| Summary: | 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. === Medicine, Faculty of === Pathology and Laboratory Medicine, Department of === Graduate
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