Abstract
purpose. To characterize the interleukin (IL)-4 receptor (IL-4R) complex in
human corneal fibroblasts.
methods. The presence of IL-4R subunit mRNAs and proteins in cultured human
corneal fibroblasts was examined by reverse transcription–polymerase
chain reaction and flow cytometry, respectively. The interaction of 125I-labeled IL-4 with specific cell surface receptors was
characterized by saturation binding and Scatchard analysis. The effects
of IL-4 on the tyrosine phosphorylation and subcellular localization of
signal transducer and activator of transcription 6 (STAT6) were
evaluated by immunoblot and indirect immunofluorescence analyses,
respectively. The concentration of eotaxin in cell culture supernatant
was measured by enzyme-linked immunosorbent assay.
results. Transcripts encoding the IL-4R components IL-4Rα, IL-2Rγc,
IL-13Rα1, and IL-13Rα2 were detected in human corneal fibroblasts;
IL-4Rα and IL-2Rγc proteins were also expressed on the cell
surface. The maximum number of IL-4 binding sites was 2.3 ×
104 per cell, and the dissociation constant for the
interaction of IL-4 with these sites was 10.1 ± 0.3 pM. IL-4
induced tyrosine phosphorylation of STAT6 as well as translocation of
this protein to the nucleus. Eotaxin release from corneal fibroblasts
stimulated by the combination of IL-4 and tumor necrosis factor-α was
inhibited by pretreatment of the cells with neutralizing antibodies to
IL-4R.
conclusions. Cultured human corneal fibroblasts express high-affinity functional
IL-4Rs on the cell surface, suggesting that these cells may contribute
to the role of IL-4 as a key mediator of allergic reactions in the
cornea.
Fibroblasts are the major cellular components of the
corneal stroma. Corneal fibroblasts contribute to the maintenance of
the normal structure of the stroma not only through their synthesis and
degradation of stromal collagen but also by secreting various bioactive
substances.
1 2 External stimuli induce the secretion from
inflammatory cells of various types of cytokines, including
proinflammatory, T helper cell (Th) type 1, and Th2 cytokines, that
affect the functions of corneal fibroblasts. We and others have shown
that interleukin (IL)-4, a Th2 cytokine, acts on corneal fibroblasts to
regulate the expression of various chemokines
3 4 5 6 and may
thereby contribute to the infiltration of inflammatory cells into the
cornea.
IL-4 plays an important role in allergic reactions by inducing
immunoglobulin isotype switching in B cells and maintaining the
production of immunoglobulin E (IgE).
7 However, IL-4 acts
not only on inflammatory cells but also on tissue-resident cells such
as epithelial cells and fibroblasts.
8 9 10 The molecular
structure of the IL-4 receptor (IL-4R) complex appears to be diverse,
with components including IL-4Rα, the common γ-chain of IL-2R
(IL-2Rγc), IL-13Rα1, and IL-13Rα2.
11 12 13 14 15 The
combination of these subunits generates multiple forms of the IL-4R
complex that are specific for different cell types, including
hematopoietic cells, tissue-resident cells, and tumor
cells.
16 17 The IL-4Rα chain is the functional subunit
in IL-4R complexes that mediate the activation of signal transducer and
activator of transcription 6 (STAT6).
18 19 20 21
Although IL-4 has been shown to affect the biological functions of
corneal fibroblasts,
3 4 5 the presence of IL-4Rs on these
cells has not been demonstrated directly. Determination of both the
structure of the IL-4R complex and the mechanism of IL-4 signal
transduction in these cells should provide insight into the
pathogenesis of allergic ocular diseases. We have now demonstrated the
expression of IL-4Rs on the surface of human corneal fibroblasts as
well as the presence of IL-4R subunit mRNAs in these cells.
Furthermore, we have characterized the kinetics of IL-4 binding and the
mechanism of IL-4 signal transduction in the corneal cells.
Eagle’s minimum essential medium (MEM), Dulbecco’s modified
Eagle’s medium (DMEM), and trypsin (0.25%) were obtained from the
Research Foundation for Microbial Diseases of Osaka University (Suita,
Osaka, Japan); fetal bovine serum from Flow Laboratories (North Ryde,
Australia); tissue culture dishes and four-chamber, polystyrene-vessel,
glass culture slides from Becton Dickinson (Franklin Lakes, NJ); HEPES,
RPMI 1640 medium, and nonenzymatic cell dissociation solution from
Gibco BRL (Grand Island, NY); bovine serum albumin (BSA),
phytohemagglutinin (PHA), and cycloheximide from Sigma (St. Louis, MO);
and human recombinant tumor necrosis factor (TNF)-α and IL-4 from
Genzyme (Cambridge, MA). A mouse monoclonal antibody to human IL-4Rα
was obtained from Beckman Coulter (Fullerton, CA), and a neutralizing
mouse monoclonal antibody to the IL-4R complex as well as normal mouse
IgG2A, mouse IgG1, and
normal rabbit IgG were from R&D Systems (Minneapolis, MN). A rat
monoclonal antibody to human IL-2Rγc was obtained from Sumitomo
Electric (Osaka, Japan), and normal rat IgG, fluorescein isothiocyanate
(FITC)–conjugated goat antibodies to mouse IgG, FITC-conjugated goat
antibodies to rat IgG, and FITC-conjugated goat antibodies to rabbit
IgG were from ICN Pharmaceuticals (Aurora, OH). Rabbit polyclonal
antibodies to STAT6 and horseradish peroxidase (HRP)–conjugated goat
antibodies to rabbit IgG were from Santa Cruz Biotechnology (Santa
Cruz, CA), and an HRP-conjugated mouse monoclonal antibody to
phosphotyrosine was from Transduction Laboratories (Lexington, KY). 125I-labeled IL-4 was from DuPont NEN (Boston,
MA), and protein G-Sepharose gel (4 Fast Flow), enhanced
chemiluminescence (ECL) immunoblot detection reagents, and
autoradiography film (Hyperfilm) were from Amersham Pharmacia Biotech
(Little Chalfont, UK). Biotinylated protein size markers were obtained
from New England Biolabs (Beverly, MA); polyacrylamide gels (7.5%)
from Daiichi Pure Chemicals (Tokyo, Japan); polyvinylidene difluoride
membranes from Millipore (Bedford, MA); protein assay reagent from
Bio-Rad (Hercules, CA); nonfat blocking solution (Block Ace)
from Dainippon Pharmaceutical (Osaka, Japan); and mounting medium
(Vectashield) from Vector Laboratories (Burlingame, CA).
Human corneas were obtained from Mid-America Transplant Service
(St. Louis, MO), Northwest Lions Eye Bank (Seattle, WA), and The Eye
Bank of Wisconsin (Madison, WI). The donors were white males and
females aged 4, 43, 50, and 70 years. Corneal fibroblasts were prepared
from the tissue remaining after corneal transplantation surgery and
were cultured as described previously.
3 4 The cells
prepared from each cornea were maintained separately in MEM
supplemented with 10% fetal bovine serum until they had achieved
approximately 90% confluence in 60-mm culture dishes. Cells in the
third to sixth passages were used in the present studies, and similar
results were obtained with the cells prepared from all four donors. The
purity of the cell cultures (∼99%) was assessed on the basis of both
the distinctive morphology of corneal fibroblasts and their reactivity
with antibodies to vimentin in immunofluorescence analysis. We also
examined the phenotype of the passaged cells by immunofluorescence
analysis with antibodies to α-smooth muscle actin (α-SMA), as
previously described.
22 The passaged corneal fibroblasts
used in this study expressed α-SMA, indicating that they were
transformed myofibroblasts.
Total RNA was extracted from human corneal fibroblasts or from
cells used as a positive control and was subjected to reverse
transcription–polymerase chain reaction (RT-PCR), as described
previously.
4 The constitutively expressed gene for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal
control. The sequences of PCR primers for IL-4Rα and IL-13Rα1
chains,
10 IL-2Rγc,
23 IL-13Rα2,
24 and GAPDH
3 were as described
previously. PHA-stimulated peripheral blood monocytes were used as a
positive control for expression of IL-4Rα, IL-13Rα1, and IL-2Rγc
genes.
23 These cells were isolated from the peripheral
blood of adult volunteers by centrifugation through a gradient
(Ficoll-Paque Plus; Pharmacia Biotech, Uppsala, Sweden) and, before
analysis, were cultured for 18 hours in RPMI 1640 medium supplemented
with 10% fetal bovine serum and PHA (5 mg/mL). Human synovial
fibroblasts used as a positive control for IL-13Rα2
mRNA
25 were obtained from Applied Cell Biology Research
Institute (Kirkland, WA) and cultured in MEM supplemented with 10%
fetal bovine serum.
Corneal fibroblasts were detached from culture dishes with the
use of nonenzymatic cell dissociation solution and then incubated for
30 minutes at 4°C in the presence of saturating concentrations of
unlabeled antibodies to human IL-4Rα or to human IL-2Rγc. The cells
were then washed, incubated for 30 minutes at 4°C with
FITC-conjugated goat antibodies to mouse IgG or FITC-conjugated goat
antibodies to rat IgG, and fixed with 1% formaldehyde in
phosphate-buffered saline (PBS). As a negative control, cells were
incubated under similar conditions with isotype-matched mouse or rat
immunoglobulin in place of the primary antibodies. Cells were then
analyzed by flow cytometry (EPICS-XL; Beckman Coulter). At least 10,000
cells were analyzed for each sample.
Cultured corneal fibroblasts (6 ×
105 cells) were suspended in assay buffer (DMEM
supplemented with 1% BSA and 20 mM HEPES), and samples were incubated
at 4°C for 2 hours in 1 mL assay buffer containing 125I-labeled IL-4 (1.56, 3.13, 6.25, 12.5, 25, or
50 pM). For determination of nonspecific binding, cells were incubated
with 125I-labeled IL-4 in the presence of a
500-fold excess of unlabeled IL-4. Cell-bound 125I-labeled IL-4 was separated from unbound
cytokine by centrifugation (1500 rpm for 3 minutes at 4°C) and was
quantitated with a gamma scintillation counter (COBRA II; Packard,
Meriden, CT). Specific binding was calculated by subtracting
nonspecific binding from total binding. The dissociation constant
(K d) and maximum number of binding
sites (B max) for IL-4 were determined
by Scatchard analysis.
The tyrosine phosphorylation of STAT6 in cultured corneal
fibroblasts was examined by immunoblot analysis, as described
previously.
26 In brief, cells were incubated at 37°C
overnight in serum-free MEM, washed with MEM containing 50 mM
Na
3VO
4, and then incubated
for various times at 37°C with MEM containing IL-4 (100 ng/mL). The
cells were washed twice and were then lysed by scraping and sonication
in 0.2 mL of an ice-cold solution containing 10 mM Tris-HCl (pH 8.0),
150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 50 mM NaF, 1% Triton X-100, 1 mM
Na
3VO
4, and 1 mM
phenylmethylsulfonyl fluoride. The lysate was centrifuged at
15,000
g for 5 minutes at 4°C, and a portion of the
resultant supernatant (60 μg protein) was mixed with protein
G-Sepharose gel (4 Fast Flow; Amersham Pharmacia Biotech) and gently
stirred for 1 hour at 4°C. After removal of the gel by
centrifugation, the supernatant was incubated at 4°C, first overnight
with antibodies to STAT6 (3.75 μg) and then, with gentle stirring,
for 1 hour with the protein G-Sepharose. The gel was separated by
centrifugation, washed, and suspended in a solution containing 250 mM
Tris-HCl (pH 6.8), 2% SDS, 30% glycerol, 0.01% bromophenol blue, and
10% β-mercaptoethanol. The samples were boiled for 5 minutes and
subjected to SDS-polyacrylamide gel electrophoresis on a 7.5% gel. The
separated proteins were transferred to a polyvinylidene difluoride
membrane, and nonspecific sites were blocked by incubation of the
membrane with a solution containing 50 mM Tris-HCl (pH 7.5), 1% BSA,
1.17% NaCl, and 0.05% Tween 20. The membrane was then incubated for 1
hour at room temperature with HRP-conjugated antibodies to
phosphotyrosine (1:2500 dilution) in Tris-buffered saline containing
0.1% Tween 20, washed in the same solution without antibodies,
immersed for 1 minute in ECL detection reagents, and exposed to
autoradiography film (Hyperfilm; Amersham Pharmacia Biotech). For
detection of STAT6 protein, the blot was reprobed with antibodies to
STAT6. Thus, the membrane was soaked overnight at 4°C in washing
buffer (20 mM Tris-HCl [pH 7.4], 2.5% nonfat blocking solution[
Block Ace; Dainippon Pharmaceuticals], and 0.1% Tween 20) and then
subjected to immunoblot analysis with anti-STAT6 (1:400 dilution in
washing buffer) and HRP-conjugated secondary antibodies (1:2000
dilution in washing buffer).
Immunostaining for STAT6 in corneal fibroblasts was performed as
described previously.
24 In brief, cell monolayers grown in
four-well chamber slides were incubated overnight at 37°C in
serum-free MEM. The cells were then incubated for 10 minutes at 37°C
with MEM in the absence or presence of IL-4 (100 ng/mL), washed twice
with PBS, and fixed with 2% paraformaldehyde in PBS. After two
additional washes, the cells were permeabilized with 1% Triton X-100
in PBS, and nonspecific adsorption of antibodies was blocked by
incubation for 30 minutes with PBS containing 2% BSA. The cells were
then incubated for 1 hour at room temperature with antibodies to STAT6
(1:200 dilution in PBS containing 2% BSA), washed, and incubated for
30 minutes at room temperature with FITC-conjugated secondary
antibodies (1:300 dilution in PBS containing 2% BSA). Cells were
finally washed, mounted (Vectashield; Vector Laboratories), and
examined with a confocal microscope (Fluoview; Olympus, Tokyo, Japan).
Corneal fibroblasts were cultured in six-well plates until
confluent and then incubated for 24 hours with serum-free MEM. The
cells were then incubated for 1 hour at 37°C with MEM containing
various concentrations of neutralizing antibodies to the IL-4R before
the addition of recombinant IL-4 (10 ng/mL) and TNF-α (10 ng/mL) and
incubation for an additional 24 hours. The amount of eotaxin released
into the culture medium was then quantified by ELISA, as
described.
3 The amount of eotaxin in the medium was
expressed in nanograms per 10
6 cells.
Data were analyzed by the Dunnett multiple comparison test
performed on computer (StatView for Windows software; ver. 5.0; SAS
Institute, Cary, NC). P < 0.05 was considered
statistically significant.
RT-PCR analysis of total RNA isolated from cultured human corneal
fibroblasts revealed the presence of an amplification product (335 bp)
corresponding to IL-4Rα mRNA
(Fig. 1) . A PCR product (448 bp) corresponding to IL-2Rγc mRNA, the amount of
which was increased after incubation of cells with the protein
synthesis inhibitor cycloheximide (10 μg/mL), was also detected. The
IL-2Rγc mRNA has previously been shown to be unstable and short
lived.
10 27 Our results are consistent with the notion
that the IL-2Rγc gene is expressed constitutively in corneal
fibroblasts. Transcripts encoding the IL-13Rα1 and IL-13Rα2 chains
were also detected in corneal fibroblasts.
We also examined whether components of the IL-4R complex are expressed
on the surface of corneal fibroblasts. Flow cytometric analysis
revealed that IL-4Rα and IL-2Rγc were indeed present on the surface
of the cultured corneal fibroblasts
(Fig. 2) . We were not able to examine the expression of IL-13Rα1 and
IL-13Rα2 proteins by flow cytometry because of the unavailability of
antibodies to IL-13R. Together, these results suggest that the IL-4R
complex, consisting of IL-4Rα, IL-2Rγc, IL-13Rα1, and IL-13Rα2,
is expressed by human corneal fibroblasts.
We next investigated whether IL-4 binds to the IL-4Rs expressed on
cultured corneal fibroblasts.
125I-labeled IL-4
was shown to bind specifically to the surface of these cells. Specific
binding was saturated at a free ligand concentration of 30 pM
(Fig. 3A) . Scatchard analysis
(Fig. 3B) revealed a single component of binding,
with a
B max of 23,400 ± 300
sites per cell and a
K d of 10.1 ± 0.3 pM (data are means ± SD of values from three independent
experiments).
STAT proteins are activated by tyrosine phosphorylation, which is
required for dimer formation, nuclear translocation, binding to DNA,
and transactivation activity, in cells treated with
cytokines.
28 To determine whether IL-4 activates STAT6 in
human corneal fibroblasts, we therefore examined the effects of this
cytokine on the tyrosine phosphorylation and cellular localization of
STAT6. Cells were incubated for various times with IL-4 (100 ng/mL),
lysed, and subjected to immunoprecipitation with antibodies to STAT6.
The immunoprecipitates were then subjected to immunoblot analysis with
antibodies to phosphotyrosine. IL-4 induced the tyrosine
phosphorylation of STAT6 in a time-dependent manner
(Fig. 4) . This effect was apparent as early as 3 minutes after exposure to IL-4
and was maximal after 10 minutes.
We examined the effect of IL-4 on the subcellular localization of STAT6
by immunofluorescence analysis. Under basal conditions, STAT6
immunofluorescence was located predominantly in the cytoplasm of
corneal fibroblasts
(Fig. 5A) . No immunofluorescence was apparent in cells stained with normal
rabbit IgG as a negative control (data not shown). Treatment of cells
with IL-4 (100 ng/mL) for 10 minutes resulted in marked translocation
of STAT6 to the nucleus
(Fig. 5B) . The tyrosine phosphorylation and
nuclear translocation of STAT6 in response to IL-4 thus suggest that
this protein is activated by IL-4 in human corneal fibroblasts.
We have previously shown that IL-4 and TNF-α induce eotaxin
release from cultured human corneal fibroblasts in a synergistic
manner.
3 We therefore finally examined the effect of
neutralizing antibodies to IL-4R on eotaxin release induced by IL-4 and
TNF-α. Pretreatment of corneal fibroblasts with neutralizing
antibodies to IL-4R inhibited in a dose-dependent manner the increase
in eotaxin release induced by the combination of IL-4 (10 ng/mL) and
TNF-α (10 ng/mL;
Fig. 6 ). No such inhibition was observed with normal mouse
IgG
2A (20 μg/mL) as a negative control (data
not shown). This effect of IL-4 on eotaxin release thus appears to be
mediated by IL-4Rs.
We have shown that cultured human corneal fibroblasts express
IL-4Rs on the cell surface and that IL-4 binds to these receptors with
high affinity (K d = 10.1 ± 0.3
pM). RT-PCR and flow cytometric analysis indicated that the corneal
fibroblasts express the IL-4Rα, IL-2Rγc, IL-13Rα1, and IL-13Rα2
chains. Furthermore, our demonstration that IL-4 induced the tyrosine
phosphorylation and nuclear translocation of STAT6, and that
neutralizing antibodies to IL-4R inhibited eotaxin release induced by
IL-4 and TNF-α, suggests that the IL-4Rs expressed by human corneal
fibroblasts are functional. Thus, IL-4, a central mediator of allergic
reactions, exerts direct effects on corneal fibroblasts. These cells
therefore probably function as important effectors in the regulation of
allergic inflammation by IL-4.
IL-4 regulates a variety of biological responses by binding to specific
IL-4Rs. These receptors are expressed by a wide range of cell types,
including T and B lymphocytes, monocytes, granulocytes, endothelial
cells, epithelial cells, and fibroblasts.
9 29 30 31 In the
present study, Scatchard analysis of the binding of IL-4 to human
corneal fibroblasts revealed a single class of high-affinity binding
sites, with a
K d of 10 pM and a
B max of 2.3 ×
10
4 sites per cell. Similar results have
previously been obtained for IL-4Rs expressed by other types of
tissue-resident cells.
9 31 Fibroblasts derived from cornea
and lung
10 exhibit a markedly greater number of IL-4
binding sites (1.4 × 10
4 to 2.3 ×
10
4 sites per cell) than do lymphoid cells and
endothelial cells (228–2150 sites per cell).
29 30
Cross-linking studies have shown that IL-4 binds to two different
receptor components, indicative of a multimeric structure for the
IL-4R.
17 The 140-kDa IL-4–binding protein (IL-4Rα) is
expressed more widely than is the 65-kDa IL-4–binding protein
(IL-2Rγc), which is a common component of other cytokine receptors.
In cells without the IL-2Rγc chain, IL-4 is thought to induce either
homodimerization of IL-4Rα or heterodimerization of IL-4Rα with
either one or two IL-13R chains (IL-13Rα1 and
IL-13Rα2).
13 17 32 We detected both IL-4Rα and
IL-2Rγc mRNAs and proteins as well as IL-13Rα1 and IL-13Rα2 mRNAs
in human corneal fibroblasts. The IL-4R complex in these cells
therefore likely consists of four components: IL-4Rα, IL-2Rγc,
IL-13Rα1, and IL-13Rα2.
STAT proteins are activated on exposure of cells to various cytokines,
growth factors, or hormones. STAT6, one of seven known mammalian
members of the STAT family, is phosphorylated and activated in response
to IL-4 or IL-13,
33 34 the latter of which also binds to
IL-4Rα. STAT6 knockout mice exhibit defects in various IL-4–mediated
functions, including induction of the expression of CD23 and major
histocompatibility complex class II genes, immunoglobulin class
switching to IgE, proliferation of B and T cells, and Th2 cell
development, demonstrating the essential role of STAT6 in IL-4
signaling.
35 36 37 In the current study, STAT6 was
phosphorylated and translocated to the nucleus in response to
stimulation of human corneal fibroblasts with IL-4, suggesting that
STAT6 contributes to IL-4R signaling in these cells. Neutralizing
antibodies to IL-4R inhibited eotaxin synthesis induced by the
combination of IL-4 and TNF-α in corneal fibroblasts. The promoter of
the eotaxin gene contains consensus binding sites for
STAT6.
38 Expression of eotaxin in corneal fibroblasts may
therefore be regulated at the transcriptional level by the IL-4R–STAT6
signaling pathway.
A clinical characteristic of vernal keratoconjunctivitis (VKC) that
distinguishes this condition from allergic keratoconjunctivitis is
serious corneal involvement. Accumulation of eosinophils in the
conjunctiva and cornea is the major pathologic change apparent in
individuals with severe allergic
keratoconjunctivitis.
39 40 41 Eotaxin is a potent and
specific chemoattractant for eosinophils,
42 and IL-4 is a
central mediator of allergic reactions.
7 We and others
have previously shown that IL-4, in the presence of the inflammatory
cytokine TNF-α, stimulates eotaxin synthesis in corneal fibroblasts
but not in corneal epithelial cells.
3 6 These various
observations have suggested that the IL-4–eotaxin axis may play a
central role in the pathogenesis of corneal disorders associated with
VKC. Indeed, the concentration of eotaxin in tear fluid has been found
to be increased in individuals with atopic keratoconjunctivitis and
correlated either with the severity of corneal damage or with the
number of eosinophils in tear fluid.
43 Furthermore, the
concentration of IL-4 in tear fluid has been shown to be higher in
individuals with VKC than in those with seasonal allergic
keratoconjunctivitis.
44 Taken together with these previous
observations, our present results suggest that therapy with agents that
specifically inhibit the IL-4R–STAT6 signaling pathway in corneal
fibroblasts represents a new approach to treatment of corneal disorders
associated with VKC.
The authors thank Shinji Kimura and Masatsugu Nakamura for
technical assistance.