The effect of the ratio of CD4+ to CD8+ T-cells on radiation-induced apoptosis in human lymphocyte subpopulations

Purpose: Apoptosis occurs spontaneously in cultured human peripheral blood lymphocytes but is enhanced by exposure to ionizing radiation. Subpopulations of lymphocytes are known to have varying radiosensitivities to radiation-induced apoptosis. The purpose of this study was to examine the radiation-induced apoptotic response of CD4+ and CD8+ T-cells incubated as a complete lymphocyte population. Materials and Methods: Using a four-colour flow-cytometry method, which measures annexin-V binding to phosphatidyl serine and propidium iodide, spontaneous and radiation-induced apoptosis was measured in the total lymphocyte fraction and in CD4+ and CD8+ T-cell subpopulations. Results: It was found that CD8+ T-cells were more sensitive to radiation-induced apoptosis than CD4+ T-cells at doses up to 2Gy. The yield of radiation-induced apoptosis in the total lymphocyte fraction decreased with increasing ratios of CD4+ to CD8+ T-cells (CD4/CD8 ratio). By manipulating the CD4/CD8 ratio within lymphocyte cultures, it was found that the CD4/CD8 ratio had a dramatic effect on the yield of spontaneous apoptosis of total lymphocytes fraction and CD4+ T-cells but not CD8+ T-cells. Conclusion: The CD4/CD8 ratio affects the apoptotic response of human lymphocytes and CD4+ T-cells

Silver-stained `comets' for the detection of radiation-induced apoptosis: Automated assay

This paper reviews the development of a bioassay that uses apoptosis (a form of cell death) to directly measure the response of cells to a physical or chemical insult. The assay described here is in mid-phase of development and has been applied to various cell lines isolated from humans and mice. Under the assay conditions, apoptotic cells present a 'signature', or morphology, distinct from other cells. The exposure of cells to ionizing radiation, UV light or some chemicals, produces a dose-dependent increase in the apoptotic fraction. The dose response is linear up to about 1 Gy of ionizing radiation. A preparation technique for the cells is used that produces a permanent record of the experiment. With automation, the statistical power of the assay is substantial since there are potentially 10 5 cells that can be examined at each data point. A prototype system is described, it consists of a brightfield microscope and a computer controlled microscope stage integrated with preliminary image analysis software. Trials indicate that 200-400 cells per minute can be assessed for 'signatures' characteristic of apoptotic and unaffected has also begun to determine if the assay can be used to predict the relative radiosensitivities of normal and tumour cells, information that could be used to assist in the planning of dosing schedules for radiotherapy

Analysis of radiation-induced apoptosis in human lymphocytes: Flow cytometry using Annexin V and propidium iodide ver

Background: The neutral comet assay was devised to measure double-stranded DNA breaks, but it has also been used to measure apoptosis based on its characteristic DNA fragmentation patterns. There is still uncertainty about the reliability of this method. By comparing the comet assay with a flow cytometry method that uses Annexin V binding to apoptotic cells, we have provided further evidence for evaluating the usefulness of the comet assay for detecting apoptosis. Methods: Apoptosis was induced in human peripheral blood mononuclear cells (PBMC) by ionizing radiation and measured using the comet assay and a flow cytometry method that measures Annexin V and propidium iodide (PI) staining. Results: The Annexin V flow cytometry assay distinguished among early apoptosis, late apoptosis, and an apoptotic or necrotic phase in which the cells were labeled with both Annexin V and PI. The comet assay detected only the latter two phases of apoptosis. Conclusions: The comet assay is a useful tool for measuring the late stages of apoptosis whereas the Annexin V assay measures higher amounts of apoptosis because it can detect cells in an earlier stage of the apoptotic pathway

Differential apoptotic response to ionizing radiation in subpopulations of human white blood cells

The purpose of this paper is to characterize the apoptotic response of various subpopulations of human white blood cells after in vitro exposure to ionizing radiation using the modified neutral comet assay (MNCA). White blood cells, isolated from human whole blood, were fractionated into granulocytes and mononuclear cells which were further separated into B-cells, natural killer (NK) cells, and CD4+ and CD8+ T-cells. The separated fractions were exposed to low doses of X-rays and then MNCA was used to measure the apoptotic fraction (AF) at different time points in irradiated and unirradiated aliquots of sorted cultures. The spontaneous AF in unirradiated control cells was the most critical determinant of whether an apoptotic response could be detected in irradiated cells. When cultured in isolation granulocytes and B-cells had the highest background AF, with NK cells having the next highest. CD4+ and CD8+ T-cells had a low, stable, spontaneous AF which gave them the highest signal-to-noise ratio. Although B-cells demonstrated the highest radiation-induced apoptotic response to 1Gy of X-rays, CD8+ T-cells were the most radiation-responsive lymphocytes due to their low spontaneous AF. By generating dose response curves for CD4+ and CD8+ T-cells, the sensitivity of the MNCA for detecting apoptosis in these two cell types was also examined