[CANCER RESEARCH 55, 3592-3597, August 15. 1995]

Production of Interleukin 8 in Adult T-Cell Leukemia Cells: PossibleTransactivation of the Interleukin 8 Gene by Human T-Cell

Leukemia Virus Type I taxNaoki Mori,1 Shuichi Murakami, Susumu Oda, Diane Prager, and Sumiya Eto

First Department of Internal Medicine, University of Occupational and Environmental Health, School of Medicine, 1-1 heigaoka. Yahalanishiku. Kitakyushu 807. Japan[N. M., S. M., S. O., S. E.J, and Department of Medicine, Division of Endocrinology and Metabolism, Cedars-Sinai Medical Center, University of California-Los Angeles School

of Medicine, Los Angeles, California 90048 [D. PJ

ABSTRACT

Interleukin 8 (II.-K) inKNA was detected in peripheral leukemic cellsobtained from adult T-cell leukemia patients, as well as in cultured humanT-cell leukemia virus type I (HTLV-I)-infected T-cell lines (HUT-102,MT-1, SALT-3, and SKT-1B). With the use of ELISA, IL-8 protein was

also detected in the culture medium of these cells and in the extracellularfluids of patients. Furthermore, we demonstrated that the HTLV-I-de-rived transactivator protein, tax, could stimulate endogenous IL-8 geneexpression in an uninfected T-cell line (Jurkat) and in a rheumatoidsynovial cell line (E-ll). Induction of IL-8 by tax at protein level was alsodemonstrated in transfected cells. We found that the IL-8 NF-KU-bindingsite specifically formed a complex with NF-KB-containing nuclear extractsfrom HTLV-I-infected T-cell lines and freshly isolated leukemic cells fromadult T-cell leukemia patients. Finally, transfection of HTLV-I tax into

Jurkat cells resulted in induction of specific binding of nuclear extracts tothe M -Kit sequence. These results suggest that the HTLV-I tax gene maytransactivate the IL-8 gene through the KB site in HTLV-I-infected cellsand that constitutive expression of the IL-8 gene may play a role inHTLV-I-associated pathogenesis.

INTRODUCTION

Most cytokines are produced upon stimulation by various agents,but constitutive expression is negligible in normal tissues. In contrast,ATL2 cells, a malignancy of CD4-positive HTLV-I-infected periph

eral T lymphocytes, have been shown to express at the mRNA leveland/or to produce various cytokines in the absence of stimulation(1-8), indicating that constitutive cytokine production may be impor

tant in autoregulatory growth stimulation of leukemic cells by autocrine or paracrine circuits. IL-8 is a newly identified potent chemo-

tactic factor for neutrophils (9) and T lymphocytes (10). In the presentpaper, we report evidence indicating that peripheral leukemic cellsobtained from ATL patients, as well as HTLV-I-infected T-cell lines,constitutively produce IL-8.

This cytokine has been shown to be produced by normal mono-cytes/macrophages, dermal fibroblasts, vascular endothelial cells, ke-

ratinocytes, and mesangial cells, as well as several types of humantumor cell lines. However, the production of IL-8 is generally notconstitutive and can be induced by several stimulants such as IL-1,tumor necrosis factor a, IFN-y, and hepatitis B virus (11, 12).HTLV-I tax can stimulate the transcription of several genes includingits own genome (13). We also found that the HTLV-I tax gene isindeed able to induce IL-8 at mRNA and protein levels in transfectedcells. Furthermore, we demonstrated that HTLV-I tax induces the

Received 2/23/95; accepted 6/9/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicale this fact.

1To whom requests for reprints should be addressed, at Department of Medicine,

Division of Endocrinology and Metabolism, Cedars-Sinai Medical Center, UCLA Schoolof Medicine, 8700 Beverly Boulevard, Los, Angeles, CA 90048.

2 The abbreviations used are: ATL. adult T-cell leukemia; HTLV-I, human T-cellleukemia virus type I; IL-8, interleukin 8; PBMC, peripheral blood mononuclear cells;RT-PCR, reverse transcriptase PCR; DEAE, diethyl aminoethyl; PMSF, phenylmethyl-

sulfonyl fluoride; EMSA, electrophoretic mobility shift assay.

IL-8/<B-binding activity in transfected cells. This study is the first toshow direct evidence for the transactivation of IL-8 gene by tax.

MATERIALS AND METHODS

Cells. All cell samples were derived from 10 patients with acute-type ATL.

All ATL patients had a high WBC count with typical ATL lymphoblasts.Morphological diagnosis was independently confirmed by immunophenotyp-

ing of isolated cells from the peripheral blood and by the demonstration bySouthern blot analysis of monoclonal integration of HTLV-I genome (data notshown). The percentage of leukemic cells determined by both CD4+ andCD25 + cells and used for each assay was greater than 90%. Leukemic cells

were isolated by standard procedures. Briefly, heparinized blood was layeredon top of lymphocyte separation medium (Litton Bionetics, Kensington, MD),and mononuclear cells were recovered from the interface, washed with PBS,and further incubated at 37°Cfor 2 h in plastic culture dishes (Falcon 3003,

Becton Dickinson, Oxnard, CA) to remove adherent cells. This solution wasremoved from the dishes, and cells were washed thoroughly with PBS. ATLsuspensions were then introduced into the 24-well culture plates (2 ml/well) atcell densities of 5 X 106/ml and cultured for 72 h at 37°Cin RPMI 1640

supplemented with 10% FCS. Culture supernatants were collected by centrif-ugation to measure the levels of released IL-8. With one of the ATL patients

(case 5), the PBMCs were further purified to obtain a highly enriched leukemicpopulation. After isolation, a more enriched ATL cell preparation was obtainedby CD4-conjugated immunomagnetic beads (Dynal, Oslo, Norway). An aliquot of these cells was analyzed by fluorescence-activated cell sorting andfound to be 98% CD4+ with <1% contamination, with cells bearing the

following markers: CDS, CD19, and CD14.The established HTLV-I-infected T-cell lines MT-1 (14), MT-2 (15), and

HUT-102 (16) were gifts from the Fujisaki Cell Center (Okayama, Japan).SALT-3 and SKT-1B (17) established from ATL patients were provided by Dr.

K. Sagawa (Kurume University, Kurume, Japan). Jurkat was provided by Dr.K. Yamamoto (Kanazawa University, Kanazawa, Japan). These cell lines weremaintained in RPMI 1640 supplemented with 10% FCS.

Human synovial E-ll cells were established from a patient with rheumatoid

arthritis by electroporation by SV40 large T antigen plasmid. They werespindle-shaped; positive for fibroblast marker FU3 antibody, which recognizesfibroblast surface antigen (18); positive for type I IL-1 receptor; and negativefor macrophage marker (CD14, HLA-DR, type II IL-1 receptor). They were

maintained in DMEM supplemented with 10% FCS.Oligonucleotide Primers. The following primers were used for gene

amplification: IL-8 sense, 5'-ATGACTTCCAAGCTGGCCGTG; IL-8antisense, S'-TTATGAATTCTCAGCCCTCTTCAAAAACTTCTC; taxsense, 5'-CCCACTTCCCAGGGTTTGGACAGA; tax antisense, 5'-CTG-TAGAGCTGAGCCGATAACGCG; ß-actin sense, 5'-ACCAACTGGGA-CGACATGGAGAA; and ß-actin antisense, 5'-GTGGTGGTGAAGCTG-

TAGCC. All primers are designed to incorporate an intron-exon boundary

to distinguish between amplification of mRNA and any contaminatinggenomic DNA.

RT-PCR. Total cellular RNA was isolated with the use of the acid guani-dinium thiocyanate-phenol-chloroform extraction method as described previ

ously (19), and quantitation of the purified RNA was performed by absorbanceat 260 nm. The integrity of the RNA was confirmed by electrophoresis underdenaturing conditions on a 1% agarose gel. One /xg of RNA was used forcDNA synthesis at 42°Cfor 60 min with 6 units of Rous-associated virus 2

reverse transcriptase and 0.2 (¿gof DNA random hexadeoxynucleotide primersin a total volume of 20 /il. Then, 6 ¡uof cDNA reaction were amplified by 31

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IL-8 PRODUCTION IN ATL CELLS

PCR cycles with 2.5 units of Taq polymerase in the presence of l /¿geach ofthe sense and antisense oligonucleotides. ß-actinprimers were used as an

internal control. To assess integrity of RNA, PCR was performed in the samecDNA. The thermal cycle profile was as follows: denaturation for 0.5 min at94°C;annealing for 1 min at 60°C,63°C,or 69°C,depending on the basecomposition of the different primers; and extension for 1 min at 72°C.Twenty

/J.1of the PCR reaction mixture (100 fil total volume reaction) were subjectedto electrophoresis in a 2% agarose visualized with the use of ethidium bromide,and gave rise to bands of the sizes expected for the primers used (IL-8, 302 bp;tax, 203 bp; ß-actin,380 bp).

Plasmids. pH2R40M and pH2Rneo plasmids (kindly provided by Dr. M.Hatanaka, Kyoto University, Kyoto, Japan) have been described previously(20). pH2Rneo (-tax) contains the SV40 sequences including the SV40

promoter and the SV40 polyadenylation signal, an R fragment of HTLV-I longterminal repeat, and neomycin-resistant gene under control of the SV40 pro

moter. pH2R40M (+tax) has Hindlll fragments that can encode tax at a

Hindlll site of pH2Rneo.DNA Transfection. Two procedures for DNA transfection were used. The

DEAE-dextran procedure was performed for transfecting Jurkat cells. Briefly,1 x IO7 cells were washed twice with serum-free RPMI 1640 and suspended

in 10 ml of transfection cocktail, consisting of 10 ¡¿gof DNA, 50 mM Tris-HCl(pH 7.2), and 2 mg of DEAE-dextran (Pharmacia, Uppsala, Sweden) inserum-free RPMI 1640 at room temperature. After 20 min, the cocktail was

replaced by 10 ml of heparin (1.5 units/ml in RPMI 1640; Sigma ChemicalCo.) to neutralize DEAE-dextran intoxication to the cells. After washing twice

with 10 ml of RPMI 1640, cells were suspended in RPMI 1640 supplemented10% PCS, plated into dishes (Falcon 3003; Becton Dickinson), cultured for 48h, and harvested.

For calcium phosphate procedure, E-ll cells were plated at a density of3 X IO6 cells/100-mm dish 24 h before transfection. Immediately before

transfection, the cells were washed with 10 ml of DMEM once and incubatedin 10 ml of DMEM supplemented with 10% PCS for 2 h. The transfection wasaccomplished by the previously reported procedure with the use of a CaPO4mammalian cell transfection kit (5 Prime —»3 Prime, Inc., Boulder, CO) (21).

Ten fig of plasmid were gently mixed with 0.5 ml of 2X HEBS [50 mMHEPES (pH 7.05)-1.5 ml Na2HPO4-10 mM KC1-0.28 M NaCl-12 mM glucose]

and 62 /il of 2 M CaCl2 in 1 ml and incubated at room temperature for 20 minto generate a fine precipitate, which was added to the cells. Sixteen h aftertransfection, the cells were incubated for 3.5 min in 15% glycerol (glycerolshock). After being washed with 5 ml of DMEM three times, cells were thenincubated in DMEM for 48 h.

Determination of IL-8 Protein in Culture Supernatants. Cells from thesame samples used for the RT-PCR studies were tested for their capacity torelease IL-8. Cell-free supernatants were assessed for IL-8 protein with the useof a commercially available ELISA kit (Toray-Fuji Bionix, Inc., Tokyo, Japan)according to the manufacturer's instructions. The lower limit of use of this

assay is 12.5 pg/ml.Preparation of Nuclear Extracts. Nuclear extracts were prepared by a

method reported previously (22-24), with modifications. After harvest,1 X IO8 cells were washed with cold PBS and incubated in 5 ml of buffer A[10 mM HEPES (pH 7.9) at 4°C-5mM MgCl2-10 mM NaCl-0.3 M sucrose-0.1

mM EGTA-0.5 mM DTT-0.5 mM PMSF] containing 1 jug/ml of each of the

protease inhibitors antipain, aprotinin, chymostatin, leupeptin, and pepstatin Aon ice for 15 min. After centrifugation, the cells were resuspended in 1 ml ofbuffer A with protease inhibitors and then Dounce homogenized (30 strokes).The homogenate was microcentrifuged for 0.5 min, and nuclei were resuspended in 0.4 ml of buffer B with protease inhibitors [20 mM HEPES (pH7.9)-5 mM MgCl,-0.3 M KC1-0.2 mM EGTA-25% glycerol-0.5 mM DTT-0.5mM PMSF] and gently rocked on a platform at 4°Cfor 30 min. After 30 minof microcentrifugation at 4°C,supernatants were removed and dialyzed against

50 volumes of buffer D [20 mM HEPES (pH 7.9)-0.1 M KC1-0.2 mM EDTA-20% glycerol-0.5 mM DTT-0.5 mM PMSF] at 4°Covernight. After 30 min ofmicrocentrifugation at 4°C,aliquots of supernatants were frozen at - 80°C,and

protein concentrations were determined with the use of a Bio-Rad assay kit.

EMSAs. EMSAs were performed essentially as described previously (24).Briefly, 5 ^ig of nuclear extracts were preincubated for 20 min at roomtemperature in 15 /il of a buffer [10 mM Tris-HCl (pH 7.5)-l mM EDTA-1 mMß-mercaptoethanol-4% glycerol-40 mM NaCl] containing i2P-labeled probe

(50 pg; approximately 20,000 cpm) and 100 ng of poly(dl-dC). The probe was

an IL-8«Boligonucleotide containing a NF-xB-binding site derived from theIL-8 promoter (—83to —69from the transcription start site of the human gene;

ctccCGTGGAATTTCCTCT). When indicated, 50 ng of unlabeled competitorDNA were added. E2F representing the P2-distal E2F-binding site of the c-mvcpromoter (-77 to -65 from the P2 start site of the mouse gene; aagtCGCT-

TGGCGGGAA) is a nonrelevant oligonucleotide (25); IL-6xB (gatcTGG-GATTTTCCCA) is an oligonucleotide containing a NF-xB binding site derived from the IL-6 promoter ( —74 to —62from the transcription start site of

the human gene; Ref. 26). DNA-protein complexes were separated in a 4%polyacrylamide gel in 0.25 X TAE buffer [6.67 mM Tris-HCl (pH 7.5)-3.3 mMsodium acetate-1 mM EDTA] and analyzed by autoradiography.

RESULTS

Expression of IL-8 and tax mRNA in HTLV-I-infected T-Cell

Lines and ATL Cells. Six cultured cell lines and 10 specimens offresh leukemic cells obtained from patients were examined by RT-PCR. Cultured cell lines were 5 HTLV-I-infected T-cell lines (MT-2,HUT-102, MT-1, SKT-1B, and SALT-3) and one acute lymphocytic

leukemia cell line (Jurkat). Ten peripheral blood specimens obtainedfrom ATL patients were also examined.

The results of RT-PCR for IL-8 mRNA are illustrated in Fig. 1. Theß-actingene was used as positive control of cDNA transcription.Among the 5 HTLV-I-infected T-cell lines, IL-8 mRNA, composed of

a band with a molecular size of 302 bp, was detected in 4 cell lines(HUT-102, MT-1, SKT-1B, and SALT-3). This band was not detectedin the HTLV-I-uninfected T-cell line, Jurkat. In 10 specimens ofperipheral leukemic cells obtained from ATL patients, IL-8 mRNA

was observed (Fig. 2). Furthermore, similar results were obtained withfresh leukemic cells in the absence of any culture (Fig. 3), indicating

J*

B

302 bpIL-8

203 bptax

380 bpß-actin

Fig. 1. Detection of IL-8 and lax mRNA in T-cell lines by RT-PCR. Reverse transcription of cellular RNA (1.0 fig tolal RNA) and PCR were carried out. One-fifth of the

PCR mixture was analyzed by ethidium bromide staining of the gel. Agarose gelelectrophoresis of DNA was amplified by IL-8 (A), tax (ß).and ß-actinprimers (C). M,molecular weight marker, Haelll digest of <J>X174.Arrow, position of the specificallyamplified DNA.

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IL-8 PRODUCTION IN ATL CELLS

M 1 2 3 4 5 6 7 9 10 11

302 bpIL-8

D I <—203 bp

tax

380 bp/3-actin

Fig. 2. Detection of IL-8 and tax mRNA in primary leukemic ATL cells. Agarose gelelectrophoresis of DNA amplified by IL-8 (A), tax (ß),and ß-actinprimers (C). Lane 1,normal T-cells; Lanes 2-11, leukemic cells from acute-type ATL patients. M. molecular

weigh! marker, Hae\\\ digest of <¡>X174.

M12345678910

302 bpIL-8

Fig. 3. RT-PCR analysis of 10 fresh leukemic ATL cells in the absence of any culture.

The case numbers refer to those of Table 2. M, molecular weight marker, Haelll digest of<)>X174.

To find out whether ATL cells secret IL-8 in vivo, IL-8 protein inthe extracts from the pleural effusion of case 8 and from the asciticfluid of case 3 was determined. As shown in Table 2, both fluidsdemonstrated IL-8 production. Thus, ATL cells from these patients, aswell as HTLV-I-infected T-cell lines, synthesize IL-8 and appear tosecret IL-8 in vivo.

HTLV-I tax Can Induce IL-8 Gene Expression. Because the taxencoded by the pX sequence of HTLV-I is a transcriptional transac-tivator, it was expected that induction of the IL-8 gene was activatedat a transcriptional level. To examine whether tax can activate IL-8gene expression, Jurkat cells were transfected either with the taxexpression plasmid (pH2R40M) or a control plasmid (pH2Rneo)because this cell line does not express mRNA for IL-8 spontaneously.Total RNA was extracted from these cells, and IL-8 gene expressionwas investigated by RT-PCR. Cells transfected with pH2R40M butnot pH2Rneo expressed IL-8 mRNA (Fig. 4). Untransfected Jurkatcells did not give any significant bands of the mRNA. As control,ß-actinprimers produced almost constant amounts of amplified DNAin all samples. This indicated that tax can specifically stimulateendogenous IL-8 gene expression in T cells.

Then, we examined the cell type specificity of the induction ofIL-8. Transfection of pH2R40M into a human synovial cell line, E-l 1,also induced IL-8 (Fig. 4). Therefore, it was concluded that thetransactivation of the IL-8 gene by tax is not restricted to T cells.

Induction of IL-8 Protein by HTLV-I tax. When the transfectedJurkat cells and E-ll cells were tested for the secretion of IL-8 byspecific ELISA, pH2R40M-transfected cells secreted a higher amountof IL-8 than did the control pH2Rneo-transfected cells or nontrans-fected cells (Table 3). Jurkat cells transfected with the pH2Rneoplasmid produced no detectable IL-8, whereas 211.7 pg/ml IL-8 couldbe detected at 48 h after transfection in Jurkat cells transfected with 10/xg of pH2R40M. Transfection into E-ll cells also induced a significant amount of the IL-8 (382.7 pg/ml) in the culture supernatants at48 h after transfection. These data indicate that transient expression of

that IL-8 mRNA expression was not a result of stimulation by components in the culture medium. Normal human peripheral blood Tcells did not express IL-8 mRNA.

Next, RT-PCR was performed to examine the expression of taxmRNA in these cells. The tax mRNA (203-bp band) was detected inall HTLV-I-infected T-cell lines and ATL cells (Figs. 1 and 2). Incontrast, the Jurkat cell line and normal human peripheral blood Tcells showed no tax expression.

IL-8 Protein in the Culture Medium of HTLV-I-infected T-CellLines and ATL Cells. To determine whether the constitutiveexpression of IL-8 mRNA was translated to protein, we examinedthe production and the release of IL-8 by HTLV-I-infected T-celllines and ATL cells by an ELISA in the culture supernatants. Asshown in Table 1, IL-8 was detected in the culture supernatants of4 HTLV-I-infected T-cell lines, in which the expression of IL-8mRNA was observed. In addition, supernatants from the PBMCderived from the ATL patients secreted high levels of IL-8 protein(Table 2). To clarify a possibility that the increased IL-8 secretionfrom the ATL-derived PBMC may result from the IL-8 productionby a contamination of other cell types in the ATL cell preparation,we reprepared a highly purified population of malignant ATL cellsby magnetic beads and examined the level of IL-8 secretion in anATL patient (case 5). The level of IL-8 secreted from the purifiedleukemic cells was 3780 pg/ml and did not differ markedly fromthat seen in unpurified population, indicating that the leukemiccells rather than contaminants (monocytes) were the source ofIL-8.

Table 1 Concentrations of IL-8 in the culture medium of HTLV-l-infected T-cell lines"

Cell lines IL-8 (pg/ml)

UninfectedJurkat

HTLV-I infectedMT-2HUT-102MT-1SALT-3SKT-1B

<12.5

<12.588.2

3461,050

136" Cells were plated at 5 X lO^/ml and cultured for 72 h in RPMI 1640 supplemented

with 10% FCS. Levels of IL-8 release/ml in the supernatants of T-cell lines were measured

by ELISA.

Table 2 Concentrations of IL-8 in the culture medium of ATL cells and in the

extracellularfluidsSampleNormal

T-cellLeukemic

cells frompatientsCase1Case2Case3Case

4CaseSCase

6Case7Case8Case9Case

10ExtracellularfluidsAscitic

fluid of case3Pleuralfluid of case 8IL-8

(pg/ml)<12.52,99512,9006,0552,8493,78038,2923,8383,8333,8543,77858034.1

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IL-8 PRODUCTION IN ATL CELLS

the tax gene results in the activation of the endogenous IL-8 gene andthe secretion of high amounts of IL-8.

NF-KB-binding Site Specifically Binds NF-KB-containing Nuclear Extracts. Activation of NF-KB by tax has been shown toinduce transcription of genes containing NF-«B-responsive elements, including IL-2, IL-2 receptor a, granulocyte-macrophagecolony-stimulating factor, and c-myc (27-30). In addition, we haverecently demonstrated that tax also induces the binding of NF-KBto the NF-KB-responsive element within the IL-6 promoter andactivates its expression (26). We first tested NF-KB-binding activity of the nuclear extract preparations from IL-8-producing cell

Jurkat 24 48 hr

M

302 bpIL-8

380 bp/8-actin

E-11 24 hr 48 hr

M12345678

^m i^m ^m ^M M* *W «M»WM

302 bpIL-8

380 bpß-actin

Fig. 4. tax induces the expression of the IL-8 gene. Jurkat and E-11 cells were

transfected with pH2Rneo (Lanes 3 and 7) or pH2R40M (Lanes 4 and 8), without DNA(Lanes 2 and 6). Total RNA was isolated from the cell lysates 24 or 48 h after transfection.Lanes I and 5, RT-PCR products from Jurkat and E-l 1 cells cultured without transfection.RT-PCR was performed with the use of the IL-8 and ß-actinprimers. M, molecular weight

marker. Hae\\\ digest of (J>X174.

Table 3 Secretion of IL-K prolein by Jurkal and E-11 cells"

Jurkat E-11

24 h 48 h 24 h 48 h

MediumMocktransfectedpH2Rnco

transfectedpH2R40M transfected<12.5

<12.5<12.5

92.9<12.5

<12.5<12.5

211.7<12.5

ND*17.1

268.621.4

22.016.1382.7

" Cells were transfected with pH2Rneo (-tax) or pH2R40M (+tax). without DNA

(Mock). These cells were cultured for 24 or 48 h. Medium represents untransfected controlcells. Supernalants were then collected and tested for IL-8 content by ELISA-specifichuman IL-8.

' ND, not done.

Competitor -

Lane

CDu°9

mse

<o

CQ14

00

6

»10 11 12

Fig. 5. NF-KB activity in HTLV-I-infected T-cells that produce IL-S. Nuclear extractsfrom T-cell lines form a specific complex with oligonucleotide IL-KtiB (50 pg; Lanes 2,6, 9, and //) in competition with excess added oligonucleotide 1L-8KB (50 ng; Lanes 4.7, 10, and 12) and IL-ÓKB(50 ng; Lanes 5 and 8). Formation of this complex is not

inhibited by addition of an unrelated oligonucleotide (E2F; 50 ng; Lane 3). Artwv,migration of the NF-KB complex, separated from the free probe.

lines SALT-3, MT-1, SKT-1B, and HUT-102. We found that theseextracts specifically bound to the IL-8/<B probe, whereas in Jurkat

cells, the levels of complex were hardly detectable (Fig. 5). Thebinding was inhibited by addition of excess unlabeled oligonucleotide IL-6«B but not by an unrelated oligonucleotide, E2F. In 3

specimens of peripheral leukemic cells obtained from ATL patients, all extracts specifically bound to the IL-8«B probe. The

levels of complex were hardly detectable in 3 specimens extractedfrom PBMC obtained from normal healthy controls (Fig. 6). Thus,these nuclear extracts from HTLV-I-infected T-cell lines and leu

kemic cells from ATL patients, in which we have demonstratedIL-8 production, recognize the IL-8«B site.

Induction of the Binding of Nuclear Extracts to the NF-KBSequence by HTLV-I tax. The HTLV-I tax responses were then

measured in Jurkat cells. Nuclear extracts were prepared from transiently transfected Jurkat cells with an expression plasmid for HTLV-I

tax (pH2R40M) or a control plasmid (pH2Rneo), incubated with alabeled oligonucleotide probe corresponding to the IL-8«Bsite, and

subjected to EMSAs. It was found that extracts prepared frompH2R40M-transfected Jurkat cells displayed induction of the complex

(Fig. 7). In contrast, the levels of complex remained unchanged afterpH2Rneo transfection. NF-KB specificity was demonstrated by elimination of complex after the addition of excess unlabeled IL-8KB orIL-ÓKBoligonucleotide, whereas no competition was obtained by anunrelated oligonucleotide, E2F. These results indicate that HTLV-I

tax is indeed able to induce specific binding of nuclear extracts to theNF-KB sequence in the IL-8 gene promoter.

DISCUSSION

IL-8 is a novel potent chemotactic cytokine for neutrophils and Tlymphocytes (9, 10). Although IL-8 is produced by diverse types ofcells, IL-8 production by normal T cells appears to be rare. Wedemonstrated in this paper that HTLV-I-infected T-cell lines and ATLcells produce constitutively IL-8 mRNA and protein.

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Competitor — — — —

Lane 1234

CDid

11 12

I

Fig. 6. NF-xB activity in leukemic cells from ATL patients. Nuclear extracts fromleukemic cells form a specific complex with oligonucleotide IL-8KB (50 pg; Lanes 4, 8,and 10) in competition with excess added oligonucleotide IL-8KB (50 ng; Lanes 6, 9, and//) and IL-ÓKB(50 ng; Lane 7). Formation of this complex is not inhibited by additionof an unrelated oligonucleotide (E2F; 50 ng; Lane 5). Arrow, migration of the NF-KB

complex, separated from the free probe.

of IL-8 in HTLV-I-infected cells. Indeed, the promoter region of IL-8

contains putative binding sites for several transcription factors, including NF-KB, CCATT/enhancer-binding protein, and AP-1 (34). Itis conceivable that tax might transactivate IL-8 promoter throughthese transcriptional factors. The IL-8«B site competed with theIL-ÓKBsequence for binding to nuclear extracts from HTLV-I-infected T-cell lines and ATL cells. Finally, transfection of tax into

Jurkat cells resulted in induction of specific binding of nuclear extracts to the NF-KB sequence. Our results indicate that tax activationis at least one mechanism by which HTLV-I activates IL-8 produc

tion. However, further functional studies on transient transfection ofIL-8-chloramphenicol acetyltransferase reporter constructs will berequired to prove that the transactivative effect of tax on the IL-8 geneis mediated through NF-KB. In addition, it is known that in the

peripheral blood cells of ATL patients, tax is not expressed or isexpressed at a very low level. Some factor(s) other than HTLV-I tax

gene (e.g., a factor associated with malignant transformation) mayenhance the expression of the IL-8 gene.

HTLV-I-infected T cells are known to produce various kinds ofcytokines. Among the cytokines reported to be produced by HTLV-I-infected T cells, IL-1, tumor necrosis factor a, and IFN--y are the

strong inducers of IL-8 (12). Our question, therefore, was whethersuch cytokines might be responsible for the induction of IL-8 produc

tion through the autocrine effect. We tested the effect of continuoustreatment with neutralizing antibodies to IL-1 and tumor necrosis

The possibility that contaminating normal cells (monocytes and Tlymphocytes) could be the source of IL-8 expression is most unlikely

for the following reasons. It has been reported that resting bloodmonocytes produce little if any IL-8 when cultured in the absence ofstimuli (31). Like monocytes, T lymphocytes produce IL-8 only after

stimulation with mitogens, albeit in considerably lower amounts thanmonocytes (32). We also showed in this paper that IL-8 mRNA andprotein were not observed in normal resting T cells. Furthermore, IL-8

mRNA was also observed in fresh uncultured ATL cells.At present, the actual mechanism of IL-8 production in ATL-

related tumor cells is not known. It seems that infection of HTLV-I

alone does not explain this phenomenon, because the ability toproduce IL-8 is quite different in 5 HTLV-I-infected T-cell lines

examined. To understand the mechanism of increased expressionof IL-8 in HTLV-I-positive T cells, we first examined whether theviral transactivator tax was involved in the IL-8 induction. Weexamined the correlation between tax expression and IL-8 expression. MT-2 was found not to produce IL-8 mRNA or protein and

thus constitutive production of tax is not implicated in the induction of IL-8 (Fig. 1 and Table 1). Honda et al. (33) reported thatHTLV-I-infected T-cell lines MT-2 and HUT-102 produced parathyroid hormone-related protein, but MT-1 did not. Like the induction of parathyroid hormone-related protein, that of IL-8 may,

therefore, be regulated by factors of either viral or cellular originother than tax in HTLV-I-infected T-cells. Nuclear extracts prepared from MT-2 cells bound to the IL-8KB site at the same levelas those from the IL-8-producing T-cell lines (data not shown).The expression of IL-8 in MT-2 cells may be negatively regulatedthrough quite different mechanisms from other HTLV-I-infectedT-cell lines.

Next, direct transactivation by the tax gene of HTLV-I was tested.IL-8 gene expression and secretion were significantly induced by taxin a similar manner to IL-2, IL-2 receptor a, granulocyte-macrophagecolony-stimulating factor, and c-myc gene expression (27-30). Thus,the transcriptional regulation of the IL-8 gene by the transactivatingprotein tax of HTLV-I may in part be responsible for the production

>°

Competitor — — —

Lane 1 2 3

m

oo

Fig. 7. HTLV-I tax induces a formed DNA-protein complex binding to the IL-8xB site.Jurkat cells were transiently transfected with pHZRneo (Lane 2) or pH2R40M (Lanes3-6). Nuclear extracts were prepared from transfected cells and incubated with 32P-

labeled oligonucleotides corresponding to the IL-SxB site. Lysates from pH2R40M-

transfected cells were incubated in the absence or presence of the unlabeled E2F (Lane 4),IL-8«B (Lane 5), and IL-oxB (Lane 6). Arrow, migration of the NF-KB complex,separated from the free probe.

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IL-8 PRODUCTION IN ATL CELLS

factor a on IL-8 production of several HTLV-I-infected T-cell lines.No suppression of IL-8 production was seen by neutralization ofcytokines in the culture supernatant (data not shown). Therefore, theautocrine mechanism is not likely to be the major cause of IL-8production in HTLV-I-infected T-cell lines.

Constitutive production of inflammatory cytokines, such as IL-8that participate in normal immune functions, may have deleteriouseffects on the host; therefore, their synthesis must be tightlycontrolled. This is the first report that clearly describes a transac-tivating function for the tax gene in the IL-8 gene of T cells orsynovial cells. Accumulating evidence has suggested that dysregu-lation of IL-8 expression may be involved in the pathogenesis ofvarious immunologically mediated diseases and malignancies.Therefore, it is important to determine the regulatory mechanismsresponsible for the constitutive expression of the IL-8 gene andhow the dysregulation of this cytokine may play a role in HTLV-I-associated pathogenesis. We believe that our findings may lead toa deeper understanding of not only this novel biological function ofthe HTLV-I tax gene but also of the role of IL-8 in HTLV-I-associated pathology.

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1995;55:3592-3597. Cancer Res   Naoki Mori, Shuichi Murakami, Susumu Oda, et al.  

taxT-Cell Leukemia Virus Type I Possible Transactivation of the Interleukin 8 Gene by Human Production of Interleukin 8 in Adult T-Cell Leukemia Cells:

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