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Differential Expression of the Melatonin Receptor
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J Immunol 1998; 160:1191-1197; ;
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Copyright © 1998 by The American Association of
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Marc J. Barjavel, Zahra Mamdouh, Nadjibe Raghbate and Ouahid
Bakouche
Differential Expression of the Melatonin Receptor in Human
Monocytes1
Marc J. Barjavel, Zahra Mamdouh, Nadjibe Raghbate, and Ouahid Bakouche2
T
he immune system interacts through its mediators with
the brain (1– 4). In return, the neuroendocrine system
modulates the immune system through its hormones
(5–7). Among the neuroendocrine factors that affect the immune system, the pineal hormone melatonin (MLT)3 appears to
be an important one (8, 9). Indeed, melatonin has been shown
to enhance the natural and acquired immunity in vivo (10), to
activate bone marrow and lymph node cells (11), to activate NK
cell activity (9, 12) and Ab-dependent cellular cytotoxicity (13),
to enhance the Ab responses in vivo (3, 10, 14), to restore the
impaired Th cell activity in immunodepressed mice (14), to
increase T cell proliferation in vivo and in vitro (14, 15), and to
activate monocytes (8, 16) and neutrophils (17). MLT has also
been shown to induce the release of lymphokines (3, 18, 19)
such as IL-1 (8, 14), TNF (14), GM-CSF (20), IL-4 (21), IL-2,
and IL-5 in vivo (18). In return, IFN-g, thymic hormones, GCSF, GM-CSF, and TNF-a influence the secretion of MLT (3,
4, 9, 16, 22, 23). MLT enhances Ag presentation by splenic
macrophages to T cells concomitant with an increase in the
expression of MHC class II molecules (14). In addition MLT
has immunoenhancing properties. Indeed, MLT in vivo increases the number of Th2 lymphocytes (18, 21), and a com-
INSERM U 294, Laboratoire d’Immunologie-Hematologie, CHU Xavier Bichat,
Paris, France
Received for publication June 12, 1997. Accepted for publication October 22, 1997.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by an INSERM grant, a grant from the Association pour
la Recherche contre le Cancer, and a grant from the Fondation pour la Recherche
Médicale.
2
Address correspondence and reprint requests to Dr. Ouahid Bakouche, INSERM U
294, Laboratoire d’Immunologie-Hematologie, CHU Xavier Bichat, 46 rue Henri
Huchard, 75018 Paris, France.
3
Abbreviations used in this paper: MLT, melatonin; GM-CSF, granulocyte-macrophage CSF; MTT, 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide; LBP,
LPS-binding protein.
Copyright © 1998 by The American Association of Immunologists
bination of MLT and IL-2, compared with IL-2 alone, increases
the number of T lymphocytes, NK cells, and eosinophils (18,
24 –26). In addition to its immunoregulatory properties,
MLT displays oncostatic properties (27–30). For instance, MLT
induces the antitumoral functions of human monocytes in vitro
(8), inhibits the growth of tumors in vivo (29), and has been
used in association with lymphokines for cancer immunotherapy in humans (16, 18, 24 –26, 31).
Besides the fact that MLT is a free radical scavenger and can act
without the presence of a receptor, the biologic activity of MLT
appears also to correlate with the expression of its receptor in
target cells. MLT receptors have been described in immune tissues
of various nonhuman species (32, 33) and in human immune cells
such as T cells (Kd 5 240 pM) (15), human Th2 lymphocytes from
bone marrow (Kd 5 346 pM) (21), human platelets (Kd 5 4 nM)
(34), human neutrophils (Kd 5 132 pM) (35), and human granulocytes (Kd 5 2 nM) (35). High and low melatonin binding sites
have been described (35–37). Also MLT receptors have been detected at plasma membrane levels, in the cytosol, and at the membranes of the nucleus and the mitochondria (38, 39). We report
here that human monocytes can be activated by melatonin to induce the release of IL-1, TNF, and IL-6. The purpose of this work
was to demonstrate the presence and the expression of MLT receptors on human monocyte cell surface. MLT receptors were
found on human monocytes with a Kd around 230 6 60 pM
(40,000 receptors/monocyte), and it seems that MLT receptor expression is sensitive to the differentiation of monocytes in vitro.
Materials and Methods
Chemicals and biologicals
LPS (a toxin derived from Escherichia coli) was purchased from Sigma
Chemical Co. (St. Louis, MO). Eagle’s MEM and sera were purchased
from M. A. Bioproducts (Walkersville, MD). RPMI 1640 was purchased
from Mediatech (Herndon, VA). The above medium components were endotoxin free as determined by the Limulus amebocyte lysate assay. Cold
melatonin (N-acetyl-5-methoxytryptamine) was purchased from Sigma
Chemical Co. (St. Louis, MO). 2-[125I]iodomelatonin (3700 kBq; 100 mCi)
and [3H]thymidine were purchased from DuPont-New England Nuclear
0022-1767/98/$02.00
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Earlier studies have shown that the pineal hormone melatonin activates human monocytes. It is reported here that melatonin
induces the secretion of IL-1, IL-6, and TNF in fresh and 1-day in vitro cultured monocytes that also express the melatonin
receptor (Kd 5 270 6 60 pM; 42,000 – 48,000 receptors/cell). However, when monocytes were cultured in vitro for 2 days, the
number of receptors decreased to 11,000 receptors/cell, with the same Kd. LPS activation of fresh or 1-day cultured monocytes did
not result in any increase in melatonin receptor number. LPS activation of 2-day cultured monocytes led to an increase in the
number of melatonin receptors, from 11,000 receptors/cell to the plateau of 42,000 to 48,000 receptors/cell. The loss of receptors
by 2-day cultured monocytes was not irreversible. Melatonin did not induce the release of IL-1, TNF, or IL-6 in monocytes
cultured in vitro for 3 days and for up to 15 days, and these long time cultured monocytes did not express the melatonin receptors
even after activation by LPS. The loss of melatonin receptors by monocytes cultured in vitro for 3 days and for up to 15 days was
irreversible. Therefore, it is shown for the first time that human monocytes express melatonin receptors. Furthermore, human
monocytes express melatonin receptors differentially depending on their state of maturation, and it appears that in vitro monocyte
differentiation and maturation negatively affect human monocyte melatonin receptor expression. The Journal of Immunology,
1998, 160: 1191–1197.
1192
HUMAN MONOCYTE MELATONIN RECEPTOR EXPRESSION
(Boston, MA). The L929 murine fibroblast cell line, the 7TD1 murine
hybridoma cell line, and the D10G4.1 T cell clone were purchased from
American Type Culture Collection (Rockville, MD). IL-1, IL-6, and TNF
were obtained from Genzyme Co. (Boston, MA).
Purification of human monocytes
Human peripheral blood monocytes were isolated from normal buffy coats
using the slightly hypertonic solution and monocyte separation medium
Nycoprep 1.068 (Life Technologies, Grand Island, NY) as previously described (8). Monocytes were purified endotoxin free and were found to be
94 to 96% pure as determined by nonspecific esterase staining of fixed
cells (8, 40, 41).
Activation of monocytes in vitro
Monocytes were incubated for 1 h at 37°C, and the nonadherent cells were
removed. The immunomodulator (LPS or melatonin) was then added to
each culture in triplicate (MEM, without serum), and the cells were incubated for 16 h at 37°C as previously described (8, 40, 41). The supernatants
(extracellular TNF, IL-1, and IL-6) were collected, clarified by centrifugation, and assayed for their TNF, IL-1, and IL-6 activities.
TNF activity was measured using the standard L929 fibroblast cell line.
TNF cytotoxicity toward L929 cells was evaluated using the MTT colorimetric assay as previously described (40, 41).
Biologic assay for IL-6
IL-6 activity was measured as previously described (42– 44) using the
7TD1 mouse IL-6-dependent hybridoma proliferation assay. This hybridoma responds mitogenically to IL-6. The proliferation was evaluated using
the MTT colorimetric assay. The 7TD1 can accurately measure 50 pg/ml
of IL-6.
Units of IL-6 activity. Samples were assayed in serial dilutions, and their
activities (units) were determined as the reciprocal of the dilutions producing 50% maximal proliferation as described.
Biologic assay for IL-1
IL-1 activity was performed using the IL-1-dependent Th cell clone
D10G4.1 (D10). D10 cells were incubated on test plates in the presence of
10-ml samples volume for 48 h at 37°C. The D10 proliferation was determined using the MTT assay (40, 41). Samples were assayed in serial dilutions, and their activities (units) were determined as the reciprocal of the
dilution producing 50% maximal proliferation (40, 41).
125
[
I]melatonin binding assay
Human monocytes or U937 or U373 cells (5 3 105 cells) were heavily
washed and incubated in HBSS buffer (Life Technologies; 1 mg/ml aprotinin and 1 mM EDTA, Ca21, MgCl2, MgSO4, and phenol red free) in the
presence of 25 to 800 pM 2-[125I]iodomelatonin concentrations (Dupont,
Wilmington, DE; 2200 Ci/mmol; 235 mCi/ml) (40). For nonspecific binding, a concentration of 0.8 mM cold melatonin was added. Incubations were
performed in 24-well polystyrene plates (Corning, Corning, NY) in a total
volume of 500 ml. After 1-h incubation at 37°C, plates were centrifuged
(15 min at 300 rpm), and the supernatants were removed with a Pasteur
pipette. Pellet cells were washed twice in 500 ml of HBSS buffer to remove
unbound melatonin. Cell were solubilized with 500 ml of NaOH (1 mM)
and 500 ml of distilled water and collected in borosilicate glass tubes (Baxter). The total 1-ml volume mixture was counted in a gamma counter (model B5002, Packard, Downers Grove, IL). Data were reported as specific
binding activity, i.e., total tracer bound minus nonspecific binding.
Binding data analysis
Before nonlinear regression analysis of binding data were available on
computer, binding data were transformed to Scatchard plots, and the binding capacity and Kd were determined using linear regression of the Scatchard plot. Linear regression assumes that the variability assay replicate y
values follow a Gaussian distribution, with a SD that does not depend on
the value of x. This assumption is rarely true with the transformed data.
Linear regression also assumes that x and y are measured independently.
This also is not always true.
Therefore, it was decided here to present the data as a hyperbola plot to
measure binding capacity and Kd, and for general understanding to display
FIGURE 1. Scatchard plot analysis of [125I]melatonin binding to human
monocytes. Fresh circulating human monocytes isolated from buffy coat
(5 3 105 cells) were incubated with [125I]melatonin (0.5–700 pM). Binding
assays were performed as described in Materials and Methods. MichaelisMentsen curve (A) and Scatchard plot (B) analysis of [125I]melatonin binding were realized.
the most important Scatchard plot, with the best and most accurate representation of the binding data remaining the hyperbola plot.
Statistical analysis
The statistical significance of difference between test groups was analyzed
by Student’s t test (two tailed); p , 0.05 was regarded as statistically
significant (40, 41).
Results
Melatonin receptor expression on fresh monocytes (Fig. 1)
To determine whether circulating monocytes express melatonin
receptor, binding experiments and Scatchard plot analysis were
performed using fresh monocytes and different concentrations of
[125I]melatonin. As shown in Figure 1, specific [125I]melatonin
binding was saturable in human monocytes and reproducible. Kd
ranges were obtained. The Kd for human monocytes was 270 6 60
pM, and human monocytes expressed an average of 41,400 6
4,500 receptors/cell. These data suggest that fresh circulating nonactivated human monocytes do express the receptor for melatonin.
Monocyte activation by melatonin (Fig. 2)
To determine whether melatonin does activate human monocytes,
human monocytes cultured for 1 day were incubated with different
concentrations of melatonin (Fig. 2, left). The monocyte activation
with MLT was determined as the ability of these MLT-activated
monocytes to secrete IL-1, IL-6, or TNF. As shown in Figure 2,
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Biologic assay for TNF
The Journal of Immunology
1193
melatonin, above a monocyte activation threshold concentration of
approximately 50 pM, was able to induce IL-1, IL-6, and TNF. A
concentration of 100 pM melatonin allowed a plateau of IL-1,
IL-6, and TNF secretion by human monocytes.
To determine whether melatonin does activate in vitro cultured
and mature monocytes, human monocytes maintained in culture in
vitro for several days were then activated with different concentrations of melatonin. As shown in Figure 2 (right), melatonin did
not induce significant secretion of IL-6, IL-1, and TNF in monocytes cultured for 3 days in vitro. Indeed, the 3-day cultured monocyte activation threshold concentration was around 80 pM for each
cytokine, and the plateau of cytokine secretion was reached for
concentrations of melatonin above 100 pM. However, the plateau
of cytokine secretion for 3-day cultured and melatonin-activated
monocytes represents only 4% of the plateau of cytokine secretion
observed for monocytes cultured for 1 day and activated by melatonin. Furthermore, this 4% cytokine secretion plateau is not statistically different from the values obtained for nonactivated monocytes (data not shown).
These data showed that melatonin does activate monocytes cultured in vitro for 1 day, but fails to activate monocytes cultured in
vitro for 3 days.
Differential expression of the melatonin receptor (Fig. 3)
Figure 2 showed that melatonin did activate monocytes cultured
in vitro for 1 day but failed to activate monocytes cultured in
vitro for 3 days. To determine whether the lack of activation of
3-day cultured monocytes by melatonin could be associated
with a lack of melatonin receptor expression, binding experiments and Scatchard plot analysis were performed using
[125I]melatonin on human monocytes cultured in vitro for different lengths of time (Fig. 3). Fresh circulating monocytes and
monocytes cultured in vitro for 1 day expressed the same number of receptors per cell, i.e., 42,000 6 4,000 for fresh monocytes and 48,000 6 2,000 for 1-day cultured monocytes. However, monocytes cultured in vitro for 2 days only expressed
11,000 6 2,000 receptors/cell, i.e., a decrease of 75% compared
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FIGURE 2. Melatonin can activate 1-day cultured monocytes. Activation of 1-day cultured monocytes by melatonin was determined by measurements
of the secretion of IL-1, IL-6, and TNF (left). Melatonin was used in a concentration range of 0.1–10 nM, and the production of cytokines was calculated
as total units per culture using a cell line bioassay specific to each cytokine and the MTT colorimetric assay (see Materials and Methods). The same type
of experiment (measurements of the IL-1, IL-6, and TNF-a secretion) is shown on the right, but the activation by melatonin was realized with 3-day cultured
monocytes.
1194
HUMAN MONOCYTE MELATONIN RECEPTOR EXPRESSION
FIGURE 3. Expression of the melatonin receptor
during different days of in vitro culture. Time course of
the expression of melatonin receptor at different days
in culture at 37°C (black histograms). For comparison,
two other cells line, U937 and U373, were represented
(white histograms). Data were expressed as the total
number of receptors per cell 6 SEM (n 5 5). The
number of receptors per cell was measured by Scatchard plot analysis using [125I]melatonin. (0 5 fresh
monocytes; 1 5 1-day in vitro cultured monocytes;
2 5 2-day in vitro cultured monocytes; 3 5 3-day in
vitro cultured monocytes; 15 5 15-day in vitro cultured monocytes).
Effect of LPS monocyte activation on monocyte melatonin
receptor expression (Figs. 4-6)
Figure 3 shows that monocytes cultured in vitro for 2 days display
a loss of 75% in melatonin receptor expression compared with
melatonin receptor expression of fresh monocytes or monocytes
cultured in vitro for 1 day. To determine whether the loss of melatonin receptor expression observed in monocytes cultured for 2
days is reversible, fresh monocytes and monocytes cultured in
vitro for either 1 or 2 days were or were not activated with 1 mg/ml
of LPS before performing binding experiments using
[125I]iodomelatonin. As shown in Figures 4 and 6, LPS activation
did not increase the number of melatonin receptors in fresh monocytes or monocytes cultured for 1 day (nonactivated fresh monocytes, 42,000 6 6,000 receptors/cell; LPS-activated fresh monocytes, 43,000 6 8,000 receptors/cell; nonactivated 1-day
monocytes, 48,000 6 7,700 receptors/cell; LPS-activated 1-day
cultured monocytes: 45,000 6 8,000 receptors/cell). However
(Figs. 5 and 6), LPS activation increased the number of melatonin
receptors per cell in monocytes cultured in vitro for 2 days from
10,800 6 1,880 to 42,800 6 9,000 melatonin receptors/cell. The
FIGURE 4. Effect of LPS activation on the expression of melatonin receptor by fresh and 1-day cultured human monocytes. The binding of [125I]melatonin on monocytes and the saturation curve were determined using different [125I]melatonin concentrations on nonactivated monocytes (fresh monocytes
(A) and 1-day cultured monocytes (B)) and LPS-activated monocytes (fresh monocytes (C) and 1-day cultured monocytes (D)). Data are expressed as the
number of receptors per cell determined for each concentration of [125I]melatonin by Scatchard plot analysis.
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with the number of receptors expressed in fresh and 1-day cultured monocytes. After 2 days of culture in vitro, monocytes did
not express melatonin receptors. We investigated whether U937
monocytic cells and U373 astrocytoma cells did express melatonin receptors. As shown in Figure 3, U373 cells expressed
12,000 6 2,000 receptors/cell, whereas U937 did not express
receptors for melatonin. These data suggest that long term culture of monocytes and/or cellular maturation/differentiation induces a loss of melatonin receptors in monocytes.
The Journal of Immunology
1195
number of melatonin receptors in LPS-activated 2-day cultured
monocytes reached a level identical with that in nonactivated fresh
monocytes (42,000 receptors/cell), LPS-activated fresh monocytes
(43,000 receptors/cell), nonactivated 1-day cultured monocytes
(48,000 receptors/cell), and LPS-activated 1-day cultured monocytes (45,000 receptors/cell). Therefore, the maximum amount of
melatonin receptors, i.e., the plateau of melatonin receptor number
per cell is around 42,000 to 48,000 receptors/cell. It is interesting
to note that LPS activation increased the number of melatonin
receptors in 2-day cultured monocytes, and that the affinity of melatonin toward these receptors remained identical (Kd 5 270 6 60
pM) in all cases, i.e., nonactivated and LPS-activated fresh monocytes and 1- or 2-day cultured monocytes. The LPS activation did
not affect the affinity of melatonin for its receptors. Therefore, it
FIGURE 6. Melatonin receptor expression on cultured monocytes. Melatonin receptors were expressed
as the maximal number of receptors per cell for nonactivated monocytes (black histograms) and for LPSactivated monocytes (white histograms). Data were
expressed as the mean 6 SEM (n 5 5); p , 0.05 vs
activated cells. 0 5 Fresh monocytes; 1 5 1-day in
vitro cultured monocytes; 2 5 2-day in vitro cultured
monocytes; 3 5 3-day in vitro cultured monocytes;
15 5 15-day in vitro cultured monocytes.
appears that LPS does increase the number of melatonin receptors
in 2-day cultured monocytes.
Effect of LPS activation on monocytes lacking melatonin
receptor expression (Fig. 6)
Our previous results showed that LPS activation can restore the
level of melatonin receptor expression in monocytes cultured in
vitro for 2 days to the level observed in fresh monocytes or in
monocytes cultured in vitro for 1 day. Also, LPS activation had no
effect on the expression of melatonin receptors by fresh or 1-day
cultured monocytes (Figs. 4 and 6). Figure 3 showed that monocytes cultured in vitro for .2 days did not express melatonin receptors. To determine whether monocytes cultured for .2 days
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FIGURE 5. Effect of LPS activation on melatonin receptor expression by 2-day cultured human monocytes. Saturation curves and binding of [125I]melatonin on monocytes were determined using different [125I]melatonin concentrations on nonactivated 2-day cultured monocytes (left) and on 2-day
LPS-activated monocytes (right). Data are expressed as the total number of receptors complexed with [125I]melatonin for the different melatonin concentrations used.
1196
and activated by LPS could express melatonin receptors, monocytes cultured in vitro for 3 and 15 days were activated by 1 mg/ml
of LPS before performing binding experiments and Scatchard plot
analysis using [125I]melatonin. Figure 6 shows that LPS activation
failed to induce the expression of melatonin receptors in 3- or
15-day cultured monocytes. Therefore, the loss of melatonin receptor expression in monocytes cultured for .2 days in vitro cannot be reversed by LPS activation.
Discussion
sites with an affinity constant that is the average between that of
high affinity sites (Kd 5 ;1–30 pM) and that of low affinity sites
(Kd 5 ;1–5 nM). MLT binding sites have also been described at
the plasma membranes (32), in the cytosol (39), at the mitochondria membranes (38), and at the nucleus membranes (38). Furthermore, Steinhilber et al. have described a nuclear receptor for MLT,
shown that MLT is a natural ligand of RZR a and b, and proposed
that ligand-induced transcriptional control could mediate physiologic functions (51). Because we used whole monocytes in our
studies, it is difficult to determine where the MLT binding sites are
in human monocytes.
Our results also show that the expression of MLT receptors in
human monocytes is correlated with the state of in vitro maturation
of the monocytes. Indeed, fresh monocytes (monocytes just purified from the blood donor) and monocytes in culture for 1 day
expressed the maximum number of MLT receptors (plateau at
40,000 receptors/cell). After 2 days in culture, the number of MLT
receptors dropped to 11,000 receptors/cell. After 3 days in culture,
the monocytes did not express MLT binding sites. In addition, it
has been shown that the maturation process of monocytes in vitro
is completed after 3 days in culture, and the circulatory monocytes
become differentiated macrophages (52). It seems from our results
that it takes 3 days of in vitro culture for monocytes to lose the
expression of MLT receptors, and it takes 3 days of in vitro culture
for monocytes to differentiate into macrophages.
It has been reported that MLT regulates its own receptor and
maintains the presence of its receptor on cells (53). Our hypothesis
is that as long as monocytes remain in contact with MLT they
express the MLT receptor. However, when monocytes are kept
away from MLT for a sufficient time and when they mature, they
lose the expression of the MLT receptors. Therefore, circulating
monocytes and freshly isolated monocytes do express MLT receptors, probably because they have been in contact with MLT in the
recent past and also because they are not yet differentiated. After
a certain time without MLT (culture in vitro for 2 days), the number of MLT receptors declines. This is an intermediary state between circulating monocytes and fully maturated monocytes in
vitro. Finally, after 3 days of in vitro culture, monocytes have been
for a sufficient time without MLT and are differentiated into mature
monocytes in vitro. They do not express MLT receptors. This loss
of MLT receptor expression in monocytes cultured in vitro for 3
days or more appears to be irreversible, since activation by LPS
did not restore the expression of MLT receptors by monocytes.
However, the decline in MLT receptor expression observed in
monocytes cultured in vitro for 2 days appears to be reversible,
since LPS activation of these monocytes restored the number of
MLT receptors per cell to its original plateau value observed for
freshly isolated monocytes (40,000 receptors/cell). The decline in
MLT receptor expression observed for monocytes cultured in vitro
for 2 days is reversible (transitory in vitro monocyte maturation
stage), whereas the loss of MLT receptor expression observed for
monocytes cultured in vitro for 3 days is irreversible. As a consequence, MLT can still induce the release of IL-1, TNF, and IL-6 in
monocytes cultured in vitro for 2 days but fail to induce the secretion of these three cytokines in monocytes cultured in vitro for
3 days. Finally, it should be noted that although the number of
MLT receptors declines when monocytes are cultured for a long
time in vitro, the affinity of these MLT receptors for MLT remains
the same.
In closing, it seems that the monocyte expression of the MLT
receptors closely depends on the state of maturation and differentiation of the monocytes and that MLT must maintain the expression of its own receptor on monocytes.
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For the first time it is reported here that human monocytes express
melatonin receptors, and the melatonin receptor expression is dependent on the monocyte state of maturation.
Indeed, we report here that MLT induces the release of IL-1,
TNF, and IL-6 in human monocytes and that human monocytes
express a single class of receptors with a Kd of approximately
270 6 80 pM. There are about 41,500 6 4,000 MLT receptors/
monocyte. The binding of MLT to the monocyte receptors was
saturable, specific, and of high affinity. The presence of the MLT
receptor on human monocytes varies with the state of differentiation of the monocytes in vitro. The major cell surface receptor for
LPS on monocytes/macrophages is CD14, and the action of LPS is
mediated and enhanced by LPS binding protein (LBP) (45). The
numbers of CD14 on the cell surface of fresh, 1-, 2-, 3-, and 15-day
cultured monocytes were the same (data not shown). Moreover,
the system CD14/LBP is involved for LPS concentrations ,10
ng/ml or equal to 10 ng/ml (45– 47); above this concentration of 10
ng/ml, LPS can activate monocyte/macrophage using a pathway
independent of CD14/LBP (46, 47). In our studies we used an LPS
concentration of 1 mg/ml well above 10 ng/ml; therefore, the system CD14/LBP was not involved. In addition, it has recently been
shown that LPS, even at concentrations ,10 ng/ml, can activate
monocytes/macrophages through a pathway independent of CD14/
LBP and the tyrosine kinases (46, 47). Therefore, the lack of melatonin receptor expression recovery by LPS in 3-day in vitro cultured monocytes was not due to deficient CD14 expression or
signal transduction.
MLT has been shown to modulate the secretion of lymphokines (3, 8, 14, 18 –21). MLT induces in vivo the secretion of
IL-4 (21), IL-5 (18), GM-CSF (20), and IL-2 (18). Interestingly,
in vitro, MLT inhibits the secretion of IL-2 and IFN-g by T
cells (48, 49). This discrepancy observed for IL-2 secretion in
vivo and in vitro is difficult to explain. MLT also induces the
secretion of IL-1 in vitro (8) and in vivo (14), and induces the
secretion of TNF-a in vivo (14). Our results show for the first
time that MLT induces the secretion of IL-6 and TNF-a by
human monocytes in vitro. These results are in agreement with
the general idea that MLT induces the secretion of lymphokines
by immune cells (3, 8, 14, 18 –21, 49).
It seems that the action of MLT on target cells is correlated with
the expression of MLT receptors by these target cells. Indeed,
MLT receptors have been described in many species (33–35) and
on human immune cells such as T lymphocytes (15), bone narrow
cells and Th2 lymphocytes (21), platelets (34), neutrophils (35),
and granulocytes (35). We report that human monocytes do express a single class of binding sites with a Kd of 270 6 60 pM.
These binding sites are specific for MLT, can be saturated, and
have the characteristics of a MLT receptor (Scatchard plot analysis). The Kd observed for the monocyte MLT receptors appeared to
correlate with that observed for most human immune cells. MLT
receptors of high and low affinity have been described; subtypes of
high affinity MLT receptors have also been reported (35, 37, 50).
It seems that in human monocytes, MLT binds to a single class of
HUMAN MONOCYTE MELATONIN RECEPTOR EXPRESSION
The Journal of Immunology
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