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The M20 IL-1 inhibitor I. Purification by preparative isoelectric focusing in free solution David Peritt a, lancu Flcchncr b, Eliyahu Okuncv ~', Pctcr Yanai ~', Tal Hal,0erin % A b r a h a m J, T r e v e s " and Vivian Barak ~ "Immunology Laboratory. Om'ology Deparlment. ifadas~ah Umlvrsiry Ilospiral. leru.~lem. L~reel. and t, hzsamte of Mu'rr~hiok~'y, I ladas~ah-I lebrew Upm'ersizy Medical Sch~nd. Jerusalem. Israel

(Received I 1 NovemberIq91,revisedreceived2(I May 1992,accepted21 May 1t.192) An interleukin-I (IL-I) inhibitor produced by the M20 myclomonocyti¢ cell line has ~ e n shown to be active in various in vitro and in vivo IL-1 induced parameters. This inhibitor has been purified from the conditioned medium by gel filtration through a Sephacryl S-3IHI column or dye ligand chromotography on Affi-Gel blue column, followed by isoeleetric focusing in free solution in the pH range 3-5 using the Rotofor cell. When gel filtration by FPLC with the Superose 12 column was used as the final step, the combined sequence of purification procedures resulted in a 1600-fold purification of the IL-I inhibitor. The purified IL-I inhibitor has a molecular weight of approximately 52 + 4 kDa and a p l of 4.15 ___ILl. By SDS-PAGE analysis the inhibitor preparation thus obtained showed the presence of two protein bands, while a few timely spaced protein bands were .seen by analytical isoclectrie focusing in ~alyacrylamide gels (pH 3-6). Some of these bands in PAGIF might correspond to different degrees of glycosylation of the inhibitory protein. Although the M2tl IL-I inhibitor has not yet been purified to homogeneity, it should be stres~d that the procedures u~d, allowed us to remove the great majority of the proteins present in the medium in which the M20 cells were cultured, and to recover in satisfactory yield the inhibitor which we consider likely to be present in the conditioned medium in subnanomolar concentrations, Key words: Liquidphase if)electricfocusing;gotofor cell; Inlerleukin-I:Inhibilor: Mych)mon~:ytlc;([lumun)

Introduction Interleukin-I (IL-I) plays a central role in immune response and inflammation, and appears to

CorrespotMell¢'e to: V. Barak, Deparlment of Oncology. Hadassah-Hebrew UniversityMedical(enler. P.O.B. 12(H~). Kiryal Iludussah.Jerusalemq112(I, Israel.Tel.: t.~72.2-776764; Fax: 972-2-434434. Abbrel'iations: IL-I, interleukin-I;IL-•, inlerleukin-6;TNF, tumor necrosisfacl~lr;IEF, isl~eleetrieft'~using; PAGIF, polyacrilamide gel i~electrtc focusing.

be involved in immuno-inflammatory d i ~ a ~ s such as rheumatoid arthritis and insulin-dependent diabetes mellitus (Bendtzen et aL, 1986; Arend and Daycr, 1990). Different mechanisms that regulate its effects have been suggested (Diharelip, 1~)91) and a variety of natural IL-1 inhibitors present in human body fluids and in supernatants of cultured human or animal cells or cell lines, have been de~ribed (Larrick, Iq89>. Recently, IL-I inhibitors derived from urine of febrile patients (Seckinger el al.. 1987h from nor* mal monucytes (Hannum et al,, 1990) and from

160 the U937 cells line (Carter et al., 1990), have been purified to homogeneity and were found to be similar or identical. These inhibitors act via IL-I receptor competition and are therefore termed receptor antagonists. Barak ctal. (1986) have described an IL-I inhibitor produced by a human m),elomonocytic cell line (M20), which specifically inhibited IL-1 induced proliferative responses of mouse thymocytes, human fibroblasts and T cells. Furthermore, in vivo IL-I induced inflammatory responses, such as fever, leukoeytosis, lymph node enlargement, or the increase in blood levels of fibrinogen and corticosterone were reduced, or abolished, following i.v. injection of the IL-I inhibitor. Similarly induced responses, induced by IL-6 or TNFa, were not affected by the inhibitor (Barak e t a l , 1991b). In this study we describe the purification of the M20 IL-I inhibitor by molecular sieving or dye ligand chromotograpby on Affi-Gel blue columns, followed by isoelectric focusing in free solution, using the Rotofor cell.

The cells were plated at 5 x 10~ thymoeytes/well in 100 V.l.The fractious to be tested for inhibitory activity were added at 25 p.l/well. The cultures were grown at 37°C in 5% CO 2 for 72 h, with the addition of I/,Ci/well of ['~H]tbymidine (specific activity 6.7 Ci/mmol) for the last 12 h, and harvested, and DNA synthesis was measured using standard techniques (Barak e t a l , 1986).

Gel filtration The concentrated sample was dialyzed against the elution buffer (PBS + 0.5 M NaCI) and applied to a Sephacryl S-300 open column (800 ml Vt and 230 ml Vo). The column was run using a peristaltic pump at a flow rate of 90 ml/h. After the volume corresponding to Vo had passed through the column, fractions of 7 ml were collected, and absorbance at 280 nm was determined. One peak, showing IL-I inhibitory activity in the bioassay, was pooled and concentrated by vacuum ultrafiltration as described above, and dialyzed against distilled water, overnight.

Affi-Gel blue chromatography Materials and methods

Tissue culture The M20 cells were grown as previously described (Barak e t a l , 1986). Briefly, the cells were grown at 37°C and 5% CO 2 in RPMI 1640 supplemented with penicillin (50 U/ml), streptomycin (50 p.g/ml), MEM vitamin solution (1:100), 2 mM L-glutamine, 1 mM sodium pytuvate (complete RPMI), with the addition of 5% FCS (Belt Haemek, Israel) for 4 days, followed by a 24 h incubation at 10o cells/ml in complete RPMI without FCS. The conditioned medium was collected and concentrated to 10 ml by vacuum ultrafiltration using dialysis tubing (cut-off 10,000 MW).

Bioassay The assay used was a modified LAF assay as described by Gery etal. (1972). Briefly, thymocytes from one-month-old C3H/HEJ mice were made into single cell suspension in complete RPML in order to establish a positive growth 2-mercaptoethanol (50 p.M), PHA (1 #g/ml) and 4-6 U/ml of rlL-1/~ were added to each well.

To remove the hulk of the BSA, which is a major protein in the tissue culture medium, 2 liters of CM were concentrated by vacuum ultrafiltration, and were passed through an Affi-Gel blue column (Bin-Rod) of 11.0 × 2.6 cm (bed volume 58 ml), previously equilibrated with 50 raM Tris buffer pH 7.5 containing 50 mM Natl. The effluent from the column was applied to the Rotofor cell as described.

Preparatice isoelectric focusing lsoelectric focusing (IEF) was performed using the Rotofor cell (Bio-Rad, Richmond CA). The concentrated sample obtained, following gel filtration or Affi-Gel blue chromotography, was mixed with 10 M urea to obtain a final concentration of 5 M. Carrier ampholytes (Bio-lyte), covering the pH range of 3-5 were added at a final concentration of 2%, and glycerol was added to obtain a final concentration of 10%. The volume of the mixture was brought to 50 ml with distilled water. To bring the sample to the running temperature (4°C), the cell was rotated for |5 rain before applying the clcctric current, The run was carried out at constant power (12 W). In a typical

161

run the initial conditions were 784 V and 15 mA. At equilibrium, after 4 h, when the run was stopped, the voitage reached 1170 V and the current was 10 mA. At the completion of the run, the pH gradient was determined on ~.he 20 harvested fractions, on aliquots di!uted 1/t0 in freshly boiled bidistilled water. To remove the carrier ampholytes before the bioa.~say, the harvested fractions, to which 0.1 mg/ml crystalline BSA was added, were dialyzed against 1 M NaCI for 24 h, followed by dialysis against RPMI 1640 for48 h. To increase the degree of purification obtained by IEF in the Rotofor cell, a second run in a narrower pH range corresponding to the fractions in which the IL-1 inhibitor had focused, was attempted. The harvested fractions obtained in the first run (pls 3.9-4.3) were pooled, diluted with glycerol (10% final concentration) and bidistilled water, and reloaded into the Rotofor cell. The run was performed as described above, lu a typical run the starting power conditions were: constant power 12 W, 1087 V and 12 mA. At the end of the run, the voltage reached 1828 V and current dropped to 7 mA. The harvested fractions were treated, as described above, for the fraetiops obtained, when only one run in the Rotofor cell was performed.

Protein concentration The determination of the protein concentration of the samples was performed using the Bradford mieroassay procedure with the Bio-Rad protein assay kit. To avoid the interference of carrier amphotytes, the samples were acidified with HCI, before adding the Coomassie brilliant blue G-250 reagent, as described by Ramagli and Rodriguez (1985). Absorbance at 595 nm was measured against a reagent blank using the Gilford 240 spectrophotometer. Crystalline BSA was used as standard.

Polyacrylamide gel isoelectricfocusing (PAGIF) Analytical isoelectrie focusing was performed in a fabric-reinforced poi~aerylamide gel (5% T and 3% C) of 150 p.m thickness, containing 5% carrier ampholytes (Servalyt), pH range 3-10. The run was performed at 4-9°C for about 3 h at 3 W constant power. 10 #1 of the samples were

applied in slots of 2 × 3,5 mm in the middle of the gel. As anolyte, a mixture of 25 mM L-aspartie acid and 25 mM L-glutamic acid was used. A mixture composed of 23 mM L-arginine, 27 mM L-lysine and 1.78 M ethylene diamine, served as eatholyte. When the run was performed with carrier ampholytes pH 3-6, the 10 p.1 samples were applied in slots of 7 × l mm in the middle of the gel. "7,re anolyte was the same mixture as described above, while a 0.27 M glyeine was used as eatholyte. The low p l calibration kit (pH 2.5-6,5) (Pharmaeia, Uppsala, Sweden)was utilized for pl markers.

SDS-PAGE Samples, heated at 95°C for 4 rain in sample buffer, were run in a 10-15% gradient SDSPAGE using the Bio-Rad Mini-Protean If ceil. The gel was run for 1 h at 200 V and was stained with Coomassie brilliant blue R-250 (Bio-Rad).

FPLC gel filtration To fractionate the mixture of proteins according to their differences in molecular dimensions, gel filtration through a Superose 12/10 column (Pharmaeia, LKB Bioteehnoiogy, Uppsala, Sweden) was used. The sample was injected into a 0.5 m] [oop and the column was run at a flow rate of 0.25 ml/min with PBS, for 40 rain. The samples were prepared for the bioassay by addition of 0.1 mg/ml BSA as a stabilizer and dialyzed against RPMI 1640 for 48 h. The column was calibrated using human hemoglobin (Sigma, St. Louis MO) and Bio-Rad Molecular weight marker kit (bovine thyroglobulin, bovine "y-globulin,chicken ovalhurain, horse myoglobin, and vitamin BI2).

Results

Preliminary fractionation of the conditioned medium by IEF in the pH range 3-10, revealed the peak of inhibitory activity at approximately pH 4,0. Therefore, subsequent runs were all conducted in the narrower pH range 3-5. When the crude conditioned medium was applied in the Rotofor cell, without a preliminary fraetionation step, the sample was first concentrated by vacuum ultrafiltration or lyophilization and dialyzed

102

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Rotofor fraction pH Fig. I. The pH I~radi~nland IL-I inhibitt)ryacti'~kyof the 20 fractions i.'~lated from the R.otoforcell. in which the crude condilioned medium was fraclionalcd, in the: prus~ncc {~f carrier ampholyles3-5. asainst distilled water to reduce the concentration of salts. Ten runs were performed under the conditions described, and the results of a typical run arc shown in Fig. I. To avoid isoelectric precipitation of the large quantity of contaminating proteins present in the conditioned medium, which might clog the screen separators and prevent electrophoretic transport, gel filtration through a Sephacryl S-30O column was used. The elution profile of the proteins, from this column is depicted in FiB. 2. The IL-I inhibitory activity appeared under a broad area, and the small peak (V,~= 524-595 ml) corresponding to a low protein content, as indi. cared by ab~rbance measurements { A 2~ n m ), WaS concentrated and dialyzed before fractionation by

ElutlOn

5.7 8,5 5~

(ml)

lines was taken f0r the fractionati0n on the Rot0For cell,

isoclcctric focusing, The pH gradient, protein concentration and IL-I inhibitory activity of the 20 fractions isolated in thc horizontal Rotofor column, are shown in Fig. 3. It can be men that the inhibitory activity was focalized in fractions 9-11, covering the pH range 4.08-4.29, with fraction 10 (pH 4.19) being the most active. A rough estimation shows that about 38% of the inhibitory activity present in the fraction from the Scphacryl S-3O0 column, which was applied to the Rotofor cell, was rccovcrcd in thcsc three fractions, while the great majority of proteins were present in fractions 13 through 17 (pF1 range 4.59-5.:34). In the bJoassay 1.875 p~g pr,~tcin of the fraction obtained from the gel filtration column, gave 57.6% inhibition. The same degree of inhibition (57.3%) was achieved with 25 ~1 of fraction number l0 (pl 4.19) obtained in the Rotofor cell.

5.g A 0

volume

Fig. 2. Profile of protein concentration and ]L-) inhibitory activityof fractionseluted fromthe Sephaci'ylS-3(Htgel filtralion column. The peak indicated between the two vertical

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.3,1

2 ~ ¢ ~ |

T | ~ 1011121~1¢151611181~20

Rototor

lraotiofl

number

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Fig, 3, Th~ pit gradi~nl and di~lrihutk)n of protein concentrations (,) and the IL-I inhibitory uclivity (h) ~f the 20 fractions

ohlained.~sing[or fr~¢tion~tionin the:Rok)forc¢[I,the active peak ~:lul~d[rom Ihe Sephac~]S-3|K)gel fillrationcolumn.

163

Since the concentration of protein in this fraction was 1.5/zg/ml, it follows that the specific activity of the inhibitor in this fraction had increased by 50-fold. To separate the contaminating proteins with a similar pl, but of different size, FPLC with the Superose 12 column was used as a final step. The results obtained, using this sequence of procedures as given in Table I, show a 1600-fold purification with a recovery of 5.4% of the inhibitory activity. The apparent molecular weight (M~, app.) of the active IL-I inhibitor, purified as described, was determined by FPLC using the Superose 12 column is 52 + 4 kDa. Similar results were previously obtained using standard gel filtration through a Sephadex G-200 column (Barak et al., 1986). As an alternatiw method to avoid overloading the Rotofor cell, Affi-Gel blue columns were used as a preliminary step to remove BSA, which is one of the major proteins present in the crude conditioned medium. This was followed by IEF in the Rotofor cell (pH range 3-5), with refractionation in the narrower pH range 3.9-4.3, in which the inhibitor had focused in the first run. Following the refractionation step, the specific activity increased 4.38-fold but the recovery dropped drastically (data not shown). in addition to the IL-I inhibitory activity, we detected the presence of some factors which increased the thymocyte proliferation induced by TABLE I PURIFICATION OF THE M20 IL-I INHIBITOR FROIvl CONDITIONED MEDIUM Step

Specific activity a (U/~g)

Purification (fold)

Yield (%)

Conditioned medium Scphacryl S-3O0 IEF pF13-5 FPLC Superose 12

O.015 I, 1.0f~ 15.9~ 24.200

l 71 1060 1613

100.0 .'0.0 7.6 5.4

I U of IL-I inhibitoris defined as the amounl of maleriat (~g) causing50% inhibitionof the proliferationin the thymocyt¢ bioassay,induced by I U of rtL-I~. h Since lL-I which is concomitantly prest:nt in the crude conditioned medium interferes with the measurement of the IL,[ inhibitor u~d in the bioagsay, its specific activity is underestimaled.

23456

78

9|~112

FiB. 4. Analysis by PAGIF (pH 3- ! O) of the crude condilioned medium and some of the fractions colkseted from the Rotofor cell. wh©n Am-Gel blue was used as a prelinlinary step. Lanes I and 5 p i mar~,ers (low pl calibration kit. Pharmacia~ lanes 2 and 3 crude conditioned medium: lane 4 feluin tO ~.g; lane 6 fraction pl 7.4; lane 7 fraction p#' 6.07; lane g fraclion p l 5.40: lane 9 pl 5.10; lane tO fraclion pl 4.66: lane It fmclion pl 4.3.5; lane 12 fzaction pl 4.12.

IL-I in the bioassay (Figs. 1 and 4). These factors, which focused at different pH values, might be heterogeneous degradation products of IL-1, since like many other cellular sources, the M20 cell produces concomitantly, both ]L-I and the IL-1 inhibitor (Barak et aL, 1986a,b), Aliqaots of selected fractions obtained from the Rotofor cell after one run (pH 3-5), were concentrated 10-50-fold by ultrafiltration (ultracent-10 filters, Bio-Rad) and anal~cd by PAGIF (Fig. 4). In the crude extract analyzed by PAGIF, it can barely be seen that proteins with a p l smaller than that of BSA, arc present in minute concentrations. The M20 IL-1 inhibitor, which has a p l smaller than that of BSA, as shown above, might therefore remain undetectable, when the crude extract is analyzed by PAGIF. When the IL-l inhibitor separated from the bulk of protein present in the crude extract, and concentrated, was analyzed by PAGIF, it showed a

164

few closely spaced protein bands of p/s, smaller than that of BSA, or the recombinant IL-i receptor antagonist (Fig. 5) (IL-Ira; a gift of Profes~r C.A. Dinarello). By SDS-PAGE analysis, the inhibitor preparation obtained, when Affi-Gcl blue chromatography was used as a preliminary step, shows the prese;,cc of two protein bands (Fig. 6). Similar results were obtained using gel filtration in the preliminary step. Amino acid sequencing revealed that the major protein band corresponds to fetuin derived from the FCS used in the tissue culture medium. To prove that the inhibitory activity is not due to fefflin or other FCS protein, medium, supplemented with 10% FCS, was fractionated by IEF pH 3-5; none of the fractions corresponding to those generated from the conditioned media showed inhibitory activity. In addition, purified fetuin (Sigma, P.O.B. 14508, St. Louis, MO 63178, USA), ranging from 10-333 pg/ml, did not have an inhibitory effect in the

1

2

3

4

Fig. O. Analysis by SDS-PAfAE of IL-I inhibitor purified by the ~quence of purifi~"alinn procedures, as described, Lanes ] and 4 MW markers (BSA, carbonic anhydrase and eytochrome c); lanes 2 and 3 the purified IL-[ inhibitor.

bioassay nor in inhibiting IL-I induced in rive rcsponses (data not shown).

Discussion •- 6.55 5.85 _

±

5.2

4,55

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let INH

3.50 IL-Ira m a r k e r s

Fig. 5. Analysis by PAC31F (pH 3-.5) of the most active ~'raclion (fruethm no. 10} from the Rotofor cell, in which set filtralion was used as a prelimin~tp/step. Lane ] BSA I() ~8; lane 2 fcluin Ifl ~.g; lane 3 IL-I inhibitor; lane 4 rcc~mbinant

1L-Ira5 ~g; lane 5 pl markers.

Although IEF in the Rotofor cell has been used as a first step in the purification of proteins, the isolation of the IL-I inhibitor from the conditioned medium required a preliminary step to remove the butk of fetuin and BSA, which are present in high concentrations in the FCS used in the tissue culture medium. Gel filtration through a Sephacryl S-300 column or dye ligand chromotography on Affi-Gel blue were used for that purpose. Due to the close MW values and elation behavior of BSA, fetuin (because of its high carbohydrate content) and the IL-1 inhibitor, the recovery of the inhibitory activity by get filtration was low, When Affi-G¢l blue columns were used, the recovery of activity was improved, but retain was not removed since it does not bind to the dye ligand. IEF in the Rotofor celt was the most efficient step in the final procedure adopted for the purification of the ]L-I inhibitor. Unfortunately, due to the p l heterogeneity of fetuin, which depends on the content of sialie acid, some quantity of fetuin copurifies with the IL-I inhibitor. We have shown that the IL-I inhibitory activity is not due to fetuin or other FCS protein, since none of the fractions obtained in the pH

165

range 3.79-4.71 from FCS fractionated by IEF, showed an inhibitory activity, From the results obtained in a neutralization assay and dot immunoblotting test, using anu-lL-lra poiyclonal antiserum, one can conclude that the M20 IL-I inhibitor is antigenically different from the IL-tra inhibitor (Barak ct al,, 1991a). We have demonstrated that the M20 IL-I inhibitor does not bind It-In in free solution nor does it affect IL-I production from monocytes. The in rive anti-inflammatory activities of the It-1 inhibitor, purified as described, are the subject of further papers (Barak et al., 1991b, 1992). Preparative isoe[ectric focusing in the horizontal Rotofor column, proved to be an effective methed for the rapid purification of the IL-I inhibitor present in the conditioned medium of the M20 cells, despite the fact that concentration of this protein is very small compared with the hulk of proteins derived from the tissue culture medium. The biological activity, of the IDI inhibitor, both in vitro and in rive, was preserved, although during the fractionation procedure used in the Rotofor cell, it was exposed to 5 M urea, and some of it at extreme pH values, before it reached its pL Further purification for the amino acid sequencing of this molecule is currently being conducted. The availability of recombinant inhibitor and its antibody will enable us to proceed further with our investigations of the immune and inflammatory mechanisms of IL-I and its antagonism.

Acknowledgements We would like to thank ImCIone Inc. New York, NY, for their support of this project. We would also like to express our appreciation to C.A. Dinarello for the IL-lra and its po[yclonal antibody and for his invaluable comments.

References Amnd, W.P. and Dayer. ].M. (19o,0) Cytokines and ¢ytokine inhihilors or anlagonists in rheumatoid a~hrilis. Atthrius Rheum. 33, 305. Barak, V., ¥amin. M.. Braun, S.. Halperln, M.. Biran. S., Milner, Y., and Treves, AJ. (1986a) Detection of different IL-J activities in human mollocytes and monocytk: lines. J. Biol. Response Modif. 5. 362. Break. V., Treves, AJ.. Yanai. P., Halpcrin. M.. Wa.~,serman, S., Biran, S., and Braun, S. (1986b) Interlcukin-I inhibitory activity secreted by a haman n~elomonocytic cell line (M20). Fur. J. ImmunoL 16, 1449. B'~rak, V., Peritt, D., FIcchncr, I., Yanai, P., Halperin, T,, Treves AJ. and Dinare]lo, C.A. (lqgla) The specific IL-I inhibitor from the human M.7.0cell line is distinct [tom the IL-I receptor antagonist. Lymldaokin¢ and C~okin¢ p c . search 10, 437. Barak. V., Perritt, D., Ftcchner, I.. Yanai, P., Hallmrin. T, Dinarello, C.A. and Treves, A.L (1991b) The M2a IL-I inhibitor prevents and ~duces rheumatoid arlhritis and IL-I induced inflammatory responses in rive. Cytokin¢ 3. 520. Barak, V., Perrin, D., Fleehner, L, Sherman, Y., Okon, E., Yanai, P., Halperin, T, and Treves, AJ. {1¢AY2)The M21t It-I inhibitor prevents onset of adjuvant arthritis. Biothcra w 4, 317. Bendtzen. K., Mandrt-Poulsen, T , Nerup, J., Nielsen, J.H., Dinar¢llo, C.A. and Svenson M. (1986) Cytotusieily of human pl 7 int~rleukin-I fur pancreatic islets of Langerhans. Science 232, 15a5. Carter, D.B., DeibeL Jr.. M.R., Dunn, C.J., et aL 11990) Purification, cloning, expression and biological characmrization of an interleukin.l receptor antagonist protein. Nature 344, 633. Dinarello, C.A. (1991) t ntedcukin-t and interlcnkln- l antagonism. Blood 77, 1627, Gew, 1. and Waksrnan, B.H, (1972) Potentiation of the 1"lymphocyte ~sponse to mitogcns. ]|. The cellular ,source of Imlcntiating mediator(s), J. Exp. Mcd. 136. 143. Hannum, C.H., Wilcox, C.J., Atcnd. W.P., et al. (1990l tntcrleukin-I receptor antagonist of a haman intedeukia-i inhibitor. ,Nature 343, 336. Larriek. J.W. (1989l Nati'~ intcrleukin-I inhibilors. Immunol. Today 10. 61. Ramagli, LA, and Rodriguez, L.V. (1085) Quantitadon of microgram amounts of protein in two.dimcnsiona[ poiyacrjlamide gel electtophoresis sample buffer. Elcctrophoresis 6, 559. Seckiuger. P., Lowcnlhal l.W.. Will[amson, K., Dayer, J.M. and MacDonald, H.R. (1987) A urine inhibitor of interleukin-I activity that blocks ligaa~ hqding. J. Immunol. 139, 1546.

The M20 IL-1 inhibitor. I. Purification by preparative isoelectric focusing in free solution.

An interleukin-1 (IL-1) inhibitor produced by the M20 myelomonocytic cell line has been shown to be active in various in vitro and in vivo IL-1 induce...
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