RESUMO
RATIONALE: Allergic asthma is characterized by reversible airway obstruction, lung inflammation, and airway hyperresponsiveness (AHR). Previous studies using leukotriene B(4) (LTB(4)) receptor 1-deficient mice and adoptive transfer experiments have suggested that LTB(4) plays a role in lung inflammation and AHR. OBJECTIVES: In this study, we used a leukotriene A(4) hydrolase (LTA(4)H) inhibitor as a pharmacological tool to directly examine the role of LTB(4) in a mast cell-dependent murine model of allergic airway inflammation. METHODS: We used the forced oscillation technique to test the effects of an LTA(4)H inhibitor dosed during the challenge phase on AHR. Lung tissue and lavage were collected for analysis. MEASUREMENTS AND MAIN RESULTS: Treatment with an LTA(4)H inhibitor improved multiple parameters encompassing AHR and lung function. Significant decreases in inflammatory leukocytes, cytokines, and mucin were observed in the lung lumen. Serum levels of antigen-specific IgE and IgG1 were also decreased. Labeled antigen uptake by lung dendritic cells and subsequent trafficking to draining lymph nodes and the lung were decreased on LTA(4)H inhibitor treatment. Provocatively, inhibition of LTA(4)H increased lipoxin A(4) levels in lung lavage fluid. CONCLUSIONS: These data suggest that LTB(4) plays a key role in driving lung inflammation and AHR. Mechanistically, we provide evidence that inhibition of LTA(4)H, affects recruitment of both CD4(+) and CD8(+) T cells, as well as trafficking of dendritic cells to draining lymph nodes, and may beneficially modulate other pro- and antiinflammatory eicosanoids in the lung. Inhibition of LTA(4)H is thus a potential therapeutic strategy that could modulate key aspects of asthma.
Assuntos
Asma/fisiopatologia , Hiper-Reatividade Brônquica/fisiopatologia , Epóxido Hidrolases/antagonistas & inibidores , Leucotrieno B4/fisiologia , Animais , Líquido da Lavagem Broncoalveolar/química , Células Cultivadas , Citocinas/análise , Imunoglobulina E/sangue , Leucotrieno B4/análise , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mucinas/análise , Ovalbumina/imunologiaRESUMO
Leukotriene A(4) hydrolase (LTA(4)H) catalyzes production of the proinflammatory lipid mediator, leukotriene (LT) B(4), which is implicated in a number of inflammatory diseases. We have identified a potent and selective inhibitor of both the epoxide hydrolase and aminopeptidase activities of recombinant human LTA(4)H (IC(50), approximately 10 nM). In a murine model of arachidonic acid-induced ear inflammation, the LTA(4)H inhibitor, JNJ-26993135 (1-[4-(benzothiazol-2-yloxy)-benzyl]-piperidine-4-carboxylic acid), dose-dependently inhibited ex vivo LTB(4) production in blood, in parallel with dose-dependent inhibition of neutrophil influx (ED(50), 1-3 mg/kg) and ear edema. In murine whole blood and in zymosan-induced peritonitis, JNJ-26993135 selectively inhibited LTB(4) production, without affecting cysteinyl leukotriene production, while maintaining or increasing production of the anti-inflammatory mediator, lipoxin (LX) A(4). The 5-lipoxygenase (5-LO) inhibitor zileuton showed inhibition of LTB(4), LTC(4), and LXA(4) production. Although zileuton inhibited LTB(4) production in the peritonitis model more effectively than the LTA(4)H inhibitor, the influx of neutrophils into the peritoneum after 1 and 2 h was significantly higher in zileuton- versus JNJ-26993135-treated animals. This difference may have been mediated by the increased LXA(4) levels in the presence of the LTA(4)H inhibitor. The selective inhibition of LTB(4) production by JNJ-26993135, while increasing levels of the anti-inflammatory mediator, LXA(4), may translate to superior therapeutic efficacy versus 5-LO or 5-LO-activating protein inhibitors in LTB(4)-mediated inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Benzotiazóis/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Hidroxiureia/análogos & derivados , Inibidores de Lipoxigenase , Piperidinas/farmacologia , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Ácido Araquidônico/farmacologia , Líquido Ascítico/efeitos dos fármacos , Líquido Ascítico/metabolismo , Benzotiazóis/metabolismo , Benzotiazóis/uso terapêutico , Cães , Orelha/patologia , Edema/patologia , Edema/prevenção & controle , Eicosanoides/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Feminino , Humanos , Hidroxiureia/metabolismo , Hidroxiureia/farmacologia , Inflamação/metabolismo , Inflamação/patologia , Inflamação/prevenção & controle , Leucotrieno B4/metabolismo , Leucotrieno C4/metabolismo , Lipoxinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Infiltração de Neutrófilos/efeitos dos fármacos , Peritonite/metabolismo , Peritonite/patologia , Peritonite/prevenção & controle , Piperidinas/metabolismo , Piperidinas/uso terapêutico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Leukotriene B4 (LTB4) is a potent activator and chemoattractant for leukocytes and is implicated in several inflammatory diseases. The actions of LTB4 are mediated by two cell surface receptors, BLT1, which is predominantly expressed in peripheral blood leukocytes, and BLT2, which is expressed more ubiquitously. Recently, BLT1 expression and LTB4-dependent chemotaxis have been reported in immature mast cells (MCs). We now show the first evidence for BLT2 mRNA expression, in addition to BLT1, in murine bone marrow-derived MCs (mBMMCs) and in a human MC line (HMC-1). Protein expression of BLT1 was confirmed by mAb staining in HMC-1 cells and shown to be predominantly intracellular. Both HMC-1 cells and mBMMCs migrated to LTB4 in a dose-dependent manner in chemotaxis assays. Migration to LTB4 could be inhibited by either a BLT1- or BLT2-selective antagonist. Significant dose-dependent migration of mBMMCs also was observed to 12-(S)-hydroxyeicosotetraenoic acid, a BLT2-selective agonist, demonstrating functional BLT2 activity in these cells. Stimulation of mBMMCs with LTB4 induced transient, dose-dependent, ERK phosphorylation and changes in Akt phosphorylation. Dose-dependent ERK phosphorylation also was observed in response to 12-(S)-hydroxyeicosotetraenoic acid, indicating signaling downstream of BLT2. Pretreatment of mBMMCs with stem cell factor significantly down-regulated expression of BLT1 and BLT2 mRNA and inhibited their migration to LTB4. This study demonstrates expression of functional LTB4 receptors, both BLT1 and BLT2, in murine and human MCs and a regulatory role for stem cell factor in their expression. These receptors may mediate recruitment and accumulation of MCs in response to LTB4 production in areas of inflammation.