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Cellular Immunology 248 (2007) 69–76
Expression of serotonin transporters by peripheral bloodmononuclear cells of rhesus monkeys (Macaca mulatta)
G.B. Yang a,*, C.L. Qiu a, P. Aye b, Y. Shao a, A.A. Lackner b,*
a State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention,
China-CDC, 27 Nanwei Road, Xuanwu District, Beijing 100050, PR Chinab Tulane National Primate Research Center, 18703 Three Rivers Road, Covington, LA 70433, USA
Received 22 July 2007; accepted 18 September 2007Available online 5 November 2007
Abstract
It has been well established that serotonin (5-hydroxytryptamine, 5-HT) plays a key role in neuro-endocrine-immune networks,mostly through its receptors and/or transporters. Although the presence of 5-HT receptor mRNAs in peripheral blood mononuclear cells(PBMCs) of rhesus monkeys has been reported, there is little information about serotonin transporter (SERT) expression by these cells.To examine SERT expression at the transcription and translation level, one-step RT-PCR, confocal microscopy and flow cytometry wereused to detect SERT mRNA and protein expression by rhesus monkey PBMCs. It was found that SERT mRNA could be detected byRT-PCR from all of the rhesus macaque PBMC RNA samples and the nucleotide sequence of the amplicons was identical to the pub-lished SERT mRNA sequence. Low level SERT immunoreactivity was also demonstrated on the surface of rhesus PBMCs by confocalmicroscopy. Almost all lymphocytes and most monocytes were positive for SERT by flow cytometry. In the 2 rhesus macaques examinedby multicolor flow cytometry, SERTbright cells were more than 84%, 94%, and 96% among CD20+, CD3+, and CD3+CD4+ lympho-cytes respectively. These data demonstrate expression of SERT by rhesus macaque PBMCs, and indicate that rhesus macaques would besuitable models to test the in vivo immune regulatory effects of 5-HT or drugs targeting SERT.� 2007 Elsevier Inc. All rights reserved.
Keywords: Serotonin transporter; Rhesus macaque; Peripheral blood mononuclear cells; Confocal microscopy; Flow cytometry
1. Introduction
Serotonin, also known as 5-hydroxytryptamine (5-HT),has been well established as a neurotransmitter in serotoner-gic neurons, an immunomodulator in various immunocytesand an endocrine, exocrine or paracrine messenger of entero-chromaffin (EC) cells [1–3]. It plays a variety of roles in dif-ferent pathophysiological processes, such as in theregulation of mood, sleep, sex, appetite, and in neuropsychi-atric disorders like depression, obsessive-compulsive disor-der, drug addiction, schizophrenia, parkinsonian disorders,Alzheimer’s disease, and eating disorders [4–10]. In addition
0008-8749/$ - see front matter � 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.cellimm.2007.09.001
* Corresponding authors. Fax: +86 1083157886 (G.B. Yang); fax: +19858716569 (A.A. Lackner).
E-mail addresses: [email protected] (G.B. Yang), [email protected] (A.A. Lackner).
to the nervous system, 5-HT also influences the function ofthe cardiovascular system, reproductive system, gastrointes-tinal system and the immune system [11–19]. At the molecu-lar level, the functions of 5-HT involve both 5-HT receptorsand transporters.
Serotonin transporter (SERT), a 12-transmembranedomain protein, is one member of a superfamily of Na+/Cl� dependent neurotransmitter transporters. It regulatesthe magnitude and duration of serotonin signaling via cellu-lar transport. The cDNAs encoding 5-HT transporters hasbeen isolated from humans and other animals [20–23].Unlike 5-HT receptor which is present in several types andsubtypes, only a single type of serotonin transporter has beenidentified [21]. The SERT of rhesus macaques has also beencloned along with 2 other monoamine transporters [24], andso the nucleotide sequence of SERT cDNA is known and thededuced amino acid sequence of SERT is available.
70 G.B. Yang et al. / Cellular Immunology 248 (2007) 69–76
Rhesus macaques have been used extensively in variousbiomedical researches including studies on behavior, alco-holism, infectious diseases and pharmacological effects ofcandidate drugs [4,25–30]. Although many of these biolog-ical processes involve the nervous system, endocrine systemand immune system, little work has examined the interac-tions between these 3 systems in rhesus monkeys. Existingwork about rhesus SERT have mainly focused on the ner-vous system, endocrine system, and reproductive systemetc., no reports have documented the expression of SERTby immunocytes [31–35]. We have recently shown thatthe EC cells in the gut mucosa were in physical contact withlymphocytes, and that rhesus PBMCs express mRNA formultiple 5-HT receptor types/subtypes, indicating likelyinteractions between the enteroendocrine cells and immu-nocytes in rhesus monkeys [36,37]. We hypothesized thatSERT would also be expressed by rhesus PBMCs, anddemonstrate this by RT-PCR, confocal microscopy andflow cytometry. This indicates that rhesus macaques wouldbe suitable models to test the in vivo immune regulatoryeffects of 5-HT or drugs targeting SERT, and provides abasis for using rhesus monkeys in the investigation of dis-eases related to both SERT function or disorders and theimmune system, such as the effects of alcoholism on AIDS.
2. Materials and methods
2.1. RNA extraction and RT-PCR
EDTA-anticoagulated whole blood samples wereobtained from healthy normal Chinese-origin rhesus mon-keys (Macaca mulatta) with no known infections or anyknown diseases. The blood was subjected to Ficoll-paquePLUS (Amersham Biosciences, Sweden) density gradientcentrifugation to isolate PBMCs. Total RNA was extractedfrom the PBMCs using RNeasy Mini Kit (Qiagen, Ger-many) according to the manufacture’s instruction. RNase-free DNase I (Qiagen, Germany) treatment was used duringthe RNA extraction to avoid DNA contamination.
For RT-PCR amplification of SERT mRNA, TakaraOne Step RNA PCR Kit (AMV) (Takara, Japan) was usedto assemble the reaction mixture following the manufac-ture’s instruction. Within each reaction system (25 ll total),2 ll of the extracted RNA was added as the template, with0.5 ll of each of the 2 oligonucleotides (Mm5HTTF, 5 0-GAACTCCTGGAACACTGGCAAC-3 0; Mm5HTTR, 5 0-ATGACAAATCCCGAAACGAAGC-3 0) as primers. Theprimers were designed using the published sequence ofrhesus macaque SERT (GenBank Accession number:AF285761). Primers for amplifying b-2 microglobulin (For-ward: 5 0-CCTTGAGGCTATCCAGCGTA-3 0; Reverse:5 0-GTTCACACGGCAGGCATACT-3 0) were used toamplify the house keeping gene as positive control.
After incubation at 50 �C for 30 min, 1 cycle of 94 �C4 min, 57 �C 1 min, 72 �C 1 min and 35 cycles of 94 �C30 sec, 57 �C 45 sec, 72 �C 45 sec followed by 72 �C 10 minfor final extension were performed using the thermal cycler
GeneAmp PCR system 9700 (Applied Biosystems, USA).Amplicons were run on 2% agarose gel and visualized underUV light. Images were captured using a gel imaging system(biostep GmbH, Germany).
For sequencing, PCR products were sent to a sequenc-ing service provider (Invitrogen, China) and weresequenced using ABI3100 genetic analyzer (Applied Bio-systems). The nucleotide sequences returned were alignedwith the published sequence AF285761 using Clustal X.
2.2. Confocal microscopy
Mouse monoclonal antibody against SERT (catalognumber AB-N09, clone 4A2.2, Advanced Targeting Sys-tems, USA), which can specifically bind to an extracellulardomain of human and rat SERT [38], was used to labelSERT on the surface of rhesus macaque PBMCs. To preparethe slides for observation, 10 ll of a 1:10 dilution of the pri-mary antibody was added to 100 ll of whole blood, after30 min to 1 h incubation time, red blood cells were lysedusing the red blood cell lysing buffer (BD, USA), alterna-tively, red blood cells were lysed using the lysing buffer beforelabeling with the first antibody. FITC labeled secondaryantibody was then added after 2 washing steps with PBS.After 30 min incubation in the dark at room temperatureand 2 final washes with PBS, stained cells were covered ona microscopic slide with anti-quench buffer. Samples pre-pared the same way except that no primary antibody (AB-N09) was added were used as negative control. After observ-ing under fluorescence microscope, stained cells were exam-ined under confocal microscope (Leica TCS-SP2) andimages were taken, as described previously [36]. Photoshopsoftware was used to compile the figures.
2.3. Flow cytometry
For flow cytometry, the same monoclonal antibody asused above was used to stain SERT on the surface ofPBMCs as described above. When other surface markersof major lymphocyte subsets (CD3-PerCP, CD4-APCand CD20-PE, monoclonal antibodies against these mark-ers were bought from BD biosciences, USA) were used,they were labeled after the completion of SERT staining.For each of the samples studied, 2 tubes (one of them withprimary antibody, another one without primary antibody)were prepared. Isotype matched antibodies were also usedto prepare the control tubes. All samples were analysedusing either a FACSCalibur or a FACSAria (BD Biosci-ences, USA). Listmode data files were analysed using eitherCellQuest or FACSDiva (BD Biosciences, USA).
3. Results
3.1. Expression of SERT mRNA by rhesus macaque PBMCs
All RNA samples were extracted from Chinese rhesusmacaque PBMCs and digestion with RNase free DNase
G.B. Yang et al. / Cellular Immunology 248 (2007) 69–76 71
was also employed to confirm no contamination of geno-mic DNA in the samples. As shown in Fig. 1, SERTmRNA could be detected in all the monkeys tested in thisstudy, although minor variation in band density can beobserved. Sequencing of the RT-PCR amplicons demon-strated that all nucleotides of the sequences were identicalto published rhesus serotonin transporter sequence,according to which the primers were designed [24].
3.2. SERT immunoreactivity on the surface of rhesus
macaque PBMCs
As shown in Fig. 2 (upper left confocal image), rhesusperipheral blood mononuclear cells could be immuno-labeled with a monoclonal antibody (AB-N09) fromAdvanced Targeting System (USA). Although the stainingsignal was weak by routine epifluorescence it was easily dis-cernable by confocal microscopy. To our knowledge, AB-N09 is a monoclonal antibody that can specifically bindSERT by both immunohistochemistry and flow cytometry,although it is not suggested to be used in Western blot. It isalso known that the binding epitope of AB-N09 is withinr376–388 of the 4th extracellular domain of rat SERTwhich is identical to that of both Human and rhesus maca-que SERT. The labeled cells were probably true SERTexpressing cells although the labeling signal was weak.
No knowledge was available as to how large a propor-tion of PBMCs was SERT positive in the peripheral bloodof any animals. It was surprising that so many cells werelabeled by AB-N09 under the confocal microscope. Flowcytometry was then performed to confirm the presence ofSERT immunoreactivity on rhesus monkey PBMCsbecause it is more sensitive than immunohistochemistry.As shown in Fig. 2 (upper right flow cytometry histogram),serotonin transporter positive cells are about up to 2 logsbrighter than and could be easily distinguished from sero-tonin transporter negative cells by flow cytometry. Further-more, most of the lymphocytes from the peripheral bloodof rhesus monkeys were positive for serotonin transporter.Besides, immunoreactivity was also found on the surface ofmonocytes by flow cytometry.
In addition to the fact that only surface staining meth-ods were used in this paper, optical sections of the confocalimages (lower confocal image in Fig. 2) of the stained cellsalso showed a peripheral distribution of fluorescencearound the cells when the optical sections were cut through
Fig. 1. Amplification of 5-HTT mRNA in PBMCs from 10 rhesus macaqumacaques by RT-PCR (1 of up to 4 independent experiments). The 1st laneamplicons from 10 monkeys: MmBJ7016, MmBJ7017, MmBJ7018, MmBJ70MmBJ7044. L12–L21 represent b-microglobulin amplicons from the 10 correspcontrols for 5-HTT and b-microglobulin amplification respectively.
the side surfaces (not through the upper or lower surfaces)of the cells. The observed staining with both confocalmicroscopy and flow cytometry should be on the surfaceof the labeled cells.
3.3. Surface level of SERT and frequency of SERTbright
leukocytes of rhesus macaques
As shown in Fig. 3 and Table 1, when whole blood sam-ples were immunolabeled with AB-N09, the level of surfaceSERT immunoreactivity represented by MFI (mean fluo-rescence intensity) was actually different between differenttypes of leukocytes. Granulocytes, not the focus of thisstudy, were positive for SERT compared with the no pri-mary antibody controls. However, the surface level ofSERT on the granulocytes was much lower compared tothat on the lymphocytes or monocytes from the same sam-ple. When the gate was set as shown in Fig. 3 to keep thebackground staining cells under 0.5% to quantify cells(SERTbright) more than 1 log left to most negative cells inthe histogram, most granulocytes were SERTmedium (withMFI between the negative and the SERTbright cells), whilemost monocytes and lymphocytes were SERTbright in thenormal rhesus macaques examined. It was also found thatmore lymphocytes were SERTbright than monocytes, andthe percentage of SERTbright cells among CD3+CD4+lymphocytes were the highest (more than 96%) among allthe lymphocyte subsets (CD20+, CD3+CD4�, andCD3+CD4+) examined in this study (Table 1). Only about3% of the granulocytes were SERTbirght cells.
4. Discussion
Serotonin is a very important molecule mediating inter-actions between the immune, endocrine and nervous sys-tems which together make up Neuro-Endocrine-Immune(NEI) networks. The NEI networks may be involved invarious pathological processes in humans [39]. We havepreviously demonstrated the expression of mRNA for mul-tiple 5-HT receptor types or subtypes by rhesus monkeyPBMCs [37]. In this study, we have expanded our knowl-edge and demonstrated the presence of SERT mRNA inrhesus monkey PBMCs by RT-PCR and sequencing. AB-N09 is a monoclonal antibody specific to human and ratSERT [38]. Using this antibody as a primary antibody,we have observed immunoreactivity to AB-N09 on rhesus
es. Showing reproducible amplification of 5-HTT in 10 Chinese rhesuson the left is the molecular weight marker. L01 to L10 represent 5-HTT19, MmBJ7020, MmBJ7030, MmBJ7031, MmBJ7035, MmBJ7037, andonding monkeys used for 5-HTT amplification. L11 and L22 are negative
Fig. 2. SERT immunoreactive positive PBMCs of rhesus macaques. Upper left confocal image: The upper left inset shows a differential interferencecontrast (DIC) image of PBMCs; the lower left image shows AB-N09 labeling (green fluorescence) on the PBMCs, the image to the right of these 2 insets isan overlay image showing most of the PBMCs in the field were positive for AB-N09 staining. The 20 lm scale is at the lower right corner in the rightimage. Upper right histogram of flow cytometry: MFI of AB-N09 stained SERT immunoreactive lymphocytes (the right histogram) were about 100 timeshigher than that of the control without primary antibody (the left histogram). Lower confocal image: Showing surface distribution of the fluorescence.Two optical sections of 3 SERT immunoreactive cells were shown. The fluorescence is mostly distributed at the periphery of the optical sections of eachcell, except for one section of the upper left cell in the first optical section, which is probably a section cutting through the cell surface. If the 2 sectionsoverlay together, the surface staining of the upper left cell would appear intracellular staining.
72 G.B. Yang et al. / Cellular Immunology 248 (2007) 69–76
monkey PBMCs by both confocal microscopy and flowcytometry (Figs. 2 and 3). We have also tried this antibodyon frozen gut mucosal tissue sections of rhesus monkeysand found that rhesus monkey colon mucosal epithelialcells are positive for AB-N09, and that some cells are dou-ble positive for CD3 and AB-N09 (data not shown). Sincethere is no difference in the amino acid sequence of theextracellular domain of SERT that bind to antibody AB-N09 between the rhesus macaque and the Human [24], itis possible that AB-N09 cross reacts with rhesus monkeySERT and that rhesus monkey PBMCs express SERT pro-teins on their surface. To our knowledge, this is the firstreport that described surface SERT on different lympho-cyte subsets. The presence of both 5-HT receptors andSERT in rhesus monkey PBMCs suggests that these ani-mals may provide an important model to assess thein vivo role of 5-HT in NEI and various disease statesinvolved in both the serotonergic and the immune system.
Most studies on SERT expression were focused on neu-rons and platelets etc. Although expression of SERT by
lymphocytes has been documented before, yet most ofthe studies focused on expression of this transporter byhuman peripheral blood lymphocytes [40–45] and only afew studies focused on lymphocytes of other animals, suchas fish [46]. It seems that expression of SERT by lympho-cytes is a conserved feature among animals and the conser-vation of SERT expression in divergent species maysuggest an important role for serotonin in lymphocyte biol-ogy and host immunology. In support of this pharmacolog-ical studies have shown that 5-HT can modulate thefunction of lymphocytes through 5-HT transporters [47–49]. This would suggest that some aspects of immune func-tion may be under the influence of 5-HT and could be mod-ulated by drugs targeting SERT. The fact that PBMCsexpress SERT may facilitate the analysis of SERT expres-sion in vivo in some disease states because PBMCs are farmore accessible than other SERT expressing cells in thebody, such as SERT expressing neurons. One might getan insight into SERT expression in the brain throughexamination of SERT expression by PBMCs [41,42]. How-
G.B. Yang et al. / Cellular Immunology 248 (2007) 69–76 73
ever, the correlation between changes in SERT expressionin PBMCs and in other SERT expressing cells is probablynecessary to be established before such analyses.
Up to now, various effects of 5-HT on almost all kindsof immunocytes, such as T and B lymphocytes, NK cells,monocytes, macrophages and dendritic cells have beendocumented [11,12,50,51]. These interactions might bemediated through 5-HT receptors or SERT or both. Thismay complicate the analysis of the mechanisms of 5-HTeffects on these cells. Knowledge about the differentialexpression of 5-HT receptors and transporters by differenttypes of immunocytes may promote such analysis andshould be obtained in future studies. There are also datashowing that the immune system have effects on the sero-tonergic system via serotonin transporters [52]. Since thewell known role of SERT is the uptake of 5-HT. Theexpression of SERT by lymphocytes may also indicatethat these cells can uptake 5-HT and play a role in 5-HT distribution in the serotonergic system of the bodyvia their migration [53]. Enterochromaffin cells in the
Fig. 3. Flow cytometric characterization of SERT+ white blood cells. Showmonocytes, lymphocytes and their major subsets (CD3+, CD4+, and CD20+).almost no cells were in the SERT+ gates in the histograms means that as expantibody AB-N09 was used (indirect labeling), most cells were in the SERT+(except for granulocytes) were highly immunoreactive to SERT when stained
gut mucosal are the largest source of 5-HT in the body,and lymphocytes can be found in close proximity or con-tact with these 5-HT producing cells [36]. It is possiblethat lymphocytes and even other immunocytes mayuptake 5-HT released by the EC cells and then transportit to other sites of the body when they migrate to othertissues or organs. This might be another mechanism thatthe immunocytes used to affect the serotonergic system.However, further in vivo studies are needed to investigatethis possibility.
Rhesus macaques have been used as the primary animalmodel for studying HIV/AIDS, which is characterized bysignificant depletion of CD4+ lymphocytes of the immunesystem. It has also been demonstrated that the level of 5-HT and its metabolites were affected in the peripheralblood and CSF of HIV/AIDS patients [54–60]. Thesechanges may be associated with the disorders in neurophys-iology, gastrointestinal physiology and reproductive physi-ology etc observed in patients living with HIV/AIDS.However, the relationship between the changes in the
ing gating strategies for analysis of SERT+ cells among granulocytes,(a) Without primary antibody AB-N09 (negative omit primary antibody),
ected there was no cells positive when AB-N09 was not used; (b) Primarygates (except for SERT+Gran) in the histograms means that most cells
with AB-N09.
Fig. 3 (continued)
Table 1Frequency and MFI of SERT immunoreactive cells among each set of leukocytes from rhesus peripheral blood
Monocyte Granulocyte Lymphocyte CD20+ CD3+CD4+ CD3+CD4� CD3�CD4�% MFI % MFI % MFI % MFI % MFI % MFI % MFI
Mm01 69.9 13842 3.0 15923 92.0 13901 87.4 12821 97.0 14492 95.2 14232 88.9 13143Mm02 74.0 14447 4.7 12534 89.8 13157 84.8 12567 96.1 13395 94.6 13492 86.0 12729Mean 72.0 14145 3.9 14229 90.9 13529 86.1 12694 96.6 13944 94.9 13862 87.5 12936
74 G.B. Yang et al. / Cellular Immunology 248 (2007) 69–76
immune system and the serotonergic system in HIV/AIDSis unknown. In this study, we found most CD4+ T lym-phocytes were SERTbright. Loss of these cells in HIV/AIDSmight affect the serotonergic system in these patients. In apreliminary study we found that the frequency and MFI ofSERTbright cells in simian immunodeficiency virus (SIV)infected macaques were higher than that in normal maca-ques (data not shown), which might be related to the lowerblood serotonin level and/or the loss of CD4+ T lympho-cytes or both. Since SIV/SHIV infection of rhesus maca-ques is the primary animal model for AIDS relatedstudies such as AIDS pathogenesis, tests of drug and can-didate AIDS vaccines; it will provide a suitable animal
model for studying the abnormalities of serotonergic sys-tem in HIV/AIDS, the treatment of such abnormalitiesand the immune effects of potential treatments.
In summary, like their counterpart in Humans,PBMCs of rhesus macaques express both 5-HT recep-tors and transporters, the molecular basis for interac-tions between serotonin and lymphocytes. This indicatethat rhesus macaques may be used to establish suitableanimal models to study the in vivo immunoregulatoryeffects of serotonin and to investigate human diseaseslike depression in HIV/AIDS, which may involve path-ological changes in both the immune system and theserotonergic system.
G.B. Yang et al. / Cellular Immunology 248 (2007) 69–76 75
Acknowledgments
Some of the work were performed in TNPRC and re-ceived help from the flow cytometry and confocal micros-copy services. Part of the work was supported by MOSTof China 973 Project 2005CB522903 and by NNSF ofChina Grant 30571750 to G.B.Y.
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