Melatonin synthesized by lymphocytes is involved in the regulation of IL-2 produ

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Evidence of MELATONIN synthesis by human

lymphocytes and its physiological significance: possible role as

intracrine, autocrine, and/or paracrine substance1

 

 

Evidence of melatonin synthesis by human lymphocytes and its

physiological significance: possible role as intracrine, autocrine,

and/or paracrine substance1

 

 

http://www.fasebj.org/cgi/content/short/18/3/537

 

ANTONIO CARRILLO-VICO*, JUAN R. CALVO*, PEDRO ABREU{dagger}, PATRICIA

J. LARDONE*, SOFÍA GARCÍA-MAURIÑO*, RUSSEL J. REITER{ddagger} and JUAN

M. GUERRERO*,2

 

* Department of Medical Biochemistry and Molecular Biology, The

University of Seville School of Medicine and Virgen Macarena Hospital,

Seville, Spain;

{dagger} Department of Physiology, School of Medicine, University of

La Laguna, Tenerife, Spain; and

{ddagger} Department of Cellular and Structural Biology, The

University of Texas, Health Science Center at San Antonio, San

Antonio, Texas, USA

 

2Correspondence: Department of Medical Biochemistry and Molecular

Biology, The University of Seville School of Medicine, Avda. Sánchez

Pizjuan 4, 41009 Seville, Spain. E-mail: guerrero

 

SPECIFIC AIMS

 

It has been historically assumed that the pineal gland is the major

source of melatonin in vertebrates. Melatonin plays a central role in

fine tuning circadian rhythms in vertebrate physiology. Additionally,

melatonin shows a remarkable functional versatility exhibiting

antioxidant, oncostatic, anti-aging, and immunomodulatory properties.

Its biosynthesis from tryptophan involves four well-defined

intracellular steps catalyzed by tryptophan hydroxylase (TPH),

aromatic amino acid decarboxylase (AADC),

serotonin-N-acetyltransferase (NAT), and

hydroxyndole-O-methyltransferase (HIOMT). This paper shows that both

resting and stimulated human lymphocytes have the necessary machinery

to synthesize melatonin as well as synthesize and release large

amounts of melatonin. Moreover, melatonin released to the culture

medium is synthesized in the cells since blocking the enzymes required

for its biosynthesis produced a significant reduction in melatonin

release. This inhibition caused decrease in IL-2 production, which was

restored by adding exogenous melatonin. These findings indicate that

human lymphoid cells are an important physiological source of

melatonin which could be involved in the regulation of the human

immune system.

 

PRINCIPAL FINDINGS

 

1. Presence of two key enzymes involved in melatonin synthesis (NAT

and HIOMT) in human peripheral blood mononuclear cells (PBMCs)

The synthesis of melatonin requires the presence of the enzymes

involved in its metabolic pathway. We investigated the presence of the

two key enzymes involved in melatonin biosynthesis in PBMCs. NAT is

the rate-limiting enzyme in melatonin synthesis, while HIOMT is the

final enzyme of the biosynthetic pathway. To determine the _expression

of NAT and HIOMT mRNA in lymphoid cells, mRNA from stimulated and

unstimulated cells was subjected to RT-PCR analysis using specific

primers to both genes. The RT-PCR amplification pattern obtained using

the NAT and HIOMT primers revealed the presence of the expected bands

in stimulated and unstimulated cells (Fig. 1 A). Southern blot

analysis performed with DIG-labeled NAT and HIOMT probes confirmed the

identity of PCR products (Fig. 1B ). To study whether the presence of

NAT and HIOMT mRNA was related with a functional response of both

enzymes, we measured NAT and HIOMT activity in these cells. This study

revealed a strong NAT and HIOMT activity in both groups of cells (Fig.

1C ).

 

 

 

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Figure 1. Presence of two key enzymes involved in melatonin

synthesis (NAT and HIOMT) in PBMCs. A) RT-PCR analysis of NAT (left)

and HIOMT (right) mRNA _expression. B) Southern blot hybridization of

the PCR products shown in panel A with the DIG-labeled specific

probes. {phi} = PCR and Southern molecular size; C = PCR reaction

without cDNA substrate used as PCR control. C) NAT and HIOMT activity.

Cells were cultured for 24 h in the presence or absence of PHA (8

µg/mL). The pineal glands from animals injected with isoproterenol

were used as positive control of NAT activity, whereas not injected

animals were used as positive control of HIOMT activity. Data are

expressed as mean values ± SE of 6 experiments performed in

triplicate. *, Significant differences between stimulated and

unstimulated cells were observed (P<0.001).

 

2. Melatonin production by PBMCs

We observed the presence of high concentrations of melatonin in the

PBMC culture supernatants using HPLC assay (Fig. 2 A). Melatonin

concentration was dependent on time and the presence of the mitogen

PHA. After 72 h incubation (Fig. 2A, B : B4) melatonin concentration

was significantly higher than at 24 h (Fig. 2A, B : B3), while in PHA

stimulated-cells, melatonin concentration was also significantly

higher than in resting cells. No melatonin was detected in medium

alone (Fig. 2A, B : B1) and insignificant containing FCS (Fig. 2A, B :

B2). Similar results were obtained when an ELISA assay was used to

measure melatonin (data not shown).

 

 

 

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Figure 2. Melatonin produced by PBMCs can be inhibited by PCPA or

HIOMT-directed antisense oligonucleotides and has biological effects

on IL-2 production. A) Melatonin production assayed by HPLC. Melatonin

was determined in RPMI 1640 medium with and without FCS and PBMC

culture supernatants. B) Representative chromatograms of melatonin.

(B1) RPMI medium. B(2) RPMI with FCS. (B3) and (B4), Melatonin formed

in the medium after 24 or 72 h of incubation. (B5) Authentic standard

of melatonin. Data are expressed as mean values ± SE of 20 experiments

performed in triplicate. *, Significant differences in melatonin

levels between 24 and 72 h (P<0.001) and between cells stimulated and

unstimulated (P<0.05). C) HIOMT-directed antisense oligonucleotides

and PCPA inhibited melatonin synthesis by PBMCs. (C1) Inhibition of

melatonin synthesis using antisense mixture to HIOMT. Cells were

incubated for 24 h with DOTAP, and a mixture of sense, random, and

antisense oligonucleotides. Data are expressed as mean values ± SE of

12 experiments performed in triplicate. *, Significant differences

between cells treated with antisense and other groups were observed

(P<0.001). (C2) Inhibitory effect of PCPA on melatonin production.

Cells were incubated in the presence or absence of PHA (8 µg/mL) and

PCPA (10-4 M) at indicated times. Data are expressed as mean values ±

SE of 12 experiments performed in triplicate. *, Significant

differences between cells treated with PCPA and untreated were

observed (P<0.05). D) Effect of PCPA on IL-2 production. Cells were

incubated for 72 h with PHA (8 µg/mL) and, where indicated, 10-8 M

melatonin or 10-4 M PCPA. Data are expressed as mean values ± SE of 20

experiments performed in triplicate. *, Significant differences

between cells treated with PCPA and other groups were observed (P<0.001).

 

3. HIOMT-directed antisense oligonucleotides and

para-cholorophenylalanine (PCPA) inhibited melatonin synthesis by

human PBMCs

Previous studies raised the question of whether melatonin in the

culture medium is synthesized by the cells or whether it was merely

released after being accumulated before lymphocytes were isolated. To

answer this question, cells were cultured in the presence of a mixture

of antisense oligonucleotides which was directed to mRNA encoding

HIOMT. Blocking HIOMT _expression resulted in a significant reduction

in melatonin concentration in the culture medium (Fig. 2C : C1).

Another method to inhibit the melatonin synthetic pathway was the

addition of PCPA, a reversible TPH inhibitor. Significant decreases in

melatonin concentration in both stimulated and unstimulated cells at

24 and 72 h were observed (Fig. 2C : C2). A decrease in melatonin

content was more obvious after HIOMT blockade than after PCPA

treatment. This is likely explained by the fact that HIOMT is the

final enzyme involved in melatonin synthesis. Moreover, when cells

were incubated with the protein synthesis inhibitor cycloheximide, the

melatonin levels fell significantly (data not shown).

 

4. Effect of PCPA on IL-2 production

Finally, to define a possible physiological role for lymphocytes

melatonin, cells were incubated in the presence of PCPA and/or

melatonin, and IL-2 production was studied. The results show that PCPA

significantly reduced IL-2 production by lymphocytes, and adding

melatonin to the medium the inhibitory effect of PCPA was reverted

(Fig. 2D ). Therefore, we propose that melatonin synthesized by the

lymphocyte could somehow contribute to regulation of its own IL-2

production.

 

CONCLUSIONS AND SIGNIFICANCE

 

In vivo models to test the immunomodulatory role of melatonin have

been widely used. Most of authors agree that pinealectomy and in vivo

models of melatonin administration clearly show the immunoenhancing

properties of the melatonin. However, when melatonin is used in vitro,

the results seem contradictory. The reasons for the apparent

contradictions are not clear but several possibilities exist. 1) The

effect of melatonin on immune cells is mediated via other tissues,

cells, hormones and/or cytokines that are not present in in vitro

studies. 2) melatonin efficiency in culture has been tested primarily

in cells fully activated. Under these conditions, immunomodulators,

including melatonin, frequently fail to achieve further activation of

immune cells. 3) presence of endogenously generated melatonin may

interfere or mask with exogenously added melatonin in in vitro

experiments.

 

In this report, we document that both in vitro cultured resting and

stimulated human lymphocytes show a strong NAT and HIOMT mRNA

_expression as well as a clear activity of both enzymes. Moreover,

these cells release large amounts of melatonin, and melatonin is

actually synthesized by the cells because the HIOMT mRNA blockage,

inhibition of TPH activity, or protein synthesis reduced melatonin

release. Taken together, these data show a new experimental approach

to demonstrate that at least, a portion of the melatonin found in the

cells is actually synthesized by the human immune system. These

results clearly demonstrate that human immune system is a source of

melatonin. Furthermore, a physiological role for melatonin produced by

human lymphocytes has been described. Thus, the inhibition of

melatonin synthesis by PCPA caused a reduction in IL-2 production and

the PCPA inhibitory effect was counteracted by exogenous melatonin.

 

This study shows for the first time that melatonin synthesized by

lymphocytes is involved in the regulation of IL-2 production, and

probably, other immune functions driven by IL-2, possibly by acting as

an intracrine, autocrine and/or paracrine substance (Fig. 3 ).

Therefore, in future studies, during in vitro experiments with

lymphocytes, a melatonin synthesis inhibitor should be added to the

culture to prevent the endogenous synthesis of melatonin by immune

cells. The development of specific inhibitors of melatonin synthesis

would be very useful in future studies of melatonin effects on the

human immune system.