Patricia S. Grutkoski

The loss of Mcl-1 expression in human polymorphonuclear leukocytes promotes apoptosis

The regulation of polymorphonuclear leukoctye (PMN) apoptosis can influence the duration of the inflammatory response. We have previously shown that PMN apoptosis is delayed by matrix adhesion and hypoxia; however, the mechanisms responsible for this delay are not well understood. Mcl‐1, an antiapoptotic Bcl‐2 family member, is present in neutrophils; therefore, we sought to characterize its localization and function as it relates to PMN apoptosis. We found that Mcl‐1 localized to the nucleus and cytoplasm and that expression levels decreased as PMN were aged in culture. Reducing available Mcl‐1 through the use of antisense oligonucleotides demonstrated that Mcl‐1 is necessary to delay apoptosis during normal PMN aging and hypoxia but is not required for suppression of apoptosis by laminin adhesion. Our results demonstrate a distinct expression pattern of Mcl‐1 and that Mcl‐1 is crucial for the delay of apoptosis initiated by certain antiapoptotic factors.

TNFalpha-induced suppression of PMN apoptosis is mediated through interleukin-8 production.

Dysregulated neutrophil (polymorphonuclear PMN) apoptosis is thought to contribute to the onset of adult respiratory distress syndrome (ARDS) in critically ill patients. Tumor necrosis factor-alpha (TNFalpha), which is present in elevated levels in the bronchoalveolar lavage fluid in patients with ARDS, is thought to play a central role in regulating PMN function in the lungs. Studies have shown that short-term culture with TNFalpha increases apoptosis yet extended culture with TNFalpha suppresses apoptosis. However, it is unclear whether this latter effect of TNFalpha is directly or indirectly mediated through production of anti-apoptotic cytokines such as interleukin (IL)-8. To investigate the role of IL-8 in TNFalpha-induced apoptosis PMN were exposed to TNFalpha (100 ng/mL) in the presence or absence of antibodies to IL-8, and the extent of apoptosis was assessed. An enzyme-linked immunoassay was used to measure levels of the anti-apoptotic cytokine IL-8, induced by TNFalpha-stimulation. Because TNFalpha may mediate its effect through various cell-signaling pathways, we next assessed the effect of kinase inhibition on the ability of TNFalpha to effect apoptosis and IL-8 production. Treatment with TNFalpha had a biphasic effect: at 4-8 h, apoptosis was increased but was markedly suppressed at 24 h (P < 0.05). PMN cultured for 24 h with TNFalpha also showed markedly increased levels of IL-8. Neutralization of IL-8 inhibited the ability of TNFalpha to suppress apoptosis (P < 0.05). Incubation of TNFalpha + p38-mitogen-activated protein kinase (MAPK) inhibitor SB202190 increased apoptosis (P < 0.01) and decreased IL-8 production to PMN control. To a lesser extent, incubation of TNFalpha with inhibitors to NF-kappaB (SN50) and PI3K (LY294002) also increased apoptosis and decreased IL-8 production (P < 0.05). These data illustrate a novel mechanism by which TNFalpha can indirectly elicit an anti-apoptotic effect via p38-MAPK induced release of the anti-apoptotic chemokine IL-8. The exploitation of such a pathway represents a potential target for regulation of PMN-mediated acute lung injury.

Interleukin-8–induced suppression of polymorphonuclear leukocyte apoptosis is mediated by suppressing CD95 (Fas/Apo-1) Fas-l interactions☆☆☆

BACKGROUND Neutrophil apoptosis is crucial in the resolution of inflammation. The role of interleukin (IL)-8 in neutrophil apoptosis has not been previously studied; we hypothesized that in addition to its role as a chemoattractant, IL-8 would regulate polymorphonuclear leukocyte (PMN) apoptosis. METHODS PMNs were adhered to plastic during hypoxia or normoxia and treated with IL-8 dosages of 0 to 1000 ng/mL. Apoptosis was assessed by cellular histology and the TUNEL assay. For receptor inhibition, blocking antibodies to IL-8 receptors in the presence of IL-8 were added. Apoptosis of PMNs treated with anti-Fas antibody +/- IL-8 was also analyzed. RESULTS After treatment with 100 ng/mL IL-8 apoptosis was decreased from an average of 39.1% 9.3%. Inhibition of IL-8RA was able to restore apoptosis to 59.4%. Western analysis showed that with IL-8, there was a marginal decrease of total Fas protein, whereas Fas ligand was increased. After incubation with an apoptosis inducing-Fas antibody plus IL-8 reduced apoptosis to 9.5%. CONCLUSIONS IL-8 not only promotes the inflammatory response by recruiting PMNs but also acts to suppress apoptosis mainly through the IL-8RA in an oxygen tension independent manner. The reduction in apoptosis is associated with changes in Fas and FasL where the presence of IL-8 suppresses the proapoptotic function of Fas-FasL interactions.

CXCR2 inhibition suppresses hemorrhage-induced priming for acute lung injury in mice

Polymorphonuclear neutrophil (PMN) extravasation/sequestration in the lung and a dysregulated inflammatory response characterize the pathogenesis of acute lung injury (ALI). Previously, we have shown that hemorrhage (Hem) serves to prime PMN such that subsequent septic challenge [cecal ligation and puncture (CLP)] produces a pathological, inflammatory response and consequent lung injury in mice. Keratinocyte‐derived chemokine (KC) and macrophage inflammatory protein‐2 (MIP‐2) are murine CXC chemokines found elevated in the lungs and plasma following Hem/CLP and have been reported by others to share a common receptor (CXCR2). Based on these data, we hypothesize that blockade of CXCR2 immediately following Hem would suppress KC and MIP‐2 priming of PMN, thereby reducing the inflammatory injury observed following CLP. To assess this, Hem mice (90 min at 35±5 mmHg) were randomized to receive 0, 0.4, or 1 mg antileukinate (a hexapeptide inhibitor of CXCRs) in 100 μl phosphate‐bufferd saline (PBS)/mouse subcutaneously, immediately following resuscitation (Ringer’s lactate‐4× drawn blood volume). Twenty‐four hours post‐Hem, mice were subjected to CLP and killed 24 h later. The results show that blockade of CXCR2 significantly (P<0.05, Tukey’s test) reduced PMN influx, lung protein leak, and lung‐tissue content of interleukin (IL)‐6, KC, and MIP‐2 and increased tissue IL‐10 levels. Plasma IL‐6 was significantly decreased, and IL‐10 levels increased in a dose‐dependent manner compared with PBS‐treated mice. A differential effect was observed in plasma levels of KC and MIP‐2. KC showed a significant reduction at the 0.4 mg antileukinate dose. In contrast, plasma MIP‐2 was significantly elevated at both doses compared with the PBS‐treated controls. Together, these data demonstrate that blockade of CXCR2 signaling attenuates shock‐induced priming and ALI observed following Hem and subsequent septic challenge in mice.

Differential Effects of Macrophage Inflammatory Chemokine-2 and Keratinocyte-derived Chemokine on Hemorrhage-induced Neutrophil Priming for Lung Inflammation: Assessment by Adoptive Cells Transfer in Mice

Prior studies have shown that hemorrhage (Hem) can serve as a priming stimulus for acute lung injury (ALI) triggered by subsequent septic challenge (cecal ligation and puncture, CLP). Furthermore, we have reported that in vivo antibody neutralization of the chemokines, macrophage inflammatory chemokine-2 (MIP-2) and keratinocyte-derived chemokine (KC), immediately after Hem appears to differentially effect the onset of ALI. However, although we hypothesize that this is due to divergent effects of MIP-2 and KC on Hem-induced neutrophil (PMN) priming, this has not been tested. To examine this hypothesis, PMN donor mice were Sham-Hem or Hem for 90 min at 35 ± 5 mmHg and were then administered anti-MIP-2 (Hem/anti-MIP2), anti-KC (Hem/anti-KC), or nonspecific immunoglobulin (Ig) G (Hem/IgG) during resuscitation (Ringers lactate = four times the amount of drawn blood volume). Twenty-four hours post-Hem, the peripheral blood PMN were purified from these donor animals and were introduced into PMN-depleted recipient mice [depleted by prior anti-Gr1 (mouse PMN-specific marker) antibody treatment]. One hour after PMN transfer, recipient mice were subjected to CLP, euthanized 24 h later, and plasma as well as lung tissue samples were collected. PMN influx was assessed by myeloperoxidase assay (MPO; &mgr;U/mg protein) and histologically (IL-6, MIP-2, KC, and IL-10 levels) by enzyme-linked immunoabsorbant assay (ELISA; ng/mg). The results show that donor PMN from Hem/IgG but not Sham-Hem mice produce increased PMN influx (↑MPO, ↑% esterase+ cells in tissue) into the lung and local tissue inflammation (↑IL-6/MIP-2, ↓IL-10) in PMN-depleted CLP recipient mice, which was attenuated in mice receiving cells from Hem/anti-MIP-2 but not Hem/anti-KC treated donors. Interestingly, although Hem/anti-MIP-2 donor PMN produced comparable effects on blood IL-6/MIP-2 levels, they were ineffective in altering the change in plasma IL-10/KC levels induce by Hem. Taken together, these data demonstrate that Hem-induced priming of PMN not only mediates ALI in the mouse, but also that this process is differentially effected by MIP2 and KC, despite the fact that both signal through CXCR2.

Pathological aspects of apoptosis in severe sepsis and shock

Today, despite the application of contemporary operative/pharmacological approaches in the treatment of the critically ill trauma/surgery patient, we are still faced with a high incidence of patients who develop sepsis and subsequent multiple organ failure. This review attempts to summarize data gathered over the last few years, from both experimental and patient settings, that not only documents the presence of apoptosis, but begins to define its contribution to the pathology of sepsis and shock, which in turn precipitate organ injury/damage.

Mechanisms of immune resolution

Initially after injury, the innate/proinflammatory and some aspects of the acquired immune response are up-regulated to maintain a defense against foreign pathogens, clear tissue debris present at the wound site, and orchestrate aspects of tissue remodeling, cell proliferation and angiogenic process, associated with the wound response. However, for proper wound healing to progress, this initial inflammatory response has to be regulated or shut down so as to allow for the reestablishment of matrix, recellularization, and tissue remodeling. Inability to properly resolve the extent of innate/acquired response at a site of injury can lead to poor wound healing, immune suppression, and recurrent infectious episodes. This review attempts to summarize information on regulatory mechanisms that are thought to be involved in controlling/resolving innate or acquired immune responses so as to provide a framework for use in thinking about the impact these processes and their manipulation may have on wound healing and its potential management.

Sepsis-induced SOCS-3 expression is immunologically restricted to phagocytes

We have shown that immune cells from septic mice exhibit a suppressed response to exogenous stimuli in vitro. The suppressors of the cytokine signaling (SOCS) family are proteins that block intracellular signaling and can be induced by inflammatory mediators. Therefore, we hypothesized that SOCS‐3 is up‐regulated in immune cells in response to a septic challenge induced by cecal ligation and puncture (CLP). Mice were subjected to CLP or sham‐CLP, and 2–48 h later, the blood, thymus, spleen, lung, and peritoneal leukocytes were harvested and examined. SOCS‐3 was undetectable in thymocytes or blood leukocytes. In contrast, SOCS‐3 was up‐regulated in the spleen, lung, and peritoneal leukocytes in a time‐dependent manner. Further examination revealed that only the macrophages and neutrophils expressed SOCS‐3. These data suggest that cytokines and bacterial toxins present during sepsis have the ability to suppress the cytokine and/or lipopolysaccharide response and the function of immune cells by up‐regulating SOCS‐3.

Paracrine suppression of apoptosis by cytokine-stimulated neutrophils involves divergent regulation of NF-kappaB, Bcl-X(L), and Bak.

Dysregulated polymorphonuclear leukocyte (PMN) apoptosis and PMN-mediated organ damage have been associated with several medical conditions such as systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), and ischemia/reperfusion injury. IL-1&bgr; and IL-8 are two cytokines that are elevated under similar conditions. Therefore, we hypothesized that PMN exposed to these cytokines would secrete factors that could affect PMN apoptosis in a cell contact-independent manner. We have previously shown that media conditioned by IL-1&bgr;-stimulated PMN (CM-IL1&bgr;) for 2 h suppressed spontaneous PMN apoptosis. Data presented here demonstrate that media conditioned by IL-8-stimulated PMN (CM-IL8) also have the ability to suppress spontaneous, as well as FasL- and TNF-&agr;-induced apoptosis. In contrast, CM-IL1&bgr; was able to suppress FasL-induced, but not TNF-&agr;-induced, apoptosis. To elucidate the mechanisms these media use to elicit their effects, we examined the expression and function of several apoptosis-related proteins. Experimental results demonstrate that both CM-IL1&bgr; and CM-IL8 have the ability to delay caspase activation, but have no effect on the expression of their upstream activator, Fas, or its ligand, FasL. Examination of several Bcl-2 family members revealed a selective regulation by each media: CM-IL1&bgr; up-regulated Bcl-XL, while CM-IL8 down-regulated Bak expression. Additionally, CM-IL1&bgr;, but not CM-IL8, promoted the activation of NF-&kgr;B, which has anti-apoptotic activity. Together, we can conclude that IL-1&bgr;- and IL-8-stimulated PMN have the ability to suppress PMN apoptosis in a paracrine manner, and that the extent and mechanism of suppression is specific for each.

Fas-Ligand Mediated Apoptosis in Severe Sepsis and Shock

Alterations in the apoptotic process in lymphoid tissues is a common condition which is encountered in the severely septic animal and critically ill patient. Here we attempt to delineate the pathological significance of these apoptotic changes and the role of Fas-FasL mediated contribution to this process.