Changes of bone turnover markers and serum PTH after night or morning administration of zoledronic acid in breast cancer patients with bone metastases.

Persistent circadian rhythm of bone turnover in bone metastatic breast cancer suggests greater skeletal retention of bisphosphonates if administered in the night. We assessed differential effects of night vs morning administration of zoledronic acid (ZA) on bone turnover. Forty-four breast cancer patients with bone metastases were randomised to receive intravenous ZA (4 mg) at 1100 or 2300 hours every 28 days for four times. Urinary concentration N-telopeptide of type-I collagen (NTX) and deoxypyridinolines, and serum C-telopeptide of type-I collagen (CTX), bone alkaline phosphatase (ALP), osteocalcin and Parathyroid hormone (PTH) was measured in the morning at baseline and after 4, 7, 14, 28, 56 and 84 days. Urinary ZA concentration was also measured. Zoledronic acid caused significant decreases of NTX and CTX (P<0.001), without any difference in percent changes between night and morning arms. Bone ALP and osteocalcin were also significantly affected by ZA (P=0.001), without any difference between arms. Parathyroid hormone significantly increased in both the arms; PTH increase was lower in the night arm (P=0.001). From the second administration onwards, urinary ZA level was significantly higher in the night arm (P<0.01). Administration of ZA at two opposite phases of the circadian cycle causes similar changes of bone-turnover marker levels, but has differential effects on the level of serum PTH.

Sport-related hyperhomocysteinaemia: a putative marker of muscular demand to be noted for cardiovascular risk.

OBJECTIVE: Regular physical activity is associated with a reduction of cardiovascular morbidity and mortality; however, evidence of unfortunate cardiovascular events accompanying elite sport involvement continues to accumulate. To date, no information is available on possible peculiarities of the cardiovascular risk profile in athletes. DESIGN: The aim of this study was to evaluate plasma homocysteine levels in a group of athletes and to search for relationship with vitamin status and other metabolic variables in order to confirm the existence of a "sport-related hyperhomocysteinaemia" and to explain its clinical significance. The study population was composed of 82 athletes (59 male and 23 female) practising different sports and 70 healthy age-matched subjects (40 male and 30 female) as a control group. Besides the general clinical and analytical determinations, the assessed variables included homocysteine, folate, vitamin B12, total and high-density lipoprotein (HDL) cholesterol, lactate dehydrogenase (LDH), creatine kinase (CPK) and interleukin-6 (IL-6). RESULTS: The prevalence of hyperhomocysteinaemia (>15 micromol/l) in athletes and controls was 47% and 15%, respectively. No correlation was found between homocysteine and any of the other investigated variables, in particular plasma folate, blood pressure, LDH, CPK, total and HDL cholesterol and IL-6. CONCLUSION: The results of this study confirm the existence of a sport-related hyperhomocysteinaemia which appears linked neither to the same variables found in the general population, nor to specific training-related variables. We suggest that it would represent an adaptation to training but the possibility of a secondary vascular damage cannot be excluded.

Autocrine down-regulation of glucocorticoid receptors by interleukin-11 in human osteoblast-like cell lines.

It has recently been suggested that interleukin (IL)-11 plays a role in the pathogenesis of glucocorticoid (GC)-induced osteoporosis. IL-11 belongs to the gp130 cytokine family, which includes also IL-6. We have previously investigated GC-IL-6 interplay, showing that GC inhibits IL-6 release and IL-6 up-regulates GC receptor (GR) numbers in the human osteoblast-like cell lines Saos-2 and MG-63, which constitutively have an opposite pattern of expression for GR, IL-11, IL-6, alkaline phosphatase and osteoprotegerin (OPG). The aim of this study was to investigate GC-IL-11 interplay in the same two cell lines. First, cells were incubated with cortisol (0.01-1 microM) for 20 h in the presence and in the absence of a known IL-11 secretagogue (IL-1beta); cell media were assayed for IL-11 by ELISA. Secondly, cells were incubated with IL-11 (0.1-100 ng/ml) or specific anti-IL-11 monoclonal antibody for 20 h, and then assayed for GR by a radioligand binding assay. Similar to IL-6, both constitutive and IL-1beta-inducible IL-11 release were dose-dependently inhibited by cortisol (P<0.01); at variance with IL-6, exogenous IL-11 dose-dependently decreased GR numbers in MG-63 cells (P<0.05), while anti-IL-11 antibody significantly increased GR numbers in both cell lines (P<0.05). IL-11-induced reduction of GR in MG-63 cells was confirmed by Western blot analysis. While exerting opposite effects on GR numbers, neither IL-6 nor IL-11 significantly modified GC-dependent inhibition of OPG release. Our data indicate that even physiological concentrations of cortisol negatively modulate IL-11 secretion and demonstrate, for the first time, an inhibitory effect of the cytokine on GR. Thus, the concept of autocrine-paracrine loops that modulate GC action and involve gp130 cytokines is corroborated. These loops could have clinical relevance for the dynamics of bone loss in patients given GC and having high concentrations of these cytokines in the bone microenvironment.

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.

Interactions between glucocorticoids and cytokines in the bone microenvironment.

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 (K(d)) 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 GRalpha (GRbeta 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.

Neuroendocrine-immune surveillance of osteosarcoma: emerging hypothesis.

Osteosarcoma is a bone-forming cancer predominantly found in children and adolescents more often than in adults. Osteosarcoma of the gnathic apparatus is relatively rare in the young population, and this condition becomes a concern of clinical dentists for predominantly the middle-aged and aging patient groups. Osteosarcomas are invaded by lymphocytes, which exhibit signs of activation. The immune processes that are engaged within the malignant bone matrix involve the production of cytokines, which regulate the process of apoptotic programmed cell death. This paper discusses the mechanisms by which apoptosis of osteosarcoma cells is modulated by the neuroendocrine-immune system, and potential physiological implications.

Effects of physiological concentrations of steroid hormones and interleukin-11 on basal and stimulated production of interleukin-8 by human osteoblast-like cells with different functional profiles.

OBJECTIVE: IL-8 is a CXC chemokine involved in the pathogenesis of articular damage in rheumatoid arthritis. Local hyperproduction of IL-8 has been suggested to play a role in subchondral bone loss, since it suppresses osteoblast activity and promotes osteoclasts recruitment. Osteoblasts are a source of IL-8; its secretion is regulated by a number of hormones and cytokines. The aim of the present study was to evaluate the single and combined effects of physiological concentrations of cortisol, 17 beta-estradiol and IL-11 upon basal and IL-1 beta-inducible production of IL-8 in two human osteoblast-like cell lines, Saos-2 and MG-63. METHODS: Cells were incubated with cortisol (0.01 to 1 microM), 17 beta-estradiol (10 to 1000 pg/ml), IL-11 (1 to 100 ng/ml), in presence or absence of IL-1 beta (10 ng/ml), for 20 h. Combinations of 17 beta-estradiol and cortisol, and of IL-11 and cortisol, were also tested. After incubation, IL-8 levels in supernatants were measured by ELISA. RESULTS: Cortisol dose-dependently inhibited spontaneous IL-8 secretion in both cell lines, although statistical significance was attained in the MG-63 cells only (P < 0.01); no effect of 17 beta-estradiol was apparent. With regard to IL-1 beta-inducible production, cortisol dose-dependently inhibited IL-8 release in both cell lines (P < 0.01); 17 beta-estradiol resulted in only a non-significant decrease in Saos-2, but not in MG-63 cells. 17 beta-estradiol did not alter the effects of cortisol in experiments involving co-incubation. IL-11 did not have any effect on spontaneous IL-8 release, but exerted a significant inhibitory effect on IL-1 beta-inducible release in MG-63 cells (P < 0.05); no additional effect was observed upon the degree of cortisol-dependent inhibition. CONCLUSION: Cortisol is a potent physiological inhibitor of IL-8 production by osteoblast-like cells. The results of the present study support the use of exogenous supplemental glucocorticoids to prevent the deleterious effects of excess IL-8. The estrogenic milieu and local concentrations of IL-11 have little if any effect on the IL-8-dependent mechanisms of disease.

Metabolic effects of single-dose pamidronate administration in prostate cancer patients with bone metastases.

BACKGROUND: Increased osteolysis usually accompanies sclerotic bone metastases from prostate cancer. This provides a rationale for the use of bisphosphonates to treat bone pain and prevent skeletal complications. METHODS: The fasting urinary levels of calcium, hydroxyproline (OHPRO), pyridinolines (PYD), deoxypyridinolines (DPYD), collagen cross-linked N-telopeptide (NTX) and the serum values of calcium, total alkaline phosphatase and relevant bone isoenzyme, bone gla protein (BGP), carboxy-telopeptide of type I collagen (ICTP) and parathyroid hormone (PTH) were determined at baseline and on the 15th, 30th, 60th and 90th days after single-dose (90 mg) pamidronate administration in 35 consecutive prostate cancer patients with bone metastases. These biochemical indices and serum interleukin 6 (IL-6) were also measured after four days in the last consecutive 17 cases. RESULTS: PYD, DPYD and NTX showed a significant decrease lasting four weeks (p<0.01, <0.01 and <0.001, respectively). OHPRO and ICTP did not change significantly. The NTX decline was greater than that of PYD and DPYD (maximum percent decrease: -71.3, -23.1 and -28.2, respectively). Bone formation markers and serum calcium did not change significantly. Serum PTH showed a rapid initial increase followed by a slow decrease (p<0.001). DPYD and NTX patterns did not correlate with changes in bone pain. As observed in the last 17 cases, the maximum osteolysis inhibition after pamidronate occurred on the fourth day after drug infusion. Serum IL-6 levels showed a short-lived decrease preceded by a transient rise on the fourth day. CONCLUSIONS: Pamidronate is able to induce a decrease in bone resorption without significantly influencing bone formation. The maximum decrease in bone resorption occurs very early. NTX is the most sensitive bone resorption marker in bisphosphonate therapy monitoring. Changes in IL-6 but not bone resorption markers may be useful in the prediction of symptomatic response.

Determinants of glucocorticoid action in the bone microenvironment.

In recent years, increased awareness and availability of proper diagnostic tools have made subclinical endocrine disease an emerging issue. The clinical impact of subtle alterations of hormonal secretion and/or action depends on the sensitivity of target cells, which shows wide interindividual variability, and within the same individual, varies among tissues and changes in particular microenvironments, e.g. when inflammation is present. Subclinical hypercortisolism is a typical example of this new approach which privileges hormonal action rather than secretion. In the present paper, we concisely review the mechanisms of genomic and non-genomic actions of glucocorticoids (GC), and the determinants of GC sensitivity, with special attention to those more investigated in relation to bone metabolism: GC receptor (GR) types, isoforms and polymorphisms, and 11beta-hydroxysteroid dehydrogenase (11beta-HSD) shuttle. Detailed knowledge of the mechanisms of GR action has opened the way to the development of novel selective GR modulators or selective GR agonists, which show promise of being efficacious for specific treatments of disease having fewer side effects. Exogenous GC excess due to the therapeutic use of GC is the most frequent cause of secondary osteoporosis. Variability of therapeutic or detrimental effects among patients and in the same patient as a function of the clinical course is nowadays recognized to depend on numerous factors. While intermediate to high doses are likely to overcome inter-individual differences in sensitivity, assessment of clinical efficacy and monitoring of adverse effects are of special importance for chronic users of low-dose GC; in these patients prediction of individual response seems still far from clinical practice. In the next future evaluation of GR polymorphisms and 11beta-HSD activities are likely to become important components of a comprehensive approach aimed to "tailor" the therapeutic strategy to the individual risk/benefit ratio.

Variations along the 24-hour cycle of circulating osteoprotegerin and soluble RANKL: a rhythmometric analysis.

UNLABELLED: The variability of serum osteoprotegerin (OPG) and soluble RANKL (sRANKL) along the 24-h cycle was assessed in 20 healthy women. No rhythmic variations of serum OPG, sRANKL or sRANKL/OPG ratio were detected as a group phenomenon. Timing of sampling is unlikely to influence the results of measurements of circulating OPG and sRANKL. INTRODUCTION: Physiological bone turnover shows diurnal variations. The aim of the study was to assess variability of OPG and sRANKL serum levels along the 24-h cycle. METHODS: Blood was collected from 20 healthy women (median age 31 years, range 25-65 years) at 4-h intervals between 08:00 and 24:00 and at 2-h intervals between 24:00 and 08:00. Serum albumin, cortisol, osteocalcin (OC), C-terminal telopeptide of type I collagen (CTX), OPG and total sRANKL were measured. Temporal variations were assessed by the COSINOR model. RESULTS: Circadian rhythms of cortisol and albumin documented a normal synchronization within the circadian structure. Serum OC and CTX showed rhythmic variations, peaking at night-time. Rhythmic variations of serum OPG, sRANKL and sRANKL/OPG ratio were not detected as a group phenomenon. On an individual basis, rhythmic changes were detected in ten patients for OPG and eight patients for sRANKL, with very small amplitudes and heterogeneous acrophases. CONCLUSIONS: The absence of consistent rhythmic variations of circulating OPG and sRANKL levels may reflect the absence of rhythmic variations of their expression in the bone microenvironment. Were this the case, the nocturnal rise of bone resorption should be accounted for by different, not RANKL/OPG-mediated factors. Since circulating OPG and sRANKL may derive from sources other than bone, rhythmicity could be masked by non-rhythmic or non-synchronized rhythmic expression in these sources. Timing of sampling is unlikely to influence the results of measurements of circulating OPG and sRANKL.

Circulating osteoprotegerin and soluble RANKL: do they have a future in clinical practice?

The discovery of the signalling system consisting of receptor activator of nuclear factor-KB ligand (RANKL), its receptor RANKL and its decoy receptor osteoprotegerin (OPG) has been a key step in the understanding of pathophysiology of the bone microenvironment and has provided pharmacological targets for new anti-resorptive drugs. The system is central to the interactions among bone, vascular and immune cells. OPG and a soluble form of RANKL (sRANKL) are present in the blood stream. The measurement of their concentrations offers insights into the regulatory mechanisms of the system and the possibility of using new markers in a number of diseases. Besides bone metabolic disorders, the list includes malignancies, rheumatic and cardiovascular diseases. However, apparent discrepancies in the outcome of studies point to a number of caveats: lack of control of pre-analytical and analytical variables; paucity of data on the metabolic removal of the molecules; the cross sectional design of most studies comparing patients with healthy subjects. Finally, circulating OPG and sRANKL derive from several sources, and their concentrations may be alterated by different coexisting pathological processes. In the absence of tissue-specific isoforms, diagnostic or prognostic significance may be suggested from data obtained in large cohorts, but is still of limited usefulness in single patients.

Differential responses of serum and salivary interleukin-6 to acute strenuous exercise.

Physical exercise is associated with elevation of serum levels of interleukin-6 (IL-6) because of its production in the muscles. The use of IL-6 measurements in saliva has been proposed in the field of immunopathology, mainly involving salivary gland disease. We evaluated the responses of serum and salivary IL-6 in two different groups of athletes submitted to different types of controlled strenuous exercise (spinning activity and maximal isokinetic test). Serum and salivary samples for IL-6 measurements, and serum samples for lactate and myoglobin determination before and after exercise, were obtained. Salivary IL-6 was measured by ELISA after dilution experiments and compared with results obtained by immunoradiometric assay. Spinning activity elicited significant increases in all the variables, and no correlation was found among the respective variations. A significant response to the isokinetic exercise was observed for serum IL-6, lactate and myoglobin only; no correlation was found between serum and salivary IL-6. Our study demonstrated that serum and salivary IL-6 responses to exercise are dissociated, possibly in relation to the lack of relationships between the systemic/muscular and the salivary routes of IL-6 production. Analytical issues that concern IL-6 measurement in saliva deserve attention, notably regarding the collection method used to absorb saliva. Concomitant monitoring of serum markers of inflammation, muscle metabolism and damage can provide information about muscle function properties and adaptations to physical effort in different types of athletes.

The overtraining syndrome in athletes: a stress-related disorder.

Physical exercise is a type of allostatic load for several endocrine systems, notably the hypothalamic-pituitary-adrenal (HPA) axis. Athletes undergoing a strenuous training schedule can develop a significant decrease in performance associated with systemic symptoms or signs: the overtraining syndrome (OTS). This is a stress-related condition that consists of alteration of physiological functions and adaptation to performance, impairment of psychological processing, immunological dysfunction and biochemical abnormalities. Universally agreed diagnostic criteria for OTS are lacking. The pituitary-adrenal response to a standardized exercise test is usually reduced in overtrained athletes. This HPA dysfunction could reflect the exhaustion stage of Selye's general adaptation syndrome. The most attractive hypothesis that accounts for the observed neuro-endocrine-immune dysregulation is the Smith's cytokine hypothesis of OTS. It assumes that physical training can produce muscle and skeletal trauma, thus generating a local inflammatory reaction. With the excessive repetition of the training stimulus the local inflammation can generate a systemic inflammatory response. The main actors of these processes are the cytokines, polypeptides that modulate HPA function in and outside the brain at nearly every level of activity. It is hoped that future research will focus on endogenous risk factors for morbidities related to the neuro-endocrine-immune adaptation to exercise.

Interleukin-6 upregulates glucocorticoid receptor numbers in human osteoblast-like cells.

Interactions between the endocrine and immune systems are well known with special regard to the hypothalamic-pituitary-adrenal axis. Little of the literature focuses on the complex effects of cytokines on tissue responsiveness to glucocorticoids (GC). In bone tissue, osteoblasts represent a valuable model for studying GC-cytokine interactions. Hence, we have studied two human osteosarcoma cell lines (Saos-2 and MG-63) with different degrees of differentiation and different constitutive IL-6 production (3.4 +/- 0.3 (mean +/- SE) and 3309 +/- 578 pg/10(6) cells). We measured glucocorticoid receptor (GR) number and affinity as a function of the exposure of cells to different amounts of IL-6. Incubation for 20 h with IL-6 at increasing concentrations up to 2000 pg/ml yielded a significant increase of GR binding sites in both cell lines. IL-6 was also able to reverse the inhibitory effect of dexamethasone (1 mumol/l) on GR in both cell lines. In MG-63 cells, expressing higher concentrations of GR, IL-6 deprivation via a specific anti-IL-6 antibody (100 ng/ml) significantly decreased GR. In Saos-2 cells, expressing lower concentrations of GR, a 40 h treatment with human IL-1 beta (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 results provide evidence that IL-6 is an autocrine positive modulator of GR number in human osteoblasts.

Inhibitory effect of physiological concentrations of cortisol but not estradiol on interleukin (IL)-6 production by human osteoblast-like cell lines with different constitutive IL-6 expression.

Estrogen deficiency and glucocorticoid excess are two well-known conditions that account for osteoporosis. Interleukin (IL)-6 plays an important role in bone resorption; both estrogens and glucocorticoids are credited with an inhibitory effect on osteoblast production of IL-6. The aim of the study was to investigate whether endogenous hormones, which lead to opposite changes in bone mass, have a common inhibitory effect upon constitutive and inducible IL-6 production by human osteoblast-like cells. We used two human osteosarcoma cell lines (MG-63 and Saos-2) with a different degree of differentiation and constitutive production of IL-6 [2587+/-536 (mean+/-SE) and 3.65+/-0.06 pg/10(6) cells, respectively]. We examined the effects of physiological and supraphysiological concentrations of 17beta-estradiol (E2) and cortisol on basal and IL-1beta-induced IL-6 release in the medium. In all experimental conditions, cellular estrogen receptors (ERs) and glucocorticoid receptors (GRs) were measured by binding assay. Both MG-63 and Saos-2 cell lines had measurable GRs (106 300+/-24 996 and 18 100+/-3215 binding sites/cell, respectively) and ERs (2197+/-377 and 1261+/-66.5 binding sites/cell, respectively). In MG-63 cells, cortisol treatment for 20 h decreased both basal and IL-1beta-induced IL-6 release in a dose-dependent manner; in Saos-2 cells the same effect was apparent for IL-1beta-induced release. Mifepristone (RU-486) did function as partial agonist and antagonist of cortisol. At variance with cortisol, E2 did not exert any effect on IL-6 secretion. Treatment with 1,25(OH)2D3 increased by 100-200% ER concentrations, but did not change ineffectiveness of E2 in modifying IL-6 production; furthermore, when E2 was combined with cortisol, there was no additive effect on cortisol-induced inhibition. The dissociation between glucocorticoid and estrogen effects observed in these human cell lines is a sufficiently robust phenomenon to raise questions about the pathogenetic role of IL-6 in osteoporosis associated with estrogen deficiency. Conversely inhibition of osteoblast production of IL-6 may offer an explanation why bone resorption is not the dominant factor in the pathogenesis of glucocorticoid-induced osteoporosis.

Interleukin-6 producing pheochromocytoma presenting with acute inflammatory syndrome.

Pheochromocytomas are tumors able to produce catecholamines and a variety of biologically active neuropeptides. We report the case of a 36-yr-old female patient with pheochromocytoma exhibiting headache, intermittent fever, thrombocytosis, and marked inflammatory signs. Nonsteroidal anti-inflammatory drugs were ineffective in lowering the body temperature, while a corticosteroid agent obtained excellent results. IL-6 was found elevated (20 pg/ml); it fell to 4.5 pg/ml 3 weeks after the adrenalectomy, in parallel to normalization of other laboratory data. The interleukin-6 (IL-6) over-production can either be ascribed directly to the tumor (as confirmed by immunohistochemistry) or indirectly accounted for by tumoral production, as a consequence of the high levels of circulating norepinephrine. To our knowledge, our paper represents the 6th case report of IL-6 secreting pheochromocytoma associated with clinical markers of inflammatory response.

Autocrine up-regulation of glucocorticoid receptors by interleukin-6 in human osteoblast-like cells.

Interleukin (IL)-6 is a bone-resorbing cytokine that acts primarily on osteoclast progenitors to stimulate both proliferation and differentiation. Glucocorticoids (GC) down-regulate IL-6 synthesis in different cell types, including osteoblasts. Given the fact that bone remodeling is a tightly controlled process, it is reasonable to think of auto-regulatory mechanisms in the bone microenvironment able to prevent excess IL-6 production. We have studied two human osteosarcoma cell lines (Saos-2 and MG-63) with different degrees of differentiation and different constitutive IL-6 production (3.4 +/- 0.2 (mean +/- SE) and 2,898 +/- 401 pg/10(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 2,000 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 microM) 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-1beta (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.

Natural killer cell activity and sensitivity to positive and negative modulation in uncomplicated obese subjects: relationships to leptin and diet composition.

BACKGROUND: Natural killer (NK) cells are a key component of innate immunity; their activity is modulated by cytokines and hormones and is influenced by diet. In obesity, a higher risk of cancer and infections has been demonstrated. Studies on NK cell activity have yielded inconsistent results; NK cell sensitivity to modulators has not been assessed before. OBJECTIVE: In this case-control study, we assessed both spontaneous NK cell activity and responsiveness to positive (interleukin (IL)-2) and negative (cortisol) modulators in uncomplicated obesity; we searched for correlations between NK cell activity and anthropometric, dietary and metabolic variables. METHODS: In all, 21 obese (six males/15 females) and 21 age- and sex-matched healthy nonobese subjects underwent clinical examination and dietary and laboratory analyses. Spontaneous and modulated NK activities of peripheral blood mononuclear cells were measured by enzyme-release cytotoxicity assay. RESULTS: Spontaneous NK cell activity was not different in obese subjects vs controls. IL-2 stimulated and cortisol inhibited NK cell activity in both populations. Cortisol-dependent inhibition was lower in the obese than in the control group (-24.4+/-2.9 vs -38.6+/-3.3%, P=0.002), but decreased sensitivity was restricted to women (P=0.0007). In obese subjects, cortisol-dependent inhibition negatively correlated with serum leptin levels (r=-0.54, P=0.02) and, in women, with body mass index (r=-0.63, P=0.01); IL-2-dependent stimulation positively correlated with dietary carbohydrates (r=0.61, P=0.005) and serum LDL levels (r=0.55, P=0.009) and negatively correlated with dietary lipids (r=-0.71, P=0.0006). CONCLUSION: Spontaneous and IL-2-inducible NK cell activity is normal in uncomplicated obesity. Sensitivity to IL-2 correlates with fat and carbohydrate intake. Sensitivity to glucocorticoids negatively correlates with serum leptin levels and is significantly diminished in obese women, in whom it correlates with body mass index.