From the
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
Interleukin 10 (IL-10)
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.
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
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
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).
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-
While a 22-fold molar excess of soluble hIL-10 receptor relative to
the
Cross-linking reactions by bifunctional cross-linker
BS
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-
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
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.
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)
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) .
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.
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).
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 pM
I-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.).
Structural Analysis of hIL-10
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 Soluble Receptor
Complex
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.
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 pM
I-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 pM
I-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).
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.
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.
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-10
soluble 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 ().
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.
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)
, bis(sulfosuccinimidyl)suberate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.