Proc. Nati. Acad. Sci. USAVol. 83, pp. 446-450, January 1986Immunology

Recombinant human tumor necrosis factor increases mRNA levelsand surface expression of HLA-A,B antigens in vascularendothelial cells and dermal fibroblasts in vitro

(monokines/lymphokines/major histocompatibility complex/interferons/HLA-DR antigen)

TUCKER COLLINS*, LYNNE A. LAPIERRE*, WALTER FIERSt, JACK L. STROMINGERt, AND JORDAN S. POBER**Departments of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115; tLaboratory of Molecular Biology, StateUniversity of Ghent, Ghent, Belgium; and tDepartment of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138

Contributed by Jack L. Strominger, September 5, 1985

ABSTRACT Recombinant human tumor necrosis factor(TNF), purified to greater than 99% homogeneity, increasessurface expression of class I major histocompatibility complex(MHC) antigens to a maximum of 9-fold on cultured humanendothelial cells (HEC) and human dermal fibroblasts (HDF).The increase is concentration dependent (peak 20-100units/ml) and time dependent (nearly maximal by 4 days);expression remains elevated in the continued presence of TNFand requires greater than 7 days to return to basal levels uponTNF withdrawal. The increase in surface expression appears toresult from increases in steady-state mRNA levels for the classI antigens, although the increase in mRNA is proportionatelygreater than for surface expression. No surface expression of ormRNA for class II MHC antigens is detectable in either controlor TNF-treated HEC or HDF. These effects are similar to thoseproduced by leukocyte or fibroblast (type I) interferons (IFNs).The protein synthesis inhibitor cycloheximide (CHX), whenadded coincidentally with type I IFNs, leads to superinductionofmRNA for class I MHC antigens and, unexpectedly, leads tothe appearance of mRNA for class II MHC antigens. CHX hasno effect by itself upon mRNA levels for class I or class II MHCantigens, nor does it modulate the increases in mRNA producedby immune (type II) IFN. Most interesting, CHX blocks theincrease in mRNA for class I MHC antigens induced by TNF.Thus TNF appears to act on MHC gene expression through anewly synthesized protein intermediate. Our results providedirect evidence that TNF can modulate gene expression innormal (untransformed) cell types and contribute to under-standing the complex nature of MHC gene regulation. Finally,they suggest that TNF may act in vivo as an immunoregulatorymolecule.

Class I major histocompatibility complex (MHC) antigensform part of the structure displayed on target cells forstimulation of and lysis by cytolytic T lymphocytes (CTL)(reviewed in ref. 1). Target cells with quantitatively greatersurface expression of class I molecules (H-2 K,D antigens inmice; HLA-A,B antigens in humans) are more potent stim-ulators and are more efficiently lysed (2). It follows thatmediators that enhance class I antigen expression on aparticular target cell may also enhance CTL-mediated lysis ofthat cell. In fact, this has been shown to be true for both typeI (leukocyte and fibroblast) and type II (immune) interferons(IFNs), the best-characterized inducers of MHC antigenexpression (3), and may contribute to both the antiviral andantitumor effects of IFNs in vivo.Tumor necrosis factor (TNF) is a 17-kDa polypeptide

mediator synthesized by activated mononuclear phagocytesthat is directly cytotoxic for certain tumor cells in vitro and

causes necrosis and regression of many solid tumors in vivo(4, 5). The mechanism of in vitro toxicity is unknown, but likethat of the related molecule lymphotoxin, it appears inde-pendent of protein synthesis and may involve degradation ofnuclear DNA (6). The mechanism(s) of in vivo tumor regres-sion are poorly understood and may or may not relate to thedirect in vitro cytotoxicity. Recent observations have sug-gested that TNF may have additional functions on culturedvascular endothelial cells (26) and other cell types (7). Herewe report that recombinant human TNF increases bothmRNA levels and surface expression of HLA-A,B antigensin normal (untransformed) human vascular endothelial cellsand dermal fibroblasts in vitro. Under these conditions, nodirect cytotoxicity is observed. The increase in mRNA levelis blocked by the protein synthesis inhibitor cycloheximide(CHX). These results have implications both for the mech-anism ofaction ofTNF and for the regulation ofMHC antigenexpression. In vivo, TNF, like IFNs, may serve to enhancethe participation ofCTL in the destruction of virally modifiedor neoplastic cells.

MATERIALS AND METHODSCells. Human umbilical vein endothelial cells were isolated

and initially cultured as described (8). Cells were seriallysubcultured on 0.1% gelatin-coated (Bactogelatin 0143-02,Difco) plastic, using endothelial cell growth factor (50-100ug/ml, a generous gift of Thomas Maciag, RevlonBiotechnology Research Center, Rockville, MD) and porcineheparin (50-100 ,g/ml, Sigma) to supplement the medium(9). Human dermal fibroblasts were isolated by JamesRheinwald (Dana Farber Cancer Institute, Boston, MA) or inour laboratory and serially subcultured, also as described(10). All cells used in these experiments were at passagelevels 5 through 15.

Mediators. Recombinant human TNF was expressed inEscherichia coli and purified to greater than 99% homoge-neity; final specific activity was 2.5 x 107 units (U)/mg asassayed for cytotoxicity on L929 cells (11). Other mediatorsused were recombinant human leukocyte (a-)IFN (stock 1 X105 U/ml of the consensus sequence from Amgen, ThousandOaks, CA), recombinant human fibroblast (,8-)IFN (stock 1 x107 U/ml) (12), and recombinant human immune (y-)IFN(stock 2 x 106 U/ml) (13). Mediators were added to theculture medium for the times and final concentrations indi-cated in the text; fresh mediator was added at each refeeding.In some experiments, CHX (20-25 ug/ml, from Sigma) wasadded as well.

Abbreviations: CHX, cycloheximide; CTL, cytolytic T lympho-cytes; HDF, human dermal fibroblasts; HEC, human endothelialcells; IFN, interferon; MHC, major histocompatibility complex;TNF, tumor necrosis factor; U, unit(s).

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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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Proc. Natl. Acad. Sci. USA 83 (1986) 447

Fluorescence Flow Cytometry. Cells were harvested andstained as previously described (14). Specifically, HLA-A,Bantigens were quantitated by staining with murine monoclo-nal antibody W6/32 (IgG2a) (15) followed by fluoresceinatedrabbit anti-mouse immunoglobulin (Miles Scientific,Naperville, IL); autofluorescence plus nonspecific stainingwas determined by using UPC10 (Walgene, Arcadia, CA) anisotype-matched myeloma protein, as the first antibody. Insome experiments murine monoclonal antibodies BBM.1(IgG2b, reactive with 832-microglobulin) and LB3.1 (IgG2b,reactive with HLA-DR antigen) (16) were used to stainreplicate samples. Fluorescently stained cell populationswere analyzed with a FACS analyzer (Becton-Dickinson).mRNA Quantitation. Cytoplasmic RNA was prepared and

analyzed by agarose gel electrophoresis and nitrocelluloseblotting as previously described (17). HLA-A,B and HLA-DR heavy chain transcripts were quantitated by using nick-translated probes pHLA2 and DRa, respectively, also aspreviously described (17).

RESULTSSurface Expression of HLA-A,B Antigens. Human endo-

thelial cells (HEC) and dermal fibroblasts (HDF) both ex-press HLA-A,B antigens in vitro. The quantity of surfaceexpression on both cell types is remarkably homogeneouswithin these uncloned cultured cell populations, as revealedby the narrow distribution of log fluorescence intensity (Fig.1), suggesting that expression is highly regulated. TNFcauses an increased expression ofHLA-A,B antigens in bothcell types (Fig. 1); comparable increases were noted for/32-microglobulin, the invariant light chain of the class I MHCantigens, but no expression of class II MHC antigens wasdetected basally or after TNF treatment (not shown). Theincrease of HLA-A,B produced by TNF at 20 U/ml is nearlymaximal by 4 days (approximately 9-fold as shown for HDFin Fig. 2) and persists at elevated levels through 7 days in thecontinued presence of TNF. Upon withdrawal of TNF, theHLA-A,B surface expression slowly declines to basal levelsover the next 7 days (not shown).The magnitude of the increased expression of HLA-A,B

antigens by HEC or HDF measured at 2 days depends uponthe concentration of TNF (shown for HEC in Fig. 3). Thereis no evidence of cytotoxicity for these normal (untrans-formed) cell types at any of the times and TNF concentra-tions used in these experiments, although there is a concen-

HEC

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FIG. 2. Cell number vs log fluorescence intensity forHDF treatedwith TNF at 20 U/ml for the number of days indicated. Staining forHLA-A,B antigens (large dots) or nonspecific staining (small dots) isas in Fig. 1. The increase in fluorescence at day 7 is 9-fold over thatat day 0.

tration-dependent decrease of replication rate for both celltypes (unpublished observations). Since fluorescence flowcytometry measures the surface expression on individualcells, the differences in cell number among the replicateculture groups does not confound this analysis. These resultswith TNF are qualitatively similar to those found previouslyfor IFNs (17).RNA Transcript Levels of HLA-A,B Antigens. IFNs have

HDF

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FIG. 1. Cell number (y axis) vs log fluorescence intensity (x axis) for HEC and HDF derived from the same donor, either control or treatedwith TNF at 100 U/ml for 3 days. Large dots represent staining for HLA-A,B antigens with antibody W6/32; small dots represent nonspecificstaining with antibody UPC10. The increase in HEC fluorescence is approximately 8-fold, whereas that for HDF is approximately 2-fold inTNF-treated cells.

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Proc. Natl. Acad. Sci. USA 83 (1986)

0 U/ml

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FIG. . Cellnumber vs log fluorescence intensity for HEC treatedfor 2 days with the concentrations of TNF indicated. Staining forHLA-A,B antigens (large dots) or nonspecific staining (small dots) isas in Fig. 1. The increase in fluorescence of cells treated with 500U/mil is about 4-fold over that of control cells.

been shown to modulate surface expression ofMHC antigensat least in part through increased transcriptional rate (18),resulting in an increased steady-state level of RNA tran-scripts (17-19). As shown in Fig. 4, TNF at 20 U/mi causesan extraordinary increase in the steady-state level of 1500-base-pair transcripts for class I heavy chains, possibly in therange of 100-fold or more. TNF, like IFN, also causes anincrease in minor transcript species that are larger than thepredominant mRNA species (see Fig. 5). These increases inclass I mRNA levels may result from increased transcriptionor changes in RNA processing or in mRNA stability or somecombination of these three processes. Transcript levelscontinue to rise throughout a 7-day time course ofTNF at 20U/ml, although the surface expression on these same cellshad reached a plateau by 4 days (compare Figs. 2 and 4). Thequantitative discrepancy between mRNA increases and in-creased surface expression is similar to that previously notedfor IFNs (17, 19).

Actions of CHX. The protein synthesis inhibitor CHX has

been used to probe the mechanisms of gene expression. Forexample, it has been reported that cells treated with type IIFNs in the presence ofCHX will synthesize mRNA relatedto the antiviral state [e.g., mRNA for (2'-5')oligoadenylatesynthetase], whereas cells treated with type II IFN will not(20). Other mRNAs may be directly induced by CHX orsuperinduced above the level of mediator alone by thecombination of CHX plus mediator. Specifically, CHX hasbeen reported to superinduce the level of class I MHCtranscripts in a tumor cell line induced by a-IFN (18). Weconfirmed this result, using recombinant a-IFN to treat HDF(assayed after 24 hr of treatment) and found that CHX alsosuperinduces class I MHC transcripts in-HDF treated with,-IFN (Fig. 5). Superinduction occurs when CHX is addedcoincident with IFN but not when added 4 hr later (notshown). CHX has no effect by itself on MHC transcripts.CHX does not superinduce class I MHC transcripts in thesecells when added coincident with y-IFN. Most significantly,addition of CHX coincident with TNF at 20 U/ml actuallyinhibits the increase of class I MHC transcripts.

Interestingly, neither type I IFNs nor TNF normallyinduces detectable levels of transcripts for class II MHCantigens. In the presence of CHX, however, the steady-statelevel of class II transcripts is increased by type I IFNs but notTNF (Fig. 5). CHX has no effect on class II transcriptsinduced by y-IFN.

DISCUSSIONTNF is a 17-kDa protein mediator produced by activatedmononuclear phagocytes (4, 5). The principal action ofTNFhas been thought to be cytotoxicity for tumor cells. Recentexperiments in our laboratory have shown that TNF actsupon cultured human endothelial cells but not dermal fibro-blasts to induce rapid (peak 4-6 hr) expression of a newsurface antigen (26). Expression of this molecule is transient,declining to basal levels by 24 hr in the absence or continuedpresence ofTNF. These endothelial-directed actions ofTNFare shared by at least one species of interleukin 1, a distinctmonokine produced in response to similar stimuli. Theexperiments presented here demonstrate that TNF alsoincreases expression of HLA-A,B antigen on bothendothelial cells and dermal fibroblasts. These effects areslower (plateau in several days) and are sustained in thepresence of TNF. This more pleiotropic action of TNF isshared by a different monokine, namely a-IFN, as well as by/3IFN and -IFN. Actions of purified preparations of inter-leukin 1 on MHC antigen expression have been inconsistent

Day0 1 4 7

FIG. 4. Cytoplasmic RNA blot for class I MHC heavy chaintranscripts from the same HDF cultures analyzed for surface antigenexpression in Fig. 2.

448 Immunology: Collins et al.

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Proc. Natl. Acad. Sci. USA 83 (1986) 449

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FIG. 5. Cytoplasmic RNA blots for HLA-A,B IHLA-DR (Lower) heavy chain transcripts fromHDF cul

for 24 hr with no mediator (control), a-IFN (1000 U/mi),U/ml), y-IFN (200 U/ml), or TNF (20 U/ml), with or v

added at 25 Ag/ml coincidently with the mediator.intensities of the HLA-A1B heavy chain transcripts (Us1.0, 4.0, 9.0, 4.5, 5.7, 2.9, 2.6, 2.6, and 1.1, respectively,by densitometric scan of a lightly exposed autoradiogr

(unpublished observations); definitive experimequire recombinant materials.TNF and type I IFNs both increase the steady

of mRNA for class I MHC antigens as wellexpression. However, type I IFNs and TNF ca

guished by their actions on cells coincidentally tthe protein synthesis inhibitor CHX. Whereas tre,type I IFN plus CHX leads to superinduction of cinterestingly, de novo appearance of class II)scripts, CHX appears to inhibit the induction causAlthough the mechanism of TNF action on the c

genes is not known, the results with CHX suggeinduces a protein, which in turn would incremessage production (through transcription or ni

processing) or stability. It is possible that TNF iralthough reprobing the time course RNA blot sh4 with a ,-IFN cDNA probe failed to demonstraof P-IFN mRNA (data not shown).The observations reported in this communi4

three major implications. First, our study of theinducing properties of type I and type II IFNs inregulation of MHC antigen expression in r

inflammatory/immune mediators is complex, in,positive and negative signals. The data suggestIFNs (i) directly (i.e., in contrast to TNF, I

synthesis of a protein mediator) increase MHCand (ii) coincidentally induce synthesis of atprotein of this effect. In this model, the inhibicould reduce production (i.e., act as a repressorate degradation of mRNA and need not act at thtas the interferon-mediated positive signal. The saddition of CHX would block synthesis of tiresulting in superinduction of class I transcriptsappearance of class II transcripts. Since CHinduce message by itself, inhibitor must not be rtutively. The lack of effect of CHX added 4 hr

suggests that the inhibitory protein must act early during theinduction process. The lack of effect ofCHX upon the actions-y-IFN suggest that (i) this mediator also acts directly to

+ increase MHC transcripts, but (ii) it does not induce synthe-4.4 v., sis of the protein inhibitor. In contrast, it has been reportedz z that transcripts related to "antiviral" effect induced by

y-IFN (but not by a- or 8-IFNs) can be inhibited by CHX(20). Thus y-IFN appears to act directly in the case ofMHCgene expression but perhaps indirectly in inducing the anti-viral state. This model predicts that a- or P3-IFN may inhibitthe ability of y-IFN to induce class II MHC transcripts(through induction of inhibitory protein) and hence surfaceexpression. Such an effect on surface expression has recentlybeen reported in murine macrophages (21). It also predictsthat similar regulatory sequences may be present in bothclass I and class II genes for induction of transcription by typeI and type II IFNs, such as those identified by Friedman andStark (22).

Second, our demonstration that TNF can increase specificmRNAs in two normal cell types is direct evidence that thismediator can increase gene expression. Furthermore, itsuggests that TNF, like interleukin 1, may prove to be apleiotropic mediator with many unsuspected activities. Therecent demonstration of Cerami and colleagues that TNFbinds to many normal cell types (23) and down-regulates

(Upper) and specific transcript levels in adipocytes (7) strongly supports[tures treated this idea. Effects of TNF on the adhesion of neutrophils to,j-IFN (1000 cultured endothelial cells have also been noted (ref. 24 andwithout CHX M. A. Gimbrone, Jr., personal communication) and theThe relative endothelial-directed effects appear to be similar to those ofqper) are 1.0, interleukin 1 (25).as measured Third, the action of TNF reported here, namely theraph. increased expression of HLA-A,B antigens, may make an

important contribution to the in vivo effects of TNF. Specif-nts will re- ically, this particular activity of TNF may enhance the

participation of the immune system, especially ofCTL, in the-state levels destruction of virally altered or neoplastic cells. It will beas surface interesting to see whether TNF (or the related mediatorn be distin- lymphotoxin) has other actions upon the cells of the immunetreated with system.atment withclass I (and, We thank Mr. Paul Cohill for expert technical assistance in cellMHC tran- culture and RNA isolation, Drs. Michael Gimbrone and Ramzied by TNF. Cotran for advice and support, Dr. Jan Tavernier for supplying thelass I MHC 3-IFN cDNA probe, and Ms. Donna Hickey for secretarial assist-st that TNF ance. This work was supported by grants from the National Institutesase class I of Health (HL36003, HL22602, and 5T32 HL07066). Research on

uclear RNA TNF is supported by Biogen. J.S.P. was a fellow of the Searleiduces IFN, Scholars Program during the initial studies reported here and is nowiown in Fig. an Established Investigator of the American Heart Association.te induction

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