S. Durant

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.

In vivo effects of catecholamines and glucocorticoids on mouse thymic cAMP content and thymolysis

In vivo administration of the beta-adrenergic agonist, isoproterenol, induced a rapid, dose-dependent increase in the mouse thymic cyclic AMP (cAMP) content. Hydrocortisone (1 mg/animal), at a concentration which by itself did not alter the cAMP content of the thymus, markedly potentiated the effect of isoproterenol (5 micrograms/animal). Isoproterenol or hydrocortisone treatment led to a significant decrease in thymic weight and an even greater decrease in thymocyte number. In addition, the simultaneous administration of both agents produced additive effects on thymic atrophy. It appears from these results that glucocorticoids and catecholamines exert a negative control on the thymic size by increasing the programmed cell death of some cell subpopulations. Thus, glucocorticoids and catecholamines, either alone or in association, may influence the immune system under physiological or pathophysiological conditions.

Glucocorticoids in the nonobese diabetic (NOD) mouse: Basal serum levels, effect of endocrine manipulation and immobilization stress

The NOD mouse is a recognized model for studying immunologically mediated insulin-dependent diabetes mellitus (IDDM). In most colonies, the disease appears with a greater preponderance in females than in males and castration alters the expression of the disease. The prevalence of diabetes may also vary depending upon environmental factors such as stress. Therefore, we measured in the NOD mouse serum glucocorticoid concentrations in basal and stress conditions. We observed in NOD as well as in C57BL/6 mice, taken as controls, a circadian rhythm of corticosterone, with females having higher values than males. After a single restraint stress, female and male NOD mice exhibit a comparable response, whereas after repeated stress, males respond significantly less than females, suggesting an adaptation phenomenon. In contrast, there is no difference in the pattern of corticosterone response of C57BL/6 females and males to both types of stress, but females always respond better than males. Moreover, whatever the stress considered, NOD mice generally exhibit a higher corticosterone response than C57BL/6 mice. The sexual dimorphism in diabetes expression in NOD mice may be related to the levels of corticosterone, a hyperglycemic hormone, in both basal and stress conditions. However, the understanding of corticosteroid effects in this model of type I IDDM is rather complex given their well known anti-inflammatory and immunosuppressive effects in other models of autoimmune diseases.

Effect of antiglucocorticoids on dexamethasone-induced inhibition of uridine incorporation and cell lysis in isolated mouse thymocytes

We have compared in isolated mouse thymocytes the action of progesterone, cortexolone, DXH (a 17-beta carboxamide derivative of dexamethasone) and RU 38486 (a new antiglucocorticoid molecule), on dexamethasone-induced inhibition of uridine incorporation and cell lysis, with the affinities of these drugs for glucocorticoid receptors. Our results show that progesterone, cortexolone and DXH which possess similar affinities for glucocorticoid receptors may exhibit variable, weak agonist and antagonist activities according to the parameter studied. RU 38486 was a potent competitor of dexamethasone and was able, when present in a 10-fold excess, to counteract almost completely the inhibitory action as well as the lytic action of 5 X 10(-8) M dexamethasone. This compound which exerts almost no agonist activity may therefore represent a useful tool to investigate the mode of action of antiglucocorticoids.

Effect of exogenous prostaglandins and nonsteroidal anti-inflammatory agents on prostaglandin secretion and proliferation of mouse embryo fibroblasts in culture

During wound healing, the positive and negative modulation of fibroblast proliferation may be due, in part, to the high prostaglandin concentration of the inflammatory exudates. In vitro, PGF2 alpha has been shown to stimulate, whereas PGE2 inhibits, the growth of different fibroblast cell lines. Therefore, we have investigated the effect of exogenous prostaglandins (PGs) and of various nonsteroidal anti-inflammatory drugs (NSAIDs) on the proliferation and the prostaglandin (PG) synthesis of normal mouse embryo fibroblasts. PGF2 alpha, 6-keto PGF1 alpha and PGE2 increase fibroblast proliferation. On the other hand, PGF2 alpha increases the synthesis of PGE2 and 6-keto PGF1 alpha while 6-keto PGF1 alpha solely inhibits PGF2 alpha release, PGE2 being inactive. The mouse embryo fibroblasts partially transform the prodrug sulindac sulfoxide in the sulfide form, which completely inhibits PG synthesis, as does indomethacin. In contrast, ibuprofen exerts a differential action, according to the type of PG measured. Among the NSAIDs tested, only sulindac (sulfoxide or sulfide) stimulates fibroblast proliferation and this effect appears independent of an alteration of PG synthesis. Therefore, in this model of normal mouse embryo fibroblasts, while endogenous prostaglandins are not involved in the control of cell proliferation, exogenous PGs have the ability to alter fibroblast growth and PG synthesis.

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.

Attempts to pharmacologically modulate prolactin levels and type 1 autoimmune diabetes in the non-obese diabetic (NOD) mouse

Prolactin (PRL) is well known for its stimulatory effects on various components of the immune response. Experimentally induced high levels of PRL have been shown to correlate with the worsening of several autoimmune diseases. In contrast, lowering PRL levels may protect from the autoimmune process. We investigated in both sexes of NOD mice a spontaneous model of autoimmune type 1 diabetes, the effects of two drugs, a dopaminergic agonist, bromocriptine (BRC, 10 mg/kg), which is assumed to inhibit PRL secretion, and a dopaminergic antagonist, metoclopramide (MCP, 5 mg/kg), which in contrast stimulates PRL secretion, on the incidence of diabetes, the severity of insulitis, and PRL and glucose levels. Chronic treatment of NOD mice with MCP slightly aggravated development of diabetes. The dopamine antagonist tended to accelerate the onset of diabetes in females and significantly increased the number of islets with peri-insulitis in both sexes. The weak deleterious effects exerted by MCP in NOD mice may be related to its stimulatory action on PRL release. Contrary to the expected results, the dopamine agonist BRC did not protect from autoimmune diabetes. In contrast, the drug appeared to accelerate diabetes onset in males and significantly increased the number of islets showing insulitis in both sexes. This study underlines the complexity of the action of BRC which in NOD mice only transiently inhibits the release of PRL. Moreover, the aggravating actions of BRC may be related to the marked hyperglycemic effect of the drug observed in male and female NOD mice.

Free fatty acid profiles in the non-obese diabetic (NOD) mouse: basal serum levels and effects of endocrine manipulation.

The non-obese diabetic mouse (NOD) is one of the few available models of spontaneous autoimmune insulin-dependent diabetes mellitus (IDDM). The authors determined the free fatty acid (FFA) levels and the concentrations and relative percentages of the various classes of FFA before the onset of diabetes in both sexes at 2 and 4 months of age and in diabetic females. A circadian rhythm of FFA concentrations was found in prediabetic mice, with lower values in the evening. Moreover, there was a sex difference in FFA concentrations in the morning, with 2-month-old females having higher concentrations than males. Sex and age-related differences were also observed in the concentrations of the various classes of FFA, with higher polyunsaturated fatty acid concentrations in 2-month-old females and increases in di- and tri-unsaturated fatty acids concentrations in both sexes with age. Hormonal manipulation such as adrenalectomy and/or castration modulated total FFA and the concentrations of the various classes of FFA in 2-month-old mice. These FFA differences between males and females should be taken into account in the onset of type I diabetes.

Role of adrenal hormones and prostaglandins in the control of mouse thymocytes lysis

The cytolytic actions of glucocorticoids and of agents increasing cyclic AMP were studied in vitro in thymocyte suspensions isolated from adrenalectomized or hydrocortisone-treated mice. Although considered as corticoresistant cells, the thymocytes isolated from hydrocortisone-treated mice were lysed to the same extent although more slowly in vitro by dexamethasone than whole thymocyte populations (i.e. corticosensitive cells). Moreover, these two cell populations were shown to contain comparable amounts of glucocorticoid receptors and to be almost equally sensitive to the metabolic effects of glucocorticoids when measured by inhibition of RNA and DNA synthesis. Studies performed with corticosensitive cells showed that prostaglandin E2, isoproterenol and dibutyrilcyclic AMP were also able to induce cell lysis and that, isoproterenol and dexamethasone exerted additive cytolytic action in vitro. In vivo experiments showed also an additive effect of steroids and isoproterenol on thymus atrophy. In contrast, cells isolated from hydrocortisone-treated animals were not sensitive to the cytotoxic action of prostaglandin E2, isoproterenol and dibutyril cyclic AMP. This difference between the two populations was not associated with any difference in the responsiveness of adenylate cyclase as determined following isoproterenol-induced accumulation of cyclic AMP. The cytolytic action of dexamethasone but also that of prostaglandin E2 and isoproterenol, could be blocked in the presence of cycloheximide, an inhibitor of protein synthesis, thus suggesting that glucocorticoids and agents increasing cyclic AMP control the synthesis of some proteins involved in the triggering of cell lysis. Among the hypotheses proposed to explain the differences between in vitro and in vivo sensitivity of lymphoid cell to glucocorticoids, it was suggested that the drug may in vivo indirectly control the viability or the proliferation of thymocytes through the release of other mediators. We have shown that in vivo injection of hydrocortisone induces an accumulation of fatty acids in the whole thymus gland but not in the isolated thymocytes. Since exogenous fatty acids exert cytolytic actions on isolated thymocytes, we suggest that glucocorticoids may exert in vivo an indirect toxic action by promoting the release of fatty acids from adipose tissue or other sources.

Cytolytic effects of dexamethasone and of agents stimulating cyclic AMP content in isolated mouse thymocytes

The cytolytic action of glucocorticoids and of agents increasing cyclic AMP were studied in corticosensitive and corticoresistant mouse thymocyte populations. Studies performed in corticosensitive cells showed that prostaglandin E2, isoproterenol and dibutyryl cyclic AMP were also able to induce cell lysis. Moreover, isoproterenol and dexamethasone exerted additive cytolytic effects. In contrast, cells isolated from hydrocortisone-treated animals were only moderately sensitive to the cytotoxic action of dexamethasone in vitro and insensitive to the action of prostaglandin E2, isoproterenol and dibutyryl cyclic AMP. Finally, we showed that the cytolytic action of dexamethasone, but also that of prostaglandin E2 and isoproterenol, could be blocked in the presence of cycloheximide, an inhibitor of protein synthesis, thus suggesting that glucocorticoids and agents increasing cyclic AMP control the synthesis of some proteins involved in the triggering of cell lysis.