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

Feyisola Awonusonu1, Suseela Srinivasan1, Jennifer Strange2, Walid Al-Jumaily1, and Margaret C. Bruce1

Departments of 1 Pediatrics and 2 Immunology, University of Kentucky Medical School, Lexington, Kentucky 40536


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have used the lipophilic, fluorescent dye Nile red and flow cytometry to identify and isolate two rat lung fibroblast subsets, lipid-containing interstitial cells (LICs) and non-LICs (NLICs) and to quantitate developmental changes in the relative percentages of these subsets. A significant decrease 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-day 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 and granularity and on light-microscopic colocalization of intracellular lipid and Hoechst-positive apoptotic bodies. Cell proliferation was compared in LICs and NLICs with both an in vitro [3H]thymidine incorporation assay and cell cycle analysis of propidium iodide-stained cells. Results of both assays indicated that on days 4-5, LICs proliferated more rapidly than NLICs. Tropoelastin and fibronectin mRNA expression, evaluated by RT-PCR, indicated that although tropoelastin mRNA levels did not differ, fibronectin mRNA levels were approximately ninefold greater in LICs. These results demonstrate the feasibility of a flow cytometric assay for the analysis of size, granularity, and intracellular lipid content of neonatal rat lung fibroblast subsets. Subsets differed substantially with respect to proliferative capacity, fibronectin mRNA expression, and incidence of apoptosis postseptation. Together with the observed changes in relative percentages of fibroblast subsets with age, these data suggest that the ratio of LICs to NLICs could be a critical determinant of fibroblast function during lung development.

fibroblast heterogeneity; flow cytometry; Nile red; lipid interstitial cell; fibronectin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ALTHOUGH ONCE CONSIDERED primarily a source of extracellular matrix necessary for structural support, the lung fibroblast is now recognized as having a far more complex role in lung function. Lung fibroblasts are a significant source of platelet-derived growth factor (9), epidermal growth factor (41, 47), insulin-like growth factors I and II (30, 46), hepatocyte growth factor (42), transforming growth factor (TGF)-beta (15, 18), and TGF-alpha (45). Specific fibroblast-derived growth factors also function as mediators of complex interactions with lung epithelial cells by influencing proliferation (keratinocyte growth factor) (10, 21) as well as differentiation and surfactant production (TGF-beta ) (48). Interactions with the immune system have also been described (34). Fibroblasts can express class II major histocompatibility complex, activate resting T lymphocytes, and synthesize and secrete interleukin (IL)-alpha (33), IL-4 (39), and IL-6 (35). Yet another aspect of the diverse role of fibroblasts is their heterogeneity in the lung (3, 24) and in other tissues such as the skin (31), periodontal ligament (11), and gingiva (19). Subpopulations can differ with respect to proliferative capacity, matrix production, and response to inflammatory cytokines during development and injury, thus adding another level of complexity to their ability to influence lung function.

Existing knowledge of the unique features of lung fibroblast subsets is, in large part, the result of extensive characterizations of rodent lung fibroblasts. These cells differ with respect to size; shape; location; presence of intracellular lipid droplets, glycogen, and myofilaments; expression of Thy 1 and glucocorticoid receptors; and production of ILs and fibroblast pneumonocyte factor (7, 17, 25, 34, 39). With few exceptions, lung fibroblast subsets produce essentially the same growth factors and extracellular matrix proteins but can differ substantially with respect to amounts produced during development and in response to injury (3, 8). Caniggia et al. (7) have shown that rat lung fibroblast subsets differ in their ability to stimulate epithelial cell proliferation and incorporation of [methyl-3H]choline into saturated phosphatidylcholine late in gestation. Fibroblast subsets from fibrotic lungs also differ with respect to proliferative capacity in vitro (16) and the amount of type I collagen produced in vivo (27).

The lipid interstitial cell (LIC), a neonatal rat lung fibroblast subset initially described by Vaccaro and Brody (44), has been of particular interest in recent years. This cell was the subject of an extensive review by McGowan and Torday (29). LICs contain abundant lipid droplets consisting primarily of triglycerides, whereas the other lung fibroblast subset, the non-LICs (NLICs), lacks lipid droplets (25). An ultrastructural comparison of 8-day with adult rat lungs by Kaplan et al. (17) suggested that lung maturation was associated with a small decrease in the relative percentage of LICs as well as a substantial decrease in the amount of lipid per cell. These authors attributed the decreased lipid content to diminishing levels of serum triglyceride; however, no explanation was provided for the decrease in the relative percentage of LICs.

Comparisons of relative abundance and specific functions of LICs and NLICs have been hampered by the absence of a technique that permits rapid quantitation, separation, and collection of these subsets. In the present study, we have used the lipophilic fluorescent dye Nile red to distinguish between LICs and NLICs and fluorescence-activated flow cytometry to quantitate LICs and NLICs over a range of fetal and postnatal ages. The relative percentage of LICs in total lung fibroblasts was found to change throughout lung development. In addition, we found that LICs undergo apoptosis postseptation, providing a logical explanation for the decrease in the relative percentage of LICs beyond day 16. Using fluorescence-activated cell sorting (FACS), we have physically separated LICs from NLICs and report herein significant differences between subsets with respect to rates of proliferation and fibronectin mRNA expression. Tropoelastin mRNA expression was essentially the same in both subsets, however.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fibroblast Isolation

Fibroblasts were isolated from the lungs of fetal and postnatal Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) on days 18-21 of gestation [embryonic day 18 (ED18) to ED21; term 22 days] and on postnatal days 2-30 following a procedure previously described in detail (5). In brief, lung tissue was minced, digested in a solution containing trypsin and collagenase for 90 min; pelleted by centrifugation (1,200 rpm for 10 min); resuspended in complete medium containing 1:1 (vol/vol) DMEM-Ham's F-12 medium, 10% fetal bovine serum, 10,000 U/100 ml of penicillin, 10 mg/ml of streptomycin, and 29.2 mg/100 ml of glutamine; and plated for 90 min. Nonadherent cells were removed from the flasks by gentle rinsing with calcium- and magnesium-free Hanks' balanced salt solution. The isolated cells were judged to be predominantly fibroblasts (>99%) based on their morphological appearance by phase-contrast microscopy and on immunohistochemical analyses. The adherent cells were immunoreactive with anti-vimentin (DAKO, Carpinteria, CA), excluding the possibility of epithelial cell contamination, but were not immunoreactive with anti-factor VIII (DAKO), indicating the absence of endothelial cells. Enzymes and tissue culture reagents were purchased from GIBCO BRL (Life Technologies, Grand Island, NY).

Quantitation and Sorting of Lipid Interstitial Fibroblasts by Flow Cytometry

Adherent fibroblasts were detached from flasks with 0.05% trypsin-0.02% EDTA and either fixed immediately in paraformaldehyde (0.5% final concentration) or resuspended in complete medium and centrifuged at 1,200 rpm for 10 min. Both fixed and viable cells were then resuspended in normal saline, filtered through nylon mesh (70 µm), and stained with Nile red, a fluorescent, lipophilic dye (Molecular Probes, Eugene, OR). The Nile red stock solution (1.0 mg/ml DMSO) was diluted in saline, then added immediately to the fibroblast suspension to achieve a final concentration of 0.1 µg Nile red/ml.

Fibroblasts were analyzed on a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA) equipped with a 15-mW argon-ion laser operated at 488 nm. A short-pass dichroic beam splitter transmitted emission < 560 nm. Nile red fluorescence was collected through a 530/30-nm band-pass filter. Forward-angle light scatter (FALS) and side-angle light scatter (SALS) measurements, estimates of cell size and cytoplasmic granularity, respectively, are expressed on a linear scale. At least 10,000 cells/sample were analyzed. In separate experiments, fibroblasts were sorted under sterile conditions at a rate of ~500 cells/s on a FACStar Plus flow cytometer equipped with a 4-W argon-ion laser tuned to 488 nm and regulated to 200 mW.

Analysis of Cellular DNA Content by Flow Cytometry

Freshly isolated fibroblasts were removed from the culture flasks by a brief trypsinization, stained with propidium iodide, and analyzed by flow cytometry as previously described (6). In brief, fibroblasts were pelleted, resuspended in PBS, fixed with ice-cold 70% ethanol, and stored at 4°C overnight. Immediately before analysis by flow cytometry, the cells were pelleted, washed with cold PBS, resuspended in 0.5 ml of propidium iodide (5 µg/ml in PBS containing 1 mg/ml of DNase-free RNase; Sigma, St. Louis, MO), and incubated for 20 min at 37°C. Samples were stored in the dark at 4°C and analyzed within 1 h on a FACS Calibur flow cytometer. The cells were excited by 488-nm light from a 15-mW argon-ion laser, and propidium iodide fluorescence was collected through a 585/42-nm band-pass filter. The fluorescence intensity (FL2) pulse area signal from a minimum of 10,000 events was analyzed with Modfit LT software (Verity Software House, Topsham, ME) to partition cells in the G0/G1, S, or G2/M phase.

Incorporation of [3H]Thymidine.

Freshly isolated, sorted LICs and NLICs and unsorted fibroblasts were seeded onto 96-well plates at a density of 2 × 105 cells/ml. Viability was determined by trypan blue exclusion. After 30 h, [3H]thymidine (Amersham, Arlington Heights, IL) was added to each well (final concentration 1.0 µCi/ml). The cells were cultured in the presence of [3H]thymidine for an additional 16 h, after which time the wells were thoroughly rinsed and the cell monolayer was removed by trypsinization. To quantitate [3H]thymidine incorporation, the cells were collected on glass filters with a FilterMate cell harvester (Packard Instrument, Meriden, CT). The filters were rinsed with distilled water, 5% trichloroacetic acid, and 70% ethanol and then dried. Radioactivity was quantitated on a Matrix Direct beta counter (Packard Instrument, Downers Grove, IL).

Light-Microscopic Quantitation of Apoptotic Bodies in Lung Fibroblasts

Freshly isolated fibroblasts were plated for 90 min; nonadherent cells were removed; and the remaining fibroblasts were air-dried for 5 min, fixed in 4% paraformaldehyde for 10 min, rinsed in PBS, and stained for 10 min with 10 µg/ml of Hoechst 33342 (Molecular Probes) to identify apoptotic bodies. The cells were then rinsed with PBS, stained with a saturated solution of oil red O in 70% ethanol for 10 min to detect intracellular lipid droplets, rinsed again in PBS, mounted in glycerol-PBS (1:2), and viewed on a Nikon Diaphot 300 microscope.

Flow Cytometric Analysis of DNA Fragmentation

DNA strand breaks were quantitated by flow cytometry (6, 22) in fibroblasts isolated from the lungs of 16- and 18-day pups. The cells were fixed in 1.0% formaldehyde at 4°C for 15 min, centrifuged, resuspended in 70% ethanol, and stored at -20°C for up to 4 days. DNA fragments were end labeled with BODIPY FL-14-dUTP (Molecular Probes) with terminal deoxynucleotidyltransferase (TdT; Boehringer Mannheim, Indianapolis, IN) as previously reported (6). In brief, fixed fibroblasts were rinsed twice in saline and incubated for 5 h at 37°C in a 50-µl reaction mixture containing 1.0 µl of TdT, 10 µl of 5× TdT buffer, 0.25 µl of BODIPY FL-14-dUTP, and 38.75 µl of water. The cells were then rinsed twice with 15 mM EDTA (pH 8.0), rinsed once with 0.1% Triton X-100 in PBS, and resuspended in 0.5 ml of a solution containing 2.5 µg/ml of propidium iodide and 0.1% RNase. After being incubated for 20 min at 37°C, the cells were immediately cooled to 4°C and evaluated by flow cytometry (FACS Calibur). DNA content was collected at FL2 area, and BODIPY fluorescence was collected through FL1. Cell aggregates and doublets were excluded. Data analysis was performed with CellQuest software (Becton Dickinson).

RT-PCR

Lung fibroblasts were homogenized with a Polytron homogenizer (Brinkman Instruments, Westbury, NY), and total RNA was extracted with TRI REAGENT LS (Molecular Research Center, Cincinnati, OH) and Phase Lock Gel tubes (5 Prime right-arrow 3 Prime, Boulder, CO) according to protocols recommended by the manufacturers. Glycogen (100 µg; Boehringer Mannheim) was used as a carrier for the precipitation of RNA. cDNA was prepared in a 2400 Perkin-Elmer Cetus (Foster City, CA) cycler with 0.4 µg of total RNA as previously described (6). PCR was performed with 1 µl of cDNA in a 50-µl reaction volume containing 2 mM MgCl2; 10 mM Tris · HCl buffer (pH 8.3); 50 mM KCl; 0.08 mM each dGTP, dATP, dTTP, and dCTP; 20 pmol of each primer; and 1.25 U of AmpliTaq DNA polymerase. Samples were heated to 94°C for 1 min 45 s to inactivate reverse transcriptase, denatured at 94°C for 30 s, annealed at 60°C for 30 s, and extended at 72°C for 1.5 min for a total of 17 (cyclophilin), 20 (tropoelastin), or 24 (fibronectin) amplification cycles. PCR products were resolved on a 12.5% polyacrylamide gel that was then stained with SYBR Gold (Molecular Probes), and ultraviolet fluorescence was quantitated on a STORM 840 with ImageQuant (Molecular Dynamics, Sunnyvale, CA). The identities of the amplified cDNAs were confirmed on an automated sequencer located in the Macromolecular Structure Analysis Facility at the University of Kentucky (Lexington).

PCR Primers

The primer pairs used to amplify tropoelastin and cyclophilin mRNAs have been previously described (5). The sequences selected for tropoelastin primers and product avoided exons known to be alternatively spliced in the rat lung (14, 36). To amplify the fibronectin 443-bp product, the 5'-forward and 3'-reverse complement primer sequences used were 5'-ACC AAC GCT CAG TCT CTA CCT CCA A-3' and 5'-TTT GAG GTT TAG CGT ATG CGG TGT C-3', respectively.

Statistical Analysis of Data

The significance of changes in the relative percentages of lipid-containing fibroblasts with increasing fetal and postnatal age was determined by a one-way ANOVA with Bonferroni correction for multiple comparisons with commercial software (Systat version 7.0, SPSS, Chicago, IL). A two-tailed pairwise Student's t-test was used to compare mRNA levels and incorporation of [3H]thymidine in LICs and NLICs. Values were considered to be significantly different when P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Flow Cytometric Identification of Lung Fibroblast Subsets

Nile red (9-diethylamino-5H-benzo[alpha ]phenoxazine-5-one) is a fluorescent, lipophilic dye used by other investigators (13, 23, 40) to detect intracellular lipid droplets in adipocytes by flow cytometry. We have used a similar approach to quantitate the percentage of total rat lung fibroblasts that contain lipid droplets (LICs) and to separate LICs from NLICs. Initial studies with ED21 lung fibroblasts conducted over a 20-fold range of Nile red concentrations demonstrated two distinct populations at a concentration of 0.1 µg Nile red/ml cell suspension. The mean fluorescence intensity of Nile red-negative fibroblasts was greater than that of unstained cells, consistent with observations by other investigators (12, 13), and is apparently due to the fact that Nile red dye is taken up by cell membrane lipids as well as by intracellular lipid droplets (Fig. 1). Although Nile red fluorescence quenched rapidly in aqueous solutions, once bound to the cellular lipid, the fluorescence intensity was relatively stable, and the difference in the mean fluorescence intensities of the two subsets remained essentially constant for 1-2 h.


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Fig. 1.   Flow cytometric analysis of intracellular lipid in freshly isolated lung fibroblasts. Cells were isolated and stained with lipophilic fluorescent dye Nile red as described in MATERIALS AND METHODS. Representative histograms are presented for fibroblasts obtained on embryonic days 18 (ED18) and 20 (ED20) and postnatal days 4, 7, 10, 23, and 30. Fluorescence intensity height (FL1-H) is plotted on a log scale. Cells in region R1 were more fluorescent due to their intracellular lipid content than cells in region R2. Relative percentage of cells in region R1 peaked on postnatal day 4, then decreased during alveolarization, remaining relatively constant thereafter.

To determine whether the relatively larger size of the lipid-filled fibroblasts was a confounding factor in the observed increased fluorescence intensity after staining with Nile red, the cells were sorted into Nile red-positive and Nile red-negative populations by flow cytometry, then stained with oil red O and examined by light microscopy. The results verified that the more brightly fluorescent population contained lipid-filled fibroblasts, whereas the less intensely fluorescent population consisted of smaller fibroblasts lacking lipid droplets. Of a total of 564 Nile red-positive cells examined, 553 (98%) stained positive for oil red O, whereas only 4% of 558 Nile red-negative cells were oil red O positive. Thus cells that stained less intensely with Nile red also failed to take up oil red O, indicating that the relative decrease in fluorescence observed in cells staining less intensely with Nile red was due not to their smaller size but to the absence of lipid droplets in these cells.

Morphological Characteristics of Nile Red-Positive and Nile Red-Negative Fibroblasts

Fibroblasts from lungs of 4-day rat pups were stained with Nile red and subjected to FACS under sterile conditions. Highly enriched populations of Nile red-positive and Nile red-negative cells were obtained by excluding from the sort cells in the region of overlap between the two peaks. Morphological differences between the two subsets were readily apparent (Fig. 2). The Nile red-negative cells were elongated and spindle shaped. Cytoplasmic extensions were seen only at opposite ends of the cells. In contrast, the Nile red-positive cells were rounded, with cytoplasmic extensions spreading in many directions. These two distinct phenotypes could also be distinguished in cultures of unsorted lung fibroblasts during the first 48 h in vitro. The Nile red-negative cells exhibited striking similarities to both the "peripheral" fibroblast subset described by Caniggia et al. (7) and the Thy 1+ subset described by Phipps et al. (34). Nile red-positive cells strongly resembled the "adherent" subset and the Thy 1- subset described by these authors.


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Fig. 2.   Light micrographs of living cultures of Nile red-positive (A) and Nile red-negative (B) fibroblasts from 6-day rats after 4 days in culture. Nile red-negative cells were long and spindle shaped with few cytoplasmic extensions, whereas Nile red-positive cells were round, often exhibiting numerous cytoplasmic extensions.

Variations in the Relative Percentages of Lipid Interstitial Fibroblasts Versus Nonlipid Interstitial Fibroblasts With Fetal and Postnatal Age

Changes in the percentage of cells that contained lipid droplets were identified over a range of fetal and postnatal ages. As early as ED18, two populations of lung fibroblasts were detected on the basis of fluorescent stain intensity. Shown in Fig. 1 are representative histograms of Nile red-stained rat lung fibroblasts obtained from ED18 through postnatal day 30. Although there was considerable overlap between fluorescence intensities of the two populations at ED18, Nile red-positive (LICs) and Nile red-negative (NLICs) fibroblasts were more readily separable into two distinct populations at ED21 and subsequent postnatal ages. The relative percentages of LICs and NLICs changed significantly with age (P < 0.0001; Fig. 3). The LIC percentage peaked at 79.0 ± 3.8% on postnatal day 4, coincident with the onset of alveolar formation, then decreased during alveolarization. A significant decrease in the percentage of LICs was seen from day 4 to days 6-7 (P < 0.007) and again from days 10-12 to day 23 (P < 0.0001).


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Fig. 3.   Flow cytometric analysis of freshly isolated fibroblasts. Values are means ± SD; n = 6 analyses for ED21, 5 analyses for 4 days (4D) postnatal, 3 analyses for 6-7 days (6-7D) postnatal, 4 analyses for 10-12 days (10-12D) postnatal, 3 analyses for 23 days (23D) postnatal, and 3 analyses for 30 days (30D) postnatal; each assay included fibroblasts obtained from lungs of 10-15 fetuses or 4-6 postnatal pups. Relative percentage of lipid-containing interstitial cells (LICs) was greater on day 4 than at subsequent ages. Percentage of LICs varied significantly over a range of fetal and postnatal ages, P < 0.0001. * Significantly less than day 4, P < 0.0001. Decrease from 6-7D to 10-12D was not significant; however, on 23D and 30D, percentage of LICs was significantly lower than at all previous ages, P < 0.0001. ** Significantly less than all previous ages, P < 0.0001.

The number of lipid droplets per cell also decreased with advancing postnatal age. Toward the end of alveolarization, days 10-12, a small shoulder was seen to the right of the NLIC peak in one of two 10-day and one of two 12-day samples. The fluorescence intensity of this population was slightly greater than that of the NLICs but less than that of the LICs. FALS and SALS measurements indicated that these cells were the same size but less granular than the LICs, suggesting that they represented LICs that had lost many of their lipid droplets. This new population, which accounted for <10% of total lung fibroblasts at these ages, was included in the LIC population.

On postnatal day 17, after the completion of alveolarization, a distinct third subset with a mean fluorescence intensity lower than that of LICs but greater than that of the NLICs was seen in each of three experiments. Flow cytometric evaluation of FALS and SALS indicated that cells with an intermediate fluorescence intensity had the same size distribution as LICs but less granularity, implying a loss of lipid droplets from LICs (Fig. 4). Light-microscopic evaluation of sorted cells indicated that fibroblasts contained in this intermediate peak contained relatively few lipid droplets compared with those in the peak with a higher mean fluorescence intensity, the LICs. By 23 days, LICs accounted for only 24.2 ± 1.6% of total lung fibroblasts. Fibroblasts were once again segregated into two subsets based on FALS and SALS estimates of size and granularity and on fluorescence intensity; however, the relative fluorescence intensities of the two subsets differed less than at earlier time points, presumably due to a decreased number of droplets per cell. No further change in percentage of LICs occurred from 23 days to 1 mo. Light-microscopic examination of sorted 30-day fibroblasts stained with oil red O indicated that only 30% of LICs contained >5 lipid droplets/cell.


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Fig. 4.   LICs lose intracellular lipid droplets postseptation. A: histogram of fluorescence intensity of 17-day fibroblasts indicates 3 populations of cells based on Nile red stain intensity. B: dot plots of forward-angle light scatter (FALS) to side-angle light scatter (SALS) for each of the 3 populations demonstrate that cells contained in region R3 are the most granular. Cells in region R2, although less granular, have the same size distribution as those in region R3. Cells in region R1 are both smaller and less granular than those in regions R2 and R3.

Cell Proliferation in NLICs Versus LICs

Freshly isolated fibroblasts obtained from lungs of 5- and 10-day rat pups were sorted into Nile red-positive and Nile red-negative populations, and the DNA content of the sorted cells was evaluated by flow cytometry to determine cell cycle distribution. For both LICs and NLICs, the percentage in the S plus G2/M phases was greater on day 5 than on day 10. At both ages, the percentage of cycling LICs exceeded the percentage cycling NLICs. On day 5, 9.4% of LICs vs. 5.6% of NLICs were in the S plus G2/M phases. By postnatal day 10, the percentage in the S plus G2/M phases decreased to 5.1 and 1.0% for LICs and NLICs, respectively.

Proliferation was also assessed in vitro in 4-day lung fibroblasts. Nile red-positive and Nile red-negative subsets were obtained by sorting under sterile conditions. Cells in the region of overlap between the Nile red-positive and Nile red-negative peaks were not included to ensure the collection of highly enriched populations of each fibroblast subset. Nile red-positive, Nile red-negative, and unsorted lung fibroblasts were cultured separately in 96-well plates in the presence of [3H]thymidine as a measure of DNA synthesis. Incorporation of [3H]thymidine, expressed as a ratio of counts per minute in LICs to counts per minute in NLICs, was fourfold greater in LICs (P < 0.02); the mean value for four experiments was 4.1 ± 1.7. Visual inspection of each of the 96-well plates confirmed that LICs proliferated much more rapidly than NLICs. Although cell numbers were not quantitated, the results of an earlier study of neonatal rat lung fibroblasts (1) demonstrated that proliferation, assessed by both [3H]thymidine incorporation and cell cycle analyses, was correlated with an increase in cell number.

Because NLICs account for ~20% of total lung fibroblasts on day 4, we anticipated that [3H]thymidine incorporation would be greater in LICs than in total lung fibroblasts. This was not the case, however. [3H]thymidine incorporation per cell was essentially the same in LICs alone as in total lung fibroblasts; the mean value for the ratio of counts per minute in LICs to counts per minute in unsorted fibroblasts was 0.96 ± 0.25 (n = 4 experiments). The fact that incorporation of [3H]thymidine in unsorted cells was essentially the same as that in LICs suggested that NLICs proliferate more rapidly in the presence of the LIC subset. To explore this possibility, a separate experiment was conducted in which LICs and NLICs were separated by flow cytometry and cultured in the presence and absence of unsorted 4-day fibroblast-conditioned medium containing 10% fetal bovine serum. Incorporation of [3H]thymidine was increased in both subsets cultured in the presence of conditioned medium; a 1.3-fold increase was seen in LICs versus a 1.4-fold increase in NLICs, suggesting that in neonatal rat lung fibroblast subsets, proliferation is under paracrine, and perhaps autocrine, control as well.

Extracellular Matrix mRNA Expression in NLICs Versus LICs

Expression of fibronectin and tropoelastin mRNAs was assessed in freshly isolated, sorted 4-day fibroblasts in three separate experiments. Total RNA was extracted immediately after sorting, and message levels were evaluated by RT-PCR. Values were normalized to cyclophilin. Although fibronectin mRNA was present in both LICs and NLICs, expression was approximately ninefold greater in LICs than in NLICs (Fig. 5). In contrast, tropoelastin mRNA expression was essentially the same in both subsets in each of three experiments (Fig. 5).


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Fig. 5.   Expression of fibronectin (FN), but not tropoelastin (TE), mRNA varies with lung fibroblast subset. Lung fibroblasts were isolated from 4-day pups, stained with Nile red, and separated by fluorescence-activated cell sorting (FACS) into Nile red-positive and Nile red-negative subsets in each of 3 separate experiments. Total RNA was extracted, reverse transcribed, and amplified for 20 (TE) or 30 (FN) cycles. PCR products were separated by PAGE and stained with SYBR Gold. TE mRNA levels were essentially the same in Nile red-positive and Nile red-negative subsets. In contrast, FN mRNA expression was ~9-fold higher in Nile red-positive than in Nile red-negative fibroblasts.

Lung Fibroblasts Undergo Apoptosis After Secondary Septal Formation

Light-microscopic identification of apoptotic bodies. Fibroblasts isolated from the lungs of 16- and 17-day rats were stained first with Hoechst 33342 followed by oil red O and examined at the light-microscopic level for evidence of nuclear fragmentation into distinct apoptotic bodies. In a total of 883 cells counted, apoptotic bodies, defined by the presence of three or more round Hoechst-positive balls of nuclear material, were detected in 28% of cells containing lipid (oil red O positive) but were not present in any of the cells that lacked lipid droplets (oil red O-negative fibroblasts). In contrast to DNA fragmentation, which occurs early in apoptosis, the formation of apoptotic bodies is an end-stage event. Thus apoptotic bodies, which are visible at the light-microscopic level for only a few hours, are likely to be seen in some fraction of those cells containing fragmented DNA (37).

Flow cytometric evaluation. In four separate experiments, DNA strand breaks in freshly isolated fibroblasts from lungs of 16- to 18-day rats were end labeled with BODIPY-conjugated dUTP, and the number of strand breaks per cell was quantitated by flow cytometry. Region R1 containing the nonapoptotic fibroblasts was defined by measuring BODIPY fluorescence in first-passage 5-day fibroblasts. The increased fluorescence intensity of the cells in region R2 was considered indicative of increased DNA strand breaks associated with apoptosis (Fig. 6A). FALS and SALS characteristics were then compared for cells in regions R1 and R2. In a representative FALS vs. SALS plot shown in Fig. 6B, the majority of the nonapoptotic cells in region R1 were small and nongranular and thus likely to be NLICs, whereas the apoptotic cells in region R2 were larger and more granular, characteristic of LICs.


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Fig. 6.   Two-dimensional frequency contour plots of apoptotic vs. nonapoptotic lung fibroblasts. A: fluorescence intensity of DNA strand breaks labeled with BODIPY-conjugated dUTP. DNA content, a measure of position in cell cycle, was determined by quantitating propidium iodide (PI) fluorescence. Fibroblasts from 16- and 18-day pups were distributed into 2 populations that differed with respect to BODIPY fluorescence intensity. Region R1 was defined on the basis of BODIPY fluorescence in a population of control (nonapoptotic) fibroblasts. Increased fluorescence of BODIPY-labeled strand breaks was seen in cells in region R2; these cells were considered to be apoptotic. B: FALS and SALS characteristics of cells from regions R1 (blue) and R2 (red). At 16 days, cells in region R2 (apoptotic) have FALS and SALS characteristics of LICs; they are generally larger and more granular than the other fibroblasts. In contrast, nonapoptotic cells in region R1 (blue) were smaller with less granularity, characteristic of NLICs. At 18 days, roughly one-half of large, granular cells were apoptotic (red), whereas remainder were normal (blue). There was no evidence of apoptosis in NLIC population. Data presented are representative of 4 separate experiments.

These differences in FALS vs. SALS characteristics were shown to be attributable to intracellular lipid content rather than to apoptosis. Apoptosis-related changes in FALS and SALS have been observed in thymocytes in which chromatin condenses into a single mass on one side of the nucleus (32). Although FALS and SALS changes have also been reported in late-apoptosis lymphocytes, similar changes are not seen in all cell types in association with apoptosis (32). Because changes in apparent size or granularity in apoptotic lung fibroblasts could have interferred with our ability to discriminate between these two phenotypes by FALS versus SALS, additional experiments were conducted on 9-day nonapoptotic cells and 17-day apoptotic cells to determine whether cells that appeared larger and more granular based on FALS and SALS characteristics were also Nile red positive and whether the smaller nongranular cells were Nile red negative. As shown in Figure 7, Nile red-positive cells were observed to be larger and more granular than the Nile red-negative cells both on day 9 when ~1% of lung fibroblasts are apoptotic (20) and on day 17 when a significant percentage of rat lung fibroblasts are apoptotic (6). These results suggest that the lung fibroblasts that undergo apoptosis after the completion of alveolarization are primarily, if not exclusively, LICs.


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Fig. 7.   Comparison of FALS and SALS characteristics of Nile red-positive and Nile red-negative lung fibroblasts obtained from 9- and 17-day rats. A: histograms of 9- and 17-day fibroblasts. Cells in region R2 were more fluorescent due to their intracellular lipid content than cells in region R1. B: dot plots of FALS vs. SALS of cells in regions R1 (green) and R2 (red). There was no evidence of either a decrease in FALS or an increase in SALS in Nile red-positive cells contained in region R2 of 17-day cells when compared with those obtained from region R2 of 9-day cells, at which age <1% of lung fibroblasts are apoptotic. Mean values for SALS were 204.86 for 9-day NLICs, 610.92 for 9-day LICs, 137.90 for 17-day NLICs, and 489.42 for 17-day LICs. For FALS, mean values were 426.61 for 9-day NLICs, 418.44 for LICs, 397.18 for 17-day NLICs, and 470.98 for 17-day LICs.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The lipophilic, fluorescent dye Nile red was initially used to detect intracellular lipid droplets in adipocytes by flow cytometry (13). Although highly soluble in organic solvents, Nile red is only negligibly soluble in aqueous solutions. In addition, the dye is highly fluorescent; the fluorescence intensity in organic solvents approaches that of rhodamine B. Both of these properties contribute to the utility of Nile red in the identification and quantitation of lipid-containing cells in a mixed population. In addition, excitation and emission spectra were shown to vary with lipid composition. Excitation and emission maxima were 549 and 628 nm, respectively, for phosphatidylcholine vesicles versus 549 and 605 nm, respectively, for phosphatidylcholine-cholesterol vesicles (12). Adipocytes containing neutral lipid droplets with excitation and emission maxima at 521 and 582 nm, respectively, could be distinguished from microsomal membranes in which phospholipid, the predominant lipid, has excitation and emission maxima of 540 and 624 nm, respectively (12). Thus by evaluating Nile red-stained adipocytes at wavelengths of 580 nm or less, the fluorescence of neutral lipid droplets is maximized, whereas the fluorescence of Nile red-binding cell membranes is minimized.

In the present study, the relative percentages of Nile red-positive and Nile red-negative rat lung fibroblasts were quantitated by flow cytometry, which facilitated documentation of the changes in the relative percentages of LICs versus NLICs during postnatal lung development. The accuracy of this method was confirmed by light-microscopic analysis of sorted cells stained with a second lipophilic dye, oil red O. This flow cytometric approach was further validated by FALS and SALS estimates of size and granularity of rat lung fibroblast subsets. In agreement with morphometric measurements reported earlier by Brody and Kaplan (4), LICs were larger and more granular than NLICs.

The use of flow cytometry to identify and separate Nile red-positive from Nile red-negative fibroblasts offers several advantages over previously reported methods for the separation of the lipid-containing subset. A commonly used protocol, initially described by Maksvytis et al. (25) and later modified by Berk et al. (2), employs a density gradient technique that separates lipid-containing fibroblasts from other cells based on the buoyant density properties of the former. A disadvantage of this technique is that the LIC fraction is only 85% pure, with macrophages accounting for the remainder of the fraction. Furthermore, as LICs begin to lose lipid in the second to third week of life, the extent to which these cells are included in the density gradient fraction will be influenced by their lipid content. In contrast, separation by flow cytometry facilitates the identification of LICs based on size and granularity as well as on lipid content, decreasing the likelihood that LICs will be excluded as intracellular lipid content decreases.

A second isolation procedure described by Caniggia et al. (7) isolated "adjacent" fibroblasts surrounding or in direct contact with epithelium from fetal lung homogenate after a relatively brief period of enzymatic digestion and "peripheral" fibroblasts not in the vicinity of the epithelium after a longer period of digestion. Although peripheral and adjacent fibroblasts appear morphologically similar to NLICs and LICs, respectively, that were isolated in the present study, the authors did not comment on the purity of the fibroblast subset populations obtained with this technique. Furthermore, observations by Brody and Kaplan (4) that NLICs and LICs were often found to be adjacent within the alveolar wall argues against the efficient separation by differential digestion.

A third isolation procedure used by Phipps and colleagues (33, 34) to separate adult mouse lung fibroblast subsets by flow cytometry was based on the differential expression of the cell surface antigen Thy 1. In the neonatal rat lung fibroblast, however, we found little correlation between the presence of this surface marker and intracellular lipid when cells sorted into Nile red-positive and Nile red-negative populations were subsequently stained with FITC-conjugated anti-Thy 1 and examined at the light-microscopic level (data not shown). Although Thy 1 expression has been used effectively by this group to separate fibroblast subsets obtained from adult mouse lung, pure subpopulations were best achieved by repeated flow cytometric separation of successive subcultures (34). In the adult mouse, the Thy 1- phenotype contains microfilaments and is similar in appearance to the LICs. Yet the Thy 1+ phenotype, which is long, spindle shaped, and similar in appearance to the NLIC, contains lipid droplets, suggesting that there are dissimilarities between neonatal rat and adult mouse lung fibroblast subsets.

Using flow cytometry to analyze a minimum of 10,000 cells in each experiment, we have observed a substantial decrease in both the relative percentage of LICs and the number of lipid droplets per cell during postnatal lung development. The possibility that LICs lost lipid droplets was addressed by Brody and Kaplan (4) and Kaplan et al. (17) in two in vivo studies in which LICs were distinguished from NLICs on the basis of specific ultrastructural characteristics, e.g., differences in size, nuclear-to-cytoplasmic ratio, location, and orientation of myofilaments. In the first study, after examining 1-, 24-, 48-, and 96-h autoradiograms from the lungs of 4-day rat pups treated with [3H]thymidine, Brody and Kaplan (4) found no evidence of any population of [3H]thymidine-labeled LICs that lost lipid over the 4-day period after labeling. Nor did they observe the appearance of a new population of LICs containing smaller amounts of lipid during this time. In a second study, however, a substantial decrease in the number of lipid droplets per cell was noted with advancing age. An ultrastructural comparison of fibroblasts from 8-day versus adult rat lungs indicated a decrease from 6.8 ± 0.8 to 0.8 ± 0.2 droplets/cell, respectively, with only a modest change in the percentage of LICs (50 vs. 38%) (17). Although data obtained in the present study support the concept that LICs lose many of their lipid droplets during lung development, we found that, in contrast to the observations of Brody and Kaplan (4) and Kaplan et al. (17), the relative percentage of LICs also decreases substantially. The percentage of LICs peaked at 79% on postnatal day 4 at the onset of alveolar formation, then decreased steadily thereafter, accounting for little more than one-half of the total lung fibroblasts toward the end of alveolarization, days 10-12. A further decrease was again noted by day 23 when only 24% of fibroblasts were LICs, but no further decrease was seen at 30 days. The differences between the two studies may be attributable to the fact that given the time-consuming nature of the ultrastructural analytic approach, relatively few cells (200-300) were examined at each of the two ages, whereas flow cytometric analysis permitted the evaluation of at least 10,000 cells/assay.

Results of prior studies failed to provide conclusive evidence in support of any of several possible explanations for the observed decrease in the relative percentage of LICs with advancing postnatal age. The data presented herein demonstrate that apoptosis, previously shown to occur in rat lung fibroblasts postseptation (6, 38), occurs primarily, if not exclusively, in LICs, providing an explanation for the decrease in the relative percentage of LICs postseptation. Although apoptosis is an ongoing process in lung cells during the canalicular, saccular, and alveolar stages of lung development (20, 37), the incidence is low in the fetal rat lung, <1% of total lung cells. On day 1, the percentage increased to 12%, then decreased to 1.5% by day 2. By postnatal day 10, only 0.6% of lung cells were apoptotic (20). Bruce et al. (6) and others (38) have reported a sharp increase in the percentage of apoptotic fibroblasts postseptation. The incidence of apoptosis, determined by flow cytometric analysis of DNA fragmentation in freshly isolated lung fibroblasts, was 51.4 ± 13.4% in 17-day, 36.9 ± 8.6% in 18-day, and 13.8 ± 5.4% in 19-day pups (6). This substantial reduction in interstitial fibroblast number appears to play an important role in the thinning of the connective tissue layer and the subsequent transition of the alveolar wall from a double- to a single-layered capillary network, a process that is an integral part of lung maturation. Although the broad emission spectra of Nile red precluded dual labeling with BODIPY, by examining FALS and SALS characteristics of BODIPY-conjugated dUTP-positive cells, we were able to identify the great majority of apoptotic cells as LICs based on their relative size and granularity.

Studies were also conducted to determine whether differences in proliferation rates might contribute to changes in the relative percentages of LICs and NLICs at the onset of alveolarization. Rates of cell proliferation in LICs and NLICs were compared by both [3H]thymidine incorporation in vitro and flow cytometric analysis of cell cycle distribution in freshly isolated fibroblast subsets. In each of these assays, LICs proliferated at a faster rate than NLICs. This differential proliferation rate is compatible with the relative increase in the percentage of LICs seen from ED21 to postnatal day 4. In contrast, Brody and Kaplan (4) reported that [3H]thymidine incorporation in vivo was ~1.8-fold greater in NLICs than in LICs on postnatal day 4. Although increased [3H]thymidine incorporation by LICs versus NLICs observed in our study could be attributable to an artifact of culture conditions and thus not accurately reflect proliferation rates in vivo, cell cycle analysis of freshly isolated cells also indicated that the percentage of cycling cells was greater for LICs than for NLICs on both postnatal days 5 and 10. Although providing an index of DNA synthesis during the relatively brief 1-h labeling time used by Brody and Kaplan, in vivo [3H]thymidine incorporation may not accurately reflect differences in proliferation under conditions where cell cycle kinetics vary. Furthermore, when viewed at the electron-microscopic level, discrimination between LICs and NLICs in thin tissue sections may not have been as accurate as was possible by flow cytometry.

Although it is not clear on the basis of these two different experimental approaches which of the two subsets proliferates faster in vivo, our results provide definitive evidence that NLICs proliferate more slowly in vitro than LICs. Enhanced proliferation of both LICs and NLICs in the presence of conditioned medium from total lung fibroblasts implies the production of mitogen(s) by one or both subsets. Furthermore, our observation that [3H]thymidine incorporation in vitro was essentially the same in LICs as in total lung fibroblasts, of which ~20% are NLICs, suggests that, in vitro, NLICs proliferate faster in the presence than in the absence of LICs. The extent to which one subset influences proliferation of the other in vivo remains to be determined.

In the postnatal lung, interstitial lung fibroblasts are a major source of elastin, a structural protein of critical importance in secondary septal development. Although both subsets synthesize elastin during lung development (29), the relative contribution of each subset has not been addressed previously. In the present study, we have shown that tropoelastin mRNA expression was essentially the same in both subsets. In contrast to tropoelastin, fibronectin mRNA expression was approximately ninefold greater in LICs. Although fibronectin production was also noted by Derdak et al. (8) to vary among Thy 1+ and Thy 1- clones, little variation in fibronectin production was seen among the original cell lines, again suggesting that distinct differences may exist between neonatal rat and adult mouse lung fibroblast subsets.

The relative abundance of the LIC fibroblast subset immediately preceding the period of rapid alveolarization and the disappearance of a substantial percentage of LICs after the completion of this process suggest a specific role for this subset during lung development in the neonatal rat. An in vitro study (43) has demonstrated that LIC triglycerides are utilized by type II epithelial cells to synthesize surfactant phospholipids (43). Lipid droplets also serve as a storage site for retinyl esters, which are converted to retinoic acid before septation (28). Recent studies have linked retinoic acid with the synthesis of tropoelastin (28) and with alveolarization (26), although the contribution of increased elastin synthesis to enhanced alveolarization remains to be delineated. The extent to which these observations are relevant to lung development in other species is unknown at present. Although lipid-containing fibroblasts have also been observed in mouse and hamster lungs, it is not at all clear that this subset exists in primates. We have been unable to identify lipid-laden fibroblasts in either human or baboon infant lungs (Awonusonu and Bruce, unpublished observations), although it is possible that fibroblasts in other species have significantly less lipid than rat lung fibroblasts and are, therefore, difficult to identify in the absence of an additional marker.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. Bruce, Dept. of Pediatrics, Neonatology Division, Univ. of Kentucky Medical School, 800 Rose St., Lexington, KY 40536 (E-mail: mbruce{at}pop.uky.edu).

Received 11 February 1999; accepted in final form 27 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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