Developmental shift in the relative percentages of lung fibroblast subsets: role of apoptosis postseptation

We have used the lipophilic, fluorescent dye Nile red and flow cytometry to identify and isolate two rat lung fibroblast subsets, lipid-containing int erstitial cells (LICs) and non-LICs (NLICs) and to quantitate developmental changes in the relative percentages of these subsets. A significant decrea se was observed in the percentage of LICs (from 79.0 +/- 3.8% on postnatal day 4 to 28.6 +/- 4.2% on day 30; P < 0.0001). To determine whether one or both subsets undergo apoptosis postseptation, fibroblasts from 16- to 18-da y rats were treated with BODIPY-conjugated dUTP to label DNA strand breaks, which were then quantitated by flow cytometry. Apoptotic cells were judged to be predominantly LICs based on flow cytometric estimates of cell size a nd granularity and on light-microscopic colocalization of intracellular lip id and Hoechst-positive apoptotic bodies. Cell proliferation was compared i n LICs and NLICs with both an in vitro [H-3]thymidine incorporation assay a nd cell cycle analysis of propidium iodide-stained cells. Results of both a ssays indicated that on days 4-5, LICs proliferated more rapidly than NLICs . Tropoelastin and fibronectin mRNA expression, evaluated by RT-PCR, indica ted that although tropoelastin mRNA levels did not differ, fibronectin mRNA levels were approximately ninefold greater in LICs. These results demonstr ate the feasibility of a flow cytometric assay for the analysis of size, gr anularity, and intracellular lipid content of neonatal rat lung fibroblast subsets. Subsets differed substantially with respect to proliferative capac ity, fibronectin mRNA expression, and incidence of apoptosis postseptation. Together with the observed changes in relative percentages of fibroblast s ubsets with age, these data suggest that the ratio of LICs to NLICs could b e a critical determinant of fibroblast function during lung development.