The oleic acid (OA) model of acute lung injury in rats is characterized by a massive and rapid influx of polymorphonuclear neutrophils (PMN) within 1 h, with a peak inflammatory response at 4 h and resolution by 72 h. We hypothesized that PMN apoptosis is involved in the resolution of OA-induced acute lung injury. To test this hypothesis, healthy adult Fischer 344 rats were given 30 μl OA in 0.1% bovine serum albumin (BSA) intravenously; controls were given BSA alone and killed at 1, 4, 24, and 72 h after OA to obtain bronchoalveolar lavage fluid (BALF) and lung tissue. Cell pellets from BALF and formalin-fixed, paraffin-embedded tissue section samples were processed for terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) to identify apoptotic cells. Propidium iodide was used to counterstain nuclei. Percentage of nuclei undergoing apoptosis was counted under a fluorescent microscope. Control rats showed only resident alveolar macrophages (AM) in the BALF with no apoptosis. At the peak of injury, 1 h and 4 h after OA injection, we observed a massive PMN response without any evidence of apoptosis. At 24 h, when the OA injury is clinically and histologically in early resolution, we observed intense apoptosis of PMN nuclei along with evidence of apoptotic bodies in the cytoplasm of AM. Some of the AM also showed apoptotic nuclei at 72 h. Similar observations were made in the lung tissue sections. The results of the TUNEL assay were confirmed by DNA ladders and electron microscopy. We conclude that apoptosis of PMN and clearance by AM is an important mechanism in resolution of OA- induced acute lung injury.
The progression from injury to acute inflammatory response is characterized by an influx of polymorphonuclear neutrophilic leukocytes (PMN). The resolution of acute inflammation depends on the timely clearance of these inflammatory cells. Emigration of PMN back into circulation does not occur after acute lung injury (1). The acute PMN response during inflammation in the lungs clears by two possible mechanisms: (1) cellular lysis and breakdown, which results in liberation of proteases and other enzymes; or (2) apoptosis and phagocytosis by macrophages and other cells.
Apoptosis (2) (a form of programmed cell death) is an active process involving gene transcription and protein synthesis that leads to an orderly sequence of events, resulting in cytoplasmic shrinking, budding, nuclear fragmentation (180- to 200-bp fragments), and formation of apoptotic bodies that are ingested by the neighboring cells (3, 4). Because the cell membrane remains intact during this process, there is no release of proinflammatory stimuli and this has been considered to be important in the resolution of inflammation (5). Grigg and colleagues (6) have shown that PMN apoptosis occurs in cells obtained from tracheal aspirates in infants recovering from respiratory distress syndrome. Cox and coworkers (7) have demonstrated that PMN apoptosis and engulfment by alveolar macrophages contributes to the resolution of bacterial lipopolysaccharide (LPS)-induced acute lung inflammation. It remains to be determined whether this process is specific to the LPS injury or common to other models of resolving lung inflammation.
Oleic acid (OA) induces lung injury that mimics the acute inflammation of the adult respiratory distress syndrome (ARDS) (8), which has been well characterized as a model for studying acute lung inflammation (8-10), but the role of apoptosis in this process has not been studied. In tissues, apoptosis and phagocytosis proceed rapidly and are completed in a matter of hours (3, 11). Visualization of small percentages of in situ stained apoptotic cells yields biologically significant data. A rate of tissue regression as rapid as 25% per day can result from apoptosis in 2% to 3% of the cells at any one time (11). The terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) assay has been shown to be a sensitive and specific method for detecting apoptosis in histologic sections (12, 13).
Based on the hypothesis that inflammatory cell apoptosis is necessary for the resolution of acute pulmonary inflammation, the purpose of this study was to determine the role of PMN apoptosis during the course of oleic acid-induced lung injury and resolution in rats.
Adult male, pathogen-free, Fisher 344 inbred rats weighing 220 to 260 g (Charles River Laboratories, Worthington, MA) were given 30 μl of OA (Sigma Chemical Co., St. Louis, MO) suspended in 270 μl 0.1% bovine serum albumin (BSA) (Sigma Chemical Co.) intravenously via a femoral vein cutdown under ketamine (15 mg/kg) (Bristol Laboratories, Syracuse, NY) anesthesia to induce acute lung injury, as detailed in our previous reports (8, 14, 15). Control animals were given an equal volume of 0.1% BSA in a similar manner.
Animals were killed at 1, 4, 24, and 72 h after administration of OA. Bronchoalveolar lavage (BAL) was performed in situ by instilling 50 ml (five 10-ml aliquots) of sterile saline via a cannula ligated in the trachea (16). Approximately 45 ml of fluid was retrieved by BAL in each animal. Total leukocyte counts were made using a standard American Optical hemocytometer (Buffalo, NY). BAL cells were obtained after centrifugation at 600 × g for 6 min, resuspended at a concentration of 1 × 106 cells/ml, and fixed in 4% neutral buffered formalin for 10 min at room temperature. One hundred fifty microliters of cell suspension was dried on a microscope slide for the TUNEL assay.
After lung lavage, the rats' partially inflated lungs were fixed in 4% neutral buffered formalin for 24 h, paraffin embedded, and processed routinely. Slides were deparaffinized in xylene and rehydrated in graded ethanol, and the protein was digested with 20 μg/ml proteinase K (Sigma Chemical Co.) for 15 min at room temperature for the TUNEL assay.
The slides were washed with two changes of phosphate-buffered saline (PBS) and the TUNEL assay (17) was performed using the Fluorescein Apoptag in situ apoptosis detection kit (Oncor, Gaithersburg, MD) according to the manufacturer's instructions. Briefly, after equilibration with the buffer provided, terminal deoxyribonucleotidyl transferase (TdT) enzyme was applied and incubated with digoxigenin-labeled dUTP for 1 h at 37°C. The reaction was then stopped using the Stop/Wash buffer provided and antidigoxigenin-fluorescein was applied for 30 min at room temperature. After being washed with PBS, the slide was counterstained with propidium iodide/Antifade (Oncor) and used for fluorescent microscopy. Appropriate controls with or without TdT enzyme were used to detect false positives.
After TUNEL staining, the slides were viewed by epifluorescence using standard fluorescein excitation at 494 nm and emission at 523 nm wavelength. An intense green signal in the nucleus indicated TUNEL-positive apoptotic cells. The absence of green nuclear emission indicated TUNEL-negative nonapoptotic cells (18). Propidium iodide was viewed with excitation at 596 nm, and a bright red emission was seen in all nuclei present, regardless of the condition of the cell. This served as the counterstain and also helped in identification of cell type (PMN or macrophage) based on the nuclear size and morphology. Percent apoptosis was calculated by number of TUNEL-positive cells × 100/total number of cells counted with propidium iodide stain. A minimum of 500 cells was counted from each sample of BAL fluid (BALF) using a Zeiss Axiovert 100 TV fluorescence microscope (Zeiss, Thornwood, NY) under oil immersion at ×630 magnification; alternatively, a minimum of 10 fields was analyzed from each sample of lung tissue after image capture using the Photometrics CCD imaging camera (Tucson, AZ).
Lung tissues and BALF cell pellets from control and OA-injured rats were fixed for 1 to 2 h in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, postfixed in 1% OsO4 0.8% potassium ferricyanide in cacodylate buffer, and stained in block with 0.5% aqueous uranyl acetate. After dehydration in ethanol, the samples were embedded in Polybed resin (Polysciences, Warrington, PA). Thin sections were cut with a diamond knife, collected on 200 mesh copper grids, stained with uranyl acetate and lead citrate, and examined in a Philips CM10 transmission electron microscope (Philips, N.V., Hillsboro, OR). Samples were examined for the presence of characteristic features of apoptosis, such as condensation of nuclear chromatin, fragmentation of nuclei, condensation of cytoplasm, and/or presence of condensed nuclear fragments in the cytoplasm of neighboring cells.
Lung tissue homogenate was suspended in lysis buffer and incubated overnight at 37°C (19). The DNA was extracted with an equal volume of 25:24:1 phenol, chloroform, and isoamyl alcohol mixture and then ethanol-precipitated for 20 h at −80°C. The DNA was washed twice (70% EtOH), air-dried, and then resuspended in RNase buffer for 1 h at 37°C. After RNase activity was quenched (56°C for 5 min), 10-μg samples of DNA (determined spectrophotometrically) were electrophoresed on a 1% agarose gel in tris-buffered ethylenediamine tetraacetic acid (50 V, 2.5 h). Electrophoresed DNA was stained with ethidium bromide (1 μg/ml) and visualized with ultraviolet irradiation.
In tissues with low apoptotic rates, a modification of the previous procedure using end labeling with 32P was used (20). Briefly, 1 μg of genomic DNA was end labeled in 30 μl of reaction buffer with 0.5 μCi of 32P-deoxycytidine triphosphate (dCTP) at room temperature for 30 min. DNA was precipitated with ethanol and resuspended in 100 μl of TBE. About 10 μl of DNA from each sample were loaded on a 1% agarose gel for electrophoresis followed by autoradiography (X-OMAT-AR; Kodak, Rochester, NY). The size of DNA fragments (180 to 200 bp) was confirmed by ladders of 123-bp molecular weight markers.
Statistical comparisons of single parameters between two groups was performed with unpaired Student's t tests (21). P < 0.05 was considered significant.
As reported previously (15, 22), administration of OA induced a rapid influx of PMN in BALF (Table 1). The absolute numbers of PMN increased sharply within 4 h, peaking around 24 h, and decreasing by 72 h after OA administration. PMN apoptosis was observed to be maximal (62 ± 3%, P < 0.0001) at 24 h after OA. AM apoptosis was seen in a small but significant percentage of cells (1 ± 0.1%, P < 0.05) about 72 h after OA.
The cells in BALF were identified as PMNs on the basis of their characteristic nucleus and smaller size compared with alveolar macrophages. The alveolar macrophages were identified on the basis of their relative larger size and distinct nuclear morphology with propidium iodide stain and fluorescent microscopy. The proportions of inflammatory cells as a percentage of BALF cells were studied at 1, 4, 24, and 72 h after OA administration and the results are shown in Figure 1. In control rats at all times, the BALF cells were predominantly (99%) alveolar macrophages (AM) with < 1% PMN (Figures 1A and 1B). The proportion of BALF cells that were PMN increased rapidly 1 and 4 h after OA administration. At 24 h after OA injury, a majority of PMN stained TUNEL-positive with the typical morphology of apoptosis (P < 0.05). At the same time, the relative proportion of PMN fell, and therefore there was a corresponding increase in the proportion of AM. By 72 h after OA injury, the relative proportion of BALF cells had reverted close to control values with some PMN and occasional AM showing apoptotic changes.
To determine whether the observations made on BALF inflammatory cells reflected the tissue response of the lung, we examined histologic sections of the lung at 1, 4, 24, and 72 h after OA-induced injury, using the TUNEL assay. Representative fields from each set of slides after fluorescent microscopy are shown in Figure 2.
The top panel in Figure 2 shows results from the BAL, and the bottom panel shows results from lung tissue. Identical fields from each sample were observed for TUNEL-positive bright green fluorescence (a, b, c) of apoptosis and propidium iodide (red fluorescence) nuclear counterstain (d, e, f ) to assess the percentage of nuclei undergoing apoptosis. No TUNEL-positive cells were seen at 4 h after OA (a in both panels of Figure 2) and these results were identical to those of the controls and 1-h samples (not shown). Maximum number of TUNEL-positive apoptotic cells were seen at 24 h after OA (b in both panels of Figure 2). Decreased numbers of TUNEL-positive cells were seen at 72 h after OA (c in both panels of Figure 2). Lung sections from control rats did not show TUNEL-positive cells (results not shown).
The fragmentation of DNA due to apoptosis was also demonstrated by the typical ladder pattern on agarose gel electrophoresis of DNA, extracted from lung tissue of OA-treated rats and untreated controls, as shown in Figure 3. Control rat lungs did not show a DNA ladder pattern even with 32P labeling, whereas typical ladder patterns were observed in specimens from OA-treated rat lungs at 24 and 72 h.
Because apoptosis has been defined in morphologic terms (12), the confirmation of this process was done by electron microscopic examination of the lung in rats treated with oleic acid at various time points. The peak of apoptosis at 24 h after OA was confirmed by finding a number of apoptotic PMN in BALF as shown in Figure 4a. The finding of apoptotic bodies ingested by macrophages in BALF, shown in Figure 4b, demonstrates the occurrence of phagocytosis that was suggested by observations with TUNEL and hematoxylin and eosin (H&E) staining.
Figure 5 shows the histopathologic changes using the H&E stains of BALF and lung tissue. Increased cellularity with acute inflammatory cells (PMN) was seen at 4 h after OA. Cellularity showed a decrease by 24 h, and was reverting back to control levels by 72 h after OA. AM with apoptotic bodies in their cytoplasm (arrows, Figure 5) could still be seen under high-power light microscopy (magnification: ×400) in these lavaged lung specimens.
About 7% to 9% of AM in BALF showed evidence of TUNEL-positive staining in their cytoplasm by 24 to 72 h after OA injury (Figure 1c). This staining represents the apoptotic PMNs that had been ingested by AM as shown in Figure 2 (bottom panel, b) and Figure 5 (arrow). The confirmation of this process was done by electron micrographic demonstration of phagosomes with apoptotic bodies in AM (Figure 4b).
OA injury is primarily due to endothelial damage, and this was seen in the OA-injured lungs as cellular swelling with electron microscopy, but no endothelial apoptosis was noted. The TUNEL staining did not identify positive staining in the endothelium.
Evidence of injury was seen with cellular and interstitial swelling and presence of fibrinous strands 24 h after OA treatment, but no morphologic changes of apoptosis were noted with electron microscopy. In the BALF, the two major cell types noted with electron microscopy were PMN and AM. The AM were twice their normal size after 24 h of injury with OA and demonstrated phagosomes with both lamellar bodies (not shown) and apoptotic bodies (Figure 4b).
We used the OA-induced lung injury model of acute inflammation that resolves spontaneously to demonstrate that (1) PMN influx is followed by PMN apoptosis between 1 and 24 h, (2) onset of PMN apoptosis correlates with histologic signs of resolution between 24 and 72 h, and (3) phagocytosis of apoptotic PMNs by macrophages helps in their clearance.
In this report of apoptosis of lung cells in the OA- induced model of acute lung inflammation, we have successfully demonstrated use of the TUNEL assay for evaluation of apoptosis in BALF and tissues with acute lung injury. Previous studies using purely light microscopic features to quantitate apoptosis (7) suffer from the drawbacks of false positives and decreased specificity. The TUNEL assay is more specific and sensitive than light microscopy for detection of apoptosis (18). The TUNEL assay of lung tissue that we have done demonstrates that PMN apoptosis also occurs in lung parenchyma and is not restricted to PMNs in the alveolar fluid. The results from DNA ladders and electron microscopy confirm the findings from the TUNEL assay.
Apoptosis as a form of programmed cell death involved in clearing up cells and tissues has been demonstrated during embryogenesis (23), during physiologic involution of organs such as the thymus (24) and ovary (25), and in adaptive response to noxious stimuli (26). We have recently demonstrated the ontogeny of apoptosis during lung development in the rat (27). The role of apoptosis in the clearance of inflammatory cells has been suggested by Savill (5) and Haslett (28) on the basis of findings in cell culture studies. Its role in resolution of granulation tissue in dermal healing was elegantly demonstrated by Desmouliere and colleagues (29), who showed that myofibroblast number in granulation tissue is reduced by apoptosis of superfluous cells during wound healing. Apoptotic bodies in healing muscle tissue suggest the role of apoptosis in clearance of inflammatory cells (30).
There have been reports about the role of apoptosis in lung injury caused by bleomycin (31, 32) and hyperoxia (33). Its role in inflammation and repair was suggested by Polunovsky and coworkers (34), who showed that apoptosis is involved during the resolution of fibrosis, but in this report, the cell types involved were not identified. Grigg and colleagues (6) showed that neutrophil apoptosis occurs in BALF cells during the resolution of neonatal respiratory distress syndrome.
Cox and coworkers (7) showed that PMN apoptosis in the LPS model of acute lung injury occurs between 6 and 72 h after injury, and they demonstrated the ingestion of apoptotic PMNs by macrophages. The question is then raised whether the PMN apoptosis and macrophage phagocytosis occur as specific responses to the LPS that was introduced intratracheally, or whether they are physiologically regulated mechanisms that occur in response to other forms of injury as well.
The OA model of pulmonary injury is well suited to answer this question because the route of administration is not intratracheal but intravenous. Moreover, it is a well-characterized model of acute lung injury and inflammation that bears similarities with ARDS, a significant clinical problem in humans (8, 9).
Interestingly, our findings of the timing of PMN influx and apoptosis are similar to those of Cox and coworkers (7). We found peak apoptotic response at 24 h after the injury. By 72 h, neutrophil numbers and percent apoptosis had decreased significantly. It is therefore reasonable to suggest that the timing of apoptosis and the resolution response are not related to a specific injurious agent or route of delivery but are more general responses during resolution of an acute inflammatory process.
It has been shown that dexamethasone inhibits neutrophil apoptosis in vitro in a dose-dependent manner (35). Our previous reports have shown that dexamethasone attenuates OA injury (15, 22) but delays the resolution of injury (15), which may well be due to its inhibition of PMN apoptosis. Conversely, it has also been shown that interleukin-10 given intratracheally during LPS-induced lung injury in rats increases PMN apoptosis and facilitates resolution of inflammation (36). These findings suggest that local or paracrine factors could modulate PMN apoptosis and thus affect resolution of lung inflammation. Identification of these factors may lead to an understanding of the triggers that determine whether an acute inflammatory response would lead to repair or progress to chronic inflammation and fibrosis.
Macrophages are known to function as scavengers of cellular material. Scavenging of debris from lytic PMN has been demonstrated in AM but has been shown to cause AM activation and release of proinflammatory cytokines (37). Phagocytosis of apoptotic PMN by AM does not activate the macrophages and is probably the optimal method of clearance of PMN (38). This process has been demonstrated to occur in vitro (39) and also in vivo in acute arthritis (40). We have shown in this study that apoptotic bodies are found in AM during the resolution of OA- induced inflammation, and this may be a common mechanism by which the superfluous and effete inflammatory cells are removed.
Kazzaz and coworkers have shown that 48 h of exposure to hyperoxia (100% oxygen) in mice resulted in increased lung cell apoptosis (33), but lung cells in culture died by necrosis with similar injury. In their study, time points other than 48 h were not reported and the specific cell types undergoing apoptosis in the lungs were not identified. As the authors speculated, it is possible that the apoptosis seen in mice may have been a “downstream phenomenon,” after the initial injury. This may be correlated with our findings of increased apoptosis 24 h after OA injury, which appears to be temporally related to the onset of the repair mechanisms. However, the factor(s) involved in this hypothetical switch from injury and inflammation to apoptosis and resolution remains to be identified.
The probable course of events that occurs during acute lung inflammation and resolution is that an initial PMN influx occurs immediately in response to the insult or injury. Upon termination of the injury, PMN influx wanes and PMN apoptosis is initiated by mechanisms hitherto unknown. This is associated with macrophage ingestion of apoptotic PMNs and effective clearance without liberation of proteases or inflammatory mediators (as suggested by Haslett and colleagues ), thus circumventing the inflammatory response. The AM themselves do not undergo stimulation or respiratory burst (41), and therefore the whole process clears effectively. Some defunct AM may themselves undergo apoptosis and clearance by neighboring cells.
It is possible that acute inflammation does not resolve either if (1) the injury persists, (2) apoptosis of neutrophils is not initiated, (3) the macrophages are not able to clear the apoptotic neutrophils, or (4) a combination of these factors occurs. Further investigations along these lines may yield vital clues to optimizing the mechanisms of healing and repair.
The authors thank Susan Kreuger and Frank Morgan of the Center for Biomedical Imaging Technology for their expert technological assistance. They are also grateful to Dr. Melinda Sanders for her help with the processing of tissue for paraffin sections, and Dr. Arthur Hand for help with electron microscopy. This study was funded in part by the University of Connecticut Health Center Faculty Research Grant and the American Lung Association Connecticut Affiliate Research Awards (N.H.).
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Abbreviations: alveolar macrophage(s), AM; bronchoalveolar lavage fluid, BALF; bovine serum albumin, BSA; deoxyuridine triphosphate, dUTP; lipopolysaccharide(s), LPS; phosphate-buffered saline, PBS; polymorphonuclear neutrophil, PMN; terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling, TUNEL.