Lina Matera

ENDOCRINE, PARACRINE AND AUTOCRINE ACTIONS OF PROLACTIN ON IMMUNE CELLS

The immune response is regulated by locally released factors, collectively referred to as cytokines. Data on the human immune system have convincingly demonstrated that the hormone prolactin (PRL), in addition to exerting its endocrine control on the immune system, acts as a cytokine in that it is released within the immune system and regulates the lymphocyte response by paracrine and autocrine mechanisms. Both lymphocyte and pituitary PRLs are under the control of immune factors. Synthesis of human PRL by lymphocytes is induced by T-cell stimuli, while increased release of PRL by the pituitary, observed in vivo after immune challenge, may be mediated by cytokines produced by monocyte-macrophages. Since hyperprolactinemia and hypoprolactinemia are both immunosuppressive, physiological levels of circulating PRL must be necessary to maintain basal immunocompetence. The effects of Cyclosporin (CsA) on IL-2 and PRL gene activation and the analysis of the intracellular signaling events downstream IL-2 and PRL receptors suggest coordinate actions of these two cytokines during T cell activation.

Modulatory effect of prolactin on the resting and mitogen-induced activity of T, B, and NK lymphocytes

Prolactin (PRL) has been shown to contribute to the development of lymphoid tissues and maintenance of physiological immune function. Here we show that the role of the hormone extends to the control of the effector phase of the immune response. In addition to triggering resting lymphocytes to cell division, the hormone can also control the magnitude of their response to polyclonal stimuli. Concentrations of PRL in the physiological range increased the [3H]thymidine, [3H]uridine, and [3H]leucine incorporation of unstimulated NK cells cultured in serum-free conditions. The same concentrations of the hormone increased the response of NK, T, and B cells to the mitogenic stimuli interleukin 2 (IL2), phytohemagglutinin (PHA), and staphylococcus aureus cowan, respectively, the effect being maximally evident in the presence of suboptimal concentrations of the mitogens. By contrast concentrations of PRL five- to tenfold the physiological levels inhibited the mitogenic response to IL2 and PHA. These data indicate a double-faceted regulatory role of this hormone in vivo.

Correlation between NK function and response to trastuzumab in metastatic breast cancer patients.

BackgroundTrastuzumab is a monoclonal antibody selectively directed against Her2 and approved for the treatment of Her2 overexpressing breast cancer patients. Its proposed mechanisms of action include mediation of antibody-dependent cellular cytotoxicity (ADCC) by triggering FcγRIII on natural killer (NK) cells. This study addresses the correlation between overall NK function and trastuzumabs clinical activity.Subjects and methodsClinical and immunological responses were assessed in 26 patients receiving trastuzumab monotherapy as maintenance management after chemotherapy (8 mg/kg load and then standard doses of 6 mg/kg every 3 weeks). Cytotoxic activity against the MHC class I-negative standard NK target K562 cell line and HER2-specific ADCC against a trastuzumab-coated Her2-positive SKBR3 cell line were assessed in peripheral blood mononuclear cells (PBMC) harvested after the first standard dose. After six months, seventeen patients were scored as responders and nine as non-responders according to the RECIST criteria, while Progression-Free Survival (PFS) was calculated during a 12 months follow-up.ResultsThe responders had significantly higher levels of both NK and ADCC activities (p < 0.05) that were not different from those of eleven normal controls. The NK activity of the non-responders was significantly (p < 0.05) lower than that of the normal controls. At twelve months, there was a marked correlation between PFS and NK activity only. PFS was significantly longer in patients with high levels of NK activity, whereas its pattern was unrelated to high or low ADCC activity.ConclusionOne of the mechanisms of action of trastuzumab is NK cell-mediated ADCC lysis of the Her2-positve target cell. We show here that its potency is correlated with the short-term response to treatment, whereas longer protection against tumor expansion seems to be mediated by pure NK activity.

Effect of prolactin on the antigen presenting function of monocyte-derived dendritic cells

Monocyte derived macrophages (Mf) and dendritic cells (DC) play critical roles at the interface between innate and adaptive immunity. Both types of cells can effectively phagocytose exogenous antigens, whereas only DC can process and present them efficiently to antigen-specific T lymphocytes. The hormone PRL is also produced by immune cells and is regarded as a key component of the neuroendocrine–immune loop and a local regulator of lymphocyte response. Its main feature is cooperation with cytokines and hemopoietins. Triggering of monocyte PRL receptors with physiological-to-supraphysiological concentrations of PRL up-regulates the GMCSF receptors, resulting in synergistic PRL-GM-CSF induced maturation of immature (i)DC. Further incubation induces increased antigen-presenting activity at the highest PRL concentrations studied (200 ng/ml). IFN-g release by allogeneic lymphocytes is dependent on T cell-triggered IL-12 release by PRL-preincubated iDC. This, in turn, may be secondary to increased DC expression of CD40 or IFN-g. The permissive action of high PRL concentrations in the antigen presenting process may be of significance in initiation of the response against major histocompatibility complex (MHC)-presented self-antigens and may explain the association of hyperprolactinemia with autoimmune diseases.

Prolactin in autoimmunity and antitumor defence

Prolactin (PRL) enhances inflammatory and antitumor responses in vitro and thus exhibits Th1-type cytokine-like effects. Evidence from experimental models indicates that inhibition of PRL release by bromocriptine downregulates immune reactions and ameliorates autoimmune diseases in which Th1 responses are predominant. A direct effect of locally produced PRL in some Th1 diseases, such as rheumatoid arthritis, supports this concept. Paradoxically, however, hyperprolactinemia can also be associated with conditions such as pregnancy, where remission of Th1-mediated diseases is known to occur in the context of a Th2-dominated milieu. This reversal of the Th1-promoting effect of PRL may be due to major changes in the levels of other hormones that can annul and/or override the PRL-mediated proinflammatory state. Nevertheless, PRL, as an immunopotentiating agent, may have a powerful therapeutic role in cancer and other immunocompromised patients.

Prolactin receptors on large granular lymphocytes: Dual regulation by cyclosporin A

Although evidence has been provided for a modulatory role of prolactin (PRL) on humoral and cell-mediated immune responses and PRL receptors have been found on T and B lymphocytes, no indications exist concerning the influence of PRL on natural killer (NK) activity nor has a structural basis for interaction been found on the NK effector cells (large granular lymphocytes, LGL). We show here that highly purified LGL express binding sites for PRL. The calculated receptor number was 660 per cell and the dissociation constant (Kd) was 3.0 X 10(-10) M. Since previous studies have reported that cyclosporin (CsA), an immunosuppressive agent used in organ transplant patients, affects the binding of PRL to T and B lymphocytes, but not to rabbit mammary gland cells, we investigated whether this compound could alter the binding of the hormone to LGL. At concentrations from 10(-7) to 10(-6), corresponding to the therapeutical range, CsA induced a complete inhibition of the PRL binding. By contrast, concentrations of CsA ranging from 10(-11) to 10(-9) increased the PRL binding to more than 100% of control levels. In addition to their antitumor role, LGL have been proposed to participate in graft versus host disease and in transplant rejection. The finding that CsA can differently affect PRL-receptor expression on LGL points to an involvement of CsA--PRL interactions in determining the output of these immune responses. In addition, these data strongly support the idea of a close relationship between the neuroendocrine and immune systems.

Cooperation of Pituitary Hormone Prolactin with Interleukin‐2 and Interleukin‐12 on Production of Interferon‐γ by Natural Killer and T Cells

Abstract: The pituitary hormone prolactin (PRL) is also produced by cells of the immune system and participates in early and late T cell activating events. We have previously shown a modulatory role of PRL during maturation of dendritic cells (DC). Production of IL‐12 by T cell receptor (TCR)‐activated DC is necessary for T cells to acquire the Th1 cytokine (i.e., IFN‐γ secreting) profile, which is associated with activation of cellular response. In a separate work, PRL has been shown to increase IFN‐γ synthesis by natural killer (NK) cells. We have extended that study by exploring the ability of PRL to induce IFN‐γ production by T and NK cells in the presence of the specific stimuli IL‐12 and IL‐2. The individual effect of PRL, IL‐12, and IL‐2 was specific for NK cells, and IL‐2 and IL‐12 were much more efficient than PRL. Cooperation of IL‐2 and PRL was observed on NK cells. IL‐2‐induced synthesis of IFN‐γ was increased by physiological concentrations of PRL but was unaffected or inhibited by high concentrations. By contrast, optimal enhancement of IL‐12‐induced IFN‐γ release was observed with T cells but not with NK cells. Unexpectedly, interaction between PRL and IL‐12 occurred only at high concentrations of PRL. These data indicate a complex role of PRL in the cytokine network and point to a revaluation of the proposed immunosuppression by stress‐related hyperprolactinemia.

Action of Pituitary and Lymphocyte Prolactin

In vivo and in vitro data combined show that prolactin (PRL) can mimic or interact with known lymphocyte cytokines and that these, in turn, can regulate PRL synthesis at the site of immune response. In contrast, pituitary PRL is under the control of both immune system products (non-cognitive stimuli) and signals to the CNS (cognitive stimuli). The role of PRL as a cytokine and as an endocrine hormone is discussed. In particular, assignment of PRL to the T helper 1 phenotype is proposed, based on its ability to enhance NK cell function, activate the interferon-regulated factor (IRF-1) transcription factor and to interact with or generate IL-2 and IFN gamma. Since hyperprolactinemia and hypoprolactinemia are both immunosuppressive, physiological levels of circulating PRL must be necessary to maintain normal immunocompetence. Moderate increases in PRL during immune stimulation of the hypothalamic-pituitary axis may counteract glucocorticoid inhibition, whereas inappropriate prolongation of PRL synthesis could lead to autoimmune diseases. Increased release of PRL by the pituitary during stress may inhibit NK cell antitumor cytotoxicity. The variety of PRL isotypes, the existence of multiple receptor subunits, and the complexity of their intracellular signaling may explain the specificity of PRL action on different target cells.

Obestatin regulates adipocyte function and protects against diet-induced insulin resistance and inflammation

The metabolic actions of the ghrelin gene‐derived peptide obestatin are still unclear. We investigated obestatin effects in vitro, on adipocyte function, and in vivo, on insulin resistance and inflammation in mice fed a high‐fat diet (HFD). Obestatin effects on apoptosis, differentiation, lipolysis, and glucose uptake were determined in vitro in mouse 3T3‐L1 and in human subcutaneous (hSC) and omental (hOM) adipocytes. In vivo, the influence of obestatin on glucose metabolism was assessed in mice fed an HFD for 8 wk. 3T3‐L1, hSC, and hOM preadipocytes and adipocytes secreted obestatin and showed specific binding for the hormone. Obestatin prevented apoptosis in 3T3‐L1 preadipocytes by increasing phosphoinositide 3‐kinase (PI3K)/Akt and extracellular signal‐regulated kinase (ERK)1/2 signaling. In both mice and human adipocytes, obestatin inhibited isoproterenol‐induced lipolysis, promoted AMP‐activated protein kinase phosphorylation, induced adiponectin, and reduced leptin secretion. Obestatin also enhanced glucose uptake in either the absence or presence of insulin, promoted GLUT4 translocation, and increased Akt phosphorylation and sirtuin 1 (SIRT1) protein expression. Inhibition of SIRT1 by small interfering RNA reduced obestatin‐induced glucose uptake. In HFD‐fed mice, obestatin reduced insulin resistance, increased insulin secretion from pancreatic islets, and reduced adipocyte apoptosis and inflammation in metabolic tissues. These results provide evidence of a novel role for obestatin in adipocyte function and glucose metabolism and suggest potential therapeutic perspectives in insulin resistance and metabolic dysfunctions.—Granata, R., Gallo, D., Luque, R. M., Baragli, A., Scarlatti, F., Grande, C., Gesmundo, I., Córdoba‐Chacón, J., Bergandi, L., Settanni, F., Togliatto, G., Volante, M., Garetto, S., Annunziata, M., Chanclón, B., Gargantini, E., Rocchietto, S., Matera, L., Datta, G., Morino, M., Brizzi, M. F., Ong, H., Camussi, G., Castaño, J. P., Papotti, M., Ghigo, E. Obestatin regulates adipocyte function and protects against diet‐induced insulin resistance and inflammation. FASEB J. 26, 3393–3411 (2012). www.fasebj.org

Influence of drug-induced apoptotic death on processing and presentation of tumor antigens by dendritic cells.

Here we have studied the effects of apoptotic cell death induced by chemotherapic agents on tumor phagocytosis by dendritic cells (DC) and presentation of the relevant antigen to T lymphocytes. Annexin‐V‐FITC (Ann‐V) and propidium iodide (PI) staining was used to assess early apoptotic (Ann‐V+/PI−) vs. late apoptotic/secondary necrotic (Ann‐V+/PI+) death after a 24 hr observation of untreated and drug‐treated gastric carcinoma cells. After treatments, the HLA‐A*0201+ tumor cell line KATO III was exposed for 24 hr to allogeneic, HLA‐related GM‐CSF, IL‐4‐driven immature (i) DC. Tumor‐loaded iDC were tested for IL‐12 release in an ELISA assay, incubated with the DC‐maturating factor TNF‐α and used as stimulators for autologous T lymphocytes. Generation of antitumor T response against KATO cells was evaluated in an anti‐MHC class I MAb‐blocked Interferon‐γ ELISPOT assay. After treatment with Cis‐platin (cis), all dying cells were in early apoptosis, whereas secondary necrosis was the prevalent death pattern observed after epirubicin (epi) and doxorubicin (doxo). Doxo and epi increased tumor expression of heat shock protein (hsp) 70 and uptake of tumor cell components by DC, whereas cis treatment had no effect on hsp70 and was associated with poor tumor uptake by DC. Significant upmodulation of IL‐12 was observed by DC that had taken up the doxo‐ and epi‐treated tumors (p< 0.005 and p< 0.01, respectively). Increased IFN‐γ release was also observed after stimulation of T lymphocytes with DC loaded with doxo‐ and epi‐treated (p< 0.02 and p< 0.005, respectively) but not with cis‐treated DC. These data show that the products of early apoptosis cannot efficiently cross‐activate MHC class I‐restricted anti‐tumor lymphocytes even in the presence of DC maturating factors, whereas secondary necrosis is associated with robust T cell response.