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UBC Theses and Dissertations

The mechanism of human monocyte activation by staphylococcal toxic shock syndrome toxin-1 (TSST-1) See, Raymond Hugh


Toxic shock syndrome (TSS) is a multisystem disease associated with staphylococcal TSS toxin—1 (TSST-1). TSST—i and the related staphylococcal enterotoxins (SE) A, B, C, D, and E have a number of biological effects on human peripheral blood mononuclear cells (PBMC), including T lymphocyte mitogenicity and induction of IL—1 and TNF from human monocytes. The aim of this thesis is to examine the molecular mechanisms of human monocyte activation by TSST—1 and SE. To gain insight into the mechanism of TSST—i and SEA activation of PBMC, receptor binding assays were performed with ¹²⁵I—labeled toxins. Scatchard analyses revealed similar numbers of receptors and dissociation constants for TSST—l and SEA on human PBMC and on purified monocytes. SEA, but not SEB, SEC, SED or SEE significantly inhibited binding of ¹²⁵I—TSST—1 to PBMC. Crosscompetition between TSST-i and SEA in binding assays suggested that they may be binding to overlapping epitopes on the same receptor. Affinity cross-linking of ¹²⁵I—labeled TSST—1 and SEA to human blood monocytes showed the presence of 2 membrane subunits consistent with the 35 kd alpha and 28 kd beta chains of human HLA—DR. The anti—HLA-DR mb, L243, inhibited radiolabeled TSST—1 and SEA binding to human monocytes and neutralized monocyte—dependent T cell mitogenicity of both toxins, adding further support that HLA—DR is the major receptor. Based on these studies and those of others who demonstrated overlapping receptor epitopes for SEA and SEB (Fraser, Nature 339:221—223, 1989) and distinct epitopes for TSST—1 and SEB (Scholl et al., J. Immunol 143:2583—2588, 1989), we postulate that SEA occupies a binding site within HLA-DR that partially overlaps with both TSST-l and SEB. The role of protein phosphorylation in the activation of normal human monocytes by TSST-l and SE was examined by two—dimensional gel electrophoresis. Examination of ³²P—orthophosphate—labeled monocytes showed that within 5 mm, TSST—1 consistently stimulated the dephosphorylation of several phosphoprotemns in a dose—dependent manner. In contrast, neither SEA nor SEB induced this dephosphorylation pattern, but instead, increased the phosphorylation of a different set of proteins. Phosphorylation patterns induced by two other monocyte agonists, PMA and bacterial LPS, demonstrated little similarity to those induced by TSST—l. Moreover, using an anti-phosphotyrosine mAb, TSST-1 and SE were shown to stimulate the tyrosine—specific phosphorylation of several cytosolic proteins that were distinct from those induced by PMA. This suggests that tyrosine phosphorylation induced by TSST—l or SEA is not mediated by activation of protein kinase C. Collectively, the data suggest that the early intracellular signal transduction pathways utilized by TSST—l, SE, LPS and PMA in monocytes are dissimilar despite common biological consequences such as lymphocyte mitogenesis and cytokine induction. TSST—1 was also tested for its ability to induce the cytokines, IL-i and TNF, from fractionated human PBMC. Highly purified monocytes alone or T lymphocytes alone did not produce IL—lB or TNFa when incubated with TSST—l for up to 72 h. However, TSST—l added to a 1:1 ratio of monocytes and T lymphocytes resulted in significant extracellular TNFa and IL—lB production at 24 h. The nature of the monocyte/T cell interaction did not involve IFN-i- but did require direct cell contact between metabolically active monocytes and T lymphocytes. Furthermore, TSST—l—mediated monocyte/T cell interaction also involved LFA—l since mAbs to this adhesion molecule significantly reduced cytokine secretion. Finally, the functional relevance of protein kinases in cytokine production by TSST—l-stirnulated monocyte/T lymphocyte co—cultures was explored. IL—lB secretion was suppressed by inhibitors of protein kinase C (H7), tyrosine kinases (genistein) and cAMP— and cGMP—dependent kinases (HA1004). In contrast, secretion of TNFa was blocked by only H7 and genistein, suggesting that induction of these two cytokines is differentially regulated. In conclusion, our data are consistent with a superantigen role for both TSST—i and SE and indicate that TSS pathogenesis occurs as a result of TSST-l interaction with both monocytes and T lymphocytes. Further studies focusing on the mechanism of cell activation by this toxin will not only enhance our knowledge of superantigens in general, but will also aid in our understanding of other bacterial toxin—mediated diseases.

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