©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Recombinant Extracellular Domain of Human Interleukin-10 Receptor (*)

Jimmy C. Tan , Serafina Braun , Hong Rong , Ruth DiGiacomo , Edward Dolphin , Samuel Baldwin , Satwant K. Narula , Paul J. Zavodny , Chuan-Chu Chou (§)

From the (1) From Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0530

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The extracellular region of the human interleukin-10 (hIL-10) receptor was expressed using a myeloma cell line and was purified to homogeneity by ligand-affinity chromatography. SDS-polyacrylamide gel electrophoresis analysis indicated that the soluble receptor is glycosylated and has an apparent molecular mass of 35,000-45,000. Under native conditions, soluble hIL-10 receptor was determined by gel filtration to be a monomeric protein. Soluble hIL-10 receptor was able to inhibit the binding of I-hIL-10 to the full-length receptor and was able to antagonize the effect of human IL-10 in cell proliferation and cytokine synthesis inhibition. The apparent dissociation constant (K ) of soluble hIL-10 receptor was determined to be 563 ± 59 pM, approximately 2- to 10-fold higher than that found on intact cells (Tan, J. C., Indelicato, S. R., Narula, S. K., Zavodny, P. J., and Chou, C.-C.(1993) J. Biol. Chem. 268, 21053-21059; Liu, [Abstract] Y., Wei, S. H.-Y., Ho, A. S.-Y., de Waal Malefyt, R., and Moore, K. W.(1994) J. Immunol. 152, 1821-1829). When hIL-10 binds [Abstract] soluble hIL-10 receptor in solution, a single complex was detected by gel filtration, and the complex was found to consist of two hIL-10 dimers and four soluble receptor monomers, suggesting that hIL-10 may induce a novel mode of oligomerization of the receptor upon binding.


INTRODUCTION

Interleukin 10 (IL-10)() is a pleiotropic cytokine secreted by B cells, T cells, and monocytic cells which exerts its effects on a variety of cell types (for reviews, see Refs. 3-6). It is, on one hand, immunosuppressive in its ability to inhibit the synthesis of Th1 T cell-derived cytokines, interferon-, IL-2, and tumor necrosis factor, which mediate the delayed-type hypersensitivity immune reactions (7) . It also inhibits the synthesis of IL-1, IL-6, IL-8, tumor necrosis factor, and IL-12 (7, 8) as well as the expression of co-stimulatory molecule B7 (9) in activated peripheral blood mononuclear cells (PBMC). On the other hand, IL-10 possesses immunostimulatory activity for the growth and differentiation of activated B cells (10, 11) , cytokine-activated T cells (12, 13) , and mast cells (14) . Furthermore, IL-10 is produced mainly by Ly-1 (B1) B cells (15) and is apparently an autocrine growth factor for malignant B1 cells (16) . Expression of hIL-10 is correlated with Epstein Barr virus-induced transformation of B cells (17, 18) . Given these biological effects of IL-10, there appears to be some conditions where antagonists to IL-10 may be desirable. Such antagonists may be useful in infections where IL-10 is involved in inhibiting macrophage-mediated immunity or in modulating B-cell transformation and malignancy (5) .

Human and mouse IL-10 receptors have been biochemically characterized (1, 2). Recently, the cDNAs of mouse and human IL-10 receptors were cloned (2, 19) . Chemical cross-linking studies indicate that radiolabeled hIL-10 is able to bind to its cognate membrane-bound receptor and form complexes (1, 2, 19) , the composition of which remains unresolved. In order to investigate further the binding characteristics of the receptor as well as develop a potential antagonist to hIL-10, we expressed and purified the extracellular region of the cloned human IL-10 receptor. We show that this soluble receptor is able to bind hIL-10 and inhibit its biological activity. From analysis of its binding, we were able to derive the stoichiometry of the ligand-receptor interaction.


MATERIALS AND METHODS

Tissue Culture

All tissue culture reagents, unless indicated otherwise, were purchased from Life Technologies, Inc. NSO (20) was maintained in Iscove's modification of Dulbecco's modified Eagle's medium (Sigma) supplemented with 2 mM glutamine, 10% fetal bovine serum, nonessential amino acids, and penicillin/streptomycin. Human peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Hypaque (Pharmacia Biotech Inc.) according to the recommended protocol and were resuspended in assay medium (RPMI 1640, 50 units of penicillin, and 50 µg of streptomycin/ml, 110 µg/ml sodium pyruvate, 0.1 mM nonessential amino acids, 5% fetal bovine serum) for cytokine synthesis inhibition assays (see below).

Antiserum

Two milligrams of the P55 22-mer peptide (RPKMAPANDTYESIFSHFREYE), corresponding to amino acids 147 to 168 of the hIL-10 receptor (2) , were provided by R. Zhang (Schering-Plough Research Institute) and used to raise rabbit antiserum by custom immunization at HRP Inc. (Denver, PA). Serum samples were tested for the presence of reactive antibodies by enzyme-linked immunosorbent assays. The antiserum was able to specifically detect the antigen peptide at dilutions as much as 1:200,000 (data not shown).

cDNA Cloning and Stable Expression

All enzymes were purchased from Boehringer Mannheim and used according to the manufacturer's instructions. Oligonucleotides used as polymerase chain reaction primers were synthesized according to the reported sequence of human IL-10 receptor cDNA (Ref. 2, GenBank data base, Accession No. U00672) using the ABI 394 DNA Synthesizer (Applied Biosystems). Primer C3628CC, 5`GCAGCGAATTCGTCGACGCCGCCACCATGCTGCCGTGCCTCGTAGTGCT3`, corresponding to the 5` end of the sense strand (nucleotides 62 to 84), contains an EcoRI and a SalI site 5` of the translation initiation codon (italicized). Primer C3629CC, 5`CACTCT-CCCTCACCGGTACCCATTGCTGTGGTACAGGTCCAAGGTC3`, corresponding to the reverse strand (nucleotides 320 to 352 of the sense strand), contains a conservative base change as underlined to create a KpnI site. Primer C3630CC, 5`GTACCACAGCAATGGGTACCGGGCCAGAGTGCGGGCTGTGGAC3`, corresponding to the sense strand (nucleotides 331 to 373), is approximately 300 bases 3` of the initiation codon and contains a conservative base change as underlined to create a KpnI site. Primer C3631CC, 5`GCTTCAGTAGCTGGATCCGAATTCTCAGTTGGTCACCGTGAAATACTGCCTGGTGAGGGAGATGC3`, corresponding to the reverse strand (nucleotides 668 to 767 of the sense strand) and the juxtamembrane region, contains a complementary stop codon (italicized), a conservative base change to create a BstEII site (underlined), and EcoRI and a BamHI sites 5` of the complementary stop codon.

Using pSW8.1, a human IL-10 receptor cDNA clone (2) as the template, polymerase chain reactions were carried out with primers C3628CC and C3629CC to produce an approximately 300-base pair fragment which corresponds to the amino-terminal peptide, and with primers C3630CC and C3631CC to produce an approximately 440-base pair fragment which corresponds to the membrane proximal half of the extracellular region. The two fragments were cloned as EcoRI-KpnI and KpnI-BamHI fragments, respectively, into pUC19 (New England Biolabs) in succession and sequenced. The IL-10 receptor extracellular domain cDNA was expressed as an EcoRI fragment using glutamine synthetase as a selectable marker in myeloma cells (21). Clones surviving selection were screened for expression of the protein by ligand dot-blotting. Briefly, 50- to 100-µl aliquots of conditioned media from viable clones were applied onto nitrocellulose filter paper using a multichamber vacuum manifold (Bio-Rad). The filter paper was then blocked with 5% non-fat dry milk and 2% bovine serum albumin in PBS for 2 h to overnight, incubated with Sephadex G-75 column fractionated I-hIL-10 (1) at 2 nM for 2 h at room temperature, washed three times with PBS, and exposed to Hyperfilm (Amersham) overnight. The intensity of the signal from the supernatant of clones correlated with inhibitory ability in receptor binding assays (data not shown). The 10 highest expressing clones were expanded into low serum (1%) medium, and conditioned medium was harvested after culturing for 7 days.

Purification of Soluble hIL-10 Receptor

A hIL-10 ligand-affinity column was prepared by cross-linking CHO-derived hIL-10 (Schering-Plough) with Affi-Gel active ester agarose (Bio-Rad) following the recommended procedure. About 7.5 ml of the agarose resin was packed in a 10-cm column (Bio-Rad) and equilibrated with phosphate-buffered saline (PBS). Conditioned medium of soluble hIL-10 receptor-producing NSO cells was passed through the column, followed by consecutive washes with 4 10 ml of PBS, 6 10 ml of 0.05% octyl glucoside in PBS, 4 10 ml of phosphate-buffered 0.5 M NaCl, and another 4 10 ml of PBS. The soluble hIL-10 receptor protein was eluted with 4 10 ml of 2 M MgCl at pH 7.5. Aliquots of the eluate fractions were analyzed with a 15% SDS-PAGE gel and silver-stained (see below). By this method, the first two fractions were identified to have greater than 90% of total eluted protein. These fractions were pooled together, buffer-exchanged into (PBS) using PD-10 columns (Pharmacia Biotech Inc.), equilibrated with PBS, and concentrated using Centricon-10 (Amicon, Inc.). The concentration of the purified protein was determined with the BCA kit (Pierce) or by amino acid composition analysis (see below).

Deglycosylation

Deglycosylation of the purified receptor was carried out by incubating the soluble receptor with endoglycanase F (PNGase F, Boehringer Mannheim) under the manufacturer's recommended conditions. Briefly, 20 µg of the soluble receptor was boiled for 10 min in the presence of 1% SDS. After bringing the sample to room temperature, it was diluted with PBS to an SDS content of 0.1%. After addition of n-octyl glucoside to 1%, the sample was treated with endoglycanase F (2 units) at 37 °C overnight. An aliquot of the deglycosylated soluble receptor was analyzed along with untreated protein and the enzyme alone by 10-20% SDS-PAGE followed by silver staining or Western blotting. For Western blotting, the SDS-gel was blotted at 150 mA for 1 h onto nitrocellulose (Schleicher and Schuell) using a semidry electroblotter (ISS Inc., Natick, MA). The blot was then blocked in 2% bovine serum albumin in PBS overnight, followed by a 2-h incubation with the anti-P55 antiserum diluted 1:200 in TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20). The blot was then washed with TBST and developed with horseradish peroxidase-conjugated goat anti-rabbit IgG (Boehringer Mannheim) and TMB Microwell peroxidase substrate (Kirkegaard & Perry Laboratories).

Chemical Cross-linking

Purified soluble hIL-10 receptor at indicated concentrations was incubated with 1 nM radioiodinated hIL-10 (1) in PBS and bovine serum albumin (0.1 mg/ml) at 4 °C for 2 h. Bifunctional cross-linker bis(sulfosuccinimidyl)suberate (BS) (Pierce) was added to a final concentration of 0.5 mM. The mixture was incubated at room temperature for 1 h. The reaction was terminated by addition of Tris-HCl (pH 7.5) to 50 mM. The cross-linked proteins were analyzed by SDS-PAGE followed by autoradiography.

Biological Assays

Ba8.1 cell proliferation assays were performed based on published procedures (2) . 15,000 cells per well were seeded in 100 µl final volume assay media (RPMI 1640 with 10% fetal calf serum, 2 mM glutamine, and 50 µM 2-mercaptoethanol) in 96-well tissue culture plates for 48 h with CHO-derived human IL-10 at 100 pM, previously determined to be the concentration of hIL-10 for half-maximal stimulation of Ba8.1 cells, and with 2-fold serial dilutions of purified soluble hIL-10 receptor starting at 400 nM. Proliferation was measured by MTS reduction using the CellTiter 96 aqueous assay system (Promega).

For cytokine synthesis inhibition assays, the assay medium consisted of RPMI 1640, 50 units/ml penicillin, 50 µg/ml streptomycin, 110 µg/ml sodium pyruvate, 0.1 mM non-essential amino acids, 5% fetal bovine serum, 80 ng/ml lipopolysaccharide (Sigma), and 10 pM hIL-10. The amount of hIL-10 was previously determined to be required for half-maximal inhibition of hIL-1 synthesis. The assay medium was added to 96-well tissue culture plates along with 10-fold serial dilutions of soluble hIL-10 receptor with a highest final concentration at 1 µM. 2 10 PBMC were then added per well in a final total volume of 200 µl. After a 6-h incubation at 37 °C, the level of hIL-1 was measured using the Quantikine System (R & D Systems, Minneapolis, MN) on the conditioned media diluted to 1:100.

Receptor Binding Assay and Scatchard Analysis

Receptor binding assays were performed as described (1) except that Ba8.1 cells (2) were used. Cells at a density of 1 10 cells per well in a 96-well v-bottom Microwell plate (Nunc, Roskilde, Denmark) were incubated with 250 pMI-hIL-10 in binding buffer (PBS, 0.1% NaN, 10% fetal bovine serum) in the presence of 2-fold serially diluted soluble hIL-10 receptor starting at 700 nM. After a 2-h incubation at 4 °C, the cells were pelleted by centrifugation at 200 g for 5 min, resuspended in 100 µl of binding buffer, layered over 200 µl of separation buffer (binding buffer with 10% glycerol) in Bio-Rad micro test tubes (Bio-Rad), and centrifuged at 300 g for 5 min. The tubes were quick-frozen in liquid nitrogen, and the cell pellets were clipped and counted in a CliniGamma counter (Pharmacia Biotech Inc.).

For equilibrium binding assays, 5 µg/ml affinity-purified soluble hIL-10 receptor in PBS was immobilized on a 96-well Nunc Maxisorp Plate (Laboratory Disposable Products, North Haledon, NJ). Two-fold serially diluted radioiodinated hIL-10 was added at a starting concentration of 8 nM in 100 µl final volume and incubated for 2 h. The wells were then washed twice with 200 µl of PBS. Labeled hIL-10 bound to the receptor was eluted with 200 µl of PBS containing 1% SDS and counted in the CliniGamma counter. Background binding was performed in parallel using the labeled protein with a 1000-fold molar excess of unlabeled hIL-10. The results were then plotted as specific counts per min bound as a function of increasing concentrations of iodinated hIL-10. Scatchard analysis was carried out by linear regression with the program EBDA (Elsevier-Biosoft, Cambridge, UK).

Structural Analysis of hIL-10Soluble Receptor Complex

Quantitation of protein was carried out by amino acid composition analysis. After gas-phase hydrolysis for 1 h at 150 °C using 6 N HCl, the samples were analyzed using a Hewlett-Packard 1090A HPLC equipped with a Chemstation. For gel filtration, human IL-10, soluble receptor, or the mixtures of the two were incubated in PBS at 25 °C for up to 90 min. The products were analyzed on a Waters (Milford, MA) 625 liquid chromatography system equipped with two connected Superose 12HR (10 300) columns (Pharmacia, Uppsala, Sweden) pre-equilibrated in 10 mM sodium phosphate buffer, pH 6.9, and 150 mM sodium chloride with a flow rate of 0.32 ml/min. The system was precalibrated with the Pharmacia gel filtration calibration kits. Eluted proteins were detected by UV absorption at 214 nm wavelength, and their molecular masses were calculated based on elution time and UV absorption using the Cricket graph software (Computer Associates International Inc., San Jose, CA). Semiquantitative amino-terminal sequencing was performed for 5 cycles using an Applied Biosystem 477A pulsed-liquid protein Sequencer.


RESULTS

Soluble hIL-10 Receptor Can Be Purified by Ligand Affinity Chromatography

As shown in Fig. 1A, ligand-affinity column purified soluble hIL-10 receptor was detected as a group of three species migrating with apparent molecular masses between 35,000 and 45,000, as analyzed by SDS-PAGE. Upon N-glycanase treatment, the receptor protein species were digested into a single one of 24,000 molecular mass, corresponding to the predicted molecular mass of the primary protein product. Both the glycosylated and deglycosylated forms were detectable by the anti-P55 antiserum (Fig. 1B), but not by the preimmune serum (data not shown), confirming the identity of the expressed protein. The deglycosylated form is recognized more strongly by the antiserum, probably reflecting the increased accessibility of the antiserum to soluble hIL-10 receptor in the absence of the carbohydrate side chains. Amino-terminal sequencing of the purified protein with 30 cycles of Edman degradation revealed a single protein sequence which was identical with the first 30 amino acids predicted for the mature polypeptide of hIL-10 receptor (data not shown, Ref. 2).


Figure 1: SDS-PAGE and Western blot of recombinant soluble hIL-10 receptor. Two sets of samples were each loaded on a separate 10-20% SDS-polyacrylamide gel in the following order: lane 1, 2 µg of untreated soluble hIL-10 receptor; lane 2, 2 µg of endoglycanase F-treated soluble hIL-10 receptor; lane 3, endoglycanase F alone (0.2 unit). Electrophoresis was carried out under reducing conditions. After electrophoresis, one gel was silver-stained as shown in A, and the other was immunoblotted and detected with anti-soluble hIL-10 receptor peptide antiserum as shown in B.



Purified Soluble hIL-10 Receptor Can Inhibit Binding of hIL-10 to the hIL-10 Receptor

When the soluble hIL-10 receptor was tested for its ability to inhibit the binding of I-hIL-10 to cellular receptor, a dose-dependent inhibition was observed (Fig. 2). Using 250 pMI-hIL-10, the half-maximal (50%) inhibitory concentration of soluble hIL-10 receptor was estimated to be 5.5 nM, approximately at a 22-fold molar excess with respect to the amount of hIL-10 present.


Figure 2: Inhibition of I-hIL-10 binding to Ba8.1 cells by soluble hIL-10 receptor. Ba8.1 cells were incubated with 250 pMI-hIL-10 in the presence of soluble hIL-10 receptor of increasing concentrations.



Purified Soluble hIL-10 Receptor Can Inhibit the Biological Activities of hIL-10

Fig. 3 shows that the purified soluble hIL-10 receptor protein can inhibit the ability of hIL-10 to induce the proliferation of Ba8.1 cells in a dose-dependent manner. The half-maximal (50%) inhibitory concentration of the soluble hIL-10 receptor was estimated to be 15 nM, about a 150-fold molar excess relative to the concentration (100 pM) of hIL-10 used. Repetition of this assay showed that a 100- to 200-fold molar excess of soluble receptor is needed for half-maximal inhibition.


Figure 3: Inhibition of hIL-10-induced proliferation by soluble receptor. Increasing amounts of soluble hIL-10 receptor were added to Ba8.1 cells in the presence of 100 pM hIL-10, and cell proliferation was assayed after 48 h with the MTS (Promega) system.



hIL-10 can inhibit the synthesis of a number of cytokines in a variety of cell types (7, 22, 23, 24) . Fig. 4 shows that the purified soluble hIL-10 receptor is able to overcome the IL-1 synthesis inhibitory activity of hIL-10 on activated mononuclear cells in a dose-responsive manner (Fig. 4). The half-maximal inhibitory dose of soluble hIL-10 receptor is around 2 nM, about a 200-fold molar excess relative to the 10 pM hIL-10 used.


Figure 4: Inhibition of the cytokine synthesis inhibitory activity of hIL-10 on human PBMC. Increasing amounts of soluble hIL-10 receptor were added to lipopolysaccharide-stimulated peripheral blood mononuclear cells (PBMCs) along with 10 pM hIL-10. After a 6-h incubation, the levels of hIL-1 were assayed using the Quantikine enzyme-linked immunosorbent assay kit.



Human IL-10 Binds to Immobilized Soluble hIL-10 Receptor

In order to determine the affinity of the soluble receptor for the radioactive ligand, saturation binding was carried out with the fractionated ligand on purified soluble hIL-10 receptor directly immobilized on a microtiter plate. The recombinant receptor bound radiolabeled hIL-10 with a K value estimated to be 563 ± 59 pM averaged from four experiments (536 pM, 473 pM, 660 pM, and 583 pM). In comparison, the K of membrane-bound receptor was measured to be 50-200 pM in the cell lines JY and MC/9 (1) and 200-250 pM in the hIL-10 receptor cDNA-transfected line Ba/F3 (2) .

Human IL-10 Forms a Multimeric Complex with Soluble Human IL-10 Receptor

Chemical cross-linking was first carried out to characterize the ligand-receptor association. Radioiodinated hIL-10 at a fixed concentration of 1 nM was incubated in the absence or presence of soluble hIL-10 receptor at increasing concentrations from 0.1 nM to 40,000 nM. BS chemical cross-linker was then added to stabilize the complexes. SDS-PAGE of the reaction mixtures followed by autoradiography revealed two major complex species (Fig. 5), one with an approximate molecular mass of 85,000 and the other slightly higher than 200,000 relative to the molecular mass standards. The high molecular mass complex was readily detectable when the molar ratio of soluble receptor to hIL-10 exceeded 10:1 (Fig. 5). Two intermediate complex species were also detected, but the signals appeared to be very weak. No complexes were detected in the absence of either the soluble receptor or the cross-linker, nor were they detected when an excess amount of unlabeled hIL-10 was added (Fig. 5).


Figure 5: Chemical cross-linking of I-hIL-10 to soluble hIL-10 receptor. Different amounts of the soluble hIL-10 receptor (SR) were incubated with a constant amount of I-hIL-10 at 1 nM, in the presence or absence of unlabeled hIL-10, as indicated. Cross-linking was performed with BS, and the samples were analyzed by SDS under reducing conditions followed by autoradiography.



To further investigate the sizes of the ligand-receptor complexes formed, hIL-10, soluble receptor, or mixtures of the two were analyzed by gel filtration chromatography. The molecular masses of the proteins were estimated based on elution time relative to those of the molecular mass standards. Human IL-10 was eluted as a 33,000 molecular mass protein (Fig. 6A), consistent with previous observations that hIL-10 forms a noncovalently linked dimer under native conditions (1, 2) . Similar to its observed molecular mass in SDS-PAGE under denaturing conditions (Fig. 1), soluble hIL-10 receptor was eluted with an estimated mass of 48,000 (Fig. 6B), indicating that native soluble hIL-10 receptor exists as a monomeric protein in solution. When hIL-10 dimer and soluble hIL-10 receptor were mixed at a molar ratio of 2:1, a complex was detected with an approximate molecular mass of 266,000 (Fig. 6C), along with another peak corresponding in mass to hIL-10 dimer. A ratio of soluble receptor to hIL-10 of 1.375 to 1 converted all the free hIL-10 dimer into the complexed form (Fig. 6D). At this ratio, the concentration of free soluble receptor is apparently not large enough to form a peak.


Figure 6: Gel filtration chromatography analysis of complex formation between hIL-10 and soluble hIL-10 receptor. A, ligand alone, 1.68 nmol; B, soluble receptor alone, 0.42 nmol; C, ligand and soluble receptor, 1.68 pmol and 0.84 pmol, respectively; D, ligand and soluble receptor, 1.68 pmol and 2.31 pmol, respectively, were incubated at room temperature for 30 min. Chromatography was performed with two connected Superose 12HR columns, as described under ``Materials and Methods.'' The system was calibrated with the Pharmacia gel filtration calibration kits which include thyroglobulin (T, 669,000), ferritin (F, 440,000), catalase (C, 232,000), aldolase (A, 158,000), bovine serum albumin (B, 67,000), ovalbumin (O, 43,000), and ribonuclease A (R, 13, 700).



Following the gel filtration, the stoichiometry of the components in the 266,000 complex was determined by semi-quantitative amino-terminal sequencing. Two sequences were observed () with an apparent molar ratio of 1:1. The higher ratio of amino acids in the first cycle is probably a result of poor recovery of histidine, which is commonly seen with Edman degradation. Based on the 1:1 ratio of hIL-10 monomer to the soluble receptor, the complex (266, 0) apparently contains two hIL-10 dimers (2 33,000) and four soluble receptor monomers (4 48,000).


DISCUSSION

In this report, we show that the extracellular region of human IL-10 receptor cDNA can be expressed in soluble form and purified by ligand affinity. The purified protein is able to bind hIL-10 and prevent hIL-10 from binding to the full-length cellular receptor. The soluble hIL-10 receptor is also able to inhibit the in vitro biological activity of hIL-10 such as growth stimulation of Ba8.1 or MC/9 cells (data not shown) and cytokine synthesis inhibition in activated monocytes. This biological antagonism of the soluble receptor is similar to that reported with the soluble IL-4 receptor (25) , the soluble IL-7 receptor (26) , the soluble erythropoietin receptor (27) , the soluble tumor necrosis factor receptor (28) , and the soluble interferon- receptor (29) in contrast to the augmentation of activity demonstrated by other soluble cytokine receptors such as the soluble IL-6 receptor (30) and the soluble ciliary neurotrophic factor receptor (31) . Under other conditions, the soluble IL-4 receptor (32) and the soluble tumor necrosis factor receptor (33) act as carrier protein agonists of their ligands as well.

While a 22-fold molar excess of soluble hIL-10 receptor relative to the I-hIL-10 was able to achieve a 50% inhibition of labeled ligand binding to Ba8.1 cells, a 100- to 200-fold molar excess of soluble hIL-10 receptor was required to achieve 50% inhibition of cell proliferation in response to hIL-10. A similar level of soluble receptor is required to neutralize the activity of hIL-10 on PBMC. This difference in inhibitory activity of the soluble hIL-10 receptor between receptor binding and cell response is not due to the radioiodination of the ligand in the receptor binding assay since our labeled hIL-10 retained greater than 50% of its biological activity (1). One explanation is that a lower level of hIL-10 is needed to elicit biological response as compared to the levels required for proportional receptor occupancy; i.e. occupancy of only a fraction of the existing receptors is sufficient for a full biological response.

Cross-linking reactions by bifunctional cross-linker BS appeared to be very efficient, since the majority of I-hIL-10 protein can cross-link into dimers in the absence or in the presence of the soluble receptor (Fig. 5). At a receptor/ligand molar ratio of 1:1, a complex of one hIL-10 dimer-one soluble receptor was formed. When the soluble receptor was added at 10 nM or higher relative to the fixed amount (1 nM) of the ligand, a higher molecular mass complex was detected (Fig. 5) which was later revealed by gel filtration to be the stable form of hIL-10soluble receptor complex under native conditions (Fig. 6). Amino-terminal sequencing of the column-purified complex further indicated a stoichiometry of two hIL-10 dimers and four soluble receptor monomers within this octameric complex ().

The ligand-induced tetramerization of the soluble receptor reported here is the first indication that the human IL-10 receptor is able to form higher order complexes upon ligand binding. The tetramer-dimer receptor-ligand complex is virtually the only complex seen by gel filtration, suggesting that receptor tetramerization may be a likely sequela of ligand binding with the intact receptor as well. It should be noted that in previous cross-linking studies with natural or recombinant cell lines the predominant bands observed are those only of monomeric receptors (1, 2) , although this may be attributed to inefficiency of cross-linking.

The interleukin-10 receptor, together with interferon receptors and tissue factor receptors, is classified as a Type II cytokine receptor family member based on structural homology (34) . Similar to soluble hIL-10 receptor, the extracellular domain of IFN- receptor exists alone in a monomeric form and oligomerizes when the cognate ligand is present (35, 36) . In addition, ligand-induced dimerization of the cellular IFN- receptor has been demonstrated under physiological conditions (35) . Whether cellular hIL-10 receptors also undergo ligand-induced oligomerization remains to be investigated. Ligand-induced oligomerization was initially found to be crucial for the signal transduction of tyrosine kinase activity-associated cytokine receptors (37, 38) . Later evidence suggested that a growing number of Type I cytokine receptors, as well as other Type II cytokine receptors, also oligomerize in response to ligand stimulation to transduce signals (39). For example, Ward et al.(40) demonstrated that IL-6, soluble IL-6 receptor, and soluble gp130 can form a ternary complex of 2 molecules of each unit.

For some cytokines, the affinities of their soluble receptors are comparable to that observed with the intact receptor, as exemplified by the IL-4 receptor (26) , the IL-7 receptor (27) , and the IL-5 receptor (41). Our plate binding results here show that the soluble receptor binds hIL-10 with a K of 563 ± 59 pM, a lower affinity than that reported using cells, 50-250 pM(1, 2, 19) . The reduced affinity may be a consequence of the inability of the immobilized soluble hIL-10 receptor to oligomerize in response to ligand treatment. It is possible that the tetramerization of receptor with two dimeric ligands contributes to increased affinity. The increased affinity of binding in intact cells may also reflect the presence of other proteins which, in association with the cloned receptor, binds with a greater affinity than with the cloned soluble receptor alone. Finally, this difference observed between the purified soluble receptor and intact receptor in cells may reflect structural alteration due to truncation of the receptor or its immobilization on a plastic surface.

  
Table: Amino-terminal sequencing of the complex of hIL-10 and soluble hIL-10 receptor (SR)

HPLC-purified protein complex was subjected to amino-terminal sequencing as described under ``Materials and Methods.'' Data were collected from five independent experiments. The ratio of moles of amino acids in each cycle is represented by mean ± S.D.



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 and reprint requests should be addressed: Dept. of Immunology, Schering-Plough Research Institute, Kenilworth, NJ 07033-0530. Tel: 908-298-3064; Fax: 908-298-3083.

The abbreviations used are: IL-10, interleukin-10; hIL-10, human IL-10; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; BS, bis(sulfosuccinimidyl)suberate.


ACKNOWLEDGEMENTS

We thank Kevin Moore for providing hIL-10 receptor cDNA and Ba8.1 cell line. We also thank Rumin Zhang for preparing the hIL-10 receptor peptide.


REFERENCES
  1. Tan, J. C., Indelicato, S. R., Narula, S. K., Zavodny, P. J., and Chou, C.-C.(1993) J. Biol. Chem. 268, 21053-21059
  2. Liu, Y., Wei, S. H.-Y., Ho, A. S.-Y., de Waal Malefyt, R., and Moore, K. W.(1994) J. Immunol. 152, 1821-1829
  3. Moore, K. W., O'Garra, A., de Waal Malefyt, R., Vieira, P., and Mosmann, T. R.(1993) Annu. Rev. Immunol. 11, 165-190 [CrossRef][Medline] [Order article via Infotrieve]
  4. Howard, M., and O'Garra, A.(1992) Immunol. Today 13, 198-200 [CrossRef][Medline] [Order article via Infotrieve]
  5. Howard, M., O'Garra, A., Ishida, H., de Waal Malefyt, R., and de Vries, J.(1992) J. Clin. Immunol. 12, 239-247 [Medline] [Order article via Infotrieve]
  6. Mosmann, T. R., and Moore, K. W.(1991) Immunol. Today 12, A49-A53 [CrossRef]
  7. de Waal Malefyt, R., Abrams, J., Bennett, B., Figdor, C. G., and de Vries, J. E.(1991) J. Exp. Med. 174, 1209-1220 [Abstract]
  8. D'Andrea, A., Aste-Amezaga, M., Valiante, N. M., Ma, X., Kubin, M., and Trinchieri, G.(1993) J. Exp. Med. 178, 1041-1048 [Abstract]
  9. Ding, L., Linsley, P. S., Huang, L. Y., Germain, R. N., and Shevach, E. M.(1993) J. Immunol. 151, 1224-1234 [Abstract/Free Full Text]
  10. Rousset, F., Garcia, E., Defrance, T., Peronne, C., Hsu, D.-H., Kastelein, R., Moore, K. W., and Banchereau, J.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1890-1893 [Abstract]
  11. Defrance, T., Vanbervliet, B., Briere F., Durand, I., Rousset, F., and Banchereau, J.(1992) J. Exp. Med. 175, 671-682 [Abstract]
  12. Suda, T., O'Garra, A., MacNeil, I., Fischer, M., Bond, M., and Zlotnik, A.(1990) Cell Immunol. 129, 228-240 [Medline] [Order article via Infotrieve]
  13. MacNeil, I., Suda, T., Moore, K. W., Mosmann, T. R., and Zlotnik, A. (1990) J. Immunol. 145, 4167-4173 [Abstract/Free Full Text]
  14. Thompson-Snipes, L., Dhar, V., Bond, M. W., Mosmann, T. R., Moore, K. W., and Rennick, D.(1991) J. Exp. Med. 173, 507-510 [Abstract]
  15. O'Garra, A., Chang, R., Go, N., Mastings, R., Haughton, G., and Howard, M.(1992) Eur. J. Immunol. 22, 711-717 [Medline] [Order article via Infotrieve]
  16. Peng, B., Mehta, N., Fernandes, H., Chou, C.-C., and Raveche, E.(1995) Leukemia Res. 19, in press
  17. Burdin, N., Peronne, C., Banchereau, J., and Rousset, F.(1993) J. Exp. Med. 177, 295-304 [Abstract]
  18. Baiocchi, R. A., Ross, M. E., Tan, J. C., Chou, C.-C., Sullivan, L., Haldar, H., Monne, M., Seiden, M. V., Narula, S. K., Sklar, J., Croce, C. M., and Caligiuri, M. A.(1995) Blood 84, 1063-1074
  19. Ho, A. S.-Y., Liu, Y., Khan, T., Hsu, D.-W., Bazan, J. F., and Moore, K. W.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11267-11271 [Abstract]
  20. Galfre, G., and Milstein, C.(1981) Methods Enzymol. 73(B), 3-46 [Medline] [Order article via Infotrieve]
  21. Bebbington C. R., Renner, G., Thomson, S., King, D., Abrams, D., and Yarranton, G. T.(1992) Bio/Technology 10, 169-175 [Medline] [Order article via Infotrieve]
  22. Wang, P., Wu, P., Anthes, J. C., Sigel, M. I., Egan, R. W., and Billah, M. M.(1994) Blood 83, 2678-2683 [Abstract/Free Full Text]
  23. Fiorentino, D. F., Zlotnik, A., Mosmann, T. R., Howard, M., and O'Garra, A.(1991) J. Immunol. 147, 3815-3822 [Abstract/Free Full Text]
  24. Bogdan, C., Vodovotz, Y., and Nathan, C.(1991) J. Exp. Med. 174, 1549-1555 [Abstract]
  25. Mosley, B., Beckmann, M. P., March, C. J., Idzerda, R. L., Gimpel, S. D., VandenBos, T., Friend, D., Alpert, A., Anderson, D., Jackson, J., Wignall, J. M., Smith, C., Gallis, B., Sims, J. E., Urdal, D., Widmer, M. B., Cosman, D., and Park, L.(1989) Cell 59, 335-348 [Medline] [Order article via Infotrieve]
  26. Goodwin, R. G., Friend, D., Ziegler, S. F., Jerzy, R., Falk, B. A., Gimpel, S., Cosman, D., Cower, S. K., March, C. J., Namen, A. E., and Park, L. S.(1990) Cell 60, 941-951 [Medline] [Order article via Infotrieve]
  27. Yet, M.-G., and Jones, S. S.(1993) Blood 82, 1713-1719 [Abstract]
  28. Schall, T. J., Lewis, M., Koller, K. J., Lee, A., Rice, G. C., Wong, G. H. W., Gatanaga, T., Granger, G. A., Lentz, R., Raab, H., Kohr, W. J., and Goeddel, D.(1990) Cell 61, 361-370 [Medline] [Order article via Infotrieve]
  29. Ozmen, L., Gribaudo, G., Fountoulakis, M., Gentz, R., Landolfo, S., and Garotta, G.(1993) J. Immunol. 150, 2698-2705 [Abstract/Free Full Text]
  30. Akira, S., Hirano, T., Taga, T., and Kishimoto, T.(1990) FASEB J. 4, 2860-2867 [Abstract]
  31. Davis, S., Aldrich, T. H., Ip, N. Y., Stahl, N., Scherer, S., Farruggella, T., DiStefano, P. S., Curtis, R., Panayotatos, N., Gascan, H., Chevalier, S., and Yancopoulos, G. D.(1993) Science 259, 1736-1739 [Medline] [Order article via Infotrieve]
  32. Fernandez-Botran, R., and Vitetta, E. S.(1991) J. Exp. Med. 174, 673-681 [Abstract]
  33. Aderka, D., Engelmann, H., Maor, Y., Brakebush, C., and Wallach, D. (1992) J. Exp. Med. 175, 323-329 [Abstract]
  34. Bazan, J. F.(1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6934-6938 [Abstract]
  35. Fountoulakis, M., Zulauf, M., Lustig, A., and Garotta, G.(1992) Eur. J. Biochem. 208, 781-787 [Abstract]
  36. Greenlund, A. C., Schreiber, R. D., Goeddel, D. V., and Pennica, D. (1993) J. Biol. Chem. 268, 18103-18110 [Abstract/Free Full Text]
  37. Heldin C.-H., Ernlund, A., Rorsman, C., and Ronnstrand, L.(1989) J. Biol. Chem. 264, 8905-8912 [Abstract/Free Full Text]
  38. Lev, S., Yarden, Y., and Givol, D.(1992) J. Biol. Chem. 267, 15970-15977 [Abstract/Free Full Text]
  39. Heldin, C.-H.(1995) Cell 80, 213-223 [Medline] [Order article via Infotrieve]
  40. Ward, L., Howlett, G. J., Discolo, G., Yasukawa, K., Hammacher, A., Moritz, R. L., and Simpson, R. J.(1994) J. Biol. Chem. 269, 23286-23289 [Abstract/Free Full Text]
  41. Devos, R., Guisez, Y., Cornelis, S., Verhee, A., Van der Heyden, J., Manneberg, M., Lahm, H.-W., Fiers, W., Tavernier, J., and Plaetinck, G.(1993) J. Biol. Chem. 268, 6581-6587 [Abstract/Free Full Text]

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