tumor human

Proc.Natl.Acad.Sci. USA

Vol. 82, pp. 6637-6641, October1985 Immunology

Purification and characterization of a human tumor necrosis factor from the LuK1I cell line

(lymphokine/antitumorfactor/cancertherapy)

BERISH Y.

RUBIN*t,

SYLVIA L. ANDERSON*, SUSAN A. SULLIVAN*, BARBARA D.

WILLIAMSONt,

ELIZABETH A. CARSWELLt, AND LLOYD J.

OLDt

*Department ofLymphokineBiology,New York BloodCenter,NewYork,NY10021;and MLaboratoryofExperimentalCancerTherapy,Memorial

Sloan-KetteringCancerCenter,NewYork,NY 10021 Contributedby LloydJ. Old, May23, 1985

ABSTRACT A factor with tumor necrosis factor (TNF) activity produced bytheLuKII humanlymphoblastoidcell line [designated TNF(LuKII)] was purified sequentially by using controlled-pore glass, lentillectin-Sepharose, andprocionred agarosechromatography, yieldingTNF with aspecificactivity of 1.5 x 107 unitsper mg ofprotein andanisoelectric point of =6.7. Purified TNF(LuKII) fractionated by NaDod- S04/PAGEunderreducingaswellasnonreducingconditions wasfoundtocontainsevenproteinbandsofMr 80,000, 70,000, 43,000,25,000, 23,000,21,000,and19,000.TheproteinsofMr 80,000and70,000couldnotbedissociated into lower molecular weight components. Peptide mapping analysis and immuno- blotting analysisrevealed that thesevenproteinbands in the purified TNF(LuKII) preparations^arerelated. After fraction- ation of TNF(LuKII) by NaDodSO4/PAGE under reducing conditions,TNFactivitywasrecoveredfrom theregionsof

M,

70,000 and 19,000-25,000. Purified human TNF(LuKII) (i) produces hemorrhagic necrosis of Meth A mouse sarcoma in thestandard in vivo mouseTNFassay;(ii)has thesamepattern ofreactivityas mouseTNF(cytotoxic/cytostatic/no effect)on apanel ofhuman cancer celllines;and(iii)has itsanticellular effectpotentiated byinterferon, alsoafeature ofmouseTNF.

Thepresenceofa tumorinhibitoryfactorin theseraof mice infected with bacillus Calmette-Guerin (BCG) and subse- quentlyinjectedwithendotoxinwasreported byCarswellet al.

(1).

Sera from these micecause necrosisandregressionof certaintumors inmiceand haveacytotoxic effectontumor cellsinvitro(1-5).Byusingsimilarmethods,afactor withthe same in vivoandin vitropropertiescanbeinduced inrats(1) andrabbits(1, 6, 7). The antitumor factorpresentin thesera of animals sensitized to BCG or otherimmunopotentiating agents, such asCorynebacterium parvumorZymosan, and then challenged with endotoxin has been termed tumor necrosis factor (TNF). Biochemical studies have indicated thatserumTNVactivity isassociated with both high molec- ular weight components (4, 8) and components in the Mr range of40,000 to 70,000(3-5, 9, 10).

Wehave recently reported that human cell lines of hem- atopoieticoriginhavethecapacity toproduceafactor with TNFactivity (11). The product ofoneofthelines (LuKII) was chosenfor detailedstudies and, according to the follow- ing criteria, TNF(LuKII) and mouse TNF have identical properties: (i) mouse L cells made resistant to mouse TNF areresistant to TNF(LuKII), and L cells made resistant to TNF(LuKII)areresistantto mouse TNF; (ii) theanticellular response ofa panelof human cell lines to TNF(LuKII) or mouseTNFisindistinguishableand can be potentiated in a synergistic fashion by interferon; and (iii) TNF(LuKII)

causes hemorrhagic necrosis of Meth A sarcomas in the standard in vivoTNFassay (11).

In the present study, we report a sequential chromato- graphic procedure for the purification ofTNF(LuKII) and describebiochemical, serological, and biological character- istics ofpurified TNF(LuKII).

MATERIALS AND METHODS

Production ofTNF(LuKII). LuKII cells (8 x 105 cellsper ml) in RPMI 1640 medium containing 8% fetal calfserum were incubated with 10 ng of mezerein per ml (L. C.

Services, Woburn, MA) for 48 hr. The cellswereseparated bycentrifugation, resuspendedinfreshRPMI medium lack- ing any proteinsupplement, and incubated for an additional 48 hr. Cellswereremovedbycentrifugation, and the culture mediawereused as the source ofTNF(LuKII).

InVitroTNFAssay.TNFassays wereperformed in 96-well microtiterplaites.Serially diluted fractionsweresterilizedby ultravioletradiation and TNF-sensitive L cellswereaddedto each wellat adensity of 2x 104cellsperwell in 100

pl.

After incubation for 2 days at 370C, the plates were examined microscopically and thepercentage of dead cellswasdeter- mined. The unitage of the sample was calculated as the reciprocalof thehighest dilution that killed50% ofthecells.

All TNF assays were run in parallel with a laboratory standard andtitersareexpressed inlaboratory units.

In Vivo TNF Assay. The standard Meth A sarcoma assay wasperformedasdescribed(11).

MonoclonalAntibodytoHumanTNF. BALB/c micewere injected with 1600 units of TNF(LuKII), with a specific activity of 1.5 x 107 units/mg. For the initial injection, TNF(LuKII) was mixed with Freund's complete adjuvant (1:1) andwasinjectedsubcutaneously. Subsequent injections were given intraperitoneally in the absence of adjuvant.

Serum antibody to TNF(LuKII) was determined by an ELISA withTNF(LuKII)boundtopolystyrene plates. After nineimmunizationsover aperiod of7months,the spleenof one mouse witha high titer ofTNF(LuKII) antibody was removed and fused with cells of the P3U1 mouse plasma- cytoma cell line. Resulting clones were screened for their ability tobindTNF(LuKII) in ELISAs. Ahybridoma(des- ignated T1-18) producing antibody reactive with TNF- (LuKII) was isolated and subcloned. Media from T1-18 hybridoma cultures served as a source of TNF(LuKII) antibody.

AffinityChromatography. Affinity chromatographyproce- dures were carried out at room temperature and column fractionswerecollected intopolypropylene tubes or bottles.

The column matrices used were controlled-pore glass 350 (Electro-Nucleonics, Fairfield, NJ), lentil lectin-Sepharose

Abbreviation: TNF,tumornecrosis factor.

tTowhomreprintrequests should be addressed.

6637 Thepublicationcostsof this articleweredefrayedinpart bypage charge payment. This article must therefore be hereby marked "advertisement"

inaccordance with18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 82(1985)

121 3 141 5 ni 7 FRACTION

lT

8

0.5

0.4

0.3 V)

-J

z

_ D

c _

0 co >-

N H

0.2< >

U-

'I

0.1 H

10

E

0

CD

Z).

z

-

z

H-

FRACTION

80,000 0.05

C~~~~~~~~~~~~~~~~

PBS PBSE5::

LOAD El E2 E3l E4

0.04 60,000

0.03-

40,000 E

0

0.02 <

20,000

0.01

0 2- 0

468 121416 FRACTION

FIG. 1. (A) Controlled-pore glass column chromatography. LuKiI culture medium (8 liters) containing200units ofTNFactivitypermlwas

appliedtoacontrolled-pore glass column (50 ml) equilibrated with phosphate-buffered saline(20 mMsodiumphosphate,pH7.0/0.15MNaCl) (PBS). The columnwaswashed with the following buffers insequence:PBS(75 ml);PBScontaining20%ethyleneglycol(vol/vol)(E1)(225 ml); PBS (120 ml)/20 mM sodium phosphate, pH 7.0/1.15 M NaCl (E2) (175 ml); PBS (50 ml)/5 mM sodium phosphate,pH 6.8 (E3) (225ml);

and 5 mM sodiumphosphate, pH 6.8/5% triethylamine(vol/vol) (E4) (150 ml). Eluted fractionswerecollectedin polypropylene bottles. The materialeluted with the E4 bufferwas collected in 50-mlaliquots. (B) Lentil lectin Sepharose column chromatography. Partially purified TNF(LuKII)(150ml) eluted from thecontrolled-pore glass columnwasloadedontoalentil lectin-Sepharose column(10ml)equilibratedwith PBS. Thecolumnwaswashed sequentially with PBS (40 ml), PBS/1 M NaCl(El)(24ml), andPBS/iMNaCl/0.2Mmethyl-ca-D-mannoside (E2) (60 ml). The material eluted with themethyl-a-D-mannoside-containing bufferwascollected in 10-mlaliquots. (C) Procionredagarose

columnchromatography. Partially purified TNF(LuKII) (60 ml) eluted from the lentil lectin-columnwasdiluted1:1withPBS and loadedonto aprocion redagarosecolumn(4 ml)equilibratedwith 20 mMsodiumphosphate, pH 6.8/0.65 M NaCl (PBS/0.5MNaCl). The columnwaswashed with thefollowing buffers insequence:PBS/0.5 M NaCl(El)(30ml); PBS/1 M NaCl (E2) (8 ml); PBS (8 ml); PBS/50%ethylene glycol(vol/vol) (E3) (8 ml); PBS (8 ml); 0.1 M Tris-HCl, pH 9.4/0.1 M NaCl (E4) (8 ml); and 0.1 M Tris HCl, pH 9.4/0.1Marginine(E5)(24 ml). The material eluted with the 0.1 M Tris HCl, pH 9.4/0.1 M arginine bufferwascollected in 4-ml aliquots.

(Pharmacia), and procion red agarose (Bethesda Research Laboratories).

Protein Determinations. Protein determinations were car-

riedoutwith the Bio-Raddyereagent(I3io-Rad) using bovine

serumalbumin as astandard.

Radioiodination ofTNF(LuKII). TNF(LuKII) was labeled with 125I using 1,3,4,6-tetrachloro-3a,6a-diphenylglycouril (Iodo-Gen, Pierce). Polypropylenetubeswerecoated with100

tug

of lodo-Gen(dissolvedinchloroform) by evaporationof the solvent. A2-mlsampleofTNF(LuKII) (50,000units/ml)with

aspecific activity of 1.5 x

107

units permg ofprotein ^was incubated for 25 min at room temperature in an lodo-Gen- coated tube containing 2 mCi of125I (1 Ci = 37 GBq). The labeledproteinwasthen separated fromthe unbound 1251 by

using a Pa column (Bio-Rad). equilibrated with phosphate- buffered salinecontaining50 ugofcytochrome cperml. The iodinated material eluted in the voidvolume of the columnwas

divided intoaliquotsandstoredat -80TC.

NaDodS04/PAGE.

NaDodSO4/PAGE was carried out in 18-cmslabgels accordingtopublishedmethods (12).

Isoelectrofocusing. Isoelectrofocusing ^was performed by using Ampholine Pagplates (pH 3.5-9.5) (LKB). The gels

wererunat30 Wfor 1.5 hr,atwhichtimethepHgradientwas

measured and thegel^wasslicedinto 18equal pieces.Thegel fractions were incubated for 18 hr in Eagle's minimum

essential medium (ME medium) containing 10% fetal calf

serumandfractionswere assayedforTNF in vitro.

Peptide Mapping Analysis. A

251I-labeled

preparation ofpu-

rifiedTNF(LuKII)wasfractionated byNaDodSO4/PAGEand individual bandslocalized byautoradiography ^werecutfrom the gel and treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin or N-a-tosyllysine chloromethyl ketone-treated chymotrypsin. Digestedfractions

wereanalyzed accordingtothe methods of Elderetal. (13).

Immunoblotting Analysis. Immunoblottingwasperformed essentially^asdescribed(14). Briefly,preparations of purified TNF(LuKII)were transferred to nitrocellulose paperover-

nightat100mA. Afterincubation of the nitrocellulosepaper in buffercontaining bovine serum albumin, the paper was

exposedfor 2 hrto40 ml ofT1-18antibody-containingculture medium. The nitrocellulose paper was then washed exten- sively and incubated overnight in 10 mM Tris-HCl, pH 7.4/0.9%NaCI/5%bovineserumalbumin/'251-labeledrabbit anti-mouseIgG.Thenitrocellulosepaperwasfurther washed andexposedto x-rayfilm.

RESULTS

Purification of TNK(LuKII). Controlled-poreglass beads bound all TNF activity from LuKII culture fluids. After washingwithseveral buffers insequence, TNFactivitywas

Table 1. Purification ofTNF(LuKII)

Load Recovery

Specific Specific

activity, activity, % -Fold

Column Units units/mg Units units/mg recovery purification

Controlled-pore glass 1.6 x 106 5.3 x 103 9.6 x 10 3.8 x 105 60 72

Lentillectin-Sepharose 9.6 x105 3.8 x 105 6.3 x 105 1.3 x 106 39 245

Procion redagarose 6.3 X 105 1 X 106 6.3 x 105 1.5 x 107 39 2830

A

LOAD PBSEl PBS E2PBSE3 E4:

1 q

16,000

-J

12,000

z

U-z H4,000

.1

I:

T 660638

immunology:

Rubin etal.

Immunology:Rubinetal.~~~~~Proc.Nati. Acad. Sci. USA 82 (1985) 6639 eluted with a 5 mM sodium

phosphate

buffer

(pH

6.8)

containing

5%

triethylamine (Fig.

lA).Theeluted TNF was

then

applied

to alentil

lectin-Sepharose

column,which was

washed first with

phosphate-buffered

saline and then with 0.02 M sodium

phosphate

buffer(pH 6.8) containing 1.15 M NaCl (buffer A).TNF

activity

waseluted from this column withbuffer A

containing

0.2 M

methyl-a-D-mannoside (Fig.

111).

All TNF

activity

bound to the lentil

lectin-Sepharose

column and 39% of the

activity

was recovered in the

methyl-a-D-mannoside-containing

buffer. (Further

washing

of the column with buffer

containing

50%

ethylene glycol

elutes

only

asmallamountof additionalTNF

activity.)

TNF from the lentil lectin column was then diluted 1:1 with

phosphate-buffered

saline and loaded onto a

procion

red agarosecolumn. The column waswashed

sequentially

with severalbuffers thatremove

protein having

noTNF

activity.

The column was then washed with 0.1 M Tris-HOl,

pH 9.4/0.1

M

arginine.

TNF

activity

waselutedwith thisbuffer,

yielding

TNFwitha

specific activity

of1.5 x

i0'

unitsper mg of

protein.

Table 1 summarizes the

purification

scheme for TNF(LuKII)with

specific

activities of the

resulting

fractions.

Biochemical Characterization of Purified TNF(LuKII).

Isoelectric

focusing

of

purified

TNF(LuKII) indicates an

isoelectric

point

of ==6.7

(Fig.

2).

"'I1-labeled

TNF(LuKII)with

a

specific activity

of 1.5 x 10 units per mg of

protein

was

analyzed by NaDodSO4/PAGE

and found to contain seven

protein

bands withMrvaluesof80,000, 70,000, 43,000,25,000, 23,000, 21,000, and 19,000

(Fig.

3). The same seven

protein

bands were observed when nonlabeled

purified

TNF(LuKII)

wasfractionated

by NaDodSO4/PAGE

andexamined

by

silver

staining.

The

proteins

ofMr 80,000and70,000wereelutedfrom the

gels

and

reanalyzed by NaDodSO4/PAGE. They migrated

once

again

to the Mr 70,000-80,000

region,

and no lower molecular

weight

componentswereobserved.Infurtherexper- iments,

purified

TNF(LuKII)wasboiled in

NaDodSO4,

urea, and

2-mercaptoethanol,

and the same characteristic ^seven bandswere found.

To determine which bands in TNF(LuKII) showed TNF

activity, parallel samples

of

purified TNF(LuKII),

one 125i1 labeled and -one unlabeled, ^were treated with 0.1%

NaDodSO4/0.

1 M

2-mercaptoethanol

and fractionated

by NaDodSO4/PAGE.

After

electrophoresis,

thelane contain-

ing

theunlabeledmaterialwascutinto

4.'4-mm

slices and the

proteins

wereelutedfrom each slice

by overnight

incubation at40Cin MEmedium

containing

fetal calfserum.The

parallel

lane

containing 1251I-labeled

TNF(LuKII) wasdried immedi-

ately

after

electrophoresis

and

protein

bandswerelocated

by autoradiography.

Asseenin

Fig.

4,TNF

activity

was recov-

ered from the

gel

at Mrvalues of

70,000

and

19,000-25,000, corresponding

to I25-labeled

protein

bands at these

posi-

1I

8 Fraction

U)

I <

z

FIG. 2. Isoelectrofocusing ofTNF(LuKII). A 60-1.d sample of purified TNF(LuKII) containing 1500 units was applied to an

ampholine gel (pH3.5-9.5).

MrXA103 1 2

70 - d

25- 23- 21 -~

19

FIG. 3. NaDodSO4/PAGE of puri- fled I'll-labeled TNF(LuKII). TNF(Lu- K11) was iodinated and fractionated by NaDodSO4/PAGE. Autoradiographs

weredevelopedfor 18 hr(lane 1)and 0.5 hr(lane 2). Thefollowing proteins pro- vided

Mr

markers: myosin(Mr, 200,000), 13-galactosidase(Mr, 130,000), phospho- rylaseb(Mr, 94,000),bovineserumalbu- min (Mr, 67,000), ovalbumin (Mr, 43,000), a-chymotrypsinogen (Mr, 25,700),

/3-lactoglobulin

(Mr, 18,400), lysozyme(Mr, 14,300), andcytochrome

c(Mr, 12,300).

tions.

TNF(LuKII) samples

that were not

exposed

to 2-

mercaptoethanol

before

NaDodSO4/PAGE

also showed TNF

activity

atMrvalues of70,000and 19,000-25,000.

To examine the

relationships

among the various

protein

bands in

purified TNF(LuKII) preparations,

two-dimensional

chymotryptic

and

tryptic peptide mapping analyses

wereper- formed. As seen in

Fig.

5a, the

chymotryptic peptide

maps demonstrate that the

proteins

ofMr 43,000, 25,000, 23,000, 21,000,and19,000arerelated and the

proteins

ofMr 80,000and 70,000 ^are related. To examine the

relationship

of the

larger

molecular

weight proteins

tothe smaller

proteins, chymotryptic digests

of theMr 70,000 and 25,000

proteins containing equal

amountsof

radioactivity

weremixed and

analyzed.

Asseenin

Fig.

5b,three of the

fragments

(termed A, B,and C)

generated by digestion

of the Mr 25,000

protein migrate

to the same

position

as three

fragments generated by digestion

of the Mr 70,000

protein.

Asimilar

analysis

wascarried^out

using trypsin

as the

proteolytic

enzyme. The results also indicate that the

seven distinct formsare

closely

related.

Furtherevidence for the

relationships

among the various

proteins

in

purified TNF(LuKII)

comesfrom

immunoblotting analysis

with Tl-18 monoclonal

antibody

to TNF(LuKII).

Fig.

6shows that the

antibody

reactswith theMr 43,000and theMr 19,000-25,000 components.

Biological

Characteristics of PurifiedTNF(LuKII).Limulus

M. 80 70 43 25-19

980

cn 70- 60 _50-

CZt340

uL 30 20 10

2 6 10 1 4 1 8 22 26 30 34 38

Fraction

FIG.4. RecoveryofTNFactivityafterNaDodSO4/PAGEfrac- tionation ofTNF(LuKII).AsampleofTNF(LuKII)containing6000 units adjusted tocontain 0.1%NaDodSO4and 0.1 M 2-mercapto- ethanolwasappliedtoa12%polyacrylamide gel.Afterelectropho- resis, thegelwassliced andactivitywaseluted andassayed. Inan

adjacent lane, I'll-labeled TNF(LuK1I)was fractionatedandauto-

radiographedtodeterminethemolecularweightof theTNF(LuKII) active fractions.

Immunology:

Rubin etal.

..nn

Proc. Natl. Acad. Sci. USA 82 (1985)

-W 25

....e

. .

x x

E

T L C

E b

25 70

B

V.-

x

*:. .a

,

f _ x

|T

L

E 25- 70

ITL

C

_

_ _

testsofpurified TNF(LuKII)indicate 25ngofendotoxinper

nil. TNF(LuKII) causes hemorrhagic necrosis of Meth A sarcomaafterintratumoralorintravenousinjectionandtotal tumorregression has been observed in some treated mice.

L-cell lines made resistant to mouse TNF or to partially purifiedTNF(LuKII)areresistanttopurified TNF(LuKII).

Withthepanelof human cell lines studiedbyWilliamsonand coworkers (11), purified TNF(LuKII) showed the same

pattern of reactivity (cytotoxic/cytostatic/no effect) as mouseTNFandpartiallypurified TNF(LuKII). Inaddition, purified TNF(LuKII) and interferon showed synergistic cytotoxic activityfor humantumorcells,similartowhathas

FIG. 5. Chymotryptic peptide mapping of12"I-la- beled proteins in purified TNF(LuKII).. 125I-abeled TNF(LuKII) was fractionated on NaDodSO4/PAGE andindividualprotein bandspresentingel sliceswere

incubatedovernight in thepresence of 50 ,ug of N-a- tosyllysine chloromethyl ketone-treated chymotrypsin

perml.Theindividualgel sliceswerethen washed with waterandsamples (10,000 cpm) of eachwerelyophilized todryness. These samples were dissolved inabuffer containing formic acid and acetic acid andwereapplied tocelluloseprecoated glass TLC platesattheorigin(x).

Electrophoresis (E) wasperformed from right toleft, followed by ascending chromatography in a buffer containing butanol, pyridine, and acetic acid. Autora- diographs ofthechymotrypticmaps of the Mr80,000, 70,000, 43,000, 25,000, 23,000, 21,000,and19,000pro-

teinsarepresented (a); (b) chymotrypticmapsofthe Mr 25,000and70,000andamixture of theMr25,000 and 70,000 proteins.

previously beenreportedforpartiallypurified TNF(LuKII) andmouseTNF(12).

DISCUSSION

Wehaverecentlydescribed theproduction, characterization, andbiologicalpropertiesofahuman factor with TNFactivity from the LuKII lymphoblastoid cell line (11). The further purification and characterization of this factor, designated TNF(LuKII), is the subject of this report.Aprotocolforthe purification of TNF(LuKII)hasbeen developedthatyields bothgoodrecoveries of TNF andmaterialwithhigh specific activity. This purification protocol allows active fractions

T LC 6110

Immunology:

Rubin etal.

0

OF

Proc. Natl. Acad. Sci. USA 82 (1985) 6641 Mrx 10-3

43 -

' FIG. 6. Immunoblotting analysis ofTNF- (LuKI1) with T1-18 mouse monoclonal anti- 19-25 body. A sample of TNF(LuKII) containing 10,000 units was fractionated by NaDod- S04/PAGE.Fractionatedproteinswere trans- ferred to anitrocellulose membrane andpro- cessedas described.

elutedfromonecolumntobeapplied either directlyorafter dilutionontothenextcolumn, thereby eliminatinganyneed for dialysis and thus avoiding the losses associated with dialysis. TNF(LuKII) has a Mr of 70,000 by gel filtration undernonreducing conditions andanisoelectricpoint of 6.7.

Examinationof purified TNF(LuKII) byNaDodSO4/PAGE under reducing as well asnonreducing conditions revealed the presence ofsevenprotein bandsranging fromMr19,000 to 80,000. The Mr 80,000and 70,000proteins could not be dissociated into smaller molecularweightcomponents even after boiling in NaDodSO4/2-mercaptoethanol/urea. Frac- tions fromNaDodSO4/PAGEwereassayed forTNFactivity andproteins in theMr70,000 and 19,000-25,000region had TNFactivity. Using peptide mapping analysis,wefoundthat the seven proteins present in purified TNF(LuKII) were related. Immunoblotting analysiswith monoclonalantibody to TNF(LuKII) showed shared determinants on the Mr 43,000 and 19,000-25,000 proteins. Antibody did notreact withthehigher molecular weight forms, even though these have been shown to be related to theMr43,000 and lower molecular weight components. This could be due to the inaccessibilityof the determinantontheMr70,000 and 80,000 species.Thus,ouranalysisindicates thatthere are anumber ofstructurallyrelated proteinsin purifiedTNF(LuKII) and that TNF activity is associated with nondissociable high molecular weight and low molecular weight forms. We conclude that thesevenproteinsinourpurifiedTNF(LuKII) areeithertheproductsofrelated genesorproductsDCofa

single genethatundergoes extensive processing.

TNF(LuKII) has the full range of biological activities associated with mouse TNF. It produces hemorrhagic ne- crosis ofMeth A sarcomain the standard TNF assay and cannotbe distinguishedfrom mouse TNF in its patternof reactivity on a large panel of human cancer cell lines. In addition, Lcells made resistantto mouseTNFareresistant toTNF(LuKII), and cells made resistanttoTNF(LuKII)are resistantto mouseTNF. Recentwork has indicated that there are surface receptors forTNF on TNF-sensitive cells(un- published data). Competitive binding studies showed that mouseTNFandTNF(LuKII)competefor thesamereceptor.

The relationship between TNF(LuKII) and lymphotoxin (15, 16) is unclear. While they share certainproperties,such astheir ability to killmouse Lcells, their affinityfor lentil lectin,theirmultipleforms, and,insomecases,their cellular origin, they differ in certain biochemical properties. Ag- garwal andcoworkers (17, 18) have observed thatlympho-

toxinfrom the RPMI 1788 lymphoblastoid cell line has a Mr valueof25,000 and 20,000 under reducing conditions, where- as TNF(LuKII) exists in several molecular weight forms, someof which donotdissociate under reducingconditions.

Granger et al. (15) have also reported that species of lymphotoxin existinhighermolecular weight forms. It seems likelythat there is afamily ofcytotoxic factors with TNF activityand thatlymphotoxinand TNF(LuKII) are members of thisfamily. This would be comparable to the interferon system in which there are three major species, all having antiviral activities, but eachbeing coded forby adifferent gene withvarying degrees of homology. As in the interferon field, the cloning of factors withTNFactivitywillprovidethe basisfordistinguishing various molecules of this family.The recentcloning ofhuman TNF (19-21)and human lympho- toxin(22) has clarified the relationship between these two molecules. RecombinantTNFandlymphotoxinaredistinct molecules ofMr 17,000 and 18,600 that share considerable sequence homology. Both kill L cells and have tumor necrosisactivity.Inlight of the observation that TNF(LuKII) exists inhigher molecular weight forms, itseemsunlikelythat allforms ofTNFhave been cloned.

This researchwassupported in part by grants from the National CancerInstitute(CA-38661 and CA-08748).

1. Carswell, E. A., Old, L.J.,Kassel, R.L., Green, S., Fiore, N. &Williamson, B. (1975) Proc. Natl. Acad. Sci. USA 72, 3666-3670.

2. Helson, L., Green, S., Carswell, E. A. & Old, L. J. (1975) Nature(London) 258, 731-732.

3. Mannel, D. N., Meltzer, M. S. & Mergenhagen, S. E. (1980) Infect.Immun.28,204-211.

4. Kull, F. C. & Cuatrecasas, P. (1981) J. Immunol. 126, 1279-1283.

5. Haranaka, K. & Satomi, N. (1981) Jpn. J. Exp. Med. 51, 191-194.

6. Matthews, N. & Watkins, J. F. (1978) Br. J. Cancer 38, 302-309.

7. Ostrove, J. M. &Gifford,G. E. (1979) Proc. Soc. Exp. Biol.

Med. 160, 354-358.

8. Green, S., Dobrjansky, A., Carswell, E. A., Kassel, R.L., Old, L.J., Fiore, N. & Schwartz, M. K. (1976)Proc. Natl.

Acad. Sci. USA 73, 381-385.

9. Matthews, N., Ryley, H. C. & Neale, M. L. (1980) Br. J.

Cancer42, 416-422.

10. Ruff, M. R. & Gifford, G. E. (1980) J. Immunol. 125, 1671-1677.

11. Williamson, B.D., Carswell, E. A., Rubin, B.Y., Prendergast, J. S. &Old, L.J. (1983)Proc.Natl.Acad. Sci.

USA80, 5397-5401.

12. Laemmli,U. K.(1970)Nature(London)227, 680-685.

13. Elder, J. H., Jensen, F. C., Bryant, M. L. & Lerner,R. A.

(1977)Nature(London) 267, 23-28.

14. Burnette,W. N.(1981) Anal. Biochem. 112, 195-203.

15. Granger, G. A., Yamamoto, R. S., Fair, D. S. & Hiserodt, J.C. (1978)Cell. Immunol.38, 388-402.

16. Rosenau,W.(1981)Int.J.Immunopharmacol. 3, 1-8.

17. Aggarwal, B.B., Moffat,B.& Harkins,R.N.(1984) J. Biol.

Chem. 259, 686-691.

18. Aggarwal, B.B., Henzel, W.J.,Moffat, B., Kohr, W. J. &

Harkins,R. N.(1985)J.Biol. Chem. 260, 2334-2344.

19. Pennica,D., Nedwin,G.E., Hayflick,J. S., Seeburg,P.H., Derynck, R., Palladino,M.A., Kohr,W.J.,Aggarwal,B. B.

&Goeddel, D.V.(1984)Nature(London)312, 724-729.

20. Shirai,T., Yamaguchi, H., Ito, H.,Todd, C.W.&Wallace, R. B.(1985)Nature (London)313, 803-806.

21. Wang, A.M., Creasey, A. A., Ladner, M.B., Lin, L.S., Strickler,J., VanArsdell,J. N., Yamamoto, R. &Mark,D. F.

(1985)Science228,149-154.

22. Gray, P. W., Aggarwal, B. B., Benton, C. V., Bringman, T. S., Henzel,W.J., Jarrett,J.A., Leung,D.W., Moffat, B., Ng, P.,Svedersky, L.P., Palladino,M.A. &Nedwin,G. E.

(1984)Nature(London)312, 721-724.

Immunology:

Rubinetal.