RESUMEN
Polymorphonuclear neutrophilic granulocytes (PMN) as cellular components of innate immunity play a crucial role in the defense against systemic Candida albicans infection. To analyze stimuli that are required for PMN activity during C. albicans infection in a situation similar to in vivo, we used a human whole-blood infection model. In this model, PMN activation 10 min after C. albicans infection was largely dependent on the anaphylatoxin C5a. Most importantly, C5a enabled blood PMN to overcome filament-restricted recognition of C. albicans and allowed efficient elimination of nonfilamentous C. albicans cph1Δ/efg1Δ from blood. Major PMN effector mechanisms, including oxidative burst, release of secondary granule contents and initial fungal phagocytosis could be prevented by blocking C5a receptor signaling. Identical effects were achieved using a humanized Ab specifically targeting human C5a. Phagocytosis of C. albicans 10 min postinfection was mediated by C5a-dependent enhancement of CD11b surface expression on PMN, thus establishing the C5a-C5aR-CD11b axis as a major modulator of early anti-Candida immune responses in human blood. In contrast, phagocytosis of C. albicans by PMN 60 min postinfection occurred almost independently of C5a and mainly contributed to activation of phagocytically active PMN at later time points. Our results show that C5a is a critical mediator in human blood during C. albicans infection.
Asunto(s)
Complemento C5a/inmunología , Hongos/inmunología , Micosis/inmunología , Neutrófilos/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígeno CD11b/metabolismo , Candida albicans/inmunología , Candidiasis/inmunología , Complemento C5a/antagonistas & inhibidores , Complemento C5a/metabolismo , Humanos , Micosis/metabolismo , Activación Neutrófila/efectos de los fármacos , Activación Neutrófila/inmunología , Neutrófilos/metabolismo , Fagocitosis/inmunología , Receptor de Anafilatoxina C5a/metabolismo , Factores de TiempoRESUMEN
Recently, we reported the phase II portion of the adaptive phase II/III PANAMO trial exploring potential benefit and safety of selectively blocking C5a with the monoclonal antibody vilobelimab (IFX-1) in patients with severe coronavirus disease 2019 (COVID-19). The potent anaphylatoxin C5a attracts neutrophils and monocytes to the infection site, causes tissue damage by oxidative radical formation and enzyme releases, and leads to activation of the coagulation system. Results demonstrated that C5a inhibition with vilobelimab was safe and secondary outcomes appeared in favor of vilobelimab. We now report the pharmacokinetic/pharmacodynamic (PK/PD) analysis of the phase II study. Between March 31 and April 24, 2020, 30 patients with severe COVID-19 pneumonia confirmed by real-time polymerase chain reaction were randomly assigned 1:1 to receive vilobelimab plus best supportive care or best supportive care only. Samples for measurement of vilobelimab, C3a and C5a blood concentrations were taken. Vilobelimab predose (trough) drug concentrations in plasma ranged from 84,846 to 248,592 ng/ml (571 to 1674 nM) with a geometric mean of 151,702 ng/ml (1022 nM) on day 2 and from 80,060 to 200,746 ng/ml (539 to 1352 nM) with a geometric mean of 139,503 ng/ml (939 nM) on day 8. After the first vilobelimab infusion, C5a concentrations were suppressed in the vilobelimab group (median 39.70 ng/ml 4.8 nM, IQR 33.20-45.55) as compared to the control group (median 158.53 ng/ml 19.1 nM, IQR 60.03-200.89, p = 0.0006). The suppression was maintained on day 8 (p = 0.001). The current PK/PD analysis shows that vilobelimab efficiently inhibits C5a in patients with severe COVID-19.
Asunto(s)
Anticuerpos Monoclonales , Tratamiento Farmacológico de COVID-19 , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Ensayos Clínicos Fase II como Asunto , Complemento C3a , Complemento C5a , Humanos , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
The history of therapeutic interventions in clinical trials for sepsis has been referred to as the "graveyard for pharmaceutical companies." That is now set to change, as research provides hope for new approaches that will be therapeutically effective in humans with sepsis.
Asunto(s)
Sepsis/tratamiento farmacológico , Animales , Antígenos CD , Apoptosis , Complemento C5a/antagonistas & inhibidores , Proteína HMGB1/fisiología , Humanos , Factores Inhibidores de la Migración de Macrófagos/fisiología , Proteína C/uso terapéutico , Receptor de Anafilatoxina C5a , Receptores de Complemento/antagonistas & inhibidores , Sepsis/etiologíaRESUMEN
Acute lung injury (ALI) in mouse lung occurs after distal airway deposition of IgG immune complexes (IgGICs), resulting in a breakdown of the vascular-airway barrier, causing intrapulmonary edema, hemorrhage, and accumulation of neutrophils [polymorphonuclear leukocytes (PMNs)] in the alveolar compartment, these changes being complement (C5a) and C5a receptor (C5aR) dependent. In this ALI model, C5aR expression (protein) was found to occur on upper (bronchial) and lower (alveolar) airway epithelial cells. An adenovirus construct (siRNA) was used to silence mRNA for C5aR in the lung. Under such conditions, C5aR protein was markedly reduced on lung epithelial cells, resulting in much reduced leakage of albumin into the lung, diminished buildup of PMNs, and lower levels of proinflammatory mediators in bronchoalveolar lavage fluids. These studies indicate that bronchial and alveolar epithelial cell C5aR is up-regulated and greatly contributes to inflammation and injury in the lung. The use of siRNA administered into the airways avoids systemic suppression of C5aR, which might compromise innate immunity. It is possible that such an intervention might be employed in humans with ALI or acute respiratory distress syndrome as well as in upper-airway inflammatory diseases, such as chronic obstructive pulmonary disease and asthma, where there is evidence for complement activation and buildup of PMNs.
Asunto(s)
Lesión Pulmonar Aguda/tratamiento farmacológico , Complejo Antígeno-Anticuerpo/inmunología , Complemento C5a/fisiología , Interferencia de ARN , ARN Interferente Pequeño/uso terapéutico , Receptor de Anafilatoxina C5a/inmunología , Animales , Bronquios/inmunología , Células Epiteliales/inmunología , Inmunoglobulina G/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Peroxidasa/metabolismo , Alveolos Pulmonares/inmunología , Receptor de Anafilatoxina C5a/genéticaRESUMEN
BACKGROUND: Severe COVID-19 is characterised by inflammation and coagulation in the presence of complement system activation. We aimed to explore the potential benefit and safety of selectively blocking the anaphylatoxin and complement protein C5a with the monoclonal antibody IFX-1 (vilobelimab), in patients with severe COVID-19. METHODS: We did an exploratory, open-label, randomised phase 2 trial (part of the adaptive phase 2/3 PANAMO trial) of intravenous IFX-1 in adults with severe COVID-19 at three academic hospitals in the Netherlands. Eligibility criteria were age 18 years or older; severe pneumonia with pulmonary infiltrates consistent with pneumonia, a clinical history of severe shortness of breath within the past 14 days, or a need for non-invasive or invasive ventilation; severe disease defined as a ratio of partial pressure of arterial oxygen to fractional concentration of oxygen in inspired air (PaO2/FiO2) between 100 mm Hg and 250 mm Hg in the supine position; and severe acute respiratory syndrome coronavirus 2 infection confirmed by RT-PCR. Patients were randomly assigned 1:1 to receive IFX-1 (up to seven doses of 800 mg intravenously) plus best supportive care (IFX-1 group) or best supportive care only (control group). The primary outcome was the percentage change in PaO2/FiO2 in the supine position between baseline and day 5. Mortality at 28 days and treatment-emergent and serious adverse events were key secondary outcomes. The primary analysis was done in the intention-to-treat population and safety analyses were done in all patients according to treatment received. This trial is registered at ClinicalTrials.gov (NCT04333420). FINDINGS: Between March 31 and April 24, 2020, 30 patients were enrolled and randomly assigned to the IFX-1 group (n=15) or the control group (n=15). During the study it became clear that several patients could not be assessed regularly in the supine position because of severe hypoxaemia. It was therefore decided to focus on all PaO2/FiO2 assessments (irrespective of position). At day 5 after randomisation, the mean PaO2/FiO2 (irrespective of position) was 158 mm Hg (SD 63; range 84-265) in the IFX-1 group and 189 mm Hg (89; 71-329) in the control group. Analyses of the least squares mean relative change in PaO2/FiO2 at day 5 showed no differences between treatment groups (17% change in the IFX-1 group vs 41% in the control group; difference -24% [95% CI -58 to 9], p=0·15. Kaplan-Meier estimates of mortality by 28 days were 13% (95% CI 0-31) for the IFX-1 group and 27% (4-49) for the control group (adjusted hazard ratio for death 0·65 [95% CI 0·10-4·14]). The frequency of serious adverse events were similar between groups (nine [60%] in the IFX-1 group vs seven [47%] in the control group) and no deaths were considered related to treatment assignment. However, a smaller proportion of patients had pulmonary embolisms classed as serious in the IFX-1 group (two [13%]) than in the control group (six [40%]). Infections classed as serious were reported in three (20%) patients in the IFX-1 group versus five (33%) patients in the control group. INTERPRETATION: In this small exploratory phase 2 part of the PANAMO trial, C5a inhibition with IFX-1 appears to be safe in patients with severe COVID-19. The secondary outcome results in favour of IFX-1 are preliminary because the study was not powered on these endpoints, but they support the investigation of C5a inhibition with IFX-1 in a phase 3 trial using 28-day mortality as the primary endpoint. FUNDING: InflaRx.
RESUMEN
IL-10 is a potent, endogenous anti-inflammatory cytokine known to decrease cytokine and keratinocyte-derived chemokine (KC) expression. Traditionally, in vivo effects of IL-10 were extrapolated from studies employing systemic antibody neutralization. As a result, divergent data regarding the protective and/or harmful roles of IL-10 have been reported. In this study, we used a lung-specific, tetracycline-inducible IL-10 overexpression-transgenic (IL-10 OE) mouse to study the effects of IL-10 overexpression on Pseudomonas aeruginosa-induced lung inflammation and corresponding survival in mice. Overexpression of IL-10 in the lung significantly increased mortality. During the early phase after infection (6-hours after infection), neutrophil recruitment as well as cytokine (TNF-alpha) and chemokine (KC) expression were significantly decreased in the IL-10 OE mice, which resulted in attenuated bacterial clearance. In contrast, overzealous production of KC and TNF-alpha intensified neutrophil infiltration and increased vascular leakage in IL-10 OE mice at the later stage of infection (24 hours after infection). Neutrophil depletion showed impaired bacterial clearance in both control and IL-10 OE mice, and further enhanced mouse mortality, whereas exogenous administration of KC reversed this finding. Our data indicate that early neutrophil recruitment is important for combating bacterial infection, and that the inhibition of neutrophil recruitment by IL-10 results in insufficient bacteria clearance in the lung, leading to excessive development of inflammation and increased mortality.
Asunto(s)
Mediadores de Inflamación/metabolismo , Interleucina-10/metabolismo , Pulmón/inmunología , Infiltración Neutrófila , Neutrófilos/inmunología , Neumonía Bacteriana/inmunología , Infecciones por Pseudomonas/inmunología , Animales , Permeabilidad Capilar , Quimiocinas/metabolismo , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Humanos , Interleucina-10/genética , Pulmón/irrigación sanguínea , Pulmón/microbiología , Ratones , Ratones Transgénicos , Neumonía Bacteriana/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The role of oxidative stress has been well appreciated in the development of sepsis-induced acute lung injury (ALI). Oxidative stress in sepsis-induced ALI is believed to be initiated by products of activated lung macrophages and infiltrated neutrophils, promptly propagating to lung epithelial and endothelial cells. This leads to tissue damage and organ dysfunction. On stimulation, neutrophils (PMNs) enable their migration machinery. The lung undergoes changes favoring adhesion and transmigration of PMNs, resulting in PMN accumulation in lung, which is a characteristic of sepsis-induced ALI. Oxidative stress turns on the redox-sensitive transcription factors (NF-kappaB, AP-1), resulting in a large output of proinflammatory cytokines and chemokines, which further aggravate inflammation and oxidative stress. During the process, transcription factor nuclear factor-erythroid 2-p45-related factor 2 (Nrf2) and heme oxygenase (HO) appear to play the counterbalancing roles to limit the propagation of oxidative stress and inflammatory responses in lung. Many antioxidants have been tested to treat sepsis-induced ALI in animal models and in patients with sepsis. However, the results are inconclusive. In this article, we focus on the current understanding of the pathogenesis of sepsis-induced ALI and novel antioxidant strategies for therapeutic purposes.
Asunto(s)
Lesión Pulmonar , Oxidantes/metabolismo , Estrés Oxidativo/fisiología , Sepsis/complicaciones , Enfermedad Aguda , Animales , Antioxidantes/uso terapéutico , Hemo Oxigenasa (Desciclizante)/fisiología , Humanos , Inflamación , Mediadores de Inflamación , Enfermedades Pulmonares/inducido químicamente , Activación de Macrófagos , Macrófagos/metabolismo , Modelos Biológicos , Factor 2 Relacionado con NF-E2/fisiología , FN-kappa B/fisiología , Activación Neutrófila , Neutrófilos/metabolismo , Oxidación-Reducción , Factor de Transcripción AP-1/fisiologíaRESUMEN
Sepsis remains a serious cause of morbidity and mortality, and the pathophysiology of the disease is not clear. The definition of the clinical manifestations of sepsis is ever evolving. This review discusses the search for effective therapeutic interventions, hurdles in translational sepsis research, and new therapies in development in current clinical trials.
Asunto(s)
Sepsis/diagnóstico , Sepsis/etiología , Sepsis/terapia , Animales , Antígenos CD/fisiología , Apoptosis , Ensayos Clínicos como Asunto , Complemento C5a/fisiología , Proteína HMGB1/fisiología , Humanos , Factores Inhibidores de la Migración de Macrófagos/fisiología , Proteína C/uso terapéutico , Receptor de Anafilatoxina C5a , Receptores de Complemento/fisiologíaRESUMEN
Excessive production of the complement activation product C5a appears to be harmful during the development of sepsis in rodents. Little is known about the role of the C5a receptor (C5aR) and its presence in different organs during sepsis. Using the cecal ligation/puncture (CLP) model in mice, we show here that C5aR immunoreactivity was strikingly increased in lung, liver, kidney, and heart early in sepsis in both control and neutrophil-depleted mice. C5aR mRNA expression in these organs was also significantly increased during sepsis. Immunohistochemical analysis revealed patterns of increased C5aR expression in parenchymal cells in all four organs following CLP. Mice injected at the start of CLP with a blocking IgG to C5aR (alphaC5aR) showed dramatically improved survival when compared with animals receiving nonspecific IgG, as did mice injected with alphaC5a. In alphaC5aR-treated mice, serum levels of IL-6 and TNF-alpha and bacterial counts in various organs were significantly reduced during CLP when compared with control CLP animals. These studies demonstrate for the first time that C5aR is upregulated in lung, liver, kidney, and heart during the early phases of sepsis and that blockade of C5aR is highly protective from the lethal outcome of sepsis.
Asunto(s)
Antígenos CD/metabolismo , Receptores de Complemento/metabolismo , Sepsis/inmunología , Animales , Anticuerpos/administración & dosificación , Antígenos CD/genética , Modelos Animales de Enfermedad , Inmunohistoquímica , Riñón/inmunología , Hígado/inmunología , Pulmón/inmunología , Depleción Linfocítica , Masculino , Ratones , Ratones Endogámicos BALB C , Miocardio/inmunología , Neutrófilos/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor de Anafilatoxina C5a , Receptores de Complemento/antagonistas & inhibidores , Receptores de Complemento/genética , Sepsis/genética , Sepsis/prevención & control , Distribución Tisular , Regulación hacia ArribaRESUMEN
Delayed neutrophil apoptosis is characteristic of sepsis and may accentuate organ injury. It has been shown that PI-3K and MAPK pathways provide survival signaling in neutrophils. In this study, we demonstrate that neutrophils isolated from septic rats are resistant to apoptosis in comparison with the cells from normal animals. In contrast to normal serum, septic sera induced strong phosphorylation of AKT and p44/42 in neutrophils obtained from normal rats, resulting in marked resistance of these cells to apoptosis. Protection from apoptosis by septic sera was abrogated completely by inhibition of PI-3K and partially diminished by MEK inhibition. Increased neutrophil survival in septic rats was associated with increased levels of Bcl-xL in neutrophils and decreased levels of Bim expression. In vivo blockade of C5a in cecal ligation and puncture rats by anti-C5a antibody markedly restored the susceptibility of neutrophils to undergo apoptosis. C5a activated AKT and p44/42 and also enhanced X-linked inhibitor of apoptosis expression in neutrophils. LPS and C5a were able to induce Bcl-xL expression. Thus, neutrophil survival signals derived from effects of septic sera could be linked to activation of ERK1/2 and PI-3K, increased antiapoptotic protein expression, and ultimately, delayed neutrophil apoptosis.
Asunto(s)
Apoptosis/inmunología , Complemento C5a/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Neutrófilos/inmunología , Sepsis/inmunología , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Caspasas/inmunología , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Complemento C5a/antagonistas & inhibidores , Complemento C5a/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/inmunología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neutrófilos/enzimología , Neutrófilos/patología , Fosfatidilinositol 3-Quinasas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Ratas , Ratas Long-Evans , Sepsis/enzimología , Sepsis/patología , Proteína Inhibidora de la Apoptosis Ligada a X/inmunología , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Proteína bcl-X/inmunología , Proteína bcl-X/metabolismoRESUMEN
During experimental sepsis in rodents after cecal ligation and puncture (CLP), excessive C5a is generated, leading to interactions with C5aR, loss of innate immune functions of neutrophils, and lethality. In the current study, we have analyzed the expression of the second C5a receptor C5L2, the putative "default" or nonsignaling receptor for C5a. Rat C5L2 was cloned, and antibody was developed to C5L2 protein. After CLP, blood neutrophils showed a reduction in C5aR followed by its restoration, while C5L2 levels gradually increased, accompanied by the appearance of mRNA for C5L2. mRNA for C5L2 increased in lung and liver during CLP. Substantially increased C5L2 protein (defined by binding of 125I-anti-C5L2 IgG) occurred in lung, liver, heart, and kidney after CLP. With the use of serum IL-6 as a marker for sepsis, infusion of anti-C5aR dramatically reduced serum IL-6 levels, while anti-C5L2 caused a nearly fourfold increase in IL-6 when compared with CLP controls treated with normal IgG. When normal blood neutrophils were stimulated in vitro with LPS and C5a, the antibodies had similar effects on release of IL-6. These data provide the first evidence for a role for C5L2 in balancing the biological responses to C5a.
Asunto(s)
Complemento C5a/fisiología , Receptor de Anafilatoxina C5a/fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos/farmacología , Ciego/cirugía , Línea Celular , Clonación Molecular , Complemento C5a/genética , ADN Complementario/genética , Expresión Génica , Humanos , Interleucina-6/sangre , Riñón/química , Ligadura , Hígado/química , Pulmón/química , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Miocardio/química , Neutrófilos/química , Neutrófilos/fisiología , Punciones , ARN Mensajero/análisis , Ratas , Ratas Long-Evans , Receptor de Anafilatoxina C5a/análisis , Receptor de Anafilatoxina C5a/genética , Receptor de Anafilatoxina C5a/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sepsis/etiología , Sepsis/inmunología , Sepsis/metabolismo , TransfecciónRESUMEN
In sepsis, apoptosis occurs in many different organs. The mediators responsible for induction of apoptosis are not clearly known, although there are some suggestions that C5a and the C5a receptor (C5aR) might be directly linked to apoptosis. In the cecal ligation/puncture (CLP) model of sepsis in rats, apoptosis occurs early in a variety of organs, especially in the thymus. We demonstrate that thymocytes from normal rats show specific, saturable, and high affinity binding of 125I-labeled recombinant rat C5a. C5a binding to thymocytes was significantly increased 3 h after CLP and also when thymocytes from normal rats were first incubated in vitro with lipopolysaccharide (LPS) or IL-6. The expression of C5aR mRNA in thymocytes was markedly increased 3, 6, and 12 h after CLP and increased similarly when normal thymocytes were first exposed to LPS or IL-6 in vitro. Thymocytes obtained 2 or 3 h after CLP and exposed in vitro to C5a, but not normal thymocytes, underwent increased apoptosis, as demonstrated by annexin-V binding, coinciding with increased activation of caspases 3, 6, and 8. These data provide the first direct evidence that in the early onset of sepsis, increased expression of C5aR occurs in thymocytes, which increases their susceptibility to C5a-induced apoptosis.
Asunto(s)
Antígenos CD/metabolismo , Apoptosis/fisiología , Receptores de Complemento/metabolismo , Sepsis/fisiopatología , Timo/metabolismo , Animales , Antígenos CD/genética , Apoptosis/efectos de los fármacos , Unión Competitiva/efectos de los fármacos , Caspasa 3 , Caspasa 6 , Caspasa 8 , Caspasa 9 , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Complemento C5a/metabolismo , Complemento C5a/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-6/farmacología , Radioisótopos de Yodo , Lipopolisacáridos/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptor de Anafilatoxina C5a , Receptores de Complemento/genética , Proteínas Recombinantes/metabolismo , Timo/citología , Timo/efectos de los fármacosRESUMEN
Complement fragment 5a (C5a)-C5a receptor (C5aR) signaling plays an essential role in neutrophil innate immunity. Blockade of either the ligand or the receptor improves survival rates in experimental sepsis. In the current study, sepsis was induced in rats by cecal ligation/puncture. Early in sepsis C5aR content on neutrophils significantly dropped, reached the nadir at 24 h after onset of sepsis, and progressively elevated thereafter. Western-blot, RT-PCR, and confocal microscopy analyses revealed that the loss and re-expression of C5aR during sepsis might be due, at least in part, to the receptor internalization and reconstitution. The reduction and reconstitution of C5aR correlate with the loss and restoration of innate immune functions of blood neutrophils (chemotaxis and reactive oxygen species production), respectively. Quantitative measurements of C5aR on blood neutrophils are highly predictive of survival or death during sepsis. These data suggest that neutrophil C5aR content represents an essential component of an efficient defense system in sepsis and may serve as a prognostic marker for the outcome.
Asunto(s)
Antígenos CD/metabolismo , Neutrófilos/inmunología , Receptores de Complemento/metabolismo , Sepsis/inmunología , Animales , Complemento C5a/biosíntesis , Modelos Inmunológicos , Pronóstico , Transporte de Proteínas , Ratas , Receptor de Anafilatoxina C5a , Sepsis/diagnóstico , Análisis de SupervivenciaRESUMEN
Experimental sepsis in rodents occurring after cecal ligation/puncture (CLP) is associated with excessive complement activation and a systemic inflammatory response. The proinflammatory mediator IL-6 has recently been shown to be an important inducer of the C5a receptor (C5aR) during sepsis. We now provide evidence that serum IL-6 production during sepsis in rats was reduced in neutrophil-depleted animals and that absence of C5aR in mice as well as antibody-blockade of C5a in rats significantly reduced serum levels of IL-6 during sepsis. Lipopolysaccharide (LPS)-induced production in vitro of IL-6 by neutrophils was significantly enhanced in the co-presence of C5a, likely due to transcriptional up-regulation of IL-6. Production of IL-6 in neutrophils by LPS was NF-kappaB dependent (but not on the presence of p50) and dependent on phosphorylation of p38-mitogen activated protein kinase (MAPK) as well as p44/p42 MAPK (ERK1/2) but not on phosphorylation of c-Jun N-terminal kinases (JNK1/2). C5a stimulation of neutrophils elicited a rapid phosphorylation of ERK1/2 and p38 MAPK. Accordingly, we suggest that induction of IL-6 after CLP is neutrophil and C5a/C5aR dependent, likely due to the ability of C5a to cause activation of ERK1/2 and p38 MAPK signaling pathways.
Asunto(s)
Complemento C5a/farmacología , Lipopolisacáridos/farmacología , Animales , Complemento C5a/antagonistas & inhibidores , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-6/biosíntesis , Interleucina-6/sangre , Interleucina-6/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , FN-kappa B/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Ratas , Receptor de Anafilatoxina C5a/antagonistas & inhibidores , Receptor de Anafilatoxina C5a/genética , Receptor de Anafilatoxina C5a/metabolismo , Sepsis/sangre , Sepsis/inmunologíaRESUMEN
Innate immune functions are known to be compromised during sepsis, often with lethal consequences. There is also evidence in rats that sepsis is associated with excessive complement activation and generation of the potent anaphylatoxin C5a. In the presence of a cyclic peptide antagonist (C5aRa) to the C5a receptor (C5aR), the binding of murine 125I-C5a to murine neutrophils was reduced, the in vitro chemotactic responses of mouse neutrophils to mouse C5a were markedly diminished, the acquired defect in hydrogen peroxide (H2O2) production of C5a-exposed neutrophils was reversed, and the lung permeability index (extravascular leakage of albumin) in mice after intrapulmonary deposition of IgG immune complexes was markedly diminished. Mice that developed sepsis after cecal ligation/puncture (CLP) and were treated with C5aRa had greatly improved survival rates. These data suggest that C5aRa interferes with neutrophil responses to C5a, preventing C5a-induced compromise of innate immunity during sepsis, with greatly improved survival rates after CLP.
Asunto(s)
Inmunidad Innata/efectos de los fármacos , Oligopéptidos/farmacología , Receptores de Complemento/antagonistas & inhibidores , Sepsis/prevención & control , Animales , Antígenos CD , Quimiotaxis de Leucocito/efectos de los fármacos , Complemento C5a/metabolismo , Complemento C5a/farmacología , Relación Dosis-Respuesta a Droga , Inflamación/inmunología , Inflamación/prevención & control , Enfermedades Pulmonares/inmunología , Enfermedades Pulmonares/prevención & control , Masculino , Ratones , Neutrófilos/citología , Neutrófilos/metabolismo , Oligopéptidos/sangre , Oligopéptidos/síntesis química , Consumo de Oxígeno/efectos de los fármacos , Cavidad Peritoneal/citología , Unión Proteica/efectos de los fármacos , Receptor de Anafilatoxina C5a , Sepsis/inmunología , Sepsis/mortalidad , Tasa de SupervivenciaRESUMEN
In recent studies, evidence has been provided for complement activation early during the onset of experimental sepsis. Excessive production of the anaphylatoxin C5a thereby appears to elicit various harmful effects. Blockade of C5a or C5a receptor (C5aR) at the start of experimental sepsis has been demonstrated to greatly improve survival in rodents. There is evidence that C5a, during the onset of sepsis, enhances the production of various proinflammatory mediators in different cell types. Besides its known, other proinflammatory effects, recent work suggested an inhibitory role of C5a for innate-immune functions of phagocytic cells (phagocytosis, reactive oxygen species production, chemotaxis) during experimental sepsis. This review article provides an overview of the important role of C5a/C5aR activation for the onset and development of sepsis.
Asunto(s)
Activación de Complemento/fisiología , Complemento C5a/fisiología , Receptor de Anafilatoxina C5a/fisiología , Sepsis/inmunología , Animales , Quimiotaxis , HumanosRESUMEN
Neutrophil trafficking in lung involves transendothelial migration, migration in tissue interstitium, and transepithelial migration. In a rat model of IgG immune complex-induced lung injury, it was demonstrated that neutrophil emigration involves regulatory mechanisms including complement activation, cytokine regulation, chemokine production, activation of adhesion molecules, and their respective counter receptors. The process is presumably initiated and modulated by the production of early response cytokines and chemokines from lung cells, especially from alveolar macrophages. TNF-alpha and IL-1 up-regulate intracellular adhesion molecule-1 (ICAM-1) and E-selectin, setting the stage for neutrophil migration through endothelium. The CXC chemokines, such as macrophage inflammatory protein (MIP)-2 and cytokine-inducible neutrophil chemoattractant (CINC), constitute chemokine gradient to orchestrate neutrophil migration in lung. Complement activation induced by IgG immune complex deposition is another important event leading to neutrophil accumulation in lung. Complement activation product C5a not only plays an important role in chemoattracting neutrophils into lung, but regulates adhesion molecules, chemokines, and cytokines expression. In addition, oxidative stress may regulate neutrophil accumulation in lung by modulation of adhesion molecule activation and chemokine production. In this review, we focus on the current knowledge of the mechanisms leading to accumulation of neutrophils during acute lung injury.
Asunto(s)
Complejo Antígeno-Anticuerpo/inmunología , Inmunoglobulina G/inmunología , Activación Neutrófila/fisiología , Neutrófilos/fisiología , Neumonía/inmunología , Animales , Citocinas/inmunología , RatasRESUMEN
C5a-C5aR signaling plays an essential role in innate immunity of neutrophils. However, excessive interaction of C5a-C5aR results in harmful effects in these cells. In sepsis, robust generation of C5a occurs; blockade of either C5a or C5aR greatly improves survival in experimental sepsis following cecal ligation and puncture (CLP). The beneficial effects derived from C5a-C5aR interaction are associated with preservation of neutrophil innate immune functions (chemotaxis, phagocytosis, respiratory burst), attenuation of the inflammatory reaction, amelioration of coagulopathy, alteration in adhesion molecule expression, and modulation of apoptosis. Following CLP, C5aR expression is significantly elevated in organs, perhaps setting the stage for C5a-induced organ dysfunction. In contrast, C5aR content on neutrophils drops significantly at early stages of sepsis and progressively increases at later time points. Re-expression of C5aR on neutrophils during sepsis appears to be associated with the functional recovery of neutrophil innate immune functions. Following CLP, there is a positive correlation between C5aR content on blood neutrophils and survival of individual animals; high levels of C5aR on neutrophils are associated with survival, whereas low levels of C5aR on neutrophils predict mortality. These data suggest that in sepsis C5a-C5aR signaling is excessive, resulting in paralysis of neutrophil function. Interception of either C5a or C5aR dramatically improves survival during experimental sepsis.
Asunto(s)
Complemento C5a/metabolismo , Receptor de Anafilatoxina C5a/metabolismo , Sepsis/metabolismo , Animales , Apoptosis , Movimiento Celular , Modelos Animales de Enfermedad , Humanos , Inflamación , Modelos Biológicos , Neutrófilos/metabolismo , Unión Proteica , Estallido Respiratorio , Transducción de SeñalRESUMEN
There is evidence in sepsis, both in rodents and in humans, that activation of the complement system results in excessive production of C5a, which triggers a series of events leading to septic shock, multiorgan failure, and lethality. In rodents following cecal ligation and puncture (CLP), which induces polymicrobial sepsis, in vivo blockade of C5a using neutralizing antibodies dramatically improved survival, reduced apoptosis of lymphoid cells, and attenuated the ensuing coagulopathy. Based on these data, it seems reasonable to consider therapeutic blockade of C5a in humans entering into sepsis and septic shock. Strategies for the development of such an antibody for use in humans are presented.
RESUMEN
BACKGROUND: Hemorrhagic shock (HS) results in oxidative stress to cells and in the induction of the inflammatory response, with an increased expression of a number of proinflammatory mediators and cytokines. We tested the ability of the nitric oxide (NO) donor sodium nitroprusside (NP) to reduce tissue injury in a rodent model of uncontrolled hemorrhagic shock. METHODS: Seventy two Sprague Dawley rats weighing 250-300 g were subjected to a model of uncontrolled hemorrhagic shock. Four groups of animals were included (n = 18 per group): sham/saline, sham/NP, shock/saline, shock/NP. Experimental design consisted of the development of hemorrhagic shock (3 ml/100 g) in a 15-min period, tail amputation (75%) and drug administration at 30 min, fluid resuscitation (FR) with Ringer's lactate (RL) solution to reach a mean arterial pressure (MAP) of 40 mmHg, a hospital phase of 60 min with hemostasis and FR with LR solution to reach a MAP of 70 mmHg, and a 3-day observation phase. Treatment at the beginning of resuscitation included either normal saline (groups 1, 3) or NP (0.5 mg/kg) (groups 2, 4). The following parameters were evaluated: fluid requirements for resuscitation, liver injury tests, liver tissue myeloperoxidase (MPO), liver histology, and 3-day survival. RESULTS: NP significantly reduced fluid requirements for resuscitation (p = 0.0001). We also observed an improved statistically significant difference in tests demonstrating hepatic injury (p = 0.0001), neutrophil infiltration as evidences by liver MPO (p <0.05), and histology studies (p = 0.001). Survival was also increased from 40% in controls to 60% with NP treatment. CONCLUSIONS: These data suggest that excess NO mediates hemorrhage-induced liver injury, and that the suppression of NO with NP may reduce the pathological consequences of severe hemorrhage, possibly by scavenging superoxide (O(2)(-)), thus limiting the production of more aggressive radicals.