Postnatal development of the bronchiolar club cells of distal airways in the mouse lung: stereological and molecular biological studies

Club (Clara) cells are nonciliated secretory epithelial cells present in bronchioles of distal pulmonary airways. So far, no information is available on the postnatal differentiation of club cells by a combination of molecular biological, biochemical, and stereological approaches in the murine lung. Therefore, the present study was designed to investigate the changes in the club cell secretory proteins (CC10, surfactant proteins A, B and D) and club cell abundance within the epithelium of bronchioles of distal airways during the postnatal development of the mouse lung. Perfusion-fixed murine lungs of three developmental stages (newborn, 15-day-old and adult) were used. Frozen, unfixed lungs were used for cryosectioning and subsequent laser-assisted microdissection of bronchiolar epithelial cells and RT-PCR analyses. High resolution analyses of the three-dimensional structures and composition of lung airways were obtained by scanning electron microscopy. Finally, using design-based stereology, the total and average club cell volume and the volume of secretory granules were quantified by light and transmission electron microscopy. Our results reveal that murine club cells are immature at birth and differentiate postnatally. Further, increase of the club cell volume and number of intracellular granules are closely correlated to the total lung volume enlargement. However, secretory granule density was only increased within the first 15 days of postnatal development. The differentiation is accompanied by a decrease in glycogen content, and a close positive relationship between CC10 expression and secretory granule abundance. Taken together, our data are consistent with the concept that the morphological and functional differentiation of club cells is a postnatal phenomenon.