The effect of hydrocortisone on the incorporation of tritiated thymidine by human blood lymphocytes cultured with phytohaemagglutinin and pokeweed mitogen

Abstract

Methods based on [³H]thymidine incorporation and morphology were used for further studies on the effect of hydrocortisone on the transformation of human lymphocytes by phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Result obtained with both methods showed inhibition of PHA stimulation in cultures preincubated with 0·01–10 μg/ml of hydrocortisone. [³H]thymidine incorporation due to PWM in vertical tube cultures was depressed by 1·0 μg/ml of hydrocortisone in cultures harvested at 48 hr, but not in similar cultures incubated for 3, 4 and 5 days. In vertical cultures 10 μg/ml of hormone depressed the level of uptake in most experiments. By contrast [³H]thymidine incorporation was enhanced in horizontal PWM cultures by 0·1 and 1·0 μg/ml of hydrocortisone, and 10 μg/ml caused stimulation or a return to the normal PWM level.

Microscopic and time-lapse observations on living cells showed that typical PWM blasts first appeared on the third day of incubation and after 5 days were numerous in corticosteroid-treated as well as in untreated cultures. Ten micrograms per millilitre of hydrocortisone increased the fragility of PWM blasts in both kinds of cultures and caused degeneration of variable numbers of blasts in 5-day-old vertical tube cultures. The depression of [³H]thymidine uptake in vertical cultures was thought to be due to a combination of enhanced toxicity of corticosteroid hormone in deep cultures, and loss of incorporated [³H]thymidine due to increased cellular fragility. Results obtained with the isotopic and morphologic methods indicated that the transformation of B lymphocytes by PWM is relatively resistant to the action of hydrocortisone in vitro.