From the Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, November 8, 2000, and in revised form, January 25, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interleukin-13 (IL-13), a predominantly
Th2-derived cytokine, appears to play a central pathological role in
asthma, atopic dermatitis, allergic rhinitis, some parasitic
infections, and cancer. We hypothesized that an IL-13 antagonist may
have profound therapeutic utility in these conditions. We, therefore,
mutagenized human IL-13 in which Glu at position 13 was substituted by
a Lys residue. This highly purified recombinant IL-13 variant,
IL-13E13K, bound with 4-fold higher affinity to the IL-13 receptor than
wild-type IL-13 but retained no detectable proliferative activity on
the TF-1 hematopoietic cell line. IL-13E13K competitively inhibited IL-13- and IL-4-dependent TF-1 proliferation. It also
inhibited IL-13-induced STAT-6 (signal transduction and activator of
transducer-6) activation in immune cells and cancer cells and reversed
IL-13-induced inhibition of CD14 expression on human primary monocytes.
These results demonstrate that high affinity binding and signal
generation can be uncoupled efficiently in a ligand receptor
interaction. These results also suggest that IL-13E13K may be a useful
antagonist for the treatment of allergic, inflammatory, and parasitic
diseases or even malignancies in which IL-13 plays a central role.
IL-131 binds to its
plasma membrane receptors on various cell types (1-10). We have
reported that IL-13Rs are overexpressed on a variety of human solid
cancer cell lines including renal cell carcinoma, AIDS-associated
Kaposi's sarcoma, ovarian carcinoma, prostate cancer, and malignant
glioma (1-9, 11-16). We have proposed that the IL-13R complex may
exist as three different types. Type I IL-13R appears to be composed of
IL-4R IL-13 regulates a variety of functions in immune cells (20-24). It has
been shown to play a prominent role in atopic dermatitis (25, 26),
allergic rhinitis (27), pulmonary asthma and related lung injury (22,
28, 29), hepatic fibrosis induced by schistosomiasis (30), and
susceptibility to Leishmania major infection (31) and
malignancies (2, 12, 32, 33). Therefore, it has been hypothesized that
blocking the effect of IL-13 can provide therapeutic benefit in these
pathological conditions.
Cytokine receptors for hematopoietic growth factors with a four
One of the difficulties in understanding the interaction between ligand
and the shared receptor subunits is that the subunit itself usually
binds its ligand with low affinity. To overcome this problem, numerous
cytokine antagonists have been generated by site-directed mutagenesis,
which has been shown to utilize heterodimeric receptor systems. These
mutants have clarified the crucial role of a particular residue in the
ligand-receptor interaction. Among them, various antagonistic muteins
including mGM-CSF (38, 39), hIL-5 (40), human IL-6 (41, 42), human
leukemia inhibitory factor (43, 44), human IL-15 (45), human and murine
IL-2, and human IL-4 (46, 47) have been produced. However, no
antagonist of murine or human IL-13 has been produced.
To produce an IL-13 antagonist, we created a mutation in the IL-13
molecule. The selection of the residue to be mutated was based on the
knowledge of mutants produced for the IL-4 family of lymphokines
(e.g. GM-CSF, IL-5, IL-4, and IL-13) (48, 49). When the
amino acid sequence of Materials--
Sequence-specific oligonucleotide primers were
synthesized at Bioserve Biotechnologies (Laurel, MD). The pET based
expression vector (Novagen, Madison, WI) was used for construction of
mutein clone. Plasmids were amplified in E. coli, DH5 Construction of Plasmids Encoding IL-13R112D and
IL-13E13K--
The mutagenesis of IL-13 gene was performed using
cDNA of wtIL-13 (20) as a template. Sense primer 5'-agg aga tat aca
tat gtc ccc agg ccc tgt gcc tcc ctc tac agc cct cag gaa gct cat tga gga-3' and antisense primer 5'-taa ttt gcc cga att cag ttg aac cgt ccc
tcg cg-3' were used to mutate Glu-13 to Lys and incorporate NdeI and EcoRI restriction enzyme sites at the 5'
and 3' termini, respectively. Construction of the expression vector for
IL-13R112D was described before (51). After subcloning the PCR
products, the fragment was restricted by NdeI and
EcoRI and inserted into an expression vector. We confirmed
the existence of mutation and restriction sites by sequencing of the plasmid.
Expression and Purification of Recombinant
Proteins--
Expression and purification of wtIL-13, IL-13 mutants,
and IL-4 was carried out by similar techniques as previously reported (51, 52). wtIL-13, IL-13 mutants, and IL-4 were produced in inclusion bodies.
Cell Proliferation Assays--
Proliferation assays were
performed as described previously (51, 53). Briefly, 1 × 104 TF-1 cells/well were cultured in 96-well plates in RPMI
with 5% fetal bovine serum. Varying concentrations of wtIL-13 or IL-4 and/or IL-13 mutein were added to the wells, and the cells were cultured for ~2 days. Tritiated thymidine (0.5 µCi) was added to
each well 6-12 h before the plates were harvested in a Skatron cell
harvester (Skatron, Inc., Sterling, VA). Glass fiber filter mats were
counted in a IL-13 Receptor Binding Studies--
wtIL-13 was labeled as
previously described (2, 51). The specific activity of radiolabeled
IL-13 was 26 µCi/µg. The equilibrium binding studies were performed
as described elsewhere (2, 51). Briefly, 5 × 105
cells in 100 µl of binding buffer were incubated at 4 °C for 2 h with 125I-IL-13 (200 or 500 pM) in the
absence or presence of various concentrations of unlabeled wtIL-13 or
IL-13 mutant. Receptor-bound 125I-IL-13 was separated from
unbound 125I-IL-13. The cell pellets were counted in a Electrophoretic Mobility Shift Assay--
EMSA was performed as
described before (7, 18, 51). After incubation with various
concentrations of wtIL-13 or IL-13 mutants for 15 min, THP-1 cells,
Ebstein-Barr virus-immortalized B cells, or KSY-1 cells were washed
with cold phosphate-buffered saline and solubilized with cold
whole-cell extraction buffer (1 mM MgCl2, 20 mM HEPES, pH 7.0, 10 mM KCl, 300 mM
NaCl, 0.5 mM dithiothreitol, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, and 20% glycerol). DNA-protein
interactions were assessed by electrophoretic mobility shift assay
using the Bandshift kit (Amersham Pharmacia Biotech) using the
32P-labeled double-stranded oligonucleotide probe (4.2 × 109 cpm/µg) SBE-1.
CD14 Regulation by IL-13--
Primary monocytes were cultured at
1 × 107 cells/ml for 48 h with 1 ng/ml wtIL-13
with or without 1 µg/ml IL-13E13K. Staining of the cells was
performed as described elsewhere (51). The fluorescence data were
collected on a FACScan/C32 (Becton Dickinson, San Jose, CA). The
results were analyzed with the CELLQuest (Becton Dickinson) program.
Protein Synthesis Inhibition Assay--
Protein synthesis
inhibition assay was performed as previously described (3, 51). In
brief, 1 × 103 PM-RCC or U251 cells/well were
cultured with various concentrations of IL-13PE38QQR incubated for
20-24 h at 37 °C, and then 1 µCi of [3H]leucine
(PerkinElmer Life Sciences) was added to each well and cultured
for an additional 4 h. For blocking experiments, wtIL-13 or IL-13
mutants and IL-13PE38QQR were added simultaneously. Finally, cells were
washed and harvested on a fiberglass filtermat, and cell associated
radioactivity was measured in a Sequence Alignment and Molecular Modeling of IL-13 Receptor CRH
Domain and IL-13--
The sequence of CRH domains of various cytokines
were aligned by the Bestfit program of GCG software (Genetics Computer
Group, Inc., Madison, WI). Helical wheel analyses were also performed using GCG software. Percent similarity and identity of extracellular domains between IL-13R
The coordinate of the CRH domain of the IL-4R Sequence Alignment of Recombinant Protein Isolation and Purification--
Recombinant
wtIL-13, IL-13E13K, and IL-13R112D in which the 112th Arg (R) residue
of IL-13 molecule was substituted for Asp (D) were expressed in
E. coli and purified from inclusion bodies as previously
described (51). After purification, each recombinant protein was
analyzed using SDS-polyacrylamide gel electrophoresis and stained with
Coomassie Blue. Each protein showed a prominent single band at ~13
kDa with purity of at least 95% (Fig.
2).
IL-13E13K Competes for the Binding of Radiolabeled
IL-13--
Binding studies were performed using U251 glioblastoma and
PM-RCC renal cell carcinoma cell lines, both of which express type I
IL-13 receptors (8). As expected, wtIL-13 displaced specific binding of
radiolabeled IL-13 (Fig. 3).
Interestingly, IL-13E13K also inhibited binding of
125I-IL-13 (Fig. 3). IL-13E13K was better in displacing
125I-IL-13 binding as compared with wtIL-13. In the
experiment shown, the EC50 (concentration causing 50%
inhibition of 125I-IL-13 binding) of wtIL-13 and IL-13E13K
on U251 cells was ~20 and 2.5 nM, respectively. On
PM-RCC, it was ~100 and 25 nM, respectively. Thus,
IL-13E13K appeared to show ~4.0-8-fold better binding avidity than
wtIL-13 in displacing 125I-IL-13 binding.
IL-13E13K Blocks Proliferative Activity of IL-13 and
IL-4--
TF-1 erythroleukemia cells proliferate in
response to IL-13 (51). We, therefore, measured proliferative activity
of wtIL-13 and IL-13E13K (Fig.
4A) either alone or in
combination of both (Fig. 4, B and C). As
expected, wtIL-13 stimulated the growth of TF-1 cells in a
concentration-dependent manner (51). In contrast, IL-13E13K
did not show any proliferative activity (Fig. 4B). This result indicated that inserting a mutation at position 13 completely suppressed its agonistic activity and that the amino acid residue at
position 13 seemed essential for the IL-13-induced proliferation of
TF-1 cells. To determine the effect of IL-13E13K on wtIL-13 induced
proliferation of TF-1 cells, we cultured cells in the presence of 1 µg/ml IL-13E13K and various concentrations of wtIL-13. Interestingly,
IL-13E13K blocked the mitogenic activity of wtIL-13 (Fig.
4B). This block of IL-13 mitogenic activity appeared to be
concentration-dependent (Fig. 4C). A
100-333-fold excess of IL-13E13K completely neutralized
wtIL-13-induced mitogenic activity. Because IL-4 has similar biological
activities to IL-13 and receptors for both cytokines share two subunits
with other (14, 18), we investigated whether IL-13E13K can also
suppress the mitogenic response induced by IL-4. As shown in Fig.
4D, IL-13E13K completely neutralized IL-4-induced mitogenic
activity.
IL-13E13K Can Neutralize the Down-regulation of CD14 Expression by
wtIL-13 on Human Primary Monocytes--
IL-13 has been shown to
down-regulate CD14 expression on monocytes (23, 51). Therefore, we
investigated whether IL-13E13K can nullify the down-regulating activity
induced by wtIL-13. As shown in Fig. 5,
wtIL-13 suppressed CD14 expression on monocytes, and IL-13E13K
completely neutralized the effect of wtIL-13. For example, IL-13
decreased the mean channel number (mean fluorescence intensity) in the
gated region from 591 to 492 (p < 0.01). IL-13E13K reversed this effect, and the mean channel number recovered to 600.
IL-13E13K Blocks Signal Transduction Induced by wtIL-13--
IL-13
has been shown to transduce signal through the Janus kinase and STAT
pathways (4, 7, 10, 13, 14, 18, 58, 59). STAT-6 is phosphorylated and
activated after IL-13 stimulation, which in turn regulates gene
transcription. We therefore tested whether IL-13E13K can block the
IL-13-induced signaling. THP-1 and Ebstein-Barr virus-immortalized B
cell lines express type II and type III IL-13 receptors, respectively,
whereas KSY-1 AIDS-associated Kaposi's sarcoma cells express type I
IL-13 receptors. In all cell types, both wtIL-13 and IL-13R112D induced
STAT-6 activation in a concentration-dependent manner.
IL-13R112D, a potent IL-13 agonist, also induced STAT-6 activation, and
it was 5-10-fold better than wtIL-13 as previously reported (51) (Fig.
6A). In sharp contrast,
IL-13E13K did not seem to stimulate STAT-6 activation even at very high
concentrations (50 ng/ml). Moreover, IL-13E13K blocked wtIL-13-induced
STAT-6 activation in the THP-1 cell line (Fig. 6B). 10 ng/ml
wtIL-13 appears to stimulate maximal activation of STAT-6; however, in
the presence of a 10-fold excess IL-13E13K, STAT-6
activation was moderately suppressed, and a 50-fold excess of IL-13E13K
almost completely blocked STAT-6 activation.
IL-13E13K Blocks Cytotoxicity Mediated by
IL-13PE38QQR--
We have previously produced a chimeric fusion
protein composed of wtIL-13 and a mutated form of
Pseudomonas exotoxin (PE38QQR) termed IL-13PE38QQR (1, 3, 6,
9, 11, 15, 17, 60). This cytotoxin is highly cytotoxic to IL-13
receptor-positive tumor cells in vitro and in
vivo (1, 3, 6, 9, 11, 15, 17, 60). IL-13PE38QQR mediates
cytotoxicity through binding to IL-13R and receptor internalization
(3); therefore, IL-13 receptor agonists and antagonists must be able to
displace the cytotoxicity of the chimeric fusion toxin. To demonstrate interaction between IL-13E13K and IL-13R, we tested whether IL-13E13K can block the cytotoxicity mediated by IL-13PE38QQR. As shown in Fig.
7A, IL-13PE38QQR mediated
cytotoxicity in a concentration-dependent manner, and
IL-13, IL-13R112D, or IL-13E13K blocked this cytotoxicity. The blocking
of cytotoxicity by IL-13, IL-13R112D, and IL-13E13K was observed in a
concentration-dependent manner in both cells studied (Fig.
7B). IL-13E13K appeared to be superior to wtIL-13 in the
neutralization of IL-13PE38QQR-induced cytotoxicity.
In this report we describe the production and characterization of
a powerful antagonist of IL-13 that was produced by site-directed mutagenesis of an amino acid in the predicted Interestingly, IL-13E13K not only suppressed IL-13-induced
proliferation of TF-1 cells, it also inhibited IL-4-induced mitogenic response in a dose-dependent manner. These results suggest
that IL-13E13K may interact with the IL-4R Various studies including the crystal structure of IL-4 and
its receptor components demonstrate that IL-4 interacts with IL-4R
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, IL-13R
1, and IL-13R
2 subunits (2, 5, 8, 14, 17-19).
Type II IL-13R is composed of IL-4R
and IL-13R
1, and this
arrangement also forms the Type II IL-4R (2, 5, 8, 14, 17, 18). In type
III IL-4R/IL-13R systems, IL-4R
, IL-13R
1, and IL-2R
subunits
are present. Whether IL-4 or IL-13 engage all three subunits
simultaneously for signaling is still not known.
-helix structure exist largely as homodimers or heterodimers (34).
For example, receptors for erythropoietin, thrombopoietin, granulocyte
colony-stimulating factor, growth hormone, prolactin, and leptin can
exist as homodimers (34). In this class of receptors, a single cytokine
binds to two identical subunits and causes receptor internalization and
signal transduction. Heterodimeric cytokine receptors generally consist
of a major cytokine binding subunit and a signaling subunit (or shared
subunit) (34). In this class of receptors, signaling subunits are often
shared with more than one cytokine. For example, gp130 receptor subunit
is shared with receptors for IL-6, IL-11, leukemia inhibitory factor,
ciliary neurotrophic factor, cardiotrophin-1, and oncostatin M. IL-2R
subunit is shared by IL-2, IL-4, IL-9, and IL-15 receptors.
The common
-subunit is shared by receptors for IL-3, IL-5, and
GM-CSF (34). Similarly, IL-4R
subunit is shared by the IL-4R and
IL-13R system (14, 35-37). In heterodimeric receptor systems, usage of
intracellular signaling mechanism(s) is generally also shared (34).
-helix A of these molecules was aligned, a
conserved structure was identified. Mutation in one of the Glu residue
in the conserved helical structure produced molecules with altered
interaction with their receptors, suggesting that this region is
critical for ligand-receptor interaction and signal transduction. For
example, when Glu-21, located in
-helix A of the mGM-CSF molecule,
was mutated to Ala, a mGM-CSF with decreased bioactivity was produced.
Similarly, when Glu-9, also located in
-helix A of the IL-4
molecule, was mutated to Lys (IL-4E9K), a dramatic inhibition in
binding to IL-4R
chain was observed (47). When amino acid residues
in
-helix A of IL-13, IL-4, mGM-CSF, and hIL-5 were aligned, Glu-13
in IL-13 was identified, which is located at the equivalent position in
IL-4, mGM-CSF, and hIL-5 (Fig. 1A). In addition, since IL-4
and IL-13 share two receptor chains with each other and Glu-9 in IL-4
molecule has been shown to interact with the IL-4R
chain, we
mutagenized Glu to Lys at this position. This molecule, IL-13E13K was
then expressed in Escherichia coli, purified, and
further characterized. We demonstrate that IL-13E13K has a 4-8-fold
higher binding affinity to IL-13R compared with wtIL-13. Interestingly,
IL-13E13K inhibited IL-13-induced signal transduction, cell
proliferation, and biological activities in many different cell types.
We conclude on the basis of these results that IL-13E13K is a novel
powerful antagonist that may have many clinical applications.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Life Technologies, Inc.), and DNA was extracted using plasmid
purification kits (Qiagen, Chatsworth, CA). Restriction endonucleases
and DNA ligase were obtained from New England Biolabs (Beverly, MA),
Life Technologies, Inc., Panvera (Madison, WI), and Roche Molecular
Biochemicals. TF-1 human erythroleukemia cell line was purchased from
ATCC (Manassas, VA). PM-RCC renal cell carcinoma cell line was
established in our laboratory (50). THP-1, human monocytic cell line,
TORY, virus-immortalized B cell, and KSY-1 AIDS-related Kaposi's
sarcoma cell line were obtained and maintained as previously described (51).
Plate counter (Wallac, Gaithersburg, MD).
counter (Wallac).
Plate counter (Wallac). The
concentration of IL-13PE38QQR at which 50% inhibition of protein
synthesis (IC50) occurred was calculated.
1 and IL-2R
subunits were 40.5 and 31.9%, respectively. These numbers indicate reasonable sequence similarity justifying the use of IL-2R
subunit as a template for modeling IL-13R
1 subunit. Conserved sequence patterns such as the
WSXWS motif and four conserved Cys residues between
-strands of IL-13R
1 and IL-2R
were perfectly aligned. The
alignment of sequences between IL-4 and IL-13 was also performed as
previously reported (20, 54). The similarity and identity of
-helix
A and D of IL-13 to the known structure of IL-4 was in a similar range,
as observed for IL-2R
and IL-13R
1 subunits. However, the
similarity of
-helix B and C could not be reasonably determined
(54).
subunit was also used
in our model that was obtained from protein data bank entry
1ILL. The model building and refinement procedures were based on
the procedure previously described in detail (55). An initial model was
built using the homology module of InsightII (Molecular Simulations
Inc., San Diego, CA). Small loops and splices were created and handled
such that the energy was kept at minimum for best model. The structures
were finally refined using the Discover program (Molecular Simulations
Inc., San Diego, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Helix A between IL-13, IL-4, mGM-CSF. and
hIL-5--
When the
-helix A of hIL-13, hIL-4, mGM-CSF, and hIL-5
were aligned, glutamic acid residues were found to be aligned perfectly in all of these molecules. In IL-13, this Glu was located at position 13 (Fig. 1A). Helical wheel
analysis of IL-13, IL-4, and mGM-CSF suggested that hydrophobic
residues clustered in one side of the
-helix (Fig. 1B).
These hydrophobic residues may be buried in the core of the molecules,
as shown for IL-4 and hGM-CSF (56, 57).
-Helix A of
hIL-5 did not show a cluster of hydrophobic residues (data not shown).
Further pattern analysis demonstrated that the aligned critical
residues are located in the other side of the hydrophobic buried
cluster. Based on these analyses and the fact that Glu in IL-4 and
mGM-CSF interact with shared receptor subunits, it is predicted that
Glu at position 13 in IL-13 molecule is the "hot residue," and it
may also interact with shared receptor subunit.
View larger version (28K):
[in a new window]
Fig. 1.
Prediction of the hot residue in the
-helix A of IL-13 molecule. A,
alignment of
-helices A between mGM-CSF, hIL-5, hIL-4, and hIL-13.
Helical residues are in bold. The structurally conserved
residues are boxed, whereas consensus buried hydrophobic
side chains are shown in reverse (white
characters in black box). *, marked Glu of mGM-CSF, hIL-5,
and hIL-4 were reported to interact with shared receptor subunits.
B, aligned
-helices of hIL-13, hIL-4, and mGM-CSF are
also shown in helical wheel projections (1 turn every 3.6 residues).
Hydrophobic and buried residues may constitute the inner core and are
indicated in a box. The aligned and proposed hot Glu are
marked with asterisk (*).
View larger version (41K):
[in a new window]
Fig. 2.
SDS-polyacrylamide gel electrophoresis
analysis of purified IL-13 and its mutants.
Approximately 500 ng of each purified cytokine was loaded per sample.
Proteins were detected using Coomassie Blue stain. M,
MultiMarkTM multicolored standard (Novex, San Diego, CA).
View larger version (23K):
[in a new window]
Fig. 3.
Competition for the binding of
125I-IL-13 by wtIL-13 and IL-13E13K. 5 × 105 PM-RCC or U251 cells/tube were incubated with 200 or
500 pM 125I-IL-13, respectively, with various
concentrations of unlabeled wtIL-13 or IL-13E13K. Data presented are
the total cell-bound 125I-IL-13, with error bars
representing the S.D. of duplicate determinations.
View larger version (36K):
[in a new window]
Fig. 4.
Proliferation of TF-1 cells induced by IL-13
or IL-13E13K. A, 10,000 TF-1 cells/well were cultured
in the presence or absence of various concentrations of wtIL-13 or
IL-13E13K for 52 - 54 h. B, the cells were cultured
with or without 1 µg/ml IL-13E13K and various concentrations of
wtIL-13 for 52 h. C, the cells were cultured with or
without 3 ng/ml wtIL-13 in the presence or absence of 300 ng/ml or 1 µg/ml IL-13E13K. D, the cells were cultured with or
without 0.1 ng/ml IL-4 in the presence or absence of 300 ng/ml or 1 µg/ml IL-13E13K. The cell proliferation was determined by
the uptake of [3H]thymidine in the dividing cells. Data
presented are the percentage of the count without cytokine stimulation
and the average of triplicate or quadruplicate samples, with
error bars representing the S.D. within a data set.
Experiments were repeated several times. E13K,
IL-13E13K.
View larger version (23K):
[in a new window]
Fig. 5.
Effect of IL-13E13K on
IL-13-induced down-modulation of CD14 expression on monocytes.
Primary elutriated monocytes (1 × 107/tube) were
cultured with or without 1 ng/ml wtIL-13 in the presence or absence of
1 µg/ml IL-13E13K for 48 h. Cells were then washed and stained
as described under "Experimental Procedures." Gated mean
fluorescence intensity (MFI) number is indicated in each
panel.
View larger version (63K):
[in a new window]
Fig. 6.
Activation of STAT-6 by IL-13 or IL-13E13K
in various cell lines. TORY, THP-1, or KSY-1
cells were incubated with 0, 1, 10, or 50 ng/ml wtIL-13 or IL-13E13K
for 15 min (A) or THP-1 cells were incubated with 0 or 10 ng/ml wtIL-13 or with 10 ng/ml wtIL-13 plus 100 or 500 ng/ml
IL-13E13K for 15 min (B). wtIL-13 and IL-13E13K were added
simultaneously. Cells were processed, and an electrophoretic mobility
shift assay was performed as described under "Experimental
Procedures."
View larger version (19K):
[in a new window]
Fig. 7.
Wild-type and mutant IL-13 block the
cytotoxic activity of IL-13PE38QQR on U251 and PM-RCC
cells. One thousand cells/well were cultured in
Leu-free media containing various concentrations of IL-13-cytotoxin
(IL-13PE38QQR) and 1 µg/ml wtIL-13 or its muteins (A) or
containing 1 ng/ml IL-13PE38QQR with or without various concentration
of wtIL-13 or its muteins (B) overnight before the addition
of 1 µCi of tritiated Leu. Cells were then incubated for 4 h and
harvested, and radioactivity was counted with a counter. The data
are the average of quadruplicate determinations, with the error
bars representing the S.D. within a data set. Experiments were
repeated several times.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix A of the IL-13
molecule. Our results demonstrate conclusively that Glu at position 13 in the IL-13 molecule is of crucial importance for the potency and
signal generation. The antagonistic activity of IL-13 mutein IL-13E13K
was determined based on (a) 4-8-fold better displacement of
125I-IL-13 binding on cancer cells, (b) the
inhibition of wtIL-13-induced proliferation of TF-1 cells,
(c) neutralization of wtIL-13-induced down-modulation of
CD14 expression in primary monocytes, (d) inhibition of
wtIL-13-induced activation of STAT-6 in monocytic cell line, and
(e) inhibition of cytotoxicity mediated by IL-13PE38QQR.
Thus, the antagonistic activities of IL-13E13K were evident in cells that expressed type I, type II, and type III IL-13 receptors.
subunit that is shared between IL-13R and IL-4R complexes (35-37). In that regard, IL-13E13K is similar to muteins of mGM-CSF and hIL-5. The mGM-CSF antagonist E21A
and the hIL-5 antagonist E13Q had mutations at the interface with
a shared signaling subunit (38-40). On the other hand, IL-4E9K, in which Glu-9 was substituted by Lys, did not result in an antagonist but was incapable of binding to purified IL-4R
-shared subunit (47).
IL-13E13K is different from hIL-6 and hIL-15 antagonists that have
mutations at
-helix D or near the C terminus of the molecule. These
antagonists were found to be very specific and could not suppress
bioactivity of the related cytokines that share a signaling subunit,
e.g. hIL-6 antagonists termed DFRD, DFFD, and DFLD did not
antagonize human oncostatin M or human leukemia inhibitory factor,
which share the gp130 subunit (41). Similarly, the antagonist of hIL-15
did not antagonize hIL-2 activity, which shares the IL-2R
subunit
with IL-15R (45).
and IL-2R
subunits (47, 56).
-Helix A of IL-4 molecule interacts with IL-4R
subunit, and
-helix D may interact with IL-2R
subunit. Because the conserved residues in the IL-2R
subunit are
well aligned with the IL-13R
1 subunit, we created a model of
interaction between the CRH domains of IL-13R
1, IL-4R
, and IL-13
(Fig. 8). This model was created based on
our hypothesis that IL-13 interacts with IL-13R
1 and IL-4R
subunits simultaneously. The model suggests that the receptor binding
interface of the IL-13 molecule is located (at least) in
-helix A
and D and that
-helix D interacts with IL-13R
1 subunit, and
-helix A may interact with IL-4R
subunit. Hot Glu in hIL-5,
mGM-CSF, and hIL-4 were proposed to interact with shared receptor
subunits (38-40, 46, 47, 61). Our model suggests that the hot Glu in
the IL-13 molecule may also interact with the IL-4R
subunit, which
is shared between the IL-13/IL-4 receptor system (Fig. 8). Future
studies involving a three-dimensional structure of IL-13 and a receptor
complex determined by x-ray crystallography will confirm these
hypotheses.
View larger version (55K):
[in a new window]
Fig. 8.
Predicted model for IL-13 interaction with
its receptors. Homology model of the CRH domain of IL-13R 1,
IL-4R
subunits, and IL-13 was created as described under
"Experimental Procedures." The three-dimensional model is shown as
a ribbon diagram. A and D indicate
-helix A and D of the IL-13 molecule, respectively. The figure was
prepared using the InsightII program.
The mechanism of antagonistic activity of IL-13 caused by single amino acid substitution is not known. This mutation may cause inappropriate aggregation of receptor subunits or intracellular signaling molecules. Alternatively, a mutation in IL-13 molecule may have created a novel surface in the ligand itself that binds the receptor and alters conformation in a manner incapable of signaling. Future studies will investigate these possibilities.
In conclusion, IL-13E13K is a powerful antagonist that may neutralize
the effect of IL-13 in various disease processes such as bronchial
asthma, allergic rhinitis, and atopic dermatitis. In addition, since
IL-13 is an autocrine growth factor for Hodgkin/Reed-Sternberg tumor
cells, it is possible that IL-13E13K may also play a significant role
in the therapy of Hodgkin's disease.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Kurt A Brorson (Center for Biologics Evaluation and Research, Food and Drug Administration) for valuable help in modeling studies, Dr. Bharat Joshi for reading this manuscript and providing radiolabeled IL-13, and Pamela Dover and Drs. S. Rafat Husain and Subhash Dhawan for reading this manuscript.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of
Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug
Administration, 29 Lincoln Dr., NIH Bldg. 29B, Rm. 2NN10, Bethesda, MD
20892. Tel.: 1-301-827-0471; Fax: 1-301-827-0449; E-mail:
puri@cber.fda.gov.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M010159200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
IL, interleukin;
IL-13, (human, otherwise noted) interleukin-13;
IL-13E13K, human
interleukin-13E13K mutein;
IL-13R112D, human interleukin-13R112D
mutein;
wtIL-13, wild-type IL-13;
STAT-6, signal transduction and
activator of transducer-6;
IL-13R, IL-13 receptor(s);
IL-4, (human
wild-type, otherwise noted in the text) interleukin-4;
IL-4R, IL-4 receptor(s);
IL-4R (also known as IL-4R
), IL-4 receptor
subunit;
IL-13R
1 (also known as IL-13R
'), IL-13 receptor
1
subunit;
IL-13R
2 (also known as IL-13R
), IL-13 receptor
subunit;
IL-2R
, human interleukin-2 receptor
subunit;
CRH
domain, cytokine receptor homology domain;
GM-CSF, granulocyte-macrophage colony-stimulating factor;
mGM-CSF, murine;
GM-CSF, hGM-CSF, human GM-CSF;
hIL-5, human IL-5.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Debinski, W., Obiri, N. I., Powers, S. K., Pastan, I., and Puri, R. K. (1995) Clin. Cancer Res. 1, 1253-1258[Abstract] |
2. |
Obiri, N. I.,
Debinski, W.,
Leonard, W. J.,
and Puri, R. K.
(1995)
J. Biol. Chem.
270,
8797-8804 |
3. |
Puri, R. K.,
Leland, P.,
Obiri, N. I.,
Husain, S. R.,
Kreitman, R. J.,
Haas, G. P.,
Pastan, I.,
and Debinski, W.
(1996)
Blood
87,
4333-4339 |
4. | Murata, T., Noguchi, P. D., and Puri, R. K. (1996) J. Immunol. 156, 2972-2978[Abstract] |
5. | Murata, T., Obiri, N. I., Debinski, W., and Puri, R. K. (1997) Biochem. Biophys. Res. Commun. 238, 90-94[CrossRef][Medline] [Order article via Infotrieve] |
6. | Husain, S. R., Obiri, N. I., Gill, P., Zheng, T., Pastan, I., Debinski, W., and Puri, R. K. (1997) Clin. Cancer Res. 3, 151-156[Abstract] |
7. | Murata, T., Obiri, N. I., and Puri, R. K. (1997) Int. J. Cancer 70, 230-240[CrossRef][Medline] [Order article via Infotrieve] |
8. | Obiri, N. I., Leland, P., Murata, T., Debinski, W., and Puri, R. K. (1997) J. Immunol. 158, 756-764[Abstract] |
9. | Maini, A., Hillman, G., Haas, G. P., Wang, C. Y., Montecillo, E., Hamzavi, F., Pontes, J. E., Leland, P., Pastan, I., Debinski, W., and Puri, R. K. (1997) J. Urol. 158, 948-953[Medline] [Order article via Infotrieve] |
10. | Murata, T., Husain, S. R., Mohri, H., and Puri, R. K. (1998) Int. Immunol. 10, 1103-1110[Abstract] |
11. |
Debinski, W.,
Obiri, N. I.,
Pastan, I.,
and Puri, R. K.
(1995)
J. Biol. Chem.
270,
16775-16780 |
12. | Obiri, N. I., Husain, S. R., Debinski, W., and Puri, R. K. (1996) Clin. Cancer Res. 2, 1743-1749[Abstract] |
13. |
Obiri, N. I.,
Murata, T.,
Debinski, W.,
and Puri, R. K.
(1997)
J. Biol. Chem.
272,
20251-20258 |
14. | Murata, T., Obiri, N. I., and Puri, R. K. (1998) Int. J. Mol. Med. 1, 551-557[Medline] [Order article via Infotrieve] |
15. | Debinski, W., Gibo, D. M., Obiri, N. I., Kealiher, A., and Puri, R. K. (1998) Nat. Biotechnol. 16, 449-453[Medline] [Order article via Infotrieve] |
16. |
Joshi, B. H.,
Plautz, G. E.,
and Puri, R. K.
(2000)
Cancer Res.
60,
1168-1172 |
17. |
Debinski, W.,
Miner, R.,
Leland, P.,
Obiri, N. I.,
and Puri, R. K.
(1996)
J. Biol. Chem.
271,
22428-22433 |
18. |
Murata, T.,
Taguchi, J.,
and Puri, R. K.
(1998)
Blood
91,
3884-3891 |
19. |
Aman, M. J.,
Tayebi, N.,
Obiri, N. I.,
Puri, R. K.,
Modi, W. S.,
and Leonard, W. J.
(1996)
J. Biol. Chem.
271,
29265-29270 |
20. | Minty, A., Chalon, P., Derocq, J. M., Dumont, X., Guillemot, J. C., Kaghad, M., Labit, C., Leplatois, P., Liauzun, P., Miloux, B., Minty, C., Casellas, P., Loison, G., Lupker, J., Shire, D., Ferrara, P., and Caput, D. (1993) Nature 362, 248-250[CrossRef][Medline] [Order article via Infotrieve] |
21. | McKenzie, A. N. J., Culpepper, J. A., Malefyt, R. D., Briere, F., Punnonen, J., Aversa, G., Sato, A., Dang, W., Cocks, B. G., Menon, S., Devries, J. E., Banchereau, J., and Zurawski, G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3735-3739[Abstract] |
22. |
Wills-Karp, M.,
Luyimbazi, J.,
Xu, X.,
Schofield, B.,
Neben, T. Y.,
Karp, C. L.,
and Donaldson, D. D.
(1998)
Science
282,
2258-2261 |
23. | Cosentino, G., Soprana, E., Thienes, C. P., Siccardi, A. G., Viale, G., and Vercelli, D. (1995) J. Immunol. 155, 3145-3151[Abstract] |
24. | Punnonen, J., Aversa, G., Cocks, B. G., McKenzie, A. N. J., Menon, S., Aurawski, G., Malefyt, R. D. W., and Vries, J. E. d. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3730-3734[Abstract] |
25. | Akdis, M., Akdis, C. A., Weigl, L., Disch, R., and Blaser, K. (1997) J. Immunol. 159, 4611-4619[Abstract] |
26. | Katagiri, K., Itami, S., Hatano, Y., and Takayasu, S. (1997) Clin. Exp. Immunol. 108, 289-294[CrossRef][Medline] [Order article via Infotrieve] |
27. | Pawankar, R. U., Okuda, M., Hasegawa, S., Suzuki, K., Yssel, H., Okubo, K., Okumura, K., and Ra, C. S. (1995) Am. J. Respir. Crit. Care Med. 152, 2059-2067[Abstract] |
28. |
Grunig, G.,
Warnock, M.,
Wakil, A. E.,
Venkayya, R.,
Brombacher, F.,
Rennick, D. M.,
Sheppard, D.,
Mohrs, M.,
Donaldson, D. D.,
Locksley, R. M.,
and Corry, D. B.
(1998)
Science
282,
2261-2263 |
29. |
Zhou, Z.,
Homer, R. J.,
Wang, Z. D.,
Chen, Q. S.,
Geba, G. P.,
Wang, J. M.,
Zhang, Y.,
and Elias, J. A.
(1999)
J. Clin. Invest.
103,
779-788 |
30. |
Chiaramonte, M. G.,
Donaldson, D. D.,
Cheever, A. W.,
and Wynn, T. A.
(1999)
J. Clin. Invest.
104,
777-785 |
31. |
Matthews, D. J.,
Emson, C. L.,
McKenzie, G. J.,
Jolin, H. E.,
Blackwell, J. M.,
and McKenzie, A. N. J.
(2000)
J. Immunol.
164,
1458-1462 |
32. |
Kapp, U.,
Yeh, W. C.,
Patterson, B.,
Elia, A. J.,
Kagi, D.,
Ho, A.,
Hessel, A.,
Tipsword, M.,
Williams, A.,
Mirtsos, C.,
Itie, A.,
Moyle, M.,
and Mak, T. W.
(1999)
J. Exp. Med.
189,
1939-1945 |
33. | Fricker, J. (1999) Mol. Med. Today 5, 463-463[CrossRef] |
34. | Nicola, N. A., and Hilton, D. J. (1999) Adv. Protein Chem. 52, 1-65 |
35. |
Izuhara, K.,
and Harada, N.
(1993)
J. Biol. Chem.
268,
13097-13102 |
36. |
Smerz-Bertling, C.,
and Duschl, A.
(1996)
J. Biol. Chem.
270,
966-970 |
37. | Wang, L., Keegan, A., Paul, W., Heidaran, M., Gutkind, J., and Pierce, J. (1992) EMBO J. 11, 4899-4908[Abstract] |
38. | Altmann, S. W., Patel, N., and Kastelein, R. A. (1995) Growth Factors 12, 251-262[Medline] [Order article via Infotrieve] |
39. |
Altmann, S. W.,
and Kastelein, R. A.
(1995)
J. Biol. Chem.
270,
2233-2240 |
40. | Tavernier, J., Tuypens, T., Verhee, A., Plaetinck, G., Devos, R., Heyden, J. V. D., Guisez, Y., and Oefner, C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5194-5198[Abstract] |
41. | Savino, R., Ciapponi, L., Lahm, A., Demartis, A., Cabibbo, A., Toniatti, C., Delmastro, P., Altamura, S., and Ciliberto, G. (1994) EMBO J. 13, 5863-5870[Abstract] |
42. | Savino, R., Lahm, A., Salvati, A., Ciapponi, L., Sporeno, E., Altamura, S., Paonessa, G., Toniatti, C., and Ciliberto, G. (1994) EMBO J. 13, 1357-1367[Abstract] |
43. |
Hudson, K. R.,
Vernallis, A. B.,
and Heath, J. K.
(1996)
J. Biol. Chem.
271,
11971-11978 |
44. |
Vernallis, A. B.,
Hudson, K. R.,
and Heath, J. K.
(1997)
J. Biol. Chem.
272,
26947-26952 |
45. |
Kim, Y. S.,
Maslinski, W.,
Zheng, X. X.,
Stevens, A. C.,
Li, X. C.,
Tesch, G. H.,
Kelly, V. R.,
and Strom, T. B.
(1998)
J. Immunol.
160,
5742-5748 |
46. | Kruse, N., Tony, H. P., and Sebald, W. (1992) EMBO J. 11, 3237-3244[Abstract] |
47. | Kruse, N., Shen, B., Arnold, S., Tony, H., Meuller, T., and Sebald, W. (1993) EMBO J. 12, 5121-5129[Abstract] |
48. |
Boulay, J.,
and Paul, W.
(1992)
J. Biol. Chem.
267,
20525-20528 |
49. | Paul, W. (1991) Blood 77, 1859-1870[Medline] [Order article via Infotrieve] |
50. | Obiri, N. I., Hillman, G. G., Haas, G. P., Sud, S., and Puri, R. K. (1993) J. Clin. Invest. 91, 88-93[Medline] [Order article via Infotrieve] |
51. |
Oshima, Y.,
Joshi, B. H.,
and Puri, R. K.
(2000)
J. Biol. Chem.
275,
14375-14380 |
52. | Kreitman, R. J., Puri, R. K., McPhie, P., and Pastan, I. (1995) Cytokine 7, 311-318[CrossRef][Medline] [Order article via Infotrieve] |
53. | Leland, P., Obiri, N., Aggarwal, B. B., and Puri, R. K. (1995) Oncol. Res. 7, 227-235[Medline] [Order article via Infotrieve] |
54. | Bamborough, P., Duncan, D., and Richards, W. G. (1994) Protein Eng. 7, 1077-1082[Abstract] |
55. | Greer, J. (1990) Proteins 7, 317-334[Medline] [Order article via Infotrieve] |
56. | Hage, T., Sebald, W., and Reinemer, P. (1999) Cell 97, 271-281[Medline] [Order article via Infotrieve] |
57. | Walter, M., Cook, W., Ealick, S., Nagabhushan, T., Trotta, P., and Bugg, C. (1992) J. Mol. Biol. 224, 1075-1085[Medline] [Order article via Infotrieve] |
58. | Murata, T., and Puri, R. K. (1997) Cell. Immunol. 175, 33-40[CrossRef][Medline] [Order article via Infotrieve] |
59. |
Murata, T.,
Noguchi, P. D.,
and Puri, R. K.
(1995)
J. Biol. Chem.
270,
30829-30836 |
60. |
Husain, S. R.,
and Puri, R. K.
(2000)
Blood
95,
3506-3513 |
61. | Kruse, N., Lehrnbecher, T., and Sebald, W. (1991) FEBS lett. 286, 58-60[CrossRef][Medline] [Order article via Infotrieve] |