Joanne Lomas

Inhibition of Fas/Fas ligand signaling improves septic survival: differential effects on macrophage apoptotic and functional capacity.

Signaling through the Fas/Fas ligand (FasL) pathway plays a central role in immune‐cell response and function; however, under certain pathological conditions such as sepsis, it may contribute to the animals or patients morbidity and mortality. To determine the contribution of FasL to mortality, we conducted survival studies by blocking Fas/FasL with Fas receptor fusion protein (FasFP) in vivo. C3H/HeN mice received FasFP or the saline vehicle (veh) immediately (0 h) or delayed (12 h), after sepsis induced by cecal ligation and puncture (CLP). Subsequently, we examined the effect of FasFP treatment (12 h post‐CLP) on macrophage apoptosis and functional capacities. Peritoneal and splenic macrophages and Kupffer cells from sham‐veh‐, CLP‐veh‐, sham‐FasFP‐, or CLP‐FasFP‐treated mice were harvested 24 h after CLP and stimulated with lipopolysaccharide (LPS) for 24 h. The results indicate that only delayed (12 h) but not 0 h administration of FasFP demonstrated a significant increase in survival. The ability of all macrophage populations to release interleukin (IL)‐6 was significantly depressed, and IL‐10 release was augmented after CLP. FasFP treatment attenuated the increased IL‐10 release in Kupffer cells. However, althogh enhanced susceptibility to LPS‐induced apoptosis could be suppressed in CLP mouse Kupffer cells by FasFP, FasFP did not change the peritoneal or splenic macrophage response. Furthermore, FasFP attenuated the elevated plasma levels of liver enzymes after sepsis. These data indicate that in vivo inhibition of Fas/FasL signaling has tissue‐specific effects on the induction of macrophage apoptosis, functional changes, and liver damage, which may contribute to the hosts ability to ward off a septic challenge.

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.

Inhibition of CD1d activation suppresses septic mortality: A role for NK-T cells in septic immune dysfunction

BACKGROUND Studies indicate that following septic insult there is development of generalized immune dysfunction in T cells, B cells and phagocytes, which is thought to contribute to morbidity and mortality. Specifically, there is a shift in the lymphocytes of septic animals toward an increased release of Th2 cytokines. NK-T cells have been shown to contribute to propagation of the Th2 response. The influence of NK-T cells on the immune response to septic challenge is poorly understood. In this study, we examine whether NK-T cells contribute to the immune dysfunction seen following the onset of polymicrobial sepsis, as produced by cecal ligation and puncture (CLP). MATERIALS AND METHODS Male 129S1/SvImJ mice were pretreated with either rat IgG (isotypic control) or monoclonal antibody to CD1d (clone 1B1) (0.5 mg), which blocks signaling/antigen presentation via the CD1d cell surface receptor, thereby, ablating the activation and differentiation of the NK-T cells. Septic survival with and without anti-CD1d (CLP/CD1d) pretreatment was assessed. Mice sacrificed 24 h after CLP were assessed for change in splenic %NK-T cell (via flourescense activated cell sector) and for splenic, hepatic, and lymphoid/macrophage production of pro-inflammatory or anti-inflammatory cytokines (via enzyme-linked immunosorbent assay). RESULTS Administration of anti-CD1d reduced septic mortality 35% at 6-10 d (n = 23 mice/group) (P <.05). There was a consistent increase in the %CD3(+) NK1.1(+) cell population (NK-T cells) in septic mice (1.706%), which was markedly suppressed by pretreatment with anti-CD1d (0.592%). IL-6 and IL-10 levels were suppressed by anti-CD1d in the spleen and blood. CONCLUSIONS Together these findings imply not only that NK-T cells may play a role in mediating the immune suppression seen in bacterial sepsis, but that inhibition of their activation promotes survival to septic challenge.

NF-kappaB activation has tissue-specific effects on immune cell apoptosis during polymicrobial sepsis.

Studies indicate that critically ill patients who succumb to sequela of sepsis/multiorgan failure, as well as septic animals, exhibit an apparently pathological increase in apoptosis (Ao) in the immune system. However, the mechanisms regulating these changes are unclear. Studies also indicate that, dependent on the cell population and the nature and/or duration of the stimuli, activation of the nuclear factor (NF)-&kgr;B can either suppress or enhance Ao. Thus, the aim of this study was to determine the contribution of NF-&kgr;B activation to the onset of Ao seen in divergent immune cell populations during sepsis, as produced by cecal ligation and puncture (CLP). To assess this, C3H/HeN mice were pretreated (for 1 h) subcutaneously with either 100 mg/kg body weight of pyrrolidine dithiocarbamate (PDTC), an NF-&kgr;B inhibitor, or with saline vehicle, prior to subjecting them to CLP or Sham-CLP (Sham). Thymocytes, phagocytes, and Peyers Patch cells were harvested 24 h later, and the extent of Ao was determined by flow cytometry. The results indicate that PDTC pretreatment had no marked effect on the increase in thymocyte or phagocyte Ao seen following CLP, but there was a significant decline in the extent of Ao observed in septic mouse Peyers patch B cells. To the extent that this was a result of NF-&kgr;B inhibition, we demonstrate by Western analysis, electrophoretic mobility shift assay (EMSA) and transfactor assay that the translocation of c-Rel to septic mouse Peyers patch B cell nuclei is attenuated by PDTC. PDTC pretreatment also markedly reduced the number of Peyers patch B cells that were producing IgA as well as attenuated the increase of proinflammatory cytokines in the blood. Interestingly, PDTC pretreatment did not restore peritoneal macrophage function or improve animal survival. Taken together, the inability of PDTC pretreatment to alter the Ao response of thymocytes or phagocytes, while inhibiting the increase in Peyers patch B cell Ao in septic mice, implies not only that the activation of NF-&kgr;B has highly tissue/cell-specific effects that must be discerned when trying to clarify the pathophysiological role of NF-&kgr;B in sepsis, but that the activation of NF-&kgr;B may contribute to the early adaptive responses required by the host to fend off septic challenge.

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.