Background-Pollens are important triggers for asthma but the mechanism of sensitisation to their proteins remains poorly understood. The intrinsic protease activity of some allergens may contribute to sensitisation by disrupting the integrity of the airway epithelial barrier. Pollens release a variety of enzymes, including proteases, upon hydration. The hypothesis that such enzymes might be able to damage airway epithelial cells was therefore tested. Methods-DiVusates from pollens of Lolium perenne (ryegrass), Poa pratensis (Kentucky bluegrass), Acacia longifolia (Sydney golden wattle), or Casuarina distyla (she-oak) were incubated with mouse tracheal epithelial cells in culture and cellular detachment was quantified using a methylene blue dye binding assay. Results-DiVusates prepared using 100 mg/ml of pollen caused detachment of 30-90% of airway epithelial cells in separate experiments. Within each experiment comparable detachment was observed with all diVusates tested, although total protein in the diVusates varied markedly between species. Viability of the cells recovered after exposure to Acacia diVusate was higher than after detachment by exposure to Lolium diVusate. Cellular detachment by all of the diVusates could be almost completely inhibited by addition of 10% serum. Aprotinin, an inhibitor of serine proteases, partially blocked activity in diVusates of Lolium pollen but not of Acacia pollen. In contrast, 1 -protease inhibitor and secretory leucocyte protease inhibitor (SLPI) were not able to block the activity of either diVusate at concentrations which inhibited cellular detachment by trypsin. Conclusions-Proteases released by pollens are able to cause detachment of airway epithelial cells from their substratum in vitro and may not be eVectively inhibited by endogenous antiproteases.

Numerous investigators have described maintenance of airway epithelial cells from various species in a differentiated state in primary culture. Because the number of cells that can be isolated from the mouse trachea is very small, published techniques are unsuitable for this species. To examine the production of growth factors by murine airway epithelial cells, the authors developed a method for culture of mouse tracheal epithelial cells from explants, in which the population of cells was expanded in the presence of epidermal growth factor and insulin-like growth factor-I, which exhibited synergistic mitogenic activity. After subculture, an essentially pure population of epithelial cells was recovered, with a yield approximately tenfold greater than reported using protease dissociation of cells from the trachea. Culture of the cells at passage 2 on a collagen gel substratum induced differentiation toward a synthetic/secretory phenotype, accompanied by marked diminution in spontaneous and mitogen-induced DNA synthesis without loss of viability. In parallel, secretion of immunoreactive transforming growth factor-beta by the epithelial cells was strikingly increased, but could be partially down-regulated in the presence of mitogenic growth factors.

We have investigated whether enhanced secretion of transforming growth factor-beta (TGF-beta) by distal respiratory epithelial cells was associated with the development of bleomycin-induced pulmonary fibrosis. Type 2 pneumocyte-enriched preparations of bronchioloalveolar epithelial cells from normal mouse lung tissue released latent TGF-beta when cultured in serum-free medium. TGF-beta in culture supernatants could be detected using a sensitive enzyme immunoassay which employed enzyme complex amplification as a reporter system, as well as by a radiolabelled receptor competition assay. Exposure to bleomycin and other potentially fibrogenic stimuli in vitro did not stimulate production of TGF-beta by the epithelial cells but release was enhanced by treatment of the cells with interferon-gamma. Type 2 pneumocyte-enriched cell preparations obtained following induction of a pulmonary inflammatory response by administration of intratracheal bleomycin to susceptible C57BL/6 mice did not demonstrate increased release of TGF-beta in culture. However, the concentration of TGF-beta in bronchoalveolar lavage (BAL) fluids was significantly elevated compared to controls at 1 and 2 weeks after bleomycin-induced injury in these mice. No such increase was detected in BAL fluids from BALB/c mice, which are resistant to the effects of bleomycin. These results provide no support for a pathogenetic role of alveolar epithelial cell-derived TGF-beta in bleomycin-induced pulmonary fibrosis. Nevertheless, elevated levels of TGF-beta in BAL fluids may provide a marker of the progression of pulmonary injury to fibrosis.

The secretion of molecular species related to epidermal growth factor (EGF) by pulmonary alveolar and parenchymal macrophages was investigated in an experimental model of pulmonary fibrosis in mice. Macrophages were isolated from cells obtained by bronchoalveolar lavage or by enzymatic disaggregation of lung tissue at intervals following induction of pulmonary injury by intratracheal injection of bleomycin. Production of EGF receptor-binding activity by these cells and concentrations of this activity in bronchoalveolar lavage fluid were measured using a radioreceptor assay. Following short-term culture under serum-free conditions, there was significantly increased production of EGF receptor-binding activity by parenchymal macrophages, which was demonstrable at 1 and 2 weeks after administration of bleomycin to susceptible C57BL/6 mice. The activity exhibited affinity for heparin and was completely blocked by an antibody to EGF. There was no such increase in production of receptor-binding activity by alveolar macrophages or in the concentration of activity in lavage fluids. Nor was there any significant increase in production of EGF receptor-binding activity by parenchymal macrophages from bleomycin-resistant BALB/c mice. These results imply that selective activation of interstitial macrophages to secrete an EGF-like growth factor may contribute to the development of pulmonary fibrosis.