The nonciliated bronchiolar epithelial (Clara) cell of the lung remains a poor characterized cell with respect to its physiologic role in the airway. A major barrier to elucidation of the Clara cell's functional significance in the lung is the great difficulty encountered in obtaining sufficient quantities of these cells for study. During the current grant period, our laboratory has developed a unique approach to this problem by establishing and characterizing an ethylnitrosourea-induced (ENU) murine lung adenoma model, whose cells possess most of the structural and known biochemical features attributed to the normal mouse Clara cell. These include ultrastructural features consistent with normal mouse Clara cells, beta-adrenergic regulation of secretion, enhanced mixed function oxidase activity, unique lectin binding patterns and preliminary evidence of shared antigens with normal Clara cells. Morphological investigations of this model clearly indicated the occurrence of a cell-cell interaction between the Clara cells of the adenoma and alveolar Type 2 cells adjacent to the tumors. Specifically, the Type 2 cells were stimulated to accumulate large quantities of surfactant-like material. This Type 2 cell alteration only occurred in relation to Clara cell adenomas and appeared directly related to age and size of the tumors. The studies detailed in this revised renewal proposal are designed to test the hypothesis that the bronchiolar Clara cell has a defined functional interaction with alveolar Type 2 cells by modulating Type 2 cell surfactant metabolism via product(s) synthesized and secreted by Clara cells. Three major approaches to testing this hypothesis are described. The first is to establish an in vitro system to allow more controlled studies of the postulated cell-cell interactions. This will be accomplished by using freshly isolated alveolar Type 2 cells or the A549 cell line that will be cocultured with Clara cells isolated from ENU-induced pulmonary adenomas. The second approach will use an alternative in vivo bioassay for the effects of Clara cell secretory product(s) using pleural implants of Clara cell tumors and/or cells in newborn or immunologically deficient nude mice. This model will reduce some of our concerns over possible alteration of parenchymal Type 2 cells by the original carcinogenic exposure. Finally, we intend to isolate, purify and biochemically characterize the composition of the Clara cell secretory product(s) and, using our existing antisera to Clara cell antigen, to define the antigenic, molecular, and functional character of the secretory substances. The application of our mouse lung Clara cell adenoma model to these long range goals offer a new and unique approach to define the Clara cell role in physiological and pathological conditions in the lung.
