Increased interleukin-8 expression by cigarette smoke extract in endothelial cells

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Abstract

It has been reported that cigarette smoking worsens alcohol-induced gastric lesions through neutrophil infiltration. We hypothesize that IL-8, a potent chemotactic factor for neutrophil is likely to be involved in this ulcerogenic process. To evaluate this phenomenon, the ability of cigarette smoke extract (CSE) to induce endothelial cell expression of IL-8 was examined. Two different fractions (ethanol or chloroform soluble extracts) of CSE with their chemical types identified showed a time- and dose-dependent increase on IL-8 secretion from ECV304 cell line. Protein kinase C (PKC) inhibitor GF109203X had no effect on IL-8 response in basal secretion and also to these stimuli. Protein tyrosine kinase (PTK) inhibitor genistein and protein kinase A (PKA) inhibitor H8 at respective concentrations significantly reduced chloroform and ethanol soluble extract-induced IL-8 expression by about 34 and 35% respectively at 8 h after incubation. It is concluded that CSE increases IL-8 release from human endothelial cells through PTK and PKA activation.

Introduction

Cigarette smoking is a major risk factor in the development of lung inflammatory diseases and cancer. Our previous study had shown that passive cigarette smoking and cigarette smoke extract worsened alcohol-induced gastric mucosal damage and the action was found to be due to the increase of neutrophil infiltration (Chow et al., 1997). It is well established that cigarette smoking can increase the incidence of cancer and invasion of tumor cells. In addition, neutrophil adhesion, infiltration and angiogenic processes are the important steps in the inflammatory process and cancer development.

IL-8 is a member of the C-X-C chemokine family which exerts chemotactic effect primarily on neutrophils (Hebert and Baker, 1993, Graves and Jiang, 1995). It has been reported that IL-8 and tumor biology have been related (Opdenakker and Van Damme, 1992). Koch et al. (1992) also suggest that IL-8 is a potent angiogenic factor. It is shown that cigarette smoke extract can induce interleukin-8 release from human bronchial epithelial cells (Mio et al., 1997). It is well known that endothelial cells play an important role in inflammatory processes and tumor development, and have the potential to secrete IL-8 in physiological conditions. However, it is unknown whether cigarette smoking can increase the secretion of IL-8 from endothelial cells. In this experiment, we aimed to examine whether cigarette smoke extract could stimulate the release of IL-8 from endothelial cell line and also investigate the possible signal transduction pathway involved in this experimental model.

Section snippets

Reagents

All chemicals and reagents were purchased from Sigma™ (Sigma Chemical Company, St Louis, USA) unless specified. Protein kinase C (PKC) inhibitor GF109203X (0.05 and 5.0 μM), protein kinase A (PKA) inhibitor H8 (0.05 and 5.0 μM), and protein tyrosine kinase (PTK) inhibitor genistein (10 and 100 μM) were bought from Calbiochem™ (San Diego, USA).

Cigarette smoke extract preparation

The commercial brand ‘Camel™’ (R.J. Reynolds, Winston-Salem, NC, USA) was used in the present study. We prepared different fractions of cigarette smoke

Effects of CSE on IL-8 expression

The ability of CSE to increase IL-8 secretion in ECV304 was evaluated by analyzing ECV304 supernatants for IL-8 using an IL-8 ELISA kit. In Fig. 1, both chloroform soluble extract (Chlo E) and ethanol soluble extract (EtOH E) showed a dose-dependent increase of IL-8 expression in ECV304 cell line. To determine the IL-8 time response to CSE, kinetic expression of IL-8 from CSE-treated ECV304 was studied at different time intervals. It was found that IL-8 levels in the supernatants of

Discussion

It is well established that IL-8, a potent leukocyte chemotactic factor, is produced in many types of cells, including monocytes, keratinocytes, lymphocytes, eosinophils, fibroblasts mesothelial cells and endothelial cells. A unique feature of IL-8 when compared to other chemotactic factors, is its specificity for neutrophils both in vitro and in vivo, and is comparatively weaker on other cell types. It is shown that endogenous IL-8 secreted by activated endothelial cells can induce the

Acknowledgements

This research was supported by a RGC grant (HKU 7277/97M) awarded by the Hong Kong Research Grant council. The authors also thanked Dr Song-Ze Ding for his technical advice on the preparation of different kinase inhibitors.

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