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Cornea  |   April 2013
Interaction Between Conjunctival Epithelial Cells and Mast Cells Induces CCL2 Expression and Piecemeal Degranulation in Mast Cells
Author Affiliations & Notes
  • Satoshi Iwamoto
    Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan
  • Yosuke Asada
    Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan
  • Nobuyuki Ebihara
    Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan
  • Kanji Hori
    Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan
  • Yoshimichi Okayama
    Division of Molecular Cell Immunology and Allergology, Advanced Medical Research Center, Nihon University Graduate School of Medical Science, Tokyo, Japan
  • Jun-ichi Kashiwakura
    Division of Molecular Cell Immunology and Allergology, Advanced Medical Research Center, Nihon University Graduate School of Medical Science, Tokyo, Japan
  • Yasuo Watanabe
    Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan
  • Satoshi Kawasaki
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Norihiko Yokoi
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Tsutomu Inatomi
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Katsuhiko Shinomiya
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Akira Murakami
    Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan
  • Akira Matsuda
    Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan
  • Correspondence: Akira Matsuda, Department of Ophthalmology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-Ku, Tokyo, 113-8431, Japan; akimatsu@juntendo.ac.jp
Investigative Ophthalmology & Visual Science April 2013, Vol.54, 2465-2473. doi:10.1167/iovs.12-10664
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      Satoshi Iwamoto, Yosuke Asada, Nobuyuki Ebihara, Kanji Hori, Yoshimichi Okayama, Jun-ichi Kashiwakura, Yasuo Watanabe, Satoshi Kawasaki, Norihiko Yokoi, Tsutomu Inatomi, Katsuhiko Shinomiya, Akira Murakami, Akira Matsuda; Interaction Between Conjunctival Epithelial Cells and Mast Cells Induces CCL2 Expression and Piecemeal Degranulation in Mast Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(4):2465-2473. doi: 10.1167/iovs.12-10664.

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

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Abstract

Purpose.: Intraepithelial mast cells are observed in giant papillae tissue samples obtained from patients with atopic keratoconjunctivitis (AKC)/vernal keratoconjunctivitis (VKC). We examined the roles of interaction between the conjunctival epithelial cells and mast cells.

Methods.: The interaction between human mast cells and conjunctival epithelial cells (HCjE) was investigated using a coculture model. Protein array analysis, ELISA, and real-time PCR were performed to test the interaction. Tissue samples (n = 6) from giant papillae were resected for therapeutic purposes, and subjected to immunohistological analysis of CCL2 expression. Recombinant CCL2 (10 ng/mL) was reacted with the cultured human mast cells and ultrastructural analysis was performed. A ragweed (RW)-induced mouse experimental allergic conjunctivitis model was used to examine ccl2 mRNA expression and mast cell morphology.

Results.: Protein array and real-time PCR analyses showed that CCL2 protein/mRNA expression was induced by mast cell–HCjE coculture. Upregulation of CCL2 mRNA was observed in mast cells, whereas in situ CCL2 expression was observed at the conjunctival epithelium of the giant papillae by immunohistochemistry. Ultrastructural analysis showed that recombinant CCL2 treatment induced piecemeal degranulation (PMD) in the mast cells. Ultrastructural analysis of tissues from the giant papillae showed PMD of mast cells within the conjunctival epithelial cells. The RW-induced experimental allergic conjunctivitis model showed increased ccl2 mRNA expression and PMD morphology in the conjunctivae.

Conclusions.: Mast cell–conjunctival epithelial cell interaction induces CCL2 expression and subsequent PMD.

Introduction
Mast cell activation and migration within and around the conjunctival epithelium is one of the histopathologic features of severe chronic allergic conjunctivitis, atopic keratoconjunctivitis (AKC), 1 and vernal keratoconjunctivitis (VKC). 2 In our study, we investigated possible interactions of mast cells and conjunctival epithelial cells using in vitro coculture models, and we found that CCL2 expression in mast cells was upregulated by coculture. A previous report showed that CCL2 could induce piecemeal degranulation (PMD) in basophils. 3 PMD and anaphylactic degranulation are known as two distinct types of mast cell degranulation. 4,5 Anaphylactic degranulation is a degranulation style with antecedent granule-to-granule and/or granule-to-plasma membrane fusions. On the other hand, gradual emptying of cytoplasmic secretory granules in the absence of granule-to-granule or granule-to-plasma membrane fusion events is observed in PMD. Previously, we reported that 34 of 168 mast cells observed in the giant papillae of eight eyes obtained from VKC patients showed PMD, whereas only 28 of the 168 mast cells had the morphology of anaphylactic degranulation. 6 These results suggested that not only anaphylactic degranulation, but also PMD might have some roles in the pathophysiology of AKC and VKC. In our study, we investigated the roles of mast cells and epithelial cells interactions, as well as the roles of PMD and CCL2 expression in mast cells, in the pathophysiology of AKC/VKC. 
Materials and Methods
Coculture Model of Mast Cells and Conjunctival Epithelial Cells
Human mast cell line LAD2 was provided by Dr Arnold Kirchenbaum (NIH) and maintained as described previously. 7 Human peripheral blood derived mast cells (p-mast) were raised and maintained as described previously. 8 The human conjunctival epithelial cell line (HCjE) was provided by Prof Ilene Gipson (Schepens Eye Research Inst., Boston, MA) and maintained as described previously. 9 Coculture models of these cells were made using Costar Transwell permeable supports (for 12-well culture dishes). 
Antibody Array Analysis and ELISA Analysis Using Culture Supernatant
For antibody array analysis, the culture supernatant was incubated with Human Inflammation Array No. 3 (Ray Biotech, Inc., Norcross, GA) according to the manufacturer's protocol. The results were visualized and quantified using an LAS 3000 image ware (Fuji Film, Tokyo, Japan). Human CCL2 ELISA was performed using a Quantikine CCL2 ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. 
Giant Papillae and Control Conjunctivae Samples
Giant papillae were resected for therapeutic purposes from 5 patients, 3 with AKC and 2 with VKC, and control conjunctival tissue was biopsied from 8 conjunctivochalasis patients during resection surgery after obtaining written informed consent as described previously. 10 Additional giant papillae were obtained from 2 AKC and 4 VKC patients for immunohistochemical analysis. All procedures were approved by the ethics committees of the Juntendo University School of Medicine and Kyoto Prefectural University of Medicine, and the study was conducted in accordance with the tenets of the Declaration of Helsinki. AKC was defined as a bilateral chronic inflammation of the conjunctiva and eyelids associated with atopic dermatitis, and VKC was defined as a chronic, bilateral, conjunctival inflammatory condition found in individuals as described previously. 11  
Reverse Transcription (RT) and Real-Time PCR
Total RNA was extracted from the cultured cells and tissues of the giant papillae using a NucleoSpin II RNA isolation kit (Macherey-Nagel GmbH & Co. KG, Duren, Germany), and cDNAs were prepared from 1 μg of total RNA using random primers and the RevaTra-Ace reverse transcriptase (both from Toyobo, Tokyo, Japan) according to the manufacturer's protocol. We used TaqMann real-time PCR probes and primers specific for human CCL2 (Hs00234140_ml) and 18SrRNA, obtained from Applied Biosystems (Assay-on-Demand gene expression products; Applied Biosystems, Inc., Foster City, CA). Real-time PCR analysis was performed on a 7500 Real-Time PCR system (Applied Biosystems). For CCL2 mRNA expression, the comparative Ct method, which uses the 18SrRNA expression in the same cDNA as a control, was used. CCL4 and ccl2 mRNA were quantified using Fast SYBR green master mix (Applied Biosystems), and the following pairs of the primers: Forward 5′-CTGTGCTGATCCCAGTGAATC-3′, reverse 5′- TCAGTTCAGTTCCAGGTCATACA-3′ (CCL4) and forward 5′-AGCAGCAGGTGTCCCAAAGAAG-3′, reverse 5′-GCACAGACCTCTCTCTTGAGCTTG-3′ (ccl2). For CCL4 and ccl2 mRNA expression, the comparative Ct method, which uses the GAPDH/gapdh expression in the same cDNA as controls, was used. 
Immunohistochemical Analysis
The specimens from the giant papillae were fixed immediately with 4% paraformaldehyde (PFA) in PBS for 3 hours. After washing with 30% sucrose in PBS, the tissues were frozen in Optimal Cutting Temperature (OCT; Sakura Finetek, Tokyo, Japan) compound using liquid nitrogen. Then, 5 μm frozen sections were made and air-dried. Immunohistochemical staining was performed according to the described previously methods. 11 A mouse anti-human CCL2 monoclonal antibody (R&D Systems) was used as a primary antibody (10 μg/mL). For mast cell staining, a rabbit anti-FcεRIβ antibody was prepared and used as described previously. 11 Alexa 488-, and 594-conjugated donkey anti-mouse IgG, and anti-rabbit IgG (all from Invitrogen Corporation, Carlsbad, CA) were used as secondary antibodies. Negative control staining was performed using isotype-matched IgG (normal mouse IgG1; BioLegend, San Diego, CA, and normal rabbit IgG; Santa Cruz Biotechnology, Santa Cruz, CA) as substitutes for the primary antibodies. A confocal laser scanning microscope (FV-1000; Olympus, Tokyo, Japan) was used for imaging. 
Ultrastructural Analysis of Human Cultured Mast Cells
P-mast cells were stimulated with 20 ng/mL recombinant human CCL2 (Peprotech, London, UK) for 3 minutes, 3 and fixed with 2.5% glutaraldehyde and postfixed with 2% osmic acid. HCjE cocultured p-mast cells also were prepared for ultrastructural analysis after 24-hour coculture experiments. For negative control, recombinant human CXCL8 (20 ng/mL for 3 minutes; Peprotech) stimulated p-mast cells, and crosslinked p-mast cells using human IgE (1 μg/mL; Chemicon/Millipore, Billerica, MA) and rabbit anti-IgE (1μg/mL; Dako Japan, Kyoto, Japan) were prepared. The samples were embedded in epoxy resin and ultrathin sections (60–80 nm) were made. The ultrathin sections then were examined using a transmission electron microscope (7000-100; Hitachi High-Technologies, Tokyo, Japan). 
Alum Ragweed (RW)–Induced Experimental Allergic Conjunctivitis
RW-induced experimental allergic conjunctivitis models were prepared as described previously using male BALB/C mice at the age of 10 to 12 weeks (SLC, Hamamatsu, Japan). 12 The expression of ccl2 mRNA was quantified for mouse conjunctival tissue 24 hours after final RW challenge. For comparison, a single-challenge RW eye drop model and 4-challenge RW eye drop model were used. Ultrastructural analysis was performed for conjunctival and eyelid samples after final RW challenge (4-challenge model). All animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Results
Mast Cell HCjE Interaction Induces CCL2 Expression in Mast Cells
The coculture experiment showed synergistic increases of CCL2 and CCL4 protein in the supernatant of the mast cell (LAD2) HCjE coculture model (Figs. 1A–C). These results also were confirmed by ELISA analysis (Fig. 1D). The LAD2 HCjE coculture model induced 2-fold higher CCL2 mRNA expression in LAD2 cells than in LAD2 cells cultured by themselves. The p-mast HCjE coculture model using also showed a significant increase of CCL2 mRNA (Fig. 2A). For CCL4 mRNA expression, only HCjE-cocultured LAD2 showed a CCL4 mRNA increase; no change was observed in HCjE-cocultured p-mast cells (Fig. 2B). 
Figure 1
 
Mast cell conjunctival epithelial cell interaction induces CCL2 expression in mast cells. Antibody arrays were used to analyze HCjE (A), LAD2 (mast cell, [B]), and LAD2 HCjE coculture (C) supernatants. Synergistic increases of CCL2 (red circles) and CCL4 protein (red arrows) in the coculture supernatant (C). Blue squares are positive control samples for array reactions. The synergistic increase of CCL2 in the supernatant of a coculture sample also was confirmed by ELISA analysis (D).
Figure 1
 
Mast cell conjunctival epithelial cell interaction induces CCL2 expression in mast cells. Antibody arrays were used to analyze HCjE (A), LAD2 (mast cell, [B]), and LAD2 HCjE coculture (C) supernatants. Synergistic increases of CCL2 (red circles) and CCL4 protein (red arrows) in the coculture supernatant (C). Blue squares are positive control samples for array reactions. The synergistic increase of CCL2 in the supernatant of a coculture sample also was confirmed by ELISA analysis (D).
Figure 2
 
Mast cell HCjE coculture induces CCL2 mRNA expression. The mast cell HCjE coculture model induced significantly increased CCL2 mRNA expression in LAD2 and p-mast cells compared to that in mast cells cultured alone (A). On the other hand, CCL4 mRNA induction was observed only with HCjE-cocultured LAD2, and no change was observed in HCjE-cocultured p-mast cells (B).
Figure 2
 
Mast cell HCjE coculture induces CCL2 mRNA expression. The mast cell HCjE coculture model induced significantly increased CCL2 mRNA expression in LAD2 and p-mast cells compared to that in mast cells cultured alone (A). On the other hand, CCL4 mRNA induction was observed only with HCjE-cocultured LAD2, and no change was observed in HCjE-cocultured p-mast cells (B).
Epithelial Cells and Infiltrating Cells in VKC/AKC Tissue Express CCL2 Protein In Situ
Immunohistologic analysis of the tissue samples from the giant papillae showed positive CCL2 immunostaining in epithelial cells (Fig. 3). FcεRIβ-immunopositive mast cells also were observed within and around CCL2-positive epithelial cells (Figs. 3B, 3C). We confirmed the specificity of the CCL2 immunostaining by using isotype-matched normal mouse IgG1 instead of the CCL2 antibody (Fig. 3F). The specificity of FcεRIβ immunostaining already was demonstrated in our previous report 11 and we always run negative controls specimens in our experiments (data not shown). CCL2-positive infiltrating cells also were observed in the substantia propria of the tissue (Fig. 4A). Higher magnification of the Figure revealed the CCL2/ FcεRIβ double-positive mast cells (arrows in Figs. 4B, 4C), as well as CCL2+/FcεRIβ− infiltrating cells (arrowheads). The results of CCL2 immunohistochemical staining are summarized in Table 1. The existence of mast cells was also verified by anti-tryptase immunostaining of adjacent sections (Supplementary Fig. S1). 
Figure 3
 
CCL2 expression in the conjunctival epithelial cells of giant papillae. Perpendicular (sagittal, [A] and [B]) and horizontal (coronal, [C] and [D]) sections of tissues from giant papillae showed positive CCL2 immunostaining (green) in epithelial cells. FcεRIβ-positive mast cells (red) also were observed within and around CCL2-positive epithelial cells (arrows in [A] and [B], arrowhead in [C]). The FcεRIβ single staining image of (C) also is shown in (D). Specificity of CCL2 immunostaining was shown by anti-CCL2 staining (E) and control mouse IgG1 antibody staining using two adjacent sections (F). Original magnification (A), (C), and (D) ×200; (B), (E), and (F) ×400.
Figure 3
 
CCL2 expression in the conjunctival epithelial cells of giant papillae. Perpendicular (sagittal, [A] and [B]) and horizontal (coronal, [C] and [D]) sections of tissues from giant papillae showed positive CCL2 immunostaining (green) in epithelial cells. FcεRIβ-positive mast cells (red) also were observed within and around CCL2-positive epithelial cells (arrows in [A] and [B], arrowhead in [C]). The FcεRIβ single staining image of (C) also is shown in (D). Specificity of CCL2 immunostaining was shown by anti-CCL2 staining (E) and control mouse IgG1 antibody staining using two adjacent sections (F). Original magnification (A), (C), and (D) ×200; (B), (E), and (F) ×400.
Figure 4
 
CCL2 expression in the substantia propria of giant papillae. CCL2 immunohistochemical staining of the substantia propria of giant papillae is shown. CCL2/ FcεRIβ double-positive mast cell (arrow) and CCL2+/ FcεRIβ- infiltrating cells (arrowheads) are observed in the substantia propria of giant papillae tissue. (B) is a high magnification image of (A), (C) is the FcεRIβ single staining image of (B). Original magnification (A) ×200, (B) and (C) ×400.
Figure 4
 
CCL2 expression in the substantia propria of giant papillae. CCL2 immunohistochemical staining of the substantia propria of giant papillae is shown. CCL2/ FcεRIβ double-positive mast cell (arrow) and CCL2+/ FcεRIβ- infiltrating cells (arrowheads) are observed in the substantia propria of giant papillae tissue. (B) is a high magnification image of (A), (C) is the FcεRIβ single staining image of (B). Original magnification (A) ×200, (B) and (C) ×400.
Table 1. 
 
Summary of CCL2 Immunostaining
Table 1. 
 
Summary of CCL2 Immunostaining
Case No. Age Sex Total IgE Specific IgE Diagnosis CCL2 Immunostaining Treatment
Epithelium Substanita Propria
1 16 F 509 Positive VKC + + Dex, CsA
2 22 M 89 Positive VKC + Dex
3 13 M 2319 Positive VKC + Dex
4 18 M 375 Positive AKC + Dex, CsA
5 21 M 1904 Positive AKC + + Dex
6 29 M 56 Positive VKC + + Dex
Significantly Higher CCL2 mRNA Expression was Observed in Tissues From Giant Papillae Than in Conjunctivochalesis Tissue Samples
Five samples from giant papillae and eight conjunctivochalasis samples (Table 2) were collected, and cDNA was prepared. Real-time PCR analysis showed significantly increased CCL2 mRNA in the samples from giant papillae compared to the conjunctivochalasis samples (Fig. 5). 
Figure 5
 
Increased CCL2 mRNA expression in the tissues from giant papillae. Five samples from giant papillae and eight control (conjunctivochalasis) samples were analyzed. Real-time PCR analysis showed significantly higher CCL2 mRNA expression in the samples from giant papillae than in the control samples. *P < 0.05, Mann-Whitney U test.
Figure 5
 
Increased CCL2 mRNA expression in the tissues from giant papillae. Five samples from giant papillae and eight control (conjunctivochalasis) samples were analyzed. Real-time PCR analysis showed significantly higher CCL2 mRNA expression in the samples from giant papillae than in the control samples. *P < 0.05, Mann-Whitney U test.
Table 2. 
 
Clinical Information for CCL2 Expression Analysis
Table 2. 
 
Clinical Information for CCL2 Expression Analysis
Age Sex Diagnosis
Control 1 69 F Conjunctivochalasis
Control 2 65 F Conjunctivochalasis
Control 3 59 M Conjunctivochalasis
Control 4 74 M Conjunctivochalasis
Control 5 69 F Conjunctivochalasis
Control 6 80 F Conjunctivochalasis
Control 7 65 F Conjunctivochalasis
Control 8 73 F Conjunctivochalasis
Case 1 21 M AKC
Case 2 18 M AKC
Case 3 12 M VKC
Case 4 32 M AKC
Case 5 29 M VKC
Mast Cells in the Giant Papillae Showed PMD and Recombinant CCL2 Could Induce PMD
Three-minute treatment of p-mast cells with recombinant CCL2 (20 ng/mL) could induce PMD (Figs. 6B, 6F) compared to naïve p-mast cells (Figs. 6A, 6E). P-mast HCjE coculture (for 24 hours) also induced PMD morphology (Figs. 6C, 6G). IgE/anti-IgE crosslinking treatment induced anaphylactic degranulation in p-mast cells (Fig. 6D). In contrast to CCL2 treatment, CXCL8 (IL-8) treatment did not induce PMD (Fig. 6H). Ultrastructural analysis of giant papillae showed that intraepithelial mast cells had PMD morphology (Fig. 7A) as well as anaphylactic degranulation morphology (Fig. 7B). 
Figure 6
 
Recombinant CCL2 stimulation could induce PMD morphology. CCL2-(20 ng/mL, for 3 minutes) stimulated p-mast cells (B, F) show PMD morphology compared to naïve p-mast cells (A, E). P-mast HCjE coculture also induced PMD morphology (C, G). Anaphylactic degranulation morphology of a p-mast cell (D) induced by IgE/anti-IgE crosslinking is shown (D). A CXCL8-stimulated (20 ng/mL, for 3 minutes) p-mast cell (H) is shown as a negative control.
Figure 6
 
Recombinant CCL2 stimulation could induce PMD morphology. CCL2-(20 ng/mL, for 3 minutes) stimulated p-mast cells (B, F) show PMD morphology compared to naïve p-mast cells (A, E). P-mast HCjE coculture also induced PMD morphology (C, G). Anaphylactic degranulation morphology of a p-mast cell (D) induced by IgE/anti-IgE crosslinking is shown (D). A CXCL8-stimulated (20 ng/mL, for 3 minutes) p-mast cell (H) is shown as a negative control.
Figure 7
 
Intraepithelial mast cells show PMD morphology in situ. Ultrastructural analysis of a giant papilla obtained from a VKC patient shows intraepithelial mast cells (M) with PMD morphology (A) and with anaphylactic degranulation morphology (B). Empty granule chambers (arrows) in a mast cell with PMD morphology, and released granules (arrowheads) and labyrinth formation (asterisk) in a mast cell with anaphylactic degranulation morphology are shown. Ep, conjunctival epithelial cells.
Figure 7
 
Intraepithelial mast cells show PMD morphology in situ. Ultrastructural analysis of a giant papilla obtained from a VKC patient shows intraepithelial mast cells (M) with PMD morphology (A) and with anaphylactic degranulation morphology (B). Empty granule chambers (arrows) in a mast cell with PMD morphology, and released granules (arrowheads) and labyrinth formation (asterisk) in a mast cell with anaphylactic degranulation morphology are shown. Ep, conjunctival epithelial cells.
Ccl2 Expression in Alum-RW Induced Mouse Experimental Allergic Conjunctivitis
Increased ccl2 mRNA expression (Fig. 8A) was observed in RW-induced allergic conjunctivitis (4-challenge RW eye drop model) compared to PBS-challenged conjunctivae. The single-challenge RW eye drop model did not show a significant ccl2 mRNA increase compared to the PBS-challenged conjunctivae. PMD morphology was observed in the conjunctival mast cells of RW conjunctivitis (Fig. 8B). 
Figure 8
 
Ccl2 expression in mouse experimental allergic conjunctivitis. Alum-RW–induced mouse experimental conjunctivitis shows increased ccl2 mRNA. (A) PMD morphology (arrow) and anaphylactic degranulation (arrowheads) are observed in the mast cells of conjunctival tissue (B, C). *P < 0.05 by Student's t-test. The nucleus of a degranulated mast cell is shown (M).
Figure 8
 
Ccl2 expression in mouse experimental allergic conjunctivitis. Alum-RW–induced mouse experimental conjunctivitis shows increased ccl2 mRNA. (A) PMD morphology (arrow) and anaphylactic degranulation (arrowheads) are observed in the mast cells of conjunctival tissue (B, C). *P < 0.05 by Student's t-test. The nucleus of a degranulated mast cell is shown (M).
Discussion
In our study, we showed that mast cell-conjunctival epithelial cell interaction induced CCL2 expression at the mRNA and at protein levels. We used LAD2 cells (a cell line) and p-mast cells (primary-cultured cells) as the source of mast cells. Initial coculture experiments, including antibody array experiments, were performed using LAD2, and confirmatory experiments were performed with primary p-mast cells due to their limited availability. Increased concentrations of CCL2 and CCL4 (MIP-1β) protein were observed in LAD2 HCjE coculture supernatant (Fig. 1), and upregulation of CCL2 mRNA and CCL4 mRNA was confirmed in LAD2 cells in the coculture model. We then tried to replicate the results using p-mast cells. CCL2 mRNA upregulation was observed in cocultured p-mast cells, but no CCL4 mRNA upregulation was observed (Figs. 2A, 2B), so we focused at the role of CCL2 for further studies. 
To examine the relevance to the pathophysiology of AKC/VKC, we next evaluated CCL2 expression in the tissues of giant papillae tissue obtained from patients. 
Immunohistochemical analysis showed CCL2-positive staining of conjunctival epithelial cells (Fig. 3). Double immunohistochemical staining with a mast cell marker (FcεRIβ) showed mast cells within and beneath the CCL2-positive conjunctival epithelial cells (Figs. 3A–C). We also found positive CCL2 immunostaining at the substantia propria of the tissue from the giant papillae (Fig. 4). Abu El-Asrar et al. found increased number of CCL2-positive staining of cells infiltrating the substantia propria of the limbal tissue of VKC. 13 Although they reported negative CCL2 expression in the conjunctival epithelium in limbal VKC tissue, we clearly detected CCL2-positive immunostaining of the conjunctival epithelium tarsal form of giant papillae (Fig. 3). Giustizieri et al. demonstrated CCL2 mRNA expression in the epithelial cells of the lesional skin of atopic dermatitis patients by in situ hybridization. 14 Gordon reported increased CCL2 expression in epidermal cells and dermal cells in a dinitrophenyl serum albumin–induced mouse passive cutaneous anaphylaxis (PCA) model, using immunohistological analysis. 15 Mercer et al. reported positive CCL2 immunostaining at the epithelial cells of human idiopathic pulmonary fibrosis tissue but not at the epithelial cells of lung tumor tissue (control tissue). 16 These three reports on CCL2 expression are supportive for our results for positive CCL2 immunostaining of epithelial cells. Since we did not examine the expression of CCL2 in the limbal VKC tissue, the reason for the difference between our results and those of Abu El-Asrar et al. 13 is unknown. We speculate that there may be a difference between the limbal and tarsal forms of VKC for the epithelial expression of CCL2. 
We found few CCL2/FcεRIβ double-positive mast cells (Fig. 4B) by immunohistochemical analysis. Although the main CCL2 mRNA-producing cells were mast cells (Fig. 2A), CCL2 protein also was secreted from mast cells as we found in culture supernatant samples (Fig. 1D). Therefore, we hypothesized that continuous CCL2 secretion from mast cells was the reason we found few CCL2/FcεRIβ double-positive mast cells. This discrepancy between abundant CCL2 mRNA expression and poor CCL2 retention in mast cells has been reported previously. 15,17  
We obtained tarsal giant papilla tissues from refractory AKC/VKC patients, all of whom were treated with topical dexamethasone eye drops for at least 4 weeks (Table 1), so treatment may have downregulated the CCL2 expression as reported previously. 18 Nonetheless, significantly higher CCL2 mRNA expression in samples from giant papillae than in conjunctival tissues obtained from conjunctivochalasis patients was observed by real-time PCR analysis (Fig. 5). 
Interestingly, the report of Gordon also showed that CCL2 expression in a PCA model was dependent on mast cells because of significantly reduced CCL2 expression in the skin of mast cell–deficient mice (W/Wv) during the PCA reaction. 15 Their results suggesting that interaction between mast cells and other components of conjunctival cells (including conjunctival epithelial cells) could upregulate CCL2 expression during allergic reactions agreed with our results in this study. 
To elucidate further the role of CCL2 protein in the pathophysiology of AKC/VKC, we examined the activation pattern of mast cells with special reference to PMD. We found that recombinant CCL2 stimulation (Figs. 6B, 6F) as well as HCjE coculture procedures (Figs. 6C, 6G) could induce PMD in cultured mast cells in vitro. We also tried to inhibit the effect of CCL2 by adding a CCR2 inhibitor (RS504393 from TOCRIS Bioscience) to the coculture model, and found partial inhibition of the PMD phenomenon (data not shown). Consistent with the results of a previous report, 19 p-mast cells stimulated with another chemokine (CXCL8) did not show PMD morphology (Fig. 6H). This result also supported the specificity of the CCL2-induced PMD phenomenon. Although we could not deny the possibility of other conjunctival epithelial cell-derived mast cell activators, CCL2 in the coculture medium had some roles in PMD. Continuous studies are ongoing in our laboratory to elucidate possible additional activators. We also found PMD in the intraepithelial mast cells of a VKC patient, showing the relevance of PMD to the pathophysiology of VKC (Fig. 7). In our previous study, 20% of the mast cells in the giant papillae samples showed the PMD morphology and 17% of the mast cells in the giant papillae samples showed anaphylactic degranulation in VKC patients. 6 These results suggested the importance of PMD and subsequent slow/persistent mediator release 20 during chronic allergic keratoconjunctivitis. The magnitude of inflammation with PMD seems to be smaller than with anaphylactic degranulation; however, the PMD reaction lasts longer without IgE crosslinking by the antigen. 5 On the other hand, the mediator release from mast cells is not long lasting in the case of anaphylactic degranulation because it needs some time to regain the cytoplasmic granules. 4  
We also confirmed Ccl2 mRNA upregulation and PMD of mast cells in the RW-induced mouse experimental allergic conjunctivitis model. After 4 RW eye drop challenges, but not after a single RW eye drop challenge, increased ccl2 mRNA expression compared to PBS-challenged control conjunctival tissue and PMD morphology were observed in the RW-challenged conjunctival tissue (Fig. 8A). This result suggested that chronic antigen stimuli were ccl2 mRNA-inducing factors. Although there are no appropriate mouse models for AKC/VKC, and RW-induced mouse experimental allergic conjunctivitis is considered to be an animal model of seasonal allergic conjunctivitis, 21 RW-induced allergic conjunctivitis can be used as a model of chronic allergic inflammation induced by repeated antigen stimuli in which eosinophil infiltration and T cell activation 22,23 are observed. 
A study by Miyazaki et al. showed that CCL2 protein was expressed in the conjunctival epithelium of mouse experimental allergic conjunctivitis and CCL2 subconjunctival injection induced mast cell degranulation. 24 Our results are consistent with their findings. They also reported that blocking the CCL2-CCR2 signaling cascade could attenuate signs and symptoms of the acute phase of experimental allergic conjunctivitis. 24 Further experiments analyzing RW-induced experimental allergic conjunctivitis using mast cell–deficient mice to clarify mast cell–conjunctival epithelial cell interactions are now ongoing. 
In conclusion, we showed that mast cell–conjunctival epithelial cell interaction could induce higher CCL2 expression and PMD in cultured human mast cells, which also was observed in situ samples of chronic allergic conjunctivitis. These results suggested that suppression of CCL2-CCR2 signaling cascades might be useful for alternative therapy for severe chronic allergic conjunctivitis. 
Supplementary Materials
Acknowledgments
The authors thank Julian M. Hopkin and Shigeru Kinoshita for their invaluable continuous support, Arnold Kirshenbaum for the LAD2 cells, and Ilene K. Gipson for the HCjE cells. 
Supported in part by grants-in-aid from MEXT Japan (Nos. 21592239 [AMa], 24592652 [AMa], and 24659768 [AMu]), from the Takeda Science Foundation (AMa), and from the Institute for Environmental and Gender-Specific Medicine, Juntendo University (AMa). 
Disclosure: S. Iwamoto, None; Y. Asada, None; N. Ebihara, None; K. Hori, None; Y. Okayama, None; J.-I. Kashiwakura, None; Y. Watanabe, None; S. Kawasaki, None; N. Yokoi, None; T. Inatomi, None; K. Shinomiya, None; A. Murakami, None; A. Matsuda, None 
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Figure 1
 
Mast cell conjunctival epithelial cell interaction induces CCL2 expression in mast cells. Antibody arrays were used to analyze HCjE (A), LAD2 (mast cell, [B]), and LAD2 HCjE coculture (C) supernatants. Synergistic increases of CCL2 (red circles) and CCL4 protein (red arrows) in the coculture supernatant (C). Blue squares are positive control samples for array reactions. The synergistic increase of CCL2 in the supernatant of a coculture sample also was confirmed by ELISA analysis (D).
Figure 1
 
Mast cell conjunctival epithelial cell interaction induces CCL2 expression in mast cells. Antibody arrays were used to analyze HCjE (A), LAD2 (mast cell, [B]), and LAD2 HCjE coculture (C) supernatants. Synergistic increases of CCL2 (red circles) and CCL4 protein (red arrows) in the coculture supernatant (C). Blue squares are positive control samples for array reactions. The synergistic increase of CCL2 in the supernatant of a coculture sample also was confirmed by ELISA analysis (D).
Figure 2
 
Mast cell HCjE coculture induces CCL2 mRNA expression. The mast cell HCjE coculture model induced significantly increased CCL2 mRNA expression in LAD2 and p-mast cells compared to that in mast cells cultured alone (A). On the other hand, CCL4 mRNA induction was observed only with HCjE-cocultured LAD2, and no change was observed in HCjE-cocultured p-mast cells (B).
Figure 2
 
Mast cell HCjE coculture induces CCL2 mRNA expression. The mast cell HCjE coculture model induced significantly increased CCL2 mRNA expression in LAD2 and p-mast cells compared to that in mast cells cultured alone (A). On the other hand, CCL4 mRNA induction was observed only with HCjE-cocultured LAD2, and no change was observed in HCjE-cocultured p-mast cells (B).
Figure 3
 
CCL2 expression in the conjunctival epithelial cells of giant papillae. Perpendicular (sagittal, [A] and [B]) and horizontal (coronal, [C] and [D]) sections of tissues from giant papillae showed positive CCL2 immunostaining (green) in epithelial cells. FcεRIβ-positive mast cells (red) also were observed within and around CCL2-positive epithelial cells (arrows in [A] and [B], arrowhead in [C]). The FcεRIβ single staining image of (C) also is shown in (D). Specificity of CCL2 immunostaining was shown by anti-CCL2 staining (E) and control mouse IgG1 antibody staining using two adjacent sections (F). Original magnification (A), (C), and (D) ×200; (B), (E), and (F) ×400.
Figure 3
 
CCL2 expression in the conjunctival epithelial cells of giant papillae. Perpendicular (sagittal, [A] and [B]) and horizontal (coronal, [C] and [D]) sections of tissues from giant papillae showed positive CCL2 immunostaining (green) in epithelial cells. FcεRIβ-positive mast cells (red) also were observed within and around CCL2-positive epithelial cells (arrows in [A] and [B], arrowhead in [C]). The FcεRIβ single staining image of (C) also is shown in (D). Specificity of CCL2 immunostaining was shown by anti-CCL2 staining (E) and control mouse IgG1 antibody staining using two adjacent sections (F). Original magnification (A), (C), and (D) ×200; (B), (E), and (F) ×400.
Figure 4
 
CCL2 expression in the substantia propria of giant papillae. CCL2 immunohistochemical staining of the substantia propria of giant papillae is shown. CCL2/ FcεRIβ double-positive mast cell (arrow) and CCL2+/ FcεRIβ- infiltrating cells (arrowheads) are observed in the substantia propria of giant papillae tissue. (B) is a high magnification image of (A), (C) is the FcεRIβ single staining image of (B). Original magnification (A) ×200, (B) and (C) ×400.
Figure 4
 
CCL2 expression in the substantia propria of giant papillae. CCL2 immunohistochemical staining of the substantia propria of giant papillae is shown. CCL2/ FcεRIβ double-positive mast cell (arrow) and CCL2+/ FcεRIβ- infiltrating cells (arrowheads) are observed in the substantia propria of giant papillae tissue. (B) is a high magnification image of (A), (C) is the FcεRIβ single staining image of (B). Original magnification (A) ×200, (B) and (C) ×400.
Figure 5
 
Increased CCL2 mRNA expression in the tissues from giant papillae. Five samples from giant papillae and eight control (conjunctivochalasis) samples were analyzed. Real-time PCR analysis showed significantly higher CCL2 mRNA expression in the samples from giant papillae than in the control samples. *P < 0.05, Mann-Whitney U test.
Figure 5
 
Increased CCL2 mRNA expression in the tissues from giant papillae. Five samples from giant papillae and eight control (conjunctivochalasis) samples were analyzed. Real-time PCR analysis showed significantly higher CCL2 mRNA expression in the samples from giant papillae than in the control samples. *P < 0.05, Mann-Whitney U test.
Figure 6
 
Recombinant CCL2 stimulation could induce PMD morphology. CCL2-(20 ng/mL, for 3 minutes) stimulated p-mast cells (B, F) show PMD morphology compared to naïve p-mast cells (A, E). P-mast HCjE coculture also induced PMD morphology (C, G). Anaphylactic degranulation morphology of a p-mast cell (D) induced by IgE/anti-IgE crosslinking is shown (D). A CXCL8-stimulated (20 ng/mL, for 3 minutes) p-mast cell (H) is shown as a negative control.
Figure 6
 
Recombinant CCL2 stimulation could induce PMD morphology. CCL2-(20 ng/mL, for 3 minutes) stimulated p-mast cells (B, F) show PMD morphology compared to naïve p-mast cells (A, E). P-mast HCjE coculture also induced PMD morphology (C, G). Anaphylactic degranulation morphology of a p-mast cell (D) induced by IgE/anti-IgE crosslinking is shown (D). A CXCL8-stimulated (20 ng/mL, for 3 minutes) p-mast cell (H) is shown as a negative control.
Figure 7
 
Intraepithelial mast cells show PMD morphology in situ. Ultrastructural analysis of a giant papilla obtained from a VKC patient shows intraepithelial mast cells (M) with PMD morphology (A) and with anaphylactic degranulation morphology (B). Empty granule chambers (arrows) in a mast cell with PMD morphology, and released granules (arrowheads) and labyrinth formation (asterisk) in a mast cell with anaphylactic degranulation morphology are shown. Ep, conjunctival epithelial cells.
Figure 7
 
Intraepithelial mast cells show PMD morphology in situ. Ultrastructural analysis of a giant papilla obtained from a VKC patient shows intraepithelial mast cells (M) with PMD morphology (A) and with anaphylactic degranulation morphology (B). Empty granule chambers (arrows) in a mast cell with PMD morphology, and released granules (arrowheads) and labyrinth formation (asterisk) in a mast cell with anaphylactic degranulation morphology are shown. Ep, conjunctival epithelial cells.
Figure 8
 
Ccl2 expression in mouse experimental allergic conjunctivitis. Alum-RW–induced mouse experimental conjunctivitis shows increased ccl2 mRNA. (A) PMD morphology (arrow) and anaphylactic degranulation (arrowheads) are observed in the mast cells of conjunctival tissue (B, C). *P < 0.05 by Student's t-test. The nucleus of a degranulated mast cell is shown (M).
Figure 8
 
Ccl2 expression in mouse experimental allergic conjunctivitis. Alum-RW–induced mouse experimental conjunctivitis shows increased ccl2 mRNA. (A) PMD morphology (arrow) and anaphylactic degranulation (arrowheads) are observed in the mast cells of conjunctival tissue (B, C). *P < 0.05 by Student's t-test. The nucleus of a degranulated mast cell is shown (M).
Table 1. 
 
Summary of CCL2 Immunostaining
Table 1. 
 
Summary of CCL2 Immunostaining
Case No. Age Sex Total IgE Specific IgE Diagnosis CCL2 Immunostaining Treatment
Epithelium Substanita Propria
1 16 F 509 Positive VKC + + Dex, CsA
2 22 M 89 Positive VKC + Dex
3 13 M 2319 Positive VKC + Dex
4 18 M 375 Positive AKC + Dex, CsA
5 21 M 1904 Positive AKC + + Dex
6 29 M 56 Positive VKC + + Dex
Table 2. 
 
Clinical Information for CCL2 Expression Analysis
Table 2. 
 
Clinical Information for CCL2 Expression Analysis
Age Sex Diagnosis
Control 1 69 F Conjunctivochalasis
Control 2 65 F Conjunctivochalasis
Control 3 59 M Conjunctivochalasis
Control 4 74 M Conjunctivochalasis
Control 5 69 F Conjunctivochalasis
Control 6 80 F Conjunctivochalasis
Control 7 65 F Conjunctivochalasis
Control 8 73 F Conjunctivochalasis
Case 1 21 M AKC
Case 2 18 M AKC
Case 3 12 M VKC
Case 4 32 M AKC
Case 5 29 M VKC
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