Irene E. Karl

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.

Caspase inhibitors improve survival in sepsis: a critical role of thelymphocyte

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 γ by 6 h. Thus, caspase inhibitors enhance immunity by preventing lymphocyte apoptosis and lymphocytes act rapidly, within 24 h, to control infection.

Apoptosis in lymphoid and parenchymal cells during sepsis : Findings in normal and T- and B-cell-deficient mice

OBJECTIVES To determine if apoptosis (programmed cell death) occurs systemically in lymphoid and parenchymal cells during sepsis. To examine the potential role of T and B cells in the apoptotic process using knockout mice deficient in mature T and B lymphocytes. DESIGN Prospective, randomized, controlled trial. SETTING Animal laboratory in a university medical setting. INTERVENTIONS Cecal ligation and puncture (CLP) (n = 34) or sham surgery (n = 13) was performed in female ND4 mice and, 15 to 22 hrs postoperatively, thymus, lung, heart, spleen, ileum, colon, liver, kidney, brain, and muscle were obtained and examined for apoptosis. A second group of mice (Rag-1) which are totally deficient in mature T and B cells also underwent CLP (n = 14) or sham surgery (n = 14) and had examination of tissues for apoptosis. MEASUREMENTS AND MAIN RESULTS Four methods with varying sensitivities and specificities were used to detect apoptosis, including: a) DNA agarose gel electrophoresis; b) terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL); c) electron microscopy; and d) light microscopy. In CLP mice, multiple methods demonstrated apoptosis in lymphocytes in thymus, spleen, ileum, colon, lung, and skeletal muscle. In addition to lymphocytes, parenchymal cells in ileum, colon, lung, and to a lesser extent, in skeletal muscle and kidney were apoptotic in CLP mice. There was no evidence of apoptosis by any method of detection in liver, brain, or heart. Results in Rag-1 mice which are deficient in T and B cells demonstrated extensive apoptosis in thymus, spleen, and ileum with less degrees of apoptosis in colon and lung. Both lymphoid cells and parenchymal cells were apoptotic. Rag-1 mice which underwent CLP did not die prematurely and there were no apparent observable differences in the physical response (tachypnea, piloerection, lethargy, etc), or intra-abdominal bowel inflammation/adhesions compared with CLP mice with normal T and B cells. CONCLUSIONS Apoptosis is an important mechanism of cell death in lymphocytes and parenchymal cells in sepsis and occurs systemically in many organs. Apoptosis may be an important cause of immunologic suppression in sepsis by inducing widespread lymphocyte depletion. Alternately, apoptosis may be beneficial to host survival by down-regulating the inflammatory response which accompanies sepsis. The degree to which parenchymal cell apoptosis is contributing to multiple organ failure cannot be determined from the present study. Findings in Rag-1 mice demonstrate that mature T and B cells and their secretory products are not necessary for apoptosis to occur during sepsis and that apoptotic cell death is not restricted to T or B cells. Apoptosis may be a key regulator of the balance between the pro- and anti-inflammatory process.

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.

Accelerated Lymphocyte Death in Sepsis Occurs by both the Death Receptor and Mitochondrial Pathways

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.

Cecal ligation and puncture (CLP) induces apoptosis in thymus, spleen, lung, and gut by an endotoxin and TNF-independent pathway.

Two challenges (intraperitoneal lipopolysaccharide (LPS) administration and cecal ligation and puncture (CLP)) and two strains of mice (LPS-normoresponder (C3H/HeN) and LPS-hyporesponder (C3H/HeJ)) were used to investigate pathways of cell injury. After intraperitoneal administration of LPS, endotoxin was absorbed into the bloodstream (HeN, 10.4 ± 9.4 × 104 EU/mL; HeJ, 14.7 ± 6.0 × 104 EU/mL), but as expected, only C3H/HeN mice produced serum tumor necrosis factor (TNF) (HeN, 2.5 ± 2.0 × 103 pg/mL; HeJ, 87.0 ± 38.7 pg/mL). Gel electrophoretic analysis of DNA extracted from six organs demonstrated the apoptotic “ladder” only in the thymus and only in the HeN mice. When the mice were challenged with CLP, both HeN and HeJ produced a small amount of serum TNF (HeN, 5.8 ± 3.5 × 102 pg/mL; HeJ, 2.2 ± 2.5 × 102 pg/mL) and both strains had very mild endotoxemia (HeN, 23.4 ± 3.8 EU/mL; HeJ, 27.9 ± 10.1 EU/mL). The DNA fragmentation pattern characteristic of apoptosis was observed not only in thymus but also in spleen, lung, and Peyers patch of gut of both strains. This organ-specific pattern was more pronounced in the thymus of HeN mice; otherwise, the organ-specific patterns were similar for HeN and HeJ mice challenged by CLP but absent in those same organs when those same mice were challenged with LPS. The data suggest the existence not only of an endotoxin-driven activation for thymic apoptosis, but also of an endotoxin-independent, TNF-independent pathway activating widespread apoptosis in the murine CLP model of sepsis.

Cell heterogeneity within the hepatic lobule: quantitative histochemistry.

This investigation was undertaken to seek quantitative information concerning the relative activity of certain enzymes in hepatic cells in various parts of the lobule. The enzymes selected for study are chiefly those with important nwtabohc roles. It was thought that such infornmtion might bring important new understandlilig of function in the liver lobule and its relationship to the localization of various types of injury. This approach has been made possible by the inicrochemical rnethodls of Lowry (4) which permit analysis of small groups of cells dissected out of frozen dried! sections of tissue. These method!s have been adlaptcd in our laboratory with the collaboration and assistance of Dr. Lowry and have been utilized in the study of liver of normal young male Sprague-Dawley rats weighing from 180 to 230 grams and! maintained! on a diet of Purina chow fed ad libitum. After eight hours of fast, the animals were sacrificed by decapitation. The liver was weighed and a small biopsy from the periphery of the left lateral lobe was droppedl immediately into liquid nitrogen. Sections were cut at 22 in a cryostat at a temperature of -25#{176}C. The tissue sections were then dehydrated! ifl vacuo at -40#{176}C. After dehydration sections were brought to room temperature in vacuo and!, in an air conditioned room, representative areas of the lobule were dissected out at a magnification of 72 x under the dissecting microscope. The areas selected included small Portal triadis surroundled by a rim

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.