Soluble IL-6 Receptor Potentiates the Antagonistic Activity of Soluble gp130 on IL-6 Responses1 Gerhard Mu¨ller-Newen,* Andrea Ku¨ster,* Ulrike Hemmann,* Radovan Keul,* Ursula Horsten,* Astrid Martens,* Lutz Graeve,* John Wijdenes,† and Peter C. Heinrich2* Soluble receptors for several cytokines have been detected in body fluids and are believed to modulate the cytokine response by binding the ligand and thereby reducing its bioavailability. In the case of IL-6, the situation is more complex. The receptor consists of two components, including a ligand-binding a-subunit (IL-6R, gp80, or CD126), which in its soluble (s) form (sIL-6R) acts agonistically by making the ligand accessible to the second subunit, the signal transducer gp130 (CD130). Soluble forms of both receptor subunits are present in human blood. Gel filtration of iodinated IL-6 that had been incubated with human serum revealed that IL-6 is partially trapped in IL-6/sIL-6R/sgp130 ternary complexes. sgp130 from human plasma was enriched by immunoaffinity chromatography and identified as a 100-kDa protein. Functionally equivalent rsgp130 was produced in baculovirusinfected insect cells to study its antagonistic potential on four different cell types. It was found that in situations in which cells lacking membrane-bound IL-6R were stimulated with IL-6/sIL-6R complexes, sgp130 was a much more potent antagonist than it was on IL-6R-positive cells stimulated with IL-6 alone. In the latter case, the neutralizing activity of sgp130 could be markedly enhanced by addition of sIL-6R. As a consequence of these findings, sIL-6R of human plasma must be regarded as an antagonistic molecule that enhances the inhibitory activity of sgp130. Furthermore, in combination with sIL-6R, sgp130 is a promising candidate for the development of IL-6 antagonists. The Journal of Immunology, 1998, 161: 6347– 6355.
A
s a multifunctional cytokine, IL-6 is involved in the regulation of complex physiologic processes, including the immune response, hematopoiesis, and the acute-phase reaction (for review, see Ref. 1). Prominent IL-6 effects are the differentiation of B and T lymphocytes (2, 3) and the induction of acute-phase protein synthesis in hepatocytes (4, 5). Since IL-6 knockout mice develop normally and appear indistinguishable from wild-type mice unless they are challenged with pathogens, this cytokine seems to play a major role in host defense (6). Dysregulation of IL-6 production is observed in many diseases, such as multiple myeloma (7), rheumatoid arthritis (8), Castleman’s disease (9), and postmenopausal osteoporosis (10). In some of these disorders, IL-6 plays a role as a causative agent, and therefore IL-6 antagonists are of great therapeutic value. IL-6 acts on target cells via a receptor consisting of two transmembrane glycoproteins. After binding of IL-6 to its specific receptor (gp80 or IL-6R), the complex of IL-6 and IL-6R triggers the dimerization of the signal-transducing receptor component gp130 (11). This leads to the activation of different signaling cascades, of which the Janus kinase (Jak)3/STAT pathway is the best understood at the moment; the cytoplasmic part of gp130 is associated
*Institut fu¨r Biochemie, Rheinisch-Westfa¨lische Technische Hochschule Aachen, Aachen, Germany; and †Diaclone, Besanc¸on, France Received for publication February 9, 1998. Accepted for publication August 11, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Deutsche Forschungsgemeinschaft (Bonn, Germany) and the Fond der Chemischen Industrie (Frankfurts/Main, Germany). 2 Address correspondence and reprint requests to Dr. Peter C. Heinrich, Institut fu¨r Biochemie, RWTH Aachen, Pauwelsstrasse 30, D-52057 Aachen, Germany. E-mail:
[email protected] 3 Abbreviations used in this paper: Jak, Janus kinase; s, soluble; MDCK, MadinDarby canine kidney; EMSA, electrophoretic mobility shift assay.
Copyright © 1998 by The American Association of Immunologists
with tyrosine kinases of the Jak family that are activated upon receptor dimerization (12). These kinases phosphorylate tyrosine residues in the membrane-distal cytoplasmic part of gp130, which in turn become binding sites for the Src homology 2 domain containing transcription factors STAT1 and STAT3/acute phase response factor (APRF) (12, 13). Recruited STATs are also tyrosine phosphorylated by the associated kinases, leading to the formation of STAT homo- and heterodimers. STAT dimers are translocated into the nucleus, where they bind to responsive DNA elements and induce target gene expression (14). Furthermore, activation of the ras/raf/mitogen-activated protein kinase pathway was observed in different cells upon IL-6 stimulation (15, 16), as well as activation of various tyrosine kinases different from Jak kinases (17, 18). The soluble form of the IL-6R (sIL-6R), which lacks the transmembrane and cytoplasmic parts, when complexed with IL-6 triggers the dimerization of gp130 and is therefore an agonistically acting molecule (19). Whereas numerous in vitro studies showed that sIL-6R acts agonistically by enhancing the effects of IL-6, the antagonistic potential of sgp130 has been described only in a single study (20). Since soluble forms of both receptor components (sIL-6R (21) and sgp130 (20)) have been detected in plasma and several biologic fluids of humans and mice, the physiologic significance of their opposite biologic effects is of great importance. To further characterize its molecular properties, we enriched sgp130 from human plasma and identified it as a monomeric 100kDa glycoprotein that is able to efficiently bind IL-6/sIL-6R complexes. To study its antagonistic potential in greater detail, rsgp130 was expressed in baculovirus-infected insect cells. The recombinant protein was purified from conditioned media and characterized with respect to its oligomerization state and its ability to form complexes with IL-6 and sIL-6R. Here we show that sgp130 is a potent IL-6 antagonist on various cell types and that its antagonistic activity is markedly enhanced in the presence of sIL-6R. 0022-1767/98/$02.00
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Materials and Methods
Gel filtration
Enzymes, proteins, Abs, chemicals, and cell culture media
Analyses of complexes formed by iodinated IL-6 in human serum were performed on a calibrated Superdex 200 16/60 gel filtration column (Pharmacia) at a flow rate of 0.85 ml/min. sIL-6R depletion of human serum was performed as described (26). 125I-labeled IL-6 (20 ng) with a specific activity of 4.7 3 104 cpm/ng was incubated with 1 ml of human serum (either normal or sIL-6R depleted) or 1 ml of a solution of BSA (50 mg/ml) in PBS for 12 h at 4°C followed by 2 h at 37°C. After centrifugation for 10 min at 10,000 3 g, the supernatant was applied to the gel filtration column, and 2.6-ml fractions were collected. Immunoprecipitations were performed using B-P4-saturated protein A-Sepharose. 125I-labeled IL-6 was quantified using a gamma counter.
Enzymes were purchased from Boehringer Mannheim (Mannheim, Germany), and protein A-Sepharose was purchased from Pharmacia (Freiburg, Germany). DMEM, DMEM/F12, RPMI 1640, Sf-900 II medium, and antibiotics were obtained from Life Technologies (Eggenstein, Germany), and FCS was obtained from Seromed (Berlin, Germany). Bolton-Hunter reagent and [a-32P]dATP were purchased from Amersham International (Little Chalfont, U.K.), and Tran[35S] label metabolic labeling reagent was purchased from ICN (Meckenheim, Germany). Recombinant human IL-6 was expressed in Escherichia coli, refolded, and purified as described by Arcone et al. (22). The specific activity was 108 units/mg of protein in the B9 cell proliferation assay (23). sIL-6R was expressed in insect cells as previously described (24). The monoclonal gp130-Abs B-T12, B-P4, and B-T2 were generated as described elsewhere (25). All other Abs were purchased from DAKO (Hamburg, Germany). The frequently used PBS buffer contained 200 mM NaCl, 2.5 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4.
sgp130 ELISA Ninety-six-well microtiter plates (F96 MaxiSorp immunoplate; Nunc, Naperville, IL) were coated overnight at room temperature with anti-sgp130 mAb B-P4 (0.5 mg/well in PBS). The plates were incubated with saturation buffer (0.1 M Tris; 20% sucrose; and 0.1% sodium acide, pH 7.7) for 2 h at room temperature. After three washes with PBS and 0.02% Tween-20, the samples or standards (in PBS and 1% BSA) and the secondary biotinylated mAb (B-T2, 50 ng/well) were incubated simultaneously for 2 h at 37°C. The standard curve was obtained by twofold serial dilutions of rsgp130. Subsequently, the plates were washed again, and streptavidin poly-horseradish peroxidase (100 ng/ml in PBS and 1% BSA) was added and incubated for 45 min at room temperature. After a final wash, substrate solution (100 mg/ml tetramethylbenzidine in 0.1 M sodium acetate, pH 5.5, and 0.003% H2O2) was added. After incubation for 30 min in the dark, the color reaction was stopped with 2 M H2SO4, and the absorbance at 450 nm was determined using an ELISA reader (SLT-Labinstruments, Gro¨dig, Austria). Affinity-purified rsgp130 from baculovirus-infected insect cells was used as a standard.
Construction of the recombinant baculoviruses and expression of rsgp130 The AccII-EcoRI fragment encoding the gp130 extracellular domains (codons 1– 606) was cut out from the vector pVL-gp130. A pair of hybridized oligonucleotides (59-AA TTC GGA (CAT)5 CAC TAG-39 and 59-G ATC CTA GTG (ATG)5 TCC G-39) encoding a glycine and six histidine residues followed by a stop codon and a BamHI 59-overhang was linked to the EcoRI site of the cDNA for soluble human gp130 and inserted into the polyhedrin locus-based baculovirus transfer vector pVL1392 using the BglII (blunt end) and the BamHI sites. Sf158 cells were cotransfected with 0.5 mg of recombinant gp130-baculovirus transfer vector and 0.125 mg of BaculoGold virus DNA as outlined in the BaculoGold transfection kit manual (Dianova, Hamburg, Germany). Single virus clones were obtained by end point dilution. Several clones were screened for expression of sgp130 by Western blotting. The selected virus clone was then amplified by infecting Sf158 cells at a multiplicity of infection less than 1. Sf158 insect cells were grown at 27°C as monolayer cultures in serum-free Sf-900 II medium. For protein expression, exponentially growing cells were infected with the rsgp130 baculovirus in suspension cultures at a multiplicity of infection from 10 to 20. Seventy-two hours after infection, the cells and cellular debris were sedimented by centrifugation, and the culture supernatants were stored at 220°C.
Immunoaffinity purification of sgp130 and rsgp130 The sgp130 mAb B-T12 was coupled to 0.5 g of CNBr-activated Sepharose CL-4B according to the protocol of the supplier (Pharmacia). Three milliliters of 1 M CaCl2 was added to 300 ml of human plasma, and after incubation at 37°C for 90 min, 10 ml of 0.5 M EDTA was added. Coagulated proteins were sedimented by centrifugation for 20 min at 14,000 3 g. The supernatant was applied to the immunoaffinity column at a flow rate of 20 ml/h. To purify rsgp130, supernatants of baculovirus-infected insect cells were loaded onto the column. After washing with 100 ml PBS/0.05% Tween 20, sgp130/rsgp130 was eluted with 0.2 M glycine buffer, pH 2.5, and immediately neutralized with 1 M Tris/HCl, pH 8.0. Pooled fractions were dialyzed against PBS, and the sgp130 concentration was determined by ELISA. Total protein concentrations were determined using the BioRad protein assay (Bio-Rad Laboratories, Richmond, CA).
Formation of ternary complexes of IL-6, sIL-6R, and sgp130 Binding experiments were performed using IL-6 radiolabeled with 125I according to the procedure of Bolton and Hunter (27), affinity-purified sIL-6R, and rsgp130 or conditioned media from Sf158 cells expressing rsgp130. All incubations were conducted at 4°C. 125I-labeled IL-6 (5.5 ng) with a specific activity of 8.3 3 104 cpm/ng was preincubated overnight with 100 ng sIL-6R in 500 ml TNET buffer (20 mM Tris-HCl, pH 7.5; 140 mM NaCl; 5 mM Na2-EDTA; 1% Triton X-100; 2 mM methionine; and 0.01% NaN3). Increasing amounts of purified rsgp130 or conditioned medium containing rsgp130 were added, and the incubation was continued for 2 h. The reaction mixtures were subjected to immunoprecipitation with the monoclonal gp130 Ab B-T12. Coprecipitated 125I-labeled IL-6 was quantified using a gamma counter.
Real-time interaction analysis For direct monitoring of ternary complex formation, the IAsys (Fisons, Cambridge, U.K.) system was used. The biosensor surface was activated using N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and subsequently incubated with gp130 mAb B-T12 (50 mg/ml in 10 mM sodium acetate, pH 4.8) for 8 min. Unbound Abs were removed by several washes with PBS containing 0.05% Tween 20 (PBS-T), and residual activated groups were blocked with 1 M ethanolamine, pH 8.5. Supernatants of baculovirus-infected insect cells expressing rsgp130 (40 ml) were diluted 5-fold with PBS-T and added to the cuvette. Subsequently, the cuvette was incubated with IL-6 (10 mg/ml in PBS-T), sIL-6R (10 mg/ml in PBS-T), and IL-6 plus sIL-6R (5 mg/ml each in PBS-T) (time intervals indicated in Fig. 4A). After each exchange of protein solution, the cuvette was rinsed with PBS-T. Binding events were monitored as an increase of the resonance angle a.
Ba/F3 proliferation assay Ba/F3-gp130 (28) cells were cultured in RPMI 1640 containing 10% FCS, plated on 96-well plates (20,000 cells/well), and stimulated with either IL-6/sIL-6R or 5% (v/v) conditioned medium from X63Ag-653 BPVmIL-3 myeloma cells (as a source of IL-3) in the presence of rsgp130 (concentrations indicated in Fig. 5). After 60 h of incubation, viable and metabolically active cells were quantified using a colorimetric assay based on the Cell Proliferation Kit II sodium 39-[A-(phenylaminocarbonyl)-3.2 4-tetratolium]-bris-(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) assay (Boehringer Mannheim).
Analysis of STAT activation in COS-7 and Madin-Darby canine kidney (MDCK)/IL-6R cells by electrophoretic mobility shift assay (EMSA) COS-7 and MDCK cells were cultured in DMEM containing 10% FCS. The cDNA of IL-6R was subcloned into the expression vector pCB6 (kindly provided by A. Le Bivic, Laboratoire de Genetique et Physiologie de De´veloppement, Faculte´ des Sciences, Marseille, France). Transfection of MDCK cells was performed by a modification of the calcium phosphate precipitation procedure described by Graham and van der Eb (29). Resistant cells growing in the presence of 0.5 mg/ml G418 (Life Technologies) for 14 days were screened for IL-6R expression by indirect immunofluorescence and binding of radioiodinated IL-6. To induce IL-6R expression, the MDCK/IL-6R cells were treated with 10 mM sodium butyrate (Sigma, St. Louis, MO) for 15 h, and COS-7, MDCK, or MDCK/IL-6R cells were incubated at 37°C for 15 min in the presence of IL-6, rsgp130, and sIL-6R (concentrations as indicated in Figs. 6 and 9). Preparation of nuclear extracts and EMSAs were performed as described (30). A mutated doublestranded oligonucleotide corresponding to the c-fos promoter (m67SIE, 59-GAT CCG GGA GGG ATT TAC GGG GAA ATG CTG-39), which provides STAT3 and STAT1 binding sites, was used as 32P-labeled probe. Protein/DNA complexes were separated on a 4.5% polyacrylamide gel
The Journal of Immunology
6349 containing 7.5% glycerol in 23 mM Tris/23 mM boric acid, pH 8.0, and 0.5 mM EDTA at 20 V/cm for 4 h. Gels were fixed in 10% (v/v) methanol, 10% (v/v) acetic acid, and 80% (v/v) water for 30 min; dried; and analyzed by autoradiography.
Induction of acute-phase protein synthesis in HepG2 cells HepG2 cells were incubated in DMEM/F12 with IL-6, rsgp130, and sIL-6R (concentrations as indicated in Fig. 7) for 18 h and metabolically pulse labeled with [35S]methionine for 3 h. Induction of the newly synthesized acute-phase protein a1-antichymotrypsin was measured in cell culture supernatants by immunoprecipitation using a rabbit anti-human a1antichymotrypsin antiserum. Immunocomplexes were precipitated with protein A-Sepharose, separated on 10% SDS-polyacrylamide gels, and visualized by autoradiography.
Western blotting Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes by a semidry blotting procedure (31). The membranes were incubated with the Ab mixtures as indicated in the figures and were processed for chemiluminescence detection as described in the enhanced chemiluminescence manual (Amersham International).
Results Establishment of an ELISA for the quantification of sgp130 To investigate the role of sgp130 in the modulation of IL-6 responses in a quantitative manner, an sgp130 ELISA was established. Several mAbs (described in Ref. 25) directed against the ectodomain of gp130 were tested for their usefulness in a sandwich ELISA. The best results regarding sensitivity and dose response were obtained by the use of B-P4 for coating of the microtiter plate and biotinylated B-T2 for the detection of sgp130. Concentrations between 100 pg/ml and 2000 pg/ml were quantified using purified rsgp130 as a standard (not shown). Plasma levels of sgp130 determined with our ELISA (320 6 22 ng/ml, n 5 6) are in good agreement with those previously reported (390 6 70 ng/ml; Ref. 20). The ELISA can also be used for the detection of sgp130 complexed with IL-6/sIL-6R (see below). IL-6 added to human serum forms a high molecular mass complex with sgp130 that depends on the presence of sIL-6R To prove the possible functional role of sgp130 in the modulation of IL-6 responses, 125I-labeled IL-6 was added to human serum, and the protein complexes formed were analyzed by gel filtration. A substantial portion of 125I-labeled IL-6 appeared in a peak covering a molecular mass range of 450 –100 kDa (Fig. 1A, closed circles, fractions 25–29). An additional smaller peak of 125I-labeled IL-6 eluted with the void volume of the column. Both peaks were not detected when 125I-labeled IL-6 was incubated with physiologic concentration of serum albumin in PBS and then subjected to gel filtration (Fig. 1A, closed triangles). The high background levels seen between fractions 22 and 31 may be due to association of 125I-labeled IL-6 with different oligomeric forms of BSA. Analysis of the serum fractions by ELISA revealed that sgp130 comigrates with the 450 –100-kDa 125I-labeled IL-6 peak (Fig. 1B, open circles). Immunoprecipitation of proteins in fractions 25–28 containing sgp130 using the sgp130 mAb B-P4 led to the coprecipitation of 125I-labeled IL-6 (Fig. 1C, fractions 25–28, solid bars) FIGURE 1. Gel filtration of human serum incubated with iodinated IL-6. A, 20 ng of 125I-labeled IL-6 (940,000 cpm) were incubated with normal human serum (F), with human serum that was sIL-6R depleted (f), or in BSA/PBS (50 mg/ml, Œ) and separated by gel filtration on a calibrated Superdex G-200 column. The measured radioactivity of the fractions is depicted as a percentage of total radioactivity to normalize for the small deviations of total radioactivity that eluted from the column in each experiment. The elution of the molecular mass marker proteins ferritin (440 kDa), aldolase (158 kDa), BSA (67 kDa), and myoglobin (17 kDa) are
indicated by arrows; V0, void volume. B, Elution profile of sgp130 in the gel filtration experiments described in A as determined by ELISA (E, normal human serum; M, sIL-6R-depleted human serum). C, The fractions of the gel filtration experiment described in (A) that contained sgp130 (25–28) and fraction 36 as a control were subjected to immunoprecipitation with the sgp130 mAb B-P4. Coprecipitated radioactivity due to association of 125Ilabeled IL-6 is depicted as a percentage of total radioactivity of the corresponding fraction (solid bars, normal human serum; gray bars, sIL-6Rdepleted human serum).
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FIGURE 2. Immunoaffinity purification of sgp130 from human plasma. A, Elution profile of sgp130 immunoaffinity chromatography. Human plasma (7 ml) was loaded onto a column of Sepharose-bound gp130 mAb B-T12. The column was rinsed with PBS-Tween, and bound proteins were eluted with 0.2 M glycine buffer, pH 2.5. Fractions of 1 ml were collected and neutralized with Tris-HCl buffer, pH 8.0. sgp130 concentrations of the fractions were determined by ELISA. B, Enriched sgp130 after immunoaffinity chromatography (50 ng) was subjected to SDS-PAGE. Proteins in lane M (marker proteins) and lane 1 were silver stained. Lane 2 shows an immunoblot analysis using the monoclonal sgp130 Ab B-P4.
indicative of a physical association of sgp130 and 125I-labeled IL-6. The highest portion of radioactivity was precipitated in the fractions corresponding to a molecular mass of 450 –200 kDa (fractions 25–27). No precipitation of 125I-labeled IL-6 with the sgp130 mAb was observed in the low molecular mass 125I-labeled IL-6 peak (Fig. 1C, fraction 36, solid bar). The same experiment was performed with serum, which was previously sIL-6R depleted by incubation with IL-6-Sepharose (Fig. 1A, closed squares). The amount of radioactivity in the 450 – 100-kDa peak was reduced from 12.5% of total radioactivity in the presence of sIL-6R to 5.3% in the absence of sIL-6R. Reduction of radioactivity was most pronounced in the fractions corresponding to a molecular mass of 450 –200 kDa. The portion of radioactivity that was precipitated with the sgp130 Ab was in the range of only 1% of total radioactivity of the corresponding fraction (Fig. 1C, gray bars). However, in the presence of sIL-6R, up to 6.6% of radioactivity was precipitated (Fig. 1C, solid bars). In the absence of sIL-6R, the sgp130 peak was moderately shifted to lower molecular mass (Fig. 1B, open squares). We conclude from these experiments that the sgp130 peak obtained after gel filtration of plasma proteins in the presence of radiolabeled IL-6 in its high molecular mass part (fractions 25–27) contains IL-6/sIL-6R/ sgp130 ternary complexes and in its low molecular mass part (fractions 27–30) consists predominantly of sgp130.
ing in a clear pattern of distinct bands (Fig. 2B, lane 1). Immunoblot analysis using the sgp130 mAb B-P4 revealed that the band of about 100 kDa corresponds to sgp130 (Fig. 2B, lane 2). This is the molecular mass expected for gp130 (130 kDa) lacking the transmembrane and cytoplasmic parts (30 kDa). Since the molecular mass calculated from the amino acid sequence of the gp130 ectodomain is 65 kDa, the remaining 35 kDa are due to glycosylation, which also explains the low intensity of this band after silver staining. Consequently, after deglycosylation with peptide-N-glycosidase F, the 100-kDa band disappeared and shifted to lower molecular mass (not shown).
Expression of human sgp130 in baculovirus-infected insect cells and characterization of the recombinant protein Since the available amount of sgp130 from human plasma was too low, we produced human rsgp130 for further studies. For this purpose, insect cells were infected with a recombinant baculovirus encoding the human gp130 ectodomain (amino acids 1– 606 followed by a polyhistidine tag; for details see Materials and Methods). After 3 days, the highest concentration of rsgp130 was observed: about 3 mg/ml as determined by ELISA. This corresponds to about 1% of total protein (250 –300 mg/ml). Furthermore, the immunoblot revealed an apparent molecular mass of about 65 kDa
Characterization of sgp130 from human plasma To characterize sgp130 from human plasma, the protein was enriched by immunoaffinity chromatography. Clotting of human plasma was induced by the addition of Ca21 ions, and precipitated fibrin was separated by centrifugation. No major loss of sgp130 was observed by the coagulation process as determined by ELISA. The clear supernatant was loaded onto a column of the anti-sgp130 mAb B-T12 immobilized to Sepharose. As shown in Fig. 2A, the sgp130 concentration was drastically reduced in the flowthrough. After a washing step, sgp130 was eluted from the column with acidic glycine buffer. The eluate of the column was immediately neutralized and used for further studies. The concentrations of total protein and sgp130 in the eluate were determined. sgp130 made up 2% of total protein, which corresponds to a 5000-fold enrichment in a single chromatographic step. The proteins of the fraction containing enriched sgp130 were separated by SDS-PAGE and visualized by silver staining, result-
FIGURE 3. Immunoaffinity purification of rsgp130 from supernatants of baculovirus-infected insect cells. Conditioned medium (20 ml; lane 1) and purified rsgp130 (100 ng; lane 2) were subjected to SDS-PAGE followed by silver staining. M, marker proteins.
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FIGURE 5. Inhibition of IL-6/sIL-6R-stimulated proliferation of Ba/ F3-gp130 cells by rsgp130. Ba/F3 cells stably transfected with gp130 were incubated with increasing amounts of rsgp130 in the presence of constant amounts of IL-3 (F) or IL-6/sIL-6R complexes (f) (10 ng/ml and 1 mg/ml, respectively) or without any cytokine (Œ). Proliferation of cells was measured after 72 h using the colorimetric XTT assay.
FIGURE 4. Interaction of rsgp130 with IL-6/sIL-6R complexes. A, Real-time interaction analysis of IL-6 and sIL-6R binding to rsgp130. The gp130 mAb B-T12 was covalently linked to the sensor surface. Proteins were added as indicated by arrowheads. Replacement of the protein solutions by PBS is indicated (*). B, Ternary complex formation of rsgp130 with 125I-labeled IL-6 and sIL-6R. 125I-labeled IL-6 (5.5 ng) was incubated with 100 ng of sIL-6R and increasing amounts of either rsgp130 or rsgp130 in the form of conditioned medium, as indicated in the figure. The complexes formed were immunoprecipitated with the monoclonal gp130 Ab B-T12, and coimmunoprecipitated 125I-labeled IL-6 was quantified by measuring of radioactivity in a gamma counter.
for rsgp130 (not shown), suggesting a much lower extent of glycosylation compared with sgp130 from human plasma. Supernatants collected 3 days postinfection were used for affinity purification of rsgp130 by the same method as described for sgp130 from human plasma. Analysis of the fractions by SDS-PAGE revealed that rsgp130 eluted as a pure protein (Fig. 3, lane 2). Purification, however, was accompanied by a marked loss of recombinant protein. Freshly prepared rsgp130 eluted from the gel filtration column as a monomer (not shown). The binding capability of rsgp130 from baculovirus-infected insect cells was demonstrated using two different approaches. In Fig.
4A, a surface plasmon resonance experiment is shown, in which binding events are measured in real time as an increase of the resonance angle a. The sgp130 mAb B-T12 was covalently linked to the sensor surface and subsequently incubated with insect cell supernatants containing rsgp130, resulting in an increase of the resonance angle due to binding of sgp130 to the Ab. Addition of IL-6 alone did not lead to any binding event, whereas addition of sIL-6R resulted in a weak increase of the resonance angle, possibly due to a very low affinity binding of sIL-6R to rsgp130. Addition of a combination of IL-6 and sIL-6R led to the expected strong binding event. Similar results were obtained by coimmunoprecipitation of 125I-labeled IL-6 (Fig. 4B). Using the sgp130 mAb BT12, IL-6 was not precipitated in the presence of rsgp130 or in the presence of sIL-6R alone. Only the combination of rsgp130 and sIL-6R led to the coprecipitation of IL-6. The amount of coprecipitated IL-6 corresponded to the increasing amounts of sgp130 used. The binding assay worked with freshly purified rsgp130, as well as with conditioned insect cell medium containing rsgp130.
FIGURE 6. Inhibition of IL-6/sIL-6R-induced STAT activation in COS-7 cells by rsgp130. COS-7 cells were stimulated for 15 min with IL-6 (10 ng/ml) and sIL-6R (1 mg/ml) in the presence of insect cell medium containing rsgp130 (1 mg/ml) or control conditioned medium (CM) as indicated in the figure. Subsequently, nuclear extracts were prepared and incubated with a 32P-labeled DNA fragment (m67SIE probe). Protein/DNA complexes were separated from unbound DNA by PAGE and visualized by autoradiography.
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FIGURE 7. Inhibition of IL-6- and IL-6/sIL-6R-induced acute-phase protein synthesis in HepG2 cells by rsgp130. HepG2 cells were stimulated for 18 h with IL-6 and sIL-6R in the presence of conditioned medium containing rsgp130 or conditioned medium from mock-transfected cells (CM). Subsequently, the cells were metabolically labeled by the addition of [35S]methionine for 4 h. The conditioned media were collected, and immunoprecipitation of a1-antichymotrypsin was performed using polyclonal a1-antichymotrypsin Abs. Bound proteins were released by the addition of sample buffer and analyzed by SDS-PAGE and fluorography.
rsgp130 efficiently inhibits IL-6/sIL-6R-induced responses on cells lacking membrane-bound IL-6R Ba/F3 cells, which constitute a pre-B cell line often used for the study of cytokine responses, grow in the presence of IL-3. After transfection with gp130, these cells become IL-6/sIL-6R responsive, but due to the lack of membrane-bound IL-6R, they do not respond to IL-6 alone. A previously established Ba/F3-gp130 cell line (28) was used to analyze the antagonistic potential of rsgp130. Ba/F3 cells were incubated with IL-3 or IL-6 and sIL-6R in the presence of increasing amounts of rsgp130. The response of the Ba/F3-gp130 cells to IL-3 was not affected by rsgp130 (Fig. 5, circles), whereas the response to IL-6/sIL-6R was suppressed in a dose-dependent manner (Fig. 5, squares). Proliferation of the cells incubated with rsgp130 alone (Fig. 5, triangles) were indistinguishable from unstimulated control cells (not shown). In the presence of 10 ng/ml IL-6 and 1 mg/ml sIL-6R, 1 mg/ml sgp130 was sufficient for the complete inhibition of the IL-6 signal. To test the influence of rsgp130 on downstream signaling events, STAT activation in COS-7 cells was analyzed. Since COS-7 cells endogenously express gp130 but no membrane-bound IL-6R, again stimulation with IL-6/sIL-6R was required. The EMSA presented in Fig. 6 shows that incubation of COS-7 cells with IL-6/sIL-6R (10 ng/ml and 1 mg/ml, respectively) resulted in a strong STAT1 activation, which is typical for this cell type (Fig. 6, lane 1). Whereas the presence of control supernatant had no significant effect on the signal intensity (Fig. 6, lane 2), STAT activation was completely abolished by the addition of supernatant
containing rsgp130 (final concentration in the assay, 1 mg/ml; Fig. 6, lane 3). Thus, on cells lacking membrane-bound IL-6R, the stimulation with IL-6/sIL-6R can efficiently be blocked by rsgp130. In the presence of rsgp130, the sIL-6R acts antagonistically on cells expressing membrane-bound IL-6R by increasing the inhibitory effect of rsgp130 Next, we investigated the antagonistic activity of rsgp130 on cells expressing membrane-bound IL-6R. These cells respond to IL-6 without the requirement of sIL-6R. HepG2 cells, a human hepatoma cell line, respond to IL-6 with the synthesis of the acutephase protein a1-antichymotrypsin, which, after metabolic labeling, can be immunoprecipitated from cell supernatants and detected by autoradiography (Fig. 7, lane 6). Here, we repeatedly observed that amounts of rsgp130 (1 mg/ml) that were sufficient to completely suppress the IL-6 signal on Ba/F3-gp130 or COS-7 cells showed only a weak antagonistic effect on HepG2 cells (Fig. 7, lane 9). Most surprisingly, sIL-6R, which normally acts agonistically on HepG2 cells (Fig. 7, lane 7), in combination with rsgp130 increased the antagonistic effect of rsgp130 (Fig. 7, lane 8). We repeated this experiment with physiologic concentrations of IL-6, sIL-6R, and sgp130. Again, an inhibitory effect of sIL-6R in the presence of sgp130 was observed (Fig. 7, lanes 1–5). Due to the lack of membrane-bound IL-6R, MDCK cells do not respond to IL-6 (Fig. 8, lane 5). After stable transfection with a cDNA encoding membrane-bound IL-6R, these cells become IL-6 responsive, resulting in a strong activation of STAT3 and STAT1 upon IL-6 stimulation (Fig. 8, lane 1). This response can be enhanced by the addition of sIL-6R (Fig. 8, lane 2). As observed on HepG2 cells, sgp130 has only a weak influence on the IL-6 response (Fig. 8, lane 3). Again, the combination of sIL-6R and sgp130 led to a drastic reduction of the IL-6 response (Fig. 8, lane 4), confirming the antagonistic activity of sIL-6R in the presence of sgp130. Taken together, these findings allow some important conclusions on the physiologic role of the naturally occurring sIL-6R and sgp130 in human plasma, as will be outlined in the discussion.
Discussion
FIGURE 8. Inhibition of IL-6-induced STAT activation in MDCK/ IL-6R cells by rsgp130 and sIL-6R. MDCK/IL-6R cells and MDCK wildtype cells used as a control were stimulated with IL-6 (10 ng/ml) in the presence of rsgp130 (1 mg/ml) and sIL-6R (1 mg/ml) for 15 min. Subsequently, nuclear extracts were prepared and incubated with a 32P-labeled DNA fragment (m67SIE probe). Protein/DNA complexes were separated from unbound DNA by PAGE and visualized by autoradiography.
The analysis of complexes formed by iodinated rIL-6 in human serum by gel filtration revealed that a considerable amount of IL-6 is found in high molecular mass complexes of 450 –100 kDa. sgp130 is also detected in the corresponding fractions, and its immunoprecipitation leads to the coprecipitation of iodinated IL-6. As previously shown by Narazaki et al. (20), by direct precipitation of serum samples and subsequent analysis by SDS-PAGE, we confirm the sIL-6R-dependent association of sgp130 with IL-6 in human serum. The peaks observed in gel filtration were relatively
The Journal of Immunology broad, which may have been caused by protein-protein interactions of unknown specificity due to the high protein concentration in human plasma. Therefore, a clear separation of free sgp130 and sgp130 complexed with IL-6/sIL-6R was not achieved. Since the applied IL-6 amounts were relatively low, a clear shift of the sgp130 peak due to complex formation was not expected. The nature of the IL-6 peak eluting with the void volume of the column, which was more pronounced in the absence of sIL-6R, did not show any sgp130 immunoreactivity in ELISA. Interestingly, May et al. (32) have described the occurrence of large amounts of endogenous IL-6 in human blood in two high molecular mass complexes. They described the IL-6 peak corresponding to the IL-6/ sIL-6R/sgp130 complexes we observed as biologically inactive (33). This is in line with our arguments that IL-6 is partially trapped and inactivated in a ternary complex with sIL-6R and sgp130. After enrichment by immunoaffinity chromatography, sgp130 appears as a monomeric protein of 100 kDa. Whether sgp130 is generated by shedding of the membrane-bound receptor or by translation of an alternatively spliced mRNA is not clear. Phorbol ester induces shedding of the IL-6R (34). Shedding of gp130, however, could hardly be detected (35). While the sIL-6R generated by alternative splicing (36) has been identified in human plasma (26), detection of the protein encoded by the alternatively spliced gp130 mRNA (37) has not been reported so far. To investigate its antagonistic activity, in vitro studies were performed with rsgp130 from baculovirus-infected insect cells. rsgp130 was purified to homogeneity in a single step by immunoaffinity chromatography. Compared with sgp130 from human plasma, the purified rsgp130 showed a markedly lower apparent molecular mass as determined by SDS-PAGE, as well as by gel exclusion chromatography. The difference in molecular mass is most probably due to a different degree of glycosylation. A similar discrepancy in glycosylation levels was observed for the sIL-6R from human plasma (70 kDa) (26) and recombinant sIL-6R from baculovirus-infected insect cells (45 kDa) (24). Since purified rsgp130 has a tendency to aggregate upon aging, the carbohydrate moiety of the soluble receptor from human blood may be required to stabilize the protein. With two different approaches, i.e., plasmon resonance and coimmunoprecipitation, we have shown that rsgp130 binds IL-6/sIL-6R complexes and is therefore suited for the study of its antagonistic activity. Maximal proliferation of our stably transfected Ba/F3-gp130 cells was achieved with 10 ng/ml (0.5 nM) IL-6 and 1 mg/ml (20 nM) sIL-6R. For half-maximal inhibition of the proliferative response (ID50), 100 ng/ml (1.4 nM) rsgp130 was sufficient (see Fig. 5). IL-6/sIL-6R-induced proliferation was completely blocked at a concentration of 1 mg/ml (14 nM) rsgp130. This is a 28-fold excess over the IL-6 concentration used in this experiment. The same concentration of rsgp130 was sufficient to inhibit the rapid IL-6/ sIL-6R-induced activation of STAT1 in COS-7 cells (Fig. 6). Presumably, rsgp130 is such a potent antagonist because it neutralizes IL-6/sIL-6R complexes by forming high affinity ternary complexes. In previous studies, IL-6 and sIL-6R variants were designed to neutralize IL-6 via the low affinity IL-6/IL-6R interaction. A concentration of at least 100 nM sIL-6R mutated in the predicted IL-6R/gp130 interface was required to achieve a partial inhibition of the IL-6 response (38). IL-6 mutated in the predicted gp130-binding sites must be applied at a 1000-fold or larger excess to efficiently inhibit IL-6 responses (39, 40). The fact that the antagonistic activity of IL-6 variants was enhanced by additional mutations strengthening the IL-6/IL-6R interaction is in line with the above arguments (40).
6353 On cells expressing membrane-bound IL-6 receptor that were stimulated with IL-6 alone, we found that rsgp130 was a much weaker antagonist. A concentration of 1 mg/ml rsgp130 was insufficient both on HepG2 and on MDCK/IL-6R cells to completely inhibit the IL-6 response, although a significant reduction was observed in both cases. Addition of sIL-6R, which in the absence of
FIGURE 9. Concentration of bioactive IL-6 in the presence of 300 ng/ml (3000 pM) sgp130 and 50 ng/ml (700 pM) sIL-6R as a function of the initial IL-6 concentration. Given the physiologic (initial) concentration of sIL-6R ([sIL-6R]i 5 0.7 nM) and the dissociation constant of the IL6/sIL-6R interaction (KD1, IL-6 1 sIL-6 % IL-6/sIL-6R), the concentration of IL-6 bound to sIL-6R can be calculated by solving Equation 1,
~@sIL-6R#i 2 @IL-6/sIL-6R#!~@IL-6#i 2 @IL-6/sIL-6R#! KD15 @IL-6/sIL-6R#
(1)
which results in Equation 2
@IL-6/sIL-6R# 5 0.5@sIL-6R#i 1 0.5@IL-6#i 1 0.5KD1 2 0.5~@sIL-6R#2i 2 2@sIL-6R#i@IL-6#i 1 2@sIL-6R#i@IL-6#i 1 @IL-6#2i 1 2@IL-6#iKD1 2 !0.5 1 KD1
(2)
Assuming that binding of one gp130 molecule is sufficient to inactivate the IL-6/sIL-6R complex, the portion of IL-6 antagonized because of ternary complex formation can be approximately calculated by solving Equation 3, in which KD2 corresponds to the dissociation constant of the interaction of sgp130 with IL-6/sIL-6R (KD2, IL-6/sIL-6R 1 sgp130 % IL-6/sIL-6R/ sgp130). KD2 5
~@IL-6/sIL-6R#i 2 @IL-6/sIL-6R/sgp130#!~@sgp130#i 2 @IL-6/sIL-6R/sgp130#! @IL-6/sIL-6R/sgp130# (3)
To solve Equation 3 to calculate the concentration of the ternary complex, the initial IL-6/sIL-6R concentration ([IL-6/sIL-6R]i) was replaced by Equation 2. The initial concentration of sgp130 ([sgp130]i) is the one measured in human blood (3 nM). Using the solved Equation 3, the concentration of ternary complexes was calculated as a function of the initial IL-6 concentration [IL-6]i. Subtraction of the concentration of IL-6 inactivated because of ternary complex formation from the initial IL-6 concentration results in the amount of bioactive IL-6. Dissociation constants used were the following: KD1, 5000 pM, and KD2, 500 pM (F); KD1, 500 pM, and KD2, 50 pM (Œ); and KD1, 50 pM, and KD2, 5 pM (f). The dashed line marks the concentration of bioactive IL-6 in the absence of the soluble receptors ([bioactive IL-6] 5 [IL-6]i); 100 pM IL-6 corresponds to 2 ng/ml.
6354 sgp130 acts as an IL-6 agonist, now potentiates the antagonistic activity of sgp130. We provide a simple explanation for this phenomenon. In cases in which HepG2 or MDCK/IL-6R cells are stimulated with IL-6 alone, the cytokine first binds to the cell surface IL-6R before it interacts either with sgp130 or with membrane-bound gp130. Here, 1 mg/ml rsgp130 is not sufficient to strongly inhibit the IL-6 response, because it has to compete with membrane-bound gp130 present in a high local concentration. Moreover, the IL-6/IL-6R complexes, due to their membrane location, have to find membrane-bound gp130 only in a two-dimensional space. When sIL-6R is added, IL-6/sIL-6R complexes can be trapped by sgp130 in the soluble high affinity ternary complexes and are thereby efficiently neutralized before they bind to the cell surface receptors. Narazaki et al. (20) observed an only 50% reduction in IL-6 responses, even at an sgp130 concentration of 2 mg/ml. Possibly, this weak inhibition is due to the low sIL-6R concentration (#75 ng/ml) that these authors have used in their experiments. What is the functional role of the naturally occurring sgp130 and sIL-6R? Since the concentration of sgp130 in human plasma (about 300 ng/ml or 3 nM; Ref. 20 and our data) is considerably higher than that of sIL-6R (about 50 ng/ml or 0.7 nM; Refs. 21 and 26), it is reasonable to assume that this soluble receptor pair has evolved to inhibit systemic IL-6 responses. Indeed, when HepG2 cells were stimulated with IL-6 in the presence of sIL-6R and rsgp130 at physiologic concentrations, sIL-6R enhanced the antagonistic effect of rsgp130 (Fig. 7). However, the effect was more pronounced at higher concentrations of IL-6, sIL-6R, and sgp130. To assess the functional role of the soluble receptor proteins more quantitatively, we performed some simple calculations applying the law of mass action (detailed in the legend to Fig. 9). In Fig. 9, the outcome is depicted as a series of graphs showing the concentration of bioactive IL-6 ([IL-6]i 2 [IL-6/sIL-6R/sgp130]) as a function of the initial IL-6 concentration ([IL-6]i) in the presence of physiologic concentrations of sgp130 and sIL-6R. If KD1 has a value of 5000 pM (Fig. 9, circles) or higher, the bioavailability of circulating IL-6 is not influenced by sIL-6R and sgp130. At a KD1 of 500 pM (Fig. 9, triangles), the concentration of bioactive IL-6 is considerably reduced to about 50% due to ternary complex formation unless the IL-6 concentration is in the range of or even exceeds the sIL-6R concentration. A KD1 of 50 pM (squares) would lead to a very strong inhibition of IL-6 responses, since more than 90% of IL-6 is trapped in ternary complexes at moderately elevated IL-6 concentrations. Using recombinant sIL-6R from baculovirus-infected insect cells, we measured a KD1 of 500 pM for the binding of iodinated IL-6 (24). A 10-fold higher affinity was measured for ternary complex formation on cells expressing IL-6R and gp130 (KD2 5 50 pM (41, 42)). Applying these dissociation constants to our mathematical model (Fig. 9, triangles) suggests that in the presence of sIL-6R and sgp130 at physiologic concentrations the systemic IL-6 response is modulated in such a way that the bioavailability of IL-6 decreases due to soluble ternary complex formation. Instead of being an agonist (19), sIL-6R of human plasma should therefore be regarded as a protein that enables sgp130 to efficiently trap IL-6 in a soluble ternary complex, thereby acting as a buffer to modulate systemic IL-6 responses. Furthermore, using the above dissociation constants, Equation 3 predicts that at the sgp130 and sIL-6R concentrations used in most of our experiments (1 mg/ml each), the amount of bioavailable IL-6 is reduced 50-fold. This corresponds to the observed strong antagonistic effects. It should be taken into consideration that due to cell activation or certain pathologic conditions, the local concentrations of sgp130 and sIL-6R may be consider-
ANTAGONISTIC ACTIVITY OF SOLUBLE gp130 ably different from the ones observed in human plasma, thus modulating the inhibitory capacity of this pair of soluble receptors. Modulation of biologic responses by antagonizing proteins is a common principle in cytokine biology. For example, the bioactivity of IL-1 can be down-regulated by a naturally occurring IL-1 receptor antagonist (43). For other soluble cytokine receptors, it has been demonstrated that they indeed play a physiologic role in the down-regulation of the response to the corresponding mediator. Very recently, a circulating leptin-binding protein has been detected in mice that is up-regulated during pregnancy. This protein efficiently neutralizes leptin and was identified as a soluble form of the leptin receptor (44). A similar functional role during pregnancy has been assigned to the soluble growth hormone receptor (45). Both receptors belong to the same family of cytokine receptors as do IL-6R and gp130. Also, for other members of the cytokine receptor family, soluble forms have been described (reviewed in Ref. 46). In each case, their physiologic roles need to be elucidated.
Acknowledgments We thank Dr. P. Kru¨ger for his help with the calculations presented in the discussion.
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