Inhibition of human lymphocyte stimulation by steroid hormones: Cytokinetic mechanisms

The steroid hormones estradiol, progesterone and testosterone, in addition to cortisol, inhibited stimulation of human peripheral blood lymphocytes by phytohaemagglutinin (PHA) and Con A. This effect upon lymphocyte transformation was assayed by three methods: quantitation of [³H]thymidine incorporation into acid precipitable material, microscopic assessment of blastic transformation and determination of the labelling index. Addition of steroid hormones at the initiation of culture resulted in a marked inhibition in all 3 parameters, which was observed with lower concentrations of cortisol than the other hormones. The inhibition was not attributable to cell death and could be partially reversed by removing hormones from the incubation medium after culture for 48-72 hours. Late addition of steroid hormones, 52 hr after addition of mitogen and 18 hr prior to pulse labelling with [³H]thymidine, also resulted in reduced [³H]thymidine incorporation, accompanied by a nearly 50% reduction in the labelling indices and only a minimal decrease in the per cent transformed cells. Inhibition of lymphocyte stimulation by steroid hormones operates by the following cytokinetic mechanisms: (1) suppressed recruitment of cells from G₀ to G₁ phase of the cell cycle, as indicated by the diminished per cent blasts; (2) inhibition of progression from G₁ phase into S phase, as evidenced by the reduced ratio [labelling index/blasts]; and, in the case of estradiol and progesterone, (3) reduced rate of DNA replication or altered intracellular [³H]thymidine specific activity, as shown by the decreased ([³H]thymidine incorporation/labelling index) ratio. Late addition of steroid hormones to stimulated cultures reduced the per cent of cells in S phase, but did not revert previously transformed cycling lymphocytes to the G₀ state.