Evidence of melatonin synthesis and release by mast cells. Possible modulatory role on inflammation

Pharmacological Research 62 (2010) 282–287

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Evidence of melatonin synthesis and release by mast cells. Possible modulatory role on inflammation M.D. Maldonado ∗ , M. Mora-Santos, L. Naji, M.P. Carrascosa-Salmoral, M.C. Naranjo, J.R. Calvo Department of Medical Biochemistry and Molecular Biology, Immunology Area, University of Seville Medical School, Avda. Sánchez Pizjuán 4, 41009 Seville, Spain

a r t i c l e

i n f o

Article history: Received 4 November 2009 Received in revised form 27 November 2009 Accepted 27 November 2009 Keywords: Melatonin Mast cells Cytokines Inflammation NAT and HIOMT

a b s t r a c t Mast cells take part of armamentarium immunologic for host defense against parasitic and bacterial infections. They are derived from bone marrow progenitors and can be activated by immunological and chemical stimuli in order to get its degranulation. The activation of mast cells generates a signalling cascade leaded to the rapid release of vasoactives and pro-inflammatory mediators. Melatonin (N-acetyl5-methoxytryptamine) is a molecule with antioxidant, cytoprotective and immunomodulatory actions. It was initially known to be produced exclusively in the pineal gland but melatonin synthesis has been found in different sites of the organism, and a major source of extrapineal melatonin is the immune system. The aim of the present study was to prove if the rat mast cell line (RBL-2H3) synthesizes and releases melatonin, also to explain its possible mechanism of action. We report that both resting and stimulated mast cells synthesize and release melatonin. We also report that the necessary machinery to synthesize melatonin is present in mast cells and that these cells showed the presence of MT1 and MT2 melatonin membrane receptors. Those results indicated that the melatonin would be able to exert a regulatory effect on inflammatory reactions mediated by mast cells. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Melatonin (N-acetyl-5-methoxytryptamine) was initially known to be produced exclusively in the pineal gland. It is derived from the amino-acid tryptophan, via synthesis of serotonin, by a series of catalyzing enzymes [1]. Subsequently its synthesis was demonstrated in other organs as for example, retina, gastrointestinal tract and lymphoid organs including bone marrow, thymus and immune competent cells [2–5]. Such a wide distribution suggests a local effect of melatonin in these tissues and cells [6]. Physiologically, the melatonin has been shown to exert immunomodulatory effects including anti-inflammatory actions. Thus, melatonin reduces tissue destruction during inflammatory processes by a number of means: (a) by its ability to directly scavenge toxic free radicals, it reduces macromolecular damage in all organs [7], (b) it prevents the translocation of nuclear factor-kappa B (NF-␬B) to the nucleus and its binding to DNA, thereby it reduces the upregulation of pro-inflammatory cytokines, as for example IL-1 and TNF-␣ [8,9] and (c) melatonin inhibits the production of adhesion molecules that promote the sticking of leukocytes to endothelial cells, reducing thus the transendothelial cell migration and edema [10–12].

Mast cells are one of the major effector cells in the immune response against parasitic and some bacterial infections [13]. They are derived from bone marrow progenitors (CFU-BM), and they are the cells that express high-affinity IgE Fc receptors (Fc RI) on their surface [14]. Activated mast cells release pro-inflammatory cytokines such as TNF-␣, IL-1, 6, 8, 13 and inflammatory mediators, including histamine, leukotrienes, serotonin, prostaglandin E2 and D2 among others. However, when mast cells are inappropriately activated by antigen or chemical exposure the resulting release of preformed inflammatory mediators and the novo synthesis of cytokines, lead to the initiation of the clinical manifestations associated with allergic inflammation and anaphylaxis [15–17]. The present study was designed to investigate the possibility that cultured rat mast cell line can synthesize melatonin. For that reason, we studied the activity of the two key enzymes in the pathway of melatonin synthesis, NAT and HIOMT. In addition, MT1 and MT2 melatonin membrane receptors expression permitted us to reflect on the possible immunomodulatory role of melatonin in the inflammatory processes generated by these cells.

2. Materials and methods 2.1. Cells and cell culture

∗ Corresponding author. Tel.: +34 954 559852; fax: +34 954 907048. E-mail address: [email protected] (M.D. Maldonado). 1043-6618/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2009.11.014

Mast cells line RBL-2H3 were maintained in DMEM supplemented with 15% heat-inactivated foetal bovine serum, 2 mM

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l-glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin and amphotericin B solution at 37 ◦ C under 5% CO2 in the air. Cells were used between passages 5 and 10. 2.2. Mast cell activation Mast cells were cultured (5 × 105 cells/ml) in plates of 24-well, with stimulation and without it. PMA 50 × 10−8 M and CI 5 × 10−7 M [phorbol-12.myristate 13-acetate plus calcium ionophore A23187 (PMACI)] for 12 h was selected to stimulate RBL-2H3. After activation, cell free culture supernatants were collected, filtered, and stored at −20 ◦ C for melatonin and biological mediators (TNF-␣ and IL-6) determinations. Likewise, the cells stimulated and unstimulated were collected from the wells, and the dry pellet was stored at −80 ◦ C for subsequent studies (measure of enzymatic activity and membrane receptors expression). 2.3. Cell viability assay using MTT The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay was used to measure cell viability on stimulated and unstimulated cells. Briefly, RBL-2H3 cells were seeded onto 96-well culture plate at a density 5 × 104 cell/100 ␮l/well. After incubation with PMACI for 12 h at 37 ◦ C, each well was washed twice with PBS to remove the medium; then 100 ␮l of MTT was added to each well, and incubation continued at 37 ◦ C for additional 4 h. After that, 100 ␮l of DMSO was added to dissolve MTT, and the absorbance at 530 nm was read on a microplate reader. The absorbance was used as a measurement of cell viability, normalized to cells incubated in control medium, which were considered 100% viable. 2.4. Cytokine analysis The levels of TNF-␣ and IL-6 contents in the culture supernatants were used to know if RBL-2H3 cells were activated well (Fig. 1). TNF-␣ and IL-6 secretion was measured using a specific ELISA (BD OptEIATM). Capture antibody anti-rat TNF-␣ and IL-6 respectively in 0.2 M sodium phosphate pH 9.0 was coated overnight to high binding microtiter plates. The plates were washed twice with phosphate-buffered saline (PBS)/0.05% Tween 20, incubated with 1% bovine serum albumin (BSA) in PBS/Tween 20 for 1 h as a blocking step, and washed again. Samples and standards (recombinant TNF-␣ and IL-6) were diluted in 1% BSA PBS/Tween 20 and incubated overnight. After being washed five times, biotinylated anti-rat TNF-␣ or IL-6 mAb was added, and bound TNF-␣ or IL6 detected using streptavidin-horseradish peroxidase conjugate, and 3,3 , 5,5 tetramethylbenzidine and hydrogen peroxide as the substrate of the enzyme. The optical densities were determined at 450 nm in an automatic microplate reader. Both the intra- and inter-assay coefficients of variation were less than 10%. 2.5. Melatonin determination Melatonin content in the culture supernatants was assayed by ELISA kit (IBL-Hamburg Diagnostic, Germany) according to manufacturers’ instructions. This assay is based on the competition principle and the microtiter plate separation. An unknown amount of antigen present in the sample and a fixed amount of enzyme labelled antigen compete for the binding sites of the antibodies coated onto the wells. After incubation the wells are washed to stop the competition reaction. Having added the p-nitro-phenyl phosphate (PNPP) substrate solution the concentration of antigen is inversely proportional to the optical density measured. The measured ODs (450 nm) of the standards are used to construct a

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calibration curve (containing 0, 3, 10, 30, 100 or 300 pg/ml melatonin) against which the unknown samples are calculated. The lower limit of the assay was 3.0 pg/ml, and the intra- and inter-essay coefficients of variance were less than 10%. Melatonin content in the culture supernatants was also assayed by high-pressure liquid chromatography with similar results. 2.6. NAT activity assay NAT activity was determined by the method of Champney et al. [18]. Each sample (RBL-2H3 cells stimulated and unstimulated) was lisated at 4 ◦ C in 0.05 M phosphate-buffered saline buffer (PBS), pH 6.8, using a cell sonicator (Sonics and Materials Inc., Danbury, CT, USA). Ten microliters of this lisate were mixed with 10 ␮l of PBS containing 40 nCi [1-14 C] acetylcoenzyme A and 5.6 mM tryptamine. The lisate was previously centrifuged (3 min, 16,000 × g), and 10 ␮l of supernatant was utilized for assay activity. The reaction was carried out for 20 min at 37 ◦ C, and was stopped by the addition of 100 ␮l 0.2 M sodium borate buffer, pH 10, and 1 ml chloroform at 4 ◦ C. The N-acetyltryptamine produced was extracted with chloroform and its radioactivity was measured by liquid scintillation spectrometry with a beta counter. NAT activity was expressed as nmol N-acetyltryptamine produced/mg protein/h. Protein content was measured following the Bradford protocol 1976 [19]. 2.7. HIOMT activity assay HIOMT activity was determined by the Champney et al. method 1984 [18] by measuring the amount of melatonin formed from N-acetylserotonin and S-adenosyl-l-methionine. Each sample (RBL-2H3 cells stimulated and unstimulated) was lisated in 0.05 M PBS, pH 6.8. Forty microliters of this lisate supernatant were mixed with 20 ␮l 0.05 M PBS, pH 7.9, containing 20 nCi S-[methyl-14C] adenosyl-l-methionine and 3 mM N-acetylserotonin. The reaction was incubated for 20 min at 37 ◦ C and was stopped by the addition of 100 ␮l 0.2 M sodium borate buffer pH 10 and 1 ml chloroform at 4 ◦ C. Synthesized melatonin was measured following extraction in 1 ml chloroform and the radioactivity by liquid scintillation spectrometry counted with a beta counter. HIOMT activity was expressed as nmol melatonin/mg protein/h. Protein content was measured following the Bradford protocol 1976 [19]. 2.8. Total RNA extraction and reverse transcription RNA was purified from cells using the High Pure RNA Isolation Kit according to the manufacturer’s instructions (Roche, Mannheim, Germany). RNA was eluted in 60 ␮l of elution buffer and quantified spectrophotometrically at 260 nm. After this, 5 ␮g of RNA were transcribed reversely in a final volume of 40 ␮l to obtain single-stranded cDNA using the following method: 5 ␮g of RNA were preincubated in 19 ␮l of RNAse-free water at 85 ◦ C for 10 min to denature it, and then rapidly chilled on ice. Then, 21 ␮l of a mixture formed by 1× RT buffer, dithiotheritol 20 mM (DTT), 2 -deoxyribonucleoside-5 -triphosphates (0.5 mM of each dNTP: dATP, dGTP, dCTP and dTTP), 40 units of Recombinant RNasin Ribonuclease Inhibitor, 0.5 ␮g Oligo (dT)15 Primer and 200 units Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) were added (all reagents from Promega, Madison, WI, USA). The reverse transcription (RT) reaction was carried out for 60 min at 42 ◦ C and heated at 94 ◦ C for 5 min to terminate the RT reaction. 2.9. Polymerase chain reaction (PCR) The cDNA was amplified in a reaction containing 5 ␮l of RT product as template DNA, 1× PCR buffer, 1 mM MgCl2 , 0.4 mM

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each deoxynucleotide, 2.5 units ECOTAQ DNA Polymerase (Ecogen, Barcelona, Spain) and 0.2 ␮M sense and antisense primers of housekeeping gene (␤-actin) and 1 ␮M sense and antisense primers of the gene in study, all this in a final volume of 25 ␮l. The template was initially denatured for 3 min at 94 ◦ C followed by different programs optimized for: MT1 cDNA (35-cycle program with 1 min of denaturation at 94 ◦ C, 1 min annealing at 60 ◦ C, 1 min extension at 72 ◦ C) and MT2 cDNA (35-cycle program with 1 min at 94 ◦ C, 1 min at 62 ◦ C, 1 min at 72 ◦ C). All programs were terminated by an extension of 5 min at 72 ◦ C. For each run of PCR a negative control was systematically added, in which water replaced cDNA. Besides, the ubiquitously expressed ␤-actin mRNA was used to monitor the quality of RNAs and the efficiency of the RT and the PCR. ␤-Actin amplification was performed simultaneously with the other genes. The primers used for the ␤-actin PCR were: 5 -TTG TAA CCA ACT GGG ACG ATA TGG-3 (sense) and 5 -GAT CTT GAT CTT CAT GGT GCT AGG-3 (antisense), obtaining a PCR product of 746 bp. Primers used for the MT1 amplification were: 5 -GCC ACA GTC TCA AGT ATG ATA GG-3 and 5 -GGT GAC AAA GTT CCT GAA GTC-3 (PCR product of 329 bp) and for the MT2 were: 5 -CCT CTA CAT CAG CCT CAT CTG GCT-3 and 5 -CTG CGA ACA TGG TTA GGA AAC TGC-3 (PCR product of 293 bp). 2.10. Southern blot After amplification, 5 ␮l PCR reaction product were separated by agarose gel electrophoresis (2%, w/v) in 1× TAE buffer (40 mM Tris-acetate,1 mM EDTA, pH 8.0) and visualized by staining with ethidium bromide (0.5 ␮g/ml) and UV illumination, using a 100 bp ladder as DNA size marker (Biotools, Madrid, Spain). Together with these, DNA Molecular Weight Marker VI DIG-labelled 0.15–2.1 kb (Roche, Mannheim, Germany) was also added as Southern size marker. Electrophoresed PCR products were transferred to a positively charged nylon transfer membrane (Amersham Biosciences, Amersham, UK) with 10× SSC as transfer solution and cross-linked to the nylon membrane using a calibrated UV light source. Blots were prehybridized at 68 ◦ C for 2 h in prehybridization buffer (5× SSC, 0.1% N-laurylsarcosyl, 0.02% SDS, 1% blocking reagent). The hybridization was performed at 60 ◦ C overnight in the same prehybridization buffer plus 25 ng/ml of labelled probe. Thereafter, blots were washed twice for 5 min in 2× SSC/0.1% SDS at room temperature and twice for 5 min in 0.1× SSC/0.1% SDS at 60 ◦ C. To detect the hybridization signal, blots were incubated for 30 min in 0.1 M maleic acid, 0.15 M NaCl, 1% blocking reagent and for 30 min with anti-DIG-AP (anti-digoxigenin conjugated to alkaline phosphatase). Finally, they were washed and incubated with the chemiluminescent substrate CSPD (Roche, Mannheim, Germany). Blots were exposed to Kodak X-OMAT AR film (Rochester, NY, USA) at room temperature and later developed. The probes used in this study were MT1 probe: 5 -TGA GTG TCA GTG TCC ATA TCA GGA ACA CGT-3 and MT2 probe 5 -TGC AGG AAT AGA TTC GCG GGT CAT ATT CT-3 . Probes were labelled with digoxigenin with an oligonucleotide tailing kit (Roche, Mannheim, Germany).

3. Results 3.1. Cell stimulation In previous pilot experiments to this study, mast cells were stimulated with immunological and chemical stimuli, observing that cell activation was greater with chemical stimuli than with the immunological one. On the other hand, the culture, incubation and handling time of mast cells was shorter with chemical stimuli than with immunological stimuli. All these factors helped us to select chemical stimuli with PMACI, for the elaboration of final experiments. To evaluate the effect of chemical stimuli on the RBL-2H3 cells, a competitive enzyme immunoassay of cytokines, which determines the levels of TNF-␣ and IL-6 contents in the culture supernatants, were performed. As shown in Fig. 1, the levels of TNF-␣ were 82% higher in stimulated than unstimulated mastocytes. Similar results were obtained for IL-6, the only difference was that the rate of IL-6 was about 68% higher with stimulation than without it. The results confirm that the cells were activated. 3.2. Cell viability Mast cells stimulated with PMACI (PMA 50 × 10−8 M and CI 5 × 10−7 M) during 12 h showed a cell viability average of the 85%. 3.3. NAT and HIOMT activity in stimulated and unstimulated mast cells The melatonin synthesis derives from the amino acid tryptophan and requires the presence of the enzymes involved in its metabolic pathway NAT (AA-NAT) and HIOMT (Fig. 2) [20,21]. To analyze if the functional response of the enzymes NAT and HIOMT been present in mast cells, both enzymes were measures. These test (Figs. 3 and 4), revealed the presence of NAT and HIOMT activity specifically in stimulated mast cells, with statistically significant values with regard to the controls (p < 0.05 for NAT and p < 0.01 for HIOMT). 3.4. Melatonin production by RBL-2H3 Fig. 5 shows as the RBL-2H3 cells line release melatonin in the culture supernatants after 12 h of incubation. The levels of melatonin were significantly higher (p < 0.001) in stimulated cells than in those that did not receive stimuli. Insignificant amount of mela-

2.11. Statistical data analysis All data are presented as the mean ± standard error of the mean (S.E.M.). All statistical procedures were performed using GraphPad InStat statistical software package program 3.0. Data were analyzed using ANOVA followed by Tukey–Kramer multiple comparisons test and Student’s paired t-test. The differences were considered to be significant at p < 0.05.

Fig. 1. Effect of chemical stimuli on the RBL-2H3 cells. Cells were cultured during12 h in the presence (stimulated) or absence (unstimulated) of PMACI. ELISA cytokine analysis for TNF-␣ and IL-6 was determined. Each bar represents the means ± SEM of six separate experiments performed in triplicate. ***Differences considered highly significant between unstimulated and stimulated cells were observed (p < 0.001).

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Fig. 4. HIOMT activity levels in RBL-2H3 cell line. Cells were cultured during 12 h in the presence (stimulated) or absence (unstimulated) of PMACI. The pineal glands were used as positive control of HIOMT activities. Data are expressed as mean ± SEM of five experiments performed in triplicate. ** Significant differences between stimulated, unstimulated and control cells were observed (p < 0.01).

Fig. 2. Biosynthetic pathway of melatonin and enzymes implied.

tonin was detected in the medium alone and in the medium plus DMSO (control vehicle). 3.5. Expression levels of two membrane receptors of melatonin (MT1 and MT2) in mast cells Membrane receptors for melatonin have been well characterized, and they likely mediate some of the many actions of melatonin [22–24]. Thus, we investigated the presence of the two melatonin membrane receptors MT1 and MT2 in RBL-2H3 cells line. To deter-

Fig. 3. NAT activity levels in RBL-2H3 cell line. Cells were cultured during 12 h in the presence (stimulated) or absence (unstimulated) of PMACI. The pineal glands were used as positive control of NAT activities. Data are expressed as mean ± SEM of five experiments performed in triplicate. * Significant differences between stimulated and control cells were observed (p < 0.05).

mine the expression of MT1 and MT2 mRNA in mast cells, we subjected mRNA from cells cultured during 12 h in the presence (stimulated) or absence (unstimulated) of PMACI to PCR analysis using specific primers to both genes. The PCR amplification pattern obtained using the MT1 primers revealed the presence of the expected band (329 bp) in unstimulated and stimulated mast cells (Fig. 6A, left). The MT2 PCR amplification pattern also revealed a single product (293 bp) in both unstimulated and stimulated mast cells (Fig. 6A, right). No band was obtained from reaction in which cDNA was omitted. To test the quality of RNA and efficiency of reverse transcription, we subjected all samples to the PCR of housekeeping gene ␤-actin. The ␤-actin primers amplified the expected 746 bp product in all samples. The Southern blot analysis performed with DIG-labelled MT1 (Fig. 6B, left) and MT2 (Fig. 6B, right) probes confirmed the identity of PCR products. These data revealed that RBL-2H3 unstimulated and stimulated cells express two well-characterized G-protein-coupled seven-transmembranedomain receptors MT1 and MT2, being greater the expression in stimulated mast cells. 4. Discussion Mast cells do not normally circulate in the blood (less than 1%) but are ordinarily distributed throughout the connective tissues, where they often lie adjacent to blood and lymphatic vessels, near or within nerves, and beneath epithelial surfaces that are exposed to the external environment [25]. Mast cells have granules that contain effector molecules which are important in host defenses [26] and play an important role in both the innate and adaptive immune responses [27,13]. The activation of these cells can be demonstrated

Fig. 5. Melatonin secretion in the RBL-2H3 culture supernatants. *** Differences considered highly significant between unstimulated and stimulated cells were observed (p < 0.001). Control mean medium alone; Control vehicle mean medium alone plus DMSO; unstimulated mean medium plus cells; Stimulated mean medium plus cells plus PMACI.

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Fig. 6. Presence of the two melatonin membrane receptors MT1 and MT2. (A) PCR analysis of MT1 (left) and MT2 (right) mRNA expression in unstimulated and stimulated RBL-2H3 cells. mRNA expression in brain homogenate was used as positive control. ␤-Actin was amplified as a housekeeping gene. PCR molecular size marker () was used (100 bp ladder), and PCR reaction without cDNA substrate was used as PCR control. (B) Southern blot hybridization of the PCR products with the DIG-labelled specific probes. Southern molecular size marker was used (DNA Molecular Weight Marker VI DIG-labelled 0.15–2.1 kb).

with both in vitro and in vivo and through immunological and chemical stimuli [25]. In this study, using an in vitro model, a strong amount of melatonin was found in the cultured rat mast cell line, being greater the concentration in stimulated than in unstimulated cells (Fig. 5). We asked if melatonin found in the cultured medium was synthesized by mast cells or merely released of the granules where previously it had been stored. To exclude the possibility that synthesized melatonin by the pineal gland in vivo might have been accumulated in the mastocytes or CFU-BM before the establishment of the cell line, we collected the mastocytes culture medium only after a 12 h incubation period. Having in mind that the half-life of melatonin is around 20 min in the rat plasma [28], the accumulated melatonin should be completely catabolized when the culture medium was collected. That is the reason why we think that released melatonin in the RBL-2H3 culture medium was synthesized by those cells. The enzymatic activity assays for NAT and HIOMT on stimulated and unstimulated mast cells indicated a clear and functional activity of both enzymes (Figs. 3 and 4), specifically in stimulated cells with significant differences. The enzymatic activity on unstimulated cells, showed a NAT activity basal greater than HIOMT. The reason why it happens is because the method used to determine NAT activity (Champney et al.) is not specific for arylkylamine-N-acetyltransferase (AA-NAT, also called serotonin-N-acetyltransferase) and it measures all activities of all N-acetyltransferase enzymes of the cells. Besides, it is logical to think that NAT activity must be greater than HIOMT activity because while the HIOMT only mediates synthesizing melatonin, NAT participates in the biosynthetic pathway of serotonin and its derivatives, including melatonin. The results of these assays suggest that rat mastocytes have the necessary machinery to synthesize melatonin and are a physiological source of it. The exact function of the local melatonin is not known at the present, but is worthwhile to note, by our results, the presence in RBL-2H3 of two well-characterized membrane receptors MT1 and MT2 (Figs. 6A and B), with greater expression in the stimulated cells. One may assume therefore, that synthesized melatonin by mast cells may provide a local protective function through an autocrine or paracrine mechanism [29]. In accordance with Galli and others authors [30–34] mast cells would be able to have a dual action in the inflammatory responses promoting or per-

haps, more surprisingly suppressing aspects of these responses. Thus, melatonin would be released of the mastocytes in the acute phase with a defensive pro-inflammatory role. After that, in a later phase, a feedback circuit would exist between the high melatonin concentrations environment and the expression of the two melatonin membrane receptors MT1 and MT2. Mast cells would actively uptake the released melatonin through its receptors by means of an autocrine mechanism, exercising anti-inflammatory effects. This theory would explain why Sutherland et al. [35] found that increased secretion of melatonin in acute bronchial asthma contributed to the worsening of symptoms and amplified airway inflammation. By contrast, Cikler et al. [31] showed that chronic melatonin treatment done before the induction of inflammatory experimental conditions reduced mast cells dermis activation and the inflammatory symptoms. In conclusion, our study revealed that cultured rat mast cell line was able to synthesize strong amounts of melatonin. In addition, mast cells showed the activity of NAT and HIOMT enzymes and expressed in its surface the melatonin membrane receptors MT1 and MT2. The functional meaning of our findings induced us to think that mast cell activation releasing melatonin and other mediators might have as many pro-inflammatory as anti-inflammatory effects, but at different stages in the development or resolution of the response. Further studies on this subject must be carried in order to establish the effects of melatonin as possible protection against activation and degranulation of mast cells. This potential function may be fruitful for the clinical and pharmacological community in the design and elaboration of new drugs to the therapy of many pathological processes where the inflammation is present. Competing interests None of the authors of this manuscript have any financial interest that has influenced the results or interpretations of this manuscript. Acknowledgements This work was supported in part by the research project from National Program I+D of Spain from Ministry of Science and Technology, number BFI 2002-03544, by Seville University

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