1 UP Cytokines & Inflammation, Institut Pasteur, 2528 rue du Dr Roux, 75015 Paris, France 2 165 Medical Sciences, 506 S. Mathews, Urbana, IL 61801, USA
Correspondence to: I. Cloëz-Tayarani; E-mail: icloez{at}pasteur.fr
Transmitting editor: T. Watanabe
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Abstract |
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Keywords: IL-1, inflammation, tumor necrosis factor
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Introduction |
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In inflammatory conditions such as thrombosis and ischemia, the activated platelets release 5-HT and this leads to an increase in its local concentrations at the inflamed region (1,2). In infectious diseases caused by Gram-negative bacteria, endotoxic lipopolysaccharide (LPS) released by bacteria may also activate the 5-HT-containing platelets by a direct mechanism (3). Released LPS also leads to translocation of platelets to the inflamed tissue and consequent increase in local concentration of 5-HT (4). Therefore, 5-HT, in addition to its possible interaction with blood cells, may interact with the inflamed tissue macrophages. The immunomodulatory role of 5-HT with respect to its stimulatory or inhibitory effects on immune cells including B, T and NK cells, and monocytes/macrophages has been well documented (59).
Numerous cytokines are mediators of infectious and inflammatory diseases such as septic shock. The balance between cytokines with pro-inflammatory properties [i.e. tumor necrosis factor (TNF)- and IL-1] and those with anti-inflammatory properties [i.e. IL-6, IL-10 and IL-1 receptor antagonist (IL-1Ra)] is important in the control of duration and severity of an inflammatory response. The presence of 5-HT at inflammatory sites suggests its possible involvement in the control of this equilibrium.
The effect of 5-HT on the production of cytokines has been the subject of only a few studies. It has been shown that 5-HT promotes IFN- production by human NK cells through a 5-HT1A receptor-mediated mechanism (10) and it suppresses the production of this cytokine by blood cells (11). On the other hand, 5-HT induces the production of IL-16, a chemotactic factor, in peripheral blood leukocytes (12) and in CD8+ T cells (13).
5-HT exerts its function through the activation of a polymorphic group of membrane receptors which represents 14 distinct receptor subtypes in humans (14). A number of 5-HT receptor subtypes including 5-HT1A (15) and 5-HT3 (16) have been reported to be present in human lymphocytes. mRNAs encoding the 5-HT1B, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT3, 5-HT6 and 5-HT7 receptor subtypes have also been detected in rat lymphocytes (17). In contrast, the presence of 5-HT receptors in monocytes has not been clearly established (18,19). We have recently reported the presence of high-affinity binding sites for 5-HT in guinea pig alveolar macrophages (20).
This study was undertaken in order to further establish the relationship between 5-HT and cytokines by studying its effect on the production of TNF-, IL-1
, IL-1ß, IL-6, IL-10 and IL-1Ra in human peripheral blood mononuclear cells (PBMC) following LPS challenge. We also investigated the nature of the 5-HT receptor involved in the specific down-regulation of LPS-induced TNF-
production and its presence in PBMC by Western blot analysis.
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Methods |
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Isolation and culture of PBMC
Approximately 450 ml of blood was obtained from healthy human volunteers. PBMC were prepared by centrifugation at 500 g for 20 min at 15°C from diluted blood 1:2 in RPMI 1640 culture medium over Ficoll gradient (MSL; Eurobio, les Ulis, France). Two volumes of diluted blood to 1 volume of MSL were used. Cells were washed by centrifugation in RPMI 1640 medium. Cell pellets were resuspended at a final concentration of 6 x 106 cells/ml of the same medium supplemented with 5% heat-inactivated normal human serum (a pool of sera from healthy volunteers) and antibiotics (penicillin 100 UI/ml and streptomycin 100 µg/ml). Aliquots of 500 µl of cell suspension were dispensed into each well of a 24-well plate and incubated at 37°C in the presence of test compounds in a 5% CO2 air incubator in a humidified atmosphere.
Stimulation of cytokine release
5-HT and 5-HT agonists were added to cells at the beginning, and then incubated for 18 h followed by an additional incubation for 6 h in the presence of 1 µg/ml LPS (smooth E. coli 0111:B4). The incubations were carried out under the above conditions. For the measurement of mRNA content, LPS was added 3 h prior to the end of the 24-h culture period. At the end of incubation, the supernatants were collected, centrifuged at 400 g for 10 min at 15°C and stored at 20°C until cytokine determination. For intracellular cytokine measurements, cell pellets were resuspended in 500 µl of RPMI medium, frozen and thawed 3 times to prepare lysates. Intracellular IL-1 and IL-1ß levels were measured by ELISA. Unless analyzed immediately, all samples were otherwise kept at 20°C until ELISA tests.
Cytokine measurements
IL-1ß, IL-10 and caspase-1 were assayed using the commercial kits DuoSets provided by R & D Systems (Abingdon, UK). Cytokine concentrations in every individual sample were determined by comparison with an internal standard according to the manufacturers instructions. Specific in-house ELISA was used for the measurements of TNF- (21), IL-6 (22) and IL-1
(23). An ELISA specific for IL-1Ra was set up using a mAb (84.1, prepared by Dr J. C. Mazie, Pasteur Institute, Paris) against a recombinant human IL-1Ra (Synergen, Boulder, CO) and a rabbit polyclonal anti-human IL-1Ra antiserum. Microtitration plates were coated with mouse monoclonal anti-human IL-1Ra (5 µg/ml in carbonate buffer, 0.05 M, pH 9.6) overnight at 4°C. After 5 cycles of washing with PBS/Tween 0.1% buffer, a protein blocking step was carried out using 2% BSA in carbonate buffer for 60 min at 37°C. After additional washings, standards of recombinant human IL-1Ra diluted in PBS/Tween 0.1%/BSA 1% were added and samples were tested after a 10-fold dilution in PBS/Tween/BSA. Incubation was performed for 2 h at 37°C. After washing, rabbit polyclonal anti-human IL-1Ra (diluted 1/375 in PBS/Tween 0.1%/BSA 1%) was added for 90 min at 37°C. After 5 cycles of washing, peroxidase-labeled goat anti-rabbit Ig (Silenus-AMRAD Biotech, Victoria, Australia; 1/2500 in PBS/Tween 0.1%/BSA 1%) was added to each well. After 1 h of incubation at 37°C, plates were washed and enzymatic activity was revealed by the addition of o-phenylenediamine substrate (Sigma; 1 mg/ml) prepared extemporaneously in citrate buffer 0.05 M, pH 5, containing 0.06% hydrogen peroxide, to each well. After 5 min incubation in the dark, the reaction was stopped by the addition of HCl 3N. Optical density was measured at 490/630 nm on microplate reader spectrophotometer (Dynex Technologies, Chantilly, VA). The lower limit of sensitivity of the assay was 40 pg.
mRNA cytokine measurements
Total RNA was isolated from PBMC in culture by using RNA-Plus reagent (Quantum, Biotechnologies, Bobigny, France) according to the manufacturers instructions. TNF- and IL-1ß gene-specific mRNA contents were measured using biotin-captured oligonucleotide probes in a microplate assay (Quantikine mRNA; R&D Systems).
Preparation of whole-cell extracts for electrophoresis
PBMC were resuspended at a final concentration of 2.5 x 106 cells/ml PBMC in RPMI 1640 medium supplemented with the same antibiotics as described above. Cells were then stimulated by the addition of LPS (1 µg/ml) for 6 h. At the end of incubation, cells were washed with ice-cold PBS. PBMC lysates were obtained by incubating the cells for 10 min at 4°C in 100 µl of ice-cold lysis buffer consisting of 50 mM TrisHCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium vanadate, 5 mM sodium pyrophosphate, 10 mM sodium ß-glycerophosphate, 50 mM sodium fluoride, 0.1% 2-mercaptoethanol, 1% Triton X-100 and a 1:100 dilution of Protease Inhibitor Cocktail Set III (Calbiochem, San Diego, CA) freshly added. The insoluble material was removed by centrifugation (14,000 g, 10 min) and the supernatant fraction was used for analysis. Protein concentration was determined according to the method of Bradford (Bio-Rad, Hercules, CA). Samples of 10 µg of protein of whole extracts were subjected to SDSPAGE.
Immunoblot
After electrophoresis, gel proteins were electrophoretically transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech, Little Chalfont, UK). After transfer, non-specific binding sites were blocked for 1 h at room temperature with 5% low-fat milk in PBS/Tween 0.1%, pH 7.4. Membranes were then treated with a 1:5000 dilution of 5-HT2A receptor antibody in blocking buffer for 1 h at room temperature. Membranes were then washed with PBS/Tween 0.1%, pH 7.4 and incubated for 1 h at room temperature with a 1:5000 dilution of secondary antibody (peroxidase-conjugated goat anti-mouse IgG; Jackson ImmunoResearch, West Grove, PA) in blocking buffer. The membranes were washed and developed using ECL (Amersham Pharmacia Biotech).
Statistical analysis
Results were expressed as means ± SEM for the indicated number of independently performed experiments. Statistical analysis was performed using the non-parametric Wilcoxon signed-rank test. Values of P < 0.05 were considered significant.
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Results |
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Comparative effect of 5-HT on extra- and intracellular levels of IL-1, IL-1ß and caspase-1 content in LPS-stimulated PBMC
IL-1 represents the major form of the cell-associated IL-1. IL-1
released from PBMC and measured upon LPS stimulation corresponds to its intracellular origin and represents almost the total amount since the extracellular level of this cytokine is very low (Fig. 3A). In contrast, IL-1ß measured in PBMC is mainly found in the extracellular environment and represents the amount released from the cells upon stimulation by LPS. The cell supernatant contains
95% of the IL-1ß, whereas the intracellular levels of this cytokine become very low once the cells are stimulated (Fig. 3B). We did not observe a significant difference between the release of intra- and extracellular forms of IL-1
when PBMC were stimulated by LPS and incubated with 5-HT at 0.01100 µM (Fig. 3A). In addition, 5-HT at the same concentrations and under the same experimental conditions did not have any effect on the amount of intracellular IL-1ß (Fig. 3B). In contrast, the extracellular levels of IL-1ß were increased under these conditions (Figs 2A and 3B).
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Effect of 5-HT on TNF- and IL-1ß mRNA production from LPS-stimulated PBMC
To further investigate the effect of 5-HT on TNF- and IL-1ß production, we analyzed its potential effect on the expression of mRNAs encoding these two cytokines. At 0.01100 µM, 5-HT did not modify TNF-
mRNA and IL-1ß mRNA production significantly (Fig. 4).
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Presence of 5-HT2A receptor protein in PBMC
To determine whether the 5-HT2A receptors are present in PBMC, we analyzed the presence of the human 5-HT2A protein in non-stimulated and LPS-stimulated PBMC using Western blotting. As shown in Fig. 6, we found that the 5-HT2A receptor protein is present in both resting and LPS-stimulated PBMC. As a positive control for these experiments, we also analyzed 5-HT2A immunoreactivity in rat brain lysate (Fig. 6).
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Discussion |
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IL-1 is a cytokine which is mainly produced and accumulated within the cells with very low amount present in the extracellular environment. In order to determine whether the increase in the extracellular level of IL-1ß was the result of an indirect effect of 5-HT possibly due to its high concentration or a normal consequence of its effect on IL-1ß production, we measured the intracellular level of IL-1
in the presence of the same concentrations of 5-HT. Indeed, if 5-HT had a damaging effect on PBMC with regard to IL-1ß production, we would expect to observe a decrease in the intracellular level of IL-1
due to cellular leakage. Our results show that the intracellular level of IL-1
remains stable regardless of the concentration of 5-HT used in these experiments. Therefore, it is reasonable to assume that 5-HT, at concentrations used in this study, does not cause any apparent damage to PBMC.
In order to further determine the role of 5-HT on TNF- and IL-1ß production, we analyzed its potential effect on the regulation of TNF-
and IL-1ß mRNA expression in human PBMC following LPS stimulation. We observed that the levels of TNF-
and IL-1ß mRNA expression were not significantly changed by 5-HT at the concentrations used in these experiments. This suggests that the regulatory effects of 5-HT on the production of TNF-
and IL-1ß occur at the post-transcriptional level since the TNF-
mRNA content in PBMC remained unchanged.
To further investigate the mechanism by which 5-HT favors the production of IL-1ß in LPS-stimulated PBMC, we analyzed its potential effect on the IL-1-converting enzyme (caspase-1). Caspase-1 cleaves the precursor form of IL-1ß leading to its mature bioactive form. Our results show that 5-HT does not have any effect on the caspase-1 level in LPS-stimulated PBMC. This suggests that the increase in IL-1ß production was not related to an increase in caspase-1 content and to a consequent increase in the maturation process of IL-1ß.
The effect of 5-HT on cytokine production by human leukocytes has not been extensively studied. There has been some reports on human monocytes (24) and it has been shown that the ratio of IFN-/IL-10 in human whole blood cells is diminished in the presence of 5-HT (11). The authors have ascribed this effect to the suppressive action of 5-HT on IFN-
production rather than to an increase in the production of IL-10, which is a cytokine with anti-inflammatory properties (11). This is in agreement with our data and indicates that 5-HT does not have a significant effect on the production of anti-inflammatory cytokines such as IL-10 and IL-1Ra in human PBMC.
Our results show that 5-HT exerts opposite effects on the production of TNF- and IL-1ß in PBMC despite the fact that these cytokines display pro-inflammatory properties and are both derived from the same cells. A similar discrepancy in the regulation of TNF-
and IL-1ß by IFN-
has been reported previously (25,26). This indicates that 5-HT does not necessarily have the same mechanism of action on the production of these cytokines. Other agents, including anti-mitotic compounds, have been shown to simultaneously display immunostimulating and immunosuppressive properties, thereby affecting IL-1ß and TNF-
production differentially (27,28).
Our results suggest that the modulatory effect of 5-HT on TNF- production occurs through the activation of the 5-HT2A receptor subtype. Indeed, ketanserin, a 5-HT2A antagonist, reversed the inhibitory effect of 5-HT on TNF-
production in PBMC, whereas DOI, a 5-HT2A/2C agonist, inhibited TNF-
production. These findings are in accordance with previous studies which showed that selective activation of 5-HT2A receptors could potentially decrease inflammatory responses (29).
The 5-HT1A receptor subtypes which are expressed by leukocytes (6,7), however, do not seem to participate in 5-HT mechanisms of action on cytokine production since 8-OH-DPAT, a selective 5-HT1A agonist, did not show any activity. In addition, NAN-190, a selective 5-HT1A antagonist, and fluoxetine, a selective inhibitor of 5-HT uptake, did not modify the 5-HT effects on TNF- (results not shown). These data indicate that neither the 5-HT1A receptors nor the 5-HT transporter, which is reported to be present in blood cells (1), are involved in the 5-HT modulatory effect on TNF-
production. Ketanserin did not reverse the 5-HT effect on IL-1ß secretion (data not shown) suggesting that the inhibitory effect of 5-HT on TNF-
and the amplifying effect on IL-1ß production are mediated via different 5-HT receptors. In contrast to the 5-HT2A receptors which possess a micromolar affinity for 5-HT, the 5-HT1A receptors bind 5-HT with a nanomolar affinity (14). Therefore, we can exclude the participation of the 5-HT1A receptors in the observed effect of 5-HT on IL-1ß production. In order to confirm this hypothesis, we tested the influence of 8-OHDPAT (a 5-HT1A agonist) and that of NAN-190 (a 5-HT1A antagonist) on the effect of 5-HT on IL-1ß production. These compounds were both ineffective (data not shown). Further experiments are needed to identify the nature of the 5-HT receptor involved in the modulation of IL-1ß production.
Our data suggest that leukocytes express the 5-HT2A receptor subtype. The presence of 5-HT2-like receptors has been confirmed on Jurkat cells by ligand-binding studies (30). mRNAs for the 5-HT2C subtype have also been detected in human resting lymphocytes (31). Using Western blot analysis in this study, we found that the 5-HT2A receptor protein is expressed both in resting and LPS-stimulated PBMC. These data indicate that such receptors may be involved in the 5-HT effect on TNF- production. Additional experiments would be required to analyze the binding characteristics of the 5-HT2A receptors in PBMC.
To extrapolate these findings to in vivo conditions, several aspects should be considered. First, the concentrations of 5-HT used in this study must be compared to those found in vivo. In healthy humans, the basal plasmatic concentration of 5-HT is low (nanomolar range) (1). In inflammatory situations such as thrombosis, however, the 5-HT concentration is considered to increase from basal levels and reach 100 µM at the immediate site of release (2). The inhibition of TNF- production from LPS-stimulated PBMC was obtained with 5-HT at 1 µM and this may be of physiological relevance. In the case of IL-1ß production, we observed a shift in the doseresponse curve since the 5-HT effect was significant only at 100 µM. This suggests that the increasing effect of 5-HT on IL-1ß production may occur only when platelet degranulation is massive, as observed in severe sepsis associated with intravascular coagulation and shock (32).
The second aspect to take into account is the kinetic parameters used in our model. As previously described in similar experimental models (24,3335), we pre-treated the cells with 5-HT before adding the stimulant. We did not observe a shift in the doseresponse curve of 5-HT when cells were incubated with 5-HT for a shorter or a longer period of time or when 5-HT and LPS were added simultaneously (data not shown).
Our experimental model may be relevant in situations such as trauma/hemorrhage and subsequent sepsis. Indeed, among the pro-coagulant factors that are released in trauma (36), thrombin can induce the secretion of 5-HT from platelets seconds before a subsequent exposure to LPS (37).
Our results suggest that 5-HT plays an important role in the regulation of immune cell function through its involvement in the control of cytokine release in human PBMC. TNF- levels are significantly elevated in immunological disorders such as septic shock. Our data support the idea that 5-HT, at physiological concentrations, may act as an anti-inflammatory mediator to down-regulate the production of TNF-
by these cells in such disorders.
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Acknowledgements |
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Abbreviations |
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8-OH-DPAT8-hydroxy-2-(di-n-propylamino)tetralin
DOI hydrochloride(2,5-dimethoxy-4-iodophenyl)-2-aminopropaneHCl
IL-1RaIL1 receptor antagonist
LPSlipopolysaccharide
NAN-1901-(2-methoxyphenyl)-4-[4-(2-phthalimmido)butyl]piperazine
PBMCperipheral blood mononuclear cell
TNFtumor necrosis factor
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References |
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