José M. Fernández-Santos

Beneficial pleiotropic actions of melatonin in an experimental model of septic shock in mice: regulation of pro-/anti-inflammatory cytokine network, protection against oxidative damage and anti-apoptotic effects.

Abstract:  Septic shock, the most severe problem of sepsis, is a lethal condition caused by the interaction of a pathogen‐induced long chain of sequential intracellular events in immune cells, epithelium, endothelium, and the neuroendocrine system. The lethal effects of septic shock are associated with the production and release of numerous pro‐inflammatory biochemical mediators including cytokines, nitric oxide and toxic oxygen and nitrogen radicals, together with development of massive apoptosis. As melatonin has remarkable properties as a cytokine modulator, antioxidant and anti‐apoptotic agent, the present study was designed to evaluate the possible protective effect of melatonin against LPS‐induced septic shock in Swiss mice. We observed that intraperitoneally (i.p.) administered‐melatonin (10 mg/kg) 30 min prior, and 1 hr after i.p. LPS injection (0.75 mg/animal) markedly protected mice from the LPS lethal effects with 90% survival rates for melatonin and 20% for LPS‐injected mice after 72 hr. The melatonin effect was mediated by modulating the release of pro‐/anti‐inflammatory cytokine levels, protection from oxidative damage and counteracting apoptotic cell death. Melatonin was able to partially counteract the increase in LPS‐induced pro‐inflammatory cytokine levels such as tumor necrosis factor‐α, IL‐12 and interferon‐γ at the local site of injection, while it increased the production of the anti‐inflammatory cytokine IL‐10 both locally and systemically. Furthermore, melatonin inhibited the LPS‐induced nitrite/nitrate and lipid peroxidation levels in brain and liver and counteracted the sepsis‐associated apoptotic process in spleen. In conclusion, we have demonstrated that melatonin improves the survival of mice with septic shock via its pleiotropic functions as an immunomodulator, antioxidant and anti‐apoptotic mediator.

Melatonin synthesized by T lymphocytes as a ligand of the retinoic acid-related orphan receptor.

Abstract:  Melatonin modulates a wide array of physiological events with pleiotropic effects on the immune system. While the relevance of specific melatonin membrane receptors has been well established for several biological functions, retinoic acid‐related orphan receptor alpha (RORα) has been suggested as a mediator of nuclear melatonin signalling by results obtained from pharmacological approaches. However, a melatonin‐mediated downstream effect cannot be ruled out, and further evidence is needed to support a direct interaction between melatonin and RORα. Here, we show that RORα is mainly located in human Jurkat T‐cell nucleus, and it is co‐immunoprecipitated with melatonin. Moreover, immunocytochemistry studies confirmed the co‐localization of melatonin and RORα. Melatonin promoted a time‐dependent decrease in nuclear RORα levels, suggesting a role in the RORα transcriptional activity. Interestingly, RORα acts as a molecular switch implicated in the mutually exclusive generation of Th17 and Treg cells, both involved in the harm/protection balance of immune conditions such as autoimmunity or acute transplant rejection. Therefore, the identification of melatonin as a natural modulator of RORα gives it a tremendous therapeutic potential for a variety of clinical disorders.

Dual effect of melatonin as proinflammatory and antioxidant in collagen-induced arthritis in rats

Abstract:  The aim of this study was to determine the effects of melatonin on proinflammatory status of rats with collagen‐induced arthritis (CIA). CIA was induced in male Wistar rats with an emulsion of type II collagen in Freunds Incomplete Adjuvant (C‐II/FIA). For 14 days, control and pinealectomized rats received a subcutaneous injection of 100 μL melatonin (30 μg) or vehicle (saline on 1% ethanol). Levels of cytokines interleukin (IL)‐1β and IL‐6 were determined in the serum, peripheral blood mononuclear cells, and joints. Levels of anti‐type II collagen antibody, nitrite/nitrate, and lipid peroxidation (LPO) were determined in the serum, joints, and brain. Treatment with melatonin significantly increased the levels of IL‐1β, IL‐6, nitrite/nitrate and LPO in joints. However, melatonin significantly reduced the levels of nitrite/nitrate and LPO in serum and brain. Moreover, CIA in pinealectomized rats presented significantly reduced levels of IL‐1β and IL‐6, titers of anti‐type II collagen antibodies, levels of nitrite/nitrate, and LPO in joints but elevated levels in serum and brain. Melatonin has been described as a proinflammatory and antioxidant agent. In a process of inflammation as CIA, melatonin acts with a markedly proinflammatory effect at local and peripheral levels maintaining its antioxidant effect only at peripheral level.

Melatonin controls experimental autoimmune encephalomyelitis by altering the T effector/regulatory balance☆

Experimental autoimmune encephalomyelitis (EAE), the experimental model for multiple sclerosis (MS), is triggered by myelin-specific Th1 and Th17 cells. The immunomodulatory activities of melatonin have been shown to be beneficial under several conditions in which the immune system is exacerbated. Here, we sought to elucidate the basis of the melatonin protective effect on EAE by characterizing the T effector/regulatory responses, particularly those of the memory cell subsets. Melatonin was tested for its effect on Th1, Th17 and T regulatory (Treg) cells in the lymph nodes and CNS of immunodominant peptide of myelin oligodendrocyte glycoprotein (pMOG)-immunized and EAE mice, respectively. The capacity of melatonin to ameliorate EAE as well as modifying both T cell response and effector/regulatory balance was surveyed. T cell memory subsets and CD44, a key activation marker involved in the EAE pathogenesis, were also examined. Melatonin protected from EAE by decreasing peripheral and central Th1/Th17 responses and enhancing both the Treg frequency and IL-10 synthesis in the CNS. Melatonin reduced the T effector memory population and its pro-inflammatory response and regulated CD44 expression, which was decreased in T effector cells and increased in Tregs. The alterations in the T cell subpopulations were associated with a reduced mononuclear infiltration (CD4 and CD11b cells) of the melatonin-treated mice CNS. For the first time, we report that melatonin protects against EAE by controlling peripheral and central T effector/regulatory responses, effects that might be partially mediated by CD44. This immunomodulatory effect on EAE suggests that melatonin may represent an effective treatment option for MS.

Functional expression of the thyrotropin receptor in C cells: new insights into their involvement in the hypothalamic‐pituitary‐thyroid axis

Thyroid C cells, or parafollicular cells, are mainly known for producing calcitonin, a hormone involved in calcium homeostasis with hypocalcemic and hypophosphatemic effects. Classically, the main endocrine activity of this cell population has been believed to be restricted to its roles in serum calcium and bone metabolism. Nonetheless, in the last few years evidence has been accumulating in the literature with regard to local regulatory peptides secreted by C cells, such as somatostatin, ghrelin, thyrotropin releasing hormone or the recently described cocaine‐ and amphetamine‐related transcript, which could modify thyroid function. As thyrotropin is the main hormone controlling the hypothalamic‐pituitary‐thyroid axis and, accordingly, thyroid function, we have examined the functional expression of the thyrotropin receptor in C‐cell lines and in thyroid tissues. We have found that rat and human C‐cell lines express the thyrotropin receptor at both mRNA and protein levels. Furthermore, incubation of C cells with thyrotropin resulted in a 10‐fold inhibition of thyrotropin‐receptor expression, and a concomitant decrease of the steady‐state mRNA levels for calcitonin and calcitonin gene‐related peptide determined by quantitative real‐time PCR was found. Finally, thyrotropin receptor expression by C cells was confirmed at protein level in both normal and pathological thyroid tissues by immunohistochemistry and immunofluorescence. These results confirm that C cells, under regulation by thyrotropin, are involved in the hypothalamic‐pituitary‐thyroid axis and suggest a putative role in local fine‐tuning of follicular cell activity.

C cells evolve at the same rhythm as follicular cells when thyroidal status changes in rats

C cells are primarily known for producing calcitonin, a hypocalcemic and hypophosphatemic hormone. Nevertheless, besides their role in calcium homeostasis, C cells may be involved in the intrathyroidal regulation of follicular cells, suggesting a possible interrelationship between the two endocrine populations. If this premise is true, massive changes induced by different agents in the activity of follicular cells may also affect calcitonin‐producing cells. To investigate the behaviour of C cells in those circumstances, we have experimentally induced two opposite functional thyroid states. We hyperstimulated the follicular cells using a goitrogen (propylthiouracil), and we suppressed thyroid hormone synthesis by oral administration of thyroxine. In both scenarios, we measured T4, TSH, calcitonin, and calcium serum levels. We also completely sectioned the thyroid gland, specifically immunostained the C cells, and rigorously quantified this endocrine population. In hypothyroid rats, not only follicular cells but also C cells displayed hyperplastic and hypertrophic changes as well as increased calcitonin levels. When exogenous thyroxine was administered to the rats, the opposite effect was noted as a decrease in the number and size of C cells, as well as decreased calcitonin levels. Additionally, we noted that the two cell types maintain the same numerical relation (10 ± 2.5 follicular cells per C cell), independent of the functional activity of the thyroid gland. Considering that TSH serum levels are increased in hypothyroid rats and decreased in thyroxine‐treated rats, we discuss the potential involvement of thyrotropin in the observed results.

Long-term melatonin administration increases polyunsaturated fatty acid percentage in plasma lipids of hypercholesterolemic rats

This study was designed to investigate the effect of melatonin on the fatty acid composition of plasma and tissue lipids. Melatonin administration to rats fed with a standard diet only increased long‐chain n‐6 polyunsaturated fatty acids (PUFA) in total plasma lipids and liver phospholipids but induced significant changes in hypercholesterolemic rats. In plasma, palmitoleic and oleic acids increased and n‐6 and n‐3 PUFA decreased in hypercholesterolemic rats; theses changes were reversed by melatonin administration. The analysis of lipid fractions revealed that only the cholesteryl ester fraction was affected by melatonin. Histological studies of the carotid artery intima revealed the appearance, in hypercholesterolemic rats, of fatty streaks produced by a mass of foam cells covered by the endothelium and by a thin layer of mononucleated cells. These changes were prevented by melatonin. We conclude that long‐term melatonin administration modifies the fatty acid composition of rat plasma and liver lipids and ameliorates the arterial fatty infiltration induced by cholesterol.

Comparative immunohistochemical study of normal, hyperplastic and neoplastic C cells of the rat thyroid gland.

Abstract. In rats, the frequency of spontaneous C-cell tumours is very high and is both age and gender dependent. The three specific stages of neoplastic progression can be distinguished into diffuse C-cell hyperplasia, focal C-cell hyperplasia and bona fide C-cell tumours. Based on this hypothetical model of human medullary thyroid carcinoma (MTC), we carried out an immunohistochemical study using different markers (calcitonin, calcitonin gene-related peptide, somatostatin and chromogranin) to verify the existence of any relationship between their expression and the successive steps of tumour development. We found a characteristic immunohistochemical staining pattern, particularly for calcitonin and somatostatin, which distinguishes C-cell tumours from both normal and hyperplastic C cells, with no differences related to the gender of the animals under study. Specifically, a considerable heterogeneity in calcitonin expression was only displayed by C-cell carcinomas, being less pronounced in C-cell adenomas. As for somatostatin, this regulatory peptide was found only in a minority of calcitonin-positive cells in normal and hyperplastic glands. However, in some C-cell adenomas and most C-cell carcinomas nearly all calcitonin-positive cells also coexpressed somatostatin. We conclude that rat C-cell neoplasms constitute a very particular tumour entity which shares many but not all immunohistochemical features with human MTC.

Ghrelin potentiates TSH-induced expression of the thyroid tissue-specific genes thyroglobulin, thyroperoxidase and sodium-iodine symporter, in rat PC-Cl3 Cells.

Ghrelin is a 28-amino-acid peptide that stimulates pituitary growth-hormone secretion and modulates food-intake and energy metabolism in mammals. It is mainly secreted by the stomach, but it is also expressed in many other tissues such as cartilage or the thyroid gland. In the present study we have analyzed by RT-PCR and using immunohistochemistry and immunofluorescence the expression and tissue distribution of ghrelin and its functional receptor (GHS-R type 1α) in thyroid cell-lines and in normal and pathological rat thyroid tissue. Additionally, by measuring the incorporation of BrdU, we have investigated if, as previously noted for FRTL-5 cells, ghrelin enhances the proliferation rate in the PC-Cl3 rat-thyrocyte cell-line. Finally, we have determined the stimulatory effect of ghrelin on TSH-induced expression of the tissue-specific key genes involved in the synthesis of thyroid hormone: thyroglobulin, thyroperoxidase and sodium-iodine symporter. Our data provide direct evidence that C-cell secreted ghrelin may be involved in the paracrine regulation of the thyroid follicular cell function.

The expression of Sam68, a protein involved in insulin signal transduction, is enhanced by insulin stimulation

Abstract. The role of Sam68, an RNA binding protein and putative substrate of the insulin receptor (IR) in insulin signaling was studied using CHO wild type (WT) cells, CHO cells overexpressing IR, and rat white adipocytes as a physiological system. In CHO-IR cells and adipocytes, Sam68 was tyrosine phosphorylated in response to insulin, and then associated with p85 phosphatidylinositol-3 kinase along with IRS-1. Sam68 was localized mainly in the nucleus of CHO-WT, and both in the nucleus and cytoplasm of CHO-IR cells, but only in the cytoplasm of rat white adipocytes. Insulin stimulation for 16h enhanced the expression of Sam68 in rat adipocytes and CHO-IR cells. Moreover, CHO-IR cells expressed more Sam68 than CHO-WT, suggesting that overexpression of the IR is enough to induce the expression of Sam68. In summary, these results demonstrate that Sam68 works as a cytoplasmic docking protein which is recruited by IR signaling and whose expression is induced by insulin stimulation, suggesting a putative role for Sam68 in insulin signal transduction.