Neutrophil-derived elastase induces TGF-beta1 secretion in human airway smooth muscle via NF-kappaB pathway.
Neutrophils are infiltrated in airways of individuals with more severe and chronic asthma, with uncertain significance. Airway smooth muscle (ASM), apart from its contractile properties, is critically involved in the pathogenesis of asthma by producing inflammatory mediators. In the present study, we investigated the impact of neutrophil-derived elastase (NE) on ASM in terms of TGF-beta1 release, and we explored the underlying mechanisms. Primary ASM cells were serum starved for 24 h before stimulation with NE (0.01-0.5 microg/ml). TGF-beta1 in supernatant was determined by ELISA and mRNA quantified by real-time RT-QPCR. NF-kappaB nuclear translocation and activation was examined by Western blotting and kappaB-2 dEGFP reporter gene assay. Association of IL-1 receptor-associated kinase (IRAK) with MyD88 was studied by co-immunoprecipitation and Toll-like receptor 4 (TLR4) determined by FACS scan and Western blotting. We demonstrated that NE enhanced TGF-beta1 release in a time-dependent manner. This induction was inhibited by actinomycin D (5 mM), cycloheximide (5 mM), and NF-kappaB inhibitors, including pyrrolidine dithiocarbamate (PDTC, 1 mM), aspirin (2.5 mM), and sodium salyicylate (2.5 mM). Stimulation with NE was rapidly followed by association of IRAK with MyD88, phosphorylation of IkappaBalpha, and nuclear translocation of p65 with increased transactivation activity. We also found that TLR4 levels were reduced upon NE treatment. These data suggest that NE upregulates TGF-beta1 gene expression and release via My88/IRAK/NF-kappaB pathway, possibly through activation of TLR4, and shed light on a potential role of neutrophils in the pathogenesis of asthma.
Lee
Kang-Yun
KY
Department of Thoracic Medicine, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, 199 Tun-Hwa N. Rd., Taipei, Taiwan.
Ho
Shu-Chuan
SC
Lin
Horng-Chyuan
HC
Lin
Shu-Min
SM
Liu
Chien-Ying
CY
Huang
Chien-Da
CD
Wang
Chun-Hua
CH
Chung
Kian Fan
KF
Kuo
Han-Pin
HP
D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism; D053592:Interleukin-1 Receptor-Associated Kinases; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D019272:Leukocyte Elastase / Q000502:physiology*; D009130:Muscle, Smooth / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D014132:Trachea / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D053773:Transforming Growth Factor beta1
D007375:Interleukin-1; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; C508960:TGFB1 protein, human; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D053592:Interleukin-1 Receptor-Associated Kinases; D017346:Protein Serine-Threonine Kinases; D019272:Leukocyte Elastase