From the
Interleukin (IL)-13 elicits a subset of the biological
activities of the related IL-4. The basis of this functional similarity
is that their specific cell-surface receptors (called IL-13R and IL-4R)
are distinct, yet are complex and share a common subunit(s). The IL-4R
primary binding subunit (called IL-4R
Adjacent genes encode the T cell-derived cytokines
IL
In this
work, we have further studied the ability of IL-13 to cross-compete for
IL-4 binding on human and monkey cell types and utilized for the first
time direct IL-13 binding analyses to characterize human IL-13R
properties. In addition, we have used anti-human IL-4R
In this work, we have further studied the structures and
relatedness of IL-4R and IL-13R. When probed with hIL-4 and the
antagonist hIL-4.Y124D, the properties of IL-4R on various human cell
types were remarkably similar. In all cases, hIL-4.Y124D bound to IL-4R
In contrast to the
homogeneity of hIL-4R that we observed on various cell types probed
with hIL-4 or hIL-4.Y124D, the ability of IL-13 to compete for hIL-4
binding to these same cells varied widely, indicating that this is not
a general property of IL-4R. The simplest notion accounting for the
above data is the existence of two IL-4R subtypes, only one of which
binds IL-13. This concept of two IL-4R subtypes was supported by our
observations of IL-13R using radiolabeled mIL-13 as a probe. IL-13R
were detected only on those cell types on which IL-13 effectively
competed for IL-4 binding. Importantly, IL-13 binding to IL-13R was
always fully competed for by hIL-4. These data suggest a model where
one type of IL-4R, the complex of IL-4R
Two studies
permitted us to define the composition of IL-13R. First, a
cross-linking study revealed a
Our characterization of IL-13R accounts for a number of
biological and receptor binding observations. Biological studies
(reviewed in Zurawski and de Vries(1994)) have revealed that IL-13
elicits only a subset of the responses of IL-4. This occurs since IL-4R
(as IL-4R
It is possible that IL-13R contains IL-2R
The anti-hIL-4R
We thank Felix Vega, Jr., for purification of
proteins, Nithya Rajan and Shane Tareni (S-P Research for providing
mAbs, Rene de Waal Malefyt for providing monocytes, Gregorio Aversa for
the Jy cell line, Sem Saeland for the NALM6 cell line, and Jean-Pierre
Galizzi and Smina Ait-Yahia for their contributions to the generation
of anti-IL-4R
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) does not by itself bind
IL-13. We show that the ability of IL-13 to partially compete for IL-4
binding to some human cell types depended on co-expression of IL-4R and
IL-13R. However, IL-13 binding was always associated with IL-4 binding.
Hyperexpression of IL-4R
on cells expressing both IL-4R and IL-13R
decreased their binding affinity for IL-4, abrogated the ability of
IL-13 to compete for IL-4 binding, and yet had no effect on IL-13R
properties. Anti-human IL-4R
monoclonal antibodies which blocked
the biological function and binding of IL-4 also blocked the function
and binding of IL-13. These data show that IL-4R
is a secondary
component of IL-13R.
-13 (
)and IL-4 (Morgan et al., 1992;
McKenzie et al., 1993a). These proteins are distantly related
at the amino acid sequence level and elicit similar proliferative
responses and morphological changes on many cell types, including
monocytes, B cells, endothelial cells, and natural killer cells.
However, unlike IL-4, IL-13 has no effect on T cells (reviewed in
Zurawski and de Vries(1994)). The overlap of these biological functions
appears to be due to the specific high affinity cell surface receptors
for IL-4 and IL-13 being distinct multisubunit complexes which share at
least one component. The direct evidence to support this notion is 1)
IL-13 can partially compete for IL-4 binding to a human cell line that
responds to both IL-13 and IL-4, but cannot compete for IL-4 binding to
a human T-cell line or to cells expressing the 130-kDa IL-4R
protein (Zurawski et al., 1993a); and 2) a mutant human IL-4
antagonist protein, which binds to IL-4R
with an affinity equal to
that of hIL-4 and binds
100-fold less well than hIL-4 to IL-4R,
also inhibits the biological action of IL-13 (Zurawski et al.,
1993a; Aversa et al., 1993). In addition, the complex nature
of IL-4R has been inferred from observations of IL-4R
-associated
proteins (Foxwell et al., 1989; Galizzi et al.,
1990a; Harada et al., 1990) and heterogeneity in IL-4 binding
(Foxwell et al., 1989; Fernandez and Vitetta, 1990, 1991) and
activation (Rigley et al., 1991) studies. Recently, the IL-2
receptor
subunit (IL-2R
) has been identified as a component
of human and mouse IL-4R that functions in signal transduction and
slightly enhances the ability of IL-4R
to bind IL-4 (Russell
et al., 1993; Kondo et al., 1993, 1994).
mAbs to
show that IL-4R
is a component of IL-13R.
Cell Culture and Reagents
Purified hIL-4
(10 units/mg), produced in Escherichia coli (Le
et al., 1988), was obtained from Schering-Plough Research
(Kennilworth, NJ). Purified hIL-13 and mIL-13 were prepared as
described by McKenzie et al. (1993b). The soluble
extracellular domain of hIL-4R
(called hIL-4R
)
was derived from Cos-7 cells transfected with a plasmid obtained by
DraIII digestion of the full-length hIL-4R
cDNA (Galizzi
et al., 1990b) and encoded a 200-amino acid protein.
hIL-4R
was purified from transfected cell culture
supernatants by hIL-4-Affi-Gel 10 chromatography as described by
Galizzi et al. (1990a). Purified phytohemagglutinin (PHA) was
from Wellcome (Dartford, Great Britain). The fluorescein
isothiocyanate-conjugated F(ab`)
fragment of goat
anti-mouse Ig was from Grub (Vienna, Austria). Phycoerythrin-conjugated
streptavidin (streptavidin-PE) was from Becton Dickinson (Mountain
View, CA). Jijoye cells, a Burkitt lymphoma; Jy, an Epstein-Barr
virus-transformed human B-cell line; Cos-7, a monkey kidney fibroblast
cell line; NALM6, a pre-B human cell line (Hurwitz et al.,
1979); L cells, a fibroblast cell line; and NS1, a myeloma cell line,
were from ATCC. Human monocytes used in the binding studies were
prepared as described by de Waal Malefyt et al.(1993).
Monocytes used for the cell surface expression studies were purified in
a standard chamber by counterflow centrifugal elutriation (J2-21
M elutriation system, Beckman Instruments) from peripheral
blood mononuclear cells isolated by Ficoll-Hypaque centrifugation. This
monocyte preparation was >90-95% pure, as determined by flow
cytometry and May Grünwald Giemsa staining. Jijoye, NS1, L cells,
tonsil B cells, and monocytes were cultured in complete culture medium
RPMI 1640 (Life Technologies, Inc.) supplemented with 10% fetal calf
serum, 2 mML-glutamine, 50 µg/ml gentamycin
(Schering-Plough). Other cells were cultured as described by Zurawski
et al. (1993a). Geneticin G418 was from Life Technologies,
Inc., or from Schering-Plough and was used at 1 mg/ml. Where not
specified, chemicals were from Sigma.
Transfection and Fluorescence-activated Sorting of L
Cells
The neomycin resistance gene was introduced into a
mammalian expression vector, pME18s (Kitamura et al., 1991),
containing the hIL-4R cDNA (pME18s neo hIL-4R). L cells, plated at
10
cells/10-cm plate the previous day, were harvested by
trypsinization and washed once with RPMI 1640, 10% fetal calf serum and
resuspended at 10
cells/ml in RPMI 1640. 25 µg of
linearized (KpnI endonuclease) plasmid (pME18s neo hIL-4R or
pME18s neo) DNA were added to 0.8 ml of cell suspension (10
cells/ml) in a 0.4-cm electroporation cuvette (Bio-Rad).
Electroporation was performed using a gene pulser (Bio-Rad) at 960 mF
and 250 V. Transfectants were selected with G418. After 1 week of
selection, 4
10
cells were harvested in
phosphate-buffered saline (PBS, 10 mM sodium phosphate, 150
mM NaCl, 3.6 mM KCl, pH 7.4) with 0.5 mM
EDTA and washed by centrifugation, and the pellet was resuspended at
10
cells/ml in PBS, 0.5 mM EDTA, 0.1% bovine serum
albumin (PEB buffer). The cell suspension was incubated (4 °C) with
0.1 µM biotinylated IL-4 in the absence or presence
(nonspecific) of 4 µM unlabeled hIL-4. After 90 min of
incubation at 4 °C, cells were washed twice by centrifugation with
PEB buffer and further incubated with a 1/5 dilution of streptavidin-PE
for 30 min at 4 °C (10
cells/ml). Then, samples were
washed twice by centrifugation and resuspended at 10
cells/ml in PEB. Fluorescence-activated cell sorting (FACS) was
on a FACS 440 (Becton Dickinson) equipped with a 5-W argon laser
running at 488 nm, 0.5 W. The 4% brightest stained cells were collected
at each sort and cultured in G418-containing culture medium until the
next sort. After four cycles of sorting, cells were cloned at 0.5
cell/well in 96-well plates. One clone (C70), selected among 300,
expressed 500,000 sites/cells, as measured by
I-hIL-4
binding.
Generation of Monoclonal Anti-IL-4R Antibodies
(mAbs)
Four BALB/C mice (Iffa Credo, Les Oncins, France) were
immunized intraperitoneally with 10 µg of hIL-4R in Freund's complete adjuvant, as described previously
(Garrone et al., 1991). At 3-week intervals, booster
immunizations (2
10 µg, 1
50 µg) were given in
Freund's incomplete adjuvant. Immune mouse spleen cells (3
10
cells) were fused with NS1 cells (ratio 5:1)
using 50% polyethylene glycol 1500 (Boehringer Mannheim) in 50
mM Hepes, pH 7.0. Then 20 96-well plates were seeded at 2
10
spleen B cells/well (100 µl) in RPMI 1640
medium containing 0.1 mM hypoxanthine, 58 µM
azaserine, 0.15 mg/ml oxaloacetate, 50 µg/ml pyruvate, 0.2 IU/ml
insulin, 10% hybridoma cloning efficiency factor (EGS, Interchim), 20%
fetal calf serum, 2 mML-glutamine, and 40 µg/ml
gentamycin. Hybridomas secreting antibodies specific for hIL-4R
were screened for their ability to stain L cells transfected with
hIL-4R
by FACscan analysis, and cloned by limiting dilution.
Positive hybridomas were produced in ascites from pristane-treated
BALB/c mice, and mAbs were purified by an anion exchange chromatography
(DEAE 5PW, Waters Associates, Millipore Corp., Milford, MA). The mAb
isotype was determined with the mouseTyper Isotyping kit (Bio-Rad).
Inhibition of
hIL-4 was labeled with NaI-hIL-4 Binding to
Cells
I using a
Tejedor-Ballesta method at the specific radioactivity of
2000-4000 cpm/mmol, as described earlier (Cabrillat et
al., 1987) or as described below for labeling mIL-13. Binding on
Jijoye cells and PHA blasts was performed as described (Galizzi et
al., 1990a). Briefly, 3
10
cells were
incubated in RPMI 1640, 1% bovine serum albumin at 4 °C in the
presence of increasing concentrations of sera, hybridoma supernatant,
or purified mAbs for 30 min. Then, 50 pM of
I-hIL-4 was added with or without 10 µM cold
hIL-4. After 4 h, samples (500 µl total) were washed twice by
centrifugation with 2 ml of binding medium at 4 °C and
radioactivity of the cell pellet was determined in a gamma counter.
Binding on all other cell types was similar, but with the modifications
previously (Zurawski and Zurawski, 1992) noted.
Biotinylation and Cross-competition between mAbs for
Binding to Cell Lines
1 mg of mAb was biotinylated as described
by Bayer (1980). 5 10
cells (C70) were incubated
with unlabeled mAb in PBS, 1% bovine serum albumin, 0.1% azide (PBA),
15 min at 4 °C. Cells were washed twice with 200 µl of PBA and
stained with 200 ng of the biotinylated anti-hIL-4R
mAbs for 15
min. After washing twice, cells were incubated with 100 µl (1/5
dilution in PBA) of streptavidin-PE, washed twice, and analyzed by
FACS.
Immunoprecipitation and Western blot
Analysis
I-labeled hIL-4R
was
prepared at a specific radioactivity of 3300 Ci/mmol by incubating 1
µg of hIL-4R
with 0.5 mCi of Na
I and
25 µl of chloramine T (10 µg/mg protein) in 150 µl of PBS
for 5 min. Free
I was separated from bound by PD10 gel
permeation (Pharmacia, Uppsala, Sweden). 10 µg of anti-mouse IgG
(Sigma) in 100 µl of PBS was coated on polyvinyl microplates (96
wells, Dynatech) at 4 °C, overnight. Plates were then saturated 1 h
at 25 °C with 300 µl of 10% fetal calf serum, PBS. After
washing with PBS (four times), wells were filled with 100 µl (10
µg/ml) of anti-hIL-4R
mAbs, 4 h at 4 °C. Wells were
incubated with 3 nM
I-hIL-4R
in
PBS, 10% fetal calf serum (100 µl). After 12 h, wells were washed,
and radioactivity was extracted with SDS-gel buffer and counted.
Samples were also analyzed by electrophoresis to confirm the presence
of hIL-4R
.
Proliferation of PHA Blasts
To test the effects of
anti-hIL-4R mAbs on PHA blast proliferation, peripheral blood
mononuclear cells purified from normal blood were cultured at 1
10
cells/ml with 1 µg/ml PHA in complete RPMI 1640
medium. After 4 days, proliferation of cells was measured by
[
H]deoxythymidine incorporation as described
previously (Garonne et al., 1991).
Biological Assays on B Lymphocytes
Functional
properties of anti-hIL-4R mAbs were also studied on B lymphocytes
purified from tonsils as described previously (Defrance et
al., 1987). Study of CD23 and surface IgM expression or
proliferation assays were previously described (Garonne et
al., 1991).
Purification of Proteins and Radiolabeling of
mIL-13
E. coli-derived hIL-4, hIL-4.Y124D, hIL-13, and
mIL-13 were purified as described previously (van Kimmenade et
al., 1988; McKenzie et al., 1993b). mIL-13 (5 µg) was
radiolabeled to a specific activity of 1525 Ci/mmol with 2.5 mCi of
NaI (IMS30, Amersham) in 100 µl of PBS and 5 µg
of Iodogen (Pierce) for 5 min followed by PD-10 gel permeation in RPMI
1640, 2% bovine serum albumin, 0.01% NaN
, 20 mM
Hepes.
Affinity Labeling of IL-4R and IL-13R
Proteins
Chemical cross-linking was performed essentially as
described by Kitamura et al.(1991). Cells were incubated with
2 nMI-hIL-13 or 1 nM
I-hIL-4 in the presence or absence of a 100-fold excess
of unlabeled hIL-13 or hIL-4. After 2 h at 4 °C, the cells were
collected by centrifugation, washed once in PBS containing 200
mM bis-sulfosuccinimidyl suberate (BS
, Pierce),
then incubated in this buffer for 30 min at 4 °C. Following
centrifugation the cell pellet was solubilized in 1% Triton X-100, 500
mM Na
EDTA, 100 mM phenylmethylsulfonyl
fluoride, 10 µg/ml leupeptin, 1 µg/ml pepstatin A, 10 µg/ml
antipapain, and analyzed on 7.5% gel SDS-polyacrylamide gel
electrophoresis followed by autoradiography.
Determination of hIL-6 Production by Human
Monocytes
Monocytes were activated in 96-well plates for 48 h
with lipopolysaccharide (LPS, 1 µg/ml), alone or in combination
with hIL-4 (0.001-100 units/ml) and/or hIL-13 (0.0001-100
ng/ml), or anti-IL-4R mAbs (20 µg/ml). After incubation,
cell-free supernatants were collected and frozen at -20 °C
until IL-6 levels were determined. Levels of hIL-6 were measured by
specific enzyme-linked immunosorbent assay (sensitivity: 600 pg/ml)
using two rat mAbs (39C3 for IL-6 coating and biotinylated 13A5 for
IL-6 detection) kindly provided by Dr. J. S. Abrams (Abrams et
al., 1992).
Immunofluorescence Analysis of Monocyte Cell Surface
Markers
After 48 h of culture, monocytes (3 10
cells/bag) were harvested from Teflon bags. For fluorescence
staining, cells were incubated with 10 µl of fluorescein
isothiocyanate-labeled mAbs: CD14, HLA-DR, HLA-DQ (Becton Dickinson),
or 10 µl of PE-labeled mAb CD23 (Serotec). After washes, the
labeled cell samples were analyzed by flow cytometry on FACscan.
Characteristics of IL-4R on Various Cell
Types
Competitive I-hIL-4 displacement studies
have defined three classes of IL-4 binding (Zurawski et al.,
1993a). 1) Cells expressing only human IL-4R
bind hIL-4 and the
biologically inactive hIL-4 Tyr
Asp mutant protein
(hIL-4.Y124D) with a similar affinity (K
10
M). This type of hIL-4
binding is not competed for by hIL-13. 2) hIL-4-responsive human SP-B21
T cells bind hIL-4.Y124D at this same affinity, but bind hIL-4
100-fold more avidly. This type of hIL-4 binding is also not
competed for by hIL-13. 3) IL-4R on hIL-4- and IL-13-responsive human
pre-myeloid erythroleukemic TF-1 cells are like those on SP-B21 cells,
except that hIL-13 can partially (a maximum of
65%) compete for
I-hIL-4 binding. We have extended these studies to other
cell types which are known to bind hIL-4. Fig. 1A shows
that IL-4R on monkey kidney Cos-7 cells had properties identical to
those described above for TF-1 cells. Human monocytes, which respond
similarly to both hIL-4 and IL-13 (McKenzie et al., 1993b;
Minty et al., 1993; de Waal Malefyt et al., 1993;
Herbert et al., 1993), had fewer IL-4R, but hIL-13 competed
for
I-hIL-4 binding similarly to that observed for IL-4R
on TF-1 and Cos-7 cells (Fig. 1B). The Epstein-Barr
virus-transformed human Jy B-cell line had IL-4R with properties
identical to SP-B21 cells, except that hIL-13 competed for only
20% of the bound
I-hIL-4 (Fig. 1C).
Collectively, these hIL-4 binding data establish that high affinity
IL-4R on various cell types are similar and that hIL-4.Y124D typically
binds to such IL-4R
100-fold less avidly than hIL-4. In contrast,
the ability of hIL-13 to compete for
I-hIL-4 binding
varies dramatically with cell type. Simplistically, these data suggest
that there are subtypes of high affinity IL-4R (i.e. those
which can also bind IL-13 and those which cannot bind IL-13). To
explore this notion, we developed a radiolabeling protocol for IL-13
which permitted direct examination of IL-13R.
Figure 1:
Competition displacement of
I-hIL-4 binding to various cell types. Various amounts of
unlabeled hIL-4 (
), hIL-4.Y124D (
), and hIL-13 (
)
were incubated for 2 h at 4 °C with 2
10
M
I-hIL-4;
I-hIL-4 bound to
cells was then determined. Panel A, Cos-7 cells, maximum was
5810 ± 314 cpm, minimum was 93 ± 51 cpm,
23% of the
bound counts were not competed for by hIL-13. Panel B,
monocytes, maximum was 702 ± 66 cpm, minimum was 73 ± 16
cpm,
23% of the bound counts were not competed for by hIL-13.
Panel C, Jy cells, maximum was 3790 ± 161 cpm, minimum
was 270 ± 25 cpm,
79% of the bound counts were not competed
for by hIL-13. Arrows on the abscissa indicate the
dose required to displace 50% of the maximal counts displaced
(IC
). Error bars represent standard deviation of
the mean (S.D., n = 2).
Properties of
IL-4R on Cos-7 cells are of the type where IL-13 partially
competes for I-IL-13 Binding to Cos-7
Cells
I-hIL-4 binding, inferring the existence of
IL-13R on these cells. This was demonstrated directly by the ability of
I-mIL-13 to bind specifically to these cells
(Fig. 2A). Specific
I-mIL-13 binding to
Cos-7 cells saturated at
5
10
M (no specific binding was observed above
10
M
I-mIL-13 due to high
nonspecific binding). Scatchard analysis of the data was most
consistent with a single class of
I-mIL-13 binding site
with K
10
M (Fig. 2A). Kinetic analysis revealed
that
I-mIL-13 associated with Cos-7 cells relatively
slowly (t
60 min, Fig. 2B).
Figure 2:
Binding of I-mIL-13 to Cos-7
cells. Panel A, Various amounts of labeled mIL-13, together
with (
) or without (
), a 500-fold excess of unlabeled
hIL-13 were added to cells and incubated for 2 h at 4 °C;
I-mIL-13 bound to cells was then determined. Analysis of
specific binding (
) by the program Ligand (Munson and Rodbard,
1980) indicated the presence of
1000 binding sites with a single
affinity class of K
1.4
10
M. Panel B, 10
M
I-mIL-13 was added to cells at 4 °C and aliquots were
removed at various times and
I-mIL-13 bound to cells was
then determined. The inset shows a natural log plot of the
data with [HR], bound
I-mIL-13 at time
t; [HReq], bound
I-mIL-13 at
equilibrium.
Characteristics of IL-13R on Various Cell
Types
Competitive I-mIL-13 displacement studies on
Cos-7 cells, TF-1 cells, and human monocytes were remarkably similar.
These data (Fig. 3, A-C) revealed that hIL-13
binds to these cells slightly more avidly than does mIL-13
(K
= 2
10
Mversus 6
10
M) and that hIL-4 is a potent (K
= 1
10
M) and
complete competitor of
I-mIL-13 binding. Cos-7 had
1000 IL-13R/cell, while TF-1 and monocytes had
70
IL-13R/cell. In similar experiments, no specific
I-mIL-13
binding was detected on NALM6 cells, a human pre B-cell line (Hurwitz
et al., 1979) on which
I-hIL-4 binding was not
inhibited by up to 10
M IL-13 (data not
shown). There was an absolute correlation between the existence of the
IL-4R subtype which interacts with IL-13 and the presence of IL-13R as
detected by IL-13 binding.
Figure 3:
Competition displacement of
I-mIL-13 binding to various cell types. Various amounts
of unlabeled hIL-4 (
), hIL-13 (
), and mIL-13 (
) were
incubated for 2 h at 4 °C with 10
M
I-mIL-13;
I-mIL-13 bound to cells was then
determined. Panel A, TF-1 cells, maximum was 496 ± 30
cpm, minimum was 243 ± 20 cpm. Panel B, monocytes,
maximum was 202 ± 16 cpm, minimum was 80 ± 12 cpm.
Panel C, Cos-7 cells, maximum was 1388 ± 71 cpm,
minimum was 367 ± 32 cpm. Using similar conditions, no specific
binding to NALM6 cells was detected. Arrows on the abscissa
indicate IC
. Error bars represent S.D. (n = 2).
Effects of IL-4R
To further explore the notion that a subtype of IL-4R can
bind IL-13, we took advantage of a transient expression system in Cos-7
cells. Previous studies demonstrated that binding of hIL-4 to Cos-7
cells hyperexpressing hIL-4R Hyperexpression on IL-4R and
IL-13R
is not competed for by IL-13
(Zurawski et al., 1993a). With the availability of a direct
probe for IL-13 binding, we could investigate the effects of expressing
various amounts of hIL-4R
on a cell type which expresses both
subtypes of IL-4R. Increasing hIL-4R
expression on Cos-7 cells
resulted in an enhanced total bound hIL-4, concomitant with decreased
apparent affinity of binding by hIL-4 (Fig. 4A). This
result is expected since additional hIL-4R
contributes to binding
hIL-4, but its affinity for hIL-4 is markedly less than that of the
endogenous IL-4R. Of interest was the observation that these changes in
IL-4R were associated with a decreased maximal ability of IL-13 to
compete for binding by hIL-4, without concomitant changes in the
potency of IL-13 competition or changes in number and affinity of
IL-13R (Fig. 4, B and C). These observations
are consistent with a model in which the ability of IL-13 to compete
for binding of IL-4 to IL-4R is determined by the relative abundance of
IL-4R subtypes and show that the IL-4R subtype which interacts with
IL-13 on Cos-7 cells is not limited by availability of IL-4R
.
Figure 4:
Effect of hyperexpression of hIL-4R
on the properties of IL-4R and IL-13R on Cos-7 cells. Panel A,
various amounts of unlabeled hIL-4 were incubated for 2 h at 4 °C
with 10
M
I-hIL-4;
I-hIL-4 bound to 10
cells was then
determined. Cells (10
) were untransfected (
) or
transfected with 5 µg (
), 15 µg (
), or 40 µg
(
) hIL-4R
expression plasmid. Panel B, various
amounts of unlabeled mIL-13 (circles) or unlabeled hIL-13
(squares) were incubated with 10
M
I-hIL-4, and
I-hIL-4 bound
to cells was then determined. Cells were untransfected (
,
) or transfected with 5 µg (
,
), 15 µg
(
,
), or 40 µg (
,
) hIL-4R
expression
plasmid. Panel C, various amounts of unlabeled hIL-13 were
incubated with 10
M
I-mIL-13, and
I-mIL-13 bound to cells was
then determined. Cells were untransfected (
) or transfected
with 5 µg (
), or 40 µg (
) hIL-4R
expression
plasmid. Arrows on the abscissa indicate
IC
.
IL-13R Cross-linking Studies
Cross-linking studies
with I-hIL-4 and various human cell lines reveal a
predominant 130-140-kDa hIL-4R
protein (reviewed in Harada
et al. (1992a)). These same studies suggested that other
proteins of
80 and
65 kDa could sometimes be found associated
with hIL-4R, and other studies show that the 64-kDa human IL-2R
protein is a component of IL-4R (Russell et al., 1993; Kondo
et al., 1993, 1994). We attempted to identify protein
components specific to IL-13R by cross-linking
I-mIL-13
bound to TF-1 and Cos-7 cells. On both cell types,
I-mIL-13 affinity-labeled a predominant protein of
65 kDa (assuming a 1:1 complex with 11.6-kDa mIL-13) and this
affinity-labeled species was not detected in the presence of excess
unlabeled hIL-13 or hIL-4 (Fig. 5). This property correlates
absolutely with the binding properties of IL-13R and contrasts with
parallel experiments utilizing
I-hIL-4 as the affinity
probe. On Cos-7 cells,
I-hIL-4 predominantly labeled a
130-140-kDa species and minor
65-kDa species which were
partially competed for by unlabeled hIL-13 and completely competed for
by unlabeled hIL-4 (Fig. 5). On TF-1 cells,
I-hIL-4
labeled 130-140-kDa,
95-kDa, and
65-kDa species which
were competed for by both unlabeled hIL-13 and hIL-4 (Fig. 5).
Taken together, these data reveal significant differences between IL-4R
and IL-13R and tentatively identified a
65-kDa IL-13R protein(s)
as the primary binding protein for IL-13. Further such work with other
cell types should reveal whether the 65-kDa protein is specific to
IL-13R.
Figure 5:
Affinity labeling of IL-4R and IL-13R
proteins on Cos-7 and TF-1 cells. Cells were incubated for 2 h at 4
°C with I-hIL-13 or
I-hIL-4 in the
presence or absence of excess unlabeled hIL-13 or hIL-4, then washed
and incubated with cross-linking agent. Solubilized cells were then
subjected to SDS-polyacrylamide gel electrophoresis and
autoradiography. Positions of the cross-linked receptor subunits are
indicated by arrows. The high molecular mass band (>200
kDa) may represent a higher order complex.
Production and Characterization of Anti-IL-4R
We developed mAbs against hIL-4R
Monoclonal Antibodies
to
aid in the further study of IL-4R and IL-13R. A 200-amino acid
extracellular portion of hIL-4R
(hIL-4R
) was used
to immunize mice. We subsequently derived several hybridomas which
produced mAbs reactive in FACS analysis to an L cell line stably
expressing hIL-4R
(C70), but were not reactive to the parental L
cell line. The mAbs varied in their reactivity to hIL-4R
as judged
by blotting, precipitation, inhibition of
I-hIL-4
binding, and cross-competition (). shows that
three of the mAbs were effective inhibitors of
I-hIL-4
binding to monocytic (monocytes), B-cell (Jijoye), and T-cell (PHA
blasts) types. As expected from their ability to inhibit binding of
hIL-4, these mAbs also abolished IL-4-induced biological effects, such
as proliferation of B and T cells and induction on B cells of low
affinity IgE receptor (Fc
II) and surface IgM (Fig. 6), as
described previously (Defrance et al., 1987; Garonne et
al., 1991).
Figure 6:
Inhibition of hIL-4-induced biological
effects by anti-hIL-4R mAbs. Panel A, peripheral blood
mononuclear cells were preactivated for 4 days with 1 µg/ml PHA and
viable blasts were recultured for 3 days with 5
10
M hIL-4 and various concentrations of
mAbs s103 (
), s456 (
), and s924 (
). Panel
B, purified tonsil B cells were cultured for 3 days with 5
µg/ml insolubilized anti-IgM and 5
10
M hIL-4 and various concentrations of the above mAbs.
The response was measured by uptake of
[
H]thymidine. Panels C and D,
purified tonsil B cells were cultured for 48 h with 2.5
10
M hIL-4 and various concentrations of
the above mAbs. The response was measured by staining cells with
specific anti-CD23 (Panel C) or anti-IgM (Panel D)
antibodies together with fluorescein isothiocyanate-labeled anti-mouse
IgG and FACscan analysis.
Anti-IL-4R
The above and previous data show that, although
hIL-4R Monoclonal Antibodies Inhibit IL-13
Binding
alone cannot bind IL-13, there appears to be an absolute
correlation between IL-4R and IL-13R. We utilized the anti-hIL-4R
mAbs to investigate a possible role for hIL-4R
in IL-13R. Two
anti-hIL-4R
mAbs tested were equipotent inhibitors of both
I-hIL-4 binding and
I-mIL-13 binding to
TF-1 cells (Fig. 7). These data revealed that hIL-4R
, which
itself cannot bind IL-13, is an important component for the binding of
IL-13 by IL-13R.
Figure 7:
Displacement by anti-hIL-4R mAbs of
I-mIL-13 and
I-hIL-4 binding to TF-1 cells.
Various amounts of unlabeled hIL-4 (
), mIL-13 (
), and
mAbs s456 (
), s103 (
), or o361 (*) were incubated for 2 h
at 4 °C with Panel A, 10
M
I-hIL-4 or Panel B, 10
M
I-mIL-13, and radioligand bound to cells
was then determined.
Anti-IL-4R
IL-13 and IL-4 elicit diverse effects on monocytic cells
(McKenzie et al., 1993b; Herbert et al., 1993; de
Waal Malefyt et al., 1993; Doherty et al., 1993)
including inhibition of LPS-stimulated synthesis of pro-inflammatory
cytokines such as IL-6 and regulation of cell surface marker expression
such as CD23, CD16, and MHC class II. We first assessed the effect of
anti-IL-4R Monoclonal Antibodies Inhibit IL-13
Action
mAbs on IL-6 production by LPS-activated human
monocytes cultured in the presence of IL-13. Fig. 8shows that
the two blocking anti-IL-4R
mAbs tested reversed the
hIL-13-induced inhibition of hIL-6 production by monocytes. This effect
was not observed with a control mAb, the nonblocking anti-IL-4R
mAb s697 (Fig. 8), or with an anti-IL-4 mAb (data not shown).
Figure 8:
Reversal by anti-hIL-4R mAbs of
hIL-13 inhibition of monocyte hIL-6 production. LPS-activated human
monocytes were incubated alone or with hIL-13 and with and without mAbs
for 48 h. Supernatants were then collected and levels of hIL-6 produced
were determined. Similar experiments (not shown) established that 5
ng/ml hIL-13 elicits a
50% maximal response and 50 ng/ml elicits a
90% maximal response. Also, blocking anti-hIL-4 mAb and non
blocking anti-hIL-4R
mAb s697 (both at 20 µg/ml) had no effect
on hIL-13 action (data not shown).
We also investigated whether anti-IL-4R mAbs reversed the
hIL-13 effects on the expression of the monocyte cell-surface markers
CD23, CD14, and MHC class II. Fig. 9shows that the
anti-IL-4R
mAbs s103 and s924 reversed in a dose-dependent fashion
the hIL-13- and hIL-4-induced increase of CD23 expression on monocytes.
No inhibition of CD23 induction was seen with the nonblocking
anti-IL-4R
mAb s697. Furthermore, an anti-hIL-4 mAb blocked CD23
expression induced by hIL-4 but did not block CD23 expression induced
by hIL-13 (data not shown). Similar results were obtained with
anti-IL-4R
mAbs when we investigated the expression of CD14 and
MHC class II molecules on monocytes in the presence of hIL-4 or hIL-13
(Fig. 10). In addition, we have previously shown that s460
reverses hIL-13 inhibition of the growth of a leukemic human B-cell
precursor (Renard et al., 1994). Together, these data show
that IL-4R
is a functionally important component of IL-13R.
Figure 9:
Reversal by anti-hIL-4R mAb of hIL-4-
and hIL-13-directed enhancement of monocyte CD23 expression. Human
monocytes were cultured with LPS in the presence of 50 units/ml hIL-4
or 50 ng/ml hIL-13 with and without various amounts (indicated in
micrograms/ml in the parentheses) of the anti-hIL-4R
mAb
s103. After 48 h the cells were stained with phycoerythrin-labeled CD23
antibody and analyzed by FACscan flow cytometry. Parallel experiments
(not shown) revealed that mAb s924 had similar though slightly less
potent effects and that the nonblocking anti-hIL-4R
mAb s697 and
blocking anti-hIL-4 mAb had no effect on hIL-13
action.
Figure 10:
Reversal by anti-hIL-4R mAb of
hIL-13-directed changes of monocyte CD14, HLA-DR, and HLA-DQ
expression. Human monocytes were cultured with LPS in the presence and
absence (medium control, left panels) of 50 ng/ml hIL-13 with
(shaded histogram in center and right
panels) and without (unshaded histograms) 20 µg/ml of
the anti-hIL-4R
mAb s456. After 48 h the cells were stained with
fluorescein isothiocyanate-labeled CD14, HLA-DR, and HLA-DQ antibodies
and analyzed by FACscan flow cytometry. Parallel experiments (not
shown) revealed that mAb s103 had effects similar to those of s456 on
CD14 and HLA-DR expression and that these effects were proportionally
greater at suboptimal levels of hIL-13.
100-fold less avidly than did hIL-4. We have previously observed
that the cloned hIL-4R
protein alone binds both hIL-4 and
hIL-4.Y124D with the lesser affinity characteristic of hIL-4.Y124D
binding to IL-4R (Zurawski et al., 1993a). These data imply
that an additional subunit associates with hIL-4R
to enhance its
affinity for hIL-4 and that this association facilitates signaling and
involves interaction with the Y124D residue of hIL-4. The hIL-2R
subunit appears to associate with hIL-4R
, although co-expression
of hIL-4R
and hIL-2R
did not enhance the affinity of
hIL-4R
to the extent expected from the above studies (Russell
et al., 1993). IL-2R
plays similar affinity-enhancing and
signaling roles in IL-4R and IL-2R (where it interacts with
IL-2R
or IL-2R
; reviewed in Minami et
al.(1993)). Additionally, mutations in similar locations, but
different side chains (Gln in IL-2, Tyr in IL-4) within the C-terminal
-helix of IL-2 and IL-4 abrogate interaction with IL-2R
(Zurawski et al., 1993a, 1993b).
+ IL-2R
, cannot
bind IL-13, while the other type of IL-4R is IL-13R.
65-kDa protein that was
specifically associated with IL-13R. It is most likely that this
65-kDa affinity-labeled protein is the primary binding subunit of
IL-13R (which we shall here refer to as IL-13R
). The
65-kDa
protein is distinct from hIL-2
, since hIL-2R
and IL-4R
are present on cells such as human SP-B21 T cells which do not bind
IL-13 (data not shown). Second, we observed that certain
anti-hIL-4R
mAbs exhibited equipotent inhibition of IL-4 and IL-13
binding and biological activity. These data show that hIL-4R
is a
component of IL-13R. Thus IL-13R is composed of IL-4R
+
IL-13R
.
+ IL-2R
) appears on a large number of cell
types, while IL-13R
, whose presence determines the ability of a
cell to bind IL-13, has a more restricted distribution. The remarkably
similar cellular responses to IL-4 and IL-13 (Zurawski and de Vries,
1994) reflect the action of the intracellular domain of the shared
IL-4R
in signaling (Harada et al., 1992b). The
hIL-4.Y124D antagonist acts against responses to both IL-4 and IL-13
(Zurawski et al., 1993a) since it binds normally to
hIL-4R
, which is a component of both IL-4R and IL-13R. The ability
of IL-13 to compete for IL-4 binding varies widely with cell type
because the prevalence of IL-4R
, IL-2R
, and IL-13R
varies. We demonstrated this directly by showing that increasing levels
of hIL-4R
decreased the maximal ability, but not the potency, of
IL-13 to compete for hIL-4 binding. This and the concomitant decrease
in the affinity of hIL-4 binding (due to increased ratio of IL-4R
to IL-2R
) had no effect on the numbers or binding properties of
IL-13R.
as well as
IL-4R
+ IL-13R
; however, recent studies (Matthews et
al., 1994) show that IL-13 responses in B cells from two X-SCID
patients with characterized IL-2R
mutations do not respond to
IL-2, but respond normally to IL-13. These same B cells respond
normally to IL-4, suggesting that IL-4 can signal via binding to
IL-13R.
mAbs which we developed will be
important reagents for further characterizations of IL-4R
abundance and distribution. Since some recognize distinct epitopes,
they can be used in solid phase assays for the detection of
hIL-4R
in biological fluids (not shown). Such soluble
forms of IL-4R
have been detected in mouse sera (Fernandez and
Vitetta, 1990, 1991; Fanslow et al., 1990) and may be
indicators of disease states such as allergy and graft rejection.
Table:
Properties of anti-hIL-4Ra mAbs
Table:
Inhibition of hIL-4 binding to cells by
anti-hIL-4R mAbs
, primary binding component of
the receptor; PE, phycoerythrin; PBS, phosphate-buffered saline; FACS,
fluorescence-activated cell sorting; MHC, major histocompatibility
complex.
mAbs.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.