International Immunopharmacology 12 (2012) 635–642

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International Immunopharmacology

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STAT6 and JAK1 are essential for IL-4-mediated suppression of prostaglandinproduction in human follicular dendritic cells: Opposing roles of phosphorylated andunphosphorylated STAT6

Whajung Cho a, Doo-Il Jeoung b, Young-Myeong Kim c, Jongseon Choe a,⁎a Department of Microbiology and Immunology, School of Medicine, Kangwon National University, Chuncheon, Gangwon, Republic of Koreab Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon, Gangwon, Republic of Koreac Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chuncheon, Gangwon, Republic of Korea

Abbreviations: FDC, follicular dendritic cell; GC, germSTAT, signal transducer and activator of transcriptionSTAT6; P-STAT6, phosphorylated STAT6.⁎ Corresponding author. Tel./fax: +82 33 250 8862.

E-mail address: jchoe@kangwon.ac.kr (J. Choe).

1567-5769/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.intimp.2012.02.012

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 January 2012Received in revised form 6 February 2012Accepted 23 February 2012Available online 7 March 2012

Keywords:Follicular dendritic cellsIL-4STAT6COX-2Prostaglandin

Prostaglandins (PGs) are emerging as important immune mediators. Since our first report on the expressionof prostacyclin synthase in the germinal centers, we have investigated production mechanisms and biologicalfunctions of PG using human follicular dendritic cell (FDC)-like cells. In the previous report, we observed thatTGF-β enhances PG production, and IL-4 prevents this upregulation. To elucidate the inhibitory mechanism ofIL-4, its effects on the key enzyme leading to PG production were analyzed in this study. IL-4 but not IL-10inhibited TGF-β-induced COX-2 expression at both mRNA and protein levels. Next the early signaling mole-cules of IL-4 were identified by siRNA technology. IL-4 induced tyrosine phosphorylation of STAT1, 3, and 6,but only JAK1-STAT6 pathway was responsible for the prevention of COX-2 augmentation and PG production.Phosphorylated STAT6 accumulated in the nucleus rapidly upon IL-4 addition, and the complete inhibition ofCOX-2 upregulation required 24 h of pretreatment with IL-4, implying that newly transcribed molecules me-diate the inhibitory signals downstream of STAT6. Interestingly, unphosphorylated STAT6 proteins were con-stitutively expressed in the nucleus, and depletion of STAT6 impaired background level expression of COX-2and PGs. Our results highlight the crucial roles of TGF-β and IL-4 in the regulation of PG production, whichlead us to suggest that T cells play an important role in FDC production of PGs.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Follicular dendritic cells (FDCs) are unique stromal cells foundnormally in the primary and secondary follicles of peripheral lym-phoid organs and ectopically in the inflamed tissues of several chronicdiseases such as rheumatoid arthritis [1]. In spite of the similarnames, FDC is different from dendritic cell (DC) in their cellular ori-gins, anatomic niches, and biological functions. FDCs derive frombone marrow mesenchymal stem cells, localize in the B cell areas oflymphoid organs, and present native antigens to B cells withoutphagocytosis [2]. In contrast, DCs originate from bone marrow hema-topoietic stem cells, establish in the T cell areas of peripherallymphoid organs, and present processed antigen peptides to T cells

inal center; JAK, Janus kinase;; U-STAT6, unphosphorylated

rights reserved.

following phagocytosis [3]. FDCs are essential components of thehumoral immune system, and the development of lymphoid tissuesand production of high affinity antibodies are severely compromisedin the absence of FDC [4,5]. However, the molecular mechanisms bywhich FDC plays the essential functions are largely unknown in partdue to the practical difficulty in isolating enough FDCs to mimic thegerminal center (GC) reactions in vitro. We have established a meth-od of preparing FDC-like primary cells, HK, from human tonsils [6]and have reported several interesting observations previously. FDCand HK cells express prostacyclin synthase [7] and have a distinctprostacyclin production mechanism [8]. Prostacyclin enhances theAPC capability of B cells by increasing CD86 expression levels [9]. Inaddition, we demonstrated that prostaglandins (PGs) produced byHK cells inhibit proliferation and apoptosis of T cells [10] and PG pro-duction from HK cells is controlled by the inhibitory effect of IL-4 onCOX-2 expression [11]. These findings suggest that FDCs also interactwith another cellular component of the GC, T cells, via PGs andcytokines.

Paying attention to the emerging concept of PGs as criticalimmune modulators [12–14], our laboratory conducted several ex-periments to understand the cellular and molecular mechanisms of

636 W. Cho et al. / International Immunopharmacology 12 (2012) 635–642

PG production regulation in the GC. We have previously demonstrat-ed that both LPS and TGF-β stimulate PG production in HK cells,which is suppressed by IL-4 [10]. As an underlying mechanism, IL-4,via the JAK1-STAT6 pathway, inhibits COX-2 expression and PG secre-tion that is stimulated with LPS [11]. Although LPS and TGF-β sharethe feature of stimulating COX expression in HK cells, they trigger in-tracellular signaling by binding distinct receptors and via uncon-nected pathways. For example, LPS signaling involves innateimmune receptors and MyD88-dependent pathway [15], whereasTGF-β signaling is associated with its cognate receptors and Smadmolecules [16]. Therefore, we reasoned that IL-4 might regulateLPS- and TGF-β-induced COX-2 expression by different mechanisms.The current results reveal that IL-4 inhibits TGF-β-induced COX-2 ex-pression and resultant PG production via the essential mediation ofJAK1 and STAT6 molecules and that other STATs are not required forthe inhibition. The critical role of STAT6 in this process is clearly dem-onstrated by the nuclear translocation and accumulation of phos-phorylated STAT6 upon IL-4 addition. In addition, we observe thatunphosphorylated STAT6 proteins are constitutively expressed inthe nucleus, and depletion of STAT6 impairs background level COX-2 expression and PG production. The distinct roles of JAK1 andSTAT6 in LPS- and TGF-β-induced COX-2 expression are discussedwith the significance of our new findings.

2. Materials and methods

2.1. Culture of HK cells

HK cells are primary cells prepared on a regular basis from humantonsils which are obtained from children undergoing tonsillectomy atAsan Medical Center (Seoul, Korea). This study was approved by theInstitutional Review Board of Asan Medical Center and written in-formed consent was obtained. HK cells at passages 5–8 were usedto ensure purity. The purity and phenotype of typical HK cells are pre-sented elsewhere [17]. They are prepared as described by Kim et al.[6] and maintained in RPMI-1640 (Irvine Scientific, Santa Ana, CA)containing 10% fetal calf serum (Hyclone, Logan, UT), 2 mML-gluta-mine (Invitrogen, Carlsbad, CA), 100 U/ml penicillin G (Sigma-Al-drich, St. Louis, MO), and 100 μg/ml streptomycin (Invitrogen). TGF-β was purchased from R&D Systems, and recombinant IL-4 and IL-10 were prepared in our laboratory [8]. IL-4-neutralizing antibodywas obtained from eBioscience (San Diego, CA).

2.2. Immunoblotting

The whole cell lysates of HK cells were subject to immunoblottingas previously described [18]. The cytosol or nuclear fractions of HKcells were obtained as follows. Cell membranes were lysed by bufferA (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.5 mM PMSF, 0.5 mMleupeptin, 0.1 mM sodium vanadate, 50 mM sodium fluoride, 0.6%NP40, pH 7.9) at 4°C for 30 s, followed by centrifugation at14,000 rpm for 5 min. The supernatants (cytosol fraction) were col-lected. The pellets were lysed by Proprep lysis buffer at 4°C for30 min and then centrifuged at 14,000 rpm for 5 min to collect the su-pernatants (nuclear fraction). The protein concentrations of the eachfraction were assayed with a BCA assay. Used antibodies were againstCOX-1, COX-2 (Cayman Chemical, Ann Arbor, MI), active and totalforms of JAK1, JAK2, JAK3, TYK2, STAT1, STAT2, STAT3, STAT5, STAT6(Cell Signaling Technology, Danvers, MA), β-actin (Sigma-Aldrich),HRP-conjugated anti-mouse IgG (Jackson Immunoresearch, WestGrove, PA), and HRP-conjugated anti-rabbit IgG (KOMA Biotech,Seoul, Korea). The membranes were incubated with SuperSignalWest Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and ex-posed to X-ray films.

2.3. Quantitative real-time PCR

After HK cells were cultured for 4 h, total RNA was purified usingeasy-BLUE™ RNA extraction kit (iNtRON Biotechnology, Seongnam,Korea). cDNA synthesis from total RNA was performed using Oligo-d(T) primers and MuLV reverse transcriptase (Roche, Indianapolis,IN). The following primers were used for PCR amplification: COX-2forward (5′-CCCGCAGTACAGAAAGTATC-3′) and reverse (5′-ATTCA-TAGGGCTTCAGCATA-3′), GAPDH forward (5′-CCCTCCAAAAT-CAAGTGGGG-3′) and reverse (5′-CGCCACAGRRRCCCGGAGGG-3′).Quantitation of cDNA was accomplished by real-time, quantitativePCR (ABI7700; Applied Biosystems, Carlsbad, CA) using iTaq™SYBR®-Green Supermix With ROX (Bio-Rad, Hercules, CA). Cyclingparameters were 53°C (COX-2) or 55°C (GAPDH) for 1 min and72°C for 1 min. Calculations of expression were normalized usingthe Ct method; Ct is the cycle number of the detection threshold.

2.4. Confocal microscopy

HK cells were cultured to 80% confluence on 18 mm roundcover slip in 12-well plate. Each well was incubated with IL-4for indicated times, fixed in 4% paraformaldehyde for 30 min,and then blocked and permeabilized with PBS solution containing10% FBS and 0.1% triton X-100. The slides were incubated withcontrol, anti-STAT6, or anti-tyrosine phosphorylated STAT6 rabbitpolyclonal antibodies (Cell Signaling Technology, Danvers, MA)and then stained with FITC-conjugated anti-rabbit IgG (Invitro-gen, Carlsbad, CA) and propidium iodide for nuclear staining.The relative distribution of fluorochromes was visualized andscanned using a Fluoview FV1000 confocal laser microscope(Olympus, Tokyo, Japan).

2.5. siRNA transfection

The siRNA duplexes used (Ambion Inc, Austin, TX) wereconstructed with the following target sequences. Control (Neg-siRNA#2, sequence not disclosed by Ambion); JAK1, sense (5′-CCAU-CACCGUUGAUGACAATT-3′), antisense (5′-UUGUCAUCAACGGUGAU-GGTG-3′); JAK2, sense (5′-CCAGCGGAAUUUAUGCGUATT-3′), antisense(5′-UACGCAUAAAUUCCGCUGGTG-3′); JAK3, sense (5′-GUAUCGUGGU-GUCAGCUAUTT-3′), antisense (5′-AUAGCUGACACCACGAUACTT-3′);TYK2, sense (5′-CAUCCACAUUGCACAUAAATT-3′), antisense (5′-UUU-AUGUGCAAUGUGGAUGCA-3′); STAT1, sense (5′-CCUACGAACAUGACC-CUAUTT-3′), antisense (5′-AUAGGGUCAUGUUCGUAGGTG-3′); STAT3,sense (5′-GGAUCUAGAACAGAAAAUGTT-3′), antisense (5′-CAUUUUCU-GUUCUAGAUCCTG-3′); STAT6, sense (5′-GGGCAAUCUGGGAUCUCA-ATT-3′), antisense (5′-UUGAGAUCCCAGAUUGCCCAT-3′). HK cells werecultured to 50–60% confluence in 100 mm plates. For each plate,40 nM of each siRNA and 24 μl Lipofectamine™ (Invitrogen) were sepa-rately diluted in 400 μl serum-free medium without antibiotics, mixedtogether, and incubated at RT for 45 min. The plates were then washedwith serum-free medium, added with 5 ml serum-free medium, andthen with the diluted solutions. The plates were incubated at 37°C for8 h, followed by the addition of a growth medium containing 10%serum. After 48 h of additional incubation, cells were used for experi-ments. The degree of gene-silencing was assayed by immunoblotting.

2.6. Enzyme immunoassay to measure prostaglandins

HK cells were cultured with TGF-β for 48 h to harvest the superna-tants. The amounts of PGE2 and 6-keto PGF1α, stable metabolite ofPGI2, were measured using enzyme immunoassay (EIA) kits as de-scribed previously [10].

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2.7. Statistical analysis

Statistical analysis and graphic presentation were carried out withGraphPad Prism 4.0. Results are presented as means of triplicates plusSEM. The statistical significance of differences was determined byStudent's t- test; Pb0.05 was considered significant.

3. Results

3.1. IL-4 represses TGF-β-induced COX-2 expression in HK cells

In order to understand the mechanism of controlling PG produc-tion in HK cells, we examined the effect of IL-4 on the key enzyme in-volved in PG generation, COX-2. Treatment of HK cells with TGF-βenhanced the expression of COX-2 but not COX-1 protein, whichwas prevented by IL-4 pretreatment (Fig. 1A). The inhibitory effectwas specific to IL-4 because such an effect was not observed withIL-10 pretreatment. Dose–response experiments revealed that IL-4exhibited the inhibitory effect from 10 U or 0.125 ng/ml of concentra-tion in a dose-dependent manner, whereas IL-10 did not modulateCOX-2 levels up to 50 ng/ml (Fig. 1B). By adding IL-4 at differenttime points before TGF-β stimulation, the duration of IL-4 stimulationthat was required to bring out the inhibitory effect was determined.The impact of IL-4 was demonstrated from the simultaneous additionwith TGF-β. Twenty-four hours of pre-incubation with IL-4 complete-ly inhibited COX-2 up-regulation, and TGF-β stimulation did not in-crease COX-2 expression beyond the background levels. Theprevention of COX-2 up-regulation indeed resulted from IL-4 treat-ment but not from a certain resistance to TGF-β in HK cells due tothe prolonged culture period, because the control cultures that weremaintained parallel for the same period without IL-4 responded nor-mally to TGF-β by increasing COX-2 (Fig. 1C). The effect of IL-4 onCOX-2 was examined at mRNA levels by real-time RT-PCR analysis.HK cells responded to TGF-β stimulation by increasing COX-2 mRNA

COX-2

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Fig. 1. IL-4 inhibits upregulation of COX-2 protein and mRNA in HK cells that is induced b(1×105 cells) were cultured in the presence or absence of IL-4 (100 U/ml) or IL-10 (50 nLPS (1 μg/ml) for 8 h. The expression levels of COX-1 and COX-2 were determined by immunin the presence of indicated concentrations of IL-4 or IL-10 for 24 h and then with TGF-β forulated with TGF-β for 8 h. Representative results of at least three reproducible experimentsIL-10 (50 ng/ml) for 24 h, followed by a further incubation with or without TGF-β for 4 h. ExSEM of four independent experiments are depicted. Asterisks indicate significant difference

about 10-fold over the media control, which was clearly inhibitedby IL-4 but not IL-10 (Fig. 1D). These results indicate that IL-4 re-presses TGF-β-induced COX-2 expression in HK cells from the tran-scription stage.

3.2. JAK1 and STAT6 mediate the inhibitory effect of IL-4

Since cytokines generally utilize the JAK-STAT pathway to trans-duce the signals coming from membrane-bound cytokine receptors,we explored which STAT proteins would mediate the inhibitory effectof IL-4 in HK cells. We have recently demonstrated the essential re-quirement for STAT6 in the inhibitory effect of IL-4 [11]. However,the previous study does not exclude the possibility that IL-4 mobilizesother STAT molecules in addition to STAT6 to display its inhibitory ef-fect. First, we examined whether HK cells express all the STAT mole-cules. All the six STATs except STAT4 were expressed in considerableamounts in HK cells. Peripheral blood mononuclear cells were used toensure the integrity of STAT4 reagents. Compared with blood mono-nuclear cells, the expression levels of STAT4 in HK cells were verylow. None of them was found in tyrosine-phosphorylated forms be-fore IL-4 stimulation, but IL-4 induced slight phosphorylation ofSTAT1 and STAT3 and strong phosphorylation of STAT6 (Fig. 2A).The weak phosphorylation of STAT1 and STAT3 was specific to IL-4stimulation since they were potently phosphorylated by IFN-α andIFN-γ. In contrast, IL-12 failed to induce phosphorylation of anySTAT molecules. Consistent with the common inhibitory effect onPG production from HK cells [10], IL-13 induced phosphorylation ofSTAT1, STAT3, and STAT6 in similar patterns to IL-4. Because weused culture supernatants that contained recombinant IL-4, weadopted an IL-4-blocking antibody in the experiment. The cytokinespecificity of IL-4 was proven by the results that IL-4-neutralizing an-tibody completely abolished the activity of IL-4 but not IL-13 (Fig. 2B).

STATs are tyrosine-phosphorylated by JAKs. To determine the JAKthat phosphorylates STATs upon IL-4 stimulation, the four JAK family

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y TGF-β. (A–C) Immunoblots of HK cells cultured with indicated stimuli. (A) HK cellsg/ml) for 24 h, followed by a further incubation with or without TGF-β (1 ng/ml) oroblotting. β-actin was used to show equal loading of lysates. (B) HK cells were cultured8 h. (C) HK cells were treated with IL-4 (100 U/ml) for the specified period before stim-are shown. (D) HK cells were cultured in the presence or absence of IL-4 (100 U/ml) orpression levels of COX-2 mRNA were assessed by a real-time RT-PCR analysis. Means±s (**pb0.01, ***pb0.001, NS, non-significant).

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Fig. 2. IL-4 induces tyrosine phosphorylation of STAT1, 3, and 6 in HK cells. (A) HK cells (3×105 cells) or peripheral blood mononuclear cells (1×105 cells) were cultured in thepresence or absence of IL-4 (100 U/ml), IL-12 (1 ng/ml), IL-13 (5 ng/ml), IFN-α (1 ng/ml), or IFN-γ (1 ng/ml) for 30 min. The effects of each cytokine on tyrosine phosphorylationof STAT proteins were evaluated by immunoblotting. (B) HK cells were stimulated with IL-4 or IL-13 for 30 min, and the effects on tyrosine phosphorylation of STAT1, STAT3, andSTAT6 were evaluated by immunoblotting. Anti-IL-4 function-blocking antibodies and IL-4 or IL-13 were pre-incubated for 30 min at 37°C before addition to HK cells. Representa-tive results of at least three reproducible experiments.

638 W. Cho et al. / International Immunopharmacology 12 (2012) 635–642

proteins were efficiently silenced by siRNA technology (Fig. 3A). In astark contrast to JAK2, JAK3, and TYK2, the silencing of JAK1 almostcompletely prevented IL-4-induced phosphorylation of STAT1,STAT3, and STAT6 (Fig. 3B), suggesting the essential role for JAK1 inIL-4 signaling in HK cells. Next we examined whether those threeSTATs are all involved in the inhibitory effect of IL-4. As shown inFig. 4, TGF-β up-regulated COX-2 expression in HK cells that weretransfected with control siRNA, which was inhibited by IL-4 almostto background levels. However, the inhibitory effect of IL-4 was notobserved when JAK1 or STAT6 were silenced, indicating the essentialrole for these proteins. In contrast, the inhibitory effect of IL-4 was vi-able in HK cells that were transfected with STAT1 or STAT3 siRNAs.This result implies that STAT1 and STAT3 may participate in other

siRNA control JAK1

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Fig. 3. IL-4-induced phosphorylation of STAT1, STAT3 and STAT6 depends on JAK1 in HK ccultured in the presence or absence of IL-4 (100 U/ml) for 30 min. (A) The efficiency of targeby immunoblotting. Representative results of at least three reproducible experiments.

activities of IL-4 than COX-2 inhibition in HK cells. It is interestingto note that TGF-β-stimulated COX-2 up-regulation was inhibitedconsistently when STAT6 was silenced in HK cells. The results that aprolonged pretreatment of HK cells with IL-4 up to 24 h resulted ina more evident inhibitory effect (Fig. 1C) suggest that IL-4 inhibitsTGF-β signals by inducing transcription of certain proteins in the nu-cleus. As an initial test of this hypothesis, we performed immunoblot-ting and confocal microscopic analyses to examine whetherphosphorylated STAT6 (P-STAT6) indeed migrates to the nucleus.STAT6 proteins were distributed in both cytoplasm and nucleus ofHK cells as unphosphorylated forms prior to IL-4 stimulation(Fig. 5A and B). STAT6 proteins became phosphorylated and were ob-served in the nucleus as early as 5 min after IL-4 addition. The levels

siRNA control TYK2siRNA control JAK3

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ells. Control, JAK1, JAK2, JAK3, or TYK2 siRNA-transfected HK cells (3×105 cells) weret depletion and (B) the effects on tyrosine phosphorylation of three STATs were assayed

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Fig. 4. JAK1-STAT6 pathway is involved in the inhibitory activity of IL-4 in TGF-β-induced COX-2 augmentation. (A–C) Immunoblots of control or target siRNA-transfected HK cells.(A) Control, JAK1, or STAT6 siRNA-transfected HK cells (1×105 cells) were cultured in the presence or absence of IL-4 (100 U/ml) for 24 h, followed by a further incubation with orwithout TGF-β (1 ng/ml) for 8 h. (B, C) Control, STAT1, STAT3, or STAT6 siRNA-transfected HK cells were cultured as described in (A). β-actin was used to demonstrate equal loadingof lysates. The results were reproducible in at least three independent experiments.

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of P-STAT6 were maintained until 30 min and then gradually de-clined. The staining by anti-STAT6 and anti-P-STAT6 was specific be-cause staining by control antibody was negative (data not shown).These results imply that STAT6 is an important transcription factormediating the inhibitory IL-4 signal in HK cells.

3.3. IL-4 suppresses PG production via JAK1-STAT6 pathway

Based upon the results that JAK1-STAT6 axis conveys IL-4 signalsin HK cells, we asked whether JAK1 and STAT6 proteins indeedwould mediate IL-4 signals leading to the ultimate suppression ofPG production. These molecules were knocked down by siRNA tech-nology, and the effects on PG production were evaluated by EIA kits.HK cells that were transfected with control siRNA responded toTGF-β by giving rise to 4.3- and 1.5-fold increases of PGE2 and 6-keto-PGF1α, respectively. This PG induction was almost completelyprevented by IL-4 pretreatment (Fig. 6), in line with the potent inhi-bition of COX-2 expression by IL-4 (Fig. 1). TGF-β stimulation of HKcells that were transfected with JAK1 siRNA also resulted in 3.5- and1.5-fold increases of PGE2 and 6-keto-PGF1α, respectively. However,IL-4 failed in inhibiting the PG induction in these cells. After transfec-tion of HK cells with STAT6 siRNA, we observed that the production ofPGE2 and 6-keto-PGF1αwas severely diminished. The concentrationsof PGE2 and 6-keto-PGF1α were only 30% and 17%, respectively, ofthose in control cells. These results are compatible with the impairedexpression of COX-2 in HK cells transfected with STAT6 siRNA (Fig. 4).Nevertheless, the addition of TGF-β gave rise to 10.3-fold and 6.2-foldincreases of PGE2 and 6-keto-PGF1α, respectively, which was unaf-fected by IL-4 pretreatment. Taken together, our data indicate thatJAK1 and STAT6 are the early signaling molecules of IL-4 leading tothe suppression of PG production in HK cells.

4. Discussion

TGF-β was used in this study to induce PG production. TGF-β is awell-known cytokine that displays pleiotropic effects on the immunecells by regulating their generation, survival, proliferation, differenti-ation, and apoptosis [16,19,20]. The cellular sources of TGF-β includeGC T cells [21] as well as Foxp3+ Treg cells, CXCR5+ T follicular helper(Tfh) cells, interleukin-10 (IL-10)-producing T regulatory-1 (Tr1)cells, DCs, stromal cells, B cells, and endothelial cells [22]. The immu-noregulatory impact of TGF-β on GC B cells has been demonstratedpreviously. For example, it is the best characterized switching factorto IgA isotypes [23,24]. Strong increase of smad1 that is involved inTGF-β signaling is observed in GC B cells [25]. TGF-β also induces ap-optosis in human centroblasts [26]. Although Lee et al. suggested aprotective role for TGF-β in the apoptosis of FDC [27], the physiolog-ical importance of TGF-β in FDC functions is unclear. Using an in vitroexperimental model containing primary FDC-like cells, we suggestedthat TGF-β induces PG production from FDC and then secreted PG in-hibits proliferation and apoptosis of T cells [10]. PG also enhances theAPC capability of B cells by increasing CD86 expression levels [9]. Theimmunostimulatory effect of TGF-β on FDC appears to be counterba-lanced by IL-4 in the manner as presented in the current investiga-tion. However, TGF-β may have a dominant effect over IL-4 on FDC,considering the findings that FDC requires pretreatment with IL-4 todisplay its inhibitory function and that the simultaneous stimulationwith TGF-β and IL-4 results in only a partial inhibitory activity. Thedominant effect of TGF-β over IL-4 was also reported in other im-mune cells [28].

We have herein shown that STAT1, 3, and 6 are tyrosine-phosphorylated upon IL-4 stimulation and the phosphorylation ofthese three molecules is abrogated by JAK1 knockdown. But the

A

B

Fig. 5. IL-4 induces nuclear translocation of phosphorylated STAT6 in HK cells. (A) HK cells (3×105 cells) were cultured in the presence or absence of IL-4 (100 U/ml) for indicatedtime periods. Collection of cytosol and nuclear fractions of cultured cells and subsequent immunoblotting were carried out as described in Materials and methods. Representative ofthree reproducible experiments. (B) HK cells (1×104 cells) were cultured in the presence of IL-4 (100 U/ml) for indicated time periods and then analyzed by a confocal microscopeafter staining with FITC-conjugated anti-STAT6 or anti-tyrosine phosphorylated STAT6 antibodies and propidium iodide. Scale bars, 50 μm. Representative results of two reproduc-ible experiments.

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phosphorylation degrees of STAT1 and STAT3 were much weakerthan that of STAT6, and knockdown of these two proteins did notaffect the inhibitory activity of IL-4 on COX-2 upregulation. These re-sults indicate that IL-4 signaling to PG regulation involves the JAK1-STAT6 pathway but does not utilize STAT1 and STAT3 proteins. Thenwhat molecule mediates IL-4 signals between STAT6 and COX-2?We do not know the identity yet, but it may be a molecule newlytranscribed through the action of phosphorylated STAT-6 (P-STAT6).Supportive of this speculation, pretreatment is required for IL-4 to ex-hibit the complete inhibition of COX-2 upregulation. In addition,P-STAT6 accumulated in the nucleus right after IL-4 stimulation. Re-garding the biological function of STAT6, it is worthwhile to notethe observations that unphosphorylated STAT6 (U-STAT6) moleculeswere dispersed in the nucleus as well as in the cytoplasm of HK cellsbefore IL-4 stimulation and that STAT6 knockdown drastically

disturbed TGF-β-stimulated COX-2 upregulation and background PGproduction. These observations imply that U-STAT6 plays a role inthe nucleus distinct from that of P-STAT6 because P-STAT6 accumu-lates in the nucleus only after IL-4 addition. U-STAT6 may contributeto the constitutive expression of COX-2 proteins at the backgroundlevels, explaining why STAT6 knockdown hampers COX-2 upregula-tion after TGF-β stimulation. This is specific to STAT6 and TGF-β-induced COX-2 upregulation since TGF-β-induced COX-2 upregula-tion is not disturbed by silencing of STAT1, STAT3, or JAK1 andSTAT6 depletion does not affect COX-2 upregulation that is inducedby LPS [11]. In contrast, JAK1 depletion disturbs LPS-stimulatedCOX-2 induction [11], suggesting that JAK1 and STAT6 appear to beinvolved in LPS- or TGF-β-stimulated PG production in distinct man-ners. TGF-β does not induce phosphorylation of STAT6 in HK cells(our unpublished observations). Taken together, P-STAT6 and

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Fig. 6. IL-4 suppresses TGF-β-stimulated prostaglandins production in HK cells via JAK1-STAT6 pathway. Control, JAK1, or STAT6 siRNA-transfected HK cells (1×104 cells) werecultured in the presence or absence of IL-4 (100 U/ml) for 24 h, followed by a further incubation with or without TGF-β (1 ng/ml) for 48 h. The amounts of PGE2 and 6-keto-PGF1α in the culture supernatants were measured by EIA. Representative results of at least three reproducible experiments. Asterisks indicate significant differences (**pb0.01,***pb0.001, NS, non-significant).

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U-STAT6 appear to have opposing roles in the expression of COX-2; P-STAT6 inhibits COX-2 expression whereas TGF-β-induced COX-2upregulation depends on the presence of U-STAT6. In line with ourspeculation, Cui et al. reported that U-STAT6 associates with p300and binds to the COX-2 GAS element, resulting in constitutive COX-2 transcription in human non-small cell lung cancer cells [29]. Thecontribution of unphosphorylated STAT3 to oncogenesis has been ob-served [30]. Our study implies that U-STATs may play significant rolesalso in normal cells.

In summary, our study provides evidence that JAK1 and STAT6may be universal mediators of IL-4 in the suppression of PG produc-tion in human FDCs. Both molecules are required for the inhibitoryactivity of IL-4 irrespective of PG production stimuli; TGF-β or LPS.Our findings reveal that IL-4 represses TGF-β- or LPS-induced COX-2expression via JAK1-STAT6 pathway, subsequently leading to the sup-pression of PG production. The cellular and molecular mechanismsrevealed here suggest that T cells play an important role in the regu-lation of PG production from FDC by timely providing counteractingcytokines, TGF-β and IL-4, in line with the recent reports on the cru-cial impact of follicular Treg on GC reactions by several investigators[31–33].

Acknowledgments

This work was supported by grants of the Korean Ministry of Edu-cation, Science and Technology (The Regional Core Research

Program/Medical & Bio-Materials Research Center and the Basic Re-search Promotion Fund No. 2011-0022077).

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