Michela Muscettola

Corticotropin-releasing hormone modulates cytokines release in cultured human peripheral blood mononuclear cells.

Immune and neuroendocrine systems interact at various levels. In particular, either cytokines activate the hypothalamus-pituitary-adrenal axis (HPA) or corticotropin-releasing hormone (CRH) induces the release of beta-endorphin from peripheral human mononuclear cells. The aim of the present study was to investigate whether CRH may affect cytokine production and activity in human peripheral blood mononuclear cells (PBMC). Primary cultures of human PBMC and monocytes were used. They were incubated in presence of different doses of synthetic human CRH. Media were collected and interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) levels were measured by ELISA, while interferon-gamma (IFN-gamma) levels were measured by bioassay. In addition, phytohemoagglutinin-induced lymphocyte proliferation was evaluated by testing [3H]thymidine incorporation in the presence of various doses of CRH. CRH significantly increased IL-6 release from PBMC (p < 0.01). The addition of CRH to PBMC significantly decreased IFN-gamma levels, in a dose dependent manner (p < 0.01). No significant effect of CRH was observed on lymphocyte proliferation or IL-1 beta production. The present results suggest a role for CRH as a paracrine mediator for human immune cells, increasing the evidence of a clear correlation between immune and neuroendocrine system.

SOMATOSTATIN AND VASOACTIVE INTESTINAL PEPTIDE REDUCE INTERFERON GAMMA PRODUCTION BY HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS

There is increasing evidence that neuropeptide modulation of the immune response is an important physiological phenomenon which involves the interaction of peptidergic neuromodulators with specific neuropeptide receptors on the plasma membrane of immune effector cells. Many studies have examined the effect of neuropeptides on mitogen-induced lymphocyte proliferation and immunoglobulin synthesis but very little is known about specific lymphokine production. In this study, we describe the effect of somatostatin (SOM) and vasoactive intestinal peptide (VIP) on interferon gamma (IFN-gamma) production by normal human peripheral blood mononuclear cells (PBMC) stimulated in vitro with polyclonal T cell activator staphylococcal enterotoxin A (SEA). Our findings provide experimental evidence that both SOM and VIP reduce the IFN-gamma production by SEA-stimulated PBMC. This reduction was time- (with maximal effect at 72 h) and dose-dependent (at doses as low as 10(-11) M with maximal effect at concentrations between 10(-9) and 10(-8) M of neuropeptides). This effect was absent in resting PBMC. The meaning of inhibitory effect of VIP and SOM on IFN-gamma production and its role in immune response in vivo are discussed.

Glucocorticoid receptors in human peripheral blood mononuclear cells in relation to age and to sport activity.

Glucocorticoid receptors (GR) are ubiquitous molecules and are present also in the hippocampus and in several other nervous and immune tissues. Peripheral blood mononuclear cells (PBMCs) are a good model for studies of GR in humans. Glucocorticoids are important for maintaining cellular and humoral homeostasis and are key mediators of neuroendocrine-immune regulatory interactions. The increase of cortisol is immunosuppressive and reduces GR concentration both in nervous and immune systems. Variation of glucocorticoids in healthy aged subjects and athletes has been shown. Prompted by these results, we have investigated in man a possible relationship between GR binding capacity in the PBMCs and age, in relation also to plasma testosterone and cortisol. The same parameters have been examined in a group of soccer players for comparison with the sedentary group. GR binding capacity was higher in younger subjects than in older ones, and lower in the group of athletes than in the younger and older sedentary subjects. In the sedentary group a negative correlation was present between GR binding capacity and age. Plasma cortisol was higher and testosterone lower in the athletes; they were negatively correlated in athletes and positively correlated in the sedentary subjects. The results for athletes agree with their lower anabolic/catabolic balance. The mechanism of reduced GR levels in relation to age and sport activity could involve a loss or an involution of receptor synthesis. However other possibilities, such as altered distribution of lymphocyte subpopulations with different receptor concentrations and with different cytokine production, cannot be excluded. Several neuroendocrine-immune interactions could be responsible for reduced GR levels with age and sport activity in man.

Effects of Formalin-Induced Pain on ACTH, Beta-Endorphin, Corticosterone and Interleukin-6 Plasma Levels in Rats

The behavioral and immunoendocrine effects of formalin-induced pain were studied in male rats following a subcutaneous injection of formalin (50 microliters; 0.1%, F01 groups, 10%, F10 groups) or sham injection (control groups). After treatment, animals were tested in a transparent open field for either 30 or 60 min and thereafter sacrificed by decapitation. Plasma was collected for adrenocorticotropic hormone (ACTH), corticosterone, beta-endorphin (beta-EP) and interleukin-6 (IL-6) determinations. Pain-evoked responses (licking, flexing, paw jerk), standard measures of activity (locomotion, rearing, olfactory exploration) and self-grooming were recorded. The higher formalin concentration induced stronger pain-evoked behavioral responses, paralleled by higher levels of ACTH, beta-EP and IL-6, but did not affect the other behavioral parameters. In contrast, the lower formalin concentration induced a marked increase in locomotion and rearing and a decrease in ACTH levels. In both formalin-injected groups, corticosterone did not differ from controls.

Gender‐related differences in erythrocyte glutathione peroxidase activity in healthy subjects

objective The antioxidant property of oestrogens may partly explain the gender differences in atherosclerotic heart disease and a reduction in the incidence of coronary heart disease, as well as mortality from cardiovascular disease in women undergoing postmenopausal oestrogen therapy. In the present study, we tested the hypothesis that the antioxidant glutathione peroxidase (GSH‐Px) erythrocyte activity is gender related and is correlated with oestradiol serum levels.

The role of liver in the catabolism of human alpha- and beta-interferon.

The susceptibility of human leukocyte (alpha), fibroblast (beta) and recombinant alpha-2-interferons to clearance by the isolated and perfused rabbit liver has been evaluated. Human leukocyte and recombinant alpha-2-interferons were stable and their initial levels were maintained in the perfusate even if they had been treated with neuraminidase, thus suggesting that alpha-interferons have no exposed sugars recognizable by hepatic binding proteins. On the other hand, native, and particularly desialylated human beta-interferon, underwent marked hepatic uptake confirming the importance of the liver as a catabolic site for glycosylated interferons.

The effect of LHRH and TRH on human interferon-gamma production in vivo and in vitro.

Accumulating evidence suggests that hypothalamic luteinizing hormone-releasing hormone (LHRH) and thyrotropin-releasing hormone (TRH) are two hypophysiotropic factors which modulate the immune response. The aim of the present study was to determine the in vivo effects of an intravenous bolus of LHRH and TRH on plasma interferon (IFN)-gamma production in five normoprolactinemic women with irregular menstrual cycles. We also determined prolactin (PRL), thyrotropin (TSH), follicle stimulating hormone (FSH), and luteinizing hormone (LH) levels before and after intravenous administration of LHRH and TRH. The results demonstrate that intravenous bolus of LHRH/TRH increases plasma IFN-gamma levels, with the maximum response 45 min after in vivo administration of hypothalamic peptides and after peak levels of adenohypophyseal hormones (PRL: 15 min; TSH: 30 min; FSH: 30 min; LH: 30 min). In order to investigate a possible direct action of hypothalamic hormones on immune cells, we also evaluated, in the same subjects, the influence of LHRH and TRH on IFN-gamma production by human peripheral blood mononuclear cells (PBMCs), collected before the intravenous administration of the peptides and stimulated in vitro with bacterial superantigen staphylococcal enterotoxin A (SEA) and concanavalin A (Con A). LHRH and TRH, separately and together, significantly enhanced in vitro IFN-gamma production by SEA- and ConA-activated PBMCs. The present results suggest that hypothalamic peptides (LHRH and TRH) directly, and/or indirectly pituitary hormones (PRL, TSH, FSH, and LH) or IL-2, have stimulatory effect on IFN-gamma producing cells and are further evidence of interactions between the neuroendocrine and immune systems.

Pulmonary catabolism of interferons: alveolar absorption of 125I-labeled human interferon alpha is accompanied by partial loss of biological activity

The catabolism of interferon was examined in isolated rabbit lungs which were ventilated and perfused with homologous blood. Natural human interferon-alpha (HuIFN-alpha) from lymphoblastoid Namalwa cells or recombinant DNA-derived HuIFN-alpha 2 were labeled with 125I, mixed with an excess of the respective cold interferons and added to the perfusion blood. Protein-bound and acid-soluble radioactivity, as well as antiviral activity, were measured at regular time intervals. During the first 3 h of perfusion, only very small fractions of the interferons disappeared from the perfusate, irrespective of whether lungs were inserted in the perfusion system. This indicated that catabolism of interferons in the pulmonary circulation was negligible. On the other hand, when the interferons were instilled into the bronchial-alveolar tree, absorption of antiviral activity differed from that of acid-precipitable protein-associated radioactivity. While most of the radioactivity was transferred into the perfusate, only 2% of antiviral activity of natural HuIFN-alpha and 30% of that of HuIFN-alpha 2 were recovered in the perfusate. In both cases acid-soluble radioactivity in the system reached about 10%. Since radioiodide, instilled in the bronchial-alveolar tree, was transported rapidly into the perfusate, this type of analysis did not help in locating the site(s) of degradation. Alveolar macrophages did not catabolize or inactivate interferons in vitro.

Rottlerin: a multifaced regulator of keratinocyte cell cycle.

Abstract:  In this study we showed that Rottlerin (also called Kamala or Mallotoxin), a natural product purified from Mallotus phillippinensis, is a potent suppressor of human keratinocytes (HaCaT cell line) proliferation. Following Rottlerin treatment, Thymidine incorporation into DNA and re‐epithelialisation in a scratch wound model was decreased. At the molecular level, Rottlerin hampered the NFkB activation process, causing loss of cyclin D1 and promoting, in a PKCδ‐dependent pathway, ERK activation, which, in turn induced the cell cycle inhibitor p21 Cip1/Kip1. The NFkB‐dependent drop in cyclin D1, along with the PKCδ/ERK‐dependent induction of p21 Cip1/Kip1, is responsible for growth arrest. These results open the way to further investigation on the Rottlerin therapeutic potential against keratinocyte hyper‐proliferative disorders.

Estrogen and μ-opioid receptor antagonists counteract the 17β-estradiol-induced licking increase and interferon-γ reduction occurring during the formalin test in male rats

&NA; Women have a higher incidence of chronic pain syndromes than men and are generally more sensitive to experimental pain. Numerous studies have shown that the female gonadal hormones, estrogens, can profoundly affect the nervous and immune systems, including mechanisms involved in pain and nociception. In the present study, we used antagonists of estrogen receptors (ER) or &mgr;‐opioid receptors (&mgr;OR) to evaluate the effects of estrogens on formalin‐induced behavioural and immune responses in male rats. After two days of priming with 17&bgr;‐estradiol or saline (i.c.v.), animals were subjected to the formalin test; 15 min prior to formalin (50 &mgr;l, 5%) or sham injection in the hind paw, animals were treated with an ER antagonist (ICI 182,780, ICI) or a &mgr;OR antagonist (&bgr;‐funaltrexamine, FNA) or saline. The spontaneous behaviours, pain‐related behaviours and interferon‐&ggr; (IFN‐&ggr;) production by peripheral blood mononuclear cells were studied in all groups. We found that central administration of estradiol increased the amount of licking of the formalin‐injected paw in the second phase of the formalin test. Whereas ICI and FNA had no effect on pain behaviour in saline‐pre‐treated animals, both antagonists reversed the estradiol‐induced increase in licking. The immune system was differently affected by formalin and estradiol treatment. Indeed, formalin injection per se decreased IFN‐&ggr; production; estradiol had no effect on sham‐injected animals but strongly reduce the decrease of IFN‐&ggr; production in formalin‐injected animals. The results demonstrate that centrally acting estrogens affect ER‐ and &mgr;OR‐mediated pain processing and influence immune function.