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(Journal of Leukocyte Biology. 2001;70:65-72.)
© 2001 by Society for Leukocyte Biology

Serotonin protects NK cells against oxidatively induced functional inhibition and apoptosis

Åsa Betten*, Claes Dahlgren{dagger}, Svante Hermodsson* and Kristoffer Hellstrand*

* Department of Virology and
{dagger} Department of Medical Microbiology & Immunology, Göteborg University, Sweden

Correspondence: sa Betten, The Phagocyte Research Laboratory, Department of Medical Microbiology & Immunology, Göteborg University, Göteborg, Sweden. E-mail: Aasa.Betten{at}microbio.gu.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
High concentrations of the neurotransmitter serotonin can be found in inflamed and ischemic peripheral tissues, but the role of serotonin in immunoregulation is largely unknown. Here we report that serotonin protected human natural-killer (NK) cells from oxidatively induced inhibition inflicted by autologous monocytes in vitro. Serotonin protected NK cells from monocyte-mediated apoptosis and suppression of cytotoxicity and maintained the activation of NK cells induced by interleukin-2 despite the presence of inhibitory monocytes. A detailed analysis of these protective effects revealed that serotonin scavenged reactive oxygen species (ROS) derived from the H2O2-myeloperoxidase (-MPO) system. Serotonin shared this scavenger activity with its precursor, 5-hydroxytryptophan (5-HTP); however, serotonin was >10-fold more potent than 5-HTP in protecting NK cells against functional inhibition and apoptosis. We propose that serotonin, by scavenging peroxidase-derived ROS, may serve to protect NK cells from oxidative damage at inflammatory sites.

Key Words: monocytes • hydrogen peroxide • scavenger • respiratory burst • myeloperoxidase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Natural-killer (NK) cells are lymphocytes endowed with constitutive cytotoxicity against malignant cells [1 ]. NK cells are assumed to participate in the nonadaptive host defense against metastatic cells and in controlling tumor growth [2 , 3 ]. The regulation of NK-cell cytotoxicity has been studied extensively, not least since knowledge about factors required for NK-cell activation may be valuable in the design of therapeutic regimens used in cancer diseases. For example, interleukin (IL)-2 and interferon-{alpha} (IFN-{alpha}) are effective activators of NK-cell-mediated cytotoxicity against malignant cells in vitro and in vivo, and these effects are believed to mediate, at least in part, the therapeutic benefit of these cytokines in neoplastic diseases [4 5 6 7 ]. NK cell activation is also assumed to contribute to the antitumor properties of IL-12, IL-15, and IL-18 in vivo in experimental tumor models and in humans [8 9 10 ].

In recent years, several investigators have reported that the tissue within or adjacent to malignant tumors is frequently subjected to oxidative stress, presumably mediated by tumor-infiltrating cells of the monocyte/macrophage lineage. The oxidative stress, which is defined as toxicity inflicted by reactive oxygen species (ROS), is assumed to contribute to the state of immunosuppression in the tumor area by inhibiting the function of NK cells and other lymphocytes of relevance to protection against neoplastic cells [11 12 13 ]. Typically, lymphocytes within or adjacent to tumors show a high degree of apoptotic cell death [14 ] and frequently display deficient cell surface expression of signal-transducing molecules [12 , 15 16 17 ]. A critical point is that IL-2 and other NK-cell-activating cytokines are ineffective in an environment of oxidative stress [12 , 18 , 19 ]. Hence, studies of the interactions between ROS-generating monocytes and NK cells may lead to the identification of compounds that can rescue NK cells from oxidative inhibition, which may be exploited therapeutically.

Serotonin is a biogenic amine, which is stored in peripheral tissues in platelets and in neurochromaffin cells of the gut mucosa [20 , 21 ]. In humans, serotonin is released from activated platelets at inflammatory sites and in ischemic tissues, and it may reach local concentrations of 100 µM at the immediate site of release [22 ]. Earlier in vitro studies have revealed that serotonin is an activator of human NK cells by regulating an interaction between NK cells and monocytes [23 24 25 ], but the mechanistic details of these activating properties are not known. Here we show that serotonin protects NK cells from monocyte-derived inhibitory and apoptosis-inducing signals conveyed by ROS; in the presence of serotonin, NK cells remain viable and functionally active and can be activated by IL-2 despite the presence of suppressive monocytes. Our data suggest that a target for serotonin is ROS derived from myeloperoxidase (MPO), a monocyte enzyme that utilizes hydrogen peroxide (H2O2) to generate toxic oxygen radicals [26 , 27 ].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Separation of mononuclear cells
Peripheral venous blood was obtained as freshly prepared leukopacks from healthy blood donors at the Blood Centre, Sahlgren’s Hospital, Göteborg, Sweden. The blood (65 mL per donor) was mixed with 92.5 mL of Iscove’s medium, 35 mL of 6% Dextran, and 7.5 mL of acid citrate dextrose. After incubation for 15 min at room temperature, the supernatant was removed and carefully layered on top of a Ficoll-Hypaque column (Lymphoprep, Nyegaard, Norway). The mononuclear cells were collected at the interface after centrifugation at 380 g for 15 min, washed twice in phosphate-buffered saline (PBS), and resuspended in Iscove’s medium supplemented with 10% human AB+ serum. During all further separation of cells, the cell suspensions were kept in siliconized test tubes (Vacuette, Greiner, Stockholm).

The mononuclear cells were further separated into lymphocytes and monocytes using the counter-current centrifugal elutriation technique, as described in detail elsewhere [18 , 28 ]. Briefly, the mononuclear cells were resuspended in elutriation buffer containing 0.5% bovine serum albumin and 0.1% EDTA in buffered NaCl and fed into a Beckman J2-21 ultracentrifuge with a JE-6B rotor (Beckman Coulter Inc., Fullerton, CA) at 2,100 rpm. A fraction with >90% monocytes was obtained at a flow rate of 18 mL/min. A lymphocyte fraction enriched for NK cells (CD3{varepsilon}-/56+ phenotype) and T cells (CD3{varepsilon}+/56- phenotype) was recovered at flow rates of 14–15 mL/min. The latter fraction consisted of CD3{varepsilon}-/56+ NK cells (45–50%), CD3{varepsilon}+/56- T cells (35–40%), CD3{varepsilon}-/56- cells (5–10%), and CD3{varepsilon}+/56+ cells (1–5%) with <3% contaminating monocytes, as judged by flow cytometry. The mixture of NK and T cells was exposed to autologous, elutriated monocytes in microplates. Lymphocytes (100,000 cells/well in 200 µL) were incubated with or without monocytes (5,000–100,000 cells/well) for 16 h at 37°C after which they were assayed for cytotoxic function, phenotype, and viability.

Target cells and microcytotoxicity assay
Cells from an NK-cell-sensitive cell line (K562) originating from patients with chronic myelogenous leukemia in blast crisis [29 ] were used as target cells. These cells (5 to 10 x106 cells/mL) were loaded with 51Cr at a concentration of 150 µCi/mL of cell suspension for 2 h at 37°C. Excess 51Cr was removed by centrifugation and resuspension of the target cells in culture medium. Finally, 10,000 51Cr-loaded target cells in 50-µL portions were added to the mononuclear cells in 96-well microplates (Nunc, Roskilde, Denmark).

The NK-cell-enriched lymphocytes and target cells were incubated in sextuplicates in microplates in a total volume of 200 µL in the presence or absence of monocytes. The compounds were added at the onset of incubation with the exception of formylmethionyl-leucyl-phenylalanine (fMLF), which was added at time t = 15 min. After incubation at 37°C for 16 h, supernatant fluids were collected by a tissue-collecting system (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and assayed for radioactivity in a {gamma}-counter. Maximum 51Cr release was determined in target cell cultures treated with Triton X-100. NK-cell cytotoxicity was calculated using the formula 100 x [(experimental 51Cr release - spontaneous release)/(maximum release - spontaneous release)] = cell lysis %.

In accordance with earlier studies [18 , 19 ], >90% of the lymphocyte cytotoxicity against K562 cells was depleted by the removal of CD56+ NK cells (by use of anti-CD56-coated beads [18 , 19 ]); in contrast, the removal of CD3+ T cells (by use of anti-CD3-coated beads) from the effector lymphocyte preparations did not significantly reduce cytotoxicity.

Determination of NADPH-oxidase activity
Monocyte superoxide production was determined using a luminol/isoluminol-amplified chemiluminescence (CL) technique [30 ]. Samples containing 500,000 elutriated monocytes in Krebs ringer glucose buffer were incubated in a six-channel Berthold Biolumat LB 9505 (Berthold Technologies Co., Wildbad, Germany) at 37°C. Release of superoxide was measured in the presence of 10 µg/mL of isoluminol and 4 U/mL of horseradish peroxidase (HRP), and the intracellular production of ROS was measured in the presence of 10 µg/mL of luminol, 20 U/mL of superoxide dismutase (SOD), and 2000 U/mL of catalase. The activity was determined without any additive, or cells were activated by the addition of 0.1 µM fMLF for induction of extracellular radical production or by 0.1 µM ionomycin for intracellular production of ROS.

H2O2 consumption
The consumption of H2O2 by serotonin or 5-hydroxytryptophan (5-HTP) in a cell-free environment was measured using a p-hydroxyphenylacetate (PHPA)-HRP fluorescence system [31 ]. PHPA was excited at a wavelength of 317 nm, and a Perkin-Elmer luminescence spectrometer (LS 50 B; Perkin-Elmer Inc., Norwalk, Connecticut) registered light emitted by oxidized PHPA at 400 nm.

Apoptosis assays
Apoptosis was detected by flow cytometry [28 ]. The fluorescein-activated cell sorter gate was set to comprise lymphocytes with a reduced forward scatter and an increased right-angle scatter characteristic of apoptosis [32 ]. Two additional methods were used for the determination of apoptosis in NK cells: analysis of DNA strand breaks by terminal deoxynucleotidyl transferase-mediated bromolated deoxyuridine triphosphate nick-end labeling (TUNEL assay) of DNA fragments (APO-BRDUTM kit, PharMingen, San Diego, CA) [19 ] and detection of extracellular expression of phosphatidyl serine [Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit I, Pharmingen] [19 ].

Detection of surface antigens
One million cells were incubated with appropriate FITC- and phycoerythrin (PE)-conjugated monoclonal antibodies (1 µL/106 cells) on ice for 30 min. The cells were washed twice in PBS, resuspended in 500 µL of sterile filtered PBS, and analyzed by flow cytometry on a FACSort with a Lysys II software program (Becton Dickinson, Stockholm, Sweden). Lymphocytes were gated on the basis of forward and right-angle scatter. The flow rate was adjusted to <200 cells s-1, and at least 10,000 cells were analyzed for each sample.

Compounds
The following compounds were used: serotonin hydrochloride, 5-HTP, luminol, isoluminol, fMLF, and PHPA (Sigma Chemical Co., St. Louis, MO); human recombinant IL-2 (Genzyme, Stockholm, Sweden); a Fas ligand inhibitor [comprising the extracellular domain of human Fas, amino acids 1 through 154, fused to the Fc portion of human immunoglobulin (Ig) G1; Kamiya Biomedical Co., Seattle, WA] [19 ]; ionomycin (Calbiochem, La Jolla, CA); MPO (kindly provided by I. Olsson, Lund, Sweden); HRP, SOD, and catalase (Bohringer-Mannheim, Mannheim, Germany); Dextran (Kabi Pharmacia, Stockholm, Sweden); acid citrate dextrose (Baxter, Deerfield, IL); 51Cr (Amersham); bovine serum albumin (ICN Biomedicals, Inc., Aurora, OH); and EDTA and H2O2 (KEBOLab, Göteborg, Sweden). All compounds were readily dissolved in Iscove’s culture medium. FITC- and PE-conjugated monoclonal antibodies (mAbs) against CD3{varepsilon}, CD56, and CD69 were purchased from Becton Dickinson. Reagents and media were regularly checked for the presence of endotoxin using the Limulus amoebocyte assay.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Monocyte and NK-cell interactions
Autologous monocytes strongly inhibited the tumor-killing activity of NK cells. In accordance with the results of earlier studies [18 , 19 , 28 ], this inhibition required the NADPH oxidase-dependent formation and release of ROS by monocytes, because (1) monocytes recovered from patients with chronic granulomatous disease, who have a defective NADPH oxidase and a deficient capacity to produce ROS [18 ], did not inhibit NK cells and (2) a scavenger of H2O2 (catalase) but not a scavenger of superoxide anion (SOD) prevented the inhibition of NK cell cytotoxicity. In addition, triggering of NADPH oxidase activity in monocytes by treatment with the chemotactic tripeptide fMLF effectively enhanced the inhibition (Fig. 1 and data not shown).



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  		Figure 1. Inhibition of NK-cell cytotoxicity by monocytes: reversal by serotonin and catalase. Eighty thousand NK-cell-enriched lymphocytes were admixed with 80,000 monocytes and assayed for cytotoxicity against K562 cells (104 cells/well) in a 16-h assay. The cell cultures were treated with culture medium (control, open bars), serotonin (100 µM; dark-gray bars), catalase (100 U/mL; light-gray bars) or serotonin plus catalase (black bars), added at the onset of incubation. Inset A: titration of monocytes [10–50% of all mononuclear cells (abscissa)] with a fixed amount of NK-cell-enriched lymphocytes (80,000/well); open circles, culture medium; closed circles, serotonin (100 µM). Inset B: dose-response experiment using serotonin concentrations of 1–100 µM (abscissa) at a monocyte-to-lymphocyte ratio of 1:1. All data are cell lysis (%) ± SE of sextuplicate determinations, and similar results were obtained in eight experiments, using blood from different blood donors. In these experiments, the NK-cell-inhibitory effect of monocytes was statistically significant at 25–50% monocytes (P<0.01), and the effect of serotonin at a 1:1 ratio between monocytes and lymphocytes was statistically significant at final serotonin concentrations of 1-100 µM (Mann-Whitney U-test, P<0.05-0.001).

 
To elucidate the mechanism(s) involved in protection of NK cells by serotonin, we first added monocytes to NK cells and monitored the degree of inhibition of cytotoxicity after treatment with or without serotonin, alone or together with catalase at a concentration (100 U/mL) that optimally reverses the monocyte-induced suppressive signal [28 ]. As shown in Figure 1 , catalase and serotonin were about equally effective in protecting NK cells against monocyte-induced inhibition. A ratio of one monocyte to three lymphocytes sufficed to significantly inhibit NK cells (Fig. 1 , inset A). The protective effect of serotonin was optimal at 100 µM, with significant protection also at 10 µM (Fig. 1 , inset B). Catalase protected NK cells dose dependently at 0.1–100 U/mL, and the combination of catalase plus serotonin at optimal concentrations did not further increase NK-cell cytotoxicity (Fig. 2 ). Serotonin did not affect NK-cell function when monocytes recovered from chronic granulomatous disease patients were added to NK cells or when serotonin was coincubated with NK cells in the absence of monocytes (data not shown).



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  		Figure 2. Protection of NK cells from monocyte-induced inhibition by catalase and serotonin. Eighty thousand NK cell-enriched lymphocytes were admixed with 80,000 monocytes and assayed for cytotoxicity against K562 cells (104 cells/well) in a 16 hr assay. The cell cultures were treated with catalase at indicated concentrations (open circles), alone or together with serotonin (100 µM; filled circles), added at the onset of incubation. Data are cell lysis % ± SE of sextuplicate determinations, and similar results were obtained in four experiments using blood from four different blood donors.

 
The lymphocyte preparation used in these experiments constituted a mixture of NK and T cells. Earlier studies have revealed that serotonin exerts regulatory effects on T-cell function [20 ]. To determine whether the T-cell content in the lymphocyte preparations influenced the effects of serotonin, we prepared a population of T-cell-depleted lymphocytes from the effector lymphocytes, using beads coated with anti-CD3 [18 , 19 ]. These lymphocytes contained 65–75% CD3-/CD56+ NK cells with <3% CD3+ T cells. Monocytes effectively inhibited the cytotoxicity of these T-cell-depleted lymphocytes, and serotonin protected NK cells from monocyte-induced inhibition (data not shown). These findings suggest that the observed inhibition of NK cells was independent of T cells, and that serotonergic effects on T cells did not influence the degree of protection of NK cells.

Role of ROS
Taken together, these data suggest that serotonin protects NK cells against monocyte-induced inhibition by interference with ROS produced by monocytes. Next we studied the effects of serotonin on NK-cell function in a model designed to simulate suppression by monocyte-derived ROS. In these experiments, monocytes were replaced by exogenously added H2O2. In accordance with earlier studies [28 ], H2O2 was found to strongly suppress NK-cell cytotoxicity. However, NK cells were not protected from the H2O2-mediated inhibition by the addition of serotonin, even at high concentrations (Fig. 3 ).



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  		Figure 3. Serotonin protects NK cells against inhibition of cytotoxicity induced by H2O2 and MPO. Eighty thousand NK cell-enriched lymphocytes were assayed for cytotoxicity against K562 cells (104 cells/well) in a 16 hr assay. The cell cultures were treated with culture medium (control, open circles), serotonin (100 µM; filled circles), MPO (open triangles), or serotonin + MPO (filled triangles). The final concentrations of H2O2 are indicated on the abscissa. All compounds were added at the onset of incubation. Similar results were obtained in seven experiments using blood from seven different blood donors; in these experiments, the protection achieved by serotonin + MPO was statistically significant as compared with MPO alone (P < 0.001; Student’s t-test). The inset shows one representative experiment out of four, where A is the control, and B the suppression of NK cell cytotoxicity induced by 150 µM H2O2 and HRP. The cell cultures were treated with culture medium (control, open bars), serotonin (100 µM; dark grey bars), HRP (light grey bars) or serotonin + HRP (filled bars). Data are cell lysis % ± SE of quadruplicate determinations.

 
Monocytes and other phagocytes can convert H2O2 into a variety of toxic radicals via peroxidases such as MPO; this enzyme catalyzes reactions in which H2O2 oxidizes halogens, leading to the formation of hypohalous acids and other toxic radicals [26 ]. We therefore monitored the suppressive effects exerted by H2O2 on NK cells in the presence of exogenously added MPO and/or serotonin [18 ]. These experiments revealed that serotonin strongly protected the NK cells from inhibition induced by the combination of H2O2 and MPO (Fig. 3) .

To determine whether serotonin protected NK cells from peroxidase-derived radicals in general or whether the protection was specific for MPO-derived products, we introduced HRP into the experimental system. Serotonin reversed the inhibition of NK-cell cytotoxicity induced by HRP plus H2O2 in a fashion similar to that observed for MPO + H2O2 (Fig. 3 , inset).

Monocyte-induced apoptosis
Earlier studies revealed that monocytes can induce programmed cell death or apoptosis in NK cells and that catalase or inhibitors of ROS formation, such as diphenyleneiodonium or histamine, afford protection against monocyte-triggered apoptosis [19 , 28 ]. Therefore, we investigated whether serotonin protected NK cells against monocyte-induced apoptosis. In these experiments, flow cytometry was used to estimate the frequency of apoptotic NK cells by gating a viable and an apoptotic lymphocyte population, based on size and granulation of cells (forward and side scatter) [19 , 28 ]. Apoptosis was confirmed by two methods: the Annexin V-binding assay and DNA fragmentation (by use of the TUNEL assay). These experiments revealed that serotonin effectively rescued NK cells from apoptosis induced by autologous monocytes as well as apoptosis induced by H2O2 plus MPO (Fig. 4A ).



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  		Figure 4. Serotonin rescues viability and inducibility of NK cells from inhibition triggered by H2O2 and MPO. Cells were prepared and treated with serotonin, MPO and H2O2 as described in the legend to Figure 3 . After incubation for 16 hrs, the frequency of apoptotic lymphocytes was determined fluorimetrically by use of an Annexin V binding assay (A). Apoptosis was confirmed by TUNEL assay and by the appearance of apoptotic morphology. The inset shows the morphological apoptosis (%) induced by H2O2 at 150 µM, and the bars represent the mean ± SE of data obtained using blood from three different blood donors; in these experiments, MPO alone protected lymphocytes from apoptosis (P<0.02), and the protection against apoptosis achieved by serotonin + MPO was statistically significant as compared with that achieved by MPO alone (P<0.02; Student’s t-test). The cells were also treated with IL-2 (100 U/ml; 16 hr), and gated viable NK cells were assayed for expression of the CD69 activation antigen (B). The inset shows the percentage of cells with CD56+/69+ phenotype at a H2O2 concentration of 150 µM and are the mean ± SE of data obtained using cells from four different blood donors; in these experiments, the enhanced CD69 expression achieved by serotonin + MPO was statistically significant as compared with that induced in cells treated with MPO alone (P<0.05; Student’s t-test).

 
The Fas ligand CD95L triggers apoptosis in many cell types after interaction with the Fas receptor (CD95), which is expressed on NK cells [33 ]. To evaluate the role of FasL-Fas interactions for the observed oxidatively induced apoptosis, we used a Fas ligand inhibitor, which comprises the extracellular domain of human Fas [amino acids (aa) 1–154), fused to the Fc portion of human IgG1. This Fas-Fc-IgG fusion protein, used at a concentration (20 µg/mL) sufficient to reduce FasL-mediated, activation-induced apoptosis in lymphocytes by >60% [34 ], did not affect MPO-induced apoptosis in NK cells, and serotonin did not alter the degree of apoptosis when incubated together with the Fas-Fc-IgG fusion protein (data not shown).

Maintenance of NK-cell activation
In the next series of experiments, we investigated whether the fraction of NK cells that remained viable after treatment with H2O2 plus MPO could be activated or instead were in a state of anergy. We used IL-2, which is a recognized inducer of the CD69 activation antigen in NK cells [19 , 35 ], and studied the acquisition of this antigen on the cell surface of viable NK cells incubated with monocytes. IL-2 induced the appearance of CD69 on >70% of NK cells, and this response was almost completely abolished by exogenous H2O2 alone or by H2O2 plus MPO. Serotonin did not maintain IL-2-induced activation in NK cells treated with H2O2 alone, but it effectively protected NK cells from inhibition induced by H2O2 plus MPO (Fig. 4B) .

Scavenger activity of serotonin
These data suggest that serotonin protects NK cells from functional inhibition and subsequent apoptosis induced by monocyte-derived ROS and specifically that serotonin rescues NK cells from oxidative damage induced by H2O2 and a peroxidase such as MPO. Earlier studies demonstrated that serotonin and structurally related compounds can be oxidized by H2O2 [36 ], but conflicting data suggest that serotonin is instead a pro-oxidant [37 38 39 ]. To establish whether serotonin is a scavenger of ROS and in particular of MPO-derived ROS, we determined ROS-induced CL after treatment with fMLF, which induces extracellular ROS generation in monocytes [40 ], or with ionomycin, which triggers intracellular ROS formation [41 ]. We also studied cell-free systems in which serotonin was coincubated with H2O2, alone or together with a peroxidase [31 ].

The extracellular fMLF-induced ROS in monocytes was strongly suppressed by serotonin (Fig. 5A and B ). To reveal whether serotonin was acting via a serotonin receptor on monocytes, we first compared the efficiency of serotonin to inhibit extracellular ROS with that of its precursor, 5-HTP, which has low activity at serotonin receptors [42 ]. Serotonin and 5-HTP were equally effective in scavenging extracellular ROS, regardless of whether ROS were generated by fMLF (Fig. 5A) or occurred spontaneously (Fig. 5C) . Neither of these compounds suppressed the intracellular radical production induced by ionomycin (Fig. 5D) .



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  		Figure 5. Effect of serotonin and its precursor 5-HTP on monocyte radical formation, measured by luminol/isoluminol-amplified CL. In experiment A, the activity was measured using 0.1 µM fMLF-treated monocytes (500,000/well) as source of extracellular radicals. Experiment B is a dose-response analysis for serotonin and 5-HTP in this experimental system. In experiment C, the scavenger properties of serotonin/5-HTP were monitored without the addition of fMLF (spontaneous radical release). Serotonin or 5-HTP did not affect intracellular radical production induced by fMLF, PMA, or the Ca2+ ionophor ionomycin; D shows an experiment using ionomycin (0.5 µM) as the inducer. Similar results were obtained in five separate experiments.

 
Second, we investigated the scavenging effect of serotonin (0.25–5µM) and 5-HTP (0.25–5 µM) in a cell-free system (H2O2-HRP/MPO-PHPA) [31 ]. Neither of the compounds consumed H2O2, measured as the amount of H2O2 available to excite PHPA, after incubation of serotonin or 5-HTP with H2O2 for 2 min followed by the addition of peroxidase (Fig. 6 ). Serotonin and 5-HTP were equally efficient in scavenging MPO/H2O2-derived ROS in this cell-free system (data not shown). Hence, although serotonin and 5-HTP were scavengers of ROS derived from H2O2 plus peroxidase, they were not scavengers of H2O2 alone.



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  		Figure 6. The scavenging activity of serotonin (5 µM; filled bars) or 5-HTP (5 µM; grey bars) was measured in a PHPA-H2O2-HRP system. To determine whether H2O2 (10 µM) was consumed by serotonin/5-HTP, HRP was added either at the onset of incubation with H2O2 or after a 2 min pre-incubation with H2O2. The ratios between the fluorescence values obtained with preincubation periods of 0 and 2 minutes, respectively are shown in the inserted table. The figure shows mean values ± SD of three different experiments.

 
NK-cell function and 5-HTP
These data demonstrated that serotonin is a potent scavenger of ROS generated by peroxidase-catalyzed reactions using H2O2 as the substrate. The scavenger activity of serotonin is likely a mechanism contributing to the protection of NK cells against monocyte-induced apoptosis and inhibition of cytotoxicity. In addition, our data revealed no differences between the scavenging efficiency of serotonin and its precursor 5-HTP, suggesting that specific serotonin receptors are not involved in the scavenging activity. Earlier studies, however, have clearly suggested the involvement of a cellular serotonin receptor mediating NK-cell regulatory properties of serotonin [24 , 25 ].

We therefore compared the effects of serotonin and 5-HTP on the cytotoxicity of NK cells against K562 cells in the presence of monocytes. Despite the similarities in scavenging oxygen radicals, there was a clear difference between the abilities of serotonin and 5-HTP to protect NK cells from the down-regulatory effects induced by monocytes. Serotonin was at least 10-fold more efficient than 5-HTP in preventing inhibition of cytotoxicity (Fig. 7A ) as well as in protecting NK cells against H2O2 plus MPO (Fig. 7B) .



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  		Figure 7. Effects of serotonin and 5-HTP on monocyte-induced or H2O2/MPO-induced inhibition of NK cells. In A, cells were prepared and analyzed as described in the legend to Figure 1 . In B, NK cell-enriched lymphocytes were treated with H2O2 (50 µM) and MPO, as decribed in the legend to Figure 3 . Data are cell lysis % ± SE of sextuplicate determinations, and similar results were obtained in five experiments using blood from five different blood donors. In these five experiments, the cytotoxicity of serotonin-treated cells was significantly superior to that of medium-treated control cells, both in the presence of monocytes and when cells were treated with H2O2/MPO, at final serotonin concentrations of 1, 5, 10, 50 and 100 µM (P<0.05-0.001, Mann-Whitney U-test). Serotonin was significantly superior to 5-HTP, both when measuring monocyte-induced and H2O2-induced inhibition of NK cells, at final compound concentrations of 10, 50, and 100 mM (P<0.02-0.01). 5-HTP significantly protected NK cells against monocyte-induced inhibition or inhibition induced by H2O2/MPO at final 5-HTP concentrations of 500 and 1000 µM (P<0.02, data not shown).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Serotonin is present at high concentrations in many types of immune or inflammatory reactions, mainly as the result of release from aggregated platelets [21 ]. In vitro studies suggest that serotonin, at concentrations ranging from 1 to 100 µM, exerts pleiotropic effects on immune effector mechanisms, including activation of signal transduction in T cells [43 ], induction of B-lymphocyte proliferation [44 ], suppression of granulocyte-mediated cytotoxicity [45 ], and inhibition of the expression of class I human leukocyte antigens on monocytes [46; for review, see ref. 20 ].

A role for serotonin in regulating NK-cell function was suggested by the findings that serotonin augments the cytotoxicity, cytokine-producing capacity, and proliferation of NK cells in the presence of autologous monocytes [23 , 24 ]. Subsequent work has revealed that serotonin reverses an NK-cell suppressive, monocyte-derived signal [6 ]. This study, aimed at elucidating the mechanism of action by which serotonin protects NK cells, used three models—one studying effects of serotonin on monocyte/NK-cell interactions, another studying effects of serotonin on NK-cell inhibition induced by exogenous ROS, and a third studying the putative scavenger properties of serotonin.

Our studies of serotonergic regulation of monocyte/NK-cell interactions confirm and extend previous findings by demonstrating that serotonin protects NK cells not only against inhibition of cytotoxicity but also against monocyte-induced apoptosis and anergy to IL-2; thus, serotonin maintained the anti-tumor cytotoxicity of NK cells, preserved the IL-2-induced acquisition of the CD69 activation antigen, and rescued NK cells from cell death by apoptosis despite the presence of ROS-producing monocytes. Serotonin was as effective as catalase, a known scavenger of H2O2, in this regard, and the combination of catalase and serotonin did not further protect NK cells.

Serotonin was also found to protect NK cells from oxidative damage induced by exogenous ROS. Serotonin did not rescue NK-cell function and viability after treatment with H2O2, but it afforded efficient protection against H2O2 in combination with a peroxidase such as MPO or HRP. In line with these findings, our data demonstrate that serotonin is a potent scavenger of ROS derived from H2O2 and a peroxidase but not of H2O2 alone.

MPO is present in monocytes and is released into the extracellular space on activation [40 ]. Earlier studies suggest that a significant part of the monocyte-induced inhibition of NK cells is dependent on the activity of the H2O2-MPO radical-generating system [28 ]. We therefore put forward the hypothesis that the consumption of ROS induced by serotonin significantly contributes to its NK-cell-protective properties; by neutralizing extracellular, peroxidase-derived toxic-oxygen products released by monocytes, serotonin preserves NK-cell function and prevents apoptosis.

The scavenger activity of serotonin can be explained either by an interaction with the peroxidase or, more likely, indirectly via neutralization by serotonin of an unidentified ROS, generated by the MPO-H2O2 system. The ability to consume MPO-H2O2-derived radicals is not unique to serotonin but is shared also by its precursor, 5-HTP. An unexpected finding was that, whereas serotonin and 5-HTP were equally effective scavengers of H2O2-peroxidase-derived radicals, serotonin was >10-fold more effective in protecting NK cells against inhibition induced by monocytes as well as by H2O2 plus MPO. The mechanism explaining this difference between serotonin and 5-HTP should be the focus of further study. Two mechanisms are proposed to account for these findings: (1) the attachment of serotonin to cellular receptors expressed on NK cells may more effectively neutralize MPO-derived ROS in direct connection with the cell membrane, or (2) serotonin triggers an unidentified intracellular signal in NK cells, which protects these cells against oxidatively induced inhibition and apoptosis.

In conclusion, our data reveal a novel immunoregulatory property of serotonin, namely to protect NK cells from oxidant stress. This mechanism may shed further light on the role of serotonin in immune and inflammatory reactions, in which serotonin may serve to neutralize ROS generated by adjacent phagocytes and thereby to preserve the viability and function of NK cells. The protection may be relevant in the host defense against neoplastic cells, since a large number of recent studies report that lymphocytes localized within or adjacent to human and murine cancer tumors frequently display signs of oxidative inhibition. Thus, intratumoral NK cells and other lymphocytes endowed with antitumor activity show reduced viability [14 ] and frequently display reduced cell surface expression of critical signal-transducing molecules [12 , 15 16 17 ]; this form of tumor-induced immunosuppression has been attributed to ROS formation by adjacent monocytes/macrophages [12 , 15 16 17 ]. Therefore, it can be speculated that serotonin or other substances with similar protective properties could be exploited therapeutically as, e.g., adjuncts to immunotherapy with IL-2 or other compounds aimed at activating lymphocyte-mediated tumor cell destruction.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Swedish Medical Research Council, the Swedish Cancer Society (Cancerfonden), the King Gustav V 80-Year Foundation, the Vårdal Foundation, and Maxim Pharmaceuticals, Inc., San Diego, CA.

We are obliged to Marie-Louise Landelius for excellent technical assistance.

Received July 5, 2000; revised February 21, 2001; accepted February 22, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Trinchieri, G. (1989) Biology of natural killer cells Adv. Immunol. 47,187-376[Medline]
  2. Whiteside, T. L., Herberman, R. B. (1994) Role of human natural killer cells in health and disease Clin. Diagn. Lab. Immunol. 1,125-133[Abstract/Free Full Text]
  3. Vujanovic, N. L., Basse, P., Herberman, R. B., Whiteside, T. L. (1996) Antitumor functions of natural killer cells and control of metastases Methods 9,394-408[Medline]
  4. Brunda, M. J., Sulich, V., Bellantoni, D. (1987) The anti-tumor effect of recombinant interferon alpha or gamma is influenced by tumor location Int. J. Cancer 40,807-810[Medline]
  5. Whiteside, T. L., Vujanovic, N. L., Herberman, R. B. (1998) Natural killer cells and tumor therapy Curr. Top. Microbiol. Immunol. 230,221-244[Medline]
  6. Hawkins, M. J. (1993) Interleukin-2 antitumor and effector cell responses Semin. Oncol. 20,52-59[Medline]
  7. Becker, J. C., Pancook, J. D., Gillies, S. D., Furukawa, K., Reisfeld, R. A. (1996) T cell-mediated eradication of murine metastatic melanoma induced by targeted interleukin 2 therapy J. Exp. Med. 183,2361-2366[Abstract/Free Full Text]
  8. Hendrzak, J. A., Brunda, M. J. (1996) Antitumor and antimetastatic activity of interleukin-12 Curr. Top. Microbiol. Immunol. 213,65-83
  9. Chapoval, A. I., Fuller, J. A., Kremlev, S. G., Kamdar, S. J., Evans, R. (1998) Combination chemotherapy and IL-15 administration induce permanent tumor regression in a mouse lung tumor model: NK and T cell-mediated effects antagonized by B cells J. Immunol. 161,6977-6984[Abstract/Free Full Text]
  10. Osaki, T., Hashimoto, W., Gambotto, A., Okamura, H., Robbins, P. D., Kurimoto, M., Lotze, M. T., Tahara, H. (1999) Potent antitumor effects mediated by local expression of the mature form of the interferon-gamma inducing factor, interleukin-18 (IL-18) Gene Ther 6,808-815[Medline]
  11. Mantovani, A., Bottazzi, B., Colotta, F., Sozzani, S., Ruco, L. (1992) The origin and function of tumor-associated macrophages Immunol. Today 13,265-270[Medline]
  12. Whiteside, T. L. (1998) Immune cells in the tumor microenvironment. Mechanisms responsible for functional and signaling defects Adv. Exp. Med. Biol. 451,167-171[Medline]
  13. Kiessling, R., Kono, K., Petersson, M., Wasserman, K. (1996) Immunosuppression in human tumor-host interaction: role of cytokines and alterations in signal-transducing molecules Springer Semin. Immunopathol. 18,227-242[Medline]
  14. Buttke, T. M., Sandstrom, P. A. (1994) Oxidative stress as a mediator of apoptosis Immunol. Today 15,7-10[Medline]
  15. Mizoguchi, H., O’Shea, J. J., Longo, D. L., Loeffler, C. M., McVicar, D. W., Ochoa, A. C. (1992) Alterations in signal transduction molecules in T lymphocytes from tumor-bearing mice Science 258,1795-1798[Abstract/Free Full Text]
  16. Otsuji, M., Kimura, Y., Aoe, T., Okamoto, Y., Saito, T. (1996) Oxidative stress by tumor-derived macrophages suppresses the expression of CD3 zeta chain of T-cell receptor complex and antigen-specific T-cell responses Proc. Natl. Acad. Sci. USA 93,13119-13124[Abstract/Free Full Text]
  17. Kono, K., Salazar-Onfray, F., Petersson, M., Hansson, J., Masucci, G., Wasserman, K., Nakazawa, T., Anderson, P., Kiessling, R. (1996) Hydrogen peroxide secreted by tumor-derived macrophages down-modulates signal-transducing zeta molecules and inhibits tumor-specific T cell- and natural killer cell-mediated cytotoxicity Eur. J. Immunol. 26,1308-1313[Medline]
  18. Hellstrand, K., Asea, A., Dahlgren, C., Hermodsson, S. (1994) Histaminergic regulation of NK cells. Role of monocyte-derived reactive oxygen metabolites J. Immunol. 153,4940-4947[Abstract]
  19. Hansson, M., Hermodsson, S., Brune, M., Mellqvist, U. H., Naredi, P., Betten, A., Gehlsen, K. R., Hellstrand, K. (1999) Histamine protects T cells and natural killer cells against oxidative stress J. Interferon Cytokine Res. 19,1135-1144[Medline]
  20. Mössner, R., Lesch, K. P. (1998) Role of serotonin in the immune system and in neuroimmune interactions Brain Behav. Immun. 12,249-271[Medline]
  21. Essman, W. B. (1978) Serotonin distribution in tissue and fluid Essman, W. B. eds. Serotonin in Health and Disease 1. Spectrum New York.
  22. Benedict, C. R., Mathew, B., Rex, K. A., Cartwright, J., Jr, Sordahl, L. A. (1986) Correlation of plasma serotonin changes with platelet aggregation in an in vivo dog model of spontaneous occlusive coronary thrombus formation Circ. Res. 58,58-67[Abstract/Free Full Text]
  23. Hellstrand, K., Hermodsson, S. (1987) Role of serotonin in the regulation of human natural killer cell cytotoxicity J. Immunol. 139,869-875[Abstract]
  24. Hellstrand, K., Hermodsson, S. (1990) Monocyte-mediated suppression of IL-2-induced NK cell activation. Regulation by 5-HT1A-type serotonin receptors Scand. J. Immunol. 32,183-192[Medline]
  25. Hellstrand, K., Czerkinsky, C., Ricksten, A., Jansson, B., Asea, A., Kylefjord, H., Hermodsson, S. (1993) Role of serotonin in the regulation of interferon-gamma production by human natural killer cells J. Interferon Res. 13,33-38[Medline]
  26. Klebanoff, S. J. (1982) Oxygen dependent defence mechanisms of phagocytes Adv. Host Def. Mech. 1,111-151
  27. Klebanoff, S. J. (1999) Oxygen metabolites from phagocytes Gallin, J. I. Snyderman, R. eds. Inflammation: Basic Principles and Clinical Correlates 1,721-768 Lippincott-Williams & Wilkins Philadelphia, PA. [Medline]
  28. Hansson, M., Asea, A., Ersson, U., Hermodsson, S., Hellstrand, K. (1996) Induction of apoptosis in NK cells by monocyte-derived reactive oxygen metabolites J. Immunol. 156,42-47[Abstract]
  29. Lozzio, C. B., Lozzio, B. B. (1975) Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome Blood 45,321-334[Abstract/Free Full Text]
  30. Dahlgren, C., Karlsson, A. (1999) Respiratory burst in human neutrophils J. Immunol. Methods 232,3-14[Medline]
  31. Hyslop, P. A., Sklar, L. A. (1984) A quantitative fluorimetric assay for the determination of oxidant production by polymorphonuclear leukocytes: its use in the simultaneous fluorimetric assay of cellular activation processes Anal. Biochem. 141,280-286[Medline]
  32. Allen, R. T., Hunter, W. J., III, Agrawal, D. K. (1997) Morphological and biochemical characterization and analysis of apoptosis J. Pharmacol. Toxicol. Methods 37,215-228[Medline]
  33. Medvedev, A. E., Johnsen, A. C., Haux, J., Steinkjer, B., Egeberg, K., Lynch, D. H., Sundan, A., Espevik, T. (1997) Regulation of Fas and Fas-ligand expression in NK cells by cytokines and the involvement of Fas-ligand in NK/LAK cell-mediated cytotoxicity Cytokine 9,394-404[Medline]
  34. Alderson, M. R., Tough, T. W., Davis-Smith, T., Braddy, S., Falk, B., Schooley, K. A., Goodwin, R. G., Smith, C. A., Ramsdell, F., Lynch, D. H. (1995) Fas ligand mediates activation-induced cell death in human T lymphocytes J. Exp. Med. 181,71-77[Abstract/Free Full Text]
  35. Lanier, L. L., Buck, D. W., Rhodes, L., Ding, A., Evans, E., Barney, C., Phillips, J. H. (1988) Interleukin 2 activation of natural killer cells rapidly induces the expression and phosphorylation of the Leu-23 activation antigen J. Exp. Med. 167,1572-1585[Abstract/Free Full Text]
  36. Schuff-Werner, P., Splettstosser, W., Schmidt, F., Huether, G. (1995) Serotonin acts as a radical scavenger and is oxidized to a dimer during the respiratory burst of human mononuclear and polymorphonuclear phagocytes Eur. J. Clin. Invest. 25,477-484[Medline]
  37. Chaplin, D. J. (1986) Potentiation of RSU-1069 tumour cytotoxicity by 5-hydroxytryptamine (5-HT) Br. J. Cancer 54,727-731[Medline]
  38. Buckman, T. D., Sutphin, M. S., Mitrovic, B. (1993) Oxidative stress in a clonal cell line of neuronal origin: effects of antioxidant enzyme modulation J. Neurochem. 60,2046-2058[Medline]
  39. Chan, T. Y., Tang, P. L. (1996) Characterization of the antioxidant effects of melatonin and related indoleamines in vitro J. Pineal Res. 20,187-191[Medline]
  40. Johansson, A., Dahlgren, C. (1992) Differentiation of human peripheral blood monocytes to macrophages is associated with changes in the cellular respiratory burst activity Cell Biochem. Funct. 10,87-93[Medline]
  41. Dahlgren, C. (1987) Difference in extracellular radical release after chemotactic factor and calcium ionophore activation of the oxygen radical-generating system in human neutrophils Biochim. Biophys. Acta 930,33-38[Medline]
  42. Peroutka, S. J., Snyder, S. H. (1983) Multiple serotonin receptors and their physiological significance Fed. Proc. 42,213-217[Medline]
  43. Aune, T. M., McGrath, K. M., Sarr, T., Bombara, M. P., Kelley, K. A. (1993) Expression of 5HT1a receptors on activated human T cells Regulation of cyclic AMP levels and T cell proliferation by 5-hydroxytryptamine. J. Immunol. 151,1175-1183
  44. Iken, K., Cheng, S., Fargin, A., Goulet, A. C., Kouassi, E. (1995) Serotonin upregulates mitogen-stimulated B lymphocyte proliferation through 5-HT1A receptors Cell. Immunol. 163,1-9[Medline]
  45. Garssadi, S. I., Regely, K., Mandi, Y., Beladi, I. (1994) Inhibition of cytotoxicity of chicken granulocytes by serotonin and ketanserin Vet. Immunol. Immunopathol. 41,101-112[Medline]
  46. Sternberg, E. M., Trial, J., Parker, C. W. (1986) Effect of serotonin on murine macrophages: suppression of Ia expression by serotonin and its reversal by 5-HT2 serotonergic receptor antagonists J. Immunol. 137,276-282[Abstract]



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