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The role of mitogen-activated protein kinases in toxic shock syndrome toxin-1 mediated signalling in THP-1 cells

University of British Columbia

Toxic shock syndrome toxin-1 (TSST-1) is a 22 kDa superantigen produced by Staphylococcus aureus and is the primary cause of toxic shock syndrome (TSS) in humans. The purpose of this project was to determine the effect of mitogen-activated protein (MAP) kinase activity in THP-1 cells prior to the recruitment of T cells. MAP kinases interact with several different kinase pathways and play important roles in many physiological processes including growth and maturation, bacterial pathogen-mediated signalling and cytokine-induced events. THP-1 cells constitutively expresses the TSST-1 binding human leukocyte antigen-DR (HLA-DR) molecule which we have shown to be upregulated with 100 U/ml IFN-γ. Initial experiments set pathophysiological conditions for TSST-1 (MN8) and lipolysaccharide (LPS) mediated signalling in pro-monocytic THP-1 cells. To induce tumor necrosis factor-α (TNF-α) release, TSST-1 (1 ng/ml) required the presence of both THP-1 and T cells in co-culture. The constitutive levels of HLA-DR on THP-1 cells were sufficient to bind to TSST-1 and present SAg to T cells in a manner which induced TNF-α release. Under these condition, TSST-1 induced the release of TNF-α within 12 hours (n=3). This TSST-1-induced TNF-α release was sustained for up to 84 hours after treatment. In contrast, LPS (1 ug/ml) was sufficient to induce the transient release (12 hours) of TNF-α from THP-1 cells alone (n=3). The addition of IFN-γ (100 U/ml) and LPS to THP-1 culture induced the sustained release of TNF-α from these cells. To screen for early MAP kinase activation in THP-1 cells alone, THP-1 cells were then treated with 1 ng/ml TSST-1, 1 nM phorbol 12- myristate 13-acetate (PMA) or RPMI 1640 and lysed within 30 minutes (n=3). Cellular proteins were separated by anion exchange chromatography and assayed for MAP kinase activity using myelin basic protein (MBP) kinase assays. Both TSST-1 and PMA induced a two-fold peak increase in THP-1 MBP kinase activity, at 30 minutes and 5 minutes respectively. The presence of other non-ERK-1/2 kinase activities in these anion exchange column eluents indicate that non-ERK-1/2 MBP kinases may also play an important role in TSST-1-induced signalling in monocytic cells. THP-1 cells were treated with TSST-1 (1 ng/ml), LPS (1 ug/ml), RPMI 1640 and a TSST-1 mutant (G31Rmut) (1 ng/ml) and lysed within 30 minutes (n=6). Extracellular-signal regulated kinase-1 (ERK-1) and ERK-2 isoforms of MAP kinase were immunoprecipitated and assayed with MBP kinase assays. When the median of areas under ERK-2 activation curves were compared, G31Rmut TSST-1 induced an ERK-2 activity profile that favored activation over deactivation (Wilcoxin signed rank test, P= 0.0313, n=6). In contrast, TSST-1 and LPS induced early ERK-2 activity profiles that favored neither activation nor deactivation. All three toxins also induced the early activation of ERK-1 but none of these profiles favored activation over deactivation. The differences between G31Rmut TSST-1 and TSST-1 induced ERK-2 activation patterns are probably due to the lowered binding affinity that G31Rmut TSST-1 has to MHC II and may reflect the physiological differences reported in cultures treated with both toxins. In comparison, the similarities in LPS and TSST-1-induced ERK-1/2 signalling may be due to the induction of common pathways in monocytic cells. In the future, it is hoped that this model can be used to study MAP kinase activation in THP-1 and T cell co-cultures.

Thesis/Dissertation

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2009-05-23

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http://hdl.handle.net/2429/8075

Pathology and Laboratory Medicine

University of British Columbia

UBCV

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