Pleural Mesothelial Cells Stimulated by Asbestos Release Chemotactic Activity for NeutrophilsIn Vitro

Abstract

The development of the pleural inflammatory response to asbestos remains poorly defined. Importantly, the role of the pleural mesothelial cell in recruitment of neutrophils to the pleural space is not known. We hypothesized that rabbit pleural mesothelial cells stimulated by asbestos fibers release chemotactic factor(s) for neutrophils. Primary cultures of rabbit pleural mesothelial cells were established, and their purity verified by the presence of keratin and hyaluronic acid mucin. Mesothelial cells in serum-free media, in the presence of 30 micrograms/ml of crocidolite asbestos, released chemotaxins for neutrophils. This activity was not dependent on the type of asbestos fiber or fiber length. It was dose-dependent until 30 micrograms/ml of asbestos. The chemotactic fractions had the ability to increase both directed and random migration of neutrophils. The chemotactic activity was not present in sonicated fractions of unstimulated mesothelial cells, nor in supernates of asbestos fibers alone. Characterization of the chemotactic activity showed that it was heat stable (56 degrees C per 30 min) and sensitive to digestion with trypsin and papain. On Sephadex G-50 chromatography, it had a molecular weight between 6,000 and 9,000. Production of the chemotactic activity was inhibitable by cycloheximide. These results demonstrate that pleural mesothelial cells can actively synthesize a protein fraction with chemotactic activity for neutrophils. Production of this mesothelial cell-derived chemotactic activity for neutrophils may play an important role in the initiation of the inflammatory response of the pleura to asbestos.

Evidence of a role for mesothelial cell-derived interleukin 8 in the pathogenesis of asbestos-induced pleurisy in rabbits.

“…Third, after confirming the findings of others (8) that incubation ofcultured rabbit pleural mesothelial cells with asbestos causes the appearance of chemotactic activity in conditioned medium (Table II, Fig. 4), we demonstrated that the bulk of this chemotactic activity is attributable to IL-8 (Fig.…”

“…In animal models, neutrophilia can be produced by introducing asbestos fibers into the airspaces (2), into the peritoneal space (34), or into the pleural spaces (4,35). Of note, in other studies (8) as well as in our own, asbestos itself did not exhibit chemotactic activity. Consequently, it is the interaction of asbestos with cells or serum that is necessary for the generation ofchemotactic activity for neutrophils.…”

“…Asbestos is known to induce the generation of several chemotactic factors for neutrophils: C5a (from serum) (36); leukotriene B4 and a 1 -kD chemotactic factor (from alveolar macrophages) (37,38); and a 6-9-kD chemotactic peptide (from pleural mesothelial cells) (8). We have now shown that asbestos directly induces production ofIL-8 by pleural mesothelial cells in vitro, and that, whether directly or indirectly, asbestos also induces the production of IL-8 in vivo.…”

“…For example, mesothelial cells have been shown to phagocytose particles including asbestos fibers (5), produce prostaglandins in response to nonspecific irritants (6), and express immune-associated antigens in response to interferon-^y (7). In a recent report by Antony et al (8), rabbit pleural mesothelial cells were shown to produce a neutrophil chemotaxin, a 6-9-kD peptide. Although the identity of the peptide was not determined, this work provided evidence that pleural mesothelial cells might be a source of asbestos-induced chemotactic activity.…”

“…Pleural mesothelial cells were harvested by instilling trypsin (0.25%, wt/vol) into the pleural spaces of anesthetized New Zealand white rabbits as described previously (8). All cells were incubated at 370C in an atmosphere of 5% CO2 in medium consisting of equal parts Dulbecco's modified Eagle's medium and RPMI-1640, containing gentamicin (0.05%, wt/vol) and heat-inactivated fetal bovine serum (10%, vol/vol) (GIBCO).…”