Alveolar macrophage apoptosis and TNF-alpha , but not p53, expression correlate with murine response to bleomycin

Luis A. Ortiz1, Kryztof Moroz2, Jing-Yao Liu2, Gary W. Hoyle1, Timothy Hammond3, Raymond F. Hamilton4, Andrij Holian4, William Banks5, Arnold R. Brody2, and Mitchell Friedman1

Sections of 1 Pulmonary Diseases, Critical Care and Environmental Medicine and 3 Nephrology, Department of Medicine, and 2 Lung Biology Program, Department of Pathology, Tulane University Medical Center, New Orleans, Louisiana 70112; 4 Division of Pulmonary and Critical Care Medicine, Department of Medicine, The University of Texas Medical School, Houston, Texas 77030; and 5 Division of Geriatrics, St. Louis University, St. Louis, Missouri 63104

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Apoptosis is considered to be a protective mechanism that limits lung injury. However, apoptosis might contribute to the inflammatory burden present in the injured lung. The exposure of mice to bleomycin (BLM) is a well-established model for the study of lung injury. BLM exposure induces DNA damage and enhances tumor necrosis factor (TNF)-alpha expression in the lung. To evaluate the importance of alveolar macrophage (AM) apoptosis in the pathogenesis of lung injury, we exposed BLM-sensitive (C57BL/6) and BLM-resistant (BALB/c) mice to BLM (120 mg/kg) and studied the induction of apoptosis [by light-microscopy changes (2, 8, 12, 24, 48, and 72 h) and annexin V uptake by flow cytometry (24 h)], the secretion of TNF-alpha (measured by ELISA), and the expression of p53 (by immunoblotting) in AM retrieved from these mice. BLM, but not vehicle, induced apoptosis in AM from both murine strains. The numbers of apoptotic AM were significantly greater (P < 0.001) in C57BL/6 mice (52.9%) compared with BALB/c mice (40.8%) as demonstrated by annexin V uptake. BLM induction of apoptosis in AM was preceded by an increased secretion of TNF-alpha in C57BL/6 but not in BALB/c mice. Furthermore, double TNF-alpha receptor-deficient mice, developed on a C57BL/6 background, demonstrated significantly (P < 0.001) lower numbers of apoptotic AM compared with C57BL/6 and BALB/c mice. BLM also enhanced p53 expression in AM from both murine strains. However, p53-deficient mice developed BLM-induced lung injury, exhibited similar lung cell proliferation (measured as proliferating cell nuclear antigen immunostaining), and accumulated similar amounts of lung hydroxyproline (65 ± 6.9 µg/lung) as did C57BL/6 (62 ± 6.5 µg/lung) mice. Therefore, AM apoptosis is occurring during BLM-induced lung injury in a manner that correlates with murine strain sensitivity to BLM. Furthermore, TNF-alpha secretion rather than p53 expression contributes to the difference in murine strain response to BLM.

tumor necrosis factor; strain susceptibility

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

APOPTOSIS, OR PROGRAMMED cell death, is the process by which the cell undergoes genetically determined death under tightly controlled circumstances (23, 40). This process is associated with distinct morphological and biochemical changes that distinguish apoptosis from necrosis (23, 40). The importance of apoptosis in the development of lung fibrosis is not completely understood. Apoptosis is considered to be a protective mechanism that allows the removal of inflammatory cells and regulates the control of mesenchymal cell proliferation in the injured lung (2, 33). Microscopic evaluation of lung tissues from patients with fibrogenic lung disease demonstrates evidence of apoptosis in areas of fibroblast proliferation, and it has been suggested that apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scarring (5, 33). Therefore, the regulation of apoptosis during tissue remodeling may be important in preventing development of pathological scarring, a classic feature of pulmonary fibrosis.

Bleomycin (BLM) therapy is associated with the development of life-threatening pneumonitis that can progress to interstitial pulmonary fibrosis (1, 18). The histological features of this BLM-induced lung fibrosis are similar to those observed in idiopathic pulmonary fibrosis, and, therefore, the BLM induction of lung injury in animals is a relevant model for the study of lung fibrosis (1, 14, 37). The mechanism(s) of BLM-induced lung injury is not well understood. The acute exposure of mice to BLM is associated with DNA strand scission in the lung (13). This effect of BLM on DNA has been shown to be associated with the induction of apoptosis (9, 11, 38). However, because the half-life of BLM in the lung is short (20), it has been suggested that the fibrotic response to BLM is mediated by the activation of cells within the lung and the release of inflammatory cytokines (3, 8, 17, 35, 37). BLM can bind to alveolar macrophages (AM), altering morphology and inducing cytokine secretion (4, 6). Among the cytokines produced by AM in response to BLM is tumor necrosis factor (TNF)-alpha . TNF-alpha has been shown to be critical in the pathogenesis of BLM-induced lung fibrosis, and measures that decrease TNF-alpha expression in the lung ameliorate BLM-induced lung fibrosis (27, 29-31).

Exposure of cells to DNA damaging agents such as BLM induces increases in the p53 protein levels (19, 26). In many instances, elevated levels of p53 lead to a temporary arrest of the cell cycle and provide an opportunity for DNA repair or induce apoptosis (26, 36). p53 may also enhance cell proliferation by regulating the expression of the proliferating cell nuclear antigen (PCNA), a DNA replication and repair protein, and concomitant expression of p53 and PCNA has been reported in fibrotic lung injury (24). The role of p53 in BLM-induced lung injury has not been studied.

In the present study, we report that the systemic exposure of mice to BLM is associated with the induction of apoptosis in AM. This induction of apoptosis in macrophages correlates with the murine strain sensitivity to BLM; i.e., apoptosis is more prominent in AM from BLM-sensitive (C57BL/6) mice. The BLM induction of apoptosis in AM is also associated with an enhanced secretion of TNF-alpha that also correlates with the murine strain sensitivity. Apoptosis is significantly decreased in double TNF receptor (TNFR)-deficient mice. BLM exposure is also associated with an enhanced p53 expression in AM. However, p53-deficient mice exposed to BLM had similar lung cell proliferation (measured as PCNA immunostaining) and collagen accumulation as did BLM-sensitive (C57BL/6) mice. These findings support our view that the induction of apoptosis in AM and the secretion TNF-alpha rather than DNA damage and p53 expression contribute to the difference in murine strain response to BLM and may play a role in the fibroproliferative response observed in the lungs of BLM-treated mice.

    MATERIALS AND METHODS
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Materials & Methods
Results
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Chemicals. Stock solutions (5 U/ml) of BLM (Blenoxane; kindly donated by Bristol-Meyers, Princeton, NJ) were prepared immediately before each experiment with endotoxin-free water. All other chemicals were of the highest grade commercially available.

Animals and treatment regimen. Specific pathogen-free, female C57BL/6 and BALB/c mice (Charles River Laboratories, Kingston, NY) weighing between 17 and 20 g (6-10 wk old) were housed at the Tulane University Medical Center vivarium in pathogen-free cabinets and provided with water ad libitum. Mice received BLM (total dose of 120 mg/kg, in 100 ml of 0.9% NaCl) or saline alone (as control) by a single intravenous injection.

In addition to these inbred murine strains, double (p55/ p75-/-) TNFR and p53 knockout mice were also exposed to BLM. The double TNFR knockout mice used in these experiments were developed at Immunex (Seattle, WA) and have been described elsewhere (27, 28). TSG-p53-deficient (p53 knockout) mice and their littermates were obtained from Genpharm International (Mountain View, CA). These animals were exposed to fibrogeneic doses of BLM (total cumulative dose of 120 mg/kg) as previously described (14). Both types of knockout mice have been developed on a BLM-sensitive background (C57BL/6). A summary of the protocol design is illustrated in Table 1.

                              
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Table 1.   Experimental design

AM collection and ex vivo cultures. At various times after BLM administration (2, 8, 12, 24, 48, and 72 h), mice were killed by pentobarbital sodium injection (120 mg/kg), the aorta was severed, and the trachea was isolated and cannulated. Lungs were lavaged with 0.9% NaCl solution, and the lavage fluid was collected on ice as previously reported (3, 6, 17). Typically, a total instillation of 4 ml (1 ml × 4) of sterile saline resulted in recovery of 3 ml of lavage fluid. Cells in the lavage fluid were isolated by centrifugation (1,000 g for 5 min) and kept on ice in Ca2+- and Mg2+-free Hanks' balanced salt solution at a concentration of 1 × 106 cells/ml (Coulter Counter, Hialeah, FL) until use. A typical lavage from lungs yielded a total of 0.5 × 106 cells per mouse that were >92% macrophages [as identified by Diff-Quik staining (Baxter Healthcare, Miami, FL)], and, therefore, lavage fluid from 10 mice was pooled at each time point to generate the number of cells necessary to carry out one experiment. Cell viability, evaluated after lavage by exclusion of trypan blue (Sigma, St. Louis, MO), was >95% for all groups. Cells were either cytocentrifuged (5 × 104 cells/slide, 4 slides per time point and condition) for the studies of apoptosis or cultured at a concentration of 5 × 105 cells/ml for up to 24 h in HEPES-buffered medium 199 containing 10% heat-inactivated FCS (Sigma) in an incubator at 37°C and 5% CO2-95% air to assess cytokine production. Cells were maintained in suspension culture by slow end-over-end tumbling in polypropylene tubes at 37°C. Incubations were terminated by pelleting the cells and collecting the supernatants. Supernatants were stored at -80°C until assayed for cytokine release.

Assay for macrophage TNF-alpha secretion. TNF-alpha was assayed in cell-free supernatants with the use of an ELISA kit (Factor-Test-X mouse TNF-alpha ; Genzyme, Cambridge, MA) following manufacturer's recommendations. The lower and higher concentrations of TNF-alpha detectable with the use of this kit were identified as 125 and 4,000 pg/ml, respectively.

Assay for the induction of apoptosis. After in vivo exposure to BLM, AM were retrieved by lung lavage as a function of time after BLM exposure (2, 8, 24, 48, and 72 h). Immediately after lavage, cells were cytocentrifuged onto positively charged slides (Fisher Biotech, Houston, TX) at a concentration of 5 × 104 cells/slide with the use of a Shandon cytospin (Shandon). A total of three slides per time point and per treatment were analyzed with an Olympus light microscope with the use of a ×400 magnification. After being air-dried, slides were stained with Diff-Quik and the cells were evaluated for microscopic evidence of apoptosis (22). The number of cells identified as exhibiting unequivocal features of apoptosis per 10 high-power fields (HPF) was computed. The apoptotic features included the loss of cell volume, the fragmentation of the nucleus (karyorrhexis), and the inclusion of apoptotic bodies. To exclude the possibility that apoptotic macrophages represent polymorphonuclear cells, cells were stained for myeloperoxidase, a polymorphonuclear-specific enzyme (2).

Evidence of apoptosis was confirmed by an additional independent assay evaluating the fluorescein-labeled annexin V binding and the exclusion of propidium iodide (PI) by AM as previously described (7, 39). AM retrieved from 10 mice were pooled for each time point, treatment condition, and murine strain. Studies were performed with an apoptosis kit (R&D Systems, Minneapolis, MN) following manufacturer's recommendations. Briefly, AM were resuspended in PBS at a concentration of 1 × 106 cells/ml and incubated in the presence of fluorescein-labeled annexin V, PI, or both at the same time. Flow cytometry analysis was performed on a Becton Dickinson FACStar flow cytometer as previously described (12). Excitation was at 488 nm (100 mW) with a Coherent 6W argon-ion laser. For each particle, emission was measured with photomultipliers with 520 ± 20 and 620 ± 35-nm band-pass filters for fluorescein. Data were collected as 5,000 event list mode files and analyzed with LYSYS II (Becton Dickinson, Mountain View, CA) software. Statistical comparisons were made both by Kolgomorov-Smirnov summation curves and comparison of means of flow cytometry distributions as previously described (41). Experiments were performed three times to guarantee reproducibility. The mean and SE represent the computation of the mean and SE achieved for each individual exposure.

DNA fragmentation in AM was assayed as described previously (22, 40). Macrophages were lavaged from the lungs of mice and lysed in 0.4 ml lysis buffer containing 200 mmol/l Tris · HCl, pH 8.0; 100 mmol/l EDTA; 1% SDS; and 50 mg/ml proteinase K (Sigma). After 4 h at 37°C, the DNA was extracted with an equal volume of water-saturated phenol-chloroform followed by chloroform-isoamyl alcohol (24:1), the aqueous phase was collected, and the cell DNA was precipitated by adding NaCl to 150 mmol/l and 2 volumes ethanol at -20°C overnight. The DNA pellet was dried under vacuum and resuspended in 0.4 ml 10 mmol/l Tris, 10 mmol/l NaCl, and 1 mmol/l EDTA (TNE buffer). RNase A (50 mg/ml; Promega, Madison, WI) was added for 5 h followed by proteinase K (120 mg), and the DNA was again extracted twice and precipitated as above. The DNA pellet was resuspended in 100 ml of TNE; separated (5 mg DNA per lane) by horizontal electrophoresis for 4 h (2 V/cm) in 1.2% agarose gel with 90 mmol/l Tris, 90 mmol/l boric acid, and 2 mmol/l EDTA, pH 8.0, as running buffer; stained with 0.5 mg/ml ethidium bromide; and visualized under ultraviolet light. Digested lambda  DNA Hind III (Bio-Rad, Hercules, CA) was used as a control for DNA fragmentation.

For the in vitro experiments, AM were lavaged from untreated animals with similar techniques and cultured in HEPES-buffered medium 199 containing 10% heat-inactivated FCS (at a concentration of 2 × 106 cells/ml) or the same medium with and without BLM (10 µM) for 24 h at 37°C and 5% CO2.

Immunoblot measurement for p53 protein levels. Immunoblots for p53 proteins were performed as previously described (15). Briefly, AM retrieved from the lung of BLM-treated mice were collected by low-speed centrifugation (2 × 106 cells). Cells were lysed in RIPA buffer [(PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS); proteinase inhibitors were added at the time of use from the following stock solution: 1) 10 mg/ml phenylmethylsulfonyl fluoride in isopropanol (used at 10 ml/ml), 2) aprotinin (used at 30 ml/ml), and 3) 100 mM sodium orthovanadate (used at 10 ml/ml)]. Cell homogenates were passaged through 21-gauge needles to disrupt DNA. Equivalent cell extracts were electrophoresed on a 10% Tris-glycine gel, semi-dry-transferred to nitrocellulose (Amersham). Membranes were incubated in blocking buffer (PBS-5% powdered milk) for 2 h at room temperature before the addition of the primary antibody, which was incubated with the membranes at 4°C overnight. The primary antibody (Boehringer Mannheim) was PAb 122, which recognizes both the wild-type and mutant p53 conformations. Signals were generated with the enhanced chemiluminescence detection system (Amersham) with a horseradish peroxidase-coupled secondary antibody (Amersham).

Immunohistochemistry. Fourteen and twenty-one days after BLM exposure, C57BL/6, BALB/c, TNFR-deficient, and p53-deficient mice were killed with pentobarbital sodium injection (120 mg/kg), the descending aorta was severed, and the hearts were perfused with cold 0.9% NaCl to remove blood. The lungs were allowed to fix in situ for 2 h by the intratracheal instillation of 10% neutral Formalin (Sigma) at a constant perfusion pressure of 30 cmH2O and were preserved in fixative for 24 h. Lung tissues were sectioned sagittally and embedded in paraffin, and sections (4-µm thick) were generated onto positively charged slides (Fisher Scientific, Pittsburgh, PA) for light microscopy and immunocytochemistry. Immunohistochemistry for PCNA was by the biotinylated second antibody method as previously described (24, 27). The primary antibody was a PCNA-specific monoclonal antibody, PC-10 (Dako, Carpinteria, CA), and the secondary antibody was biotinylated goat anti-mouse IgG (Jackson Immunoresearch, Bar Harbor, ME). After the slide was incubated with streptavidin-horseradish peroxidase, detection was with diaminobenzidine. The immunostaining was assessed qualitatively by evaluating the immunostaining in 300 cells in the lungs of control or BLM-treated mice and expressing the result as the percentage of positively stained cells. Immunostaining specificity was determined by substituting nonspecific IgG for the primary antibody. Positive controls were performed by using mouse intestine.

Statistics. Data are shown as means ± SE. The number of replicate experiments used for a given experiment is denoted by n. Each in vivo and in vitro experiment was performed by pooling AM to the specified concentration. An average of 0.5 × 106 cells were retrieved per mouse, and, therefore, cells from 10 mice were pooled at each time point. For each experiment, statistical analysis was performed by a three-way ANOVA with strain, time, and treatment as independent variables. The analysis was computed with the use of a statistical package (BMDP, Los Angeles, CA) followed by Duncan's multiple range test (42). For the flow cytometry studies, statistical comparisons were made by both Kolgomorov-Smirnov summation curves and comparison of means (obtained from n = 3) of flow cytometry distributions as previously described (41).

    RESULTS
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Abstract
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Materials & Methods
Results
Discussion
References

AM apoptosis after in vivo exposure to BLM. After BLM treatment or vehicle treatment alone as control, AM retrieved by lung lavage from both the BLM-sensitive (C57BL/6) mouse strain or the BLM-resistant (BALB/c) strain appeared viable, as assessed by trypan blue exclusion (average trypan blue exclusion >95% for all time points). No differences in macrophage number or viability were found between the treatment groups and the strain groups.

Vehicle treatment did not result in any evidence of induction of apoptosis in AM from either C57BL/6 or BALB/c animals (Fig. 1A). In contrast, characteristic findings of apoptosis (loss of cytoplasm, nuclear fragmentation, formation of apoptotic bodies) were found in AM retrieved from both C57BL/6 and BALB/c mice following an in vivo exposure to BLM. A representative example is shown in Fig. 1B. Because apoptotic macrophages resemble the morphology of polymorphonuclear cells, we performed immunostaining against polymorphonuclear cell-specific myeloperoxidase to confirm that the cells being identified were indeed AM. Polymorphonuclear cells are clearly stained in their cytoplasm, whereas cells demonstrating the morphological characteristics of apoptosis are not, suggesting that the apoptotic cells were indeed AM. A representative example is illustrated in Fig. 1C.


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Fig. 1.   Bleomycin (BLM) induction of apoptosis in alveolar macrophages. Light microscopy of alveolar macrophages (AM) after in vivo exposure to BLM. See text for complete description. A: photomicrograph of AM from BLM-sensitive (C57BL/6) mice after exposure to saline. Cell exhibits normal morphology, and no evidence of apoptosis is found. B: AM retrieved from BLM-sensitive (C57BL/6) mice after exposure to BLM. AM demonstrate microscopic evidence of apoptosis. Arrows indicate nuclear fragmentation and loss of cytoplasm ("shrinkage"). C demonstrates presence of apoptotic features in cells that stain negative for myeloperoxidase, suggesting a nonmyeloid origin for these cells (alveolar macrophages).

The in vivo exposure to BLM resulted in a greater number of apoptotic AM from C57BL/6 mice compared with BALB/c mice (Fig. 2). Features of apoptosis in AM retrieved from BALB/c mice were identified in 2 cells/10 HPF 24 h after BLM exposure. No further significant increases in the number of apoptotic cells were found at 48 and 72 h postexposure compared with controls. In contrast, a significantly greater number of apoptotic cells were identified in AM from C57BL/6 mice 24 and 48, but not 72, h (11, 6, and 2 cells/10 HPF, respectively) after BLM exposure compared with cells retrieved from control treated mice (P < 0.05).


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Fig. 2.   Differences in numbers of apoptotic AM in bronchoaveolar lavage for BLM-sensitive (S) and BLM-resistant (R) murine strains. Lungs were lavaged 24, 48, and 72 h after in vivo exposure to saline alone as control (C) or to 120 mg/kg BLM (B). Number of AM exhibiting microscopic features of apoptosis was counted as described in MATERIALS AND METHODS. HPF, high-power field. * P < 0.05, BLM-treated animals after BLM exposure compared with control treated animals. dagger  P < 0.05, resistant vs. sensitive strains at similar time points.

To further evaluate the induction of apoptosis, after an in vivo exposure to BLM, we studied the binding of fluorescein-labeled annexin V and the exclusion of PI by AM. AM retrieved from vehicle-treated animals from both strains demonstrated minimal (<7% of the cells stained positive) binding of annexin V and universally excluded PI (<1% of the cells demonstrated PI uptake), suggesting that the cells (>90%) were viable. A representative fluorescence three-dimensional histogram of vehicle-treated AM is illustrated in Fig. 3A. Each panel in Fig. 3 depicts a frequency histogram generated from data on 5,000 individual cells. The number of cells per channel is in the third dimension. The horizontal axes depict fluorescein-labeled annexin V binding and PI fluorescence on a log scale of 1,024 channels. Compared with vehicle-treated mice, BLM exposure resulted in a significant (P < 0.001) increase in the number of apoptotic AM as determined by increased annexin V binding in both BLM-sensitive (C57BL/6) (Fig. 3B) and BLM-resistant (BALB/c) mice (Fig. 3C), but the magnitude of the increase in apoptosis, as reflected by the number of annexin-positive AM, was significantly greater (P < 0.001) in C57BL/6 mice (52.9%) compared with BALB/c mice (40.8%) [D/S (n) = 5.64, D = 0.11]. Representative fluorescence dot plots are illustrated in Fig. 3, B and C. BLM treatment also resulted in an increased induction of necrosis in macrophages compared with vehicle-treated mice, but there were no significant differences between strains (P > 0.05).


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Fig. 3.   Flow cytometry colocalization of annexin V binding and propidium iodine uptake for quantitation of apoptotic and necrotic AM populations. Horizontal axes, fluorescence of fluorescein-conjugated annexin V and propidium iodide uptake; vertical axes, number of cells per channel. A: control. Staining of AM from a control treated C57BL/6 mouse defines levels of nonspecific binding and autofluorescence. B: BLM-sensitive (C57BL/6) mice exposed to BLM. A large population of apoptotic AM is characterized by annexin V binding without propidium iodine uptake (lower middle), and a smaller necrotic population is characterized by both annexin V binding and propidium iodide uptake (right). C: AM from BLM-resistant (BALB/c) mice exposed to BLM. See text for details. D: AM from double (p55/p75-/-) tumor necrosis factor (TNF) receptor knockout (TNFR ko) mice exposed to BLM.

AM apoptosis after in vitro exposure to BLM. To further examine the capacity of BLM to induce apoptosis in AM, we analyzed characteristic DNA fragmentation of apoptosis after an in vitro exposure to BLM. AM from both murine strains were isolated and then exposed in vitro to BLM (10 µM) (or medium alone as control) for 24 h. After in vitro exposure, DNA was isolated and subjected to agarose electrophoresis as described. A representative gel is depicted in Fig. 4. AM from BLM-sensitive (C57BL/6) and BLM-resistant (BALB/c) mice demonstrated DNA laddering following an in vitro exposure to BLM (Fig. 4, lanes d and e). No evidence of DNA damage was found in AM exposed to medium alone (Fig. 4, lanes b and c). AM from C57BL/6 mice demonstrated significantly larger numbers of apoptotic cells (40 ± 1 cells/10 HPF) than macrophages retrieved from BALB/c mice (15 ± 1 cells/10 HPF) (P < 0.05, n = 3). AM stained negatively for myeloperoxidase (data not shown).


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Fig. 4.   BLM induction of DNA fragmentation in AM. Figure illustrates electrophoresis of DNA isolated from AM after in vitro exposure to culture medium or BLM (10 mM) as described in text. Lane a represents Hind III digested lambda  DNA. Lanes b and c represent DNA isolated from AM from BLM-resistant (b) and BLM-sensitive (c) mice after exposure to medium alone. Lanes d and e represent DNA extracted from AM from BLM-resistant (d) and BLM-sensitive (e) mice after exposure to BLM.

TNF-alpha release by AM after BLM exposure. The amounts of TNF-alpha produced by murine AM retrieved by lung lavage from BLM-sensitive (C57BL/6) and BLM-resistant (BALB/c) strains after in vivo exposure to BLM are shown in Fig. 5. The release of TNF-alpha in AM obtained from BALB/c mice was not increased compared with saline-treated animals 2, 8, or 24 h after BLM exposure (Fig. 5A). There was a statistically significant difference (P < 0.5) between control exposed C57BL/6 mice and C57BL/6 mice at 2 and 8 h after BLM exposure (Fig. 5B).


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Fig. 5.   TNF-alpha production by murine AM from BLM-resistant (BALB/c) and BLM-sensitive (C57BL/6) mice after in vivo exposure to BLM; n = 4. TNF-alpha production by murine AM from BLM-resistant (A) and BLM-sensitive (B) mice after in vivo exposure to BLM (120 mg/kg) or saline as control. TNF-alpha was measured by ELISA as described in MATERIALS AND METHODS. AM from BLM-sensitive, but not from BLM-resistant, mice produced significant (* P < 0.05) amounts of TNF-alpha 2 and 8 h after exposure to BLM compared with controls.

AM apoptosis in double TNFR-deficient mice. To further evaluate the role of TNF-alpha in the induction of apoptosis in AM, we exposed BLM-resistant double TNFR-deficient mice to BLM. The induction of apoptosis was measured by annexin V uptake and PI exclusion in AM retrieved from these animals by flow cytometry techniques. Vehicle treatment had no effect on the induction of apoptosis (Fig. 3A). In contrast, BLM exposure resulted in a significant increase (P < 0.001) in annexin V binding in 34% of the AM retrieved from these animals (Fig. 3D). BLM treatment also resulted in increased PI uptake by 11% of the cells examined (Fig. 3D). The number of apoptotic AM in TNFR knockout mice was significantly lower (P < 0.001) compared with either BLM-sensitive [52.9%; D/S (n) = 7.83, D = 0.16] or BLM-resistant [40.8%; D/S (n) = 4.78, D = 0.10] mice.

Effect of BLM exposure on p53 expression by murine AM. To further study the mediators involved in the BLM induction of apoptosis in AM, we studied the expression of p53 in these cells after an in vivo murine exposure to BLM. The p53 anti-oncoprotein was detected by Western blotting of AM proteins with a monoclonal anti-p53 antibody. A distinct p53 band that comigrated with authentic p53 and reacted with p53 antibodies (p122) was seen only in the BLM-treated animals. Both murine strains responded to BLM exposure with an increase in the p53 signal (Fig. 6). This enhanced expression of p53 was maximal 24 h after BLM exposure and could be identified in AM 72 h after the original murine exposure to BLM. Densitometric analysis of these immunoblots demonstrated that although AM from both murine strains reacted to BLM with significant increases in p53 levels, a greater increase in p53 levels in AM isolated from C57BL/6 mice was found compared with those in the BALB/c mice (P < 0.05; Fig. 6).


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Fig. 6.   p53 expression in murine AM from BLM-resistant (BALB/c) and BLM-sensitive (C57BL/6) mice after BLM exposure. p53 was detected by immunoblotting as described in MATERIALS AND METHODS. AM from both murine strains reacted to BLM exposure with an increase in p53 signal (A). B illustrates magnitude of the increase in p53 in AM, expressed as a percentage over control (100%), after BLM exposure. * P < 0.05 vs. control.

PCNA immunostaining and hydroxyproline accumulation in BLM-treated mice. To further study the role of the p53 protein in the pathogenesis of BLM-induced lung injury, we exposed p53 knockout mice to fibrogenic doses of BLM and compared their response to BLM-sensitive (C57BL/6) mice. BLM treatment resulted in the development of subpleural areas of pneumonitis in both p53-deficient (knockout) and C57BL/6 (BLM-sensitive) mice 21 days after BLM exposure (data not shown). BLM exposure resulted in significant (P < 0.001) hydroxyproline accumulation in the lungs of both p53 (Table 2) and C57BL/6 (Table 2) mice compared with control treated animals (Table 2). The hydroxyproline accumulation for the BLM-treated mice was similar in both the p53 and the C57BL/6 mice (Table 2).

                              
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Table 2.   Bleomycin-induced lung injury in C57BL/6 and p53-deficient mice

Minimal PCNA immunostaining was observed in the lungs of control treated mice (Fig. 7). In contrast, BLM exposure induced an increased immunostaining for PCNA in the subpleural areas of pneumonitis in both C57BL/6 and p53-deficient mice (Fig. 7). This immunostaining signal was specific because it was eliminated by substituting mouse IgG for the primary antibody (Fig. 7). No qualitative difference in the immunostaining for PCNA could be appreciated between the two murine strains. This observation contrasted with an attenuated immunostaining for PCNA observed in the lungs of BALB/c (data not shown) and double TNFR-deficient mice, which are resistant to BLM-induced lung injury (Fig. 7).


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Fig. 7.   Proliferating cell nuclear antigen (PCNA) immunostaining in lungs of BLM-treated mice. Fourteen days after exposure to BLM, lungs of mice were fixed by intratracheal perfusion of Formalin and processed for immunohistochemistry as described in MATERIALS AND METHODS. A: minimal PCNA immunostaining is detected in lungs of BLM-sensitive (C57BL/6) mice exposed to vehicle. B and C: PCNA immunostaining (see boxes, magnified in insets at top right) is greatly enhanced in subpleural areas of BLM-induced lung injury in C57BL/6 (B) and p53-deficient (C) mice 14 days after exposure to BLM. This enhanced PCNA immunostaining is not observed in lungs of double TNFR-deficient mice (D). E: positive control for the immunostaining using mouse intestine. F: specificity of PCNA immunostaining is demonstrated by use of a nonspecific IgG that abolish the PCNA immunostaining in lungs of C57BL/6 mice treated with BLM. PL, pleura; TB, terminal bronchi. Bars = 20 µm.

    DISCUSSION
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Abstract
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Materials & Methods
Results
Discussion
References

It has been reported that apoptosis is central to the pathogenesis of fibrotic lung disease because it contributes to the perpetuation of inflammation after lung injury (32, 33). This persistent inflammatory reaction could lead to the pathological scarring of pulmonary fibrosis (32). Excessive apoptosis contributing to the development of lung injury and fibrosis has been reported by Hagimoto and associates (9), who demonstrated that chronic ligation of the Fas antigen in murine lung, achieved by the repeated nebulization of an anti-Fas antibody, is associated with the development of inflammation and apoptosis in alveolar epithelial cells and the accumulation of hydroxyproline. The same authors also reported the rapid appearance of apoptosis in bronchial and alveolar epithelial cells of mice following intratracheal injection of BLM (10). This BLM induction of apoptosis in the murine lung was biphasic, showing an early and a late phase. The early induction of apoptosis was short-lived and resolved within 24 h after the BLM exposure (10). In contrast to this, apoptosis reappeared in the lung of BLM-treated animals 7 days after the BLM exposure and was still detectable 14 days later (10).

In the present study, we found that a systemic exposure to BLM is associated with the induction of apoptosis in AM, a cell type that has been considered to play a fundamental role in the pathogenesis of BLM-induced lung injury (1, 3, 6, 29). This BLM induction of apoptosis in AM is in accordance with the murine strain sensitivity to BLM, i.e., a larger number of apoptotic macrophages can be identified in the lungs of BLM-sensitive (C57BL/6) mice compared with BLM-resistant (BALB/c) mice. The number of AM that demonstrated microscopic characteristics of apoptosis in lung lavages of BLM-treated mice was small (5-10% of the total number of macrophages isolated from BLM-sensitive mice 24 h after BLM exposure). However, the life of an apoptotic cell is very short [30 min from onset to complete cell breakup (23)], and studies based on microscopic analysis alone would be likely to underestimate the number of apoptotic AM during lung injury. To clarify this issue, we also studied annexin V binding to the AM cell surface. The capacity of annexin V to bind to the cell membrane occurs early in the life of an apoptotic cell because the exposure of phosphatidylserine residues in the outer cell membrane are obvious (7, 39). Therefore, this technique may identify an apoptotic cell before it exhibits microscopic features of apoptosis (7, 39). Using this technology, we found that increased numbers of macrophages (53% in BLM-sensitive mice) were apoptotic in the lungs of BLM-treated animals.

The mechanisms by which BLM induces apoptosis in AM are not completely understood. The capacity of BLM to damage DNA and induce apoptosis is directly related to its ability to reach the cell cytoplasm (38). BLM binding sites that may facilitate BLM transport have been described on AM (3). BLM complexes with ferrous ions and molecular oxygen between pairs of DNA and induces single- and double-strand DNA breakages (1, 18, 34). The capacity of the cell to repair these single- and double-strand DNA breaks is an important determinant of resistance to BLM (1, 18). DNA strand scission can, in a direct manner, induce apoptosis or stimulate the activation of endonucleases that can induce programmed cell death (11, 34). Supporting this idea are data suggesting that ZnCl2, which can inhibit endonuclease activity, is able to prevent BLM induction of apoptosis in human AM (11). As demonstrated in the present study (Fig. 4), BLM is able to induce DNA fragmentation in AM retrieved from both BLM-sensitive (C57BL/6) and BLM-resistant (BALB/c) mice. Previous studies have suggested that the amount of DNA damage observed in the lungs of mice after a BLM exposure is similar in both of these murine strains (13). In the present study, we found that the induction of apoptosis observed in AM from C57BL/6 mice was greater compared with that observed in BALB/c mice, suggesting that DNA damage alone does not account for this observed difference in the induction of apoptosis.

Exposure of cells to DNA damaging agents induces increases in p53 protein levels (26). p53 has been shown to be a key mediator of the cellular response to DNA damage, and p53 expression is a first line of defense in maintaining genomic integrity (36). In the present study, AM from both murine strains reacted to BLM with an enhanced p53 expression. Because p53 promotes DNA integrity by arresting the cell cycle (promoting DNA repair) or inducing apoptosis, these data suggest that DNA repair is taking place in the AM retrieved from BLM-treated mice. It has been shown, using in vitro incubation of lung slices with BLM, that the exposure is associated with activation of poly(ADP-ribose) polymerase, a nuclear enzyme that facilitates DNA repair. This DNA repair activity appears to be greater in C57BL/6 mice where lung expression of p53 is greater in response to BLM (16).

To further evaluate the importance of p53 in the pathogenesis of BLM-induced lung injury, we exposed p53-deficient mice raised in a C57BL/6 background to BLM and compared their response to the BLM-sensitive (C57BL/6) mice. Compared with C57BL/6 mice, p53-deficient mice accumulate equivalent amounts of lung hydroxyproline and demonstrated an equivalent fibroproliferative response as evaluated by an enhanced PCNA immunostaining. In contrast, we found attenuation in PCNA immunostaining in the lungs of BLM-resistant (BALB/c) and double TNFR-deficient mice, which are resistant to BLM-induced lung fibrosis. These data suggest that the enhanced PCNA expression and the fibroproliferative response observed in response to BLM are not mediated by increased levels of p53 protein.

In addition to inducing DNA damage, BLM binding to the AM is associated with an altered capacity to upregulate and secrete inflammatory cytokines (3, 6, 17, 35). Among these cytokines, TNF-alpha has been found to play a fundamental role in the pathogenesis of BLM-induced lung injury and fibrosis (29, 30, 37). Blocking TNF-alpha expression in the lung of BLM-treated mice with the administration of neutralizing antibodies or soluble TNF receptor results in a significant reduction in BLM-induced lung fibrosis (30, 31). The production of TNF-alpha during BLM-induced lung injury has been described as a relatively late event, observed 5-15 days after the endotracheal instillation of BLM (29, 30). In addition, in two separate studies using TNFR-deficient mice, we have shown that these animals are protected from the fibrogenic effects of both BLM (27) and asbestos (21). In the present study, we found that the AM can increase the release of TNF-alpha as early as 2 h after BLM exposure (Fig. 5). This enhanced secretion of TNF-alpha by the AM after BLM correlated with the murine strain sensitivity to BLM and with the induction of apoptosis (Figs. 2, 3, and 5). These results are in agreement with previous reports suggesting that TNF-alpha is a major contributor to the murine strain difference in response to BLM (27, 29).

To further correlate TNF-alpha secretion by the AM with the induction of apoptosis, we exposed double TNFR-deficient mice to BLM. AM retrieved from these animals, which are resistant to the inflammatory and fibrogenic effects of BLM (27), demonstrated a significantly lower level of apoptosis than that observed in wild-type mice. These data, as well as those showing decreased numbers of apoptotic macrophages in BLM-resistant (BALB/c) mice (Figs. 2 and 3) and the absence of an enhanced TNF-alpha secretion after BLM exposure in these cells, suggest that the interaction of TNF-alpha with its receptors plays an important role in the induction of apoptosis by BLM. It has been demonstrated that the overexpression of TNF-alpha in the murine lung is associated with an inflammatory and fibrotic response that is similar to the one described after BLM exposure (25).

In summary, in the present study, we found that the systemic exposure of mice to BLM is associated with the induction of apoptosis in AM. This induction of apoptosis in macrophages correlates with the murine strain sensitivity to BLM and was associated with an enhanced secretion of TNF-alpha . Furthermore, the importance of TNF-alpha as a mediator for BLM induction of apoptosis in AM is suggested by the data demonstrating that apoptosis was significantly decreased in double TNFR-deficient mice. BLM exposure was also associated with an enhanced p53 expression in AM. However, a direct role for p53 in the pathogenesis of BLM-induced lung injury was not found because p53-deficient mice reacted to BLM exposure with similar lung cell proliferation and collagen accumulation. Further studies addressing AM apoptosis in p53-deficient mice will be necessary to understand the contribution of AM apoptosis in the pathogenesis of fibrotic lung injury. These models of BLM-resistant mice and TNFR-deficient animals will aid in our ongoing efforts to define the mechanisms that mediate interstitial pulmonary fibrogenesis.

    ACKNOWLEDGEMENTS

These studies were supported by National Heart, Lung, and Blood Institute Grant HL-03569 and National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-51392.

    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: L. A. Ortiz, Section of Pulmonary Diseases, Critical Care and Environmental Medicine, Dept. of Medicine SL9, Tulane Univ. Medical Center, New Orleans, LA 70112-2699.

Received 12 June 1998; accepted in final form 8 September 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Chandler, D. B. Possible mechanisms of bleomycin-induced fibrosis. Clin. Chest Med. 11: 21-30, 1990[Medline].

2.   Cox, G., J. Crossley, and Z. Xing. Macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary inflammation in vivo. Am. J. Respir. Cell Mol. Biol. 12: 232-237, 1995[Abstract].

3.   Denholm, E. M., and S. H. Phan. The effects of bleomycin on alveolar macrophage growth factor secretion. Am. J. Pathol. 134: 355-363, 1989[Abstract].

4.   Denholm, E. M., and S. H. Phan. Bleomycin binding on alveolar macrophages. J. Leukoc. Biol. 48: 519-523, 1990[Abstract].

5.   Desmoulière, A., M. Redard, I. Darby, and G. Gabbiani. Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar. Am. J. Pathol. 146: 56-66, 1995[Abstract].

6.   Everson, P. M., and D. B. Chandler. Changes in distribution, morphology, and tumor necrosis factor-alpha secretion of alveolar macrophage subpopulations during the development of bleomycin-induced pulmonary fibrosis. Am. J. Pathol. 140: 503-512, 1992[Abstract].

7.   Fadok, V. A., D. R. Voelker, P. A. Campbell, J. J. Cohen, D. L. Bratton, and P. M. Henson. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148: 2207-2216, 1992[Abstract/Free Full Text].

8.   Giri, S. N., D. M. Hyde, and M. A. Hollinger. Effect of antibody to transforming growth factor beta  on bleomycin induced accumulation of lung collagen in mice. Thorax 48: 959-966, 1993[Abstract].

9.   Hagimoto, N., K. Kuwano, H. Miyazaki, R. Kunitake, M. Fujita, M. Kawasaki, Y. Kaneko, and N. Hara. Induction of apoptosis and pulmonary fibrosis in mice in response to ligation of Fas antigen. Am. J. Respir. Cell Mol. Biol. 17: 271-278, 1997.

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

11.   Hamilton, R. F., L. Li, T. B. Felder, and A. Holian. Bleomycin induces apoptosis in human alveolar macrophages. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L318-L325, 1995[Abstract/Free Full Text].

12.   Hammond, T. G., R. R. Majewski, K. Schell, and L. W. Morrissey. Pulse processing resolves multiple populations of endosomes in two in vivo systems. Cytometry 14: 411-420, 1993[Medline].

13.   Harrison, J. H., D. G. Hoyt, and J. S. Lazo. Acute pulmonary toxicity in bleomycin: DNA scission and matrix protein mRNA levels in bleomycin-sensitive and -resistant strains in mice. Mol. Pharmacol. 36: 231-238, 1989[Abstract].

14.   Harrison, J. H., and J. S. Lazo. High dose continuous infusion of bleomycin in mice: a new model for drug-induced pulmonary fibrosis. J. Pharmacol. Exp. Ther. 243: 1185-1194, 1987[Abstract].

15.   Hermeking, H., and D. Eick. Mediation of c-Myc-induced apoptosis by p53. Science 265: 2091-2093, 1994[Medline].

16.   Hoyt, D. G., and J. S. Lazo. Murine strain differences in acute lung injury and activation of poly(ADP-ribose) polymerase by in vitro exposure of lung slices to bleomycin. Am. J. Respir. Cell Mol. Biol. 7: 645-651, 1992[Medline].

17.   Jordana, M., C. Richards, L. B. Irving, and J. Gauldie. Spontaneous in vitro release of alveolar-macrophage cytokines after intratracheal instillation of bleomycin in rats. Am. Rev. Respir. Dis. 137: 1135-1140, 1988[Medline].

18.   Jules-Elyse, K., and D. A. White. Bleomycin-induced pulmonary toxicity. Clin. Chest Med. 11: 1-20, 1990[Medline].

19.   Kastan, M. B., O. Onyekwere, D. Sidransky, B. Vogelstein, and R. Craig. Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 51: 6304-6311, 1991[Abstract].

20.   Lazo, J. S., and E. T. Pham. Pulmonary fate of [3H] bleomycin A2 in mice. J. Pharmacol. Exp. Ther. 228: 13-18, 1984[Abstract].

21.  Liu, J.-Y., D. Brass, G. W. Hoyle, and A. R. Brody. TNF-alpha receptor knockout mice are protected from the fibroproliferative effects of inhaled asbestos fibers. Am. J. Pathol. In press.

22.   Lotem, J., E. J. Cragoe, and L. Sachs. Rescue from programmed cell death in leukemic and normal myeloid cells. Blood 78: 953-960, 1991[Abstract].

23.   Majno, G., and I. Joris. Apoptosis, oncosis, and necrosis. An overview of cell death. Am. J. Pathol. 146: 3-15, 1995[Abstract].

24.   Mishra, A., J.-Y. Liu, A. R. Brody, and G. F. Morris. Inhaled asbestos fibers induce p53 expression in the rat lung. Am. J. Respir. Cell Mol. Biol. 16: 479-485, 1997[Abstract].

25.  Miyazaki, Y., T. Tashiro, Y. Higuchi, M. Setoguuchi, S. Yamamoto, H. Nagai, M. Nasu, and P. Vassalli. Expression of osteopontin in a macrophage cell line and in transgenic mice with pulmonary fibrosis resulting from the lung expression of a tumor necrosis factor-alpha transgene. Ann. NY Acad. Sci. 160: 334-341.

26.   Nelson, W., and M. C. Kastan. DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways. Mol. Cell. Biol. 14: 1815-1823, 1994[Abstract].

27.  Ortiz, L., J. A. Lasky, R. Hamilton, A. Holian, G. W. Hoyle, W. A. Banks, J. Peschon, A. R. Brody, and M. Friedman. Expression of TNF and the necessity for TNF receptors in bleomycin-induced lung injury in mice. Exp. Lung Res. In press.

28.   Peschon, J. J., D. S. Torrance, K. L. Stocking, M. B. Glaccum, C. Otten, C. R. Willis, K. Charrier, P. J. Morrissey, C. B. Ware, and K. M. Moheler. TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation. J. Immunol. 160: 943-952, 1998[Abstract/Free Full Text].

29.   Phan, S. H., and S. L. Kunkel. Lung cytokine production in bleomycin-induced pulmonary fibrosis. Exp. Lung Res. 18: 29-43, 1992[Medline].

30.   Piguet, P. F., M. A. Collart, G. E. Grau, Y. Kapanci, and P. Vassalli. Tumor necrosis factor/cachectin plays a key role in bleomycin-induced pneumopathy and fibrosis. J. Exp. Med. 170: 655-663, 1989[Abstract].

31.   Piguet, P. F., and C. Vesin. Treatment by recombinant soluble TNF receptor of pulmonary fibrosis induced by bleomycin or silica in mice. Eur. Respir. J. 7: 515-518, 1994[Abstract/Free Full Text].

32.   Polonovsky, V., and P. Bitterman. Regulation of cell population size. In: The Lung: Scientific Foundation (2nd ed.), edited by R. G. Crystal, J. B. West, E. R. Weibel, and P. J. Barnes. Philadelphia, PA: Lippincott-Raven, 1997, p. 133-153.

33.   Polonovsky, V., B. Chen, C. Henke, D. Snover, D. Ingbar, and P. Bitterman. Role of mesenchymal cell death in lung remodeling after injury. J. Clin. Invest. 92: 388-397, 1993[Medline].

34.   Rusnak, J. M., P. G. Thierry, D. G. Hoyt, Y. Kondo, J. C. Yalowich, and J. S. Lazo. Genesis of discrete higher order DNA fragments in apoptotic human prostatic carcinoma cells. Mol. Pharmacol. 49: 244-252, 1996[Abstract].

35.   Scheule, R. K., R. C. Perkins, R. Hamilton, and A. Holian. Bleomycin stimulation of cytokine secretion by the human alveolar macrophage. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L386-L391, 1992[Abstract/Free Full Text].

36.   Smith, M. L., I. T. Chen, Q. Zhan, I. Bae, C. Y. Chen, T. M. Gilmer, M. B. Kastan, P. M. O'Connor, and A. J. Fornace, Jr. Interaction of the p53-regulated protein Gadd 45 with proliferating cell nuclear antigen. Science 266: 1376-1380, 1994[Medline].

37.   Smith, R. E., R. M. Strieter, S. H. Phan, and S. L. Kunkel. C-C chemokines: novel mediators of pro-fibrotic inflammatory response to bleomycin challenge. Am. J. Respir. Cell Mol. Biol. 15: 693-702, 1996[Medline].

38.   Tounekti, O., G. Pron, J. Belehradek, and L. Mir. Bleomycin, an apoptosis-mimetic drug that induces two types of cell death depending on the number of molecules internalized. Cancer Res. 53: 5462-5469, 1993[Abstract].

39.   Vermes, I., C. Haanen, H. Steffens-Nakken, and C. Reutelingsperger. A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled annexin V. J. Immunol. Methods 184: 39-51, 1995[Medline].

40.   Wyllie, A. H., J. F. R. Kerr, and A. R. Currie. Cell death: the significance of apoptosis. Int. Rev. Cytol. 68: 251-356, 1980[Medline].

41.   Young, I. T. Proof without prejudice: use of the Kolgomorov-Smirnov test for the analysis of histograms from flow systems and other sources. J. Histochem. Cytochem. 25: 935-941, 1977[Abstract].

42.   Zar, J. H. Biostatistical Analysis. Englewood Cliffs, NJ: Prentice-Hall, 1984.


Am J Physiol Lung Cell Mol Physiol 275(6):L1208-L1218
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