[Skip to Content]
Access to paid content on this site is currently suspended due to excessive activity being detected from your IP address 54.163.129.96. Please contact the publisher to request reinstatement.
Sign In
Individual Sign In
Create an Account
Institutional Sign In
OpenAthens Shibboleth
[Skip to Content Landing]
Download PDF
Figure 1. Gut Epithelial Apoptosis in Sepsis From Pseudomonas aeruginosa Pneumonia
Image description not available.
Gut epithelium of a mouse with P aeruginosa pneumonia (A-C) compared with that of a sham-operated mouse (D-F) stained for active caspase 3 and counterstained with hematoxylin (A, D); the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay, counterstained with hematoxylin (B,E); and hematoxylin-eosin (C,F). Arrowheads identify apoptotic cells. Original magnification ×400.
Figure 2. Bcl-2 and Intestinal Apoptosis in Sepsis From Pseudomonas aeruginosa Pneumonia
Image description not available.
Gut epithelium of an FVB/N mouse (A,B) and an Fabpl-Bcl-2 mouse (C,D) with P aeruginosa sepsis stained for active caspase 3 and counterstained with hematoxylin (A,C [original magnification ×400]) and hematoxylin-eosin (B,D [original magnification ×200]). Arrowheads identify apoptotic cells.
Figure 3. Survival 7 Days After Induction of Sepsis
Image description not available.
Survival curve was based on 25 Fabpl-Bcl-2 mice and 26 FVB/N mice, each of which received 40 µL of intratracheal Pseudomonas aeruginosa.
1.
Angus DC, Linde-Zwirble WT, Lidicker J.  et al.  Epidemiology of severe sepsis in the United States.  Crit Care Med.2001;29:1303-1310.
2.
Murphy SL. Deaths: final data for 1998.  Natl Vital Stat Rep.2000;48:1-105.
3.
Carrico CJ, Meakins JL, Marshall JC, Fry D, Maier RV. Multiple-organ-failure syndrome.  Arch Surg.1986;121:196-208.
4.
Jiang J, Bahrami S, Leichtfried G.  et al.  Kinetics of endotoxin and tumor necrosis factor appearance in portal and systemic circulation after hemorrhagic shock in rats.  Ann Surg.1995;221:100-106.
5.
Wang W, Smail N, Wang P, Chaudry IH. Increased gut permeability after hemorrhage is associated with upregulation of local and systemic IL-6.  J Surg Res.1998;79:39-46.
6.
Baker JW, Deitch EA, Li M, Berg RD, Specian RD. Hemorrhagic shock induces bacterial translocation from the gut.  J Trauma.1988;28:896-906.
7.
Mainous MR, Ertel W, Chaudry IH, Deitch EA. The gut: a cytokine-generating organ in systemic inflammation?  Shock.1995;4:193-199.
8.
Magnotti LJ, Xu DZ, Lu Q, Deitch EA. Gut-derived mesenteric lymph.  Arch Surg.1999;134:1333-1340.
9.
Grotz MR, Deitch EA, Ding J, Xu D, Huang Q, Regel G. Intestinal cytokine response after gut ischemia: role of gut barrier failure.  Ann Surg.1999;229:478-486.
10.
Maloney JP, Halbower AC, Fouty BF.  et al.  Systemic absorption of food dye in patients with sepsis.  N Engl J Med.2000;343:1047-1048.
11.
Unno N, Fink MP. Intestinal epithelial hyperpermeability.  Gastroenterol Clin North Am.1998;27:289-307.
12.
Wilmore DW, Smith RJ, O'Dwyer ST, Jacobs DO, Ziegler TR, Wang XD. The gut: a central organ after surgical stress.  Surgery.1988;104:917-923.
13.
Hassoun HT, Kone BC, Mercer DW, Moody FG, Weisbrodt NW, Moore FA. Post-injury multiple organ failure: the role of the gut.  Shock.2001;15:1-10.
14.
O'Boyle CJ, MacFie J, Mitchell CJ, Johnstone D, Sagar PM, Sedman PC. Microbiology of bacterial translocation in humans.  Gut.1998;42:29-35.
15.
Doig CJ, Sutherland LR, Sandham JD, Fick GH, Verhoef M, Meddings JB. Increased intestinal permeability is associated with the development of multiple organ dysfunction syndrome in critically ill ICU patients.  Am J Respir Crit Care Med.1998;158:444-451.
16.
Koo DJ, Zhou M, Jackman D.  et al.  Is gut the major source of proinflammatory cytokine release during polymicrobial sepsis?  Biochim Biophys Acta.1999;1454:289-295.
17.
Baue AE. MOF/MODS, SIRS: an update.  Shock.1996;6(suppl 1):S1-S5.
18.
Hotchkiss RS, Swanson PE, Freeman BD.  et al.  Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction.  Crit Care Med.1999;27:1230-1251.
19.
Hotchkiss RS, Swanson PE, Cobb JP, Jacobson A, Buchman TG, Karl IE. Apoptosis in lymphoid and parenchymal cells during sepsis.  Crit Care Med.1997;25:1298-1307.
20.
Hiramatsu M, Hotchkiss RS, Karl IE, Buchman TG. Cecal ligation and puncture (CLP) induces apoptosis in thymus, spleen, lung, and gut by an endotoxin and TNF-independent pathway.  Shock.1997;7:247-253.
21.
Coopersmith CM, Chang KC, Swanson PE.  et al.  Overexpression of Bcl-2 in the intestinal epithelium improves survival in septic mice.  Crit Care Med.2002;30:195-201.
22.
Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death.  Cell.1993;74:609-619.
23.
Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival.  Science.1998;281:1322-1326.
24.
Hengartner MO, Horvitz HR. C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian proto-oncogene bcl-2.  Cell.1994;76:665-676.
25.
Nicholson DW. From bench to clinic with apoptosis-based therapeutic agents.  Nature.2000;407:810-816.
26.
Coopersmith CM, O'Donnell D, Gordon JI. Bcl-2 inhibits ischemia-reperfusion-induced apoptosis in the intestinal epithelium of transgenic mice.  Am J Physiol.1999;276:G677-G686.
27.
Hotchkiss RS, Dunne WM, Swanson PE.  et al.  Pseudomonas aeruginosa pneumonia induces profound lymphocyte but not bronchial epithelial apoptosis.  Science.2002;294:1783.
28.
Starke JR, Edwards MS, Langston C, Baker CJ. A mouse model of chronic pulmonary infection with Pseudomonas aeruginosa and Pseudomonas cepacia Pediatr Res.1987;22:698-702.
29.
Yu P, Martin CM. Increased gut permeability and bacterial translocation in Pseudomonas pneumonia-induced sepsis.  Crit Care Med.2000;28:2573-2577.
30.
Gavrieli Y, Sherman Y, Ben Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.  J Cell Biol.1992;119:493-501.
31.
Wheeler AP, Bernard GR. Treating patients with severe sepsis.  N Engl J Med.1999;340:207-214.
32.
Moore FA, Moore EE, Poggetti R.  et al.  Gut bacterial translocation via the portal vein.  J Trauma.1991;31:629-636.
33.
Sun Z, Wang X, Deng X.  et al.  The influence of intestinal ischemia and reperfusion on bidirectional intestinal barrier permeability, cellular membrane integrity, proteinase inhibitors, and cell death in rats.  Shock.1998;10:203-212.
34.
Sun Z, Wang X, Wallen R.  et al.  The influence of apoptosis on intestinal barrier integrity in rats.  Scand J Gastroenterol.1998;33:415-422.
35.
Abreu MT, Palladino AA, Arnold ET, Kwon RS, McRoberts JA. Modulation of barrier function during Fas-mediated apoptosis in human intestinal epithelial cells.  Gastroenterology.2000;119:1524-1536.
36.
Magnotti LJ, Upperman JS, Xu DZ, Lu Q.  et al.  Gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock.  Ann Surg.1998;228:518-527.
37.
Hotchkiss RS, Chang KC, Swanson PE.  et al.  Caspase inhibitors improve survival in sepsis: a critical role of the lymphocyte.  Nat Immunol.2000;1:496-501.
38.
Hotchkiss RS, Tinsley KW, Swanson PE.  et al.  Prevention of lymphocyte cell death in sepsis improves survival in mice.  Proc Natl Acad Sci U S A.1999;96:14541-14546.
Preliminary Communication
April 3, 2002

Inhibition of Intestinal Epithelial Apoptosis and Survival in a Murine Model of Pneumonia-Induced Sepsis

Author Affiliations

Author Affiliations: Departments of Surgery (Drs Coopersmith and Buchman, Mssrs Stromberg and Amiot), Anesthesiology (Mr Davis and Dr Hotchkiss), Pathology (Dr Dunne), and Medicine (Dr Karl), Washington University School of Medicine, St Louis, Mo.

JAMA. 2002;287(13):1716-1721. doi:10.1001/jama.287.13.1716
Context

Context Increased intestinal epithelial apoptosis is present in both human autopsy studies and animal models of sepsis. Whether altering gut apoptosis decreases mortality in sepsis induced by pathogenic bacteria outside the gut is unknown.

Objective To determine if decreasing levels of intestinal cell death improves survival in a murine model of Pseudomonas aeruginosa pneumonia–induced sepsis.

Design and Materials Prospective study in which transgenic mice that overexpress the antiapoptotic protein Bcl-2 in their intestinal epithelium (n = 25) and control littermates (n = 26) were subjected to intratracheal injection of P aeruginosa.

Main Outcome Measures Survival at 7 postoperative days, compared between the 2 groups. Secondary outcomes included quantification of gut epithelial apoptosis.

Results Survival in transgenic mice that overexpress Bcl-2 in the intestinal epithelium was 40% (10/25) compared with 4% (1/26) in control littermates 7 days after intratracheal injection of P aeruginosa (P = .001), with differences in survival noted within 24 hours of surgery. Overexpression of Bcl-2 was associated with a decrease in gut epithelial apoptosis demonstrated by active caspase 3 staining, the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay, and hematoxylin-eosin staining.

Conclusions In this murine model, inhibiting gut epithelial apoptosis by overexpression of Bcl-2 was associated with a survival advantage in P aeruginosa pneumonia–induced sepsis. These results suggest that intestinal epithelial apoptosis may play a role in sepsis-related mortality.

Sepsis is the leading cause of mortality in critically ill patients nationwide. A recent analysis estimated that 750 000 individuals develop sepsis annually and more than 210 000 die of the disease.1 The national hospital cost associated with care of patients with severe sepsis is $16.7 billion.1 The rate of death from sepsis has increased more than 90% in the last 20 years.2 Previous studies have implicated alterations of intestinal function as critical to the development of sepsis,315 leading to the theory that the gut represents the "motor" of the systemic inflammatory response.3,6,7,12,13,16,17

Gut epithelial apoptosis is increased in human autopsy studies and animal models of sepsis.1821 An autopsy study comparing samples from multiple organ systems in 20 patients who died in the surgical intensive care unit of sepsis and multiple organ dysfunction with those from 16 critically ill, nonseptic patients demonstrated increased intestinal epithelial apoptosis in septic patients.18 The gut epithelium represented 1 of only 2 cell types (along with lymphocytes) in septic patients with prominent apoptosis. Mice that underwent cecal ligation and puncture (CLP), a murine model of ruptured appendicitis, had increased gut epithelial apoptosis compared with controls.19

In other studies, septic transgenic mice that overexpress the antiapoptotic protein Bcl-22225 in their intestinal epithelium had increased survival following CLP compared with their septic control littermates that underwent the same procedure.21 These studies suggest that decreasing gut epithelial apoptosis is associated with a survival advantage in sepsis of intestinal origin but do not evaluate whether gut epithelial apoptosis plays a role in mortality in sepsis caused by pathogenic bacteria focused outside the gut. In this study, we examined survival in transgenic mice that had Pseudomonas aeruginosa pneumonia–induced sepsis.

METHODS
Transgenic Mice

Strain FVB/N transgenic mice containing nucleotides −596 to +21 of a rat fatty acid binding protein (Fabpl) linked to human Bcl-2 (a gift from Jeffrey I. Gordon, MD, Washington University, St Louis, Mo) were generated and genotyped using polymerase chain reaction protocols detailed elsewhere.26Fabpl-Bcl-2 animals have no detectable abnormalities when aged to 18 months and appear phenotypically identical to nontransgenic littermates.21,26 All studies complied with National Institutes of Health guidelines for the use of laboratory animals and were approved by the Washington University Animal Studies Committee.

Pneumonia Model

A total of 103 six- to eight-week-old mice had a midline cervical incision performed under halothane anesthesia. 2729 Each animal received an intratracheal injection of 40 µL of a P aeruginosa solution, after which the mouse was held vertically for 10 seconds to enhance delivery into the lung. The incision was closed in 2 layers. Sham-operated mice were handled identically, but had intratracheal injection of 40 µL of 0.9% NaCl.

Microbiologic Preparation

The ATCC 27853 strain of P aeruginosa was grown overnight in trypticase soy broth with constant shaking. A 10-mL volume of the culture medium was placed in a 50-mL tube and centrifuged for 10 minutes at 6000g. The resulting pellet was resuspended in an equal volume of saline and centrifuged again. The final density of the inoculum was adjusted to 0.3 A600nm, corresponding to a cell density ranging between 5 × 108 and 1 × 109 colony-forming units (CFUs)/mL as determined by serial dilution and colony counts. Based on the cell density and the volume injected intratracheally, the dose of P aeruginosa administered to each animal was between 20 million and 40 million CFUs per injection.

Quantification of Apoptosis

For quantification of apoptosis, 21 mice (12 nontransgenic and 9 transgenic) were euthanized 24 hours after injection with P aeruginosa. Each animal's entire small intestine was immediately removed. The intestine was opened along the length of its cephalocaudal axis, washed in 10% buffered formalin (to remove luminal contents) and then fixed in the same solution.

Apoptotic cells were identified using 3 complementary techniques: active caspase 3 staining, the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay,30 and hematoxylin-eosin (H&E) staining. Serial sections were scored for apoptosis by a single investigator. For active caspase 3 staining, a minimum of 100 well-oriented crypt-villus units were scored per section. Well-oriented was defined as a crypt sectioned parallel to the crypt-villus axis with Paneth cells at the crypt base and an unbroken epithelial column extending to the villus tip. For H&E staining and the TUNEL assay, a minimum of 10 random fields were evaluated in each sample. Cells undergoing apoptosis were identified by characteristic morphology including nuclear fragmentation (karyorrhexis) and cell shrinkage with condensed nuclei (pyknosis) on H&E-stained sections and by immunohistochemical detection of positive cells using the TUNEL assay. In comparing animals subjected to intratracheal injection with either P aeruginosa or 0.9% NaCl, identifying information on sections was obscured, and the slides were extensively shuffled by an investigator different from the person scoring for apoptosis (P.E.S.). When scoring on all slides was entirely complete, the numerical code (devised by C.M.C.) was broken to reveal the identity of all slides counted.

Staining for active caspase 3 was performed as previously described.18,21 Briefly, paraffin-embedded tissues were dewaxed and rehydrated. Endogenous peroxidase activity was blocked by incubating in 3% H2O2 in methanol at 23°C for 15 minutes. Sections were then microwaved in citrate buffer (pH 6.0) for 9 minutes to facilitate antigen retrieval. Polyclonal rabbit antiactive caspase 3 was applied, diluted 1:100 in phosphate-buffered saline for 60 minutes at 23°C (Cell Signaling Technology Inc, Beverly, Mass), followed by a secondary biotinylated goat anti–rabbit antibody for 30 minutes (1:200) (Vector Laboratories, Burlingame, Calif). Slides were then incubated with VECTASTAIN ABC-AP (Vector Laboratories), developed with alkaline phosphatase substrate solution, and counterstained with hematoxylin.

Tissue sections were stained for the TUNEL assay using a commercially available kit according to manufacturer specifications (Roche Diagnostics Corp, Indianapolis, Ind). After rinsing, a streptavidin-biotin complex (VECTASTAIN ABC, Vector Laboratories) was applied at 23°C for 30 minutes. After rinsing, metal-enhanced diaminobenzidine (Pierce Chemical Co, Rockford, Ill) was added, and slides were counterstained with hematoxylin.

Survival Studies

The survival studies were conducted in a separate group of mice (n = 51) in the following fashion: offspring of FVB/N and heterozygous Fabpl-Bcl-2 mice were weaned at 3 weeks of age. Prior to identifying the genotype of each of these animals, the tails of mice in the resulting litters (up to 5 mice per cage of approximately 50% transgenic and 50% nontransgenic animals as would be expected by mendelian genetics) were uniquely marked by circumferential inscription with indelible ink, 1 to 5 circles per mouse. Each animal was subsequently genotyped using polymerase chain reaction techniques described above.

Three weeks later, an investigator (P.E.S.) blinded to the identity of the mice performed intratracheal injections of 40 µL of P aeruginosa in both age-matched and sex-matched Fabpl-Bcl-2 mice and FVB/N littermates. Injections were done sequentially according to the tail marks (animals with 1 mark were injected first, followed by animals with 2 marks, etc). The identifying tail marks were made prior to genotyping of the animals and the investigator was unaware of the identity of any animal injected. Mice were allowed free access to food and water throughout the course of the experiment. Animal survival was recorded for 7 days postoperatively.

Bacteriologic Analysis

In a different group of animals (n = 12) than those used for either quantitation of apoptosis or survival curves, mesenteric lymph node and spleen were removed 16 hours following intratracheal instillation of P aeruginosa, then weighed and homogenized in glass tissue grinders containing 1 mL of sterile phosphate-buffered saline. Blood (100 µL) was diluted 1:10 in sterile phosphate-buffered saline. Serial dilutions of homogenate and blood were then cultured on blood agar (total aerobes) and MacConkey (gram-negative facultative aerobes) plates. Plates were incubated at 37°C and examined 24 hours later.

Cytokine Determination

At 2, 16, or 24 hours after intratracheal injection with P aeruginosa solution, whole blood was drawn by cardiac puncture. Animals in the cytokine experiments were separate from those used in either apoptosis quantitation or survival experiments, although the bacteriologic analysis described above was performed on the same subgroup of animals examined here at 16 hours after bacterial injection. Blood was centrifuged for 5 minutes at 6000g to separate out plasma. Concentrations of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interferon γ (IFN-γ), and IL-10 were measured by enzyme-linked immunosorbent assay using commercially available kits (R&D Systems, Minneapolis, Minn) according to manufacturer specifications.

Statistical Analysis

Data analysis was performed using Prism v3.0 (GraphPad Software, San Diego, Calif). Differences in group survival were analyzed using the χ2 test. Comparisons of apoptosis levels, bacteriologic data, and cytokine levels between transgenic and nontransgenic animals were analyzed using the t test. A P value of less than .05 was considered statistically significant.

RESULTS
Gut Epithelial Apoptosis

Mice that received P aeruginosa were judged to be septic by the presence of bacteremia and objective clinical findings (eg, tachypnea, lethargy) while sham-operated animals that received 0.9% NaCl had no gross abnormalities. There was no evidence of wound infection in any animal studied. Animals that received intratracheal bacteria had a 50% mortality 24 hours postoperatively, while no animals that received saline died. Septic animals that received intratracheal injection of P aeruginosa (n = 8) had significantly greater numbers of active caspase 3 cells/100 crypts (mean, 71.4 [SEM, 1.6]) (Figure 1A), compared with sham-operated animals that received intratracheal saline (n = 4) that had 10.8 (1.4) active caspase 3 cells/100 crypts (Figure 1D; P<.001). Similar results were qualitatively obtained using both the TUNEL assay and H&E staining (Figure 1B, C, E, F).

Bcl-2 Overexpression and Intestinal Apoptosis

Transgenic mice that overexpress human Bcl-2 under control of the Fabpl promoter have detectable levels of this antiapoptotic protein in all epithelial lineages of the intestine, without expression in the surrounding gut-associated lymphoid tissue.21,26 Following intratracheal injection of P aeruginosa, and compared for gut epithelial apoptosis as described above, transgenic Fabpl-Bcl-2 mice (n = 9) had a decrease in mean (SEM) intestinal cell death—23.9 (2.0) active caspase 3 cells/100 crypts in Fabpl-Bcl-2 animals vs 71.4 (1.6) active caspase 3 cells/100 crypts in nontransgenic septic littermates (n = 8) (Figure 2A and C; P<.001). Similar qualitative results were seen with H&E staining and the TUNEL assay (Figure 2B and D; also, data not shown).

Intestinal Bcl-2 and Survival

Survival experiments were performed on 3 separate days using 8 to 10 mice per group (total: 25 Fabpl-Bcl-2 mice and 26 FVB/N mice). P aeruginosa pneumonia–induced sepsis was highly lethal in wild-type mice, with only 1 (4%) animal surviving 5 days. Among Fabpl-Bcl-2 mice, 10 (40%) survived for 7 days (Figure 3, P<.005). The survival curves began diverging within the first postoperative day, and nearly all mortality in both groups occurred within the first 3 days of the study.

Septicemia and Bacterial Translocation

The majority of both transgenic and nontransgenic mice were still alive 16 hours postoperatively. However, a substantial portion of wild-type animals injected with P aeruginosa died within 24 hours.

All animals in both groups had P aeruginosa bacteremia and had organisms detectable in their spleens. Nontransgenic animals (n = 7) had the greatest mean (SEM) organism burden in their blood—2 274 000 (1 497 000) CFU/mL. Although Fabpl-Bcl-2 mice (n = 3) had a 120-fold decrease in bacterial counts (19 000 (8185) CFU/mL), there was no statistical difference between groups, probably due to the small sample size and large variation of P aeruginosa counts in the blood of nontransgenic animals (range, 20 000-10 million CFU/mL). A similar trend was seen in splenic cultures, with mean (SEM) 629 300 (8185) CFU/g in nontransgenic animals compared to 29 970 (19 040) CFU/g in septic transgenic animals.

Mesenteric lymph node cultures obtained 16 hours after injection with P aeruginosa and tested for bacterial translocations had low levels of P aeruginosa in the mesenteric lymph nodes of all mice tested (n = 12 animals), but evidence of other enteric organisms in 2 animals—2600 CFU/g in a nontransgenic animal and 560 CFU/g in a transgenic animal, both of which grew Klebsiella pneumoniae.

Cytokine Analysis

Plasma from transgenic and nontransgenic animals was measured for the proinflammatory cytokines TNF-α, IL-6, and IFN-γ, and the anti-inflammatory cytokine IL-10 at 2, 16, and 24 hours after intratracheal injection of P aeruginosa. All cytokines were either undetectable or present at low levels at the 2-hour time point (transgenic: n = 4; nontransgenic: n = 7). Levels of each cytokine increased by 16 hours in both groups of animals. There was a nonsignificant trend toward higher quantities of all cytokines in nontransgenic animals compared with Fabpl-Bcl-2 mice, especially for IL-10 measurements, with mean (SEM) levels of 233 (76) pg/mL in nontransgenic animals (n = 6) vs 105 (46) pg/mL in transgenic animals (n = 4). Smaller differences were observed in levels of TNF-α (179 [61] pg/mL for nontransgenic vs 99 [16] pg/mL for transgenic) and IL-6 (3344 [750] pg/mL for nontransgenic vs 2461 [379] pg/mL for transgenic).

By 24 hours postinjection, cytokine levels were relatively static or decreasing in all animals. The sole exception was TNF-α in wild-type animals (n = 4), which had a greater than 2-fold increase compared with septic animals of the same genotype at 16 hours, increasing to a level of 376 (145) pg/mL. Levels of TNF-α in Fabpl-Bcl-2 mice (n = 4) were minimally changed (158 [35] pg/mL]) at the 24-hour time point.

COMMENT

In this study, sepsis induced by P aeruginosa pneumonia resulted in an increase in gut epithelial apoptosis. Although sepsis originated from a pulmonary source, decreasing intestinal epithelial cell death by overexpression of Bcl-2 was associated with improved survival.

The results described herein suggest that decreasing intestinal cell death with Bcl-2 may play a role in improving survival for sepsis that originates from an extra-abdominal source. This finding may be clinically significant because approximately 40% of septic episodes in humans begin in the lung.1,31

While this study shows an association between increased Bcl-2 and survival in sepsis, we cannot conclude that a decrease in gut epithelial apoptosis is directly responsible for the decrease in mortality observed in transgenic animals. Although the role of Bcl-2 as an antiapoptotic protein has been demonstrated,2225 it is possible that a separate effect of the transgene that we are unable to detect is responsible for the survival benefit conferred. If, however, the lower death rate in transgenic animals is due to an alteration in gut epithelial apoptosis, the mechanisms that underlie this link must be further clarified.

One possibility we tested for was the presence of bacterial translocation. However, the microbiologic data demonstrate that bacterial translocation is not the predominant mechanism of the survival benefit conferred by Bcl-2. Although low levels of P aeruginosa were present in the mesenteric lymph nodes of all animals examined 16 hours postoperatively, only 2 animals demonstrated evidence of enteric organisms at this time point. In addition, although all animals were bacteremic, no enteric organisms were cultured from the blood of either transgenic or nontransgenic animals.

Our results are consistent with human studies that challenge the role of bacterial translocation in the origins of sepsis,32 but differ from a recent study by Yu and Martin29 that demonstrated 67% of rats subjected to intratracheal injection of P aeruginosa had evidence of translocation of intestinal bacteria to the mesenteric lymph system. However, that study examined translocation 40 hours after surgery, whereas the cultures in our study were obtained 24 hours earlier. It is possible that had the animals in our study been cultured 40 hours postoperatively, a similar increase in bacterial translocation may have been observed. This would be of little physiologic significance since 75% of nontransgenic animals and 50% of transgenic animals did not survive 48 hours.

Even though bacterial translocation does not seem to be responsible for the survival benefit conferred by Bcl-2, it is possible that altered intestinal permeability plays a role in a potential link between gut epithelial apoptosis and survival. Yu and Martin29 have shown that sepsis from P aeruginosa pneumonia is associated with decreased gut barrier function. This is consistent with rat studies that show that gut permeability is increased in intestinal ischemia/reperfusion.33 Increased gut epithelial apoptosis is also associated with gut hyperpermeability in animal models.34 Importantly, treatment with the caspase inhibitor z-VAD in an intestinal cell line prevented both gut epithelial apoptosis and barrier dysfunction.35 Since translocation of gut-derived factors other than intact bacteria to the mesenteric lymph has been shown to be detrimental in animal models of critical illness,32,36 additional studies are needed to assess the role of Bcl-2 on gut barrier function.

The cellular signaling pathways responsible for increased gut apoptosis in sepsis are unclear. In this study, apoptotic cells were present in nearly every crypt in pneumonia from P aeruginosa, whereas they are a more focal phenomenon in CLP21; this may be attributable to the differing mortalities between the models.

The role cytokines play in the relationship between Bcl-2 overexpression and survival is incompletely answered by this study. Multiple studies have shown alterations in cytokine levels of intestinal4,5,7,9,11,15,16 or immune37,38 origin in critical illness. The data presented here, however, fail to show any statistically significant differences in cytokine levels between transgenic and nontransgenic animals. Potential explanations for this include alterations in cytokine levels not examined for this study and true differences in local mediator levels not adequately detected by measuring systemic blood levels.

Our study has several limitations. It is possible that even if alterations in gut cell death are responsible for the effect presented in this murine study, decreasing intestinal apoptosis would therefore not be beneficial to septic patients in the intensive care unit. To have clinical applicability, an approach must be developed to decrease gut epithelial apoptosis after a patient becomes septic. While caspase inhibitors administered via intraperitoneal injection improve survival in CLP,37,38 it is unclear what role the intestine plays in this response, and no data exist on gut-directed anticaspase therapy. The role intestinal apoptosis plays in either gram-positive or fungal septicemia also is not known, since both this study and earlier work on CLP used gram-negative bacteria in murine models of sepsis. In addition, animals in this study were not given antibiotics, which is standard treatment for patients with pneumonia, sepsis, or both.31 Although both transgenic and nontransgenic animals were bacteremic, it is possible that antibiotics would have improved survival disproportionately in the nontransgenic group.

In summary, similar to other animal models and human studies of critical illness, monomicrobial sepsis induced by P aeruginosa pneumonia is associated with an increase in gut epithelial apoptosis. Overexpression of Bcl-2 was associated with reduced apoptosis in the intestinal epithelium and conferred a survival advantage, with 40% of transgenic mice surviving their septic insult. Gut epithelial apoptosis may play a role in sepsis-related mortality, and reduction of intestinal cell death may represent a potential therapeutic approach toward improving survival in sepsis.

References
1.
Angus DC, Linde-Zwirble WT, Lidicker J.  et al.  Epidemiology of severe sepsis in the United States.  Crit Care Med.2001;29:1303-1310.
2.
Murphy SL. Deaths: final data for 1998.  Natl Vital Stat Rep.2000;48:1-105.
3.
Carrico CJ, Meakins JL, Marshall JC, Fry D, Maier RV. Multiple-organ-failure syndrome.  Arch Surg.1986;121:196-208.
4.
Jiang J, Bahrami S, Leichtfried G.  et al.  Kinetics of endotoxin and tumor necrosis factor appearance in portal and systemic circulation after hemorrhagic shock in rats.  Ann Surg.1995;221:100-106.
5.
Wang W, Smail N, Wang P, Chaudry IH. Increased gut permeability after hemorrhage is associated with upregulation of local and systemic IL-6.  J Surg Res.1998;79:39-46.
6.
Baker JW, Deitch EA, Li M, Berg RD, Specian RD. Hemorrhagic shock induces bacterial translocation from the gut.  J Trauma.1988;28:896-906.
7.
Mainous MR, Ertel W, Chaudry IH, Deitch EA. The gut: a cytokine-generating organ in systemic inflammation?  Shock.1995;4:193-199.
8.
Magnotti LJ, Xu DZ, Lu Q, Deitch EA. Gut-derived mesenteric lymph.  Arch Surg.1999;134:1333-1340.
9.
Grotz MR, Deitch EA, Ding J, Xu D, Huang Q, Regel G. Intestinal cytokine response after gut ischemia: role of gut barrier failure.  Ann Surg.1999;229:478-486.
10.
Maloney JP, Halbower AC, Fouty BF.  et al.  Systemic absorption of food dye in patients with sepsis.  N Engl J Med.2000;343:1047-1048.
11.
Unno N, Fink MP. Intestinal epithelial hyperpermeability.  Gastroenterol Clin North Am.1998;27:289-307.
12.
Wilmore DW, Smith RJ, O'Dwyer ST, Jacobs DO, Ziegler TR, Wang XD. The gut: a central organ after surgical stress.  Surgery.1988;104:917-923.
13.
Hassoun HT, Kone BC, Mercer DW, Moody FG, Weisbrodt NW, Moore FA. Post-injury multiple organ failure: the role of the gut.  Shock.2001;15:1-10.
14.
O'Boyle CJ, MacFie J, Mitchell CJ, Johnstone D, Sagar PM, Sedman PC. Microbiology of bacterial translocation in humans.  Gut.1998;42:29-35.
15.
Doig CJ, Sutherland LR, Sandham JD, Fick GH, Verhoef M, Meddings JB. Increased intestinal permeability is associated with the development of multiple organ dysfunction syndrome in critically ill ICU patients.  Am J Respir Crit Care Med.1998;158:444-451.
16.
Koo DJ, Zhou M, Jackman D.  et al.  Is gut the major source of proinflammatory cytokine release during polymicrobial sepsis?  Biochim Biophys Acta.1999;1454:289-295.
17.
Baue AE. MOF/MODS, SIRS: an update.  Shock.1996;6(suppl 1):S1-S5.
18.
Hotchkiss RS, Swanson PE, Freeman BD.  et al.  Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction.  Crit Care Med.1999;27:1230-1251.
19.
Hotchkiss RS, Swanson PE, Cobb JP, Jacobson A, Buchman TG, Karl IE. Apoptosis in lymphoid and parenchymal cells during sepsis.  Crit Care Med.1997;25:1298-1307.
20.
Hiramatsu M, Hotchkiss RS, Karl IE, Buchman TG. Cecal ligation and puncture (CLP) induces apoptosis in thymus, spleen, lung, and gut by an endotoxin and TNF-independent pathway.  Shock.1997;7:247-253.
21.
Coopersmith CM, Chang KC, Swanson PE.  et al.  Overexpression of Bcl-2 in the intestinal epithelium improves survival in septic mice.  Crit Care Med.2002;30:195-201.
22.
Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death.  Cell.1993;74:609-619.
23.
Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival.  Science.1998;281:1322-1326.
24.
Hengartner MO, Horvitz HR. C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian proto-oncogene bcl-2.  Cell.1994;76:665-676.
25.
Nicholson DW. From bench to clinic with apoptosis-based therapeutic agents.  Nature.2000;407:810-816.
26.
Coopersmith CM, O'Donnell D, Gordon JI. Bcl-2 inhibits ischemia-reperfusion-induced apoptosis in the intestinal epithelium of transgenic mice.  Am J Physiol.1999;276:G677-G686.
27.
Hotchkiss RS, Dunne WM, Swanson PE.  et al.  Pseudomonas aeruginosa pneumonia induces profound lymphocyte but not bronchial epithelial apoptosis.  Science.2002;294:1783.
28.
Starke JR, Edwards MS, Langston C, Baker CJ. A mouse model of chronic pulmonary infection with Pseudomonas aeruginosa and Pseudomonas cepacia Pediatr Res.1987;22:698-702.
29.
Yu P, Martin CM. Increased gut permeability and bacterial translocation in Pseudomonas pneumonia-induced sepsis.  Crit Care Med.2000;28:2573-2577.
30.
Gavrieli Y, Sherman Y, Ben Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.  J Cell Biol.1992;119:493-501.
31.
Wheeler AP, Bernard GR. Treating patients with severe sepsis.  N Engl J Med.1999;340:207-214.
32.
Moore FA, Moore EE, Poggetti R.  et al.  Gut bacterial translocation via the portal vein.  J Trauma.1991;31:629-636.
33.
Sun Z, Wang X, Deng X.  et al.  The influence of intestinal ischemia and reperfusion on bidirectional intestinal barrier permeability, cellular membrane integrity, proteinase inhibitors, and cell death in rats.  Shock.1998;10:203-212.
34.
Sun Z, Wang X, Wallen R.  et al.  The influence of apoptosis on intestinal barrier integrity in rats.  Scand J Gastroenterol.1998;33:415-422.
35.
Abreu MT, Palladino AA, Arnold ET, Kwon RS, McRoberts JA. Modulation of barrier function during Fas-mediated apoptosis in human intestinal epithelial cells.  Gastroenterology.2000;119:1524-1536.
36.
Magnotti LJ, Upperman JS, Xu DZ, Lu Q.  et al.  Gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock.  Ann Surg.1998;228:518-527.
37.
Hotchkiss RS, Chang KC, Swanson PE.  et al.  Caspase inhibitors improve survival in sepsis: a critical role of the lymphocyte.  Nat Immunol.2000;1:496-501.
38.
Hotchkiss RS, Tinsley KW, Swanson PE.  et al.  Prevention of lymphocyte cell death in sepsis improves survival in mice.  Proc Natl Acad Sci U S A.1999;96:14541-14546.
×