Nerve growth factor secretion by human lung epithelial A549 cells in pro- and anti-inflammatory conditions

doi:10.1016/S0014-2999(01)01280-8

European Journal of Pharmacology

Volume 428, Issue 3, 12 October 2001, Pages 365-369

https://doi.org/10.1016/S0014-2999(01)01280-8 Get rights and content

Abstract

Nerve growth factor (NGF) has recently been presented as a possible effector of inflammation and bronchial hyperresponsiveness. However, the production of NGF in human airways as well as the regulation of its expression by inflammatory cytokines and glucocorticoids have received little attention. A549 epithelial cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum, and starved for 24 h. The effect of the pro-inflammatory cytokine interleukin-1β (1–30 U/ml), and of the glucocorticoid dexamethasone (1 μM) on NGF secretion was studied and quantified by enzyme-linked immunosorbent assay (ELISA). In addition, NGF production within the cells was visualized by immunocytochemistry. Under basal conditions, A549 cells produced and secreted NGF (12.6±2.0 pg/ml). Stimulation by interleukin-1β for 24 h induced a dose-dependent increase in NGF production (maximal at 10 U/ml with 59.6±3.5% increase, P<0.05). Dexamethasone (1 μM) markedly reduced the constitute NGF secretion by 44.9% (7.0±2.1 pg/ml, P<0.001). In addition, the interleukin-1β-stimulated NGF secretion was inhibited to approximately the same low level (8.5±2.5 pg/ml, P<0.001). In conclusion, we here report that human airway A549 epithelial cells are capable of producing NGF. This production is positively regulated by the pro-inflammatory interleukin-1β, and negatively regulated by dexamethasone.

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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The airway epithelium has also now been shown to be a major source of NGF and BDNF as demonstrated by their elevated levels in the lung of mice following repeated allergen challenges (Iwasaki, 2007) and in bronchial biopsies of asthmatics (Iwasaki, 2007). As evidenced by in vitro experiments, NGF expression is dramatically increased following stimulation of cultured human airway epithelial cells with proinflammatory cytokines such as IL-1β and TNF-α (Pons et al., 2001). Moreover, vascular changes were shown to occur simultaneously with epithelial remodeling, especially so in chronically inflamed tissues.
Allergic inflammation is the result of a specific pattern of cellular and humoral responses leading to the activation of the innate and adaptive immune system, which, in turn, results in physiological and structural changes affecting target tissues such as the airways and the skin. Eosinophil activation and the production of soluble mediators such as IgE antibodies are pivotal features in the pathophysiology of allergic diseases. In the past few years, however, convincing evidence has shown that neurons and other neurosensory structures are not only a target of the inflammatory process but also participate in the regulation of immune responses by actively releasing soluble mediators. The main products of these activated sensory neurons are a family of protein growth factors called neurotrophins. They were first isolated in the central nervous system and identified as important factors for the survival and differentiation of neurons during fetal and postnatal development as well as neuronal maintenance later in life. Four members of this family have been identified and well defined: nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3, and neurotrophin 4/5. Neurotrophins play a critical role in the bidirectional signaling mechanisms between immune cells and the neurosensory network structures in the airways and the skin. Pruritus and airway hyperresponsiveness, two major features of atopic dermatitis and asthma, respectively, are associated with the disruption of the neurosensory network activities. In this chapter, we provide a comprehensive description of the neuroimmune interactions underlying the pathophysiological mechanisms of allergic and inflammatory diseases.
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The inflammatory cytokines TNF-α and IL-1β, which were used in the present study as stimulants, have been detected at high levels in the PF of patients with endometriosis, and they may be important in the pathogenesis and maintenance of endometriosis (13). These inflammatory cytokines have been reported to up-regulate NGF expression in other cell types such as fibroblasts, epithelial cells, adipocytes, and smooth muscle cells (41–48). In the present study, inflammatory cytokines also up-regulated NGF in immortalized hEEC lines.
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Tumor necrosis factor-α and IL-1β induced NGF mRNA and protein and increased NGF bioactivity in the culture medium. These activities were inhibited by DNG in a hEEC line that stably expresses PR. In contrast, in an hEEC line that constitutively expresses faint levels of PR, no inhibitory effect of DNG on NGF mRNA was detected. The NGF mRNA was also inhibited in hEEC lines that express only PR-A or only PR-B.
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However, it is difficult to characterize the major source for NGF-induced AHR. Although many NGF-producing cells are involved in pathogenesis of allergic asthma,16,17 airway epithelium is commonly believed to be the main source and plays a key role in airway inflammation and remodeling by releasing inflammatory mediators and growth factors.17,18 NGF derived from epithelium cells promoted the survival of eosinophils, which initiated and maintained the allergic response.19
Airway hyperresponsiveness is the hallmark of allergic asthma and caused by multiple factors. Nerve growth factor (NGF), a neurotrophin, is originally known for regulation of neural circuit development and function. Recent studies indicated that NGF contributes to airway hyperresponsiveness and pathogenesis of asthma. The objective of this study is to develop a small interfering RNA against NGF to attenuate airway hyperresponsiveness and further elucidate the underlying mechanism. In a murine model of allergic asthma, the ovalbumin-sensitized mice were intratracheally delivered small interfering RNA against NGF or administered an inhibitor targeting NGF receptor, tropomyosin-related kinase A, as a positive treatment control. In this study, knockdown NGF derived from pulmonary epithelium significantly reduced airway resistance in vivo. The levels of NGF, proinflammatory cytokines and infiltrated eosinophils in airway were decreased in small interfering RNA against NGF group but not in tropomyosin-related kinase A inhibitor and mock siRNA group. Furthermore, induction of neuropeptide (substance P) and airway innervation were mediated by NGF/tropomyosin-related kinase A pathway. These findings suggested that NGF targeting treatment holds the potential therapy for antigen-induced airway hyperresponsiveness via attenuation of airway innervation and inflammation in asthma.
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Neurotrophins are involved in inflammatory pathways influencing several cells in healthy states and in diseases such as bronchial asthma. Recent studies have shown that nerve growth factor (NGF) is expressed in various non-neuronal cells. Furthermore, little is known about the different origins and regulation of NGF. In the present study, the expression of NGF and its regulation by dexamethasone was investigated in cultured human smooth muscle cells derived from umbilical veins (HSMC) and human iliacal arteries (HISMC).
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Nerve growth factor secretion by human lung epithelial A549 cells in pro- and anti-inflammatory conditions

Abstract

Introduction

Section snippets

Cell culture

NGF secretion under basal culture conditions

Effect of interleukin-1β on NGF secretion

Discussion

The expanding role of nerve growth factor: from neurotrophic activity to immunologic disease

Allergy

Nerve growth factor: a neurotrophin with activity on cells of the immune system

Microsc. Res. Tech.

Inflammatory mediators in asthma: an update

Pharmacol. Rev.

Release of interleukin-8, interleukin-6, and colony-stimulating factors by upper airway epithelial cells: implications for cystic fibrosis

Am. J. Respir. Cell Mol. Biol.

Expression of RANTES in human airway epithelial cells: effect of corticosteroids and interleukin-4, -10 and-13

Immunology

Circulating nerve growth factor levels are increased in human with allergic diseases and asthma

Proc. Natl. Acad. Sci. U. S. A.

Cytokines in symptomatic asthmatic airways

J. Allergy Clin. Immunol.

Nerve growth factor induces expression of certain cytokine genes and bcl-2 in mast cells: potential role in survival promotion

J. Biol. Chem.

Stretch-activated signaling of nerve growth factor secretion in bladder and vascular smooth muscle cells from hypertensive and hyperactive rats

J. Cell Physiol.

Fluticasone and budesonide inhibit cytokine release in human lung epithelial cells and alveolar macrophages

Allergy

Evaluation of human astrocytoma and glioblastoma cell lines for nerve growth factor release

Neurochem. Int.

Release of nerve growth factor from human airway epithelial cells

Am. J. Respir. Crit. Care Med.

Nerve growth factor expression by human airway smooth muscle cells in culture. Effect of the pro-inflammatory cytokine IL-1β

Fund. Clin. Pharmacol.

Nerve growth factor enhances survival and cytotoxic activity of human eosinophils

J. Haematol.

p38 MAP kinase regulates TNF alpha-, IL-1 alpha- and PAF-induced RANTES and GM-CSF production by human bronchial epithelial cells

Clin. Exp. Allergy

IL-1beta induces eotaxin gene transcription in A549 airway epithelial cells through NF-kappaB

Am. J. Physiol.

2.5S nerve growth factor enhances survival, phagocytosis, and superoxide production of murine neutrophils

Blood

Local increase in the number of mast cells and expression of nerve growth factor in the bronchus of asthmatic patients after repeated inhalation of allergen at low dose

Clin. Exp. Allergy

Nerve growth factor: from neurotrophin to neurokine

Trends Neurosci.