Inflammation, Vol. 28, No. 4, August 2004 ( C© 2004)

Different Effects of Melatonin on ExperimentalGranulomatous Inflammation

Nadir Ernesto de la Rocha,1 Alejandra Ester Rotelli,2 Teresita Guardia,2

and Lilian Eugenia Pelzer2,3

Abstract—Several experiments have detected that melatonin exerts a marked influence on the in-flammatory process. In the present study we evaluated the effect of exogenous melatonin on theexperimental granulomatous tissues in rat. Our data show that subcutaneous administration of dif-ferent doses of melatonin given daily during 6 days at 9:00 h do not have significant action onthe granuloma weight. On the other hand melatonin (4 mg/kg) administered at 17:00 h showed aremarkable action pro-inflammatory. However, this effect was abolished when the animals receivedpreviously higher doses of melatonin (40 mg/kg) at 9:00 h.

KEY WORDS: melatonin; inflammatory response; chronic inflammation; granuloma test.

INTRODUCTION

The inflammatory reactions are characterized by an in-crease of vascular permeability with diffusion of rich fluidin serum proteins, which forms the exudate, and the pro-cess is followed by a cellular migration into the injuredsite. These pathological processes are characterized by amultifrequency time structure with prominent circadian,ultradian, and circannual rhythms in cellular proliferationand cellular function.

The number of circulating blood cells shows circa-dian rhythms in all cell lines, while those in the red cellare of low amplitude, the circadian rhythms of circula-tion lymphocytes and granulocytes are high amplitude (1).These granulocytes are involved in large numbers in in-flammatory reactions.

1Becario del Colegio de Farmaceuticos de la Provincia de Buenos Aires,Argentina.

2Catedra de Farmacologıa, Facultad de Quımica, Bioquımica y Farma-cia, Universidad Nacional de San Luis, Chacabuco y Pedernera, SanLuis, Argentina.

3To whom correspondence should be addressed at Farmacologıa, Fac-ultad de Quımica, Bioquımica y Farmacia, Universidad Nacional deSan Luis, Chacabuco y Pedernera (5700), San Luis, Argentina. E-mail:lpelzer@unsl.edu.ar

On the other hand, granuloma formation occursin response to chronic inflammatory stimuli such asinjection or presence of inert material. The granu-loma term means any organized nodular aggregation ofmononuclear inflammatory cells or collection of mod-ified macrophages, usually surrounded by a rim oflymphocytes and often containing multinucleated giantcells.

Several factors such as interleukin-1 (IL-1), tumornecrosis factor (TNF), interferon gamma (INF-γ ), nitricoxide (NO), and active species of oxygen are involved inthe initiation and maintenance of these lesions.

The pineal gland and its major hormone melatoninare responsible for the modulations of biological rhythmsin mammals (2). Although, a relationship between thepineal organ and the immune system has been suspectedfor decades, only in the last years convincing evidenceon the existence of such interaction (3, 4) has been ac-cumulated . Chen et al. (5) reported that administrationof melatonin enhanced significantly the thymocyte pro-liferation and IL-2 production decrease. Other authors,showed the existence of specific binding sites for mela-tonin in human lymphocytes (6, 7), chick splenocytes (8),rat thymus membranes (9), and other reports indicate thatrole of melatonin in lymphoproliferative diseases would

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190 de la Rocha et al.

be more complex (10). Therefore, melatonin interferes inthe chronic process.

Previous results in our laboratory showed that admin-istration of melatonin (4 mg kg−1) had anti-inflammatoryactivity on acute oedema induced by carrageenan in rats(11). Thus, the aim of this study was to evaluate the ef-fect of melatonin administered at different hour of the dayon the granulomatous inflammation induced by cottonpellet.

MATERIAL AND METHODS

Animals

Female Wistar rats (130–150 g) was used. They wererandomly assigned to different groups in individual cagesand a period of 3 days was allowed for adaptation.

Animals were provided with food (standard chowdiet, manufacture) and water ad libitum and were main-tained during the experiment under natural condition oflight and temperature.

Drugs

Dexamethasone and melatonin were purchased fromSigma Chemical Co, St. Louis, MO, USA.

Cotton Pellet Granuloma in Rats

Experimental granuloma was induced in rats, accord-ing to the method of Meier et al. (12).

On day 1 each animal was anaesthetized with etherand a small piece of sterile cotton (50 mg) was introducedinto the dorsal area.

Melatonin was dissolved in 100% ethanol and di-luted in saline solution to a final concentration of 1%ethanol and it was administered to the test group animals.Dexamethasone was dissolved in the same delivery sys-tem and it was given to standard group animals. Controlgroup received only the vehicle. The compounds wereadministered 0.3 mL to each animal. All groups wereinjected daily for 6 days, starting on the first day of thepellet implantation. At day 7, the rats were killed by cervi-cal dislocation, and granulomas, thymus and spleens weredissected out and immediately weighed.

The increase in the pellet weight was consideredas granuloma tissue deposit. The inhibition percentageor increase percentage of granuloma tissue weight wascalculated for each animal group in comparison with thecontrol group.

Thymus and spleen weights were compared with thecontrol group.

Experiment I

Eighteen rats previously implanted with a cotton pel-let were divided in control group, standard group, and testgroup of six each and they received daily a subcutaneousinjection of vehicle (0.3 mL), dexamethasone (2 mg/kg),and melatonin (4 mg/kg), respectively at 9:00 h.

Experiment II

Eighteen rats previously implanted with a cotton pel-let were divided in control group, standard group, and testgroup of six each and they received daily a subcutaneousinjection of vehicle (0.3 mL), dexamethasone (2 mg/kg),and melatonin (4 mg/kg), respectively at 17:00

Experiment III

Eighteen rats previously implanted with a cotton pel-let were divided in control group, standard group, and testgroup of six each and they received daily a subcutaneousinjection of vehicle (0.3 mL), dexamethasone (2 mg/kg),and melatonin (40 mg/kg), respectively at 9:00 h.

In the afternoon, at 17:00 h, they received a subcu-taneous injection of vehicle (0.3 mL), dexamethasone (2mg/kg), and melatonin (4 mg/kg), respectively.

Experiment IV

On the basis of results obtained we evaluated theeffect of different doses of melatonin administered in themorning.

Fifty rats (group of five each) previously implantedcotton pellet, received during 6 days an injection of ve-hicle (0.3 mL)—the control group and the test groups:melatonin 10 µg/kg, 200 µg/kg, 4 mg/kg, 8 mg/kg, 12mg/kg, 25 mg/kg, 50 mg/kg, 100 mg/kg, and 200 mg/kgat 9 h, respectively.

Statistical Analysis

Data obtained from the pharmacological experimentare expressed as mean ± SD. Differences between thecontrol and the experimental groups were tested for sig-nificance using Dunnett’s test. A probability of p < 0.05was considered significant and a probability of p < 0.01was considered very significant.

Effects of Melatonin on Experimental Granulomatous Inflammation 191

Fig. 1. Effect of melatonin and dexamethasone on granuloma weightat different administration time. Values represent means ± SD of gran-uloma weights (n = 6 animals). Numbers on each column representthe inhibition percent (−) or the increase percent (+) with respect tocontrol group. Dunnett’s test ∗p < 0.01 compared with control group.

RESULTS

Effect of Different Treatment of Melatoninon Granuloma Weight

Experiment I: Effect of Melatonin Administeredat 9.00 h on Cotton Pellet Granuloma

Melatonin (4 mg/kg) treatment during 6 days did notinduce any further modification on the granuloma.

Dexamethasone exhibited an important inhibitoryactivity of 47.08% p < 0.01 (Fig. 1).

Experiment II: Effect of Melatonin Administeredat 17.00 h on Cotton Pellet Granuloma

The daily administration of melatonin (4 mg/kg) at17.00 h showed a significant increase on the granulomaweight of 40.32% p < 0.01 compared with the controlgroup.

Dexamethasone exhibited an important inhibitoryactivity of 45.97% p < 0.01 (Fig. 1).

Experiment III: Effect of Melatonin Administered at9.00 h and 17.00 h on Cotton Pellet Granuloma Weight

Melatonin administration during 6 days at 9.00 h(40 mg/kg) and 17:00 h (4 mg/kg) did not produce dif-ference statistically significant on the granuloma weightbetween test and control group.

Dexamethasone the standard anti-inflammatory drugin this experiment inhibited significantly the granuloma-tous tissue (61.82%, p < 0.01, see Fig. 1).

Experiment IV: Effect of Different Melatonin Doses Ad-ministered at 9.00 on Cotton Pellet Granuloma Weight

No melatonin doses administered at 9:00 h influ-enced significantly on the granulomatous tissues (data notshown).

Effect of Different Treatment of Melatoninon Lymphoid Organ Weight

In the experiments 1, 2, 3, and 4 the influence ofmelatonin on the thymus and spleen weight were ana-lyzed.

The mean value weight did not differ significantly af-ter any treatment with respect to the control group (Table 1show data of experiments 1, 2, and 3). Treatment withdexamethasone induced a significant decrease in the lym-phoid organ weight.

DISCUSSION

Evidence shows that cells of immune system aredeeply influenced by hormones and neurotransmitter. Sev-eral reports point to the fact that hormones can operate atmore refined levels on specific cell functions. Glucocorti-coids are crucial mediators of endocrine immune interac-tions. In general, they exert inhibitory effect on immunefunction, but it should be emphasized that they displaysome stimulatory effects too (13–15).

The pineal gland and its main hormonal productmelatonin has often been ignored by many clinicians; inthe last three decades tremendous advances in the under-standing of the biochemistry and physiology of the pinealgland have occurred (16). At present, there is much evi-dence about the influence of the pineal gland, which acts asa photoneuroendocrine transducer organ and participatesin the regulation of the diurnal rhythm of the immunesystem (17), even though several authors showed con-tradictory results. Bindoni and Cambria (18), Rella andLapin (19) observed that absence of the pineal gland stim-ulated the proliferation of immunocompetent cell. Othersauthors claiming the opposite, Barat and Csaba (20), andMaestroni (21) proposed the use of melatonin orally asimmunotherapeutic agent especially in states of immun-odeficiency. The same author (22, 23) revealed that mela-tonin could indeed augment the primary and secondaryantibody response to sheep red blood cells (SRBC) andthat this effect was exerted only when melatonin was in-jected daily in the late afternoon at a dosage ranging from

192 de la Rocha et al.

Table 1. Effect of Melatonin and Dexamethasone on Thymus and Spleen Weight

Experiment N Treatment Group Thymus weight (mg) Spleen weight (mg)

Experiment 1 (administration at 9:00 h) 6 Vehicle Control 467.5 ± 45 841.6 ± 1976 Melatonin Test 474 ± 107 918 ± 175.66 Dexamethasone Standard 154 ± 47∗ 340 ± 63.5∗

Experiment 2 (administration at 17:00 h) 6 Vehicle Control 467.5 ± 80 876.6 ± 1756 Melatonin Test 468 ± 76.6 973 ± 1396 Dexamethasone Standard 151.6 ± 38∗ 379 ± 73∗

Experiment 3 (administration at 9:00 h and 17:00 h) 6 Vehicle Control 441.4 ± 90.6 798.4 ± 1266 Melatonin Test 423.4 ± 64 902 ± 1616 Dexamethasone Standard 61 ± 17.8∗ 213.9 ± 54.2∗

Note. Values represent means ± SD of thymus and spleen weights. Dunnett’s test ∗p < 0.01 compared with control group.

0.01 to 10 mg/kg; the same doses administered in themorning were ineffective and doses as high as 200 mg/kgproved to be immunosuppressive (21). On the other hand,melatonin can interact with specific binding sites in cellsfrom lymphoid organs from humans and rodents, i.e.,blood lymphocytes, thymus, and spleen (24). The expo-sure to high exogenous melatonin levels may cause de-sensitization by downregulation of receptor-binding sites(25). Lopez-Gonzalez et al. (8) demonstrated that the spe-cific binding 125-I iodomelatonin to the human lympho-cytes was dependent on time and that was inhibited byincreasing concentrations of this hormone.

On experimental granuloma a circadian rhythm hasbeen observed, the acrophase occurs during the lightphase and the vascular permeability around the granu-lomatous lesion was higher at 12:00 h than at 24:00 h;these rhythmic variations was eliminated by pinealec-tomy (26). In chicken, it has been documented that thecircadian rhythm in granulocyte number was modifiedor abolished by pinealectomy, while daily melatonin in-jections restored the rhythm (27). Other advances inthe knowledge of the pineal gland have suggested thatmelatonin may stimulate the cellular immune responsemainly by promoting the release of interleukin-2 from Thelper lymphocyte through a direct stimulation of spe-cific melatoninergic receptor expressed on T lymphocytecell surface (28, 29). The pathogenesis of granuloma-tous inflammations has a great variety of mechanisms. Inthese complex process was demonstrated that IL-2 con-tribute to the increase on T lymphocytes. The physiolog-ical role of melatonin-binding sites in thymus and spleenremains unclear. However, several authors have showna physiological link between pineal gland and thymus.It was shown that the pineal extracts administration inlong-term experiments resulted in an increase of thymusweight accompanied by lymphoid cell hyperplasia of thisgland (30).

Our results showed that different doses of melatonindaily administered at 9:00 h did not affect the granulomaformation. In contrast, melatonin administered in the af-ternoon (17:00 h) showed to be a stimulatory agent ofchronic inflammation. These different effects could beexplained in term that this hormone could be interactingwith specific binding sites in cells from lymphoid organsor lymphocytes which have a different sensitivity at dif-ferent times during the 24-h cycle, which is exposed byLopez-Gonzalez (8).

When we administered melatonin (40 mg/kg) in themorning together with melatonin 4 mg/kg in the after-noon, we did not observe an increase in granulomatoustissue like the one produced when melatonin (4 mg/kg)was administered in a single daily dose in the afternoon.It is probably because exposure to high exogenous mela-tonin levels may cause desensitization by downregulationof receptor-binding sites, according to the reports of Car-dinali (25) and Lopez-Gonzalez (8).

On the basis of our results, we concluded that mela-tonin possibly could modify chronic inflammatory pro-cess like granuloma depending principally on the time itis administered.

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