Zulma S. Ferreira

The Immune-Pineal Axis: A Shuttle between Endocrine and Paracrine Melatonin Sources

The time course of the innate immunological response involves a pro-inflammatory phase followed by an anti-inflammatory phase. Pro-inflammatory responses serve as a defense against several stressor conditions, and sequential processes that shut down these responses are necessary to avoid exacerbation or the development of chronic diseases. In the present review, we put together recent data that show that the pineal gland is a player in bidirectional control of the inflammatory response. Healthy organisms stay in standby mode, ready to react. The nocturnal melatonin surge impairs the rolling and adherence of leukocytes to endothelial layers, limiting cell migration, and stimulates nocturnal production of IL-2 by T helper lymphocytes, exerting an immunostimulatory effect. Otherwise, the release of TNF-α from activated macrophages suppresses the nocturnal melatonin surge, allowing a full cell migration and inhibiting IL-2 production. In sequence, activated mononuclear and polymorphonuclear cells produce melatonin in a paracrine manner at the site of injury, which scavenges free radicals and collaborates to resolve the inflammatory response. The sequential diminution of TNF-α production is followed by the recovery of the nocturnal melatonin surge and IL-2 production. In summary, the immune-pineal axis, implicated in the sequential involvement of the melatonin produced by the pineal gland and immune-competent cells, is an integral participant of the innate immune response.

Effect of TNF-α on the melatonin synthetic pathway in the rat pineal gland: basis for a ‘feedback’ of the immune response on circadian timing

Abstract:  A retino‐hypothalamic‐sympathetic pathway drives the nocturnal surge of pineal melatonin production that determines the synchronization of pineal function with the environmental light/dark cycle. In many studies, melatonin has been implicated in the modulation of the inflammatory response. However, scant information on the feedback action of molecules present in the blood on the pineal gland during the time course of an inflammatory response is available. Here we analyzed the effect of tumor necrosis factor‐α (TNF‐α) and corticosterone on the transcription of the Aa‐nat, hiomt and 14‐3‐3 protein genes in denervated pineal glands of rats stimulated for 5 hr with norepinephrine, using real‐time reverse transcription‐polymerase chain reaction. The transcription of Aa‐nat, a gene encoding the key enzyme in melatonin biosynthesis, together with the synthesis of the melatonin precursor N‐acetylserotonin, was inhibited by TNF‐α. This inhibition was transient, and a preincubation of TNF‐α for more than 24 hr had no detectable effect. In fact, a protein(s) transcribed, later on, as shown by cycloheximide, was responsible for the reversal of the inhibition of Aa‐nat transcription. In addition, corticosterone induced a potentiation of norepinephrine‐induced Aa‐nat transcription even after 48 hr of incubation. These data support the hypothesis that the nocturnal surge in melatonin is impaired at the beginning of an inflammatory response and restored either during the shutdown of an acute response or in a chronic inflammatory pathology. Here, we introduce a new molecular pathway involved in the feedback of an inflammatory response on pineal activity, and provide a molecular basis for understanding the expression of circadian timing in injured organisms.

Corticosterone modulates noradrenaline-induced melatonin synthesis through inhibition of nuclear factor kappa B

Abstract:  In chronically inflamed animals, adrenal hormones exert a positive control on the secretion of melatonin by the pineal gland. In this paper, the mechanism of corticosterone as a modulator of melatonin and N‐acetylserotonin (NAS) was determined. Rat pineal glands in culture, stimulated for 5 hr with noradrenaline (10 nm), were previously incubated with corticosterone (1.0 nm–1.0 μm) for 48 hr in the presence or absence of the glucocorticoid receptor (GR) antagonist, mifepristone (1.0 μm), the proteasome inhibitor, N‐acetyl‐leucinyl‐leucinyl‐norleucinal‐H (ALLN, 12.5 μm) or the antagonist of the nuclear factor kappa B (NFκB), pyrrolidinedithiocarbamate (PDTC, 12.5 μm). Corticosterone potentiated noradrenaline‐induced melatonin and NAS production in a bell‐shaped manner. The increase in NAS (12.9 ± 2.7, n = 6 versus 34.3 ± 8.3 ng per pineal) and melatonin (16.3 ± 2.0, n = 6 versus 44.3 ± 12.9 ng per pineal) content induced by 1 μm corticosterone was blocked by mifepristone, and mimicked by ALLN and PDTC. The presence of GRs was shown by [3H]‐dexamethasone binding (0.30 ± 0.09 pmol/mg protein) and corticosterone inhibition of NFκB nuclear translocation was demonstrated by electromobility shift assay. Therefore, corticosterone potentiates noradrenaline‐induced melatonin and NAS production through GR inhibition of NFκB nuclear translocation. To the best of our knowledge, this is the first time that this relevant pathway for passive and acquired immune response is shown to modulate melatonin production in pineal gland.

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.

Intracellular Peptides as Natural Regulators of Cell Signaling

Protein degradation by the ubiquitin proteasome system releases large amounts of oligopeptides within cells. To investigate possible functions for these intracellularly generated oligopeptides, we fused them to a cationic transactivator peptide sequence using reversible disulfide bonds, introduced them into cells, and analyzed their effect on G protein-coupled receptor (GPCR) signal transduction. A mixture containing four of these peptides (20–80 μm) significantly inhibited the increase in the extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Subsequently, either alone or in a mixture, these peptides increased luciferase gene transcription in AT1R CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. These peptides without transactivator failed to affect GPCR cellular responses. All four functional peptides were shown in vitro to competitively inhibit the degradation of a synthetic substrate by thimet oligopeptidase. Overexpression of thimet oligopeptidase in both CHO-S and HEK293 cells was sufficient to reduce luciferase activation triggered by a specific GPCR agonist. Moreover, using individual peptides as baits in affinity columns, several proteins involved in GPCR signaling were identified, including α-adaptin A and dynamin 1. These results suggest that before their complete degradation, intracellular peptides similar to those generated by proteasomes can actively affect cell signaling, probably representing additional bioactive molecules within cells.

DAILY VARIATION OF CONSTITUTIVELY ACTIVATED NUCLEAR FACTOR KAPPA B (NFKB) IN RAT PINEAL GLAND

In mammals, the production of melatonin by the pineal gland is mainly controlled by the suprachiasmatic nuclei (SCN), the master clock of the circadian system. We have previously shown that agents involved in inflammatory responses, such as cytokines and corticosterone, modulate pineal melatonin synthesis. The nuclear transcription factor NFKB, detected by our group in the rat pineal gland, modulates this effect. Here, we evaluated a putative constitutive role for the pineal gland NFKB pathway. Male rats were kept under 12 h:12 h light-dark (LD) cycle or under constant darkness (DD) condition. Nuclear NFKB was quantified by electrophoretic mobility shift assay on pineal glands obtained from animals killed throughout the day at different times. Nuclear content of NFKB presented a daily rhythm only in LD-entrained animals. During the light phase, the amount of NFKB increased continuously, and a sharp drop occurred when lights were turned off. Animals maintained in a constant light environment until ZT 18 showed diurnal levels of nuclear NFKB at ZT15 and ZT18. Propranolol (20 mg/kg, i.p., ZT 11) treatment, which inhibits nocturnal sympathetic input, impaired nocturnal decrease of NFKB only at ZT18. A similar effect was observed in free-running animals, which secreted less nocturnal melatonin. Because melatonin reduces constitutive NFKB activation in cultured pineal glands, we propose that this indolamine regulates this transcription factor pathway in the rat pineal gland, but not at the LD transition. The controversial results regarding the inhibition of pineal function by constant light or blocking sympathetic neurotransmission are discussed according to the hypothesis that the prompt effect of lights-off is not mediated by noradrenaline, which otherwise contributes to maintaining low levels of nuclear NFKB at night. In summary, we report here a novel transcription factor in the pineal gland, which exhibits a constitutive rhythm dependent on environmental photic information. (Author correspondence: rpmarkus@usp.br)

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.

Endogenous glucocorticoids control neutrophil mobilization from bone marrow to blood and tissues in non-inflammatory conditions

We have shown that endogenous glucocorticoids control neutrophil mobilization in the absence of inflammation. In this study the role of the glucocorticoid receptor (GR) in the physiological control of neutrophil mobilization was investigated, focusing on the specific mechanisms for mature neutrophils in bone marrow, circulating neutrophils and endothelial cells.

Presence of P2‐purinoceptors in the rat pineal gland

1 The effects of noradrenaline, ATP, adenylyl‐imidodiphosphate (AMP‐PNP), adenosine, α,β‐methylene‐ATP and the P2‐purinoceptor antagonist, suramin on N′‐acetyl‐5‐hydroxytryptamine production were studied in cultured denervated rat pineal glands. 2 Noradrenaline (3 nm – 1 μm) increased N′‐acetyl‐5‐hydroxytryptamine production as measured both in the gland and the culture medium. 3 In noradrenaline (10 nm)‐stimulated pineal glands, ATP (0.03 nm – 1 mm) or AMP‐PNP (0.1 μm – 1 mm) increased N′‐acetyl‐5‐hydroxytryptamine production in a concentration‐dependent manner. 4 α,β‐Methylene‐ATP at the concentration of 0.1 mm, but not 3 μm, attenuated the enhancement by ATP (0.1 mm) of noradrenaline (10 nm)‐induced N′‐acetyl‐5‐hydroxytryptamine production. 5 Suramin (0.1 mm) blocked the potentiating effect of ATP (0.1 mm), but not the potentiating effect of adenosine (0.1 mm) in glands incubated with noradrenaline (10 nm). 6 These findings suggest that the rat pineal gland possesses P2‐purinoceptors which when stimulated potentiate the effect of noradrenaline but do not, by themselves, induce an increase in N′‐acetyl‐5‐hydroxytryptamine production.

Vigabatrin: Clinical Evidence Supporting Rational Polytherapy in Management of Uncontrolled Seizures

Summary: Monotherapy is the policy for management of patients with epilepsy. With increasing knowledge of the biology of epilepsy and of the modes of action of antiepileptic drugs (AEDs), this concept must be reevaluated. When monotherapy fails to control seizures, subsequent treatment should be based on “rational pharmacology,” taking into consideration the mode of action of the drugs, to provide improved efficacy with maintained tolerance and ease of administration. Introduction of vigabatrin (VGB) as a new AED calls for just such a reevaluation. VGB is an enzyme‐activated irreversible inhibitor of γ‐aminobutyric acid (GABA)‐transaminase that increases brain and cerebrospinal (CSF) GABA concentrations in animals and humans. It has limited efficacy in the classic animal seizure screening tests, but in many clinical studies has halved the incidence of seizures in ∼50%of patients, especially those with partial epilepsies. We evaluated the efficacy of VGB in “socially integrated and active outpatients” as a likely subset to demonstrate any advantage of rational polytherapy. The criteria for this evaluation included the effects on seizure frequency, patient tolerability, and cognitive performance in a battery of psychometric tests. Fourteen of the 19 patients (73%) completing the study had >50% réduction in seizure frequency, and 10 of 19 (52%)had >70% réduction in seizure frequency. Tolerability appeared good; somnolence was the most frequent adverse event. Three patients complained of a worsening of their seizures, 1 with an increase in frequency and 2 with development of myoclonic jerks not previously reported. No deleterious effect of VGB on cognitive function was noted, and performance in late recognition tests showed significant improvement by the end of the maintenance period, which may have been due to the réduction in seizure frequency. The efficacy of VGB in this study in controlling seizures as compared with results of earlier published studies of refractory epilepsy may be related to selection of patients with more benign partial epilepsy, but does support the need for further studies in such patients to evaluate the hypothesis of rational polytherapy.