|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3884-3891
Interleukin-13 Receptor   But Not Chain: A
Functional Component of Interleukin-4 Receptors
By
Takashi Murata,
Jun Taguchi, and
Raj K. Puri
From the Laboratory of Molecular Tumor Biology, Division of Cellular
and Gene Therapy, Center for Biologics Evaluation and Research, Food
and Drug Administration, Bethesda, MD.
 |
ABSTRACT |
In hematopoietic cells, interleukin-2 receptor (IL-2R)  chain
(termed c) is shown to be a component of the IL-4R
system, whereas in nonhematopoietic cells, c is absent
and it is not a component of the IL-4R system. Here, we show that the
IL-13R   chain (termed IL-13R) but not the IL-13R
chain (termed IL-13R) can substitute for c and,
thus, IL-13R forms a novel component of the IL-4R system.
This conclusion was drawn on the basis of chemical cross-linking,
immunoprecipitation, the ability of IL-13R but not IL-13R
to augment IL-4 binding affinity, and the requirement of
IL-13R for IL-4-induced STAT6 activation in Chinese hamster
ovary (CHO) cells transfected with various receptor subunits.
Cotransfection of IL-4 receptor p140 (termed IL-4R ) with
c or IL-13R increased IL-4 binding affinity
and allowed for STAT6 activation in response to IL-4. However,
cotransfection of all three chains did not further increase IL-4
binding or alter the extent of STAT6 activation suggesting that all
three chains together do not seem to participate in IL-4 function.
Instead, IL-4R heterodimerizes with c or
IL-13R and mediates STAT6 activation. Cotransfection of
IL-4R with IL-13R neither increased IL-4 binding affinity nor
allowed for STAT6 activation in response to IL-4 indicating that
IL-13R does not convert binding affinity nor transmit signals for
IL-4. Because IL-4 phosphorylates JAK1 and JAK2 tyrosine kinases in
nonhematopoietic cells, we investigated whether JAK1 and JAK2 are
required for IL-4-induced STAT6 activation in various transfectants.
Cotransfection experiments with different chains of IL-4R and
kinase-deficient JAK1 and JAK2 mutants in CHO cells showed that JAK1
and JAK2 are required for optimal activation of STAT6 in the
transfectant but only partially in the
c transfectant. Taken together, our results show that
IL-13R is a novel functional component of the IL-4R system
and that JAK1 and JAK2 mediate IL-4-induced optimal activation of
STAT6 in nonhematopoietic cells.
| |
INTRODUCTION |
INTERLEUKIN-4 (IL-4) is a growth and
differentiation factor for human B- and T-lymphocytes.1 In
contrast to its growth stimulatory effects on lymphocytes, IL-4 has
growth inhibitory effects on many human carcinoma cell
lines.2,3 The receptors for IL-4 have been shown to be
expressed on a variety of cell types,4-6 and the effects of
IL-4 involve IL-4 signaling through its receptors.
The structure of IL-4 receptor (IL-4R) has been extensively
investigated; however, the exact structure is still
unknown.1 The primary subunit of IL-4R was identified as a
140-kD protein, originally termed IL-4R.7 However, based
on similarities in the extracellular domain (WSXWS motif and four
cysteine residues at the fixed location) and the long intracellular
domain between IL-4R and chains of receptors for IL-3, IL-5, and
granulocyte-macrophage colony-stimulating factor (GM-CSF), we have
recently proposed to rename this chain IL-4R.8,9 This
recommendation was also based on the basis of its similarity with the
IL-2R chain, which, like IL-4Rp140, binds IL-2 but does not transmit
signal on its own.10 The second subunit of
the IL-4R system was shown to be the IL-2R chain (termed
c),10,11 and recently we8,12 and
other groups13 have proposed that the 60- to 70-kD protein form of IL-13R may also participate in mediating IL-4 effects and,
thus, may constitute the third subunit of the IL-4R system (termed
IL-4R).8
Recently, two different types of human IL-13 receptor chains were
cloned. The IL-13R chain cloned from the human RCC cell line, Caki-1,
is an approximately 70-kD protein and has a 50% homology to IL-5R
on DNA level (termed here, IL-13R).14 On the other hand,
Aman et al15 have cloned another type of IL-13R from the
human T-cell leukemia virus-1 (HTLV-1)-infected MT-2 cell
line by using the sequence of the murine IL-13R cDNA (termed here,
IL-13R).16 Unlike Caki-1 IL-13R (IL-13R), MT-2
IL-13R (IL-13R) has no homology with any other cytokine
receptors similar to cloned mouse IL-13R.
To directly examine which IL-13R chains are required for functional
IL-4R, we reconstituted the IL-4R systems by transfection of two
different IL-13R chains ( and ), IL-4R, or
c chains into Chinese hamster ovary (CHO-K1)
cells. We then investigated their binding characteristics to IL-4,
subunit structure of IL-4R by cross-linking and immunoprecipitation,
and signal transduction mechanisms in response to IL-4.
| |
MATERIALS AND METHODS |
Materials.
Recombinant human IL-4 was kindly provided by Schering Corporation
(Kenilworth, NJ). Recombinant IL-13 was purified as
described.30 Polyclonal antibodies against c-Myc and
c antibodies were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Antibody for IL-4R chain (P7) was
provided by Immunex Corp (Seattle, WA). Horseradish peroxidase
(HRP)-conjugated antimouse or rabbit IgG antibodies and streptoavidine
HRP were obtained from Amersham (Arlington Heights, IL).
Cells.
The CHO-K1 cell line was obtained from American Type Culture Collection
(Rockville, MD). Cells were cultured in a modified Eagle's minimum
essential medium (AMEM) with 10 mmol/L HEPES, antibiotics, and 10%
fetal bovine serum (FBS).
Plasmids and genes.
Human IL-4Rp140 () chain cDNA7 was kindly provided by Dr
M. Widmer of Immunex Corp. Human IL-13R15 and
IL-2R10 cDNA were cloned into pME18S mammalian
expression vector. To epitope-tag the IL-13R with a c-Myc
tag, new BamH1 sites were created at the C-terminus of
IL-13R cDNA and it was cloned into the BamH1 site of
CS+MT plasmid (derived from CS2 plasmid with Myc
tag).17 IL-13R with c-Myc tag cDNA was recloned
into the EcoR1 site of PME18S. The plasmids containing wild
type JAK1 or its mutant (pME18SJAK1 and pME18SJAK1 delta)18
were provided by Dr S. Watanabe (University of Tokyo Medical Science,
Tokyo, Japan).19 JAK2 expression vector, pBOSJAK2, and
kinase-deficient JAK2 expression vector, pBOSJAK2 D VIII, were provided
by Dr D.M. Wojchowski (Pennsylvania State University, University Park,
PA).20
Transient transfection of DNA.
Individual plasmid DNA or a combination of multiple plasmid DNAs (6 µg/60-mm dish or 12 µg/100-mm dish) were transfected into semiconfluent CHO-K1 cells by Lipofectamine (GIBCO-BRL, Gaithersburg, MD) according to the manufacturer's instructions. Briefly, CHO-K1 cells (1 × 106/60-mm dish or 3 × 106/100-mm dish) were incubated with the DNA Lipofectamine
mixture for 5 hours in Opti-MEM (GIBCO-BRL). The medium was then
changed to AMEM with 10% FBS and incubated for 48 hours.
Radioreceptor binding assay.
Recombinant human IL-4 was labeled with 125I (Amersham
Research Product, Arlington Heights, IL) by the IODO-GEN iodination
reagent (Pierce, Rockford, IL) according to the manufacturer's
instructions. The IL-4 equilibrium binding studies were performed by
the method previously described.2,8 Briefly, 1 × 106 cells in 100 µL binding buffer (RPMI 1640 containing
0.2% human serum albumin and 10 mmol/L HEPES) were incubated for 2 hours with 100 to 200 pmol/L 125I-IL-4 with or without
unlabeled IL-4 (50 nmol/L) or IL-13 (200 nmol/L) at 4°C. Cell-bound
125I-IL-4 was separated from unbound
125I-IL-4 by centrifugation through a cushion of phthalate
oils. Pelleted cells were counted on a counter. To determine
binding affinity, transfected cells were incubated with 100 pmol/L of 125I-IL-4 with or without various concentrations of
unlabeled IL-4. Scatchard data was analyzed by the
LIGAND program (Provided by Dr P. Munson, National
Institutes of Health).21
Affinity cross-linking of 125I-IL-4 to its receptor.
Transfected cells (5 × 106) were incubated with
125I-IL-4 in the presence or absence of excess unlabeled
IL-4 or IL-13 for 2 hours at 4°C. Bound 125I-IL-4 was
cross-linked to IL-4R with disuccinimidyl suberate (DSS; Pierce) at a
final concentration of 2 mmol/L for 20 minutes. The cells were then
lysed at 4°C with modified radio immunoprotein assay (RIPA) buffer (1% NP-40, 300 mmol/L NaCl, 50 mmol/L Tris [pH
7.4], 1 mg/mL leupeptin, 1 mg/mL pepstatin A, 2 mg/mL aprotinin, 20 mmol/L phenylmethyl sulfonyl fluoride [PMSF], 1 mmol/L Na-vanadate, 25 mmol/L Na-F, 10 mmol/L Na-pyrophosphate, and 1 mmol/L EDTA). The
resulting lysate was analyzed by electrophoresis through sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 7%). The gel was
dried and exposed to radiograph film for 4 days at  80°C.
For immunoprecipitation, 125I-IL-4/IL-4R cross-linked
complex was immunoprecipitated from the lysate for 2 hours at 4°C
by incubating with protein A (G) sepharose beads, which had been
preincubated with anti-IL-4R (P7), c, or c-Myc
antibodies. The resulting conjugate was washed five times with lysing
buffer, resuspended with reducing buffer, boiled for 5 minutes, and
analyzed by 8% SDS-PAGE as described previously.
Electrophoretic mobility shift assay (EMSA).
After incubation with IL-4 (50 ng/mL) for 10 minutes, cells were washed
with cold phosphate-buffered saline (PBS) and solubilized with cold
whole cell extraction buffer (1 mmol/L MgCl2, 20 mmol/L HEPES pH 7.0, 10 mmol/L KCl, 300 mmol/L NaCl, 0.5 mmol/L
dithiothreitol, 0.1% NP-40, 1 mmol/L PMSF, 1 mmol/L
Na3VO4, and 20% glycerol). DNA protein
interactions were assessed by EMSA by using the Bandshift kit from
Pharmacia (Piscataway, NJ). Briefly, 50 µg of sample proteins were
incubated for 20 minutes at room temperature with 1 ng of
32P-labeled double stranded oligonucleotide probe SBE1
(signal transduction and activator of transcription
[STAT]-binding element;
5-gatcGCTCTTCTTCCCAGGAACTCAATG-3;3-CGAGAAGAAGGGTCCTTGAGTTACagct-5) from the region flanking the transcription start site of the human sIL-1R antagonist gene that is necessary for response to IL-4 alone22 in binding buffer [10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl, 0.5 mmol/L dithiothreitol (DTT), 10%
glycerol, 0.05% NP-40, 0.05 mg/mL poly (dI-dC)2]. A
10× loading dye was added to samples that were then applied to a
4% nonreducing polyacrylamide gel and run at 150 V for 2 hours. Gels
were dried for 2 hours and autoradiographed overnight four 4 days at
70°C. In some experiments, antimouse STAT6 (M-20) or
antihuman STAT6 (S-20) rabbit polyclonal IgG (both from Santa Cruz
Biotechnology, Santa Cruz, CA) were included in the reaction mixture
for "supershift" assay.
| |
RESULTS |
125I-IL-4 binding assay on CHO-K1 cells transfected with
IL-13R, IL-13, IL-4R, and c.
Previously, we proposed that IL-4R shares at least one chain of IL-13R
in nonhematopoietic cancer cell lines.8,12 To examine directly the subunit structure of IL-4R, we introduced the cDNA of the
IL-4R chain into the CHO-K1 cell line along with recently cloned
IL-13R chains (IL-13R and ).14,15 As shown
in Fig 1A, 125I-IL-4 bound to
IL-4R-transfected CHO-K1 cells, but it did not bind to cells
transfected with IL-13R or c alone. The IL-4 binding to IL-4R-transfected cells was specifically inhibited by a
50-fold mol/L excess of unlabeled IL-4. However, IL-13 did not inhibit
this binding. Interestingly, although IL-13R- and c-transfected cells did not bind to
125I-IL-4, cotransfection of IL-4R with these chains
induced a high affinity binding (Fig 1B). In the case of
transfectants, 125I-IL-4 binding to its receptor was
inhibited by a 50-fold mol/L excess of unlabeled IL-4 and also
partially by IL-13 (Fig 1A, upper panel). On the other hand, in
c-transfected cells, IL-4 bound with high-affinity
125I-IL-4 binding was only inhibited by IL-4 and not by
IL-13. In CHO-K1 cells transfected with all three
c chains, 125I-IL-4 also bound
with high affinity and this binding was completely blocked by unlabeled
IL-4 and partially by IL-13 (Fig 1A, lower panel). These data suggest
that IL-13R allows interaction of IL-13 with IL-4R, and when
it is absent, such as in the case of the IL-4R and c
transfectant, IL-13 does not interact with IL-4R. 125I-IL-4 also did not bind to cells transfected with
IL-13R alone. However, when it was coexpressed with the IL-4R
chain, the IL-4 binding was similar to that seen in cells transfected
with alone. Again, only IL-4 completely inhibited the binding of
radiolabeled IL-4 whereas IL-13 did not inhibit this
binding.
View larger version (26K):
[in this window]
[in a new window]
View larger version (15K):
[in this window]
[in a new window]
| Fig 1.
125I-IL-4 binding to CHO-K1 cells
transfected with IL-13R, IL-4R, and
c. cDNA for various receptor chains (2 µg/chain) was transfected in CHO-K1 cells (1 × 106) by
using Lipofectamine reagent for 48 hours. For IL-4 binding assay, 1 × 106 cells were incubated with 100 pmol/L of
125I-IL-4 with or without a 200-fold molar excess of
unlabeled IL-4 or IL-13. Binding assays were performed on two different
occasions. Cell bound radioactivity was determined as described in
Materials and Methods. (A) To determine binding affinity, transfected
cells were incubated with 100 pmol/L of 125I-IL-4 with or
without various concentrations of unlabeled IL-4. Scatchard data were
analyzed by the LIGAND program (B). In four experiments, the binding
data were fitted with only one site model.
|
|
We also analyzed the binding affinity of IL-4R in CHO-K1 cells
transfected with IL-4R, IL-4R plus IL-13R or , or
c by Scatchard analysis using the LIGAND
program. This program allowed fitting of our data in only a one-site
model. In four separate experiments, the data did not fit in a two-site
model. As shown in Fig 1B, IL-4 bound to IL-4R-transfected cells
with high to intermediate affinity (Kd = 0.69 nmol/L); however, when
this chain was cotransfected with IL-13R or c,
the affinity was approximately threefold higher (Kd = 0.21 nmol/L or
0.20 nmol/L, respectively) compared with -transfected cells. In the
cells transfected with , , and c chains
(not shown), the affinity was similar to - or
c-transfected cells (Fig 1B). We also transfected the IL-13R chain14 into CHO cells; however, the IL-13R
chain did not bind to 125I-IL-4 (Fig 1A), and
cotransfection with IL-4R did not appear to modulate IL-4R binding
affinity (Fig 1B). These results suggest that IL-13R but not
IL-13R is a novel component of the IL-4R system and it forms a
high-affinity IL-4 receptor with IL-4R as c does.
Affinity cross-linking of 125I-IL-4 to CHO-K1 cells
transfected with IL-13, IL-4R, and c.
To visualize 125I-IL-4 binding to different chains, we
cross-linked 125I-IL-4 to IL-4 receptors on CHO-K1 cells
transfected with IL-4R alone or cotransfected with IL-13,
c, or both. As shown in Fig
2, 125I-IL-4 cross-linked to a major protein of
approximately 155 kD in the IL-4R chain transfectant (; lane 1).
A faint, broad, approximately 70-kD band was also detected (lane 1, white arrow). After the subtraction of the size of IL-4 (15 kD), these
proteins were estimated to be 140 kD and approximately 55 kD. These
data suggest that the 140-kD protein is a transcript of human IL-4R cDNA and the approximately 55-kD band may be a degradation product of
IL-4R.23 These bands completely disappeared when the
cells were coincubated with 200-fold mol/L excess of unlabeled IL-4 (lane 2), and this binding was not displaced by 200-fold mol/L excess
of IL-13 (lane 3). No cross-linking of radiolabeled IL-4 was seen in
cells transfected with vector alone (data not shown). In the
IL-13R and the IL-4R transfectant (), an
additional broad band was seen at 80 to 100 kD along with a 155-kD band
(lane 4, white arrowhead). This band corresponded to the
IL-13R chain (65-85 kD). These bands completely disappeared
when cross-linking was performed in the presence of 200 mol/L excess of
unlabeled IL-4 (lane 5). These bands also partially disappeared when
cross-linking was performed in the presence of 200 mol/L excess of
unlabeled IL-13 (lane 6). In the IL-4R and c
transfectant (c), an additional, approximately 80-kD
band (c, approximately 65 kD, arrowhead), was observed
along with a 155-kD band (lane 7). These bands completely disappeared
when unlabeled IL-4 (lane 8) but not unlabeled IL-13 (lane 9) was
added. In the triple chain transfectant
(c), there were two bands at 155 kD and
approximately 80 kD as in the c transfectants (lane
10). As observed with binding data, a 200-mol/L excess of IL-4
completely displaced 125I-IL-4 binding (lane 11); however,
IL-13 only partially displaced 125I-IL-4 binding to these
cells (lane 12).
View larger version (108K):
[in this window]
[in a new window]
| Fig 2.
Affinity cross-linking of IL-4R in transfected CHO-K1
cells. Two micrograms of cDNA for IL-4R (lanes 1, 2, and 3),
IL-4R plus IL-13R (lanes 4, 5, and 6), IL-4R plus
IL-2Rc (lanes 7, 8, and 9), and all three chains
(, , and c; lanes 10, 11, and 12) were
transfected to CHO-K1 cells. Transfected cells (5 × 106)
were incubated with 125I-IL-4 in the absence (lanes 1, 4, 7, and 10) or presence of excess unlabeled IL-4 (lanes 2, 5, 8, and 11)
or IL-13 (lanes 3, 6, 9, and 12) for 2 hours at 4°C. Bound
125I-IL-4 was cross-linked to IL-4R with (DSS). The cells
were then lysed at 4°C with modified RIPA buffer. The resulting
lysate was analyzed by electrophoresis through an SDS-PAGE (7%) gel.
The gel was dried and exposed to radiograph film for 4 days at
80°C. The molecular weight markers are shown on the left. The
positions of different receptor chains are indicated (IL-4R, black
arrow; IL-13R, white arrowhead; IL-2Rc, black
arrowhead; and approximately 55 kD, white arrow).
|
|
Immunoprecipitation of IL-4R, IL-13R, and
c chains.
To unequivocally show whether IL-4R interacts/associates with
IL-13R, we immunoprecipitated 125I-IL-4
cross-linked proteins from the four combinations of transfectants (,
, c, and c)
by using specific antibodies for appropriate chains. Because an
antibody for IL-13R is not commercially available, we made an
expression plasmid for the IL-13R chimera protein with a
c-Myc epitope tag at C-terminus. The transfectants were cross-linked
with 125I-IL-4 and lysed as described previously. The cell
lysates were incubated with Protein A (G)-sepharose beads conjugated
with anti-IL-4R, -c-Myc, or -c antibody, and
immunoprecipitants were electrophoresed on 8% SDS-PAGE. As shown in
Fig 3, when immunoprecipitated with the
IL-4R antibody (P7), a sharp 155-kD band and a diffuse and faint
approximately 70-kD band were detected in chain transfectants (lane
1). In transfectants, an additional diffuse broad 110- to
130-kD band was detected (lane 2). Because this IL-13R chain has six repeats of c-Myc tag, the size of this protein is larger than
that of IL-13R in affinity cross-linking data. In
c transfectants, an additional dark, approximately
80-kD band was detected (lane 3), which corresponded to the
c chain. In c transfectants, a 155-kD band was detected along with additional, approximately 130-kD,
80-kD, and 70-kD bands, although additional bands were faint and
diffuse (lane 4). When c-Myc antibody was used for IL-13R immunoprecipitation, a 155-kD and a 130-kD doublet were detected in
only and c transfectants
(lane 6 and 8). On the other hand, no band was observed in and
c transfectants (lane 5 and 7). All four combinations
of transfectants were also immunoprecipitated by anti-c
antibody to show c interactions with IL-4R. A 155-kD and an approximately 80-kD protein was detected in c
and c transfectants (lane 11 and 12),
whereas no band was detected in and transfectants
(lane 9 and 10). These results indicate that IL-13R
associates with IL-4R as c does. Moreover, in triple
chain transfectants (c), two types of
heterodimers ( and c) are probably
induced, but there is no evidence for heterotrimer
(c).
View larger version (86K):
[in this window]
[in a new window]
| Fig 3.
The IL-13R chain associates with IL-4. cDNA
for IL-4R (lanes 1, 5, and 9), IL4R plus IL-13R (lanes
2, 6, and 10), IL-4R plus IL-2Rc (lanes 3, 7, and
11), and all three chains (, , and c; lanes
4, 8, and 12) was transfected to CHO-K1 cells. Transfected cells were
incubated with 1 nmol/L of 125I-IL-4.
125I-IL-4/IL-4R cross-linked complex was
immunoprecipitated from the cell lysate at 4°C by incubating with
protein A (G) sepharose beads that had been preincubated with
anti-IL-4R (P7), c, or c-Myc antibodies. The
resulting complex was washed five times with lysing buffer, resuspended
with reducing buffer, and analyzed by 8% SDS-PAGE as described
previously. The molecular weight markers are shown on the left. The
position of different receptor chains is indicated (IL-4R, black
arrow; IL-13R, white arrowhead; IL-2Rc, black
arrowhead; and approximately 55 kD, white arrow).
|
|
Activation of STAT6 in response to IL-4 in IL-4R chain transfectants.
To determine whether and heterodimer complexes are
biologically functional, we analyzed STAT activity in response to IL-4
in various transfectants. It has been shown that IL-4 activates only
STAT6 protein in various cell types.8,24-26 As shown in Fig 4A, single chain transfectants of
, c, or vector alone did not activate
STAT6-DNA binding activity (lanes 1, 2, and 4), whereas in some
experiments, in chain transfectants, STAT6 was activated at low
levels (lane 3). The activation of STAT6 in response to IL-4 was
optimal when the chain was cotransfected with (lane 5).
As expected, STAT6 activation was also seen in c
(lane 6) and c (lane 8) transfectants. We
also investigated whether IL-13R can induce STAT6-DNA binding in
response to IL-4. Any combination of the IL-13R chain failed to
generate a STAT6-DNA binding complex (lane 9-12). These results suggest
that combination is sufficient to activate STAT6 protein
in a comparable manner with the c or
c combination.
View larger version (93K):
[in this window]
[in a new window]
View larger version (88K):
[in this window]
[in a new window]
| Fig 4.
Cotransfection of the IL-13R but not the
IL-13R chain with the IL-4R chain is sufficient to reconstitute
STAT activation in response to IL-4. CHO-K1 cells were transfected with
various chains and then incubated with IL-4 (50 ng/mL) for 10 minutes, washed with cold PBS, and solubilized with cold whole cell extraction buffer. Fifty micrograms of sample proteins were incubated for 20 minutes at room temperature with 1 ng of 32P-labeled SBE1
probe in binding buffer. Then, samples were loaded on a 4% nonreducing
polyacrylamide gel and run at 150 V for 2 hours (A). For supershift
assay, antimouse STAT6 (anti m-STAT6) or antihuman STAT6 (anti h-STAT6)
rabbit polyclonal IgG was included in the reaction mixture before
electrophoresis. The gel was dried and analyzed by autoradiography (B).
|
|
To confirm whether IL-4-induced SBE1-binding complex contains STAT6,
an antibody supershift assay was performed. We used two different STAT6
antibodies because cross-reactivity of antibodies to CHO cell-derived
STAT6 was not known. Antibody to mouse STAT6 caused a small shift,
whereas antibody to human STAT6 caused a substantial shift in the
electrophoretic mobility of the SBE1-binding activity (Fig 4B). These
data confirm that the IL-4-induced SBE-1-binding complexes indeed
contain STAT6 molecule.
JAK1 and JAK2 are required for the activation of STAT6 in response to
IL-4 in but not chain transfectants.
We have previously reported that JAK1 and JAK2 are phosphorylated and
activated in response to IL-4 in nonhematopoietic cells that do not
express c, whereas JAK3 is phosphorylated in response to
IL-4 in immune cells that do express
c.8,24,26 To show whether JAK1, JAK2, or
both, are required for STAT6 activation in response to IL-4 in the
cells that express or c chains, we
cotransfected wild type JAK1 (wtJAK1), JAK2 (wtJAK2), or both or
kinase-deficient JAK1 ( JAK1), JAK2 (JAK2), or both with
or c chains into CHO-K1 cells. STAT6
activation was then assessed by EMSA. In transfectants,
IL-4 induced activation of STAT6 when either wtJAK1, wtJAK2, or both
were coexpressed (Fig 5A, lanes 1-6). When
JAK1 or JAK2 were coexpressed with chains, the
activation of STAT6 in response to IL-4 was inhibited (Fig 5, lanes 7 and 9). However, when chains were cotransfected with both
JAK1 and JAK2 the STAT6 activation was completely blocked (lane
11). On the other hand, in cells transfected with c
and JAK1, JAK2, or both, the activation of STAT6 in response to
IL-4 was only partially inhibited (Fig 5B, lanes 7, 9, and 11). These
results suggested that both JAK1 and JAK2 tyrosine kinases are required
for optimal activation of STAT6 in response to IL-4 in the cells that
express chains of the IL-4R system. However, in cells
that express c chains of the IL-4R system, JAK2 is
not required whereas JAK1 is partially required for STAT6 activation by
IL-4.
View larger version (83K):
[in this window]
[in a new window]
View larger version (86K):
[in this window]
[in a new window]
| Fig 5.
JAK1 and JAK2 are required for the activation of STAT6 in
response to IL-4 in the cells that express IL-13R and
IL-4R chains of IL-4R. Wild type JAK1 (wtJAK1), JAK2 (wtJAK2), or
both, and kinase-deficient JAK1 ( JAK1), JAK2 (JAK2), or both,
were cotransfected with (A) the IL-4R chain and the IL-13R
() or (B) the IL-4R chain and c ()
as described in Materials and Methods. After 48 hours, the cells were
stimulated by IL-4 (50 ng/mL) and lysed in lysing buffer. Fifty
micrograms of sample proteins were incubated for 20 minutes at room
temperature with 0.5 to 1 ng of 32P-labeled SBE1 probe. The
samples were loaded on a 4% nonreducing polyacrylamide gel and run at
150 V for 2 hours. The gel was dried and analyzed by autoradiography.
|
|
| |
DISCUSSION |
In this report, we show that the IL-4R () and the
IL-13R () chains can induce high affinity binding
to IL-4 as does the + c complex, although the
chain by itself does not bind IL-4. By using chemical
cross-linking studies, we also show that the chain is
associated with the chain in response to IL-4. Thus, the IL-4R
chain can form a complex with either the chain or
c, but whether all three chains form a trimeric complex
is not clear. Because antibody to anti-c-Myc-tagged IL-13R
or c did not immunoprecipitate all three chains, our
data suggest that IL-4 does not seem to cross-link to all three chains
simultaneously.
In contrast to the participation of IL-13R, IL-13R did not
seem to participate in the formation of the IL-4R complex. IL-13R chain transfectants neither bound 125I-IL-4 nor increased
binding affinity to IL-4 when coexpressed with IL-4R. These results
suggest that IL-13R is not a component of the IL-4R system.
We have previously reported that in nonhematopoietic cancer cells, a
high number of IL-4R are expressed.2,3,8,23 Although the
c chain is not expressed in these cells, IL-4R is still
functional and STAT6 protein is activated in response to
IL-4.8 IL-13R was also expressed in these cells and IL-13
inhibited 125I-IL-4 binding to its
receptor.8,25 Thus, we hypothesized that part of IL-13R may
be a functional component for the IL-4R system. Our current data
confirm this hypothesis.
Two types of patterns were observed when excess of IL-4 or IL-13 were
included to compete for 125I-IL-4 binding or cross-linking
in various transfectants. In the first pattern, IL-4 completely blocked
the binding of 125I-IL-4, whereas IL-13 did not inhibit
125I-IL-4 binding in cells expressing alone, ,
or c. In the second pattern, IL-13 significantly
inhibited binding and cross-linking of 125I-IL-4, although
not completely, in cells expressing or
c. The mechanism of partial inhibition of
125I-IL-4 binding by IL-13 is not completely clear. We
believe that in the presence of all three chains, IL-4 has an option to
form a complex with either IL-4R and c or IL-4R
and IL-13Rchains. Because IL-13R does not bind to
IL-13 in the absence of IL-4R9,12,15,16 and because
IL-4R is most likely sequestered in the complex with
c to form a high affinity IL-4 receptor, IL-13 is unable to block 125I-IL-4 binding completely. Similarly, in
IL-4R and IL-13R transfectant IL-4 will have higher
affinity than IL-13. Thus, IL-4 will completely block but IL-13 will
partially block 125I-IL-4 binding. Finally, the lack of
competition of IL-4 binding by IL-13 in , , or
c transfectants can be explained at least partly by
the lack of binding of IL-13 to the or c chain
(Murata and Puri, unpublished data). These data agree with our previous report in which IL-13 did not compete for the binding of IL-4 in Raji
and MLA144 cell lines.12
It is of interest to note that IL-4R heterodimerization is quite
different from the IL-2R or IL-3R system. In these receptors, IL-2R
or IL-3R has no binding affinity to IL-2 or IL-3 respectively; however, when complexed with the IL-2R or IL-3R chain it can form a high-affinity receptor. Moreover, chains are important for
their signal transduction. On the other hand, the IL-4R chain has an
intermediate affinity by itself and oligomerization with IL-13R or c only causes a modest twofold to
threefold increase in binding affinity to IL-4 (Fig 1B). Moreover, in
c transfectants, receptor affinity was not
significantly different from that of the or
c transfectants. These results suggest that the
accessory molecules ( and c) for IL-4R are not
critical for increasing the IL-4 receptor affinity.
To show whether the IL-4R and IL-13R chain complex was
effective in STAT6 activation, we examined STAT6 activation in various transfectants. Our data indicate that the IL-4R chain by itself can
bind to IL-4 and when overexpressed alone can cause modest activation
of STAT6 protein. These data are consistent with recent reports that
showed that homodimerization of the IL-4R chain can cause STAT6
activation.27,28 The activation of the STAT6 protein was
robust when the chain was cotransfected with the chain
and this activation level was similar to that caused by the
c combination. Recently, we have observed that the
chain is always expressed along with the IL-4R chain in
cells that lack c expression29 and STAT6 is
activated in response to IL-4 without c in some
nonhematopoietic cancer cell lines.8,25 These results
confirm that the IL-13R chain is a functional component of
the IL-4R system and that the chain requires either or
c for STAT6 activation. Unlike the chain,
transfection of chain along with the chain did not cause
activation of STAT6 protein indicating that IL-13R is not a
functional component of the IL-4R complex.
It is still unknown why oligomerization is used for the
functional IL-4R system in nonhematopoietic cells. The signaling pattern by both combinations of chains (+ or
+c) in response to IL-4 is identical except for the
JAK kinases used. In nonhematopoietic cells that lack c,
JAK1, JAK2, and Tyk2 are phosphorylated and activated in response to
IL-4. On the other hand, JAK3 is phosphorylated instead of JAK2 in
hematopoietic cells that express c. We showed here that
JAK1 and JAK2 are required for STAT6 activation in response to IL-4 in
the cells that express chains of IL-4R but are not
required in the cells that express c chains (Fig 5).
Interestingly, the substrates for these JAK kinases (STAT6 and
IRS-1/IRS-2) are phosphorylated and activated in both cases. Thus, it
is possible that STAT6 and IRS-1/IRS-2 pathways are common for the
function of IL-4R in hematopoietic and nonhematopoietic cells. It is
also possible that other unknown pathway(s) of signaling may contribute to specific functions of IL-4. Alternatively, the
heterodimer is used by both the IL-4 and the IL-13 receptor system and
the c heterodimer is used by only the IL-4 receptor
system as shown in T cells. In T cells, IL-13 does not compete for IL-4
binding, and these cells do not express IL-13R nor do they respond to
IL-13. Additional investigations are required to show the role of the two different types of IL-4R system.
In summary, we have reconstituted a functional IL-4R in CHO-K1 cells by
transfecting two different IL-13R chains ( and ), IL-4R, and c chains. We provide experimental evidence
for the first time that IL-13R, but not IL-13R can
associate with the IL-4R chain, and heterodimerization
is sufficient to elicit STAT6 activation in response to IL-4. Moreover,
both JAK1 and JAK2 are required for optimal activation of STAT6 in
response to IL-4 in the cells that express the IL-13R chain
along with IL-4R but only partially in the cells that express
c. These results suggest that the IL-13R
chain is a functional component of the IL-4R system, and that may, in
part, be responsible for the redundant biological response of IL-4 and
IL-13.
| |
FOOTNOTES |
Submitted June 25, 1997;
accepted January 9, 1998.
Address reprint requests to Raj K. Puri, MD, PhD, Laboratory of
Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologic Evaluation and Research, FDA, NIH-Building 29B,
Room 2NN10, 29 Lincoln Drive, MSC 4555, Bethesda, MD 20892-4555.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr G. Johnson for reading the manuscript, Dr N.I. Obiri for
labeling IL-4 and reading the manuscript, Dr Waldemar Debrinski for
IL-13, Dr T. Tomoda for providing PME18S and CS+MT plasmids, Dr W. Leonard for IL-13R and c cDNA, Dr D.M.
Wojchowski for pBOSJAK2 and pBOSJAK2VIII, Dr S. Watanabe for
plasmids for JAK1 and mutant JAK1, Dr Pasual Ferrara for IL-13R, Dr
Ray Donnelly for SBE1 probe, Dr S.R. Husain for helpful comments on
this manuscript and Ms P. Dover for excellent technical assistance.
| |
REFERENCES |
1.
Paul WE:
Interleukin-4: A prototypic immunoregulatory lymphokine.
Blood
77:1859,
1991[Free Full Text]
2.
Obiri NI,
Hillman G,
Haas GP,
Sudha S,
Puri RK:
Expression of high affinity Interleukin 4 receptors on human renal carcinoma cells and inhibition of tumor growth in vitro by Interleukin 4.
J Clin Invest
91:88,
1993
3.
Obiri NI,
Siegel J,
Varricchio F,
Puri RK:
Expression of high affinity IL-4 receptors on human melanoma, ovarian and breast carcinoma cells.
Clin Exp Immunol
95:148,
1994[Medline]
[Order article via Infotrieve]
4.
Lowenthal JW,
Castle BE,
Christiansen J,
Schreurs J,
Arai N,
Hoi P,
Takabe Y,
Howard M:
Expression of high affinity receptors for murine interleukin 4 (BSF-1) on hematopoietic and nonhematopoietic cells.
J Immunol
140:456,
1988[Abstract]
5.
Park LS,
Friend D,
Sassenfeld HM:
Characterization of the human B cell stimulatory factor 1 receptor.
J Exp Med
166:476,
1987[Abstract/Free Full Text]
6. Ohara J: Interleukin 4 Molecular structure and biochemical
characteristics, biological function and receptor expression, in Cruse
JM, Lewis REJ (eds): The Year in Immunology. Immunoregulatory Cytokines
and Cell Growth (vol 5). Basel, Switzerland, Basel Karger, 1989
7.
Idzerda RL,
March CJ,
Mosley B,
Lyman SD,
Bos TV,
Gimpel SD,
Din WS,
Grabstein KH,
Widmer MB,
Park LS,
Cosman D,
Beckmann MP:
Human interleukin 4 receptor confers biological responsiveness and defines a novel receptor superfamily.
J Exp Med
171:861,
1990[Abstract/Free Full Text]
8.
Murata T,
Noguchi PD,
Puri RK:
Receptors for interleukin (IL)-4 do not associate with the common chain, and IL-4 induce the phosphorylation of JAK2 tyrosine kinase in human colon carcinoma cells.
J Biol Chem
270:30829,
1995[Abstract/Free Full Text]
9.
Obiri NI,
Leland P,
Murata T,
Debinski W,
Puri RK:
The IL-13 receptor structure differs on various cell types and may share more than one component with IL-4 receptor.
J Immunol
158:756,
1997[Abstract]
10.
Kondo M,
Takeshita T,
Ishi N,
Nakamura M,
Watanabe S,
Arai K,
Sugamura K:
Sharing of interleukin-2 (IL-2) receptor chain between receptors for IL-2 and IL-4.
Science
262:1874,
1993[Abstract/Free Full Text]
11.
Russell SM,
Keegan AD,
Harada N,
Nakamura Y,
Noguchi M,
Leland P,
Friedmann MC,
Miyajima A,
Puri RK,
Paul WE,
Leonard WJ:
Interleukin-2 receptor gamma chain: A functional component of the interleukin-4 receptor.
Science
262:1880,
1993[Abstract/Free Full Text]
12.
Obiri NI,
Debinski W,
Leonard WJ,
Puri RK:
Receptor for interleukin 13. Interaction with interleukin 4 by a mechanism that does not involve the common gamma chain shared by receptors for interleukins 2, 4, 7, 9, and 15.
J Biol Chem
270:8797,
1995[Abstract/Free Full Text]
13.
Lin J-X,
Migone T-S,
Tsang M,
Friedmann M,
Weatherbee JA,
Zhgou L,
Yamauchi A,
Bloom ET,
Mietz J,
John S,
Leonard WJ:
The role of shared receptor motif and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15.
Immunity
2:331,
1995[Medline]
[Order article via Infotrieve]
14.
Caput D,
Laurent P,
Kaghad M,
Lelias J-M,
Lefort S,
Vita N,
Ferrara P:
Cloning and characterization of specific Interleukin (IL)-13 binding protein structurally related to the IL-5 receptor chain.
J Biol Chem
271:16921,
1996[Abstract/Free Full Text]
15.
Aman MJ,
Tayebi N,
Obiri NI,
Puri RK,
Modi WS,
Leonard WJ:
cDNA cloning and characterization of the human interleukin-13 receptor chain.
J Biol Chem
271:29265,
1996[Abstract/Free Full Text]
16.
Hilton DJ,
Zhang JG,
Metcalf D,
Alexander WS,
Nicola NA,
Willson TA:
Cloning and characterization of a binding subunit of the interleukin 13 receptor that is also a component of the interleukin 4 receptor.
Proc Natl Acad Sci USA
93:497,
1996[Abstract/Free Full Text]
17.
Turner DL,
Weintraub H:
Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate.
Genes Dev
8:1434-1447,
1994[Abstract/Free Full Text]
18.
Watanabe S,
Kubota H,
Sakamoto KM,
Arai K-I:
Characterization of cis-acting sequences and trans-acting signals regulating early growth response 1 and c-fos promoters through the granulocyte-macrophage colony-stimulating factor receptor in BA/F3 cells.
Blood
89:1197,
1997[Abstract/Free Full Text]
19.
Watanabe S,
Arai K-I:
Roles of the JAK-STAT system in signal transduction via cytokine receptors.
Curr Opin Genet Dev
6:587,
1996[Medline]
[Order article via Infotrieve]
20.
Zhuang H,
Patel SV,
He TC,
Sonsteby SK,
Niu Z,
Wojchowski DM:
Inhibition of erythropoietin-induced mitogenesis by a kinase-deficient form of Jak2.
J Biol Chem
269:21411,
1994[Abstract/Free Full Text]
21.
Munson PJ,
Rodbard D:
LIGAND: A versatile computerized approach for characterization of ligand-binding systems.
Anal Biochem
107:220,
1980[Medline]
[Order article via Infotrieve]
22.
Ohmori Y,
Smith MF Jr,
Hamilton TA:
IL-4 induced expression of Il-1 receptor antagonist gene is mediated by STAT6.
J Immunol
157:2058,
1996[Abstract]
23.
Obiri NI,
Puri RK:
Characterization of interleukin-4 receptors expressed on human renal cell carcinoma cells.
Oncol Res
6:419,
1995
24.
Murata T,
Puri RK:
Comparison of IL-13 and IL-4 induced signalling in EBV-immortalized human B cells.
Cell Immunol
175:33,
1997[Medline]
[Order article via Infotrieve]
25.
Murata T,
Obiri NI,
Puri RK:
Human ovarian carcinoma cell lines express IL-4 and IL-13 receptors: Comparison between IL-4 and IL-13 induced signal transduction.
Int J Cancer
70:230,
1997[Medline]
[Order article via Infotrieve]
26.
Quelle FW,
Shimoda K,
Thierfelder W,
Fischer C,
Kim A,
Ruben SM,
Cleveland JL,
Pierce JH,
Keegan AD,
Nelms K,
Paul WE,
Ihle JN:
Cloning of murine Stat6 and human Stat6, Stat proteins that are tyrosine phosphorylated in responses to IL-4 and IL-3 but are not required for mitogenesis.
Mol Cell Biol
15:3336,
1995[Abstract]
27.
Lai SY,
Molden J,
Liu KD,
Puck JM,
White MD,
Goldsmith MA:
Interleukin-4-specific signal transduction events are driven by homotypic interactions of the interleukin-4 receptor alpha subunit.
EMBO J
15:4506,
1996[Medline]
[Order article via Infotrieve]
28.
Kammer W,
Lischke A,
Moriggl R,
Groner B,
Ziemiecki A,
Gurniak CB,
Berg LJ,
Friedrich K:
Homodimerization of interleukin-4 receptor alpha chain can induce intracellular signaling.
J Biol Chem
271:23634,
1996[Abstract/Free Full Text]
29.
Murata T,
Obiri NI,
Debinski W,
Puri RK:
Structure of IL-13 receptor: Analysis of subunit composition in cancer and immune cells.
Biochem Biophys Res Commun
238:90,
1997[Medline]
[Order article via Infotrieve]
30.
Debinski W,
Obiri NJ,
Pastan I,
Puri RK:
A novel chimeric protein composed of IL-13 and Pseudomonas Endotoxin is highly cytotoxic to human carcinoma cells expressing receptors for IL-13 and IL-4.
J Biol Chem
270:16775,
1995[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
B. H. Joshi, P. Leland, A. Calvo, J. E. Green, and R. K. Puri
Human Adrenomedullin Up-regulates Interleukin-13 Receptor {alpha}2 Chain in Prostate Cancer In vitro and In vivo: A Novel Approach to Sensitize Prostate Cancer to Anticancer Therapy
Cancer Res.,
November 15, 2008;
68(22):
9311 - 9317.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. K. Konstantinidis, S. J. Barton, I. Sayers, I. A. Yang, J. L. Lordan, S. Rorke, J. B. Clough, S. T. Holgate, and J. W. Holloway
Genetic association studies of interleukin-13 receptor {alpha}1 subunit gene polymorphisms in asthma and atopy
Eur. Respir. J.,
July 1, 2007;
30(1):
40 - 47.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Simard, M.-L. Ricketts, S. Gingras, P. Soucy, F. A. Feltus, and M. H. Melner
Molecular Biology of the 3{beta}-Hydroxysteroid Dehydrogenase/{Delta}5-{Delta}4 Isomerase Gene Family
Endocr. Rev.,
June 1, 2005;
26(4):
525 - 582.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Kawakami, K. Kawakami, M. Kioi, P. Leland, and R. K. Puri
Hodgkin lymphoma therapy with interleukin-4 receptor-directed cytotoxin in an infiltrating animal model
Blood,
May 1, 2005;
105(9):
3707 - 3713.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Kawakami, M. Kawakami, and R. K. Puri
Specifically targeted killing of interleukin-13 (IL-13) receptor-expressing breast cancer by IL-13 fusion cytotoxin in animal model of human disease
Mol. Cancer Ther.,
February 1, 2004;
3(2):
137 - 147.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Kawakami, M. Kawakami, S. R. Husain, and R. K. Puri
Effect of Interleukin (IL)-4 Cytotoxin on Breast Tumor Growth after in Vivo Gene Transfer of IL-4 Receptor {alpha} Chain
Clin. Cancer Res.,
May 1, 2003;
9(5):
1826 - 1836.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Wood, M. J. Whitters, B. A. Jacobson, J. Witek, J. P. Sypek, M. Kasaian, M. J. Eppihimer, M. Unger, T. Tanaka, S. J. Goldman, et al.
Enhanced Interleukin (IL)-13 Responses in Mice Lacking IL-13 Receptor {alpha} 2
J. Exp. Med.,
March 17, 2003;
197(6):
703 - 709.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A.-L. Andrews, J. W. Holloway, S. M. Puddicombe, S. T. Holgate, and D. E. Davies
Kinetic Analysis of the Interleukin-13 Receptor Complex
J. Biol. Chem.,
November 22, 2002;
277(48):
46073 - 46078.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Kawakami, K. Kawakami, V. A. Stepensky, R. A. Maki, H. Robin, W. Muller, S. R. Husain, and R. K. Puri
Interleukin 4 Receptor on Human Lung Cancer: A Molecular Target for Cytotoxin Therapy
Clin. Cancer Res.,
November 1, 2002;
8(11):
3503 - 3511.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. H. Joshi, K. Kawakami, P. Leland, and R. K. Puri
Heterogeneity in Interleukin-13 Receptor Expression and Subunit Structure in Squamous Cell Carcinoma of Head and Neck: Differential Sensitivity to Chimeric Fusion Proteins Comprised of Interleukin-13 and a Mutated Form of Pseudomonas Exotoxin
Clin. Cancer Res.,
June 1, 2002;
8(6):
1948 - 1956.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. O. Rahaman, P. Sharma, P. C. Harbor, M. J. Aman, M. A. Vogelbaum, and S. J. Haque
IL-13R{alpha}2, a Decoy Receptor for IL-13 Acts As an Inhibitor of IL-4-dependent Signal Transduction in Glioblastoma Cells
Cancer Res.,
February 1, 2002;
62(4):
1103 - 1109.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Fukuda, Y. Fujitsu, N. Kumagai, and T. Nishida
Characterization of the Interleukin-4 Receptor Complex in Human Corneal Fibroblasts
Invest. Ophthalmol. Vis. Sci.,
January 1, 2002;
43(1):
183 - 188.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Kawakami, M. Kawakami, P. Leland, and R. K. Puri
Internalization Property of Interleukin-4 Receptor {alpha} Chain Increases Cytotoxic Effect of Interleukin-4 Receptor-targeted Cytotoxin in Cancer Cells
Clin. Cancer Res.,
January 1, 2002;
8(1):
258 - 266.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Kawakami, M. Kawakami, P. J. Snoy, S. R. Husain, and R. K. Puri
In Vivo Overexpression of IL-13 Receptor {alpha}2 Chain Inhibits Tumorigenicity of Human Breast and Pancreatic Tumors in Immunodeficient Mice
J. Exp. Med.,
December 17, 2001;
194(12):
1743 - 1754.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Blease, C. Jakubzick, J. M. Schuh, B. H. Joshi, R. K. Puri, and C. M. Hogaboam
IL-13 Fusion Cytotoxin Ameliorates Chronic Fungal-Induced Allergic Airway Disease in Mice
J. Immunol.,
December 1, 2001;
167(11):
6583 - 6592.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. H. Joshi, P. Leland, A. Asher, R. A. Prayson, F. Varricchio, and R. K. Puri
In Situ Expression of Interleukin-4 (IL-4) Receptors in Human Brain Tumors and Cytotoxicity of a Recombinant IL-4 Cytotoxin in Primary Glioblastoma Cell Cultures
Cancer Res.,
November 1, 2001;
61(22):
8058 - 8061.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Kawakami, M. Kawakami, B. H. Joshi, and R. K. Puri
Interleukin-13 Receptor-targeted Cancer Therapy in an Immunodeficient Animal Model of Human Head and Neck Cancer
Cancer Res.,
August 1, 2001;
61(16):
6194 - 6200.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. C. LAPORTE, P. E. MOORE, S. BARALDO, M.-H. JOUVIN, T. L. CHURCH, I. N. SCHWARTZMAN, R. A. PANETTIERI Jr, J.-P. KINET, S. A. SHORE, and S. A. SHORE
Direct Effects of Interleukin-13 on Signaling Pathways for Physiological Responses in Cultured Human Airway Smooth Muscle Cells
Am. J. Respir. Crit. Care Med.,
July 1, 2001;
164(1):
141 - 148.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Debinski, B. H. Joshi, G. E. Plautz, and R. K. Puri
Correspondence re: B. H. Joshi et al., Interleukin-13 Receptor {alpha} Chain: A Novel Tumor-associated Transmembrane Protein in Primary Explants of Human Malignant Gliomas. Cancer Res., 60: 1168-1172, 2000.
Cancer Res.,
July 1, 2001;
61(14):
5660 - 5660.
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Kawakami, J. Taguchi, T. Murata, and R. K. Puri
The interleukin-13 receptor {alpha}2 chain: an essential component for binding and internalization but not for interleukin-13-induced signal transduction through the STAT6 pathway
Blood,
May 1, 2001;
97(9):
2673 - 2679.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Umeshita-Suyama, R. Sugimoto, M. Akaiwa, K. Arima, B. Yu, M. Wada, M. Kuwano, K. Nakajima, N. Hamasaki, and K. Izuhara
Characterization of IL-4 and IL-13 signals dependent on the human IL-13 receptor {alpha} chain 1: redundancy of requirement of tyrosine residue for STAT3 activation
Int. Immunol.,
November 1, 2000;
12(11):
1499 - 1509.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Zaitsu, Y. Hamasaki, M. Matsuo, A. Kukita, K. Tsuji, M. Miyazaki, R. Hayasaki, E. Muro, S. Yamamoto, I. Kobayashi, et al.
New induction of leukotriene A4 hydrolase by interleukin-4 and interleukin-13 in human polymorphonuclear leukocytes
Blood,
July 15, 2000;
96(2):
601 - 609.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Kawakami, P. Leland, and R. K. Puri
Structure, Function, and Targeting of Interleukin 4 Receptors on Human Head and Neck Cancer Cells
Cancer Res.,
June 1, 2000;
60(11):
2981 - 2987.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Oshima, B. H. Joshi, and R. K. Puri
Conversion of Interleukin-13 into a High Affinity Agonist by a Single Amino Acid Substitution
J. Biol. Chem.,
May 5, 2000;
275(19):
14375 - 14380.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Gingras and J. Simard
Induction of 3{beta}-Hydroxysteroid Dehydrogenase/ Isomerase Type 1 Expression by Interleukin-4 in Human Normal Prostate Epithelial Cells, Immortalized Keratinocytes, Colon, and Cervix Cancer Cell Lines
Endocrinology,
October 1, 1999;
140(10):
4573 - 4584.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
D. Ford, C. Sheehan, C. Girasole, R. Priester, N. Kouttab, J. Tigges, T. C. King, A. Luciani, J. W. Morgan, and A. L. Maizel
The Human B Cell Response to IL-13 Is Dependent on Cellular Phenotype as Well as Mode of Activation
J. Immunol.,
September 15, 1999;
163(6):
3185 - 3193.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. L. Watson, A.-M. White, E. M. Campbell, A. W. Smith, J. Uddin, T. Yoshimura, and J. Westwick
Anti-Inflammatory Actions of Interleukin-13 . Suppression of Tumor Necrosis Factor-alpha and Antigen-Induced Leukocyte Accumulation in the Guinea Pig Lung
Am. J. Respir. Cell Mol. Biol.,
May 1, 1999;
20(5):
1007 - 1012.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
R. K. Puri
Toxicologic Pathology of Cytokines, Cytokine Receptors, and Other Recombinant Human Proteins: Development of a Recombinant Interleukin-4-Pseudomonas Exotoxin for Therapy of Glioblastoma
Toxicol Pathol,
January 1, 1999;
27(1):
53 - 57.
[Abstract]
[PDF]
|
|
|
|
|
|
|
Y. Oshima and R. K. Puri
Characterization of a Powerful High Affinity Antagonist That Inhibits Biological Activities of Human Interleukin-13
J. Biol. Chem.,
April 27, 2001;
276(18):
15185 - 15191.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract