Life Sciences, Vol. 48, pp. 2557-2562 Printed in the U.S.A.
Pergamon Press
SEROTONIN INHIBITION OF TUMOR NECROSIS FACTOR-(z SYNTHESIS BY HUMAN MONOCYTES Eduardo Arzt*, Mbnica Costas, S. Finkielman* and Vfctor E. Nahmod Departamento de Sustancias Vasoactivas, Instituto de Investigaciones M6dicas, Facultad de Medicina, Univers~dad de Buenos Aires, Donato Alvarez 3150, 1427 Buenos Aires, Argentina. *Members of the National Research Council (CONICET) Argentina. (Received in final form April 24, 1991)
Summarv Serotonin inhibited in a concentration dependent way (10.3 M to 10-1°M) the LPS induced Tumor Necrosis Factor-~ synthesis both, when added to the monocyte cultures from the beginnmg and when added together w~th the activating stimulus 8 hours before the end of the culture. The inhabitory effect was specifically blocked by the 5-HT~ and 5-HT2 serotonin antagonist methysergide and the 5-HT2 receptor antagonist ketanserin. This indicates that only the 5-HT2 receptor family (5-HT2 or 5-HTlc ) may be involved in the inhibitory effect. Serotonin seems to play an important immunomodulatory role =n macrophage functions. An extensive number of observations point out the neuroendocnne regulation of immunological functions. Particularly serotonin was studied for its ability to influence immune responses =n wtro, and in vivo. Systemic administration of serotonin or its precursor, 5-hydroxytryptophan to mice, 30-60 rain before immunization, resulted in a dose-dependent suppression of both, the IgM and the IgG plaque-forming cell response to sheep red blood cells (1,2). Also, in vivo treatment of sensitized mice with several antagonists of the serotonin type 2 receptor (5-HT2), i.e., ketanserin, ritanserm and LY53857, inhibited the delayed-type hipersensitivity effector T cell function (3). In vitro serotonin (10.3 to 10.7 M) inhibited through the 5-HT~ receptor the mitogen-stimulated peripheral blood mononuclear cell proliferation and interferon-? (IFN-y) production (4). Serotonin did not inhibit interleukin-2 (IL-2) synthesis but decreased the expression of the iL-2 receptor (5). Receptors for serotonin have been described in human and munne lymphocytes (6,7), in mouse pulmonary alveolar macrophages (8) and the 5-HT 1 receptor in murine lymphocytes and the 5-HT2 in murine macrophages (9). Several functional actions of serotonin were also reported on macrophages. The indoleamine modulates IFN-7 ~nduced phagocytosis (10) and suppresses monocyte la expression, specifically through the 5-HT2 receptor (11). Monocytes cultured in vitro produce tumor necrosis factor-~ (TNF-cc) when stimulated by hpopolysaccharide (LPS) (12,13). Although it was reported that glucocorticoids suppress (14,15) and bradykinin stimulates (16) in vitro human and murine TNF-c~ production, relatively little is known about the factors, particularly neuroendocrine, involved in the modulation of the monocyte TNF-c~ synthesis. Methods Peripheral blood mononuclear leukocytes (PBML) were obtained by densi~ gradient centrifugation on FicolI-Hypaque (Pharmacia Fine Chemicals, Sweden) by the standard procedure 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
2558
Serotonin Inhibits TNF-~ Synthesis
Vol. 48, No. 26, 1991
(17) employing buffy coats from normal donors. Cells from a single donor were used in each experiment and each study was performed with separate cells of at least four different healthy donors. PBML were plated (0.2 ml/well) in flat-bottom multiwell plates (Nunc, Denmark) m the RPMI 1640 culture medium (M.A. Bioproducts, USA) supplemented with 5% fetal bovine serum (FBS) (Gibco, Grand Island, NY) at a cell density of 4x106 cells/ml. The cultures were incubated at 37°C in a wet atmosphere of 5% CO 2 and 95% air After 120 mm. of incubation the nonadherent mononuclear cells were aspirated. The monolayers were washed three times w~th Hank's balanced salt solution, and fresh FBS supplemented medium was added. Cells were highly enriched in monocytes (>95%) and contaminating cells were almost exclusively lymphocytes. Purity of cell populations was assessed by classical immunofluorescence and cytotox~c~ty tests using IOM1 and lOT7 monoclonal antibodies (Immunotech, France). Monocyte enriched cells were cultured as descnbed above for 48 h. LPS (Sigma Chemicals, St. Louis, MO) (1 or 10 ug/ml) was added to the cultures for the last 8 h. and serotonin (Sigma Chemicals) at the concentrations indicated in each experiment added either at beginning of the cultures or s~multaneously with LPS as indicated. Incubation and further experimental procedures were performed in the dark to avoid photosensitive drug degradation The long incubation period and the presence of serotonin in the fetal serum do not disturb in the expenments carried out w~th macrophages, as it was demostrated companng the effects of serotonm added during 48 h and 5 days on the la expression on macrophages in cultures with an without fetal serum (11). Ketanserin (a kind gift from Johnson & Johnson, Argentina) and methysergide (Sandoz, Argentina), both at 10 .6 M and 107 M, were added 15 mm. prior to serotonm addition to cells in order to block serotonin receptors as ~ndicated under results TNF ~n the monocyte supernatants was determined by a cytotox~city assay based on the L-929 cell line as descnbed elsewhere (18). Briefly, monolayers of a continuous hne of mouse fibroblasts (L-929) were incubated in 96 well microtiter dishes for 24 h. at 37°C, 5% CO 2 with Mem (Gibco, USA), supplemented with 2% fetal bovine serum. After this period, the medium was removed and replaced by fresh medium, 2% serum and actinom~cin-D (S~gma Chemicals) at 1 ug/ml. Serial two-fold dilutions of test samples and an international TNF standard (gently prowded by Dr. E. Falcoff, Paris, France) were then plated m a volume of 100 ul. After 24 h. of incubation at 37°C 5% CO 2, the samples were removed, and the plates were stained with 0.5% gentian violet in 70% methanol. One umt of TNF activity is defined as the amount required to get a 50% lys~s of the L-929 target cells. For TNF characterization mixtures of serial dilutions of positive TNF test samples and a constant dilution of ant~-human TNF-c~ serum (gently provided by Dr. E. Falcoff) were incubated for 1 h at 37°C After the incubation period, the residual TNF was titrated in the TNF assay prewously described. TNF-c~ titration without antiserum was done m parallel Serotonin, at the experimental concentrations, dDd not affect the TNF-c~ b~oassay when added to the L-929 cells Student's t test was applied to data to evaluate the significance of found diferences. Results Serotonin inhibited TNF-c~ production induced by LPS (1 ug/ml) when added from the beginning of the culture (FIgure 1). The dose response curve shows that serotonin inhibits TNF-c~ synthesis between 10-3 M and 10 -l° M The maximal inhLbitory effect is observed with 10 .3 M serotonin (20% of control values), with shght changes of the degree of inhibition until 10 -l° M (45% of control values) including some intermediate variations (Figure 1) The same inhibitory pattern was observed when LPS 10 ug/ml was (data not shown). The inhibitory effects were not due to toxicity of cells, as determined by trypan blue exclusion, was over 98% was always the alfa type as it was comDletely neutrahzed with
used to stimulate monocytes of the drug, as the viabihty in all cases. TNF produced human TNF-o~ antiserum.
When serotonin was added together w~th LPS to the cultures a significant inhibition was still observed (Figure 2). We cannot assert the differences among the data obtained when serotonm was added at the beginning of the culture with the corresponding value when serotonin was added with LPS because although they are significantly different (p
Pergamon Press
SEROTONIN INHIBITION OF TUMOR NECROSIS FACTOR-(z SYNTHESIS BY HUMAN MONOCYTES Eduardo Arzt*, Mbnica Costas, S. Finkielman* and Vfctor E. Nahmod Departamento de Sustancias Vasoactivas, Instituto de Investigaciones M6dicas, Facultad de Medicina, Univers~dad de Buenos Aires, Donato Alvarez 3150, 1427 Buenos Aires, Argentina. *Members of the National Research Council (CONICET) Argentina. (Received in final form April 24, 1991)
Summarv Serotonin inhibited in a concentration dependent way (10.3 M to 10-1°M) the LPS induced Tumor Necrosis Factor-~ synthesis both, when added to the monocyte cultures from the beginnmg and when added together w~th the activating stimulus 8 hours before the end of the culture. The inhabitory effect was specifically blocked by the 5-HT~ and 5-HT2 serotonin antagonist methysergide and the 5-HT2 receptor antagonist ketanserin. This indicates that only the 5-HT2 receptor family (5-HT2 or 5-HTlc ) may be involved in the inhibitory effect. Serotonin seems to play an important immunomodulatory role =n macrophage functions. An extensive number of observations point out the neuroendocnne regulation of immunological functions. Particularly serotonin was studied for its ability to influence immune responses =n wtro, and in vivo. Systemic administration of serotonin or its precursor, 5-hydroxytryptophan to mice, 30-60 rain before immunization, resulted in a dose-dependent suppression of both, the IgM and the IgG plaque-forming cell response to sheep red blood cells (1,2). Also, in vivo treatment of sensitized mice with several antagonists of the serotonin type 2 receptor (5-HT2), i.e., ketanserin, ritanserm and LY53857, inhibited the delayed-type hipersensitivity effector T cell function (3). In vitro serotonin (10.3 to 10.7 M) inhibited through the 5-HT~ receptor the mitogen-stimulated peripheral blood mononuclear cell proliferation and interferon-? (IFN-y) production (4). Serotonin did not inhibit interleukin-2 (IL-2) synthesis but decreased the expression of the iL-2 receptor (5). Receptors for serotonin have been described in human and munne lymphocytes (6,7), in mouse pulmonary alveolar macrophages (8) and the 5-HT 1 receptor in murine lymphocytes and the 5-HT2 in murine macrophages (9). Several functional actions of serotonin were also reported on macrophages. The indoleamine modulates IFN-7 ~nduced phagocytosis (10) and suppresses monocyte la expression, specifically through the 5-HT2 receptor (11). Monocytes cultured in vitro produce tumor necrosis factor-~ (TNF-cc) when stimulated by hpopolysaccharide (LPS) (12,13). Although it was reported that glucocorticoids suppress (14,15) and bradykinin stimulates (16) in vitro human and murine TNF-c~ production, relatively little is known about the factors, particularly neuroendocrine, involved in the modulation of the monocyte TNF-c~ synthesis. Methods Peripheral blood mononuclear leukocytes (PBML) were obtained by densi~ gradient centrifugation on FicolI-Hypaque (Pharmacia Fine Chemicals, Sweden) by the standard procedure 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
2558
Serotonin Inhibits TNF-~ Synthesis
Vol. 48, No. 26, 1991
(17) employing buffy coats from normal donors. Cells from a single donor were used in each experiment and each study was performed with separate cells of at least four different healthy donors. PBML were plated (0.2 ml/well) in flat-bottom multiwell plates (Nunc, Denmark) m the RPMI 1640 culture medium (M.A. Bioproducts, USA) supplemented with 5% fetal bovine serum (FBS) (Gibco, Grand Island, NY) at a cell density of 4x106 cells/ml. The cultures were incubated at 37°C in a wet atmosphere of 5% CO 2 and 95% air After 120 mm. of incubation the nonadherent mononuclear cells were aspirated. The monolayers were washed three times w~th Hank's balanced salt solution, and fresh FBS supplemented medium was added. Cells were highly enriched in monocytes (>95%) and contaminating cells were almost exclusively lymphocytes. Purity of cell populations was assessed by classical immunofluorescence and cytotox~c~ty tests using IOM1 and lOT7 monoclonal antibodies (Immunotech, France). Monocyte enriched cells were cultured as descnbed above for 48 h. LPS (Sigma Chemicals, St. Louis, MO) (1 or 10 ug/ml) was added to the cultures for the last 8 h. and serotonin (Sigma Chemicals) at the concentrations indicated in each experiment added either at beginning of the cultures or s~multaneously with LPS as indicated. Incubation and further experimental procedures were performed in the dark to avoid photosensitive drug degradation The long incubation period and the presence of serotonin in the fetal serum do not disturb in the expenments carried out w~th macrophages, as it was demostrated companng the effects of serotonm added during 48 h and 5 days on the la expression on macrophages in cultures with an without fetal serum (11). Ketanserin (a kind gift from Johnson & Johnson, Argentina) and methysergide (Sandoz, Argentina), both at 10 .6 M and 107 M, were added 15 mm. prior to serotonm addition to cells in order to block serotonin receptors as ~ndicated under results TNF ~n the monocyte supernatants was determined by a cytotox~city assay based on the L-929 cell line as descnbed elsewhere (18). Briefly, monolayers of a continuous hne of mouse fibroblasts (L-929) were incubated in 96 well microtiter dishes for 24 h. at 37°C, 5% CO 2 with Mem (Gibco, USA), supplemented with 2% fetal bovine serum. After this period, the medium was removed and replaced by fresh medium, 2% serum and actinom~cin-D (S~gma Chemicals) at 1 ug/ml. Serial two-fold dilutions of test samples and an international TNF standard (gently prowded by Dr. E. Falcoff, Paris, France) were then plated m a volume of 100 ul. After 24 h. of incubation at 37°C 5% CO 2, the samples were removed, and the plates were stained with 0.5% gentian violet in 70% methanol. One umt of TNF activity is defined as the amount required to get a 50% lys~s of the L-929 target cells. For TNF characterization mixtures of serial dilutions of positive TNF test samples and a constant dilution of ant~-human TNF-c~ serum (gently provided by Dr. E. Falcoff) were incubated for 1 h at 37°C After the incubation period, the residual TNF was titrated in the TNF assay prewously described. TNF-c~ titration without antiserum was done m parallel Serotonin, at the experimental concentrations, dDd not affect the TNF-c~ b~oassay when added to the L-929 cells Student's t test was applied to data to evaluate the significance of found diferences. Results Serotonin inhibited TNF-c~ production induced by LPS (1 ug/ml) when added from the beginning of the culture (FIgure 1). The dose response curve shows that serotonin inhibits TNF-c~ synthesis between 10-3 M and 10 -l° M The maximal inhLbitory effect is observed with 10 .3 M serotonin (20% of control values), with shght changes of the degree of inhibition until 10 -l° M (45% of control values) including some intermediate variations (Figure 1) The same inhibitory pattern was observed when LPS 10 ug/ml was (data not shown). The inhibitory effects were not due to toxicity of cells, as determined by trypan blue exclusion, was over 98% was always the alfa type as it was comDletely neutrahzed with
used to stimulate monocytes of the drug, as the viabihty in all cases. TNF produced human TNF-o~ antiserum.
When serotonin was added together w~th LPS to the cultures a significant inhibition was still observed (Figure 2). We cannot assert the differences among the data obtained when serotonm was added at the beginning of the culture with the corresponding value when serotonin was added with LPS because although they are significantly different (p
