RESUMO
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.
Assuntos
Anticorpos Monoclonais , Tratamento Farmacológico da COVID-19 , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Ensaios Clínicos Fase II como Assunto , Complemento C3a , Complemento C5a , Humanos , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
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.
RESUMO
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.
Assuntos
Complemento C5a/imunologia , Fungos/imunologia , Micoses/imunologia , Neutrófilos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígeno CD11b/metabolismo , Candida albicans/imunologia , Candidíase/imunologia , Complemento C5a/antagonistas & inibidores , Complemento C5a/metabolismo , Humanos , Micoses/metabolismo , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/imunologia , Neutrófilos/metabolismo , Fagocitose/imunologia , Receptor da Anafilatoxina C5a/metabolismo , Fatores de TempoRESUMO
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.
RESUMO
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.
Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Complexo Antígeno-Anticorpo/imunologia , Complemento C5a/fisiologia , Interferência de RNA , RNA Interferente Pequeno/uso terapêutico , Receptor da Anafilatoxina C5a/imunologia , Animais , Brônquios/imunologia , Células Epiteliais/imunologia , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peroxidase/metabolismo , Alvéolos Pulmonares/imunologia , Receptor da Anafilatoxina C5a/genéticaRESUMO
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.
Assuntos
Mediadores da Inflamação/metabolismo , Interleucina-10/metabolismo , Pulmão/imunologia , Infiltração de Neutrófilos , Neutrófilos/imunologia , Pneumonia Bacteriana/imunologia , Infecções por Pseudomonas/imunologia , Animais , Permeabilidade Capilar , Quimiocinas/metabolismo , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Humanos , Interleucina-10/genética , Pulmão/irrigação sanguínea , Pulmão/microbiologia , Camundongos , Camundongos Transgênicos , Pneumonia Bacteriana/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Hepatite/prevenção & controle , Circulação Hepática/efeitos dos fármacos , Doadores de Óxido Nítrico/uso terapêutico , Nitroprussiato/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Choque Hemorrágico/tratamento farmacológico , Animais , Avaliação Pré-Clínica de Medicamentos , Hidratação , Hepatite/etiologia , Hepatite/fisiopatologia , Soluções Isotônicas/administração & dosagem , Soluções Isotônicas/uso terapêutico , Fígado/irrigação sanguínea , Fígado/patologia , Masculino , Modelos Biológicos , Necrose , Óxido Nítrico/fisiologia , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/análise , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/fisiopatologia , Ressuscitação , Lactato de Ringer , Choque Hemorrágico/complicações , Choque Hemorrágico/fisiopatologia , Método Simples-CegoRESUMO
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.
Assuntos
Animais , Masculino , Ratos , Choque Hemorrágico/tratamento farmacológico , Circulação Hepática/efeitos dos fármacos , Doadores de Óxido Nítrico/uso terapêutico , Hepatite/prevenção & controle , Nitroprussiato/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Avaliação Pré-Clínica de Medicamentos , Doadores de Óxido Nítrico/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Hidratação , Hepatite , Soluções Isotônicas , Fígado , Modelos Biológicos , Necrose , Nitroprussiato/farmacologia , Óxido Nítrico/fisiologia , Peroxidase/análise , Ratos Sprague-Dawley , Traumatismo por Reperfusão , Ressuscitação , Choque Hemorrágico , Método Simples-CegoRESUMO
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.
Assuntos
Lesão Pulmonar , Oxidantes/metabolismo , Estresse Oxidativo/fisiologia , Sepse/complicações , Doença Aguda , Animais , Antioxidantes/uso terapêutico , Heme Oxigenase (Desciclizante)/fisiologia , Humanos , Inflamação , Mediadores da Inflamação , Pneumopatias/induzido quimicamente , Ativação de Macrófagos , Macrófagos/metabolismo , Modelos Biológicos , Fator 2 Relacionado a NF-E2/fisiologia , NF-kappa B/fisiologia , Ativação de Neutrófilo , Neutrófilos/metabolismo , Oxirredução , Fator de Transcrição AP-1/fisiologiaRESUMO
Tumor necrosis factor (TNF)-alpha is a major cytokine produced by alveolar macrophages in response to pathogen-associated molecular patterns such as lipopolysaccharide. TNF-alpha secretion is regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation occurs by modulation of TNF-alpha mRNA stability via the binding of tristetraprolin (TTP) to the adenosine/uridine-rich elements found in the 3'-untranslated region of the TNF-alpha transcript. Phosphorylation plays important roles in modulating mRNA stability, because activation of p38 MAPK by lipopolysaccharide stabilizes TNF-alpha mRNA. We hypothesized that the protein phosphatase 2A (PP2A) regulates this signaling pathway. Our results show that inhibition of PP2A by okadaic acid or small interference RNA significantly enhanced the stability of TNF-alpha mRNA. This result was associated with increased phosphorylation of p38 MAPK and MAPK-activated kinase 2 (MK-2). PP2A inhibition increased TTP phosphorylation and enhanced complex formation with chaperone protein 14-3-3. TTP physically interacted with PP2A in transfected mammalian cells. A functional consequence of TTP-14-3-3 complex formation appeared to be protection of TTP from dephosphorylation by inhibition of the binding of PP2A to phosphorylated TTP. Mutation of the MK-2 phosphorylation sites of TTP did not influence TNF-alpha adenosine/uridine-rich element binding and did not alter the increased TNF-alpha 3'-untranslated region-dependent luciferase activity induced by PP2A-small interference RNA silencing. Our data indicate that, although phosphorylation stabilizes TNF-alpha mRNA, PP2A regulates the mRNA stability by modulating the phosphorylation state of members of the p38/MK-2/TTP pathway.
Assuntos
Proteínas 14-3-3/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Estabilidade de RNA , RNA Mensageiro/metabolismo , Tristetraprolina/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/metabolismo , Camundongos , Ácido Okadáico/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosforilação , Proteína Fosfatase 2 , RNA Interferente Pequeno/farmacologiaRESUMO
C5a, one of the most potent inflammatory peptides, induces its inflammatory functions by interacting with C5a receptor (C5aR) that belongs to the rhodopsin family of seven-transmembrane G protein-coupled receptors. C5a/C5aR signaling has been implicated in the pathogenesis of many inflammatory and immunological diseases such as sepsis and acute lung injury. Widespread upregulation of C5aR has been seen at both the protein level and transcriptional level under pathological conditions. Here, we show that C5aR gene expression can be specifically suppressed by siRNA, both in vitro and in vivo. A panel of chemically siRNA oligonucleotides was first synthesized to identify the functional siRNA sequences. The short hairpin RNAs (shRNAs) were also designed, cloned, and tested for the silencing effects in C5aR transfected cells. The effective shRNA expression cassettes were then transferred to an adenovirus DNA vector. ShRNA-expressing adenoviruses were intratracheally administered into mouse lung, and a significant in vivo silencing of C5aR was obtained four days after administration. Thus, C5aR shRNA-expressing adenoviruses appear to be an alternative strategy for the treatment of complement-induced disorders.
RESUMO
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.
Assuntos
Apoptose/imunologia , Complemento C5a/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Neutrófilos/imunologia , Sepse/imunologia , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Caspases/imunologia , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Complemento C5a/antagonistas & inibidores , Complemento C5a/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neutrófilos/enzimologia , Neutrófilos/patologia , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Ratos Long-Evans , Sepse/enzimologia , Sepse/patologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/imunologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteína bcl-X/imunologia , Proteína bcl-X/metabolismoRESUMO
Neutrophil accumulation in the lung plays a pivotal role in the pathogenesis of acute lung injury during sepsis. Directed movement of neutrophils is mediated by a group of chemoattractants, especially CXC chemokines. Local lung production of CXC chemokines is intensified during experimental sepsis induced by cecal ligation and puncture (CLP), as reflected by rising levels of MIP-2 and cytokine-induced neutrophil chemoattractant-1 in bronchoalveolar lavage fluids. Alveolar macrophages are primed and blood neutrophils are down-regulated for production of MIP-2 and cytokine-induced neutrophil chemoattractant production in response to LPS and C5a. Under these conditions of stimulation, activation of MAPKs (p38, p42/p44) occurs in sham neutrophils but not in CLP neutrophils, while under the same conditions phosphorylation of p38 and p42/p44 occurs in both sham and CLP alveolar macrophages. These data indicate that, under septic conditions, there is impaired signaling in neutrophils and enhanced signaling in alveolar macrophages, resulting in CXC chemokine production, and C5a appears to play a pivotal role in this process. As a result, CXC chemokines increase in lung, setting the stage for neutrophil accumulation in lung during sepsis.
Assuntos
Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Sepse/imunologia , Sepse/metabolismo , Transdução de Sinais/imunologia , Sequência de Aminoácidos , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Ceco , Movimento Celular/imunologia , Quimiocina CXCL2 , Quimiocinas CXC/biossíntese , Quimiocinas CXC/sangue , Quimiocinas CXC/metabolismo , Complemento C5a/antagonistas & inibidores , Complemento C5a/farmacologia , Ligadura , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/patologia , Masculino , Dados de Sequência Molecular , Neutrófilos/patologia , Punções , Ratos , Ratos Long-Evans , Receptor da Anafilatoxina C5a/biossíntese , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/fisiologia , Sepse/patologiaRESUMO
The lung inflammatory response caused by intratracheal deposition of IgG immune complexes (IC) includes the production of IL-6, which signals through activation of STAT transcription factors. Recently, suppressor of cytokine signaling 3 (SOCS3) has been shown to be a key negative regulator of IL-6/gp130/Jak/STAT3 signal transduction. Although SOCS3 has been implicated in several inflammatory diseases, very little is known regarding its activation and its function in the lung during acute inflammation. Our previous study showed that IL-6/STAT3 activation was triggered in lungs after intrapulmonary deposition of IgG IC in rats. In the current study, we sought to determine whether SOCS3 is playing a regulatory role in the lung inflammatory response. SOCS3 induction occurred during development of inflammation in the IgG IC model of lung injury. Overexpression of SOCS3 in lung using a recombinant adenovirus encoding murine SOCS3 resulted in substantial increases in lung vascular permeability and lung myeloperoxidase, together with enhanced levels of TNF-alpha, MIP-2, and keratinocyte-activated cytokine in bronchoalveolar lavage fluids. SOCS3 overexpression in lungs led to overproduction of bronchoalveolar lavage IL-6, but not IL-10, in this inflammatory model. We further show that activation of STAT3 was inhibited by SOCS3 overexpression as well as by anti-IL-6 treatment during IgG IC-induced lung injury, as determined by EMSA. In vitro, SOCS3 overexpression abrogated IL-6-induced activation of STAT3 in lung epithelial cells. These findings suggest SOCS3 is an important regulator of lung inflammatory injury after deposition of IgG IC.
Assuntos
Adenoviridae/genética , Adjuvantes Imunológicos/administração & dosagem , Complexo Antígeno-Anticorpo/administração & dosagem , Imunoglobulina G/administração & dosagem , Pulmão/imunologia , Pulmão/patologia , Proteínas Supressoras da Sinalização de Citocina/biossíntese , Proteínas Supressoras da Sinalização de Citocina/genética , Doença Aguda , Adjuvantes Imunológicos/biossíntese , Adjuvantes Imunológicos/genética , Animais , Complexo Antígeno-Anticorpo/metabolismo , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Linhagem Celular , Retroalimentação Fisiológica/genética , Retroalimentação Fisiológica/imunologia , Imunoglobulina G/metabolismo , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-6/antagonistas & inibidores , Interleucina-6/biossíntese , Contagem de Leucócitos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , NF-kappa B/fisiologia , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/administração & dosagemRESUMO
The complement activation product, C5a, is a potent inflammatory peptide with a broad spectrum of biological functions. Plasma levels of C5a are increased in sepsis, accompanied by increased content of C5a receptor (C5aR) in various organs. In the mouse and rat models of sepsis (cecal ligation and puncture, CLP), C5a blockade by anti-C5a antibody, anti-C5aR antibody or use of a C5aR antagonist (C5aRa) significantly improved survival in CLP animals. C5a blockade in sepsis attenuated the systemic inflammatory response syndrome (SIRS) by reducing plasma levels of IL-6 and decreasing bacteria counts in blood and organs. Anti-C5a treatment in CLP rodents markedly attenuated sepsis-induced defects in the coagulation/fibrinolytic system, while liver and kidney functions were remarkably preserved in contrast to CLP animals not receiving anti-C5a in which multi-organ failure occurs. In CLP rats treated with anti-C5a, thymus atrophy was diminished and thymocyte apoptosis was inhibited. Defective neutrophil functions (chemotaxis, phagocytosis, respiratory burst) caused by sepsis were significantly improved in CLP rats treated with anti-C5a. These data suggest during CLP-induced sepsis C5a has very harmful consequences and that its blockade might be a promising therapeutic strategy for the treatment of humans with sepsis. This review will summarize the beneficial effects of anti-C5a treatment in the rodent model of sepsis and will introduce the most recent patents on this line of research.
Assuntos
Complemento C5a/antagonistas & inibidores , Sepse/tratamento farmacológico , Animais , Humanos , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/prevenção & controle , Neutrófilos/efeitos dos fármacos , Patentes como Assunto , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Sepse/complicações , Sepse/mortalidade , Sobrevida , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológicoRESUMO
The complement system not only represents an effective innate immune mechanism of host defense to eradicate microbial pathogens, but it is also widely involved in many forms of acute and chronic inflammatory diseases including sepsis, acute lung injury, ischemia-reperfusion injury, and asthma, to give just a few examples. The complement-activated product, C5a, displays powerful biological activities that lead to inflammatory sequelae. C5a is a strong chemoattractant and is involved in the recruitment of inflammatory cells such as neutrophils, eosinophils, monocytes, and T lymphocytes, in activation of phagocytic cells and release of granule-based enzymes and generation of oxidants, all of which may contribute to innate immune functions or tissue damage. Accumulating data suggest that C5a provides a vital bridge between innate and adaptive immune functions, extending the roles of C5a in inflammation. Herein, we review human and animal data describing the cellular and molecular mechanisms of C5a in the development of inflammatory disorders, sepsis, acute lung injury, ischemia-reperfusion injury, and asthma.
Assuntos
Complemento C5a/imunologia , Inflamação/imunologia , Animais , Asma/imunologia , Coagulação Sanguínea/imunologia , Ativação do Complemento , Complemento C5a/antagonistas & inibidores , Humanos , Hipersensibilidade/imunologia , Imunidade Inata , Pulmão/imunologia , Lesão Pulmonar , Modelos Imunológicos , Neutrófilos/imunologia , Receptor da Anafilatoxina C5a/imunologia , Traumatismo por Reperfusão/imunologia , Sepse/imunologia , Transdução de SinaisRESUMO
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.
Assuntos
Complemento C5a/fisiologia , Receptor da Anafilatoxina C5a/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Ceco/cirurgia , Linhagem Celular , Clonagem Molecular , Complemento C5a/genética , DNA Complementar/genética , Expressão Gênica , Humanos , Interleucina-6/sangue , Rim/química , Ligadura , Fígado/química , Pulmão/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Miocárdio/química , Neutrófilos/química , Neutrófilos/fisiologia , Punções , RNA Mensageiro/análise , Ratos , Ratos Long-Evans , Receptor da Anafilatoxina C5a/análise , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse/etiologia , Sepse/imunologia , Sepse/metabolismo , TransfecçãoRESUMO
There is mounting evidence that apoptosis plays a significant role in tissue damage during acute lung injury. To evaluate the role of the apoptosis mediators Fas and FasL in acute lung injury, Fas (lpr)- or FasL (gld)-deficient and wild-type mice were challenged with intrapulmonary deposition of IgG immune complexes. Lung injury parameters ((125)I-albumin leak, accumulation of myeloperoxidase, and wet lung weights) were measured and found to be consistently reduced in both lpr and gld mice. In wild-type mice, lung injury was associated with a marked increase in Fas protein in lung. Inflamed lungs of wild-type mice showed striking evidence of activated caspase-3, which was much diminished in inflamed lungs from lpr mice. Intratracheal administration of a monoclonal Fas-activating antibody (Jo2) in wild-type mice induced MIP-2 and KC production in bronchoalveolar lavage fluids, and a murine alveolar macrophage cell line (MH-S) showed significantly increased MIP-2 production after incubation with this antibody. Bronchoalveolar lavage fluid content of MIP-2 and KC was substantially reduced in lpr mice after lung injury when compared to levels in wild-type mice. These data suggest that the Fas/FasL system regulates the acute lung inflammatory response by positively affecting CXC-chemokine production, ultimately leading to enhanced neutrophil influx and tissue damage.
Assuntos
Quimiocinas CXC/metabolismo , Inflamação/patologia , Pulmão/patologia , Glicoproteínas de Membrana/metabolismo , Receptor fas/metabolismo , Animais , Apoptose , Western Blotting , Lavagem Broncoalveolar , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Linhagem Celular , Quimiocina CXCL2 , Quimiocinas/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Proteína Ligante Fas , Imunoglobulina G/química , Pulmão/imunologia , Pulmão/metabolismo , Lesão Pulmonar , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Tamanho do Órgão , Permeabilidade , Peroxidase/metabolismoRESUMO
Sepsis is associated with extensive complement activation, compromising innate immune defenses, especially in neutrophils (PMN). Recently, a second C5a receptor (C5L2) was detected on PMN without evidence of intracellular signaling. The current study was designed to determine changes in C5L2 in blood PMN during sepsis. In vitro exposure of PMN to C5a, but not to fMLP, led to reduced content of C5L2. Following cecal ligation and puncture-induced sepsis in rats, PMN demonstrated a time-dependent decrease in C5L2. In vivo blockade of C5a during experimental sepsis resulted in preservation of C5L2. Similarly, PMN from patients with progressive sepsis showed significantly reduced C5L2 expression (n = 26), which was virtually abolished in patients who developed multiorgan failure (n = 10). In contrast, sepsis survivors exhibited retention of C5L2 (n = 12/13). The data suggest that C5L2 on PMN diminishes during sepsis due to systemic generation of C5a, which is associated with a poor prognosis.
Assuntos
Proteínas de Membrana/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores de Complemento/metabolismo , Sepse/imunologia , Sepse/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Bloqueadores/farmacologia , Ceco , Complemento C5a/antagonistas & inibidores , Complemento C5a/metabolismo , Complemento C5a/farmacologia , Humanos , Ligadura , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/biossíntese , Camundongos , Dados de Sequência Molecular , Neutrófilos/imunologia , Neutrófilos/metabolismo , Punções , Ratos , Ratos Long-Evans , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/biossíntese , Receptor da Anafilatoxina C5a/química , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/química , Receptores de Complemento/antagonistas & inibidores , Receptores de Complemento/biossíntese , Sepse/mortalidade , Choque Séptico/imunologia , Choque Séptico/metabolismo , Choque Séptico/mortalidadeRESUMO
There is evidence that C5a and macrophage migration inhibitory factor (MIF) both play important roles in experimental sepsis. Humans with sepsis also show elevated levels of both mediators in the blood. Regulation of MIF during sepsis is poorly understood. We now demonstrate that neutrophil depletion greatly reduced serum MIF levels in rats and mice during the onset of sepsis after cecal ligation and puncture. In vitro, C5a induced MIF release from rat and mouse neutrophils. In vivo blockade of C5aR or absence of C5aR led to significantly reduced MIF generation during the onset of sepsis. C5a-induced release in vitro of MIF from neutrophils appeared to be due to up-regulation of MIF in cytoplasmic granules of neutrophils via activation of the protein kinase B signaling pathway together with involvement of PI3K. Our data suggest that C5a plays a role in enhancing MIF release from neutrophils in vitro and during sepsis. These findings represent a previously unrecognized function of C5a and neutrophils in the appearance of MIF in sepsis.