RESUMO
Two functions have been assigned to properdin; stabilization of the alternative convertase, C3bBb, is well accepted, whereas the role of properdin as pattern recognition molecule is controversial. The presence of nonphysiological aggregates in purified properdin preparations and experimental models that do not allow discrimination between the initial binding of properdin and binding secondary to C3b deposition is a critical factor contributing to this controversy. In previous work, by inhibiting C3, we showed that properdin binding to zymosan and Escherichia coli is not a primary event, but rather is solely dependent on initial C3 deposition. In the present study, we found that properdin in human serum bound dose-dependently to solid-phase myeloperoxidase. This binding was dependent on C3 activation, as demonstrated by the lack of binding in human serum with the C3-inhibitor compstatin Cp40, in C3-depleted human serum, or when purified properdin is applied in buffer. Similarly, binding of properdin to the surface of human umbilical vein endothelial cells or Neisseria meningitidis after incubation with human serum was completely C3-dependent, as detected by flow cytometry. Properdin, which lacks the structural homology shared by other complement pattern recognition molecules and has its major function in stabilizing the C3bBb convertase, was found to bind both exogenous and endogenous molecular patterns in a completely C3-dependent manner. We therefore challenge the view of properdin as a pattern recognition molecule, and argue that the experimental conditions used to test this hypothesis should be carefully considered, with emphasis on controlling initial C3 activation under physiological conditions.
Assuntos
Complemento C3b/metabolismo , Properdina/metabolismo , Células Cultivadas , Ativação do Complemento , Granulócitos/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Neisseria meningitidis , Peptídeos Cíclicos/farmacologia , Peroxidase/metabolismo , SoroRESUMO
Sepsis is an infection-induced systemic inflammatory syndrome, potentially causing organ failure. We previously showed attenuating effects on inflammation, thrombogenicity and haemodynamics by inhibiting the Toll-like receptor co-factor CD14 and complement factor C5 in a porcine Escherichia coli-induced sepsis model. The present study explored the effect on organ inflammation in these pigs. Tissue samples were examined from the combined treatment group (n = 8), the positive (n = 8) and negative (n = 6) control groups after 4h of sepsis. Inflammatory biomarkers were measured using ELISA, multiplex and qPCR analysis. Combined inhibition of C5 and CD14 markedly attenuated IL-1ß by 31-66% (P < 0.05) and IL-6 by 54-96% (P < 0.01) in liver, kidney, lung and spleen; IL-8 by 65-100% in kidney, lung, spleen, and heart (P < 0.05) and MCP-1 by 46-69% in liver, kidney, spleen and heart (P < 0.05). Combined inhibition significantly attenuated tissue factor mRNA upregulation in spleen (P < 0.05) and IP-10 mRNA upregulation in four out of five organs. Finally, C5aR mRNA downregulation was prevented in heart and kidney (P < 0.05). Combined inhibition of C5 and CD14 thus markedly attenuated inflammatory responses in all organs examined. The anti-inflammatory effects observed in lung and heart may explain the delayed haemodynamic disturbances observed in septic pigs receiving combined inhibition of C5 and CD14.
Assuntos
Complemento C5/antagonistas & inibidores , Infecções por Escherichia coli/terapia , Escherichia coli/imunologia , Receptores de Lipopolissacarídeos/imunologia , Insuficiência de Múltiplos Órgãos/terapia , Sepse/terapia , Animais , Células Cultivadas , Quimiocina CCL2/metabolismo , Modelos Animais de Doenças , Infecções por Escherichia coli/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/imunologia , Inflamação/terapia , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Insuficiência de Múltiplos Órgãos/microbiologia , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Sepse/imunologia , SuínosRESUMO
Combined inhibition of complement and CD14 is known to attenuate bacterial-induced inflammation, but the dependency of the bacterial load on this effect is unknown. Thus, we investigated whether the effect of such combined inhibition on Escherichia coli- and Staphylococcus aureus-induced inflammation was preserved during increasing bacterial concentrations. Human whole blood was preincubated with anti-CD14, eculizumab (C5-inhibitor) or compstatin (C3-inhibitor), or combinations thereof. Then heat-inactivated bacteria were added at final concentrations of 5 × 10(4) -1 × 10(8) /ml (E. coli) or 5 × 10(7) -4 × 10(8) /ml (S. aureus). Inflammatory markers were measured using enzyme-linked immunosorbent assay (ELISA), multiplex technology and flow cytometry. Combined inhibition of complement and CD14 significantly (P < 0.05) reduced E. coli-induced interleukin (IL)-6 by 40-92% at all bacterial concentrations. IL-1ß, IL-8 and macrophage inflammatory protein (MIP)-1α were significantly (P < 0.05) inhibited by 53-100%, and the effect was lost only at the highest bacterial concentration. Tumour necrosis factor (TNF) and MIP-1ß were significantly (P < 0.05) reduced by 80-97% at the lowest bacterial concentration. Monocyte and granulocyte CD11b were significantly (P < 0.05) reduced by 63-91% at all bacterial doses. Lactoferrin was significantly (P < 0.05) attenuated to the level of background activity at the lowest bacterial concentration. Similar effects were observed for S. aureus, but the attenuation was, in general, less pronounced. Compared to E. coli, much higher concentrations of S. aureus were required to induce the same cytokine responses. This study demonstrates generally preserved effects of combined complement and CD14 inhibition on Gram-negative and Gram-positive bacterial-induced inflammation during escalating bacterial load. The implications of these findings for future therapy of sepsis are discussed.
Assuntos
Complemento C3/imunologia , Complemento C5/imunologia , Escherichia coli/imunologia , Inflamação/imunologia , Receptores de Lipopolissacarídeos/imunologia , Staphylococcus aureus/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Carga Bacteriana/imunologia , Antígeno CD11b/sangue , Antígeno CD11b/imunologia , Complemento C3/antagonistas & inibidores , Complemento C5/antagonistas & inibidores , Citocinas/sangue , Citocinas/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Granulócitos/imunologia , Granulócitos/metabolismo , Temperatura Alta , Humanos , Inflamação/sangue , Inflamação/prevenção & controle , Receptores de Lipopolissacarídeos/sangue , Monócitos/imunologia , Monócitos/metabolismo , Peptídeos Cíclicos/imunologia , Peptídeos Cíclicos/farmacologiaRESUMO
Combined inhibition of CD14 and complement, two main inducers of the inflammatory response, have proved particularly effective in attenuating Gram-negative bacteria-induced inflammation. Approaching possible clinical relevance, we investigated the effect of such inhibition in a post-challenge setting. Human whole blood was anti-coagulated with lepirudin. Anti-CD14, compstatin (C3 inhibitor) and the combination thereof were added 5 min prior to or 5, 15 or 30 min after adding Escherichia coli. Total incubation time with Escherichia coli was 120 min. Cytokines, myeloperoxidase (MPO) and the terminal complement complex (TCC) were measured using multiplex technology and ELISA. Delayed combined inhibition significantly attenuated the inflammatory response. IL-1ß, IL-8 and TNF-α were significantly inhibited in the range of 20-40%, even when adding the inhibitors with up to 30 min delay. IL-6 was significantly inhibited with 15 min delay, and MIP-1α and MPO with 5 min delay. Complement activation (TCC) was blocked completely at each time point compstatin was added, whereas the cytokines and MPO increased steadily between the time points. The combined regimen was significantly more effective than single inhibition in the pre-challenge setting. The attenuation of Escherichia coli-induced inflammation in a post-challenge setting suggests a potential therapeutic window for this treatment in sepsis.
Assuntos
Sangue/imunologia , Complemento C3/imunologia , Escherichia coli/imunologia , Imunoterapia , Receptores de Lipopolissacarídeos/metabolismo , Sepse/imunologia , Anticorpos Bloqueadores/farmacologia , Sangue/efeitos dos fármacos , Ativação do Complemento/efeitos dos fármacos , Complemento C3/antagonistas & inibidores , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Citocinas/metabolismo , Combinação de Medicamentos , Sinergismo Farmacológico , Escherichia coli/metabolismo , Temperatura Alta , Humanos , Imunidade Inata/efeitos dos fármacos , Imunização , Mediadores da Inflamação/metabolismo , Receptores de Lipopolissacarídeos/imunologia , Peptídeos Cíclicos/farmacologia , Peroxidase/metabolismo , Sepse/terapiaRESUMO
CD14 is a key recognition molecule of innate immune responses, interacting with several TLRs. TLR signaling cross-talks extensively with the complement system, and combined CD14 and complement inhibition has been proved effective in attenuating inflammatory responses. Pig models of human diseases have emerged as valuable tools to study therapeutic intervention, but suitable neutralizing Abs are rare. Undesired Fc-mediated functions, such as platelet activation and IL-8 release induced by the porcine CD14-specific clone Mil2, limit further studies. Therefore, an inert human IgG2/IgG4 hybrid C region was chosen for an rMil2. As revealed in ex vivo and in vivo pig experiments, rMil2 inhibited the CD14-mediated proinflammatory cytokine response similar to the original clone, but lacked the undesired Fc-effects, and inflammation was attenuated further by simultaneous complement inhibition. Moreover, rMil2 bound porcine FcRn, a regulator of t1/2 and biodistribution. Thus, rMil2, particularly combined with complement inhibitors, should be well suited for in vivo studies using porcine models of diseases, such as sepsis and ischemia-reperfusion injury. Similarly, the recombinant anti-human CD14 IgG2/4 Ab, r18D11, was generated with greatly reduced Fc-mediated effects and preserved inhibitory function ex vivo. Such Abs might be drug candidates for the treatment of innate immunity-mediated human diseases.
Assuntos
Imunoglobulina G/uso terapêutico , Imunoterapia , Inflamação/imunologia , Inflamação/terapia , Receptores de Lipopolissacarídeos/imunologia , Animais , Anticorpos , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-Idiotípicos/uso terapêutico , Antígenos de Diferenciação/imunologia , Linhagem Celular , Ativação do Complemento/imunologia , Células HEK293 , Humanos , Imunoglobulina G/imunologia , Receptores de IgG/imunologia , Sus scrofaRESUMO
Complement and the TLR family constitute two important branches of innate immunity. We previously showed attenuating effects on inflammation and thromogenicity by inhibiting the TLR coreceptor CD14 in porcine sepsis. In the present study, we explored the effect of the C5 and leukotriene B4 inhibitor Ornithodoros moubata complement inhibitor (OmCI; also known as coversin) alone and combined with anti-CD14 on the early inflammatory, hemostatic, and hemodynamic responses in porcine Escherichia coli-induced sepsis. Pigs were randomly allocated to negative controls (n = 6), positive controls (n = 8), intervention with OmCI (n = 8), or with OmCI and anti-CD14 (n = 8). OmCI ablated C5 activation and formation of the terminal complement complex and significantly decreased leukotriene B4 levels in septic pigs. Granulocyte tissue factor expression, formation of thrombin-antithrombin complexes (p < 0.001), and formation of TNF-α and IL-6 (p < 0.05) were efficiently inhibited by OmCI alone and abolished or strongly attenuated by the combination of OmCI and anti-CD14 (p < 0.001 for all). Additionally, the combined therapy attenuated the formation of plasminogen activator inhibitor-1 (p < 0.05), IL-1ß, and IL-8, increased the formation of IL-10, and abolished the expression of wCD11R3 (CD11b) and the fall in neutrophil cell count (p < 0.001 for all). Finally, OmCI combined with anti-CD14 delayed increases in heart rate by 60 min (p < 0.05) and mean pulmonary artery pressure by 30 min (p < 0.01). Ex vivo studies confirmed the additional effect of combining anti-CD14 with OmCI. In conclusion, upstream inhibition of the key innate immunity molecules, C5 and CD14, is a potential broad-acting treatment regimen in sepsis as it efficiently attenuated inflammation and thrombogenicity and delayed hemodynamic changes.
Assuntos
Proteínas de Artrópodes/farmacologia , Proteínas de Transporte/farmacologia , Complemento C5/antagonistas & inibidores , Leucotrieno B4/antagonistas & inibidores , Receptores de Lipopolissacarídeos/imunologia , Sepse/imunologia , Animais , Antitrombina III/biossíntese , Pressão Arterial/efeitos dos fármacos , Pressão Arterial/imunologia , Antígeno CD11b/biossíntese , Escherichia coli/imunologia , Infecções por Escherichia coli/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/imunologia , Hemodinâmica/efeitos dos fármacos , Imunidade Inata , Inflamação/tratamento farmacológico , Inflamação/imunologia , Interleucina-10/biossíntese , Interleucina-1beta/biossíntese , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Contagem de Leucócitos , Receptores de Lipopolissacarídeos/metabolismo , Neutrófilos/citologia , Peptídeo Hidrolases/biossíntese , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Sus scrofa , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Sepsis is an infection-induced systemic inflammatory response syndrome. Upstream recognition molecules, like CD14, play key roles in the pathogenesis. The aim of the present study was to investigate the effect of systemic CD14 inhibition on local inflammatory responses in organs from septic pigs. Pigs (n = 34) receiving Escherichia coli-bacteria or E. coli-lipopolysaccharide (LPS) were treated with an anti-CD14 monoclonal antibody or an isotype-matched control. Lungs, liver, spleen, and kidneys were examined for bacteria and inflammatory biomarkers. E. coli and LPS were found in large amounts in the lungs compared to the liver, spleen, and kidneys. Notably, the bacterial load did not predict the respective organ inflammatory response. There was a marked variation in biomarker induction in the organs and in the effect of anti-CD14. Generally, the spleen produced the most cytokines per weight unit, whereas the liver contributed the most to the total load. All cytokines were significantly inhibited in the spleen. Interleukin-6 (IL-6) was significantly inhibited in all organs, IL-1ß and IP-10 were significantly inhibited in liver, spleen, and kidneys, and tumor necrosis factor, IL-8, and PAI-1 were inhibited only in the spleen. ICAM-1 and VCAM-1 was significantly inhibited in the kidneys. Systemic CD14-inhibition efficiently, though organ dependent, attenuated local inflammatory responses. Detailed knowledge on how the different organs respond to systemic inflammation in vivo, beyond the information gained by blood examination, is important for our understanding of the nature of systemic inflammation and is required for future mediator-directed therapy in sepsis. Inhibition of CD14 seems to be a good candidate for such treatment.
Assuntos
Escherichia coli/imunologia , Inflamação/imunologia , Receptores de Lipopolissacarídeos/imunologia , Sepse/imunologia , Suínos/imunologia , Estruturas Animais/imunologia , Estruturas Animais/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Biomarcadores/metabolismo , Quimiocina CXCL10/imunologia , Quimiocina CXCL10/metabolismo , Escherichia coli/metabolismo , Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Interleucinas/imunologia , Interleucinas/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/imunologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Sepse/metabolismo , Suínos/metabolismo , Molécula 1 de Adesão de Célula Vascular/imunologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Evidence suggests that adjunctive treatment with intravenous immunoglobulin preparations enriched with IgA and IgM reduce mortality in sepsis. The mode of action of polyvalent immunoglobulin is complex, including neutralization of toxins and modulation of complement activation and cytokine formation toward an anti-inflammatory profile. In this study we explored the effect of Pentaglobin, containing IgG, IgA and IgM, on the initial inflammatory reaction as well as on hemodynamics, using a well characterized and standardized porcine model of sepsis. Anesthetized and mechanically ventilated pigs, mean weight 14.9 kg, were allocated into two groups of 8 animals, receiving either Pentaglobin or saline, before sepsis was induced by intravenous Escherichia coli infusion. Five negative controls received saline only. All animals were observed for 4 h under extensive invasive monitoring. Pentaglobin significantly (p < 0.05) attenuated IL-1ß formation by 38% at the end of the experiment, and markedly increased (p < 0.05) the formation of IL-10 at 60 min. TNF-α, IL-6, IL-8 and expression of the cell surface marker wCD11R3 were lower in the Pentaglobin group, but the differences were not significant. The serum concentration of LPS was three times higher in the Pentaglobin group (p < 0.005), indicating binding of LPS to Pentaglobin. Complementary in vitro experiments showed a higher binding affinity for IgM and IgA to LPS than for IgG. LPS-induced formation of IL-6 was significantly (p < 0.05) attenuated by Pentaglobin in an in vitro whole blood model. In conclusion, Pentaglobin decreased the key inflammasome IL-1ß molecule in an E. coli-model of pigs sepsis.
Assuntos
Infecções por Escherichia coli/tratamento farmacológico , Imunoglobulina A/farmacologia , Imunoglobulina M/farmacologia , Imunoglobulinas Intravenosas/farmacologia , Interleucina-1beta/antagonistas & inibidores , Sepse/tratamento farmacológico , Animais , Complexo Antígeno-Anticorpo/sangue , Biomarcadores/sangue , Escherichia coli/efeitos dos fármacos , Escherichia coli/imunologia , Infecções por Escherichia coli/sangue , Infecções por Escherichia coli/imunologia , Hemodinâmica/efeitos dos fármacos , Imunoglobulina A/sangue , Imunoglobulina M/sangue , Imunoglobulinas Intravenosas/sangue , Inflamação/prevenção & controle , Interleucina-10/sangue , Interleucina-10/imunologia , Interleucina-1beta/sangue , Interleucina-1beta/imunologia , Interleucina-6/sangue , Interleucina-6/imunologia , Interleucina-8/sangue , Interleucina-8/imunologia , Lipopolissacarídeos/sangue , Ligação Proteica , Sepse/sangue , Sepse/imunologia , Suínos , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Properdin is well known as an enhancer of the alternative complement amplification loop when C3 is activated, whereas its role as a recognition molecule of exogenous pathogen-associated molecular patterns and initiator of complement activation is less understood. We therefore studied the role of properdin in activation of complement in normal human serum by zymosan and various Escherichia coli strains. In ELISA, microtiter plates coated with zymosan induced efficient complement activation with deposition of C4b and terminal complement complex on the solid phase. Virtually no deposition of C4b or terminal complement complex was observed with mannose-binding lectin (MBL)-deficient serum. Reconstitution with purified MBL showed distinct activation in both readouts. In ELISA, normal human serum-induced deposition of properdin by zymosan was abolished by the C3-inhibiting peptide compstatin. Flow cytometry was used to further explore whether properdin acts as an initial recognition molecule reacting directly with zymosan and three E. coli strains. Experiments reported by other authors were made with EGTA Mg²âº buffer, permitting autoactivation of C3. We found inhibition by compstatin on these substrates, indicating that properdin deposition depended on initial C3b deposition followed by properdin in a second step. Properdin released from human polymorphonuclear cells stimulated with PMA did not bind to zymosan or E. coli, but when incubated in properdin-depleted serum this form of properdin bound efficiently to both substrates in a strictly C3-dependent manner, as the binding was abolished by compstatin. Collectively, these data indicate that properdin in serum as well as polymorphonuclear-released properdin is unable to bind and initiate direct alternative pathway activation on these substrates.
Assuntos
Via Alternativa do Complemento/imunologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/imunologia , Properdina/fisiologia , Zimosan/fisiologia , Adulto , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Humanos , Masculino , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Properdina/metabolismo , Ligação Proteica/imunologia , Especificidade por Substrato/imunologiaRESUMO
Experimental evidence suggests that C inhibition and more particularly combined inhibition of C and the TLR coreceptor CD14 may be of therapeutic benefit in sepsis and other inflammatory conditions. A barrier to the testing and further development of many inhibitors is that their activity is species specific. Pig is a relevant species for experimental models of human disease, and this study undertakes a comprehensive comparison of the inhibitory efficacy of the C5 inhibitor Ornithodoros moubata C inhibitor (OmCI) in human and porcine whole blood ex vivo models of Escherichia coli-induced sepsis. The effect of OmCI on complement activity in pigs undergoing E. coli sepsis was also examined. Porcine and human serum, and whole blood anticoagulated with lepirudin, was incubated with E. coli and the effect of OmCI investigated. The ex vivo results were virtually identical in pig and human. OmCI completely ablated the activity of all three C pathways at 0.64 µM. E. coli-induced C activation and expression of CD11b (wCD11R3 in the pig), was abolished ex vivo at 0.32 µM OmCI. Combining anti-CD14 and OmCI reduced the formation of IL-8 and TNF-α more potently than the single inhibitors. OmCI also efficiently bound E. coli-induced leukotriene B(4) in pig and human plasma. In support of our ex vivo findings, in vivo the activity of all C pathways was inhibited at 0.6 mg OmCI/kg pig. In conclusion, OmCI efficiently inhibited pig and human C activation, has accompanying anti-inflammatory effects and is a promising candidate inhibitor for further in vivo studies of sepsis.
Assuntos
Complemento C5a/antagonistas & inibidores , Proteínas Inativadoras do Complemento/fisiologia , Ornithodoros/imunologia , Animais , Complemento C5a/metabolismo , Proteínas Inativadoras do Complemento/uso terapêutico , Via Alternativa do Complemento/imunologia , Via Clássica do Complemento/imunologia , Modelos Animais de Doenças , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/prevenção & controle , Feminino , Humanos , Masculino , Projetos Piloto , Proteínas e Peptídeos Salivares/fisiologia , Proteínas e Peptídeos Salivares/uso terapêutico , Sepse/imunologia , Sepse/prevenção & controle , SuínosRESUMO
Bradykinin (BK) is regarded as an important mediator of edema, shock, and inflammation during sepsis. In this study, we evaluated the contribution of BK in porcine sepsis by blocking BK and by measuring the stable BK metabolite, BK1-5, using anesthetized pigs. The effect of BK alone, the efficacy of icatibant to block this effect, and the recovery of BK measured as plasma BK1-5 were first investigated. Purified BK injected intravenously induced an abrupt fall in blood pressure, which was completely prevented by pretreatment with icatibant. BK1-5 was detected in plasma corresponding to the doses given. The effect of icatibant was then investigated in an established model of porcine gram-negative sepsis. Neisseria meningitidis was infused intravenously without any pretreatment (n = 8) or pretreated with icatibant (n = 8). Negative controls received saline only. Icatibant-treated pigs developed the same degree of severe sepsis as did the controls. Both groups had massive capillary leakage, leukopenia, and excessive cytokine release. The plasma level of BK1-5 was low or nondetectable in all pigs. The latter observation was confirmed in supplementary studies with pigs undergoing Escherichia coli or polymicrobial sepsis induced by cecal ligation and puncture. In conclusion, icatibant completely blocked the hemodynamic effects of BK but had no beneficial effects on N. meningitidis-induced edema, shock, and inflammation. This and the fact that plasma BK1-5 in all the septic pigs was virtually nondetectable question the role of BK as an important mediator of porcine sepsis. Thus, the data challenge the current view of the role of BK also in human sepsis.
Assuntos
Antagonistas dos Receptores da Bradicinina , Bradicinina/análogos & derivados , Bradicinina/metabolismo , Bradicinina/farmacologia , Sepse/metabolismo , Animais , Bradicinina/uso terapêutico , Edema/tratamento farmacológico , Edema/microbiologia , Inflamação/tratamento farmacológico , Inflamação/microbiologia , Neisseria meningitidis/patogenicidade , Sepse/tratamento farmacológico , Choque/tratamento farmacológico , Choque/microbiologia , SuínosRESUMO
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
OBJECTIVE: To dissect the in vivo responses to lipopolysaccharide compared with nonlipopolysaccharide structures of whole meningococci. DESIGN: Comparative experimental study. SETTING: University hospital with an animal intensive care unit and laboratory. SUBJECTS: Twenty-four anesthetized healthy Norwegian landrace pigs of 30 kg (+/- 2.5 kg) grouped into two test groups and one control group. INTERVENTIONS: Exponentially increasing numbers of Neisseria meningitidis H44/76 (NmLPS+) or a knockout mutant of H44/76 completely lacking lipopolysaccharide (NmLPS-) were infused intravenously to the pigs. MEASUREMENTS AND MAIN RESULTS: Physiological and hematologic parameters were continuously recorded and biochemical analyses were performed in batch after completion. Systemic vascular resistance, cardiac index and lactate changed significantly more in the NmLPS+ than in the NmLPS- group (p < .05). Mean pulmonary artery pressure increased early in the NmLPS+ and late in the NmLPS- group, but finally reached equally high values. Capillary leakage (fluid requirement, plasma albumin loss, organ wet/dry ratio) was more prominent in the NmLPS+ group (p < .05). Leukocytes were depleted in a highly lipopolysaccharide-dependent manner (p < .001). Thrombin-antithrombin complexes and plasminogen activator inhibitor-1 increased 2.5 to five times more in the NmLPS+ group (p < .05). Maximum cytokine concentrations in plasma were markedly higher in the NmLPS+ group (p < .05): tumor necrosis factor-alpha (40 times), interleukin-1beta (40 times), interleukin-6 (13 times), and interleukin-10 (four times). Interleukin-12 increased only in the NmLPS+ group. CONCLUSION: This large animal model, which simulates human disease well, confirms the potency of lipopolysaccharide but provides clear evidence that nonlipopolysaccharide molecules induce cardiovascular and hematologic changes quite similar to those caused by lipopolysaccharide. In general, 10- to 20-fold higher doses of the lipopolysaccharide-deficient mutant were required to induce the same degree of pathophysiological changes. Endotoxic activity of Gram-negative bacteria should no longer be attributed solely to the activity of lipopolysaccharide.
Assuntos
Lipopolissacarídeos/farmacologia , Infecções Meningocócicas/etiologia , Neisseria meningitidis/imunologia , Sepse/etiologia , Animais , Fatores de Coagulação Sanguínea/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Modelos Animais de Doenças , Hematócrito , Interleucinas/metabolismo , Infecções Meningocócicas/metabolismo , Infecções Meningocócicas/fisiopatologia , Sepse/metabolismo , Sepse/fisiopatologia , Albumina Sérica/metabolismo , Suínos , Fator de Necrose Tumoral alfa/metabolismo , Resistência Vascular/efeitos dos fármacosRESUMO
Inhibition of the inappropriate and excessive inflammatory response has been a main issue in sepsis-related research. Historically, TNF-alpha and IL-1 beta have been postulated as key mediators in sepsis, but selective inhibition of these cytokines has failed in clinical trials. Recently it was found that inhibition of upstream recognition by complement and CD14 could efficiently reduce Escherichia coli (E. coli)-induced inflammation. An ex vivo model with lepirudin-anticoagulated human whole blood was used to explore the significance of selective inhibition of TNF-alpha and IL-1 beta in E. coli-induced inflammation. The effect of TNF-alpha, IL-1 beta, complement and CD14 on the inflammatory response was assessed by adding highly specific neutralizing agents to these mediators. Proinflammatory cytokines, expression of CD11b and oxidative burst were measured. The controls included relevant isotype-matched immunoglobulins and peptides. Selective inhibition of TNF-alpha or IL-1 beta had no impact on E. coli-induced release of proinflammatory cytokines, CD11b-upregulation or oxidative burst. In contrast, the combined inhibition of complement and CD14 virtually abolished these responses. These data suggest that both TNF-alpha and IL-1 beta are downstream mediators and as single mediators play a limited role within the complex inflammatory reactions induced by E. coli.
Assuntos
Escherichia coli/imunologia , Inflamação/imunologia , Interleucina-1beta/imunologia , Fator de Necrose Tumoral alfa/imunologia , Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais/farmacologia , Antígeno CD11b/sangue , Citocinas/sangue , Etanercepte , Humanos , Imunoglobulina G/farmacologia , Inflamação/sangue , Inflamação/prevenção & controle , Mediadores da Inflamação/sangue , Infliximab , Interleucina-1beta/sangue , Interleucina-1beta/genética , Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Explosão Respiratória/imunologia , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/genéticaRESUMO
Sepsis is a severe infection-induced systemic inflammatory syndrome. Inhibition of downstream inflammatory mediators of sepsis, e.g., TNF-alpha, has failed in clinical trials. The aim of this study was to investigate the effects of inhibiting CD14, a key upstream innate immunity molecule, on the early inflammatory and hemostatic responses in a pig model of gram-negative sepsis. The study comprised two arms, whole live Escherichia coli bacteria and E. coli lipopolysaccharide (LPS) (n=25 and n=9 animals, respectively). The animals were allocated into treatment (anti-CD14) and control (IgG isotype or saline) groups. Inflammatory, hemostatic, physiological, and microbiological parameters were measured. The proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and IL-8, but not the anti-inflammatory cytokine IL-10, were efficiently inhibited by anti-CD14. Furthermore, anti-CD14 preserved the leukocyte count and significantly reduced granulocyte enzyme matrix metalloproteinase-9 release and expression of the granulocyte membrane activation molecule wCD11R3 (pig CD11b). The hemostatic markers thrombin-antithrombin III complexes and plasminogen activator inhibitor-1 were significantly attenuated. Anti-CD14 did not affect LPS or E. coli DNA levels. This study documents that CD14 inhibition efficiently attenuates the proinflammatory cytokine response and granulocyte activation and reverses the procoagulant state but does not interfere with LPS levels or bacterial counts in E. coli-induced sepsis.-Thorgersen, E. B., Hellerud, B. C., Nielsen, E. W., Barratt-Due, A., Fure, H., Lindstad, J. K., Pharo, A., Fosse, E., Tønnessen, T. I., Johansen, H. T., Castellheim, A., Mollnes, T. E. CD14 inhibition efficiently attenuates early inflammatory and hemostatic responses in Escherichia coli sepsis in pigs.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Escherichia coli/patogenicidade , Receptores de Lipopolissacarídeos/imunologia , Sepse/tratamento farmacológico , Sepse/imunologia , Animais , Escherichia coli/genética , Feminino , Citometria de Fluxo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Sepse/induzido quimicamente , Sepse/microbiologia , Suínos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Complement activation plays an important role in human pathophysiology. The effect of classical pathway activation is largely dependent on alternative pathway (AP) amplification, whereas the role of AP for the down-stream effect of mannan-induced lectin pathway (LP) activation is poorly understood. In normal human serum specific activation of LP was obtained after exposure to a wide concentration range of mannan on the solid phase. Reaction mechanisms in this system were delineated in inhibition experiments with monoclonal antibodies. Direct mannose-binding lectin (MBL) independent activation of AP was not observed even at high mannan concentrations since addition of the inhibiting anti-MBL mAb 3F8 completely abolished generation of the terminal C5b-9 complex (TCC). However, selective blockade of AP by anti-factor D inhibited more than 80% of TCC release into the fluid phase after LP activation showing that AP amplification is quantitatively responsible for the final effect of initial specific LP activation. TCC generation on the solid phase was distinctly but less inhibited by anti-fD. C2 bypass of the LP pathway could be demonstrated, and AP amplification was also essential during C2 bypass in LP as shown by complete inhibition of TCC generation in C2-deficient serum by anti-fD and anti-properdin antibodies. In conclusion, the down-stream effect of LP activation depends strongly on AP amplification in normal human serum and in the C2 bypass pathway.
Assuntos
Via Alternativa do Complemento/imunologia , Mananas/imunologia , Lectina de Ligação a Manose/imunologia , Adolescente , Adulto , Anticorpos Monoclonais/imunologia , Complemento C2/imunologia , Complemento C4b/imunologia , Fator D do Complemento/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Humanos , Masculino , SoroRESUMO
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
The objective of this study was to establish a porcine analog of human meningococcal sepsis for pathophysiological investigations and possible future therapy in severe sepsis. Heat-killed Neisseria meningitidis was continuously infused in sublethal concentrations into 10 anesthetized 30-kg pigs (sepsis group). The dose was doubled every 30 min. Six pigs received saline only (control group). The changes described in the succeeding paragraphs were observed in the sepsis group but not in the control group. MAP was aimed to be kept normal by fluid infusion but declined after 3 h in parallel with a decrease in systemic vascular resistance. Pulmonary arterial pressure increased considerably after 30 to 45 min. A massive plasma extravasation was shown by increased hematocrit and a 50% reduction in plasma albumin content. Fluid accumulated in lungs, muscles, and jejunum, as shown by increased wet-dry ratios. Peak inspiratory pressures and fraction of inspired oxygen had to be increased. The cytokines TNF-alpha, IL-1beta, IL-6, IL-8, IL-10, and IL-12 increased markedly. Neutrophils fell to zero-levels, and platelets were markedly reduced. Thrombin-antithrombin complexes increased notably after 120 min. This is the first large animal model of sepsis using whole Neisseria meningitidis. The model simulates well central aspects of human meningococcal sepsis and could be used for future interventional studies.
Assuntos
Infecções Meningocócicas/microbiologia , Infecções Meningocócicas/patologia , Choque Séptico/microbiologia , Choque Séptico/patologia , Animais , Modelos Animais de Doenças , Feminino , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Masculino , Infecções Meningocócicas/metabolismo , Neisseria meningitidis/fisiologia , Distribuição Aleatória , Choque Séptico/metabolismo , Suínos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
We recently showed that acidosis is protective during hypoxia and detrimental during reoxygenation. We hypothesized that the detrimental effect of acidosis during reoxygenation was due to a negative effect on mitochondrial function. Human postmitotic NT2-N neurons were exposed to 3 h of hypoxia and glucose deprivation and then reoxygenated for 0, 1, 4, 9, or 21 h. The detrimental effect of acidotic reoxygenation on metabolic activity was evident already after 1 h of reoxygenation, when MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] reduction (percentage of normoxic controls) was significantly higher in cells reoxygenated with neutral compared with acidotic medium both after acidotic hypoxia (83+/-26% versus 67+/-27%, p=0.006) and after neutral hypoxia (51+/-12% versus 41+/-7%, p=0.005). Hypoxanthine, a marker of cellular energy failure, increased more with acidotic compared with neutral reoxygenation both after acidotic hypoxia (after 21 h: 7.7+/-2.7 versus 3.1+/-1.9 microM, p<0.001) and after neutral hypoxia (10.4+/-2.6 versus 7.9+/-2.8 microM, p=0.001). During hypoxia and reoxygenation, there was an earlier reduction in the activity of complex IV compared with complexes II+III, and the ratio between these complexes fell during the first hour of reoxygenation. The reduction in complex IV activity was alleviated with acidotic hypoxia. Acidosis during reoxygenation, however, had no effect on the activity of either complex IV or complexes II+III. We conclude that acidosis during hypoxia increases neuronal survival and preserves complex IV activity. Acidosis during reoxygenation has an early detrimental effect on metabolic activity, but this is not mediated through an effect on the mitochondrial complexes IV or II+III.
Assuntos
Acidose/fisiopatologia , Hipóxia Celular , Glucose/metabolismo , Hipóxia-Isquemia Encefálica , Neurônios/metabolismo , Reperfusão , Animais , Bovinos , Linhagem Celular , Humanos , Mitocôndrias/metabolismo , Complexos Multienzimáticos , Neurônios/citologia , Oxigênio/metabolismoRESUMO
Studies on primary cultures of newborn rodent neurons have suggested that neuronal death induced by unconjugated bilirubin (UCB) is mainly apoptotic in nature. We exposed a human teratocarcinoma-derived cell line, NT2-N neurons, to different concentrations of UCB and albumin at a 1.5 molar ratio and used multiple, independent measures of cell damage to evaluate neuronal injury after 6, 24, and 48 h. Low doses of UCB (0.66, 2, and 5 microM) induced a moderate loss of 3-4[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) cleavage accompanied by delayed morphologic changes consistent with apoptosis (2 and 5 microM). Moderate concentrations of UCB (10 and 25 microM) resulted in early (6 h) necrosis in a subset of neurons, while remaining neurons underwent progressive impairment of MTT cleavage and increasing lactate dehydrogenase (LDH) release accompanied by predominantly delayed apoptosis. High concentrations of UCB (100 microM) induced severe impairment of MTT cleavage, extensive LDH release, and morphologic changes consistent with necrosis within 6 h. Used as a positive control for apoptosis, 2 microM STS induced progressive impairment of MTT cleavage and morphologic changes consistent with apoptosis over the entire observation period. DNA electrophoresis at 48 h was compatible with apoptosis both after treatment with STS and UCB concentrations Assuntos
Apoptose
, Bilirrubina/metabolismo
, Neurônios/metabolismo
, Linhagem Celular
, Linhagem Celular Tumoral
, Núcleo Celular/metabolismo
, Corantes/farmacologia
, DNA/metabolismo
, Fragmentação do DNA
, Relação Dose-Resposta a Droga
, Corantes Fluorescentes/farmacologia
, Humanos
, L-Lactato Desidrogenase/metabolismo
, Necrose
, Poli(ADP-Ribose) Polimerases/metabolismo
, Sais de Tetrazólio/farmacologia
, Tiazóis/farmacologia
, Fatores de Tempo