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(The FASEB Journal. 2007;21:708-719.)
© 2007 FASEB

Multiple triggers of cell death in sepsis: death receptor and mitochondrial-mediated apoptosis

Katherine C. Chang*, Jacqueline Unsinger*, Christopher G. Davis*, Steven J. Schwulst{dagger}, Jared T. Muenzer{ddagger}, Andreas Strasser§ and Richard S. Hotchkiss*,{dagger},§,1

Departments of
* Anesthesiology,

{dagger} Surgery,

{ddagger} Pediatrics, and

§ Medicine, Washington University School of Medicine, St. Louis, Missouri, USA; and

|| Department of Immunology, Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia

1Correspondence: Department of Anesthesiology, 660 S. Euclid, St. Louis, MO 63110, USA. E-mail: hotchkir{at}msnotes.wustl.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Lymphocyte apoptosis plays a central role in the pathophysiology of sepsis. Lymphocyte apoptosis was examined in mice with defective death receptor pathways due to transgenic expression of a dominant negative mutant of Fas-associated death domain (FADD-DN) or Bid–/– and in mice with defective mitochondrial-mediated pathways due to loss of Bim–/–, Puma–/–, or Noxa–/–. FADD-DN transgenic and Bid–/– mice had significant albeit incomplete protection, and this protection was associated with increased survival. Surprisingly, splenic B cells were also protected in FADD-DN mice although transgene expression was confined to T cells, providing evidence for an indirect protective mechanism. Bim–/– provided virtually complete protection against lymphocyte apoptosis whereas Puma–/– and Noxa–/– mice had modest or no protection, respectively. Bim–/– mice had improved survival, and adoptive transfer of splenocytes from Bim–/– mice into Rag 1–/– mice demonstrated that this was a lymphocyte intrinsic effect. The improved survival was associated with decreased interleukin (IL) -10 and IL-6 cytokines. Collectively, these data indicate that numerous death stimuli are generated during sepsis, and it therefore appears unlikely that blocking a single "trigger" can inhibit apoptosis. If siRNA becomes practical therapeutically, proapoptotic proteins would be potential targets.—Chang, K. C., Unsinger, J., Davis, C. G., Schwulst, S. J., Muenzer, J. T., Strasser, A., Hotchkiss, R. S. Multiple triggers of cell death in sepsis: death receptor and mitochondrial-mediated apoptosis.


Key Words: endotoxin • necrosis • cytokines


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
SEPSIS IS A HEALTH CARE PROBLEM OF ENORMOUS importance and remains the most common cause of mortality in most intensive care units (1 , 2) . A major pathological process in sepsis is apoptotic death of immune effector cells, including lymphocytes and dendritic cells (3 4 5 6 7 8 9) . Three independent autopsy studies of adult, pediatric, and neonatal patients who died of sepsis showed profound apoptosis-induced depletion of CD4+ T cells and B cells (3 , 10 , 11) . Thus, apoptotic death of immune effector cells is a uniform response that occurs in all age groups during sepsis. The elimination of large numbers of lymphocytes and dendritic cells severely compromises the ability of the host to sustain an effective immune response. The central role of apoptosis in sepsis is further highlighted by numerous studies demonstrating that prevention of apoptosis improves survival in clinically relevant animal models of sepsis (12 13 14 15 16 17) .

Apoptotic death in mammals can proceed by two distinct pathways that ultimately converge to activate executioner caspases (18 19 20 21 22) (Fig. 1 ). The death receptor pathway involves activation of members of the tumor necrosis factor (TNF) receptor (TNF-R) family with an intracellular death domain including Fas, TNF-R1, DR3, and the receptors for TRAIL (23 24 25) . After ligand binding to death receptors, the death domain recruits (directly or, in the case of TNF-R1, indirectly via TRADD) an adaptor protein called Fas-associated death domain (FADD). FADD can then recruit procaspase-8 to the "death-inducing signal complex," thereby causing its activation (23 , 24) . Caspase-8 then activates caspase-3 and other executioner caspases (caspase-6 and caspase-7) that mediate the systematic demolition of the cell.


Figure 1
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  		Figure 1. Model for regulation of lymphocyte apoptosis during sepsis. The two cell death pathways and the interconnection of these pathways via truncated Bid (tBid) are presented. This study investigated interruption of the cell death pathways by five unique mutations that caused specific defects in the cell death pathways (illustrated in red).

The second apoptotic death pathway is termed the "intrinsic," "mitochondrial-mediated," or "Bcl-2-regulated" pathway (19 20 21 , 26 27 28) (Fig. 1) . A myriad of diverse stress stimuli cause activation of BH3-only proapoptotic members of the Bcl-2 family that include, for example, Bim, Puma, and Noxa (20 , 29 30 31 32) . These BH3-only proteins bind to and neutralize the antiapoptotic members of the Bcl-2 family, such as Bcl-2, Bcl-xL, or Mcl-1, thereby unleashing the proapoptotic multi-BH domain Bcl-2 family members Bax and Bak (20) . It is significant that three separate laboratories have reported that transgenic mice overexpressing Bcl-2 in immune effector cells have decreased apoptosis and improved survival in sepsis (12 , 15 , 31) . Bim is the most widely studied BH-only protein; it is required for death due to cytokine withdrawal and deregulated calcium flux, death of autoreactive T cells, and is critical to the termination of an acute T cell immune response to viral infection (20 , 33 34 35) .

In certain types of cells there may be cross-talk between the death receptor and mitochondrial-mediated pathways (18 , 23 , 24) (Fig. 1) . Bid is a proapoptotic BH3-only member of the Bcl-2 family that is essential for Fas-induced apoptosis of hepatocytes (32 , 36) . Bid is cleaved and thereby activated by active caspase-8 to form truncated Bid (tBid) (36) . Although the exact mechanism is uncertain, tBid acts either directly or indirectly to activate Bax and Bak and thereby stimulate mitochondrial release of apoptogenic compounds such as cytochrome c, resulting in mitochondrial-mediated apoptosis and amplification of the death receptor-mediated pathway (36) .

The purpose of the present study was to use specific transgenic and gene knockout mice to define the molecular pathways of lymphocyte apoptosis in sepsis (Fig. 1) . Delineation of the pertinent pathways may help identify the particular death-inducing stimuli and provide insight into possible therapy. Specifically, mice that express a dominant negative mutant of FADD (FADD DN) in their T cells were used to probe the role of the death receptor pathway. Bim–/–, Puma–/–, and Noxa–/– mice were used to examine the role of the mitochondrial pathway, and Bid–/– mice were used to examine possible cross-talk between the death receptor and mitochondrial-mediated pathways. In addition to examining effects on cell death, we also investigated the impact of inhibition of apoptosis on mortality and on the levels of key pro- and anti-inflammatory cytokines/chemokines that have been linked to clinical outcome in sepsis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Transgenic and gene knockout mice
FADD-DN transgenic mice, Puma–/– mice, and Noxa–/– mice were developed at the Walter and Eliza Hall Institute of Medical Research (WEHI, Parkville, Melbourne, Australia) by one of the authors (A.S.); earlier publications detail the methods used to generate these various constructs (37 , 38) . Bim–/– mice were also developed at the WEHI by two of the authors (P.B. and A.S.) and have been characterized extensively (29) . FADD-DN transgenic mice were generated using the lck proximal promoter; therefore, only T cells were defective in FADD activity (37) . Bid–/– mice were developed by and were a gift from Dr. Stanley Korsmeyer (32) . Mice were genotyped using tail snips and polymerase chain reaction as detailed previously. All mice were either generated on a C57BL6 background or had been backcrossed onto this background for 9–11 generations. C57BL/6 mice and Rag 1–/– mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and used as appropriate controls where indicated. Wild-type (WT) and genetically modified mice were age and sex matched for all studies.

Cecal ligation and puncture (CLP) model of sepsis
All animal studies were approved by the Washington University Animal Studies Committee (St. Louis, MO, USA). Mice weighing 18–26 g (8–12 wk of age) were housed for at least 5 days prior to use. The CLP model was used to induce intra-abdominal peritonitis, as described (12 13 14) . Mice were anesthetized with halothane and a midline abdominal incision was made. The cecum was mobilized, ligated below the ileocecal valve, and punctured twice with a 25-gauge needle. The abdomen was closed in two layers and the mice were injected subcutaneously (s.c.) with 2.0 ml of 0.9% saline. Sham-operated mice were handled in the same manner except the cecum was not ligated or punctured. For survival studies, both WT and mutant mice underwent CLP as described. A single dose of imipenem (25 mg/kg body wt) was administered s.c. ~5–6 h postoperatively. Survival was recorded for 7 days.

Adoptive transfer study
Splenocytes (7x107 cells) were obtained from unmanipulated Bim–/– or control C57BL/6 mice by gently dissociating the spleen using a 70 µ filter. The cells were washed and injected retro-orbitally into halothane-anesthetized Rag 1–/– mice. Two days after adoptive transfer of the cells, the Rag 1–/– mice underwent CLP and survival was recorded for 7 days.

Detection of lymphocyte apoptosis: flow cytometry
At 20–22 h postsurgery, sham-operated and CLP mice were killed, and spleens and thymi were removed and dissociated by gently pressing through a 70 µ filter. Cells were washed and stained with fluorochrome-conjugated antibodies to cell subset-specific surface markers (CD3 and CD20; BD PharMingen, San Diego, CA, USA) to identify T and B cells, respectively, as described (39) . To detect lymphocyte apoptosis by flow cytometry, at least two independent methods (i.e, detection of active caspase-3 and TUNEL staining) were utilized as complementary methods, as described previously. Cells were first fixed in 1% paraformaldehyde for 30 min at room temperature before repeat washing. Cells were then permeabilized with 90% methanol on ice for 30 min.

Detection of active caspase-3 via flow cytometry
Active caspase-3 was quantitated per the manufacturer’s recommendations using antibodies to the cleaved fragment of caspase-3 (Cell Signaling catalog #9661; Danvers, MA, USA); this rabbit anti-mouse antibody does not recognize the procaspase-3 form. A secondary PE-labeled donkey anti-rabbit IgG antibody was used to detect the primary antibody (39) .

Detection of DNA strand breaks via TUNEL
Apoptosis was quantitated by the TUNEL method using a commercially available Apo-BrdU Kit from Phoenix Flow Systems, Inc. (San Diego, CA, USA) per the manufacturer’s instructions. A secondary antibiotin PE-labeled antibody was used for detecting BrdU-labeled strand breaks.

Detection of active caspase-8
Paraformaldehyde fixed cells were stained for active caspase-8 using a rabbit antiactive caspase-8 antibody generously provided by Dr. Don Nicholson (Merck Research Laboratories, Rahway, NJ, USA) as described (39) . The primary antibody was used at a concentration of 1:500. After washing, a PE-labeled donkey anti-rabbit IgG antibody (1:300 concentration) was used to detect the primary antibody.

Conventional bright-field microscopy of hematoxylin and eosin (H&E) -stained tissue sections
Tissue specimens of thymi and spleens were obtained at 20–22 h postsurgery and fixed overnight in 10% buffered formalin. Tissue sections were then processed and stained by H&E in the Gastrointestinal Core Laboratory facility at the Washington University School of Medicine. Specimens were examined via bright-field microscopy using a Nikon Eclipse E600 as a nonquantitative confirmatory method to flow cytometry. Apoptotic thymocytes and splenocytes exhibit characteristic findings of nuclear compaction (pyknosis) and fragmentation (karyorrhexis). These morphological features are readily apparent on bright-field light microscopy. A minimum of 6 sham and 10 CLP samples were examined for each strain of mice. A minimum of five to seven random fields were evaluated for each organ section (x200 and x400 magnification).

Determination of cytokine levels
Approximately 20–22 h postsurgery, mice were anesthetized with halothane and blood was obtained by cardiac puncture in heparinized syringes. Plasma was obtained by centrifugation and stored at –80oC. Levels of cytokines in plasma were quantitated using BD FACS Array and the Inflammation Kit per the manufacturer’s recommendations. Samples were run in duplicate. The lower limits of detection were IL-10 (17.5 pg/ml), IL-6 (5 pg/ml), MCP-1 (22.7 pg/ml), TNF-{alpha} (7.3 pg/ml), IL-12p70 (10.7 pg/ml), and IFN-{gamma} (2.5 pg/ml).

Statistical analysis
Data are reported as mean ± SEM. Data were analyzed with the statistical software program PRISM (GraphPad, San Diego, CA, USA). Data comparing two groups were analyzed using Student’s t test; plasma cytokine data were analyzed by nonparametric Mann-Whitney t test. One-way ANOVA with Tukey’s multiple comparison post-test was used to compare three or more groups. Survival studies were analyzed using {chi}2. Significance was accepted at P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Lymphocyte apoptosis is decreased in FADD-DN transgenic mice and is associated with reduced levels of active caspase-8
Compared with WT mice with sepsis, FADD-DN transgenic mice had a slight decrease (~20–25%) in sepsis-induced apoptosis in thymocytes as evaluated by active caspase 3 and TUNEL assays (Fig. 2 A, B). Splenic CD3+ T cells of FADD-DN transgenic mice had a greater decrease (~50%) in sepsis-induced apoptosis compared with WT mice as determined by active caspase-3 and TUNEL assays (Fig. 2C ). Surprisingly, B cells of FADD DN mice also had an ~40% decrease in sepsis-induced apoptosis compared with WT mice (Fig. 2C , Fig. 3 ), even though the FADD-DN protein is only expressed in the T cells of these mice (37 , 40) . To confirm that the death receptor pathway was inhibited in FADD-DN transgenic mice, staining for active caspase-8 was performed. During receptor-mediated cell death, caspase-8 binds to and is activated by FADD. Flow cytometric evaluation of active caspase-8 in lymphocytes from sham-operated and septic mice showed a marked increase in active caspase-8 in WT but not in FADD-DN transgenic mice (Fig. 4 ).


Figure 2
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  		Figure 2. Sepsis-induced lymphocyte apoptosis detected by active caspase-3 or TUNEL analysis is reduced in FADD-DN transgenic mice. Apoptosis was evaluated in WT and FADD-DN transgenic mice that had been sham-operated or undergone cecal ligation and puncture (CLP). CD3+ thymocytes, splenic T cells, and CD20+ splenic B cells were examined by active caspase-3 staining and TUNEL staining. Apoptosis was decreased in both T and B cells of FADD-DN transgenic mice with sepsis compared with WT mice with sepsis; *P < 0.05 CLP WT vs. CLP FADD-DN transgenic. n = 10 sham-operated and 15 septic WT mice; n = 12 sham-operated and 16 septic FADD-DN transgenic mice. Data represent mean ± SEM.


Figure 3
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  		Figure 3. Flow cytometric TUNEL analysis of splenocyte apoptosis in WT and FADD-DN transgenic mice that had been sham-operated or subjected to CLP. Approximately 20 h after sham operation or CLP, mice were killed and splenocytes were harvested for quantitation of apoptotic cells. This figure demonstrates apoptotic B cells, identified by staining with antibody to CD20, that are undergoing apoptosis as detected by the TUNEL technique. The upper right quadrant of each of the four images details the percentage of splenic B cells undergoing apoptosis. n = 1 mouse for each flow diagram.


Figure 4
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  		Figure 4. Sepsis-induced lymphocyte apoptosis in WT and FADD-DN transgenic mice detected by staining for active caspase-8. Apoptosis was evaluated in splenocytes of WT and FADD-DN transgenic mice that had been subjected to sham surgery or CLP. Apoptosis was quantitated by staining for active caspase-8. *P < 0.05. n = 4 sham and 8 CLP WT mice, and n = 4 sham and 7 septic FADD-DN transgenic mice. Data are mean ± SEM.

Microscopic examination of H&E-stained tissue sections substantiated the flow cytometry findings. In WT mice with sepsis, >50% of thymocytes in the cortex exhibited characteristic apoptotic features of nuclear pyknosis and karyorrhexis (Fig. 5 , top panel). Spleens of WT mice also demonstrated extensive apoptosis, with focal regions in white pulp (regions rich in B cells) and peri-arteriolar lymphoid sheaths (regions rich in T cells) exhibiting extensive cell death of >5–10 cells per high-powered field (x400) (Fig. 5 , middle panel). Thymic sections of FADD-DN transgenic mice with sepsis also revealed extensive apoptosis but there did appear to be a slight decrease in apoptosis compared with WT mice (data not shown). Splenic sections of FADD-DN transgenic mice had a more readily observable decrease in apoptosis compared with WT mice with sepsis. Protection was observed in both peri-arteriolar lymphoid sheath (a T cell-rich area) (Fig. 5 , middle panel) and in white pulp (a B cell-rich area; data not shown).


Figure 5
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  		Figure 5. Sepsis-induced lymphocyte apoptosis in WT, FADD-DN transgenic, and Bim–/– mice detected by morphological examination of tissue sections. WT or FADD-DN transgenic mice were sham-operated or subjected to CLP and tissues were harvested 20–22 h later for H&E staining. Demonstration of extensive apoptosis in thymocytes from a septic WT mouse compared with a sham-operated WT mouse. Thymocytes in the sham-operated mouse appear normal (top left panel) whereas virtually the entire field of thymocytes in the CLP-operated WT mouse demonstrates classical hallmarks of apoptosis including shrunken, compacted, and fragmented nuclei (top right panel). Peri-arteriolar lymphoid sheath (a T cell-rich area) demonstrated rare apoptotic lymphocytes in the FADD-DN transgenic mouse with sepsis (middle left panel), whereas numerous apoptotic splenocytes were seen in the WT mouse with sepsis (middle right panel). Circles identify apoptotic cells. Thymus from Bim–/– mouse that had CLP shows few apoptotic cells (bottom left panel) whereas the thymus from WT mouse with CLP shows massive apoptosis (bottom right panel). x400.

Lymphocyte apoptosis is slightly decreased in septic Bid–/– mice
Bid–/– mice demonstrated a slight but statistically significant decrease (~25–30%) in apoptosis of thymic CD3+ T cells as assessed by both active caspase-3 and TUNEL methods (Fig. 6 A, B). Similarly, data for splenic CD3+ T cells showed a slight but statistically significant decrease in apoptosis by both active caspase 3 and TUNEL assay (Fig. 6C ). The results for splenic B cells were inconsistent; there was a small decrease in B cell apoptosis by active caspase 3 but not by TUNEL assay (Fig. 6C ). Light microscopic examination of thymi and spleens from Bid–/– mice with sepsis were not immediately distinguishable from comparable specimens from WT mice. This failure to observe a readily detectable difference on light microscopy may be due to the fact that differences in the degree of apoptosis were not marked in the two groups of mice. Flow cytometry is a preferred method to detect small changes quantitatively.


Figure 6
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  		Figure 6. Sepsis-induced lymphocyte apoptosis detected by active caspase-3 or TUNEL in Bid–/– mice. Apoptosis was evaluated ~20 h after CLP or sham surgery by immunohistochemical staining for active caspase-3 and TUNEL. Bid–/– mice had a slight decrease in sepsis-induced apoptosis of CD3+ thymocytes and spleen cells compared with septic WT mice by staining for active caspase-3 and by TUNEL staining, P < 0.05. Splenic B cell apoptosis in sepsis in Bid–/– mice was equivocal with no decrease compared with septic WT mice detected by analysis of active caspase-3, but a slight decrease was detected by TUNEL, P < 0.05. n = 10 sham-operated or 11 septic WT mice; n = 7 sham-operated and n = 12 septic Bid–/– mice. Data expressed as mean ± SEM.

Lymphocyte apoptosis is almost totally prevented in septic Bim–/– mice
In contrast to the WT mice that had a dramatic increase in lymphocyte apoptosis in sepsis, Bim–/– mice had essentially complete protection in thymi and spleens as determined by active caspase-3 and TUNEL assays (Fig. 7 , 8 ). The protection was evident for both T and B cells. Microscopic examination of H&E-stained thymic and splenic tissue sections from WT septic mice showed extensive apoptosis as described before. In contrast, thymic and splenic sections from septic Bim–/– mice were almost indistinguishable from sham-operated mice and showed only a slight increase in apoptosis above baseline (Fig. 5 , lower panel).


Figure 7
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  		Figure 7. Sepsis-induced lymphocyte apoptosis in Bim–/– mice assessed by staining for active caspase-3 and TUNEL staining. WT or Bim–/– mice were subjected to sham surgery or CLP and were euthanized ~20–22 h postsurgery. Lymphoid cells were harvested and stained for active caspase-3 or analyzed by TUNEL staining. Note the decrease in sepsis-induced apoptosis in thymocytes and splenocytes from Bim–/– mice vs. WT mice (P<0.01). n = 6 sham-operated and 11 septic WT mice and n = 9 sham-operated and 15 septic Bim–/– mice. Data expressed as mean ± SEM.


Figure 8
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  		Figure 8. Flow cytometric analysis of thymocyte apoptosis in WT and Bim–/– mice that had been sham-operated or subjected to CLP. Apoptosis was determined in CD3+ thymocytes via staining for active caspase-3. Note the large increase in apoptotic CD3+ T cells in the WT mouse with sepsis (CLP) vs. the sham-operated WT mouse, 29.7% vs. 3.7%, respectively. In contrast, the Bim–/– mouse with sepsis had no increase in apoptosis compared with the sham-operated Bim–/– mouse. n = 1 mouse for each flow diagram.

Lymphocyte apoptosis is decreased in Puma–/– but not Noxa–/– mice
There was an ~50% decrease in apoptosis in CD3+ thymocytes from Puma–/– mice with sepsis vs. WT mice with sepsis as determined by both active caspase-3 and TUNEL assays (Fig. 9 A). Similarly, loss of Puma–/– conferred marked protection in CD3+ T and CD20+ B cells in spleens (Fig. 9B, C ). No protection against sepsis-induced apoptosis was observed in Noxa–/– mice (Fig. 10 A–C).


Figure 9
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  		Figure 9. Sepsis-induced apoptosis in Puma–/– mice assessed by staining for active caspase-3 or TUNEL staining. Apoptosis was evaluated by staining for active caspase-3 and TUNEL staining in WT and Puma–/– mice subjected to sham surgery or CLP. Puma–/– mice had decreased thymocyte and splenocyte apoptosis as assessed by staining for active caspase-3 and TUNEL analysis. Note that only 5 Puma–/– mice were available for analysis. Therefore, duplicate analyses were performed for these mice. Sepsis-induced apoptosis was decreased in cells from Puma–/– mice with sepsis vs. WT mice with sepsis, *P < 0.05. n = 1 sham-operated Puma–/– and n = 4 CLP Puma–/– mice. n = 2 sham-operated WT mice and 4 CLP WT mice. Data expressed as mean ± SEM.


Figure 10
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  		Figure 10. Sepsis-induced lymphocyte apoptosis in Noxa–/– mice as assessed by staining for active caspase-3 and TUNEL analysis. Apoptosis was evaluated by staining for active caspase-3 and TUNEL analysis in WT and Noxa–/– mice after sham surgery or CLP. There was no decrease in sepsis-induced apoptosis in thymocytes or splenocytes from Noxa–/– mice compared with WT mice with sepsis. Note that only 5 Noxa–/– mice were available. Therefore, duplicate analyses were performed for these mice. n = 1 sham-operated; n = 4 CLP Noxa–/– mice; n = 2 sham and 4 CLP WT mice. Data expressed as mean ± SEM.

Animal survival is improved in FADD-DN transgenic, Bid–/–, and Bim–/– mice with sepsis
Survival studies were conducted in FADD-DN transgenic, Bid–/–, and Bim–/– mice. Note that there were insufficient numbers of Puma–/– and Noxa–/– mice to conduct survival studies in these two groups. The survival in all three groups of genetically altered mice (i.e, FADD-DN transgenic, Bim–/–, and Bid–/– mice with sepsis) was markedly superior to that observed in WT mice with sepsis (Fig. 11 A–C).


Figure 11
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  		Figure 11. Survival of mutant mice subjected to CLP. CLP was performed in the various cohorts of genetically modified mice and WT mice and survival was recorded for 7 days. A) FADD-DN transgenic mice, B) Bim–/– mice, and C) Bid–/– mice had a marked improvement in survival compared with WT mice with sepsis. n = 8 WT and n = 9 FADD-DN transgenic mice in panel A; n = 15 WT and n = 16 Bim–/– mice in panel B, n = 16 WT and n = 16 Bid–/– mice in panel C. D) Adoptive transfer of splenocytes (7x107 cells/mouse) from Bim–/– or WT mice into Rag 1–/– mice was performed. Two days later, Rag 1–/– mice underwent CLP and survival was followed for 7 days. Note the improved survival in Rag 1–/– mice that received splenocytes from Bim–/– mice. n = 16 Rag 1–/– mice injected with spleen cells from Bim–/– mice and n = 17 Rag 1–/– mice injected with spleen cells from WT mice.

Adoptive transfer of cells from Bim–/– mice improves survival in Rag 1–/– mice
To determine whether the improved survival observed in the Bim–/– mice vs. WT mice was due to effects of Bim deletion on cells of the hematopoietic system vs. other cell types, (e.g., gastrointestinal epithelial cells, which also have an accelerated rate of apoptosis during sepsis), adoptive transfer of splenocytes (7x107 cells) from Bim–/– mice into Rag 1–/– mice was performed. The control group consisted of Rag 1–/– mice adoptively transferred with splenocytes from WT C57BL/6– mice. Mice receiving splenocytes from Bim–/– mice had a 56% survival whereas mice receiving splenocytes from WT mice had an 18% survival (P<0.02) (Fig. 11D ).

FADD DN, Bid–/–, and Bim–/– mice have decreased serum levels of pro- and anti-inflammatory cytokines
Plasma cytokines/chemokines were quantitated at 20–22 h postsurgery in sham-operated and CLP mice (Fig. 12 ). Sham-operated WT and transgenic/knockout mice had low to undetectable levels of the cytokines/chemokines examined. IFN-{gamma} and IL-12p70 were below the limit of detection in both sham-operated and CLP mice and are not depicted here. WT mice with sepsis had robust increases in proinflammatory TNF-{alpha}, IL-6, and MCP-1 as well as a marked increase in anti-inflammatory IL-10. In contrast, FADD-DN transgenic, Bid–/–, and Bim–/– mice had much less of an increase in these four cytokines/chemokines.


Figure 12
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  		Figure 12. Plasma cytokine concentrations. Mice were subjected to sham or CLP surgery and 20–22 h later were anesthetized with halothane. Note the marked decrease in TNF-{alpha}, IL-6, and IL-10 in FADD-DN transgenic, Bid–/–, and Bim–/– mice with sepsis compared with WT mice with sepsis. n = 5 sham-operated and n = 7 CLP FADD-DN transgenic mice vs. n = 5 sham-operated and n = 7 WT CLP. n = 5 sham-operated and n = 5 CLP Bid–/– mice vs. n = 5 sham-operated and n = 5 WT CLP mice. n = 6 sham-operated and n = 4 CLP Bim–/– mice; n = 9 sham-operated and n = 7 WT CLP mice. Samples were run in duplicate and cytokine levels are expressed in pg/ml. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Apoptosis of immune effector cells is a hallmark of sepsis in all age groups (3 , 10 , 11) . Animal studies have shown that prevention of lymphocyte apoptosis improves survival in sepsis. Moreover, clinical studies of patients with sepsis demonstrate that the degree of apoptosis of circulating lymphocytes correlates with sepsis severity (7 , 39) . A key question in this field has been the nature of the apoptogenic stimuli (8 , 41) . The present results showing that both the death receptor pathway and the mitochondrial-mediated pathway mediate lymphocyte apoptosis strongly suggest that no one trigger of cell death is responsible. Rather, it appears that numerous independent stimuli are driving lymphocyte apoptosis.

The findings from the FADD-DN transgenic mice indicate that activation of the death receptor pathway occurs in sepsis and is at least partially responsible for the extensive lymphocyte apoptosis in sepsis. A natural question arises: What is the death ligand that is activating the death receptors? Some potential death receptors and their ligands have been considered as candidates for this function. Previous studies have shown that inhibiting TNF-TNF receptor signaling did not decrease lymphocyte apoptosis in sepsis (42) . Additional work from our laboratory detected no decrease in sepsis-induced lymphocyte apoptosis in TNF p55/p75 receptor knockout mice, Trail knockout mice, or blockade by DR3 (unpublished observations). Although Fas does play a role in sepsis-induced apoptosis in hepatocytes and in selected gastrointestinal-associated lymphoid tissues (5 , 6) , Fas deficiency (in lpr mutant mice) did not reduce apoptosis in thymocytes or splenocytes in sepsis (43) . The failure of the ability to decrease lymphocyte apoptosis by knocking out any one death receptor pathways and the significant amount of inhibition seen in the FADD-DN transgenic mice lead us to speculate that there is a redundant role for death ligands and their receptors in this process. We speculate that knocking out one death receptor or its ligand does not significantly decrease sepsis-induced lymphocyte apoptosis, but inhibition of multiple death receptor or their ligands (as occurs by blocking FADD function) will block lymphocyte apoptosis in sepsis (Fig. 1) .

Although our data strongly suggest that apoptotic death proceeds by both the death receptor and mitochondrial pathways, the findings also indicate a preeminent role for the latter. The fact that Bim–/– mice had essentially complete protection against sepsis-induced thymocyte and splenocyte apoptosis is consistent with similar studies from Bcl-2 transgenic mice showing virtually complete protection as well (12 , 13) . Light microscopic examination of H&E-stained thymi and spleens from sham-operated and septic Bim–/– mice were almost indistinguishable. Given the essentially complete protection provided by blockade of mitochondrial-mediated apoptosis, why did blockade of the death receptor pathway also offer protection? These results suggest that cross-talk exists between the two prototypical apoptotic pathways, and data from the Bid–/– mice are consistent with this concept (Fig. 1) . This tBid-mediated cross-talk between the two pathways has been established for hepatocytes in which Bid–/– mice are protected against anti-Fas antibody-induced apoptosis (32) . There is less certainty about cross-talk between the death receptor pathway and the mitochondrial pathway in lymphocytes; so far, tBid has not been definitively shown to impact on the death of these cells (20 , 23 , 24) . Our data indicate that under certain pathological conditions, such as sepsis, cross-talk between the two pathways does play a significant role in lymphocytes. Our findings on Bid–/– mice agree with work from the laboratory of Ayala, who observed similar protection against sepsis-induced lymphocyte apoptosis (44) .

It is likely there are a number of important and interactive factors that are responsible for the improvement in animal survival in the FADD-DN transgenic, Bid–/–, and Bim–/– mouse strains. One potential reason for the beneficial effect is the decrease in lymphocyte apoptosis. Apoptosis results in loss of key immune effector cells, including lymphocytes and dendritic cells, and thereby reduction in their effector functions (e.g., cytolysis, opsonization) (4 , 8 , 45) . In addition, uptake of apoptotic cells can lead to anergy or a TH2 immune profile in surviving immune effector cells (46 47 48) . These two factors could be critical in compromising the ability of the host to eliminate the microorganisms.

Another potential mechanism for the improved survival of septic FADD-DN transgenic and bid or bim knockout mice is the effect of these mutations on circulating cytokines and chemokines. Survival outcome during sepsis is critically affected by a proper balance between the pro- and anti-inflammatory host responses (9) . An early unbridled proinflammatory cytokine response that is not moderated by the anti-inflammatory response can lead to excess tissue injury and early death. Alternatively, an excessive anti-inflammatory response can result in failure of the host to mount an effective antimicrobial response, thereby leading to uncontrolled infection and death. In this regard, the proinflammatory cytokines TNF-{alpha} and IL-6 were found to be markedly elevated in septic WT mice. Excessively increased levels of IL-6 have been shown to be an important predictor of death in sepsis in both mice and humans (49 , 50) . The concentration of circulating IL-6 was significantly reduced in the FADD-DN transgenic, Bid–/–, and Bim–/– mice compared with septic WT mice. Also potentially important was the decrease in levels of IL-10 in these animals. IL-10 is potently immunosuppressive and increased IL-10 has been correlated with worsened mortality in both animal models of sepsis and in human clinical studies (51) . One likely explanation for the decreased levels of IL-10 in the FADD-DN transgenic as well as the bid and bim knockout mice vs. WT mice is the increased apoptosis in the latter. Uptake of apoptotic cells induces a TH2 phenotype with increased IL-10 secretion by the scavenging phagocytic cells (46 47 48) .

Besides preventing sepsis-induced lymphocyte apoptosis, knockout of bim or bid may also decrease cell death in other organs during sepsis. For example, gastrointestinal epithelial cell apoptosis is a prominent feature of sepsis that may result in bacterial translocation across the gut and may affect morbidity and mortality (52) . The finding that adoptive transfer of splenocytes from Bim–/– mice into Rag 1–/– mice conferred a survival advantage during CLP compared with adoptive transfer of splenocytes from WT mice strongly supports the notion that it is the effect of bim deletion on the immune system that is central to the survival advantage in sepsis. Moreover, we did not detect a significant difference in sepsis-induced intestinal epithelial cell apoptosis between WT and Bim–/– mice (unpublished data).

Another novel finding in the present study is the effect of deletion of BH3-only proteins on sepsis-induced lymphocyte apoptosis. Previous work on other Bcl-2 family members from our laboratory has shown that overexpression of Bcl-2 or Bcl-xL provides nearly complete protection against sepsis-induced lymphocyte apoptosis (12 , 13) . In contrast, Bax–/– mice displayed normal levels of lymphocyte apoptosis in sepsis (12) , indicating that Bax and Bak have largely overlapping functions in this process. Bim, Puma, and Noxa are members of the BH3-only proapoptotic subgroup of the Bcl-2 family and are thought to act by binding to and inhibiting the antiapoptotic function of their prosurvival relatives (e.g., Bcl-2, Bcl-xL, Mcl-1) (Fig. 1) . The mechanisms whereby these individual proteins become activated are unique to each family member (53 , 54) . Although there is no clear explanation for the differential protective effect of loss of Bim, Puma, or Noxa on sepsis-induced apoptosis, the present results are analogous to work from Erlacher et al., who reported that loss of Puma and Bim but not loss of Noxa provided protection from {gamma}-radiation and glucocorticoid-induced lymphocyte apoptosis in vivo (30) . The two studies differ, however, in that loss of Puma provided greater protection against {gamma}-radiation-induced lymphocyte apoptotic death than did loss of Bim, whereas loss of Bim provided greater protection against sepsis-induced apoptosis than did loss of Puma (30) . It is known that {gamma}-radiation induces DNA damage with resultant activation of p53; thus, loss of Puma (p53 up-regulated modulator of apoptosis) would be expected to provide significant protection against this type of injury. Although Puma deficiency can provide protection against both p53-dependent as well as p53-independent death stimuli, previous work from our laboratory has shown that sepsis-induced apoptosis of thymocytes but not splenocytes is significantly but incompletely decreased in p53–/– mice (43) . Thus, it appears likely there are several mechanisms (both p53-dependent and p53-independent) that are inducing death of immune cells in sepsis. Also, the ability to block sepsis-induced lymphocyte apoptosis may be affected by the stage of cell maturation, cytokine milieu, and/or other coexisting stimuli.

One of the puzzling findings was the protection afforded to B cells in the septic FADD-DN transgenic mice. Our group has consistently noted that protection of splenic T cells against apoptosis (by overexpression of antiapoptotic proteins using an lck promoter) confers a limited protection to splenic B cells event though the B cells do not themselves express this antiapoptotic protein (12 , 56) . Similarly, we have observed protection of T cells when only B cells express an antiapoptotic protein (13) . This cross protection has been termed the bystander effect. A number of investigators have observed similar paracrine effects in apoptosis (57 , 58) . Rathwell and Goodnow speculated that imbalances in the cytokine milieu generated in microenvironments of cells may be essential in determining the life vs. death decision of the neighborhood (57) . Green has commented that the survival of a cell is dependent on the availability of factors produced by other cells, often belonging to distinct lineages (59) . Therefore, T cells may be making some factor that is essential for B cell survival and vice versa, thus explaining why transgenic expression of FADD-DN in T cells causes enhanced survival of B cells during sepsis.

Findings from the present study add further support to the concept that inhibition of apoptosis is a rational therapeutic approach to sepsis. Blockade of apoptosis by all three mutations resulted in improved survival. These findings closely mirror data showing that overexpression of the antiapoptotic proteins Bcl-2 or Bcl-xL decreases sepsis mortality (12 , 56) . Work from the laboratory of Ayala and associates has shown that knocking down expression of Fas or caspase-8 using siRNA results in decreased apoptosis and improved survival in sepsis (17) . Weaver and associates demonstrated that antiretroviral protease inhibitors decreased lymphocyte apoptosis and improved sepsis survival even if administered up to 4 h after onset of sepsis (60) .

Limitations
The purpose of this work was to define the molecular pathways of apoptosis. A second issue relates to the role of apoptosis in the morbidity and mortality of sepsis. The adoptive transfer studies in the Bim–/– mice do confirm that the immune cells are essential for improved survival. Nevertheless, it is important to reiterate that the decrease in sepsis-induced lymphocyte apoptosis observed in the mutant mice is likely only one factor among several that are responsible for the improved survival observed in these transgenic and knockout mice. The fact that the Bid–/– mice, which had only a modest decrease in sepsis-induced lymphocyte apoptosis, had a marked improvement in sepsis survival that was comparable to the improved survival in the Bim–/– mice, which had essentially complete protection against sepsis-induced lymphocyte apoptosis, suggests that other yet-to-be-identified factors are also playing a role. For example, Wei and associates reported that Bid–/– mice have decreased renal failure and delayed death after ischemia/reperfusion injury compared with WT mice (55) . It is probable that many physiological changes are present in the transgenic and knockout mice that are contributing to the improved survival in sepsis.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
In summary, numerous death stimuli are generated during sepsis and it is unlikely that blocking a single mediator or trigger will prevent the profound immune suppression that is a defining feature of this syndrome. There is activation of both the death receptor and mitochondrial death pathways, with the latter being preeminent. A small extent of tBid-mediated cross-talk between the two death pathways exists. Prevention of apoptosis in one cell type may confer a survival benefit to neighboring cells. Knockout of proapoptotic molecules may represent a novel therapeutic strategy in sepsis.


   ACKNOWLEDGMENTS
 
We thank the Washington University Digestive Diseases Research Morphology Core Laboratory for their expert help with hematoxylin- and eosin-stained tissue sections. We are grateful to Professors J. M. Adams and S. Cory (Walter and Eliza Hall Institute of Medical Research) and to Dr. K. Roth (University of Alabama, Birmingham) for gifts of transgenic and gene knockout mice. Dr. P. Bouillet provided many helpful comments on the manuscript. This work was supported by National Institutes of Health Grants GM44118, GM55194, by the Alan A. and Edith L. Wolff Foundation (to R.H.), and by the NHMRC Leukemia and Lymphoma Society of America and the JDRF/NHMRC (to A.S.)

Received for publication July 2, 2006. Accepted for publication August 22, 2006.


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