Characterization of the Interleukin-4 Receptor Complex in Human Corneal Fibroblasts

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. 

Methods

Materials

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 IgG_2A, mouse IgG_1, 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). 

Preparation and Culture of Human Corneal Fibroblasts

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. 

RT-PCR Analysis of IL-4R Subunit mRNAs

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. 

Flow Cytometric Analysis of Cell Surface Expression of IL-4R Subunits

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. 

Assay of IL-4 Binding

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. 

Immunoprecipitation and Immunoblot Analysis of STAT6 Phosphorylation

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 Na₃VO₄, 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 Na₃VO₄, and 1 mM phenylmethylsulfonyl fluoride. The lysate was centrifuged at 15,000g 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). 

Immunofluorescence Staining of STAT6

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). 

Eotaxin ELISA

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 10⁶ cells. 

Statistical Analysis

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. 

Results

Expression of the IL-4R in Human Corneal Fibroblasts

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. 

Binding of IL-4 to the Surface of 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). 

IL-4–Induced Phosphorylation and Translocation of STAT6 in Corneal Fibroblasts

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. 

Inhibition of IL-4–Promoted Eotaxin Release by Neutralizing Antibodies to IL-4R

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. 

Discussion

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 × 10⁴ 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 × 10⁴ to 2.3 × 10⁴ 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. 

 Submitted for publication January 23, 2001; revised July 26, 2001; accepted August 13, 2001.

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Figure 1.

 

RT-PCR analysis of IL-4R gene transcripts in human corneal fibroblasts. Total RNA isolated from cultured human corneal fibroblasts (CF) and from cells used as a positive control (P) was subjected to RT-PCR analysis with primers specific for IL-4Rα, IL-13Rα1, IL-13Rα2, or IL-2Rγc mRNAs. GAPDH mRNA served as a control for the quality of cDNA synthesis. For detection of IL-2Rγc mRNA, cells were incubated in the absence (−) or presence (+) of cycloheximide (CHX) at a concentration of 10 μg/mL before isolation of RNA. The positive control for IL-13Rα2 was human synovial fibroblasts, and that for the other IL-4R chains was PHA-stimulated human monocytes. No products were detected in the absence (−) of reverse transcription (RT). Right: Sizes of the specific amplification products; left: molecular size standards. Data are representative of three independent experiments, and identical results were obtained with corneal fibroblasts derived from three additional donors.

Figure 2.

 

Flow cytometric analysis of the expression of IL-4Rα and IL-2Rγc on the surface of human corneal fibroblasts. Intact cells were subjected to immunostaining with monoclonal antibodies to human IL-4Rα (A) or to human IL-2Rγc (B), as indicated by the hatched curves. Control staining with isotype-matched monoclonal antibodies was also performed (open curves). Data are representative of three independent experiments.

Figure 3.

 

Characterization of IL-4 binding to IL-4Rs on the surface of corneal fibroblasts. (A) Saturation curve for ¹²⁵I-labeled IL-4 binding. Cells were incubated with various concentrations of ¹²⁵I-labeled IL-4 for 2 hours at 4°C in the absence (total binding; ○) or presence (nonspecific binding; ▴) of a 500-fold excess of unlabeled IL-4. Specific binding (•) was calculated by subtracting nonspecific binding from total binding. (B) Scatchard plot of specific ¹²⁵I-labeled IL-4 binding generated from the data in (A). The line of best fit was determined by linear regression. Data are representative of three independent experiments.

Figure 4.

 

Effect of IL-4 on the tyrosine phosphorylation of STAT6 in human corneal fibroblasts. (A) Cells were incubated for the indicated times with IL-4 (100 ng/mL), lysed, and subjected to immunoprecipitation with antibodies to STAT6. The resultant precipitates were subjected to immunoblot analysis with antibodies to phosphotyrosine. Arrow: Phosphorylated STAT6 (pSTAT6). (B) The blot in (A) was reprobed with antibodies to STAT6 to confirm equal application of samples to the gel. Arrow: STAT6 protein. The positions of molecular size standards are indicated. Data are representative of three independent experiments.

Figure 5.

 

Effect of IL-4 on the subcellular localization of STAT6 in human corneal fibroblasts. Cells were incubated in the absence (A) or presence (B) of IL-4 (100 ng/mL) for 10 minutes, fixed, and subjected to immunocytochemical analysis with antibodies to STAT6. Data are representative of three independent experiments. Scale bar, 100 μm.

Figure 6.

 

Effect of neutralizing antibodies to IL-4R on eotaxin release induced by the combination of IL-4 and TNF-α in corneal fibroblasts. Cells were preincubated for 1 hour with the indicated concentrations of a neutralizing monoclonal antibody to IL-4R and were then incubated for 24 hours in the additional presence of IL-4 (10 ng/mL) and TNF-α (10 ng/mL). The amount of eotaxin released into the culture medium was then determined by ELISA. Data are means ± SEM of quadruplicate determinations from a representative experiment. * P < 0.0001, (Dunnett test) versus the value for cells incubated in the absence of antibody to IL-4R.

The authors thank Shinji Kimura and Masatsugu Nakamura for technical assistance.