Nerve growth factor secretion by human lung epithelial A549 cells in pro- and anti-inflammatory conditions
Introduction
The neurotrophin nerve growth factor (NGF) is well known for triggering nerve survival and growth (for review: Levi-Montalcini et al., 1996, Aloe et al., 1997). NGF has also a profound effect on cells of the immune system, many of which are important in the development of bronchial inflammation and asthma-related symptoms (for review: Aloe et al., 1999, Olgart and Frossard, 2000, Olgart and Frossard, 2001a). For example, NGF modulates immunological and inflammatory processes by increasing the number of mast cells in peripheral tissues (Aloe et al., 1997), by promoting mast cell differentiation (Matsuda et al., 1991), and by enhancing survival and/or activation of mast cells (Bullock and Johnson, 1996), neutrophils (Kannan et al., 1991), lymphocytes (Otten et al., 1989) and eosinophils Hamada et al., 1996, Solomon et al., 1998. NGF also enhances mediator release from various inflammatory cells including mast cells (for review: Olgart and Frossard, in press).
Recent studies suggest that NGF may play an important role in asthma. Increased levels of the neurotrophin are found in the serum of patients with allergic diseases and asthma (Bonini et al., 1996). Increased levels of NGF protein are also present in the bronchoalveolar lavage fluid of patients after segmental allergen challenge (Virchow et al., 1998). As well, enhanced levels of NGF mRNA are reported in the bronchial mucosa after repeated inhalation of allergen at low dose (Kassel et al., in press). In allergic rhinitis also, increased levels of NGF are found in the nasal lavage fluid after allergenic provocation (Sanico et al., 1999). NGF may be synthesised and secreted by structural cells, in particular fibroblasts Yoshida et al., 1992, Olgart and Frossard, 2001b and smooth muscle cells from various origins Clemow et al., 2000, Ueyama et al., 1993, Freund et al., 2001. However, little is known about secretion and regulation of NGF expression by airway epithelial cells.
In the present study, we have hypothesised that A549 epithelial cells may be a source of NGF, and have studied the regulation of NGF secretion by the pro-inflammatory and asthma-associated cytokine, interleukin-1β. Since NGF expression is known to be regulated by glucocorticoids Emmett et al., 1997, Olgart and Frossard, 2001b and since glucocorticoids generally resolve airway inflammation in asthma and allergic respiratory diseases, we have also studied the effect of the anti-inflammatory glucocorticoid dexamethasone on the constitutive and interleukin-1β-stimulated secretion of NGF by A549 cells.
Section snippets
Cell culture
A549 human lung epithelial cells (American Type Culture Collection, Rockville, MD, USA) were cultured in Dulbecco's modified Eagle's medium supplemented with F-12 (DMEM/F-12), foetal bovine serum (10%), penicillin (100 U/ml) and streptomycin (100 μg/ml) (all products from Gibco BRL, Cergy Pontoise, France). Culture was carried out in 75-cm2 flasks (Costar, Cambridge, MA, USA) in a humidified chamber at 37 °C in air containing 5% CO2 with a medium changed every other day. Confluent cells were
NGF secretion under basal culture conditions
A549 cells in culture produced NGF constitutively. A 12.6±2.0 pg/ml NGF protein secretion was measured in culture supernatants after a 24-h period. This constitutive production of NGF was confirmed in immunocytochemical studies, showing NGF-immunoreactive staining in the cytoplasm of A549 cells (Fig. 1).
Effect of interleukin-1β on NGF secretion
NGF protein secretion was significantly enhanced after a 24-h stimulation with interleukin-1β (P<0.05) (Fig. 2). This effect was dose-dependent (1–30 U/ml). Increase in NGF was maximal at 10
Discussion
Our study clearly shows that airway epithelial cells in culture are capable of producing and secreting NGF and that this secretion is increased in response to the pro-inflammatory and asthma-associated cytokine interleukin-1β. The glucocorticoid dexamethasone decreases both the constitutive and the interleukin-1β-induced secretion of NGF by A549 cells.
The demonstration of NGF secretion by the human A549 airway epithelial cells is in agreement with findings showing that structural cells of the
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