Experimental and Molecular Pathology 87 (2009) 54–58

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Experimental and Molecular Pathology

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Interleukin-10 and interferon-γ up-regulate the expression of B-cell activating factorin cultured human promyelocytic leukemia cells

Lin Zhou ⁎,1, Renqian Zhong ⁎,1, Wanying Hao, Hao Wang, Xiaoxia Fan, Lingzhen Zhang, Qingmei MiDepartment of Laboratory Medicine, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shaghai 200003, PR China

⁎ Corresponding authors. Fax: +86 21 33110236.E-mail addresses: gracelin_zhou@yahoo.com.cn (L. Z

(R.Q. Zhong).1 These authors contributed equally to this article.

0014-4800/$ – see front matter. Crown Copyright © 20doi:10.1016/j.yexmp.2009.04.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 December 2008Available online 22 April 2009

Keywords:CytokinesB-cell activating factorHuman promyelocytic leukemia cellsGenesPromoter

B-cell activating factor (BAFF) is a potent cell-survival factor, expressed in many hematopoietic cells, for B-cell maturation and activation. It is involved in pathogenesis of autoimmune disorders and B-cellmalignancies. Although BAFF is produced by interferon-γ (IFN-γ) and interleukin-10 (IL-10) in monocytes,the mechanisms of the modulation of BAFF production and expression under normal and pathologicconditions have not been completely elucidated. In this study, we examined the effects of severalinflammatory cytokines on BAFF expression in cultured human promyelocytic leukemia (HL-60) cells both atthe transcriptional and posttranscriptional level. Incubation of the cells with IL-10 and IFN-γ elevated theexpression of membrane-bound and soluble forms of BAFF. A similar increase in BAFF-specific mRNA wasnoted in cultured cells. Unexpectedly, interleukin-4 (IL-4) treatment hardly affected BAFF expression at themRNA and protein levels. Transcriptional regulation was examined in cultures transfected with a humanBAFF promoter/reporter gene (chloramphenicol acetyltransferase) construct. IL-10 and IFN-γ elicitedmarked enhancement of the human BAFF promoter activity. Collectively, these results demonstrated that IL-10 and IFN-γ both regulate BAFF expression and synthesis in human promyelocytic leukemia cell cultures,and the activation occurs at the transcriptional level.

Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved.

Introduction

B-cell activating factor (BAFF) has emerged since 1999 as one of thecritical factors controllingB-cellmaturation, tolerance andmalignancy. Itexists both as a type II membrane-bound protein (mBAFF) and as asoluble protein (sBAFF) (Moore et al., 1999; Ng et al., 2005). BAFF exertsits effects by binding to three receptors, known as B-cell maturationantigen (BCMA), transmembrane activator and calcium modulator andcyclophilin ligand interactor (TACI) andBAFFreceptor (BAFF-R) (Grossetal., 2000; Thompson et al., 2001). Reports in literature suggest that BAFFplays a key role in the pathogenesis of B-cell-dependent autoimmunedisorders. BAFF has also implicated in various B-cell malignancies, suchas myeloma and lymphoma (Cancro, 2006; Sutherland et al., 2006).

A number of cell types have the capacity to express BAFF, includingnot only monocytes, macrophages, and dendritic cells (DCs), but alsohematopoietic tumor cell lines and malignant B cells. Initial reportshave shown that BAFF was produced by monocytes upon stimulationwith interferon-γ or interleukin-10. Thus, interleukin-4 slightly inhibitsthe expression of BAFF inmonocytes (Nardelli et al., 2000; Scapini et al.,2003). In general, the mechanisms of the regulation of BAFF expressionby these factors are not well delineated. Furthermore, it is not known if

hou), rqzhong@yahoo.com

09 Published by Elsevier Inc. All rig

these inflammatory cytokines also modulate BAFF expression andproduction in human promyelocytic leukemia (HL-60) cells in culture.

In this study, we investigated the regulation of BAFF expressionboth at the mRNA and protein levels in response to IL-10, IFN-γ, andIL-4 in HL-60 human leukemia cells. Specifically, we have examinedthese inflammatory cytokines for their effects on human BAFFpromoter activity in HL-60 cells.

Materials and methods

Cell culture

The human HL-60 promyelocytic leukemic cell line was main-tained in RPMI-1640 medium supplemented with 10% heat-inacti-vated fetal calf serum (FCS), penicillin and streptomycin (Gibco BRL,Rockville, MD, USA) at 37 °C in a humidified atmosphere of 5% CO2 and95% air. The cells cultured in passages 3–8 were used for study.

Effects of cytokines

HL-60 cells were seeded at 2×105/well in a 6-well plate (Corning,New York, USA) in 2ml RPMI-1640. The cells were incubated until 60–80% confluence was reached. The medium was then replaced withfresh culture medium without FCS 24 h before adding the cytokines.Thereafter, recombinant human (rh) cytokines including rhIL-10 (100ng/ml), rhIFN-γ (5 ng/ml) and rhIL-4 (100 ng/ml), purchased from R

hts reserved.

55L. Zhou et al. / Experimental and Molecular Pathology 87 (2009) 54–58

and D Systems (Minneapolis, MN, USA), were added to duplicatecultures, respectively. The dosages of cytokines used were similar tothose in the published data and were confirmed in our preliminarystudy, and they demonstrated that concentration was very importantin stimulating or inhibiting cell proliferation with least toxicity(Nardelli et al., 2000). Control cells incubated in parallel receivedculturemediumwithout the cytokines. After incubation for 24, 48, and72 h, the cells were ready for BAFF expression measurement.

Flow cytometry for membrane-bound BAFF

Cultured cells in various conditions were washed with Hank'sbalanced salt solution (HBSS) and resuspended in phosphate bufferedsolution (PBS, 0.1 mol/L, pH 7.4). According to the manufacturer'sinstructions, 20 μl phycoerythrin (PE)-conjugated anti-human BAFFantibody (eBioscience, San Diego, CA, USA) was added to the cellsuspension and the cells were placed at 4 °C for 20 min. Cells werewashed, resuspended in fixing buffer, and analyzed using a flowcytometer and associated Cell Quest software (both from BeckmanCoulter, Fullerton, CA, USA). For all cells, two fluorescence values wereobtained and averaged.

ELISA for soluble BAFF

Cell-free supernatants from 3 days' cultures in the presence orabsence of cytokines were harvested. A sandwish ELISA assay was usedto quantify soluble BAFF protein as previously described withmodifica-tion assay (Zhou et al., 2007). Briefly, 96-well Nunc-Immuno ELISAplates (Nunc, Roskilde, Denmark) coated 2 μg/ml monoclonal anti-Human BAFF antibody (PeproTech, Rocky Hill, NJ, USA) were incubatedovernight at 4 °C and then blocked with 1% BSA (Hua Mei Biotech,Beijing, China). Supernatant samples or standard protein: recombinanthuman BAFF (PeproTech, Rocky Hill, NJ, USA), were diluted, added induplicate and incubated for 4 h at 37 °C. Biotinylated anti-Human BAFFpolyclonal antibody (PeproTech, Rocky Hill, NJ, USA) and streptavidinHRP (Serotec, Raleigh, NC, USA) was added in sequential steps. Colordevelopment achieved in the dark using a substrate reagent pack (R andDSystems,Minneapolis,MN,USA)was stoppedwith 1MH2SO4 at roomtemperature. Plates were read at 450-nm absorbance using Model 550microplate reader system for ELISA (Bio-Rad, Hercules, CA, USA).

Real-time PCR for BAFF mRNA

Messenger RNAwas extracted and isolated from cultured cells usinga Qiagen Rneasy minikit (Qiagen, Hilden, Germany). The concentrationand purity of the extracted RNA were determined by the OD260/OD280

ratiousingUV light spectrophotometry (DU640, Beckman, Fullerton, CA,USA). Real-time PCR for BAFF mRNA was carried out as describedpreviously (Zhou et al., 2007). Briefly, the extracted RNA obtained fromcultured cells in various conditionswas amplified in duplicate using ABIPRISM 7000 Sequence Detection Systems (PE Biosystems, Foster City,CA, USA). And a standard curve was created for each plate using serialplasmid standard (pMD18-T-BAFF) dilutions. With the help of thestandard curve, threshold cycle (Ct) values, indicating the cycle atwhichamplification enters the exponential phase of PCR, were used todetermine the corresponding mRNA quantities in each sample. Cellsampleswith a Ct valuegreater than40were excluded from the analysis.Analysis was performed using ABI PRISM 7000 SDS Software (PEBiosystems, Foster City, CA, USA). Results were normalized for internalcontrol gene, glyceraldehydes-3-phosphate-dehydrogenase (GAPDH),steady-state expression.

Cloning of the human BAFF promoter

A healthy volunteer donor was recruited from the blood bank atChangzheng Hospital, Shanghai, China. Genomic DNA from this

individual was purified from peripheral blood leukocytes using theQIAamp blood kit (Qiagen, Hilden, Germany). The 1020 bp fragment ofthe human BAFF promoter containing sequences from −1349 to−329 bp was obtained by routine PCR with this genomic DNA as thetemplate, using a high-fidelity PCR kit (Qiagen, Hilden, Germany)and a PE-9600 PCR amplifier (PE Biosystems, Foster City, CA, USA).Two gene-specific primers were designed from the published humanBAFF promoter sequence (GenBank accession number: AB073224):5′-AGCCTGGGTCTGGAGTTC-3′ and 5′-ACTTGGAGTTTGGATTGGC-3′.After purification, the PCR product was cloned into the pMD18-TVector (Promega, San Luis Obispo, CA, USA). Sequence analysisconfirmed that the plasmid pMD18-T-BAFF-promoter overlapped thepromoter region of the human BAFF gene.

Construction of plasmids

Plasmid pCAT3-enhancer (Promega, San Luis Obispo, CA, USA), apromoterless vector that contains SV40 enhancer and chlorampheni-col acetyltransferase (CAT) as the reporter gene, was used as therecombinant plasmid backbone. The putative promoters in fiveconstructions, phBAFF1, phBAFF2, phBAFF3, phBAFF4 and phBAFF5,corresponded to sequences−489 to−329 bp,−719 to 329 bp,−929to−329 bp,−1122 to−329 bp, and−1349 to−329 bp, respectively,in the human BAFF promoter regionwith the same 3′ ends. They wereobtained by PCR with plasmid pMD18-T-BAFF-promoter as thetemplate.

Antisense primers, 5′-AGCCTACTCGAGACTTGGAGTTTGGATTGGC-3′,were the same for the five putative promoters.

The sense primers for PCR were as follows:

phBAFF1: 5′-ATGTCTACGCGTTCCTGCCTTATCCTGATGG-3′,phBAFF2: 5′-ATGTCTACGCGTGAGAATCAGCCTTGCCTC-3′,phBAFF3: 5′-ATGTCTACGCGTAAGGAAAACATGCATAAAC-3′,phBAFF4: 5′-ATGTCTACGCGTACCACATGGCTTCATAAG-3′, andphBAFF5: 5′-ATGTCTACGCGT AGCCTGGGTCTGGAGTTC-3′.

The sense and antisense primers containedMluI and XhoI adaptorsthat were 6 bases away from the 5′ end of the primers. Routine PCRwas performedwith a high-fidelity PCR kit (Qiagen, Hilden, Germany)and a PE-9600 PCR amplifier (PE Biosystems, Foster City, CA, USA). ThePCR products were digested with MluI and XhoI and ligated to theMluI–XhoI linearized pCAT3 vector with T4 DNA ligase (Promega, SanLuis Obispo, CA, USA). The ligation mixtures were transformed tocompetent Escherichia coli (JM109), and the ampicillin-resistantpositive clones were further identified by small-scale restrictionenzyme digestion and DNA sequencing (ABI 377). The correct cloneswere amplified, and recombinant plasmids were extracted andpurified with a plasmid isolation kit (Qiagen, Hilden, Germany). Thepurity and yield rate were determined by ultraviolet (UV) spectro-photometer (DU640, Beckman, Fullerton, CA, USA).

Promoter transfection and cytokines treatment

DNA transfection was performed with FuGENE 6 transfectionreagent (Roche Applied Science, Mannheim, Germany) according tothe manufacturer's instruction. Briefly, the day before transfection,HL-60 cells were seeded at 5×105/well in a 6-well plate (Corning,New York, USA) in 2ml RPMI-1640. The cells were incubated until 60–80% confluence was reached. The medium was then replaced withfresh culture medium without FCS shortly before adding thetransfection reagent. Two micrograms of each plasmid, phBAFF1,phBAFF2, phBAFF3, phBAFF4, phBAFF5, and pCAT3 basic-vector,together with 1 μg of pSV β-gal (Promega, San Luis Obispo, CA, USA)as an internal standard were cotransfected. The CAT expressionplasmid, pCAT3-Control (Promega, San Luis Obispo, CA, USA), was alsotransfected as a CAT expression positive control. Twenty-four hours

Fig. 2. Effects of cytokines on BAFF secretion from the HL-60 cells. The amount ofreleased BAFF from the HL-60 cells was determined by ELISA in the medium after 24 hto72 h of cytokines treatment. ⁎, Pb0.05.

56 L. Zhou et al. / Experimental and Molecular Pathology 87 (2009) 54–58

after the transfection, cells were starved for 4 h, then culture mediumwas replaced by fresh medium containing 10% FCS. Cytokinesincluding IFN-γ 5 ng/ml, IL-10 100 ng/ml or IL-4 100 ng/ml wereadded to duplicate cultures, respectively. Control cells incubated inparallel received culture mediumwithout the cytokines. Another 24 hlater, the transfections were terminated and ready for reporter genedetection.

Determination of CAT and β-galactosidase

Forty-eight hours after initial transfection, cells were washed withprecooled phosphate buffered solution (PBS, 0.1 mol/L, pH 7.4) andlysed with lysis buffer (Roche Applied Science, Mannheim, Germany).Aliquots of cell extracts were made for protein determination in a UVspectrophotometer (Du 640, Beckman). CAT was measured by ELISA(Roche Applied Science, Mannheim, Germany) and the β-galactosi-dase enzyme activity analysis method (Promega, San Luis Obispo, CA,USA).

Statistical analysis

Results were expressed as mean±SD. Data were analyzed by thepaired or unpaired t-test, ANOVA and linear regression using SAS 6.12software. Theprobability values less than0.05were consideredsignificant.

Results

Effects of cytokines on membrane-bound BAFF in HL-60 cells

HL-60 human leukemia cells were treated with rhIL-10 (100 ng/ml), rhIFN-γ (5 ng/ml), and rhIL-4 (100 ng/ml) for 24, 48, and 72 h,respectively. Flow cytometry analysis for the levels of cell surface-associated BAFF (mBAFF) in the cultures showed that mBAFFexpression was increased by IL-10 after 24 h, and reached a peak at48 h (Pb0.05). These phenomena were also detected by IFN-γstimulation under theses conditions (Fig. 1). In contrast, IL-4treatment failed to change the expression of mBAFF on HL-60 cells.

Effects of cytokines on soluble BAFF in HL-60 cells

To investigate the effects of these inflammatory cytokines on BAFFsecretion, we tested the levels of soluble BAFF (sBAFF) in the culturemedia by ELISA. As shown in Fig. 2, IFN-γ treatment caused over 2-foldincrease in sBAFF production, while IL-10 treatment induced less than

Fig.1. Expression of membrane-bound BAFF (mBAFF) on the HL-60 cells. Following 24 hto 72 h treatment with cytokines, levels of mBAFF expression on the HL-60 cells wereanalyzed by flow cytometry. Mean fluorescence values were normalized with theisotype control. ⁎, Pb0.05.

a 2-fold increase in BAFF release. In addition, IL-4 treatment slightlyinhibited the BAFF level found in the culture media (Pb0.05).

Effects of cytokines on BAFF mRNA in HL-60 cells

To determine whether the changes in BAFF protein levels resultedfrom the regulation of endogenous BAFF transcripts, real-time PCRwas conducted with mRNA from the cultures stimulated with thecytokines for 24–72 h. After incubation for 24 h, IL-10 produced thehighest amount of BAFF-specific mRNA in HL-60 cells in the GAPDHadjustment methods (Fig. 3). Similar upregulation of BAFF mRNAlevels could be observed in cultures following IFN-γ stimulation(Pb0.05). Unexpectedly, IL-4 treatment did not affect BAFF mRNAlevels in HL-60 cells.

Cloning and construction of the human BAFF promoter

The nucleotide sequences of the human BAFF promoter have beenpreviously reported (Kawasaki et al., 2002). Therefore, we cloned a1020 bp fragment of the 5′-flanking region of the human BAFF gene(−1349 to −329 bp). Fig. 4 shows the correct digestion of the seriesof CAT constructs with 5′ end points from −1349 bp to−329 bp thatwere prepared to analyze sequences potentially important forregulation of BAFF gene expression. The putative promoters in theconstructions were 160 bp, 390 bp, 600 bp, 793 bp and 1020 bp,corresponding to −489 to −329 bp, −719 to 329 bp, −929 to−329 bp,−1122 to−329 bp, and−1349 to−329 bp, respectively, ofthe human BAFF promoter gene. DNA sequencing indicated that the

Fig. 3. BAFF mRNA levels in the HL-60 cells. HL-60 cells were cultured for 24 h to 72 hwith or without cytokines. The levels of endogenous BAFF transcripts were detected byquantitative PCR, and shown relative to the expression of the internal control gene.⁎, Pb0.05.

Fig. 4. Electrophoresis of five constructs digested with MluI and XhoI. M1, M2: widerange DNA marker (TaKaRa Biotech, Dalian, China), Lane 1: phBAFF1 (160 bp), Lane 2:phBAFF2 (390 bp), Lane 3: phBAFF3 (600 bp), Lane 4: phBAFF4 (793 bp), Lane 5:phBAFF5 (1020 bp). Five recombinant plasmids containing serial 5′-deleted flankingsequences of human BAFF gene as putative promoter were digested with MluI and XhoIat 37 °C for 2 h. The digested DNAs were fractionated on 15 g/L agarose gel showingvector DNA (4.3 kb) and insertion promoters with different sizes.

57L. Zhou et al. / Experimental and Molecular Pathology 87 (2009) 54–58

inserted sequences of the putative promoters were the same as thosepublished in GenBank (accession number AB073224).

Effects of cytokines on BAFF promoter activity

To explore whether IL-10, IFN-γ, and IL-4 upregulated BAFF geneexpression at the transcriptional level, we have examined the effectsof cytokines on human BAFF promoter activity. Transient transfectionsof the HL-60 cells with a human BAFF promoter/chloramphenicolacetyltransferase (CAT) construct, following by the addition of IL-10(100 ng/ml) 24 h later, resulted in an approximately 2.1-fold elevationof the CAT activity. Similarly, a significant, up to 4.2-fold, increase inthe promoter activity was observed in human HL-60 cells at 24 h ofincubation of with IFN-γ (5 ng/ml) after transfection (Fig. 5). ThoughIL-4 treatment had no effect on promoter activity of human BAFF gene.IL-4 at 100 ng/ml slightly inhibited both IFN-γ-induced and IL-10-induced upregulation of human BAFF promoter activity.

Localization of BAFF promoter regions responsive to IL-10

In order to localize regions within human BAFF promoter that areresponsive to stimulation by IL-10 in the HL-60 cells, the effects of IL-10on the CATactivity in cell transfectedwith promoter deletion constructs

Fig. 5. Effects of cytokines on human BAFF promoter activity. HL-60 cells were transfected“Materials and methods”. Cells were incubated with cytokines, and CAT activity was determgroup of no cytokine treatment.

with 5′ end points at −489 bp, −719 bp, −929 bp, −1122 bp, and−1349 bp were examined. Incubation with IL-10 for 24 h resulted in agreater than 2-fold increase (Pb0.05) in CAT activity driven by theconstructs with 5′ end points at −929 bp, −1122 bp, and −1349 bp,suggesting that IL-10-responsive elements were located within theproximal region of the promoter 3′ to the−929 bp end point (Fig. 6). Incontrast, IL-10 did not stimulate the CAT activity driven by the−719 bppromoter. These results indicated that IL-10-responsive sequences arelocated between−929 and −719 bp of human BAFF promoter.

Discussion

BAFF, belonging to the tumor necrosis factor (TNF) family, has beenmapped to human chromosome 13q34. It is a 285-amino-acidglycoprotein originally expressed as a type II membrane-boundprotein, and then cleaved at a putative furin consensus site andreleased as a soluble protein (Mackay et al., 2003; Batten et al., 2000).The soluble form of BAFF is a critical survival/maturation factor forperipheral B cells and this activity is mediated through its threespecific receptors (Binard and Youinou, 2007; Novak et al., 2004).Overexpression of BAFF has been linked to autoimmune disease andaspects of B-cell neoplasia (Collins et al., 2006; Binard et al., 2008; Taiet al., 2006).

BAFF is restrictedly expressed by cells of myeloid origin. There isevidence that several inflammatory cytokines are involved in theregulation of BAFF expression by monocytes. IFN-γ is among thestrongest cytokines stimulating BAFF expression, while IL-4 isbelieved to be a slight inhibitor. International studies have confirmedthat IFN-γ and IL-10 were able to increase BAFF mRNA expression inmonocytes (Litinskiy et al., 2002; Moon et al., 2006; Fu et al., 2006).However, the regulatory mechanisms responsible for themaintenanceof normal BAFF mRNA expression are not completely understood.Relatively little is known of the modulation of BAFF production andexpression by these cytokines in cultured human promyelocyticleukemia (HL-60) cells. In this context, we have investigated theeffects of cytokines including IFN-γ, IL-10, and IL-4 on the expressionof BAFF in HL-60 human leukemia cells both at the transcriptional andposttranscriptional level.

Following cytokines treatment, we found that IFN-γ and IL-10increased the expression of mBAFF and sBAFF. Further, IFN-γ and IL-10were both confirmed to be the stimulator of BAFF mRNA expression.The enhancement ofmRNA levels reached amaximal value after a 24 hexposure to the cytokines. And the expression of mBAFF showed amaximal level of induction after 48 h of treatment. The results werepartly similar to those reported in monocytes (Zhou et al., 2007). Thisfinding illustrates that, the enhancement of IL-10 and IFN-γ arecoordinately regulated. In this regard, IL-10 might up-regulate BAFFprotein levels through a transcriptional or posttranscriptional

with BAFF promoter constructs (phBAFF1.02) and pCAT3 basic-vector as described inined 24-h later. Data are representative of three experiments. ⁎, Pb0.05 compared to

Fig. 6. Regulation of the human BAFF promoter activity by IL-10. HL-60 cells weretransfected with the−489 bp,−719 bp,−929 bp,−1122 bp, and−1349 bp end pointconstructs, and pCAT3 basic-vector. Following a 24-h incubation with or without IL-10,CAT activity in the cell extracts was determined as described in “Materials andmethods”. Data are representative of three experiments. ⁎, Pb0.05 compared withcontrol of no cytokine treatment.

58 L. Zhou et al. / Experimental and Molecular Pathology 87 (2009) 54–58

mechanism that is sensitive to IFN-γ treatment. We suggest thereforethat IL-10 first promotes the expression of BAFF gene, secondly resultsin production of mBAFF, and sBAFF is then derived from it.

However, we observed that IL-4 treatment failed to change thelevels of BAFF mRNA and mBAFF except for the amounts of sBAFF inthe cultures. Thus, IL-4 was able to down-regulate the concentrationof sBAFF found in the culture. The data suggest that there might beadditional regulatory steps involved in IL-4 treatment. It has beenproposed that sBAFF is produced by enzymatic processing of themembrane-bound protein. Although this cytokine is not sufficient toinhibit the expression of BAFF gene and the synthesis of mBAFF in HL-60 cells. It might alter the concentration or activity of proteolyticenzyme, and therefore down-regulate BAFF secretion.

In order to investigate whether the BAFF-inducing capacities ofcytokines are associated with their transcriptional regulation, weexamined the effect of cytokines on the expression of BAFF promoterconstruct. To date, only one separate promoter has been identified forthe human BAFF gene, and the nucleotide sequences of this BAFFpromoter have been reported (Kawasaki et al., 2002). Thus, we have,in a first step, cloned a 1020 bp fragment of the human BAFF promotercontaining sequences from −1349 to −329 bp and analyzed itspromoter activity. Secondly, using promoter–reporter assays, wefound that the transcriptional activity of BAFF promoter constructwas enhanced by IFN-γ and IL-10, but was non-significantly inhibitedby IL-4. IL-4 can decrease the upregulation of IFN-γ and IL-10 on thischimeric construct. That is, in cultured HL-60 cells, IL-10 and IFN-γcould influence the expression of BAFF through promoter activation.Consistent with these results, it has recently reported that PKA/CREBwas involved in the IFN-γ-induced BAFF expression (Kim et al., 2008).To further localize regions within the human BAFF promoter that areresponsive to stimulation by IL-10 in the HL-60 cells, we examined theeffects of IL-10 on CAT activity driven by various lengths of the 5′-flanking sequences of the human BAFF gene. Our data demonstratedthat IL-10-responsive sequences are both located between −929 and−719 bp from the transcription start site.

In conclusion, we confirm that IL-10 and IFN-γ both regulate BAFFexpression and synthesis in HL-60 cultures, and the activation occur at

the transcriptional level. Thus, IL-4 could inhibit BAFF secretion andslightly suppress the enhancement of human BAFF promoter activityby IFN-γ and IL-10 in the HL-60 cells. However, which signalingpathway was involved in the IL-10-induced BAFF expression needs tobe solved in future studies. Our in vitro studies also raise thepossibility to investigate the molecular mechanisms of BAFF synthesisand expression in other hematopoietic tumor cell lines in the diseasestate.

Acknowledgments

This study was supported by the grants from Shanghai Science andTechnology Development Foundation (07JC14070), and the Out-standing Young Scientist Foundation of Shanghai ChangzhengHospital Project.

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