0 1989 by The American Society for Blochemistry and Molecular Biology, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 264, No. 20, Issue of July 15, pp. 11966-11973 1989

Printed in L~.s.A.

Purification and Biologic Characterization of a Specific Tumor Necrosis Factor a Inhibitor*

(Received for publication, December 27, 1988)

Philippe Seckinger, Sylvie Isaaz, and Jean-Michel DayerS From the Division of Immunology and Allergy, Hans Wikdorf Laboratory, H6pital Cantonal Uniuersitaire, 1211 Geneua4, Switzerland

The purpose of the present investigation was to pu- rify a urine-derived tumor necrosis factor a inhibitor (TNFa INH) and to characterize its mechanism of ac- tion. For the purification procedure, urine was concen- trated and TNFa INN purified by ion-exchange chro- matographies, gel filtration, TNFa affinity column, and reverse-phase chromatography. The TNFa INH migrates with an apparent M, of -33,000 when esti- mated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis run under both reducing and nonre- ducing conditions. Elution of TNFa INH activity from the gel yields also a -33,000-Da inhibitory fraction. Besides inhibiting TNFa-induced cytotoxicity in L929 cells in the presence of actinomycin D, the TNFa INH impeded in a dose-dependent manner prostaglandin EP production and expression of cell-associated interleu- kin-1 by human dermal fibroblasts. Therefore, TNFa INH is active on both actinomycin D-treated and un- treated cells. In contrast to TNFa, TNFB-induced cy- totoxicity was only slightly affected by the inhibitor. This specificity was confirmed by the fact that it af- fected neither interleukin- l a nor interleukin- 18 bio- logic activities. The mechanism of action of TNFa INH involves blocking of la6I-TNFa binding to the promon- ocytic cell line U937. Moreover, preincubation of “‘1- TNFa with TNFa INH increased binding inhibition, suggesting an interaction between TNFa and the inhib- itor.

Tumor necrosis factor a (TNFa),l also called “cachectin,” is a peptide originally identified for its ability to cause hem- orrhagic necrosis of tumors and cytotoxicity in certain cell lines (1). It is mainly produced by monocyte macrophages and participates in a wide range of biologic activities (2). In the inflammatory process, TNFa stimulates both in vitro and in

~

* This work was supported in part by the Swiss National Science Foundation, Grant 3.400.0.86 by a grant from GLAXO, and by the Foundation “Centre de recherches mbdicales Carlos et Elsie de Reu- ter.” The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘uduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

~

$ To whom reprint requests should be addressed. The abbreviations used are: TNFa, tumor necrosis factor a; IL-

1, interleukin-1; rhIL-la, recombinant human IL-la; PGE2, prosta- glandin Ea, rhIL-lp, recombinant human IL-l@; TNFP, tumor necro- sis factor @; TNFa INH, tumor necrosis factor inhibitory activity; rhTNFa, recombinant human TNFa; PBS, phosphate-buffered sa- line; MEM, minimal essential medium; DMEM, Dulbecco’s modified Eagle’s minimal essential medium; PHA, phytohemagglutinin; OD, optical density; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography; LAF, lymphocyte-activating factor.

uiuoproduction of interleukin-1 (IL-1) (3) with which it shares several biologic activities, i.e. prostaglandin EP (PGE2) and collagenase production by human dermal fibroblasts and syn- ovial cells (4) and induction of fever by stimulating hypothal- amic PGE, synthesis (3). TNFcv also induces a cell-associated form of IL-1 (5) and is a common mediator of cachexia, shock, and anemia (2,6-9).

A closely related protein, TNFP (lymphotoxin), is also cytotoxic for some tumor cells and in almost all experimental systems has properties similar to TNFa, presumably due to the binding to a common receptor. The two genes encoding for TNFa and -B are closely related with cognate proteins sharing 34% homology in their amino acid sequence (10-12).

Despite the fact that TNFa is capable of killing certain neoplastic cells in uiuo, therapeutic use may be complicated by the diversity of its activities, suggesting that both agonists and antagonists may have functional utility (13, 14).

In the context of inhibition of inflammation, cytotoxicity, and fever, we originally reported that urine of some febrile patients contains a TNFa inhibitory activity (TNFa INH) when tested in a cytotoxicity assay on the TNF-susceptible cell line L929 (15). Primary characterization indicated an apparent molecular weight of 35,000-60,000 and a PI range of 5.5-6.1. This TNFa INH was distinct from the “23,000-Da” IL-1 inhibitor, also reported in our laboratory (16, 17). We now report the purification of TNFa INH and its specificity to block TNFa-induced biologic activities in various target cells. The TNFa INH specificity for TNFB or IL-1 was investigated as well as its mechanism of action that involves ligand binding inhibition.

MATERIALS AND METHODS

Reagents and Media-Phosphate-buffered saline (PBS), fetal calf serum, penicillin, streptomycin, glutamine, minimal essential medium (MEM), Dulbecco’s modified Eagle’s minimal essential medium (DMEM), and RPMI 1640 were obtained from Gibco (Paisley, Scot- land). Phytohemagglutinin (PHA) was purchased from Wellcome Research Laboratories (Beckenham, Kent, England). Recombinant human TNFa (rhTNFa) with a specific activity of 9.6 X 10’ units/ mg (18), rhIL-la and rhIL-lp with an identical specific activity of 1.5 X IO7 units/mg (19,20) were produced in Escherichia coli (Biogen SA, Geneva, Switzerland). Natural human lymphotoxin was a gift of Dr. E. D. Amento (Genentech Corp., San Francisco, CA). Goat polyclonal antibody to rhTNFa was provided by Dr. R. Ulevitch (Scripps Clinic, La Jolla, CA) and rabbit polyclonal antibody to rhIL- la from Biogen SA (Geneva, Switzerland).

Cell Culture-The human cell line U937, was maintained in RPMI 1640 medium supplemented with 100 wg/ml streptomycin, 100 units/ ml penicillin, 1% glutamine, and 10% fetal calf serum. The murine fibroblast cell line L929 was maintained by passage in Eagle’s MEM supplemented with 100 pg/ml penicillin, 100 units/ml streptomycin, 1% glutamine, and 10% heat-inactivated fetal calf serum. All cultures were performed in 96-well microplates and cultured at 37 “C in 595% C02/air in humidified atmosphere.

TNFa INH Bioassay-Prior to bioassay, all specimens were steri-

11966

A Human Tumor Necrosis Factor a Inhibitor 11967

lized using a 0.22 pm membrane filter (SLG S 025BS, Millipore, Bedford, MA). TNFa INH activity was measured in an assay of cytotoxicity using a TNF-susceptible cell line L929. Cells were seeded at 25,000 cells/well and cultured for 24 h. Cells were incubated with actinomycin D (1 pg/ml) (Sigma), and urine specimens were tested in the presence of various concentrations of rhTNFa ranging from 1 to 5 ng/ml. After 18 h of incubation, media were removed and cell lysis was determined by staining the plates with 0.5% crystal violet. Dye uptake was calculated by using an automated Micro-ELISA autoreader (MR 700; Dynatech Laboratories Inc., Guernsey, Channel Islands, U.K.). The blank was determined by killing all L929 cells with 50 pg/ml phorbol myristate acetate. Low values of dye uptake measured by the OD at 570 nm correspond to a high cell mortality induced by rhTNFa, and high values of optical density correspond to an increased cell viability. The percent of inhibition was calculated by using the formula:

TNFa INH (%) = 100

(OD actinomycin D + rhTNFol + TNFa INH) - (OD actinomycin D + rhTNFa)

(OD actinomycin D) - (OD actinomycin D + rhTNFa) 1

Optical density values from cells cultured with actinomycin D alone correspond to 100% of inhibition, and optical density from cells cultured with actinomycin D and rhTNFa correspond to 0% of inhibition.

Urine Collection and TNFa INH Purification-Urine (30 liters), collected from untreated febrile patients (r38.5 "C) devoid of urinary infections, was concentrated by Amicon ultrafiltration apparatus, molecular size cutoff -5000 Da, HI0 P5-20 cartridge (Amicon). Post- Amicon concentrate (600 ml) was salt-fractionated with 40-80% of ammonium sulfate, the pellet dissolved in 120 ml of 10 mM Tris-HCl, pH 8.0, containing 2 mM EDTA, and stored at -70 "c.

Ammonium sulfate-precipitated urine (120 ml) was chromato- graphed on a DEAE-Sephadex anion-exchange chromatography col- umn (2.6 X 20 cm) (Pharmacia, Uppsala, Sweden) equilibrated in a 10 mM Tris-HC1 buffer, pH 8.0, containing 2 mM EDTA. Bound material was eluted with the equilibration buffer containing 0.8 M NaCl. After testing in the L929 assay, inhibitory fractions (8 ml) were pooled (160 ml) and dialyzed against 4 X 2 liters of 10 mM sodium acetate buffer, pH 5.0, and loaded onto a Sulphopropyl- Sephadex cation-exchange column (0.8 X 15 cm) (Pharmacia, Upps- ala, Sweden) equilibrated in the same sodium acetate buffer. Bound material was eluted with the equilibration buffer containing 0.5 M NaC1. Inhibitory fractions (7.5 ml) were concentrated 20-fold by Amicon (molecular size cutoff -10,000 Da) and then gel-filtrated on a Sephadex S-200 column (2.6 X 100 cm) (Pharmacia, Uppsala, Sweden) equilibrated with 50 mM Tris-HC1, pH 7.4, buffer containing 100 mM NaCl at a flow rate of 27 ml/h. Fractions (9 ml) were collected and tested for TNFa INH activity. The column was calibrated with dextran blue (2,000,000 Da), bovine serum albumin (67,000 Da), ovalbumin (43,000 Da), a-chymotrypsinogen A (25,000 Da), and RNase (13,500 Da). This purified material was used for the biologic activities and referred to as TNFa INH.

TNFa column affinity was prepared as indicated by the manufac- turers. Briefly, 1 mg of rhTNFa was coupled to Mini Leak Agarose (Kem En Tec, Biotechnology, Corp., Denmark) in 0.8 M potassium phosphate buffer, pH 8.6. The remaining active groups were blocked by incubation in 0.1 M ethanolamine-HCI, pH 8.5, buffer. The gel was washed three times in 50 ml of 50 mM Tris-HC1, pH 7.4, buffer containing 100 mM NaC1. The TNFa INH fractions (15 ml) from the previous step were chromatographed on the affinity column. Bound material was eluted with a 0.2 M glycine-HC1, pH 3.5, buffer. The eluted fractions (1 ml) were immediately adjusted to pH 7.0 by addition of 5-40 ~l of 1 M Tris. The fractions containing TNFa INH were lyophilized and subjected to reverse-phase chromatography.

Reverse-phase FPLC Chromatography-Lyophilized affinity col- umn-bound proteins were dissolved in 2 ml of 0.1% trifluoroacetic acid (Fluka, Buchs, Switzerland), and loaded onto a ProRPC reverse- phase FPLC column (5 X 20 cm) (Pbarmacia) equilibrated in 0.1% trifluoroacetic acid. Bound proteins were eluted with a 0-100% ace- tonitrile gradient in 0.1% trifluoroacetic acid at a flow rate of 0.3 ml/ min. To each fraction (0.75 ml) 10 pl of 0.5 M NH4HC03 was added. Eluted material was immediately lyophilized and then dissolved in 1 ml of 10 mM Tris-HC1, pH 7.4, containing 2 mM EDTA.

SDS-PAGE was performed as described by Laemmli (21). Samples

were loaded onto a 15% polyacrylamide gel with a 3% stacking gel, and gels were silver-stained as described by MerriI et al. (22). The molecular weight markers (Bio-Rad) were bovine serum albumin (66,200 Da), ovalbumin (45,000 Da), carbonic anhydrase (31,000 Da), soybean trypsin inhibitor (21,500 Da). Samples run under nonreduc- ing conditions were tested for biologic activity. Two-millimeter gel slices were eluted by overnight incubation at room temperature in 200 pl of Tris-HC1, pH 7.4, containing 2 mM EDTA and then tested for the presence of TNFa INH at 1:lO dilution on L929 cells in the presence of a final concentration of 0.15 ng/ml rhTNFa.

Purification of IL-1 Inhibitor (IL-1 INHI-The urinary IL-1 INH was purified as described previously (16). Briefly urine of febrile patients (>38.5 "C) was concentrated by ultrafiltration and am- monium sulfate precipitation. The pellet was chromatographed on DEAE-Sephadex anion-exchange chromatography (Pharmacia, Uppsala, Sweden), hydroxyapatite (Bio-Rad), and finally gel-filtrated using an Ultrogel AcA 54 column (2.6 X 100 cm, LKB, Bromma, Sweden). Fractions eluting between 18,000 and 25,000 Da were used for the biologic studies.

PGE2 Production by Dermal Fibroblosts-Cells were seeded at a concentration of 20,000 cells/well and cultured for 48 h. Cells were then stimulated with buffer alone or rhTNFa at concentrations ranging from 0.5 to 5 ng/ml, and TNFa INH studied at three dilutions (1:20,1:50, and 1:80) in DMEM-supplemented medium. After 72 h of incubation, PGE, production was measured in fibroblast supernatants by radioimmunoassay, using an antiserum to PGEz provided by Dr. L. Levine (Brandeis University, Waltham, MA) (23).

Induction of Cell-associated IL-I-Induction of cell-associated IL- 1 was carried out in dermal fibroblasts as described previously (5). Briefly, 2 days before induction of cell-associated IL-1, the fibroblasts were plated in 96-well flat bottom plates at a density of 20,000 cells/ well and stimulated 24 h later by various concentrations of rhTNFa ranging from 5 to 100 ng/ml in DMEM-supplemented culture me- dium. After 24-h incubation in the presence of rhTNFa and/or TNFa INH at three dilutions (1:20, 1:50, and 1:80), the medium was re- moved, cells washed five times with 200 p1 of PBS, and treated with 1% paraformaldehyde (w/v) at room temperature for 15 min. Excess of paraformaldehyde was removed, and fixed cells were washed five times with 200 ~1 of PBS again. Unstimulated paraformaldehyde- fixed fibroblasts were used as control. Cell-associated IL-1 induced by rhTNFa was then tested in the lymphocyte proliferation assay (LAF) (16). In a control experiment rhTNFa was preincubated with goat antisera raised against rhTNFa for 1 h at 37 "C at 1:50 and 1:500 dilutions. When added to fibroblasts the final dilutions were 1:lOO and 1:1,000.

Cell-associated I L - l / L A F Assay-IL-1 activity of paraformal- dehyde-fixed cells was assayed with the C3H/HeJ mouse thymocyte proliferation assay (16). Briefly, thymocytes (1.5 X lo6 cells/well, plated in 96-well flat-bottom plates) were costimulated for 72 h with PHA (1 pg/ml) in the presence of paraformaldehyde-fixed fibroblasts. In a control experiment, after stimulation with rhTNFa, paraform- aldehyde-fixed fibroblasts were preincubated for 1 b at 37 "C with anti-rhIL-la rabbit antiserum (1:1000 dilution) before addition to thymocytes. The final dilution of the antiserum on thymocytes was 1:2000. Specificity of cell-associated IL-1 was also tested by addition at 1:20 dilution on thymocytes of the IL-1 INH that blocks both soluble and cell-associated IL-1.2 Cells were cultured in quadruplicate for 3 days, and 6 h before end of culture, cells were pulsed with ['HI thymidine (1 pCi/well) harvested onto glass fiber filters, and [3H] thymidine uptake was counted on a scintillation counter. Specificity of TNFa INH versus IL-I was investigated in the standard IL-1/LAF assay costimulated with PHA and various concentrations of either rhIL-la or rhIL-10 (16).

Binding Assay for 1Z51-TNFa-rhTNFa was iodinated by using the iodogen method (24). The specific activity of 1251-TNFa was 2.2 X 10'

by SDS-PAGE. Aliquots of lo6 U937 cells were incubated at 4 "C for cpm/ng and produced a single band with a M, = 17,000 when analyzed

2 h in a final volume of 200 pl of U937 culture medium containing 0.04% sodium azide and 0.5 ng of lZ6I-TNFa. Binding inhibition was performed by addition of various dilutions of TNFa INH (1:20,1:200, and 1:2,000). Nonspecific binding was measured in the presence of a 100-fold excess of unlabeled rhTNFa, and free radioactivity was separated from bound '251-TNFa by centrifugation through an oil

P. Seckinger, M.-T. Kaufmann, and J.-M. Dayer, submitted for publication. An interleukin-1 inhibitor affects both cell-associated interleukin-1-induced T cell proliferation and/or PGE$collagenase production by human dermal fibroblasts and synovial cells.

11968 A Human Tumor Necrosis Factor CY Inhibitor mixture as described previously (25). Cell-bound lZ6I-TNFa was then counted in a y-counter (LKB, Bromma, Sweden). The percent of binding inhibition was determined by using the formula:

[ cpm in the presence of TNFa INH

100 x 1- - cpm nonspecific binding

cpm of total binding - cpm nonspecific binding 1 Reversibility of lZ5I-TNFa Binding to U937”Binding inhibition

was further characterized by preincubating for 1 h at room tempera- ture the U937 cells in culture medium in the presence of either lZ5I- TNFa alone or 1261-TNFa in the presence of a 100-fold excess of cold rhTNFa (nonspecific binding). Cells were then washed three times in 50 ml of PBS at 4 “C. The cells incubated with 1Z51-TNFa alone were divided into two batches and incubated with TNFa INH (1:20 dilution) and with buffer alone, respectively. All aliquots were further incubated at either 4 or 37 “C, and aliquots of total binding, nonspe- cific binding, and binding in the presence of TNFa INH were deter- mined at various intervals.

RESULTS

Purification of the TNFa INH-After Amicon concentra- tion and ammonium sulfate precipitation, the material of

urine origin, tested for inhibition of TNFa-induced cytotox- icity, was first chromatographed on a DEAE-Sephadex anion- exchange column, then a Sulphopropyl-Sephadex cation-ex- change column as reported under “Materials and Methods’’ (data not shown). The inhibitory material was Amicon-con- centrated and gel-filtrated on a Sephacryl S-200 column. TNFa INH, eluting with an apparent molecular mass of 35,000 to 60,000 Da (Fig. lA), was used for both biologic characterization and further purification. Protein content in the undiluted TNFa INH is -60-80 pg/ml. Since TNFa INH was found to bind TNFa (see below), further purification was performed by using an activated Mini Leak Agarose column linked to rhTNFa. Sephacryl S-200 fractions (32-38) were adsorbed on rhTNFa, and bound material was eluted from the gel with a glycine buffer at pH 3.50. As seen in Fig. lB, TNFa INH elutes in one single peak when tested in the L929 assay of cytotoxicity. However, these inhibitory fractions contain still two major bands when analyzed on silver-stained SDS-PAGE (data not shown). The affinity-purified material

FIG. 1. Purification of TNFa INH. Panel A, elution profile of Sephacryl S- 200 chromatography. 200 mg of protein from Sulphopropyl-Sephadex inhibitory fractions was applied to the Sephacryl S-200 column, calibrated, and run as de- scribed under “Materials and Methods.” Column fractions (9 ml) were sterilized and tested at 150 dilution against rhTNFa (1.0 ng/ml) in the presence of actinomycin D (1 pg/ml) in the L929 assay of cytotoxicity. Bars represent, as in panek B and C , cell lysis measured in the presence of actinomycin D (0) and actinomycin D plus rhTNFa (a), respec- tively. Values represent cell lysis meas- ured by dye uptake at 570 nm (o”-o). Protein elution is recorded at A 280 nm (-). Panel B, elution profile of TNFa affinity column. TNFa INH was ad- sorbed on rhTNFa cross-linked to acti- vated Mini Leak Agarose beads and eluted as described under “Materials and Methods.” Column fractions were tested at the dilution and rhTNFa concentra- tions used in panel A. Panel C, elution profile of reverse-phase chromatogra- phy. Inhibitory fractions from panel B were loaded (2 ml) onto a ProRPC re- verse-phase FPLC column and run as described under “Materials and Meth- ods.’’ Column fractions were lyophilized, resuspended in 10 mM Tris-HC1, pH 7.4, containing 2 mM EDTA and tested as in panel A , but against 5 ng/ml rhTNFa. Dotted lines represent percent of aceto- nitrile. DB, dextran blue; BSA, bovine serum albumin; OA, ovalbumin; aCT, a- chymotrypsinogen A; q5 red, phenol red.

Fraction number

E 0

I

r - 1 oo 8

#

A #

1 5 10 15 25 30

80 - 0.08

h

40 5 - 0.04 8 N

20 - 0.02

Fraction number

A Human Tumor Necrosis Factor a Inhibitor 11969

was lyophilized and purified on a reverse-phase column (Fig. IC). Bioactivity directed against TNFa elutes in one major peak between 65 to 70% of acetonitrile. When proteins of the inhibitory fractions were analyzed on SDS-PAGE and then silver-stained, one single polypeptide migrating with an ap- parent M, = 33,000 was revealed under reducing and nonre- ducing conditions (Fig. 2). The proteins from a gel run under nonreducing conditions were eluted as described under “Ma- terials and Methods,” and each slice was tested for TNFa INH activity (Fig. 2). The activity directed against rhTNFa migrates with an apparent M, identical to the 33,000 band of the silver-stained band on the gel run under reducing condi- tions.

Inhibition of TNFa-induced PGEZ Production by Dermal Fibroblasts-The activity of TNFa INH was studied at dif- ferent dilutions for its ability to block TNFa-induced PGEz production by dermal fibroblasts. Dose-dependent PGE, pro- duction was observed when adding rhTNFa to the target cells in three different experiments (Table I). The biologic activity of rhTNFa was inhibited by adding TNFa INH at three different dilutions. At 1:80 dilution of TNFa INH the inhib- itory activity was partially overcome by increasing rhTNFa concentrations. Addition of TNFa INH alone led to a slight increase of PGE, basal level, and this phenomenon was de- pendent on the dilution of TNFa INH fraction. Contrary to the 150 dilution, the differences in PGE2 production at 1:20

% TNFa INH

A B 0 10 20 30 40 I I

21 +

M.W. Markers (kD) ? FIG. 2. SDS-PAGE analysis of purified TNFa INH. SDS-

PAGE was run under both reducing (lane A ) and nonreducing (lane B ) conditions and silver stained as described under “Materials and Methods.” For elution of biologic activity, a gel containing nonboiled proteins was run under nonreducing conditions and cut in 2-mm gel slices. Proteins were eluted by overnight incubation in 200 pl of 10 mM Tris-HC1, pH 7.4, containing 2 mM EDTA. Fractions were tested at 1 : lO dilution against 0.15 ng/ml rhTNFa, as described in Fig. 1, panel A ; percent of inhibition (%) was determined as reported under “Materials and Methods.”

and 1:80 dilutions are statistically significant (p < 0.05) as assessed by Wilcoxon’s rank sum test. PGE, production in the presence of both rhTNFa and TNFa INH was never lower than values observed with TNFa INH alone, ruling out the possibility that a cytotoxic phenomenon of the semipuri- fied material is involved in the decreased PGEz production. These data suggest that the TNFa INH is capable of blocking TNFa-induced biologic activity without actinomycin D.

Inhibition of TNFa-induced Cell-associated IL-I-TNFa has been found to induce the expression of cell-associated IL- 1. We have investigated if the TNFa INH is also effective in blocking the induction of cell-associated IL-1 induced by TNFa and the specificity by using a specific goat anti- rhTNFa serum. rhTNFa induces a dose-dependent cell-as- sociated IL-1 production up to a concentration of 50 ng/ml of rhTNFa as measured by [3H]thymidine uptake in thymocytes (Fig. 3). Addition of TNFa INH alone during the stimulation of cell-associated IL-1 did not affect IL-1 expression. The biologic activity of rhTNFa was inhibited by addition of TNFa INH at three different dilutions, and inhibition was partially overcome by increasing rhTNFa concentrations when TNFa INH dilution was 1:80 (Fig. 3A). The specificity of inhibition was tested by preincubation for 1 h of rhTNFa with goat anti-rhTNFa serum at 37 “C prior to addition to dermal fibroblasts. The polyclonal antiserum abolished the induction of cell-associated IL-1 in a dose-dependent manner (Fig. 3B) , whereas a nonimmune goat antiserum was not effective (data not shown).

Specificity of TNFa-induced Cell-associated IL-l-Previ- ously we found an IL-1 INH to be capable of blocking cell- associated IL-1 induction of T cell proliferation. IL-1 INH activity was studied in the IL-1/LAF assay in order to assess specificity of the cell-associated molecule induced by rhTNFa. Complete inhibition of costimulated thymocyte proliferation was observed in the presence of IL-1 INH (Fig. 4). Addition of IL-1 INH to unstimulated paraformaldehyde-fixed fibro- blasts did not affect basal level [3H]thymidine incorporation. Specificity of TNFa-induced cell-associated IL-1 in fibro- blasts was also demonstrated by blocking thymocyte prolif- eration with a polyclonal antiserum raised against rhIL-la which neutralizes biologic activities of rhIL-la only without affecting those induced by rhIL-10 (Fig. 4). In contrast, ad- dition of a nonrelevant rabbit antiserum at the same final dilution was not effective (data not shown). Thus, cell-asso- ciated IL-1 expression can be regulated either at its level of induction, by TNFa INH or an appropriate antiserum to TNFa, or at its level of biologic activity, by IL-1 INH or an appropriate antiserum to IL-1.

Specificity of TNFa INH for rhTNFa, TNFP, and rhIL-1- TNFa and -0 are two different peptides encoded by two different genes that share similar biologic activities and the

TABLE I TNFa INH inhibits TNFa-induced PGEz production by fibroblasts

Three different experiments were carried out with the same strain of fibroblasts. Buffer or TNFa INH (see “Materials and Methods”) was incubated at various dilutions in the presence or absence of various concentrations of rhTNFa. PGEz production by cultured human dermal fibroblasts was measured after 3 days. Values represent tridicate means of the three cultures f S.E. ( n = 3).

Concentration of rhTNFa on human fibroblasts

PGE, production by human dermal fibroblasts in dilution of TNFa INH on fibroblasts

None 1:80 1:50 1:zo pglml wlml

0 50.6 f 7.4 88.8 f 5.6 103.0 f 8.9 111.0 f 9.4 160.0 f 14.1 126.0 f 9.3 115.9 f 6.6 113.9 f 7.1 331.7 f 28.4 217.2 f 10.7 156.7 f 10.7 115.3 f 21.3 381.7 f 19.6 257.2 -C 13.7 253.1 f 21.2 221.6 f 16.0

500 2000 5000

11970 A Human Tumor Necrosis Factor a Inhibitor - ql 0 ’30

A TNFaINH B TNFa Ab

I I TL G “ d ; 50

1w 0 5 50 100

fhTNFa concentralion added to dermal fibrcblasts (ngml)

FIG. 3. TNFa INH regulates cell-associated IL-1 expres- sion. Panel A, TNFa INH blocks induction of cell-associated IL-1. Human dermal fibroblasts were stimulated for 24 h with various concentrations of rhTNFa in the absence (0) or presence of three different dilutions of TNFa INH (A, 1:20; A, 150; and B, 1:80). Cells were fixed and tested for biologic activity as described under “Mate- rials and Methods.” Values represent means f S.E. ( n = 4) of [3H] thymidine incorporation in thymocytes in the presence of PHA. Panel B, TNFa antibody (Ab) blocks induction of cell associated IL-1. Cell- associated IL-1 was induced in the same conditions as described in panel A. Anti-rhTNFa was preincubated with rbTNFa (V, 1:100; and V, 1:lOOO) prior to cell addition. Cells were fixed and tested for biologic activity as described under “Materials and Methods.” Values represent means 5 S.E. (n = 4) of [3H]thymidine incorporation in thymocytes in the presence of PHA.

“ 1 .

0 5 50 100

0 fhTNFa concentration added to dermal fibroblasts (ns/mU

FIG. 4. Specificity of cell-associated rhTNFa-induced activ- ity. Specificity of cell-associated IL-1 was investigated by addition to PHA costimulated thymocytes of either rabbit anti-human recom- binant IL-1 (V) at 1:200 final dilution or IL-1 INH at 1:20 final dilution (V). Values represent means _+ S.E. (n = 4) of [3H]thymidine incorporation in thymocytes in the presence of PHA.

same receptor. We investigated whether the TNFa INH ex- hibits some specificity for either one or the other cytokine. Addition of increasing concentrations of either rhTNFa or TNFP to actinomycin D-treated L929 resulted in a dose- dependent cytotoxicity (Fig. 5 ) . However, in our L929 cyto- toxic assay, the specific activity of TNFP was two to three times higher than that of rhTNFa since half-maximal cyto- toxicity induced by TNFB was obtained at concentrations ranging between 20 and 50 pg/ml, whereas rhTNFa half- maximal cytotoxicity was obtained at concentrations between 50 and 100 pg/ml. Addition of TNFa INH alone to actino-

. . . . . , , 0 20 50 1w 250 5w 1250 2500 0 20 50 l o o 250 5w 1250 2500

TNFamcanbatiMls (pp‘ml) TNFp mcerWationr (pp’ml)

FIG. 5. Specificity of TNFa INH. Panel A, cell cytotoxicity was induced either in the absence (0) or presence (B) of 1:20 dilution of TNFa INH and increasing concentrations of rhTNFa, as described in Fig. L4. Panel B, cell cytotoxicity was induced either in the absence (0) or presence (B) of 1:20 dilution of TNFa INH and increasing concentrations of TNFb as described in Fig. 1A. Values represent means f S.E. ( n = 3) of cell lysis measured by dye uptake at 570 nm.

0 0 l o o 2 0 0 500 1 M o 0 0 loo 200 500 1 M o

e

1

l o o 2w 5w

fh IL-1 (pg/ml)

FIG. 6. Effect of TNFa INH on IL-l/LAF activity. Panel A, effect of TNFa INH on rhIL-la-induced thymocyte proliferation in the presence of PHA. Panel B, effect of TNFa INH on rhIL-l@- induced thymocyte proliferation in the absence of PHA. Values represent means f S.E. ( n = 4) of [3H]thymidine incorporation in thymocytes. Open symbols represent proliferative response to PHA and rhIL-la and PHA and rhIL-lB in the absence of TNFa INH. Closed symbols represent thymocyte proliferation in response to the same stimuli in the presence of TNFa INH at 150 dilution.

tion of TNFa INH to rhTNFa resulted in the inhibition of cytotoxicity, this inhibition being partially overcome by in- creasing rhTNFa. At 1:20 dilution, TNFa INH reversed cy- totoxicity by about 90 and 50% when rhTNFa concentrations were 100 and 1250 pg/ml, respectively. Contrasting with the effect observed on rhTNFa, the same dilution of TNFa INH reversed cytotoxicity by about 21 and 1% when induced by 100 and 1250 pg/ml of TNFP, respectively.

Since TNFa and both IL-la and -P share several biologic properties, we examined whether the TNFa INH would block rhIL-la and rhIL-lB. Therefore, the activity of the TNFa INH was tested in the IL-l/LAF assay when induced by either form of rhIL-1. As shown in Fig. 6, a dose-response of [3H]thymidine incorporation was observed in up to 200 pg/ ml concentrations of either rhIL-la and $3. Addition of TNFa INH at a dilution that inhibits cytotoxicity of rhTNFa did not affect thymocyte proliferation, proving the inhibition to be specific for TNFa only.

Effect of TNFa INH on 1251-TNFa Binding to U937”TNFa INH blocks a variety of TNFa-dependent bioactivities both on actinomycin D-treated and untreated cells. The mecha-

mycin D did not affect cell viability (data not shown). Addi- nism of action of TNFa INH was investigated by utilizing the

A Human Tumor Necrosis Factor a Inhibitor 11971

U937 cell line that expresses a high number of TNF receptors. Fig. 7 shows that the specific binding of lZ5I-TNFa to U937 cells was inhibited at 4 "C by 100, 80, and 35% by the three respective dilutions of TNFa INH (1:20, 1:200, and 1:2000). In contrast, a control fraction from the same gradient but devoid of TNFa INH activity was inactive when tested at the same final dilution. In addition, binding inhibition was in- creased to 90 and 60% when '251-TNFa was preincubated with TNFa INH at dilutions of 1:200 and 1:2000, respectively, prior to cell addition. These data suggest that TNFa INH acts by binding to the ligand and not to the receptor. Prein- cubation of U937 with TNFa INH prior to '251-TNFa addition did not increase binding inhibition (data not shown). Binding inhibition was further characterized by preincubating U937

b""

t -""~""""""""~ "-1."

A

1/20 11200 1moo

TNFa INH dilution

FIG. 7. TNFa INH binding inhibition. TNFa INH or nonin- hibitory fractions were incubated at three different dilutions in the presence of lZ5I-TNFa with the U937 cell line. Aliquots (lo6 cells) were incubated for 2 h at 4 "C with a 1 ng/ml concentration of lZ5I- TNFa in the presence of TNFa INH (M, C - 4) or control fractions (A-A, A- - -A). Values representing percent of binding inhibition were corrected for nonspecific binding in the presence of a 100-fold excess of unlabeled rhTNFa. Closed symbols refer to a 30- min preincubation of TNFa INH at 20 "C with "'I-TNFa in U937 culture medium prior to cell addition.

0 30 60 120 240

Time alter TNFa INH addtion (min)

FIG. 8. TNFa INH dissociates l2'1-TNFa from its receptor. U937 cells were preincubated with '*'I-TNFa, washed, and incubated at either 4 "C (M) or 37 "C (C - 4) in the presence or absence of TNFa INH, as reported under "Materials and Methods." At times indicated, cell-associated radioactivity was measured and percent of specific binding determined. 100% for each time corresponds to the value obtained without addition of TNFa INH, normalized by sub- straction of the corresponding nonspecific binding value.

FIG. 9. Lack of proteolytic activity of TNFa INH. '251-TNF was incubated for 1 h at room temperature without TNFa INH ( A ) or with TNFa INH at three dilutions ( B , 1:20, C, 1:200, D, 1:2000) (see "Materials and Methods"). The different incubation conditions were analyzed on a 15% SDS-PAGE, followed by an autoradiogram for 24-h exposure.

for 1 h in the presence of 12'I-TNFa as described under "Materials and Methods." Fig. 8 illustrates that cell surface- bound '251-TNFa dissociation is faster in the presence of TNFa INH than in its absence. The phenomenon is time- and temperature-dependent.

There still remained the hypothesis that TNFa INH has a proteolytic activity for rhTNFa. Therefore, '251-TNFa was incubated for 1 h at room temperature in the presence of PBS or three different dilutions of TNFa INH (1:20, 1:200, and 1:2000). As seen in Fig. 9, lZ5I-TNFa produced a single band both in the absence and presence of TNFa INH when ana- lyzed by SDS-PAGE and autoradiographed.

DISCUSSION

We have found that urine of some febrile patients contained a TNFa INH (15). The TNFa INH was purified by Amicon concentration, ammonium sulfate precipitation, DEAE-Seph- adex, Sulphopropyl-Sephadex ion-exchange chromatogra- phies, and Sephadex S-200 gel filtration. TNFa INH eluted with an apparent molecular mass of 35,000-60,000 Da and was further purified using an affinity column and reverse- phase chromatography. A protein with an apparent M, = 33,000 was seen on silver-stained SDS gel run under both reducing and nonreducing conditions. Biologic activity mi- grating also with an apparent M , = 33,000 was eluted from a gel run under nonreducing conditions. The apparent dissocia- tion between biological activity (retardation on the gel) and protein staining shown on Fig. 2 may be explained by the fact that prior to loading on the stained gel the TNFa INH was boiled. This step was omitted when protein had to be eluted, hence the lower migration rate. The discrepancy between the molecular mass after gel filtration and SDS-PAGE may be attributed to TNF fragments in the urine that bind to the inhibitor. The molecular mass of TNFa INH as measured by SDS-PAGE differs also from the 14,700-Da protein encoded by the E3 transcription unit of group C adenoviruses which prevents TNFa cytolysis of adenovirus-infected cells (26).

TNFa INH is active on both actinomycin D-treated and untreated cells. Indeed, not only TNFa-induced cytotoxicity but also the induction of PGEz and cell-associated IL-1 in dermal fibroblasts can be inhibited. This rules out the possi- bility that the inhibition is due to an actinomycin D-binding protein. When added alone to dermal fibroblasts TNFa INH induces a dose-dependent increase in the basal level of PGE, production. It has to be established whether TNFa INH purified to homogeneity also interferes with the basal level of PGE, production or whether this phenomenon may be attrib-

11972 A Human Tumor Necrosis Factor a Inhibitor

uted to the partial purification of TNFa INH used in this assay, as other molecules with some IL-1-like activities may still be present. The inhibition of TNFa-induced PGEz pro- duction deserves our attention since PGE, plays a role in both immune and inflammatory responses. Thus, it may in partic- ular increase vascular permeability, edema, cause fever, and sensitize pain receptors (27-29). However, regulation may be more complex since PGEz can exert negative feedback regu- lation by decreasing both IL-1 and TNFa release (30). In contrast, blocking of TNFa-induced collagenase may be ben- eficial since the latter, produced by dermal fibroblasts and human synovial cells, contributes significantly to tissue de- struction (4).

We found that cell-associated IL-1 induces PGE, and/or collagenase production by dermal fibroblasts and synovial cells. Cell-associated IL-1 may regulate tissue destruction at the local level through a direct cell-cell contact. We also found that IL-1 INH blocks cell-associated IL-1-induced biologic activities and may therefore play a role in the regulation of tissue destruction.' It would be of interest to investigate whether different fibroblast cell lines produce one or both forms of IL-1 in response to TNFa. Biologic activity of TNFa- induced dermal fibroblasts could be neutralized by a specific antibody to rhIL-la alone. In contrast it was reported that TNFa induces IL-la and -P mRNA in human diploid FS-4 fibroblasts (5). This does not rule out the possibility that results similar to ours are obtained at the protein level. In addition, the difference between the observation made by Le et al. (5) and ours could be accounted for by the strain of fibroblasts studied. TNFa INH differs unequivocally from the IL-1 INH (molecular mass 18,000-25,000 Da, PI 4.5-4.7) also present in the urine of febrile patients, and the latter inhibitor does not interfere with TNFa biologic activity (16, 17). Why urine contains such immunosuppressive molecules has still to be elucidated. TNFa INH blocks the induction of cell-asso- ciated IL-1 so that IL-1 may be regulated by distinct inhibitors at its level of induction (TNFa INH) and that of its biologic activities (IL-1 INH).

Both genes, TNFa and -@, have been cloned (18, 31-35), both molecules compete for the same receptor and have sim- ilar biologic activities (10-12, 34). Different pathological sit- uations can induce the production of TNFa without inducing that of TNFB (35). This might imply that their genes are under different controls (36). We therefore investigated whether TNFa INH affects both forms of TNF, as in the case of IL-1 INH that blocks both IL-la and -0. Since TNFa and -@ used do not have the same specific activity, we compared levels of cytotoxicity in the presence and absence of TNFa INH at different concentrations. Addition at 1:20 final dilu- tion of TNFa INH reversed almost all cytotoxicity of rhTNFa. In contrast, TNFP was slightly affected by the TNFa INH.

TNF shares many biologic activities with IL-1, including the induction of fever, adrenocorticotropic hormone release, enhancement of B cell proliferation, and growth stimulation for thymocytes (3,36-38). Contrary to IL-1 for which different inhibitors have been described (39), little is known about TNFa inhibitor(s). The present investigation shows that at least one inhibitor exists that, as in the case of IL-1, regulates TNFa-induced biologic activities. The TNFa INH is specific for TNFa and does not interfere with IL-1-induced biologic activity.

Due to the wide range of TNFa INH biologic activity we investigated whether it interfered with ligand binding, using the promonocytic cell line U937. Binding of '251-TNFa to its receptor was inhibited in a dose-dependent manner by addi-

tion of TNFa INH. Preincubation of the TNFa INH with U937 cells did not result in an increased binding inhibition, as is the case with IL-1 INH, suggesting that the TNFa INH does not bind to the cell surface. In contrast, preincubation of '261-TNFa with the TNFa INH prior to cell addition resulted in an increased binding inhibition due to an inter- action between ligand and TNFa INH. The TNFa INH may recognize a specific structure on TNFa not present on TNFP, which would explain why the latter was less affected. The TNFa INH that regulates TNFa-induced biologic activities is more than a simple carrier protein, since it can dissociate bound '251-TNFa from its receptor; the physiologic meaning of this phenomenon remains to be elucidated. Our data em- phasize the importance of the ligand-receptor interaction with respect to the biologic activity. Dissociation of the TNFa- receptor complex reverses binding and consequently internal- ization of TNFa required for its cytotoxic activity, as has been demonstrated in the following experiments. (a) Inhibi- tion of ligand binding by a specific antiserum was found to block TNFa-induced cytotoxicity (40). ( b ) A lack of cytolysis was observed following microinjection of TNFa directly into cells or cell nuclei proving the essential role of ligand binding (41). The increased dissociation at 37 "C when compared to 4 "C is relevant since the free receptor has a half-life of 2 h on the cell surface, and this is shortened to 30 min when TNF binds to its receptor (42). Thus, TNFa binding and internal- ization is in competition with dissociation of TNFa from its receptor induced by TNFa INH.

Other polypeptides have been found associated with pro- teins that regulate their biologic activity. Thus, transforming growth factor P1 (TGFP,) has been described complexed with fibronectin (43) and a,-macroglobulin (44) which binds the free form of TGFP1. The interaction with a2-macroglobulin inhibits the binding of TGFPl to its receptor. Subsequently, a latent high molecular weight complex of TGF& composed of a carrier protein and two TGF& precursor molecules cross- linked by disulfide bonds have been described (45, 46). The TNFa INH does not bind through disulfide bonds because it involves a competitive mechanism of action.

In the binding inhibition experiments, 40-50% of binding inhibition was observed at 1:2000 final dilution on cells, although it was no longer effective in any assay. This also suggests that TNFa INH present in urine may be altered during renal excretion, or that it is present in high amounts in urine. The existence at least in uitro of a TNFa-inhibitory mechanism, in analogy to IL-1(16,17), suggests a high degree of complexity of pathological situations which may be the result either of the induction of proinflammatory cytokines or the lack of inhibitor production.

In conclusion, our data show that urine of febrile patients contains a TNFa INH with a molecular mass of 33,000 Da. It is active against actinomycin D-treated cells, as measured by inhibition of TNFa-induced cytotoxicity on L929 cells, and untreated cells, as measured by inhibition of TNFa- induced PGE, production on dermal fibroblasts. TNFa INH shows specificity for TNFa and does not affect rhIL-la or -P, and TNFP only to a small extent. TNFa INH blocks the different biologic activities induced by TNFa by interfering with the interaction of the ligand and its receptor. In addition, the TNFa-receptor complex shows a faster dissociation con- stant in the presence of TNFa INH than in its absence. It may therefore be an important novel specific "anti-cytokine," considering its inhibitory properties.

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