©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interleukin-6 (IL-6) Antagonism by Soluble IL-6 Receptor Mutated in the Predicted gp130-binding Interface (*)

Anna Laura Salvati (1), Armin Lahm (2), Giacomo Paonessa (1), Gennaro Ciliberto (1)(§), Carlo Toniatti (1)

From the (1) Departments of Genetics and (2) Biocomputing, Istituto di Ricerche di Biologia Molecolare P. Angeletti, Via Pontina Km. 30.600, 00040 Pomezia, Rome, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Interleukin-6 (IL-6) triggers the formation of a high affinity receptor complex constituted by the ligand-binding subunit IL-6 receptor (IL-6R) and the signal-transducing chain gp130. Since the cytoplasmic region of IL-6R is not required for signal transduction, soluble forms of IL-6R (sIL-6R) show agonistic properties because they are still able to originate IL-6sIL-6R complexes, which in turn associate with gp130. A three-dimensional model of the human IL-6IL-6Rgp130 complex has been constructed and verified by site-directed mutagenesis of regions in shIL-6R (where ``h'' is human) anticipated to contact hgp130, with the final goal of generating receptor variants with antagonistic properties. In good agreement with our structural model, substitutions at Asn-230, His-280, and Asp-281 selectively impaired the capability of shIL-6R to associate with hgp130 both in vitro and on the cell surface, without affecting its affinity for hIL-6. Moreover, the multiple substitution mutant A228D/N230D/H280S/D281V expressed as a soluble protein partially antagonized hIL-6 bioactivity on hepatoma cells.


INTRODUCTION

Interleukin-6 (IL-6)() elicits a variety of biological responses on different target cells (1) . While the physiological production of IL-6 regulates B-cell proliferation and maturation, T-cell activation, and the production of acute-phase proteins in liver during inflammation (2) , the disregulated production of this cytokine is believed to play a crucial role in the pathogenesis of multiple myeloma, post-menopausal osteoporosis, and autoimmune diseases (3, 4, 5) .

IL-6 stimulates responsive cells by promoting the sequential association of two transmembrane glycoproteins with distinct functional properties (6) . The subunit of the IL-6 receptor (IL-6R) specifically recognizes the cytokine at low affinity (10M) (7) . The complex formed by IL-6 and IL-6R is in turn able to interact with the extracellular region of the chain, namely gp130, which has no intrinsic IL-6 binding properties, but is required for the generation of high affinity (10 to 10M) IL-6-binding sites (8) . While the integrity of the intracytoplasmic region of gp130 is absolutely required for IL-6-dependent signal transduction (9), the intracellular region of the IL-6R chain is dispensable (10). As a result, soluble forms of IL-6R, constituted by the entire extracytoplasmic region of the protein, do not behave as IL-6 antagonists, but still bind to the cytokine and mediate its function through interaction with membrane-bound gp130 (11) .

Random mutagenesis of hIL-6R identified distinct sets of residues that are either involved in hIL-6 binding or in hgp130 recognition (12). These findings suggest that it might be possible to generate receptor variants with antagonistic instead of agonistic properties by introducing mutations that still allow formation of the hIL-6shIL-6R complex, but selectively impair its association with hgp130 in a high affinity receptor complex. Such engineered molecules might be of potential therapeutic value for the treatment of diseases in which hIL-6 plays a pathogenetic role (1, 2, 3, 4, 5) .

Deciphering which are the contact surfaces between hIL-6R and hgp130, but also between hIL-6 and its receptors, can be attempted, on a predictive base, by molecular modeling. A suitable template for initiating modeling of the hIL-6hIL-6Rhgp130 complex is the crystallographic structure of hGH bound to the extracellular region of its homodimeric receptor (13) . This is supported by the following observations. First, structural predictions strongly suggest that hIL-6 shares a common four-helix bundle topology with hGH (14, 15) . Second, hGHbp, hIL-6R, and hgp130 all belong to the hematopoietin family of receptors, characterized by the presence of a conserved 220-amino acid-long region called the cytokine-binding domain (CBD), which is responsible for the interaction with the cytokine and is predicted to fold as a tandem repeat of immunoglobulin-like barrels (16) . Third, the hGH system shows rather close functional analogy to the hIL-6 system since the generation of the biologically active hGHh(GHbp) complex proceeds through the association of an initial hGHhGHbp complex with a second hGHbp molecule (17, 18) . The x-ray structure of the hGHh(GHbp) complex revealed that the hormone serves as a bridging ligand that contacts the two receptors with distinct binding sites (named sites 1 and 2) located on opposite sides of the molecule (13) . Receptor dimerization is further stabilized by a direct interaction between the C-terminal subdomains of their respective CBDs (13) .

We have recently reported the construction of a three-dimensional model of hIL-6 and its superimposition onto the hGH structure in the context of the h(GHbp) receptor complex; this approach allowed us to identify a patch of amino acid residues on hIL-6 involved in the interaction with hgp130 (15, 19) . In this paper, we present a further advancement of the model, which describes the structure of the complex of hIL-6 bound to the CBDs of hIL-6R and hgp130. The model also predicts residues in hIL-6R involved in the interaction with hgp130. Their mutagenesis gave rise to shIL-6R variants with reduced binding to the extracellular portion of hgp130 both in vitro and on the cell surface. Finally, when the mutant showing the most impaired interaction with hgp130 in vitro was tested on cells, it partially inhibited hIL-6 activity.


EXPERIMENTAL PROCEDURES

Molecular Modeling

Modeling of the hIL-6R and hgp130 CBDs was initiated by inscribing their amino acid sequences, according to a preliminary sequence alignment against hGHbp, onto the structural template provided by the corresponding hGHbp molecules within the hGHh(GHbp) complex (Protein Data Bank code 2HHR) (15, 20) . This alignment was based on conserved residues such as the four cysteines predicted to form disulfide bridges, prolines in the interdomain linker, the WSXWS motif (21) , and the pattern of hydrophobic residues within the strand regions. Assignment of buried and solvent-accessible positions was accomplished using a complete model of the hGHh(GHbp) complex generated from the available C- coordinates with the WHATIF software package (22) . In a subsequent step, unfavorable contacts and cavities within the hydrophobic core of the two subdomains were minimized by optimization of side chain conformations, and where necessary, the packing was improved, shifting parts of the sequence within the strands regions. The connecting loop regions containing the majority of insertions and deletions were modeled either manually or using the loop search procedure as implemented in the INSIGHT software package (23) . For generation of the AB2 loop variants of hIL-6R, we used either the available x-ray structures of hGHh(GHbp) (Protein Data Bank code 2HHR) (13) and tenascin (Protein Data Bank code 1TEN) (24) , the latter superimposed manually onto the hIL-6R CBD model, or structurally similar segments from an immunoglobulin constant domain (Protein Data Bank code 1FDL) (25) and the staphylococcal nuclease (Protein Data Bank code 2SMN) (26) , selected from the Protein Data Bank protein structure data base (20) , through the loop search procedure of INSIGHT (23) . Amino acid sequences for the receptor chains correspond to the SWISSPROT data bank codes GHR_HUMAN (hGHbp) and IL6R_HUMAN (hIL-6R) or to code A36337 (hgp130) from the PIR protein sequence data bank.

Plasmid Construction and Site-directed Mutagenesis of shIL-6R

shIL-6R cDNA was obtained as a 5`-EcoRI-3`-XbaI fragment by polymerase chain reaction using the hIL-6R complete cDNA as a template (27) . The 3`-primer was designed in order to introduce an artificial TAG stop codon at amino acid 324 preceded by a sequence coding for six histidines. The fragment generated was then introduced into the expression vector pcDNAI (Invitrogen) to obtain plasmid pC6FRH. Plasmid pC6FRH was used as a template to obtain receptor mutants by polymerase chain reaction (28, 29) . All the mutant cDNAs were cloned into pcDNAI. Their identity was verified by sequencing. For the expression of soluble IL-6 receptors in insect cells, the coding sequences of histidine-tagged wild-type shIL-6R and Mut-4 were excised from pcDNAI and introduced as a 5`-BamHI-3`-XbaI-filled fragment into pBlueBacIII baculovirus transfer vectors (Invitrogen) previously restricted with BamHI-HindIII blunt-ended to obtain plasmids pBsIL-6RH and pBMut-4H.

shgp130 cDNA was obtained as a 5`-HindIII-3`-EcoRV fragment coding for a soluble form of human gp130 (amino acids 1-605) by polymerase chain reaction using plasmid pBS130BES (27) as a template. The 3`-oligonucleotide was engineered in order to add a c-myc tag immediately upstream of the stop codon (30) . The amino acid sequence of the C terminus of shgp130 was the following: EFEEQKLISEEDL-Stop. To express the protein in insect cells, the cDNA was inserted as a 5`-HindIII-filled-3`EcoRV fragment into pBlueBacIII to obtain the transfer plasmid pBsGPm.

Cytokine Production and Purification

Recombinant human IL-6 and recombinant human OM were produced and purified as described previously (31, 32) . The specific bioactivity of hIL-6 was 2 10 units/mg of protein as assessed by 7TD1 cell growth assay (33) . I-IL-6 (900-1200 Ci/mmol) was purchased from Amersham Corp.

Expression of shIL-6R Mutants in COS-7 Cells

COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum plus glutamine and antibiotics at 5% CO. Cells (2.5 10) were seeded in 100-mm tissue culture dishes and transfected with 2 µg of the various shIL-6R expression vectors using the DEAE-dextran technique (34) . 16 h after transfection, cells were replated in 100-mm dishes and grown in complete medium at 37 °C. After 72-96 h, the medium was collected, centrifuged, and used for coimmunoprecipitation experiments and binding analysis. To monitor the expression level of each mutant, 2.5 10 transfected COS-7 cells were replated in 35-mm dishes and, 48 h after transfection, metabolically labeled with [S]methionine for 4 h. The supernatants were immunoprecipitated with anti-human IL-6R monoclonal antibody I6R1/9.G11 (27) and protein A-Sepharose 4 Fast Flow (Pharmacia Biotech Inc.).

Binding Analysis

Unlabeled ligand competition assays were performed essentially as described previously (27) . Briefly, appropriate amounts of cell supernatants, previously determined in titration experiments and to which imidazole was added up to 5 mM (final concentration), were mixed with 20-40 pMI-IL-6 and increasing concentrations of unlabeled cytokine. The total volume of the binding reaction was 500 µl. Under equilibrium conditions, 40 µl of Ni-nitrilotriacetic acid-agarose (QIAGEN Inc.) were added, and incubation was prolonged for 1 h. Bound ligand (``resin-associated'') was separated from free ligand (supernatant) by centrifugation through a cushion of 30% sucrose in PBS. All steps were performed at 4 °C. Normalization of the amount of receptor used in binding experiments was achieved as follows. 10% of the transfected cells were used to monitor the production of each receptor by immunoprecipitation of metabolically labeled supernatants and densitometric analysis of gels similar to the one shown in Fig. 2 , using Image software Version 1.22 (National Institutes of Health, Bethesda, MD). The remaining 90% of the cells from the same transfection were used as a source of unlabeled receptor in binding experiments. 30 ± 10 µl of culture medium were utilized in the case of wild-type shIL-6R. For each mutant, the quantity of supernatant used was adjusted for its expression level with respect to wild-type shIL-6R. The total volume of the binding reaction was kept constant (500 µl) by adding supernatant from untransfected COS-7 cells. In the case of Mut-5 to Mut-7, the culture medium was also concentrated 5-10-fold using Centricon-10 (Amicon, Inc.). The final concentration of I-IL-6 was 400-800 pM. The apparent affinity of the various soluble hIL-6 receptors for hIL-6 was determined after Scatchard transformation of the results (27) . Analysis of binding data and curve fitting was done using UltraFit software (Biosoft).


Figure 2: Expression level of shIL-6R mutants in the medium of transiently transfected COS-7 cells. COS-7 cells transfected with expression plasmids carrying wild-type (W.T.) or mutant (Mut-1 to Mut-8) cDNA encoding shIL-6R were metabolically labeled with [S]methionine. 250 µl of conditioned supernatants were immunoprecipitated with 1 µg of anti-hIL-6R monoclonal antibody I6R1/9.G11 and protein A-Sepharose. Immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. In control experiments, cells were transfected with the empty expression vector (controllane). One representative experiment is shown.



I-IL-6 Binding on A375 Cells

Confluent monolayers of A375 human melanoma cells grown in 24-well tissue culture dishes were preincubated with 500 µl of binding buffer (Dulbecco's modified Eagle's medium plus 50 mM Hepes, pH 7.2, 3% bovine serum albumin, 0.2% sodium azide) for 45 min at 23 °C and then washed with PBS. Normalized aliquots of conditioned COS-7 cell medium were added to each well in duplicate, together with 1 nMI-IL-6, and incubated for 90 min at 23 °C (see also legend to Fig. 4 ). Normalization was performed by determining for each receptor the quantity of conditioned medium capable of retaining 6 fmol of I-IL-6 on Ni-nitrilotriacetic acid-agarose as described under ``Binding Analysis.'' The total volume of the binding reaction was kept constant (200 µl) throughout different wells. Nonspecific binding was measured in the presence of a 200-fold molar excess of unlabeled IL-6. At the end of the incubation time, monolayers were washed twice with cold binding buffer and solubilized with 200 µl of 1 N NaOH. Cell-bound radioactivity was measured in a -counter.


Figure 4: Mut-1 to Mut-4 are impaired in their association with membrane-bound gp130. Confluent monolayers of human A375 cells (3 10 cells/well) in 24-well tissue culture plates were incubated for 90 min with 1 nMI-hIL-6 alone (column1) or in combination with equivalent amounts of wild-type (W.T.; column2) and mutant (columns 4-7) receptors. After washing steps, cell-bound radioactivity was determined as described under ``Experimental Procedures.'' In a competition experiment (column3), cells were incubated with 1 nMI-hIL-6, wild-type shIL-6R, and 1 µM unlabeled OM. Data are expressed as specific binding. One representative experiment is shown. For details, see also ``Experimental Procedures.''



Production of Soluble Receptor Chains in Insect Cells

Production of soluble hIL-6 receptors in insect cells was achieved using the MaxBac baculovirus expression system (Invitrogen) following the manufacturer's instruction with minor modifications. High Five cells (Invitrogen) were used for protein production. The culture supernatants were harvested at 36 h post-infection, dialyzed against PBS, and directly loaded on a Ni-nitrilotriacetic acid-agarose column. After washing steps with PBS, 8 mM imidazole, both wild-type shIL-6R and Mut-4 were eluted in PBS, 80 mM imidazole. Purified proteins were dialyzed against PBS. Receptor purity was estimated to be >90% as judged by silver staining of SDS-polyacrylamide gel. Soluble gp130 was produced in insect cells following the same procedure. For in vivo labeling of shgp130, High Five cells were infected with the shgp130 recombinant virus. 24 h later, cells were incubated for 1 h in methionine-free Grace's medium and metabolically labeled with 100 µCi/ml [S]methionine in the same medium for 4 h at 27 °C. The supernatant, containing S-shgp130, was harvested and used in immunoprecipitation experiments. Coimmunoprecipitation of shgp130 and shIL-6R was performed at 4 °C as described (15, 27) .

Nuclear Extract Preparation and Electrophoretic Mobility Shift Assays

Nuclear extracts from HepG2 cells were prepared as described (35) . For gel retardation analysis, nuclear extracts were incubated with a P-labeled double-stranded oligonucleotide containing the serum-inducible element of c-fos in its superactive form as described (36) . DNA-protein complexes were loaded on 5% polyacrylamide gel containing 0.5 Tris borate/EDTA and 2.5% glycerol and electrophoresed at 130 V for 3 h. The gels were dried and autoradiographed.

Induction of Haptoglobin Synthesis and Protein Determination

HepG2 human hepatoma cells were grown in minimum essential medium with nonessential amino acids (Life Technologies, Inc.) supplemented with 10% fetal calf serum. Induction of haptoglobin synthesis was achieved essentially as described (37) with minor modifications. HepG2 cells were plated in 24-well cell culture plates at a density of 1 10 cells/cm and left to reach confluence. Confluent monolayers were washed with PBS, starved for 1 h in minimum essential medium without fetal calf serum and nonessential amino acids, and subsequently treated for 20 h in 250 µl of serum-free minimum essential medium with hIL-6 and its soluble receptors. The amount of haptoglobin secreted in the culture medium was monitored by an enzyme-linked immunosorbent assay developed at the Istituto di Ricerche di Biologia Molecolare P. Angeletti by Dr. R. Laufer. 96-Well microtiter plates (Nunc Maxisorp) were coated with goat polyclonal serum anti-human haptoglobin (Sigma) (100 µl in each well of a 1:1000 dilution in 50 mM sodium carbonate, pH 9.4). Following a blocking step with 200 µl of TBSMT (5% non-fat dry milk in Tris-buffered saline, 0.05% Tween 20), 50 µl of an appropriate dilution (1:50 or 1:100) of cell culture medium in TBSMT were added to each well and incubated for 1 h at room temperature. Plates were washed, and 50 µl of rabbit polyclonal serum anti-human haptoglobin (Sigma) (1:1000 dilution in TBSMT) were added. After 1 h of incubation, wells were washed, and bound haptoglobin was revealed by sequential incubation with alkaline phosphatase-conjugated donkey polyclonal serum anti-rabbit IgG (Pierce) (1:1000 dilution in TBSMT) and enzyme substrate. Absorbance at 405 nm was quantitated using a microplate reader (Labsystems Multiskan). Human haptoglobin (Fluka) was used as a reference reagent to obtain a standard curve. Dilutions of cell culture media were chosen in order to obtain absorbance values in the range of linearity of the standard curve.

RESULTS

Computer-assisted Modeling of the Human IL-6IL-6Rgp130 Complex

As a first step toward the modeling of the hIL-6 receptor complex, we aligned the CBDs of human IL-6R and gp130 with that of hGHbp (Fig. 1A), taking advantage of conserved residues such as the WSXWS motif, prolines in the interdomain linker, and the four cysteines predicted to form disulfide bridges. The sequences of the CBDs of hIL-6R and hgp130 were thus inscribed onto the structural templates from the hGHh(GHbp) complex, where the hGHbp that binds hGH at site 1 (helix D) corresponds to hIL-6R, and the hGHbp bound at site 2 (helixes A and C) corresponds to hgp130. In a subsequent step, the packing of buried side chains was optimized, and finally, the loop regions including insertions and deletions were modeled (for details, see ``Experimental Procedures''). The resulting final alignment (shown in Fig. 1C) reflects the most satisfactory internal packing. Two important observations can be made. 1) Deletions and insertions occur only within loop regions; and 2) the relative orientation of the two subdomains is likely to be identical in hGHbp, hIL-6R, and hgp130 because there are no insertions or deletions between subdomains 1 and 2, and also the PXPP motif at the beginning of subdomain 2 is conserved. Therefore, as in both hIL-6R and hgp130 CBDs the strand structure and subdomain orientation were predicted to be identical to those of hGHbp, we were encouraged to combine their three-dimensional models with that of hIL-6 (15) and to orient the three molecules as in the hGHh(GHbp) complex, with hIL-6R corresponding to hGHbp1 and hgp130 corresponding to hGHbp2. The final model, shown in Fig. 1B, predicts that, similar to the hGHh(GHbp) complex (13) , the dimerization interface between hIL-6R and hgp130 is constituted by the AB2 loop and the beginning of the E2 strand of their CBDs (for nomenclature, see legend to Fig. 1). While mutations in the E2 strand of hIL-6R have already been reported to decrease the interaction of the hIL-6hIL-6R complex with gp130 (12) , the sequence alignment shown in Fig. 1C predicts that the AB2 loops of both hIL-6R and hgp130 are shorter than that of hGHbp by four residues, pointing toward a possible different role of these regions in the two systems. To test our model, we decided to generate targeted mutations in the AB2 loop of hIL-6R either individually or in combination with substitutions in the E2 strand.


Figure 1: Sequence alignment of the CBDs of hIL-6R, hgp130, and hGHbp and schematic view of the computer-predicted model of the human IL-6IL-6Rgp130 complex. A, comparison of the extracellular domain topology of GHbp (left), IL-6R (middle), and gp130 (right). The central element, the common CBD with two subdomains (openboxes), is present in IL-6R and gp130 together with additional domains. FNIII, fibronectin III. B, schematic MOLSCRIPT (52) representation of the three-dimensional model of hIL-6 interacting with the CBDs of hIL-6R and hgp130. The three components of the complex are oriented analogously to the hGHh(GHbp) complex structure (13), with hIL-6 serving as a bridging ligand enabling hIL-6R and hgp130 to dimerize. The predicted interface between the two receptors is shown in more detail, with hIL-6R residues 228 and 230 (AB2 loop) and residues 280 and 281 (E2 strand) indicated as spheres. C, multiple sequence alignment of hIL-6R, hgp130, and hGHbp (see ``Experimental Procedures''). strand regions are boxed. Regions implied by the hGHh(GHbp) complex x-ray structure (13) to be involved in cytokine recognition (loop regions AB, EF, and BC2 and the interdomain linker) are indicated (#) in addition to regions participating in receptor dimerization (E2 strand and AB2 loop) (*).



Mutagenesis of the Presumptive hIL-6Rhgp130 Dimerization Interface

We generated a double substitution of residues 280-281 in the E2 strand, which, as previously mentioned, were already shown to be required for hIL-6hIL-6R binding to hgp130 (12) . According to our model, these residues correspond to hGHbp Tyr-200 and Ser-201, both of which are involved in hGHbp homodimer formation (13) . His-280 was changed to Ser and Asp-281 to Val. These substitutions were predicted to be compatible with the conformational constraints and unable to establish specific contacts with the neighboring gp130 residues Lys-241, Thr-285, and/or Glu-275.

With respect to the AB2 loop, hIL-6R mutants previously generated at the end of the loop (Arg-232 and Trp-233) showed no decrease in biological activity (12) . We decided therefore to mutate Asn-230 either singly (Mut-2) or in combination with Ala-228 (Mut-3) (). To facilitate charge repulsion with the predicted facing Asp-288 in hgp130, both residues were substituted with Asp. We also constructed a mutant carrying all four substitutions (A228D/N230D/H280S/D281V, Mut-4) (). Of the remaining residues, Pro-231 was not mutated because its conservation in the hIL-6R sequence from various species (data not shown) suggested that it plays an important structural role in the conformation of the loop. Arg-229 was also excluded because it corresponds to Gly in murine IL-6R (38); since murine IL-6R is able to functionally interact with human gp130 (10) , this position is unlikely to be an important contact point.

In addition, two shorter AB2 loop variants (Mut-6 and Mut-7), carrying the equivalent regions of the tenth domain of tenascin (24) or part of an immunoglobulin constant domain (25) , respectively, were generated with the idea to eliminate altogether AB2 loop contact points with gp130 (). Two other IL-6R mutants, containing longer versions of the AB2 loop taken either from hGHbp (13) or from an unrelated protein (26) , were also generated (Mut-5 and Mut-8, respectively). Both variants were predicted to lead to steric clashes and/or charge repulsion ().

Expression Level and hIL-6 Binding Properties of shIL-6R Mutants

All mutants were generated in the context of a cDNA encoding a soluble form of human hIL-6R, with a C-terminal stretch of six histidine residues immediately upstream of an artificial termination codon introduced at amino acid 324. Wild-type and mutant cDNAs were expressed in COS-7 cells (see ``Experimental Procedures'').

To verify the production of soluble hIL-6 receptors, the medium of metabolically labeled transfected cells was immunoprecipitated with the non-neutralizing anti-hIL-6R monoclonal antibody I6R1/9.G11 (27) , which is directed against the N-terminal Ig-like domain of hIL-6R.() As shown in Fig. 2, a protein with an apparent molecular mass of 53 kDa was immunoprecipitated from the culture medium of transfected cells. The signal was specific since it was not detected when an empty expression vector was used (Fig. 2, controllane). Mut-5 to Mut-7 were constantly produced in lower amounts, suggesting that the corresponding mutations might affect either stability or secretion. On the contrary, the expression level of the other five variants was comparable to that of the wild type.

We next determined the affinity of the various shIL-6R variants for hIL-6 using a technique that exploits C-terminal histidine tagging (27). A fixed amount of labeled cytokine was incubated with the histidine-tagged soluble receptor, and self-competition was carried out by adding increasing concentrations of unlabeled cytokine. Under equilibrium conditions, a metal-chelating resin was added; receptor-bound (resin-associated) counts were separated from free radioactivity by centrifugation. The values obtained were plotted in a typical self-displacement curve, and the apparent affinity was determined after Scatchard transformation of the data (27) . Using this technique, an apparent Kvalue for IL-6 of COS-7-produced wild-type shIL-6R was obtained (2.0 ± 1 10M) (), which is close to that previously determined with standard techniques (39) .

Point mutations in the E2 strand and the AB2 loop do not substantially affect the affinity for hIL-6, except for Mut-1, where a slight but reproducible decrease (from 2.0 to 4.3 nM) was observed (). Interestingly, when substitutions in the E2 strand are present together with mutations in the AB2 loop (Mut-4), full hIL-6 binding activity is restored (). On the contrary, all variants in which the complete AB2 loop had been substituted (Mut-5 to Mut-8) displayed a strongly decreased ability to interact with the cytokine (). Since the receptor mutants with the weakest apparent affinity for hIL-6, namely Mut-5 to Mut-7, were the same mutants that showed the lowest levels of expression (compare Fig. 2and ), we believe that the substitution of the entire AB2 loop might cause severe and unpredicted conformational changes, which alter folding and/or stability of the molecule.

Substitution Mutants in Both the AB2 Loop and the E2 Strand Have Impaired Interaction with hgp130

We selected the mutants showing a hIL-6 binding affinity similar to that of wild-type shIL-6R and evaluated their interaction with shgp130 by coimmunoprecipitation in the presence of hIL-6 using suitable monoclonal antibodies (15, 27, 40, 41) . S-Labeled shgp130 (produced in insect cells; see ``Experimental Procedures'') was incubated with I-hIL-6 (as an internal standard for immunoprecipitation) and aliquots of transfected COS-7 cell culture medium containing either native or mutant receptors. After in vitro binding, hIL-6shIL-6Rshgp130 complexes were immunoprecipitated with anti-hIL-6R monoclonal antibody I6R1/9.G11 and resolved by SDS-polyacrylamide gel electrophoresis (see also legend to Fig. 3 , A and B). The results are shown in Fig. 3A. As expected, substitutions at the beginning of the putative E2 strand (Mut-1) strongly reduced the association of the hIL-6shIL-6R complex with the shgp130 molecule (Fig. 3A, lane4). However, also the single mutation at position 230 in the AB2 loop (Mut-2) had decreased interaction with shgp130 (lane2), which was substantially unaffected by the addition of a second negatively charged residue (Mut-3) in near proximity (lane3). Finally, the quadruple mutant Mut-4 displayed the lowest ability to coimmunoprecipitate S-shgp130, thus suggesting an additive effect of the two pairs of mutations located in different regions of the receptor (lane5).


Figure 3: shIL-6R mutants (Mut-1 to Mut-4) have a reduced capability to coimmunoprecipitate S-shgp130. A represents the results of a coimmunoprecipitation assay. Conditioned COS-7 culture medium containing either wild-type shIL-6R (W.T.; lane1) or selected mutants (Mut-1 (lane4), Mut-2 (lane2), Mut-3 (lane3), and Mut-4 (lane5)) was mixed with 2 µg of anti-hIL-6R monoclonal antibody I6R1/9.G11 and 30 µl (packed volume) of protein A-Sepharose for 4 h at 4 °C. After washing steps, the Sepharose beads were incubated for 12 h with 5 nMI-hIL-6 and 100 µl of S-shgp130. Immunoprecipitates were electrophoresed on SDS-12% polyacrylamide gel. The dried gel was subjected to autoradiography at -80 °C (exposure time = 36 h). B shows the portion of the gel (the same as in A) corresponding to migrated I-hIL-6 after 1 h of exposure at room temperature.



We next tested receptor variants for their ability to interact with membrane-bound gp130. Binding experiments were performed on A375 human melanoma cells, which are known to display more hgp130 molecules than hIL-6R on their surface (42) . Specific binding of I-hIL-6 to A375 monolayers was strongly enhanced in the presence of wild-type shIL-6R (Fig. 4). The increase was competed by excess unlabeled OM, an IL-6-related cytokine that directly binds, at low affinity (K 10M), hgp130 and competes for its association with the hIL-6hIL-6R complex (27) . On the contrary, only a minor increase in specific binding was detected when cells were challenged with I-hIL-6 plus receptor derivatives (Fig. 4). Interestingly, Mut-1 and Mut-4 were the most defective variants also in this assay.

Mut-4 Is a hIL-6 Antagonist

The results shown above indicated Mut-4 as the receptor mutant with the most affected interaction with hgp130 and full hIL-6 binding activity. To study its biological activity, the production of Mut-4 and of wild-type shIL-6R was scaled up by using the baculovirus expression system. Both His-tagged proteins were produced at high levels (>5 mg of protein/liter of medium) and purified from the supernatant of infected High Five insect cells (see ``Experimental Procedures'').

The baculovirus-produced receptors were first tested in coimmunoprecipitation experiments. The use of purified proteins allowed us to quantify the amount of receptor used and to compare the behavior of the wild type and Mut-4 over a wide range of hIL-6 concentrations. The results, shown in Fig. 5, were in good agreement with those previously obtained with COS-7 cell-produced receptors. Interestingly, along the entire hIL-6 curve, the amount of bound gp130 was lower for Mut-4 than for wild-type shIL-6R, and this was maintained even at the highest cytokine concentration (100 nM).


Figure 5: Evaluation of the ability of wild-type shIL-6R and Mut-4 produced in insect cells to coimmunoprecipitate S-shgp130. 2 µg of purified wild-type shIL-6R (W.T.; lanes 1-5) or purified Mut-4 (lanes 6-10), both produced in insect cells, were mixed with 100 µl of S-shgp130 and increasing concentrations of hIL-6. Immunoprecipitations with 5 µg of anti-hIL-6R monoclonal antibody I6R1/9.G11 were performed as described in the legend to Fig. 3.



To verify the potential antagonism of Mut-4 for hIL-6 activity, we first tested its ability to interfere with early steps in the IL-6 signal transduction pathway. The assay we used was the activation of transcription factor APRF/STAT3 in human hepatoma cells (35, 36) . Acquisition of DNA binding by APRF is dependent on tyrosine phosphorylation and takes place within minutes of stimulation (43, 44) .

Human HepG2 cells were treated with increasing concentrations of hIL-6 (from 2 to 200 pM) together with a fixed amount of wild-type and mutant receptors (100 nM). As shown in Fig. 6A, we observed that while shIL-6R increased the sensitivity of the cells to hIL-6 (compare lanes 2-4 with 5-7), Mut-4 not only lacked agonistic activity, but instead inhibited IL-6-dependent APRF activation (compare lanes 2-4 with 8-10). To test if antagonism was specific for hIL-6, Mut-4 was added to HepG2 cells induced with OM, which is also known to efficiently induce APRF phosphorylation and binding (43) . As shown in Fig. 6B, Mut-4 did not affect signaling by OM.


Figure 6: Mut-4 specifically inhibits hIL-6-dependent APRF activation in HepG2 human hepatoma cells. A shows the hIL-6 antagonistic activity of Mut-4 on HepG2 cells compared with the agonistic properties of wild-type shIL-6R. HepG2 cells were treated for 15 min with increasing concentrations of hIL-6 alone (lanes 2-4) or together with a fixed amount of purified wild-type shIL-6R (W.T.; lanes 5-7) or purified Mut-4 (lanes 8-10). 5 µg of nuclear extracts were used to monitor the binding of APRF to the serum-inducible element probe by electrophoretic mobility shift assay (see ``Experimental Procedures''). The dried gel was exposed for 36 h. B demonstrates that Mut-4 does not inhibit OM-dependent APRF activation in HepG2 cells. Nuclear extracts were prepared from HepG2 cells treated (15 min) with 200 pM IL-6 or OM alone (lanes2 and 5, respectively) or in combination with 100 nM wild-type shIL-6R (lanes3 and 6) or Mut-4 (lanes4 and 7). Electrophoretic mobility shift assay was performed as described for A, but the gel was exposed for only 12 h.



We next tested whether Mut-4 might inhibit hIL-6 ability to induce the production and secretion of the acute-phase protein haptoglobin in the same cells. HepG2 cells were treated for 20 h with IL-6 (200 pM) alone or in combination with increasing concentrations of wild-type and mutant receptors. At the end of the incubation time, the medium was collected and assayed for the presence of secreted haptoglobin (see ``Experimental Procedures''). As expected, the addition of wild-type shIL-6R to hIL-6 induced a dose-dependent increase (Fig. 7). On the contrary, Mut-4 antagonized hIL-6 bioactivity in a dose-dependent fashion, with a 50% inhibition achieved at the highest concentration tested (100 nM) (Fig. 7). Also in this case, the effect was specific since neither the native nor the mutant receptor interfered with the OM-induced production of haptoglobin (data not shown).


Figure 7: Mut-4 down-modulates hIL-6-induced synthesis of haptoglobin in HepG2 cells. HepG2 cells were cultured for 20 h with 200 pM hIL-6 and the indicated amounts of either wild-type (W.T.) shIL-6R (blackbars) or Mut-4 (shadedbars). In control experiments, cells were either untreated (whitebar) or incubated with 200 pM hIL-6 alone (hatchedbar). The amount of secreted haptoglobin was measured with an enzyme-linked immunosorbent assay as described under ``Experimental Procedures.'' Means ± S.D. of three separate experiments are shown. No effect on haptoglobin production was observed when cells were challenged with soluble receptors only (data not shown).



DISCUSSION

In this paper, we present a computer-assisted three-dimensional model of the interaction between hIL-6 and its two receptor chains, based on the x-ray structure of the hGHh(GHbp) complex (13), and use it to selectively introduce mutations into the hIL-6R chain that modify its biochemical and biological properties. Residues in both the putative AB2 loop and the E2 strand of the hIL-6R CBD are identified as being involved in the interaction with hgp130. To date, residues in the E2 strand and the AB2 loop in the receptor-receptor interface have been demonstrated to be involved at the structural level only for homodimeric hGHbp (13) . This finding has been recently confirmed, from the functional point of view, by the isolation of a natural mutant of hGHbp (D152H) unable to homodimerize (45). Our results demonstrate that the same regions are also involved in stabilizing a heterodimeric receptor (hIL-6Rhgp130). It is thus tempting to speculate that participation of the AB2 loop and the E2 strand in receptor association is a common rule for both homodimeric complexes, like those assembled by GH, erythropoietin, and granulocyte colony-stimulating factor, and heterodimeric receptors, like those for IL-3, IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor (16).

Interestingly, combining substitutions in the E2 strand and the AB2 loop as it was done generating variant Mut-4 does not totally abolish interaction with gp130, in line with its being only a partial antagonist. There are several explanations to this finding. First, not having tested all the possible substitutions at positions 280 and 281 and not having mutated all residues of the AB2 loop, we cannot exclude that a more systematic approach would lead to a full antagonist like the one recently discovered for hGHbp (45) . In this latter case, however, one has to consider that, the hGH receptor being a homodimer, mutation of Asp-152 in the center of the interface between the two hGHbp molecules (13) simultaneously affects both receptor chains, which in our case would correspond to mutagenizing hIL-6R and hgp130 at the same time. Another important difference arises from the fact that hgp130, in contrast with homodimeric hGHbp, is able to interact with a variety of receptors such as leukemia inhibitory factor receptor and ciliary neurotrophic factor receptor and probably also with the specific OM and IL-11 receptor (6) . This suggests that the gp130 interface might be more flexible and able to establish several low specific contacts with its dimerizing partner receptor, rendering the dimerization interface tolerant to mutations. Finally, our model for the hIL-6hIL-6Rhgp130 complex is compatible with the results obtained in several mutagenesis studies on both hIL-6 and hIL-6R (12, 46, 47), but is probably incomplete given the observation that hgp130 undergoes a covalent dimerization as a final step in the formation of biologically active receptor complexes (48) . The possibility is thus open that hIL-6R might bind two distinct hgp130 chains via separate surfaces and that our mutagenesis has only identified one of them. This idea is in line with the identification by site-directed mutagenesis of two distinct sites on hIL-6 that are both responsible for the interaction with hgp130 (19, 49) . Further mutagenesis of hIL-6, hIL-6R, and hgp130 and the determination of the stoichiometry of the receptor complex in vitro for wild-type and mutant molecules will be required to clarify this important issue.

Disregulated production of hIL-6 has been proposed as playing a pathogenetic role in the development of multiple myeloma and autoimmune diseases (1, 3, 4, 5) . Generating hIL-6 antagonists is thus believed to be a potentially useful therapeutic approach. In this framework, the properties of Mut-4 open up new possibilities of blocking IL-6 activity. Initial attempts to counteract IL-6 in vivo both in murine models and in human clinical trials with the use of neutralizing monoclonal antibodies gave rise sometimes to enhanced IL-6 effects as a consequence of the decreased clearance of the cytokine complexed with antibodies (50, 51) . Although the mode of action of soluble IL-6R antagonists should be identical to that of monoclonal antibodies, it cannot be ruled out that IL-6soluble IL-6R complexes might be cleared from the body with kinetics faster than that of immune complexes.

Mut-4 partially antagonizes both hIL-6-induced early signaling, monitored as APRF activation (15-min assay), and biological activity, assessed as induction of haptoglobin production (20-h assay). However, antagonism was more pronounced in the short-term assay. Since we have measured different biological responses that are not known to be linked to each other, it is possible that the transcription of the haptoglobin gene is not strictly dependent on APRF activation. Alternatively, we cannot exclude that the lower efficiency of long-term inhibition is due to a slow release of hIL-6 from preformed complexes with Mut-4. The generation of shIL-6R mutants with a completely abolished binding to hgp130 and the analysis of their biological activity should demonstrate if the strategy outlined in this paper can be effectively used to fully inhibit hIL-6 in vivo.

  
Table: List of amino acid substitutions introduced in the context of predicted AB2 loop and E2 strand mutants (Mut-1 to Mut-8) and binding affinity of shIL-6R variants for hIL-6

Mutated residues are in boldface. The wild-type sequence is shown for comparison. For each mutant, where no change is indicated, the wild-type sequence is present. The apparent affinity for hIL-6 of each soluble receptor produced in COS-7 cells was determined as described under ``Experimental Procedures.'' The results shown are the means of at least two separate experiments.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 39-691093-204; Fax: 39-691093-654.

The abbreviations used are: IL-6, interleukin-6 (the prefix ``h'' represents human); IL-6R, IL-6 receptor (the prefixes ``h'' and ``s'' represent human and soluble, respectively); hgp130, human gp130; shgp130, soluble human gp130; hGH, human growth hormone; hGHbp, hGH-binding protein; CBD, cytokine-binding domain; OM, oncostatin M; PBS, phosphate-buffered saline; APRF, acute-phase response factor.

C. Toniatti, unpublished observations.


ACKNOWLEDGEMENTS

The enzyme-linked immunosorbent assay for the detection of haptoglobin in HepG2 culture medium was conceived and optimized by Dr. R. Laufer, whose help we gratefully acknowledge. We also thank Dr. A. Tramontano, Dr. M. Sollazzo, and J. Clench for critically reading the manuscript; P. Neuner for the synthesis of oligonucleotides; and M. Emili for graphical work.


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