Lung fibroblasts are known to be heterogeneous. The relative contributions of the lipid interstitial fibroblast (LIF) and the non-lipid interstitial fibroblast (NLIF) to the process of alveolar formation and elastin synthesis have not yet been delineated. The aim of this study was to seperate the two fibroblast populations and to determine the relative percentage of each population in fetal (E21) and postnatal(days, 10-12, 17 and 23) rat lungs. Density gradient centrifugation techniques have been used to separate LIF; however NLIF cannot be purified using this approach. The lipophilic fluorescent dye Nile Red was used to quantitate, by flow cytometry (FACStar), the percentage of LIF vs. NLIF in fetal and neonatal rat lungs before, during and after alveolar development. Lungs were removed from fetal (E-21) and postnatal (days 4, 10-12, 17 and 23) rats; lung fibroblasts were isolated by enzymatic digestion and differential adhesion. The cells were then released by trypsinization, fixed in 1% paraformaldehyde, and sorted on a FACStar. Fluorescence was detected through a 530/30 nM filter. The validity of this separation technique was confirmed by light microscopic evaluation of the sorted cells. The results indicate that LIFs constitute 69.6±6.8%(mean±sd) of total lung fibroblasts in the fetal lung at 21 days gestation (n=5). At the onset of the alveolar septal formation, postnatal day 4 (n=4), the percentage of LIFs increased to 79.0±3.8%. Towards the end of septation, postnatal days 10-12 (n=3), the percentage of LIFs decreased to 54.4±10.3%. On days 17 and 23, the percentages of LIFs were further decreased to 25.5±4.2 and 24.2±1.6 respectively. We have previously shown that rat lung fibroblasts undergo apoptosis from postnatal days 16-19. This study suggest that apoptosis occurs primarily in the LIF population. Although the roles of each of the lipids contained in the LIF remains to be determined, the presence of high concentration of retinoids in the lipid droplets supports other studies which suggest a role for these morphogens in the formation of alveoli.
