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Am J Physiol Lung Cell Mol Physiol 287: L730-L742, 2004; doi:10.1152/ajplung.00120.2004
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Fas/FasL-mediated apoptosis in perinatal murine lungs

Monique E. De Paepe,1,2,3 Quanfu Mao,2,3 Michelle Embree-Ku,3 Lewis P. Rubin,1,4,5 and Francois I. Luks1,6

1Program in Fetal Medicine, Departments of 2Pathology and 4Pediatrics, Women and Infants Hospital, Providence 02905; and Departments of 3Pathology and Laboratory Medicine, 5Pediatrics, and 6Surgery, Brown Medical School, Providence, Rhode Island 02905

Submitted 1 April 2004 ; accepted in final form 19 May 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 murine 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 <0.1% 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 apoptosis-related 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 involvement of the Fas/FasL system in perinatal type II cell apoptosis. We have found that postcanalicular lung remodeling in mice is associated with a marked rise in type II cell apoptosis occurring synchronously with upregulation of Fas and FasL expression. Direct Fas activation of fetal and postnatal type II cells by anti-Fas antibody resulted in a dramatic increase of apoptosis in vitro and in vivo in wild-type animals. Conversely, caspase blockade in newborn mice resulted in a significant decrease of pulmonary apoptosis. The combined evidence strongly implicates the Fas/FasL system as a critical developmental regulator of perinatal type II cell apoptosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and Tissue Processing

Breeding colonies were established from homozygous matings of inbred C57BL/6J ("wild type") mice and Fas-deficient lpr mice bred onto the same genetic background (B6.MRL-Faslpr; Jackson Laboratories, Bar Harbor, ME). Pregnancy (day 1) was ascertained by the presence of a postcoital vaginal plug. The patterns of apoptosis and Fas/FasL expression were studied in fetal and newborn mice between gestational day 17 (E17; term = E20) and postnatal day 5 (P5; P1 = day of birth). Pregnant dams were euthanized by intraperitoneal pentobarbital overdose, and the fetuses were removed via hysterotomy. Newborn mice were also euthanized with pentobarbital. All animal experiments were conducted in accordance with institutional guidelines for the care and use of laboratory animals.

Body weights and wet lung weights were recorded. Lungs were snap-frozen in liquid nitrogen and stored at –80°C for molecular analyses. For morphological studies, fetal lungs were immersion fixed in freshly prepared 4% paraformaldehyde in PBS, pH 7.4. The lungs of newborn mice were formalin fixed by tracheal instillation at a constant pressure of 20 cmH2O. After overnight fixation, the lungs were dehydrated in graded ethanol solutions, embedded in paraffin, and stained with hematoxylin and eosin.

Administration of Fas-Activating Jo-2 Antibody

The in vivo effects of direct Fas-activation on type II cell apoptosis were studied in fetal and newborn mice between E19 and P5. Anti-Fas antibody (clone Jo-2, BD Bioscience; San Jose, CA) was injected into newborn pups (20 µg ip) and pregnant dams (60 µg ip). Control mice received an equal amount of isotope-matched hamster IgG (BD Bioscience). In some newborn pups, 30 µg/animal of the broad-spectrum caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F (ZVAD-fmk; Enzyme System Products, Livermore, CA) was injected 30 min before administration of Jo-2.

Administration of Caspase Inhibitor

The effect of inhibition of Fas-dependent apoptosis was studied by injection of newborn mice (P1) with the caspase inhibitor ZVAD-fmk. Three doses of ZVAD-fmk (20 µg ZVAD-fmk in 1% DMSO in sterile normal saline solution) were injected intraperitoneal at 3-h intervals, and the animals were euthanized 3 h after the last doses. Control littermates were injected with normal saline containing 1% DMSO or remained untreated.

Type II Cell Isolation

Alveolar type II cells were isolated from fetal and newborn mice by a modification of the methods described by Corti et al. (9) and Rice et al. (37). Briefly, mice were anesthetized with Nembutal (0.5 ml ip). The abdominal cavity was opened, and the mice were exsanguinated when the inferior vena cava was severed. The lungs were perfused with 10–20 ml 0.9% saline via the right ventricle. The trachea was cannulated, Dispase (3 ml; Fisher, Cincinnati, OH) was rapidly instilled, and the lungs were dissected and incubated in 1 ml of Dispase for 45 min at 25°C. Lung tissue was subsequently transferred to a 60-mm culture dish containing 7 ml of HEPES-buffered DMEM (Sigma) and 100 U/ml DNase I, and parenchyma was gently teased from the bronchi. The cell suspension was filtered through 100- and 40-µm strainers and nylon gauze (20 µm). Cells were collected by centrifugation at 130 g for 8 min at 4°C and placed on prewashed 100-mm tissue culture plates coated with CD45 and CD32 (BD PharMingen, San Diego, CA). After 2 h of incubation at 37°C, type II cells were gently panned from the plate and collected by centrifugation. Type II cells were resuspended in culture medium (HEPES-buffered DMEM, 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin). The dishes were coated with Matrigel (BD Bioscience). The medium was changed after the first day of culture. After 48 h, the cells were exposed to the experimental conditions detailed in RESULTS.

Analysis of Apoptosis

TUNEL labeling. Localization and quantification of apoptotic cells was accomplished with terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick-end labeling (TUNEL), as previously described (12, 13). Negative controls for TUNEL labeling consisted of omission of the transferase enzyme. For quantification of TUNEL signals, a minimum of 25 high-power fields were viewed per sample, and the number of apoptotic nuclei per total number of nuclei [apoptotic index (AI)] was recorded. To estimate the rates of type II cell apoptosis, TUNEL labeling was combined with immunohistochemical detection of type II cells using an anti-surfactant protein B (SP-B) antiserum, kindly provided by J. Whitsett (University of Cincinnati, OH), as described (12, 13). Negative controls consisted of omission of the primary antibody.

Genomic DNA size analysis. The presence of internucleosomal DNA cleavage, characteristic of apoptosis, was investigated by DNA size analysis. For DNA extraction, frozen lung samples were homogenized and incubated in digestion buffer (100 mM NaCl; 10 mM Tris·Cl, pH 8; 25 mM EDTA, pH 8; 0.5% SDS; and 0.1 mg/ml proteinase K). After overnight lysis at 50°C, the samples were extracted with phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated with ammonium acetate/ethanol. Nucleosomal ladders were detected using a sensitive ligase-mediated PCR-based system (ApoAlert LM-PCR Ladder Assay Kit; Clontech Laboratories, Palo Alto, CA). Amplified products were visualized by electrophoresis in 1.2% agarose ethidium bromide gels.

Western blot analysis of caspase-3 cleavage. Processing and cleavage of procaspase-3 were assayed by Western blot analysis of whole lung lysates. Protein lysates (20 µg) were size fractionated by NuPAGE Bis-Tris (4–12%) gel electrophoresis (Novex; San Diego, CA), and transferred to polyvinylidene difluoride membranes. Western blots were hybridized with a polyclonal anti-caspase 3 antibody (Cell Signaling Technology, Beverly, MA). Secondary goat antibody was conjugated with horseradish peroxidase, and blots were developed with an ECL detection assay (Amersham Pharmacia Biotech, Piscataway, NJ). Band intensity was expressed as the integrated optical density of relevant bands, normalized to the integrated optical density of actin bands. Specificity controls included preincubation of antibody with blocking peptide.

Analysis of Expression of Apoptosis-Related Genes

RNase protection assay. RNase protection assay (RPA) was used to detect and quantify Fas/FasL-related mRNAs in lung tissue. Total cellular RNA was isolated from murine lungs according to the method of Chomczynski and Sacchi (7). Subsequently, RPA was performed using the RiboQuant RNase Protection Assay kit with the mAPO-3 mouse apoptosis multiprobe template set (BD Biosciences) containing DNA templates for caspase 8, FasL, Fas, FADD, FAP, FAF, and TNF-related apoptosis-inducing ligand and constitutively expressed L32. Specific antisense cRNA probes were synthesized using [{alpha}-32P]UTP in an in vitro transcription reaction, and 10-µg samples of total RNA were hybridized for 16 h at 56°C. After RNase and proteinase K treatment, the RNA:RNA duplexes were heat denatured and resolved on a 5% denaturing polyacrylamide gel. Dried gels were exposed to X-AR film (Kodak, Rochester, NY) at –70°C. The resulting bands were scanned and quantified using PhotoShop and NIH Image software. Band intensities were normalized to that of L32 in the same reaction.

Western blot analysis. Fas/FasL protein levels were evaluated by Western blot analysis of cell or whole lung homogenates using polyclonal rabbit anti-Fas (M-20, Santa Cruz Biotechnology, Santa Cruz, CA) and monoclonal anti-FasL (BD Transduction Laboratories, Lexington, KY) antibodies, as described in detail elsewhere (12).

Immunohistochemical analysis. The cellular localization of Fas and FasL proteins was studied by the streptavidin-biotin immunoperoxidase method using the antibodies described above. Immunoreactivity was detected with 3,3'-diaminobenzidine tetrachloride. Specificity controls consisted of omission of primary antibody and/or preabsorption with blocking peptide, which abolished all immunoreactivity.

Data Analysis

Values are expressed as means ± SD, or, where appropriate, as means ± SE. The significance of differences between experimental and control groups was determined with the unpaired Student's t-test or the nonparametric Mann-Whitney U-test where indicated. For multiple-group comparisons, one-way ANOVA with a post hoc Scheffé's test was used. The significance level was set at P < 0.05. Statview software (Abacus, Berkeley, CA) was used for all statistical work.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Perinatal Lung Remodeling

Fetal and neonatal mouse lungs underwent dramatic architectural and cellular remodeling between E17 and P5, characterized by decreasing cellularity and increasing complexity of the alveolar septa, combined with progressive transformation of the epithelial lining of the air spaces from clear (glycogen rich) cuboidal cells to more eosinophilic-differentiated type II cells and attenuated type I cells (Fig. 1).



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  		Fig. 1. Morphology of murine lungs between embryonic day 17 (E17) and postnatal day 5 (P5). Representative photomicrographs of perinatal murine lung between E17 and P5. A: fetal lungs at E17 are densely cellular and show interspersed primitive airway structures lined by clear columnar epithelium (late pseudoglandular stage). B: fetal lung at E19, showing decreased cellularity and thickness of the alveolar septa combined with transformation of the epithelial lining of the air spaces from clear cuboidal (glycogen rich) cells to flattened and eosinophilic cells (canalicular-early saccular stage). C: newborn lung at P1 showing large, simple "primary saccules" with smooth, relatively cellular walls (advanced saccular stage). D: at P3, the size of the primary saccules is slightly larger, and the previously smooth saccule wall is modified by the development of very low "secondary crests" (late saccular stage). E: by P5, primary saccules become subdivided into alveoli by the rapid elongation of secondary crests, heralding the beginning of true alveolarization (early alveolar stage). Arrows indicate secondary crests forming on the walls of the primary saccules (hematoxylin and eosin staining).

 
Temporal Pattern of Apoptosis in Perinatal Murine Lungs

TUNEL assay. At E17, pulmonary apoptotic activity, estimated by TUNEL labeling, was low and mainly localized to peribronchial and perivascular mesenchymal cells (Fig. 2A). A twofold increase in TUNEL labeling was seen between E17 and E19 (P < 0.02), followed by a further marked postnatal surge at P1 and P3, reaching levels fivefold higher than at E17 (P < 0.001 on P1 and P3 vs. E17) (Fig. 2B). The perinatal wave of pulmonary apoptosis was associated with a shift from mesenchymal to alveolar epithelial apoptosis (Fig. 2B). Between P3 and P5, the pulmonary apoptotic activity returned to lower levels (Fig. 2C). TUNEL positivity, expressed as AI relative to the mean AI on E17, is shown in Fig. 2.



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  		Fig. 2. Terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick-end (TUNEL) labeling of perinatal murine lungs. Top, TUNEL labeling of murine lungs at E17 (A), P1 (B), and P5 (C). TUNEL positivity was low and chiefly localized to peribronchial and perivascular cells at E17. Numerous TUNEL-positive nuclei were noted on P1, whereas apoptotic activity was again lower by P5. Omission of transferase enzyme abolished all FITC labeling (not shown). Arrows indicate TUNEL-positive nuclei (TUNEL-FITC labeling). Bottom, apoptotic index (AI, number of TUNEL-positive nuclei per total number of nuclei). Values represent fold increase of mean AI ± SE relative to E17. At least 4 animals were studied per time point. *P < 0.02 vs. E17; **P < 0.001 vs. E17.

 
To determine the apoptotic rate of type II cells, we combined TUNEL labeling with anti-SP-B immunohistochemical analysis in a double immunofluorescence assay. During fetal life, virtually no type II cell apoptosis was observed. Instead, nearly all apoptotic activity was limited to SP-B-negative, central, and interstitial mesenchymal cells (Fig. 3A). Postnatally, however, >60% of the TUNEL positivity was associated with adluminal or intraluminal SP-B-positive alveolar epithelial type II cells (Fig. 3B). Although TUNEL-positive type II cells were detected throughout the peripheral lung parenchyma, there appeared to be a predilection for the most distal air spaces, which are the zones of most active alveolar remodeling. By P5, the apoptotic activity of type II cells had returned to low levels (Fig. 3C). Bronchiolar Clara cell apoptosis was negligible at all time points. As shown in Fig. 3, the AI of type II cells (percentage of TUNEL-positive type II cells) showed a more than tenfold increase between E17 and P1 (P < 0.001). The AI of type II cells remained high until P3, but declined to prenatal levels by P5 (0.3%).



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  		Fig. 3. TUNEL labeling combined with anti-surfactant protein B (SP-B) immunohistochemistry. Top, perinatal murine lungs at E17 (A), P1 (B), and P5 (C). Arrows in A indicate apoptotic interstitial SP-B-negative cells. Arrows in B and C indicate apoptotic SP-B-positive type II cells. Omission of transferase enzyme and anti-SP-B antibody abolished all staining (not shown). Bottom, type II cell AI. Values represent mean AI ± SE of SP-B-positive cells, expressed as a percentage. *P < 0.05 vs. E17; **P < 0.01 vs. E17.

 
DNA size analysis. Oligonucleosomal DNA cleavage, a hallmark of apoptosis, was assayed using a sensitive PCR-based detection method. Genomic DNA was prepared from whole lung homogenates of fetal and newborn mice between E17 and P5. Fragmentation of DNA into multiples of 180–200 bp, resulting in the characteristic nucleosomal ladder pattern, was clearly seen at all ages from E19 onward (Fig. 4). The intensity of the DNA ladder was greatest shortly after birth (P1), suggesting increased pulmonary apoptotic activity.



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  		Fig. 4. DNA size analysis by ligation-mediated PCR. Lane 1, DNA size ladder. Lane 2, calf thymus DNA (control, C). The remaining lanes represent DNA samples from murine lungs between E17 and P5.

 
Spatiotemporal pattern of Fas/FasL expression in perinatal murine lungs. The mRNA levels of Fas/FasL-related genes were assayed using multiprobe RPA. Analysis of relevant genes (Fas, caspase 8, and FADD), normalized to the constitutively expressed housekeeping gene L32, is shown in Fig. 5. Levels of Fas mRNA showed a threefold increase between E17 and E19 (P < 0.02). The fetal-to-newborn transition between E19 and P1 was characterized by a robust additional increase of Fas mRNA levels, to reach levels 14-fold higher than at E17 (P < 0.001 vs. E19). The postnatal upregulation of Fas mRNA was transient, followed by a decrease by P3 and P5. Weak FasL bands were identified at P3, whereas at all other time points no FasL expression could be detected (not shown). The mRNA levels for other Fas/FasL-associated genes, such as FADD and caspase-8, were low in fetal lungs but showed a ~50% increase at P3 (Fig. 5).



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  		Fig. 5. RNase protection assay (RPA) analysis of Fas/Fas ligand (FasL)-related mRNA expression. Top, RPA analysis of Fas mRNA expression in lung lysates of mice between E17 and P5. The housekeeping gene L32 is included to assess relative RNA loading equality. Bottom, densitometry of Fas, Fas-associated death domain (FADD), and caspase-8 mRNA expression. Values represent mean integrated optical density (IOD) of the gene of interest, normalized to the IOD of constitutively expressed L32 mRNA (IOD/IOD L32), relative to the mean IOD/IOD L32 ratio at E17. At least 4 animals were studied per time point. *P < 0.02 vs. E17; **P < 0.001 vs. E19.

 
Fas and FasL protein expression was studied by Western blot analysis of whole lung lysates. Levels of immunoreactive Fas showed a robust, 15-fold increase between E17 and P1, as measured by densitometric analysis (P < 0.005 at P1 vs. E17, Fig. 6). By P5, Fas protein levels had returned to lower levels (Fig. 6). The levels of FasL protein were more than threefold higher after birth than at E17 (P < 0.05 at P1 vs. E17; Fig. 6). Preincubation of the primary antibodies with blocking peptide abolished immunoreactivity, confirming the authenticity of the bands (data not shown).



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  		Fig. 6. Western blot analysis of Fas/FasL protein expression. A: Western blot analysis of Fas/FasL protein expression in lung lysates of mice between E17 and P5. Appropriately sized bands were detected for FasL (37 kDa) and Fas (45 kDa). Actin (42 kDa) served as internal loading control. B: densitometry of Fas (top) and FasL (bottom) Western blot analyses. Values are means ± SE. At least 4 animals were studied per time point. *P < 0.005 vs. E17; **P < 0.05 vs. E17.

 
The cellular distribution of Fas and FasL proteins was determined by immunohistochemical analysis. Fas and FasL proteins were co-localized to bronchial epithelial cells and alveolar epithelial cells morphologically consistent with type II cells. The staining pattern was combined cytoplasmic and membranous in both cell types (Fig. 7).



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  		Fig. 7. Immunohistochemical analysis of Fas and FasL proteins. Representative micrographs of mouse lungs at E19 showing immunostaining for Fas (A) and FasL (B), immunolocalized to alveolar epithelial cells morphologically consistent with type II cells and bronchial epithelial cells in a combined cytoplasmic and membranous staining pattern. br, Bronchus (immunostaining by ABC method, hematoxylin counterstain).

 
Direct Fas activation in vitro. The synchrony between Fas/FasL upregulation and increased type II cell apoptosis strongly implicates the Fas/FasL system as regulator of perinatal type II cell apoptosis. To determine whether the Fas receptor of perinatal type II cells is functional in transducing apoptotic signals, we studied the effect of an agonist, the Fas cross-linking antibody Jo-2, on the apoptotic activity of isolated fetal (E18) and postnatal (P1/P2) type II cells in vitro. The purity of type II cell preparations, assessed by modified Papanicolaou stain (15), was typically >90% (Fig. 8). Cell viability, assessed by Trypan blue exclusion, was >95%. The type II cell phenotype was maintained throughout the 2- to 3-day culture period, as confirmed by electron microscopy and Western blot analysis of surfactant protein expression in type II cell lysates after 3 days of culture. In addition, the cells showed persistent Fas expression, as determined by Western blot analysis (not shown).



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  		Fig. 8. Effect of direct Fas activation on perinatal type II cell apoptosis in vitro. Morphology of murine fetal type II cells (E18–19) after 24 h exposure to culture media as seen by phase contrast microscopy (A) and light microscopy after modified Papanicolaou staining (B). A typical aggregate of isolated type II cells containing numerous characteristic cytoplasmic granules (lamellar bodies) is shown here. TUNEL labeling (A, C, E, G, I) and DAPI staining (B, D, F, H, J) of fetal (E18) murine type II cells exposed to (C and D): anti-Fas Jo-2 antibody, 20 µg/ml, 5 h (E and F): anti-Fas Jo-2 antibody, 20 µg/ml, 24 h (G and H): isotype-matched IgG, 20 µg/ml, 5 h (I and J): culture media only. Similar results were obtained with postnatal type II cells, although both the baseline (IgG and media) and Jo-2-induced apoptotic activity tended to be higher. K: apoptotic activity (TUNEL labeling) of fetal and postnatal type II cells exposed to culture media, IgG, anti-Fas Jo-2 antibody, or anti-Fas antibody with the broad-spectrum caspase inhibitor Z-val-Ala-Asp (OMe)-CH2 (ZVAD-fmk) for 5 h. Values are expressed as fold increase over apoptotic activity of IgG-exposed cells of same age. Results represent means ± SE of experiments performed at least in triplicate. *P < 0.02 vs. IgG; **P < 0.05 vs. Jo-2.

 
We previously determined that incubation with Jo-2 (20 µg/ml) induced apoptosis in a murine lung epithelium-derived cell line. In the present study, we used Jo-2 at this same concentration to test its effect on the apoptotic activity of fetal and postnatal primary murine type II cells. Exposure of isolated type II cells to Jo-2 antibody (20 µg/ml; 5 h) resulted in a more than fourfold increase in TUNEL activity compared with cells exposed to equal concentration of isotype-matched hamster IgG (Fig. 8). More prolonged (24 h) exposure of cells to Jo-2 antibody resulted in a significant loss of confluence, and residual cells displayed very high levels of apoptotic activity (Fig. 8). The baseline apoptotic activity of postnatal type II cells tended to be higher than that of fetal type II cells. However, in both age groups, Jo-2 treatment induced a fourfold increase of TUNEL positivity (Fig. 8). Pretreatment of the cells with the broad-spectrum caspase inhibitor ZVAD-fmk significantly diminished the apoptotic activity after subsequent incubation with Jo-2 (Fig. 8) (P < 0.05 vs. Jo-2).

Direct Fas activation in vivo. To verify the validity of the in vitro observations, we investigated the effect of in vivo administration of Jo-2 antibody on type II cell apoptosis in fetal and newborn mice.

Newborn C57BL/6J pups (P1, P3, and P5, 20 µg ip) and pregnant dams (E19, 60 µg ip) were injected with Jo-2 antibody and euthanized 5 h later. Control animals were injected with equal amounts of hamster IgG. Jo-2 treatment did not affect the behavior or survival of newborn pups within the 5-h time period studied. In contrast, Jo-2-treated pregnant dams appeared lethargic and moribund. In newborn pups and pregnant dams, moderate to marked necrotic/apoptotic liver injury was noted (not shown). Fetuses of Jo-2-treated dams were alive and showed only minimal focal light microscopic evidence of apoptotic liver injury.

Apoptotic activity. From as early as E19, the lungs of Jo-2-exposed mice could be distinguished from those of IgG-treated control mice by the presence of relatively large numbers of free-floating cells within the air spaces. On the basis of their granular cytoplasm, their rounded to cuboidal shape, and their presence within the alveoli, these cells were morphologically consistent with detached and apoptotic type II cells (Fig. 9). Quantification and localization of apoptotic cells were performed with the use of TUNEL labeling (Fig. 9). At E19, the pulmonary apoptotic activity of Jo-2-exposed fetuses was more than threefold higher than that of control fetuses (P < 0.05). Similarly, the apoptotic rates of Jo-2-exposed newborn pups were two- to threefold higher than control IgG-treated littermates (P < 0.05, Fig. 9). Apoptotic activity expressed as relative TUNEL positivity compared with the TUNEL positivity of control lungs at E19 is shown in Fig. 9. The apoptotic activity in Jo-2-exposed animals (pre- or postnatal) was chiefly localized to detached cells within the air spaces and alveolar epithelial cells still lining the air spaces. The apoptotic activity of bronchial epithelial cells and interstitial cells was similarly low in control and Jo-2 fetuses. The Jo-2 experiments were duplicated in Fas-deficient lpr pups at P3 and P5. The pulmonary apoptotic activity of Jo-2-treated lpr pups, determined by TUNEL labeling, was similar to that of IgG-treated lpr pups (Fig. 9).



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  		Fig. 9. Effect of direct Fas activation on pulmonary apoptosis in vivo. A–D, top: murine lungs at E19, exposed to IgG (A, C) or Jo-2 (B, D). E–H: murine lungs at P3, exposed to IgG (E, G) or Jo-2 (F, H). I, J: murine lungs at P1, exposed to Jo-2 (I) or caspase inhibitor ZVAD-fmk, followed by Jo-2 (J). K and L: lungs of Fas-deficient lpr mice at P3, exposed to IgG (K) or Jo-2 (L). Arrows in B and F indicate detached apoptotic nuclear fragments. (A, B, E, and F: hematoxylin and eosin stain, others: TUNEL-FITC labeling). M: pulmonary apoptotic activity (TUNEL labeling) of fetal and postnatal mice exposed to IgG, anti-Fas Jo-2 antibody, or anti-Fas antibody with ZVAD-fmk for 5 h. Values are expressed as the fold increase over apoptotic activity of IgG-exposed lungs at E19. Results represent means ± SE of at least 3 animals per time point. *P < 0.05 vs. IgG; **P < 0.02 vs. IgG; #P < 0.05 vs. Jo-2; ##P < 0.02 vs. Jo-2 wild type.

 
To confirm the involvement of Fas-dependent signaling in Jo-2-induced cell death, we studied the effect of caspase inhibition on the pulmonary apoptotic activity of Jo-2-exposed animals. Newborn pups (P1) were injected with the broad-spectrum caspase inhibitor ZVAD-fmk 30 min before Jo-2 injection, whereas control pups were injected with the vehicle DMSO. Preadministration of caspase inhibitor almost completely abolished the effect of Jo-2 on pulmonary apoptosis, consistent with the involvement of the caspase cascade in Jo-2-induced apoptosis (Fig. 9). DMSO alone did not affect Jo-2-inducible apoptotic activity (not shown).

Effect of Jo-2 administration on caspase-3 cleavage. Processing and cleavage of caspase-3, the main executioner of the apoptotic machinery, in Jo-2 lungs was assessed by Western blot analysis using an antibody specific for the cleavage products of caspase-3. Consistent with Fas-mediated cell death, Jo-2 caused cleavage of procaspase-3 and increased levels of the immunoreactive 17- and 19-kDa active subunits of caspase-3. In contrast, levels of the caspase split products were significantly lower in control lung homogenates (Fig. 10). Levels of immunoreactive caspase-3 split products were similar in Jo-2-treated and IgG-treated Fas-deficient lpr pups (Fig. 10).



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  		Fig. 10. Western blot analysis of caspase-3 cleavage. Western blot analysis of caspase processing in lung lysates of wild-type (WT) and Fas-deficient lpr mice after administration of Jo-2 or IgG. In WT mice, induction of apoptosis by Jo-2 correlated with increased levels of the 17- and 19-kDa subunits of caspase-3. Jo-2 administration in lpr mice did not result in increased levels of the caspase subunits.

 
Effects of caspase inhibition on perinatal lung apoptosis. To further demonstrate the functional involvement of Fas/FasL-signaling, we studied the effects of caspase inhibition on perinatal lung apoptosis. Postnatal animals at P1 were injected with three consecutive 20-µg doses of broad-spectrum caspase inhibitor ZVAD-fmk to inhibit systemic caspases for at least 9 h during the expected window of elevated type II cell apoptosis. The 20 µg-dose of ZVAD was based on the determination that this concentration is effective in preventing Jo-2-induced apoptosis and, therefore, should suffice to inhibit physiological Fas-mediated apoptosis.

The study of the effect of ZVAD-fmk on lung apoptosis was limited to those animals in which administration of the caspase inhibitor resulted in a significant (>50%) reduction of liver apoptosis to assure effective absorption and systemic action of the inhibitor. ZVAD-fmk was effective in reducing the apoptotic activity of the liver, as judged by TUNEL assay of liver sections, in 7/10 (70%) of P1-P2 pups studied (Fig. 11). The lungs of these seven pups appeared similar to those of DMSO-treated littermates on routine hematoxylin-and-eosin analysis (not shown). However, TUNEL labeling revealed a significant fourfold decrease in pulmonary apoptotic activity in lungs of ZVAD-treated animals (P < 0.05); DMSO alone did not affect apoptosis in lungs or liver (Fig. 11). Whereas residual TUNEL positivity after ZVAD-fmk treatment was occasionally seen in alveolar epithelial cells, most residual apoptotic cells were interstitial/mesenchymal.



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  		Fig. 11. Effect of administration of the caspase inhibitor ZVAD-fmk on pulmonary apoptosis in vivo. Top, TUNEL labeling of lungs (A and B) and liver (C and D) of pups at P1 treated with repeated injections of DMSO (A and C) or ZVAD-fmk (B and D). ZVAD-fmk injection resulted in a significant reduction of apoptotic activity in both organs (TUNEL-FITC labeling). Bottom, pulmonary apoptotic activity (TUNEL labeling) of mice at P1 exposed to DMSO or ZVAD-fmk. Values are expressed as the fold increase over pulmonary apoptotic activity of untreated control mice at P1. Results represent means ± SE of at least 3 animals per time point. *P < 0.05 vs. DMSO.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have determined the spatiotemporal patterns of apoptosis and Fas/FasL-related gene expression in perinatal murine lungs from late pregnancy through the first 5 postnatal days. During this time interval, roughly equivalent to 22–40 wk gestation in humans, murine lungs undergo marked architectural remodeling and progress from late pseudoglandular through early alveolar stages of development. We found that this perinatal transition was associated with a 10-fold increase, and subsequent decline, in type II cell apoptosis. The high apoptotic activity of type II cells, also observed in rabbits (12, 13), strongly suggests apoptotic elimination of surplus type II cells during perinatal alveolar remodeling is a naturally occurring and developmentally relevant event.

We further determined that the perinatal increase in type II cell apoptosis coincided with a robust upregulation of Fas and FasL expression. Both Fas and FasL immunolocalized to alveolar epithelial type II and bronchial epithelial cells. This striking synchrony, also observed in rabbits (12), suggests that the Fas/FasL system may be a critical mediator of postcanalicular type II cell apoptosis. Fas-mediated apoptosis has been shown to play a role in developmental apoptosis in several other cell types, including neurons (28, 36), osteoclasts (48), hematopoietic progenitor cells (31), lymphocytes (11), and testicular germ cells (5).

To investigate the functional importance of the Fas/FasL system in the regulation of perinatal type II cell apoptosis, we used the murine model to test the effect of mouse-specific Fas-activating and Fas-inhibiting reagents on perinatal pulmonary apoptosis. We first determined the in vitro and in vivo effect of Fas activation using the cross-linking anti-Fas antibody Jo-2. We found that exposure of isolated fetal and postnatal murine type II cells to this known Fas activator resulted in a significant fourfold increase in apoptosis that was abrogated by preincubation with a broad-spectrum caspase inhibitor. These results indicate that ligation of Fas in late gestation and neonatal type II cells mediate cell-specific apoptosis. Although the Fas mRNA and protein levels are higher in postnatal type II cells than in fetal type II cells, the relative increase in apoptotic activity after Jo-2 induction was similar in fetal and postnatal cells. We speculate that the lack of higher levels of apoptosis in postnatal type II cells may be due to saturation of Fas-dependent downstream signaling pathways.

These in vitro observations were extended by testing the effect of Jo-2-administration on lung apoptosis of fetal and newborn mice in vivo. Intratracheal (16) or intranasal (30) instillation of Jo-2 antibody, as well as inhalation of aerosolized antibody (33), are known to induce apoptosis in alveolar type II cells in adult mice. Because these methods of Jo-2-administration are not technically suited for fetal and newborn mice, we opted instead for intraperitoneal Jo-2-administration. Intraperitoneal administration of anti-Fas antibody to adult mice at doses similar to those used in the present study has been shown to induce massive hepatocyte apoptosis, resulting in fulminant liver failure and death (21, 33, 35). In addition, lesser degrees of Jo-2-induced apoptosis have been described in various other organs, including the kidney, ovary, spleen, thymus, skin, and intestine (17, 21, 39).

We determined that intraperitoneal administration of Jo-2 antibody to pregnant dams resulted in a marked increase of type II cell apoptosis in late-gestation fetuses, in striking contrast with the normally low levels of apoptotic activity at that age. Similarly, systemic Jo-2 administration to newborn pups resulted in a three- to fourfold increase in the already high levels of type II cell apoptosis.

It is unclear why, in our experience, intraperitoneal Jo-2 injection induces lung apoptosis in fetal and newborn mice, while this has not been reported in adult animals. Several explanations can be suggested to explain this apparent discrepancy. First, fetal and newborn type II cells may be inherently more sensitive than adult type II cells to direct Fas-activation. Second, lung apoptosis may have existed in adult animals but may have been masked by the more striking liver pathology because cell death may be difficult to detect unless specifically searched for by specialized techniques (such as TUNEL). Finally, the difference may be explained by age-dependent tolerance of mice to systemic Jo-2 administration. In accord with observations by Ogasawara et al. (35) and Nishimura et al. (33), we found that newborn pups tolerate intraperitoneal Jo-2 administration better than adult mice. It is conceivable that the longer survival of newborn pups after Jo-2 administration allowed sufficient time for lung apoptosis to develop, whereas adult mice succumb to liver failure before lung involvement becomes manifest.

Several approaches were taken to establish that the Jo-2 effect was Fas-receptor mediated. First, we confirmed that Jo-2-induced pulmonary apoptosis was mediated by processing and cleavage of caspase-3, the pivotal Fas-dependent executioner caspase. We subsequently demonstrated that Jo-2 induction of pulmonary (and hepatic) apoptosis was abrogated both in vivo and in vitro by prior administration of the caspase inhibitor ZVAD-fmk. Finally, and most conclusively, the Fas specificity of the Jo-2 effect was confirmed by the absence of apoptosis induction in Fas-deficient lpr pups. Similarly, in Jo-2-induced liver injury, anti-Fas antibody-mediated hepatocyte apoptosis is caspase-3 dependent, preventable by injections with ZVAD-fmk, and absent in adult lpr mice (47) (24, 35, 38).

In contrast with its effect on type II cells, Jo-2 did not increase the apoptotic activity of other pulmonary cell types such as bronchial epithelial cells or interstitial stromal cells. Lung fibroblasts are resistant to anti-Fas antibody-mediated apoptosis, and antiapoptotic proteins, such as X-chromosome-linked inhibitor of apoptosis and FADD-like IL-1{beta}-converting enzyme-like inhibitor protein have been implicated in preventing Fas-mediated apoptosis in these cells (43). The explanation for resistance of bronchial epithelial cells to direct Fas activation, despite relatively high Fas protein expression, remains undetermined.

Our findings indicate that perinatal type II cells are exquisitely susceptible to Fas activation. Even the usually nonapoptotic fetal type II cells can be triggered to undergo premature apoptosis, which can reach levels normally seen only during early postnatal life. Several recent reports (20, 27) have described the occurrence of apoptosis (epithelial and interstitial) during the acute/subacute stages of lung disease of the premature newborn bronchopulmonary dysplasia. It is tempting to speculate that more prolonged Fas activation in premature lungs may contribute to the increased alveolar epithelial apoptosis described in early bronchopulmonary dysplasia. Analogously, the Fas/FasL system has been implicated as a critical mediator of type II cell apoptosis in clinical and experimental adult lung injury models that are characterized by epithelial damage and alveolar epithelial type II cell apoptosis (4, 18, 19, 25, 26, 29, 30, 34).

In vitro and in vivo Jo-2 administration has demonstrated that the Fas receptor of perinatal type II cells is capable of transducing apoptotic signals, thus supporting a functional involvement of the Fas/FasL system in perinatal type II cell apoptosis. To verify the in vivo relevance of the Fas/FasL system, we assessed the effect of inhibition of Fas/FasL signaling on perinatal pulmonary apoptosis. When ZVAD-fmk, a broad-spectrum activated caspase inhibitor, was administered to newborn mice during the anticipated wave of pulmonary apoptosis, we observed a significant reduction in alveolar epithelial apoptosis. ZVAD-fmk administration did not completely abolish all pulmonary apoptotic activity. We speculate that some of the TUNEL-positive nuclei, particularly the more fragmented ones, may represent cells that had entered apoptosis prior to caspase inhibition (the clearance rates of apoptotic cells within the pulmonary airspaces are unknown). However, it is also possible that alternative, caspase-independent death pathways are utilized in perinatal lung cells.

These Fas-mediated pulmonary effects in perinatal mice need reconciliation with the apparent absence of a pulmonary phenotype in Fas- and FasL-deficient mouse strains. Mutant Fas-deficient lpr mice and FasL-deficient gld mice (3, 42) develop a range of phenotypic anomalies involving the lymphoid system, including lymphadenopathy, splenomegaly, and autoantibody production (1, 2, 8, 32, 42, 46). The lungs of lpr and gld mice appear structurally normal and their perinatal pulmonary apoptotic activity is similar to wild-type animals (M. E. De Paepe, unpublished observations). We suspect that the lack of a pulmonary phenotype in Fas/FasL-deficient animals may be due (at least in part) to the presence of alternate apoptotic signaling pathways in developing lungs. Of note, gene deletion models involving different downstream effectors of Fas-mediated apoptosis have not been particularly informative about the role of the Fas/FasL system in late-gestation lung development. FADD-null mice die in utero before E11.5 due to cardiac failure and abdominal hemorrhages (49). Caspase-8-null mice also die during embryogenesis due to aberrant cardiac development (44). The embryonic lethality of FADD- and caspase-8-null mice indicates that these key signal transducers, immediately downstream from Fas/FasL, are essential in development but precludes their use to determine the role of Fas/FasL in late fetal and neonatal lung development.

In summary, we have demonstrated that perinatal type II cell apoptosis in mice coincides with upregulation of Fas and FasL expression. The Fas receptor of perinatal type II cells is susceptible to Fas activation. This Fas-dependent perinatal pulmonary apoptosis is a caspase-mediated process. The combined evidence strongly implicates the Fas/FasL system as a pivotal developmental regulator of postcanalicular type II cell apoptosis. The mechanisms regulating Fas and FasL expression in a developmental context remain to be determined. We speculate that physiological regulators of the fetal-to-newborn transition, such as glucocorticoids, mechanical distension (stretch), and oxygen tension may contribute to the postcanalicular regulation of Fas/FasL expression. We expect that the developmentally dependent timing of these cellular and molecular events will facilitate future studies aimed at elucidating the developmental regulation of perinatal type II cell apoptosis using specific gene-targeting strategies.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by American Lung Association Research Grant RG-042-N (to M. E. De Paepe) and National Institutes of Health Grant P20-RR-18728 (to M. E. De Paepe and L. P. Rubin).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. E. De Paepe, Women and Infants Hospital, Dept. of Pathology, 101 Dudley St., Providence, RI 02905 (E-mail: mdepaepe{at}wihri.org)

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. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
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 RESULTS
 DISCUSSION
 GRANTS
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