Alkaline Phosphatase: A Marker of Alveolar Type II Cell Differentiation

Abstract In an effort to identify type II cells by a method independent of staining phospholipid inclusions, we evaluated a histochemical technique for alkaline phosphatase activity in normal rat lung, in freshly isolated type II cells, and in primary culture of type II cells. In the adult rat alveolus, alkaline phosphatase staining selectively identified type II cells, although nonciliated bronchiolar (Clara) cells and loose perivascular connective tissue also stained for alkaline phosphatase activity. In cell suspensions of type II cells and other dissociated lung cells, alkaline phosphatase staining correlated closely with the modified Papanicolaou technique and was particularly useful in distinguishing type II cells from alveolar macrophages. To determine if alkaline phosphatase was related to the differentiated phenotype of type II cells, we studied conditions known to affect other type II cell functions. When type II cells were cultured on plastic substrata, the intensity of alkaline phosphatase staining decreased with increasing time in culture. To quantitate the apparent decrease in alkaline phosphatase activity, we used a biochemical assay to study the expression of alkaline phosphatase by type II cells. The specific activity of alkaline phosphatase in type II cells declined with increasing time in tissue culture on plastic substrata. Alkaline phosphatase activity was maintained, however, by culturing cells on Englebreth-Holm-Swarm (EHS) tumor matrix. Cells that had reduced levels of alkaline phosphatase activity following 48 h of culture on plastic substrata could be “rescued” by removing them from the plastic substratum and reculturing them for 48 h on EHS matrix. Alkaline phosphatase activity was also increased by culturing type II cells in the presence of cAMP or sodium butyrate. By examining preparations of fetal rat lung, we found low alkaline phosphatase levels early in gestation and an increase at the end of gestation. The developmental peak of alkaline phosphatase activity occurred two days before term in the rat. Alkaline phosphatase expression by type II cells appears to be regulated in concert with the synthesis of the phospholipid and apoprotein components of pulmonary surfactant in both adult type II cells in primary culture and in the fetal lung. While its physiologic function remains unknown, we postulate that alkaline phosphatase expression represents a marker of differentiated function of type II Cells.