ELEVATED EXPRESSION OF CCL5/RANTES IN ADULT T-CELL LEUKEMIACELLS:POSSIBLE TRANSACTIVATION OF THE CCL5 GENE BY HUMANT-CELL LEUKEMIA VIRUS TYPE I TAXNaoki MORI

1*, Alan M. KRENSKY2, Koichi OHSHIMA

3, Mariko TOMITA1, Takehiro MATSUDA

1,4, Takao OHTA4, Yasuaki YAMADA

5,Masao TOMONAGA

6, Shuichi IKEDA7 and Naoki YAMAMOTO

8

1Division of Molecular Virology and Oncology, Graduate School of Medicine, University of the Ryukyus, Nishihara, Japan2Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA3Department of Pathology, School of Medicine, Fukuoka University, Fukuoka, Japan4Division of Child Health and Welfare, Faculty of Medicine, University of the Ryukyus, Nishihara, Japan5Division of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan6Department of Hematology, Molecular Medicine Unit, Atomic Bomb Disease Institute, Nagasaki University School of Medicine,Nagasaki Japan7Department of Internal Medicine, Sasebo City General Hospital, Sasebo, Japan8Department of Molecular Virology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan

HTLV-I is the etiologic agent of ATL and of tropical spasticparaparesis/HTLV-I-associated myelopathy. Infiltration ofvarious tissues by circulating leukemic cells and HTLV-I-infected T cells is a characteristic of ATL and HTLV-I-asso-ciated inflammatory diseases. Chemokines play importantroles in migration and tissue localization of various lympho-cyte subsets. Here, we report the highly frequent expressionof CCL5 (RANTES) in ATL and HTLV-I-infected T-cell lines.Among various human T-cell lines, those infected withHTLV-I selectively expressed the CCL5 gene and secretedCCL5. Furthermore, CCL5 was expressed by leukemic cellsin peripheral blood and lymph nodes from patients with ATL.Inducible expression of HTLV-I transcriptional activator Taxin a human T-cell line Jurkat, up-regulated CCL5 mRNA andinduced CCL5 secretion. Analysis of the CCL5 promoterrevealed that this gene is activated by Tax, via the activationof NF-�B, whose responsive element, R(A/B), is located atpositions �71 to �43 relative to the putative transcriptionstart site. Aberrant expression of CCL5 by HTLV-I-infectedT cells may impact on the pathophysiology of HTLV-I-asso-ciated diseases.© 2004 Wiley-Liss, Inc.

Key words: HTLV-I; ATL; CCL5; Tax; NF-�B

HTLV-I is an etiologic agent for ATL, a unique malignancy ofmature CD4-positive T cells and other chronic inflammatory dis-eases such as tropical spastic paraparesis/HTLV-I-associated my-elopathy and HTLV-I-associated uveitis.1–6 The 40 kDa viraltransactivator protein Tax triggers viral transcription as well asinduction of cellular genes involved in cellular proliferation andantiapoptotic signals. Tax can also repress cellular promoters, suchas �-polymerase, p53 and c-Myb.7 These pleiotropic effects of Taxare essential for HTLV-I transformation of human T lymphocytes.Infiltration by leukemic cells of various organs, such as lymphnodes, liver, spleen, lungs, skin and intestinal tract, is a frequentmanifestation of ATL. This type of cell infiltration often posesserious clinical problems for ATL patients, affecting the diseaseprofile and prognosis. Various HTLV-I-associated inflammatorydiseases are also commonly characterized by infiltration of lym-phocytes, including HTLV-I-infected T cells, into target organssuch as central nervous system and eyes.8 Tissue infiltration byATL cells and HTLV-I-infected T cells is most likely a sequentialmultistep process, which may be attributable to biologic propertiesof the cells, such as the deregulated expression of certain chemo-kines, chemokine receptors and adhesion molecules. Deregulatedadhesion molecule expression may enhance ATL cell interactionswith endothelial cells. After transendothelial migration, ATL cellsand HTLV-I-infected T cells encounter the extracellular matrix,where they must pass through the basement membrane and migrateinto the interstitial matrix. Recently, we showed that overexpres-

sion of MMP-9 in HTLV-I-infected T cells may be in part respon-sible for the invasiveness of ATL cells.9

Chemokines have been divided into the 2 major subfamilies onthe basis of the arrangement of the 2 N-terminal cysteine residues,CXC and CC, depending on whether the first 2 cysteine residueshave an amino acid between them (CXC) or are adjacent (CC). Thefunctions of chemokines include attracting blood leukocytes tosites of inflammation,10,11 regulating leukocyte maturation, traf-ficking and homing and the development of lymphoid tissues.12,13

All chemokines transduce signals through 7 transmembrane Gprotein-coupled receptor. It is thus conceivable that chemokinesand their receptors play important roles in tissue infiltration ofATL cells and HTLV-I-infected T cells. Previously, it has beenshown that increased surface expression of CCR7, the sharedreceptor for CCL21 (SLC) and CCL19 (ELC), which are bothexpressed in the secondary lymphoid organs,14,15 correlates wellwith lymphoid organ involvement in ATL.16 Recent study also hasdemonstrated highly frequent expression of CCR4 in HTLV-I-infected T-cell lines and fresh ATL cells.17 In addition, HTLV-I-infected T-cell lines express SDF-1 and CXCR4, a receptor forSDF-1, and migrate toward SDF-1.18

CCL5 (RANTES) belongs to the CC chemokine subfamily. It isa potent chemotactic agent for memory T lymphocytes and mono-cytes,19 and is expressed after cellular activation in fibroblasts, T

Abbreviations: ATL, adult T-cell leukemia; EMSA, electrophoretic mo-bility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;HTLV-I, human T-cell leukemia virus type I; IKK, I�B kinase; IL, inter-leukin; MIP, macrophage inflammatory protein; MMP, matrix metallopro-teinase; NIK, NF-�B-inducing kinase; PBMCs, peripheral blood mononu-clear cells; RFLAT, RANTES factor of late activated T lymphocytes;RT-PCR, reverse transcription-PCR; SDF, stromal cell-derived factor;SDS, sodium dodecyl sulfate; TK, thymidine kinase.

Grant sponsor: Japan Society for the Promotion of Science; Grantnumber: 14570988

*Correspondence to: Naoki Mori, Division of Molecular Virology andOncology, Graduate School of Medicine, University of the Ryukyus, 207Uehara, Nishihara, Okinawa 903-0215, Japan. Phone: 81-98-895-1130.�Fax: 81-98-895-1410. E-mail: n-mori@med.u-ryukyu.ac.jp

Received 18 September 2003; Revised 2 February 2004; Accepted 6February 2004

DOI 10.1002/ijc.20266Published online 4 May 2004 in Wiley InterScience (www.interscience.

wiley.com).

Int. J. Cancer: 111, 548–557 (2004)© 2004 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

cells, monocytes, endothelial cells and certain epithelial cells. Inthe present study, we show that among various human T-cell lines,those infected with HTLV-I preferentially express CCL5, and theexpression is at least in part mediated by a transcriptional trans-activator, Tax. Importantly, CCL5 expression was also markedlyincreased in ATL cells in peripheral blood and lymph nodes. Thesedata support the notion that aberrant expression of CCL5 mayimpact on the pathophysiology of ATL and HTLV-I-associatedinflammatory diseases.

MATERIAL AND METHODS

Cell linesHuman leukemic T-cell lines (Jurkat, MOLT-4 and CCRF-

CEM) and HTLV-I-infected T-cell lines (MT-2,20 MT-4,21 C5/MJ,22 SLB-1,23 HUT-1025 and MT-124) were cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal bo-vine serum (HyClone Laboratories, Inc., Logan, UT), 100 U/mlpenicillin and 100 �g/ml streptomycin. JPX-9 and JPX/M (kindlyprovided by Dr. M. Nakamura, Tokyo Medical and Dental Uni-versity, Tokyo, Japan) are subclones of Jurkat cells expressingeither Tax wild type or a nonfunctional Tax mutant, respectively,under the control of the metallothionein promoter.25,26

Patient samplesThe diagnosis of ATL was based on clinical features, hemato-

logic findings and the presence of anti-HTLV-I antibodies inpatient sera. Monoclonal HTLV-I provirus integration into theDNA of leukemic cells was confirmed by Southern blot hybrid-ization in all cases (data not shown). PBMCs from healthy volun-teers or patients with acute-type (cases 1, 3 and 4) or chronic-type(cases 2 and 5) ATL and lymph nodes samples from patients withATL were analyzed. Mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation (Pharmacia LKB, Uppsala, Swe-den) and washed with PBS. Each patient sample contained morethan 90% leukemic cells at the time of analysis. All samples wereobtained after informed consent was received.

Plasmids and transfectionsA series of expression vectors based on the human �-actin

promoter for HTLV-I Tax (p�MT-2 Tax) and its mutants (TaxM22 and Tax 703) were described previously.27,28 I�B��N29 andI�B��N30 (kindly provided by Dr. D.W. Ballard, Vanderbilt Uni-versity School of Medicine, Nashville, TN) are deletion mutants ofI�B� and I�B� lacking the N-terminal 36 amino acids and 23amino acids, respectively. The kinase-deficient K44M IKK�,K44A IKK� and KK429/430AA NIK mutants (kindly provided byDr. R. Geleziunas, Merck and Company, Inc., West Point, PA)have been described previously.31 A series of CCL5 promoterpGL2-luciferase reporter constructs described previously32,33 wereused to map Tax-responsive regions. Transient transfections wereperformed in Jurkat cells by electroporation using 5 � 106 cellsand 5 �g of appropriate reporter and effector plasmids. To nor-malize transfection efficiencies, a TK promoter-driven Renillaluciferase plasmid (pRL-TK, 1 �g; Promega, Madison, WI) wascotransfected as an internal control plasmid. After 24 hr, trans-fected cells were collected by centrifugation, washed with PBS andlysed in reporter lysis buffer (Promega). Lysates were assayed forreporter gene activity with the dual luciferase reporter assay sys-tem (Promega). Luciferase activities were normalized based on theRenilla luciferase activity from pRL-TK.

RT-PCRTotal cellular RNA was extracted with Trizol (Life Technolo-

gies, Gaithersburg, MD) according to the protocol provided by themanufacturer, and the amount of total RNA was determined bymeasuring the absorbance at 260 nm. First-strand cDNA wassynthesized from 1 �g total cellular RNA in a 20-�l reactionvolume using an RNA PCR kit (Takara Shuzo, Kyoto, Japan) withrandom primers. Thereafter, cDNA was amplified for 35 cycles forCCL5, 30 cycles for MMP-9 and 28 cycles for Tax and �-actin.

The oligonucleotide primers used were as follows: for CCL5,sense, 5�-CCGTGCCCACATCAAGGAGTATTT-3� and anti-sense, 5�-CCAGCCTGGGGAAGGTTTTTGTAA-3�;34 for Tax,sense, 5�-CCCACTTCCCAGGGTTTGGACAGA-3� and anti-sense, 5�-CTGTAGAGCTGAGCCGATAACGCG-3�; for MMP-9,sense, 5�-CGCAGACATCGTCATCCAGT-3� and antisense, 5�-GGATTGGCCTTGGAAGATGA-3�; and for �-actin, sense, 5�-GTGGGGCGCCCCAGGCACCA-3� and antisense, 5�-CTCCT-TAATGTCACGCACGATTTC-3�. Product sizes were 311-bp forCCL5, 203-bp for Tax, 406-bp for MMP-9 and 548-bp for �-actin.Cycling conditions were as follows: denaturing at 94°C for 45 sec(for CCL5) or for 30 sec (for Tax and �-actin) or 95°C for 60 sec(for MMP-9), annealing at 57°C for 90 sec (for CCL5), 60°C for30 sec (for Tax and �-actin) or 62°C for 60 sec (for MMP-9) andextension at 72°C for 60 sec (for MMP-9) or for 90 sec (for CCL5,Tax and �-actin). The PCR products were fractionated on 2%agarose gels and visualized by ethidium bromide staining.

Northern blot analysisTotal RNA (20 �g) was subjected to electrophoresis through a

formaldehyde-agarose gel and transferred to a nylon filter. Filterswere prehybridized (in 0.5 M sodium phosphate, 0.1% bovineserum albumin, 7% SDS, 100 �g/ml salmon testis DNA and 100�g/ml yeast RNA) for 2 hr at 65°C. Hybridization was then carriedout in prehybridization buffer overnight containing the following[�-32P]-radiolabeled probes: cDNA of HTLV-I Tax and GAPDH.Radiolabeled probes were generated using a Megaprime DNALabeling System (Amersham, Arlington Heights, IL).

Western blot analysisCells were lysed in a buffer containing 62.5 mM TRIS-HCl (pH

6.8), 2% SDS, 10% glycerol, 6% 2-mercaptoethanol and 0.01%bromophenol blue. Samples were subjected to electrophoresis onSDS-polyacrylamide gels followed by transfer to a polyvinylidenedifluoride membrane and probing with polyclonal antibody toCCL5 (Santa Cruz Biotechnology, Santa Cruz, CA) and monoclo-nal antibody to actin (NeoMarkers, Fremont, CA). The bands werevisualized with the enhanced chemiluminescence kit (AmershamBiosciences, Piscataway, NJ).

CCL5 measurementThe CCL5 content in the culture supernatants was measured by

ELISA (BioSource International, Inc., Camarillo, CA). Cells weresuspended in fresh culture medium at a concentration of 1�105/ml and cultured for 72 hr. The supernatants were then col-lected after centrifugation and stored at 80°C until assayed forCCL5 by ELISA. The concentration of CCL5 was determinedusing a standard curve obtained with recombinant CCL5.

Flow cytometryCells were washed with cell WASH (Becton Dickinson Immu-

nocytometry Systems, San Jose, CA) and incubated for 30 minwith phycoerythrin-labeled mouse monoclonal antibody againstCCR5 (clone 3A9, Pharmingen, San Diego, CA) or control mouseIgG2a (Coulter Immunotech Co, Marseille, France). Cells wereanalyzed on a FACS Caliber (Becton Dickinson) after gating onforward and side scatter to exclude debris and clumps, usingCellQuest software.

Nuclear extracts preparation and EMSANF-�B binding activity to �B elements was examined by

EMSA, as described previously.35 In brief, 5 �g of nuclear extractswere preincubated in a binding buffer containing 1 �g poly-deoxy-inosinic-deoxy-cytidylic acid (Pharmacia, Piscataway, NJ) fol-lowed by addition of [�-32P]-labeled oligonucleotide probes con-taining �B elements (approximately 50,000 cpm). These mixtureswere incubated for 15 min at room temperature. The DNA proteincomplexes were separated on a 4% polyacrylamide gel and visu-alized by autoradiography. To examine the specificity of the �Belement probes, we preincubated unlabeled competitor oligonucle-otides with nuclear extracts for 15 min before incubation with

549ELEVATED CCL5 EXPRESSION IN ATL

probes. The probes or competitors used were prepared by anneal-ing the sense and antisense synthetic oligonucleotides as follows:A site NF-�B of the CCL5 gene, 5�-tcgaATTTTGGAAACTC-CCCTTAGG-3�; B site NF-�B of the CCL5 gene, 5�-tcgaCT-TAGGGGATGCCCCTCAACTG-3� and a consensus AP-1 bind-ing site, 5�-gatcCGCTTGATGAGTCAGCCGGAA-3�. Under-lined sequences represent the NF-�B or AP-1 binding sites. Toidentify NF-�B proteins in the DNA protein complex revealed byEMSA, we used antibodies specific for various NF-�B familyproteins, including p65, p50, c-Rel and p52 (Santa Cruz Biotech-nology), to elicit a supershift DNA protein complex formation.These antibodies were incubated with the nuclear extracts for 45min at room temperature before incubation with radiolabeledprobes.

ImmunohistochemistryA portion of each lymph node, which was stored at 80°C, was

examined using monoclonal antibodies to CCL5 (Pharmingen) andCCR5 (Dako, Copenhagen, Denmark). Paraffin sections were cutand dried overnight at 37°C, dewaxed in xylene and rehydratedusing serial concentrations of ethanol. Heat-mediated antigen re-trieval was performed. The sections were washed in PBS andpreincubated with 10% normal horse serum for the monoclonalantibodies. Slides were then incubated with the primary antibodyand exposed to streptavidin/AP (1:100) (Dako) for an additional 30min. Slides were then stained with naphthol AS-BI phosphate(Sigma Chemicals Co., St. Louis, MO) in 100 ml of 0.2 M PBS(pH 8.2) containing 4% hydrochloric acid and 4% nitric acid.

RESULTS

CCL5 expression in HTLV-I-infected T-cell linesTo determine whether CCL5 expression is deregulated after

HTLV-I infection of T cells, we examined mRNA levels of CCL5in several HTLV-I-infected or uninfected cell lines. The results ofRT-PCR analysis of CCL5 mRNA levels in several T-cell lines areshown in Figure 1a. CCL5 transcripts (311 bp) were detected in all6 HTLV-I-infected T-cell lines (MT-2, MT-4, C5/MJ SLB-1,HUT-102 and MT-1) but were not detected in the uninfected T-celllines (Jurkat, MOLT-4 and CCRF-CEM). CCL5 protein expres-sion was also studied by Western blotting. As shown in Figure 1b,CCL5 was upregulated in HTLV-I-infected T-cell lines. We nextmeasured secretion of CCL5 in the culture supernatants fromHTLV-I-infected and uninfected T-cell lines by ELISA. As shownin Figure 1c, CCL5 was secreted at very high levels by all 6HTLV-I-infected T-cell lines. In contrast, CCL5 was not secretedin uninfected cell lines. CCL5, thus, was not only expressed butalso secreted by HTLV-I-infected T-cell lines. These results dem-onstrate that CCL5 is selectively expressed in HTLV-I-infectedT-cell lines. The HTLV-I-encoded transactivator protein, Tax,activates expression of a number of cellular genes. Thus, to ex-amine whether CCL5 expression correlated with Tax expression,we measured Tax mRNA levels in these T-cell lines by Northernblot analysis. We found that Tax-related mRNA (pX 2.1 kb) wereexpressed strongly in the MT-2, MT-4, SLB-1 and HUT-102 celllines, weakly expressed in C5/MJ and not expressed at all in MT-1.Hybridization with GAPDH probe confirmed comparable RNAloading in these lanes (Fig. 1a). Furthermore, we analyzed theexpression of Tax mRNA by RT-PCR. The expression of TaxmRNA was detected in all HTLV-I-infected T-cell lines, withrelatively low levels found in MT-1 cells, but not in uninfectedT-cell lines. Consistent with Northern blot analysis, Tax proteinwas detected by immunoblot analysis in the 5 HTLV-I-infectedT-cell lines but not in MT-1.36 Thus, there was no obvious corre-lation between the levels of CCL5 expression and secretion, andTax expression among HTLV-I-infected T-cell lines.

CCL5 expression in leukemic cells from ATL patientsTo assess the relevance of our findings, we analyzed the secre-

tion of CCL5 in primary blood cells from ATL patients. As shownin Figure 2a, CCL5 was secreted at high levels by leukemic cells

from ATL patients. In contrast, CCL5 was hardly secreted byPBMCs from healthy volunteers. However, we cannot exclude thepossibility that in vitro cultivation affects the induction of CCL5expression in ATL cells. Therefore, we further carried out immu-nological staining of CCL5 using lymph nodes biopsy samplesfrom ATL patients. Representative results are shown in Figure 2b.CCL5 expression in ATL cases was detected in ATL cells. Toensure the specificity of monoclonal antibody, we performed thestaining with control mouse IgG. No staining could be detectedwith control mouse IgG (data not shown). Furthermore, we per-formed RT-PCR analysis on CCL5 and Tax expression in freshATL cells without cultivation. As shown in Figure 2c, strongCCL5 signals were consistently observed for ATL cells from allpatients. In contrast, a faint CCL5 signal was observed for normalperipheral blood lymphocytes. The Tax transcript was detectablein ATL cells. Collectively, these results indicate that CCL5 isupregulated in ATL cells derived from patients.

Tax induces CCL5 gene expression and secretion in T cellsBecause Tax is known to induce a number of cellular genes, we

next examined whether Tax itself caused up-regulation of CCL5gene expression. To investigate the effects of Tax expression on

FIGURE 1 – Constitutive expression of CCL5 in HTLV-I-infectedT-cell lines. (a) Northern blot analysis of HTLV-I mRNA expressionand RT-PCR analysis of CCL5 and Tax mRNA levels in variousHTLV-I-infected and uninfected T-cell lines. Total RNA was preparedfrom the indicated T-cell lines. Predominant HTLV-I mRNA speciesof 2.1, 4.2 and 8.5 kb were detected in MT-2, MT-4, C5/MJ, SLB-1and HUT-102 cell lines (lanes 1–5). GAPDH and �-actin expressionserved as controls. (b) Western blot analysis of CCL5 expression inHTLV-I-infected T-cell lines. Protein extracts were separated by SDS-16% (for CCL5) or -10% (for actin) polyacrylamide gel electrophore-sis, transferred to a membrane and blotted with either a specificpolyclonal anti-CCL5 antibody or anti-actin antibody (as a proteinloading control). (c) Measurements of CCL5 secreted in the culturesupernatants of human T-cell lines by ELISA. Indicated cell lines wereplated at 1 � 105/ml and culture supernatants were collected after 72hr.

550 MORI ET AL.

CCL5 gene expression, we used JPX-9, which stably carries Taxexpression plasmid, pMAXneo, in which expression of Tax isinducible by the addition of CdCl2.25,26 The level of expression ofTax mRNA in these cells was determined by Northern blot anal-ysis, and expression of the CCL5 gene was assayed by RT-PCR.As shown in Figure 3a, the addition of CdCl2 (20 �M) to theculture medium of JPX-9 cells induced the expression of Taxwithin 5 hr, which persisted until 72 hr after treatment. pMAXneocontains an intron with splice donor and acceptor sites in the Taxcoding region.26 The 2 hybridized bands correspond to spliced andunspliced Tax mRNA, respectively. A concomitant increase in theexpression of CCL5 within 10 hr of treatment with CdCl2 wasobserved in JPX-9 cells. This rapid kinetics of CCL5 induction likeMMP-9 gene indicates that CCL5 activation did not involve the

humoral factors such as cytokines whose production is induced byTax. Although we measured the contents of IL-1� and tumornecrosis factor-� in the culture supernatants of JPX-9 by CdCl2treatment for 10 hr by using ELISA, no cytokine production wasseen (data not shown). The induction of CCL5 could not beattributed to CdCl2 treatment because CCL5 expression was notinduced in JPX/M cells, expressing a nonfunctional Tax mutantprotein, after treatment with CdCl2 (Fig. 3b). These results indicatethat Tax itself is capable of causing elevated expression of theCCL5 gene in Jurkat cells. Furthermore, we measured secretion ofCCL5 in culture supernatants of JPX-9 cells with or without CdCl2treatment (Fig. 3c). Thus, consistent with the ability of Tax toinduce the transcription of the CCL5 gene, the actual CCL5 che-mokine is secreted by Tax-expressing cells.

FIGURE 2 – CCL5 secretion and expression in ATL cells. (a) Measurements of CCL5 secreted in the culture supernatants of PBMCs fromhealthy volunteers and leukemic cells from ATL patients by ELISA. Indicated cells were plated at 1 � 105/ml and culture supernatants werecollected after 72 hr. (b) Immunohistochemical findings of the lymph node biopsy from a patient with ATL. CCL5 exhibited positive stainingin ATL cells. The original magnification was �200 (c) RT-PCR analysis for expression of CCL5 and Tax in normal peripheral bloodlymphocytes and primary ATL cells.

551ELEVATED CCL5 EXPRESSION IN ATL

Tax transactivation of the CCL5 promoter in T cellsActually, CCL5 expression is regulated both translationally and

transcriptionally.37–39 We investigated whether Tax-mediated up-regulation of CCL5 gene expression could directly enhance theactivity of its promoter. Jurkat cells were transiently transfectedwith a reporter gene construct containing the 195 nucleotides ofthe CCL5 upstream regulatory sequences (pGL2-R). Coexpressionof Tax caused a 6.2-fold elevation in the activity of this CCL5-driven reporter construct (Fig. 4b), suggesting that Tax activatesCCL5 gene at transcriptional level. The CCL5 promoter has been

subdivided into 5 regions (A–E) based upon deletion studies andreporter gene assays (Fig. 4a).32,33,40 The A site is very importantfor the promoter activity since point mutations or nonsense nucle-otide replacement decreases the activity by 90%.40,41 The A sitecontains NF-�B-like characteristics (GGAAACTCCC), especiallywhen analyzed on the opposite DNA strand (GGGAGTTTCC),and binds both NF-�B family proteins and RFLAT-1 (also namedKruppel-like factor, KLF-1338).40,42 The B site also contains aconsensus for a nearly palindromic NF-�B-like binding site(GGGGATGCCCC).40 The C site and E site bind nuclear proteinsthat are strongly induced between days 3 and 5 after activation ofnormal peripheral blood T cells.33 To test the relative contributionsof these 4 regions to Tax-mediated activation of CCL5, plasmidswith deletions in these regions of the CCL5 promoter were trans-fected (Fig. 4b). Deletion of either the A site, the B site or bothsites abolished Tax-mediated activation of this reporter construct.When the C or E sites were deleted, Tax-mediated induction wasnot significantly impaired, indicating that these sites mainly do notaffect Tax-mediated CCL5 induction in T cells. Thus, only the Aand B sites, but not the E and C sites, are major Tax-responsiveelements within the CCL5 transcriptional regulatory sequences.This was confirmed with a reporter gene construct containing only3 A/B sites. Coexpression of Tax significantly induced CCL5 geneexpression when pGL2-SV40-(AB)3-luciferase construct was usedfor the transfection.

Tax can stimulate transcription through distinct transcriptionfactors, including cAMP-responsive element-binding protein andNF-�B. Next, through the use of 2 additional Tax mutants thatselectively retain the ability to activate the cAMP-responsive ele-ment within the HTLV-I long terminal repeat (M22) or NF-�B(703),27,28 we investigated whether Tax-mediated activation ofNF-�B was required for induction of the CCL5 promoter in Tcells. Whereas wild-type Tax and the 703 mutant, which couldactivate NF-�B, increased CCL5-driven reporter gene activity, nosignificant activation of this reporter was observed with the M22mutant (Fig. 4c). Therefore, NF-�B activation contributes to acti-vation of the CCL5 promoter by Tax.

Dominant interfering components of the NF-�B pathway inhibitTax-mediated transactivation of CCL5 gene expression

We next examined whether Tax-mediated activation of CCL5gene expression involves signal transduction components inNF-�B activation. The activation of NF-�B requires phosphoryla-tion of 2 conserved serine residues of I�B� (Ser-32 and Ser-36)and I�B� (Ser-19 and Ser-23) within their N-terminal domain.43

Phosphorylation leads to ubiquitination and the 26S proteasome-mediated degradation of I�Bs, thereby releasing NF-�B from thecomplex to translocate to the nucleus and activate genes.43 Ahigh-molecular weight complex, IKK complex, which is com-posed of 2 catalytic subunits, IKK� and IKK�, and a regulatorysubunit, IKK, phosphorylates I�Bs.44 Recent studies indicatedthat members of the mitogen-activated protein kinase kinase ki-nase protein kinase family mediate physiologic activation ofIKK.45 These kinases include NIK46 and mitogen-activated proteinkinase/extracellular signal-regulated kinase kinase 1.47 We nextexamined whether Tax-mediated transactivation of CCL5 geneexpression involves signal transduction components in NF-�Bactivation. Dominant interfering mutants of I�B� and I�B� andkinase-deficient mutants of IKK�, IKK� and NIK were tested fortheir ability to inhibit Tax-mediated activation of CCL5-drivenreporter gene. Expression of these various inhibitory mutants in-hibited the induction of the CCL5 promoter activation by Tax.There was no effect of these mutants on the basal activity of theCCL5 promoter, indicating that these mutants effects are specificto Tax-induced transcriptional activation (Fig. 4d). These datademonstrate that signaling components, NIK and IKK, involved inthe activation of NF-�B are necessary for Tax transactivation ofthe CCL5 promoter.

FIGURE 3 – Induction kinetics of the CCL5 gene and secretion inJPX-9 cells treated with CdCl2. (a) Total RNA samples were preparedfrom CdCl2-treated JPX-9 cells at the indicated time points (0–72 hr).The expression of Tax in the extracted RNA was analyzed by Northernblot analysis, while the expression of CCL5 and MMP-9 was analyzedby RT-PCR analysis. GAPDH served as a loading control for Northernanalysis, whereas �-actin served as an internal control in the RT-PCRprocedure. (b) The expression of CCL5 mRNA was not induced inJPX/M cells treated with CdCl2. (c) Induction of CCL5 secretion inJPX-9 cells after CdCl2 induced Tax expression. JPX-9 cells inocu-lated at 5 � 105/ml were treated with or without 20 �M CdCl2 for theindicated durations. CCL5 secreted in the culture supernatants wasquantified by using ELISA.

552 MORI ET AL.

Tax induces binding of NF-�B family proteins to the NF-�Belements of the CCL5 promoter

Because the deletional analyses of the CCL5 promoter indicatedthat Tax activated transcription through the A and B sites, it wasimportant to identify the nuclear factors that bind to these sites.JPX-9 cells were incubated with CdCl2, and at different timepoints after challenge, nuclear protein extracts were prepared andanalyzed for NF-�B DNA binding activity. As shown in Figure 5a,a complex formed with the A and B oligonucleotide probes wasinduced in JPX-9 cells within 24 hr after addition of CdCl2 (toppanel). This binding activity was reduced by the addition of coldprobe but not by an oligonucleotide containing a consensus AP-1

sequence (Fig. 5b, lanes 2, 3, 9 and 10). In contrast, CdCl2 did notinduce the complex formation in JPX/M cells (Fig. 5a, bottompanel). Next, we characterized the Tax-induced complexes identi-fied by the CCL5 A and B probes. These complexes were super-shifted or reduced by the addition of anti-p50 or anti-p65 antibod-ies (Fig. 5b, lanes 4, 5, 11 and 12), suggesting that Tax-inducedCCL5 NF-�B binding activity is composed of p50 and p65.Therefore, Tax induces CCL5 gene expression, at least in partthrough the induced binding of p50 and p65 to NF-�B sites withineither the A or B site in the CCL5 promoter region.

Binding of NF-�B family proteins to the Tax-responsiveelements within the CCL5 upstream regulatory sequences

Since we have shown that HTLV-I-infected T-cell lines expresssignificantly more CCL5 mRNA than do uninfected T-cell lines,we sought to determine whether HTLV-I-infected T-cell linesbetter exhibited NF-�B DNA binding activity. Using the NF-�B-like regions within A/B sites derived from the CCL5 promoter asprobes in EMSA, we observed clear shifted bands when theseprobes were incubated with nuclear extracts from HTLV-I-in-fected T-cell lines but not with nuclear extracts from uninfectedcells (Fig. 5c). These shifted complexes were specific to theNF-�B sequence because complex formations were reduced by theaddition of excess cold probe and the typical NF-�B sequence ofthe IL-2 receptor � enhancer but not by an oligonucleotide con-taining a consensus AP-1 sequence (data not shown). These resultsindicate that the increased activity of NF-�B binding may play akey role in the observed activation of the CCL5 gene in HTLV-I-infected T-cell lines. Thus, HTLV-I infection induces CCL5 geneexpression, at least in part, through the induced binding of p50 andp65 NF-�B family members to the �B elements within A/B sitesof the CCL5 promoter, and this effect is at least in part dependenton Tax.

Expression of CCR5 on HTLV-I-infected T-cell lines andinfiltrating ATL cells in lymph nodes

To test the hypothesis of a CCL5 autocrine and/or paracrineloop, we examined whether HTLV-I-infected T-cell lines expressits high-affinity receptor, CCR5. Using a panel of 9 human T-celllines, we examined the surface expression of CCR5 by flow

FIGURE 4 – Tax transactivates the CCL5 promoter mainly throughthe NF-�B pathway in T cells. (a) Diagram of regulatory regions in the195 CCL5 promoter and their relative to transcription start site �1.(b) Deletional analysis of the cis-elements required for Tax-inducedCCL5 promoter activity. Luciferase reporter constructs (5 �g) derivedfrom the CCL5 promoter were cotransfected with pH�APr-1-neo(Tax) and �-actin-Tax (�Tax) into Jurkat cells. pRL-TK (1 �g) wasalso cotransfected as an internal control plasmid. Open and solid barsrepresent luciferase activity of pH�APr-1-neo and �-actin-Tax-trans-fected Jurkat cells, respectively. A-, B-, A/B-, E- and C-: reporter geneconstructs derived from pGL2-R-luciferase with either the A, B, A/B,E or C site deleted. (AB)3: pGL2-SV40-(AB)3-luciferase. Luciferaseactivities were normalized based on the Renilla luciferase activityfrom pRL-TK. The activities are expressed relative to that of cellstransfected with pGL2-R and pH�APr-1-neo (left panel) or pGL2-SV40-(AB)3-luciferase and pH�APr-1-neo (right panel), which wasdefined as 1. (c) Jurkat cells were transfected with HTLV-I Tax (TaxWT), Tax M22, Tax 703 or pH�APr-neo vector and a luciferasereporter construct containing the 195 CCL5 promoter (pGL2-R).The results are expressed as fold induction relative to the basal levelmeasured in cells transfected with the empty vector (pH�APr-neo). (d)Functional effects of I�B� and I�B� dominant interfering mutants andkinase-deficient IKK�, IKK� and NIK mutants on Tax-induced acti-vation of the CCL5 promoter. The indicated effector plasmids werecotransfected with pGL2-R. Open bar represents luciferase activity ofI�B� and I�B� mutants and kinase-deficient IKK�, IKK� and NIKmutants without Tax. Solid bars represent luciferase activity of thesemutants in the presence of Tax. The activities are given relative to theactivity of empty vector (pCMV4) without Tax, which was defined as1. Data are mean � SD values of 3 independent experiments.

553ELEVATED CCL5 EXPRESSION IN ATL

cytometry. The results are shown in Figure 6a. Three HTLV-I-infected T-cell lines (MT-4, C5/MJ and MT-1) were clearly pos-itive, whereas all uninfected T-cell lines were negative. We nextcarried out immunologic staining of CCR5 using lymph nodes ofpatients with ATL. Representative results are shown in Figure 6b.Lymph nodes samples from patients with ATL were indeed clearlystained positive for CCR5. To ensure the specificity of monoclonalantibody, we performed the staining with control mouse IgG. No

staining could be detected with control mouse IgG (data notshown). Thus, some HTLV-I-infected T-cell lines and infiltratingATL cells were consistently found to express CCR5.

DISCUSSION

ATL is a malignancy of mature T cells and is particularlynotorious for its highly frequent invasion into various organs.8Furthermore, infiltration of lymphocytes, including HTLV-I-in-fected T cells, into affected tissues such as the central nervoussystem and eyes is commonly observed in HTLV-I-associatedinflammatory diseases and may be a critical step in the develop-ment of the diseases.8 The expression of chemokines has beenanalyzed in HTLV-I-infected T cells, since they are cruciallyinvolved in migration and tissue localization of various lympho-cyte subpopulations. HTLV-I-infected T-cell lines and primaryATL cells are known to express and secrete constitutively severalchemokines. These include CXC chemokines (IL-8,34,48 inter-feron-inducible protein-1034 and SDF-118) and CC chemo-kines (monocyte chemoattractant protein-1,49 MIP-1�,34,50,51 MIP-1�,34,50,51 and I-30952). Among the CC chemokine subfamily,CCL5 is known to have a strong chemoattractant activity formemory T lymhocytes.19 The major findings of the present studywere the following: i) the expression of CCL5 in HTLV-I-infectedT-cell lines as well as primary ATL cells, ii) that the viral proteinTax is responsible for the expression of CCL5 through NF-�Bpathway and iii) the expression of CCR5 of HTLV-I-infectedT-cell lines and ATL cells. Thus, CCL5 induced by Tax may be afactor responsible for infiltration of ATL cells and HTLV-I-in-fected T cells into tissues affected in ATL and HTLV-I-associatedinflammatory diseases. In agreement with our findings, Baba etal.34 showed constitutive expression of CCL5 in HTLV-I-infectedT-cell lines. However, they reported that CCL5 mRNA expressionwas not induced by Tax using JPX-9 cells.34 Why these results aredivergent is unclear at this time. We observed induction of CCL5both at the mRNA and protein levels in JPX-9 cells. Furthermore,Miyazato et al.53 have demonstrated elevated expression levels ofCCL5 in lungs of transgenic mice and the correlation between itsexpression and the severity of histopathological changes in thelung. CCL5 may contribute to the accumulation of T lymphocytesin the lungs, leading to inflammation of the lungs in HTLV-Icarrier.

CCL5 is expressed relatively late after activation of peripheralblood T cells by antigen or mitogens but is rapidly induced innormal fibroblasts and epithelial cells by tumor necrosis factor-�and IL-1�, suggesting that different control mechanisms mayregulate CCL5 transcriptional activation.32 Among the multipleregulatory domains identified in the CCL5 upstream promoter are2 binding sites for the NF-�B transcription factors, located 78 to42 upstream from the transcription initiation site.40,41 Inductionof NF-�B plays an important role in CCL5 gene activation bystimuli such as phorbol myristate acetate-ionomycin, proinflam-matory cytokines or anti-CD3 and anti-CD28 antibodies;41 further-more, NF-AT-like and a CD28RE-like motifs also serve as NF-�Bbinding sites.41 The interferon regulatory factor-3 also plays a rolein the regulation of CCL5 gene expression.54 The Tax-responsiveelements within the 5� regulatory sequences of the CCL5 genewere localized in A/B sites. Deletion of the NF-�B sites in thisregion resulted in loss of Tax responsiveness, indicating that CCL5regulation by Tax is mediated by NF-�B sites. Experiments usingTax mutants indicated that NF-�B pathway is required for the fullactivation of CCL5 promoter by Tax. These findings suggest thatNF-�B activation may be a necessary prerequisite for increasedT-cell CCL5 activation in response to HTLV-I infection. In ourstudy, we identified the signaling components NIK and IKKs aslikely participants in Tax-mediated CCL5 activation. Activation ofthe NF-�B pathway by Tax has been extensively investigated. Insupport of our findings, Tax expression has been shown to promotephosphorylation and activation of IKK� and IKK� by increasingthe activity of the upstream kinase NIK.31

FIGURE 5 – Tax-dependent binding of NF-�B family proteins to theTax-responsive elements. (a) Time course of NF-�B activation inJPX-9 cells treated with CdCl2 evaluated by EMSA. Nuclear extractsfrom JPX-9 or JPX/M cells, treated with or without CdCl2 (20 �M) forthe indicated time periods, were mixed with either A or B [32P]-labeledprobes. (b) Sequence specificity of NF-�B binding activity and char-acterization of NF-�B proteins that bound to the NF-�B binding siteswithin A/B sites of the CCL5 gene. Competition assays were per-formed with nuclear extracts from JPX-9 cells treated with CdCl2 for72 hr. Where indicated, 100-fold excess amounts of each specificcompetitor oligonucleotide (lanes 2, 3, 9 and 10) were added to thereaction mixture with labeled probe A (lanes 1–7) or B (lanes 8–14).Supershift assay of NF-�B DNA binding complexes in the samenuclear extracts was also performed. Where indicated, appropriateantibodies were added to the reaction mixture before the addition of[32P]-labeled probes (lanes 4–7 and 11–14). (c) Binding of nuclearproteins from HTLV-I-infected T-cell lines to the NF-�B A or B probederived from the CCL5 promoter. Nuclear extracts were prepared fromHTLV-I-infected (lanes 1–6 and 10–15) and uninfected T-cell lines(lanes 7–9 and 16–18) and incubated with either A or B [32P]-labeledprobe.

554 MORI ET AL.

While other members of the chemokine family are expressed asearly genes, CCL5 mRNA undergoes late up-regulation duringT-cell activation. The presence of RFLAT-1 in the nuclei of T cellsmay explain the unique kinetics of CCL5 expression in T lympho-cytes.42 RFLAT-1 binds the A site of the CCL5 promoter with highspecificity.42 Northern blot analysis was performed using RNA

from several T-cell lines. RFLAT-1 was expressed in all T-celllines tested, irrespective of the HTLV-I infection (data not shown).Although HTLV-I-infected T cells that constitutively express highlevels of CCL5 are mature T cells, NF-�B, but not RFLAT-1, inCCL5 expression is involved. This transcriptional regulation of theCCL5 gene appears unique to HTLV-I-infected T cells.

FIGURE 6 – HTLV-I-infected T cells express CCR5. (a) Flow cytometric analysis on surface expression of CCR5 in HTLV-I-infected T-celllines. (b) Immunohistochemical staining of CCR5 in a lymph node of a patient with ATL. The original magnification was �200.

555ELEVATED CCL5 EXPRESSION IN ATL

We may also stress the possibility of Tax-independent mecha-nisms operating for constitutive expression of CCL5. The expres-sion of Tax in ATL cells can be detected only by RT-PCR, asobserved in an ATL-derived cell line, MT-1. A trace amount ofTax might be sufficient for the induction of CCL5 in ATL cells andMT-1 cells. Alternatively, in the absence of Tax, ATL cells andMT-1 cells might maintain high CCL5 expression. Recent studieshave shown that leukemic cells from several ATL patients possessmutation and truncation of the Tax coding region, which inactivateits functions.55,56 These results indicate that Tax is dispensable forthe late stage of ATL leukemogenesis. CCL5 was still expressed inanother ATL-derived cell line, TL-OmI that does not express Taxat all by RT-PCR (data not shown). Interestingly, the NF-�Bpathway is constitutively activated despite low level of Tax ex-pression in both ATL cells and MT-1 cells.9,35 TL-OmI cells alsoexhibits constitutive activation of NF-�B.35 Thus, Tax is not theonly mechanism for constitutive expression of CCL5 in HTLV-I-infected T cells. Rather, the NF-�B activation might be necessaryfor constitutive expression of CCL5.

To test the hypothesis of a CCL5 autocrine and/or paracrineloop, we studied HTLV-I-infected T cells for its high affinityreceptor, CCR5 expression. In our study, we have shown that someHTLV-I-infected T-cell lines express CCR5 on their surface. Thisis in good agreement with previous studies.51 MIP-1� and MIP-1�produced by ATL cells have been reported to activate leukocytefunction-associated antigen-1 in an autocrine manner, resulting inincreased adhesion of ATL cells to human umbilical vein-derivedendothelial cells. The high affinity receptor for MIP-1� andMIP-1� is CCR5. These results suggest that the autocrine CCR5/chemokines signaling may work in ATL cells. We observed weakdirectional chemotaxis of HTLV-I-infected T cells toward CCL5,suggesting that high expression of CCL5 could be involved innodular proliferation of ATL cells in multiple tissues in vivo (datanot shown). However, it should be noted that not all HTLV-I-infected T-cell lines are CCR5 positive. We investigated the ex-pression of CCR5 in ATL cells in lymph nodes from patients withATL by immunohistochemical staining. CCR5 was found in theATL cell infiltrates of 10 of 15 ATL cases (data not shown). Itremains to be seen whether there are any clinical differencesbetween CCR5 positive and negative ATL cases. Although weexamined the surface expression of CCR5 in peripheral bloodsamples from 2 patients with ATL by flow cytometry, CCR5-positive cells were only 5% and 14% of ATL cells from these 2

cases, respectively. The infiltrating ATL cells in lymph nodes mayexpress CCR5 at higher levels.

A number of chemokine receptor associates with the Th1 andTh2 phenotypes have been reported.57 CCR5 has associated withTh1 phenotype. Furthermore, Th1 cells have been shown to pro-duce many more chemokines including CCL5 than Th2 cells.58

These observations suggest that ATL cells may be phenotypicallysimilar to Th1. However, CCR4 that is strongly upregulated inT-cell populations polarized to the Th2 phenotype is highly ex-pressed in ATL cells.17 The expression patterns of chemokinereceptors in ATL is unique.

Recently, we have observed the elevated expression of MMP-9in HTLV-I-infected T-cell lines and ATL cells, and concluded thatMMP-9 actively contributes to invasiveness of ATL cells.9 Also, itwas previously demonstrated that CCL5 induces MMP-9 in Tcells.59 Through this action, CCL5 may modulate invasiveness ofATL cells. On the other hand, Seki et al.60 have reported that theelevated levels of CCL5 was observed in bronchoalveolar lavagefluid of HTLV-I carriers. Thus it is tempting to speculate that inHTLV-I-associated inflammatory diseases, CCL5 may be an im-portant chemokine. Based on the biological activities attributed tochemokines, including a role in inflammation, leukocyte recircu-lation and other mechanisms of host defense, an understanding thechemokine mechanisms of action will be beneficial for treatmentof various inflammatory and noninflammatory disease states.Many efforts to elucidate the complex and intriguing chemokinenetworks have been made recently. It is not yet completely clearthe role of chemokines in the pathology of HTLV-I-associateddiseases. Animal models with cells derived from patients withHTLV-I-associated diseases may provide important clues to betterunderstand the role of chemokines and design novel therapeuticstrategies for the treatment of HTLV-I-associated diseases.

ACKNOWLEDGEMENTS

We are deeply indebted to the many patients with ATL and thecontrol subjects who donated blood and lymph nodes for thesestudies. We thank Dr. K. Matsumoto, Dr. D.W. Ballard and Dr. R.Geleziunas for providing expression vectors for HTLV-I Tax andthe Tax mutants, for deletion mutants of I�Bs and for the kinase-deficient K44M IKK�, K44A IKK� and KK429/430AA NIKmutants. We also thank Dr. M. Nakamura for providing JPX-9 andJPX/M and Fujisaki Cell Center, Hayashibara Biomedical Labo-ratories (Okayama, Japan) for providing Jurkat, C5/MJ, HUT-102and MT-1 cell lines.

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