RESUMO
Thrombin activation of C5 connects thrombosis to inflammation. Complement research in whole blood ex vivo necessitates anticoagulation, which potentially interferes with the inflammatory modulation by thrombin. We challenged the concept of thrombin as an activator of native C5 by analyzing complement activation and C5 cleavage in human whole blood anticoagulated with Gly-Pro-Arg-Pro (GPRP), a peptide targeting fibrin polymerization downstream of thrombin, allowing complete endogenous thrombin generation. GPRP dose-dependently inhibited coagulation but allowed for platelet activation in accordance with thrombin generation. Spontaneous and bacterial-induced complement activation by Escherichia coli and Staphylococcus aureus, analyzed at the level of C3 and C5, were similar in blood anticoagulated with GPRP and the thrombin inhibitor lepirudin. In the GPRP model, endogenous thrombin, even at supra-physiologic concentrations, did not cleave native C5, despite efficiently cleaving commercially sourced purified C5 protein, both in buffer and when added to C5-deficient serum. In normal serum, only exogenously added, commercially sourced C5 was cleaved, whereas the native plasma C5 remained intact. Crucially, affinity-purified C5, eluted under mild conditions using an MgCl2 solution, was not cleaved by thrombin. Acidification of plasma to pH ≤ 6.8 by hydrochloric or lactic acid induced a C5 antigenic change, nonreversible by pH neutralization, that permitted cleavage by thrombin. Circular dichroism on purified C5 confirmed the structural change during acidification. Thus, we propose that pH-induced conformational change allows thrombin-mediated cleavage of C5 and that, contrary to previous reports, thrombin does not cleave plasma C5 in its native form, suggesting that thrombin cleavage of C5 may be restricted to certain pathophysiological conditions.
Assuntos
Complemento C5 , Trombina , Coagulação Sanguínea , Ativação do Complemento , Fibrina , HumanosRESUMO
The mechanism of action of recombinant IgG2/4 antibodies involves blocking of their target without the induction of effector functions. Examples are eculizumab (Soliris®), which is used clinically to block complement factor C5, as well as anti-human CD14 (r18D11) and anti-porcine CD14 (rMIL2) produced in our laboratory. So far, no proper IgG2/4 control antibody has been available for controlled validation of IgG2/4 antibody functions. Here, we describe the design of a recombinant control antibody (NHDL), which was generated by combining the variable light (VL) and heavy (VH) chains from two unrelated specificities. NHDL was readily expressed and purified as a stable IgG2/4 antibody, and showed no detectable specificity toward any putative antigen present in human or porcine blood. The approach of artificial VL/VH combination may be adopted for the design of other recombinant control antibodies.
Assuntos
Anticorpos Monoclonais/genética , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Proteínas Recombinantes de Fusão/genética , Animais , Anticorpos Monoclonais/metabolismo , Terapia Biológica , Proteínas Sanguíneas/metabolismo , Epitopos/metabolismo , Humanos , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Camundongos , Placebos , Engenharia de Proteínas , SuínosRESUMO
The aim of the study was to investigate the role of complement factor 5 (C5) in reactions elicited by plasma separation using blood from a C5-deficient (C5D) individual, comparing it to C5-deficient blood reconstituted with C5 (C5DR) and blood from healthy donors. Blood was circulated through an ex vivo plasma separation model. Leukocyte CD11b expression and leukocyte-platelet conjugates were measured by flow cytometry during a 30-min period. Other markers were assessed during a 240-min period. Granulocyte and monocyte CD11b expression did not increase in C5D blood during plasma separation. In C5DR samples granulocytes CD11b expression, measured by mean fluorescence intensity (MFI), increased from 10481 ± 6022 (SD) to 62703 ± 4936, and monocytes CD11b expression changed from 13837 ± 7047 to 40063 ± 713. Granulocyte-platelet conjugates showed a 2.5-fold increase in the C5DR sample compared to the C5D sample. Monocyte-platelet conjugates increased independently of C5. In the C5D samples, platelet count decreased from 210 × 109 /L (201-219) (median and range) to 51 × 109 /L (50-51), and C3bc increased from 14 CAU/mL (21-7) to 198 CAU/mL (127-269), whereas TCC formation was blocked during plasma separation. In conclusion, up-regulation of granulocyte and monocyte CD11b during plasma separation was C5-dependent. The results also indicate C5 dependency in granulocyte-platelet conjugates formation.
Assuntos
Transtornos das Proteínas Sanguíneas/metabolismo , Antígeno CD11b/metabolismo , Complemento C5/deficiência , Granulócitos/metabolismo , Monócitos/metabolismo , Plasma/química , Plaquetas/metabolismo , Transtornos das Proteínas Sanguíneas/sangue , Transtornos das Proteínas Sanguíneas/genética , Antígeno CD11b/genética , Feminino , Humanos , MasculinoRESUMO
We report a pediatric patient with atypical hemolytic uremic syndrome due to a C3 gain-of-function mutation diagnosed in infancy. She was treated from the start with a constant dose of 300 mg eculizumab every second week from the onset and followed by routine complement analyses for six years. Her complement system was completely inhibited and the dose interval was prolonged from 2 to 3 weeks without alteration of the dose and the complement activity continued to be completely inhibited. Blood samples taken immediately before, immediately after, and between eculizumab doses were analyzed for eculizumab-C5 complexes and percentage of total complement activity, using the Wieslab® test, and compared to a pool of sera from 20 healthy controls. The patient exhibited complete complement inhibition at all three time-points and had no free circulating C5 suggesting there was complete binding to eculizumab. She has now been treated for six years with full complement blockade. We suggest therefore that analysis of complement activity using the Wieslab® test is useful for evaluating the effect of eculizumab when dose intervals are prolonged.
RESUMO
The complement system has obtained renewed clinical focus due to increasing number of patients treated with eculizumab, a monoclonal antibody inhibiting cleavage of C5 into C5a and C5b. The FDA approved indications are paroxysmal nocturnal haemoglobinuria and atypical haemolytic uremic syndrome, but many other diseases are candidates for complement inhibition. It has been postulated that eculizumab does not inhibit C5a formation in vivo, in contrast to what would be expected since it blocks C5 cleavage. We recently revealed that this finding was due to a false positive reaction in a C5a assay. In the present study, we identified expression of a neoepitope which was exposed on C5 after binding to eculizumab in vivo. By size exclusion chromatography of patient serum obtained before and after infusion of eculizumab, we document that the neoepitope was exposed in the fractions containing the eculizumab-C5 complexes, being positive in this actual C5a assay and negative in others. Furthermore, we confirmed that it was the eculizumab-C5 complexes that were detected in the C5a assay by adding an anti-IgG4 antibody as detection antibody. Competitive inhibition by anti-C5 antibodies localized the epitope to the C5a moiety of C5. Finally, acidification of C5, known to alter C5 conformation, induced a neoepitope reacting identical to the one we explored, in the C5a assays. These data are important for interpretation of complement analyses in patients treated with eculizumab.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Ativação do Complemento/efeitos dos fármacos , Complemento C5/imunologia , Complemento C5a/imunologia , Complemento C5b/imunologia , Anticorpos Monoclonais Humanizados/metabolismo , Síndrome Hemolítico-Urêmica Atípica/sangue , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Síndrome Hemolítico-Urêmica Atípica/imunologia , Cromatografia em Gel , Ativação do Complemento/imunologia , Complemento C5/metabolismo , Complemento C5a/metabolismo , Complemento C5b/metabolismo , Inativadores do Complemento/metabolismo , Inativadores do Complemento/uso terapêutico , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Epitopos/metabolismo , Hemoglobinúria Paroxística/sangue , Hemoglobinúria Paroxística/tratamento farmacológico , Hemoglobinúria Paroxística/imunologia , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Avaliação de Resultados em Cuidados de Saúde , Ligação Proteica/imunologiaRESUMO
RATIONALE: Antiphospholipid syndrome (APS) in pregnancy may trigger the life-threatening catastrophic antiphospholipid syndrome (CAPS). Complement activation is implicated in the pathogenesis, and inhibition of complement factor C5 is suggested as an additional treatment option. PATIENT CONCERNS, DIAGNOSIS AND INTERVENTIONS: We present a pregnant patient treated with the C5-inhibitor eculizumab due to high risk of developing devastating APS-related complications. The complement inhibitory effects of the treatment were examined both in the patient and the premature infant. OUTCOMES: Complement activity in the mother recovered considerably faster than anticipated; however, no new thrombosis or CAPS developed during the last week of pregnancy or postpartum. Blood sampling from the umbilical vein and artery, and from the infant after delivery showed low complement activity; however, only 0.3% of the eculizumab concentration detected in the mother, consistent with low placental passage of eculizumab. LESSONS: The data underscore the importance of close monitoring of complement inhibition and individualizing dosage regimens in pregnant patients receiving eculizumab. We document how traditional functional complement activity tests cannot assess the effect of eculizumab in premature infants due to the very low levels of complement factors detected in this infant born in gestational week 33. Only trace amounts of eculizumab passed the placenta. In conclusion, complement C5 inhibition might be a safe candidate treatment option for APS during pregnancy and delivery, and additionally, enables prolongation of pregnancy with important weeks.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Síndrome Antifosfolipídica/tratamento farmacológico , Complemento C5/antagonistas & inibidores , Complicações Hematológicas na Gravidez/tratamento farmacológico , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/farmacocinética , Cesárea , Proteínas do Sistema Complemento/metabolismo , Feminino , Humanos , Recém-Nascido , Gravidez , Adulto JovemRESUMO
BACKGROUND: Fulminant meningococcal sepsis, characterized by overwhelming innate immune activation, mostly affects young people and causes high mortality. This study aimed to investigate the effect of targeting two key molecules of innate immunity, complement component C5, and co-receptor CD14 in the Toll-like receptor system, on the inflammatory response in meningococcal sepsis. METHODS: Meningococcal sepsis was simulated by continuous intravenous infusion of an escalating dose of heat-inactivated Neisseria meningitidis administered over 3 h. The piglets were randomized, blinded to the investigators, to a positive control group (n = 12) receiving saline and to an interventional group (n = 12) receiving a recombinant anti-CD14 monoclonal antibody together with the C5 inhibitor coversin. RESULTS: A substantial increase in plasma complement activation in the untreated group was completely abolished in the treatment group (p = 0.006). The following inflammatory mediators were substantially reduced in plasma in the treatment group: Interferon-γ by 75% (p = 0.0001), tumor necrosis factor by 50% (p = 0.01), Interleukin (IL)-8 by 50% (p = 0.03), IL-10 by 40% (p = 0.04), IL-12p40 by 50% (p = 0.03), and granulocyte CD11b (CR3) expression by 20% (p = 0.01). CONCLUSION: Inhibition of C5 and CD14 may be beneficial in attenuating the detrimental effects of complement activation and modulating the cytokine storm in patients with fulminant meningococcal sepsis.
RESUMO
Complement C5 inhibitor eculizumab treatment in atypical hemolytic uremic syndrome is effective, but associated with high costs. Complement inhibition monitoring in these patients has not been standardized. In this study we evaluated novel functional assays for application in routine follow-up. We documented that the Wieslab® complement screen assay showed a sensitivity of 1-2% of C5 activity by adding purified C5 or normal human serum to a C5 deficient serum. All the patient samples obtained during the treatment course, were completely blocked for terminal complement pathway activity for up to four weeks after the eculizumab infusion. Levels of complexes between eculizumab and C5 were inversely correlated to the complement activity (p=0.01). Moreover, titrating serum from eculizumab-treated patients into normal serum revealed that eculizumab was present in excess up to four weeks after infusion. Thus, we demonstrate sensitive, reliable and easy-performed assays which can be used to design individual eculizumab dosage regimens.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Inativadores do Complemento/uso terapêutico , Monitoramento de Medicamentos/instrumentação , Kit de Reagentes para Diagnóstico , Adolescente , Adulto , Síndrome Hemolítico-Urêmica Atípica/imunologia , Criança , Pré-Escolar , Ativação do Complemento/imunologia , Relação Dose-Resposta a Droga , Monitoramento de Medicamentos/métodos , Feminino , Humanos , Masculino , Reprodutibilidade dos Testes , Adulto JovemRESUMO
Eculizumab is a humanized IgG2/4 chimeric anti-complement C5 antibody used to treat patients with paroxysmal nocturnal hemoglobinuria (PNH) or atypical hemolytic uremic syndrome. The aim of this study was to evaluate whether or not the complement activity in newborns from pregnant women who receive eculizumab is impaired. A novel eculizumab-C5 complex (E-C5) specific assay was developed and revealed that two newborns carried only 6-7% of the E-C5 detected in their eculizumab-treated PNH mothers. Serum from the pregnant women completely lacked terminal complement pathway activity, whereas the complement activity in the serum of the newborns was completely normal. Data from the pregnant women and their newborns were compared with that of healthy age-matched female controls and healthy newborns, as well as a non-treated pregnant woman with PNH and her newborn. These all showed normal complement activity without detectable E-C5 complexes. Furthermore, absence of eculizumab or E-C5 in the newborn could not be explained by lack of eculizumab binding to the neonatal Fc receptor (FcRn), as eculizumab bound strongly to the receptor in vitro. In conclusion, despite binding to FcRn neither eculizumab nor E-C5 accumulates in fetal plasma, and eculizumab treatment during pregnancy does not impair the complement function in the newborn.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Ativação do Complemento/efeitos dos fármacos , Proteínas do Sistema Complemento/imunologia , Exposição Materna , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Complexo Antígeno-Anticorpo/sangue , Complexo Antígeno-Anticorpo/imunologia , Estudos de Casos e Controles , Complemento C5/antagonistas & inibidores , Complemento C5/imunologia , Feminino , Hemoglobinúria Paroxística/sangue , Hemoglobinúria Paroxística/tratamento farmacológico , Hemoglobinúria Paroxística/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Recém-Nascido , Gravidez , Ligação Proteica/imunologia , Receptores Fc/metabolismoRESUMO
The importance of the complement system in clinical medicine has become evident during the last decades and complement therapeutics has now reached the clinic. Thus, there is an increased interest in and need for assays to evaluate complement activity and dysfunction. Pathologically increased complement activation can indirectly be evaluated by quantification of complement components, but in order to exactly measure such activation, assays for quantification of products formed during activation are required. Progress in this field is hampered by lack of standardization. Therefore, members of the International Complement Standardization Committee, a joint initiative of the International Complement Society and the International Union of Immunological Societies (IUIS), prepared a defined standard for application in assays for complement activation products. We here report on the production and properties of this International Complement Standard #2 (ICS#2). ICS#2 was made from a pool of sera from healthy blood donors (ICS#1) that was activated with a combination of heat-aggregated IgG and zymosan, and subsequently stabilized by adding EDTA and nafamostat mesylate. The protocol was optimized to make the standard applicable in the following activation product assays: C1rs-C1-inhibitor complexes, C4a, C4bc, C4d, Bb, C3bBbP, C3a, C3bc, C3dg, C5a and the soluble terminal C5b-9 complement complex (SC5b-9, TCC). ICS#2 was defined as containing 1000 complement activation units (CAU)/mL for all activation products measured. All activation products were stable after 10 times thawing and freezing and most of the activation products were stable during storage at 4°C for up to 21 days. ICS#2 was produced large-scale and is considered a valuable tool for standardization, calibration and reference control for complement activation assays, providing the necessary prerequisite for quality assessments between complement laboratories.
Assuntos
Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Ensaio de Imunoadsorção Enzimática/normas , Proteínas do Sistema Complemento/uso terapêutico , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Padrões de ReferênciaRESUMO
The initial interaction of Gram-negative bacteria with erythrocytes and its implications on leukocyte phagocytosis and oxidative burst in human whole blood were examined. Alexa-labeled Escherichia coli, wild-type H44/76 N. meningitidis and the H44/76lpxA lipopolysaccharide (LPS)-deficient mutant were incubated with whole blood using lepirudin as anticoagulant which has no adverse effects on complement. Bacteria free in plasma, bound to erythrocytes or phagocytized by granulocytes and monocytes were quantified using flow cytometry. The effects of the C3 inhibitor compstatin, a C5a receptor antagonist (C5aRa) and a complement receptor 1 (CR1)-blocking antibody (3D9) were examined. Most bacteria (80%) immediately bound to erythrocytes. The binding gradually declined over time, with a parallel increase in phagocytosis. Complement inhibition with compstatin reduced erythrocyte binding and bacterial C3 opsonization. In contrast, the C5aRa efficiently reduced phagocytosis, but did not affect the binding of bacteria to erythrocytes. The anti-CR1 blocking mAb dose-dependently reduced bacterial binding to erythrocytes to nil, with subsequent increased phagocytosis and oxidative burst. LPS had no effect on these processes since similar results were obtained using an LPS-deficient N. meningitidis mutant. In vivo experiments in a pig model of sepsis showed limited binding of bacteria to erythrocytes, consistent with the facts that erythrocyte CR1 receptors are absent in non-primates and that the bacteria were mainly found in the lungs. In conclusion, complement-dependent binding of Gram-negative bacteria to erythrocyte CR1 decreases phagocytosis and oxidative burst by leukocytes in human whole blood.
Assuntos
Eritrócitos/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Leucócitos/imunologia , Fagocitose/imunologia , Receptores de Complemento 3b/metabolismo , Animais , Separação Celular , Proteínas do Sistema Complemento , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Eritrócitos/metabolismo , Escherichia coli/imunologia , Escherichia coli/metabolismo , Citometria de Fluxo , Imunofluorescência , Infecções por Bactérias Gram-Negativas/metabolismo , Humanos , Leucócitos/metabolismo , Neisseria meningitidis/imunologia , Neisseria meningitidis/metabolismo , Explosão Respiratória , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse/imunologia , Sepse/metabolismo , SuínosRESUMO
BACKGROUND: Exposing blood to artificial surfaces results in an inflammatory response, including complement activation and cytokine release. The aim of this investigation was to study complement-dependency and independency in artificial surface-induced inflammation in human whole blood from a patient with a genetic deficiency of complement factor 5 (C5). METHODS: Whole blood from a C5-deficient patient, C5 protein reconstituted blood, and blood from a control subject was used. The complement inhibitor compstatin (C3 inhibitor) and a C5a receptor antagonist were used to block complement. Blood was circulated in closed loops of polyvinyl chloride tubing. Leukocyte CD11b expression and release of granule enzymes (myeloperoxidase, elastase, lactoferrin), cytokines (interleukins, chemokines, and growth factors; n = 27) as well as complement activation were measured after incubation. RESULTS: In C5-deficient blood, there was no formation of the terminal complement complex, as opposed to reconstituted or control blood. Release of granule enzymes was partly dependent on C3, revealed by a compstatin-dependent effect in C5-deficient blood, and partly C5a-dependent as evident from the reconstitution and control blood. The chemokines interleukin-8 and monocyte chemoattractant protein-1 were also highly complement dependent, the effect being C5a-mediated, whereas platelet-derived and vascular endothelial growth factors were partly complement dependent. Interferon-γ increased in a complement-independent manner, whereas the rest of the cytokines did not respond to the surface. Leukocyte expression of CD11b was only marginally increased in deficient blood exposed to the surface, whereas reconstitution induced a considerable, C5a-dependent increase, comparable with that of the control. CONCLUSIONS: The polyvinyl chloride surface induced a defined inflammatory response, which largely depended on C5.
Assuntos
Materiais Biocompatíveis/efeitos adversos , Complemento C5/deficiência , Complemento C5/imunologia , Inflamação/sangue , Inflamação/imunologia , Degranulação Celular/imunologia , Quimiocinas/sangue , Ativação do Complemento , Citocinas/sangue , Exposição Ambiental/efeitos adversos , Citometria de Fluxo , Humanos , Técnicas In Vitro , Neutrófilos/imunologiaRESUMO
Complement component C5 is crucial for experimental animal inflammatory tissue damage; however, its involvement in human inflammation is incompletely understood. The responses to gram-negative bacteria were here studied taking advantage of human genetic complement-deficiencies--nature's own knockouts--including a previously undescribed C5 defect. Such deficiencies provide a unique tool for investigating the biological role of proteins. The experimental conditions allowed cross-talk between the different inflammatory pathways using a whole blood model based on the anticoagulant lepirudin, which does not interfere with the complement system. Expression of tissue factor, cell adhesion molecules, and oxidative burst depended highly on C5, mediated through the activation product C5a, whereas granulocyte enzyme release relied mainly on C3 and was C5a-independent. Release of cytokines and chemokines was mediated to varying degrees by complement and CD14; for example, interleukin (IL)-1beta and IL-8 were more dependent on complement than IFN-gamma and IL-6, which were highly dependent on CD14. IL-1 receptor antagonist (IL-1ra) and IFN-gamma inducible protein 10 (IP-10) were fully dependent on CD14 and inversely regulated by complement, that is, complement deficiency and complement inhibition enhanced their release. Granulocyte responses were mainly complement-dependent, whereas monocyte responses were more dependent on CD14. Notably, all responses were abolished by combined neutralization of complement and CD14. The present study provides important insight into the comprehensive role of complement in human inflammatory responses to gram-negative bacteria.
Assuntos
Proteínas do Sistema Complemento/deficiência , Proteínas do Sistema Complemento/genética , Inflamação/genética , Inflamação/imunologia , Adolescente , Adulto , Estudos de Casos e Controles , Adesão Celular/imunologia , Ativação do Complemento , Complemento C2/deficiência , Complemento C2/genética , Complemento C5/deficiência , Complemento C5/genética , Escherichia coli/imunologia , Feminino , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Negativas/patogenicidade , Humanos , Imunidade Inata/genética , Técnicas In Vitro , Inflamação/etiologia , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Modelos Imunológicos , Monócitos/imunologia , Monócitos/microbiologia , Neisseria meningitidis/imunologia , Fagocitose , Explosão Respiratória/imunologia , Tromboplastina/biossínteseRESUMO
In an in vitro whole blood model of artificial surface-induced inflammation, we have studied the contribution of leukocyte populations in the synthesis of inflammatory mediators. This was done by depleting the blood of specific cell types using magnetic beads coated with monoclonal antibodies against leukocyte surface antigens. Synthesis of interleukin 8 (IL-8) was highly dependent on CD15+ cells and was reduced by 80% when these cells were removed from the blood. Correspondingly, IL-8 production showed a high correlation with the concentration of granulocytes (r = 0.77, p < 0.0001). Synthesis of monocyte chemoattractant protein 1 (MCP-1) was dependent on CD14+ cells and was reduced by 35% when these cells were removed from the blood. Correspondingly, MCP-1 production correlated with the concentration of monocytes (r = 0.39, p < 0.0001). Synthesis of leukotriene B4 (LTB4) was highly dependent on CD15+ cells and was reduced by 75% when these cells were removed from the blood. Correspondingly, LTB4 production correlated strongly with the granulocyte concentration (r = 0.54, p < 0.0001). As expected, complement activation was not affected by cell depletion and did not correlate with the concentration of any of the cell types. Thus, artificial surface-induced IL-8 and LTB4 synthesis was almost exclusively granulocyte dependent. However, MCP-1 synthesis was mainly a product of monocytes, although granulocytes and other subpopulations may partly contribute. (c) 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2005.
Assuntos
Quimiocina CCL2/biossíntese , Granulócitos/efeitos dos fármacos , Interleucina-8/biossíntese , Leucotrieno B4/biossíntese , Monócitos/efeitos dos fármacos , Cloreto de Polivinila/farmacologia , Ativação do Complemento , Granulócitos/citologia , Humanos , Técnicas Imunoenzimáticas , Técnicas In Vitro , Monócitos/citologiaRESUMO
BACKGROUND: Contact between blood and artificial surfaces induces an inflammatory response including activation of leukocytes and platelets, as well as complement and other plasma cascade systems. In the present study we investigated the roles of complement and surface modification in polyvinyl chloride-induced synthesis of eicosanoids (arachidonic acid metabolites). METHODS: Human whole blood was incubated in rotating loops of polyvinyl chloride or heparin-coated polyvinyl chloride tubing for 4 hours. Plasma concentrations of the eicosanoids leukotriene B4, prostaglandin E2 and thromboxane B2 were quantified. RESULTS: Polyvinyl chloride induced a substantial increase in leukotriene B4, prostaglandin E2, and thromboxane B2. Inhibition of complement activation by the complement factor 3 binding peptide compstatin or blockade of the complement factor 5a receptor with a specific antagonist significantly and specifically inhibited the synthesis of leukotriene B4, whereas thromboxane B2 and prostaglandin E2 synthesis were apparently complement independent. The increase in all three mediators was significantly reduced by the heparin coating. Indomethacin abolished the increase of the cyclooxygenase products prostaglandin E2 and thromboxane B2, but had no effect on the increase of the lipoxygenase product leukotriene B4, consistent with the specificity of indomethacin for the cyclooxygenase and confirming the specificity of complement inhibition. CONCLUSIONS: Polyvinyl chloride-induced increase in all three eicosanoids was attenuated by heparin coating, whereas complement inhibition selectively reduced the synthesis of leukotriene B4.
Assuntos
Proteínas Inativadoras do Complemento/efeitos dos fármacos , Dinoprostona/biossíntese , Heparina/farmacologia , Leucotrieno B4/biossíntese , Tromboxano B2/biossíntese , HumanosRESUMO
BACKGROUND: Contact between blood and artificial surfaces induces an inflammatory response including activation of leukocytes and platelets, as well as complement and other plasma cascade systems. In the present study we investigated the roles of complement and surface modification in polyvinylchloride-induced cytokine production. METHODS: Human whole blood was incubated in rotating loops of polyvinylchloride or heparin-coated polyvinylchloride tubing for 4 hours. Plasma concentrations of the cytokines tumor necrosis factor alpha, interleukin (IL) 1 beta, IL-6, IL-8, IL-10, and monocyte chemoattractant protein 1 (MCP-1) were quantified. RESULTS: Polyvinylchloride induced a substantial increase in IL-8 and MCP-1, which was abolished by cycloheximide, indicating that they were synthesized during incubation. Interleukin 8 synthesis was completely complement-dependent since it was abolished by neutralizing antibodies to factor D and complement factor 5, as well as by a complement factor 5a receptor antagonist. Monocyte chemoattractant protein 1 synthesis was reduced by approximately half the amount by the complement inhibitors. Heparin-coated polyvinylchloride efficiently prevented synthesis of both IL-8 and MCP-1. Addition of recombinant human complement factor 5a to blood incubated in heparin-coated polyvinylchloride restored IL-8 and MCP-1 production completely and partly, respectively. In contrast to IL-8 and MCP-1, tumor necrosis factor alpha, IL-1 beta, interleukin 6 and IL-10 increased only marginally. A minor but significant increase in IL-1 beta was complement-dependent, whereas a similar increase in IL-10 was completely prevented by heparin-coated polyvinylchloride. No significant changes were observed for tumor necrosis factor alpha and IL-6. CONCLUSIONS: Polyvinylchloride induced a marked increase in IL-8 and MCP-1, in contrast to a marginal increase in tumor necrosis factor alpha, IL-1 beta, IL-6, and IL-10. The increase in IL-8 and MCP-1 was prevented by heparin-coated polyvinylchloride. Interleukin 8 production was totally complement-dependent and mediated by complement factor 5a.
Assuntos
Anticoagulantes/farmacologia , Materiais Revestidos Biocompatíveis , Ativação do Complemento/efeitos dos fármacos , Proteínas do Sistema Complemento/fisiologia , Citocinas/biossíntese , Heparina/farmacologia , Sangue , Ponte Cardiopulmonar/efeitos adversos , Ponte Cardiopulmonar/instrumentação , Quimiocina CCL2/biossíntese , Quimiocina CCL2/sangue , Materiais Revestidos Biocompatíveis/efeitos adversos , Complemento C5a/farmacologia , Cicloeximida/farmacologia , Citocinas/sangue , Humanos , Inflamação/induzido quimicamente , Interleucina-1/biossíntese , Interleucina-1/sangue , Interleucina-10/biossíntese , Interleucina-10/sangue , Interleucina-6/biossíntese , Interleucina-6/sangue , Interleucina-8/biossíntese , Interleucina-8/sangue , Ativação Linfocitária/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Cloreto de Polivinila/efeitos adversos , Cloreto de Polivinila/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/biossínteseRESUMO
BACKGROUND: Exposure of blood to artificial surfaces, as in cardiopulmonary bypass, induces an inflammatory response involving complement, leukocyte and platelet activation. To elucidate the specific role of complement in this process, studies were performed on blood circulated in polyvinyl chloride tubing in the absence and presence of complement inhibitors. Parallel experiments were performed with heparin-coated polyvinyl chloride tubing, which is known to prevent complement and cell activation. METHODS: A novel experimental model was used, based on human whole blood anticoagulated with lepirudin. Complement activation products, myeloperoxidase, lactoferrin, and thrombospondin were quantified in enzyme immunoassays. Leukocyte CD11b expression and leukocyte-platelet conjugates were detected by flow cytometry. RESULTS: Increased levels of C3 activation products, alternative pathway convertase, and the terminal SC5b-9 complex, combined with unchanged levels of C1rs-C1-inhibitor complexes and marginal changes in C4 activation demonstrated that complement was activated through the alternative pathway. Granulocyte and monocyte CD11b expression and granulocyte-platelet conjugate formation were efficiently attenuated by blocking either factor D, C3, C5, or C5a receptor. In contrast, monocyte-platelet conjugate formation and release of myeloperoxidase, lactoferrin, and thrombospondin were not reduced by complement inhibition. Heparin-coated polyvinyl chloride tubing efficiently reduced all inflammatory markers studied, except for C1rs-C1-inhibitor complexes, which increased, consistent with the enhancing effect of heparin on C1-inhibitor function. This effect did not, however, reduce fluid-phase classic pathway activation induced by heat-aggregated immunoglobulin G. CONCLUSIONS: Leukocyte and platelet activation in response to artificial materials occur by mechanisms that vary in their dependence on complement. Heparin coating precludes both the complement-dependent and complement-independent reactions.
Assuntos
Ponte Cardiopulmonar/instrumentação , Materiais Revestidos Biocompatíveis , Ativação do Complemento , Heparina/farmacologia , Leucócitos/fisiologia , Ativação Plaquetária/fisiologia , Cloreto de Polivinila , Antígenos CD11/análise , Antígeno CD11b/análise , Ponte Cardiopulmonar/efeitos adversos , Via Alternativa do Complemento , Proteínas do Sistema Complemento/efeitos dos fármacos , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Técnicas In Vitro , Cadeias alfa de Integrinas/análise , Lactoferrina/análise , Modelos Biológicos , Peroxidase/análise , Ativação Plaquetária/efeitos dos fármacos , Trombospondinas/análiseRESUMO
Complement plays an essential role in inflammation and tissue damage. However, it is largely unknown to what extent the system acts as a primary inducer of secondary mediator systems in the inflammatory network of human whole blood. Here we describe a novel in vitro model using the thrombin-specific hirudin analog lepirudin as anticoagulant, which, in contrast to heparin, did not interfere with complement activation. The model was used to study the role of complement in Escherichia coli-induced inflammatory responses. Granulocyte and monocyte oxidative burst was complement dependent as it was reduced by 85% and 70%, respectively, by the C3 [corrected] binding peptide compstatin. A similar reduction was found by inhibition of C5, C5a, and C5a receptor (C5aR). Furthermore, anti-CR3 antibodies were as efficient as the C5aR antagonist in reducing granulocyte oxidative burst, whereas blocking CD14 or C3aR had no effect. Up-regulation of granulocyte CR3 was virtually abolished by a C5aR antagonist. Opsonization and phagocytosis was completely inhibited by blocking of C5aR or CR3, whereas blocking of the FcgammaRs (CD16, CD32, CD64) had no effect. In contrast to oxidative burst and phagocytosis, cytokine secretion was largely complement independent. Thus, anti-CD14 abolished tumor necrosis factor-alpha, interleukin-6 (IL-6), and IL-10 secretion, whereas IL-8 was equally inhibited by anti-CD14 and compstatin. In conclusion, the present model is particularly useful for studying complement as part of the inflammatory network. The results emphasize a crucial role for C5a-C5aR interaction in E coli-induced up-regulation of CR3 and the subsequent oxidative burst and phagocytosis. Complement inhibition may have therapeutic implications in oxidative burst-induced tissue damage.