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We hypothesized that spleen microarray gene expression profiles analyzed with contemporary pathway analysis software would provide molecular pathways of interest and target genes that might help explain the effect of bcl-2 on improving survival during sepsis. Two mouse models of sepsis, cecal ligation and puncture and tracheal instillation of Pseudomonas aeruginosa, were tested in both wild-type mice and mice that overexpress bcl-2. Whole spleens were obtained 6 h after septic injury. DNA microarray transcriptional profiles were obtained using the Affymetrix 430A GeneChip, containing 22,690 elements. Ingenuity Pathway Analysis software was used to construct hypothetical transcriptional networks that changed in response to sepsis and expression of the bcl-2 transgene. A conservative approach was used wherein only changes induced by both abdominal and pulmonary sepsis were studied. At 6 h, sepsis induced alterations in the abundance of hundreds of spleen genes, including a number of proinflammatory mediators (e.g., interleukin-6). These sepsis-induced alterations were blocked by expression of the bcl-2 transgene. Network analysis implicated a number of bcl-2-related apoptosis genes, including bcl2L11 (bim), bcl-2L2 (bcl-w), bmf, and mcl-1. Sepsis in bcl-2 transgenic animals resulted in alteration of RNA abundance for only a single gene, ceacam1. These findings are consistent with sepsis-induced alterations in the balance of pro- and anti-apoptotic transcriptional networks. In addition, our data suggest that the ability of bcl-2 overexpression to improve survival in sepsis in this model is related in part to prevention of sepsis-induced alterations in spleen transcriptional responses.

To determine whether and by which pathway (via the death receptor or mitochondrial mediated pathway) lymphocyte apoptosis occurs in pneumonia and to determine if increased bronchial epithelial cell apoptosis occurs in pneumonia. Prospective randomized study in a university research laboratory. Male C57BL/6 mice (n = 30). Animals received an intratracheal injection of Streptococcus pneumoniae or Pseudomonas aeruginosa to induce gram-positive or gram-negative pneumonia, respectively and were killed 24, 30, or 48 h later. Presence of pneumonia was confirmed via gross visual examination of lungs and by histology. Lymphocyte apoptosis in spleen and thymus was analyzed by flow cytometry for active caspases 3, 8, and 9 and by immunohistochemical (IHC) staining for active caspase 3 and DNA strand breaks. Respiratory epithelial cell apoptosis was assessed by IHC. Histologically, pneumonia was present in all bacteria-treated animals but none in sham-treated mice. Extensive lymphocyte apoptosis in spleen and thymus was documented by characteristic morphological changes on hematoxylin and eosin staining and by IHC staining in both S. pneumonia and P. aeruginosa infection. Flow cytometry confirmed IHC and showed apoptotic lymphocytes positive for active caspases 3, 8, and 9 in both thymi and spleens in both infections. In contrast to the extensive lymphocyte apoptosis, only rare scattered apoptotic changes were seen in respiratory epithelial or endothelial cells in pneumonia due to either organism. Increased lymphocyte but not bronchial cell apoptosis occurs in both gram-positive and gram-negative pneumonia and probably involves both the extrinsic and intrinsic pathway.

Apoptosis is a key pathogenic mechanism in sepsis that induces extensive death of lymphocytes and dendritic cells, thereby contributing to the immunosuppression that characterizes the septic disorder. Numerous animal studies indicate that prevention of apoptosis in sepsis improves survival and may represent a potential therapy for this highly lethal disorder. Recently, novel cell-penetrating peptide constructs such as HIV-1 TAT basic domain and related peptides have been developed to deliver bioactive cargoes and peptides into cells. In the present study, we investigated the effects of sepsis-induced apoptosis in Bcl-x(L) transgenic mice and in wild-type mice treated with an antiapoptotic TAT-Bcl-x(L) fusion protein and TAT-BH4 peptide. Lymphocytes from Bcl-x(L) transgenic mice were resistant to sepsis-induced apoptosis, and these mice had a approximately 3-fold improvement in survival. TAT-Bcl-x(L) and TAT-BH4 prevented Escherichia coli-induced human lymphocyte apoptosis ex vivo and markedly decreased lymphocyte apoptosis in an in vivo mouse model of sepsis. In conclusion, TAT-conjugated antiapoptotic Bcl-2-like peptides may offer a novel therapy to prevent apoptosis in sepsis and improve survival.

Sepsis is the leading cause of death in surgical intensive care units and is a major cause of morbidity and mortality in neonatal and medical intensive care units. The Centers for Disease Control and Prevention estimates that, in the United States alone, approximately 500,000 people develop sepsis and 175,000 people die each year. Sepsis is a growing problem; its incidence has tripled from 1972 to 1992. Recently, apoptosis has been identified as an important mechanism of cell death in animal models of sepsis and endotoxemia. During sepsis, there is extensive apoptotic death of lymphocytes and gastrointestinal epithelial cells. The extensive apoptotic death of lymphocytes is likely an important cause of the profound immunosuppression that is a hallmark of patients with sepsis. The apoptosis of gastrointestinal epithelial cells may compromise the integrity of the bowel wall, resulting in translocation of bacteria or endotoxins into the systemic circulation. The potential importance of apoptosis in the pathophysiology of sepsis is illustrated by results from animal models that demonstrate that blocking lymphocyte apoptosis improves survival in sepsis. A variety of strategies to inhibit apoptosis may ultimately provide an effective therapy for this highly lethal disorder.

Objectives: The aim of this study was to determine the effects of acute lung injury on the gut epithelium and examine mechanisms underlying changes in crypt proliferation and apoptosis. The relationship between severity and timing of lung injury to intestinal pathology was also examined., Design: Randomized, controlled study., Setting: University research laboratory., Subjects: Genetically inbred mice., Interventions: Following induction of acute lung injury, gut epithelial proliferation and apoptosis were assessed in a) C3H/HeN wild-type and C3H/HeJ mice, which lack functional Toll-like receptor 4 (n = 17); b) C57Bl/6 mice that received monoclonal anti-tumor necrosis factor-alpha or control antibody (n = 22); and c) C57Bl/6 wild-type and transgenic mice that overexpress Bcl-2 in their gut epithelium (n = 21). Intestinal epithelial proliferation and death were also examined in animals with differing degrees of lung inflammation (n = 24) as well as in a time course analysis following a fixed injury (n = 18)., Measurements and Main Results: Acute lung injury caused decreased proliferation and increased apoptosis in crypt epithelial cells in all animals studied. C3H/HeJ mice had higher levels of proliferation than C3H/HeN animals without additional changes in apoptosis. Anti-tumor necrosis factor-alpha antibody had no effect on gut epithelial proliferation or death. Overexpression of Bcl-2 did not change proliferation despite decreasing gut apoptosis. Proliferation and apoptosis were not correlated to severity of lung injury, as gut alterations were lost in mice with more severe acute lung injury. Changes in both gut epithelial proliferation and death were apparent within 12 hrs, but proliferation was decreased 36 hrs following acute lung injury while apoptosis returned to normal., Conclusions: Acute lung injury causes disparate effects on crypt proliferation and apoptosis, which occur, at least in part, through differing mechanisms involving Toll-like receptor 4 and Bcl-2. Severity of lung injury does not correlate with perturbations in proliferation or death in the gut epithelium, and acute lung injury-induced changes in intestinal epithelial proliferation persist longer than those in apoptosis.

Background: Sepsis, iron loading, and aging cause independent increases in gut epithelial and splenic apoptosis. It is unknown how their combination will affect apoptosis and systemic cytokine levels., Materials and Methods: Hfe-/- mice (a murine homologue of hemochromatosis) abnormally accumulate iron in their tissues. Aged (24-26 months) or mature (16-18 months) Hfe-/- mice and wild type (WT) littermates were subjected to cecal ligation and puncture (CLP) or sham laparotomy. Intestine, spleen, and blood were harvested 24 h later and assessed for apoptosis and cytokine levels., Results: Gut epithelial and splenic apoptosis were low in both aged septic and sham Hfe-/- mice, regardless of the amount of iron in their diet. Mature septic WT mice had increased apoptosis compared to age-matched sham WT mice. Mature septic Hfe-/- mice had similar levels of intestinal cell death to age-matched septic WT mice but higher levels of splenic apoptosis. Apoptosis was significantly lower in septic aged Hfe-/- mice than septic mature Hfe-/- animals. Interleukin-6 was elevated in septic aged Hfe-/- mice compared to sham mice., Conclusions: Although sepsis, chronic iron dysregulation, and aging each increase gut and splenic apoptosis, their combination yields cell death levels similar to sham animals despite the fact that aged Hfe-/- mice are able to mount an inflammatory response following CLP and mature Hfe-/- mice have elevated sepsis-induced apoptosis. Combining sepsis with two risk factors that ordinarily increase cell death and increase mortality in CLP yields an apoptotic response that could not have been predicted based upon each element in isolation.

Patients with sepsis are immune compromised, as evidenced by their failure to clear their primary infection and their propensity to develop secondary infections with pathogens that are often not particularly virulent in normal healthy individuals. A potential mechanism for immunosuppression in sepsis is lymphocyte apoptosis, which may occur by either a death receptor or a mitochondrial-mediated pathway. A prospective study of blood samples from 71 patients with sepsis, 55 nonseptic patients, and 6 healthy volunteers was undertaken to quantitate lymphocyte apoptosis and determine cell death pathways and mechanisms of apoptosis. Apoptosis was evaluated by flow cytometry and Western blotting. Lymphocyte apoptosis was increased in CD4 and CD8 T cells, B cells (CD20), and NK cells (CD56) in septic vs nonseptic patients. Samples taken sequentially from 10 patients with sepsis showed that the degree of CD3 T cell apoptosis correlated with the activity of his/her sepsis. In septic patients, apoptotic lymphocytes were positive for active caspases 8 and 9, consistent with death occurring by both mitochondrial-mediated and receptor-mediated pathways. In support of the concept that both death pathways were operative, lymphocyte apoptosis occurred in cells with markedly decreased Bcl-2 (an inhibitor of mitochondrial-mediated apoptosis) as well as cells with normal concentrations of Bcl-2. In conclusion, apoptosis occurs in a broad range of lymphocyte subsets in patients with sepsis and correlates with the activity of the disease. Lymphocyte loss occurs by both death receptor and mitochondrial-mediated apoptosis, suggesting that there may be multiple triggers for lymphocyte apoptosis.

Both aging and sepsis independently increase splenic and gut epithelial apoptosis. Sepsis-induced apoptosis in either cell type is also associated with increased mortality in young mice. We sought to determine whether age alters sepsis-induced splenic and gut epithelial cell death. Young (2 months) and aged (22 months) male ND4 mice were subjected to either single-puncture cecal ligation and puncture (CLP) with a 23-gauge needle or sham laparotomy. Apoptosis was assessed 24 hours later in the spleen and gut epithelium by active caspase 3 and hematoxylin and eosin staining. Aged septic mice had increased splenic apoptosis compared with either young septic animals or aged sham animals (15 vs. 7 vs. 5 apoptotic cells/high-powered field, P < 0.05). Similarly, aged septic animals had an elevation in gut epithelial cell death compared with either young septic or aged sham mice (33 vs. 16 vs. 6 apoptotic cells/100 contiguous crypts, P < 0.05). Elevated intestinal cell death was not associated with changes in either gut proliferation or cell division. To verify that the increase in splenic apoptosis seen in septic aged animals was not strain specific, double-puncture CLP with a 25-gauge needle or sham laparotomy was performed on young (4 months) or aged (24 months) C57BL/6 male mice. Similar to results seen in outbred animals, aged septic animals in this inbred strain had increased splenic apoptosis compared with either young septic animals or aged sham animals (23 vs. 7 vs. 4 apoptotic cells/ high powered field, P < 0.05). These results indicate that although infection and aging each independently cause an increase in splenic and gut epithelial apoptosis, their combination leads to a disproportionate increase in cell death in these rapidly dividing cell populations,and potentially plays a role in the marked increase in mortality seen with aging in sepsis.

Sepsis induces extensive death of lymphocytes that may contribute to the immunosuppression and mortality of the disorder. The serine/threonine kinase Akt is a key regulator of cell proliferation and death. The purpose of this study was to determine whether overexpression of Akt would prevent lymphocyte apoptosis and improve survival in sepsis. In addition, given the important role of Akt in cell signaling, T cell Th1 and Th2 cytokine production was determined. Mice that overexpress a constitutively active Akt in lymphocytes were made septic, and survival was recorded. Lymphocyte apoptosis and cytokine production were determined at 24 h after surgery. Mice with overexpression of Akt had a marked improvement in survival compared with wild-type littermates, i.e., 94 and 47% survival, respectively, p < 0.01. In wild-type littermates, sepsis caused a marked decrease in IFN-gamma production, while increasing IL-4 production >2-fold. In contrast, T cells from Akt transgenic mice had an elevated production of IFN-gamma at baseline that was maintained during sepsis, while IL-4 had little change. Akt overexpression also decreased sepsis-induced lymphocyte apoptosis via a non-Bcl-2 mechanism. In conclusion, Akt overexpression in lymphocytes prevents sepsis-induced apoptosis, causes a Th1 cytokine propensity, and improves survival. Findings from this study strengthen the concept that a major defect in sepsis is impairment of the adaptive immune system, and suggest that strategies to prevent lymphocyte apoptosis represent a potential important new therapy.

Objectives: Despite having dysregulated iron metabolism, critically ill patients may receive exogenous iron for the treatment of anemia. Iron is associated with increased tissue apoptosis and may facilitate bacterial growth. We hypothesized that exogenous iron administration given after the onset of sepsis would lead to increased mortality rate. To discriminate between elevated cell death and bacterial overgrowth as potential mediators of mortality, we examined gut epithelial and lymphocyte apoptosis and systemic bacterial counts in animals given iron supplementation after the onset of sepsis., Design: Prospective, randomized, controlled study., Setting: Animal laboratory in a university medical center., Subjects: Male C57BL/6 mice, 6-10 wks old., Interventions: C57BL/6 mice were subjected to cecal ligation and puncture (CLP), a well-accepted model of intra-abdominal sepsis, followed by daily subcutaneous injections of either 1 mL of iron dextran (5 mg/mL) or 0.9% NaCl for a total of five doses. Animals (n = 78) were followed for survival for 8 days. Separate cohorts (n = 76) were killed 24 or 48 hrs after cecal ligation and puncture or sham laparotomy and were assayed for gut epithelial and splenic apoptosis as well as for quantitative blood cultures., Measurements and Main Results: Eight-day survival was 7% in animals that received iron and 26% in mice that received 0.9% NaCl (p < .005). Iron supplementation after cecal ligation and puncture increased apoptosis by both active caspase 3 and hematoxylin and eosin staining in both the intestinal epithelium and spleen at 24 hrs (p < .05). Iron supplementation after sham laparotomy did not cause mortality or elevated apoptosis. Quantitative blood cultures revealed no detectable differences between septic animals that received iron and those that received 0.9% NaCl., Conclusions: High-dose iron supplementation with iron dextran after the onset of sepsis significantly increases mortality rate in this animal model. Iron-induced mortality may be mediated by an increase in gut epithelial and splenic apoptosis, whereas severity of bacteremia does not appear to play a causative role.

Genetically identical mice have a heterogeneous response to antibiotic therapy in sepsis, with only a subset deriving therapeutic benefit. We sought to determine whether the severity of a septic insult correlates with the survival benefit conferred by antibiotics. We also sought to determine whether antibiotics given 12 h after injury alter survival in animals predicted to die based upon high interleukin (IL)-6 levels drawn 6 h earlier. Adult male ND4 mice (n = 363) were subjected to double-puncture cecal ligation and puncture (CLP) with a 19-, 21-, or 23-gauge needle. Animals were randomized to receive imipenem or 0.9% NaCl every 12 h after CLP for 5 days. Ten-day survival was 16%, 26%, and 52%, respectively, for untreated animals. Antibiotics decreased the absolute risk of death 17% to 23% regardless of injury severity. In a separate cohort, mice (n = 37) were subjected to single or double-puncture CLP with a 21-gauge needle. IL-6 levels were assayed 6 h postoperatively and mice were followed for survival. Levels greater than 14,000 pg/mL were identified as predicting a 100% mortality (7/7 animals dead). A third set of mice (n = 94) then underwent double-puncture CLP with either 21-, 23-, or 25-gauge needle and had IL-6 levels measured in a similar fashion. Animals were randomized to receive imipenem or 0.9% NaCl beginning 12 h postoperatively (6 h after IL-6 levels were drawn) and continued for 5 days or until death. Although antibiotics decreased mortality overall, all animals with IL-6 levels greater than 14,000 pg/mL (n = 13) died, regardless of whether they received antibiotics or the gauge of needle used. These results indicate that antibiotics improve outcome in murine sepsis, regardless of injury severity. Furthermore, there is a threshold IL-6 level that can be identified 6 h after sepsis above which animals are destined to die, and antibiotic treatment does not alter their outcome.

Gut epithelial apoptosis is increased in human studies and animal models of noninfectious inflammation and sepsis. Elevated intestinal cell death appears to be physiologically significant in sepsis. Previous studies demonstrate that overexpression of the antiapoptotic protein Bcl-2 in the gut epithelium of transgenic mice is associated with improved survival from Pseudomonas aeruginosa pneumonia and cecal ligation and puncture. The functional significance of elevated gut apoptosis in noninfectious inflammation has not been examined. We hypothesized that intestinal apoptosis would be detrimental to survival in noninfectious critical illness. To address this issue, acute lung injury (ALI) was induced with intratracheal injection of lipopolysaccharide (LPS, 800 microg) in wild-type (WT) FVB/N mice and transgenic mice that overexpress Bcl-2 in their intestinal epithelium. Guts were harvested at 12, 24, 48, and 72 h and assessed for apoptosis by both hematoxylin and eosin and active caspase-3 staining in 100 contiguous crypts. ALI increased gut epithelial apoptosis 12 h after LPS instillation compared with shams (P < 0.01), whereas overexpression of Bcl-2 decreased intestinal apoptosis compared with WT animals with ALI when assayed by active caspase-3 (P < 0.05). Plasma levels of tumor necrosis factor alpha, interleukin (IL)-6, and IL-10 were similar between WT and transgenic animals with ALI, both of which had elevated IL-10 levels at 12 h and elevated IL-6 levels at 24 h compared with sham animals. In a separate experiment, transgenic and WT animals with ALI were followed for mortality to determine whether gut overexpression of Bcl-2 conferred a survival advantage. Survival at 10 days was 73% in WT animals (n = 33) and 65% in Bcl-2 animals (n = 23, P = ns). These results indicate that while gut epithelial apoptosis is elevated in multiple models of critical illness, prevention of intestinal cell death by overexpression of Bcl-2 is associated with a disparate survival effect between sepsis and noninfectious inflammation.

Objective: To investigate the presence and extent of chondrocyte apoptosis following impact load of articular cartilage in an in vivo model., Design: An in vivo animal model, using a pendulum device and New Zealand White rabbits, was designed to study the effects of impact load on the development of chondrocyte apoptosis. Animals were placed into either a High Impact group or a Low Impact group, and the right medial femoral condyle was impacted with a single impact load. A sham operation was performed on the left limb, and this cartilage served as the control., Setting: Academic medical center., Participants: New Zealand White rabbits (3 months)., Intervention: Impact load to the right medial femoral condyle., Main Outcome Measures: Three different methods were used to assess the presence and extent of chondrocyte apoptosis: 1) light microscopy (hematoxylin and eosin and terminal dUTP nick end labeling staining); 2) transmission electron microscopy; and 3) fluorescent microscopy with Hoechst 33342 staining. Secondary outcome measures included determination of the magnitude of impact force and time to peak force., Results: Light microscopy demonstrated chondrocytes with changes consistent with apoptosis including condensed nuclei, deep eosinophilic cytoplasmic staining, and vacuolization within the impacted specimens. Terminal dUTP nick end labeling staining-stained specimens had a high degree of positively stained cells (60%) in both injured and uninjured specimens. Transmission electron microscopy of the impacted specimens demonstrated numerous chondrocytes with changes characteristic of apoptosis, including nuclear and cellular fragmentation, volume shrinkage, and cytoplasmic vacuolization. Eleven percent of the cells in the High Impact group had changes consistent with apoptosis, versus 3% for the low impact group and <1% for the sham specimens. The High Impact group received a statistically significant greater stress than the Low Impact group. Impact group (P < 0.05), and the average time to peak force was 0.021 seconds for each impact group., Conclusions: The current data strongly indicate that in vivo chondrocyte apoptosis can be stimulated by the application of a single, rapid impact load and that the extent of chondrocyte apoptosis is related to the amount of load applied. The contribution chondrocyte apoptosis makes to the development of posttraumatic arthritis following joint injury or intra-articular fracture still remains to be determined.

Dendritic cells are a phenotypically diverse group of APC that have unique capabilities to regulate the activity and survival of B and T cells. Although proper function of dendritic cells is essential to host control of invading pathogens, few studies have examined the impact of sepsis on dendritic cells. The purpose of this study was to determine the effect of sepsis on splenic interdigitating dendritic cells (IDCs) and follicular dendritic cells (FDCs) using a clinically relevant animal model. Immunohistochemical staining for FDCs showed that sepsis induced an initial marked expansion in FDCs that peaked at 36 h after onset. The FDCs expanded to fill the entire lymphoid zone otherwise occupied by B cells. Between 36 and 48 h after sepsis, there was a profound caspase 3 mediated apoptosis induced depletion of FDCs such that only a small contingent of cells remained. In contrast to the initial increase in FDCs, IDC numbers were decreased to approximately 50% of control by 12 h after onset of sepsis. IDC death occurred by caspase 3-mediated apoptosis. Such profound apoptosis induced loss of FDCs and IDCs may significantly compromise B and T cell function and impair the ability of the host to survive sepsis.

Objectives: To evaluate whether the up-regulation in sepsis-induced gut epithelial apoptosis is balanced by an increase in intestinal proliferation and to assess mechanisms affecting the gut's regenerative response to overwhelming infection., Design: Prospective, randomized, controlled study., Setting: Animal laboratory in a university medical center., Interventions: Mice were subjected to intratracheal injection of Pseudomonas aeruginosa and killed between 1.5 and 24 hrs after induction of pneumonia-induced sepsis to assess for gut epithelial proliferation and cell division and for apoptosis. Animals were compared with sham-operation controls, septic transgenic mice that overexpress Bcl-2 throughout their small intestinal epithelium, and septic p53-/- mice., Measurements and Main Results: Proliferation and cell division were assessed by measuring S-phase and M-phase cells in intestinal crypts. The number of S-phase cells showed a progressive decline at all time points measured, with a 5-fold decrease in proliferation between control animals and septic mice 24 hrs after intratracheal injection of pathogenic bacteria (p <.0001). In contrast, cells in M-phase remained constant for the first 12 hrs after the onset of sepsis, but increased nearly 50% at 24 hrs after instillation of P. aeruginosa (p <.005). Both the decrease in S-phase cells and the increase in M-phase cells were partially suppressible in Bcl-2 overexpressors, but cellular proliferation and division were similar between septic p53-/- and p53+/+ mice. Crypt apoptosis was increased at all time points, with maximal death occurring between 12 and 24 hrs., Conclusions: Sepsis from P. aeruginosa pneumonia induces a p53-independent decrease in gut epithelial proliferation. Despite an increase in sepsis-induced intestinal apoptosis, there is no compensatory increase in intestinal epithelial proliferation, and there is evidence of a cell cycle block with an accumulation of cells in M-phase. Decreasing gut apoptosis by overexpression of Bcl-2 is associated with a partial reversal of the effect of sepsis on the cell cycle.

Lymphocyte rolling velocity is determined largely by interactions between leukocyte alpha(4)-integrin (CD49d) and L-selectin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in mesenteric postcapillary venules and Peyer's patch high endothelial venules (HEVs). The role of these interactions in other tissue sites of lymphocyte emigration is not known. With the use of real-time intravital confocal microscopy, we found that rolling velocities of T lymphocytes in the murine mesenteric lymph node (MLN) HEV also depend on L-selectin and CD49d. However, in the murine spleen, rolling velocities of T lymphocytes are not influenced by the loss of L-selectin and CD49d. With the use of FITC-dextran and TIE2-GFP mice, we further defined the microvascular compartments of the spleen and showed that adherence of T cells is localized to regions in the white pulp that are not lined by endothelial cells and have shear rates similar to bone marrow sinusoids. These results establish that T cell trafficking to the spleen differs from trafficking to other secondary lymphoid organs and suggest that the mechanical properties of the blood-filtering role of the spleen are important in T cell accumulation in the organ.

In sepsis, both necrotic and apoptotic cell death can occur. Apoptotic cells induce anergy that could impair the host response, whereas necrotic cells cause immune activation that might result in enhanced antimicrobial defenses. We determined whether adoptive transfer of apoptotic or necrotic cells impacted survival in a clinically relevant sepsis model. We also evaluated the effects of adoptive transfer of apoptotic or necrotic cells on the prototypical TH1 and TH2 cytokines IFN-gamma and IL-4, respectively. C57BL6/J mice had adoptive transfer of apoptotic (irradiated) or necrotic (freeze thaw) splenocytes. Controls received saline. Apoptotic cells greatly increased mortality, whereas necrotic splenocytes markedly improved survival, P < or = 0.05. The contrasting effects that apoptotic or necrotic cells exerted on survival were mirrored by opposite effects on splenocyte IFN-gamma production with greatly decreased and increased production, respectively. Importantly, either administration of anti-IFN-gamma antibodies or use of IFN-gamma knockout mice prevented the survival benefit occurring with necrotic cells. This study demonstrates that the type of cell death impacts survival in a clinically relevant model and identifies a mechanism for the immune suppression that is a hallmark of sepsis. Necrotic cells (and likely apoptotic cells) exert their effects via modulation of IFN-gamma

Differing antibiotic regimens can influence both survival and the inflammatory state in sepsis. We investigated whether the addition and/or type of antimicrobial agent could effect mortality in a murine model of Pseudomonas aeruginosa pneumonia-induced sepsis and if antibiotics altered systemic levels of cytokines. FVB/N mice were subjected to intratracheal injection of pathogenic bacteria and were given gentamicin, imipenem, or 0.9% NaCl 2 h after surgery, which continued every 12 h for a total of six doses. Survival at 7 days (n = 24 in each group) was 100% for mice given gentamicin, 88% for mice given imipenem, and 8% for sham mice treated with 0.9% NaCl (P < 0.0001). Systemic interleukin (IL) 6 levels were assayed 6 h postoperatively on all mice to see if they were predictive of outcome. Plasma IL-6 levels above 3,600 pg/mL were associated with a 100% mortality, levels under 1,200 pg/mL were associated with a 100% survival, and levels between 1,200 and 3,600 pg/mL had no utility in predicting mortality. In a separate experiment, mice were sacrificed at 3, 6, 12 or 24 h after instillation of P. aeruginosa and were assayed for levels of TNF-alpha, IL-6, IL-10, and IL-12. Significant alterations in the proinflammatory cytokines TNF-alpha and IL-6 were present at all time points except 3 h between mice treated with antibiotics and sham controls. In contrast, statistically significant differences in the anti-inflammatory cytokine IL-10 were present between the groups only at 6 h, and levels of IL-12 were similar at all time points. These results indicate that both gentamicin and imipenem increase survival at least 10-fold in a model of pneumonia-induced monomicrobial sepsis, and this is predominantly associated with a down-regulation of proinflammatory cytokines.

Sepsis is the leading cause of morbidity and mortality in critically ill patients in many intensive care units. The pathophysiology of organ failure and death in patients with sepsis remain elusive. This review focuses on recent advances in our understanding of the mechanisms of cell death in sepsis, the types of cells that are dying and the consequences on immunity. Extensive apoptotic death results in immune cell depletion and may compromise the ability of the patient to eradicate the primary infection and predispose to secondary nosocomial infections. Peripheral circulating lymphocyte apoptosis is also increased in patients with sepsis and correlates with the severity of the disease. In addition, recent evidence indicates that uptake of apoptotic cells impairs the immune function of surviving cells and contributes to immunosuppression. This new understanding of sepsis may lead to novel therapeutic approaches including pharmacological agents that block apoptosis.

Objective: DNA microarrays allow genome-wide assessment of changes in relative messenger RNA abundance and thus can be used to monitor changes in gene expression. The aim of this series of experiments was to gain experience in sepsis gene expression profiling in a well-accepted model of murine polymicrobial abdominal sepsis and begin characterizing (in the parlance of genomics) the sepsis "transcriptome.", Design: Prospective animal study., Setting: University-based animal research facility.SUBJECTS C57BL/6 mice., Interventions: After induction of general anesthesia, cecal ligation and puncture were performed to induce peritonitis and polymicrobial sepsis. The control group had sham laparotomy only. Three samples of spleen and liver were collected from septic and sham animals at 24 hrs after laparotomy. Changes in expression were measured for 588 annotated mouse genes by using a commercially available complementary DNA microarray kit., Measurements and Main Results: Broad-scale gene expression profiles were characterized for septic liver and spleen and compared with sham controls. The analytical tools used included commercially available software packages and a novel analysis program. Very little overlap was observed in the septic gene expression profiles of these two organs. Most of the genes identified have previously been linked to regulation of the inflammatory response; importantly, however, some have not. In addition, hierarchical cluster analysis showed that cecal ligation and puncture at 24 hrs induced coordinate expression of genes that alter cell signaling and survival pathways in spleen, consistent with previously published reports of sepsis-induced splenocyte apoptosis. The current limitations of microarray analysis as reflected in these studies are also discussed., Conclusions: Microarray technology provides a powerful new tool for rapidly analyzing tissue-specific changes in gene expression induced by sepsis in animal models. To our knowledge, these data constitute the first report on the use of microarrays to determine the sepsis transcriptome.

Objective: To discuss a potential role for endothelial cell apoptosis in the pathogenesis of sepsis., Data Sources: Studies published in biomedical journals and studies from the authors' laboratory., Study Selection: In vitro and in vivo studies of endothelial cell apoptosis in endotoxin and sepsis models., Data Extraction and Synthesis: Relevant studies that investigate the role of apoptosis in endotoxemia and sepsis are presented. The divergent results of the different studies and the potential reasons for the discrepant findings are presented. The importance of apoptosis in sepsis and the potential impact on endothelial cells and organ function are highlighted., Conclusions: Apoptosis is an important mechanism of lymphocyte and gastrointestinal epithelial cell death in sepsis. Although abundant in vitro studies indicate that endothelial cell apoptosis can occur in response to certain pathogenic organisms (e.g., Rickettsia rickettsii), data documenting endothelial cell apoptosis in in vivo models of sepsis are lacking. Because endothelial cells that undergo apoptosis detach from the vessel basement membrane, enter the circulation, and are rapidly cleared, it may be difficult to detect endothelial cell apoptosis in in vivo models of sepsis. The impact of endothelial cell apoptosis in sepsis may either be detrimental or beneficial to host survival, depending on the particular pathogen.

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.

Dendritic cells (DCs) are a group of APCs that have an extraordinary capacity to interact with T and B cells and modulate their responses to invading pathogens. Although a number of defects in the immune system have been identified in sepsis, few studies have examined the effect of sepsis on DCs, which is the purpose of this study. In addition, this study investigated the effect of sepsis on macrophages, which are reported to undergo apoptosis, and MHC II expression, which has been noted to be decreased in sepsis. Spleens from 26 septic patients and 20 trauma patients were evaluated by immunohistochemical staining. Although sepsis did not decrease the number of macrophages, sepsis did cause a dramatic reduction in the percentage area of spleen occupied by FDCs, i.e., 2.9 +/- 0.4 vs 0.7 +/- 0.2% in trauma and septic patients, respectively. The number of MHC II-expressing cells, including interdigitating DCs, was decreased in septic, compared with trauma, patients. However, sepsis did not appear to induce a loss of MHC II expression in those B cells, macrophages, or DCs that were still present. The dramatic loss of DCs in sepsis may significantly impair B and T cell function and contribute to the immune suppression that is a hallmark of the disorder.

Objectives: The aim of this study was to determine whether decreasing intestinal epithelial apoptosis in sepsis would alter mortality rates. The roles of the antiapoptotic protein Bcl-2 and the "executioner" protease caspase-3 in sepsis-induced gut cell death also were evaluated., Design: Prospective, randomized, controlled trial., Setting: Animal laboratory in an academic medical center., Interventions: Transgenic mice that overexpress Bcl-2 throughout the small intestinal epithelium (n = 23) and littermate controls (n = 27) were subjected to cecal ligation and puncture (CLP) and followed for 8 days to assess survival. A second group of transgenic (n = 15) and littermate animals (n = 15) were subjected to CLP and were killed between 16 and 48 hrs postoperatively to assess for intestinal apoptosis and active caspase-3 staining., Measurements and Main Results: Survival of transgenic animals was 83% 8 days after CLP compared with 44% for littermate controls (p < .005). Survival curves between the two groups of animals began diverging within 24 hrs. Overexpression of Bcl-2 was associated with a significant decrease in apoptosis between 16 and 24 hrs post-CLP (p < .05) as well as decreased staining for active caspase-3., Conclusions: Decreasing intestinal epithelial cell death via overexpression of Bcl-2 improves survival in septic mice. The gut may play a central role in the pathophysiology of sepsis.

Intravital microscopy has provided many insights into cellular interactions in various secondary lymphoid tissues. Because this technique allows for the visualization of cellular movement in real-time, it has been very powerful. However, until now, it has been difficult to apply this technique to the spleen. We report a technique that utilizes the Nikon RCM-8000 scanning laser, confocal microscope that allows for visualization of cellular movement in real-time in the rodent spleen. Using fluorescently labeled high molecular weight dextran or monoclonal antibodies, we are able to visualize fluorescently labeled cells rolling, tethering, and adhering in the spleen. In addition, we show that the majority of blood flow to the spleen remains within the white pulp nodules, as do most transferred erythrocytes at early time points. This is the first report of intravital microscopy of the spleen using a method that allows for easy identification of transferred cells.

Patients with sepsis have impaired host defenses that contribute to the lethality of the disorder. Recent work implicates lymphocyte apoptosis as a potential factor in the immunosuppression of sepsis. If lymphocyte apoptosis is an important mechanism, specific subsets of lymphocytes may be more vulnerable. A prospective study of lymphocyte cell typing and apoptosis was conducted in spleens from 27 patients with sepsis and 25 patients with trauma. Spleens from 16 critically ill nonseptic (3 prospective and 13 retrospective) patients were also evaluated. Immunohistochemical staining showed a caspase-9-mediated profound progressive loss of B and CD4 T helper cells in sepsis. Interestingly, sepsis did not decrease CD8 T or NK cells. Although there was no overall effect on lymphocytes from critically ill nonseptic patients (considered as a group), certain individual patients did exhibit significant loss of B and CD4 T cells. The loss of B and CD4 T cells in sepsis is especially significant because it occurs during life-threatening infection, a state in which massive lymphocyte clonal expansion should exist. Mitochondria-dependent lymphocyte apoptosis may contribute to the immunosuppression in sepsis by decreasing the number of immune effector cells. Similar loss of lymphocytes may be occurring in critically ill patients with other disorders.

The traditional approach to the study of biology employs small-scale experimentation that results in the description of a molecular sequence of known function or relevance. In the era of the genome the reverse is true, as large-scale cloning and gene sequencing come first, followed by the use of computational methods to systematically determine gene function and regulation. The overarching goal of this new approach is to translate the knowledge learned from a systematic, global analysis of genomic data into a complete understanding of biology. For investigators who study shock, the specific goal is to increase understanding of the adaptive response to injury at the level of the entire genome. This review describes our initial experience using DNA microarrays to profile stress-induced changes in gene expression. We conclude that efforts to apply genomics to the study of injury are best coordinated by multi-disciplinary groups, because of the extensive expertise required.

Sepsis induces lymphocyte apoptosis and prevention of lymphocyte death may improve the chances of surviving this disorder. We compared the efficacy of a selective caspase-3 inhibitor to a polycaspase inhibitor and to caspase-3-/- mice. Both inhibitors prevented lymphocyte apoptosis and improved survival. Caspase-3-/- mice shared a decreased, but not total, block of apoptosis. The polycaspase inhibitor caused a very substantial decrease in bacteremia. Caspase inhibitors did not benefit RAG-1-/- mice, which had a > tenfold increase in bacteremia compared to controls. Adoptive transfer of T cells that overexpressed the anti-apoptotic protein Bcl-2 increased survival. T cells stimulated with anti-CD3 and anti-CD28 produced increased interleukin 2 and interferon gamma by 6 h. Thus, caspase inhibitors enhance immunity by preventing lymphocyte apoptosis and lymphocytes act rapidly, within 24 h, to control infection.

To determine whether iron-laden tissue subsequently stimulated to produce the stress ("heat shock") response-sustained injury, hindlimbs of male ND4 mice were injected with iron salts, hemin, or hemoglobin. The stress response was induced with sodium arsenite or with heat. Ulcers appeared at the injection site. Tissues were analyzed by three distinct techniques-electron microscopy, TUNEL stain, and agarose gel electrophoresis of low molecular weight DNA-which collectively suggest that the tissue injury is, at least in part, the consequence of accelerated apoptosis. The data suggest that the toxicity of free iron is amplified by induction of the stress (heat shock) response to signal a programmed response. This model and mechanism may have implications in pathological processes ranging from the cutaneous wounds of venous stasis disease to the tissue failure of multiple organ dysfunction.

Objective: Apoptosis is a cellular suicide program that can be activated by cell injury or stress. Although a number of laboratory studies have shown that ischemia/reperfusion injury can induce apoptosis, few clinical studies have been performed. The purpose of this study was to determine whether apoptosis is a major mechanism of cell death in intestinal epithelial cells and lymphocytes in patients who sustained trauma, shock, and ischemia/ reperfusion injury., Design: Intestinal tissues were obtained intraoperatively from 10 patients with acute traumatic injuries as a result of motor vehicle collisions or gun shot wounds. A control population consisted of six patients who underwent elective bowel resections. Apoptosis was evaluated by conventional light microscopy, laser scanning confocal microscopy using the nuclear staining dye Hoechst 33342, immunohistochemical staining for active caspase-3, and immunohistochemical staining for cytokeratin 18., Setting: Academic medical center., Patients: Patients with trauma or elective bowel resections., Measurements and Main Results: Extensive focal crypt epithelial and lymphocyte apoptosis were demonstrated by multiple methods of examination in the majority of trauma patients. Trauma patients having the highest injury severity score tended to have the most severe apoptosis. Repeat intestinal samples obtained from two of the trauma patients who had a high degree of apoptosis on initial evaluation were negative for apoptosis at the time of the second operation. Tissue lymphocyte apoptosis was associated with a markedly decreased circulating lymphocyte count in 9 of 10 trauma patients., Conclusions: Focal apoptosis of intestinal epithelial and lymphoid tissues occurs extremely rapidly after injury. Apoptotic loss of intestinal epithelial cells may compromise bowel wall integrity and be a mechanism for bacterial or endotoxin translocation into the systemic circulation. Apoptosis of lymphocytes may impair immunologic defenses and predispose to infection.

Injury will equal or surpass communicable disease in the year 2020 as the number one cause of lost disability-adjusted life-years worldwide. The major cause of "late death" after trauma is organ dysfunction, commonly as a complication of shock or sepsis. The pathophysiology of injury-induced organ dysfunction is poorly characterized but has been linked to systemic inflammation as a result of infection (either obvious or occult) or massive tissue injury (systemic inflammatory response syndrome, SIRS). Subsequent complications of organ dysfunction, including death, may also stem from immunosuppression characteristic of what has been called the counter-regulatory anti-inflammatory response syndrome (CARS). At the cellular level, injurious stimuli trigger adaptive stress responses that include changes in gene expression. Multiple organ dysfunction syndrome (MODS) is the summation of these stress responses to severe systemic injury, integrated at the cellular, organ, and host levels. We hypothesize that a complete understanding at the molecular level of the stress responses induced by injury will aid in the development of therapeutic strategies for treating MODS in the critically ill surgical patient. This paper reviews recent data from our Cellular Injury and Adaptation Laboratory relevant to our understanding of MODS pathophysiology, particularly as it relates to stress-induced cell death by apoptosis. Our data suggest that inhibition of stress-induced apoptosis may improve survival after severe injury.

Objective: The lymphocyte is a principal mediator of the inflammatory response, and lymphocyte depletion via apoptosis may be an important mechanism of modulating inflammation. Increased oxygen consumption occurs during sepsis and results in the generation of reactive oxygen species. Although reactive oxygen species initiate apoptosis in many biological systems, their role in controlling lymphocyte apoptosis during sepsis is unclear. The objective of this study was to better characterize the role of oxidative stress in precipitating lymphocyte apoptosis during sepsis and to specifically define the role of the CuZn superoxide dismutase (SOD) enzyme complex, a major antioxidant defense, in modulating this process., Design: Prospective, randomized, controlled study., Setting: Research laboratory at an academic medical center., Subjects: Mice that were either genetically normal or that were deficient in or overexpressed the enzyme CuZn SOD., Interventions: Mice from each genetic group were randomized to no manipulation (control), sham surgery, or cecal ligation and puncture. Mice were killed 18-24 hrs after study entry, and the thymi and spleen were removed for analysis of apoptosis., Measurements and Main Results: Lymphocyte apoptosis was assessed by three independent methods: light microscopy, fluorescent terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling, and DNA gel electrophoresis. Comparisons were performed using standard parametric statistical tests. Lymphocyte apoptosis was present in mice after CLP but not in control mice or in mice after sham surgery (p < .05). Mice completely lacking CuZn SOD developed significantly more lymphocyte apoptosis than did either partially CuZn SOD-deficient or genetically normal mice (p < .05). This apoptosis was more pronounced in the thymus than the spleen and, within the thymus, more prominent in the cortex than medulla (p < .05 for all). In contrast, mice that overexpressed CuZn SOD did not differ in the amount of apoptosis after CLP compared with genetically normal mice (p = NS for all)., Conclusions: Oxidative stress occurs in sepsis and appears to be one stimulus for the development of lymphocyte apoptosis, a process that is partly regulated by CuZn SOD. However, we were unable to demonstrate that overexpression of this enzyme suppressed lymphocyte apoptosis, suggesting that either other antioxidant defenses or other pathways independent of oxidative stress may mediate lymphocyte elimination in this syndrome.

Sepsis induces extensive apoptosis of lymphocytes, which may be responsible for the profound immune suppression of the disorder. Two potential pathways of sepsis-induced lymphocyte apoptosis, Fas and p53, were investigated. Lymphocyte apoptosis was evaluated 20-22 h after sepsis by annexin V or DNA nick-end labeling. Fas receptor-deficient mice had no protection against sepsis-induced apoptosis in thymocytes or splenocytes. p53 knockout mice (p53-/-) had complete protection against thymocyte apoptosis but, surprisingly, had no protection in splenocytes. p53-/- mice had no improvement in sepsis survival compared with appropriately matched control mice with sepsis. We conclude that both p53-dependent and p53-independent pathways of cell death exist in sepsis. This differential apoptotic response of thymocytes vs splenocytes in p53-/- mice suggests that either the cellular response or the death-inducing signal is cell-type specific in sepsis. The fact that p53-/- lymphocytes of an identical subtype (CD8-CD4+) were protected in thymi but not in spleens indicates that cell susceptibility to apoptosis differs depending upon other unidentified factors.

Sepsis induces extensive lymphocyte cell death that may contribute to immune depression and morbidity/mortality in the disorder. bcl-2 is a member of a new class of oncogenes that prevents cell death from an array of noxious stimuli. Transgenic mice that overexpress BCL-2 in T lymphocytes are resistant to sepsis-induced T cell apoptosis, and mortality was decreased in sepsis. The purpose of this study was to identify key initiator and executioner "caspases" involved in sepsis-induced lymphocyte apoptosis and to determine if BCL-2 acts prior to caspase activation. Thymi were removed 5-22 h post-cecal ligation and puncture (CLP) or sham surgery. Apoptosis was evaluated in thymocytes by annexin-V FITC labeling and flow cytometry. Caspase-1 activity was determined by western blot analysis of the procaspase protein and p20 subunit of the activated caspase; activities of caspases -2, -6, and -9 were determined by colorimetric assays using specific substrates conjugated to a color reporter molecule. Caspase-3 activity was determined both by western blot and by a fluorogenic assay in which a fluorescent compound was generated. Thymocytes from CLP mice had markedly increased apoptosis and activation of caspases -2, -3, -6, and -9 in comparison with thymocytes of sham-operated mice. Caspase-1 was not activated. BCL-2 prevented sepsis-induced thymocyte apoptosis and inhibited activation of all caspases. We conclude that sepsis causes activation of multiple caspases and that BCL-2 acts upstream as an inhibitor of caspase activation. The pattern of caspase activation suggests a mitochondrial mediated pathway.

Sepsis induces extensive lymphocyte apoptosis, a process which may be beneficial to host survival by down-regulating the inflammatory response or, alternatively, harmful by impairing host defenses. To determine the beneficial vs. adverse effects of lymphocyte apoptosis in sepsis, we blocked lymphocyte apoptosis either by N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl) fluoromethyl ketone (z-VAD), a broad-spectrum caspase inhibitor, or by use of Bcl-2 Ig transgenic mice that selectively overexpress the antiapoptotic protein Bcl-2 in a lymphoid pattern. Both z-VAD and Bcl-2 prevented lymphocyte apoptosis and resulted in a marked improvement in survival. z-VAD did not decrease lymphocyte tumor necrosis factor-alpha production. Considered together, these two studies employing different methods of blocking lymphocyte apoptosis provide compelling evidence that immunodepression resulting from the loss of lymphocytes is a central pathogenic event in sepsis, and they challenge the current paradigm that regards sepsis as a disorder resulting from an uncontrolled inflammatory response. Caspase inhibitors may represent a treatment strategy in this highly lethal disorder.

Background: Nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS or NOS2) has been implicated in the hypotension, organ failure, and death that complicate sepsis. To avoid the confounding effects and limitations of iNOS inhibitors, we used iNOS gene "knockout" mice to examine the effect of inducible NO production in a model of polymicrobial abdominal sepsis treated with antibiotics. We hypothesized that iNOS gene deficiency would significantly alter outcome., Methods: C57BL6 wild-type (control) and congenic iNOS knockout mice were studied concurrently. Under halothane anesthesia, the ceca were ligated with 4-0 silk suture and punctured twice with a 26-gauge needle (cecal ligation and puncture, CLP). Survival was followed for 7 days, after which necropsies were performed in surviving animals. In an accompanying study examining the acute effects of sepsis, organ injury at 18 hours after CLP as determined by histology and the degree of cell death by apoptosis were examined with the use of hematoxylin and eosin (H&E) and TUNEL staining and two-channel fluorescence-activated cell sorter (FACS) analysis., Results: Sham laparotomy produced no lethality in either knockout (n = 3) or wild-type (n = 3) animals. Compared with survival in controls (n = 20), survival after CLP in iNOS knockout mice (n = 21) was significantly decreased (P < .01 at 2 days, P = .080 at 7 days, Mantel-Haenszel log-rank test). CLP-induced apoptotic cell death was significantly less in the thymus of iNOS knockout mice compared with wild-type mice., Conclusions: We conclude that iNOS gene function provides a survival benefit in septic mice and is associated with increased sepsis-induced thymocyte apoptosis. To our knowledge, this is the first survival study examining the effect of iNOS gene deficiency in a clinically relevant model of sepsis.

Objectives: The purpose of this study was to determine whether apoptosis is a major mechanism of cell death in patients with sepsis. The activities of caspase-3 and the antiapoptotic protein, BCL-2, were investigated also., Design: A prospective study of 20 patients who died of sepsis and multiple organ dysfunction was performed. The control group of 16 patients consisted of critically ill, nonseptic patients who were evaluated either prospectively (7) or retrospectively (9). In addition, normal colon sections from seven patients who had bowel resections were included. Apoptosis was evaluated in hematoxylin and eosin-stained specimens by deoxyuridine triphosphate nick end-labeling (TUNEL) and by DNA gel electrophoresis., Setting: Two academic medical centers., Patients: Critically ill patients., Measurements and Main Results: In septic patients, apoptosis was detected in diverse organs by all three methods with a predominance in lymphocytes and intestinal epithelial cells. Hematoxylin and eosin-stained specimens from septic patients demonstrated at least focal apoptosis in 56.3% of spleens, 47.1% of colons, and 27.7% of ileums. Indirect evidence of lymphocyte apoptosis in septic patients included extensive depletion of lymphocytes in white pulp and a marked lymphocytopenia in 15 of 19 patients. Hematoxylin and eosin from nonseptic patients' tissues revealed a low level of apoptosis in one patient only. The TUNEL method increased in positivity with a delay in tissue fixation and was highly positive in many tissues from both septic and nonseptic patients. Immunohistochemical staining for active caspase-3 showed a marked increase in septic vs. nonseptic patients (p < .01), with >25% to 50% of cells being positive focally in the splenic white pulp of six septic but in no nonseptic patients., Conclusions: We conclude that caspase-3-mediated apoptosis causes extensive lymphocyte apoptosis in sepsis and may contribute to the impaired immune response that characterizes the disorder.

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 marked increases in TNF-alpha, IL-1beta, and IL-10 in thymic tissue in sepsis in the three species of mice, and the increase in TNF-alpha 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 against cell death in sepsis. Lymphocyte death may be detrimental in sepsis by compromising host defense.

Ca2+ is a critical intracellular second messenger, but few studies have examined Ca2+ signaling in whole organs. The amplitude and frequency of Ca2+ oscillations encode important cellular information. Using laser scanning confocal microscopy in the indo 1 acetoxymethyl ester dye-loaded rat liver, we investigated the effect of various Ca2+ agonists that act at distinct mechanistic sites on Ca2+ signaling. Perfusion with suprathreshold doses of arginine vasopressin (AVP) (2-20 nM) caused a single Ca2+ wave that originated in the pericentral vein region and spread centrifugally to the periportal area. Lower doses of AVP (0.2-2 nM) caused multiple Ca2+ waves and Ca2+ oscillations. Perfusion with ATP (1. 4-17.5 microM) caused rapid transient elevations in intracellular free Ca2+ concentration ([Ca2+]i) occurring in isolated hepatocytes or groups of hepatocytes throughout the lobule and were of shorter duration than those due to AVP. Also in contrast to AVP, there was no specific anatomic location within the hepatic lobule that was more susceptible to ATP. Thapsigargin and cyclopiazonic acid did not cause a Ca2+ wave but rather produced a uniform and fairly simultaneous increase in [Ca2+]i in all hepatocytes in the lobule. Perfusion with 14 microM ryanodine produced a single transient spike in [Ca2+]i in a small number (<2%) of hepatocytes. Dantrolene, an inhibitor of Ca2+ release, reduced the increased [Ca2+]i occurring after AVP. Insight into the mechanism of action of these Ca2+-active compounds on Ca2+ signaling in the intact liver is provided.

Sepsis is a major cause of death in intensive care units. Clinically, sepsis induces a number of physiologic and metabolic abnormalities, including decreased myocardial contractility and decreased plasma ionized calcium. There is debate about the proper therapy of hypocalcemia in sepsis because calcium administration may worsen cell function by causing intracellular Ca2+ overload. We investigated the effect of Ca2+ administration on myocardial systolic and diastolic function in an extensively utilized rat model of sepsis, i.e., the cecal ligation and puncture model (CLP). Approximately 24 h after CLP or sham surgery, rats were anesthetized and myocardial function assessed in vivo by a left ventricular Millar catheter and simultaneous two-dimensional guided M-mode echocardiography. Septic rats had a 28% decrease in peak left ventricular developed pressure, a 30% decrease in +dP/ dt, and a 23% decrease in -dP/dt (p < 0.05). Plasma ionized Ca2+ was decreased in septic compared with that in sham rats: 4.9 +/- 0.9 and 5.6 +/- 0.01 mg/dl, respectively (p < 0.05). CaCl2 improved both systolic and diastolic function and there was no evidence of adverse effects of Ca2+ even at supraphysiologic levels. Surprisingly, correction of decreased afterload in septic rats, using the pure alpha-agonist phenylephrine, caused normalization of all indices of cardiac contractility, indicating that the presumed decrease in cardiac function was due entirely to an effect of the decreased afterload to "unload" the left ventricle. We conclude that Ca2+ administration is not detrimental to cardiac function in the rat CLP model. Although the rat CLP model is widely utilized and reproduces many of the clinical hallmarks of sepsis, it does not cause intrinsic myocardial depression and, therefore, it may not be an appropriate model to investigate the clinical cardiac dysfunction that occurs in patients with sepsis.

Objectives: To determine a mechanism by which nitric oxide alters induction of stress-induced endothelial cell apoptosis in vitro. Apoptosis is a form of cellular suicide that has been implicated in the pathogenesis of multiple organ dysfunction syndrome., Design: Prospective, controlled trial., Setting: Research laboratory of a large, academic medical center., Subjects: Cultured primary porcine aortic endothelial cells., Interventions: Cells were treated with a range of doses of agents that either spontaneously generate nitric oxide (S-nitroso-N-acetyl-D,L-penicillamine [SNAP] or (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate [DETA-NO]) or block nitric oxide production (Nomega-methyl-L-arginine [L-NMA]). The ability of these agents to alter the rate of cell death by apoptosis (induced by the sequence stimuli lipopolysaccharide [LPS] followed by sodium arsenite) was measured. Mechanistic studies included examining the ability of: a) nitric oxide "donors" to alter nuclear factor kappa B (NF-kappaB) DNA binding activity and the level of IkappaBalpha accumulation; and b) a stable cyclic guanosine monophosphate (cGMP) analog (8-bromo-cGMP) to mimic the effect of nitric oxide donors., Measurements and Main Results: The sequence LPS/sodium arsenite increased the rate of endothelial cell apoptosis (47.4%, p< .05 vs. control), as measured by fluorescent-activated cell scanning using annexin V/propidium iodide staining. DETA-NO generated nitric oxide (as indicated by an increase in the concentration of the stable end-products of nitric oxide metabolism) and decreased the rate of endothelial cell apoptosis (20.6% at a dose of 2 mM, p=.0001 vs. control). DETA-NO also decreased NF-kappaB DNA binding activity and the apparent accumulation of its endogenous inhibitor, IkappaBalpha. The 8-bromo-cGMP did not mimic the effects of nitric oxide donors (DETA-NO) on apoptosis., Conclusions: These data suggest that exogenous nitric oxide can block stress-induced endothelial cell apoptosis in vitro. The mechanistic studies are consistent with our hypothesis that inhibitors of NF-kappaB DNA binding activity are associated with protection against apoptosis-inducing stimuli. The results do not support a role for cGMP in mediating the protective effect of DETA-NO in our model.

Background: Apoptosis, a mechanism of cell death prominent in critical illnesses including disseminated inflammation and multiorgan dysfunction syndrome, is characterized by morphologic changes including cell shrinkage, condensation of organelles, blebbing, and chromatin fragmentation. These phenomena suggest substantial changes in cytoskeletal structure. We hypothesized that stress-induced apoptosis in endothelial cells is, in part, a consequence of a critical cytoskeletal rearrangement., Methods: Porcine aortic endothelial cells in culture, surrogates for the microvasculature in vivo, were exposed sequentially to Escherichia coli endotoxin (25 micrograms/mL; 18 hours) to induce the inflammatory response and then to sodium arsenite (160 mumol/L; 120 minutes) to induce the heat-shock response, a well-characterized model of stress-induced apoptosis. Laser confocal micrographs of fluorescein isothio-cyanate-labeled phalloidin-stained cells were analyzed to calculate the border fractal dimension of the cytoskeleton. Other cells were exposed to cytochalasin D, a fungal metabolite, which interferes with polymerization of actin from its globular to its filamentous form, and similarly were analyzed with respect to fractal dimension, viability (neutral red assay), and manner of death (annexin V fluorescence-activated cell scanning analysis)., Results: Induction of the inflammatory or heat-shock responses caused subtle and distinct rearrangement of the actin cytoskeleton. When these stimuli were applied in sequence, a synergistic interaction led to profound cytoskeletal collapse. Reversal of the sequence did not induce the cytoskeletal disruption. Cytochalasin D alone induced a dose-dependent cytoskeletal collapse indistinguishable from that caused by the acute phase-heat shock sequence that caused cell death by apoptosis. The effect of lower doses of Cytochalasin D could be potentiated by subsequent induction of the heat-shock response., Conclusions: Sequential stresses that mimic pathophysiologic "two-hit" stimuli induce a characteristic fractal rearrangement of the actin cytoskeleton. Because cytochalasin D-induced rearrangement of this cytoskeleton produced apoptosis indistinguishable from the stress-induced apoptosis, we conclude that the cytoskeletal rearrangement is likely a critical event in the pathway to apoptosis. This disruption of intracellular interconnections mirrors endotoxin-induced disruption in signals among organs and supports the mechanistic hypothesis that multiorgan dysfunction syndrome generally reflects disruption of signals and connections at several levels of biologic organization.