Rosa Gabriella Masera

Inhibition by cortisol of human natural killer (NK) cell activity.

The effects of cortisol on the natural killer (NK) activity of human peripheral blood mononuclear (PBM) cells were studied in vitro using a direct 4-h 51Cr-release assay and K 562 cell line as a target. Preincubation for 20 h of PBM cells drawn from healthy donors with 1 X 10(-8) to 1 X 10(-5) M cortisol resulted in a significant decrease of NK cell activity. The magnitude of the suppression was directly related to the steroid concentration and inversely related to the number of effector cells. Cortisol was able to minimize the enhancement of NK cytotoxicity obtainable in the presence of immune interferon (IFN-gamma). A significantly higher suppression was achieved after sequential exposure of PBM cells to cortisol and equimolar levels of prostaglandin E2 (PgE2). The concomitant incubation with theophylline and isobutyl-methylxanthine failed to enhance the cortisol-induced suppression, whereas PgE2-dependent inhibition significantly increased after exposure of PBM cells to methyl-xanthines. The inhibitory effect of cortisol was partially or totally prevented by the concomitant incubation with equimolar amounts of 11-deoxycortisol and RU 486 but not of progesterone. Treatment of NK effectors with a monoclonal anti-human corticosteroid-binding globulin (CBG) antibody produced an enhancement of the spontaneous NK activity and a partial suppression of cortisol-mediated effects. Our results suggest that endogenous glucocorticoids play a role in the regulation of NK cell-mediated cytotoxicity. Since the effect of cortisol was additive to that of PgE2 and was not changed by phosphodiesterase inhibitors, it is conceivable that the hormone acts at a level different from the adenylate cyclase-phosphodiesterase system. Data obtained with the use of antiglucocorticoids and the anti-CBG antibody are compatible with a role both of high-affinity glucocorticoid receptors and of CBG in mediating cortisol action on the human NK cell activity.

Modulation by cytokines of glucocorticoid action.

ABSTRACT: Glucocorticoids (GC) are potent modulators of the inflammatory response. Their effects serve to down‐regulate the inflammatory response and are mediated by genomic pathways that follow the interaction with specific receptors (glucocorticoid receptors, GR). Interleukin (IL)‐1, IL‐2, and IL‐6 are able to increase GC secretion by enhancing synthesis and release of CRH and ACTH. Cytokine effects upon steroidogenesis also occur at the adrenal level. The role of cytokines as modulators of GR has received scarce attention. IL‐1 has been shown to up‐regulate GR mRNA expression in hypothalamic CRH secreting cells. On the other hand, macrophage migration inhibitory factor (MIF), a T‐cell product inducible by inflammatory substances including other cytokines, counterregulates GC action within the immune system. Besides immunocytes and neurons, bone cells are a sensitive target for GC and cytokines. We have found that IL‐2 and IL‐6 up‐regulate remarkably the number of GR binding sites and the expression of GR mRNA in peripheral blood mononuclear cells and in osteoblast‐like Saos‐2 cells. Available data suggest that inflammatory cytokines have both direct and indirect effects on GC action at the target level. Autocrine‐induced transcription of GR in immunocytes and/or osteoblasts could be a mechanism that restrains excess cytokine production.

Interactions between Glucocorticoids and Cytokines in the Bone Microenvironment

Abstract: Cytokines belonging to the so‐called interleukin‐6 (IL‐6) or gp130 cytokine family, notably IL‐6 and IL‐11, are known as pro‐resorptive cytokines, in that they promote osteoclastogenesis. Glucocorticoid (GC)‐induced osteoporosis is admittedly the most frequent secondary osteoporosis. The pathogenesis still has many unresolved issues. Although the effects of GCs on cytokine production and recognition have been extensively studied, little is known about the effects of cytokines on GC action at the target level. We have focused on the effects of IL‐6 and IL‐11 on specific binding by type II GC receptors (GRs) in two human osteoblast‐like cell lines (Saos‐2 and MG‐63) that have remarkably different constitutive expression of these cytokines and GRs as well. We have provided evidence that IL‐6 upregulates GR binding sites, while IL‐11 downregulates these sites, as determined by radioligand binding assay and Scatchard analysis. GR affinity (Kd) did not change after exposure to both cytokines. A number of experiments were consistent with the view that in human osteoblast‐like cells, cytokines of the IL‐6 family have autocrine modulatory effects on GRα (GRβ is a variant that does not bind specifically in our method). Complex effects of GCs on the system(s) of proinflammatory/anti‐inflammatory cytokines and conversely of these cytokines on GC action could account for the dynamics of bone loss in patients given GCs and conceivably having high concentrations of these cytokines in the bone microenvironment.

Corticostatins/defensins inhibit in vitro NK activity and cytokine production by human peripheral blood mononuclear cells

Corticostatins/defensins are a family of cationic peptides recently isolated from phagocytotic cells of the myeloid lineage. Natural killer (NK) cells are spontaneously cytotoxic large granular lymphocytes that are involved in immunosurveillance against cancer and infections. Their activity is modulated by hormones of the hypothalamic-pituitary-adrenal axis. We wished to determine whether two human corticostatins/defensins, HP-1 and HP-4, are able to change in vitro the spontaneous NK activity of human peripheral blood mononuclear cells (PBMC) and the responses either to the stimulatory cytokines immune interferon (IFN-gamma) or interleukin 2 (IL-2) and to the inhibitory hormone cortisol. NK cell activity was measured in a 4-h direct cytotoxicity assay with K562 cells as a target. HP-1 and HP-4 (10 (-8) -10 (-9) M) significantly inhibited the spontaneous and cytokine-inducible NK activity, and increased the cortisol-dependent inhibition. Radioimmunoassay of HPLC purified fractions obtained from sonicated NK cells showed HP-1 in the two cell preparations examined. We also evaluated the effects of HP-1 and HP-4 (10 (-8) M -10(-9) M) upo IFN-gamma and interleukin 6 (IL-6) production by PBMC stimulated with phytohemagglutinin (PHA) or concanavalin A (ConA). IFN-gamma was titrated with the biological assay using WISH cells as indicators and vescicular stomatitis virus (VSV) as the challenge virus. IL-6 was measured using an enzyme amplified sensitivity immunoassay. Both HP-1 and HP-4 significantly reduced cytokine production. Our data indicate that corticostatins/defensins are novel modulators of lymphocyte functions in vitro. Their immunodepressing properties could add complexity and plasticity to hypothalamic-pituitary-adrenal-cytokine circuits in vivo.

Mental deterioration correlates with response of natural killer (NK) cell activity to physiological modifiers in patients with short history of Alzheimer's disease

Natural killer (NK) cell activity of peripheral blood mononuclear (PBM) cells was measured in 16 subjects with mild to moderate senile dementia of Alzheimers type (sDAT) chosen for short history of disease and no medication, and in 17 age- and sex-matched controls. Levels of cytotoxicity at baseline and after PBM cell exposure to modifiers either negative (cortisol 10(-6) M) or positive (rIL-2 650 IU/ml and rIFN-gamma 100 UI/ml, respectively) were related to indices of hypothalamic-pituitary-adrenal (HPA) function and Gottfries Bråne Rating Scale (GBS) score for mental deterioration. Spontaneous NK cell activity was not significantly different in sDAT subjects vs controls. In vitro inhibition by cortisol was lower in sDAT (P<0.05); cytokine-induced changes were greater (rIL-2, P<0.02; rIFN-gamma, P<0.05). Percent negative or positive variations from baseline significantly correlated with GBS scores (P<0.05 or less). Serum cortisol and cortisol/DHEAS molar ratio at 0800 h were significantly higher in sDAT (P<0.05 and P<0.02, respectively). Cortisol/DHEA ratio positively correlated with GBS scores (P<0.02). Moreover, the ratios of incremental area of response ACTH/cortisol and beta-endorphin/cortisol after 1 microg/kg ovine-corticotrophin-releasing hormone (o-CRH) positively correlated with percent increase of NK cell activity after rIL-2 (P<0.01). Data indicate that patients with mild cognitive impairment and short history of sDAT show abnormal responsiveness of NK cell activity to physiological modifiers while maintaining normal spontaneous activity. Furthermore, data are compatible with partial glucocorticoid resistance at the immune level. Progressing sDAT longitudinal studies are needed to address: i) the clinical applicability of these abnormalities as prognostic factors; ii) the role played by pro-opiomelanocortin (POMC)-derived peptides and adrenal androgens in the control of NK cell activity.

Autocrine Up-Regulation of Glucocorticoid Receptors by Interleukin-6 in Human Osteoblast-Like Cells

6 cells, respectively). We measured the expression of glucocorticoid receptor (GR) in terms of specific binding sites after exposure of cells to different amounts of IL-6. Incubation for 20 hours with IL-6 at increasing concentrations up to 2000 pg/ml yielded significant increase of GR binding sites in both cell lines. IL-6 was also able to revert the inhibitory effect of dexamethasone (1 μM) on GR in both cell lines. In MG-63 cells, that express higher concentrations of GR, IL-6 deprivation via a specific anti-IL-6 antibody (100 ng/ml) significantly decreased GR, as it was noticed, although to a lesser degree, using a specific anti-IL-6 receptor antibody. In Saos-2, cells that express lower concentrations of GR, a 40-hour treatment with human IL-1β (10 ng/ml) significantly increased both IL-6 production and GR. This latter effect was completely abolished by co-treating the cells with the anti-IL-6 antibody. Our data are consistent with an autocrine up-regulation of GR expression by IL-6 in human osteoblast-like cells. This phenomenon, which is also relevant to paracrine cell-to-cell communication, subserves a feedback loop in the bone microenvironment that restrains excess inducible IL-6 production. In patients having high levels of IL-6 and given GCs, it could offer an additional explanation for the biphasic pattern of bone loss in the course of therapy.

Hypothalamic-pituitary-adrenal axis function, psychopathological traits, and natural killer (NK) cell activity in anorexia nervosa.

To evaluate the role of Hypothalamic-Pituitary-Adrenal (HPA) hormones and psychoneuroendocrine modulation on NK cell activity in Anorexia Nervosa (AN) we studied in 24 patients and 20 sex- and age-matched healthy controls, the spontaneous NK activity of peripheral blood mononuclear (PBM) cells and the susceptibility in vitro to cortisol or immune interferon or interleukin-2. NK cytotoxicity of PBM cells was measured in a direct non-radiometric 4h cytolytic assay using K562 cells as targets. HPA axis function was evaluated by IV ovine Corticotropin Releasing Hormone (o-CRH) administration. We did not find clear-cut abnormalities of NK cytotoxicities either in basal conditions or after exposure to challengers. The extent of cortisol-dependent inhibition was comparable in patients and controls. Significant inverse and direct correlations were found respectively between the spontaneous NK cell activity and baseline serum cortisol at 0800 h (r = -0.5; p < .02), and between IL-2 dependent boosting of NK cell cytotoxicity and ACTH, beta-endorphin or cortisol responses after o-CRH, expressed as areas under the curve (AUC) (r = 0.46, p < .05; r = 0.46, p < .05; and r = -0.48, p < .05, respectively). Correlations observed with AUC ratios yielded more significant results (r = 0.62; p < .01 and r = 0.51; p < .05 respectively). These data suggest a role for Proopiomelanocortin (POMC) derived peptides in the regulation of NK cell activity in AN, and multifaceted relationships between this particular immune function, on the one hand, and certain patterns of HPA axis function on the other.

Interleukin 2 up-regulates glucocorticoid receptor number in human peripheral blood mononuclear cells and the osteosarcoma cell line saos-2 in vitro

Glucocorticoids are well-recognized modulators of immunocytes and osteoblasts via specific receptor-mediated mechanisms. We have evaluated the in vitro effect of interleukin 2 (IL-2) on the expression of glucocorticoid receptors (GRs) in peripheral blood mononuclear cells (PBMCs) obtained from healthy donors and osteoblast-like Saos-2 cells. Aliquots of PBMC or Saos-2 cells were incubated for 20 h in the presence or absence of recombinant human IL-2 (100 IU/mL) at 37 degrees C. After incubation, a [3H]dexamethasone radioligand-binding assay and Scatchard analysis were used to determine GR-binding parameters in both cell populations. Saos-2 cells basally express higher numbers of GR than PBMCs. After IL-2, a significant increase in GR number was found for both PBMCs and Saos-2 cells. The relative increase was higher in Saos-2 cells; in PBMCs, the apparent affinity fell to almost half. These data represent an additional piece of evidence that cytokine and steroid hormones may act in a complementary way to regulate specific cell functions.

The expanding field of hypothalamic-pituitary-adrenal modulation of human natural killer cell activity.

There is ample evidence that the neuroendocrine and the immune systems communicate with each other. For a long time, hormones have been known to affect immune function. More recently, an increasing number of neurotransmitter and neuropeptide molecules that are released from central and peripheral nerve endings were demonstrated to have regulatory effects on immune cells.’,’ Conversely, immune cells release soluble factors that influence neuroendocrine mechanisms, notably those controlling the function of the hypothalamic-pituitary-adrenal (HPA) ~ y s t e m . ~ ~ The concept has emerged that neuroendocrine-immune interactions are essential to maintaining immune efficiency and to preventing diseases related to altered immunosurveillance. Relationships between HPA hormones and immunity have received special attention due to the importance of these hormones in stress response and the observed abnormalities of their secretory patterns in psychiatric and autoimmune diseases as Excess activity of the HPA system has been suggested to occur also in cognitive disorders, namely, senile or Alzheimer’s dementia.* Generally speaking, hyperactivity involves abnormal production of glucocorticoids and hypothalamic corticotropin-releasing hormone (CRH), and resistance to the inhibitory

Studies on the mechanism of cortisol inhibition of human natural killer cell activity: effects of calcium entry blockers and calmodulin antagonists

The role of Ca2+ in mediating the inhibition by glucocorticoids of human natural killer (NK) activity was investigated using Ca2+ entry blockers (verapamil and its desmethoxy-derivatives LU46973 and LU47093) and calmodulin antagonists (pimozide and two naphthalenesulfopamide derivatives, W-7 and W-13). Peripheral blood mononuclear (PBM) cell preparations were incubated for 20 h with 1 x 10(-6) M cortisol and these agents in various combinations (concentration range: 1 x 10(-7) - 1 x 10(-5) M) and then assayed in a direct 4-h cytolytic assay using 51Cr-labeled K 562 target cells. Exposure to cortisol led to a significant reduction of NK cell activity (about 50% with respect to the spontaneous activity). Ca2+ entry blockers displayed per se a dose-dependent depressive effect on cytotoxicity and gave significant enhancement of cortisol-dependent inhibition. Calmodulin antagonists were per se minimally effective but clearly amplified the cortisol-mediated inhibition. Raising extracellular Ca2+ by CaCl2 or intracellular Ca2+ by the ionophore A23187 yelded an appreciable reduction of these effects. Our data are compatible with the view that extracellular and intracellular Ca2+ play a role in the control of human NK cell activity. Moreover, it is conceivable that the mechanisms involved in glucocorticoid inhibition of NK cell activity involve Ca2+-dependent pathways.