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Title: The effects of thapsigargin on lymphocyte activation.
Author: Fenton, Mandy.
Awarding Body: University of Sussex
Current Institution: University of Sussex
Date of Award: 1995
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Thapsigargin is a sesquiterpene lactone which was isolated from the plant Thapsia gargantica. It has been used in studies investigating intracellular calcium pools and calcium signalling as it causes a rise in intracellular calcium concentration. It inhibits the endoplasmic/sarcoplasmic reticulum family of calcium-ATPases and allows calcium to leak out of intracellular stores followed by a sustained influx of extracellular calcium. Porcine lymphocytes are used throughout this thesis due to their ready availability and as they have been used in this laboratory for many years as a model for human lymphocyte activation. In this study, the effects of thapsigargin on the activation of porcine and human peripheral blood mononuclear cells in vitro was studied and compared with those of the calcium ionophore, ionomycin and an alternative endoplasmic/sarcoplasmic reticulum calcium-ATPase inhibitor, 2,5-di-(tert-butyl)-1,4-benzohydroquinone. The drugs were also used in combination with the phorbol ester, phorbol myristate acetate, used to activate protein kinase C, and the immunosuppressive drug, FK506, which inhibits calcium dependent T cell activation. Thapsigargin was found to raise intracellular calcium concentration in both porcine and human cells in a concentration-dependent manner but was more effective in human than in porcine cells. In porcine cells, thapsigargin raised intracellular calcium concentration to a similar level as the mitogenic lectin conconavalin A but not to as high a level as ionomycin or 2,5-Di-(tent-butyl)-1,4-benzohydroquinone. In human cells, thapsigargin was as effective as both ionotnycin and 2,5-Di-(tert butyl)-1,4-benzohydroquinone. Neither phorbol myristate acetate nor FK506 had any effect on the rise in cytoplasmic calcium concentration induced by thapsigargin in porcine cells. As expected, all three of the drugs used to raise intracellular calcium concentration in combination with phorbol myristate acetate were found to be mitogenic for both human and porcine cells. They induced DNA synthesis at 48 hours post stimulation in a concentration dependent manner. Thapsigargin was found to induce DNA synthesis at a lower cytoplasmic calcium concentration than either of the alternative calcium mobilisers in porcine cells. FK506 inhibited the DNA synthesis induced by ionomycin/ phorbol myristate acetate and 2,5-Di-(tert-butyl)-1,4-benzohydroquinone/phorbol myristate acetate in both human and porcine cells and DNA synthesis induced by thapsigargin/phorbol myristate acetate in human cells. However, FK506 enhanced DNA synthesis in porcine cells that had been exposed to an antagonistic combination of thapsigargin and phorbol myristate acetate. This result was surprising as FK506 is expected to inhibit calciumdependent activation via the inhibition of the calcium-dependent protein phosphatase, calcineurin. On further investigation it was found that the porcine cells incubated with thapsigargin, phorbol myristate acetate and FK506 were able to proliferate in the absence of measurable levels of the cytokine interleukin 2. Furthermore, calcineurin activation was inhibited in these cells. It was also found that combinations of thapsigargin, phorbol myristate acetate and/or FK506 did not affect activation of some of the protein kinases believed to be involved in T cell activation in any different way from the alternative mitogenic combinations used in this study. The effects of thapsigargin and other mitogens on apoptosis in porcine and human peripheral blood mononuclear cells were also investigated. lt was found that a very high proportion of resting porcine cells in culture were apoptotic and that there were fewer apoptotic cells in proliferating cultures. There were fewer apoptotic cells in resting human cell cultures and all combinations of mitogenic or immunosuppressive drugs increased the proportion of apoptotic human cells. In addition, DNA synthesis was occurring in only a small proportion of the population of stimulated human cells regardless of the mitogen(s) used. The data presented show that the effects of thapsigargin on cytoplasmic calcium concentration and/or the effects of phorbol myristate acetate on protein kinase C activation cannot entirely account for the results and that the activation of porcine peripheral blood mononuclear cells does not represent an entirely accurate model of human lymphocyte activation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: T cells Biochemistry