RESUMO
The chemokine CXCL10 is produced by many inflammatory cells found in the diseased lung and has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). The present study demonstrates elevated CXCL10 protein in the lungs of COPD patients, which appears histologically in airway smooth muscle (hASM). In primary cultured hASM cells taken from normal donors, CXCL10 protein expression was induced by IFN-gamma and TNF-alpha, cytokines reported as elevated in COPD, and a synergistic response was obtained when they were combined. TNF-alpha stimulation of hASM enhanced accumulation of CXCL10 mRNA, indicating regulation at the transcriptional level, while IFN-gamma stimulation resulted in a smaller accumulation of CXCL10 mRNA. When these cytokines were applied simultaneously, an additive effect was obtained. TNF-alpha-induced CXCL10 expression in hASM was dependent on NFkappaB activation, and a salicylanilide NFkappaB inhibitor blocked the CXCL10 expression. In contrast, IFN-gamma stimulation resulted in transient NFkappaB activation, and the inhibitor had little effect on CXCL10 expression. When these cytokines were added simultaneously, NFkappaB was activated earlier and lasted longer, and the effect was blocked by the inhibitor. These data demonstrate a potential active role for hASM in pulmonary inflammatory diseases such as COPD by producing CXCL10.
Assuntos
Quimiocinas CXC/biossíntese , Interferon gama/farmacologia , Pulmão/imunologia , Músculo Liso/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Células Cultivadas , Quimiocina CXCL10 , Sinergismo Farmacológico , Humanos , Inflamação/imunologia , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/patologia , Receptores CXCR3 , Receptores de Quimiocinas/metabolismo , Sistema Respiratório/anatomia & histologiaRESUMO
Recent advances in our understanding of the role of cytokine networks in inflammatory processes have led to the development of novel biological agents for the treatment of chronic inflammatory diseases. At the present time, significant efforts are focused on characterizing the complex signal transduction cascades that are activated by these cytokines and, in turn, regulate their expression. The transcription factor NF-kappaB is a pivotal regulator of the inducible expression of key proinflammatory mediators, and activated NF-kappaB has been observed in several debilitating inflammatory disorders, including rheumatoid arthritis and osteoarthritis. In light of its central role in inflammation, the identification of inhibitors of NF-kappaB should provide novel therapeutics for the treatment of chronic joint disease.
Assuntos
Artrite/tratamento farmacológico , NF-kappa B/antagonistas & inibidores , Animais , Artrite/patologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Humanos , NF-kappa B/fisiologia , Osteoartrite/tratamento farmacológico , Osteoartrite/patologiaRESUMO
Demonstration that IkappaB kinase 2 (IKK-2) plays a pivotal role in the nuclear factor-kappaB-regulated production of proinflammatory molecules by stimuli such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 suggests that inhibition of IKK-2 may be beneficial in the treatment of rheumatoid arthritis. In the present study, we demonstrate that a novel, potent (IC(50) = 17.9 nM), and selective inhibitor of human IKK-2, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1), inhibits lipopolysaccharide-induced human monocyte production of TNF-alpha, IL-6, and IL-8 with an IC(50) = 170 to 320 nM. Prophylactic administration of TPCA-1 at 3, 10, or 20 mg/kg, i.p., b.i.d., resulted in a dose-dependent reduction in the severity of murine collagen-induced arthritis (CIA). The significantly reduced disease severity and delay of disease onset resulting from administration of TPCA-1 at 10 mg/kg, i.p., b.i.d. were comparable to the effects of the antirheumatic drug, etanercept, when administered prophylactically at 4 mg/kg, i.p., every other day. Nuclear localization of p65, as well as levels of IL-1beta, IL-6, TNF-alpha, and interferon-gamma, were significantly reduced in the paw tissue of TPCA-1- and etanercept-treated mice. In addition, administration of TPCA-1 in vivo resulted in significantly decreased collagen-induced T cell proliferation ex vivo. Therapeutic administration of TPCA-1 at 20 mg/kg, but not at 3 or 10 mg/kg, i.p., b.i.d., significantly reduced the severity of CIA, as did etanercept administration at 12.5 mg/kg, i.p., every other day. These results suggest that reduction of proinflammatory mediators and inhibition of antigen-induced T cell proliferation are mechanisms underlying the attenuation of CIA by the IKK-2 inhibitor, TPCA-1.
Assuntos
Amidas/uso terapêutico , Antiasmáticos/uso terapêutico , Artrite Experimental/tratamento farmacológico , Citocinas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Tiofenos/uso terapêutico , Trifosfato de Adenosina/metabolismo , Amidas/farmacologia , Animais , Antiasmáticos/farmacologia , Artrite Experimental/induzido quimicamente , Artrite Experimental/metabolismo , Artrite Experimental/prevenção & controle , Ligação Competitiva , Proliferação de Células/efeitos dos fármacos , Quimiocinas/metabolismo , Colágeno , Citocinas/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Quinase I-kappa B , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Tiofenos/farmacologia , Fator de Transcrição RelA , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Marine natural products provide a rich source of chemical diversity that can be used to design and develop new, potentially useful therapeutic agents. We report here that scytonemin, a pigment isolated from cyanobacteria, is the first described small molecule inhibitor of human polo-like kinase, a serine/threonine kinase that plays an integral role in regulating the G(2)/M transition in the cell cycle. Scytonemin inhibited polo-like kinase 1 activity in a concentration-dependent manner with an IC(50) of 2 microM against the recombinant enzyme. Biochemical analysis showed that scytonemin reduced GST-polo-like kinase 1 activity in a time-independent fashion, suggesting reversibility, and with a mixed-competition mechanism with respect to ATP. Although scytonemin was less potent against protein kinase A and Tie2, a tyrosine kinase, it did inhibit other cell cycle-regulatory kinases like Myt1, checkpoint kinase 1, cyclin-dependent kinase 1/cyclin B, and protein kinase Cbeta2 with IC(50) values similar to that seen for polo-like kinase 1. Consistent with these effects, scytonemin effectively attenuated, without chemical toxicity, the growth factor- or mitogen-induced proliferation of three cell types commonly implicated in inflammatory hyperproliferation. Similarly, scytonemin (up to 10 microM) was not cytotoxic to nonproliferating endotoxin-stimulated human monocytes. In addition, Jurkat T cells treated with scytonemin were induced to undergo apoptosis in a non-cell cycle-dependent manner consistent with its activities on multiple kinases. Here we propose that scytonemin's dimeric structure, unique among natural products, may be a valuable template for the development of more potent and selective kinase inhibitors used for the treatment of hyperproliferative disorders.