Modulation by cytokines of glucocorticoid action.

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.

Natural killer cell activity in the peripheral blood of patients with Cushing's syndrome.

BACKGROUND: Natural killer (NK) cells are CD3(-)CD16(+)CD56(+) bone-marrow-derived lymphocytes mediating first-line defence by direct cytotoxicity against various types of target cells without prior immunization. NK cell activity is positively regulated by immune interferon (IFN-gamma); among hormones, glucocorticoids are potent in vitro and in vivo inhibitors, whereas ACTH and beta-endorphin in many experimental circumstances enhance NK cytotoxicity. DESIGN: We measured NK cytotoxicity of peripheral blood mononuclear cells (PBMC) obtained at 0800h and 2000h from 26 patients with Cushing's syndrome (12 pituitary-dependent, 12 adrenal-dependent and two dependent on ectopic ACTH secretion). In vitro responsiveness to IFN-gamma or cortisol was also tested. METHODS: NK activity was measured in a 4-h direct cytotoxicity assay using K562 cells as targets. Plasma ACTH, serum and urinary free cortisol were concomitantly measured with commercially available kits. RESULTS: Spontaneous activity and responsiveness to IFN-gamma or cortisol were significantly greater in 15 age- and sex-matched controls than in Cushing's patients at 0800h. In pituitary-dependent Cushing's patients, plasma ACTH correlated positively with mean levels of spontaneous NK activity (r=0.64, P<0.05) and negatively with cortisol-dependent percentage inhibition (r=-0.69, P<0.02). In adrenal-dependent Cushing's patients, a negative correlation was observed between levels of spontaneous NK activity and urinary free cortisol (r=-0.67, P<0.02). CONCLUSIONS: Our data indicate that excess endogenous glucocorticoids affect spontaneous NK cell activity and responsiveness to exogenous IFN-gamma or cortisol. The differential patterns observed between pituitary-dependent and adrenal-dependent groups are compatible with a positive immunomodulatory role of pituitary pro-opiomelanocortin-derived peptides that effectively counterbalance, at least partially, glucocorticoid immunosuppression.

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.

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.

Altered circadian rhythms of natural killer (NK) cell activity in patients with autoimmune rheumatic diseases.

Natural Killer (NK) cells are a lymphocyte subset actively involved in cytotoxicity against tumor-transformed and virus-infected cells; they are a reliable model for the study of neuroendocrine-immune interactions. In previous works we demonstrated that in healthy subjects NK activity of peripheral blood mononuclear cells (PBMC) and susceptibility to endogenous modifiers display statistically validated circadian rhythms. In rheumatoid arthritis (RA) and in other autoimmune rheumatic diseases abnormalities of the circadian rhythm of serum cortisol and altered levels of NK cell activity have been reported. We evaluated the circadian pattern of NK cell activity in 7 hospitalized patients with autoimmune rheumatic diseases (4 RA, 1 scleroderma, 2 mixed connective tissue disease). Temporal variations of in vitro responses to either positive recombinant (immune interferon, r IFN-gamma IFN-gamma: 650 IU/ml; recombinant interleukin-2, r IL-2 IL-2: 100 IU/ml) or negative (cortisol: 10(-6) M) modifiers were also studied. Blood was drawn at 4h intervals for 24 h, starting at 0800. PBMC preparations were immediately separated and incubated for 20h in the presence or absence of modifiers. NK activity was assessed with a direct non-radiometric 4h cytolytic assay, using K 562 cells as targets. Significant circadian variations of spontaneous NK activity were documented only in women with RA, with a peak in the evening hours and a minimum in the night or in the early morning (p < 0.05, PR 51.5%, phi 1829). Population-mean cosinor analysis did not yield detection of significant circadian variations of in vitro responsiveness to modifiers.(ABSTRACT TRUNCATED AT 250 WORDS)

[The autocrine-paracrine modulation of natural killer activity in man by peptides of the corticostatin-defensin family]. / Modulazione autocrino-paracrina dell'attività natural killer nell'uomo da parte di peptidi della famiglia delle corticostatine-defensine.

Corticostatins (CS)-defensins are a family of peptides recently isolated from neutrophils and cells of myeloid lineage. They have been termed CS in that members of the family inhibit ACTH-induced steroidogenesis, and defensins in that they are highly effective as enhancers of intracellular killing of pathogens. Natural killer (NK) cells are an immunocyte subset whose cytotoxic activity is modulated by lymphokines and hormones. Recent evidence suggests a myeloid origin for these cells. We evaluated whether two human CS-defensins, HP-1 and HP-4, are able to modulate in vitro spontaneous NK cell activity of human peripheral blood mononuclear (PBM) cells and in vitro susceptibility to the stimulatory effect by immune interferon (IFN-gamma) or interleukin 2 (IL-2) and to the inhibitory effect of cortisol. PBM cells were incubated for 20 h with HP-1 or HP-4 and IFN-gamma or IL-2 or cortisol. NK cell activity was measured in a 4-h direct cytotoxicity assay (K562 cells as a target). We also searched for CS-defensins in NK-enriched cell preparations by means of HPLC separation of the supernatant obtained from sonicated cells. HP-1 and HP-4 significantly inhibited both spontaneous and lymphokine-inducible NK cell activity, and potentiated cortisol-dependent inhibition. Radioimmunoassay on HPLC purified fractions demonstrated the presence of HP-1 in NK-enriched cell preparations. Our data indicate that HP-1 and HP-4 are negative modulators of NK cell cytotoxicity and that autocrine/paracrine mechanisms are conceivably involved. HP-1 production by NK cells may be viewed as additional support for the thesis of the myeloid origin of these immune effectors.

Interplay in vitro between ACTH, beta-endorphin, and glucocorticoids in the modulation of spontaneous and lymphokine-inducible human natural killer (NK) cell activity.

Release of pro-opiomelanocortin (POMC)-derived peptides and glucocorticoids characterizes the activation of the hypothalamic-pituitary-adrenal (HPA) axis and represents a major adaptive response to stress. Both glucocorticoids and POMC-derived hormones are known to be crucial modifiers of the immune response. Natural killer (NK) cells are a lymphocyte subset deeply involved in immunosurveillance. Cortisol, the most important glucocorticoid hormone in humans, is a well-established inhibitor, whereas the two lymphokines, immune interferon (IFN-gamma) and interleukin-2 (IL-2), are important physiological stimulators. In the present study, physiological as well as superphysiological concentrations of two POMC-derived peptides, ACTH and beta-endorphin, were shown not only to affect in vitro spontaneous and lymphokine-inducible NK activity of peripheral blood mononuclear (PBM) cells, but also to modify cortisol-mediated inhibition. NK activity was measured in a 4-h cytotoxic assay using the cell line K562 as a target, after prior incubation with ACTH (10(-8)-10(-12) M) and beta-endorphin (10(-8)-10(-14) M) in the presence or absence of cortisol (10(-6) M), IFN-gamma (325 IU/ml), and IL-2 (25 IU/ml). ACTH was ineffective in changing spontaneous NK activity at all concentrations, whereas beta-endorphin enhanced NK cytotoxicity (p < .02). The concomitant exposure of PBM cells to the two POMC-derived peptides and IFN-gamma or IL-2 significantly enhanced the lymphokine-induced boosting of NK activity. Moreover, ACTH and beta-endorphin were able to significantly reduce the cortisol-dependent inhibition (p < .05). These data are compatible with the hypothesis that POMC-derived peptides have a role in the modulation of NK cell activity. It seems likely that in cases of activation of the HPA axis, ACTH and beta-endorphin may effectively counteract the negative effects of glucocorticoids on NK cell activity, and prevent, at least in some instances, any overshooting of the glucocorticoid-dependent effect on immune cells.

Effects of CRH, ACTH and the corticostatin HP-4 on the spontaneous NK cell activity and susceptibility to endogenous modifiers.

Natural killer (NK) cells are mononuclear leukocytes which are thought to play an important role in immunosurveillance; in the elderly a progressive reduction occurs both of their spontaneous activity and of their responsiveness to positive modulators such as immune interferon (IFN-y) and interleukin-2 (IL-2). NK cells represent also a reliable model to study immunomodulatory properties of the hypothalamic-pituitary-adrenal (HPA) system, since cortisol (F) is a well defined inhibitor of their activity whereas proopiomelanocortin (POMC)-derived peptides may counteract this effect. Corticostatins (CS)-defensins are a family of peptides recently purified from cells capable of phagocytic activity; they are able to inhibit the steroidogenic activity of ACTH and to enhance internalization and/or killing of intracellular pathogens. We have investigated the effects in vitro of corticotropin releasing hormone (CRH), ACTH and CS HP-4 on human NK cell activity. Peripheral blood mononuclear (PBM) cells from healthy donors were incubated with CRH (10(-14)-10(-11) M). ACTH (10(-12)-10(-8) M) and HP-4 (10(-10)-10(-8) M) in the presence or absence of F (10(-6) M) or IFN-y (325 IU//ml) or IL-2 (25 IU/ml). NK cell activity was measured in a 4-h cytotoxic assay using K562 cells as a target. CRH was able to significantly reduce the spontaneous and IL-2-induced NK activity and to significantly potentiate the F-dependent inhibition. ACTH was per se ineffective on the spontaneous NK activity, but was able to augment the enhancing effects of IFN-y and IL-2, and to reduce the degree of inhibition obtainable with the glucocorticoid. HP-4 was able to enhance the F-dependent inhibition of PBM preparations. Our results are consistent with an immunomodulatory role for CRH, ACTH and CS, specially in conditions of high concentrations of glucocorticoids. Since in elderly has been demonstrated a condition of hypercortisolism, we suggest that complex steroid-peptides interactions are involved in the net effect of HPA axis on immune functions in senescence, and that such interactions involve paracrine/autocrine CS.