RESUMEN
The p38 mitogen-activated protein kinase (MAPK) plays a critical role in the activation of inflammatory cells. Therefore, we investigated the antiinflammatory effects of a respirable p38alpha MAPK antisense oligonucleotide (p38alpha-ASO) in a mouse asthma model. A potent and selective p38alpha-ASO was characterized in vitro. Inhalation of aerosolized p38alpha-ASO using an aerosol chamber dosing system produced measurable lung deposition of ASO and significant reduction of ovalbumin (OVA-)-induced increases in total cells, eosinophils, and interleukin 4 (IL-4), IL-5, and IL-13 levels in bronchoalveolar lavage fluid, and dose-dependent inhibition of airway hyperresponsiveness in allergen-challenged mice. Furthermore, inhaled p38alpha-ASO markedly inhibited OVA-induced lung tissue eosinophilia and airway mucus hypersecretion. Quantitative polymerase chain reaction analysis of bronchoalveolar lavage fluid cells and peribronchial lymph node cells showed that p38alpha-ASO significantly reduced p38alpha MAPK mRNA expression. Nose-only aerosol exposure of mice verified the p38alpha-ASO-induced inhibition of OVA-induced pulmonary eosinophilia, mucus hypersecretion, and airway hyperresponsiveness. None of the effects of the p38alpha-ASO were produced by a six-base mismatched control oligonucleotide. These findings demonstrate antisense pharmacodynamic activity in the airways after aerosol delivery and suggest that a p38alpha MAPK ASO approach may have therapeutic potential for asthma and other inflammatory lung diseases.
Asunto(s)
Asma/prevención & control , Proteína Quinasa 14 Activada por Mitógenos/administración & dosificación , Oligonucleótidos Antisentido/administración & dosificación , Administración por Inhalación , Aerosoles , Animales , Asma/fisiopatología , Hiperreactividad Bronquial , Líquido del Lavado Bronquioalveolar , Masculino , Ratones , Ratones Endogámicos BALB C , Moco/metabolismo , Ovalbúmina/inmunología , Reacción en Cadena de la Polimerasa , Eosinofilia Pulmonar/prevención & controlRESUMEN
CC chemokine receptor 1 (CCR1) has been implicated in inflammation. The present study examined the signaling mechanisms that mediate GM-CSF/IL-10-induced synergistic CCR1 protein expression in monocytic U937 cells. GM-CSF alone markedly increased both the mRNA and protein expression of CCR1. IL-10 augmented GM-CSF-induced CCR1 protein expression with no effect on mRNA expression. PD098059 and U0126 (two MEK inhibitors), and LY294002 (a PI3K inhibitor) inhibited GM-CSF/IL-10-induced CCR1 gene and protein expression. PD098059, U0126, and LY294002 also attenuated chemotaxis of GM-CSF/IL-10-primed U937 cells in response to MIP-1alpha. Immunoblotting studies show that GM-CSF alone induced ERK2 phosphorylation; whereas, IL-10 alone induced p70(S6k) phosphorylation in U937 cells. Neither cytokine when used alone induced PKB/Akt phosphorylation. Combined GM-CSF/IL-10 treatment of U937 cells induced phosphorylation of ERK2, p70(S6k), and PKB/Akt. PD098059 and U0126 completely abrogated ERK2 phosphorylation; whereas, LY294002 completely blocked PKB/Akt and p70(S6k) phosphorylation. Our findings indicate that IL-10 may potentiate GM-CSF-induced CCR1 protein expression in U937 cells via activation of PKB/Akt and p70(S6k).
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interleucina-10/farmacología , Monocitos/inmunología , Proteínas Serina-Treonina Quinasas , Receptores de Quimiocina/biosíntesis , Quimiocina CCL3 , Quimiocina CCL4 , Quimiotaxis de Leucocito , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Humanos , Proteínas Inflamatorias de Macrófagos/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monocitos/efectos de los fármacos , Células Mieloides/efectos de los fármacos , Células Mieloides/inmunología , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , ARN Mensajero/biosíntesis , Receptores CCR1 , Receptores de Quimiocina/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Células U937RESUMEN
Airway remodeling is one of the major hallmarks of asthma. The present study examined the effects of tyrosine kinase inhibitors on thrombin-induced guinea pig ASM cell proliferation, in comparison with inhibitors of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K). The ASM cells expressed smooth muscle alpha-actin and myosin, and responded to thrombin by increasing cytosolic Ca(+2). Thrombin (1-10 U/ml) induced [(3)H]thymidine incorporation into ASM cells. Tyrphostin 47, a broad-spectrum tyrosine kinase inhibitor, PP2, a Src-specific inhibitor, and piceatannol, a Syk-selective inhibitor, significantly attenuated thrombin-induced [(3)H]thymidine incorporation. In addition, the tyrosine kinase inhibitors significantly reduced thrombin-induced cyclin D(1) expression in ASM cells. PD098059 and U0126, two MAPK kinase inhibitors, and LY294002, a PI3K inhibitor, significantly blocked thrombin-induced [(3)H]thymidine incorporation and cyclin D(1) expression in ASM cells. Our data show that inhibitors of Src and, probably Syk, can modulate thrombin-induced ASM cell proliferation, which may have therapeutic potential for asthma.