Journal of Neuroimmunology 159 (2005) 113 – 118 www.elsevier.com/locate/jneuroim
Peritoneal macrophages express the serotonin transporter M.L. Rudda, A.N. Nicolasa, B.L. Browna, K. Fischer-Stengerb, J.K. Stewarta,* a
Department of Biology, Virginia Commonwealth University, Richmond, VA 23284, United States b Department of Biology, University of Richmond, Richmond, VA 23173, United States Received 9 July 2004; received in revised form 6 October 2004; accepted 14 October 2004
Abstract Although it is known that macrophages take up serotonin, a specific monoamine transporter has not been identified in macrophages. In this study, mRNA coding for the serotonin transporter (SERT) was detected with the reverse transcription–polymerase chain reaction (RTPCR) in recruited mouse peritoneal macrophages. Sequencing confirmed the identity of the RT-PCR product to mouse SERT mRNA. SERT protein was detected by Western blotting. Macrophage activation with lipopolysaccharide had no effect on expression of SERT mRNA or protein. Consistent with expression of a functional SERT, specific uptake of 3H-serotonin in macrophages was sodium dependent and inhibited by fluoxetine (IC50 6.9 nM) and desipramine (IC50 32 nM) but not by nisoxetine or reserpine. D 2004 Elsevier B.V. All rights reserved. Keywords: Macrophages; Serotonin transporter; Dopamine transporter
1. Introduction Serotonin (5HT) modulates macrophage functions including phagocytosis, acid phosphatase activity, production of superoxide anion and H2O2, and interactions with T cells (Jackson et al., 1985; Sternberg et al., 1987; Mo¨ssner and Lesch, 1998; Freire-Garabal et al., 2003; Kondomerkos et al., 2003a,b). Also, there is evidence that 5HT is present in macrophages (Essman, 1978) and that macrophages synthesize melatonin, which is derived from 5HT (Martins et al., 2004). Jackson et al. (1988) demonstrated saturable uptake of 5HT by peritoneal macrophages and found that 5HT transport in a mixed population of splenocytes is blocked by fluoxetine, an inhibitor of the 5HT transporter (SERT). It is unclear, however, if peritoneal macrophages express one or more of the neuronal monoamine transporters. The goal of this study was to clarify whether the SERT is expressed in peritoneal macrophages. Based on evidence for expression of the dopamine transporter (DAT) in some im-
* Corresponding author. Tel.: +1 804 828 0650; fax: +1 804 828 0503. E-mail address:
[email protected] (J.K. Stewart). 0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2004.10.013
mune cells (Gordon and Barnes, 2003), we also investigated expression of DAT mRNA in macrophages. Definitive identification of monoamine transporters in macrophages is important, because the uptake mechanism potentially limits effects of extracellular monoamines on macrophages, and specific regulators of the transporters (both endogenous and pharmacological) may modulate macrophage functions.
2. Materials and methods 2.1. Cells Female CBA/J mice (6–8 weeks of age) were obtained from Harlan (Indianapolis, IN) and housed under supervision of the Institutional Animal Care and Use Committee at Virginia Commonwealth University. Macrophages were recruited with a single intraperitoneal injection of 0.5 ml of Complete Freund’s Adjuvant diluted 1:1 in Dulbecco’s Phosphate-Buffered Saline (Sigma, St. Louis, MO). Fourteen days later, the mice were euthanized with CO2. Trunk blood was collected into heparinized tubes for preparation of platelets (see below). Peritoneal exudate cells were harvested
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in ice-cold Hanks’ Balanced Salt Solution (HBSS; Invitrogen, Carlsbad, CA), centrifuged at 500g for 10 min at 4 8C, washed twice in HBSS (centrifuged after each wash) and resuspended in complete RPMI (cRPMI) consisting of RPMI 1640 (Invitrogen) supplemented with 10% heat inactivated fetal bovine serum, 1% l-glutamine, 1% non-essential amino acids, 1% minimal essential medium vitamins, 100 units/ml penicillin and 100 Ag/ml streptomycin. Cells were plated in cRPMI in either 25-cm2 tissue culture flasks (approximately 10–30106 cells/flask) for subsequent extraction of RNA or protein or 12-well tissue culture plates at a density of 3106 cells/well for assay of 3H-5HT uptake. All cells were allowed to adhere for 2 h after plating, and non-adherent cells were discarded. Adherent cells (N99% macrophages) were washed with cRPMI and maintained in fresh medium for the times designated in each experiment. Cells used for extraction of RNA or protein were treated with vehicle (medium alone) or 50 ng/ml lipopolysaccharide (LPS; E. coli-serotype 055:B5 from Sigma) and incubated for the times indicated in the figure legends. Murine bone marrow derived mast cells obtained from Dr. John Ryan, Department of Biology, Virginia Commonwealth University, were maintained as previously described (Freeman et al., 2001) in cRPMI containing 5 ng/ml interleukin-3 and 50 ng/ml stem cell factor. The COS-1 kidney fibroblast cell line was obtained from American Type Culture Collection (ATCC # CRL-1650) and maintained at 37 8C in a 5% CO2 atmosphere in ATCC # 30-2002 DMEM containing 4 mM l-glutamine, 4.5 g/l glucose and 1.5 g/l sodium bicarbonate, and supplemented with 10% heat inactivated fetal bovine serum, 100 units/ml penicillin and 100 Ag/ml streptomycin. Mouse platelets were prepared as described by others (Lagadec et al., 2003). Whole blood mixed with heparin (20 units/ml) was centrifuged at 125g for 25 min at 25 8C. The platelet rich plasma from 11 mice was pooled and centrifuged at 18,000g for 20 min at 4 8C to yield a crude platelet pellet. 2.2. Western blotting All cells used for Western blotting, with the exception of mast cells, were lysed with 250–700 Al of ice cold buffer containing 0.05 M Tris (pH 7.5), 0.3 M NaCl, 2 mM EDTA, 0.5% (or 1.0% for platelets) Triton-X 100, 2 Ag/ml leupeptin, 1 Ag/ml aprotinin and 0.2 mM phenylmethylsulfonylfluoride. Approximately 5106 mast cells were pelleted by centrifugation (500g for 15 min at 4 8C), then lysed in 120 Al ice-cold buffer containing 100 mM Tris (pH 7.2), 150 mM NaCl, 1.5 mM MgCl2, 10% glycerol, 0.5% Triton-X and a complete protease inhibitor cocktail tablet (Roche Diagnostic, Mannheim, Germany) for 40 min on ice. The protein lysates were centrifuged at 20,000g for 20 min at 48 C. Protein concentrations of the supernatants were determined with the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA).
Proteins (50 Ag) were separated by SDS-PAGE on 10% polyacrylamide gels (Bio-Rad Laboratories), transferred by electroblotting to nitrocellulose membranes and incubated overnight at 4 8C in a blocking solution of Tris-buffered saline (TBS=10 mM Tris, 0.15 M NaCl, pH 7.4) containing 5% dry milk. Membranes were incubated for 1 h with rabbit anti-SERT polyclonal antibody (Chemicon, Temecula, CA) diluted 1:200 in TBS containing 0.1% Tween-20 and 0.2% dry milk (TBST), washed three times with TBST and incubated for 1 h with HRP-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:7500 in TBST, then membranes were washed four times with TBST. Enhanced chemiluminescence (Amersham Biosciences, UK) was used to visualize immunoreactive proteins. Optical density of bands was measured with the Eagle Eye II Image Analyzer (Stratagene, La Jolla, CA). 2.3. RNA isolation and reverse transcription–polymerase chain reaction (RT-PCR) Total RNA from approximately 20 million cells or mouse medulla/pons (positive control) was extracted with TRI-LS (Molecular Research Center Cincinnati, OH) according to procedures suggested by the manufacturers. RNA samples were stored at 70 8C for b1 month. Three micrograms of total RNA were reverse-transcribed with oligo (dT) primers and the Superscriptk Preamplification System for First Strand cDNA Synthesis (Invitrogen). Negative controls in each assay included a tube without RNA and samples without reverse transcriptase. PCR primers were designed with Primer3 (http://wwwgenome.wi.mit.edu/cgi-bin/primer3 _www.cgi) and purchased from Integrated DNA Technologies (Coralville, IA). Primer sequences and NCBI accession numbers for the mouse sequences were: SERT # AF013604—sense 5VTGTCTTGGTTCTATGGAATCACTC-3V (nucleotides 1536–1559) and anti-sense 5V-CGTTCACAGACCTAATCTAAACCT-3V (complementary to nucleotides 2178–2201); DAT # AF109072—sense 5V-CCTTCTGTATGTGGTCGTGGT-3V (nucleotides 1670–1690) and anti-sense 5V-TCTTGACGTGTTGGTTTCCTT-3V (complementary to nucleotides 2001–2021); h-actin # M12481—sense 5VAAGGTGTGATGGTGGGAATGG-3V (nucleotides 44–64) and anti-sense 5V-GGCGTGAGGGAGAGCATAG-3V (complementary to nucleotides 426–444). To distinguish genomic DNA contamination from the smaller mRNA products, primers were designed to span at least one intron. The predicted cDNA product sizes were: 665 bp (SERT), 351 bp (DAT) and 401 bp (h-actin). PCR was performed with 2–4 Al of the RT reaction products, specific forward and reverse primers (4 AM final concentration), and PCR reagents (Invitrogen) as previously described (Andreassi et al., 1998). The cDNA was amplified 38 cycles for the target sequence and 25 cycles for h-actin. In preliminary experiments, these cycle numbers were determined to give linear amplification of the targets or h-
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Fig. 1. SERT mRNA in peritoneal macrophages. Ethidium-bromide stained gels (1% agarose) are shown. DNA size markers are shown at the left (bp=base pairs). Recruited macrophages were treated with vehicle (V) or 50 ng/ml LPS and incubated for 6 h. Negative controls included a tube without RNA (no RNA) and samples without reverse transcriptase (no RT). Mouse medulla/pons (M/P) was the positive control. Three micrograms of total RNA were reverse transcribed, and 2 Al of the RT reaction were amplified 38 cycles for SERT or DAT and 25 cycles for h-actin. Expected sizes of the RT-PCR products were h-actin 401, SERT 665 and DAT 391 bp. MeanFS.E. density ratios of SERT/h-actin RT-PCR products were 0.87F0.02 in vehicle-treated macrophages and 0.81F0.02 in LPS-treated macrophages in six to eight flasks of macrophages per treatment group ( PN0.05).
actin. All cycles were 15 s, 94 8C; 15 s, 56 8C; 30 s, 72 8C. PCR products were separated by electrophoresis on 1.0% agarose gels and stained with ethidium bromide. Products were visualized and optical density was measured with the Eagle Eye II Image Analyzer (Stratagene). 2.4. Sequencing of the RT-PCR product Following visualization of the PCR product, 5 Al of each amplicon were treated with 2 Al of ExoSapk (USB, Cleveland, OH) and held for 15 min at 37 8C followed by 15 min at 80 8C. Subsequent sequencing reactions included 1 Al of the treated PCR product, 2 Al of ET-ROX terminator mix (Amersham Biosciences, Piscataway, NJ), 0.5 Al of 10 AM primer and 2.5 Al of deionized sterile water for a total reaction volume of 6 Al. The sequencing reaction was performed on the MJ Research thermal cycler for 40 cycles with the thermal profile: 20 s denaturing at 95 8C, 15 s annealing at 52 8C and 1 min extension at 60 8C. Reactions were performed for both forward and reverse primers. Sequencing reactions were cleaned with Amersham’s MontageSeqk plates and analyzed on the MegaBace1000k Sequencer (Amersham). Base scoring was conducted with Sequence Analyzerk (Amersham). Forward and reverse sequences for each PCR product were compared with pairwise BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). 2.5. Uptake of 3H-5HT The uptake assay is a modification of methods of Horschitz et al. (2003). Immediately prior to the assay, cells in 12-well plates were washed twice at room temerature with 1 ml of sucrose Hepes (SH) uptake buffer containing 140
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mM NaCl (or LiCl in selected experiments), 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 10 mM Hepes (pH 7.4, adjusted with KOH) and 320 mM sucrose. Macrophages were preincubated for 5 min in 800 Al of SH uptake buffer containing 250 AM ascorbic acid, 10 AM pargyline and vehicle or various concentrations of unlabeled 5HT (Sigma) or transport inhibitors including fluoxetine (a gift from Eli Lilly, Indianapolis, IN), desipramine (US.P.C.), nisoxetine (Tocris, Ellisville, MO) and reserpine (Regis Chemical, Moron Grove, IL). Radiolabeled 5HT (3H-5HT) (5H[G-3H]T creatine sulfate 15–18 Ci/mmol; Amersham Biosciences) was added at a final concentration of 25 nM, and incubation at room temperature was continued for 5 min (except in time course experiments). All incubations were conducted at room temperature. The uptake buffer was removed and cells were washed three times with 2 ml SH buffer. Buffer was removed and the cells were lysed with 800 Al of 0.2 N NaOH containing 1% SDS. The lysate was transferred to 10 ml of Scintisafe scintillation fluid (Fisher Scientific, Fair Lawn, New Jersey), the wells were washed with an additional 600 Al of the NaOH/SDS, and the wash was transferred to the scintillation fluid. Radioactivity was measured with a Beckman LS6000IC scintillation counter. 2.6. Data analyses An unpaired t-test was used to compare the protein or mRNA density ratios of SERT/h-actin in macrophages treated with vehicle or LPS. Analysis of variance (ANOVA) and Tukey’s multiple comparison test were used to evaluate effects of transport inhibitors or LiCl on 3H-5HT uptake. IC50 values were calculated with Prism software (Graphpad, San Diego, CA). Differences were considered statistically significant at Pb0.05.
Fig. 2. Immunoreactive SERT in peritoneal macrophages assayed by Western blotting. Total protein (50 Ag per lane) was separated by SDSPAGE on 10% polyacrylaminde gels. Molecular size standards are shown at the left. Protein samples extracted from platelets (P) or bone marrow derived mast cells (M) were positive controls. COS-1 kidney fibroblasts (C) did not express SERT protein. Recruited macrophages were treated with vehicle (V) or 50 ng/ml LPS and incubated for 24 h. MeanFS.E. density ratios of SERT/h-actin protein were 0.27F0.02 in vehicle-treated macrophages and 0.31F0.02 in LPS-treated macrophages in three to five samples of each treatment group ( PN0.05).
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3. Results 3.1. SERT mRNA in macrophages RT-PCR indicated that peritoneal macrophages express mRNA coding for the SERT but not the DAT (Fig. 1). The sequence of the SERT PCR product has been submitted to GenBank (accession # AY765218). Sequencing confirmed identity to the mouse serotonin transporter mRNA (accession #’s AF013604 and X66119.1) and differed from accession # NM_010484 by two bases. Absence of the DAT PCR product suggests that peritoneal macrophages do not express the DAT and is consistent with our observations that washed adherent macrophages have little if any contamination with nonadherent lymphocytes, which express the DAT (Gordon and Barnes, 2003). No effects of LPS on macrophage expression of SERT or DAT mRNA were evident (Fig. 1). Comparison of density
Fig. 4. (A) Effects of fluoxetine on specific uptake of 3H-5HT in recruited peritoneal macrophages. Recruited peritoneal macrophages were plated at a density of 3106 cells/well. Macrophages were preincubated for 5 min with increasing concentrations of fluoxetine. After addition of 25 nM 3H-5HT, incubation continued for 5 min. Bars represent mean values FS.E. of three to four replicates. *Pb0.01, **Pb0.001 compared to control (O) with no inhibitor. (B) Kinetics of fluoxetine inhibition of 3H-5HT uptake in recruited peritoneal macrophages. Each data point is the meanFS.E. of three to four replicates. IC50=6.9 nM.
ratios of SERT/h-actin PCR products in six two eight samples of vehicle- and LPS-treated macrophages from different mice confirmed that LPS activation did not change the quantity of SERT mRNA (Fig. 1 legend). 3.2. Immunoreactive SERT in macrophages Fig. 3. 3H-5HT uptake in recruited peritoneal macrophages. Recruited peritoneal macrophages were plated at a density of 3106 cells/well. (A) Cells were incubated with 25 nM 3H-5HT+vehicle (total uptake) or 25 nM 3 H-5HT+25 AM unlabeled 5HT (non-specific uptake). Points on the curve represent mean values FS.E. of three to four replicates. (B) Bars represent the meanFS.E. specific uptake (total uptake–non-specific uptake in the presence of 25 AM unlabeled 5HT). Macrophages were incubated for 5 min with 3H-5HT. C=control incubation with no additives, LiCl=incubation in sodium-free buffer containing LiCl. RES and RESV=macrophages preincubated for 5 min with either 1 AM reserpine or the reserpine vehicle (0.1% glacial acetic acid), then 3H-5HT was added and incubation continued for 5 min. Data are mean values FS.E. of three to four replicates. **Pb0.001 compared to control (C).
Western blotting demonstrated an immunoreactive SERT protein at approximately 70 kDa (Fig. 2). Measurement of density ratios of SERT/h-actin protein revealed no effects of LPS on the quantity of transporter protein measured 24 h after treatment (Fig. 2 legend). 3.3. Uptake of 3H-5HT in macrophages Based on our observations that LPS activation was not necessary for macrophage expression of SERT mRNA and protein, recruited macrophages with no further activation
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were used for 5HT uptake assays. Total macrophage uptake of 25 nM 3H-5HT was linear up to 20 min (Fig. 3A). Nonspecific uptake of 3H-5HT measured in the presence of 25 AM unlabeled 5HT was approximately 40% of total uptake. Specific uptake of 3H-5HT (total-non-specific) measured over 5 min was sodium dependent, as indicated by replacement of NaCl with LiCl in the uptake buffer (Fig. 3B). To verify that uptake was not due to vesicular monoamine transporters (VMAT), macrophages were incubated with 1 AM reserpine, which inhibits both isoforms of the VMAT. There were no effects of reserpine on specific uptake of 3H-5HT in macrophages (Fig. 3B). A concentration-dependent decrease in specific 3H-5HT uptake was observed after incubation with the selective SERT inhibitor fluoxetine (Fig. 4A). Fluoxetine inhibited specific 3H-5HT uptake with an IC50 of 6.9 nM (Fig. 4B). The IC50 values for desipramine, an inhibitor of both the norepinephrine transporter (NET) and the SERT, was 32 nM and the IC50 for the selective NET inhibitor nisoxetine was N10 AM (data not shown).
4. Discussion These findings provide compelling evidence for expression of a functional SERT in mouse peritoneal macrophages. Our detection of SERT mRNA and immunoreactive SERT protein and the observation of a low IC50 for the SERT inhibitor fluoxetine are consistent with expression of SERT activity. Furthermore, the IC50 of 6.9 nM for fluoxetine and 32 nM for desipramine are close to K i values reported for the human SERT (Barker and Blakely, 1998). Inhibition of 3 H-5HT uptake by replacement of extracellular NaCl with LiCl verified sodium-dependence, a characteristic of the SERT and other neuronal type monoamine transporters located in the plasma membrane. These findings do not exclude other mechanisms for monoamine transport in macrophages. Balter and Schwartz (1977) observed macrophage uptake of norepinephrine that was not decreased by metanephrine, an inhibitor of nonneuronal type-II transport mechanisms, or cocaine, an inhibitor of the SERT, DAT and NET. Additionally, they found that only a portion of the macrophage uptake of norepinephrine is specific (Balter and Schwartz, 1977), which is consistent with our observations that approximately forty percent of the total 3H-5HT uptake was not inhibited by either high concentrations of unlabeled 5HT (10 AM) or replacement of NaCl with LiCl. Although we did not observe changes in expression of SERT mRNA and protein in macrophages stimulated with LPS, it is conceivable that LPS treatment alters SERT activity. LPS-activation of macrophages stimulates production of cytokines, including interleukin-1h and tumor necrosis factor-a, and both have been shown to increase SERT activity (Ramamoorthy et al., 1995; Mo¨ssner et al., 1998). Further investigation of SERT
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function in LPS-activated macrophages is needed to clarify this issue.
Acknowledgements This study was supported by NSF grant MCB 0131419 and the Jeffress Memorial Trust (grant J-623). The authors thank Dr. John Ryan of the Department of Biology, Virginia Commonwealth University for bone marrow derived mast cells used as a positive control for the SERT protein.
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