CS1 (SLAMF7) inhibits production of proinflammatory cytokines by activated monocytes (PDF Download Available)
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15Baylor Health Care System8.28University of North Texas HSC at Fort Worth20.68University of North Texas HSC at Fort Worth36.12University of North Texas HSC at Fort WorthAbstractObjective and design:
CS1 (CRACC, CD319, SLAMF7) is a member of the Signaling Lymphocyte Activation Molecule family expressed on immune cells mediating host defense. CS1 is a self-ligand and has both activating and inhibitory functions in Natural Killer cells. However, the function of CS1 in human monocytes is currently unknown. The objective of this study was to evaluate the control of CS1 surface expression in activated monocytes and to assess the effect of CS1 triggering on proinflammatory cytokine production by monocytes.
Material, methods and treatment:
Human monocytes were isolated from PBMC of healthy volunteers by magnetic depletion method or FACS sorting. The monocytes were cultured with or without LPS (1 μg/ml) in the presence or absence of various pharmacological inhibitors to inhibit NF-кB and PI3K signaling pathways. The cells were stimulated with anti-CS1 antibody or isotype control. Total RNA was extracted and RT-PCR was performed using specific primers for CS1 and EAT-2. Cell supernatants were collected and cytokine levels (TNF-α and IL-12p70) were determined by sandwich ELISA.
Our study revealed that adherent or LPS-activated monocytes express CS1, and CS1 induction is via NF-кB and PI3K pathways. Importantly, cross-linking CS1 resulted in reduced production of proinflammatory cytokines TNF-α and IL-12p70 by LPS-activated monocytes.
Conclusions:
Our study demonstrated that CS1 plays an inhibitory role in human monocytes to control proinflammatory immune responses.Discover the world's research14+ million members100+ million publications700k+ research projectsFigures
ORIGINAL RESEARCH PAPERCS1 (SLAMF7) inhibits production of proinflammatory cytokinesby activated monocytesJong R. Kim oNathan C. Horton oStephen O. Mathew oPorunelloor A. MathewReceived: 14 March 2013 / Accepted: 3 May 2013 / Published online: 22 May 2013?Springer Basel 2013AbstractObjective and design CS1 (CRACC, CD319, SLAMF7)is a member of the Signaling Lymphocyte ActivationMolecule family expressed on immune cells mediating hostdefense. CS1 is a self-ligand and has both activating andinhibitory functions in Natural Killer cells. However, thefunction of CS1 in human monocytes is currentlyunknown. The objective of this study was to evaluate thecontrol of CS1 surface expression in activated monocytesand to assess the effect of CS1 triggering on proinflam-matory cytokine production by monocytes.Material, methods and treatment Human monocytes wereisolated from PBMC of healthy volunteers by magneticdepletion method or FACS sorting. The monocytes werecultured with or without LPS (1 lg/ml) in the presence orabsence of various pharmacological inhibitors to inhibitNF-rB and PI3K signaling pathways. The cells werestimulated with anti-CS1 antibody or isotype control. TotalRNA was extracted and RT-PCR was performed usingspecific primers for CS1 and EAT-2. Cell supernatantswere collected and cytokine levels (TNF-aand IL-12p70)were determined by sandwich ELISA.Results Our study revealed that adherent or LPS-acti-vated monocytes express CS1, and CS1 induction is viaNF-rB and PI3K pathways. Importantly, cross-linking CS1resulted in reduced production of proinflammatory cyto-kines TNF-aand IL-12p70 by LPS-activated monocytes.Conclusions Our study demonstrated that CS1 plays aninhibitory role in human monocytes to control proinflam-matory immune responses.Keywords CS1 ?Monocytes ?Cytokines ?SLAM familyIntroductionMonocytes, a subset of circulating white blood cells, arecontinuously generated from monoblasts in the bone mar-row [1,2]. In steady state, monocytes circulate through thebloodstream for approximately 1–3 days, and reach theperipheral tissues for the supply of macrophages and den-dritic cells [3]. As a major component of innate immunecells, monocytes can detect various immunological threatsmainly by expressing multiple toll-like receptors, includingTLR-1, TLR-2, TLR-4, TLR-5, TLR-6, TLR-7, and TLR-8[4–7]. After recognizing these danger signals, monocytesrespond by producing pro-inflammatory cytokines, such asTNF-a, IL-1, IL-6, IL-8, IL-12, IL-15, and IL-18 [8,9], andanti-inflammatory cytokines, such as IL-10 and TGF-b[10,11]. Therefore, monocytes need to fine-tune the delicatebalance between pro- and anti-inflammatory immuneresponses. This can be achieved by regulating the expres-sion of activating or inhibitory receptors. For example,FccRIIa acts as an activating receptor, whereas FccRIIbacts as an inhibitory receptor [12–15].Signaling Lymphocyte Activation Molecules (SLAM)family receptors are expressed on hematopoietic cells, butnot on non-hematopoietic cells, and play an important rolein immune regulation [16,17]. They consist of six corereceptors: SLAM (SLAMF1, CD150), CD229 (SLAMF3,Responsible Editor: John Di Battista.J. R. Kim ?N. C. Horton ?S. O. Mathew ?P. A. Mathew (&)Department of Molecular Biology and Immunology and Institutefor Cancer Research, University of North Texas Health ScienceCenter at Fort Worth, 3500 Camp Bowie Blvd.,Fort Worth, TX , USAe-mail: porunelloor.mathew@unthsc.eduInflamm. Res. (–772DOI 10.-013-0632-1 Inflammation Research123
Ly-9), 2B4 (SLAMF4, CD244), CD84 (SLAMF5), NTB-A(SLAMF6; Ly108 in mouse), and CS1 (SLAMF7, CD319,CRACC) [18]. SLAM family members are characterizedby an extracellular domain containing two or four immu-noglobulin-like domains. The cytoplasmic tails of thesereceptors contain immunoreceptor tyrosine-based switchmotifs (ITSM) which associate with SLAM-associatedprotein (SAP) and/or Ewing’s sarcoma-associated tran-script 2 (EAT-2) adaptor proteins. SAP was first identifieddue to its mutation in patients with X-linked lymphopro-liferative syndrome, distinguished by a fatal response toEpstein–Barr virus infection [19–21]. SLAM familyreceptors interact with SAP or EAT-2 with varying affin-ities. Engagement of SLAM family receptors can be eitheractivating or inhibitory depending on the availability ofadaptor proteins [22,23].Human CS1 is expressed on NK cells, a subset of CD8?T lymphocytes, mature dendritic cells, and activated Bcells [24–27]. CS1 is a self-ligand, like most of the SLAMfamily members, exhibiting homophilic interactions [28].However, CS1 is unique amongst the SLAM familymembers as it does not signal through SAP but only EAT-2[25,29]. In NK cells, CS1 acts to activate the PI3K andphospholipase Ccsignaling pathways, resulting in activa-tion of NK cell function [30]. However, recent workutilizing CS1 knockout mice reveals that CS1 can play bothactivating and inhibitory functions in NK cells, with theactivating signals conveyed dependent on EAT-2 expres-sion [23]. We have previously reported that CS1 inducesproliferation and production of autocrine cytokines inhuman B lymphocytes [31]. Furthermore, recent clinicaltrials have focused on the use of a CS1 monoclonal anti-body in treatment for multiple myeloma (MM), a type ofhuman plasma cell tumor over-expressing CS1. Treatmentwith monoclonal anti-CS1 antibody inhibits myeloma celladhesion to bone marrow stromal cells and induces anti-body-dependent cellular cytotoxicity [32–34]. Ourprevious studies on human systemic lupus erythematosus(SLE) patients demonstrated an association of CS1 high-expressing plasmablasts with active disease [35]. Thus,CS1 has a wide distribution as well as varying functionsacross immune
however, no study hasyet to investigate expression or function of CS1 in humanmonocytes.While strongly toxic against bacteria, monocytes canalso elicit tissue destruction, resulting in inflammatorydiseases. Thus, understanding the regulation of monocyteresponses is vital in further comprehending monocytefunction in immune responses and inflammatory diseases.In this study, we investigated the expression and functionof CS1 in human monocytes. We found that LPS-activatedmonocytes express CS1, via induction of the NF-rB andPI3K pathways. CS1 engagement results in reducedsecretion of proinflammatory cytokines by LPS-activatedmonocytes that do not express EAT-2. This parallels neg-ative regulation by CS1 of NK and T cells lacking EAT-2.Materials and methodsHuman peripheral blood mononuclear cell isolationHuman venous blood was collected from healthy volun-teers from the University of North Texas Health ScienceCenter (UNT-HSC) with prior approval from the InternalReview Board of UNT-HSC. Written informed consentswere obtained from each healthy individual. Blood wascollected into tubes containing ethylene-diamine-tetra-acetic acid (EDTA), and human peripheral blood mono-nuclear cells (PBMCs) were prepared by density gradientcentrifugation on Histopaque-1077 (Sigma Chemicals, St.Louis, MO, USA). Remaining red blood cells were lysedby adding ACK lysis buffer.Human monocyte isolationHuman monocytes were isolated by magnetic depletionmethod using monocyte isolation kit (Miltenyi Biotec, CA,USA) or positive selection using FACS sorting. Briefly,total PBMCs were incubated with human FcR blockingreagent, biotin-conjugated cocktail antibodies against CD3,CD7, CD16, CD19, CD56, CD123, and CD235a, and anti-biotin Microbeads. After applying the cell suspension to amagnetic column, the negative fraction was collected andthe cell number was determined. Alternatively, totalPBMCs were stained with anti-CD14-antibody (cloneM5E2, BD Biosciences, San Jose, CA, USA) and sorted byCytopeia InFlux Cell Sorter in the Flow Cytometry andLaser Capture Microdissection Core Facility at UNT-HSC.Above 90 % were CD14-positive after both purificationmethods.Immunofluorescence microscopyMagnetically enriched monocytes were added in polysty-rene chamber slide pre-coated with 0.01 % poly-L-lysine(SigmaAldrich, St. Louis, MO, USA) and were cultured for30 min at 37 °CinaCO2incubator. Then, cells werestained with CD14-FITC, CS1-PE (clone 235614; R&DSystems, Minneapolis, MN, USA), and DAPI (Vectashieldwith D Vector Labs, Burlingame, CA, USA). Cellswere analyzed on an Olympus AX70 Fluorescent Micro-scope (Flow Cytometry and Laser Capture MicrodissectionCore Facility at UNT-HSC).766 J. R. Kim et al.123
Human monocyte cultureMagnetically enriched monocytes or FACS-sorted mono-cytes were cultured in RPMI/10 % FCS with or withoutLPS (SigmaAldrich). Then 1 ml of monocytes (1 9106cells/ml) was cultured in a 5-ml polypropylene round-bottom tube in the presence or absence of LPS (1 lg/ml)for the indicated time periods. By avoiding normal tissueculture dishes, adhesion of monocytes to the surface ofculture dish was minimized. For pharmacological inhibitortreatments, the sorted monocytes were pre-treated withindicated inhibitors for 30 min at 37 °C, then LPS wasadded to the culture. Inhibitors and concentrationsemployed were 10 lM SB lM PD98059,25 lM LY294002, and 1 lM Bisindolylmaleimide I. Allinhibitors were purchase from Calbiochem, (La Jolla, CA,USA) and dissolved in DMSO. For co-engagementexperiments, 100 ul of monocytes (1 9106cells/ml) werecultured in the 96-cell culture plate with 162.1 anti-CS1agonist antibody or isotype control antibody at the con-centration of 10 lg/ml. Then, goat anti-mouse IgGsecondary antibody and LPS were added to the culture.THP-1 cell cultureTHP1 cells were maintained in RPMI 1640 supplementedwith 10 % FBS (Gembio, West Sacramento, CA, USA),2 mM glutamine, 100 U/mol penicillin, 100 U/mol strep-tomycin, 10 mM HEPES, and 10 mM non essential aminoacids at 37 °C in a humidified 5 % CO2/95 % air envi-ronment. For LPS-mediated THP-1 activation, lowerconcentrations of cells (5 x 105cells/ml) were used forCS1 induction study. THP-1 cells were cultured in thepresence or absence of LPS for 18 h.Flow cytometryAfter LPS-mediated activation of monocytes or THP-1cells, cells were centrifuged and washed with 19PBS.Activated monocytes were stained with CD14-FITC, CS1-PE, and 7-AAD. After washing, samples were run in theCytomics FC500 (Beckman Coulter, CA, USA).Reverse transcriptase polymerase chain reaction(RT-PCR) analysisAfter culturing with LPS, activated monocytes were lysedin 200 ll of RNAstat-60. Total RNA was extracted, and1lg of RNA was reverse transcribed using Superscript IIRT (Invitrogen Life Technologies, CA, USA) in the pres-ence of a primer mixture of Random hexamer and oligo dT.After RT reaction, the concentration of cDNA was mea-sured and the cDNA was diluted with 19RT buffer togenerate 100 ng/ll of cDNA template. The primersequen CS1 primers (forward, 50-CCTCCC ATG GTC CTC CTG TG-30; reverse, 50-GAG ACTTAG GGG AGT GCA CTG CTG-30), SAP primers (for-ward, 50-GAC GCA GTG GCT GTG TAT-30; reverse, 50-TCA TGG GGC TTT CAT TTC AGG CAG AC-30), andEAT-2 primers (forward, 50-ATG GAT CTG CCT TACTAC CAT G-30; reverse, 50-TCA AGG CAA GAC ATCCAC ATA ATC-30). Each mRNA level was normalized tothe expression of GAPDH (human GAPDH primers: for-ward, 50-ATGACATCAAGAAGGTGGTG-30; reverse, 50-CATACCAGGAAATGAGCTTG-30).Sandwich ELISA assay for TNF-aand IL-12p70After culture, supernatants were collected and cytokinelevels were determined using Opt EIA cytokine ELISA kits(BD Biosciences, CA, USA). Briefly, 96-well ELISAplates were precoated with capture antibody overnight at4°C. After 1 h blocking, 100 ll of undiluted sample ortenfold diluted samples together with serially dilutedcytokine standards were added. After 2 h incubation atroom temperature (RT) and several washings, biotin-con-jugated detection antibodies plus HRP-conjugatedstreptavidin were added. After extensive washing, thesubstrate solution was added to each well and incubated for30 min at RT in the dark. After adding 50 ll of stopsolution, the optical density at 450 nm was 570 nm was used as a correction wavelength.Statistical analysisData were analyzed by Mann–Whitney Utest. A pvalue ofless than 0.05 was considered as significant.ResultsCS1 is expressed in activated monocytesAdherence to glass or plastic is a well-known characteristicof human monocytes, and has been used as an easy isola-tion method of human monocyte populations [36].However, surface adherence of monocytes leads to acti-vation and differentiation of monocytes [37]. It wasoriginally reported that human monocytes do not expressCS1, while monocyte-derived dendritic cells do express it[25]. To investigate CS1 expression in adherent monocytes,CD14?monocytes were purified and cultured for 30 minat 37 °C. Cells were then stained with CD14-FITC, CS1-PE, and DAPI and imaged. Figure 1a shows that some ofCD14-positive monocytes do express CS1. Interestingly,the surface expression of CS1 is restricted to adherentActivated Monocytes 767123
monocytes that have large and flat morphology, but not toloosely adherent monocytes that are small and circular inform. Next, human monocytes were isolated by negativedepletion from PBMCs obtained from blood drawn fromfive healthy volunteers. Monocytes were cultured with LPSfor 18 h in a polypropylene tube to minimize surfaceadhesion. RT-PCR analysis found that CS1 mRNA is notexpressed in freshly isolated monocytes but expressed inLPS-activated monocytes (Fig. 1b). Next, flow cytometryanalysis was performed using freshly isolated monocytesand LPS-activated monocytes. Consistent with RT-PCRdata, freshly isolated monocytes did not express CS1 on thesurface, whereas LPS-activated monocytes clearly did so(Fig. 1c). This LPS-induced CS1 expression was alsoobserved in THP-1, a human monocytic cell line (data notshown). Next, we determined whether CS1 can be inducedafter other TLR ligation. Human monocytes are known toexpress TLR-2, TLR-4, TLR-5, and TLR-7/8. As seen inFig. 1d, CS1 was induced by all TLR ligands tested. Col-lectively, our results obtained by RT-PCR, flow cytometry,and immunofluorescence microscopy clearly indicate thathuman activated monocytes, such as adherent monocytesor LPS-activated monocytes, express CS1.Induction of CS1 surface expression is dependenton the NF-rB pathway and involves Src family,PI3, and p38 MAP kinasesAfter confirming CS1 expression in activated monocytes,we next sought to identify the transcription factor that isABCRelative cell countLog fluorescence intensityDLPS Pam3CSK4 Flagellin R848NoneLog fluorescence intensityRelative cell countFig. 1 Induction of CS1expression in activated humanmonocytes. aEnrichedmonocytes were cultured for30 min at 37 °C. Cells werestained with CD14-FITC, CS1-PE, and DAPI. CS1 is expressedin adherent monocytes whichhave a large, flat morphology(arrowhead) but not expressedin semi-adherent monocyteswhich are small and circular inmorphology (arrow). Onerepresentative data arerepresented out of twoindependent experiments.bHuman monocytes wereisolated from five healthyindividuals by magneticnegative depletion methods.Fresh monocytes were directlyanalyzed or cultured in thepresence of LPS for 18 h. CS1mRNA expression was analyzedby RT-PCR. GAPDH was usedas a housekeeping control gene.cFreshly sorted monocytes orLPS-activated monocytes (18 h)were analyzed for the surfaceexpression of CS1 by flowcytometry. dSorted monocyteswere cultured in the presence orabsence of LPS (TLR-4 ligand),Pam3CSK4 (TLR-2 ligand),Flagellin (TLR-5 ligand), andR848 (TLR-7/8 ligand) for 18 h,and CS1 induction was analyzedby flow cytometry768 J. R. Kim et al.123
important for the induction of CS1 expression. Bothmonocyte adherence and TLR pathways are known toactivate NF-rB, a transcription factor critical for monocytefunction. When purified monocytes were pre-treated for30 min with pyrrolidine dithiocarbamate (PDTC), aninhibitor of NF-rB, surface expression of CS1 was almostcompletely inhibited (Fig. 2a). We next sought to identifymolecules involved in the signaling cascade leading to CS1surface expression. Human monocytes were pre-treated for30 min with SB203580, LY294002, Bisindolylmaleimide,or PD98059, inhibitors of p38 mitogen-associated proteinkinase (MAPK), phosphatidylinositol 3 kinase (PI3K),phosphokinase C (PKC), and MAPK/ERK kinase 1(MEK1), respectively. Cells were then cultured in thepresence or absence of LPS for 18 h and CS1 surfaceexpression was determined. As shown in Fig. 2b, CS1induction in LPS-activated monocytes was reduced by pre-treatment with LY294002, indicating that the PI3K path-way is involved in LPS-mediated CS1 induction. Again,these results were confirmed with THP-1 cells (data notshown).Cross-linking of CS1 down modulates productionof proinflammatory cytokines by LPS activatedmonocytesMonocytes play an important role in host defense and arecapable of producing a wide variety of cytokines [3]. Sincemonocytes are known to play an important role in theinduction of inflammation, which can both facilitateclearance of infection but also exacerbate disease, under-standing the regulation of monocyte responses is extremelyimportant. Thus, we investigated how CS1 engagementalters the function of monocytes. Since previous reportshave demonstrated that CS1 function is dependent on EAT-2 expression, we first evaluated levels of mRNA for EAT-2in LPS-activated monocytes by RT-PCR. As seen inFig. 3a, two out of five donors expressed EAT-2 inmonocytes with a detectable level. In contrast, after LPStreatment, EAT-2 expression was no longer detectable inany of the five donors by RT-PCR. In NK and T cells, CS1plays an inhibitory role when EAT-2 is not expressed [21].In order to discover whether CS1 acts as an inhibitoryreceptor, we investigated the production of proinflamma-tory cytokines when monocytes were coengaged with LPSand anti-CS1 agonist monoclonal antibody. CD14?sortedhuman monocytes were cultured for 18 h without LPS,with LPS plus control antibody, or with LPS plus an anti-CS1 agonist antibody. In Fig. 3b, levels of TNF-aand IL-12p70 in culture supernatants were measured by sandwichELISA. When CS1 was engaged on the surface of LPS-activated monocytes, TNF-aproduction was significantlyreduced compared to the control antibody. Likewise, levelsof IL-12p70 were also significantly reduced when com-pared to the control antibody with LPS and monocytescultured without LPS. Interestingly, CS1 coengament withANone LPS LPS+PDTCCS1-PE CS1-PE CS1-PELog fluorescence intensityRelative cell countBLog fluorescence intensityRelative cell count010010110 210 3010010110 210 3010010110 210 3010010110 210 3010010110 210 3LPS LPS+LYLPS+SB LPS+Bis LPS+PDFig. 2 Induction of CS1 surface expression on LPS-activated mono-cytes is dependent on the NF-rB and involves the PI3K pathway.aSorted monocytes were pre-treated with or without pyrrolidinedithiocarbamate (PDTC), an NF-rB inhibitor, and activated with LPSfor 18 h. Then, the cells were analyzed for CS1 surface expression byflow cytometry. One representative data out of five independentexperiments are shown. bSorted monocytes were precultured for30 min with indicated inhibitors. Cells were then cultured in thepresence of LPS for 18 h and CS1 surface expression was analyzed byflow cytometry. SB (SB203580, p38 MAPK inhibitor), LY(LY294002, PI3K inhibitor), Bis (Bisindolylmaleimide I, PKCinhibitor), or PD (PD98059, MEK1 inhibitor) were used in thisstudy. One representative data out of three independent experimentsare shownActivated Monocytes 769123
LPS also resulted in reduced production of IL-10 in freshmonocytes, whereas it showed a very minimal down-modulating effect on IL-10 production in already activatedmonocytes (data not shown). This kind of bimodal inhibi-tory effect on IL-10 production was only observed in CS1,but not in other SLAM family receptors, including SLAM.Thus, CS1 negatively regulates activated monocytes, mostlikely due to lack of EAT-2 expression.DiscussionMonocytes represent an important population of whiteblood cells that circulate throughout the body via thebloodstream and enter tissue at sites of infection. Recentadvances in depleting neutrophils but not monocytes inmice emphasize the important role of monocytes in pro-tective immune responses against several pathogens [38–41]. While playing a protective role against foreigninvaders, monocytes can also play a detrimental role ininflammatory diseases and contribute to tissue destructionin chronic inflammation. Therefore, the function ofmonocytes needs to be tightly regulated. In NK cells, it iswell known that delicate balances between activating andinhibitory function are maintained by upregulating ordownregulating surface receptors. As innate immune cells,monocyte might also share similar mechanisms with NKcells to maintain those balances.Acting as self-ligands, SLAM family receptors enablehomotypic cell-to-cell interactions, resulting in SAP- orEAT-2-dependent signal transductions. Moreover, accu-mulating evidence supports the idea that SLAM familyreceptors can function as cell adhesion receptors [42–45].In human monocytes, 2B4 and CD84 are known to behighly expressed on the surface [46,47]. However, in theLPS-activated monocytes, the surface expressions of CD84and 2B4 are markedly downregulated [48]. Also, Farinaet al. [49]. reported that SLAM is a unique marker forTLR-activated monocytes. They found that SLAMexpression is transiently upregulated and is p38 MAPK-dependent, but is independent of NF-rB. These dynamicregulations of SLAM family receptors in LPS-activatedmonocytes suggest that SLAM family receptors may playan important role in monocyte functions.CS1 is a typical receptor of the SLAM family, exhibit-ing homophilic recognition and expression on a variety ofimmune cell types, yet unique by exclusively interactingwith only EAT-2. Previous studies on CS1 in our labora-tory and others have determined its function in NK, T, andB cells [24,25,31,50]. Moreover, HuLuc63, a humanizedFig. 3 LPS-activated monocytes do not express EAT-2 and cross-linking of CS1 down-modulates the production of proinflammatorycytokines by LPS-activated monocytes. aHuman monocytes wereisolated from five healthy individuals by magnetic negative depletionmethods. Monocytes were directly analyzed or cultured in thepresence of LPS for 18 h. EAT-2 mRNA expression were analyzed byRT-PCR with the housekeeping gene GAPDH. bSorted CD14?human monocytes were cultured for 18 h with or without LPS. Cellswere stimulated with anti-CS1 agonist antibody to cross-link CS1 orwith isotype control antibody. The production of TNF-aand IL-12p70 were measured by sandwich ELISA. Accumulated data of fiveindependent experiments are depicted in the histograms.Asteriskindicates significant differences between the groups770 J. R. Kim et al.123
anti-CS1 monoclonal antibody, is currently in phase IIIclinical trials for treatment of MM [32–34]. Thus, CS1 is areceptor of particular importance in immune cells due to itsfunctional control of several cell types and applications innovel immunotherapies.To date, CS1 expression in activated monocytes hasonly received brief mention, and has yet to be completelystudied [32]. Therefore, the purpose of our study was toconfirm the expression of CS1 on monocytes and toinvestigate the regulation of CS1 induction and the effectsof CS1 ligation on the function of monocytes. We firstdetermined the conditions in which CS1 expression wasinduced in monocytes. CS1 mRNA and protein expressionwas induced upon monocyte activation, either by adher-ence to tissue culture dishes or by TLR-ligation. This wasconfirmed in Fig. 1a–d by RT-PCR, flow cytometry anal-ysis, and immunofluorescence microscopy of activatedmonocytes. These results add CS1 to SLAM familyreceptors that are dynamically regulated in TLR-activatedmonocytes, including 2B4, CD84, and SLAM.We also determined that CS1 surface expression isdependent on the signaling events of the PI3K and NF-rBpathways (Fig. 2). This was interesting because SLAM isknown to be induced by p38 MAPK-dependent but NF-rB-independent pathways in LPS-activated monocytes.Finally, we examined the expression of EAT-2 adaptormolecules by RT-PCR in activated monocytes to predictthe function of CS1. In fact, we used exactly the samedonors for CS1 and EAT-2 mRNA amplification inFigs. 1b and 3a. Interestingly, we clearly observed thatEAT-2 is downregulated or non-expressed in LPS-acti-vated monocytes. In resting NK cells, 2B4 interacts withEAT-2, but, in activated NK cells, 2B4 interacts with SAP[51]. Conversely, CS1 interacts with SAP in resting NKcells, but, in activated NK cells, CS1 interacts with EAT-2[30,52]. Therefore, we were able to predict that CS1 mightplay an inhibitory role in LPS-activated monocytes in theabsence of EAT-2. As seen in Fig. 3b, CS1 ligation indeedsignificantly reduced expression of TNF-aand IL-12p70,both proinflammatory cytokines released upon monocyteactivation. Taken together, we demonstrated that CS1 isexpressed in activated monocytes, and that the expressionof CS1 on activated monocytes is mediated by PI3K- andNF-rB-dependent pathways. Finally, CS1 play as aninhibitory role by reducing the production of proinflam-matory cytokines by LPS-activated monocytes. Thesefindings provide new insights into how SLAM familyreceptors contribute to innate immunity.Acknowledgments Flow cytometry was performed in the FlowCytometry and Laser Capture Microdissection Core Facility at TheUniversity of North Texas Health Science Center. This study wassupported by UNT Health Science Center Seed grant G67704.References1. Kumar S, Jack R. 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