Confocal fluorescent intravital microscopy of the murine spleen.

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.

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.

Injury in the era of genomics.

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.

Injection of iron compounds followed by induction of the stress response causes tissue injury and apoptosis.

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.

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

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.

Role of CuZn superoxide dismutase in regulating lymphocyte apoptosis during sepsis.

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.

p53-dependent and -independent pathways of apoptotic cell death in sepsis.

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.

Caspases -2, -3, -6, and -9, but not caspase-1, are activated in sepsis-induced thymocyte apoptosis.

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.

Caspase inhibitors improve survival in sepsis: a critical role of the lymphocyte.

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.

Molecular biology of multiple organ dysfunction syndrome: injury, adaptation, and apoptosis.

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.

Prevention of lymphocyte cell death in sepsis improves survival in mice.

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.

Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction.

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.

Inducible nitric oxide synthase (iNOS) gene deficiency increases the mortality of sepsis in mice.

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.

Overexpression of Bcl-2 in transgenic mice decreases apoptosis and improves survival in sepsis.

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.

Effect of Ca2+ agonists in the perfused liver: determination via laser scanning confocal microscopy.

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.

Reversal of hypocalcemia and decreased afterload in sepsis. Effect on myocardial systolic and diastolic function.

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.