↗
PubMed1991

FUT-175 as a potent inhibitor of C5/C3 convertase activity for production of C5a and C3a.

We examined the inhibitory effect of FUT-175 on the C3/C5 convertase activity of the cobra venom factor-derived enzyme CVF,Bb by measuring C5b6-mediated reactive lysis of unsensitized guinea pig erythrocytes and by measuring directly the released fragments C3-des-Arg and C5a-des-Arg. In this study, we showed that the concentration of 4.5 X 10(-6) M of FUT-175 caused 50% inhibition of C5 convertase activity of CVF,Bb in reactive hemolysis assays, and that 4.0 X 10(-6) M FUT-175 caused 50% inhibition of the production of C3a and C5a generated by the C3/C5 convertase activity of CVF,Bb.

Animals↗
PubMed1990

Three-dimensional structure of porcine C5adesArg from 1H nuclear magnetic resonance data.

Two-dimensional nuclear magnetic resonance spectra of porcine C5adesArg (73 residues) have been used to construct a list of 34 hydrogen bonds, 27 dihedral angle constraints, and 151 distance constraints, derived from nuclear Overhauser effect data. These constraints were used in restrained molecular dynamics calculations on residues 1-65 of C5a, starting from a folded structure modeled on the crystal structure of a homologous protein, C3a. Forty-one structures have been calculated, which fall into three similar families with few violations of the imposed constraints. Structures in the most populated family have a root-mean-square deviation from the average structure of 1.02 A for the C alpha atoms, with good definition of the internal residues. There is good agreement between the calculated structures and other nuclear magnetic resonance data. The structure is very similar to that recently reported for human C5a [Zuiderweg et al. (1989) Biochemistry 28, 172-185]. Some biological implications of these structures are discussed.

Amino Acid Sequence↗
PubMed1990

Hypersensitivity reactions during haemodialysis: role of complement fragments and ethylene oxide antibodies.

IgE-antibodies against ethylene oxide (EtO) and activation of the complement system have been suspected as causative factors for acute hypersensitivity reactions at the onset of haemodialysis. The present study was conducted to determine which of the two mechanisms is mainly responsible for the occurrence of reactions. According to clinical criteria, 13 of the 129 patients studied were identified as having suffered from at least one episode of acute hypersensitivity. Seven of them experienced severe symptoms, including five with elevated circulating serum EtO antibodies. Of the remaining six patients who experienced moderate symptoms, only one had elevated serum EtO antibodies, and of the 114 patients not exhibiting symptoms, two had enhanced serum concentrations of EtO antibodies. In-vivo complement activation was determined during haemodialysis by measurement of both C3adesarg and C5adesarg fragments. No differences in the onset levels and the degree of complement activation were found between patients suffering from severe, moderate, or no hypersensitivity reactions. In-vitro activation of the complement cascade by zymosan, measured in plasma samples obtained from patients prior to dialysis, revealed no differences in the extent of C3adesarg generation. Finally, we determined the activity of the C3a- and C5a-inactivating enzyme, carboxypeptidase N1 (CN1), in plasma samples of patients both with and without hypersensitivity reactions. No difference in the CN1 activity between the two groups of patients was found. These data lead us to conclude that sensitisation to ethylene oxide is responsible for the majority of severe hypersensitivity reactions in haemodialysis patients. However, a small subgroup of patients having anaphylactoid reactions displayed neither elevated serum EtO antibodies with excessive complement activation nor a tendency towards reduced inactivation of anaphylatoxins.

Adolescent↗
PubMed1989

Analysis of the interaction of human C5a and C5a des Arg with human monocytes and neutrophils: flow cytometric and chemotaxis studies.

C5a and C5a des Arg are potent complement-derived mediators that bind receptors on peripheral blood leukocytes and tissue-specific cellular elements to elicit and amplify inflammatory and immunomodulatory reactions. To study the interactions of C5a and C5a des Arg with these cells, fluorescein conjugates of these ligands were prepared by a new technique and their binding to monocytes, neutrophils, platelets, and endothelial cells was studied with flow cytometry. Fluoresceinated C5a produced neutrophil myeloperoxidase release and chemotaxis and also bound rabbit anti-C5a antibody much like native anaphylatoxin; likewise, fluoresceinated C5a des Arg demonstrated retention of biologic and antigenic activities. Both fluorescein-conjugated C5a and C5a des Arg bound to monocytes and neutrophils in a concentration-dependent, saturable, and homogeneous manner, but 10- to 15-fold higher concentrations of C5a des Arg were required to attain saturable binding of these leukocytes. Ligand binding was specifically inhibited by native purified human C5a in a concentration-dependent manner, while it was unaffected by C3a or N-formyl-methionyl-leucyl-phenylalanine-lysine. There was no evidence of a C5a receptor-negative subpopulation of monocytes or neutrophils. Moreover, comparative binding experiments with leukocytes from multiple normal volunteers showed that a greater percentage of monocytes than neutrophils bound C5a at less than saturable concentrations of ligand (P less than 0.05, 0.5 to 5.0 nM). A representative half-maximal binding of fluorescein-conjugated C5a (C5a des Arg) binding to monocytes and neutrophils was 1.2 nM (30 nM) and 2.6 nM (68 nM), respectively. In contrast, fluorescein-conjugated C5a did not specifically bind to human platelets or umbilical vein endothelial cells.

Blood Platelets↗
PubMed1991

The priming effects of the products of stimulated mononuclear cells on the response of neutrophils to C5a des arg.

Certain recombinant human cytokines have been shown to enhance polymorphonuclear leucocyte (PMN) responses to subsequent stimulation. Mononuclear cells (MNC) from normal healthy individuals were stimulated for 5 h with 1 micrograms/ml bacterial lipopolysaccharide (LPS) in order to induce production and secretion of inflammatory cytokines into the surrounding medium. These mononuclear cell conditioned media (MNCM) were then used to prime PMN isolated from healthy volunteers. Preincubating the PMN with MNCM for 15 min at 4 degrees C followed by washing and warming to 37 degrees C caused a 344% increase (n = 26) in the rate of superoxide anion production in response to zymosan-activated serum (ZAS), a source of C5a des arg. This effect could not be reproduced with recombinant human forms of interleukin 1 beta (Il-1 beta) or granulocyte-macrophage-colony stimulating factor (GM-CSF), although, with the latter, there was some effect when the preincubation stage was carried out for 60 min at 37 degrees C. Only recombinant human tumour necrosis factor-alpha (rh-TNF-alpha) gave a similar PMN priming effect to that seen with MNCM. This effect could not be reversed by washing away either the MNCM or rh-TNF-alpha. The priming effect could be markedly reduced (74.8%, n = 6) by employing the use of polyclonal antibody to TNF-alpha in the preincubation step; assaying for TNF-alpha in these MNCMs showed that the degree of priming corresponded to the amount of TNF-alpha present. Rh-TNF-alpha alone appeared to have very little direct stimulatory effect on respiratory burst activity. The results show that TNF-alpha produced by LPS stimulated MNC after 5 h binds to a PMN surface receptor in the cold and warming of the cells to 37 degrees C allows for an immediate and dramatic response to ZAS stimulation. This suggests that TNF-alpha is the important cytokine upregulating PMN responses to other physiological mediators, including C5a des arg during the early phases of an inflammatory reaction.

Cells, Cultured↗
PubMed1985

Inflammatory oedema induced by synergism between calcitonin gene-related peptide (CGRP) and mediators of increased vascular permeability.

The potent vasodilator calcitonin gene-related peptide (CGRP, human synthetic), when mixed with histamine and injected intradermally in the rabbit, induced a marked potentiation of local oedema. CGRP also potentiated oedema induced by other mediators of increased microvascular permeability in the rabbit; bradykinin, platelet-activating factor (Paf), C5a des Arg, N-formylmethionyl-leucyl-phenylalanine (FMLP) and leukotriene B4 (LTB4). Substance P alone, or mixtures of substance P and CGRP, failed to induce oedema in rabbit skin. In rat skin, however, substance P induced oedema and this was potentiated by CGRP. CGRP had a protracted potentiating action following intradermal injection in the rabbit. The time for half loss of activity for CGRP was 40.1 +/- 7.5 min compared to 18 +/- 1 min for prostaglandin E2 (PGE2). No loss of potentiating activity was detected after incubation of CGRP in rabbit plasma or blood for 60 min. We postulate that endogenous CGRP, if released locally from nerve endings, could have a marked enhancing effect on oedema induced by other mediators in an inflammatory reaction.

Animals↗
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

Role of granulocytes and C5a in myocardial response to zymosan-activated serum.

Although previous studies have demonstrated that complement (C)5a causes myocardial ischemia and mechanical dysfunction, the cardiac response of endogenously produced C5a and C5a des-Arg in zymosan-activated serum (ZAS) and the critical role of granulocytes in this process are poorly understood. Therefore, we compared the coronary and cardiac effects of ZAS and purified C5a and investigated the role of leukocyte adhesion-promoting receptors (i.e., CD11/CD18). Like purified C5a, ZAS (0.5 ml) significantly reduced coronary artery blood flow and regional segment shortening, whereas coronary venous granulocyte concentration and myocardial lactate extraction were significantly decreased. A monoclonal antibody (MoAb) to C5a/C5a des-Arg attenuated ZAS-induced cardiac alterations. Three minutes of continuous infusion of C5a or ZAS induced sustained decreases in coronary venous granulocyte concentrations, although coronary flow and segment shortening returned to control levels after 2 min. Another MoAb, IB4, directed against CD18, significantly inhibited ZAS-induced granulocyte extraction and associated cardiac effects. Thus, cardiac dysfunction occurs after activation of the complement cascade with zymosan resulting in extraction of granulocytes mediated by the CD18 adherence glycoprotein. Furthermore, intramyocardial retention of granulocytes appears necessary for the initial and full ZAS-induced cardiac dysfunction.

Anaphylatoxins↗
PubMed1990

The influence of the geometry of the dialyzer and the composition of the dialysate in activating the complement system.

During hemodialysis there is a complex interaction between the patient and the extracorporeal circuit that activates the complement system, among others. To better understand the influence of the dialyzer geometry and the dialysate composition, we compared hollow fiber versus parallel plate dialyzers and acetate versus bicarbonate dialysates and their role in the production of C3a, C4a and C5a. There was no significant difference in the plasmatic levels of these anaphylotoxins and their des-Arg derivates, as measured by RIA, in either dialyzer. The same was true when the dialysate in question had a different composition. We thus concluded that neither the geometric configuration of the dialyzer nor the composition of the dialysate influence their biocompatibility as regards the activation of the complement system, and that the differences that have been described shall have to be explained in another manner or assessed by methods other than those used in this study.

Adult↗