RESUMO
Tumor necrosis factor α-converting enzyme (TACE) is responsible for the shedding of cell surface TNF. Studies suggest that reactive oxygen species (ROS) mediate up-regulation of TACE activity by direct oxidization or modification of the protein. However, these investigations have been largely based upon nonphysiological stimulation of promonocytic cell lines which may respond and process TACE differently from primary cells. Furthermore, investigators have relied upon TACE substrate shedding as a surrogate for activity quantification. We addressed these concerns, employing a direct, cell-based fluorometric assay to investigate the regulation of TACE catalytic activity on freshly isolated primary human monocytes during LPS stimulation. We hypothesized that ROS mediate up-regulation of TACE activity indirectly, by activation of intracellular signaling pathways. LPS up-regulated TACE activity rapidly (within 30 min) without changing cell surface TACE expression. Scavenging of ROS or inhibiting their production by flavoprotein oxidoreductases significantly attenuated LPS-induced TACE activity up-regulation. Exogenous ROS (H(2)O(2)) also up-regulated TACE activity with similar kinetics and magnitude as LPS. H(2)O(2)- and LPS-induced TACE activity up-regulation were effectively abolished by a variety of selective p38 MAPK inhibitors. Activation of p38 was redox-sensitive as H(2)O(2) caused p38 phosphorylation, and ROS scavenging significantly reduced LPS-induced phospho-p38 expression. Inhibition of the p38 substrate, MAPK-activated protein kinase 2, completely attenuated TACE activity up-regulation, whereas inhibition of ERK had little effect. Lastly, inhibition of cell surface oxidoreductases prevented TACE activity up-regulation distal to p38 activation. In conclusion, our data indicate that in primary human monocytes, ROS mediate LPS-induced up-regulation of TACE activity indirectly through activation of the p38 signaling pathway.
Assuntos
Proteínas ADAM/biossíntese , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Monócitos/enzimologia , Oxidantes/farmacologia , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteína ADAM17 , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Peróxido de Hidrogênio/metabolismo , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Monócitos/citologia , Oxidantes/metabolismo , Regulação para Cima/fisiologiaRESUMO
Margination and activation of monocytes within the pulmonary microcirculation contribute substantially to the development of acute lung injury in mice. The enhanced LPS-induced TNF expression exhibited by Gr-1(high) compared with Gr-1(low) monocytes within the lung microvasculature suggests differential roles for these subsets. We investigated the mechanisms responsible for such heterogeneity of lung-marginated monocyte proinflammatory response using a combined in vitro and in vivo approach. The monocyte subset inflammatory response was studied in vitro in mouse peripheral blood mononuclear cell-lung endothelial cell coculture and in vivo in a two-hit model of intravenous LPS-induced monocyte margination and lung inflammation in mice, by flow cytometry-based quantification of proinflammatory genes and intracellular phospho-kinases. With LPS stimulation in vitro, TNF expression was consistently higher in Gr-1(high) than Gr-1(low) monocytes, markedly enhanced by coculture with endothelial cells, and abrogated by p38 MAPK inhibitors. Expression of IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) was only detectable under coculture conditions, was substantially higher in Gr-1(high) monocytes, and was attenuated by p38 inhibition. Consistent with these differential responses, phosphorylation of p38 and its substrate MAPK-activated protein kinase 2 (MK2) was significantly higher in the Gr-1(high) subset. In vivo, p38 inhibitor treatment significantly attenuated LPS-induced TNF expression in "lung-marginated" Gr-1(high) monocytes. LPS-induced p38/MK2 phosphorylation was higher in lung-marginated Gr-1(high) than Gr-1(low) monocytes and neutrophils, mirroring TNF expression. These results indicate that the p38/MK2 pathway is a critical determinant of elevated Gr-1(high) subset responsiveness within the lung microvasculature, producing a coordinated proinflammatory response that places Gr-1(high) monocytes as key orchestrators of pulmonary microvascular inflammation and injury.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/imunologia , Microvasos/imunologia , Monócitos/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/fisiopatologia , Animais , Linhagem da Célula , Técnicas de Cocultura , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Citometria de Fluxo , Inflamação/imunologia , Inflamação/fisiopatologia , Interleucina-6/análise , Interleucina-6/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/farmacologia , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
TNF plays a crucial role in the pathogenesis of acute lung injury. However, the expression profile of its two receptors, p55 and p75, on pulmonary endothelium and their influence on TNF signaling during lung microvascular inflammation remain uncertain. Using flow cytometry, we characterized the expression profile of TNF receptors on the surface of freshly harvested pulmonary endothelial cells (PECs) from mice and found expression of both receptors with dominance of p55. To investigate the impact of stimulating individual TNF receptors, we treated wild-type and TNF receptor knockout mice with intravenous TNF and determined surface expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1) on PECs by flow cytometry. TNF-induced upregulation of all adhesion molecules was substantially attenuated by absence of p55, whereas lack of p75 had a similar but smaller effect that varied between adhesion molecules. Selective blockade of individual TNF receptors by specific antibodies in wild-type primary PEC culture confirmed that the in vivo findings were due to direct effects of TNF receptor inhibition on endothelium and not other cells (e.g., circulating leukocytes). Finally, we found that PEC surface expression of p55 dramatically decreased in the early stages of endotoxemia following intravenous LPS, while no change in p75 expression was detected. These data demonstrate a crucial in vivo role of p55 and an auxiliary role of p75 in TNF-mediated adhesion molecule upregulation on PECs. It is possible that the importance of the individual receptors varies at different stages of pulmonary microvascular inflammation following changes in their relative expression.
Assuntos
Células Endoteliais/metabolismo , Pulmão/fisiologia , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologia , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Animais , Selectina E/metabolismo , Endotoxemia/fisiopatologia , Molécula 1 de Adesão Intercelular/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumonia/fisiopatologia , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
The specialized role of mouse Gr-1(high) monocytes in local inflammatory reactions has been well documented, but the trafficking and responsiveness of this subset during systemic inflammation and their contribution to sepsis-related organ injury has not been investigated. Using flow cytometry, we studied monocyte subset margination to the pulmonary microcirculation during subclinical endotoxemia in mice and investigated whether marginated monocytes contribute to lung injury in response to further septic stimuli. Subclinical low-dose i.v. LPS induced a rapid (within 2 h), large-scale mobilization of bone marrow Gr-1high monocytes and their prolonged margination to the lungs. With secondary LPS challenge, membrane TNF expression on these premarginated monocytes substantially increased, indicating their functional priming in vivo. Zymosan challenge produced small increases in pulmonary vascular permeability, which were markedly enhanced by the preadministration of low-dose LPS. The LPS-zymosan-induced permeability increases were effectively abrogated by pretreatment (30 min before zymosan challenge) with the platelet-activating factor antagonist WEB 2086 in combination with the phosphatidylcholine-phospholipase C inhibitor D609, suggesting the involvement of platelet-activating factor/ceramide-mediated pathways in this model. Depletion of monocytes (at 18 h after clodronate-liposome treatment) significantly attenuated the LPS-zymosan-induced permeability increase. However, restoration of normal LPS-induced Gr-1high monocyte margination to the lungs (at 48 h after clodronate-liposome treatment) resulted in the loss of this protective effect. These results demonstrate that mobilization and margination of Gr-1high monocytes during subclinical endotoxemia primes the lungs toward further septic stimuli and suggest a central role for this monocyte subset in the development of sepsis-related acute lung injury.