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Basic Science Aspects

BCL-2 Inhibits Gut Epithelial Apoptosis Induced by Acute Lung Injury in Mice but Has No Effect On Survival

Husain, Kareem D.*; Stromberg, Paul E.*; Javadi, Pardis*; Buchman, Timothy G.*; Karl, Irene E.; Hotchkiss, Richard S.; Coopersmith, Craig M.*

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doi: 10.1097/01.shk.0000094559.76615.1c
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Abstract

INTRODUCTION

Alterations in the gut epithelium have been demonstrated to play a central role in critical illness, leading to the gut's frequent characterization as the `motor` of the systemic inflammatory response (1–6). Although numerous abnormalities in the gut have been described in shock and sepsis, we have recently proposed that increased gut epithelial apoptosis is a potential mechanism underlying its physiologic importance in critical illness (7,8). Gut epithelial apoptosis is elevated in numerous human and animal studies of both noninfectious inflammation and sepsis (7–14), although the importance of this observation is incompletely understood.

The potential functional significance of preventing gut epithelial apoptosis in sepsis was demonstrated in experiments that compared survival between wild-type (WT) mice and transgenic mice that overexpress the antiapoptotic protein Bcl-2 in their intestinal epithelium. Bcl-2 transgenic animals had a 10-fold improvement in survival in Pseudomonas aeruginosa pneumonia (7) and a 2-fold improvement in survival in cecal ligation and puncture (CLP) (8). Because preventing apoptosis was associated with decreased mortality in a highly lethal (4% survival) model of monomicrobial pulmonary infection and a less lethal (44% survival) model of polymicrobial intraabdominal infection, elevated gut apoptosis appears to be detrimental to survival in sepsis.

The significance of gut epithelial apoptosis in noninfectious inflammation remains unclear. We therefore used a well-accepted model of acute lung injury (ALI) (15–21) that results in 30% mortality to see 1) whether a model of noninfectious inflammation that causes mortality would cause increased gut apoptosis, 2) whether overexpression of gut Bcl-2 would decrease intestinal cell death, and 3) whether overexpression of gut Bcl-2 would confer a survival advantage.

MATERIALS AND METHODS

Animals

Transgenic FVB/N mice containing nucleotides −596 to +21 of a rat fatty acid binding protein (Fabpl) linked to human Bcl-2 (a generous gift from Jeffrey I Gordon, Washington University, St Louis, MO) were generated as described elsewhere (11). Transgenic mice were mated to WT littermates and genotyped using polymerase chain reaction protocols previously described (11). Fabpl-Bcl-2 FVB/N mice have no detectable abnormalities when aged to 18 months and are grossly identical to WT littermates (11). Mice were maintained on a 12-h light-dark schedule in a specific pathogen-free environment and received standard laboratory mouse chow and water ad libitum. All studies were approved by the Washington University Animal Studies Committee and were in accordance with National Institutes of Health guidelines for the use of laboratory animals.

ALI model

Six- to 12 week-old mice underwent a midline neck incision followed by blunt dissection and strap muscle incision as previously described (7,17,22). Animals received an intratracheal injection of 800 μg of lipopolysaccharide (LPS) (from Escherichia coli 055:B5; Sigma, St. Louis, MO) diluted in 50 μL of 0.9% NaCl and were then held upright vertically for 10 s to enhance delivery into the lungs. The 800-μg dose was chosen secondary to pilot studies, which revealed that this led to approximately 30% mortality. Anesthesia was induced with 5% halothane and maintained with 3% halothane.

After incision closure, mice were injected subcutaneously with 1 mL 0.9% NaCl to compensate for fluid loss during surgical manipulation. Sham animals were handled identically but received an intratracheal injection of 50 μL 0.9% NaCl alone. Lung injury was assessed in hematoxylin and eosin (H&E)-stained lung sections from sham, WT, and transgenic Fabpl-Bcl-2 mice sacrificed at 12 or 24 h (n = 3 per group per timepoint) by inflammatory infiltration, changes in alveolar integrity, and airspace hemorrhage. All experiments were conducted by an investigator (K.D.H.) blinded to whether an animal was receiving LPS or 0.9% NaCl and whether an animal carried the Bcl-2 transgene.

Assessment of apoptosis

To assess the tissue distribution of apoptosis induced by ALI, mice were euthanized by cervical dislocation under halothane anesthesia 12 or 24 h after LPS administration or sham manipulation, followed by immediate removal and fixation (except as described below) of their small intestine, lungs, spleen, thymus, heart, liver, kidney, and brain in 10% buffered formalin for 24 h. Before fixation, lungs were inflated with 1 mL 10% buffered formalin via intratracheal injection, and intestines were opened along the length of their cephalocaudal axes and washed in 0.9% NaCl to remove luminal contents. After fixation, all organs were placed in tissue cassettes in 70% ethanol for sectioning. Two additional sets of animals were sacrificed 48 or 72 h after ALI, with removal and fixation of the intestine alone in an identical fashion to that described above.

Apoptotic cells were identified by both active caspase-3 and H&E staining. On H&E-stained sections, apoptosis was identified by morphological identification of cells with nuclear fragmentation (karyorrhexis) and cell shrinkage with condensed nuclei (pyknosis). For functional assessment of cell death, active caspase-3 staining was performed as previously described (7–9). Briefly, paraffin-embedded sections were heated at 60°C for 10 min and then incubated at 23°C in 3% H2O2 in methanol for 10 min to block endogenous peroxidase activity. Slides were then microwaved in citrate buffer (pH 6.0) for 10 min at 89°C to facilitate antigen retrieval, followed by incubation with polyclonal rabbit anti-active caspase-3 (1:100; Cell Signaling, Beverly, MA) for 60 min at 23°C in a humidified chamber. Sections were then incubated at 23°C with secondary biotinylated goat anti-rabbit antibody for 30 min (1:200; Vector Laboratories, Burlingame, CA) followed by VECTASTAIN ABC (Vector Laboratories) for the same length of time, both at 23°C. Development was performed using metal-enhanced DAB solution, followed by counterstaining with hematoxylin.

Apoptosis in organs other than the intestine was identified in five random high-power fields per slide. Gut apoptosis was quantified in 100 contiguous crypts, in well-oriented crypt-villus units. “Well-oriented” was defined as having a single crypt parallel to the crypt-villus axis with Paneth cells at the base and an unbroken epithelial column extending to the villus tip. Apoptosis was quantified in all tissues by an investigator (P.E.S.) blinded to sample identity.

Cytokine and endotoxin determination

Mice were euthanized 12 (for cytokines) or 24 h (for cytokine and endotoxin determination) after intratracheal administration of LPS or 0.9% NaCl, and whole blood was drawn from the inferior vena cava. Blood was centrifuged for 5 min at 6000 g to separate out plasma. Concentrations of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-10 were measured by enzyme-linked immunosorbent assay using commercially available kits (R&D Systems, Minneapolis, MN) according to manufacturer specifications. For analysis of TNF-α and IL-10, 10 μL of plasma per mouse was used, whereas 5 μL was used to measure IL-6 levels. The presence or absence of endotoxemia was evaluated using the commercially available LAL Pyrotell get-clot kit (Associates of Cape Cod, Woods Hole, MA) according to manufacturer specifications.

Survival studies

Survival experiments comparing WT and transgenic mice subjected to ALI were conducted in a separate cohort of age-matched and sex-matched mice by an investigator (K.D.H.) blinded to the presence or absence of the Bcl-2 transgene. Mice were allowed free access to food and water throughout the course of the experiment. Animal survival was recorded for 10 days after LPS injection.

Statistical analysis

Data comparing apoptosis levels and cytokine levels were analyzed using one way analysis of variance followed by Bonferonni's multiple comparison test. Survival curves were evaluated using the χ2 test. All data was analyzed on the statistical program Prism 3.0 (GraphPad Software, San Diego, CA) and are presented as mean ±SEM. A P value <0.05 was considered to be significant.

RESULTS

ALI and apoptosis

Qualitative multiorgan survey of active caspase-3 and H&E-stained slides from FVB/N mice subjected to ALI and sham controls (n = 3 both groups at 12 and 24 h) demonstrated elevated gut epithelial apoptosis at 12 h and increased lymphocytic apoptosis in spleen and thymus at both 12 and 24 h (Fig. 1 and data not shown). No gross differences in apoptosis were detected in lungs, heart, liver, kidney, or brain at either timepoint (data not shown).

F1-8
Fig. 1:
Gut epithelial apoptosis 12 h after ALI. Active caspase-3 (A and B) and H&E-stained sections (C and D) of sham FVB/N mouse (A and C) and animal injected with 800 μg of LPS (B and D). Arrows point to apoptotic cells.

Quantitative analysis was then performed comparing gut epithelial apoptosis between sham animals (n = 4) and WT animals subjected to ALI (Fig. 2). Increased intestinal cell death was observed at 12 h (n = 8) by both active caspase-3 (8 ± 2 apoptotic cells/100 crypts vs. 21 ± 3 apoptotic cells/100 crypts, P < 0.01) and H&E (10 ± 1 apoptotic cells/100 crypts vs. 33 ± 5 apoptotic cells/100 crypts, P < 0.01). No difference in gut apoptosis was detected 24 (n = 10), 48 (n = 4) or 72 h (n = 3) after ALI.

F2-8
Fig. 2:
Quantification of gut epithelial apoptosis. Number of apoptotic cells/100 crypts by active caspase-3 (A) and H&E-stained sections (B) in sham mice and FVB/N animals given intratracheal injection of 800 μg of LPS 12, 24, 48, or 72 h earlier. Asterisks represent P values < 0.01.

Bcl-2 prevents gut epithelial apoptosis

Transgenic mice that overexpress Bcl-2 in their intestinal epithelium had decreased gut apoptosis when subjected to ALI 12 h earlier (n = 8, Figs. 3 and 4) when assayed by active caspase-3 (21 ± 3 apoptotic cells/100 crypts vs. 12 ± 2 apoptotic cells/100 crypts, P < 0.05) but not H&E-staining (33 ± 5 apoptotic cells/100 crypts vs. 20 ± 3 apoptotic cells/100 crypts, P > 0.05). Bcl-2 did not alter gut apoptosis 24 (n = 9) or 48 h (n = 4) after ALI (Fig. 4). Gut overexpression of Bcl-2 had no distant effect on splenic apoptosis, which was similar in both transgenic and WT animals 12 h after ALI (n = 4, data not shown).

F3-8
Fig. 3:
Effect of Bcl-2 on gut epithelial apoptosis after ALI. Active caspase-3 (A and B) and H&E-stained sections (C and D) of WT mouse (A and C) or transgenic mouse that overexpresses Bcl-2 in its intestinal epithelium (B and D) subjected to ALI 12 h earlier. Arrows point to apoptotic cells.
F4-8
Fig. 4:
Quantification of Bcl-2's effect on gut epithelial apoptosis. Number of apoptotic cells/100 crypts by active caspase-3 (A) and H&E-stained sections (B) in WT FVB/N mice (dark columns) and Fabpl-Bcl-2 transgenic mice (hatched columns) given intratracheal injection of 800 μg of LPS 12, 24, or 48 h earlier. Asterisks represent P values < 0.05.

Lung, cytokine, and endotoxin analysis in transgenic and WT mice

To examine whether preventing gut apoptosis had distant effects, H&E-stained sections of lungs from sham animals and both Bcl-2 overexpressors and FVB/N mice subjected to ALI at 12 and 24 h (n = 3 per timepoint per group) were examined for evidence of lung injury (of note, these transgenic animals do not overexpress Bcl-2 in their lungs). In sham animals, alveolar architecture was maintained in the typical lacy pattern and the airspaces were clear (Fig. 5A). In contrast, there was an intense inflammatory response with numerous polymorphonuclear cells in the lungs of WT animals given intratracheal LPS as well as considerable loss of alveolar architecture and hemorrhage in the airways with a fibrinous exudate (Fig. 5B). Lungs from transgenic mice that overexpress Bcl-2 in their intestinal epithelium showed similar inflammatory infiltration and loss of alveolar architecture to that seen in WT mice (Fig. 5C). There were no obvious differences at 12 or 24 h in either transgenic or WT mice.

F5-8
Fig. 5:
Lung histology in sham, WT, and Bcl-2 transgenic mice with ALI. H&E-stained sections from lungs of sham FVB/N mice (A) and WT (B) or Fabpl-Bcl-2 (C) mice given animals given 800 μg of LPS 12 h earlier.

Plasma levels from sham animals (n = 3–4) and transgenic and WT mice subjected to ALI (n = 4-6 per group) were also measured for the pro-inflammatory cytokines TNF-α and IL-6, as well as the anti-inflammatory cytokine IL-10 at 12 or 24 h (Fig. 6). TNF-α levels were not statistically different compared with sham in either WT or transgenic Bcl-2 overexpressors, although a trend toward increased levels were noted at both 12 h and 24 h (Fig. 6A). IL-6 levels were elevated at 24h in both Bcl-2 overexpressors and their FVB/N littermates compared with sham mice (Fig. 6B). Il-10 levels were elevated at 12h in both WT and transgenic animals compared with sham and in WT animals at 24 h (Fig. 6C). No statistically significant differences were noted between transgenic and WT animals. All WT animals (n = 4) were observed to have detectable endotoxin in their bloodstream 24 h following intratracheal LPS injection.

F6-8
Fig. 6:
TNFα, IL-6, and IL-10 levels 12 and 24 h after ALI. TNFα (A), IL-6 (B), and IL-10 (C) levels in sham, WT and Fabpl-Bcl-2 mice at 12 h (dark columns) and 24 h (hatched columns). Asterisks represent P values < 0.05.

Bcl-2 and survival

To test the functional significance of Bcl-2's ability to decrease gut epithelial apoptosis, a survival study was performed comparing transgenic (n = 23) and WT (n = 33) animals subjected to ALI. After 10 days, 73% of WT and 65% of Fabpl-Bcl-2 mice were still alive (Fig. 7, P = ns). Although mortality occurred later in Bcl-2 transgenic mice (posthoc analysis showed there was a statistically significant difference in survival at day 3), final survival was similar between the groups, with transgenic deaths occurring three days after the final mortality in WT animals.

F7-8
Fig. 7:
Effect of Bcl-2 on survival in ALI. Ten-day survival is similar between WT (n = 33) and Fabpl-Bcl-2 (n = 23) animals given intratracheal injection of 800 μg of LPS.

DISCUSSION

This study demonstrates that an animal model of ALI increases gut epithelial apoptosis 12 h after injury and that overexpression of gut Bcl-2 decreases intestinal cell death to levels similar to sham animals. However, Bcl-2 fails to confer a survival advantage, demonstrating that unlike in sepsis, there is no detectable direct association between gut overexpression of this antiapoptotic protein and mortality in this model.

These results expand our understanding of the complex role gut apoptosis plays in critical illness. Although gut epithelial apoptosis has been shown to be elevated in numerous studies of both noninfectious inflammation and sepsis (7–14), the importance of this observation is unclear. Previous data from our laboratory has shown that preventing apoptosis by overexpression of Bcl-2 is associated with a survival benefit in sepsis from both P. aeruginosa pneumonia and CLP (7,8). Although this study shows a similar decrease in gut apoptosis from intestinal overexpression of Bcl-2 in ALI, the functional significance of apoptosis appears to be disparate between infection and noninfectious inflammation since Bcl-2 improves survival in sepsis but not ALI.

To understand whether the distribution of apoptosis was similar between ALI and sepsis, we performed a multi-tissue survey for the presence of programmed cell death, where we found the gut epithelium and lymphocytes were the only two cell types where an increase in apoptosis was noted. This is similar to findings in human autopsy studies of sepsis (9), and murine models of CLP (23) and P. aeruginosa pneumonia (7,22) although apoptosis in the gut and lymphocytes has always previously been detected at 24 h as opposed to the 12 h noted in this study. Of note, the appearance of apoptosis in ALI in this study is distinct from a rabbit model of ALI using acid aspiration (24) where increased apoptosis was noted in lung but not small intestinal epithelium, 8 h after injury (lymphocytes were not examined). The significance of apoptosis in different cell types at a different timepoint in a different model in a different species is unclear.

Although the mechanisms underlying Bcl-2's disparate survival effect on ALI and sepsis remain to be elucidated, understanding the mortality advantage conferred in sepsis may provide clues to why a similar effect does not occur in noninfectious inflammation. The sepsis survival benefit is not limited to the gut, but also exists in lymphocytes, where decreased cell death via Bcl-2 overexpression (25) or caspase inhibition (26) is associated with decreased mortality. Immunosuppression represents a potential explanation of this phenomenon. Adoptive transfer of apoptotic lymphocytes decreases survival in CLP and is associated with a decreased Th1 phenotype (27). It is unclear whether the immunosuppression associated with apoptosis occurs in noninfectious inflammation and if gut apoptosis is indirectly functionally important via immunomodulation. More detailed examination of the immune status of animals with ALI and whether altering apoptosis effects immunocompetence may help explain the differential effect of gut cell death between sepsis and ALI.

The cytokine data presented herein provides some insights into the inflammatory state seen with this model. Plasma cytokine levels were similar between WT and Fabpl-Bcl-2 mice. This is similar to findings that TNF-α and IL-10 levels in spleen and thymus are similar following CLP between mice that overexpress Bcl-2 in lymphocytes and nontransgenic littermates, despite the fact that Bcl-2 improves survival in this model (28). Together, these data indicate that altering cellular apoptosis is not sufficient to affect systemic cytokine levels, although the impact of changing cell death on the local microenvironment is unknown.

The impact of intratracheal LPS on systemic cytokines is also incompletely understood. While numerous investigators have reported inflammatory profiles in LPS-induced ALI (24–26), nearly all studies focus on inflammatory mediator measurements in bronchoalveolar lavage fluid (BALF) and do not mention plasma levels. Of the few studies examining plasma cytokine levels, conflicting results have been reported. Miller et al. reported an increase in plasma (and BALF) TNF-α, IL-6, and IL-10 in rats with this injury, with early peaks and levels approaching baseline by 12–24 h (21). In contrast, Jiang et al. observed no increase in serum TNF-α levels in immunocompromised guinea pigs given intratracheal LPS compared with controls (20). The pattern of cytokines in our murine model differs from both of these published results. IL-6 levels were elevated in this study at 24 h whereas IL-10 levels were elevated at 12 h. Given our findings that endotoxin was detectable in the blood 24 h after LPS injection, we expected that serum cytokines would be elevated early. However, the fact that IL-6 levels continued to be elevated at 24 h argues that this is not a model of “pure” endotoxemia, but a model of ALI with accompanying systemic inflammation. This statement is based on the fact the cytokine profiles in FVB/N mice that receive intratracheal or intraperitoneal LPS are distinct. For instance IL-6 levels at 24 h are 1140 pg/ml in this study but are 68 pg/ml at the same time point in mice that receive intraperitoneal LPS (29).

These results suggest ALI caused by primary lung injury causes a continued pro-inflammatory state, at least 24 h after injury occurs. The fact that ALI induces systemic inflammation supports Slutsky's hypothesis that the lung plays a critical role in the origin of MODS (30) because primary pulmonary injury in our study caused secondary gut epithelial and immune apoptosis as well as elevation of systemic cytokines. However, in a different model of acid aspiration in rabbits, ALI alone did not cause increased cell death in distant tissues outside of the lung (24). The addition of injurious mechanical ventilation to ALI was necessary to cause increased small intestinal villus and kidney (and decreased lung) apoptosis in this model. We believe it is highly likely that there is no single “motor” of multiorgan dysfunction syndrome. Although we believe our previous data on P. aeruginosa pneumonia and CLP demonstrate an important role for gut apoptosis in animal models of sepsis, there are clearly important interactions between (at a minimum) the gut, the immune system, the lung, and the type of mechanical ventilation received, and alterations in any of these has the potential to have systemic effects which can either improve or worsen systemic inflammation.

Although this study gives new insight into the relationships among gut epithelial apoptosis, survival, and the inflammatory response in ALI, it has a number of limitations. The conclusion that preventing intestinal cell death has a different functional significance in noninfectious critical illness and sepsis is based upon two sepsis models and a single inflammatory model in a single strain of mice at a single age. It is not clear whether the results in either form of critical illness has generalized applicability. Further, it assumes the models have similar mortalities and that animals have similar levels of gut apoptosis. Because neither of these is true, comparisons between them are more difficult to interpret. WT mortality in our previous results in P. aeruginosa pneumonia and CLP was between 56 and 96% whereas the mortality in this study was 28%. It is possible that preventing gut apoptosis only has a survival effect at higher mortalities, and that intestinal cell death may even be adaptive at lower mortalities. It is also possible that a threshold of apoptosis must be induced by critical illness for it to have a detrimental effect. For instance, gut apoptosis is greater than three times more common in Pseudomonas aeruginosa pneumonia than in intratracheal LPS, and in fact, overexpression of Bcl-2 in pneumonia only decreases intestinal cell death to levels seen in WT animals subjected to ALI.

It is also possible that our findings result solely from the effects of endotoxemia. We believe this is unlikely in light of the substantial cytokine differences seen between intratracheal and intraperitoneal LPS, outlined above. Further, it is unlikely that the presence of gut epithelial apoptosis is due to a direct effect of LPS in light of data that shows the gut has low levels of expression of Toll-like receptor 4 (TLR4), which is required for recognition of LPS (31,32). It is unknown whether a high dose of LPS such as was used in these experiments has a direct toxic effect on the gut since distal small bowel and colonic TLR4 levels are increased in a model of pharmacologically-induced colitis (33). However, because endotoxin is clearly present in the blood of all animals 24 h after LPS injection, it is possible that this study is actually examining “pure” endotoxemia as opposed to a combination of ALI and endotoxemia. Because both ALI and endotoxemia represent noninfectious inflammatory insults, our conclusions regarding apoptosis and survival in infection versus inflammation are not altered by the relative contributions of ALI and endotoxemia.

In summary, similar to murine models of sepsis, ALI induces gut epithelial apoptosis, and overexpression of Bcl-2 prevents intestinal cell death. However, unlike in sepsis, preventing gut apoptosis does not confer a survival advantage in ALI. The mechanisms underlying this disparity require further study.

ACKNOWLEDGMENTS

The authors thank Isaiah R. Turnbull, Cheryl A. Woolsey, and Daniel M. Amiot, II for technical assistance.

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Keywords:

Cytokines; tumor necrosis factor; interleukin-6; interleukin-10; caspase-3; lipopolysaccharide; crypts; transgenic

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