Human B cells stimulated through both their
immunoglobulin and CD40 receptors up-regulate 745 ± 51 interleukin (IL)-13 ligand binding sites with an affinity of 0.91 ± 0.08 nM within 24 h. IL-13 binds primarily to
the IL-13R
1 with subsequent sequestration of the IL-4R
into the
complex. IL-13R
1 may also be found in those receptors capable of
binding IL-4.
chain (
c) participates in receptors
capable of binding IL-4 but is not found in association with bound
IL-13. Dimeric receptors composed of the IL-4R
complexed with either
the IL-13R
1 or
c occur simultaneously within defined B cell populations. mRNAs for all receptor constituents are
increased subsequent to immunoglobulin stimulation alone, while maximal expression of IL-13R
1 is more dependent upon co-stimulation of immunoglobulin and CD40 receptors. mRNA levels for IL-13R
1 vary over a wider range subsequent to surface stimulation than other receptor components. Although
c is not bound to IL-13 in
B cells under the conditions evaluated, it may influence IL-13 binding by competing with IL-13R
1 for association/sequestration with the
IL-4R
chain. IL-13R
2 does not participate in the IL-13 receptor that is up-regulated upon activation of quiescent tonsillar B lymphocytes, although mRNA for the protein may be found in the centroblastic fraction of tonsillar cells.
 |
INTRODUCTION |
Receptor structure for
IL-41 and IL-13 (1-5) has
been studied in diverse cell types including those of lymphoid,
epithelial, and mesenchymal derivation (6-13). Several configurations
for the IL-4 receptor have been postulated based on variations in the
smaller molecular weight component of a 140-kDa/70-kDa multimeric complex. In T cells, the 70-kDa entity has definitively been shown to
be
c, a protein that is shared among several receptor
types, including those for IL-2, IL-7, IL-9, and IL-15 (8, 14-19). A
70-kDa protein, distinct from
c, has been proposed as
part of a second variant of the IL-4 receptor. This moiety has been reported to also function as part of the IL-13 receptor (9, 11-13, 20,
21). Several reports of the cloning of distinct 70-kDa IL-13 binding
chains strengthen the proposed role of this component(s) in a shared
IL-4/IL-13 receptor configuration (22-26). The 70-kDa human protein
encoded by the cDNA isolated by Aman et al. (23), by
Miloux et al. (25), and by Gauchat et al. (26),
originally based on the murine cDNA isolated by Hilton et
al. (22), is herein referred to as IL-13R
1; the human protein encoded by the cDNA isolated by Caput et al. (24) is
referred to as IL-13R
2.
The receptor capable of binding IL-13 appears to vary structurally and
functionally depending upon the cell of origin (6, 11-13, 27). Human
cell populations that bind IL-13 with high affinity include several
renal cell carcinoma lines, the glioma line U251, and the ovarian
cancer cell line IGROV-1 (13, 28). It has recently been hypothesized
that in these cell lines isodimers of the IL-13 binding proteins may
result in the reported high affinity IL-13 binding (28). Multiple cell
types including the ovarian cancer cell line PA1, an epidermal
carcinoma line A431, a primate fibroblast line COS-7, and normal human
endothelial cells bind both IL-4 and IL-13, although some cell types
preferentially bind IL-4. It has been suggested from a combination of
functional and structural studies that these populations possess IL-13
receptors composed of an IL-13-specific binding component in
conjunction with IL-4R
(13). Evaluation of the erythroleukemia cell
line TF-1 suggests that the
c protein may participate as
part of a functional IL-13 receptor. A potential role for
c has also been suggested from studies evaluating
phorbol dibutyrate and ionomycin-stimulated B cells exposed to high
IL-13 concentrations (29). Whether
c acts directly as a
binding element or as part of the signal transduction mechanism in
these cells remains to be definitively determined.
Evaluation of the IL-13 receptor on human B cells has mainly focused
upon Epstein-Barr virus-transformed cell populations as well as
functional studies on B cells derived from X-SCID patients (21, 28,
30). Examination of IL-13 receptor-related proliferation has recently
been extended to cells derived from patients with non-Hodgkin's
lymphoma as well as partially purified B cells from reactive lymph
nodes (31). In the present series of experiments, normal, nonvirally
transformed, purified mature human B lymphocytes have been directly
evaluated both structurally and functionally. Prior examination of
receptor binding on Epstein-Barr virus-transformed or freshly isolated
B cells has been hindered by relatively low binding of radiolabeled
native IL-13 in part secondary to low receptor numbers (10, 11, 13).
Herein, these difficulties have been partially overcome by utilization
of a mutant IL-13 moiety that can be labeled to high specific activity
in association with specific external activation conditions that
substantially up-regulate ligand binding components (12). In addition,
we report the initial utilization of antibodies specific for the IL-13R
1 binding proteins with direct identification of those elements involved in cytokine binding and receptor coaggregation to
permit a detailed, direct assessment of receptor structure on normal
lymphoid cells.
 |
MATERIALS AND METHODS |
Cytokines and Reagents--
Recombinant human IL-2 (rhIL-2) was
obtained from Genzyme Corp. (Boston, MA), and rhIL-4, rhIL-6, rhIL-10,
rhIL-13, rhIL-15, and recombinant human tumor necrosis factor-
were
procured from PeproTech Inc. (Rocky Hill, NJ). F(ab')2
fragments of rabbit anti-human IgM antibody, specific for the µ chain, were obtained from Dako Corp. (Carpinteria, CA) and used at 10 µg/ml. Coupled rabbit anti-human IgM antibody, µ chain-specific,
was acquired from Irvine Scientific (Santa Anna, CA) and used in
proliferation experiments at 7.5 µg/ml. An anti-CD40 mouse monoclonal
antibody (32) was derived and purified from the G28-5 cell line
(American Type Culture Collection, Bethesda, MD). Anti-CD40 antibody
was used at 5 µg/ml in specified assays. Goat anti-human
and
anti-human
antibodies were purchased from Biosource International
(Camarillo, CA). Rabbit anti-human
and anti-human
antibodies
were obtained from Dako Corp. Anti-human
and anti-human
antibodies were covalently coupled to activated Immunobead Matrix from
Irvine Scientific (Santa Anna, CA) and used at 1.6 µg/ml. Polyclonal
antisera to a synthetic peptide derived from the deduced sequence of
IL-13R
1 was prepared in collaboration with Zymed, Inc. (San
Francisco, CA). The carboxyl-terminal peptides 379-397 of the deduced
IL-13R
1 sequence were utilized. [Phe43]IL-13-GYGY was
prepared as described previously (12). Na125I and
125I-labeled IL-4 (125I-IL-4) were purchased
from NEN Life Science Products. Anti-CD45FITC/CD14PE, anti-CD3FITC/CD19PE, anti-CD20FITC, and the isotype control
IgG1FITC/IgG1PE were obtained from Immunotech
(Westbrook, ME). FITC-conjugated anti-IgD, anti-IgM, and anti-IgG
antibodies were acquired from Caltag Laboratories (San Francisco, CA).
Anti-CD38PE and anti-CD44FITC were from Becton-Dickinson (San Jose,
CA).
Isolation of Tonsillar B Lymphocytes--
Tonsillar human B
lymphocytes,
98% pure, were prepared as described previously (33).
For specific experiments, tonsillar B lymphocytes were fractionated on
Percoll (Amersham Pharmacia Biotech) density gradients and separated
essentially as described (34) with the exception that Percoll mix
solution was substituted for Hanks' balanced salt solution. The
density of cells prepared in this manner may be categorized as high
(>1.094 g/ml of Percoll solution), moderate (>1.089 g/ml), or low
(>1.082 g/ml). Phenotypic analysis, performed on a minimum of 2 × 105 cells/evaluation, utilized a Becton-Dickinson
FACScan. High density tonsillar B lymphocytes are characterized
phenotypically as the following: 1 ± 1% CD3+,
99 ± 2% CD19+, 7 ± 2%
CD38bright+, 93 ± 2% CD38dim+, 85 ± 4% CD44+, 53 ± 6% IgD+, 15 ± 12% IgG+, and 65 ± 5% IgM+; moderate
density cells as 0 ± 0% CD3+, 99 ± 1%
CD19+, 23 ± 4% CD38bright+, 77 ± 4% CD38dim+, 66 ± 6% CD44+, 36 ± 5% IgD+, 35 ± 13% IgG+, and 54 ± 10% IgM+; and low density cells as 0 ± 0%
CD3+, 100 ± 0% CD19+, 56 ± 8%
CD38bright+, 44 ± 8% CD38dim+, 21 ± 2% CD44+, 8 ± 3% IgD+, 41 ± 14% IgG+, and 31 ± 12% IgM+.
Measurement of Proliferation--
Cell proliferation, as
measured by thymidine incorporation, was performed as described
previously (33). The S.E. values of triplicates within an individual
experiment were invariably less than 10%. The percentage of cells in S
phase was determined by propidium iodide staining and flow cytometric
analysis as described by Look et al. (35).
Radiolabeling and Binding of
[Phe43]IL-13-GYGY--
Biologically functional
125I-labeled [Phe43]IL-13-GYGY
(125I-IL-13, 60-100 µCi/µg) was prepared by methods
essentially as described previously (12). Modifications to the
procedure included a stepwise addition of four 2-µl aliquots of 30 nM chloramine T at 1-min intervals and fractionation of the
labeled product via Sephadex G-25 chromatography. Ligand affinity was
evaluated utilizing a two-step paradigm. Initially, the binding of the
radiolabeled ligand was determined to evaluate effects introduced by
the labeling procedure. The dissociation constant
(Kd) of the labeled ligand was then used to
calculate the affinity of the native ligand according to the procedures
of Munson et al. using Ligand software (36). The receptor
number and binding affinity were determined from the Scatchard and
displacement analyses of 3-5 replicate experiments. The receptor
competitions for specific cytokines were performed in RPMI medium
containing 0.25% bovine serum albumin with 2 nM
125I-labeled IL-4 and 30 nM
125I-labeled [Phe43]IL-13-GYGY as indicated
in the figure legends. The total binding reaction volume was 35 µl or
0.3 ml as indicated in figure legends.
Affinity Cross-linking of Receptor Complexes--
Activated B
lymphocytes were removed from culture; washed twice with RPMI 1640, 0.25% bovine serum albumin; and subsequently incubated with
radioiodinated IL-4 or IL-13-GYGY for 30 min at 37 °C or for 2-3 h
at 4 °C. The cells were incubated in the absence or presence of
excess unlabeled ligand. Reactions were stopped by the addition of cold
phosphate-buffered saline containing 0.1% azide and 100 µM Na3VO4. Receptor cross-linking
was performed by incubation of washed cells in 1 mM
disuccinimidyl suberate or BS3, at 14 or 4 °C,
respectively, for 20-30 min. The washed cells were resuspended in
lysis buffer (50 mM HEPES, pH 7.5, 0.5% Brij 96, 50 mM NaCl, 50 mM NaF, 5 mM EDTA, 1 mM Na3VO4, 2 mM PMSF, 10 mM aprotinin, 10 µg/ml leupeptin) incubated for 15-30
min on ice, and centrifuged for 15 min at 14,000 × g
at 4 °C. Supernatants were either boiled in Laemmli buffer and
analyzed by 7-7.5% SDS-PAGE or were further immunoprecipitated with
appropriate antibodies.
Immunoprecipitation of Cross-linked Receptor
Complexes--
Solubilized samples to be immunoprecipitated were
exposed overnight, at 4 °C, with either 10 µg of purified,
polyclonal rabbit anti-human receptor component antibody
(anti-IL-3R
1, anti-IL-4R
) or 3 µl of rat monoclonal anti-human
c ascites (Tugh4 kindly provided by Dr. Sagamura). The
anti-IL-4R
utilized was a rabbit polyclonal antiserum against a
C-terminal peptide corresponding to amino acids 801-820 of the mature
protein (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The
anti-IL-13R
1 antibody was prepared as described above.
Immunoprecipitated complexes, isolated with protein G-Sepharose beads,
were resolved by 7% SDS-PAGE.
Evaluation of mRNA Expression--
IL-13R
1, IL-13R
2,
IL-4R
, and
c mRNA levels were assessed using
RT-PCR with primers derived from published sequences (see below) (19,
23, 24, 37, 38). B lymphocytes (107) were harvested, and
total RNA was prepared using Trizol® (Life Technologies, Inc.) reagent
or RNeasy spin columns (Qiagen). 2-5 µg of total RNA were used as a
template for oligo(dT)-primed cDNA synthesis using a cDNA cycle
kit (InVitrogen, Carlsbad, CA or Life Technologies, Inc.).
cDNA quality and quantity were standardized using RT-PCR
for glyceraldehyde-3-phosphate dehydrogenase and/or
-actin (CLONTECH). A hot start technique was employed in
50-µl reactions with GeneAmp® buffer (Perkin-Elmer), 1.5 mM MgCl2, 200 µM GeneAmp
deoxynucleotide triphosphates (Perkin-Elmer), 5-10 µCi of
[
-32P]dCTP (Amersham Pharmacia Biotech),
and 1.5 units of ampliTaq® DNA polymerase (Perkin-Elmer). For
semiquantitative reactions, 30 cycles (94 °C for 1 min; 59 °C for
1 min; 72 °C for 1.5 min) of amplification were performed, and a
minimum of three dilutions were evaluated for each sample. Nested
reactions were performed using 5 µl of the primary reaction as
template for the secondary reaction. PCR products were separated by
electrophoresis on 2% agarose or 10% acrylamide gels, stained with
ethidium bromide, photographed, dried, and autoradiographed. Band
intensity was quantitated and presented in relative arbitrary
units.
For quantitative competitive PCR, cloned competitive substrates were
prepared from cDNA as described using the indicated primers (39). A
constant known amount of competitive substrate was added to each PCR
reaction, and the amount of cDNA was evaluated in reference to the
intensity of the competitive band. Amplicon number was calculated based
on the concentration of the competitive substrate. Sequences of the
primer pairs utilized are as shown in Table
I.
 |
RESULTS |
Comparative Efficacy of Cytokines in Stimulating the Proliferation
of Quiescent B Cells--
Mature human B cells were evaluated for
their proliferative response to IL-13. Without exogenous stimulation,
98% of these cells were in G0/G1. B
lymphocytes were stimulated with either insolubilized
anti-immunoglobulin M (anti-µ) and/or soluble anti-CD40 antibodies in
the presence or absence of individual cytokines including IL-2, IL-4,
IL-6, IL-13, IL-15, and tumor necrosis factor-
. Stimulation of B
cells by ligation of both the antigen and CD40 receptors in combination
with a cytokine provided the strongest proliferative response. The
relative mitogenic efficacy, dependent upon dual surface receptor
ligation, was IL-4
IL-13. These cytokines showed a consistent
proliferative effect when utilized at
10
9
M. IL-2 and IL-15 produced significant effects only at
concentrations
10
8 M. Similar rank orders
of proliferative efficacy were seen when cells were solely stimulated
by either ligation of the immunoglobulin receptor or CD40 receptor
alone followed by cytokine exposure, with soluble anti-CD40 being the
least effective stimulus.
Expression of IL-4 and IL-13 Receptors as a Function of Surface
Stimulation--
Fig. 1,
A-F, presents binding curves and Scatchard analyses for
radiolabeled IL-4 and IL-13. The affinity constants given in the legend
to Fig. 1 were used to calculate native ligand binding values. Table
II shows the affinity and binding site
determinations for native IL-4 and IL-13 as a function of surface
stimulation. Anti-µ stimulation alone resulted in the appearance of
1498 ± 224 IL-4 sites/cell of high affinity with a
Kd of 41 ± 10 pM. After
cross-linking both surface receptors, surface immunoglobulin and CD40,
1398 ± 151 IL-4 receptor sites/cell were found to have a
Kd of 37 ± 7 pM. Examining B cells
stimulated with anti-µ alone, the Kd and site
number for IL-13 could not be reliably calculated due to the low level
of specific binding, which resulted in high coefficients of variation.
In the presence of both anti-immunoglobulin and anti-CD40 antibodies,
745 ± 51 IL-13 binding sites/cell were found with an intermediate
affinity of 0.91 ± 0.08 nM.

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Fig. 1.
Binding parameters for radiolabeled IL-4 and
IL-13 on normal high density B lymphocytes. High density B
lymphocytes were cultured with soluble anti-µ at 10 µg/ml with and
without anti-CD40 at 5 µg/ml in bulk culture. After 24-36 h of
culture, cells (4-6 × 106/assay point), in
duplicate, were incubated for 2 h at 4 °C with 2-fold serial
dilutions of labeled IL-4 ranging from 1.0 nM to 8.0 pM in a final volume of 0.3 ml. The binding curve
(A) and the Scatchard analysis (B) for anti-µ
stimulation alone, and the binding curve (C) and Scatchard
analysis (D) for anti-µ and anti-CD40 stimulation revealed
1864 ± 172 sites/cell with an affinity of 193 ± 35 pM and 1600 ± 11 sites/cell with an affinity of
133 ± 14 pM, respectively. The data presented are
representative of three separate experiments analyzed by the Ligand
software. Activated high density B lymphocytes were incubated, in
duplicate, at 4 °C with 2-fold serial dilutions of labeled
[Phe43]IL-13-GYGY. Those cells stimulated with anti-µ
alone demonstrated minimal binding, which did not allow for reliable
calculations of the affinity and site number. The binding curve
(E) and the Scatchard analysis (F) for anti-µ
and anti-CD40 stimulation revealed 1028 ± 138 sites/cell with an
affinity of 3.8 ± 0.43 nM. The results are
representative of three experiments for each group analyzed by the
Ligand software.
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Table II
IL-4 and IL-13 binding parameters
Quiescent B lymphocytes activated for 24-36 h with anti-µ with or
without anti-CD40 were analyzed for 125I-IL-4 and
125I-IL-13 binding in the presence of unlabeled IL-4 and IL-13.
Using the previously determined affinity for the iodinated cytokines in
Fig. 1, Kd was determined from displacement data
using a constant concentration of 125I-IL-4 (95 pM) and
125I-IL-13 (30 nM) in the presence of 2-fold serial
dilutions of unlabeled native IL-4 and IL-13. The values given are
for native cytokine.
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|
Differential Receptor Competition as a Function of Cytokine
Exposure--
Fig. 2A
demonstrates that 125I-IL-4 binding was completely
inhibited by unlabeled IL-4 and that unlabeled IL-13 competed for approximately 50% (control lanes, no preincubating cytokines) of such
binding (n = 3). Conversely, as seen in Fig.
2B, unlabeled IL-13 completely blocked radiolabeled IL-13
binding, and unlabeled IL-4 displaced 87% of the ligand
(n = 4) (control lanes, no preincubating cytokines).
Neither radiolabeled IL-4 nor IL-13 were displaced by excess unlabeled
IL-2 (Fig. 2, A and B, control lanes,
respectively). The actual binding assays depicted in Fig. 2,
A and B, were conducted with an incubation period
of 1-2 h at 4 °C, conditions associated with minimal receptor
internalization/down-regulation. However, incubation of cells with
ligand at 37 °C is associated with such internalization/down-regulation (40, 41). To determine the efficacy of
each cytokine to occupy and/or internalize IL-4 and IL-13 receptors,
high density cells were stimulated in culture with anti-µ and
anti-CD40 antibodies in the presence or absence of unlabeled ligands.
After the cultures were maintained at 37 °C for 24-36 h, the cells
were tested for radiolabeled ligand binding and for competition of that
binding by an unlabeled growth factor. As seen in Fig. 2A,
preincubation at 37 °C with IL-2 failed to modulate subsequent
125I-IL-4 binding, while unlabeled IL-4 resulted in
95%
reduction in specific 125I-IL-4 binding. Exposing cells to
IL-13 during the 37 °C incubation phase resulted in a 60% decrease
in 125I-IL-4 binding. In experiments that measured
125I-IL-13 sites (Fig. 2B), preincubation at
37 °C with either the homologous ligand or IL-4 resulted in near
elimination of subsequent 125I-IL-13 binding, while
preincubation with IL-2 induced no measurable change in binding.

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Fig. 2.
125I-Labeled IL-4 and
125I-labeled IL-13 binding on high density B lymphocytes in
the presence or absence of native cytokines. High density B
lymphocytes were cultured with soluble anti-µ at 10 µg/ml and
anti-CD40 at 5 µg/ml in bulk culture in the absence (filled
bars) or presence of native IL-2 (diagonally hatched
bars), IL-4 (cross-hatched bars), or IL-13
(unfilled bars). After a 24-36-h incubation, cells (5 × 106) were incubated in duplicate at 4 °C with
1-2 × 105 dpm of 125I-IL-4
(A) or 2-3 × 105 dpm of
125I-[Phe43]IL-13-GYGY (B) with or without a
100-200-fold molar excess of IL-2, IL-4, or IL-13. Data shown are the
mean ± S.E. from a representative experiment. Results from
replicate experiments indicated the same findings. The additional
unfilled bar in panel A in the section concerning
cells preincubated with IL-13, represents specific radiolabeled IL-4
binding after a 2.0-min treatment at pH 3.0 to remove any residual
cytokine still bound following preincubation and cell washing.
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The observed reduction in radiolabeled IL-4 binding subsequent to IL-13
preincubation at 37 °C could be due to IL-13 occupancy of shared
receptor elements, IL-13 down-regulation/internalization of shared
receptor components, or steric hindrance of radiolabeled IL-4 binding
by IL-13 bound to its specific receptor. To rule out the blocking
effect of residually bound cytokine to either a shared or contiguous
receptor, cells were washed after the 24-36-h incubation and exposed
for 2 min to RPMI 1640 medium (10% fetal calf serum) adjusted to pH
3.0. Such acidic incubation releases surface-bound ligand without loss
of cell viability (40). These treated cells were tested for
125I-IL-4 binding in comparison with cells that had been
washed and not exposed to acidic conditions. As seen in Fig.
2A (IL-13 preincubation, pH 3.0-treated columns), such a
comparison of 125I-IL-4 binding both before and after
residual cytokine (IL-13) release revealed that 10-15% of the
reduction in binding seen after the 24-36-h incubation with IL-13
could be accounted for by receptor competition/blockade by IL-13, while
the remainder of the IL-13 effect on IL-4 binding was due to actual
receptor down-regulation. This latter result suggests that most of the IL-13 competitive effect can be accounted for by a sharing/utilization of common receptor elements.
Experiments were next directed at specifically quantitating the
variations in site number due to homologous or heterologous ligand
pre-exposure. Quiescent B cells were stimulated with either anti-immunoglobulin and/or anti-CD40 antibody in the presence of IL-13.
Under these conditions, IL-13 binding should be quantitatively reduced
as previously demonstrated. Measurement of IL-4 binding sites on those
cells incubated overnight with IL-13 after dual surface ligation with
anti-immunoglobulin and anti-CD40 indicated that the number of
measurable IL-4 binding sites was reduced by approximately 50% to
702 ± 2 sites (Table III) per cell,
compared with those cells not treated with IL-13 during the culture
period (approximately 1400 IL-4 sites, refer to Table II). Furthermore, the sites exhibited a high affinity with a Kd of
22 ± 14.6 pM, similar to those cells not treated with
IL-13 where the Kd for IL-4 was 37 pM
(refer to Table II). In those cells stimulated with anti-immunoglobulin
antibody alone, which do not possess detectable high affinity IL-13
sites yet do possess high affinity IL-4 receptors, incubation overnight
with IL-13 did not reduce the measurable number of IL-4 binding
sites or alter their affinity.
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Table III
IL-4 binding to cells preincubated with native IL-13
Quiescent B lymphocytes activated for 24-36 h with anti-µ with or
without anti-CD40 and IL-13 (10 ng/ml) were analyzed for
125I-IL-4 binding in the presence of unlabeled native IL-4.
Using the previously determined affinity for the iodinated cytokine in
Fig. 1, Kd was determined from displacement data
acquired using a constant concentration of 125I-IL-4 in the
presence of 2-fold serial dilutions of unlabeled native IL-4. The
values given are for native IL-4.
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Determination of Protein Constituents of the IL-4 and IL-13
Receptors--
Activated B cells were incubated with radiolabeled
ligand either for 2-3 h at 4 °C or for 30 min at 37 °C. At
4 °C, ligand binding takes place (Fig.
3A), while at 37 °C both
ligand binding and receptor protein coaggregation occur (Fig.
3B). The electrophoretic profile of the whole cell lysates
for IL-4 specifically bound at 4 °C demonstrated a predominant band
at 150-160 kDa, consistent with a 140-kDa binding protein bound with
labeled ligand. A less intense 85-kDa band, consistent with a 70-kDa
protein bound with labeled ligand, is also seen (n = 5). Fig. 3A additionally demonstrates that
125I-IL-13 bound predominantly to a band of 80 kDa,
consistent with a binding protein of 70 kDa complexed with labeled
ligand. There was faint binding detected to a higher molecular weight
complex of 150 kDa indicative of a 140-kDa protein associated with the labeled ligand. Fig. 3B reveals that at 37 °C the ligand
binding/coaggregation band pattern for 125I-IL-4 replicates
that seen at 4 °C. The pattern seen with IL-13 at 37 °C differs
from that seen at 4 °C in that a band corresponding to a protein of
140 kDa coaggregates with the 70-kDa protein, the predominant moiety
seen at the lower temperature.

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Fig. 3.
Radioiodinated ligand binding and
cross-linking to activated B lymphocytes. After 24-36 h of
activation, B lymphocytes were incubated with radioiodinated IL-4 or
IL-13-GYGY in the absence ( ) or presence (+) of excess cold ligand
for 2 h at 4 °C (A) or 30 min at 37 °C
(B). Receptor complexes were biochemically cross-linked with
BS3, and cell lysates were separated on 7.5%
SDS-PAGE.
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The identity of the binding proteins was investigated using Western
analysis and immunoprecipitation. Experiments examined the contribution
of the common
c protein to receptors reactive with
either IL-4 or IL-13. Eighteen-hour activated B cells were exposed to
either native IL-4 or IL-13 for 30 min and subsequently immunoprecipitated with either anti-IL-4 or anti-IL-13 antibody, respectively. Following resolution of the immunoprecipitate on 7.5%
SDS-PAGE and transfer to membranes, the samples were blotted with an
affinity-purified polyclonal antibody prepared against the
c C terminus (Santa Cruz Biotechnology). Immunological
recognition of this protein as a 70-kDa band, as seen in Fig.
4, was demonstrated in those activated
cells incubated with IL-4 and immunoprecipitated with anti-IL-4. The
c protein was not identifiable in those
immunoprecipitates derived from activated cells exposed to IL-13 and
immunoprecipitated with a polyclonal antibody to IL-13.

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Fig. 4.
Identification of c as part of
the IL-4 receptor and its absence in the IL-13 receptor.
Immunochemical identification of receptor binding chains was undertaken
following immunoprecipitation of the receptor components as they are
complexed with either native IL-4 or IL-13. Cells stimulated for
18 h with either anti-µ alone or anti-µ with anti-CD40 were
exposed to cytokine for 30 min at 37 °C and then immunoprecipitated
with either anti-IL-4 or anti-IL-13. Following resolution of the
immunoprecipitated proteins on 7.5% PAGE and subsequent transfer to
membranes, the samples were blotted with anti- c
antibodies. Immunological recognition of c occurred with
activated cells incubated with IL-4 and immunoprecipitated with IL-4.
The c protein was not identifiable in immunoprecipitates
derived from cells stimulated with IL-13 and immunoprecipitated with
anti-IL-13.
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Experiments next utilized immunoprecipitation to evaluate the presence
of IL-4R
, IL-13R
1, or
c within the ligand-binding complexes formed with either IL-4 or IL-13. Activated B cells were
exposed to either radiolabeled IL-4 or IL-13-GYGY at 37 °C for 30 min, cross-linked, and immunoprecipitated, and proteins were separated
by 7.5% SDS-PAGE. As seen in Fig.
5A, the anti-IL4R
antibody
immunoprecipitated a two-band complex of 140 and 70 kDa in activated
cells incubated with radiolabeled IL-4 at 37 °C. The relative band
intensity was similar to that seen previously in that the predominant
band was 140 kDa, with 70 kDa being of lesser intensity. The
anti-IL-4R
immunoprecipitate of activated cells exposed to
radiolabeled IL-13 at 37 °C also revealed a two-band profile but
with a predominance of the 70-kDa moiety. This is in contradistinction
to the cross-linked whole cell lysate at 4 °C (refer to Fig.
3A), which reveals a predominant component of 70 kDa without
substantial presence of a 140-kDa protein. The anti-IL-13R
1
polyclonal antiserum immunoprecipitated a two-band profile from cells
exposed to either radiolabeled IL-4 or IL-13 (refer to Fig.
5B). Therefore, exposure of cells to either IL-4 or IL-13 at
37 °C for 30 min resulted in the immunoprecipitation of complexes
that are composed of both the IL-4R
and the IL-13R
1. Activated
cells were also immunoprecipitated with anti-
c after a
37 °C incubation period with radiolabeled cytokine. For IL-4-exposed cells, this procedure resulted in a radiolabeled immunoprecipitate of
140 and 70 kDa for incubations with cytokine ranging from 5 to 30 minutes. In contrast, anti-
c immunoprecipitation of
IL-13-exposed cells did not yield any radiolabeled immunoprecipitate at
any incubation time point tested (data not shown).

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Fig. 5.
Immunoprecipitation of radiolabeled receptor
complexes. After 24-36 h of activation, B lymphocytes were
incubated with radiolabeled ligand at 37 °C for 30 min in the
absence ( ) or presence (+) of excess cold ligand. Receptor complexes
were biochemically cross-linked with BS3, and cell lysates
were immunoprecipitated with either anti-IL-4R (A) or
anti-IL-13R 1 (B).
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mRNA Expression for Receptor Constituents--
Experiments
next evaluated message levels for the protein elements of the IL-4 and
IL-13 receptors. Fig. 6 shows
semiquantitative RT-PCR with dilutions of cDNA. These experiments
permit evaluation of changes of a specific message that occur as a
function of stimulation but do not permit evaluation of relative
changes between receptor components. IL-13R
1 message was
substantially up-regulated as a function of ligation of both the
immunoglobulin and CD40 receptors. The IL-4R
and
c
were up-regulated as a function of activation through the
immunoglobulin receptor. Semiquantitative evaluation seen in Fig. 6,
A-C, reveals that dual surface ligation does not lead to
further significant increases in these message levels. The time course
for message up-regulation reveals that within 6-12 h subsequent to
cell activation, message for all constituents had been increased (data
not shown).

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Fig. 6.
Effects of co-stimulation on receptor
mRNA expression. High density B lymphocytes were unstimulated
or activated by anti- / or by anti- / and anti-CD40 for
24 h. Equal quantities of isolated RNA were used as a template for
single-stranded cDNA synthesis. Samples were subsequently utilized
undiluted (lanes 1, 4, and 7), diluted
1:10 (lanes 2, 5, and 8) and 1:100
(lanes 3, 6, and 9) for IL-4R and
c. For IL-13R 1, samples were utilized undiluted
(lanes 1, 4, and 7), diluted 1:7
(lanes 2, 5, and 8), and diluted 1:50
(lanes 3, 6, and 9). All samples were
subjected to a 30-cycle amplification protocol. Films were scanned and
quantified into relative, arbitrary units for each message component
separately. The sample with the greatest absorbance was given a value
of 1000 units, and all other dilutions of the specific message
component were compared with that value.
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More precise quantitations of mRNA levels were next undertaken
using competitive PCR. Examination of Table
IV reveals that IL-13R
1 shows a
consistent increase in message amount with maximal values following
dual surface ligation. Interestingly, both
c and the
140-kDa moieties show significant change subsequent to anti-immunoglobulin stimulation alone.
c shows an
additional small increase in message with dual surface ligation. The
mRNA abundance of these different species indicates that the
140-kDa component is apparently in limiting amounts.
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Table IV
Competitive PCR quantitation of mRNA for receptor-specific proteins
The number of amplicons was determined using a Life Technologies, Inc.
SuperscriptTM cDNA synthesis kit. The efficiency of cDNA
synthesis using this kit is at least 1 log greater than with a similar
product from Invitrogen (Carlsbad, CA). This was particularly evident
in terms of the IL-4R , where the calculated differences approached 2 logs. These results are in agreement with data derived from RNase
protection assays (RiboquantTM; Pharmingen, San Diego, CA) (data not
shown).
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In addition to the IL-13R
1, a second IL-13 binding protein has been
identified and termed IL-13R
2 (24). To evaluate its presence within
the lymphoid populations isolated from the tonsil, cells were
fractionated into high density quiescent cells and two levels of lower
density cells corresponding to populations inclusive of centrocytes and
centroblasts (42, 43). Cells were either unstimulated or stimulated by
anti-immunoglobulin alone or with anti-CD40. After 24 h, cells
were collected, and RNA was isolated and evaluated by standard RT-PCR
for the presence of message for IL-13R
1 or IL-13R
2. IL-13R
1
was present in all fractions studied and varied as a function of cell
activation (data not shown). Utilization of a standard RT-PCR revealed
the presence of message for IL-13R
2 only in that cellular fraction corresponding to the lowest density cells, which contains the centroblastic population. The high density (i.e. those
quiescent lymphocytes utilized in the present receptor studies) and
moderate density fractions (centrocytic cells) failed to demonstrate
the presence of mRNA for IL-13R
2 by either standard or nested
RT-PCR (data not shown).
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DISCUSSION |
Depending on the specific mode of surface activation, the
proliferation of mature human B cells induced by IL-13 is either equal
to or less in magnitude than that induced by IL-4. The differential effectiveness of IL-4 and IL-13 noted in the present studies was most
pronounced in cells on which only the immunoglobulin receptor had been
ligated. Surface stimulation with anti-immunoglobulin alone results in
up-regulation of a high affinity IL-4 receptor, while IL-13 binding
under these conditions is minimal. Dual surface ligation by
anti-immunoglobulin and anti-CD40 up-regulates a second receptor type
that binds and transmits signals from both IL-4 and IL-13. It therefore
seems plausible that the quiescent human B cell may be induced to
express two forms of IL-4 receptor that differ in their ability to bind
and/or respond to IL-13. Results from both thymidine incorporation and
Scatchard analyses favor a model in which both receptor types coexist
on an individual cell. Coexistence on the same cell would require a
receptor configuration in which both the
c chain of the
IL-4 receptor and the specific IL-13 binding chain simultaneously
compete for the shared 140-kDa binding chain. This model is compatible
with (a) the binding of the cytokines in the presence or
absence of the specific IL-13 binding component, (b) the
results of immunoprecipitation and Western analyses, and (c)
the observation that quiescent density-fractionated cells stimulated by
both anti-immunoglobulin and anti-CD40 antibodies respond similarly to
either cytokine. In addition, differential sequestration of IL-13
receptor constituents has been suggested by studies examining cells
lacking the cytoplasmic domain of the IL-13R
1 (44). It is
interesting to note that studies on cells derived from human X-SCID
patients that lack functional
c demonstrate proliferative sensitivity to either IL-4 or IL-13 (21, 30). These data
and others suggest that the
c protein is a sufficient although not a necessary component for B cell proliferation in response
to IL-4 and that an IL-4 receptor composed of the 140-kDa binding unit
and the IL-13-specific binding chain is sufficient for B cell
proliferation to either IL-4 or IL-13.
IL-4 binds to its receptor with approximately 50 pM
affinity and associates with both 140- and 70-kDa receptor components at 4 and at 37 °C, with more radiolabeled IL-4 complexed with the
140-kDa IL-4R
. In contrast, IL-13 binds to its receptor with at
least 10-fold lower affinity and is bound principally to the 70-kDa
IL-13R
1 at 4 °C. IL-13 binding to the 140-kDa protein is minimal
unless the activated cells are exposed to ligand at 37 °C. Even at
37 °C, IL-13 shows more association with the 70-kDa chain than the
140-kDa component. These data are consistent with a model wherein IL-4
binds predominantly to the 140-kDa IL-4R
, which subsequently
coaggregates with a 70-kDa component that can be either
c or IL-13R
1. Conversely, IL-13 predominantly binds to the IL-13R
1, which subsequently coaggregates with the 140-kDa IL-4R
. Whether this differential utilization of receptor components underlies the variation in affinity noted above requires further investigation (45).
IL-13 does not appear to bind to and/or coaggregate with the
c component in human B cells stimulated as described
herein. The absence of
c in the human IL-13 receptor has
been suggested for several human cell types including cell lines
derived from renal cell carcinoma, glioma, and epidermoid carcinoma
(13).
c has been implicated in an IL-13 receptor
described for the TF-1 cell line derived from a human erythroleukemia
(46). The role of
c in Epstein-Barr virus-transformed B
cells is less clear, but a direct or indirect interaction of this
protein with the IL-13 receptor complex has been suggested (10, 13).
Functional studies on endothelial cells and human B cells derived from
X-SCID patients have demonstrated that these cells are able to
functionally respond to IL-13 in the absence of detectable
c expression (6, 21, 30, 47). In contradistinction,
human B cells activated with phorbol dibutyrate and ionomycin
demonstrate rapid coaggregation of the 140-kDa IL-4R
and the
c protein after exposure to high concentrations of
IL-13. This latter result has been used to argue that IL-13 utilizes
c in its receptor complex (29). These experiments with
ionomycin and phorbol dibutyrate-stimulated B cells do not distinguish
between initial binding events and downstream effects subsequent to
IL-13 binding. In the present work, the absence of the
c
protein in the B cell IL-13 receptor was demonstrated by its specific
absence from the IL-13 binding complex. This was assessed both by
immunoprecipitation of receptor ligand complexes with anti-IL-13 with
subsequent blotting for
c and by evaluation of
radiolabeled IL-13 in anti-
c immunoprecipitates. As
mentioned above, it appears from experiments presented herein and
elsewhere that some form of competition exists between the IL-13R
1
and
c for aggregation with the IL-4R
(44, 45).
Whether this is simply a function of which ligand interacts with the
cell surface remains to be determined. Furthermore,
c
may indirectly affect IL-13 binding, since IL-13 receptor affinity is
altered in those transfected cell populations overexpressing
c (24, 45).
In the present series of experiments, the role of IL-13R
1 in the
IL-13 receptor has been confirmed utilizing antisera specific for its C
terminus. These are the first such studies to demonstrate the actual
presence of this protein in a functional receptor apart from the
assembled receptors in transfected cell populations. The observation
confirming the presence of IL-13R
1 in mature B cells and the
relative paucity/absence of IL-13R
2 in mature lymphoid cells is of
substantial interest. Future studies establishing the identity of that
cellular phenotype in the centroblast fraction of the tonsillar B
lymphocytes that contains both forms of the IL-13 receptor binding
proteins should provide further essential information about the
functional significance of each receptor element.
We thank the staff at the Blackstone Valley
Surgicenter for diligent help in procuring tonsil specimens. We also
thank Dr. Sagamura for the Tugh4 antibody, Anna Chromiak and Andrea
Luciani for excellent technical assistance, and Linda Mulzer for
excellent manuscript preparation.