November 2011
Volume 52, Issue 12
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Cornea  |   November 2011
Role of the IL-6 Classic- and Trans-Signaling Pathways in Corneal Sterile Inflammation and Wound Healing
Author Affiliations & Notes
  • Nobuyuki Ebihara
    From the Departments of Ophthalmology and
  • Akira Matsuda
    From the Departments of Ophthalmology and
  • Shinji Nakamura
    the Division of Biomedical Imaging Research, Juntendo University School of Medicine, Tokyo, Japan.
  • Hironori Matsuda
    Immunology, and
  • Akira Murakami
    From the Departments of Ophthalmology and
  • Corresponding author: Nobuyuki Ebihara, Department of Ophthalmology, Juntendo University School of Medicine, 3-1-3, Hongo, Bunkyo-ku, Tokyo 113-8431, Japan; ebihara@juntendo.ac.jp
Investigative Ophthalmology & Visual Science November 2011, Vol.52, 8549-8557. doi:10.1167/iovs.11-7956
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      Nobuyuki Ebihara, Akira Matsuda, Shinji Nakamura, Hironori Matsuda, Akira Murakami; Role of the IL-6 Classic- and Trans-Signaling Pathways in Corneal Sterile Inflammation and Wound Healing. Invest. Ophthalmol. Vis. Sci. 2011;52(12):8549-8557. doi: 10.1167/iovs.11-7956.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To investigate the role of the IL-6 classic- and trans-signaling pathways in corneal sterile inflammation and wound healing.

Methods.: To assess the production of inflammatory molecules by corneal fibroblasts treated with supernatant derived from necrotic corneal epithelial cells, the authors used an antibody array. Expressions of membrane IL-6 receptor (mIL-6R) and soluble IL-6R (SIL-6R) by fibroblasts and epithelial cells were detected with flow cytometry and RT-PCR. Expressions of signal transducer and activator of transcription 3 (STAT3), vascular endothelial growth factor (VEGF), and monocyte chemotactic protein-1 (MCP-1) by fibroblasts stimulated with IL-6 alone or IL-6/SIL-6R were determined by ELISA. The effect of IL-6 or IL-6/SIL-6R on epithelial cell migration was investigated in vitro by the scratch assay, whereas expressions of IL-6R and S100 A4 in the corneas of mice were detected by immunohistochemistry after incision of the corneal stroma.

Results.: IL-1 derived from necrotic corneal epithelial cells induced the production of IL-6 by corneal fibroblasts. mIL-6R and SIL-6R mRNAs were expressed by both types of cells, although IL-6R protein at the cell surface was expressed only by epithelial cells. Expression of gp130 was detected in both types of cells. Activation of the IL-6 trans-signaling pathway induced the phosphorylation of STAT3, resulting in an increase of VEGF and MCP-1 production by corneal fibroblasts. Activation of the IL-6 classic-signaling pathway promoted the migration of corneal epithelial cells. IL-6R expression was also detected in activated fibroblasts and basal cells of the epithelium during the processes of wound healing in vivo.

Conclusions.: The IL-6 classic- and trans-signaling pathways have an important role in corneal sterile inflammation and wound healing.

When cells die in vivo, a strong inflammatory response is initiated, including the rapid migration of neutrophils (followed by monocytes) into injured tissues. However, the mechanism that activates inflammation in response to cellular injury is not fully understood. There is evidence that necrotic cells release various endogenous “danger” molecules, such as high-mobility group box 1 protein (HMGB1), IL-1α, IL-33, heat shock protein 60 (HSP60), uric acid, S100 proteins, DNA, adenosine triphosphate, and β-defensin 2. 1 5 Among these danger signals, IL-1α has been investigated to assess its role in sterile inflammation of the cornea. Wilson et al. 6 8 reported that IL-1α/β are expressed by intact corneal epithelial cells and released into the corneal stroma after mechanical injury. Hong et al. 9 found that IL-1α has a crucial role in inflammatory cell infiltration into the cornea after epithelial scrape injury in rabbit. Recently, Stapleton et al. 10 examined the administration of a topical soluble IL-1 receptor antagonist on the infiltration of bone marrow–derived cells after corneal epithelial scrape injury in a mouse model, demonstrating that the IL-1 receptor antagonist dramatically suppressed the migration of CD11-b–positive monocytes into the corneal stroma. Therefore, IL-1α may be a master regulator of sterile inflammation, which occurs in response to injury of the cornea. 11 However, the mechanisms by which sterile corneal inflammation and wound healing are related to cell death have not been sufficiently investigated. Endogenous danger molecules released from necrotic corneal epithelial cells not only have a chemotactic effect on inflammatory cells, but also upregulate the production of chemokines by corneal fibroblasts. 8,9,12 In this study, we revealed that IL-1 derived from necrotic corneal epithelial cells induced the production of IL-6 and soluble IL-6 receptor (SIL-6R) by corneal fibroblasts. 
IL-6 and SIL-6R are pleiotropic molecules that regulate inflammation and the immune response. Many researchers have found significantly increased concentrations of IL-6 and SIL-6R in the tear fluid of patients with Sjögren syndrome and vernal keratoconjunctivitis compared with the levels in healthy subjects. 13 17 Also, elevated levels of SIL-6R and IL-6 have been detected in the aqueous humor and vitreous fluid of patients with uveitis. 18 These findings suggest that IL-6 and SIL-6R may play an important role in several ocular inflammatory diseases. IL-6 is also known to be involved in wound healing, for example, IL-6–deficient mice show delayed cutaneous wound healing. 19 21 Nishida and colleagues 22 25 reported that IL-6 stimulates the migration of corneal epithelial cells both in vitro and in vivo. IL-6 is not involved in cell growth and differentiation, but makes a contribution to stem cell niche characteristics. 26,27  
Two different signaling pathways for IL-6 have been described. In the classic-signaling pathway, IL-6 binds to membrane-bound IL-6R (mIL-6R), leading to dimerization and activation of the signal-transducing protein glycoprotein 130 (gp130). On the other hand, in the trans-signaling pathway, IL-6 binds to SIL-6R and the IL-6/SIL-6R complex activates gp130. This trans-signaling pathway has some important biological effects. In particular, it enlarges the spectrum of targets for IL-6 because cells that do not express membrane-bound IL-6R can still be stimulated by the IL-6/SIL-6R complex. The level of ubiquitously expressed gp130 protein is believed to be relatively constant for all cells, whereas expression of IL-6R varies between different cell types. Through both the classic- and trans-signaling pathways, IL-6 activates signal transducer and activator of transcription 3 (STAT3) by phosphorylation of this molecule. 28 31  
Two mechanisms for generation of SIL-6R have been described in humans. First, SIL-6R can be formed via limited proteolysis of the membrane-bound receptor, a process called shedding. The second mechanism that can generate the soluble receptor is translation from alternatively spliced mRNA lacking the coding region for the transmembrane domain (DS-SIL-6R: differential SIL-6R splicing). 32,33 Recently, Sugaya et al. 17 revealed that corneal epithelial cells produced SIL-6R by shedding and alternatively spliced mRNA. 
Although IL-6, SIL-6R, and STAT3 have been recognized as inflammatory mediators in the wound healing process, their role in corneal sterile inflammation and wound healing has not been adequately investigated. Therefore, we performed this study to assess the role of the IL-6 classic- and trans-signaling pathways in both sterile corneal inflammation and corneal wound healing. 
Materials and Methods
Antibodies and Ligands
Mouse anti-human IL-6R and gp130 monoclonal antibodies (mAb) were purchased for flow cytometry (R&D Systems, Minneapolis, MN). Goat anti-mouse IL-6R polyclonal Ab (pAb) (R&D Systems) and rabbit anti-S100 A4 pAb (Thermo Fisher Scientific, Fremont, CA) were used in primary antibodies for immunohistochemistry. Other products used were as follows: FITC-conjugated donkey anti-goat IgG pAb (Santa Cruz Biotechnology, Santa Cruz, CA), biotinylated rabbit anti-mouse pAb (CosmoBio, Tokyo, Japan) (these antibodies were used in second antibodies for immunohistochemistry); recombinant human IL-1α, IL-6, and SIL-6R (PeproTech Inc., Rocky Hill, NJ); and recombinant human IL-1 receptor antagonist (ProSpec-TechnoGene Ltd., Rehovot Science Park, Rehovot, Israel). 
Cell Culture
In experiments using human primary keratocytes and corneal epithelial cells (ScienCell Research Laboratories, Carlsbad, CA), keratocytes were primarily cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS). The keratocytes showed transdifferentiation into corneal fibroblasts. Corneal epithelial cells (grown in FCS-free Epimedium; ScienCell Research Laboratories) were also used in primary culture. 
Inducing Necrosis of Corneal Epithelial Cells
According to a previous report, 34 necrosis was induced by three cycles of freezing and thawing. Corneal epithelial cells cultured with serum-free DMEM were frozen (−80°C) for 20 minutes and thawed (37°C) for 20 minutes over three cycles. The supernatant obtained from necrotic corneal epithelial cells was used in this study. IL-6, SIL-6R, and MCP-2 were not detected in this supernatant by ELISA (data not shown). 
Antibody Array
Culture supernatant obtained from corneal fibroblasts was analyzed with an antibody array (RayBio; Human Inflammation Antibody III kit; RayBiotech Inc., Norcross, CA) according to the manufacturer's instructions. Corneal fibroblasts were grown to subconfluence in DMEM containing 10% FCS, washed twice with PBS, and then incubated in serum-free DMEM for 24 hours with or without the supernatant of necrotic corneal epithelial cells (final concentration: 20%) or recombinant IL-1α (30 ng/mL). The culture supernatant was then harvested for antibody array analysis. To estimate the effect of IL-1 receptor antagonist, corneal fibroblasts were incubated in serum-free DMEM for 24 hours with the supernatant of necrotic corneal epithelial cells and IL-1 receptor antagonist (100 ng/mL), after which the culture supernatant was harvested for antibody array analysis. 
Enzyme-Linked Immunosorbent Assay
To detect vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), and phosphorylated STAT3 (Try705) in the supernatant of corneal fibroblasts, we used ELISA kits according to the manufacturer's instructions (Quantikine; R&D Systems). Corneal fibroblasts were grown to subconfluence and then incubated in serum-free DMEM for 24 hours with or without exposure to IL-6, SIL-6R, or IL-6/SIL-6R. The supernatant was then harvested for ELISA. 
Preparation of RNA and RT-PCR
We examined the expression of two isoforms of IL-6R by using different primers for reverse transcription (RT)-PCR. 17 Total RNA was obtained from cultured corneal fibroblasts and epithelial cells (Nacleo Spin RNA II; Macherey-Nagel GmbH, Düren, Germany). RT-PCR was performed to examine the expression of mIL-6R and DS-SIL-6R, as described previously, using an mIL-6R sense primer (5′-cattgccattgttctgaggttc-3′), a DS-SIL-6R sense primer (5′-gcgacaagcctcccaggttc-3′), and a shared IL-6R antisense primer (5′-gtgccacccagccagctatc-3′). For mIL-6R, 40 cycles were performed with annealing at 60°C for 45 seconds. For DS-SIL-6R, 40 cycles were performed with annealing at 65°C for 45 seconds. PCR products were detected by electrophoresis on 1.5% agarose gel containing ethidium bromide, and bands were visualized under UV light. 
Flow Cytometry
To detect the expression of IL-6R and gp130 protein by corneal fibroblasts and epithelial cells, we used flow cytometry (FACScan; BD Biosciences, San Jose, CA). After being washed with buffer, 106 cells were treated with Fc-block (BD Biosciences) for 15 minutes and then were incubated with mAbs targeting human IL-6R or gp130 or with isotype control mouse IgG (BD PharMingen, San Diego, CA) for 1 hour at room temperature. The cells were then washed twice with the buffer and incubated for 30 minutes with FITC-conjugated anti-mouse IgG. Finally, the cells were washed twice more with the buffer and analyzed. To gate out dead cells, staining with a kit containing propidium iodide was performed according to the manufacturer's instructions (BD Biosciences). 
In Vitro Scratch Assay
Corneal epithelial cells were grown to confluence on six-well plates. A uniform wound was made in each plate using a 200-μL pipette tip, after which the plates were washed with PBS and incubated in Epimedium with or without IL-6, SIL-6R, or IL-6/SIL-6R. The wound area (immediately and 16 hours after creation) was observed and photographed. Experiments were done in duplicate, with three to five plates for each condition. 
Animal Protocols
Female C57 BL6 mice (10 to 12 weeks old) were used in this experiment, and were handled according to the ARVO statement on the Use of Animals in Ophthalmic and Vision Research. The mice were given an intraperitoneal (IP) injection of ketamine (50 mg/kg) and xylazine (5 mg/kg) for deep anesthesia and a drop of 1% pro-paracaine for local anesthesia before creating stromal injury of the cornea. Stromal injury was then created by incision of the cornea with a blade while viewing the eye through an operating microscope. Twenty-four hours after corneal injury, the animals were euthanized with an overdose of pentobarbital (100 mg/kg, administered IP), after which the eyes were harvested and fixed in paraffin. 
Immunohistochemistry
Paraffin specimens were cut into 4-μm sections, air dried, fixed in cold acetone for 10 minutes, and then washed in PBS. Next, the sections were blocked by incubation with 3% BSA, after which goat anti-mouse IL-6R pAb (1:50 dilution) was added and the slides were allowed to stand overnight at 4°C. After three washes in PBS, the slides were incubated for 40 minutes with FITC-conjugated donkey anti-goat IgG pAb (1:100 dilution) at room temperature. After being washed three more times with PBS, the slides were incubated for 2 hours with rabbit anti-S100 A4 pAb (ready-to-use) at room temperature. After three washes in PBS, the slides were incubated for 40 minutes with biotinylated goat anti-rabbit pAb (1:300 dilution) at room temperature. After being washed in PBS, the slides were incubated for 40 minutes with fluorescent dye–conjugated (Alexa Fluor 594; Invitrogen) streptavidin. Counterstaining was done with 4′,6′-diamidino-2-phenindole (DAPI). The slides were then inspected by the use of a laser confocal microscope (TCS SP5; Leica Microsystems, Tokyo, Japan). 
Statistical Analysis
Results were expressed as the mean ± SE. Differences were evaluated by Student's t-test using analytical software (Excel; Microsoft, Redmond, WA). 
Results
Chemokine and Cytokine Production by Corneal Fibroblasts Is Stimulated by Supernatant of Necrotic Corneal Epithelial Cells
Representative arrays analyzing culture supernatant obtained from corneal fibroblasts are shown in Figure 1. We estimated the mean optical intensity of positive spots from the culture supernatants (Fig. 2). Corneal fibroblasts cultured with serum-free medium constitutively produced IL-8, MCP-1, RANTES (Regulated upon Activation, Normal T Expressed, and presumably Secreted), and TIMP-2 (Fig. 1a). Moreover, production of IL-6, MCP-2, and SIL-6R was induced and production of IL-8, MCP-1, and RANTES was enhanced when corneal fibroblasts were cultured with serum-free medium containing the supernatant of necrotic corneal epithelial cells (Fig. 1b). Among these molecules, IL-6 was most strongly induced by the supernatant of necrotic corneal epithelial cells (Fig. 2). 
Figure 1.
 
Antibody array of culture supernatants from corneal fibroblasts stimulated by the supernatant of necrotic corneal epithelial cells. Culture supernatant from corneal fibroblasts was analyzed with an antibody array (Human Inflammation Antibody III kit; Ray Biotech Inc.). Representative arrays analyzing culture supernatant obtained from corneal fibroblasts are shown in (a). Cells cultured with serum-free medium. (b) Cells cultured with serum free medium containing the supernatant of necrotic corneal epithelial cell. (c) Cells cultured with serum free medium containing the supernatant of necrotic corneal epithelial cells and human IL-1 receptor antagonist (IL-1RA: 100 ng/mL).
Figure 1.
 
Antibody array of culture supernatants from corneal fibroblasts stimulated by the supernatant of necrotic corneal epithelial cells. Culture supernatant from corneal fibroblasts was analyzed with an antibody array (Human Inflammation Antibody III kit; Ray Biotech Inc.). Representative arrays analyzing culture supernatant obtained from corneal fibroblasts are shown in (a). Cells cultured with serum-free medium. (b) Cells cultured with serum free medium containing the supernatant of necrotic corneal epithelial cell. (c) Cells cultured with serum free medium containing the supernatant of necrotic corneal epithelial cells and human IL-1 receptor antagonist (IL-1RA: 100 ng/mL).
Figure 2.
 
Estimation of the mean optical intensity of positive spots from the culture supernatants. Corneal fibroblasts constitutively produced IL-8, MCP-1, RANTES, and TIMP-2. When cells were cultured with the supernatant of necrotic corneal epithelial cells, the production of IL-6, MCP-2, and SIL-6R was induced and that of IL-8, MCP-1, and RANTES was enhanced. Treatment with an IL-1RA almost completely inhibited the production of IL-6, MCP-2, and SIL-6R and partially inhibited the production of IL-8, MCP-1, and RANTES.
Figure 2.
 
Estimation of the mean optical intensity of positive spots from the culture supernatants. Corneal fibroblasts constitutively produced IL-8, MCP-1, RANTES, and TIMP-2. When cells were cultured with the supernatant of necrotic corneal epithelial cells, the production of IL-6, MCP-2, and SIL-6R was induced and that of IL-8, MCP-1, and RANTES was enhanced. Treatment with an IL-1RA almost completely inhibited the production of IL-6, MCP-2, and SIL-6R and partially inhibited the production of IL-8, MCP-1, and RANTES.
Next, we investigated which factor in the supernatant of necrotic corneal epithelial cells induced the production of IL-6, MCP-2, and SIL-6R by corneal fibroblasts. Treatment with an IL-1R antagonist almost completely inhibited the production of these molecules, indicating that IL-1 derived from necrotic corneal epithelial cells induced the production of IL-6, MCP-2, and SIL-6R by corneal fibroblasts. On the other hand, the IL-1R antagonist partially inhibited production of IL-8, MCP-1, and RANTES by corneal fibroblasts cultured with the supernatant of necrotic corneal epithelial cells, indicating that other danger molecules also enhanced the production of these chemokines (Figs. 1c, 2). 
Influence of IL-1α on IL-6 Production by Corneal Fibroblasts
The mean optical density of positive spots obtained from the culture supernatants of cells treated with IL-1α (30 ng/mL, 24 hours) was compared with that of spots from the supernatants of untreated cells (Fig. 3), revealing that stimulation of fibroblasts with IL-1α induced the production of IL-6, MCP-2, and SIL-6R. Among these molecules, IL-6 was most strongly induced by IL-1α. 
Figure 3.
 
Antibody array of culture supernatants from corneal fibroblasts stimulated by recombinant IL-1α. Culture supernatant from corneal fibroblasts treated with recombinant human IL-1α (30 ng/mL) was analyzed with a commercial antibody array. Representative arrays analyzing culture supernatant are shown: (a) Cells cultured with serum-free medium. (b) Cells cultured with serum-free medium with recombinant IL-1α (30 ng/mL). The mean optical intensity of positive spots was estimated from the culture supernatants. Corneal fibroblasts constitutively produced IL-8, MCP-1, RANTES, and TIMP-2. When cells were treated with IL-1α, the production of IL-6, SIL-6R, and MCP-2 was induced. Among these molecules, IL-6 was most strongly induced by IL-1α.
Figure 3.
 
Antibody array of culture supernatants from corneal fibroblasts stimulated by recombinant IL-1α. Culture supernatant from corneal fibroblasts treated with recombinant human IL-1α (30 ng/mL) was analyzed with a commercial antibody array. Representative arrays analyzing culture supernatant are shown: (a) Cells cultured with serum-free medium. (b) Cells cultured with serum-free medium with recombinant IL-1α (30 ng/mL). The mean optical intensity of positive spots was estimated from the culture supernatants. Corneal fibroblasts constitutively produced IL-8, MCP-1, RANTES, and TIMP-2. When cells were treated with IL-1α, the production of IL-6, SIL-6R, and MCP-2 was induced. Among these molecules, IL-6 was most strongly induced by IL-1α.
IL-6R and gp130 Expression by Corneal Fibroblasts
Corneal fibroblasts showed the expression of mRNAs for mIL-6R and DS-SIL-6R by RT-PCR (Fig. 4). However, when cell surface expression of mIL-6R and gp130 proteins on corneal fibroblasts was examined by flow cytometry, expression of mIL-6R was not detected, but gp130 was detected. This result indicates that corneal fibroblasts showed little or no cell surface expression of mIL-6R. 
Figure 4.
 
Expression of IL-6R and gp130 on primary cultured corneal fibroblasts. Corneal fibroblasts showed strong expression of the mRNAs for mIL-6R (280 bp) and DS-SIL-6R (278 bp) by RT-PCR. Next, the expression of mIL-6R and gp130 on corneal fibroblasts by flow cytometry was examined. mIL-6R was not expressed, but gp130 was expressed on the cell surface of corneal fibroblasts (black line, control mAb; green line, anti-IL-6R mAb or anti-gp130 mAb).
Figure 4.
 
Expression of IL-6R and gp130 on primary cultured corneal fibroblasts. Corneal fibroblasts showed strong expression of the mRNAs for mIL-6R (280 bp) and DS-SIL-6R (278 bp) by RT-PCR. Next, the expression of mIL-6R and gp130 on corneal fibroblasts by flow cytometry was examined. mIL-6R was not expressed, but gp130 was expressed on the cell surface of corneal fibroblasts (black line, control mAb; green line, anti-IL-6R mAb or anti-gp130 mAb).
IL-6R and gp130 Expression by Corneal Epithelial Cells
Corneal epithelial cells showed the expression of mRNAs for mIL-6R and DS-SIL-6R by RT-PCR (Fig. 5). When cell surface expression of mIL-6R and gp130 proteins on corneal epithelial cells was examined by flow cytometry, both proteins on corneal epithelial cells were detected almost equally. 
Figure 5.
 
Expression of IL-6R and gp130 on primary cultured corneal epithelial cells. Corneal epithelial cells showed strong expression of the mRNAs for mIL-6R (280 bp) and DS-SIL-6R (278 bp) by RT-PCR. Next, the expression of mIL-6R and gp130 on corneal epithelial cells by flow cytometry was examined. Both mIL6R and gp130 were expressed on the cell surface of corneal epithelial cells (black line, control mAb; green line, anti-IL-6R mAb or anti-gp130 mAb).
Figure 5.
 
Expression of IL-6R and gp130 on primary cultured corneal epithelial cells. Corneal epithelial cells showed strong expression of the mRNAs for mIL-6R (280 bp) and DS-SIL-6R (278 bp) by RT-PCR. Next, the expression of mIL-6R and gp130 on corneal epithelial cells by flow cytometry was examined. Both mIL6R and gp130 were expressed on the cell surface of corneal epithelial cells (black line, control mAb; green line, anti-IL-6R mAb or anti-gp130 mAb).
Expression of Phosphorylated STAT3 by Corneal Fibroblasts
We examined the influence of IL-6, SIL-6R, or IL-6/SIL-6R on phosphorylation of STAT3 in corneal fibroblasts. Figure 6 reveals that IL-6 alone increased STAT3 phosphorylation at a high dose, whereas SIL-6R alone did not induce the phosphorylation of STAT3. On the other hand, the IL-6/SIL-6R significantly increased STAT3 phosphorylation. 
Figure 6.
 
Expression of phosphorylated STAT3 by corneal fibroblasts. The influence of IL-6, SIL-6R, or IL-6/SIL-6R on phosphorylation of STAT3 was examined by ELISA. IL-6 alone increased STAT3 phosphorylation at a high dose (100 ng/mL), whereas SIL-6R alone did not induce the phosphorylation. IL-6/SIL-6R significantly increased STAT3 phosphorylation.
Figure 6.
 
Expression of phosphorylated STAT3 by corneal fibroblasts. The influence of IL-6, SIL-6R, or IL-6/SIL-6R on phosphorylation of STAT3 was examined by ELISA. IL-6 alone increased STAT3 phosphorylation at a high dose (100 ng/mL), whereas SIL-6R alone did not induce the phosphorylation. IL-6/SIL-6R significantly increased STAT3 phosphorylation.
VEGF and MCP-1 Production by Corneal Fibroblasts
When we examined the production of VEGF by corneal fibroblasts, IL-6 or SIL-6R alone did not induce VEGF production. On the other hand, the IL-6/SIL-6R significantly induced VEGF production (Fig. 7A). We also examined MCP-1 production by corneal fibroblasts. We found that IL-6 or SIL-6R alone slightly induced the production of MCP-1, whereas IL-6/SIL-6R significantly induced its production (Fig. 7B). 
Figure 7.
 
VEGF and MCP-1 production by corneal fibroblasts. The production of VEGF and MCP-1 by corneal fibroblasts was examined by ELISA. IL-6 or SIL-6R alone did not induce VEGF production. The IL-6/SIL-6R significantly induced VEGF production (A). IL-6 or SIL-6R alone slightly induced the production of MCP-1, whereas the IL-6/SIL-6R significantly induced its production (B).
Figure 7.
 
VEGF and MCP-1 production by corneal fibroblasts. The production of VEGF and MCP-1 by corneal fibroblasts was examined by ELISA. IL-6 or SIL-6R alone did not induce VEGF production. The IL-6/SIL-6R significantly induced VEGF production (A). IL-6 or SIL-6R alone slightly induced the production of MCP-1, whereas the IL-6/SIL-6R significantly induced its production (B).
Expression of Phosphorylated STAT3 by Corneal Epithelial Cells
We examined the effect of IL-6, SIL-6R, or IL-6/SIL-6R on phosphorylation of STAT3 in corneal epithelial cells. As shown in Figure 8, IL-6 alone and the IL-6/SIL-6R significantly increased the expression of phosphorylated STAT3 in corneal epithelial cells, but SIL-6R alone did not increase its expression. 
Figure 8.
 
Expression of phosphorylated STAT3 by corneal epithelial cells. The influence of IL-6, SIL-6R, or IL-6/SIL-6R on phosphorylation of STAT3 was examined by ELISA. IL-6 or IL-6/SIL-6R increased STAT3 phosphorylation as dose dependent, whereas SIL-6R alone did not induce the phosphorylation.
Figure 8.
 
Expression of phosphorylated STAT3 by corneal epithelial cells. The influence of IL-6, SIL-6R, or IL-6/SIL-6R on phosphorylation of STAT3 was examined by ELISA. IL-6 or IL-6/SIL-6R increased STAT3 phosphorylation as dose dependent, whereas SIL-6R alone did not induce the phosphorylation.
Corneal Epithelial Cell Migration in the Scratch Assay
The scratch assay was done to assess in vitro migration of cultured corneal epithelial cells stimulated by IL-6, SIL-6R, or IL-6/SIL-6. Figure 9 shows that IL-6 or IL-6/SIL-6R significantly induced cell migration compared with that by untreated cells. Surprisingly, SIL-6R alone also induced the migration of these cells. 
Figure 9.
 
Corneal epithelial cell migration in the scratch assay. The scratch assay was done to assess in vitro migration of cultured corneal epithelial cells stimulated by IL-6, SIL-6R, or IL-6/SIL-6R. A uniform wound was made in each plate using a 200-μL pipette tip. The wound area was observed immediately and at 16 hours after creation. IL-6, SIL-6R, or IL-6/SIL-6R significantly induced cell migration compared with that by untreated cells.
Figure 9.
 
Corneal epithelial cell migration in the scratch assay. The scratch assay was done to assess in vitro migration of cultured corneal epithelial cells stimulated by IL-6, SIL-6R, or IL-6/SIL-6R. A uniform wound was made in each plate using a 200-μL pipette tip. The wound area was observed immediately and at 16 hours after creation. IL-6, SIL-6R, or IL-6/SIL-6R significantly induced cell migration compared with that by untreated cells.
Immunohistochemistry
We observed the expression of IL-6R and S100 A4 in corneal fibroblasts at 24 hours after corneal injury. S100 A4 is the marker of activated corneal fibroblasts. 35 Corneal fibroblasts at and nearby the site of injury were stained with an antibody against IL-6R together with S100 A4 antibody. Immunostaining for IL-6R was also recognized in basal cells of the epithelium near the site of injury. There was no immunostaining for IL-6R and S100 A4 in keratocytes or corneal epithelial cells of the intact mouse cornea (data not shown). These immunohistochemical analyses (Fig. 10) for mouse corneal wound healing model showed IL-6R expression on activated fibroblasts. 
Figure 10.
 
Expression of IL-6R and S100 A4 in mouse corneal wound healing model. Stromal injury was created by incision of the center of the cornea with a blade. Twenty-four hours after corneal injury, the expression of IL-6R and S100 A4 was observed by immunohistochemistry (a). Immunostaining for IL-6R (green) was observed in corneal fibroblasts at and nearby the site of injury. Immunostaining for IL-6R (green) was also recognized in basal cells of the epithelium near the site of injury and stratified epithelial cells at the site of injury (b). Immunostaining for S100 A4 (red), which was the marker of activated corneal fibroblasts, was observed in corneal fibroblasts at and nearby the site of injury (c). Green fluorescence from IL-6R and red fluorescence from S100 A4 identified activated corneal fibroblasts as expressing IL-6R (yellow) when the images were superimposed.
Figure 10.
 
Expression of IL-6R and S100 A4 in mouse corneal wound healing model. Stromal injury was created by incision of the center of the cornea with a blade. Twenty-four hours after corneal injury, the expression of IL-6R and S100 A4 was observed by immunohistochemistry (a). Immunostaining for IL-6R (green) was observed in corneal fibroblasts at and nearby the site of injury. Immunostaining for IL-6R (green) was also recognized in basal cells of the epithelium near the site of injury and stratified epithelial cells at the site of injury (b). Immunostaining for S100 A4 (red), which was the marker of activated corneal fibroblasts, was observed in corneal fibroblasts at and nearby the site of injury (c). Green fluorescence from IL-6R and red fluorescence from S100 A4 identified activated corneal fibroblasts as expressing IL-6R (yellow) when the images were superimposed.
Discussion
This study showed that culture supernatant derived from necrotic corneal epithelial cells induced the production of IL-6, MCP-2, and SIL-6R by corneal fibroblasts, whereas an IL-1R antagonist inhibited the production of these molecules almost completely. In addition, recombinant IL-1α induced the production of IL-6, MCP-2, and SIL-6R. These results demonstrate that IL-1 derived from necrotic corneal epithelial cells stimulates corneal fibroblasts via IL-1R, resulting in the production of IL-6, MCP-2, and SIL-6R. Necrotic corneal epithelial cell supernatant also enhanced the production of IL-8, MCP-1, and RANTES, whereas the IL-1R antagonist partially inhibited their production, suggesting that other endogenous danger molecules influenced production of these chemokines in addition to IL-1. Among these various molecules, IL-6 may be the key molecule for sterile inflammation of the cornea and corneal wound healing. Sotozono et al. 36 showed that IL-1α and IL-6 levels are dramatically elevated in the regenerating epithelium of the mouse cornea during the early stage of recovery from an alkali burn. Biswas et al. 37 demonstrated that herpes infection of the cornea induced the expression IL-1α and IL-6 in mice. These results indicate that chemical injury or viral infection of cornea induces the release of IL-1α from damaged corneal epithelial cells, enhances IL-6 production by corneal fibroblasts, and promotes inflammatory cell infiltration into the corneal stroma. 
Cellular responses to IL-6 or the IL-6/SIL-6R complex are determined by the level of IL-6R expression. The level of ubiquitously expressed gp130 protein is believed to be relatively constant for all cells, whereas expression of IL-6R varies between different cell types. Cells that do not have surface expression of IL-6R can be stimulated only by the IL-6/SIL-6R complex (the trans-signaling pathway) and are insensitive to IL-6 alone. Cells with fewer IL-6R molecules on their surface than gp130 molecules can respond to IL-6, and this response can be enhanced by the IL-6/SIL-6R complex. Cells that show balanced surface expression of IL-6R and gp130 also respond to IL-6, but this response is not altered by the IL-6/SIL-6R complex. 28 31 In this study, IL-6R protein was not detected on the surface of corneal fibroblasts, although RT-PCR detected mIL-6R at the mRNA level. In contrast, gp130 protein was expressed on the cell surface. These results indicate that corneal fibroblasts express fewer (or no) IL-6R molecules on the cell surface than gp130 molecules. Therefore, corneal fibroblasts responded to IL-6/SIL-6R and showed phosphorylation of STAT3 (the trans-signaling pathway). Our results were similar to those of another group. 17 On the other hand, expression of IL-6R and gp130 proteins on the surface of corneal epithelial cells was detected by flow cytometry. Therefore, corneal epithelial cells could respond to IL-6 alone by phosphorylation of STAT3 (the classic-signaling pathway), and this response was not altered by exposure of cells to IL-6/SIL-6R. 
Recently, IL-6 has been suggested to play a role in corneal neovascularization. In the rat cornea micropocket assay, IL-6 was shown to induce corneal angiogenesis. 38 The present study indicated that stimulation by IL-6/SIL-6R leads to activation of STAT3 and the increased production of VEGF in corneal fibroblasts, whereas IL-6 alone did not have such an effect. One possible explanation for these results was that corneal fibroblasts express more gp130 molecules than mIL-6R molecules. 
The first cells to participate in the inflammatory response related to wound healing are neutrophils, which infiltrate tissues and act as first line of defense against microorganisms. These neutrophils then undergo apoptosis and become an important source of SIL-6R. Recently, Chalaris et al. 39 demonstrated that shedding of SIL-6R from neutrophils was induced by apoptosis and that SIL-6R then promoted the trans-signaling pathway to regulate the attraction of monocytes involved in the clearance of apoptotic neutrophils. In this study, MCP-1 secretion was observed when corneal fibroblasts were treated with IL-6 alone, but the release of MCP-1 was significantly greater when cells were stimulated with IL-6/SIL-6R. Addition of SIL-6R alone also promoted a slight, but significant, increase of MCP-1 production, which suggests that IL-6 produced by corneal fibroblasts may bind to SIL-6R, resulting in activation of the IL-6/SIL-6R trans-signaling pathway. Thus, the SIL-6R/IL-6 complex may contribute to recruitment of monocytes into the corneal stroma, resulting in the prevention of tissue damage from excessive inflammation by clearance of damaged/dead neutrophils, 40,41  
Previously, Nishida et al. 22 25 reported that IL-6 stimulates the migration of corneal epithelial cells both in vitro and in vivo. IL-6/STAT3 signaling also regulates human biliary epithelial cell migration and wound healing in vitro. 42 In the present study, IL-6 promoted the migration of corneal epithelial cells in the scratch assay, and IL-6/SIL-6R also induced migration of corneal epithelial cells in the same way as IL-6 alone. These results indicate that corneal epithelial cells show similar surface expression of IL-6R and gp130. Therefore, corneal epithelial cells responded to IL-6, and this response was not altered by addition of IL-6/SIL-6R. Surprisingly, however, SIL-6R itself promoted the migration of corneal epithelial cells. This raises the possibility that IL-6 produced by corneal epithelial cells bound to SIL-6R, resulting in the activation of these cells via the trans-signaling pathway. Furthermore, in our mouse corneal wound healing model, the expression of IL-6R and S100 A4 was recognized in corneal fibroblasts near the site of injury, revealing that activated fibroblasts express IL-6R. IL-6R expression was also recognized on basal cells near the site of injury. Therefore, IL-6 produced by corneal fibroblasts and IL-6R expression by activated fibroblasts and basal cells of the corneal epithelium may be important for corneal wound healing. 
In conclusion, IL-1 derived from necrotic corneal epithelial cells induced the production of IL-6 by corneal fibroblasts. Activation of the IL-6 trans-signaling pathway induced the phosphorylation of STAT3, resulting in an increase of VEGF and MCP-1 production by corneal fibroblasts. Activation of the IL-6 classic-signaling pathway promoted the migration of corneal epithelial cells. IL-6R expression was also detected in activated fibroblasts and basal cells of the corneal epithelium during the processes of wound healing in vivo. These results emphasize the role of the IL-6 classic- and trans-signaling pathways in sterile inflammation of the cornea and corneal wound healing. 
Footnotes
 Disclosure: N. Ebihara, None; A. Matsuda, None; S. Nakamura, None; H. Matsuda, None; A. Murakami, None
The authors thank Saori Ito and Ai Miyazaki for secretarial and technical assistance. 
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Figure 1.
 
Antibody array of culture supernatants from corneal fibroblasts stimulated by the supernatant of necrotic corneal epithelial cells. Culture supernatant from corneal fibroblasts was analyzed with an antibody array (Human Inflammation Antibody III kit; Ray Biotech Inc.). Representative arrays analyzing culture supernatant obtained from corneal fibroblasts are shown in (a). Cells cultured with serum-free medium. (b) Cells cultured with serum free medium containing the supernatant of necrotic corneal epithelial cell. (c) Cells cultured with serum free medium containing the supernatant of necrotic corneal epithelial cells and human IL-1 receptor antagonist (IL-1RA: 100 ng/mL).
Figure 1.
 
Antibody array of culture supernatants from corneal fibroblasts stimulated by the supernatant of necrotic corneal epithelial cells. Culture supernatant from corneal fibroblasts was analyzed with an antibody array (Human Inflammation Antibody III kit; Ray Biotech Inc.). Representative arrays analyzing culture supernatant obtained from corneal fibroblasts are shown in (a). Cells cultured with serum-free medium. (b) Cells cultured with serum free medium containing the supernatant of necrotic corneal epithelial cell. (c) Cells cultured with serum free medium containing the supernatant of necrotic corneal epithelial cells and human IL-1 receptor antagonist (IL-1RA: 100 ng/mL).
Figure 2.
 
Estimation of the mean optical intensity of positive spots from the culture supernatants. Corneal fibroblasts constitutively produced IL-8, MCP-1, RANTES, and TIMP-2. When cells were cultured with the supernatant of necrotic corneal epithelial cells, the production of IL-6, MCP-2, and SIL-6R was induced and that of IL-8, MCP-1, and RANTES was enhanced. Treatment with an IL-1RA almost completely inhibited the production of IL-6, MCP-2, and SIL-6R and partially inhibited the production of IL-8, MCP-1, and RANTES.
Figure 2.
 
Estimation of the mean optical intensity of positive spots from the culture supernatants. Corneal fibroblasts constitutively produced IL-8, MCP-1, RANTES, and TIMP-2. When cells were cultured with the supernatant of necrotic corneal epithelial cells, the production of IL-6, MCP-2, and SIL-6R was induced and that of IL-8, MCP-1, and RANTES was enhanced. Treatment with an IL-1RA almost completely inhibited the production of IL-6, MCP-2, and SIL-6R and partially inhibited the production of IL-8, MCP-1, and RANTES.
Figure 3.
 
Antibody array of culture supernatants from corneal fibroblasts stimulated by recombinant IL-1α. Culture supernatant from corneal fibroblasts treated with recombinant human IL-1α (30 ng/mL) was analyzed with a commercial antibody array. Representative arrays analyzing culture supernatant are shown: (a) Cells cultured with serum-free medium. (b) Cells cultured with serum-free medium with recombinant IL-1α (30 ng/mL). The mean optical intensity of positive spots was estimated from the culture supernatants. Corneal fibroblasts constitutively produced IL-8, MCP-1, RANTES, and TIMP-2. When cells were treated with IL-1α, the production of IL-6, SIL-6R, and MCP-2 was induced. Among these molecules, IL-6 was most strongly induced by IL-1α.
Figure 3.
 
Antibody array of culture supernatants from corneal fibroblasts stimulated by recombinant IL-1α. Culture supernatant from corneal fibroblasts treated with recombinant human IL-1α (30 ng/mL) was analyzed with a commercial antibody array. Representative arrays analyzing culture supernatant are shown: (a) Cells cultured with serum-free medium. (b) Cells cultured with serum-free medium with recombinant IL-1α (30 ng/mL). The mean optical intensity of positive spots was estimated from the culture supernatants. Corneal fibroblasts constitutively produced IL-8, MCP-1, RANTES, and TIMP-2. When cells were treated with IL-1α, the production of IL-6, SIL-6R, and MCP-2 was induced. Among these molecules, IL-6 was most strongly induced by IL-1α.
Figure 4.
 
Expression of IL-6R and gp130 on primary cultured corneal fibroblasts. Corneal fibroblasts showed strong expression of the mRNAs for mIL-6R (280 bp) and DS-SIL-6R (278 bp) by RT-PCR. Next, the expression of mIL-6R and gp130 on corneal fibroblasts by flow cytometry was examined. mIL-6R was not expressed, but gp130 was expressed on the cell surface of corneal fibroblasts (black line, control mAb; green line, anti-IL-6R mAb or anti-gp130 mAb).
Figure 4.
 
Expression of IL-6R and gp130 on primary cultured corneal fibroblasts. Corneal fibroblasts showed strong expression of the mRNAs for mIL-6R (280 bp) and DS-SIL-6R (278 bp) by RT-PCR. Next, the expression of mIL-6R and gp130 on corneal fibroblasts by flow cytometry was examined. mIL-6R was not expressed, but gp130 was expressed on the cell surface of corneal fibroblasts (black line, control mAb; green line, anti-IL-6R mAb or anti-gp130 mAb).
Figure 5.
 
Expression of IL-6R and gp130 on primary cultured corneal epithelial cells. Corneal epithelial cells showed strong expression of the mRNAs for mIL-6R (280 bp) and DS-SIL-6R (278 bp) by RT-PCR. Next, the expression of mIL-6R and gp130 on corneal epithelial cells by flow cytometry was examined. Both mIL6R and gp130 were expressed on the cell surface of corneal epithelial cells (black line, control mAb; green line, anti-IL-6R mAb or anti-gp130 mAb).
Figure 5.
 
Expression of IL-6R and gp130 on primary cultured corneal epithelial cells. Corneal epithelial cells showed strong expression of the mRNAs for mIL-6R (280 bp) and DS-SIL-6R (278 bp) by RT-PCR. Next, the expression of mIL-6R and gp130 on corneal epithelial cells by flow cytometry was examined. Both mIL6R and gp130 were expressed on the cell surface of corneal epithelial cells (black line, control mAb; green line, anti-IL-6R mAb or anti-gp130 mAb).
Figure 6.
 
Expression of phosphorylated STAT3 by corneal fibroblasts. The influence of IL-6, SIL-6R, or IL-6/SIL-6R on phosphorylation of STAT3 was examined by ELISA. IL-6 alone increased STAT3 phosphorylation at a high dose (100 ng/mL), whereas SIL-6R alone did not induce the phosphorylation. IL-6/SIL-6R significantly increased STAT3 phosphorylation.
Figure 6.
 
Expression of phosphorylated STAT3 by corneal fibroblasts. The influence of IL-6, SIL-6R, or IL-6/SIL-6R on phosphorylation of STAT3 was examined by ELISA. IL-6 alone increased STAT3 phosphorylation at a high dose (100 ng/mL), whereas SIL-6R alone did not induce the phosphorylation. IL-6/SIL-6R significantly increased STAT3 phosphorylation.
Figure 7.
 
VEGF and MCP-1 production by corneal fibroblasts. The production of VEGF and MCP-1 by corneal fibroblasts was examined by ELISA. IL-6 or SIL-6R alone did not induce VEGF production. The IL-6/SIL-6R significantly induced VEGF production (A). IL-6 or SIL-6R alone slightly induced the production of MCP-1, whereas the IL-6/SIL-6R significantly induced its production (B).
Figure 7.
 
VEGF and MCP-1 production by corneal fibroblasts. The production of VEGF and MCP-1 by corneal fibroblasts was examined by ELISA. IL-6 or SIL-6R alone did not induce VEGF production. The IL-6/SIL-6R significantly induced VEGF production (A). IL-6 or SIL-6R alone slightly induced the production of MCP-1, whereas the IL-6/SIL-6R significantly induced its production (B).
Figure 8.
 
Expression of phosphorylated STAT3 by corneal epithelial cells. The influence of IL-6, SIL-6R, or IL-6/SIL-6R on phosphorylation of STAT3 was examined by ELISA. IL-6 or IL-6/SIL-6R increased STAT3 phosphorylation as dose dependent, whereas SIL-6R alone did not induce the phosphorylation.
Figure 8.
 
Expression of phosphorylated STAT3 by corneal epithelial cells. The influence of IL-6, SIL-6R, or IL-6/SIL-6R on phosphorylation of STAT3 was examined by ELISA. IL-6 or IL-6/SIL-6R increased STAT3 phosphorylation as dose dependent, whereas SIL-6R alone did not induce the phosphorylation.
Figure 9.
 
Corneal epithelial cell migration in the scratch assay. The scratch assay was done to assess in vitro migration of cultured corneal epithelial cells stimulated by IL-6, SIL-6R, or IL-6/SIL-6R. A uniform wound was made in each plate using a 200-μL pipette tip. The wound area was observed immediately and at 16 hours after creation. IL-6, SIL-6R, or IL-6/SIL-6R significantly induced cell migration compared with that by untreated cells.
Figure 9.
 
Corneal epithelial cell migration in the scratch assay. The scratch assay was done to assess in vitro migration of cultured corneal epithelial cells stimulated by IL-6, SIL-6R, or IL-6/SIL-6R. A uniform wound was made in each plate using a 200-μL pipette tip. The wound area was observed immediately and at 16 hours after creation. IL-6, SIL-6R, or IL-6/SIL-6R significantly induced cell migration compared with that by untreated cells.
Figure 10.
 
Expression of IL-6R and S100 A4 in mouse corneal wound healing model. Stromal injury was created by incision of the center of the cornea with a blade. Twenty-four hours after corneal injury, the expression of IL-6R and S100 A4 was observed by immunohistochemistry (a). Immunostaining for IL-6R (green) was observed in corneal fibroblasts at and nearby the site of injury. Immunostaining for IL-6R (green) was also recognized in basal cells of the epithelium near the site of injury and stratified epithelial cells at the site of injury (b). Immunostaining for S100 A4 (red), which was the marker of activated corneal fibroblasts, was observed in corneal fibroblasts at and nearby the site of injury (c). Green fluorescence from IL-6R and red fluorescence from S100 A4 identified activated corneal fibroblasts as expressing IL-6R (yellow) when the images were superimposed.
Figure 10.
 
Expression of IL-6R and S100 A4 in mouse corneal wound healing model. Stromal injury was created by incision of the center of the cornea with a blade. Twenty-four hours after corneal injury, the expression of IL-6R and S100 A4 was observed by immunohistochemistry (a). Immunostaining for IL-6R (green) was observed in corneal fibroblasts at and nearby the site of injury. Immunostaining for IL-6R (green) was also recognized in basal cells of the epithelium near the site of injury and stratified epithelial cells at the site of injury (b). Immunostaining for S100 A4 (red), which was the marker of activated corneal fibroblasts, was observed in corneal fibroblasts at and nearby the site of injury (c). Green fluorescence from IL-6R and red fluorescence from S100 A4 identified activated corneal fibroblasts as expressing IL-6R (yellow) when the images were superimposed.
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