Epithelial differentiation is a determinant in the production of eotaxin-2 and -3 by bronchial epithelial cells in response to IL-4 and IL-13

Abstract

The composition of the airway epithelium is dynamic and epithelial differentiation is regulated by endogenous mediators as well as inhaled substances. In atopic asthma the differentiation of the epithelium is altered. Various studies have addressed the ability of cultured airway epithelial cells to release the eosinophil-attractant chemokines eotaxin, eotaxin-2 and eotaxin-3 using epithelial cell lines or poorly differentiated primary cells. Since little is known about the role of the epithelial differentiation state in the response of epithelial cells to stimuli that increase production of mediators such as the eotaxins, we analyzed the effect of differentiation state on the production of the eotaxins. In particular, we investigated the effects of the Th2 cytokines IL-4 and IL-13 on eotaxin-2 and -3 production by primary human bronchial epithelial cells and examined whether their production is affected by epithelial cell differentiation using both submerged and air–liquid interface (ALI) cultures. The results show that both IL-4 and IL-13 increase eotaxin-2 and -3 mRNA expression and protein release in submerged- and ALI-cultures. Moreover, epithelial differentiation in ALI-cultures appeared an important determinant in the regulation of eotaxin-2 and -3. Mucociliary differentiation of the epithelial cells was induced by culture in the presence of a high concentration of retinoic acid (RA), whereas low concentrations of RA resulted in a flattened squamous epithelial phenotype. Mucociliary differentiated ALI-cultures expressed and released more eotaxin-3 upon stimulation with IL-4/IL-13, whereas eotaxin-2 production was predominantly found in squamous differentiated ALI-cultures. TNFα reduced IL-4-induced eotaxin-2 release in submerged cultures but not in ALI-cultures; no effects on eotaxin-3 synthesis were observed. The results indicate that epithelial differentiation is an important determinant in Th2 cytokine-induced eotaxin-2 and -3 release by airway epithelial cells. These findings may provide new insights into the role of airway epithelial differentiation and Th2 cytokines in the pathogenesis of inflammatory lung disorders such as asthma.

Introduction

Asthma is a chronic condition characterized by variable airflow obstruction, airway hyperresponsiveness and chronic inflammation. This inflammation is particularly dominated by the infiltration of eosinophils and T lymphocytes into the airways (Bousquet et al., 2000). Increased numbers of eosinophils are present in bronchial lining fluid, mucosa and peripheral blood and are indicated to correlate with disease severity and airway hyperresponsiveness (Bousquet et al., 1990). These findings implicate eosinophils as the critical effector cells in the pathogenesis of asthma. Currently a wide range of molecules involved in eosinophil recruitment, survival and activation have been described, including the family of eotaxins. In humans, the family of eotaxins consists of three members, i.e. eotaxin (CCL11), eotaxin-2 (CCL24) and eotaxin-3 (CCL26). They all act via the CCR3 receptor, which is expressed by various cell types including eosinophils (Forssmann et al., 1997, Kitaura et al., 1999), and have comparable functional properties. Eotaxin mRNA and protein levels are increased in both tissue and bronchoalveolar lavage fluid of patients with asthma after allergen challenge (Brown et al., 1998). The levels of eotaxin were found to correlate with eosinophilia and impaired lung function in asthmatics (Lilly et al., 2001, Nakamura et al., 1999). In addition to eotaxin, also increased expression of eotaxin-2 and -3 by epithelial cells in asthmatics as compared to healthy controls has been reported (Berkman et al., 2001, Komiya et al., 2003). A limited number of studies suggest that the three eotaxins differentially contribute to the recruitment of eosinophils. Mice studies demonstrated that neutralization or knocking out the eotaxin gene fails to result in a strong attenuation of eosinophilia indicating that an additional mechanism is involved (Rothenberg et al., 1997). Studies in humans showed increased levels of eotaxin and eotaxin-2 in stable asthmatics when compared to healthy controls, whereas eotaxin-3 was only significantly increased after allergen challenge (Berkman et al., 2001) suggesting a role for eotaxin-3 in the allergen-induced eosinophil recruitment. Results from our own laboratory suggested that both eotaxin-2 and -3 are involved in eosinophil recruitment following allergen exposure in asthmatics (Ravensberg et al., 2005). Finally, injection of allergen into the skin of atopic subjects revealed that eotaxin-2 expression correlated with the late-phase influx of eosinophils (Ying et al., 1999). Whereas these observations point to a role of eotaxins in asthma, data about the regulation of eotaxins is limited and predominantly based on mRNA levels. Studies using airway epithelial cell lines have shown the induction of eotaxin expression upon stimulation with TNFα and IL-1β (Lilly et al., 1997). In addition, synergistic effects of TNFα and IL-4 on eotaxin release have been reported (Atasoy et al., 2003, Matsukura et al., 1999). Studies using epithelial cells have shown that IL-4 and IL-13 also induce eotaxin-2 and eotaxin-3 mRNA and protein release in primary bronchial epithelial cells (Komiya et al., 2003), and in the human A549 alveolar type II epithelial-like cells (Abonyo et al., 2005).

Airway epithelium is increasingly recognized as an important regulator of inflammatory processes in the lung through its ability to produce a range of cytokines, chemokines, and other inflammatory mediators. The pseudostratified bronchial epithelium is composed of various cell types including basal cells, mucus producing goblet cells, ciliated cells and Clara cells (Corrin, 2000, Danel, 1996). Both in vivo and in vitro studies have demonstrated that the composition of the airway epithelium is dynamic and airway epithelial differentiation is regulated by cytokines, growth factors, and other endogenous mediators as well as inhaled substances (e.g. cigarette smoke and microbial products). Most in vitro studies examining the regulation of chemokines and cytokine release by airway epithelial cells are performed either using epithelial cell lines or submerged cultures of primary bronchial epithelial cells. Submerged cultured cells are limited in their ability to develop into fully differentiated pseudostratified epithelial layers. This is at least in part explained by the fact that: (i) they are cultured on plastic surfaces; (ii) not at an air–liquid interface. Several groups, including our own, have now developed an air–liquid interface culture system consisting of polarized cells growing on an extracellular matrix with a pseudostratified mucociliary histology that mimics many of the properties of bronchial epithelium in vivo (de Jong et al., 1993, Gray et al., 1996). However, so far little attention has been paid to the functional consequences of epithelial differentiation regarding chemokine and cytokine release.

In the present study we investigated the effect of IL-4 and IL-13 on the regulation of eotaxin, eotaxin-2 and eotaxin-3 expression by primary bronchial epithelial cells in both poorly (submerged) and well-differentiated air–liquid interface airway epithelial cell cultures. For the air–liquid interface cultures, both well-differentiated mucociliary cultures and squamous differentiated cultures were used.