Sepsis in Decreases Apoptosis and Improves Survival Overexpression

Overexpression of Bcl-2 in Transgenic Mice Decreases Apoptosis and Improves Survival in Sepsis

This information is current as Richard S. Hotchkiss, Paul E. Swanson, C. Michael Knudson, of September 26, 2021. Katherine C. Chang, J. Perren Cobb, Dale F. Osborne, Kimberly M. Zollner, Timothy G. Buchman, Stanley J. Korsmeyer and Irene E. Karl J Immunol 1999; 162:4148-4156; ;

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Overexpression of Bcl-2 in Transgenic Mice Decreases Apoptosis and Improves Survival in Sepsis1

Richard S. Hotchkiss,2* Paul E. Swanson,† C. Michael Knudson,‡ Katherine C. Chang,* J. Perren Cobb,§ Dale F. Osborne,¶ Kimberly M. Zollner,† Timothy G. Buchman,§ Stanley J. Korsmeyer,ሻ and Irene E. Karl¶

In sepsis there is extensive apoptosis of lymphocytes, which may be beneficial by down-regulating the accompanying inflammation. Alternatively, apoptosis may be detrimental by impairing host defense. We studied whether Bcl-2, a potent antiapoptotic protein, could prevent lymphocyte apoptosis in a clinically relevant model of sepsis. Transgenic mice in which Bcl-2 was overexpressed in T cells had complete protection against sepsis-induced T lymphocyte apoptosis in thymus and spleen. Surprisingly, there was also a decrease in splenic B cell apoptosis in septic Bcl-2 overexpressors compared with septic HeJ and HeOuJ mice. There were Downloaded from marked increases in TNF-␣, IL-1␤, and IL-10 in thymic tissue in sepsis in the three species of mice, and the increase in TNF-␣ and IL-10 in HeOuJ mice was greater than that in Bcl-2 mice. Mitotracker, a mitochondrial membrane potential indicator, demonstrated a sepsis-induced loss of membrane potential in T cells in HeJ and HeOuJ mice but not in Bcl-2 mice. Importantly, Bcl-2 overexpressors also had improved survival in sepsis. To investigate the potential impact of loss of lymphocytes on survival in sepsis, Rag-1؊/؊ mice, which are totally deficient in mature T and B cells, were also studied. Rag-1؊/؊ mice had decreased survival compared with immunologically normal mice with sepsis. We conclude that overexpression of Bcl-2 provides protection http://www.jimmunol.org/ against cell death in sepsis. Lymphocyte death may be detrimental in sepsis by compromising host defense. The Journal of Immunology, 1999, 162: 4148–4156.

epsis is the leading cause of death in the surgical intensive ample, loss of intestinal epithelial cells in sepsis may lead to in- care unit and a major cause of morbidity and mortality in creased bacterial translocation, thus worsening infection. S neonatal and medical intensive care units. The Center for The proto-oncogene bcl-2 was discovered by translocation anal- Disease Control estimates that each year approximately 500,000 ysis of follicular B lymphomas. The bcl-2 gene family is now people develop sepsis, and 175,000 die in the United States alone known to contain both pro- and antiapoptotic members (10, 11).

(1). Sepsis is a growing problem; its incidence has tripled from The protein product Bcl-2 prevents apoptotic cell death from a by guest on September 26, 2021 1972 to 1992 (1). Recently, apoptosis has been identified as an wide array of noxious stimuli, including hypoxia, serum and important mechanism of cell death in animal models of sepsis and growth factor withdrawal, ionizing irradiation, glucocorticoids, endotoxemia (2–6). In this setting, lymphocytes are the predomi- and calcium ionophores (11, 12). Because of the known protective nant type of cells undergoing apoptotic death. However, parenchy- ability of Bcl-2 to prevent cell death in a variety of conditions, we mal cells, including intestinal and lung epithelial cells and vascular investigated its possible role in apoptotic cell death occurring in endothelial cells, also have increased apoptotic cell death in certain sepsis. Transgenic mice overexpressing Bcl-2 in T lymphocytes models of sepsis (6–8). The potential beneficial or adverse effects were made septic, and thymus and spleen were examined for the of apoptosis on sepsis are unknown. Sepsis is a subset of the sys- degree of apoptotic cell death. The genetic background strain for temic inflammatory response syndrome, and apoptosis could be the Bcl-2 overexpressors was the C3H HeJ mouse. This strain of beneficial to the host by eliminating many of the lymphocytes that mouse is resistant to the lethal effects of endotoxin and recently has produce proinflammatory cytokines, such as TNF or IL-1 (7, 9). been demonstrated to have extensive apoptosis in multiple organs Alternatively, apoptosis may be detrimental in sepsis by depleting in an intra-abdominal model of sepsis (3, 13). To eliminate the lymphocytes, with resultant compromise of the host defenses potential confounding effects of endotoxin resistance, an additional against the invading organisms (7, 9). Also, if apoptosis of paren- strain of nonendotoxin-resistant mice, i.e., C3H HeOuJ mice, very chymal cells is extensive, organ dysfunction may occur. For ex- similar in genetic background to C3H HeJ mice, was used as a second control group in all studies. Departments of *Anesthesiology, †Pathology, ‡Laboratory Medicine, §Surgery, and A second member of the bcl-2 gene family is the proapoptotic ¶Medicine, and the Howard Hughes Medical Institute, Washington University Bax. Targeted disruption of Bax blocks cell death after trophic School of Medicine, St. Louis, MO 63110 factor deprivation and during development (14, 15). The protein Received for publication September 28, 1998. Accepted for publication January Bax forms heterodimers with Bcl-2, and it is postulated that a 13, 1999. preset ratio of Bcl-2/Bax determines the survival or death of cells The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance following an apoptotic stimulus (11, 15). To investigate the po- with 18 U.S.C. Section 1734 solely to indicate this fact. tential role of Bax in the apoptotic cell death occurring during 1 This work was supported by the National Institutes of Health (Grants GM44118 and sepsis, Bax-deficient mice were examined also. GM55194) and the Alan A. and Edith L. Wolff Foundation. Cytokines are important mediators of sepsis, and the apoptotic cell 2 Address correspondence and reprint requests to Dr. Richard S. Hotchkiss, Depart- death program can be triggered by TNF-␣ (16). Bcl-2 has been shown ment of Anesthesiology Research Unit, Washington University School of Medicine, Campus Box 8054, 660 S. Euclid Ave., St. Louis, MO 63110. E-mail address: to decrease T cell activation and block cytokine production in Jurkat [email protected] T cells by impairing DNA binding and nuclear translocation of the

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 4149 nuclear factor of activated T cells (NFAT)3 (17). Therefore, one po- tered 1 h after CLP (see survival studies for details of antibiotic selec- tential mechanism for the protective effect of Bcl-2 in sepsis would be tion and administration). to decrease cytokines. To address this question, proinflammatory Quantification of apoptosis (TNF-␣ and IL-1␤) and anti-inflammatory (IL-10) cytokines were Apoptosis was quantified by four independent methods with varying measured in thymi and spleens in sham and septic mice. sensitivity and specificity as described previously (7). Sham or CLP In addition to possible effects on cytokines, Bcl-2 may decrease mice were killed 18–22 h postsurgery, and thymus and spleen were apoptosis by preventing the loss of mitochondrial membrane po- removed rapidly. A portion of the thymus and spleen was frozen in tential (MMP) (18, 19). A current theory concerning the apoptotic liquid nitrogen and stored at Ϫ80°C for later use in DNA agarose gel death program maintains that loss of MMP allows leakage of cy- electrophoresis and cytokine determination. A second portion of the tissue samples was placed in 10% neutral buffered paraformaldehyde tochrome c into the cytosol, which triggers apoptosis. Bcl-2 local- for hematoxylin and eosin and TUNEL staining. A third portion of the izes to the outer mitochondrial membrane and is postulated to thymi and spleens was used for flow cytometry. block cytochrome c release by an unknown mechanism. The flu- Fluorescent TUNEL orescent indicator Mitotracker Red and flow cytometry were employed in isolated thymocytes to examine MMP in sepsis and de- At 16–22 h after CLP or sham surgery, mice were killed, and fluorescent termine potential effects of Bcl-2 on preservation of membrane TUNEL was performed as described previously (7, 13) with minor modifications. Paraffin-embedded tissue slices were dewaxed and rehydrated. potential (20, 21). The present experiments were performed pri- Using an apoptosis detection kit (Boehringer Mannheim, Indianapolis, IN) marily to test the hypothesis that Bcl-2 can prevent sepsis-induced cells were permeabilized by microwave treatment in citrate buffer and lymphocyte apoptosis. Survival studies also were performed to de- DNA labeled with dUTP fluorescein via action of terminal deoxynucleo- Ј termine whether prevention of lymphocyte apoptosis was benefi- tidyl transferase, which attaches the dUTP at the 3 -hydroxyl DNA ends Downloaded from (fluorescent TUNEL). Apoptotic nuclei have nicks (strand breaks) in their cial or detrimental to host survival in sepsis. DNA that are labeled with the fluorescein dUTP. In some cases, a second DNA counterstain, i.e., Hoechst 33342, was used to label nonapoptotic Materials and Methods nuclei. Tissue sections were examined at ϫ400 magnification by fluores- Transgenic mice cence microscopy, and a minimum of three random fields were evaluated. The percent area of the field that was positively labeled for apoptosis (flu- Mice that selectively overexpress Bcl-2 in T lymphocytes using the lck- orescein stained) was calculated using an image analysis program (Meta- proximal promoter were generated and backcrossed to C3H-HeJ mice for morph, Universal Imaging Corp., West Chester, PA). The percent area was http://www.jimmunol.org/ Ͼ10 generations as described previously (22). Also as described, all trans- divided by the area of an average cell to obtain the number of TUNEL- genic lines demonstrated Bcl-2 protein in the thymus, spleen, and lymph positive cells. nodes (both mesenteric and inguinal) (22). Bcl-2 protein was higher in Light microscopy of hematoxylin- and eosin-stained specimens thymus than spleen or lymph nodes, consistent with the percentage of T cells in these organs (22). Mice homozygous for the gene causing Bcl-2 Hematoxylin- and eosin-stained specimens were examined by a pathologist overexpression were mated to C3H HeJ mice, and a tail blood sample was (P.E.S.) blinded to sample identity who identified apoptosis based on char- obtained from offspring to verify overexpression of Bcl-2 in T lymphocytes acteristic cellular morphologic changes, including compacted/condensed by PCR analysis (23). The genetic background of the Bcl-2 overexpressors nuclei (pyknosis) and/or nuclear fragmentation (karyorrhexis). The speci- is C3H HeJ, a species of mouse that is highly resistant to the lethal effects mens were graded at ϫ400 magnification for the percentage of cells dem- of endotoxin (24). To avoid potential confounding influence of the endo- onstrating apoptotic features. A minimum of three random fields were eval- by guest on September 26, 2021 toxin resistance, a second group of endotoxin-sensitive mice, C3H HeOuJ, uated in each sample. also served as a control group. C3H HeOuJ mice have a very similar genetic background to the C3H HeJ mice but are endotoxin sensitive. C3H DNA agarose gel electrophoresis HeJ and C3H HeOuJ mice were obtained from The Jackson Laboratory DNA agarose gel electrophoresis was performed as described previously (Bar Harbor, ME) and were age and sex matched with the Bcl-2 transgenic (7, 13) with minor modifications. Tissue was homogenized in digestion mice. buffer, i.e., DNAzol (Molecular Research, Cincinnati, OH). Following cen- The Bax-deficient mice were generated as described previously (15), trifugation, the top layer was transferred to a second tube, and absolute and the genotype was verified by PCR analysis (23). Normal littermates, ethanol was added. After a second centrifugation, the precipitate was i.e., non-Bax-deficient mice were used as controls. Four Bax-deficient mice washed with 95% ethanol, and DNA dissolved in TE buffer (pH 8.0). DNA and four normal littermates were available for study. Therefore, tissue sam- (7.5 ␮g) was pipetted onto a 2% agarose gel containing either ethidium ple analysis was conducted, but animals were not available for survival bromide or Sybr Green (Molecular Probes, Eugene, OR), and electrophore- studies. sis was performed. Sepsis model: cecal ligation and puncture (CLP) Flow cytometry: cell phenotyping and mitochondrial membrane Mice weighing 17–25 g were housed for at least 3 days before manipula- potential tions. The CLP model of sepsis was used to induce intra-abdominal peri- Tissue sections (10–200 mg) from thymi or spleens were gently glass tonitis as described previously (7, 13). The CLP model was selected be- ground to dissociate the cells that were washed twice in PBS with 1% BSA cause it results in a model that reproduces many of the clinical hallmarks and 0.01% sodium azide. Residual RBC were lysed by hypotonic lysis in of sepsis that occur in patients (25). Previous studies in our laboratory ice-cold ammonium chloride. The cells were resuspended in PBS, and the include positive blood cultures for polymicrobial organisms (aerobic and desired fluorescent indicators were added. The degree of apoptotic cell anaerobic bacteria) from CLP mice but not sham-operated mice (7). The death was quantified using a commercially available, fluorescein-labeled, presence of live bacterial organisms is an important component of the annexin V product (Apoptosis Detection Kit, R&D Systems, Minneapolis, model because many drugs that inhibit the inflammatory component of the MN) as previously described (26). Mouse T and B cells were identified septic response impair the ability of the host to fight pathogens. The details using cychrome-labeled anti-CD3 and phycoerythrin-labeled anti-CD19 of the CLP mouse model in our laboratory have been described previously Abs, respectively (PharMingen, San Diego, CA). Identification of apopto- (7, 13). In the CLP group, a midline laparotomy was performed using tic cells by annexin V and cell phenotyping were performed simultaneously halothane anesthesia. The cecum was mobilized, ligated with 4-0 silk su- using three-color analysis and flow cytometry. Assessment of MMP was ture below the ileocecal valve, and punctured once with a 23-gauge needle. performed using Mitotracker Red (catalogue no. M-7513, Molecular The abdomen was closed in two layers, and the mice were injected with 1.0 Probes) (20, 21). Cells were incubated in 100 nM Mitotracker Red at 37°C ml of 0.9% saline s.c. Sham-operated mice were handled identically, for1hinthedark. Flow cytometric analysis (50,000 events/sample) was but the cecum was not ligated or punctured. Antibiotics were adminis- performed on FACSCalibur (Becton Dickinson, San Jose, CA). Cell debris was electronically gated out based on the forward light scatter.

3 Abbreviations used in this paper: NFAT, nuclear factor of activated T cells; MMP, Cytokine determination mitochondrial membrane potential; CLP, cecal ligation and puncture; Rag-1, recom- bination-activating gene-1; TUNEL, terminal deoxynucleotidyl transferase-mediated Tissue concentrations of TNF-␣, IL-1␤, and IL-10 were measured by dUTP-nick end labeling. ELISA using commercially available kits (Genzyme, Cambridge, MA) as 4150 Bcl-2 PREVENTS APOPTOSIS AND IMPROVES SURVIVAL Downloaded from http://www.jimmunol.org/

FIGURE 1. A–C, Fluorescent TUNEL. Thymus tissue sections from sham and septic mice were examined by the fluorescent TUNEL method. Nuclei that appear bright green are labeled by fluorescein-conjugated dUTP and are positive for apoptosis. To estimate the number of TUNEL-positive cells, the TUNEL-positive area was determined by an image analysis program Metamorph (Universal Imaging). Next the TUNEL-positive area was divided by the averaged area of a single cell to get the estimated number of TUNEL-positive cells. A, Septic HeJ (147 nuclei positive); B, septic Bcl-2 (three nuclei positive); C, septic HeOuJ (129 nuclei positive). Magnification, ϫ400. The data are representative of HeJ (n ϭ 17), HeOuJ (n ϭ 6), and Bcl-2 (n ϭ 13) mice. TUNEL and blinded light microscopy experiments comparing HeJ and Bcl-2 mice were conducted three times, while experiments comparing HeOuJ and Bax-deficient mice were performed once. D–F, Hematoxylin and eosin (H&E) staining. Thymic tissue sections from sham and septic mice were stained by guest on September 26, 2021 by H&E and examined at ϫ400 magnification. D, Septic HeJ; E, septic Bcl-2; F, septic HeOuJ. Note the shrunken (pyknotic) and fragmented (karyor- rhectic) nuclei in HeJ and HeOuJ mice but their absence from Bcl-2 mice. The slides were evaluated by a pathologist who was blinded to sample identity. A minimum of three random fields were evaluated, and the percentage of cells demonstrating apoptotic features was estimated. Note that Ͼ25% of cells in septic HeJ and HeOuJ thymi appear apoptotic. The thymocytes in the septic Bcl-2 mouse appear normal.

previously described (27). Approximately 10–15 mg of tissue samples tory. Both groups of mice underwent CLP, and survival was recorded as were homogenized in a tissue grinder containing 1 ml of lysis buffer (PBS previously stated. containing 2 mM PMSF and 1 mg/ml of aprotinin, leupeptin, and pepstatin This study was conducted in accordance with the National Institutes of A) as described by Clark et al. (28). Analysis was performed with 100 ␮l Health guidelines on the use of laboratory animals and with approval of the of standard (diluted in lysis buffer) or 10, 50, or 100 ␮l of tissue homog- Washington University animal studies committee. enate. Each sample was run in duplicate, and a portion of the sample was analyzed for protein. Data are expressed as picograms per milligrams of Statistical analysis protein. For each assay a standard curve was generated and, based on Data are reported as the mean Ϯ SEM. Data were analyzed using a sta- replicates of the measured absorbance, demonstrated an average coefficient tistical software program RS/1 from BBN Software (Cambridge, MA). Ͻ of variance of 10%. Recovery of spiked samples, i.e., containing known Differences in group survival and DNA agarose gels were determined using ␣ ␤ amounts of TNF- , IL-1 , or IL-10, was about 90% (data not presented). Fischer’s exact p test. All other data were analyzed using one-way analysis Յ Survival studies in sepsis of variance. Significance was accepted at the p 0.05. Additional groups of mice, C3H HeJ, C3H HeOuJ, and Bcl-2, from over- Results expressor strains underwent CLP and were included in survival studies. Fluorescent TUNEL The methods for the survival studies in the mouse CLP model have been described previously (29). Briefly, an investigator (R.S.H.) blinded to the Tissue sections were examined at ϫ400 magnification. Thymi and identity of the mice performed CLP in the various groups of mice. Ap- spleens from sham-operated Bcl-2, HeJ, and HeOuJ mice had approximately 1 h after CLP, the mice received metronidazole (35 mg/kg; an proximately two to five cells positively stained for apoptosis, with antibiotic with an anaerobic bacterial spectrum of action) and ceftriaxone many fields entirely negative. Therefore, these tissues were clas- (50 mg/kg; an antibiotic with a Gram-negative bacterial spectrum of action). The antibiotics were repeated every 12 h for 48 h and then discon- sified as negative for apoptosis. tinued. The mice were allowed free access to food and water, and survival Thymi from septic HeJ (n ϭ 17), HeOuJ (n ϭ 6), and Bax- was recorded for 6 days. deficient (n ϭ 4) mice had many fields in which approximately To further evaluate the impact of lymphocyte apoptosis in sepsis, an 10–15% of cells were positively labeled for apoptosis (Fig. 1, A additional sepsis survival study was conducted in Rag-1Ϫ/Ϫ mice. Rag- 1Ϫ/Ϫ mice are homozygous for the Rag-1Ϫ/Ϫ Tm1 Mom mutation and and C, and Fig. 2) and were indistinguishable from each other. It produce no mature T or B cells (30). Rag-1Ϫ/Ϫ mice and their matched was not possible to count individual nuclei in thymi because many normal controls (C57B46ϫ129) were obtained from The Jackson Labora- apoptotic cells were confluent or overlapping (Fig. 1, A and C). The Journal of Immunology 4151

were: HeJ, 63.6 Ϯ 16.9; OuJ, 76.5 Ϯ 25.7; and Bax-deficient, 121.2 Ϯ 30.8. Thymi from Bcl-2 mice with sepsis (n ϭ 13) had 9.1 Ϯ 1.6 cells per high powered field positively labeled for apoptosis, with many areas entirely negative for apoptosis (Figs. 1B and 2). Bcl-2 thymi were statistically different from all other groups ( p Ͻ 0.05). Spleens from HeJ (n ϭ 13), HeOuJ (n ϭ 6), Bcl-2 overexpressor septic mice (n ϭ 13), and Bax-deficient mice (n ϭ 4) had less apoptotic labeling than did thymi from the same animal. The numbers of TUNEL-positive cells in spleens per HPF from HeJ, HeOuJ, and Bax-deficient mice were 18.6 Ϯ 3.2, 19.8 Ϯ 4.0, and 18.3 Ϯ 3.3, respectively. Spleens from Bcl-2 mice had an average of 8.2 Ϯ 1.0 cells TUNEL-positive/high powered field, and this was statistically different from the values in HeJ and HeOuJ spleens ( p Ͻ 0.05). Conventional light microscopy of hematoxylin- and eosin-stained specimens

Histologic examination of specimens was performed by a pathol- Downloaded from ogist (P.E.S.) who was blinded to sample identity. A minimum of three random fields were examined at ϫ400 magnification, and the percentage of cells demonstrating apoptotic features was recorded. Cells were judged to be apoptotic if they exhibited the characteristic findings of shrunken, compacted nuclei (pyknosis) and/or nu-

clear fragmentation (karyorrhexis). Thymi and spleens from sham- http://www.jimmunol.org/ operated HeJ, HeOuJ, and Bcl-2 overexpressor mice appeared normal, and Ͻ1% of cells exhibited apoptotic features (n ϭ 3–5 mice/group). Thymi from septic HeJ (n ϭ 6; Fig. 1D), HeOuJ (n ϭ 6; Fig. 1F), and Bax-deficient (n ϭ 4) mice revealed extensive apoptosis, which was more prominent in cortical than in medullary regions (Fig. 1, D and F). In selected regions, the percentage of cells with apoptotic features was Ͼ25% (Fig. 1). There was no readily apparent difference in the degree of apoptosis in thymi in these three strains of mice. Thymi from septic Bcl-2 mice were by guest on September 26, 2021 identical with those from sham-operated mice, with Ͻ1% of cells showing evidence of apoptosis (Fig. 1E), and this was statistically different from other groups ( p Ͻ 0.05). Spleens from septic HeJ (n ϭ 6), HeOuJ (n ϭ 6), and Bax- deficient (n ϭ 4) mice had less extensive apoptosis than thymi from these mice. Approximately 2–5% of cells had pyknotic or fragmented nuclei. In some regions of the spleen as many as 10% of cells were apoptotic. In contrast to the thymus, spleens from septic Bcl-2 mice (n ϭ 10) were not obviously different from those from the other groups of mice, and approximately 2–5% of cells had characteristic changes of pyknosis and/or karyorrhexis. How- ever, direct comparison of spleens from septic Bcl-2 mice to FIGURE 2. Fluorescent TUNEL. Representative black and white im- spleens from HeJ and HeOuJ mice did reveal a difference in the ages from thymus tissue sections of septic mice were labeled using the anatomic localization of apoptosis. Apoptosis was present through- fluorescent TUNEL method (to identify apoptotic nuclei) and were coun- out the spleens of septic HeJ and HeOuJ mice, including the area terstained with Hoechst 33342, which stains all nuclei (apoptotic and no- surrounding the periarteriolar lymphoid sheath, which is the T cell- napoptotic). Note the large number of apoptotic nuclei in HeJ, HeOuJ, and rich area. In contrast, there was a noted lack of apoptosis in the Bax thymus but minimal apoptosis in Bcl-2 thymus. Images obtained using periarteriolar lymphoid sheath in the septic Bcl-2 mice. a laser scanning confocal microscope at ϫ400. The TUNEL and corre- sponding Hoechst images were obtained from the identical microscopic DNA agarose gel electrophoresis field of view using two different excitation frequencies and emission filters. DNA agarose gels from thymi and spleens of sham-operated mice had no evidence of DNA fragmentation, i.e., ladder formation by the electrophoresis procedure (n ϭ 3–5 mice/group). Thymi from Therefore, to estimate the number of TUNEL-positive cells, the septic HeJ (n ϭ 10), HeOuJ (n ϭ 6), and Bax-deficient (n ϭ 4) TUNEL-positive area first was determined by an image analysis mice had 80, 67, and 100% degrees of positive ladder formation, program, Metamorph (Universal Imaging, West Chester, PA). a characteristic feature of apoptosis. In contrast, none of the thymi Next, the total TUNEL-positive area was divided by the averaged from Bcl-2 (n ϭ 12) mice had ladder formation, and this was area of a single cell to determine the estimated number of TUNEL- significantly different from all other groups ( p Ͻ 0.05). An exam- positive cells. Employing this method, the numbers of TUNEL- ple of HeJ and Bcl-2 is shown in Fig. 3. The spleens from the HeJ positive cells per high powered field (ϫ400) in the various group (n ϭ 12), HeOuJ (n ϭ 6), and Bcl-2 (n ϭ 11) mice had 33, 50, and 4152 Bcl-2 PREVENTS APOPTOSIS AND IMPROVES SURVIVAL

Tissue cytokine analysis In thymi, sepsis caused a marked increase in both the proinflammatory cytokines TNF-␣ (about three- to sixfold) and IL-1␤ (two- to ninefold) as well as the anti-inflammatory cytokine IL-10 (1.3- to 2-fold) in all three species of mice ( p Ͻ 0.05; Table III). The increases in TNF-␣ and IL-10 in sepsis were greater in HeOuJ thymi than in Bcl-2 thymi ( p Ͻ 0.05), but were not different from those in HeJ thymi. The increase in cytokine concentrations in spleens was not as great as that which occurred in the thymi. In spleens, there was a 74% increase in TNF-␣ in septic vs sham HeJ mice ( p Ͻ 0.05) and a 116% increase in IL-10 in septic vs sham FIGURE 3. DNA agarose gels. Thymus samples from sham and septic HeOuJ mice ( p Ͻ 0.05). Cytokine concentrations did not increase Bcl-2 and HeJ mice were homogenized in DNAzol and prepared for elec- in septic vs sham spleens of Bcl-2 mice (Table III). trophoresis as described in Materials and Methods. Markers are reference commercial DNA markers (n ϭ 1 sham HeJ, 1 sham Bcl-2, 4 septic HeJ, Survival study and 5 septic Bcl-2 mice). The DNA agarose gel experiment is representa- The numbers of mice in the various group were as follows: 15 HeJ, tive of two studies, both of which confirmed the protective effect of Bcl-2 20 HeOuJ, and 9 Bcl-2 homozygous expressors, and 13 Bcl-2 het- in thymus. erozygous expressors. The Bcl-2 homozygous and heterozygous expressors had very similar survival curves; therefore, their data Downloaded from 27% positive incidence of DNA ladder formation; these values were combined. There were no deaths in any group at 24 h (Fig. 5). were not statistically significantly different. At 48 and 72 h, some of the HeJ and HeOuJ mice, but no Bcl-2 mice, had died. At 72 h there was a statistical difference between Flow cytometry: apoptosis and MMP Bcl-2 and both groups ( p Ͻ 0.05). At the end of the 6-day period, Results of flow cytometry in thymic T cells from sham-operated there were 26, 55, and 73% survivals in HeJ, HeOuJ, and Bcl-2 HeJ, HeOuJ, and Bcl-2 mice revealed an approximately 6% inci- mice, respectively. There was a statistically significant improve- http://www.jimmunol.org/ dence of apoptosis, which was not statistically different among the ment in long term, i.e., 6-day survival in Bcl-2 mice compared various groups (n ϭ 3–5 mice/group; Table I). T cells from thymi with HeJ mice ( p Ͻ 0.05) but not compared with HeOuJ mice. of septic HeJ and HeOuJ mice had 127 and 690% increases in There was also an improved survival in HeOuJ mice vs HeJ mice apoptosis, respectively, compared with their matched sham con- at 6 days ( p Ͻ 0.05). Ϫ/Ϫ trols ( p Ͻ 0.01). In contrast, there was no increase in T cell ap- Nineteen Rag-1 mice and 24 matched, immunologically optosis in sepsis in Bcl-2 mice (Table I). competent, control mice underwent CLP. At the end of 6 days, Ϫ/Ϫ Splenic T and B cells in septic HeJ and HeOuJ mice had a there was a 5.3% survival in Rag-1 mice vs a 45.8% survival in matched controls, and this difference was significant ( p Ͻ 0.05; marked increase in apoptosis compared with those in sham mice by guest on September 26, 2021 (Table I). The degree of apoptosis was more severe in T cells Fig. 6). (ϳ34% of T cells apoptotic) vs B cells (ϳ17.7% of B cells apoptotic) in HeJ mice ( p Ͻ 0.05). This difference in the degree of T Discussion vs B cell apoptosis did not occur in HeOuJ mice. There was no Perhaps the most important finding of the present study was the increase in splenic T cell apoptosis in septic Bcl-2 vs sham Bcl-2 remarkable ability of Bcl-2 to protect against the lymphocyte cell mice (Table I). There was a 42% increase in apoptosis in B cells death occurring in the highly complex and clinically relevant in Bcl-2 mice with sepsis compared with that in sham-operated model of sepsis. There was complete protection against apoptosis Bcl-2 mice, but this increase was not statistically significant. There in thymocytes and splenic T cells during sepsis, a finding that was a statistically significant increase in B cell apoptosis in sepsis corresponds to the T cell-restricted native overexpressor pheno- in HeJ mice (150%) and HeOuJ mice (259%; p Ͻ 0.05). type in these animals. The ability of Bcl-2 overexpression to pre- Thymic T cells in HeJ and HeOuJ mice, but not in Bcl-2 mice, vent lymphocyte cell death was surprising because of previous demonstrated a loss in MMP with sepsis (Table II and Fig. 4). The studies in our laboratory demonstrating a lack of efficacy of TNF-␣ loss in MMP in HeJ and HeOuJ T cells in sepsis was noted in a receptor blockade (13) and transgenic models including Fas recep- subset of cells by a decrease in fluorescence intensity, while the tor knockout and perforin knockout mice (our unpublished obser- majority of T cells maintained MMP (Fig. 4). vations). Indeed, the ability of Bcl-2 to prevent cell death in sepsis

Table I. Percent apoptotic cells determined by ﬂow cytometry a

HeJ sham HeOuJ sham Bcl-2 sham HeJ clp HeOuJ clp Bcl-2 clp

Thymus T cells 6.9 Ϯ 1.2 (4) 6.3 Ϯ 1.2 (4) 5.1 Ϯ 0.8 (5) 15.6 Ϯ 1.2* (6) 50.1 Ϯ 4.2*⌬ (6) 6.2 Ϯ 0.9 (10) Spleen B Cells 7.0 Ϯ 0.5 (4) 10.0 Ϯ 2.0 (4) 7.1 Ϯ 1.2 (5) 17.7 Ϯ 0.8* (6) 35.9 Ϯ 2.6*⌬ (6) 10.0 Ϯ 0.8 (10) Spleen T Cells 9.6 Ϯ 1.2 (4) 11.4 Ϯ 4.4 (4) 8.4 Ϯ 1.4 (5) 33.9 Ϯ 4.8* (6) 29.2 Ϯ 5.2* (6) 9.3 Ϯ 0.8 (10)

a Values expressed as mean Ϯ SEM. The percent of T or B cells that were undergoing apoptosis were determined using annexin V and flow cytometry as described in Materials and Methods. Cecal ligation and puncture (clp) caused an increase in p Ͻ 0.05 clpϾsham for matched strains. The change ,ء ;apoptosis in T and B cells in HeJ and HeOuJ mice but not in Bcl-2 mice in the percent apoptosis in T or B cells in clp HeOuJ mice was greater than the percent apoptosis in T or B cells of HeJ mice. The number of mice in each group is identified in parenthesis. The flow cytometry study was conducted twice. The Journal of Immunology 4153

Table II. Percent Thymus T cells with decreased MMP a

Thymus T Cells

HeJ sham HeOuJ sham Bcl-2 sham HeJ clp HeOuJ clp Bcl-2 clp

2.3 Ϯ 0.4† (4) 7.1 Ϯ 0.6 (4) 5.3 Ϯ 1.6 (4) 28.4 Ϯ 2.2* (6) 18.9 Ϯ 2.6* (6) 5.4 Ϯ 1.5 (6)

a The percent of thymic T cells with decreased MMP were determined using flow cytometry and the fluorescent indicator Mitotracker as described in Materials and Methods. There was a loss of MMP with sepsis in T cells from HeJ and HeOuJ but clpϾsham for matched strain, p Ͻ 0.05. Septic Bcl-2 was less than both clp HeJ and HeOuJ, p Ͻ 0.05. See ,ء .not in Bcl-2 mice Fig. 5 for representative flow data. †, Values are mean Ϯ SEM and expressed as pg/mg protein. The number of mice in each group is identified in parenthesis. This experiment was conducted twice and results of the two studies were combined. is extraordinary given the complex pathophysiologic nature of this decrease in B cell apoptosis in septic Bcl-2 mice as well. The 42% disorder and the failure of the previous transgenic models to block increase in splenic B cell apoptosis in septic vs sham Bcl-2 mice apoptosis. An array of interlocking pathways and mediators is re- did not achieve statistical significance and was much less than the cruited in sepsis, and a partial list includes cytokines, complement, marked increases in B cell apoptosis in HeJ (150%) and HeOuJ leukotrienes, oxygen radicals, and coagulation factors. The inhib- (259%) mice ( p Ͻ 0.05). There is no ready explanation for this itory effect of Bcl-2 on cell death in sepsis implies that it acts in a protective effect in B cells, although histologic examination may

ﬁnal common pathway of apoptosis. Studies conducted on isolated provide a clue. As is apparent by both light and ﬂuorescence mi- Downloaded from cells have shown that Bcl-2 has a wide-ranging capability to block croscopy, apoptotic cell death in sepsis occurred in islands or clus- cell death from an array of noxious stimuli, including gamma ir- ters (Fig. 1, A, C, D, and F, and Fig. 2). This focal anatomic pattern radiation, hypoxia, growth factor withdrawal, and oxidative stress suggests a potential paracrine effect, and recent studies indicate (12, 22). The present study is important because it demonstrates that such a phenomenon exists in apoptosis. Su et al. demonstrated that Bcl-2 has a protective effect in a clinically relevant in vivo that paracrine apoptosis occurred in Jurkat T cells and was medi-

model. ated by shedding of FAS ligand (31). Therefore, if T splenocyte http://www.jimmunol.org/ Although the Bcl-2 transgenic mice used in this study had over- apoptosis is decreased in the spleen, neighboring B cells may also expression of Bcl-2 limited to T cells, there was also an apparent be protected. by guest on September 26, 2021

FIGURE 4. MMP. Suspensions of thymic cells were prepared from sham or septic mice as described in Materials and Methods. The cells were incubated with Mitotracker Red (Molecular Probes), which is a sensitive indicator of MMP. There is a decrease in the ﬂuorescent intensity of Mitotracker Red (x- axis), with loss of MMP. Note the increase in the percentage of thymocytes from septic HeJ mice with decreased MMP (27.3% in HeJ septic vs 3.4% sham). No loss of MMP occurred in Bcl-2 septic mice. The arrow in HeJ septic identiﬁes the subset of T cells of cells. This %27.3 ,ء .with decreased MMP experiment was performed on two occasions and demonstrated reproducible results. 4154 Bcl-2 PREVENTS APOPTOSIS AND IMPROVES SURVIVAL

Table III. Tissue cytokines a

Tissue Cytokines

HeJ sham HeOuJ sham Bcl-2 sham HeJ clp HeOuJ clp Bcl-2 clp

Thymus n 6 57155 13 TNF-␣ 218.4 Ϯ 46.3␥ 268 Ϯ 50.9 256.5 Ϯ 50.3 1010.4 Ϯ 93.9* 1657.4 Ϯ 221.7*† 803.7 Ϯ 78.1* IL-1␤ 231 Ϯ 27.5 329.2 Ϯ 102 206.2 Ϯ 20.6 742.3 Ϯ 139.1* 3024.1 Ϯ 1036.8* 739.0 Ϯ 234.3* IL-10 605.2 Ϯ 110.0 1423.2 Ϯ 362.1 614.5 Ϯ 61.5 1613.2 Ϯ 313.1* 2438.8 Ϯ 419.4*† 811.7 Ϯ 179.9* Spleen n 9 47196 14 TNF-␣ 171.1 Ϯ 21.2 176.1 Ϯ 17.2 166.8 Ϯ 22.2 297.8 Ϯ 24.5* 369.3 Ϯ 64.7 279.5 Ϯ 39.2 IL-1␤ 52.6 Ϯ 6.5 32.7 Ϯ 4.2 68.7 Ϯ 12.3 78.3 Ϯ 5.6 61.2 Ϯ 15.3 99.3 Ϯ 10.7 IL-10 200 Ϯ 28.3 110.5 Ϯ 13.7 230.3 Ϯ 63.1 263.2 Ϯ 27.3 238.7 Ϯ 37.3* 250.2 Ϯ 20.4

TNF-␣, IL-1␤, and IL-10 were ,ء .a Tissue cytokines were measured using a commercially available ELISA kit as described in Materials and Methods increased in clp vs sham thymi in all three groups of mice for matched strain, p Ͻ 0.05. Note that the sepsis-induced increase in cytokines in thymi were much greater than in spleen. ‡, Values are mean Ϯ SEM and expressed as pg/mg protein. The number of mice in each group is identified. The tissues were obtained from three separate experiments and then analyzed together at one time. Downloaded from Another important implication of this study relates to the role of tionship of Bcl-2 to HeJ mice (all five control mice died of endo- apoptosis in the pathophysiology of sepsis. Investigators postu- toxin (ϳ50 mg/kg), but no Bcl-2 mouse died). However, the fact lated that apoptosis might be beneficial by causing elimination of that Bcl-2 mice had improved survival compared with HeOuJ mice lymphocytes, which are a major source of cytokines in sepsis. A (mice that are endotoxin sensitive) on day 3 also supports a partial decrease in cytokines could reduce the uncontrolled systemic in- protective effect of Bcl-2 compared with that strain as well. The flammatory component of sepsis that contributes to organ injury increase in septic mortality in Rag-1Ϫ/Ϫ mice that lack mature T or (7, 9, 13). Alternatively, apoptosis of lymphocytes may be detri- B cells compared with that in their immunologically competent http://www.jimmunol.org/ mental in sepsis by compromising the ability of the host to combat controls with sepsis verifies the important role of lymphocytes in infection (7, 9). Overexpression of Bcl-2 prevented T cell death host defense in sepsis and strengthens the conclusion that Bcl-2 and caused improvement in 3-day survival in Bcl-2 transgenic may be protective in sepsis by preventing loss of lymphocytes. mice compared with that in both HeJ and HeOuJ strains. The pro- Recent evidence from the clinical arena also indicates the critical tective effect of Bcl-2 had decreased by day 6 in Bcl-2 compared nature of lymphocytes in the battle against sepsis. Rajan and Sleigh with HeOuJ, but not compared with HeJ. It is important to em- reported that patients in whom the circulating lymphocyte count phasize that the most appropriate strain of mice for comparison decreased for a 3-day duration had a greatly increased risk of nos-

with Bcl-2 transgenic mice is HeJ mice, the genetic strain from ocomial sepsis (32). by guest on September 26, 2021 which Bcl-2 mice are derived. The Bcl-2 promoter was expressed The particular model of sepsis used to examine therapeutic efficacy in HeJ mice, and the Bcl-2 homozygous overexpressors are mated is of great importance in study design (25). We selected the CLP (currently Ͼ12 generations) to other normal HeJ mice (22, 23). model because it is believed to reflect the conditions that occur in Studies in our laboratory confirmed that the Bcl-2 overexpressors patients (25). Importantly, live bacterial pathogens can be cultured retain the endotoxin resistance present normally in HeJ mice (our from blood and peritoneal fluid in mice that have undergone CLP (7). unpublished observations), thus supporting the close genetic rela- In the present study broad spectrum antibiotics were employed in the

FIGURE 5. Survival curve: percent survival of mice following CLP. Fifteen HeJ, 20 HeOuJ, and 22 Bcl-2 overexpressors had CLP and received i.p. antibiotics (metronidazole and ceftriaxone) 1 h after surgery as described in Materials and Methods. Survival was recorded for 6 days. ⌬,On FIGURE 6. Survival curve: percent survival of mice following CLP. day 3, Bcl-2 had improved survival compared with both HeOuJ and HeJ Nineteen Rag-1 mice and 24 matched immunologically competent control At the end ,ء .On day 6, Bcl-2 had improved survival compared with HeJ mice underwent CLP as described in Materials and Methods ,ء .(p Յ 0.05) (p Ͻ 0.05). ϩ, On day 6, HeOuJ had improved survival compared with of 6 days, there was a 5.3% survival in Rag-1 mice and a 45.8% survival HeJ, (p Ͻ 0.05). The survival experiment was conducted in one large in matched controls, and this difference was significant (p Ͻ 0.05). The study. survival study was conducted in one large study. The Journal of Immunology 4155 treatment of septic mice to enhance the clinical relevance by prolong- the latter relative to the former. Additional evidence against the ing the time course of the sepsis. The longer time course (6 days) hypothesis that Bcl-2 protects by decreasing cytokines is provided probably allowed for activation and recruitment of lymphocytes in the from our previous study in which administration of a TNF receptor body’s defense against invading pathogens. blocker failed to decrease apoptosis in the identical mouse CLP The resistance of HeJ mice to endotoxin is due to a mutation in model (13). However, it is possible that Bcl-2 prevented the pro- a single gene on chromosome 4 and is related partially to a de- duction of other unmeasured cytokines, e.g., IL-6 or that the effect creased production of circulating cytokines, e.g., TNF, in response of Bcl-2 on cytokine production occurred at an earlier time point to endotoxin challenge (13). Interestingly, in some models of sep- in the course of disease. sis, HeJ mice are more susceptible to death than are mice of the The present findings are consistent with the current hypothesis endotoxin-sensitive parent strain (24). The findings of extensive that Bcl-2 prevents apoptosis by preserving MMP (18, 19). Thy- lymphocyte apoptosis in thymus and spleen of septic HeJ mice mocytes in which Bcl-2 was overexpressed had no loss of MMP confirm the results of our previous study, indicating that endotoxin with sepsis, whereas a loss in MMP did occur in a subset of cells is not a sine qua non for apoptosis in sepsis (13). from septic HeJ and HeOuJ mice (Fig. 4 and Table II). The loss of The failure of Bax deficiency to prevent lymphocyte cell death MMP is one of the common manifestations of the apoptotic pro- in this sepsis model is important given the purported interrelation- cess and occurs in diverse types of cells regardless of the stimulus ship between the antiapoptotic Bcl-2 and the proapoptotic Bax (18, 19). This mitochondrial alteration is thought to mark the (11). Bax is known to heterodimerize with Bcl-2 and a preset ratio “point of no return” and may be a key mechanism (18). It is spec- of Bcl-2/Bax is thought to be critical in determining survival in ulated that the loss of MMP is due to the opening of the mito- certain, but not all, types of cells following specific apoptotic stim- chondrial permeability pores. Subcellular localization studies dem- Downloaded from uli (11). Bax-deficient mice should have demonstrated decreased onstrate that Bcl-2 has a patchy distribution at the contact sites sepsis-induced apoptosis in thymi and spleens, but no protection between the outer and inner mitochondrial membranes (33). Most was seen. Recently, Knudson and Korsmeyer used a genetic ap- studies indicate that Bcl-2 and other antiapoptotic Bcl-2 family mem- proach with gain-and-loss-of-function models of Bcl-2 and Bax bers must localize to mitochondria to prevent apoptosis (18). The (23). They determined that two characteristic physical features of mechanism by which Bcl-2 prevents loss of MMP is unknown.

Bcl-2-deficient mice, i.e., apoptosis and thymic hypoplasia, are In conclusion, transgenic mice overexpressing Bcl-2 in T cells http://www.jimmunol.org/ largely absent in mice that are deficient in Bax. In contrast, over- had complete protection against T lymphocyte apoptosis and par- expression of Bcl-2 still decreased apoptosis in the absence of Bax. tial protection of B lymphocytes in sepsis. A subset of T cells in These investigators concluded that while an in vivo competition septic HeJ and septic HeOuJ mice had loss of MMP, and this was exists between Bax and Bcl-2, each family member is able to prevented by Bcl-2 overexpression. The mechanism of the protec- regulate apoptosis independently. The present results in a sep- tive effect of Bcl-2 is unlikely to be due to regulation of cytokine sis-induced apoptosis model further support the concept that production, but may be related to preservation of MMP. The im- Bcl-2 may function independently of Bax to regulate cell death proved survival in Bcl-2 transgenic mice compared with HeJ and in some circumstances (23). HeOuJ mice and the decreased survival in the Rag-1Ϫ/Ϫ mice con- An intriguing question that follows from the present study is the sidered together suggest that lymphocyte apoptosis is sepsis is det- by guest on September 26, 2021 role of the different lymphocyte subsets in sepsis. Preliminary rimental to host survival. studies in transgenic mice in which Bcl-2 is overexpressed in B lymphocytes show complete protection against sepsis-induced ap- References optosis in B cells (our unpublished observations). In addition, 1. Stone, R. 1994. Search for sepsis drugs goes on despite past failures. Science these mice also have improved survival in sepsis compared with 264:365. wild-type controls with sepsis (our unpublished observations). 2. Grigg, J. M., J. S. Savill, C. Sarraf, C. Haslett, and M. Silverman. 1991. 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