The involvement of diesel exhaust particles (DEPs) in respiratory diseases was evaluated by studying their effects on two in vitro models of human airway epithelial cells. The cytotoxicity of DEPs, their phagocytosis, and the resulting immune response were investigated in a human bronchial epithelial cell line (16HBE14o−) as well as in human nasal epithelial cells in primary culture. DEP exposure induced a time- and dose-dependent membrane damage. Transmission electron microscopy showed that DEPs underwent endocytosis by epithelial cells and translocated through the epithelial cell sheet. Flow cytometric measurements allowed establishment of the time and dose dependency of this phagocytosis and its nonspecificity with different particles (DEPs, carbon black, and latex particles). DEPs also induced a time-dependent increase in interleukin-8, granulocyte-macrophage colony-stimulating factor, and interleukin-1β release. This inflammatory response occurred later than phagocytosis, and its extent seems to depend on the content of adsorbed organic compounds because carbon black had no effect on cytokine release. Furthermore, exhaust gas posttreatments, which diminished the adsorbed organic compounds, reduced the DEP-induced increase in granulocyte-macrophage colony-stimulating factor release. These results suggest that DEPs could 1) be phagocytosed by airway epithelial cells and 2) induce a specific inflammatory response.
The culture of rabbit tenocytes could be a useful model in the study of the physiopathology and pharmacotoxicology of tendons. This work was undertaken to examine the in vitro behavior of tenocytes form juvenile rabbit Achilles tendons. We report observations of the morphological and biological characteristics of primary culture and subsequent passages of rabbit tendon cells cultured in monolayer. Data obtained by electron microscopy and growth curves were complementary. After 36 passages, the generation time of tenocytes did not change and no sign of senescence could be seen. Primary culture and the first passages retained the expression of tenocyte differentiated functions, synthesis of type I collagen and decorin. Cell growth behavior was not modified upon passaging. However, when subcultured, tenocytes displayed a modulated phenotype.
The internalization of conidia of the opportunistic fungus Aspergillus fumigatus by primary cell cultures of nonprofessional phagocytes was investigated. This study is the first to show that A. fumigatus conidia were able to be engulfed by tracheal epithelial, alveolar type II, and endothelial cells. Aspergillus fumigatus is an opportunistic pathogen responsible for various respiratory diseases in normal hosts and severe invasive infections in neutropenic patients (2). The common route of infection is via inhalation of the infective particles, the conidia, from the environment. Due to their small size, the conidia can reach the lower parts of the lung, where they adhere to epithelial cells before infection develops. Alveolar macrophages and neutrophils are known to phagocytose and kill Aspergillus conidia (14) and are the main lung defense against Aspergillus infection. A mechanism for Aspergillus conidia to escape from professional phagocytes could be to be internalized by lung epithelial and endothelial cells and then to colonize the respiratory tissue and initiate vascular diffusion. In this paper, we show that incubation of A. fumigatus conidia with epithelial and endothelial cells results in internalization of conidia by both cell types. Rabbit tracheal epithelial (TE) cells (1, 9), rat alveolar type II (ATII) cells (6, 16), and human umbilical cord endothelial cells (EC) (15) were obtained and cultured as previously described. The A. fumigatus strain used in this study was G10, a pathogenic spontaneous nitrate reductase mutant of strain CBS 144-89 (12). Conidia were collected from 1-week-old culture at 25ЊC on 2% malt agar. Then they were resuspended in cell culture medium without fetal calf serum, added to epithelial cell cultures to obtain a conidium/cell ratio of 5:1, and incubated for 0.5 to 6 h at 37ЊC (15, 16). At the end of the incubation, cells were washed free of nonadherent conidia, recovered, and processed for electron microscopy by standard techniques (1, 6, 15). TE cells. The tracheobronchial epithelial (TE) cells are the first cells of the respiratory tract to encounter inhaled pathogens. Therefore, we examined the ability of A. fumigatus to enter epithelial cells of the trachea. Primary cultures of tracheal explants reconstituted an epithelial stratified outgrowth composed of basal cells, ciliated cells, and nonciliated cells; the nonciliated cells of the outgrowth did not contain secretory granules and do not secrete polysaccharides (mucins) (1). After 6 h of incubation, conidia did not adhere to the ciliated cells, since the ciliary beat removed them from the outgrowth surface, and most of the conidia were concentrated outside the
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