Mast cells play a detrimental role in IgE-dependent allergic reactions. In contrast, a protective function for mast cells has been proposed on the basis of some worm infection models. No reports exist on the in vivo significance of these cells in bacterial infections. Here we use congenitally mast-cell-deficient W/Wv mice and normal +/+ littermates to analyse the role of mast cells in a model of acute septic peritonitis (caecum ligation and puncture (CLP)). Following CLP, W/Wv mice showed a significantly increased mortality compared to +/+ mice. The selective reconstitution of W/Wv mice with cultured +/+ mast cells substantially protected them from the lethal effects of CLP, whereas an anti-tumor-necrosis-factor (TNF) antibody injected immediately after CLP completely suppressed this protection. Our results reveal a previously unrecognized protective role of mast cells and mast-cell-derived TNF in acute bacterial peritonitis.

Identification of new therapeutic targets for the management of septic shock remains imperative as all investigational therapies, including anti-tumor necrosis factor (TNF) and anti-interleukin (IL)-1 agents, have uniformly failed to lower the mortality of critically ill patients with severe sepsis. We report here that macrophage migration inhibitory factor (MIF) is a critical mediator of septic shock. High concentrations of MIF were detected in the peritoneal exudate fluid and in the systemic circulation of mice with bacterial peritonitis. Experiments performed in TNFalpha knockout mice allowed a direct evaluation of the part played by MIF in sepsis in the absence of this pivotal cytokine of inflammation. Anti-MIF antibody protected TNFalpha knockout from lethal peritonitis induced by cecal ligation and puncture (CLP), providing evidence of an intrinsic contribution of MIF to the pathogenesis of sepsis. Anti-MIF antibody also protected normal mice from lethal peritonitis induced by both CLP and Escherichia coli, even when treatment was started up to 8 hours after CLP. Conversely, co-injection of recombinant MIF and E. coli markedly increased the lethality of peritonitis. Finally, high concentrations of MIF were detected in the plasma of patients with severe sepsis or septic shock. These studies define a critical part for MIF in the pathogenesis of septic shock and identify a new target for therapeutic intervention.

SummaryBecause mice are more resistant than humans to the pathogenic effects of bacterial toxins, we used D-Galactosamine-(D-Gal) sensitized mice as a modal system to evaluate potential toxic shock symptoms triggered by the superantigcn staphylococcal enterotoxin B (SEB). We show that similar to endotoxin (lipopolysaccharide) [LPS], the exotoxin SEB causes lethal shock within 8 h in D-Gal-sensitized mice, inducing 100% and about 50% lethality with 20 and 2 ~g SEB, respectively. The lethal shock triggered by the superantigcn SEB is mediated by T cells, a conclusion based on the observation that T cell repopulation of SCID mice conferred sensitivity to SEB. Since CSA also conferred protection, the role of T ceU-derived lymphokines in mediating lethal shock was evaluated. Within 30-60 min after SEB injection, serum tumor necrosis factor (TNF) levels peaked, followed immediately by interleukin-2 (IL-2). Serum-borne lymphokines were detected weU in advance of signs of T ceU activation, as assessed by IL-2 receptor expression of SEB-reactive VB8 + T ceUs. Passive immunization with anti-TNF-a/~-neutralizing monodonal antibody also conferred protection, indicating that it is TNF which is critical for initiating toxic shock symptoms. Taken together, this study defines basic differences between endotoxin (LPS)-and cxotoxin (SEB)-mediated lethal shock, in that the former is mediated by macrophages and the latter by T calls. Yet the pathogcnesis distal to the lymphokine/cytokine-producing cells appears surprisingly similar in that TNF represents a key mediator in inducing shock.

Sepsis, sepsis-induced hyperinflammation and subsequent sepsis-associated immunosuppression (SAIS) are important causes of death. Here we show in humans that the loss of the major reactive oxygen species (ROS) scavenger, glutathione (GSH), during SAIS directly correlates with an increase in the expression of activating transcription factor 3 (ATF3). In endotoxin-stimulated monocytes, ROS stress strongly superinduced NF-E2–related factor 2 (NRF2)–dependent ATF3. In vivo, this ROS-mediated superinduction of ATF3 protected against endotoxic shock by inhibiting innate cytokines, as Atf3−/− mice remained susceptible to endotoxic shock even under conditions of ROS stress. Although it protected against endotoxic shock, this ROS-mediated superinduction of ATF3 caused high susceptibility to bacterial and fungal infections through the suppression of interleukin 6 (IL-6). As a result, Atf3−/− mice were protected against bacterial and fungal infections, even under conditions of ROS stress, whereas Atf3−/−Il6−/− mice were highly susceptible to these infections. Moreover, in a model of SAIS, secondary infections caused considerably less mortality in Atf3−/− mice than in wild-type mice, indicating that ROS-induced ATF3 crucially determines susceptibility to secondary infections during SAIS.

SummaryThe influence of endogenous and exogenous tumor necrosis factor (TNF) on metastasis was investigated in an experimental fibrosarcoma metastasis model. A single intraperitoneal injection of recombinant human (rh) TNF or recombinant mouse (rm) TNF into mice 5 h before intravenous inoculation of methylcholanthrene-induced fibrosarcoma cells (CFS1) induced a significant enhancement of the number of metastases in the lung. Dose responses of rmTNF and rhTNF demonstrated a stronger metastasis-augmenting effect by rmTNF compared with rhTNF. This effect was time dependent, as administration of rmTNF 5 h before or 1 h but not 24 h after tumor cell inoculation caused an increase of tumor cell colony formation on the lung surface, suggesting an influence of TNF on the vascular adhesion and diapedesis of tumor cells. Since tumor-bearing mice showed an enhanced ability to produce TNF after endotoxin injection compared to control mice, tumor-bearing mice were treated with anti-mTNF antibodies. Neutralization of endogenous tumor-induced TNF led to a significant decrease of the number of pulmonary metastases. Histological analysis of micrometastases in the lung on day 5 by silver staining of proteins associated with nucleolar organizer regions revealed more metastatic loci and augmented proliferative activity of the tumor cells after rmTNF pretreatment of mice. However, no direct effect of rmTNF on the proliferation rate of tumor cells was seen in vitro. These findings suggest that low doses of endogenous TNF or administered TNF during cytokine therapy might enhance the metastatic potential of circulating tumor cells.

Mast cells are thought to contribute significantly to the pathology and mortality associated with anaphylaxis and other allergic disorders. However, studies using genetically mast cell–deficient WBB6F1-KitW/KitW-v and congenic wild-type (WBB6F1-+/+) mice indicate that mast cells can also promote health, by participating in natural immune responses to bacterial infection. We previously reported that repetitive administration of the c-kit ligand, stem cell factor (SCF), can increase mast cell numbers in normal mice in vivo. In vitro studies have indicated that SCF can also modulate mast cell effector function. We now report that treatment with SCF can significantly improve the survival of normal C57BL/6 mice in a model of acute bacterial peritonitis, cecal ligation and puncture (CLP). Experiments in mast cell–reconstituted WBB6F1-KitW/KitW-v mice indicate that this effect of SCF treatment reflects, at least in part, the actions of SCF on mast cells. Repetitive administration of SCF also can enhance survival in mice that genetically lack tumor necrosis factor (TNF)-α, demonstrating that the ability of SCF treatment to improve survival after CLP does not solely reflect effects of SCF on mast cell– dependent (or –independent) production of TNF-α. These findings identify c-kit and mast cells as potential therapeutic targets for enhancing innate immune responses.

Migration from sites of antigen encounter to lymphoid organs is essential to the strong immunogenic function of dendritic cells (DC). In the skin, migration proceeds through dermal lymphatic vessels and is regulated in an incompletely understood way by inflammatory mediators. We studied the effects of tumor necrosis factor ␣ (TNF-␣) and interleukin-1␤ (IL-1␤) in mouse skin organ cultures by direct enumeration of migrating DC and by immunohistochemistry.

Properdin is a positive regulator of complement activation so far known to be instrumental in the survival of infections with certain serotypes of Neisseria meningitidis. We have generated a fully backcrossed properdin-deficient mouse line by conventional gene-specific targeting. In vitro, properdin-deficient serum is impaired in alternative pathway-dependent generation of complement fragment C3b when activated by Escherichia coli DH5α. Properdin-deficient mice and wild-type littermates compare in their levels of C3 and IgM. In an in vivo model of polymicrobial septic peritonitis induced by sublethal cecal ligation and puncture, properdin-deficient mice appear immunocompromised, because they are significantly impaired in their survival compared with wild-type littermates. We further show that properdin localizes to mast cells and that properdin has the ability to directly associate with E. coli DH5α. We conclude that properdin plays a significant role in the outcome of polymicrobial sepsis.