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Inflammation ISSN 0360-3997Volume 35Number 1 Inflammation (2012) 35:1-10DOI 10.1007/s10753-010-9283-3

Stimulation of FasL Induces Productionof Proinflammatory Mediators ThroughActivation of Mitogen-Activated ProteinKinases and Nuclear Factor-κB in THP-1CellsSang-Min Lee, Eun-Ju Kim, KyounghoSuk & Won-Ha Lee

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Stimulation of FasL Induces Production of ProinflammatoryMediators Through Activation of Mitogen-Activated ProteinKinases and Nuclear Factor-κB in THP-1 Cells

Sang-Min Lee,1 Eun-Ju Kim,1 Kyoungho Suk,2 and Won-Ha Lee1,3

Abstract—FasL is a member of the tumor necrosis factor (TNF) superfamily involved in the variousimmune reactions such as activation-induced cell death, cytotoxic effector function, and establish-ment of immune privileged sites through its interaction with Fas. On the other hand, FasL is knownto transmit a reverse signal that serves as a T cell co-stimulatory signal. However, the role of FasL-mediated reverse signaling in macrophage function has not been investigated. In order to investigatethe presence of FasL-mediated signaling in macrophages, the human macrophage-like cell lineTHP-1 was analyzed after treatment with FasL ligating agents such as recombinant Fas:Fc fusionprotein or anti-FasL monoclonal antibody. Stimulation of FasL induced the expression of proinflam-matory mediators such as matrix metalloproteinase-9, TNF-α, and IL-8. The specificity of the reactionwas confirmed by the transfection of the FasL-specific siRNAs, which suppressed FasL expression aswell as the production of proinflammatory mediators. Utilization of various inhibitors of signalingadaptors and ELISA-base nuclear factor (NF)-κB binding assay demonstrated that the signaling initiatedfrom FasL is mediated by mitogen-activated protein kinases including extracellular signal-regulatedkinase, p38, and c-Jun N-terminal kinase which induce subsequent activation of NF-κB. These dataindicate that membrane expression of FasL and its interaction with its counterpart may contribute to theinflammatory activation of macrophages during immune reactions or pathogenesis of chronic inflam-matory diseases.

KEY WORDS: macrophage; FasL; TNFSF; inflammation; signal transduction.

INTRODUCTION

Members of the tumor necrosis factor superfamily(TNFSF) are involved in the regulation of variousimmune reactions and pathogenesis via stimulation oftheir cognate receptors. The membrane-bound form ofTNFSF has been shown to be able to transduceactivation signals when they are stimulated. A majorityof cases of this reverse signaling have been reported in Tcells as a co-stimulatory signal. Stimulation ofTRANCE, which is originally known to stimulate its

receptor RANK on the surface of myeloid cells, withRANK:Fc fusion protein augmented IFN-γ secretion viaa p38 mitogen-activated protein kinase (MAPK)-dependent pathway [1]. Reverse signaling has also beenreported to be mediated by CD40L [2], CD30L [3],LIGHT [4], TRAIL [5], and FasL [6] in T cells.Recently, however, the existence of reverse signaling inmacrophage has been reported in case of GITRL [7],BAFF [8], and APRIL [9].

FasL (CD178) is well characterized for its role inthe induction of apoptosis within the immune systemthrough its receptor Fas. In contrast to Fas, which isconstitutively expressed in many cell types, FasLexpression is restricted to several cell types includingCD4+ T helper 1 cells, activated CD8+ cells, naturalkiller cells, and monocytes (reviewed in [10]). Inactivated T cells, the upregulation of FasL expressionlevels and subsequent interaction of it with Fas triggers

1 School of Life Sciences and Biotechnology, Kyungpook NationalUniversity, Daegu, 702-701, South Korea

2 Department of Pharmacology, School of Medicine, KyungpookNational University, Daegu, 702-701, South Korea

3 To whom correspondence should be addressed at School of LifeSciences and Biotechnology, Kyungpook National University, Daegu,702-701, South Korea. E-mail: [email protected]

0360-3997/12/0100-0001/0 # 2010 Springer Science+Business Media, LLC

Inflammation, Vol. 35, No. 1, February 2012 (# 2010)DOI: 10.1007/s10753-010-9283-3

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apoptotic dell death called activation-induced cell death(AICD) [11]. Fas/FasL interaction is one of the two mainpathways that are responsible for the cytotoxic effectorfunction of natural killer cells and CD8+ cytotoxic Tlymphocytes [12]. Furthermore, constitutive expressionof FasL on cells or tissues of immune-privileged sitesand some tumor cells results in the suppression ofimmune responses against them [13, 14]. In addition tothe death-promoting activity, reverse signaling throughFasL has been implicated in T cell activation as a co-stimulatory signal. In CD8+ T cells, FasL-mediatedsignaling was shown to be required for the optimalproliferation [15, 16]. In CD4+ T cells, FasL engagementinhibited cell proliferation, cell-cycle progression, andIL-2 secretion [17]. Transfection of a construct for theexpression of a fusion protein containing N-terminalmyristylation motif (required for the membranelocalization) and the intracellular part of FasL intoJurkat T cells mimicked the costimulatory effect ofFasL [18, 19]. FasL-mediated co-stimulation of T cellsrequired the proline-rich intracellular domain of FasLand transmitted through lipid raft. In addition, the signaltransmission was associated with phosphorylation ofFasL itself and signaling adaptors such as AKT,extracellular signal-regulated kinase (ERK) 1/2, c-JunN-terminal kinase (JNK), and activation of transcriptionfactors such NFAT and AP-1 [18–20].

Although the co-stimulatory action of FasL hasbeen well documented in T cells, its role in macrophagefunctions has not been investigated. The expressionpattern and the role of FasL in processes associated withinflammation have been investigated in the humanmacrophage-like cell line, THP-1. Stimulation of FasLusing specific monoclonal antibody (mAb) or recombi-nant Fas:Fc fusion protein regulated the production ofproinflammatory mediators and phagocytic activity. Themolecular mechanism of FasL-induced cellular activa-tion was investigated. In an effort to find out animmunomodulatory molecule that can regulate themacrophage activation, the effect of immune receptorexpressed on myeloid cells (IREM)-1 triggering in FasL-mediated macrophage activation was investigated.IREM-1 is known to have inhibitory action throughmultiple intracellular immunoreceptor tyrosine-basedinhibitory motifs (ITIMs), which interacts with SH2-containing tyrosine phosphatase (SHP)-1 [21–23]. SHP-1 has been demonstrated to inhibit cellular signals thatinvolve PI3K, Janus kinase 2, STATs, MAPKs, ERK,and NF-κB [24, 25]. Finally, the effect of an ITIM-containing synthetic peptide that mimics the inhibitory

action of IREM-1 in FasL-mediated cellular activationwas investigated.

MATERIALS AND METHODS

Monoclonal Antibodies and Reagents

Anti-FasL (clone NOK-1) mAb was purchasedfrom Biolegend (San Diego, CA, USA). Fas:Fc fusionprotein and anti-BAFF mAb were from R&D Systems(Minneapolis, MN, USA). IREM-1-specific mAbs weregenerated in our laboratory (clones 2–3.6D and 2–1.7B)as described previously [22]. Polyclonal antibodies toERK (p42/44 MAPK), phospho-ERK (Thr202/Tyr204),JNK, p38, and phospho-p38 and mAb to phospho-JNK(G9) were purchased from Cell Signaling (Danvers, MA,USA). Antibodies against NF-κB p65/p50 and p52subunits came from Santa Cruz (Santa Cruz, CA,USA) and Cell Signaling, respectively. Mouse IgG1

was from BD Biosciences (HI111; San Jose, CA, USA).Human IgG, rottlerin, Ro-31-8425, Gö6976, SB203580,LY294002, JNK inhibitor I (JNK-I1), a cell-permeablefusion protein containing 20 AA of the JNK-bindingdomain of islet-brain and HIV-TAT48–57 [26], and itsnegative control containing only HIV-TATwere obtainedfrom Calbiochem International Inc. (La Jolla, CA,USA). Lipopolysaccharide (LPS), phorbol myristicacetate (PMA), N-tosyl-L-phenylalanine chloromethylketone (TPCK), ethyl pyruvate, and sulfasalazine werepurchased from Sigma (St. Luis, MO, USA). U0126 waspurchased from Cell Signaling. Fusion peptide contain-ing ITIM of IREM-1 (AA201–210) and HIV-TAT48–57

(named as TAT-YADL), its Y205F substitution mutant(named as TAT-FADL), and negative control containingonly HIV-TAT was custom-designed and synthesized byPeptron Inc. (Daejeon, Korea). The sequences for TAT,TAT-YADL, and TAT-FADL are GRKKRRQRRR,G R K K R R Q R R R - G D L C YA D LT L , a n dGRKKRRQRRR-GDLCFADLTL, respectively. TheTHP-1 cell lines were obtained from the American TypeCulture Collection (Rockville, MD, USA).

SiRNA Transfection

FasL-specific siRNAs (a pool of three target-specific 20–25 nt siRNAs) and control siRNA (a randombase sequence with no known specificity to humangenes) were purchased from Santa Cruz (Santa Cruz,CA, USA). SiRNAs were transfected into the THP-1cells using DharmaFECT (Dharmacon Inc.), according

2 Lee, Kim, Suk, and Lee


to a protocol provided by the manufacturer. Down-regulation of cell surface FasL was measured at 3, 5, and10 days after transfection.

ELISA-Based Measurement of NF-κB BindingActivity

NF-κB binding activity was measured following apreviously described method [27]. Briefly, streptavidin-coated 96-well culture plates were used to immobilizebiotin-labeled double-stranded oligonucleotides contain-ing a consensus NF-κB binding site (5′cacagttgaggg-gactttcccaggc3′; 0.02 nm/well). Whole cell lysates ornuclear lysates were then added into the NF-κB oligoplates and incubated at room temperature for 1 h withmild agitation in 100 μl/well of PBS. Utilization ofeither whole cell lysate or nuclear lysate resulted in thesame results. The plates were then sequentially incu-bated with antibodies specific to NF-κB subunits, HPR-labeled secondary antibody, and tetramethylbenzidine(chromogen). Absorbance (450–540 nm) was thenmeasured, and the values were normalized by subtract-ing the background values. For blocking, cell lysateswere pre-incubated with 1.0 nm/sample of double-stranded oligonucleotides containing wild-type NF-κBbinding sequence or a mutant sequence (5′cacagttgaggc-cactttcccaggc3′) before adding them to the NF-κB oligoplates.

Gelatin Zymogram, ELISA, and Western BlotAnalysis

THP-1 cells (1×106/ml) were activated by addingantibodies and fusion proteins to the medium inRPMI1640 supplemented with 0.1% FBS. The culturesupernatants were collected 24 h after activation, and thegelatin zymogram analyses for the measurement ofmatrix metalloproteinase (MMP)-9 activity and ELISAfor the quantification of cytokine concentrations wereperformed as described previously [28, 29]. Western blotanalysis was performed as described previously [28, 29].

Phagocytosis Assay and Flow Cytometry

Zymosan opsonization was performed by treatingzymosan tagged with Alexa Fluor 594 with one tenthvolume of zymosan A opsonizing reagent (Invitrogen,Eugene, OR, USA) at 37°C for 1 h. For the measure-ment of the phagocytic activity, cells were pre-treated for1 μg/ml of Fas:Fc for 30 min and then incubated with30 μg/ml of opsonized-zymosan-594 for 3 h. The

percentage of cells associated with zymosan wasmeasured using flow cytometry analysis. Flow cytom-etry analysis was performed using FACS-calibur (Bec-ton-Dickinson, Mountain View, CA, USA). For the flowcytometric analysis of cell surface antigens, cells (5×105) were pelleted and incubated with 0.3 μg offluorescence-labeled primary or secondary antibodies in30 μl of FACS solution (a PBS containing 0.5% BSAand 0.1% sodium azide) for 20 min on ice. Forbackground fluorescence, the cells were stained with anisotype-matching control antibody. The fluorescenceprofiles of 2×104 cells were collected and analyzed.

Statistical Analysis

Statistical significance of differences was evaluatedby means of a two-sided Student’s t test, assuming equalvariances. Differences were considered significant whenp<0.05.

RESULTS AND DISCUSSION

THP-1 Cells Express FasL and Its Expression LevelsIncrease after Activation of the Cells

In order to test the expression patterns of FasL onthe surface of THP-1 cells, flow cytometry analysis wasperformed. Moderate basal expression levels of FasLwere detected in THP-1 cells (Fig. 1a). The expressionlevels of FasL were reported to be induced by activationin T lymphocytes [10]. In order to test whether it is thecase in monocytic lineage cells, THP-1 cells werestimulated with LPS or PMA. As a ligand of Toll-likereceptor 4 (TLR4), LPS induces potent inflammatoryactivation in various cell types including macrophages[30]. PMA is a well-known stimulator of protein kinaseC (PKC) and induces macrophage differentiation inmonocytic cells [31]. When THP-1 cells were stimulatedwith these agents, the expression levels of FasLincreased slightly after 24 h and increased further after48 h (Fig. 1a, b).

Stimulation of FasL Induces Productionof Inflammatory Mediators such as MMP-9 and IL-8in THP-1 Cells

For the stimulation of FasL, Fas:Fc fusion proteinwas used to stimulate THP-1 cells. As shown in Fig. 2a,treatment with Fas:Fc induced the expression of proin-flammatory mediators such as TNF-α and IL-8 in a

3FasL-Mediated Inflammatory Activation in THP-1 Cells


dose-dependent manner. In contrast, treatment of thecells with human IgG (which was used as a negativecontrol for the Fc portion of the fusion protein) failed toaffect cellular responses. In order to confirm that thestimulation of FasL induces production of these inflam-matory mediators, THP-1 cells were then stimulatedwith anti-FasL mAb, which cross-link cell surface FasLand subsequently trigger its activation. As shown inFig. 2b, anti-FasL treatment induced expression of bothMMP-9 and IL-8. Treatment of the cells with mouse IgG(which represent the negative control for the anti-FasLmAb) failed to induce cellular responses. For theconfirmation of the role of FasL in the inflammatoryactivation of THP-1 cells, FasL-specific siRNAs(siFasL) were transfected into THP-1 cells. Transfectionof siFasL, but not the control siRNA, completelyabolished the surface expression of FasL (Fig. 3a). ThesiFasL-transfected cells failed to respond to the anti-FasL mAb, while the cells transfected with controlsiRNA responded (Fig. 3b). As positive controls, cellswere stimulated with LPS or anti-BAFF mAb. BAFF isanother member of TNFSF which also generates reversesignaling [8]. Treatment of the cells with these agentsinduced IL-8 and MMP-9 expression in both controlsiRNA and siFasL transfected cells. MMP-9 is a matrix-

degrading enzyme regulated mainly by NF-κB, and itsexpression levels are upregulated by inflammatorychanges in macrophages while MMP-2 expression levelsdo not. These results demonstrate that the cellularresponsiveness to agents that ligate other cell surfacemolecules was not affected by the siFasL transfection.These data also demonstrate that the activation of thecell surface FasL can generate certain signaling thatresults in the production of proinflammatory mediators.

FasL-Mediated Cellular Activation Requiresthe Involvement of MAPKs and NF-κB

The cellular signaling adaptors involved in FasL-mediated activation of THP-1 cells were then inves-tigated. As shown in Fig. 4, all three PKC inhibitorsfailed to block the FasL-mediated expression of IL-8. Incontrast, FasL-mediated expression of IL-8 was signifi-cantly suppressed by MAPK inhibitors. SB203580,U0126, and JNK inhibitor (JI) were used as the specificinhibition of p38, ERK1/2, and JNK MAPK, respec-tively. Activation MAPK, especially activation ofERK1/2, is well known to be associated with inflamma-tory activation of macrophages [7, 32, 33]. Activation ofMAPKs usually leads to the activation of NF-κB, whichis the major transcription factor required for theexpression of cytokines, MMPs, and cell adhesionmolecules. When FasL was stimulated in the presenceof NF-κB inhibitors such as TPCK [34], ethyl pyruvate[35], and sulfasalazine [36], the expression of IL-8 wasblocked by all these agents (Fig. 4a). In contrast, FasL-mediated induction of IL-8 expression was not affectedby LY294002, which is a phosphoinositide kinase-3(PI3K) inhibitor, or SU6656, which is a Src kinaseinhibitor. These data indicate that FasL-mediated inflam-matory activation in THP-1 cells requires the activitiesof MAPK and NF-κB.

Since inhibitor assay demonstrated the involvementof MAPKs in the FasL-mediated induction of IL-8expression, the activation of MAPKs was directly testedin THP-1 cells after stimulation of FasL. As shown inFig. 4b, stimulation of FasL resulted in the phosphor-ylation of ERK1/2 and p38 as early as 3 min afterstimulation. Phosphorylation of JNK was detected at20 min after stimulation (Fig. 4b).

In order to investigate the FasL-mediated activationof NF-κB at the functional level, ELISA-based NF-κBbinding assay was performed. The amount of DNA-bound NF-κB was detected with antibodies specific forp65, Rel-B, p50, and p52 subunits of NF-κB. NF-κB

Fig. 1. THP-1 cells express FasL which can be upregulated by cellularactivation. a THP-1 cells were stimulated with 1 μg/ml of LPS or100 nM of PMA for 48 h. Cells were then stained with FasL-specificmAb, and the fluorescence profile (empty area) was compared withbackground levels (stained with isotype-matching mouse IgG, filledarea). b THP-1 cells were treated as in a for indicated time periods.The mean fluorescence intensity (MFI) values were measured and co-mpared with control (without any treatment). These experiments wererepeated twice with essentially the same results.



binding activity was induced by the activation of FasL,but the time points were different for each subunit(Fig. 5). The amount of p65 subunit peaked at 30 minafter stimulation while that of p50 and p52 peaked at 20

and 40 min, respectively. The binding of Rel-B was notdetected after FasL stimulation (data not shown). Themajor form of NF-κB is heterodimer composed of p65(or Rel-B) and p50 (or p52). Homodimeric form of NF-κB containing p50 is also possible in some cases. Itappears that different forms of NF-κB dimer aresequentially activated after stimulation of FasL inTHP-1 cells. The binding was inhibited by pre-incuba-tion of cell lysates with consensus NF-κB bindingsequences but not with mutant sequences (Fig. 5),indicating that the interaction was specific. The induc-tion of NF-κB binding activity was significantlydecreased in cells treated with MAPK inhibitors butnot with the PI3K inhibitor (Fig. 5d), which demon-strates the requirement of MAPKs in the FasL-mediatedactivation of NF-κB activity.

Stimulation of FasL Results in the Suppressionof Phagocytic Activity in THP-1 Cells

Previously, stimulation of membrane-bound formof BAFF resulted in the inhibition of phagocytic activityin THP-1 cells [8]. This inhibitory activity was possiblethrough suppression of Rho-family GTPase activity,which had been reported to be required for actinassembly at nascent phagosomes and the subsequentinternalization of IgG-opsonized particles [37]. Whenphagocytosis of opsonized zymosan was measured inTHP-1 cells after treatment with Fas:Fc, phagocyticactivity was blocked in a dose-dependent manner

Fig. 2. Treatment of THP-1 cells with Fas:Fc or anti-FasL mAb induces the expression of proinflammatory cytokines. a Cells were stimulated with1 μg/ml of LPS; 0.3, 1, and 3 μg/ml of Fas:Fc; or 3 μg/ml of human IgG. Culture supernatants were collected 24 h after activation, and cytokine levelswere measured using ELISA. b THP-1 cells were stimulated with 1 μg/ml of LPS; 0.3, 1, and 3 μg/ml of anti-FasL mAb; or 3 μg/ml of mouse IgG.Culture supernatants were collected 24 h after activation for the measurement of MMP-9/-2 activity using gelatin zymogram and IL-8 concentrationsusing ELISA. Data points are represented as mean ± SD for triplicate measurements. These experiments were repeated twice with essentially the sameresults. *p<0.05 when compared with control samples and ***p<0.001. Con no treatment control.

Fig. 3. Stimulation of FasL induces the expression of MMP-9 and IL-8. a THP-1 cells were transfected with FasL-specific siRNA or controlsiRNA. FasL expression levels were tested using flow cytometry. bCells in a were stimulated with 1 μg/ml of LPS (L), anti-BAFF mAb(B), anti-FasL mAb (F), or mouse IgG (M). Culture supernatants werecollected 24 h after activation for the measurement of MMP-9 activityand IL-8 concentrations. Data points are represented as mean ± SD fortriplicate measurements. These experiments were repeated twice withessentially the same results. c No treatment control.



(Fig. 6). As a control, THP-1 cells were stimulated witha fusion protein containing an Fc portion and theextracellular portion of glucocorticoid-induced TNFreceptor family-related protein (GITR). GITR is anothermember of TNF receptor superfamily that can interactwith and induce reverse signaling through GITRL [7].However, the phagocytic activity was not affected byGITR:Fc fusion protein (Fig. 6).

Inflammatory activation of macrophages results inthe production of cytokines as well as enhancement ofits phagocytic activity. It is interesting that the stimula-tion of FasL led to the induction of cytokine secretion

while suppressing phagocytosis of opsonized zymosan.Although these two processes are associated withinflammatory activation of macrophages, the signalingpathways are different. In case of cytokine production,MAPK-mediated activation of NF-κB is an essentialprocess, while phagocytic process requires mobilizationof cellular cytoskeleton through activation of varioussignaling adapters including src kinase, Rho GTPase,PI3K, and PLC [37, 38]. It is likely that activation ofFasL induces MAPK-mediated activation of NF-kBwhile suppressing the activation of signaling adapter(s)that are associated with phagocytosis.

Fig. 4. FasL-mediated cellular signaling is mediated by MAPKs and NF-κB. a THP-1 cells were pretreated with 1 μM of Rottlerin (Rt), Gö6976(Go), Ro31-8425 (Ro), 5 μM of SB203580 (SB), 10 μM of U0126 (U), JNK inhibitor (JI), negative control of JNK inhibitor (JI-), 5 mM of ethylpyruvate (EP), 1 mM of sulfasalazine (SZ), 1 μM TPCK (TP), 20 μM of LY294002 (LY), 1 μM of SU6656 (SU), or 0.01% DMSO (VC vehiclecontrol) for 30 min. Cells were then treated with 1 μg/ml of Fas:Fc for 24 h. Culture supernatants were then collected for the measurement of IL-8concentration using ELISA. Data points are represented as mean ± SD for triplicate measurements. ***p<0.001 when compared with samples treatedwith Fas:Fc. b THP-1 cells were treated with 1 μg/ml of anti-FasL mAb. Cell lysates were obtained at indicated times after activation for Western blotanalysis using antibodies specific for ERK1/2, p38, and JNK and their phosphorylated forms. Experiments were repeated twice for a and three timesfor b with essentially the same results.



Treatment of THP-1 Cells with Anti-IREM-1 mAbor a Synthetic Peptide that Contains the ITIMof IREM-1 Blocks FasL-Mediated Inductionof Proinflammatory Mediator Expression throughSuppression of NF-κB Activation

The expression of FasL on the surface of THP-1cells and the proinflammatory changes associated withFasL stimulation suggest that FasL-mediated macro-phage activation may play a role during normal immuneresponses or pathologic conditions such as chronicinflammatory diseases. It is also possible that FasL-mediated signaling works in concert with other proin-flammatory cytokines for a synergistic activation ofmacrophages. Since excessive activation of macro-phages leads to the cell or tissue damage in chronicinflammatory diseases, it is required to regulate exces-sive activation of macrophages for the prevention of the

damages. IREM-1, an inhibitory receptor expressed onmyeloid cells including macrophages [39–41], is acandidate molecule that can suppress overactivation ofmacrophages. IREM-1 contains five ITIMs in its intra-cellular domain [42] and triggering of IREM-1 leads tothe inhibition of various activation processes in myeloidcells [21–23]. It has been demonstrated that ligation ofIREM-1 with specific mAb inhibited BAFF-mediatedcellular activation in THP-1 cells [22]. Since both BAFFand FasL induced similar signaling pathways involvingMAPK and NF-κB, it was expected that IREM-1 mighthave inhibitory effect on FasL-mediated macrophageactivation. When IREM-1 was cross-linked with specificmAb, Fas:Fc-induced expression of IL-8 expression wasblocked in THP-1 cells (Fig. 7a).

Since IREM-1 exhibits its inhibitory effect throughinteraction ITIMs, a synthetic fusion peptide containingan ITIM of IREM-1 and HIV-TAT48–57 (named as TAT-

Fig. 5. FasL-mediated cellular signaling activates DNA binding activity of NF-κB through MAPK. a–c THP-1 cells were stimulated with 1 μg/ml ofanti-FasL mAb for indicated times. Cell lysates were then obtained and utilized for ELISA-based NF-κB binding assay using mAbs against p65 (a),p50 (b), or p52 (c) subunit. For blocking experiment, cell lysates were pre-incubated with 50-fold excess of wild type (WT) or mutant (Mut) form ofconsensus NF-κB binding sequence. d THP-1 cells were pre-treated with 5 μM of SB203580 (SB), 10 μM of U0126 (U), JNK inhibitor (JI), negativecontrol of JNK inhibitor (JI-), 20 μM of LY294002 (LY), or 0.01% DMSO (VC) for 30 min. Cells were then treated with 1 μg/ml of anti-FasL mAbfor additional 30 min. Cell lysates were used for the ELISA-based NF-κB binding assay. Data points are represented as mean ± SD for quintuplicatemeasurements. These experiments were repeated twice with essentially the same results. #p<0.05 when compared with no treatment control; ##p<0.01; ###p<0.001. ***p<0.001 when compared with corresponding samples without any competitor. &&&p<0.001 when compared with anti-FasL-treated samples.



YADL) was then synthesized in order to test whether itmimics the inhibitory action of IREM-1. TAT-YADL is ashort (20 amino acids-long) peptide containing a 10 AAfragment representing the first ITIM of IREM-1 intra-cellular region (AA201–210: GDLCYADLTL) fused withHIV-TAT48–57 which was required for rapid internal-ization of the peptide from the culture medium into thecells [26]. When THP-1 cells were stimulated throughFasL in the presence of TAT-YADL, the expression ofboth MMP-9 and IL-8 was blocked (Fig. 7b). Incontrast, incubation of the cells with TAT peptide failedto have the inhibitory effect. These inhibitory effects ofIREM-1 and TAT-YADL are believed to be mediated byits association with SHP-1, a protein tyrosine phospha-tase that has been shown to interact with Y205 of IREM-1 [21–23].

In order to demonstrate that the synthetic peptidesuppresses FasL-mediated cellular activation by block-ing NF-κB activation, ELISA-based NF-κB bindingassay was performed using cells that had been stimu-lated with anti-FasL in the presence or absence of TAT-YADL. Pretreatment of the cells with TAT-YADL, butnot with TAT-FADL which has tyrosine to phenylalaninesubstitution mutation, abolished the FasL-mediatedactivation of NF-κB binding activity (Fig. 7c). Thesedata indicate that ITIM containing synthetic peptidesmay be useful for the regulation of inflammatoryactivities of macrophages, which are usually overacti-vated in chronic inflammatory diseases.

Current data provide the first experimental evidenceshowing the presence of reverse signaling mediated byFasL in THP-1 cells, the human macrophage-like cellline. Stimulation of FasL induced the expression of

Fig. 6. Stimulation of FasL results in the suppression of phagocyticactivity. THP-1 cells were pretreated with 0.3, 1, and 3 μg/ml of Fas:Fc; 3 μg/ml of GITR:Fc (G); or human IgG (H) for 30 min and thenincubated with 30 μg/ml of opsonized-zymosan-594 for 3 h. The per-centage of cells that phagocytozed the zymosan particle was measuredusing flow cytometry.

Fig. 7. Treatment with ITIM containing synthetic peptides results inthe blockage of FasL-mediated activation of THP-1 cells. a Cells werepretreated with 1 μg/ml of anti-IREM-1 mAb (IR) or mouse IgG (M)for 30 min and then treated with 1 μg/ml of Fas:Fc. Culture superna-tants were collected 24 h after activation for the measurement of IL-8concentrations. b Cells were pre-treated with 5 μM of TAT, TAT-YA-DL, or TAT-YASL for 30 min and then stimulated with 1 μg/ml of Fas:Fc. Culture supernatants were collected 24 h after activation for themeasurement of MMP-9 activity and IL-8 concentrations. c THP-1cells were pre-treated with 5 μM TAT, TAT-YADL, or TAT-FADLfor 30 min. Cells were then treated with 1 μg/ml of anti-FasL mAbfor additional 30 min. Cell lysates were then collected and used forthe ELISA-based NF-κB binding assay using mAb against p65 subunit.Data points are represented as mean ± SD for triplicate measure-ments. These experiments were repeated twice with essentially thesame results. ***p<0.001 when compared with samples treated with Fas:Fc or anti-FasL mAb.



proinflammatory mediators through a signaling pathwaythat requires the activation of MAPK that leads to thesubsequent activation of NF-κB. Additionally, ligationof FasL resulted in the suppression of phagocyticactivity in THP-1 cells. These activities of FasL, inassociation with various other mediators that are knownto regulate macrophage inflammation, may regulatemacrophage inflammatory responses during normalimmune reactions or chronic inflammatory diseases.Current data further demonstrate the inhibitory actionof IREM-1 and a synthetic peptide containing an ITIMdomain of IREM-1 in FasL-mediated macrophageactivation. Since this synthetic peptide appeared toinhibit the action of multiple activating agents, it maydemonstrate to be useful for the suppression of macro-phages in various inflammatory conditions in the future.

ACKNOWLEDGEMENTS

This work was supported by the Korea ResearchFoundation Grant funded by the Korean Government(KRF-2008-313-C00647).

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