RESUMO
Efficient clearance of apoptotic cells from the lung by alveolar macrophages is important for the maintenance of tissue structure and function. Lung tissue from humans with emphysema contains increased numbers of apoptotic cells and decreased levels of vascular endothelial growth factor (VEGF). Mice treated with VEGF receptor inhibitors have increased numbers of apoptotic cells and develop emphysema. We hypothesized that VEGF regulates apoptotic cell clearance by alveolar macrophages (AM) via its interaction with VEGF receptor 1 (VEGF R1). Our data show that the uptake of apoptotic cells by murine AMs and human monocyte-derived macrophages is inhibited by depletion of VEGF and that VEGF activates Rac1. Antibody blockade or pharmacological inhibition of VEGF R1 activity also decreased apoptotic cell uptake ex vivo. Conversely, overexpression of VEGF significantly enhanced apoptotic cell uptake by AMs in vivo. These results indicate that VEGF serves a positive regulatory role via its interaction with VEGF R1 to activate Rac1 and enhance AM apoptotic cell clearance.
Assuntos
Apoptose , Pulmão/metabolismo , Macrófagos Alveolares/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Anticorpos Monoclonais , Linhagem Celular , Doxiciclina/administração & dosagem , Doxiciclina/farmacologia , Enfisema/imunologia , Enfisema/metabolismo , Humanos , Indóis/administração & dosagem , Indóis/farmacologia , Células Jurkat , Pulmão/citologia , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pirróis/administração & dosagem , Pirróis/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/imunologiaRESUMO
BACKGROUND: Alcohol abuse increases the risk for acute respiratory distress syndrome (ARDS). Efferocytosis, the clearance of apoptotic cells, is important in the resolution of inflammation and is regulated by RhoA and rho kinase (ROCK) activation. The effects of alcohol on pulmonary Rho pathway activation and efferocytosis have not been determined. We hypothesize that acute and chronic alcohol exposure impair pulmonary efferocytosis, leading to heightened inflammation during ARDS. METHODS: For in vivo experiments, C57BL/6 mice received either a single intraperitoneal injection of alcohol or chronic ethanol-in-water for 8 weeks prior to intratracheal instillation of apoptotic cells or lipopolysaccharide (LPS). Bronchoalveolar lavage (BAL) was performed for cells counts, calculation of the phagocytic index (PI), and Rho activity measurements. For in vitro studies, primary alveolar macrophages were cultured in alcohol (25-100 mM) and then co-cultured with apoptotic cells. RhoA activity was determined following alcohol exposure, and the PI was determined before and after treatment with the ROCK inhibitor, Y27632. RESULTS: Acute alcohol exposure was associated with impaired efferocytosis. Following LPS exposure, acute alcohol exposure was also associated with increased BAL neutrophils. Chronic alcohol exposure alone did not alter efferocytosis. However, following exposure to LPS, chronic alcohol exposure was associated with both impaired efferocytosis and increased BAL neutrophils. In vitro alcohol exposure caused a dose-dependent decrease in efferocytosis. Despite the fact that RhoA activity was decreased by alcohol exposure and RhoA inhibition did not alter the effects of alcohol on efferocytosis, treatment with the Rho kinase inhibitor, Y27632, reversed the effects of alcohol on efferocytosis. CONCLUSIONS: Acute alcohol exposure impairs pulmonary efferocytosis, whereas exposure to chronic alcohol is only associated with impaired efferocytosis following LPS-induced lung injury. Both forms of alcohol exposure are associated with increased alveolar neutrophil numbers in response to LPS. The acute effects of alcohol on efferocytosis appear to be mediated, at least in part, by RhoA-independent activation of ROCK. Further studies are needed to dissect the differences between the effects of acute and chronic alcohol exposure on efferocytosis and to determine the effects of alcohol on alternative activators of ROCK.
Assuntos
Apoptose/fisiologia , Etanol/efeitos adversos , Fagocitose/efeitos dos fármacos , Pneumonia/fisiopatologia , Síndrome do Desconforto Respiratório/fisiopatologia , Amidas/farmacologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Etanol/administração & dosagem , Feminino , Lipopolissacarídeos , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose/fisiologia , Pneumonia/induzido quimicamente , Piridinas/farmacologia , Síndrome do Desconforto Respiratório/induzido quimicamente , Síndrome do Desconforto Respiratório/metabolismo , Proteínas rho de Ligação ao GTP/análise , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTPRESUMO
RATIONALE: Cigarette smoke (CS) is the primary cause of chronic obstructive pulmonary disease (COPD), an effect that is, in part, due to intense oxidant stress. Clearance of apoptotic cells (efferocytosis) is a critical regulator of lung homeostasis, which is defective in smokers and in patients with COPD, suggesting a role in disease pathogenesis. OBJECTIVES: We hypothesized that CS would impair efferocytosis through oxidant-dependent activation of RhoA, a known inhibitor of this process. METHODS: We investigated the effect of CS on efferocytosis in vivo and ex vivo, using acute, subacute, and long-term mouse exposure models. MEASUREMENTS AND MAIN RESULTS: Acute and subacute CS exposure suppressed efferocytosis by alveolar macrophages in a dose-dependent, reversible, and cell type-independent manner, whereas more intense CS exposure had an irreversible effect. In contrast, CS did not alter ingestion through the Fc gamma receptor. The inhibitory effect of CS on apoptotic cell clearance depended on oxidants, because the effect was blunted in oxidant-resistant ICR mice, and was prevented by either genetic or pharmacologic antioxidant strategies in vivo and ex vivo. CS inhibited efferocytosis through oxidant-dependent activation of the RhoA-Rho kinase pathway because (1) CS activated RhoA, (2) antioxidants prevented RhoA activation by CS, and (3) inhibitors of the RhoA-Rho kinase pathway reversed the suppressive effect of CS on apoptotic cell clearance in vivo and ex vivo. CONCLUSIONS: These findings advance the hypothesis that impaired efferocytosis may contribute to the pathogenesis of COPD and suggest the therapeutic potential of drugs targeting the RhoA-Rho kinase pathway.
Assuntos
Apoptose , Macrófagos Alveolares/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Fumar/fisiopatologia , Proteínas rho de Ligação ao GTP/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Humanos , Macrófagos Alveolares/imunologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Neutrófilos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/imunologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Transdução de Sinais/efeitos dos fármacos , Fumar/imunologia , Fator de Necrose Tumoral alfa , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/efeitos dos fármacos , Proteína rhoA de Ligação ao GTPRESUMO
Efficient removal of apoptotic cells is essential for resolution of inflammation. Failure to clear dying cells can exacerbate lung injury and lead to persistent inflammation and autoimmunity. Here we show that TNFalpha blocks apoptotic cell clearance by alveolar macrophages and leads to proinflammatory responses in the lung. Compared with mice treated with intratracheal TNFalpha or exogenous apoptotic cells, mice treated with the combination of TNFalpha plus apoptotic cells demonstrated reduced apoptotic cell clearance from the lungs and increased recruitment of inflammatory leukocytes to the air spaces. Treatment with intratracheal TNFalpha had no effect on the removal of exogenous apoptotic cells from the lungs of TNFalpha receptor-1 (p55) and -2 (p75) double mutant mice and no effect on leukocyte recruitment. Bronchoalveolar lavage from mice treated with TNFalpha plus apoptotic cells contained increased levels of proinflammatory cytokines IL-6, KC, and MCP-1, but exhibited no change in levels of anti-inflammatory cytokines IL-10 and TGF-beta. Administration of TNFalpha plus apoptotic cells during LPS-induced lung injury augmented neutrophil accumulation and proinflammatory cytokine production. These findings suggest that the presence of TNFalpha in the lung can alter the response of phagocytes to apoptotic cells leading to inflammatory cell recruitment and proinflammatory mediator production.
Assuntos
Apoptose/efeitos dos fármacos , Pulmão/patologia , Pneumonia/etiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Inflamação/etiologia , Inflamação/fisiopatologia , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Leucócitos/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Macrófagos Alveolares/citologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/metabolismo , Fagocitose , Pneumonia/fisiopatologia , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Receptores Tipo II do Fator de Necrose Tumoral/fisiologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Cystic fibrosis (CF) is caused by mutated CF transmembrane conductance regulator (CFTR) and is characterized by robust airway inflammation and accumulation of apoptotic cells. Phagocytosis of apoptotic cells (efferocytosis) is a pivotal regulator of inflammation, because it prevents postapoptotic necrosis and actively suppresses release of a variety of proinflammatory mediators, including IL-8. Because CF is associated with accumulation of apoptotic cells, inappropriate levels of IL-8, and robust inflammation, we sought to determine whether CFTR deficiency specifically impairs efferocytosis and its regulation of inflammatory mediator release. Here we show that CFTR deficiency directly interferes with efferocytosis by airway epithelium, an effect that is not due to altered binding of apoptotic cells to epithelial cells or altered expression of efferocytosis receptors. In contrast, expression of RhoA, a known negative regulator of efferocytosis, is substantially increased in CFTR-deficient cells, and inhibitors of RhoA or its downstream effector Rho kinase normalize efferocytosis in these cells. Impaired efferocytosis appears to be mediated through an amiloride-sensitive ion channel, because amiloride restores phagocytic competency in CFTR-deficient cells. Finally, ineffective efferocytosis in CFTR-deficient cells appears to have proinflammatory consequences, because apoptotic cells enhance IL-8 release by these cells, but not by wild-type controls. Therefore, in CF, dysregulated efferocytosis may lead to accumulation of apoptotic cells and impaired regulation of the inflammatory response and, ultimately, may suggest a new therapeutic target.
Assuntos
Apoptose , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Mediadores da Inflamação/metabolismo , Interleucina-8/metabolismo , Fagocitose , Actinas/metabolismo , Amilorida/farmacologia , Animais , Western Blotting , Células Cultivadas , Células Epiteliais/metabolismo , Eritrócitos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Receptores de IgG/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Fibras de Estresse , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
We developed a 1-hour field enzyme immunoassay (EIA) for detecting antibody to Sin Nombre virus in deer mice (Peromyscus maniculatus). The assay specificity and sensitivity were comparable to those of a standard EIA. This test will permit identification of rodents with antibody to this and perhaps other hantaviruses.