Staphylococcus aureus: the toxic presence of a pathogen extraordinaire.

Staphylococcus aureus is a facultative, Gram-positive coccus well known for its disease-causing capabilities. In particular, methicillin and vancomycin resistant strains of S. aureus (MRSA and VRSA, respectively) isolated globally represent daunting medical challenges for the 21(st) Century. This bacterium causes numerous illnesses in humans such as food poisoning, skin infections, osteomyelitis, endocarditis, pneumonia, enterocolitis, toxic shock, and autoimmune disorders. A few of the many virulence factors attributed to S. aureus include antibiotic resistance, capsule, coagulase, lipase, hyaluronidase, protein A, fibronectin-binding protein, and multiple toxins with diverse activities. One family of protein toxins is the staphylococcal enterotoxins (SEs) and related toxic shock syndrome toxin-1 (TSST-1) that act as superantigens. There are more than twenty different SEs described to date with varying amino acid sequences, common conformations, and similar biological effects. By definition, very low (picomolar) concentrations of these superantigenic toxins activate specific T-cell subsets after binding to major histocompatibility complex class II. Activated T-cells vigorously proliferate and release proinflammatory cytokines plus chemokines that can elicit fever, hypotension, and other ailments which include a potentially lethal shock. In vitro and in vivo models are available for studying the SEs and TSST-1, thus providing important tools for understanding modes of action and subsequently countering these toxins via experimental vaccines or therapeutics. This review succinctly presents the pathogenic ways of S. aureus, with a toxic twist. There will be a particular focus upon the biological and biochemical properties of, plus current neutralization strategies targeting, staphylcoccocal superantigens like the SEs and TSST-1.

The flavonoid baicalin inhibits superantigen-induced inflammatory cytokines and chemokines.

Excessive release of proinflammatory cytokines mediates the toxic effect of superantigenic staphylococcal exotoxins (SE). Baicalin, a flavone isolated from the Chinese herb Scutellaria baicalensis Georgi and used in China to treat infectious diseases, inhibited SE-stimulated T-cell proliferation (by 98%) and production of interleukin 1beta, interleukin 6, tumor necrosis factor, interferon gamma, monocyte chemotactic protein 1, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta mRNA and protein by human peripheral blood mononuclear cells. These data suggest that baicalin may be therapeutically useful for mitigating the pathogenic effects of SE by inhibiting the signaling pathways activated by superantigens.

Serum cytokine levels and antibody response to influenza vaccine in the elderly.

Cytokines play critical roles in regulating the antibody response to vaccines. We sought to understand the role of endogenous cytokines in the determination of antibody production in the elderly, a group of subjects known to have a lower response rate to vaccination. We found that in a healthy elderly group, only 52% of whom responded to the influenza vaccine, endogenous levels of interleukin 6 (IL-6), IL-10 and gamma interferon (IFNgamma) did not differ statistically significantly between responders and non-responders (responders: n = 27, IL-6 = 293 +/- 101 pg/ml, IL-10 = 882 +/- 240 pg/ml; nonresponders: n = 26, IL-6 = 223 +/- 71 pg/ml, P = 0.57, IL-10 = 445 +/- 148 pg/ml, mean +/- SE, P = 0.14, respectively, and undetectable IFNgamma). Serum levels of these three cytokines were not changed significantly four weeks after vaccination (P < 0.05 for IL-6 and P < 0.01 for IL-10). In addition, there were also no age-dependent differences in serum IL-6 and IL-10 levels.

Suppression of endotoxin- and staphylococcal exotoxin-induced cytokines and chemokines by a phospholipase C inhibitor in human peripheral blood mononuclear cells.

Excessive release of proinflammatory cytokines from cells stimulated with lipopolysaccharide (LPS) or staphylococcal exotoxin (SE) mediates the pathophysiologic manifestations of septic shock. Tricyclodecan-9-yl (D609), an inhibitor of phosphatidylcholine-specific phospholipase C, suppressed LPS- or SE-induced cytokines and chemokines in human peripheral blood mononuclear cells. These data suggest a potential role for D609 in the treatment of septic shock.

Pentoxifylline inhibits ICAM-1 expression and chemokine production induced by proinflammatory cytokines in human pulmonary epithelial cells.

Airway epithelium participates in inflammatory reactions by producing chemokines and expressing cell-surface adhesion molecules which aid in the selective recruitment of effector cells. Previous studies showed that proinflammatory cytokines, interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF alpha), induce surface expression of intercellular adhesion molecule 1 (ICAM-1) and the production of the chemokines interleukin 8 (IL-8) and monocyte chemoattractant protein (MCP-1) on pulmonary epithelial cell lines in vitro. In this study, the dose response of four cytokines, IL-1alpha, IL-1beta, TNF alpha and TNF beta, in inducing ICAM-1 expression and production of IL-8 and MCP-1 on pulmonary A549 epithelial cells was examined. Both IL-1alpha and IL-1beta induced ICAM-1 expression and IL-8 and MCP-1 production at lower doses than TNF alpha or TNF beta. Pentoxifylline, an anti-inflammatory agent used to treat vascular diseases, was tested for its ability to inhibit the activation of airway epithelial cells by these cytokines. Pentoxifylline completely inhibited the surface expression of ICAM-1 and the production of IL-8 and MCP-1 by cytokine-activated epithelial cells. As elevated levels of chemokines are often present in bronchial lavage fluids of patients suffering from various acute respiratory diseases, pentoxifylline may be useful for preventing the rapid development of immune reactions leading to lung injury.

Coordinate suppression of superantigen-induced cytokine production and T-cell proliferation by a small nonpeptidic inhibitor of class II major histocompatibility complex and CD4 interaction.

Proinflammatory cytokines mediate the toxic effect of superantigenic staphylococcal exotoxins (SE). TJU103, a small nonpeptidic molecule that blocks the interaction between major histocompatibility complex class II and CD4 molecules inhibited SE-stimulated T-cell proliferation (by 92%) and production of tumor necrosis factor, interleukin 1beta, interleukin 6, and gamma interferon (by 66, 56, 76, and 72%, respectively) by human peripheral blood mononuclear cells. These data suggest that TJU103 may be useful for mitigating the pathogenic effects of SE.

Immune response to staphylococcal superantigens.

Staphylococcal exotoxins, staphylococcal enterotoxins A-E (SEA-SEE), and toxic shock syndrome toxin- (TSST-1) are potent activators of the immune system and cause a variety of diseases in humans, ranging from food poisoning to shock. These toxins are called superantigens because of their ability to polyclonally activate T cells at picromolar concentrations. Superantigens bind to both MHC class II molecules and specific Vbeta regions of the T cell receptor, leading to the activation of both antigen-presenting cells and T lymphocytes. These interactions lead to excessive production of proinflammatory cytokines and T cell proliferation, causing clinical symptoms that include fever, hypotension, and shock. Recent studies suggest that staphylococcal superantigens may also be involved in the pathogenesis of arthritis and other autoimmune disorders. This review summarizes the in vitro and in vivo effects of staphylococcal enterotoxins and TSST-1, recent progress with the use of transgenic knockout mice to identify key mediators and receptors involved in superantigen-induced shock, and therapeutic agents to mitigate the toxic effects of staphylococcal superantigens.

A method for correcting for the variability of inhibitory effects of soluble human interleukin 1 receptor II measured by different ELISAS.

Seven ELISAs were developed by using several combinations of anti-human IL-1beta antibodies for detecting interleukin 1beta (IL-1beta) in cell culture supernatants. These ELISAs have different sensitivities in detecting standard preparations of recombinant human IL-1beta (WHO reference standard) compared with conventional preparations of IL-1beta produced by stimulated human peripheral blood mononuclear cells. The observed differences were attributed to differences in epitope specificity of the various monoclonal antibodies used and the heterogeneity of IL-1beta secreted into culture supernatants. The presence of soluble IL-1 receptor type I did not alter the levels of IL-1beta detected by these ELISAs. However, soluble IL-1 receptor type II interfered with the detection of IL-1beta to different degrees in these ELISAs. A method involving standarization by means of separate measurement of the amount of receptor and its inhibitory effect in the IL-1beta ELISA, yields consistent estimates of the correct IL-1beta levels.

Pentoxifylline inhibits superantigen-induced toxic shock and cytokine release.

Tumor necrosis factor alpha (TNF-alpha) is a critical cytokine that mediates the toxic effects of bacterial superantigens like staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin 1 (TSST-1). Pentoxifylline, an anti-inflammatory agent that inhibits endotoxemia and lipopolysaccharide (LPS)-induced release of TNF-alpha, was tested for its ability to inhibit SEB- and TSST-1-induced activation of human peripheral blood mononuclear cells (PBMCs) in vitro and toxin-mediated shock in mice. Stimulation of PBMCs by SEB or TSST-1 was effectively blocked by pentoxifylline (10 mM), as evidenced by the inhibition of TNF-alpha, interleukin 1beta (IL-1beta), gamma interferon (IFN-gamma), and T-cell proliferation. The levels of TNF-alpha, IL-1alpha, and IFN-gamma in serum after an SEB or TSST-1 injection were significantly lower in mice given pentoxifylline (5.5 mg/animal) versus control mice. Additionally, pentoxifylline diminished the lethal effects and temperature fluctuations elicited by SEB and TSST-1. Thus, in addition to treating endotoxemias, the cumulative in vitro and in vivo data suggest that pentoxifylline may also be useful in abrogating the ill effects of staphylococcal enterotoxins and TSST-1.

Induction of CC chemokines in human peripheral blood mononuclear cells by staphylococcal exotoxins and its prevention by pentoxifylline.

We investigated the inflammatory processes that might be associated with the arthrogenic activity of Staphylococcus aureus, the principal causative agent of bacterial arthritis. Human peripheral blood mononuclear cells (PBMC) were stimulated with the staphylococcal toxic shock syndrome toxin-1 (TSST-1) or enterotoxin B (SEB) and the production of chemokines was examined. Both TSST-1 and SEB induced high levels (ng/mL) of MIP-1alpha, MIP-1beta, and MCP-1. The induction of these chemokines occurred mostly by direct stimulation of PBMC with staphylococcal exotoxins (SE), without requiring the intervention of IL-1 and TNF-alpha. The production of SE-induced chemokines was blocked partially by anti-DR and anti-CD2 antibodies. Cell separation revealed monocytes as the cell source of these chemokines. However, addition of purified T cells amplified the levels of chemokine produced, suggesting that cognate interaction of SE bound on antigen-presenting cells with T cells also contributes to chemokine production. The activation and recruitment of leukocytes by these chemokines may contribute to the pathophysiology of septic arthritis caused by staphylococci in humans through tissue injury and the recruitment of T lymphocytes, perhaps also initiating autoimmune responses. Pentoxifylline, an anti-inflammatory agent, completely inhibited the production of these chemokines.

Variability in the sensitivity of nine enzyme-linked immunosorbant assays (ELISAs) in the measurement of human interleukin 6.

ELISAs employing several combinations of polyclonal and monoclonal anti-human IL-6 antibodies for the detection of interleukin 6 (IL-6) in cell culture supernatants were developed and compared. They were found to have differing sensitivities in the measurement of standard preparations of recombinant human (rh) IL-6 (WHO reference standard) as well as conventional preparations of IL-6 produced by stimulated human peripheral blood mononuclear cells. Thus, an ELISA that was optimal for detecting rhIL-6 standard was suboptimal for detecting IL-6 in cell culture supernatants. The presence of soluble IL-6 receptor (sIL-6R) or the signal-transducing gp130 molecule or serum did not alter the levels of IL-6 detected by these ELISAs. Therefore, the observed variability in sensitivity of the assays may be due to differences in epitope specificity of the various monoclonal antibodies used and the heterogeneity of IL-6 secreted into culture supernatants.

Interleukin-8 production by human monocytic cells in response to staphylococcal exotoxins is direct and independent of interleukin-1 and tumor necrosis factor-alpha.

Staphylococcal exotoxins have been implicated as major virulence factors responsible for toxic shock syndrome. To elucidate further the cellular mechanisms contributing to shock, human monocytic cells were stimulated with the staphylococcal toxic shock syndrome toxin-1, enterotoxin A, or enterotoxin B, and the production of the chemokine interleukin-8 (IL-8) was examined. All three exotoxins were potent inducers of IL-8. IL-8 induction occurred rapidly, within 2 h after stimulation, independent of IL-1 and tumor necrosis factor-alpha produced by these cells. This study suggests that IL-8 is one of the earliest mediators produced by monocytic cells in direct response to staphylococcal exotoxins. IL-8 may serve as the first proinflammatory signal for neutrophil recruitment to tissue and may contribute to staphylococcal superantigen-mediated shock and multiorgan failure characteristic of toxic shock.

Lack of IL-1 receptor antagonistic activity of the capsular F1 antigen of Yersinia pestis.

Because capsular F1 antigen of Yersinia pestis is reported to share sequence homology with interleukin-1 receptor antagonist (IL-1ra), we investigated the potential IL-1 receptor antagonistic activity of F1 on human endothelial cells (EC). The biological activities of IL-1 receptor antagonist (IL-1ra) or IL-1ra-like molecule were measured by its ability to suppress the IL-1beta-mediated induction of adhesion molecules (ICAM and ELAM) on EC and of IL-6 secretion by these cells. Two different, purified, immunogenic and biologically active preparations of F1, at concentrations up to 10-fold higher than that of IL-1ra, did not exhibit any inhibitory activities of IL-1ra. These F1 preparations also did not activate peripheral blood mononuclear cells to produce IL-4 or IL-10, cytokines which might downregulate pro-inflammatory cytokine production in response to infection. Thus, even though there is a high degree of similarity between F1 antigen and IL-1ra in three-dimensional structure by computer modeling and sequence homology, our work indicates that F1 antigen of Y. pestis does not have IL-1ra-like activity.

Differential immune responses to staphylococcal enterotoxin B mutations in a hydrophobic loop dominating the interface with major histocompatibility complex class II receptors.

Bacterial superantigens, such as staphylococcal enterotoxin B (SEB), can trigger acute pathologic effects in humans. A hydrophobic loop on the surface of SEB and other bacterial superantigens, centered around a conserved leucine (L45) residue, is essential for binding to class II major histocompatibility complex molecules. Single residue changes of wild type SEB, designated Q43P, F44P, or L45R, resulted in nonlethal proteins at a dose equivalent to 30 murine LD50 of SEB. Relative to SEB, the mutant proteins did not elevate serum concentrations of proinflammatory cytokines in mice and caused minimal proliferation of human lymphocytes. Anti-SEB titers of mice immunized with Q43P, F44P, L45R, or SEB were similar and protected 77%-100% of animals against a lethal SEB challenge. Levels of toxin-specific IgG1, IgG2a, IgG2b, and IgG3 in mice immunized with SEB, Q43P, or F44P were equivalent, but animals immunized with L45R had significantly elevated levels of IgG2a and IgG2b. Vaccines against staphylococcal superantigens should focus on this critical leucine residue.

Detection of interleukin-6 and interleukin-2 in serum of rhesus monkeys exposed to a nonlethal dose of staphylococcal enterotoxin B.

The immune response to a nonlethal dose of aerosolized staphylococcal enterotoxin B (SEB) was studied in nonhuman primates to define the potential human host response to a nonlethal exposure of SEB on the battlefield. Serum levels of the cytokines interleukin 2 (IL-2) and interleukin 6 (IL-6) increased significantly (p < 0.01) in six juvenile rhesus monkeys 4 hours after receiving a nonlethal, inhaled dose of SEB. The mean (+/-SD) peak serum levels of IL-2 and IL-6 were 63 +/- 39 units/ml and 514 +/- 234 pg/ml, respectively, post-SEB treatment. Tumor necrosis factor, known to be associated with SEB-mediated lethal toxic shock, was undetectable in all samples. gamma-Interferon concentrations were also elevated, but not significantly [p < 0.089]. Hence, elevated levels of IL-2 and IL-6 might be used as a serological marker for a nonlethal, incapacitating exposure to SEB.

Clostridium perfringens enterotoxin lacks superantigenic activity but induces an interleukin-6 response from human peripheral blood mononuclear cells.

We investigated the potential superantigenic properties of Clostridium perfringens enterotoxin (CPE) on human peripheral blood mononuclear cells (PBMC). In contrast to the findings of a previous report (P. Bowness, P. A. H. Moss, H. Tranter, J. I. Bell, and A. J. McMichael, J. Exp. Med. 176:893-896, 1992), two different, biologically active preparations of CPE had no mitogenic effects on PBMC. Furthermore, PBMC incubated with various concentrations of CPE did not elicit interleukin-1, interleukin-2, gamma interferon, or tumor necrosis factor alpha or beta, which are cytokines commonly associated with superantigenic stimulation. However, CPE did cause a dose-related release of interleukin-6 from PBMC cultures.

Staphylococcal enterotoxin B mutants (N23K and F44S): biological effects and vaccine potential in a mouse model.

Superantigens produced by Staphylococcus aureus can cause food poisoning and toxic shock syndrome. The biological activities and vaccine potential of mutant staphylococcal enterotoxin B (SEB) proteins, N23K and F44S, were studied in a lipopolysaccharide-potentiated mouse model. Although 10 micrograms of SEB per mouse is equivalent to 30 LD50, the same intraperitoneal dose of either mutant protein was nonlethal and did not elevate serum levels of tumor necrosis factors (TNF). N23K, F44S, and SEB were serologically identical in an enzyme-linked immunosorbent assay with polyclonal anti-SEB. Immunization with alum containing N23K, F44S, or SEB elicited an anti-SEB response that protected 80-87% of the mice against a 10 micrograms SEB challenge. Controls lacking an anti-SEB titer did not survive. Pooled sera from immunized mice effectively blocked SEB-induced T-cell proliferation in vitro. Naive mice survived a lethal SEB challenge when given pooled antisera 1, 2, or 4 h later, whereas the antisera failed to protect animals when administered 6 or 8 h after the toxin. Lethality at the later times was consistent with increased serum levels of TNF observed 6 h after SEB injection. These studies suggest that the N23K and F44S mutant proteins of SEB are less biologically active than the wild-type toxin, yet retain epitopes useful for eliciting a protective antibody response.

Evidence for protein kinase C pathway in the response of human peripheral blood mononuclear cells to cholera toxin.

Cholera toxin (CT) is a potent mucosal adjuvant and is widely used for vaccine studies in animal models. However, there have been few studies that describe the immunomodulating effects of CT on cells of the human immune system. In this study, the immunomodulatory properties of CT on human peripheral blood mononuclear cells (PBMC) were examined to gain insights to its effects on cells of the human immune system. CT induced production of immunostimulating (IL-1 beta and IL-6) and immunosuppressive (IL-10) cytokines by PBMC. However, the dose-response curve of its cytokine-inducing activity did not correlate well with the concentrations of intracellular cAMP generated by varying doses of CT. the CT mode of action on human PBMC, regarding induction of these cytokines, was clarified by the use of inhibitors of adenyl cyclase, protein kinase A (PKA), and protein kinase C (PKC). 2',3'-Dideoxyadenosine, which inhibits adenyl cyclase activity, reduced IL-1, IL-6, and IL-10 levels by 29, 15, and 28% respectively. HA1004, an inhibitor of PKA, reduced the IL-1 and IL-6 levels by 29 and 27%, respectively. The PKC inhibitor, H7, completely blocked the induction of all three cytokines by CT, suggesting a cAMP-independent mode of action for CT on human PBMC. These observations suggest that CT induces immunomodulating cytokines from human PBMC via the PKC pathway.

Detection of adhesion of superantigen-activated T lymphocytes to human endothelial cells by ELISA.

A sensitive ELISA using monoclonal antibodies (mAb) reactive with surface molecules specific for various leukocytes was devised to measure the adhesion of these cells to cultured monolayers of human umbilical vein endothelial cells. Superantigens, staphylococcal enterotoxin B or toxic shock syndrome toxin 1 were used to activate human peripheral blood mononuclear cells. The extent of adhesion of these cells to endothelial cells was assayed by measuring the optical density produced by a complex of peroxidase-labeled streptavidin, biotin-conjugated F(ab')2 antimouse Ig and monoclonal antibody specific for leukocytes on fixed leukocytic cells that had adhered to endothelial cells. This method was fast and sensitive, and because the detection is by a specific marker on the cell of interest, it can be used in preparations of unseparated mixtures of cells. An increase in adhesion of superantigen-activated CD4+ and CD8- T lymphocytes to endothelial cells may contribute to the pathologic mechanism of superantigens.