F. Homo

Calcium and A23187-induced cytolysis of mouse thymocytes

Abstract The cytotoxic effects of ionophore A 23187 were studied in parallel with its action on calcium uptake in isolated mouse thymocytes. Under conditions where the cells were preincubated in a calcium-containing medium prior to ionophore treatment a close relationship could be observed between the extent of cell lysis and the stimulation of calcium uptake in the presence of A 23187 . In addition, increasing concentrations of calcium ions in the incubation medium lead to a pronounced decrease of cell viability and to a stimulation of calcium uptake suggesting that calcium is critical for cell survival.

Glucocorticoid sensitive and resistant cell populations in the mouse thymus

Abstract We have studied in parallel the effects of dexamethasone on in vitro incorporation of [ 3 H]-uridine and [ 3 H]-thymidine and on cell viability in thymocyte subpopulations isolated by centrifugation on a discontinuous bovine serum albumin gradient from intact, adrenalectomized and hydrocortisone-treated mice. In cell populations isolated from intact and adrenalectomized animals, the steroid induces both inhibition of precursor incorporation and a decrease in cell viability, whereas cells isolated from hydrocortisone-treated mice show marked inhibition of precursor incorporation but no cell death after 24 h. Our present results suggest that differences in steroid sensitivity are not directly related to variations in cell receptor content but may be associated with cell differentiation.

Glucocorticoid receptors and their functions in lymphocytes.

Abstract Most, if not all. the effects of glucocorticoid hormones on lymphoid tissue are thought to be mediated by an initial step of interaction of the steroid with specific cytoplasmic receptors. We have presented results, obtained using whole cell suspensions of mouse thymocytes. showing however that the number of receptors does not accurately reflect the steroid sensitivity of the target cell: both inhibitory effects of steroid on nucleic acid precursor incorporation and steroid-induced cell lysis are not obligatorily related to receptor levels but more likely to cell differentiation. We have therefore also investigated the mechanism of the immunosuppressive effects of steroids and the role of various factors which could be involved in the proliferation or lysis of mouse thymocytes. Dexamethasone and 25-OH cholesterol are the most potent agents, capable of inhibiting cholesterol biosynthesis in non stimulated thymocytes. Although the effect of dexamethasone. which is blocked by actinomycin D. appears to be mediated through receptor occupancy, the action of 25-OH cholesterol is likely to be due to an inhibition of HMG COA reductase activity. Mitogen induced lymphoblastic transformation of thymocytes is blocked by low concentration of dexamethasone but also by sex steroids at higher concentrations. These compounds, which exert immunosuppressive effects in vivo, inhibit concanavalin A induced transformation at concentrations above 10−5 M, at which concentration they also elicit a rapid and transient decrease of uridine uptake in non stimulated thymocytes. In addition, calcium, which has been considered to play an important role in the mechanism of steroid-induced lysis, appears highly toxic by itself.

Human lymphocyte subpopulations: Effects of glucocorticoids in vitro

Abstract Glucocorticoid receptor levels and steroid induced inhibition of nucleic acid precursors incorporation have been determined in circulating lymphocytes of healthy donors on subpopulations isolated by immunoabsorbant column and density gradient centrifugation. The number of steroid binding sites and the affinities of the receptors for the tracer are not significantly different in B and T + Null cells fractions. [ 3 H]-uridine incorporation tends to be in general more sensitive to steroid action in B cells than in T + Null cells, whereas the situation is reversed for [ 3 H]-thymidine.

Early effect of steroids on 45calcium uptake by mouse thymocytes

Abstract The effects of various steroids on 45 calcium uptake were investigated in parallel with the viability of mouse thymocytes. Dexamethasone, a synthetic compound with glucocorticoid activity, induced a rapid increase in membrane permeability to calcium. This effect was still measurable using 10 −7 M dexamethasone and appeared specific for compounds with glucocorticoid potency. In addition, calcium efflux from prelabeled cells was not altered in the presence of dexamethasone, indicating an increased total cell concentration. It is therefore suggested that calcium ions play a role in steroid-induced cell lysis.

Effect of glucocorticoids on cholesterol synthesis in isolated mouse thymocytes

Abstract Glucosteroids, as well as oxygenated derivatives of cholesterol, have been demonstrated to inhibit cholesterol biosynthesis in stimulated lymphocytes and Hela cells. We have therefore measured the effect of various steroids on the incorporation of [ 3 H]-acetate into digitonin-precipitable material in mouse thymocytes. Dexamethasone, induces a marked inhibition of cholesterol synthesis (40–50% inhibition after 6 h incubation in the presence of 10 −6 M dexamethasone). This inhibitory effect appears specific of glucocorticoids as the other steroids tested (testosterone, estradiol, progesterone) are minimally effective. Moreover, the decrease in cholesterol synthesis produced by dexamethasone is abolished in the presence of Actinomycin D suggesting that the effects of steroids on cholesterol synthesis are mediated via glucocorticoid receptor occupancy and macromolecular synthesis. In contrast, human circulating lymphocytes and corticoresistant thymocytes do not show a significant inhibition of cholesterol synthesis in the presence of dexamethasone. As the rate of cholesterol synthesis is considered to represent a major factor controlling cell proliferation, our results suggest that part of the effects of glucosteroids on cell growth could result from an inhibition of cholesterol synthesis.

Effect of dexamethasone on non-esterified fatty acid metabolism in isolated mouse thymocytes

Abstract It has been postulated that the cytolytic action exerted by glucocorticoids on lymphoid cells could be the consequence of a steroid-induced accumulation within the cells of cytotoxic compounds, namely free fatty acids. In order to investigate this possibility we have determined in isolated mouse thymocytes the action of dexamethasone on cell fatty acid content and on both fatty acid turnover and de novo fatty acid synthesis. Our results show that neither fatty acid content nor fatty acid turnover is significantly modified during in vitro incubation in the presence of steroid. In addition, dexamethasone induces a marked inhibition of the incorporation of [ 3 H]-acetate into lipids, including free fatty acid pools. It appears therefore that, in vitro , the cytolytic effect of glucocorticoids is not due to free fatty acid accumulation. However, in vivo injection of hydrocortisone leads to an increase of fatty acid content within the whole thymus gland. As we have also demonstrated a cytotoxic action of exogenous fatty acids on isolated thymocytes, we suggest that steroids could exert an indirect action on lymphoid cells by promoting fatty acid release from other sources.

Effect of 25-hydroxycholesterol and dexamethasone on sterol synthesis in isolated mouse thymocytes

Abstract Oxygenated derivatives of cholesterol, such as 25-OH-cholesterol, are potent inhibitors of cholesterol synthesis in various cell types. Although the main site of 25-OH-cholesterol action appears to be mediated by the inhibition of the enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, one of the key steps in the pathway of cholesterol formation, there is no indication on the molecular mechanism leading to enzyme inhibition. We have therefore compared in isolated mouse thymocytes the mode of action of 25-OH-cholesterol to that of dexamethasone which has also been demonstrated to block cholesterol synthesis. 25-OH-cholesterol induced a rapid, dose dependent, inhibition of [ 3 H]acetate incorporation into the sterol fraction. This effect was specific for the cholesterol biosynthetic pathway and, in contrast to dexamethasone, did not affect either lipid synthesis or RNA formation. Unlike glucocorticoid action, the effect of 25-OH-cholesterol was not suppressed in the presence of actinomycin D and did not appear to be mediated via an interaction with specific receptors. It thus appears that the mechanism of action of 25-OH-cholesterol on cholesterol biosynthesis is not comparable to that of steroid hormones.

Glucocorticoid receptors and hormonal responsiveness of human blood and bone-marrow cells in non-lymphocytic leukemia

Abstract In man, acute non lymphoid leukemia (ANLL) bas been shown to respond in an extremely variable manner to glucocorticoid therapy. In addition to glucocorticoids. other agents such as β-adrenergic agents and prostaglandins are known to modulate the proliferation and differentiation of granulocyte monocyte colonies (CFU-C). We have measured in vitro on the blood and bone-marrow cells from three patients with ANLL several parameters including glucocorticoid receptors, the percentage of cells in the S phase of the cell cycle (using pulse cytophotometry) and the effect of drugs on nucleoside incorporation, cell number and viability. Our results led to the following conclusions: (i) The examination of blood and bone-marrow samples of a given patient does not necessarily give similar results in terms of response to drugs and the level of glucocorticoid receptors, (ii) in the same patient, the effect of any agent varies greatly throughout the incubation period and is also variable from one subpopulation to another, (iii) Prostaglandin E 2 markedly enhanced the level of [ 3 H]-thymidine incorporation in the patients tested. As steroid-induced prostaglandin production has been recently implicated in the differentiation of myeloid cells, this may explain the stimulatory action of glucocorticoids which has been observed both in vivo and in vitro in ANLL patients.

In vitro hormonal responsiveness of human blood and bone marrow cells in non-lymphocytic leukemia

Abstract Glucocorticoid, β-adrenergic agents and prostaglandin E 2 are all known to modulate the proliferation and differentiation of granulocyte-monocyte colonies (CFU-C). However, in man, acute non lymphoid leukemia (ANLL) has been shown to respond in a highly variable manner to glucocorticoid therapy. Therefore, we have measured in vitro on blood and bone-marrow cells of two patients with ANLL several parameters including glucocorticoid receptors, percentage of cells in the S phase of the cell-cycle, effects of drugs on nucleoside incorporation, cell number and viability. Our results led to the following conclusions: (i) the examination of blood and bone marrow samples in a given patient does not necessarily give similar results in terms of response to drugs and levels of glucocorticoid receptors, (ii) in the same patient, the effect of any agent varies greatly throughout the incubation period and is also variable from one cell subpopulation to another, (iii) prostaglandin E 2 markedly enhances the level of [ 3 H]-thymidine incorporation in the two patients tested. As steroid-induced prostaglandin production has been recently implicated in the differentiation of myeloid cells, this may explain the stimulatory action of glucocorticoids demonstrated both in vivo and in vitro in ANLL patients.