Kevin W. Tinsley

Sepsis-Induced Apoptosis Causes Progressive Profound Depletion of B and CD4+ T Lymphocytes in Humans

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.

Depletion of Dendritic Cells, But Not Macrophages, in Patients with Sepsis

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.

Rapid onset of intestinal epithelial and lymphocyte apoptotic cell death in patients with trauma and shock.

ObjectiveApoptosis 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. DesignIntestinal 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. SettingAcademic medical center. PatientsPatients with trauma or elective bowel resections. Measurements and Main ResultsExtensive 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. ConclusionsFocal 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.

Adoptive transfer of apoptotic splenocytes worsens survival, whereas adoptive transfer of necrotic splenocytes improves survival in sepsis

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-γ 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 ≤ 0.05. The contrasting effects that apoptotic or necrotic cells exerted on survival were mirrored by opposite effects on splenocyte IFN-γ production with greatly decreased and increased production, respectively. Importantly, either administration of anti-IFN-γ antibodies or use of IFN-γ 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-γ

Sepsis Induces Apoptosis and Profound Depletion of Splenic Interdigitating and Follicular Dendritic Cells

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 ∼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.

Overexpression of Bcl-2 in the intestinal epithelium improves survival in septic mice.

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.

Akt Decreases Lymphocyte Apoptosis and Improves Survival 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-γ production, while increasing IL-4 production >2-fold. In contrast, T cells from Akt transgenic mice had an elevated production of IFN-γ 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.

Characterization of the Systemic Loss of Dendritic Cells in Murine Lymph Nodes During Polymicrobial Sepsis

Dendritic cells (DCs) play a key role in critical illness and are depleted in spleens from septic patients and mice. To date, few studies have characterized the systemic effect of sepsis on DC populations in lymphoid tissues. We analyzed the phenotype of DCs and Th cells present in the local (mesenteric) and distant (inguinal and popliteal) lymph nodes of mice with induced polymicrobial sepsis (cecal ligation and puncture). Flow cytometry and immunohistochemical staining demonstrated that there was a significant local (mesenteric nodes) and partial systemic (inguinal, but not popliteal nodes) loss of DCs from lymph nodes in septic mice, and that this process was associated with increased apoptosis. This sepsis-induced loss of DCs occurred after CD3+CD4+ T cell activation and loss in the lymph nodes, and the loss of DCs was not preceded by any sustained increase in their maturation status. In addition, there was no preferential loss of either mature/activated (MHCIIhigh/CD86high) or immature (MHCIIlow/CD86low) DCs during sepsis. However, there was a preferential loss of CD8+ DCs in the local and distant lymph nodes. The loss of DCs in lymphoid tissue, particularly CD8+ lymphoid-derived DCs, may contribute to the alterations in acquired immune status that frequently accompany sepsis.

Endothelial cell apoptosis in sepsis.

ObjectiveTo discuss a potential role for endothelial cell apoptosis in the pathogenesis of sepsis. Data SourcesStudies published in biomedical journals and studies from the authors’ laboratory. Study selectionIn vitro and in vivo studies of endothelial cell apoptosis in endotoxin and sepsis models. Data Extraction and SynthesisRelevant 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. ConclusionsApoptosis 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.

Induction of chondrocyte apoptosis following impact load

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.