Elsevier

Life Sciences

Volume 74, Issue 20, 2 April 2004, Pages 2479-2492
Life Sciences

Human neutrophil-derived elastase induces airway smooth muscle cell proliferation

https://doi.org/10.1016/j.lfs.2003.07.059Get rights and content

Abstract

Neutrophils and their derived elastase are abundant in chronic inflammatory responses of asthma. This study aimed to investigate the mitogenic effect of elastase on airway smooth muscle (ASM) cells and the implicated signal transduction pathway. Near confluent cultured human ASM cells were treated with human neutrophil elastase (HNE, 0.01 to 0.5 μg/ml) or vehicle for 24 hours with or without extracellular signal-regulated kinase (ERK) inhibitor (PD98059, 30 μM), p38 kinase inhibitor (SB203580, 10 μM) or elastase inhibitor II (100 μg/ml). The ASM cell numbers were counted by a hemocytometer and DNA synthesis was assessed by flowcytometry. Western blots analysis for the expression of ERK, p38 and cyclin D1 was determined. HNE dose-dependently increased ASM cell numbers and the percentage of cells entering S-phase of cell cycle. This response was abolished by neutrophil elastase inhibitors and attenuated by PD98059, but not SB203580. HNE increased ERK phosphorylation and cyclin D1 expression. Pretreatment with PD98059 significantly inhibited elastase-induced cyclin D1 activity. The increased ASM cellular gap and cell shape change by proteolytic activity of HNE may be contributory to ERK activation and therefore cell proliferation. Our results demonstrate that HNE is mitogenic for ASM cells by increasing cyclin D1 activity through ERK signaling pathway.

Introduction

Asthma is characterized by chronic airway inflammation leading to reversible bronchoconstriction, epithelial cell damage, mucus plugging, enhanced neural reflexes and infiltration of eosinophils, macrophages and lymphocytes (Djukanovic et al., 1990). Structural changes in the airways wall such as increased smooth muscle content and subepithelial fibrosis associated with matrix deposition are also present Ebina et al., 1993, Roche et al., 1989 and may contribute to persistent airway obstruction as well as bronchial hyperresponsiveness. It is now known that airway smooth muscle (ASM) cells in asthma are also capable of responding to cytokines or bradykinin to release other inflammatory mediators, in particular chemokines such as RANTES, interleukin (IL)-8 John et al., 1997, John et al., 1998, Pang and Knox, 1998, eotaxin Chung et al., 1999, Pang and Knox, 2001, monocyte chemotactic protein (MCP)-1, MCP-2, and MCP-3 (Pype et al., 1999). In addition, hyperplasic and hypertrophy ASM may synthesize epidermal growth factors (Amishima et al., 1998) and upregulate nitric oxide synthase (Patel et al., 1999) and cyclooxygenase (especially COX-2) to release a variety of mediators which act as an autocrine or paracrine to influence ASM cell proliferation Vlahos and Stewart, 1999, Hamad et al., 1999.

Airway neutrophilia remains apparent in severe asthma patients even after treatment with high doses of corticosteroids Jatakanon et al., 1999, Lamblin et al., 1998. In vitro, corticosteroids paradoxically enhance neutrophil survival by reducing apoptosis, and therefore, corticosteroid therapy may exacerbate neutrophil activity in vivo (Sampson, 2000). The role of neutrophils in pathogenesis of asthma is yet unknown. Recent research indicates that the sequestrated neutrophils in asthmatic airways release transforming growth factor beta (Chu et al., 2000), and are closely associated with the pathogenesis of airway remodeling in asthma. Neutrophils are equipped with an array of preformed compounds that are stored in the various types of neutrophil granules, and that may be released upon stimulation. Proteinases released from neutrophils have been contributory to mucus metaplasia, epithelial cell metaplasia, matrix deposition by fibroblast activation, growth factor release, and matrix metalloproteinase (MMP) activation Fischer and Voynow, 2000, Nadel et al., 1999, Van Wetering et al., 1997, Chetty et al., 1995, Nakamura et al., 1992, Vliagoftis et al., 2000. Our recent report demonstrates that adherence process mediated via interaction between β2-integrins and intercellular adhesion molecule–1 (ICAM-1) may stimulate neutrophils to release serine proteinases (Kuo et al., 2000). Among the neutrophil-derived proteinases, elastase level is elevated in the sputum in chronic asthmatics, ranging from 0.1-50 μg/ml (Vignola et al., 1998). The role of sequestrated neutrophils and their derived elastase in development of airway remodeling in asthmatic airways deserve elucidation.

The mitogen-activated protein kinases (MAPK) are important signal transduction pathways from the cell membrane to the nucleus. Three subgroups of mammalian MAPK have been molecularly characterized, including extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase (Davis, 1994). Most mitogens activate receptor-related tyrosine kinase and downstream via the ERK pathway to regulate cell cycles. The other two pathways, JNK and p38 MAPK are responsive to stress stimulation, such as cytokines, osmolarity, and UV radiation (Paul et al., 1997). Activation of ERK pathway appears to be the major candidate second messenger pathways for G-protein-coupled receptor (GPCR)- and tyrosine kinase (RTK)-coupled receptor to stimulate ASM proliferation Hershenson and Abe, 1999, Orsini et al., 1999, Hirst et al., 2000. Cyclin D1 is most closely linked to cell-cycle progression through G1 phase and commitment of cells to enter S phase (Xiong et al., 1997). Immunoneutralization and antisense studies have demonstrated that cyclin D1 is capable of shortening the G1 phase of the cell cycle, indicating that cyclin D1 is rate-limiting in G1 phase progression Jiang et al., 1993, Quelle et al., 1993, Resnitzky et al., 1994. Catalytic activation of ERKs has been shown to regulate cyclin D1 expression in ASM cells (Ramakrishnan et al., 1998). This study is therefore to investigate the effect of human neutrophil-derived elastase (HNE) on ASM cell proliferation and the possible implicated signal transduction pathway in relation to cyclin D1 activity.

Section snippets

Preparation of cultured human ASM cells

Human ASM cells were obtained from Clonetics, (San Diego, CA) and prepared according to the manufacturer's instruction. Cells were grown in smooth muscle basal medium (SMBM) with 5% fetal bovine serum (FBS), insulin (5 μg/ml), epidermal growth factor (EGF, 0.5 ng/ml), fibroblast growth factor (FGF, 2 ng/ml), gentamicin (50 ng/ml), amphotericin B (50 ng/ml). Cells were maintained in Falcon culture flasks and incubated (37 °C; 5% CO2) until monolayer confluence was reached. They were harvested

Effects of HNE on ASM cell proliferation

HNE induced human ASM cell proliferation in a dose-dependent fashion, and significantly at the concentration of 0.05 μg/ml (2.6 ± 0.1 ×104 cells, n = 5, p < 0.05), 0.1 μg/ml (2.9 ± 0.1 ×104 cells, n = 5, p < 0.01), and 0.5 μg/ml (4.0 ± 0.2 ×104 cells, n = 5, p < 0.01) compared with the control (1.8 ± 0.3 ×104 cells, n = 5) (Fig. 1). This effect was attenuated by pretreatment with neutrophil elastase inhibitor II (100 μg/ml) (Fig. 1) or a protease inhibitor cocktail (500 μM AEBSF, HCl, 150 nM

Discussion

ASM cell proliferation contributes to airway remodeling as a consequence of chronic airway inflammation in asthma. The responsible growth factors and cytokines or their encoding mRNAs for ASM cell proliferation have been detected in BAL fluid or sputum obtained from asthmatic subjects Redington et al., 1997, Miadonna et al., 1997. HNE is also found in the sputum of patients with asthma (Vignola et al., 1998). Our study has demonstrated that HNE dose-dependently increases human ASM cell number.

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