Neutrophil elastase induces IL-8 synthesis by lung epithelial cells via the mitogen-activated protein kinase pathway.
The sequestration of neutrophils in the lung and the release of proinflammatory mediators, including neutrophil elastase, are responsible for sepsis-induced microvascular permeability and alveolar epithelial cell damage. To assess the underlying mechanism, human neutrophil elastase (0.01-0.5 microg/ml) was added to cultured A549 epithelial cells in the presence or absence of inhibitors. IL-8 was analyzed by ELISA or by RT-PCR to measure the IL-8 synthesis capacity. Mitogen-activated protein kinase (MAPK) activity was detected by Western blot analysis. Neutrophil elastase dose-dependently increased IL-8 release from cultured A549 epithelial cells. Pretreatment with a specific elastase inhibitor, elastase inhibitor II (at 0.5, 5, and 50 microg/ml), dose-dependently inhibited neutrophil elastase-induced IL-8 release. The activities of MAPK, p38, and extracellular signal-regulated kinase (ERK) were upregulated by neutrophil elastase. Nuclear transcriptional factor-kappa B (NF-kappaB) and activator protein 1 (AP-1) were also activated. These responses were significantly inhibited by elastase inhibitor II. A specific inhibitor of p38 MAPK (SB203580) and an NF-kappaB inhibitor (pyrrolidine dithiocarbamate), but not an ERK inhibitor (PD 98059), significantly inhibited neutrophil elastase-induced IL-8 release and mRNA expression. The specific tyrosine kinase inhibitor, genistein, and the protein kinase C (PKC) inhibitor, Ro 31-8220, also inhibited IL-8 release and mRNA expression as well as p38 and NF-kappaB activation. There was no significant effect by the protein kinase A inhibitor, H-89, on neutrophil elastase-induced IL-8 synthesis or p38 MAPK activation. Our results indicate that neutrophil elastase activates p38 MAPK which upregulates NF-kappaB and AP-1 activities, thus inducing IL-8 mRNA expression and protein synthesis. Tyrosine kinase and PKC are implicated in neutrophil elastase activation of the MAPK pathway.
Chen
Hao-Cheng
HC
Department of Thoracic Medicine II, Chang Gung Memorial Hospital, 199 Tun Hwa N. Road, Taipei 10507, Taiwan, ROC.
Lin
Horng-Chyuan
HC
Liu
Chien-Ying
CY
Wang
Chun-Hua
CH
Hwang
Tritium
T
Huang
Tzu-Ting
TT
Lin
Chien-Huang
CH
Kuo
Han-Pin
HP
D002448:Cell Adhesion; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism*; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D052002:NF-kappa B p50 Subunit; D009504:Neutrophils / Q000201:enzymology*; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism*; D051996:Transcription Factor RelA; D014157:Transcription Factors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D016209:Interleukin-8; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D051996:Transcription Factor RelA; D014157:Transcription Factors; D020928:Mitogen-Activated Protein Kinases; D019272:Leukocyte Elastase