Fas ligand expression coincides with alveolar cell apoptosis in late-gestation fetal lung development

Monique E. De Paepe1, Lewis P. Rubin2, Craig Jude1, Anne M. Lesieur-Brooks1, David R. Mills1, and Francois I. Luks3

1 Department of Pathology and 3 Division of Pediatric Surgery, Rhode Island Hospital, and 2 Department of Pediatrics, Women and Infants' Hospital, Brown University School of Medicine, Providence, Rhode Island 02903


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Apoptosis plays a central role in the cellular remodeling of the developing lung. We determined the spatiotemporal patterns of the cell death regulators Fas and Fas ligand (FasL) during rabbit lung development and correlated their expression with pulmonary and type II cell apoptosis. Fetal rabbit lungs (25-31 days gestation) were assayed for apoptotic activity by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and DNA size analysis. Fas and FasL expression were analyzed by RT-PCR, immunoblot, and immunohistochemistry. Type II cell apoptosis increased significantly on gestational day 28; the type II cell apoptotic index increased from 0.54 ± 0.34% on gestational day 27 to 3.34 ± 1.24% on day 28, P < 0.01 (ANOVA). This corresponded with the transition from the canalicular to the terminal sac stage of development. The day 28 rise in epithelial apoptosis was synchronous with a robust if transient 20-fold increase in FasL mRNA and a threefold increase in FasL protein levels. In contrast, Fas mRNA levels remained constant, suggestive of constitutive expression. Fas and FasL proteins were immunolocalized to alveolar type II cells and bronchiolar Clara cells. The correlation of this highly specific pattern of FasL expression with alveolar epithelial apoptosis and remodeling implicates the Fas/FasL system as a potentially important regulatory pathway in the control of postcanalicular alveolar cytodifferentiation.

programmed cell death; organogenesis; rabbit; type II cell; surfactant


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DURING ORGANOGENESIS, the fetal lung develops from a semisolid to a saccular organ that is capable of sustaining air exchange at birth. This distal airway cellular remodeling is attributable in part to apoptosis ("programmed" or "caspase-mediated" cell death) of mesenchymal and epithelial cells (7, 20, 33, 34).

The signaling pathways that mediate fetal lung and fetal type II cell apoptosis are unknown. Research on the mechanism of alveolar epithelial apoptosis in the setting of acute lung injury has recently focused on the role of the Fas/Fas ligand (FasL) system in the regulation of alveolar epithelial cell turnover. The Fas/FasL system is a widely distributed apoptosis signal transduction pathway in which ligand-receptor interaction triggers cell death (28, 29). Fas (FasR, APO-1, CD95) is a type I transmembrane receptor protein belonging to the tumor necrosis factor/nerve growth factor receptor superfamily (29, 37). Activation of Fas by binding to cross-linking antibodies or to the natural Fas ligand (FasL, CD95L), a type II transmembrane protein belonging to the tumor necrosis factor family of ligands (17, 23, 35), activates intracellular caspases and culminates in apoptosis (8, 36). The Fas system is involved in maintaining cell homeostasis in various systems, including maintenance of peripheral T and B cell tolerance, cell-mediated cytotoxicity, and control of immune-privileged sites, as well as the regulation of physiological epithelial cell turnover (10, 28).

Although initial emphasis was placed on their expression in lymphoid cells, Fas and FasL are also expressed in certain nonlymphoid tissues including lung (9, 10, 17, 35, 37, 39) where they may play a role in controlling epithelial cell homeostasis. The Fas/FasL system is activated in various models of clinical and experimental lung injury that are associated with alveolar epithelial apoptosis (2, 12, 13, 25, 30). Furthermore, experimental activation of the Fas/FasL system has been shown to result in type II cell apoptosis both in vivo (9, 12, 34) in murine lungs and in vitro (9, 38) in alveolar epithelial type II cell lines.

Based on the role of the Fas/FasL system in experimental and clinical models of alveolar epithelial apoptosis in adult lungs, we speculated that Fas activation may be implicated in the activation of apoptotic remodeling of the developing pulmonary acinus. We previously reported that fetal lung apoptosis in rabbits, as detected by in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL), follows a characteristic temporal pattern (7). Furthermore, with a combination of immunohistochemistry for surfactant-associated proteins and TUNEL, we determined that alveolar epithelial type II cells undergo significant apoptotic activity toward the end of gestation (7). These observations formed the basis for an experimental model to explore the molecular regulation of late-gestation alveolar remodeling.

In this study, we determined the spatiotemporal patterns of Fas and FasL expression in fetal rabbit lungs between 25 and 31 days gestational age (dGA; term is 31 dGA). During this time period, which spans the late pseudoglandular, canalicular, and terminal sac stages of development, fetal rabbit lungs undergo progressive architectural and cellular alterations that include mesenchymal involution and cytodifferentiation of the developing alveolar epithelium from immature, glycogen-rich epithelial cells to fully differentiated type I and type II cells (5, 6). In addition, we confirmed the characteristic temporal pattern of apoptotic activity by DNA size analysis and defined the precise time course of type II cell apoptosis.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and Tissue Processing

Time-mated New Zealand White rabbits (Millbrook Farm, Amherst, MA) were obtained 17-19 days into gestation. Between days 25 and 31, the rabbits underwent laparotomy under general anesthesia with intravenous thiopental sodium (20 mg/kg), and the fetuses were dissected via hysterotomy. The fetal left lung was immersion fixed in freshly prepared 4% paraformaldehyde in PBS, pH 7.4. After overnight fixation at 21°C, the lung was dehydrated in graded ethanol solutions and embedded in paraffin. The right lung was snap-frozen in liquid nitrogen and stored at -80°C for the molecular analyses. All procedures and protocols in this experiment were approved by the Rhode Island Hospital Animal Care and Use Committee and complied with standard principles of laboratory animal care and experimentation.

Analysis of Apoptosis

Analysis of DNA fragmentation. Apoptosis was confirmed by analysis of oligonucleosomal DNA cleavage. 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 of proteinase K). After overnight lysis at 50°C, the samples were extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and purified with ammonium acetate-ethanol. To detect the nucleosomal ladders generated during apoptosis, we used a PCR-based system (ApoAlert LM-PCR ladder assay kit; Clontech Laboratories, Palo Alto, CA) according to the manufacturer's instructions. Amplified products were visualized by electrophoresis in 1.2% agarose ethidium-bromide gels.

Analysis of type II cell apoptosis. To determine the incidence of type II cell apoptosis, we combined TUNEL with immunohistochemical detection of type II cells with anti-rabbit surfactant protein A (SP-A) antiserum, which was kindly provided by Dr. K. Sueishi (First Department of Pathology, Kyushu University, Fukuoka, Japan) (1). For TUNEL, we used the in situ cell death detection kit (Boehringer, Mannheim, Germany) according to the manufacturer's instructions. Briefly, sections were dewaxed and rehydrated according to standard protocols, incubated with 20 µg/ml of proteinase K (Sigma, St. Louis, MO) in 10 mM Tris · HCl (pH 7.5) for 15 min at 37°C, then rinsed and incubated with the TUNEL reaction mixture for 60 min at 37°C. In negative controls for TUNEL, the transferase enzyme was omitted.

After TUNEL, the sections were incubated with polyclonal anti-rabbit SP-A antiserum. Bound anti-SP-A antibody was visualized with the use of biotinylated rabbit anti-goat IgG antiserum (Sigma) followed by incubation with Texas Red-conjugated streptavidin (Zymed Laboratories, San Francisco, CA). The samples were washed in buffer and mounted on coverslips with the use of aqueous mounting medium. In negative controls for anti-SP-A staining, the primary antibody was omitted. To further validate the results of the double-immunostaining procedures, sequential sections were independently stained for TUNEL and SP-A with the same chromogens as in the double-staining procedure. For quantitation of positive TUNEL signals, a minimum of 50 high-power fields were viewed per animal. Results are expressed as the type II cell apoptotic index, i.e., the fraction (%) of TUNEL-positive type II cells of total type II cells. The quantitative analyses were performed by an observer blinded to the gestational age of the animals.

Analysis of Fas and FasL Expression

RT-PCR analysis. A semiquantitative RT-PCR technique was used to evaluate the expression profiles of Fas and FasL during fetal lung development. Total cellular RNA was isolated from fetal lungs according to the method of Chomczynski and Sacchi (4) and quantitated by measuring absorbance at 260 nm. cDNA was prepared by RT of 2 µg of RNA sample in a 20-µl reaction volume containing 10 mM deoxynucleotide triphosphates, 0.5 µg/µl of oligo(dT) (GIBCO BRL, Life Technologies, Grand Island, NY), 0.1 M dithiothreitol, 5× first-strand buffer (GIBCO BRL), 34 U/µl of RNase inhibitor, and 100 U/µl of Moloney murine leukemia virus reverse transcriptase (GIBCO BRL). The cDNAs were diluted to 100 µl and subjected to PCR with the following primer sets: Fas forward (F), 5'-TTG CTG TCA GCC NTG TCC TC-3'; Fas reverse (R), 5'-TGC ACT TGG TAT TCT GGG TC-3' (product size 209 bp); FasL F, 5'-GGA ATG GGA AGA CAC ATA TGG AAC TGC-3'; FasL R, 5'-CAT ATC TGG CCA GTA GTG CAG TAA TTC-3'(237 bp); beta -actin F, 5'-AGG CAT CCT GAC CCT GAA GTA C-3'; beta -actin R, 5'-TCT TCA TGA GGT AGT CTG TCA G-3'(389 bp); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) F, 5'-ACC ACG GTG CAC GCC ATC AC-3'; and GAPDH R, 5'-TCC ACC ACC CTG TTG CTG TA-3' (450 bp). The Fas PCR primers were derived from published mouse sequence (13) with minor modifications. The FasL PCR primers (generously provided by Dr. Kim Boekelheide, Brown University) were designed from rat sequence (GenBank accession no. U03470) and optimized for rabbit with the conditions described.

PCR amplifications were performed in a 50-µl reaction volume containing 5 µl of cDNA, 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 1.5 or 2.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, and 1.25 U of Taq Gold DNA polymerase (PerkinElmer, Foster City, CA). After an initial denaturation at 94°C for 10 min, amplification was performed in a PTC-200 thermal cycler (MJ Research) for 35 cycles under the following conditions: 20 s of denaturation at 94°C, 30 s of annealing at 50°C (Fas) or 55°C (FasL), and 40 s of extension at 72°C. The last cycle was followed by a final extension for 7 min at 72°C. Amplification of GAPDH or beta -actin mRNA was used to control for template concentration loading. Initial experiments determined that cDNA amplification was linear within 30 to 40 cycles of PCR for the genes of interest as well as for beta -actin and GAPDH (data not shown). The PCR products were electrophoretically separated in 2% NuSieve 1:1 agarose gels, washed in double-distilled H2O, and transferred to a Nytran filter (Schleicher and Schuell, Keene, NH).

Amplified Fas, FasL, and beta -actin cDNA fragments were inserted into the pCRII vector with TA cloning (Invitrogen, San Diego, CA). Identity of cloned Fas and FasL fragments was verified by sequencing. These cDNAs were subsequently used as hybridization probes in Southern blots of PCR products. Resulting signals were estimated densitometrically with NIH Image software (National Institutes of Health, Bethesda, MD) and are expressed as the integrated optical density of FasL-beta -actin bands. Control reactions that lacked RT products were performed to exclude sample contamination.

Western blot analysis of FasL protein. FasL protein levels were evaluated with Western blot analysis with SDS-polyacrylamide gels. Frozen lung tissues were homogenized with a tissue grinder, solubilized in lysis buffer (T-PER tissue protein extraction reagent; Pierce, Rockford, IL) in the presence of protease inhibitors (Boehringer), and centrifuged at 16,000 g at 4°C for 5 min. Supernatant protein was quantified with a Coomassie blue assay (Pierce).

Fifty micrograms of protein were analyzed by NUPage BISTris (MOPS) (4-12%) gel electrophoresis (Novex, San Diego, CA) and transferred to nitrocellulose membranes where they were incubated first in blocking solution (ECL kit, Amersham) for 4 h at room temperature and then incubated with a polyclonal antibody against FasL (N-20, 1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. Blots were then incubated with biotinylated rabbit anti-goat IgG (1:50,000 in blocking solution) for 1 h at room temperature followed by incubation with streptavidin-conjugated horseradish peroxidase (1:1000 in blocking solution) for 1 h at room temperature. Bands were detected with enhanced chemiluminescence, and signals were estimated densitometrically. Specificity controls included preincubation of anti-FasL antibody with blocking peptide.

Fas and FasL immunohistochemistry. The streptavidin-biotin immunoperoxidase method was used to study the expression of Fas and FasL protein. Anti-Fas antibody (M-20, Santa Cruz Biotechnology) was used at 1:1,000 dilution, and the anti-FasL antibody was used at 1:500 dilution. Immunoreactivity was revealed with 3,3'-diaminobenzidine tetrachloride (DAB), which yielded a dark brown precipitate. Controls of specificity consisted of omission of the primary antibody and/or preabsorption with FasL blocking peptide, which abolished all immunoreactivity.

Data Analysis

All values are means ± SE. The significance of differences between multiple experimental groups was evaluated by ANOVA, followed by the modified t-test according to Bonferroni with the use of Statview computer software (Abacus, Berkeley, CA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Apoptosis

Oligonucleosomal DNA cleavage was assayed with PCR-mediated detection. Fragmentation of DNA into multiples of 180-200 bp, resulting in characteristic DNA ladders, was detected on each dGA from 26 to 31 (Fig. 1). The apoptotic activity of type II cells between 27 and 31 dGA was analyzed by combined SP-A immunohistochemistry and TUNEL, as previously described (7). SP-A immunoreactivity was seen in alveolar type II cells and bronchiolar Clara cells as early as 27 dGA. Colocalization of SP-A and TUNEL positivity in type II cells was readily visualized between 28 and 30 dGA both in cells attached to the alveolar wall and in detached cells within the alveolar lumina (Fig. 2). The apoptotic index of SP-A-positive type II cells (attached and intraluminal combined) increased from 0.54 ± 0.34% at 27 dGA to 3.34 ± 1.24% at 28 dGA after which it gradually decreased until 31 dGA (Fig. 3). Rare apoptotic activity was seen in SP-A-negative interstitial cells (Fig. 2). Less than 0.1% of SP-A-positive bronchiolar (Clara) cells were TUNEL positive. Negative control samples for TUNEL, which lacked the transferase enzyme, were negative for FITC-labeled nuclei. Similarly, omission of the anti-SP-A antibody resulted in complete abolition of Texas Red cytoplasmic fluorescence signals (data not shown).


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 1.   DNA size distribution analysis by ligation-mediated PCR. Left lane, DNA size ladder; second lane, calf thymus DNA (control). The remaining lanes represent DNA samples from fetal rabbit lung between 26 and 31 days gestational age (dGA; C26-C31). Apo, apoptosis.



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 2.   Combined surfactant protein A (SP-A) immunohistochemistry and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) in fetal rabbit lung at 28 (A-C) and 27 (D) dGA. A: SP-A positivity is seen in alveolar type II cells and bronchiolar epithelial cells. TUNEL positivity (yellow-green) is seen in several type II cells (arrows). Bronchiolar epithelium (bottom) does not show apoptotic activity. B: colocalization of SP-A immunoreactivity (red cytoplasm) and TUNEL positivity (yellow-green nucleus) in alveolar type II cells. C: several intraluminal apoptotic type II cells characterized by combined SP-A and TUNEL positivity. D: TUNEL-positive apoptotic interstitial cells (arrows). A-D, TUNEL (FITC-conjugated nuclei) combined with SP-A staining (Texas Red cytoplasm). Original magnification, ×200 (A) and ×400 (B-D).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3.   Type II cell apoptotic index (type II AI). Values are means ± SE; n = at least 4 animals/time point. * P < 0.01 vs. 27 dGA (ANOVA).

Alveolar cells with the morphological characteristics of type II cells (large cuboidal cells with ample cytoplasm that protrude into the alveolar space) were invariably immunoreactive for SP-A regardless of TUNEL positivity. These observations indicate that type II cells retain their SP-A antigenicity even while undergoing apoptosis. Similarly, the vast majority of intraluminal TUNEL-positive nuclei had sufficient attached cytoplasm to allow immunohistochemical identification of the cells as type II cells.

Fas and FasL Expression

Semiquantitative RT-PCR was performed on fetal lung RNA samples obtained between 25 and 31 dGA to evaluate the expression levels of FasL and Fas mRNA. The designed Fas- and FasL-specific primers amplified the cDNA products of the predicted size for Fas (209 bp) and FasL (236 bp) (Fig. 4). Figure 4A demonstrates that Fas mRNA levels did not significantly change between 25 and 31 dGA. This observation may suggest that Fas is constitutively expressed in the fetal lung. In contrast, FasL expression levels showed striking alterations during fetal development. The FasL PCR product was low between 25 and 27 dGA, showed an abrupt and transient spike at 28 dGA, and returned to the lower level between 29 and 31 dGA (Fig. 4B). Densitometric analysis of Southern blots prepared with the RT-PCR products revealed that FasL message increased ~20-fold from 27 to 28 dGA (Fig. 4C). The identity of the Fas and FasL RT-PCR products was confirmed by sequence analysis of the amplified fragments.


View larger version (49K):
[in this window]
[in a new window]
 
Fig. 4.   RT-PCR analysis of Fas and Fas ligand (FasL) mRNA. A: Fas mRNA DNA size ladder (123 bp) is shown in the first lane. Next seven lanes (C25-C31) show representative samples of fetal rabbit lung between 25 and 31 dGA. Lane at far right (M), maternal lung. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. B: Southern blot analysis of FasL RT-PCR products. Lanes C25-C31, representative samples of fetal rabbit lung between 25 and 31 dGA. Kid, fetal kidney; Liv, fetal liver. beta -Actin was used as the internal control. C: densitometry of FasL RT-PCR products. IOD, integrated optical density. Values are means ± SE of at least 3 animals studied. * P < 0.001 vs. 27 dGA (ANOVA).

To further study the production of FasL in fetal lungs, whole lung protein lysates were examined by Western blot analysis with the use of a polyclonal anti-FasL antibody. Immunoblotting revealed the presence of a band with a molecular mass of ~40-42 kDa (Fig. 5A), which corresponded to the transmembrane form of FasL. Immunoreactive FasL was significantly greater at 28 dGA than at 26 and 30 dGA, confirmed by densitometric quantitation (Fig. 5B). Preincubation of the anti-FasL antibody with blocking peptide abolished immunoreactivity, confirming that the bands represent authentic FasL (data not shown).


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 5.   A: Western blot analysis for FasL protein expression in lung lysates between 26 and 30 dGA. An appropriately sized 42-kDa band was detected, with highest intensity at day 28 (D28) of gestation. B: densitometry of FasL protein Western blot. Values are means ± SE. At least 3 animals from each experimental group were studied per time point. * P < 0.05 vs. 26 dGA (ANOVA).

Immunohistochemistry of fetal lung sections was performed to localize FasL and Fas protein. During early pulmonary development (25-27 dGA), FasL protein was localized in primitive bronchial epithelium. With increasing architectural and cellular maturation, FasL protein was detected in bronchial and bronchiolar Clara cells in a cytoplasmic- and, to a lesser extent, a membrane-associated pattern. In addition, the majority of epithelial type II cells stained for FasL in a combined cytoplasmic and membrane pattern (Fig. 6A). Apical Fas protein staining was detected in Clara cells and in alveolar type II cells (Fig. 6C). Interstitial cells (fibroblasts, endothelial cells, and macrophages) did not exhibit Fas or FasL immunoreactivity. Preadsorption of anti-FasL antibody with specific blocking peptide (Fig. 6B) and omission of the anti-Fas antibody (Fig. 6D) abolished all immunostaining.


View larger version (151K):
[in this window]
[in a new window]
 
Fig. 6.   Immunohistochemical analysis of Fas and FasL protein with the avidin-biotin complex (ABC) method, 3,3'-diaminobenzidine tetrachloride (DAB) detection, and hematoxylin counterstain. A: FasL immunostaining in fetal rabbit lung at 28 dGA. FasL protein (brown) is seen in bronchiolar Clara cells and alveolar type II cells. B: negative control sample of A. Preadsorption of anti-FasL antibody with blocking peptide abolished all immunoreactivity. C: Fas immunostaining in fetal rabbit lung at 28 dGA. Fas immunoreactivity is seen in Clara cells and type II cells. D: negative control of C. No immunostaining was observed after omission of the primary anti-Fas antibody.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we confirm and extend the previous observations of De Paepe et al. (7) that fetal lung development in rabbits is characterized by a specific spatiotemporal pattern of apoptotic activity. While low during early fetal development (25-27 dGA; late pseudoglandular to canalicular stage), apoptotic activity increases dramatically at 28 dGA, heralding the transition from the canalicular to the terminal sac stage. Furthermore, a progressive shift from mesenchymal to epithelial apoptosis is seen during fetal lung development (7). During the late pseudoglandular and canalicular stages, apoptosis appears to be restricted to interstitial cells and likely contributes to mesenchymal involution and thinning of the alveolar septa. From 28 dGA on, most of the apoptotic activity is detected in epithelial cells, either attached or free-floating within the air spaces.

Using a combination of TUNEL and SP-A immunohistochemistry, we detected significant apoptotic activity in type II cells during the postcanalicular phases of fetal lung development; apoptotic rates were more than 3% around 28 dGA. These findings corroborate the observations of Kresch et al. (20), who studied the ontogeny of apoptosis in developing rat lungs and detected epithelial cell apoptosis from the canalicular stage of development onward. The relatively high prevalence of fetal type II cell apoptosis detected in rats and rabbits, concomitant with a gradual decrease of cell proliferation (5, 6), suggests that type II cell deletion by apoptosis may be a physiologically significant event in late-gestation lung remodeling. The actual type II cell loss resulting from a 3% apoptotic index cannot be accurately estimated because neither the duration of the detection span by TUNEL nor the clearance rates of apoptotic fetal rabbit type II cells is known. However, it seems likely that fetal type II cell apoptosis is biologically relevant because the values we obtained for the apoptotic indexes for type II cells are about 10-fold higher than those seen in other tissues in which apoptosis effects important changes (e.g., the developing nervous system) (3).

The specific and predictable time course of lung and type II cell apoptosis during fetal development that is seen in rabbits but not in rats (20, 33) provides an excellent model for elucidation of the molecular regulation of the death-signaling mechanisms involved. Several observations lead us to propose the Fas/FasL system as a pivotal mediator of late-gestation fetal type II cell apoptosis. First, we have demonstrated that the elements of the Fas death signaling pathway are present in fetal type II cells. Previous studies (9, 13, 39) have shown that adult murine type II cells and type II cell lines express Fas and that type II cell apoptosis can be induced by cross-linking agonistic anti-Fas antibodies both in vivo and in vitro (9, 12, 34, 38). Herein, we provide immunohistochemical evidence that fetal type II cells express Fas protein as well. Second, the timing of increased type II cell apoptosis correlated precisely with upregulation of FasL expression. Whereas Fas may be constitutively expressed, a robust increase of FasL protein and mRNA levels was seen at 28 dGA, synchronous with increased apoptosis and remodeling. Interestingly, the period of FasL upregulation was quite transitory. Within 24 h, both FasL mRNA and protein levels had returned to near baseline levels. Although FasL upregulation was short-lived, type II cell apoptosis continued, albeit at slightly lower levels, after 28 dGA. This observation may indicate that other proapoptotic death signals participate in developmental airway remodeling.

The finding of Fas receptors in fetal type II cells, in association with upregulation of FasL expression at the time of increased type II cell apoptosis, suggests that the Fas/FasL system may be a pivotal mediator of postcanalicular airway epithelial cell apoptosis. By analogy, the Fas/FasL system has been implicated in the regulation of homeostasis of other progenitor epithelial cells such as testicular germ cells (22), hepatocytes (15), and keratinocytes (32). It is uncertain which cell type is the sole or predominant source of upregulated FasL contributing to fetal type II cell apoptosis. We found FasL protein to be immunolocalized in type II cells and Clara cells, raising the possibility of both autocrine and paracrine regulation of type II cell death. Western blot analysis revealed increased levels of a 40- to 42-kDa FasL protein at 28 dGA, corresponding to the size of the membrane-associated form of FasL, which might favor autocrine interactions. However, further studies are required to test whether the FasL upregulation is associated with altered levels of the smaller soluble form of FasL as well.

The time-specific upregulation of FasL mRNA and protein during fetal development is highly suggestive of a tightly regulated transcriptional or possibly posttranscriptional regulation of the FasL gene. The mystery of the control of FasL gene transcription in other organ systems is only slowly beginning to unravel. Previous investigations have focused on the transcriptional regulation of induced FasL expression in activated T cells by the transcription factors nuclear factor-kappa B (NF-kappa B), nuclear factor of activated T cells (NFAT), activator protein-1 (AP-1), and egr (16, 18, 19, 21, 23, 24, 27, 29). Much less is known about the regulatory factors controlling constitutive or induced expression in nonlymphoid organs, although recent studies implicate Sp-1 (26), AP-1 (31), and hitherto uncharacterized elements (40). In this context, the study of transcriptional regulation of FasL in postcanalicular rabbit lungs may provide a useful model for studying the regulation of constitutive (25-27 dGA and 30-31 dGA) as well as induced (28-29 dGA) expression.

Intense immunoreactivity for both Fas and FasL was also seen in nonciliated bronchiolar Clara cells. Previous reports have localized Fas mRNA (13) and FasL protein and mRNA (11) to adult murine Clara cells where the Fas/FasL system is thought to play an immunomodulatory role (11). Paradoxically, we found that apoptotic activity of fetal Clara cells was negligible despite the intense coexpression of both receptor and ligand, possibly implying a protective antiapoptotic mechanism in these cells. Similar findings have been reported in human proximal airway epithelial cells where constitutive coexpression of ligand and receptor does not result in high apoptotic rates either (14).

Fetal interstitial cells (fibroblasts, macrophages, and endothelial cells) did not express Fas or FasL protein, which corroborates findings in adult murine lungs (9, 11, 12, 13). Nevertheless, apoptotic activity in interstitial mesenchymal cells was relatively high, especially during the pseudoglandular and canalicular stages of development. These findings suggest the possible role of pathways other than the Fas/FasL system in the mesenchymal involution and apoptosis of early fetal, and possibly also embryonal, morphogenesis.

In summary, we have demonstrated that postcanalicular lung and type II cell apoptosis is associated with transient upregulation of FasL expression. The restricted spatiotemporal pattern of FasL expression, synchronous with apoptosis and architectural and/or cellular remodeling in the developing lung, strongly suggests that the Fas/FasL system may play a unique role in the regulation of the fate of fetal type II cells. Further studies are required to confirm the functional role of the Fas/FasL system and to elucidate the molecular mechanisms underlying Fas-mediated postcanalicular type II cell apoptosis. Studies of lung development in FasL-deficient mice, to our knowledge not yet reported, may be extremely valuable in this regard. The role of the Fas/FasL system in the type II cell loss seen in pathological perinatal lung diseases such as infant respiratory distress syndrome and bronchopulmonary dysplasia remains to be determined.


    ACKNOWLEDGEMENTS

We thank Drs. Cynthia Jackson and Kim Boekelheide for helpful discussions and Ci Lin Sun for diligent technical assistance.


    FOOTNOTES

This study was supported in part by American Lung Association Research Grant RG-159-N.

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: Monique_DePaepe{at}brown.edu).

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.

Received 11 February 2000; accepted in final form 11 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Asabe, K, Toki N, Hashimoto S, Suita S, and Sueishi K. An immunohistochemical study of the expression of surfactant apoprotein in the hypoplastic lung of rabbit fetuses induced by oligohydramnios. Am J Pathol 145: 631-639, 1994[Abstract].

2.   Bardales, RH, Xie SS, Schaefer RF, and Hsu SM. Apoptosis is a major pathway responsible for the resolution of type II pneumocytes in acute lung injury. Am J Pathol 149: 845-852, 1996[Abstract].

3.   Barres, BA, Hart IK, Coles HS, Burne JF, Voyvodic JT, Richardson WD, and Raff MC. Cell death and control of cell survival in the oligodendrocyte lineage. Cell 70: 31-46, 1992[ISI][Medline].

4.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 62: 156-159, 1987.

5.   De Paepe, ME, Johnson BD, Papadakis K, and Luks FI. Lung growth response after tracheal occlusion in fetal rabbits is gestational age-dependent. Am J Respir Cell Mol Biol 21: 65-76, 1999[Abstract/Free Full Text].

6.   De Paepe, ME, Johnson BD, Papadakis K, Sueishi K, and Luks FI. Temporal pattern of accelerated lung growth after tracheal occlusion in the fetal rabbit. Am J Pathol 152: 179-190, 1998[Abstract].

7.   De Paepe, ME, Sardesai MP, Johnson BD, Lesieur-Brooks AM, Papadakis K, and Luks FI. The role of apoptosis in normal and accelerated lung development in fetal rabbits. J Pediatr Surg 34: 863-871, 1999[ISI][Medline].

8.   Enari, M, Talanian RV, Wong WW, and Nagata S. Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis. Nature 380: 723-726, 1996[ISI][Medline].

9.   Fine, A, Anderson NL, Rothstein TL, Williams MC, and Gochuico BR. Fas expression in pulmonary alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 273: L64-L71, 1997[Abstract/Free Full Text].

10.   French, LE, Hahne M, Viard I, Radlgruber G, Zanone R, Becker K, Muller C, and Tschopp J. Fas and Fas ligand in embryos and adult mice: ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover. J Cell Biol 133: 335-343, 1996[Abstract].

11.   Gochuico, BR, Miranda KM, Hessel EM, De Bie JJ, Van Oosterhout AJ, Cruikshank WW, and Fine A. Airway epithelial Fas ligand expression: potential role in modulating bronchial inflammation. Am J Physiol Lung Cell Mol Physiol 274: L444-L449, 1998[Abstract/Free Full Text].

12.   Hagimoto, N, Kuwano K, Miyazaki H, Kunitake R, Fujita M, Kawasaki M, Kaneko Y, and Hara N. Induction of apoptosis and pulmonary fibrosis in mice in response to ligation of Fas antigen. Am J Respir Cell Mol Biol 17: 272-278, 1997[Abstract/Free Full Text].

13.   Hagimoto, N, Kuwano K, Nomoto Y, Kunitake R, and Hara N. Apoptosis and expression of Fas/Fas ligand mRNA expression in bleomycin-induced pulmonary fibrosis in mice. Am J Respir Cell Mol Biol 16: 91-101, 1997[Abstract].

14.   Hamann, KJ, Dorscheid DR, Ko FD, Conforti AE, Sperling AI, Rabe KF, and White SR. Expression of Fas (CD95) and FasL (CD95L) in human airway epithelium. Am J Respir Cell Mol Biol 19: 537-542, 1998[Abstract/Free Full Text].

15.   Hiramatsu, N, Hayashi N, Katayama K, Mochizuki K, Kawanashi Y, Kasahara A, Fusamoto H, and Kamada T. Immunohistochemical detection of Fas antigen in liver tissue of patients with chronic hepatitis C. Hepatology 19: 1354-1359, 1994[ISI][Medline].

16.   Holtz-Heppelmann, CJ, Algeciras A, Badley AD, and Paya CV. Transcriptional regulation of the human FasL promoter-enhancer region. J Biol Chem 273: 4416-4423, 1998[Abstract/Free Full Text].

17.   Itoh, N, Yonehara S, Ishii A, Yonehara M, Mizushima S, Sameshima M, Hase A, Seto Y, and Nagata S. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66: 233-243, 1991[ISI][Medline].

18.   Kasibhatla, S, Brunner T, Genestier L, Echeverri F, Mahboubi A, and Green DR. DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-kappa B and AP-1. Mol Cell 1: 543-551, 1998[ISI][Medline].

19.   Kasibhatla, S, Genestier L, and Green DR. Regulation of Fas-ligand expression during activation-induced cell death in T lymphoctes via nuclear factor-kappa B. J Biol Chem 274: 987-992, 1999[Abstract/Free Full Text].

20.   Kresch, MJ, Christian C, Wu F, and Hussain N. Ontogeny of apoptosis during lung development. Pediatr Res 43: 426-431, 1998[Abstract].

21.   Latinis, KM, Norian LA, Eliason SL, and Koretzky GA. Two NFAT transcription factor binding sites participate in the regulation of CD95 (Fas) ligand expression in activated human T cells. J Biol Chem 272: 31427-31434, 1997[Abstract/Free Full Text].

22.   Lee, J, Richburg JH, Younkin SC, and Boekelheide K. The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology 138: 2081-2088, 1997[Abstract/Free Full Text].

23.   Lynch, DL, Watson ML, Alderson MR, Baum PR, Miller RE, Tough T, Gibson M, Davis-Smith T, Smith CA, Hunter K, Bhat D, Din W, Goodwin RG, and Seldin MF. The mouse Fas-ligand gene is mutated in gld mice and is part of a TNF family gene cluster. Immunity 1: 131-136, 1994[ISI][Medline].

24.   Matsui, K, Fine A, Zhu B, Marshak-Rothstein A, and Ju ST. Identification of two NF-kappa B sites in mouse CD95 ligand (Fas ligand) promoter: functional analysis in T cell hybridoma. J Immunol 161: 3469-3473, 1998[Abstract/Free Full Text].

25.   Matute-Bello, G, Liles WC, Steinberg KP, Kiener PA, Mongovin S, Chi EY, Jonas M, and Martin TR. Soluble Fas ligand induces epithelial cell apoptosis in humans with acute lung injury (ARDS). J Immunol 163: 2217-2225, 1999[Abstract/Free Full Text].

26.   McClure, RF, Heppelmann CJ, and Paya CV. Constitutive Fas ligand gene transcription in Sertoli cells is regulated by Sp1. J Biol Chem 274: 7756-7762, 1999[Abstract/Free Full Text].

27.   Mittelstadt, PR, and Ashwell JD. Role of egr-2 in upregulation of Fas ligand in normal T cells and aberrant double negative lpr and gld T cells. J Biol Chem 274: 3222-3227, 1999[Abstract/Free Full Text].

28.   Nagata, S. Apoptosis by death factor. Cell 88: 355-365, 1997[ISI][Medline].

29.   Nagata, S, and Golstein P. The Fas death factor. Science 267: 1449-1456, 1995[ISI][Medline].

30.   Nomoto, Y, Kuwano K, Hagimoto N, Kunitake R, Kawasaki M, and Hara N. Apoptosis and Fas/Fas ligand mRNA expression in acute immune complex alveolitis in mice. Eur Respir J 10: 2351-2359, 1997[Abstract/Free Full Text].

31.   Pyrzynska, B, Mosieniak G, and Kaminska B. Changes of the trans-activating potential of AP-1 transcription factor during cyclosporin A-induced apoptosis of glioma cells are mediated by phosphorylation and alterations of AP-1 composition. J Neurochem 74: 42-51, 2000[ISI][Medline].

32.   Sayama, K, Yonehara S, Watanabe Y, and Miki Y. Expression of Fas antigen in keratinocytes in vivo and induction of apoptosis in cultured keratinocytes. J Invest Dermatol 103: 330-334, 1994[Abstract].

33.   Scavo, LM, Ertsey R, Chapin CJ, Allen L, and Kitterman JA. Apoptosis in the development of rat and human fetal lungs. Am J Respir Cell Mol Biol 18: 21-31, 1998[Abstract/Free Full Text].

34.   Schittny, JC, Djonov V, Fine A, and Burri PH. Programmed cell death contributes to postnatal lung development. Am J Respir Cell Mol Biol 18: 786-793, 1998[Abstract/Free Full Text].

35.   Suda, T, Takahashi T, Golstein P, and Nagata S. Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 75: 1169-1178, 1993[ISI][Medline].

36.   Vaux, DL, and Strasser A. The molecular biology of apoptosis. Proc Natl Acad Sci USA 93: 2239-2244, 1996[Abstract/Free Full Text].

37.   Watanabe-Fukunaga, R, Brannan CI, Itoh N, Yonehara S, Copeland NG, Jenkins NA, and Nagata S. The cDNA structure, expression and chromosomal assignment of the mouse Fas antigen. J Immunol 148: 1274-1279, 1992[Abstract/Free Full Text].

38.   Wen, LP, Madani K, JA, Fahrni Duncan SR, and Rosen GD. Dexamethasone inhibits lung epithelial cell apoptosis induced by IFN-gamma and Fas. Am J Physiol Lung Cell Mol Physiol 273: L921-L929, 1997[Abstract/Free Full Text].

39.   Xerri, L, Devilard E, Hassoun J, Mawas C, and Birg F. Fas ligand is not only expressed in immune privileged human organs but is also coexpressed with Fas in various epithelial tissues. Mol Pathol 50: 87-91, 1997[Abstract].

40.   Zhang, J, Ma B, Marshak-Rothstein A, and Fine A. Characterization of a novel cis-element that regulates Fas ligand expression in corneal endothelial cells. J Biol Chem 274: 26537-26542, 1999[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 279(5):L967-L976
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society