Responses of well-differentiated nasal epithelial cells exposed to particles: Role of the epithelium in airway inflammation
Introduction
Individual pollutants, including diesel exhaust particles (DEP), which are the major contributors to particulate air pollution in cities, have been identified in a number of epidemiological studies as causing adverse health effects, including respiratory and cardiovascular diseases, particularly in persons with pre-existing lung disease (Atkinson et al., 2001, Pope et al., 1992). Experimental exposure to DEP causes an inflammatory response in the airways of healthy human volunteers (Diaz-Sanchez et al., 2000, Salvi et al., 1999). Moreover, deposition of atmospheric particles in the lung results in a systemic inflammatory response that includes stimulation of the bone marrow to increase production and release of polymorphonuclear leukocytes into the circulation (Tan et al., 2000, van Eeden et al., 2001). Current knowledge of the underlying physiopathological mechanisms that link particulate air pollution and cardiopulmonary diseases remains very limited. Many studies suggest that the airway epithelium plays a crucial role in initiating and augmenting pulmonary inflammatory defense mechanisms by synthesizing and releasing a variety of cellular mediators that can cause differentiation, chemotaxis and activation of inflammatory cells. Numerous in vitro studies have shown that particles, namely DEP, residual oil fly ash (ROFA) and particulate matter (PM), stimulate airway epithelial cells to increase the secretion of proinflammatory cytokines such as GM-CSF, IL-8, IL-6, IL-1β, RANTES, TNF-α, LIF and MIP-3α (Boland et al., 1999, Carter et al., 1997, Fujii et al., 2001, Kawasaki et al., 2001, Reibman et al., 2003), as well as amphiregulin, a ligand of the epidermal growth factor receptor (EGFR) (Blanchet et al., 2004). Previous results from our laboratory suggested that cytokine release resulted from the phagocytosis of particles and the activation of transduction pathways (MAPK) and transcription factors like NF-κB, which control transcription of cytokine genes (Boland et al., 1999, Boland et al., 2000, Bonvallot et al., 2000). These results were obtained mainly from experiments with immortalized human airway epithelial cell lines and primary cultures of human nasal and bronchial cells grown submerged on plastic. However, the airway epithelium differs greatly from these cultures in its morphology and in the distribution of differentiation markers. A fully differentiated normal airway epithelium is a highly polarized pseudostratified structure consisting of a tight columnar layer, comprising ciliated and secretory cells, supported by basal cells. For this reason, we chose to use the air–liquid interface (ALI) culture system in which cells seeded at low density first actively divide and then differentiate under the influence of ALI and retinoic acid (Million et al., 2001). The reconstituted epithelium obtained is pseudostratified, containing both secreting and ciliated cells, with spatially restricted apical and basolateral domains. Similar though not identical to the human airway epithelia in vivo, it may be regarded as a model for the mucociliary epithelia. Moreover, it enables the study of the vectorialization of secretions, an important parameter for understanding the relationship of the epithelium to adjacent tissues (smooth muscle cells, fibroblasts and endothelial cells of microvessels). Thus, this system offers the opportunity of answering the question: to what extent might the non-injured airway epithelium be a direct effector of local and systemic inflammation in response to contact with airborne particles? In the present study, primary ALI cultures of well-differentiated human nasal epithelial (HNE) cells, representative of the intact upper airway epithelium, were apically exposed to DEP, commonly used as a model of urban particles, and to urban Paris PM2.5. We chose to examine the effects of particles on several markers of inflammation: (i) secretion of cytokines IL-1 β and TNF-α, multifunctional cytokines of the acute inflammatory response; GM-CSF and IL-6, which are known to activate leukocytes; and IL-8, which is a powerful chemoattractant for inflammatory cells; and (ii) expression of ICAM-1 on the epithelial cell surface. Indeed, this molecule is considered to play a critical role in the attachment and migration of immune and inflammatory cells into and through the airway mucosa. We also evaluated the effects of particles on secretion of the growth factor amphiregulin: previous works in our laboratory have shown that amphiregulin secretion is increased in 16HBE cells after particle exposure. This growth factor could be involved in repair processes and airway remodeling. Moreover, we investigated intracellular ROS production and particle uptake by epithelial cells as both events are known to contribute to cellular activation leading to the inflammatory response.
Section snippets
Cell culture
Primary cultures of human nasal epithelial cells were made up according to Million et al. (2001). Human nasal turbinates (kindly supplied by Prof. Jacques Soudant, Service ORL, CHU La Pitié-Salpêtrière de Paris, France, and by Prof. Philippe Herman, Service ORL, CHU Lariboisière de Paris, France) were obtained from patients undergoing turbinectomy. Protocol was approved by the review and ethics committees of the hospitals. Briefly, turbinates were washed with Dulbecco's modified Eagle's medium
Cultures and treatments
When nasal epithelial cells in ALI differentiate through the addition of retinoic acid to the medium, secretory cells appear first, followed by ciliated cells, and the proportions of both cell types increase with time (Million et al., 2001). Fig. 1A shows ciliated cells at the surface of a differentiated culture. After confluence and after the creation of an air–liquid interface, a gradual increase in transepithelial resistance (TER) is observed, which results from the formation of tight
Discussion
Our growing understanding of the ability of particles to stimulate gene expression and production of soluble factors is derived primarily from studies on submerged primary and line cultures of airway epithelial cells grown on plastic, under conditions that result in poorly differentiated proliferating cell populations. As the physiology of transformed cells or primary cultures of poorly differentiated cells may differ from that of well-differentiated cells, we used the ALI culture system in
Acknowledgments
We thank Prof. Philippe Herman and Prof. Jacques Soudant for providing us with human nasal turbinates and Jerri Bram for English corrections. We acknowledge Karine Andréau and Armelle Baeza-Squiban for their helpful suggestions. This work has been supported by Primequal Grant II:01-J/2003.
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