Proinflammatory cytokines mediate the toxic effect of superantigenic staphylococcal exotoxins (SE). Doxycycline inhibited SE-stimulated T-cell proliferation and production of cytokines and chemokines by human peripheral blood mononuclear cells. These results suggest that the antibiotic doxycycline has anti-inflammatory effects and is therapeutically useful for mitigating the pathogenic effects of SE.Staphylococcal toxic shock syndrome toxin 1 (TSST-1) and the structurally related exotoxins are bacterial exotoxins that bind directly to major histocompatibility complex class II molecules on antigen-presenting cells (1,5,8,18,23) and activate T cells expressing specific V␤ elements (7). These toxins are called superantigens because of their ability to polyclonally stimulate large populations of T cells (1,4,7,14). Thus, staphylococcal exotoxins (SE) are potent activators of the immune system and cause a variety of diseases in humans, including food poisoning, toxic shock, and autoimmune diseases (1,2,6,12,14,22). Their interactions with cells of the immune system result in massive production of proinflammatory cytokines and chemokines (1,4,15,17). The cytokines tumor necrosis factor alpha (TNF-␣), interleukin-1 (IL-1), and gamma interferon (IFN-␥) are key mediators in superantigen-induced toxic shock (1, 21). Both TNF-␣ and IL-1 have potent immunostimulating activities and act synergistically with IFN-␥ to enhance immune reactions and promote tissue injury (16). Consequently, these cytokines are pathogenic at high concentrations in vivo and are responsible for fever and toxic shock induced by SE (13,14,18,19).Doxycycline is a broad-spectrum antibiotic widely used for infections caused by both gram-negative and gram-positive microorganisms. It acts as a bacteriostatic agent and is highly effective against many microorganisms, including Staphylococcus aureus, Streptococcus pyogenes, Bacillus anthracis, and Yersinia pestis. Doxycycline belongs to the tetracycline antibiotic family, the members of which have been shown to have other biological actions independent of their antimicrobial effects (10). Doxycycline inhibits phorbol-12-myristate-13-acetate-mediated matrix metalloproteinase 8 (MMP-8) and MMP-9 in human endothelial cells (11). Doxycycline also decreases elastin degradation and reduces MMP activity in a model of aneurismal disease (3). More recently, doxycycline was shown to inhibit the production of IL-1␤ in lipopolysaccharide-treated corneal epithelial cultures to an extent comparable to that achieved by corticosteroids (25). In vivo, doxycycline protected mice from lethal endotoxemia by downregulating cytokine and nitrate secretion in blood (20). This study was undertaken to determine the modulatory effect of doxycycline on staphylococcal superantigen-induced T-cell activation and cytokine production from human peripheral blood mononuclear cells (PBMC).Purified SEB and TSST-1 were obtained from Toxin Technology (Sarasota, Fla.). The endotoxin content of these preparations was Ͻ1 ng of endotoxin/mg of protei...

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Bacterial exotoxins and endotoxins both stimulate proinflammatory mediators but the contribution of each individual toxin in the release of mediators causing lethal shock is incompletely understood. This study examines the cytokine response and lethality of mice exposed to varying doses of staphylococcal enterotoxin B (SEB) or lipopolysaccharide (LPS) and their combinations. In vivo, SEB alone induced moderate levels of IL-2 and MCP-1 and all mice survived even with a high dose of SEB (100 μg/mouse). LPS (80 μg/mouse) caused 48% lethality and induced high levels of IL-6 and MCP-1. SEB induced low levels of TNFα, IL-1, IFNγ, MIP-2, and LPS synergized with SEB in the expression of these cytokines and that of IL-6 and MCP-1. Importantly, the synergistic action of SEB and LPS resulted in lethal shock and hypothermia. ANOVA of cytokine levels by survival status of SEB-plus-LPS groups revealed significantly higher levels of TNFα, IL-6, MIP-2, and MCP-1 in nonsurvivors measured at 8 hours. Significantly higher levels of IFNγ and IL-2 were observed at 21 hours in nonsurvivors of toxic shock compared to those in survivors. Overall, synergistic action of SEB and LPS resulted in higher and prolonged levels of these key cytokines leading to toxic shock.

Staphylococcal enterotoxins are potent activators for human T cells and cause lethal toxic shock. Rapamycin, an immunosuppressant, was tested for its ability to inhibit staphylococcal enterotoxin B (SEB)-induced activation of human peripheral blood mononuclear cells (PBMC) in vitro and toxin-mediated shock in mice. Stimulation of PMBC by SEB was effectively blocked by rapamycin as evidenced by the inhibition of tumor necrosis factor alpha (TNF-␣), interleukin 1␤ (IL-1␤), IL-6, IL-2, gamma interferon (IFN-␥), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1␣ (MIP-1␣), MIP-1␤, and T-cell proliferation. In vivo, rapamycin protected 100% of mice from lethal shock, even when administered 24 h after intranasal SEB challenge. The serum levels of MCP-1 and IL-6, after intranasal exposure to SEB, were significantly reduced in mice given rapamycin versus controls. Additionally, rapamycin diminished the weight loss and temperature fluctuations elicited by SEB.Staphylococcal exotoxins are among the most common etiological agents that cause toxic shock syndrome (28-30, 38, 44). The disease is characterized by fever, hypotension, desquamation of skin, and dysfunction of multiple organ systems (8,38,41). These toxins bind directly to the major histocompatibility complex (MHC) class II molecules on antigen-presenting cells and subsequently stimulate T cells expressing specific V␤ elements on T-cell receptors (9,15,24,29,35,42). Staphylococcal enterotoxin B (SEB) and the distantly related toxic shock syndrome toxin 1 are also called superantigens because they induce massive proliferation of T cells (29). In vitro and in vivo studies show that these superantigens induce high levels of various proinflammatory cytokines, and these potent mediators cause lethal shock in animal models (1,6,22,27,37,39,45,51,55). SEB also causes food poisoning (4, 21, 52) and is a potential bioterrorism threat agent, as humans are extremely sensitive to this superantigen, especially by inhalation (28). There is currently no effective therapeutic treatment for SEB-induced shock except for the use of intravenous immunoglobulins (11). Various in vitro experiments identified inhibitors to counteract the biological effects of SEB, only some of which were successful in ameliorating SEB-induced shock in experimental models (1,(25)(26)(27)51).Rapamycin is a relatively new FDA-approved drug used to prevent graft rejection in renal transplantation, as it shows less nephrotoxicity than do calcineurin inhibitors (14,40,43,48). Recent studies reveal other uses in animal models of cancer (23, 34), diabetic nephropathy (36), bleomycin-induced pulmonary fibrosis (31), liver fibrosis (5), and tuberous sclerosis (32). Rapamycin binds intracellularly to FK506-binding proteins, specifically FKBP12; the rapamycin-FKBP12 complex then binds to a distinct molecular target called mammalian target of rapamycin (mTOR) (reviewed in reference 48). Rapamycin inhibits mTOR activity, prevents cyclin-dependent kinase activation, and affects G 1 -to-S-pha...

The superantigenic staphylococcal enterotoxins are important virulence factors and contribute to various diseases, including food poisoning and toxic shock. Dexamethasone, an anti-inflammatory agent, attenuated staphylococcal enterotoxin B (SEB)-induced hypothermia and serum proinflammatory cytokines and improved survival from 0% to 86% in a lethal mouse model of SEB-mediated shock.Staphylococcal enterotoxin B (SEB) and related superantigenic toxins are potent activators of the immune system and cause a myriad of maladies, ranging from food poisoning to potentially life-threatening toxic shock (13,17,21,24). These toxins bind directly to the major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (17,20,22) and stimulate T cells via specific V␤ regions of the T-cell receptors (TCR) (5, 17), resulting in activation of both monocytes/macrophages and T lymphocytes. The specific interaction of these microbial toxins with multiple cell types in the host leads to excessive production of proinflammatory cytokines, chemokines, and tissue factor, causing clinical symptoms that include fever, hypotension, and shock (13,18,24). Two key inflammatory cytokines, interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-␣), are direct mediators of fever, hypotension, and shock (15). Additionally, gamma interferon (IFN-␥) from superantigen-activated T cells acts synergistically with IL-1 and TNF-␣ to enhance immune reactions and tissue injury.At present, there is no available therapeutic for treating staphylococcal exotoxin-induced shock except for the use of intravenous immunoglobulins (6). Most therapeutic strategies for experimental animal models of SEB-induced shock have targeted proinflammatory cytokines, as there is a strong correlation between toxicity and increased serum levels of these inflammatory mediators (4,11,16,19,26). These therapeutics include cytokine inhibitors and signal transduction inhibitors that target SEB-induced cellular activation pathways (10, 16). Experimental approaches aimed at disrupting toxin interactions with receptors and costimulatory molecules on macrophages and lymphocytes have also been used by different laboratories (2, 9, 10, 28). Thus, bispecific chimeric inhibitors, composed of the DR␣1 domain of MHC class II and the V␤ domain of the TCR connected by a flexible linker, were designed to bind SEB competitively and prevent binding to MHC class II of antigen-presenting cells and the TCR on T cells (9).Conserved peptides corresponding to residues 150 to 161 of SEB can act as an antagonist and prevent SEA-, SEB-, or toxic shock syndrome toxin 1-induced lethal shock in mice when given intravenously 30 min after an intraperitoneal (i.p.) toxin dose (2). This segment of SEB is not associated with the classically defined MHC class II or TCR binding domains, but it may block costimulatory signals necessary for T-cell activation. However, a subsequent study of these peptides indicates that they are ineffective inhibitors of SEB-induced effects both in vitro and in viv...