Neutrophil elastase induces IL-8 synthesis by lung epithelial cells via the mitogen-activated protein kinase pathway

Abstract

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.

Effects of Urinary Trypsin Inhibitor on Lipopolysaccharide-Induced Acute Lung Injury in Rabbits

“…However, LPS-induced neutrophils infiltration of lung was attenuated in the Pre-UTI high group. Previous studies reported that UTI inhibits LPS-induced production of IL-8 in vascular endothelial cells [21], neutrophil elastase-induced IL-8 gene expression [22,23], expression of intercellular adhesion molecule-1, endothelial cell adhesion molecule-1 on endothelial cells, and transendothelial migration of neutrophils stimulated by IL-8 [24]. Also, UTI not only inactivated elastase secreted by neutrophils, but also suppressed the production and secretion of elastase from neutrophils which play an important role of diapedesis and extravasation of granulocytes [21,25,26].…”

Antiplatelet Activities of Anthrax Lethal Toxin Are Associated with Suppressed p42/44 and p38 Mitogen‐Activated Protein Kinase Pathways in the Platelets

“…The "vehicle" was dimethyl sulfoxide, PBS, or both, or, in different experiments, a solvent control. The doses of reagents used were as follows: thrombin, 0.5 U/mL; collagen, 5 mg/mL; plateletactivating factor (PAF), 0.1 mmol/L; AA, 100 mmol/L; PD98059, 40 mmol/L [36,37]; SB203580, 40 mmol/L [38,39]; and SP600125, 3 mmol/L. Detection of agonist-induced platelet surface P-selectin expression by flow-cytometric analysis and ELISA.…”

THERMAL INJURY-INDUCED PRIMING EFFECT OF NEUTROPHIL IS TNF-α AND P38 DEPENDENT

“…Recent studies of the mitogen-activated protein kinase (MAPK) superfamily provide a framework for investigating the response of neutrophil to various stimulators (4). The MAPK can be categorized into 5 subfamilies: the ERK1/2, the p38, the JNK, the ERK5, and the ERK3/4 subfamilies (5).…”