Aoptosis and activation of lymphocytes: a flow cytometic study

Flow cytometry is now a widespread methodology for investigations of the immune system. The aim of this thesis was to examine the use of flow cytometry for the study of lymphocyte activation and, in particular, its suitability for the study of apoptosis. Firstly, investigations entailed the assessment of flow cytometric techniques for the analysis of lymphocyte activation. Multiparametric procedures combining DNA content analysis with other parameters, such as protein content and surface membrane antigens, were investigated. Secondly, DNA content analysis was assessed for the flow cytometric study of apoptosis and necrosis, as the distinction between these two forms of cell death is now recognised as being important in understanding mechanisms regulating cell survival. Recent reports have indicated that DNA of apoptotic cells has decreased fluorescence compared with that of viable cells, when stained with propidium iodide (Pl). This phenomenon was investigated in several models of apoptosis and one of necrosis using a detergent lysis method for the simultaneous analysis of DNA content and ninety degree light scatter (90LS). Lastly, this method was then applied to the study of apoptosis of lymphocytes from individuals infected with human immunodeficiency virus (HIV). In the evaluation of simultaneous DNA and protein content analysis, it was demonstrated that a cell lysis technique was superior to one using ethanol fixation to permeabilise the cells for staining. The inclusion of RNase in the staining solution improved staining, demonstrated by lower coefficient of variance (CV) values of GJG1 peaks. In addition it was shown that the use of blocking agents, such as colcemid or tritiated thymidine, which cause an accumulation of cells in the late stages of the cell cycle, could be useful to identify responsive cells. In the evaluation of a detergent lysis technique for the analysis of DNA content, it was shown that staining was affected by RNase and incubation temperature, but not Triton-Xl00 concentration. It was demonstrated that it was possible to use this method for the study of apoptosis. The characteristic ladder pattern of apoptosis was observed following electrophoresis of DNA extracted from flow cytometric sorted populations with decreased PI staining. In the evaluation of simultaneous DNA content and surface membrane antigen analysis, it was shown that performing cell fixation and DNA staining after membrane antigen staining, prevented a notable decrease in fluorescence of membrane antigen staining. Significant differences in the PI staining pattern and a shift in 90LS were observed when apoptotic death occurred at different stages of the cell cycle. Importantly, such differences only allowed accurate quantification of apoptosis when it occurred in GofG1. While necrosis could be distinguished from apoptosis when examined during its early stages, a similar staining pattern to that found with apoptosis was observed when necrosis was examined during its latter stages. The results indicate that the measurement of DNA staining cannot be exclusively relied upon to detect apoptosis occurring in all models. However it is useful in the investigation of this process when the death occurs in GofG1, as the method offers a rapid means for quantification. Lymphocytes from HIV-infected individuals, cultured in a serum free system, underwent significant levels of apoptosis, as demonstrated by flow cytometry. After 24 h in culture, on average, 22.5% of lymphocytes died when cultured with anti-CD3 antibody OKT3, and 25.3% when cultured without OKT3. Apoptosis was significantly reduced (p=0.000) when human recombinant interleukin-2 (IL-2) was included in the culture system (to 14.9% when cultured with OKT3 and to 12.8% when cultured without OKT3). A number of recent reports has proposed apoptosis to be the mechanism involved in the depletion of CD4 T -cells which occurs in HIV infection. These results provide further evidence that CD4 T -cells are depleted by an apoptotic process in HIV infection. Furthermore, they show that IL-2 may be important in the control of this cell loss. The findings of this study reported in the thesis have highlighted the analytical benefits gained by using flow cytometry to investigate lymphocyte activation and apoptosis. The ability to simultaneously examine several cellular features in a rapid, precise manner has enabled further insights into these two important mechanisms and their role within the immune system