Allan Munck

Glucocorticoids and Immune Function

This chapter discusses the role of glucocorticoids and immune function. The effects of glucocorticoids on the effector functions of monocytes and macrophages are quite complicated. Mononuclear phagocytes are activated by IFN-γ to a heightened state of activation that results in more efficient recognition and killing of pathogens and tumor cells. Specific changes that accompany macrophage activation include increased expression of receptors for the Fc portion of immunoglobulin G (IgG), increased capacity for production of reactive oxygen metabolites, and an increased ability to kill tumor cells. In some cases, IFN-γ can completely reverse the inhibitory effects of glucocorticoids. Crude preparations of cytokines containing IFN-γ could activate human alveolar macrophages to inhibit the growth of the Legionnaires disease bacillus, even in cultures containing 1 μ cortisol. Lower levels of glucocorticoid either have no effect or enhance monocyte and macrophage function. The relative concentrations of glucocorticoid and IFN-γ are key determinants of the ability of macrophages to ingest and kill infectious agents or autologous red blood cells.

Glucocorticoids and Stress: Permissive and Suppressive Actions

Protection against stress by glucocorticoids is discussed in relation to their permissive and suppressive actions. Evidence from the last decade is summarized regarding the physiological nature of the suppressive actions, and the hypothesis that they prevent stress-activated defense mechanisms from overshooting and damaging the organism. Support for this hypothesis has come from observations on how endogenous or administered glucocorticoids control inflammatory and immune responses, protect in endotoxic and hemorrhagic shock, regulate central nervous system responses to stimuli, and moderate many defense reactions through suppression of cytokines and other mediators. Studies showing that glucocorticoids permissively induce receptors for several mediators that they suppress have led to a model in which stimulated activity of a mediator system is increased permissively through induction of mediator receptors and decreased through suppression of mediator production.

Prostaglandin inhibition of T-cell proliferation is mediated at two levels

Abstract While numerous investigators have established that E-series prostaglandins inhibit proliferation of lectin- or antigen-activated T cells, the mechanism by which this effect is mediated remained poorly defined. It was recently shown that T-cell replication is mediated by a soluble factor (T-cell growth factor, TCGF), produced by antigen- or lectin-stimulated T cells. Thus, inhibition of T-cell replication by prostaglandins could be controlled either at the level of TCGF production or at the subsequent step of actual proliferation. We have found that differentiated cytotoxic T lymphocytes harvested from TCGF-dependent culture were, indeed, significantly sensitive to as little as 1 n M PGE 1 or PGE 2 ; 1000 n M PGE 1 or PGE 2 reduced TCGF-dependent T-cell proliferation by greater than 43% in all cases. In addition, PGE 1 and PGE 2 suppressed lectin-induced production of TCGF to remarkably similar concentration-dependent levels. Thus, the prostaglandin-E-mediated suppression of lectin-initiated T-cell proliferation could be traced to an inhibition of both the production and action of the T-cell-specific mitogen, TCGF. Since TCGF is produced by one T-cell subpopulation and acts on different T-cell subpopulations including both T helper and T cytotoxic cells, the consequences of prostaglandin-E inhibition of TCGF may have broad effects on cellular and humoral immune responses.

Glucocorticoid-receptor complexes and the earliest steps in the action of glucocorticoids on thymus cells

Abstract Cortisol and other glucocorticoids added at physiological concentrations to rat thymus cells in vitro at 37°C begin to inhibit glucose transport after about 15 min. This effect corresponds to effects observed in vivo and probably is in good part responsible for the catabolic actions of glucocorticoids on lymphoid tissue. From a variety of experiments we have concluded that cortisol initially stimulates synthesis in the nucleus of a specific form of RNA that. after an intermediate step, by 15 min initiates synthesis of a protein that inhibits glucose transport. The first step in cortisol action is formation of specific cortisol receptor complexes. At 37°C this process is complete by 7 min, by which time the complexes are localized largely in the nucleus. At 3°C, however, most of the complex appears in the supernatant when cells are broken by osmotic shock and nuclei spun down. This non-nuclear bound complex we refer to as “cytoplasmic”. On warming the cells to 37°C, [3H]-cortisol bound in the cytoplasmic complex becomes bound in the nucleus within l min, probably by transfer of the cytoplasmic complex in loto to a nuclear acceptor site. Isolated cytoplasmic and nuclear receptors bind glucocorticoids specifically, becoming saturated over the physiological range of glucocorticoid concentrations. The isolated cytoplasmic receptor has a half-life at 3°C of about 2 h. Saturating concentrations of cortisol or other glucocorticoids increase this value to more than 20 h. Both α and β sides of the steroid appear to interact with the binding site. which probably consists of a hydrophobic pocket with polar groups that form hydrogen bonds. The principal driving forces for formation of the hormone-receptor complex are probably hydrophobic interactions, the hydrogen bonds conferring specificity to the interactions. Cortisol-eytoplasmic receptor complexes are transformed by brief warming at 25°C into complexes with high affinity for nuclei. These latter complexes become rapidly associated with isolated nuclei at 3°C. Two alternative functions are proposed for the hormone in this temperature-sensitive transformation: one is that hormone binding displaces the equilibrium of the receptor towards a form with high affinity for nuclei; the other assumes that the cytoplasmic receptor is in a non-equilibrium state, and that hormone binding accelerates its transformation to the equilibrium high-affinity form. Protein synthesis does not appear to bo necessary to replace receptors that are transferred to the nucleus in the presence of hormone. ATP or some related substance, however, does seem to be required, it is proposed that ATP generates the normal cytoplasmic receptor from a pre-cursor. Furthermore, it is suggested that the precursor may be the form in which the receptor leaves the nuclear site, so that in the presence of hormone there is a continuously operating receptor cycle, dependent for energy on ATP.

Glucocorticoid receptor phosphorylation: overview, function and cell cycle-dependence.

All steroid hormone receptors are phosphorylated and undergo hormone-induced hyperphosphorylation. Most phosphorylated residues identified so far are serines in the N-terminal domain. Other residues and domains may also be phosphorylated, e.g. the estrogen receptor is phosphorylated on tyrosine in the hormone-binding domain. Many sites lie in consensus sequences for proline-directed, cell cycle-associated kinases. In some receptors hyperphosphorylation is induced by hormone antagonists as well as agonists, and leads to new phosphorylated sites. With glucocorticoid receptors, hyperphosphorylation is specific for glucocorticoid agonists, follows receptor activation and produces no new sites. Rate studies suggest that hyperphosphorylation is due to accelerated phosphorylation rather than delayed dephosphorylation. Evidence to date indicates that steroid hormone receptor phosphorylation serves not as an on-off switch but modulates function more subtly. Mutations of phosphorylated sites to alanine have been found to decrease activity by 0 to 90%, depending on mutated site, cell type, reporter gene and hormone concentration. With glucocorticoid receptors, some alanine mutants are up to 75% less active in hormone-induced transactivation of certain reporter genes. They are also inactive in hormone-induced repression of transcription of their own gene and down regulation of the receptor protein. Furthermore, they are much less sensitive to degradation. Both basal phosphorylation and hormone-dependent hyperphosphorylation of these receptors are cell cycle-dependent, basal phosphorylation being low in S phase and high in G2/M and hyperphosphorylation the reverse, suggesting a causal relation to the cell cycle-dependence of glucocorticoid activity reported with several cell lines. Hyperphosphorylation appears to be regulated by basal phosphorylation through negative charge in the N-terminal domain, which in S phase is relatively low and permits hyperphosphorylation, but in G2/M is relatively high and blocks hyperphosphorylation.

Studies on the mode of action of glucocorticoids in rats II. The effectsin vivo andin vitro on net glucose uptake by isolated adipose tissue

Abstract 1. Injection of adrenalectomized, fasted rats with cortisol, corticosterone and deoxycorticosterone reduces the net glucose uptake by subsequently isolated epididymal adipose tissue. Deoxycorticosterone is less active than the other two compounds, which are about equally active. The effect can be observed within 30 min of injection. Normal rats, fed or fasted, and hypophysectomized, adrenalectomized rats respond in the same way. 2. The same steroids in vitro have similar actions, with relative activities comparable to those displayed in vivo . Significant effects are observed at all concentrations down to 9·10 −8 M . Below 10 −6 M , the effects occur mainly after the incubation has proceeded for 2.5 h. Adipose tissue from alloxan-diabetic, adrenalectomized rats appears to respond in the same way. 3. It is suggested that these findings: (a) account in part for the changes in carbohydrate metabolism which take place during the first hours after injection of rats with glucocorticoids; (b) show that epididymal adipose tissue is a direct target for the action of glucocorticoids; (c) can account qualitatively for the known effects of glucocorticoids on adipose tissue, in particular, for the permissive actions with respect to free fatty acid release. 4. These findings are considered to provide direct evidence in support of the hypothesis that some of the major effects of glucocorticoids on extrahepatic tissues may be secondary to a decrease in glucose utilization.

EFFECT OF GLUCOCORTICOIDS IN VIVO AND IN VITRO ON NET GLUCOSE UPTAKE AND AMINO ACID INCORPORATION BY RAT-THYMUS CELLS.

Abstract 1. 1. The general hypothesis that glucocorticoids may act on certain extrahepatic tissues via a direct, rapid inhibition of glucose utilization, has been tested using incubated suspensions of rat-thymus cells. 2. 2. It has been found that net glucose uptake by cells obtained from rats injected 2 h previously with cortisol (approx. 1 mg per 100 g rat wt.) is significantly lower than that of control cells. 3. 3. Cortisol in vitro, at concentrations down to 10−7M, significantly decreases net glucose uptake by such cell preparations during the first hour of incubation, and at 10−6M (the only concentration tested) decreases the incorporation of leucine into protein during the second hour. Deoxycorticosterone is less active than cortisol with respect to inhibition of both glucose uptake and amino acid incorporation. 4. 4. These findings are taken to show that a direct, rapid physiological action of glucocorticoids is to decrease glucose uptake by thymus tissue. 5. 5. From the results of incubations with various concentrations of glucose, it appears that the inhibition of amino acid incorporation produced by cortisol is not simply a consequence of decreased availability of glucose to the cell.

IN-VITRO GLUCOCORTICOID STUDIES FOR PREDICTING RESPONSE TO GLUCOCORTICOID THERAPY IN ADULTS WITH MALIGNANT LYMPHOMA

Neoplastic tissues from 28 adults with malignant lymphoma were examined for glucocorticoid receptors and in-vitro sensitivity to glucocorticoids. The patients were then treated with desamethasone for 5--14 days. 13 patients achieved at least a partial remission, and 15 had no significant tumour response. Lymphoma cells from patients who responded had more glucocorticoid-receptor sites per cell and greater in-vitro sensitivity as measured by glucocorticoid inhibition of incorporation of leucine and uridine than did tumour cells from non-responders. Study of tumour glucocorticoid receptors and glucocorticoid sensitivity in vitro may allow selection of those patients with lymphoma who should receive glucocorticoids as part of combination chemotherapy.

Kinetics of glucocorticoid-receptor complexes in rat thymus cells

Abstract Using cortisol and dexamethasone, we have studied the kinetic behavior of glucocorticoid receptors in intact rat thymus cells at 37°C. As reported previously, translocation of the cortisol-receptor complexes from “cytoplasmic” to nuclear-bound form takes about 1 min. At 26°C this time is doubled. Consistent with these observations and with the current view that formation of nuclear-bound complex must be preceded by formation of cytoplasmic complex, on addition of hormone to cells at 37°C the initial time-course of formation of cytoplasmic complex precedes that of nuclear complex by about 30 s. After cells have achieved a steady state with [ 3 H]-dexamethasone, however, a “chase” of unlabelled dexamethasone lowers nuclear levels of [ 3 H]-dexamethasone several minutes before cytoplasmic levels, suggesting formation of nuclear complex by direct reaction of hormone with nuclear-bound receptors. Following depletion of cytoplasmic receptor by incubation with a high concentration of dexamethasone and then sudden lowering of steroid concentration by dilution, the time-course of replenishment of cytoplasmic receptors appears to lag significantly behind the time-course of removal of nuclear-bound steroid. The lag is roughly the same magnitude as the time-constant of dissociation of the glucocorticoid used, and is much shorter with cortisol. The pathway of replenishment thus may be different from simple reversal of the pathway of depletion of cytoplasmic receptor, and may depend on the dissociation rate or affinity constant of the steroid.

Activated and non-activated glucocorticoid-receptor complexes in rat thymus cells: Kinetics of formation and relation to steroid structure☆

Abstract Previously we showed that when glucocorticoids initially enter rat thymus cells incubated at 37°C. non-activated hormone-receptor complexes are formed within 15 s and are then rapidly replaced by activated complexes. Activated and non-activated forms were identified in cytosols from the cells by DEAE-cellulose chromatography, where they are eluted with progressively higher salt concentrations in what are referred to as Peaks I and II. On DNA-cellulose columns dexamethasone-receptor complexes in cytosols that on DEAE-cellulose give mainly Peak II (the non-activated complex, referred to as Complex II) are not bound significantly: they are eluted at the lowest salt concentration along with free steroid, from which they can be separated by binding to hydroxyapatite. This DNA-cellulose peak is referred to as Peak a. Complexes in cytosols that give only Peak I on DEAE, however, on DNA separate into two components. One of these (Complex Ia) is not bound significantly and appears in Peak a. The other (Complex Ib, presumably the true activated complex) appears in a later Peak b. These three complexes have been measured by first adsorbing Ib with DNA-cellulose, then separating Complexes Ia and II. which are not adsorbed on DNA. by means of a DEAE-cellulose column. When [3H]-dexamethasone is added to cells at 37°C. II is formed within 15 s and is rapidly replaced by Ib, in agreement with our earlier results. Complex Ia accounts for 15–20% of total complexes at all times, and is also present after 120 min at 0°C. Various steroids have been compared on DNA-cellulose after 30 min at 37°C. when the amount of Complex II is negligible. The ratio of Ib to Ia is highest for dexamethasone and triamcinolone acetonide, intermediate for cortisol and corticosterone and lowest for cortexolone (which seems to form only Ia), roughly in proportion to intrinsic glucocorticoid activity. Complex Ia may thus be a third normal form of extranuclear glucocorticoid-rcceptor complex, but the possibility remains that it is an artifact formed after cells are broken. The relation of Complexes Ia, Ib and II to glucocorticoid activity is discussed.