Proc. Nati. Acad. Sci. USAVol. 84, pp. 4557-4561, July 1987Immunology

Induction and regulation of mRNA encoding 26-kDa protein inhuman cell lines treated with recombinant human tumornecrosis factor

(cytotoxicity/interleukin 6/interleukin 1/y interferon/B-cell stimulatory factor 2)

P. DEFILIPPI*t, P. POUPART*, J. TAVERNIERt, W. FIERS§, AND J. CONTENT*¶*D6partement de Virologie, Institut Pasteur du Brabant, B 1180 Bruxelles, Belgium; tBiogent, B 9000 Gent, Belgium; and §Laboratory of Molecular Biology,State University of Ghent, B 9000 Gent, Belgium

Communicated by J. Brachet, February 17, 1987

ABSTRACT A 26-kDa protein, originally described inhuman fibroblasts superinduced for interferon f3 (IFN-13)production, and termed IFN-f32 by other investigators, isinduced by cycloheximide and by a 22-kDa, interleukin 1(IL-1)-related factor. Although the structure and sequence ofthe corresponding gene show nonhomology with the IFN-f3gene, the gene is identical to that of B-cell stimulatory factor 2,a human interleukin, and displays a very potent growth anddifferentiation factor activity for B lymphocytes. In this workwe show that IL-1,8 and tumor necrosis factor (TNF) stronglyinduce the 26-kDa protein in FS-4 fibroblasts and in sometransformed cell lines. Addition of cycloheximide to recombi-nant (r)IL-l and rTNF further enhances the level of 26-kDa-protein mRNA. We determined the kinetics of induction andthe amounts of rTNF and rIL-1j3 required for optimal induc-tion of this mRNA in FS-4 cells and in HeLa H21 cells and foundthat rIL-1f3 is a more efficient inducer of 26-kDa proteinmRNA than is TNF. By analyzing the inducibility of the 26-kDaprotein gene by rTNF and rIL-1/3 in a series of transformed celllines that differ in their sensitivity to the cytotoxic action ofTNF, we report a direct correlation between the 26-kDa proteinmRNA expression and the resistance of these cells to thecytotoxic effect of TNF.

When human fibroblast cells are induced to produce inter-feron (IFN)-p by treatment with poly(I)-poly(C) in the pres-ence of cycloheximide (CHX), synthesis of a protein of 26kDa (1), earlier described as IFN-02 (2), is stimulated.Induced epithelial cells (3) and peripheral blood lymphocytes(4) also express the 26-kDa-protein mRNA. Further, the genecoding for the 26-kDa protein is also induced by a highlypurified interleukin 1ip (IL-1p3)-related protein obtained fromcon A-stimulated human leukocytes (5), later demonstratedto be identical to IL-1f3 (6). Although it has been claimed thatthe 26-kDa protein has a weak IFN-like activity-neutralizedby anti-IFN-p8 sera-the biological function of this moleculeremained mysterious until the recent report of an identicalcDNA sequence coding for an interleukin, active on B-lymphocytes (7-9), and having a potent growth factor activityon the same cells (10).Tumor necrosis factor (TNF) is a cytokine secreted by

macrophages and/or monocytic cell lines upon stimulation-e.g., by lipopolysaccharide (LPS). It was discovered orig-inally in the sera of mice treated with bacillus Calmette-Guerin (BCG), followed by an endotoxin challenge for a fewhours, a substance that causes hemorrhagic necrosis, or, insome cases, complete regression of certain transplantedtumors in mice (11). In the last few years human TNF cDNAhas been cloned, sequenced, and efficiently expressed in

Escherichia coli by several groups (12-14). The biologicalactivities exerted by TNF are very broad. In vitro recombi-nant (r)TNF has divergent effects on the cell physiology,being either cytocidal and cytostatic on some transformedcell lines and inactive on others (15), or growth-promoting innormal diploid human fibroblast cells (16, 17).Because in multiple instances TNF displays activities

exerted mainly by IL-1 (18), it was of interest to checkwhether TNF also induced the 26-kDa-protein gene in humanfibroblast cells and, if so, whether it acts as a growth factor(10) or, conversely, as an antimitogenic effector appearingwhen cell growth is stimulated (19, 20). When comparing therTNF and rIL-10-mediated induction and regulation of the26-kDa-protein gene in several cell lines differing in theirsensitivity to the cytotoxic effect of TNF, we observed adirect correlation between inducibility of the gene encoding26-kDa protein by IL-103, or TNF, and its mitogenic versuscytotoxic effect in these cells.

MATERIALS AND METHODSCells were treated with 99% pure human rTNF (13), humanrIL-1f3, human rIFN-y (21) or rIFN-a2c in tissue culturemedium containing 2% newborn calf serum.

Hybridization and Probe. Hybridization was for 4 hr at 600(5) in the presence of 10% (wt/vol) PEG 6000 (22). The26-kDa-protein cRNA probe was prepared by transcribingeither T3 or SP6 plasmid constructions covering 800 basepairs (bp) from the 5' end of the cDNA clone 26K-7 to the 3'distal Sau3AI site (7) in the presence of 80 ,uCi (a-32P)UTP(Amersham; 800 Ci/mmol; 1 Ci = 37 GBq) and used at 10-15x 10, cpm/ml.

RESULTS

Induction of 26-kDa-Protein mRNA in Human Cell LinesTreated with rTNF or rIL-j3. In initial experiments, the26-kDa-protein mRNA level has been studied by dot hybrid-ization on cytoplasmic extracts of foreskin fibroblasts FS-4with a cRNA probe transcribed on the cDNA coding for the26-kDa protein. A typical experiment is presented in Fig. 1A.The rTNF treatment (30 ng/ml for 6 hr) enhances the level of26-kDa-protein mRNA more than 5-fold above the back-ground level of untreated culture. Because we had previouslyobserved a strong induction of the 26-kDa-protein mRNA by

Abbreviations: IFN, interferon; r, recombinant; IL-1p, interleukin1,; TNF, tumor necrosis factor; CHX, cycloheximide; rIFn-al2,recombinant human IFNa2c.tPresent address: Universitd di Torino, Cattedra Biologia Generale,Facoltd di Medicina e Chirurgia, Via Santena Sbis, 1-10126 Torino,Italy.STo whom reprint requests should be addressed at: Institut Pasteurdu Brabant, Rue Engeland, 642, B 1180 Bruxelles, Belgium.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. rTNF and rIL-1/3-mediated induction of26-kDa protein inFS4 cells. (A) Dot blot hybridization of cytoplasmic extracts. Cellswere induced for 6 hr, and cytoplasmic extracts were prepared andprocessed on Hybond filter (Amersham) as described (36) andhybridized. Lane a, untreated FS-4 cells; lane b, rTNF-treated cells(30 ng/ml); lane c, rIL-lp-treated cells (100 ng/ml); and lane d,IFN-a-treated cells (103 units/ml). All treatments were for 6 hr. (B)RNA blot analysis of total RNA (3) from induced cells. The b-d lanescorrespond to 5 ,ug of total cytoplasmic RNA. Lane a, marker = 2,ug of poly(A)+ RNA extracted from MG-63 fibroblast cells super-induced for IFN-,8 production (3). Lane b, untreated FS-4 cells; lanec, IFN-a (103 units/ml); lane d, rTNF-treated cells (30 ng/ml). Thetreatments were for 15 hr.

a natural IL-1 (5), it was important to check whether thisearlier result was really reflecting the effect of IL-1 or thepresence of trace levels of TNF in the leukocyte-derivedpreparations (23). A treatment with 100 ng of rIL-1 per mlconfirmed unequivocally our earliest conclusion (5) thatIL-1,8 induces 26-kDa-protein mRNAper se because, indeed,it enhanced the mRNA level 5- to 10-fold more than did TNF.Conversely, IFN-a (103 units/ml for 6 hr) does not signifi-cantly affect the basal level of 26-kDa-protein mRNA. RNAblot analysis of total RNA extracted from untreated andrTNF-induced cells (30 ng/ml) after 15 hr of treatment (Fig.1B) confirmed the high level of induction of the 26-kDa-protein mRNA in TNF-treated fibroblasts FS-4 and demon-strated that this mRNA migrates in agarose gel as a singleband of about 1.3 kilobases (kb) (3). A poly(A)+ mRNApreparation extracted from MG-63 cells (derived from anosteosarcoma), superinduced for IFN-f3 production, wasused as a positive control (1, 3). No detectable level of26-kDa-protein mRNA was observed in untreated FS-4 cells,which means that 26-kDa mRNA is not constitutively ex-pressed in normal fibroblast cells. Because 26-kDa mRNA isstrongly induced by IL-1 and TNF in MG-63 cells (Fig. 2A),another fibroblastic cell line that is resistant to the cyto-lytic/cytostatic effect ofTNF (15), we tested whether in thesecells this mRNA was also appropriately translated into26-kDa protein and its derivatives (1). In Fig. 2B we showthat, indeed, two immunoprecipitable proteins of apparentmolecular mass 22 kDa and 27 kDa were expressed andsecreted by the MG-63 cells; these proteins have previouslybeen described as processed products of the primary 26-kDapolypeptide first observed by cell-free translation of mRNAfrom induced fibroblasts (1).

Considering the possible involvement of the 26-kDa pro-tein in growth regulation, cytoplasmic dot-hybridizationanalysis of this mRNA was extended to a series of fivedifferent human-transformed cell lines, differing in theirsensitivity to TNF cytolytic/cytostatic effects (15). A clearTNF-mediated 26-kDa protein mRNA induction was ob-served in MG-63 and HeLa 21 (Table 1), two transformed celllines characterized by a relatively low sensitivity to treatmentwith TNF alone. The extent of induction of 26-kDa-proteinmRNA in TNF-treated HeLa H21 cells was three to fivetimes less pronounced than that obtained in TNF-treatedMG-63 cells, as quantified by cytoplasmic dot hybridizationof parallel cultures treated with 30 ng of rTNF per ml for 15

A a b c B d e f

FIG. 2. (A) rTNF- and rIL-1j-mediated induction of 26-kDamRNA. Dot blot hybridization of cytoplasmic extracts. Lanes a-c,cytoplasmic RNA from MG-63 cells; lane a, untreated cells; lane b,rTNF-treated cells (30 ng/ml); lane c, rIL-1p-treated cells (100ng/ml). All treatments were for 15 hr. (B) Immunoprecipitation of26-kDa protein synthesized by MG-63 cells. MG-63 cells weretreated as described below, washed, and labeled with 30 uCi of[35S]methionine per ml (800 Ci/mmol, Amersham) (1) for 2 hr.Culture medium was immunoprecipitated with 5 ,1u of goat serumdirected against partially purified IFN-P [which also contains anti-26-kDa-protein antibodies (1)], analyzed by electrophoresis on a 13%polyacrylamide gel, and fluorographed at -70°C for 20 hr. Lane d,Mr markers; lane e, untreated MG-63 cells; lane f, MG-63 cellstreated with both rIL-1P (100 ng/ml) and CHX (50 ,ug/ml) for 6 hr;during the last 2 hr ofincubation actinomycin D (2 ,ug/ml) was added.Arrowheads, 22-kDa and 27-kDa polypeptides derived from the26-kDa protein (1).

hr and RNA blot analysis (data not shown). Interestingly,both 26-kDa inducibility and the resistance to the cytolyticaction of TNF (15) are abrogated when HeLa H21 cells arepretreated with IFN-y (Fig. 4, next section).

Conversely, the cervix carcinoma line HeLa D98/AH2 andthe breast carcinoma lines BT-20 and MCF-7, for which 50%cytolysis occurs at very low TNF concentration, did notreveal any detectable expression of the 26-kDa-proteinmRNA after treatment with rTNF, in a concentration rangevarying from 0.3 ng/ml to 3 ,g/ml rTNF (Table 1). Fig. 3shows that even in the presence of 100 ng of rIL-1i3 per ml noinduction was observed in these cells. Because a chromo-somal rearrangement could be responsible for the loss ofexpression of the 26-kDa-protein gene in these tumoral celllines, it was of interest to evaluate the inducibility of26-kDa-protein mRNA by other inducers. As expected fromprevious results (3) showing that a protein synthesis inhibitorsuch as cycloheximide (CHX) is a good inducer of the26-kDa-protein mRNA, we found, indeed, that in HeLaD98/AH2 cells, this mRNA is well expressed by treating cellswith 30 ,ug of CHX per ml for 7 hr (Fig. 3).

Influence of IFN-y Treatment on 26-kDa-Protein mRNAExpression. Several TNF-mediated activities are stronglyinfluenced by other cytokines. A remarkable synergismbetween rTNF and rIFN-y in the in vitro and in vivocytotoxic/cytostatic action of TNF on tumor cells has beenreported (15, 24); however, the results concerning themitogenic effect of TNF in the presence of IFN-y are lessconsistent (17, 25). In order to determine whether TNF- andIL-1-mediated expression of 26-kDa-protein mRNA in ourexperimental conditions could be influenced by IFN-y,parallel cultures of FS-4 and HeLa H21 cells were pretreatedwith IFN-y (103 units/ml) for 15 hr, IFN-y was washed off,and the cells were rinsed once with phosphate-buffered salineand further incubated in the presence of TNF (30 ng/ml) orIL-1 (100 ng/ml) for 15 hr.

In HeLa H21 cells, whose cytotoxic response is strictlydependent on concurrent treatment with IFN-y, remarkably,the IFN-y-pretreated cells failed to accumulate 26-kDa-protein mRNA after an additional TNF treatment (Fig. 4,

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Table 1. 26-kDa-protein mRNA induction in different transformed and nontransformed humancell lines

50% growth inhibition assay* 26-kDa mRNA inductionCell line TNF sensitivity, ng/ml rTNF (30 ng/ml) rIL-1,i (100 ng/ml)

Cervix carcinomaHeLa H21 90 + +++HeLa D98/AH2 3 - -

Breast carcinomaBT-20 1.2 - -MCF-7 2.4 - -

OsteosarcomaMG-63 >90 ++ +++

Diploid fibroblastFS-4 >90 ++ +++

*This column was modified from ref. 15, table 1; sensitivity is now expressed in ng of rTNF per mlrequired to reduce cell survival to 50%; >90 means that no 50% inhibition could be obtained at rTNFconcentration up to 90 ng/ml. mRNA is compared with induced control: +, 3- to 5-fold; + +, 10-fold;+ + +, >10-fold, as determined by densitometric scan analysis of dot hybridization autoradiograms.All treatments were done for 18 hr in parallel confluent cultures.

lanes g-i). Similar observations were made on culturestreated simultaneously with both IFN-y and rTNF for 15 hr(data not shown). This effect is specific to TNF and was notobserved with IL-1, which is not cytolytic on these cellsunder the same conditions (Fig. 4, lanes g, 1, m). On thecontrary for FS-4 cells, although IFN-y is not a good inducerof 26-kDa-protein mRNA, in pretreated cells it clearly en-hanced the accumulation of the 26-kDa mRNA protein byrTNF, or even more strongly by rIL-1,B (Fig. 4, lanes a-f).

Kinetics of26-kDa-Protein mRNA Expression in Cells Treat-ed with rTNF or rIL-1,. These results suggest, at least inIFN-y-pretreated H21 cells, a differential effect of IL-1 andTNF on the gene encoding the 26-kDa protein. To gain moreinformation on the expression of the 26-kDa-protein mRNA,we compared its induction by IL-1 and TNF in FS-4 and inHeLa H21 cells because their different biological response toTNF is accompanied by a difference in 26-kDa-proteinmRNA accumulation.To measure the kinetics of 26-kDa-protein mRNA accu-

mulation in response to continuous treatment with rTNF or

rIL-lp, parallel cultures of FS-4 and HeLa H21 cells weretreated with 30 ng of rTNF per ml and 100 ng of rIL-1, perml, respectively, for different periods. Dot hybridization on

4.

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FIG. 3. 26-kDa mRNA induction in HeLa D98/AH2 cells. Dotblot hybridization of cytoplasmic extracts. The hybridization signalswere measured by scanning at 500 nm in a Beckman model DV-8densitometer; -fold induction, densitometer units induced/densitom-eter units uninduced-background level for untreated culture isnormalized to 1-fold induction. Lane a, untreated cells; lane b-f,rTNF treatments; lane b, 0.3 ng/ml; lane c, 3 ng/ml; lane d, 30 ng/ml;lane e, 300 ng/ml; lane f, 3 ,g/ml; lane g, rIL-1,8 (100 ng/ml); laneh, CHX (30 ,ug/ml); lane i, CHX (30 ,ug/ml) + rTNF (30 ng/ml). Allthe treatments were for 7 hr.

cytoplasmic extracts of these cultures showed that in HeLaH21 cells the kinetics of mRNA accumulation were similarfor both inducers; maximal level was reached within 5 hr oftreatment, remained fairly constant up to 15 hr and then, evenin the presence of the inducers, decreased to uninduced levelwithin 24 hr (Fig. 5 Upper). Conversely, in FS-4 cells therIL-lp-mediated 26-kDa-protein mRNA maximal inductionwas very early (2-5 hr of treatment) and remained constantfor up to 48 hr, whereas the rTNF-mediated response wasslower and reached a maximum only after 15 hr of treatment(Fig. 5 Upper).Moreover, although TNF and IL-1f3 share several common

biological actions (18, 26, 27), induction of cells may requiredifferent amounts of these monokines (26). At optimal time ofinduction (15 hr), maximal increase in 26-kDa mRNA was

observed by treating FS-4 cells with 3-30 ng of TNF per ml(Fig. 5 Lower); higher concentrations (300 ng/ml) onlyslightly increase this value. A very similar curve was ob-served with rIL-ip, although a shift towards the lowerconcentration range indicates a 3-fold higher "inducing"specific activity than that of rTNF. Using the same experi-mental conditions, this latter phenomenon was even more

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FIG. 4. Effect of IFN-y treatment on 26-kDa-protein mRNAaccumulation in FS-4 cells and in HeLa H21 cells. Dot blothybridization of cytoplasmic extracts. The treatments were asdescribed in Results. Cells were pretreated with rIFN-y (103units/ml) for 15 hr, rinsed once with phosphate-buffered saline, andtreated with rTNF (30 ng/ml) or rIL-lB (100 ng/ml) for 15 hr.Hybridization signals were measured and normalized as in Fig. 3.Lanes a-f, FS-4 cells; lane a, untreated cells; lane b, rTNF; lane c,rIFN-y--wash-rTNF; lane d, rIL-1,3; lane e, rIFN-y--wash-rIL-1p; lane f, rIFN-y. Lanes g-n, HeLa H21 cells; lane g, untreatedcells; lane h, rTNF; lane i, rIFN-y-wash-rTNF; lane 1, rIL-1,3;lane m, rIFN-y--wash-rIL-13; lane n, rIFN-y.

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FIG. 5. Kinetics of 26-kDa-protein mRNA accumulation (Upper)and dose-response experiments (Lower) in FS-4 and HeLa H21 cellstreated with either rTNF or rIL-1p. Dot blot hybridization ofcytoplasmic extracts. The hybridization signals were measured, andthe values were normalized as indicated in Fig. 3. (Upper) Cells wereincubated for the indicated time with rTNF (30 ng/ml) or rIL-1, (100ng/ml). (Lower) Cells were incubated for 15 hr with rTNF or rIL-1,.*, FS-4 cells + rTNF; o, FS-4 cells + rIL-13; *, HeLa H21 cells +rTNF; o, HeLa H21 cells + rIL-1,3.

remarkable in HeLa H21 cells, where the inducing activity ofrIL-1,8 was almost 30-fold higher than that of rTNF, 1 ng ofrIL-1,8 per ml yielding maximal 26-kDa-protein mRNAexpression.CHX Effect of TNF and IL-l-Mediated 26-kDa Protein.

Because it was reported that TNF can stimulate IL-1 pro-duction in macrophages (28) and because we cannot excludefrom the kinetics described above the occurrence of such aphenomenon here, we investigated whether the TNF-medi-ated induction of 26-kDa-protein mRNA is a primary re-sponse by testing its requirement for protein synthesis. It hasbeen shown that CHX alone induces the 26-kDa mRNA andthat this effect is additive with IL-1-mediated induction (3, 5).Fig. 6 shows that treatment of parallel cultures of FS-4 andHeLa H21 cells for 6 hr with CHX (30 ,g/ml) induced 10- to30-fold 26-kDa-protein mRNA. The same result was obtainedin HeLa D98/AH2 cells (Fig. 3). In FS-4 and in HeLa H21cells combinations of the CHX treatment with either rTNF(30 ng/ml) or rIL-1P (100 ng/ml) showed that the twomonokines have at least additive, ifnot synergistic, effects onCHX induction of the 26-kDa-protein mRNA level.

a b c d e f g h i I m

FIG. 6. Effect of CHX on the TNF and rIL-1,8 accumulation ofmRNA in FS-4 and HeLa H21 cells. Dot blot hybridization ofcytoplasmic extracts. Cells were treated for 6 hr with CHX (30Ag/ml) with or without rTNF (30 ng/ml) or rIL-1f3 (100 ng/ml).Lanes a-e, FS-4 cells; lanes a, untreated cells; b, rTNF; c, CHX; d,CHX + rTNF; e, rIL-1f3. Lanes f-m, HeLa H21 cells; lanes f,untreated cells; g, rTNF; h, CHX; i, CHX + rTNF; 1, rIL-1,8; m,CHX + rIL-10.

DISCUSSIONIL-1,8- and TNF-Mediated Induction of 26-kDa-Protein

mRNA: Comparison of Their Specific Induction Activities. Itis intriguing that many of the effects ofTNF on various cellscan also be seen after treatment with IL-1 (18, 26, 27),although the specific activity can vary considerably betweenthe cytokines (26). Our results confirm this view, showingthat rIL-1P is a more efficient 26-kDa-protein mRNA inducerthan is TNF. We observed, indeed, that 26-kDa protein wasinduced to a different extent in normal FS-4 fibroblasts andin transformed HeLa H21 cells after treatment with thesemonokines (Fig. 1); in both cell lines, although optimalinduction of this mRNA was obtained with similar kineticsfor the two monokines (Fig. 5), lower concentrations by afactor of almost 20-30 of rIL-1/3 were required than of rTNF(Fig. 5). Although TNF induced IL-1 in some types of cells(28), the effects seen are probably mediated directly by TNFbecause the effects are not abrogated by CHX treatment (Fig.6) [even if, in this case, CHX itself is a potent inducer (3)].Because, at least in endothelial cells (27) and in 3T3-L1adipocytes cells (29), TNF does not compete with IL-1 for thesame cell surface receptor, we suggest that the secondmessenger(s) involved in nuclear activation of specific genesmight be identical for TNF or for IL-1. The difference inspecific activities could be related either to the type ofreceptor-molecule interactions or to a different intracellulardistribution of the second messenger(s) molecules. Never-theless, whether the 26-kDa mRNA accumulation observedis the result of transcriptional activation of the 26-kDa-protein gene, or of some post-transcriptional stabilizationmechanisms, or of both (30, 31) has still to be elucidated.

Influence of IFN-y Treatment on 26-kDa-Protein mRNAExpression. IFN-y amplifies TNF activities on different celllines, either by enhancing the in vitro cytotoxic action ofTNFon tumoral cells (15) or by counteracting the mitogenicstimulation of fibroblast cells (17, 25).When HeLa H21 cells are exposed to IFN-y before TNF

treatment, they fail to express the 26-kDa-protein mRNA.Considering that this cell line becomes sensitive to TNFcytotoxic action only in the presence ofIFN-y (15), this resultconfirms the correlation between the 26-kDa inducibility andthe resistance to TNF-mediated cytotoxicity (Table 1). Thispoint is reinforced by the fact that IFN-y abrogates only theTNF-mediated induction of 26-kDa mRNA in HeLa 21 cellsand not the effect of IL-1 on these cells. This strongspecificity might reflect the distinct biological effects ofTNFon tumor cells (15, 24).

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The Biological Role of 26-kDa Protein in TNF/IL-1p Activ-ity. Although the precise function of this protein has beendebated (1), it has been reported that this protein, also namedIFN-032, shows IFN-like properties, such as antiviral activityand stimulation of expression of both 2-5A synthetase andHLA genes (8)-it is clear now that this protein is aninterleukin, with potent growth factor activity for B lympho-cytes (10). One question raised by our observations iswhether the growth-stimulating activity of TNF or IL-1 onfibroblasts is mediated by way of the 26-kDa protein. Weshow that the cell lines where TNF or IL-1 fail to elicit26-kDa-protein mRNA expression are characterized by ahigh response to TNF-mediated cytotoxic action (15 andTable 1). The data analysis suggests a correlation linking theTNF-mediated expression of the 26-kDa-protein gene inhuman cells and the cellular resistance to TNF cytotoxicity.Although no causal relationship presently exists, at least twointerpretations ofthe role of the 26-kDa protein (if any, in thissystem) are possible. (i) 26-kDa-protein induction could actas a mediator of TNF mitogenic activity thus extending itsgrowth-factor activity to new cell lineages. Future experi-ment will tell if this interleukin has multiple functions, or if itserves mainly as a link between various tissues and theimmune system. (ii) Alternatively, or additionally, the 26-kDa protein may act as a negative growth factor, constitutinga feed-back loop damping, or limiting, the mitogenic effect ofTNF when it occurs. This implies that on different cell typesthe 26-kDa protein could exert opposite functions as de-scribed previously for TGF-P (32). These possible alterna-tives can well be tested experimentally, as recombinant26-kDa protein and specific antibodies are now becomingavailable.When these studies were finished, TNF-mediated 26-kDa-

protein induction in fibroblast cells was reported to be relatedto the control of mitogenic action ofTNF (8, 19); these resultsare based on the increase of mitogenic response to TNF in thepresence of anti-IFN-P serum. A role of IFN-p8-like mole-cules in the autocrine control of cell growth, cell cycleprogression, and terminal differentiation has been proposedin several systems (33, 34). In PDGF-induced mitogenesis theinduction of IFN-,8 and 2-5A synthetase may attenuate theinitial growth response to growth factor (20). The nature ofthe IFN-,B-type molecule(s) implicated in feed-back controlof cell proliferation has not yet been ascertained. That theso-called IFN-A2 could be the 01-related molecule secreted inthe terminal differentiation of human hematopoietic cells hasbeen excluded (35). We do not presently know whether26-kDa-protein induction by TNF in some treated cells isrequired for, secondary to, or completely unrelated to itsactivity on cell growth. In the second case, the molecularbasis for a dual cell-specific activity could be investigated bycomparing specific cell surface receptors on various celltypes. Furthermore, the 26-kDa mRNA modulation in re-sponse to TNF treatment of tumor cells in vitro may help topredict the TNF response in vivo.

We thank Dr. P. Vandenbussche for help in densitometric scans,Dr. G. Haegeman for helpful discussion, Drs. E. De Clercq and A.Billiau for providing us with the goat anti-INF-f3 antisera, and Drs.A. Shaw and P. Wingfield (Biogen, Geneve) for a gift of rIL-13. Wealso thank Boehringer Ingelheim for a gift of IFN-a2,. We furtheracknowledge the help of Mrs. J. Herinckx for typing the manuscript.This work was supported by Grant 2.9006.79 of the Fund for JointBasic Research (Belgium) to J.C. and a grant from the Fonds voorGeneeskundig Wetenschappelijk Onderzoek and the General Sav-ings and Retirement Fund (A.S.L.K.) to W.F. P.D. was a Fellow ofthe Fondation Rose et Jean Hoguet.

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