MOLECULAR AND CELLULAR BIOLOGY, June 1993, p. 3831-38370270-7306/93/063831-07$02.00/0Copyright X 1993, American Society for Microbiology

Regulation of the Interleukin-l,B (IL-1lB) Gene byMycobacterial Components and Lipopolysaccharide Is

Mediated by Two Nuclear Factor-IL6 MotifsYIHONG ZHANG AND WILLIAM N. ROM*

Division ofPulmonary and Critical Care Medicine, Departments ofMedicine and Environmental Medicine,and Chest Service, Bellevue Hospital Center, New York University Medical Center,

550 First Avenue, New York, New York 10016

Received 2 December 1992/Returned for modification 6 January 1993/Accepted 8 March 1993

The cytokines interleukin-113 (IL-10) and tumor necrosis factor alpha (TNF-a) are released by mononuclearphagocytes in vitro after stimulation with mycobacteria and are considered to mediate pathophysiologic events,including granuloma formation and systemic symptoms. We demonstrated that the Mycobacterium tuberculosiscell wall component lipoarabinomannan (LAM) is a very potent inducer of IL-1I gene expression in humanmonocytes and investigated the mechanism of this effect. We localized the LAM-, lipopolysaccharide (LPS)-,and TNF-ca-inducible promoter activity to a -1311+15 (positions -131 to +15) DNA fragment of the IL-11gene by deletion analysis and chloramphenicol acetyltransferase assay. Within this DNA fragment, there weretwo novel 9-bp motifs (-90/-82 and -40/-32) with high homology to the nuclear factor-IL6 (NF-IL6) bindingsite. Site-directed mutagenesis demonstrated that the two NF-IL-6 motifs could be independently activated byLAM, LPS, or TNF-a and that they acted in an orientation-independent manner. DNA mobility shift assayrevealed specific binding of nuclear protein(s) from LAM-, LPS-, or TNF-a-stimulated THP-1 cells to theNF-EL6 motifs. We conclude that the two NF-IL6 sites mediate induction of IL-11" in response to the stimuliLAM, LPS, and TNF-cx.

Tuberculosis is characterized by granuloma formationwith caseation necrosis and systemic symptoms of weightloss, fever, and night sweats (8). Animal models demonstratecytokines in the granulomas, and in vitro experiments withmononuclear phagocytes have established that Mycobacte-rum tuberculosis stimulates the release of cytokines (5, 6,25, 33, 40, 41). Interleukin-lp (IL-1,B) and tumor necrosisfactor alpha (TNF-a) are the two major inflammatory medi-ators released in vitro by mycobacteria (5, 13). IL-l1 ischemotactic for lymphocytes and can activate CD4+ helper-inducer T lymphocytes, causing them to proliferate andrelease gamma interferon (IFN--y) (22). IL-1 and IFN--ystimulate macrophages to release TNF-a, which can alsostimulate the release of IL-113 (28, 35). Studies of recombi-nant IL-1 impregnated in ethyl vinyl disks revealed that IL-1could directly induce a granulomatous response in juxtapo-sition to the implanted disks (17, 27). Pulmonary granulomasinduced in mice by the intratracheal injection of antigen-coated beads were excised, and aqueous extracts of thegranulomata contained high levels of IL-1 (26). Studies byKunkel and colleagues demonstrated that IL-lp is the dom-inant cytokine in early schistosomal granuloma develop-ment, followed temporally by TNF-a (27). Thus, IL-1 maybe more important to the early cellular recruitment ofinflammatory cells and their activation, while TNF-a maycontribute to the continued organization of the maturegranulomatous lesion. Activation of the IL-1i gene isachieved by a number of stimuli, including lipopolysaccha-ride (LPS) from gram-negative bacteria, phorbol esters,silica, and other bacterial products (18, 30, 36, 43).Although mycobacteria do not possess LPS, the cell wall

of M. tuberculosis contains a complex lipid glycoprotein

* Corresponding author.

called lipoarabinomannan (LAM) which shares many phys-icochemical properties with LPS (5, 12). The cell wallbackbone of LAM is complemented by phenolic glycolipids,mycolic acids, arabinogalactan, peptidoglycan, and manno-phosphoinositides. However, the most antigenic and conse-quently biologically active component is LAM (10). Inaddition to stimulating cytokine release, LAM inhibitsIFN-y activation of macrophages and protein-antigen pro-cessing by antigen-presenting cells and can suppress T-lym-phocyte proliferation in vitro in response to mitogens andspecific antigens (5, 32, 39). Barnes and colleagues demon-strated that the phosphoinositol component anchoring LAMto the cell wall was critical for LAM's ability to stimulatecytokine release, because a deacylated LAM lacking thiscomponent was unable to stimulate cytokine mRNA expres-sion (5). In addition, Chatterjee and colleagues demonstratedthat LAM from the attenuated strain H37Ra stimulatedsignificantly more TNF-ot in vitro from murine peritoneal orbone marrow macrophages than LAM from the more viru-lent Erdman strain (12). The LAM from the Erdman strainwas shown to have mannose moieties capping the arabinanside chains, in contrast to the LAM from strain H37Ra (11).

In order to further dissect the host immune response totuberculosis, we have studied the mechanism of cytokinegene regulation in mononuclear phagocyte cells after stimu-lation with the M. tuberculosis component, the purified cellwall, LAM. We isolated a 1.145-kb fragment of the 5' IL-13genomic DNA and evaluated the responsiveness of thisfragment to stimulation with LAM and LPS. Interestingly,two copies of nuclear factor-IL6 (NF-IL6) binding sites werethe major cis-acting elements for stimulation with LAM,LPS, or TNF-a, as demonstrated by deletion analysis andsite-directed mutagenesis studies. In addition, the binding ofnuclear proteins to the NF-IL6 site was enhanced by thesestimuli.

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MATERIALS AND METHODS

Materials. 055 LPS was purchased from Sigma. Recom-binant human TNF-a (specific activity, 4.8 x 107 U/ml) wassupplied by M. Tsujimoto, Suntory Institute for BiomedicalResearch, Osaka, Japan. LAM was from a laboratory atten-uated strain of M. tuberculosis. H37Ra was provided by P.Brennan, Colorado State University, Ft. Collins. Culturefiltrate from an M. tuberculosis Erdman strain that was freeof LAM by sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) and Western blot (immunoblot)after anion-exchange and molecular size chromatographywas provided by P. Brennan. A Mycobacterium bovis 65-kDa recombinant heat shock protein (HSP-65kD) was pro-vided by R. Van der Zee, National Institute of Public Healthand Environmental Protection, Bilthoven, The Netherlands.The LAM had been eluted through Detoxi-Gel with sterile,pyrogen-free water and was stored in pyrogen-free vialsfrom which any contaminating LPS had been removed. Onlypyrogen-free water was used in reconstitution of this mate-rial. Evaluation of tuberculosis reagents, including LAM, forthe presence of gram-negative bacterial endotoxin was donewith an amebocyte lysate assay (E-Toxate kit; Sigma).

Cell culture. Human myelomonocytic leukemic cell lineTHP-1 cells were purchased from the American Type Cul-ture Collection, Rockville, Md. THP-1 cells were maintainedin RPMI 1640 supplemented with 10% fetal bovine serum.Peripheral blood mononuclear cells (PBMs) from purifiedprotein derivative-negative healthy volunteers were isolatedby centrifugation over Ficoll-Hypaque (Flow Laboratories),washed, and seeded onto the plastic surface of a 175-cm2flask in RPMI 1640 with 10% fetal bovine serum for 2 h at37°C. The flask was washed three times with RPMI 1640 toremove nonadherent cells, and the monocyte-enriched pop-ulation (>90% PBMs) was detached by scraping with arubber policeman.

Isolation ofRNA and Northern (RNA) blot analysis. HumanPBMs were stimulated for 5 h, collected by centrifugation,and lysed by addition of 5.5 M guanidinium isothiocyanatebuffer. Cytoplasmic RNA was isolated through CsCl2 gradi-ent ultracentrifugation. Equal amounts of the extracted RNAwere fractionated by electrophoresis through a 1% agar-ose-6% formaldehyde denaturing gel, transferred onto anitrocellulose filter (BA 85; Schleicher and Schuell), andbaked at 80°C for 2 h. The baked filter was incubated in 40 mlof prehybridization solution {50% formamide, 0.5% SDS,lOx Denhardt's solution, 2.5% herring sperm DNA, and 4xSSPE [lx SSPE is 0.18 M NaCI, 10 mM NaPO4, and 1 mMEDTA (pH 7.7)]} at 42°C for 6 to 12 h. An IL-11 cDNA probe(S. Gillis, Immunex, Seattle, Wash.), a TNF-ot cDNA probe(Genentech, South San Francisco, Calif.), and a pHe7cDNA probe were radiolabeled with [a-32P]dCTP (specificactivity, 3,000 Ci/mmol; New England Nuclear) by nicktranslation. Hybridization was carried out at 42°C for 10 h.The filter was then washed in 2x SSC (lx SSC is 0.15 MNaCl plus 0.015 M sodium citrate)-0.5% SDS at roomtemperature for 20 min and then was washed in 0.1xSSC-0.5% SDS at 65°C for 30 min. Autoradiography wasperformed at -70°C.

Screening of human genomic library and plasmid construc-tion. A human genomic library (Clontech) was screened byusing human IL-13 cDNA and the synthetic oligonucleotidesequence of IL-11 as probes. Eight positive clones werepicked up, and one of the eight was used for subcloning. AnIL-1p DNA fragment from positions -1130 to +15 (-1130/+15 fragment) was cut out with restriction enzymes XbaI

and TaqI, and subcloned into a polylinker site in front of thechloramphenicol acetyltransferase (CAT) structural gene onthe plasmid vector pTK(-).CAT (from Herbert Samuels,New York University Medical Center). A series of deletionmutant fragments were created by restriction enzyme cuttingfrom the 5' end, namely, Sau3 (-680), Styl (-490), DraI(-313), HindIII (-131), and DdeI (-69) fragments.

Transient transfection of human suspension cultures andassay of CAT activity. THP-1 cells were transfected withserially constructed plasmids by the DEAE-dextran method(19). Briefly, 107 cells were washed in STBS solution (25 mMTris [pH 7.4], 137 mM NaCl, 5 mM KCl, 0.6 mM Na2HPO4,0.7 mM CaCl2, 0.5 mM MgCl2) and were transfected with 10,ug of CsCl2-purified plasmids in 1 ml of STBS solutioncontaining 500 ,ug of DEAE-dextran per ml at 37°C for 90min. The transfected cells were then washed with STBSsolution and incubated in complete medium for 24 h in theabsence or presence of inducing agents. The transfectedcells were lysed by three cycles of freeze-thawing, and equalamounts of protein from different cell extracts were assayedfor CAT activity (21). The protein concentration was deter-mined with Bio-Rad reagents. For the CAT assay, 100 ,ug ofprotein was incubated with 0.1 ,Ci of [14C]chloramphenicol-250 mM Tris (pH 7.5)-360 ,ug of acetyl coenzyme A per ml ina total volume of 170 RI at 37°C for S h. The reactions werestopped by addition of 0.5 ml of cold ethyl acetate. Afterextraction with ethyl acetate, the upper layer was saved,dried, and spotted onto a thin-layer chromatography plate.After being developed in organic solution (95% chloroformand 5% methanol), the plate was air-dried and exposed toX-ray film.

Preparation of nuclear extracts and DNA mobility shiftassay. Cell extracts were prepared from THP-1 cells by usingthe method first described by Dignam et al. (16). Cultures(approximately 5 x 108 cells) were lysed by homogenizationin buffer A (10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.9], 1.5 mM MgCl2, 10 mMKCl, 0.5 mM dithiothreitol [DTT], 0.5 mM phenylmethylsul-fonyl fluoride [PMSF]) and centrifuged at 1,000 x g for 10min at 4°C. The nuclei were washed with buffer A once. Thenuclear proteins were extracted in buffer C (20 mM HEPES[pH 7.9], 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2mM EDTA, 0.5 mM DTT, 0.5 mM PMSF) on ice for 30 minwith shaking. The extracted nuclear proteins were dialyzedagainst buffer D (20 mM HEPES [pH 7.9], 20% glycerol, 0.1M KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF) for 5h and were frozen in liquid nitrogen in aliquots.The DNA mobility shift assay was carried out as described

by Sen and Baltimore (37). The DNA probes were end-labeled by using polynucleotide kinase in the presence of 100,Ci of [-y-32P]ATP. Probes (10,000 cpm) were incubated with2 ,g of nuclear extracts under binding conditions [of 10 mMTris (pH 7.5), 50 mM NaCl, 1 mM EDTA, 0.1 mM DPT, 2 ,ugof poly(dI-dC), and 10% glycerol in a total volume of 20 ,ul atroom temperature for 15 min, and then the probes wereelectrophoresed on a 4% polyacrylamide gel. The gel wasanalyzed by autoradiography.

RESULTS

Expression of IL-1,B and TNF-ca genes after stimulation withM. tuberculosis. Exaggerated quantities of the cytokinesIL-1, and TNF-ot are released after stimulation of mononu-clear phagocytes in vitro with components purified from M.tuberculosis. We have demonstrated that PBMs stimulatedfor 5 h with LAM, HSP-65kD, or culture filtrate upregulate

MOL. CELL. BIOL.

IL-13 GENE REGULATION BY NF-1L6 MOTIFS 3833

A.1 2 3 4 5

1.6kb -* -hi

B.1 2 3 4 5

AP- 1

+-1130

1.7kb -k j "

NF-kB NF-IL-6OCTAIoITI

IAP-1SRE TATA

-313 | +15

C.IL-16 TNFt

1 2 3 4 5

1kb i fI

pHe7

FIG. 1. Northern blot analysis of IL-lp and TNF-a levels inPBMs after stimulation. (A) Analysis of steady-state IL-lp mRNAlevels. Human PBMs were isolated as described in Materials andMethods and were stimulated for 5 h with the following stimuli (bylane): 2, TNF-a (10 ,ug/ml); 3, LAM (100 ng/ml); 4, HSP-65kD (100ng/ml); 5, culture filtrate (100 ng/ml). Lane 1 was the control. (B)TNF-a mRNA. Lanes are as in panel A. (C) pHe7 housekeepinggene in mRNA. Lanes are as in panel A.

IL-10 and TNF-cx mRNA, as detected by Northern analysis(Fig. 1A and B, respectively). Equal amounts of total RNAwere placed in each lane, as demonstrated by the pHe7housekeeping gene (Fig. 1C). The LAM and HSP-65kD were

similar in their stimulation of IL-13 and TNF-a mRNA; incontrast, the culture filtrate from the Erdman strain of M.tuberculosis was less potent. All three stimuli were evalu-ated for LPS contamination by the amebocyte lysate assayand were found to contain <5 pg/,ug of test reagent in theseveral lots tested.

Analysis of IL-11 promoter region by deletion analysis. Inorder to investigate the molecular mechanism involved in theactivation of IL-1i gene expression, we initiated experi-ments aimed at the identification of DNA sequences respon-sible for the regulation of the IL-1, gene by LAM, LPS, orTNF-a. A 1,145-bp IL-13 DNA fragment (-1130/+15) was

isolated from a human genomic library and cloned into thepTK(-).CAT plasmid. This DNA fragment contains severalsequences resembling known regulatory elements, as sche-matically illustrated in Fig. 2. To identify cis-acting elementsresponsive to LAM, LPS, or TNF-a, four IL-1i DNAfragments with deletions from the 5' end were fused to theCAT reporter gene. Activation of the IL-13 promoter regionwas determined by measuring CAT activities in THP-1 cellstransfected with these constructs after stimulation with thedifferent reagents.Our results showed that CAT activity gradually increased

as deletion advanced from positions -1130 to -313 and to-131 (Fig. 3). After further deletion to position -69, whichresulted in the removal of a sequence (-90/-82) that sharedhigh homology with the NF-IL6 binding site, there was onlyone-half to three-fourths the increase in CAT activity overthat of the control after stimulation. The data suggest thatmajor positive regulating elements on the IL-13 gene inresponse to stimulation by LAM, LPS, or TNF-a are located

-69- +15FIG. 2. Summary of deletion analysis of the 5'-flanking region of

the human IL-11 gene. Serial deletion mutants of the IL-113 DNA(-1130/+15) were created by using several restriction endonu-cleases and were cloned into the polylinker sites of plasmidpTK(-).CAT as described in Materials and Methods. Abbrevia-tions: SRE, serum response element; AP-1, activating protein 1;NF-kB, binding site for nuclear binding protein KB; NF-IL6, bindingsites for nuclear binding protein(s) to the IL-6 gene; OCTA, octamerbinding site.

between positions -131 and +15 because removal of thefragment (-1130/-131) did not decrease CAT activity. CATactivity actually increased after deletion of this fragment.This phenomenon may be due to derepression by removal ofsome negative regulatory element(s) within this DNA frag-ment (-1130/-131). Interestingly, the increase in CAT ac-tivity of stimulated versus unstimulated cells after deletionfrom positions -131 to -69 actually was smaller, stronglysuggesting that a positive regulatory element or elementsexisted between positions -131 and -69 (Fig. 3). Finally,the 84-bp DNA fragment from positions -69 to +15 stillshowed a significant increase in CAT activity after stimula-tion, suggesting that this 84-bp-long DNA sequence alsocontains a positive regulatory element or elements in re-

sponse to LAM, LPS, or TNF-o.LAM, LPS, and TNF-at have common positive regulatory

elements. There are two sequences on IL-1 that share theNF-IL-6 consensus sequence T(T/G)NNGNAA(T/G) (31).They are 5'-TTGTGAAAT-3' (-90/-82) and 5'-TTlTTGAAAG-3' (-40/-32). To test whether the two sequences are thepositive regulatory element(s), we performed site-directedmutagenesis of the IL-10 DNA fragment (Fig. 4). The

-1130 +15

C LAM LPS TNFtt C

.1 l. .-W

-313 +15 -131 +15

LAM LPS TNFtt C LAM LPS TNFt C

-69 +15

LAM LPS TNF.

1 5.3 4.7 2.9 1 4.8 4.5 4.0 1 4.2 4.0 6.7 1 3.1 3.0 2.8

FIG. 3. Deletion analysis of the 5'-flanking region of the IL-13gene. THP-1 cells were transiently transfected with four plasmidconstructs of the IL-1i promoter region, as indicated by thenumbers on the top. After stimulation with LAM (500 ng/ml), LPS(500 ng/ml), or TNF-ac (10 ng/ml), cytosolic extracts from thetransfected THP-1 cells were assayed for CAT activity. The foldincrease over control (C) activity is given below each lane.

-1+15

VOL. 13, 1993

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3834 ZHANG AND ROM

-1

131 j -- -- -- 1 5

NF-IL56 TATA

C LAM LPS TNF(i

--1 r -1

-1F31- X- 15-131TT[ I-I- AT 15

NF-IL-6 TATA NF-IL-6 TATA

C LAM LPS TNFQ

10-40 4

5-3--- -3I 3- -- 5 13-31 ----NF.- I-_ -69 -69 - ;NF-I-L-6 13

C LAM LPS TNFtt

pTK.CAT

C LAM LPSTNF(v

:.,

1 4.9 4.1 4.0 1 5.2 5.4 5.9 1 1.1 1.0 0.8

FIG. 4. Analysis of the IL-1i promoter region by site-directedmutagenesis. Three plasmids constructed with one NF-IL6 mutationor double NF-IL6 site mutations were tested by transient transfec-tion and CAT assays in THP-1 cells as shown on the top. The boxeslabeled X represent the mutated NF-IL6 motifs, and the foldincrease over control (C) activity is given below each lane.

mutations were made as follows: (i) the NF-IL6 sequence(-90/-82) was mutated to 5'-ACGTAAAAQ-3'; (ii) thesecond NF-IL6 sequence (-40/-32) was mutated to 5'-CCTTAAAA--3'. Three kinds of mutations were carried out bymutating either one of the NF-IL6-like sites and leaving theother one intact and mutating both NF-IL6-like sites. Themutants were sequenced to confirm the correct mutation.Induction of CAT activity by LAM, LPS, or TNF-a wasstriking, with mutation of the 5' NF-IL6 site leaving the 3'site intact (Fig. 4, left panel) or mutation of the 3' NF-IL6site leaving the 5' NF-IL6 site intact (Fig. 4, middle panel).After mutation of both NF-IL6 sites, inducibility of theIL-13 promoter by LAM, LPS, or TNF-a was abolished(Fig. 4, right panel). The inducibility of CAT activity on thewild-type -131/+ 15 DNA fragment is shown in Fig. 3. Theseexperiments suggested that the two NF-IL6 sites on theIL-lp gene functioned as positive regulatory sequences inresponse to the induction with LAM, LPS, or TNF-a. Eachof the NF-IL6 sites functioned independently of the otherbecause a single mutation did not affect CAT activity.NF-IL6 motifs on the IL-13 gene act in an orientation-

independent manner. Experiments were conducted with theDNA fragments containing the 5' NF-IL6 or 3' NF-IL6motifs subcloned into the polylinker sites of the plasmidpTK.CAT in either orientation. THP-1 cells were trans-fected with these constructs and were stimulated with LAM,LPS, or TNF-ac as described above. Stimulation of the-131/-69 fragment (containing the -90/-82 NF-IL6 site)with any of the stimuli revealed enhanced CAT activity witheither orientation of the DNA fragment (Fig. 5, left andmiddle panels). There was no CAT activity induced afterstimulation of the pTK(-).CAT plasmid vector alone (Fig. 5,right panel). Similar results were obtained with a -68/-31DNA fragment containing the 3' NF-IL6 site (-40/-32).Enhanced CAT activity was observed after any of the stimuliwith the DNA fragment in either orientation (Fig. 6, left andmiddle panels), and no CAT activity was induced afterstimulation of the pTK.CAT plasmid vector alone (Fig. 6,right panel).LAM, LPS, and TNF-a enhance interaction between nu-

clear protein(s) and the NF-IL6 sequence. To determinewhether the NF-IL6 sequence interacts specifically with

1 6.5 6.4 6.0 1 4.8 4.3 2.9 1 0.9 0.7 0.7

FIG. 5. The cis-acting element (-90/-82) that mediates induc-tion with LAM, LPS, and TNF-a functions in front of a heterolo-gous thymidine kinase promoter in an orientation-independent man-ner. The DNA fragment from positions -131 to -69 which containsthe NF-IL6 sequence (-90/-82) was subcloned into the polylinkersites of pTK.CAT in either orientation. As a control, the pTK.CATvector was included. The fold increase over unstimulated cells isgiven below each lane.

nuclear proteins, THP-1 cultures were treated with LAM,LPS, or TNF-a, and activation of nuclear protein binding toNF-IL6 sequences was tested by the DNA mobility shiftassay. Nuclear proteins isolated from stimulated THP-1 cellswere incubated with the end-labeled synthetic double-stranded 5' or 3' NF-IL6 probes in the presence or absenceof specific or nonspecific competitors. After treatment with

5 '3- 68 NNF-IL-6 - -31

C LAM LPS TNFIx

3 - 5-31 NF-IL-6 -68

C LAM LPS TNFu C

pTK.CAT

LAM LPS TNFet

I

I6.9; 6.

1 6.9 6.3 7.0 1 4.0 3.8 3.0 1 0.9 1.1 0.7

FIG. 6. The NF-IL6 sequence (-40/-32) functions in front of aheterologous thymidine kinase promoter in an orientation-indepen-dent manner. The DNA fragment from positions -68 to -31 whichcontains the NF-IL6 sequence (-40/-32) was subcloned into thepolylinker sites of pTK.CAT in either orientation. The pTK.CATvector is a control (C), and the fold increase over activity ofunstimulated cells is given below each lane.

MOL. CELL. BIOL.

IL-1, GENE REGULATION BY NF-1L6 MOTIFS 3835

A. LAM

1 2 3 4

es0

B. LPS

5 6 7 8

.4

C. TNFi,

9 10 11 12

0

A. LAM B. LPS C. TNFoc12 3 4 5 6 7 8 9 10 11 12

FIG. 8. Induction of the binding of nuclear protein(s) to the 3'NF-IL6 sequence as analyzed by DNA mobility shift assay. Lanesare identical to those in Fig. 7, with the 3' NF-IL6 sequence used asan end-labeled probe.

mm~~~~~~~~~~~~~~~FIG. 7. Induction of the binding of nuclear protein(s) to the 5'

NF-IL6 sequence as analyzed by DNA mobility shift assay. Stimu-lated THP-1 cell nuclear extracts were incubated with 32p- end-labeled synthetic 5' NF-IL6 probe (10,000 cpm) in the absence or

presence of competitors under the binding conditions described inMaterials and Methods. Lanes: 1, 5, and 9, unstimulated nuclearextracts; 2, 6, and 10, LAM-, LPS-, and TNF-a-stimulated nuclearextracts, respectively; 3, 7, and 11, LAM-, LPS-, and TNF-a-stimulated nuclear extracts, respectively, in the presence of a

100-fold molar excess of unlabeled synthetic mutant NF-IL6 se-

quence; 4, 8, and 12, LAM-, LPS-, and TNF-a-stimulated nuclearextracts, respectively, in the presence of a 100-fold molar excess ofunlabeled synthetic wild-type NF-1L6 DNA.

LAM, LPS, or TNF-a, a dramatically enhanced binding ofnuclear protein(s) to the 5' NF-IL6 site was observed (Fig. 7,lanes 2, 6, and 10, respectively). To test whether the bindingwas specific, competition experiments were carried out witha 100-fold molar excess of cold wild-type or mutated NF-IL6probe. The protein-DNA interaction was still present aftercompetition experiments with a 100-fold molar excess ofcold mutated NF-IL6 probe (Fig. 7, lanes 3, 7, and 11).However, binding of nuclear protein(s) to the NF-IL6 sitewas abolished by competition performed with 100-fold ex-cess cold wild-type NF-IL6 probe (Fig. 7, lanes 4, 8, and 12)after stimulation by LAM, LPS, or TNF-a, respectively.Similar results were obtained by using end-labeled 3' NF-IL6 probe (Fig. 8, same lanes as in Fig. 7). These datastrongly suggested that LAM, LPS, or TNF-a activated theIL-1f gene by enhancing the interaction between the NF-IL6 sequence and its binding protein(s).

DISCUSSION

IL-1lB appears to be an important cytokine in the hostresponse to M. tuberculosis in that large quantities can bereleased in vitro after stimulation of mononuclear phago-cytes (5, 13, 41). We demonstrated that LAM can increasethe IL-11 and TNF-a mRNA level in PBMs, and thisincrease was greater than the increase elicited by a LAM-free culture filtrate from the Erdman strain. The HSP-65kD

protein was equipotent compared with LAM in stimulatingIL-11 and TNF-a gene expression and is a cytosolic proteinimportant for protein assembly, folding, and transport (38).Interestingly, LAM was the most potent stimulus of IL-13mRNA, followed by HSP-65kD, TNF-a, and the culturefiltrate. Because LAM also stimulates TNF-a protein andmRNA, the release of TNF-a could amplify the stimulus forIL-10.We concentrated on investigating the mechanism by

which IL-1i gene expression is regulated by the mycobac-terial cell wall component LAM at the level of transcription.By comparing the action of LAM to that of LPS, we showedthat both LAM and LPS activate the IL-1l3 gene throughNF-IL6 binding sequences that function as positive regula-tory elements. TNF-a, a well-known activator of the IL-1,Bgene, shared a similar mechanism with LAM or LPS inIL-1i gene activation in human myelomonocytic THP-1cells (28). The two NF-IL6 sites functioned independently inresponse to LAM, LPS, or TNF-a stimulation becausemutation of either of them did not interfere with the functionof the other. An NF-KB site located at fragment -295/-286can be activated by a variety of stimuli, including LPS,TNF-oa, and IL-1, and very likely LAM, because LAMactivated NF-KB independently on the IL-6 gene (44).LAM or LPS induced nuclear proteins that bound to the

NF-IL6 wild-type target sequence specifically, as demon-strated by the DNA mobility shift assay. These resultssuggest that binding of the LAM-, LPS-, or TNF-a-inducedtranscription factors to the NF-IL6 sites contributed to thetranscriptional activation of the IL-1,B gene.

In both gel mobility shift assays with 5'- and 3'-end-labeled NF-IL6 probes, the stimulated nuclear proteins wereshown to compete with wild-type NF-IL6, and the TNF-a-stimulated nuclear proteins acted similarly, as shown in Fig.8. This report also demonstrates that LPS from gram-negative bacteria and LAM from mycobacteria functionthrough similar mechanisms: they both activate NF-IL6elements on the IL-1B gene. Thus, the NF-IL6 sequencemay be an important mycobacterial or bacterial responseelement and may be relevant to other host cytokines (1, 23).In fact, we have demonstrated that LAM activates NF-IL6sites on the IL-6 gene and enhances interactions betweennuclear proteins and the NF-IL6 element (44).The IL-1, gene spans a region of 7.5 kb with a coding

region divided into seven exons (4, 7, 31). Although there are

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3836 ZHANG AND ROM

two forms of IL-1 (IL-loL and IL-1,B) encoded by twoseparate genes with only 26% similarity in their amino acidsequences and 45% similarity in their nucleotide sequences,they bind to the same cell surface receptors and haveoverlapping biological activities (1, 4, 7, 14, 31). Interest-ingly, IL-lao has two NF-IL6 motifs as well (located at-199/191 and -147/-139) and an NF-KB (-749/-740) site(20). Pro-IL-10 is an inactive 31-kDa precursor that iscleaved by IL-10-converting enzyme between Asp-116 andAla-117, releasing the 153 COOH-terminal amino acids thatconstitute the mature cytokine (4, 9, 31).

Previous investigators have reported that LPS and theimmediate-early genes of human cytomegalovirus (CMV IE)activated promoter/enhancer elements on the 5'-flankingregion of IL-1,B and that progressive deletions of a -1,097/+14 sequence localized CMV IE-responsive elements to a-131/+14 DNA fragment similar to the one we isolated (15,24). Crump and colleagues described a 15-fold increase inCAT activity with this fragment after cotransfection inTHP-1 cells with a plasmid containing the CMV IEl gene(15). In this regard, the sequence between positions -131and +15 is an important positive regulatory element on theIL-lp gene and the two NF-IL6 motifs may contribute to thepromoter activity within this region. IL-11 mRNA in humanimmunodeficiency virus type 1 (HIV-1)-infected mononu-clear phagocytes increases 8- to 15-fold after LPS stimula-tion in comparison with non-HIV-1-infected cells that weresimilarly stimulated, suggesting synergism in transcriptionalactivation by the two stimuli (42). HIV-1 tat, a majorregulatory protein of HIV-1, may be the mediator of theseeffects.The NF for IL-6 expression (NF-IL6) has been cloned,

and the full-length cDNA encodes a 345-amino-acid proteinwhose C-terminal portion reveals a high degree of homologyto the C/EBP family of liver-specific transcriptional factors(2, 3). Also, NF-IL6 binds to transcriptional regulatoryregions in TNF-ot, IL-8, and granulocyte colony-stimulatingfactor, suggesting a role for NF-IL6 in inflammation and theacute phase response. Under Northern blot analysis, avariety of tissues, especially lung, expressed NF-IL6 mRNAafter LPS stimulation, and this pattern correlated with IL-6induction. The best fit for a consensus sequence for NF-IL6among species was found, and the C/EBP family was T(T/G)NNGNAA(T/G) (2). Using the NF-IL6 cDNA clone fromAkira and colleagues (2), we did not detect this specificmRNA in stimulated THP-1 cells, consistent with the con-cept that there is a family of nuclear binding proteins thatbind to the NF-IL6 consensus sequence. The proteins NF-KB-pSO and NF-IL6 may interact by using common leucinezipper motifs to enhance transcription of cytokine and othergenes involved in inflammation (29). As macrophages differ-entiated to more mature forms, there was a striking increasein NF-IL6 expression (34). Thus, we propose that LAMfrom M. tuberculosis activates NF-IL6, triggering a cytokinecascade of IL-1i, IL-6, IL-8, and TNF-ao release attractingmacrophages, lymphocytes, and neutrophils to sites of in-flammation; stimulating cell-cell adhesion; and differentiat-ing macrophages into the epithelioid and Langerhans giantcells forming a typical granuloma.Macrophages are major participants in immune defense

against M. tuberculosis (5, 8, 10, 13, 40). Cytokines pro-duced by macrophages play important roles in manifesta-tions and pathological events of tuberculosis, especiallygranuloma formation and systemic symptoms. To under-stand the mechanism(s) of how the expression of these

cytokine genes is regulated is important for future efforts tomodulate the host's response to M. tuberculosis infection.

ACKNOWLEDGMENTS

We thank Jan Vilcek for critical review of the manuscript, LauraSternberg for editorial assistance, and Patrick Brennan for LAM.We also thank the Aaron Diamond Foundation and grant M01

RR00096-2851 for support.

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