I. Luks. Fas/FasL-mediated apoptosis in perinatal murine lungs. Postcanalicular lung development is characterized by a time-specific increase in alveolar epithelial type II cell apoptosis. We have previously demonstrated that, in fetal rabbits, developmental type II cell apoptosis coincides with transient upregulation of the cell death regulator Fas ligand (FasL). The aims of this study were 1) to determine the spatiotemporal patterns of pulmonary apoptosis and Fas/FasL gene expression in the

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... rine model [embryonic day 17 (E17) through postnatal day 5 (P5)], and 2) to investigate the functional involvement of the Fas/FasL system by determining the effect of Fas activation and inhibition on perinatal pulmonary apoptosis. The apoptotic activity of alveolar epithelial type II cells, determined by combined TUNEL labeling and anti-surfactant protein B immunohistochemistry, showed a dramatic increase during the perinatal transition (type II cell apoptotic index Ͻ0.1% at E17, 1.5% at P1-P3, and 0.3% at P5). This timing of enhanced type II cell apoptosis coincided with a robust 14-fold increase in Fas mRNA and protein levels and a threefold increase in FasL protein levels; both Fas and FasL immunolocalized to type II and bronchial epithelial cells. In vitro and in vivo exposure of fetal and postnatal murine type II cells to anti-Fas antibody induced a fourfold increase in apoptotic activity that was prevented by administration of a broad-spectrum caspase inhibitor; the pulmonary apoptotic activity of Fas-deficient lpr mice remained unchanged. Conversely, administration of a caspase inhibitor to newborn mice (P1) resulted in marked diminution of pulmonary apoptotic activity. These combined findings strongly implicate the Fas/FasL system as a critical regulator of perinatal type II cell apoptosis. The developmental time dependence of apoptosis-related events in the murine model should facilitate investigations of the regulation of perinatal pulmonary apoptotic gene expression. programmed cell death; lpr; CD95; lung development DURING PERINATAL development, the distal lung parenchyma undergoes a series of orchestrated morphological and functional changes required for optimizing postnatal gas exchange. This process is initiated in utero during the transition from the pseudoglandular to the canalicular and saccular stages of lung development and is completed after birth during the alveolar stage. Postcanalicular lung development is characterized by a coordinated thinning of the interstitium, expansion of the alveolar septa, and a significant reduction in the number of alveolar type II cells (6, 22) . The loss of alveolar type II cells in postcanalicular lungs traditionally has been attributed to terminal differentiation of type II cells into type I cells, but recent observations suggest that apoptosis (programmed cell death) may be an important contributor to perinatal type II cell homeostasis as well (reviewed in Ref. 14). We previously demonstrated in fetal rabbits that periods of marked architectural and cellular remodeling of the alveolar septa during the transition from a canalicular to saccular lung are associated with a precisely timed, dramatic increase in type II cell apoptosis (12, 13). Others have shown a similar type II cell apoptosis in perinatal rats (23, 40) . Although the precise biological role of type II cell apoptosis in perinatal lung remodeling remains uncertain, its choreographed occurrence across mammalian species suggests elimination of "surplus" type II cells is an important facet of lung development. The effector pathways regulating perinatal type II cell apoptosis remain undetermined. We have previously shown that, in fetal rabbit lungs, the timing of increased type II cell apoptosis coincides precisely with a robust upregulation of pulmonary expression of the cell death regulator Fas-ligand (FasL) (12). Fas (Apo-1, CD95) is a member of a family of specialized transmembrane proteins called death receptors that belong to the TNF receptor superfamily. Stimulation of Fas by its physiological ligand, FasL, or by Fas-activating antibodies results in the recruitment of two key signaling proteins, the adapter protein Fas-associated death domain (FADD) and the initiator cysteine protease caspase-8 to form a death-inducing signaling complex. Proteolytic autoactivation of death-inducing signaling complex results in activation of the effector caspases, including the key effector caspase, caspase-3. Activated caspase-3 cleaves DNA repair enzymes, cellular and nuclear structural proteins, endonucleases, and many other cellular constituents, culminating in cell death (10, 41, 45) . Previous reports concerning the prevalence and regulation of apoptosis during lung development have focused on rat (23, 40) and rabbit (12, 13) models. The murine model offers important practical and genetic advantages over these species, including the commercial availability of Fas-activating and Fas-inhibiting reagents effective in mice, the existence of spontaneous mutant Fas/FasL-deficient mouse lines, and the adaptability for genetically modified animals either lacking or overexpressing key apoptotic signaling molecules. A thorough description of the time course of apoptosis and apoptosisrelated gene expression is required to exploit these advantages. The goals of this study were 1) to determine the spatiotemporal patterns of apoptosis and Fas/FasL gene expression in perinatal murine lungs and 2) to determine the functional Address for reprint requests and other correspondence: M. E. De Paepe, Women and Infants Hospital,