Severe bloody diarrhea and subsequent serious post-diarrheal illnesses, including the hemolytic uremic syndrome and central nervous system complications, may develop following infections with Shiga toxin (Stx)-producing bacteria. The cytotoxic actions of Stxs destroy the microvasculature of organs, preventing function. A role for the cytokines tumor necrosis factor-alpha (TNF-[alpha]) and interleukin-1 beta (IL-1[beta]) in exacerbating disease may lie in their ability to up-regulate the Stx receptor, Gb3, on endothelial cell surfaces. A main source of proinflammatory cytokines is the macrophage, thus leading us to utilize the monocytic/macrophage-like cell line, THP-1, as a model for cytokine production in Stx pathogenesis. In addition to treating THP-1 cells with purified Stx1, cells were also treated with lipopolysaccharides (LPS), since bacterial LPS are known to be potent inducers of cytokines, and may be present during infection. Undifferentiated THP-1 cells are sensitive to Stx1 and do not produce TNF-[alpha] or IL-1[beta], while differentiated THP-1 cells, a better model for resident tissue macrophages, are less sensitive to Stx1 and produce TNF-[alpha] and IL-1[beta]. Prolonged expression of TNF-[alpha] mRNA over a 12 h time course experiment led us to inquire whether the extended elevation of transcripts involved Stx1induced mRNA stability. Our data suggest that the presence of Stx1 increases the stabilities of TNF-[alpha] and IL-1[beta] transcripts. In contrast to TNF-[alpha], the level of secreted IL-1[beta] protein does not correlate with the level IL-1[beta] mRNA, suggesting an alteration of post-translational processing and/or secretion of IL-1[beta]. Differentiated THP-1 cells produce chemokines in response to Stx1 and/or LPS treatments. Chemokines may enhance the destruction of tissue cells during an infection by mediating an inflammatory cell influx. Comparison of cytokine and chemokine mRNA and protein kinetics suggests that the regulation of expression may differ between individual cytokines and chemokines. Extension of experimental time courses demonstrated THP-1 cell sensitivity to killing by Stx1, especially in the presence of LPS. Further experiments revealed that undifferentiated and differentiated THP-1 cells were induced to undergo apoptosis following treatment with Stx1, LPS, and Stx1+LPS, and that caspase activation was involved. Collectively, these results allowed us to propose a model of the role of macrophages in Stx1 pathogenesis.
| Identifer | oai:union.ndltd.org:TEXASAandM/oai:repository.tamu.edu:1969/1038 |
| Date | 15 November 2004 |
| Creators | Harrison, Lisa Margaret |
| Contributors | Tesh, Vernon L., McMurray, David N., Samuel, James E., Miranda, Rajesh |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Electronic Dissertation, text |
| Format | 2761574 bytes, 229220 bytes, electronic, application/pdf, text/plain, born digital |
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