Eduardo Koji Tamura

TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway

Abstract:  Nuclear factor‐kappa B (NFKB), a pivotal player in inflammatory responses, is constitutively expressed in the pineal gland. Corticosterone inhibits pineal NFKB leading to an enhancement of melatonin production, while tumor necrosis factor (TNF) leads to inhibition of Aa‐nat transcription and the production of N‐acetylserotonin in cultured glands. The reduction in nocturnal melatonin surge favors the mounting of the inflammatory response. Despite these data, there is no clear evidence of the ability of the pineal gland to recognize molecules that signal infection. This study investigated whether the rat pineal gland expresses receptors for lipopolysaccharide (LPS), the endotoxin from the membranes of Gram‐negative bacteria, and to establish the mechanism of action of LPS. Here, we show that pineal glands possess both CD14 and toll‐like receptor 4 (TLR4), membrane proteins that bind LPS and trigger the NFKB pathway. LPS induced the nuclear translocation of p50/p50 and p50/RELA dimers and the synthesis of TNF. The maximal expression of TNF in cultured glands coincides with an increase in the expression of TNF receptor 1 (TNFR1) in isolated pinealocytes. In addition, LPS inhibited the synthesis of N‐acetylserotonin and melatonin. Therefore, the pineal gland transduces Gram‐negative endotoxin stimulation by producing TNF and inhibiting melatonin synthesis. Here, we provide evidence to reinforce the idea of an immune‐pineal axis, showing that the pineal gland is a constitutive player in the innate immune response.

Melatonin inhibits LPS-induced NO production in rat endothelial cells

Abstract:  Endothelial cells produce NO by activation of constitutive nitric oxide synthase (NOS) and transcription of inducible NOS (iNOS). We have previously shown that melatonin, in the nanomolar range, inhibits activation of constitutive NOS, and in the present paper, we evaluated whether it could interfere with the expression of iNOS, which is activated by lipopolysaccharide (LPS), a major component of gram‐negative bacteria cell walls. Primary cultures of rat endothelial cells were loaded with fluorescent probe for NO detection. Nuclear factor kappa B (NF‐κB) translocation in endothelial cells elicited by LPS was measured by electromobility shift assay, and the vasodilation of aortic rings was accessed by recording isometric contraction. Melatonin in a micromolar but not in a nanomolar range inhibits the NO production induced by LPS. This effect is not dependent on the activation of G protein‐coupled melatonin receptors. The nuclear NF‐κB translocation is a process necessary for iNOS transcription, and melatonin also inhibits its translocation. LPS induced vasodilation only in endothelium‐intact aortic rings, and melatonin (10 μm) inhibits the vasodilation. Here, we show that concentrations compatible with nocturnal melatonin surge (nm) did not interfere with the activity of iNOS. Considering that micromolar melatonin concentrations could be locally achieved through production by activated immune competent cells, extra‐pineal melatonin could have a protective effect against tissue injury. We propose that melatonin blocked the LPS‐induced vasodilation by inhibiting the NF‐κB pathway. Finally, we propose that the effect of melatonin on vascular reactivity is one of the mechanisms that underlies the protective effect of this indolamine against LPS.

NF-κB drives the synthesis of melatonin in RAW 264.7 macrophages by inducing the transcription of the arylalkylamine-N-acetyltransferase (AA-NAT) gene.

We demonstrate that during inflammatory responses the nuclear factor kappa B (NF-κB) induces the synthesis of melatonin by macrophages and that macrophage-synthesized melatonin modulates the function of these professional phagocytes in an autocrine manner. Expression of a DsRed2 fluorescent reporter driven by regions of the aa-nat promoter, that encodes the key enzyme involved in melatonin synthesis (arylalkylamine-N-acetyltransferase), containing one or two upstream κB binding sites in RAW 264.7 macrophage cell lines was repressed when NF-κB activity was inhibited by blocking its nuclear translocation or its DNA binding activity or by silencing the transcription of the RelA or c-Rel NF-κB subunits. Therefore, transcription of aa-nat driven by NF-κB dimers containing RelA or c-Rel subunits mediates pathogen-associated molecular patterns (PAMPs) or pro-inflammatory cytokine-induced melatonin synthesis in macrophages. Furthermore, melatonin acts in an autocrine manner to potentiate macrophage phagocytic activity, whereas luzindole, a competitive antagonist of melatonin receptors, decreases macrophage phagocytic activity. The opposing functions of NF-κB in the modulation of AA-NAT expression in pinealocytes and macrophages may represent the key mechanism for the switch in the source of melatonin from the pineal gland to immune-competent cells during the development of an inflammatory response.

Glia-Pinealocyte Network: The Paracrine Modulation of Melatonin Synthesis by Tumor Necrosis Factor (TNF)

The pineal gland, a circumventricular organ, plays an integrative role in defense responses. The injury-induced suppression of the pineal gland hormone, melatonin, which is triggered by darkness, allows the mounting of innate immune responses. We have previously shown that cultured pineal glands, which express toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1), produce TNF when challenged with lipopolysaccharide (LPS). Here our aim was to evaluate which cells present in the pineal gland, astrocytes, microglia or pinealocytes produced TNF, in order to understand the interaction between pineal activity, melatonin production and immune function. Cultured pineal glands or pinealocytes were stimulated with LPS. TNF content was measured using an enzyme-linked immunosorbent assay. TLR4 and TNFR1 expression were analyzed by confocal microscopy. Microglial morphology was analyzed by immunohistochemistry. In the present study, we show that although the main cell types of the pineal gland (pinealocytes, astrocytes and microglia) express TLR4, the production of TNF induced by LPS is mediated by microglia. This effect is due to activation of the nuclear factor kappa B (NF-kB) pathway. In addition, we observed that LPS activates microglia and modulates the expression of TNFR1 in pinealocytes. As TNF has been shown to amplify and prolong inflammatory responses, its production by pineal microglia suggests a glia-pinealocyte network that regulates melatonin output. The current study demonstrates the molecular and cellular basis for understanding how melatonin synthesis is regulated during an innate immune response, thus our results reinforce the role of the pineal gland as sensor of immune status.

Melatonin inhibits nitric oxide production by microvascular endothelial cells in vivo and in vitro

We have previously shown that melatonin inhibits bradykinin‐induced NO production by endothelial cells in vitro. The purpose of this investigation was to extend this observation to an in vivo condition and to explore the mechanism of action of melatonin.

Melatonin inhibits endothelial nitric oxide production in vitro

Abstract:  Endothelial cell function is a major player on the regulation of both vascular tonus and permeability. Activation of nitric oxide synthase (NOS) by bradykinin is one physiological pathway for the well‐known vascular relaxation mediated by endothelial‐derived nitric oxide (NO). In this study we investigated if melatonin, which is known to modulate endothelial cell function and NO production in other tissues, is able to impair bradykinin‐induced NO production in vitro. Rat microvascular endothelial cells were incubated with fluorescent dyes to detect either NO or Ca2+. In addition, cGMP levels were measured by enzyme immunoassay. We found that while bradykinin (1–100 nm) increased both cytosolic Ca2+ and NO production, melatonin (1 nm) abolished this NO production but not cytosolic Ca2+ elevation. N‐acetylserotonin (0.1 and 1 nm) had the same effect, while the selective agonist for MT3 receptors (5‐MCA‐NAT, 1 nm) had no effect. Moreover, nonselective and MT2‐selective antagonists did not alter the effect of melatonin, suggesting that it is not mediated by MT melatonin receptors. A possible direct inhibition of calmodulin was also discarded as melatonin did not mimic the effect of calmidazolium on cytosolic Ca2+. Melatonin also abolished cGMP production induced by 1 μm bradykinin, indicating that the NO downstream effect is impaired. Thus, here we show that melatonin reduces NO production induced by bradykinin by a mechanism upstream to the interaction of Ca2+–calmodulin with NOS. Moreover, this effect might be the basis of the diurnal variation in endothelial cell function.

Long-Lasting Priming of Endothelial Cells by Plasma Melatonin Levels

Background Endothelial cells are of great interest for cell therapy and tissue engineering. Understanding the heterogeneity among cell lines originating from different sources and culture protocols may allow more standardized material to be obtained. In a recent paper, we showed that adrenalectomy interferes with the expression of membrane adhesion molecules on endothelial cells maintained in culture for 16 to 18 days. In addition, the pineal hormone, melatonin, reduces the adhesion of neutrophils to post-capillary veins in rats. Here, we evaluated whether the reactivity of cultured endothelial cells maintained for more than two weeks in culture is inversely correlated to plasma melatonin concentration. Methodology/Principal Findings The nocturnal levels of melatonin were manipulated by treating rats with LPS. Nocturnal plasma melatonin, significantly reduced two hours after LPS treatment, returned to control levels after six hours. Endothelial cells obtained from animals that had lower nocturnal melatonin levels significantly express enhanced adhesion molecules and iNOS, and have more leukocytes adhered than cells from animals that had normal nocturnal levels of melatonin (naïve or injected with vehicle). Endothelial cells from animals sacrificed two hours after a simultaneous injection of LPS and melatonin present similar phenotype and function than those obtained from control animals. Analyzing together all the data, taking into account the plasma melatonin concentration versus the expression of adhesion molecules or iNOS we detected a significant inverse correlation. Conclusions/Significance Our data strongly suggest that the plasma melatonin level primes endothelial cells “in vivo,” indicating that the state of the donor animal is translated to cells in culture and therefore, should be considered for establishing cell banks in ideal conditions.

Molecular Basis for Defining the Pineal Gland and Pinealocytes as Targets for Tumor Necrosis Factor

The pineal gland, the gland that translates darkness into an endocrine signal by releasing melatonin at night, is now considered a key player in the mounting of an innate immune response. Tumor necrosis factor (TNF), the first pro-inflammatory cytokine to be released by an inflammatory response, suppresses the translation of the key enzyme of melatonin synthesis (arylalkylamine-N-acetyltransferase, Aanat). Here, we show that TNF receptors of the subtype 1 (TNF-R1) are expressed by astrocytes, microglia, and pinealocytes. We also show that the TNF signaling reduces the level of inhibitory nuclear factor kappa B protein subtype A (NFKBIA), leading to the nuclear translocation of two NFKB dimers, p50/p50, and p50/RelA. The lack of a transactivating domain in the p50/p50 dimer suggests that this dimer is responsible for the repression of Aanat transcription. Meanwhile, p50/RelA promotes the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide, which inhibits adrenergically induced melatonin production. Together, these data provide a mechanistic basis for considering pinealocytes a target of TNF and reinforce the idea that the suppression of pineal melatonin is one of the mechanisms involved in mounting an innate immune response.

Inhibitory effects of Solidago chilensis Meyen hydroalcoholic extract on acute inflammation

AIM OF THE STUDY Alcoholic or hydroalcoholic preparations of the plant Solidago chilensis Meyen (Asteraceae) are employed in popular medicines to treat inflammation. The anti-inflammatory effects of the hydroalcoholic extract of aerial parts of the plant (93% ethanol) were investigated and the main components of the extract were identified. MATERIALS AND METHODS Ear oedema was induced in male Wistar rats by topical application of the chloroform fraction of latex-extract from Euphorbia milii. Leukocyte mobilisation was quantified after air-pouch inflammation evoked by oyster glycogen. Leukocyte-endothelial interactions and mast cell degranulation were quantified by intravital microscopy. The extract itself was characterised via HPLC-DAD-MS and HPLC-MS/MS. RESULTS Topical (12.5-50mg/kg) or intraperitoneal (25 or 50mg/kg) administrations of the extract reduced ear oedema formation (>25% reduction). Intraperitoneal applications of 25mg/kg of extract inhibited the migration of polymorphonuclear cells into the inflamed cavity (about 50%). In addition, the rolling behaviour and adherence of circulating leukocytes to postcapillary venules of the mesentery network was diminished (50%), but the mast cell degranulation in the perivascular area was not affected. The major components of the extract were identified as caffeoylquinic acid derivatives and the flavonoid rutin. CONCLUSIONS The data presented herein show local and systemic anti-inflammatory effects of the hydroalcoholic extract of aerial parts of Solidago chilensis, and implicate the inhibition of leukocyte-endothelial interactions as an important mechanism of the extracts action.

Melatonin synthesis in human colostrum mononuclear cells enhances dectin‐1‐mediated phagocytosis by mononuclear cells

Many cells in the organism besides pinealocytes, synthesize melatonin. Here, we evaluate both the mechanism of zymosan‐induced melatonin synthesis and its autocrine effect in human colostral mononuclear cells. The synthesis of melatonin was induced by activation of the transcription factor nuclear factor kappa B (NF‐κB), as either the blockade of the proteasome or the binding of NF‐κB to DNA inhibits zymosan‐induced melatonin synthesis. As observed in RAW 264.7 lineage cells, the dimer involved is RelA/c‐Rel. Melatonin plays a direct role in mononuclear cell activity, increasing zymosan‐induced phagocytosis by stimulating MT2 melatonin receptors and increasing the expression of dectin‐1. This role was confirmed by the blockade of melatonin receptors using the competitive antagonist luzindole and the MT2‐selective partial agonist 4P‐PDOT. In summary, we show that melatonin produced by immune‐competent cells acts in an autocrine manner, enhancing the clearance of pathogens by increasing phagocyte efficiency. Given that these cells are present in human colostrum for 4 or 5 days after birth, this mechanism may be relevant for the protection of infant health.