http://informahealthcare.com/ipi ISSN: 0892-3973 (print), 1532-2513 (electronic) Immunopharmacol Immunotoxicol, 2014; 36(5): 371–377 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/08923973.2014.953957

RESEARCH ARTICLE

Caffeic acid phenethyl ester inhibits the inflammatory effects of interleukin-1b in human corneal fibroblasts Jae-Wook Yang1*, Won-Kyo Jung2*, Chang-Min Lee3*, Sung Su Yea4, Yung Hyun Choi5, Gi-Young Kim6, Dae-Sung Lee7, Giyoun Na8, Sae-Gwang Park8, Su-Kil Seo8, Jung Sik Choi9, Young-Min Lee9, Won Sun Park10, and Il-Whan Choi8 1

Department of Ophthalmology, Busan Paik Hospital, College of Medicine Inje University, Busan, Republic of Korea, 2Department of Biomedical Engineering, Center for Marine-Integrated Biomedical Technology (BK21 Plus), Pukyong National University, Busan, Republic of Korea, 3Department of Molecular Microbiology and Immunology, Warren Alpert School of Medicine, Providence, RI, USA, 4Department of Biochemistry, College of Medicine Inje University, Busan, Republic of Korea, 5Department of Biochemistry, College of Oriental Medicine, Dongeui University, Busan, Republic of Korea, 6Laboratory of Immunobiology, Department of Marine Life Sciences, Jeju National University, Jeju, Republic of Korea, 7Marine Biodiversity Institute of Korea, Sejong, Republic of Korea, 8Department of Microbiology, College of Medicine Inje University, Busan, Republic of Korea, 9 Department of Internal Medicine, Busan Paik Hospital, College of Medicine Inje University, Busan, Republic of Korea, and 10Department of Physiology, Kangwon National University School of Medicine, Chuncheon, Republic of Korea Abstract

Keywords

Context: Expression of various inflammatory mediators in corneal fibroblasts contributes to corneal inflammation. Objective: The purpose of this study was to assess the possible effects of caffeic acid phenethyl ester (CAPE) on the expression of inflammatory mediators during an inflammatory response in human corneal fibroblasts. Materials and methods: The levels of interleukin (IL)-6, monocyte chemotactic protein (MCP)-1, and intercellular adhesion molecule-1 (ICAM-1) from IL-1b-exposed human corneal fibroblasts were measured with enzyme-linked immunosorbent assays (ELISA). The regulatory mechanisms of CAPE on cellular signaling pathways were examined using Western blot and electrophoretic mobility shift assays. A functional validation was carried out by evaluating the inhibitory effects of CAPE on neutrophil and monocyte migration in vitro. Results: CAPE inhibited the expression of IL-6, MCP-1 and ICAM-1 induced by the proinflammatory cytokine IL-1b in corneal fibroblasts. The activation of AKT and NF-kB by IL-1b was markedly inhibited by CAPE, whereas the activity of mitogen-activated protein kinases (MAPKs) was not affected. CAPE significantly suppressed the IL-1b-induced migration of differentiated (d)HL-60 and THP-1 cells. Discussion: These anti-inflammatory effects of CAPE may be expected to inhibit the infiltration of leukocytes into the corneal stroma in vivo.

AKT, caffeic acid phenethyl ester, chemotaxic migration, corneal inflammation, nuclear factor-kB

Introduction The cornea is a clear dome-shaped window covering the front of the eyeball that covers the iris and the pupil. Transparency and avascularity make the cornea a very unique tissue. In the unperturbed cornea, keratocytes are the major cellular constituent of the corneal stroma. These cells are normally quiescent and play a key role in the maintenance of corneal transparency. Keratocytes play crucial roles in structural

*These authors contributed equally to this work. Address for correspondence: Il-Whan Choi, Department of Microbiology, College of Medicine Inje University, Bokji-ro 75, Busanjin-gu, Busan, Republic of Korea. Tel: +82 51 890 6461. Fax: +82 51 891 6004. E-mail: [email protected] Won Sun Park, Department of Physiology, Kangwon National University School of Medicine, 1 Kangwondaehak-gil, Chuncheon, Republic of Korea. Tel: +82 33 242 8834. E-mail: [email protected]

History Received 12 December 2013 Revised 7 August 2014 Accepted 8 August 2014 Published online 25 August 2014

support, inflammation, and wound healing. However, when the cornea sustains damage through trauma, infections, contact lenses, or chemical injury, loss of corneal clarity, visual impairment, and even blindness due to scarring, melting, or vascularization can result1,2. Many microorganisms cause infective corneal ulcers. Among them are bacteria, fungi, viruses, protozoa, and chlamydia. These immunopathologic processes are modulated by expression of adhesion molecules and the secretion of regulatory factors such as cytokines and chemokines, which are produced by both the resident cells and the infiltrating immune cells in the cornea3–5. Keratocytes transform into corneal fibroblasts in response to stromal injury6–8. Therefore, corneal fibroblasts may function as sentinel cells in the defense of the cornea against various external stimuli. Pro-inflammatory cytokines such as tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6), and chemokines

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Figure 1. The chemical structure of caffeic acid phenethyl ester (CAPE).

such as IL-8, granulocyte colony-stimulating factor, and monocyte chemoattractant protein-1 (MCP-1), as well as adhesion molecules such as intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule-1 (VCAM-1) are expressed by corneal fibroblasts during corneal inflammation9–12. Among these, IL-6 is a multipotent proinflammatory cytokine involved in a broad variety of ocular inflammatory diseases and wound healing process13–15. Enhanced production of IL-6 by corneal fibroblasts plays a role in corneal neovascularization as well as promoting inflammatory cell infiltration into the corneal stroma. MCP-1 is responsible for the directed migration of monocytes/ macrophages and activated T cells to areas of injury and for inflammation in pathological and physiological conditions16–18. The changes in the level of MCP-1 expression in corneal fibroblasts may modify the process of corneal wound healing or may influence the normal functions of these cells. ICAM-1 is a cell surface glycoprotein and a ligand for lymphocyte function-associated antigen-1 (integrin, LFA-1), a receptor found on leukocytes19. ICAM-1 is important in transendothelial migration of leukocytes through blood vessels and retention of the leukocytes in the inflamed tissue by means of firm attachment to the extracellular matrix20. Under these conditions, this factor enhances immune responses and contributes to inflammatory corneal diseases. Together these factors induce the chemotaxis and activation of various leukocytes and play important roles in the pathogenesis of corneal stromal inflammation including corneal ulcer. Caffeic acid phenethyl ester (CAPE, empirical formula C17H16O4, Figure 1), a potent flavonoid antioxidant, is the active component of propolis (honeybee resin). Various investigators have demonstrated that CAPE has antiviral21, antitumoral22, anti-inflammatory23, antioxidant24, neuroprotective25, antiatherosclerotic26, and immunomodulatory properties27 in diverse systems. Although CAPE has demonstrated a broad spectrum of biological pharmacological activities, there have been no reports on the effects of CAPE on the corneal stromal inflammation. In the present study, we investigated the anti-inflammatory effects of CAPE and its effects on intracellular signaling pathways of action, and the resulting effects on the production of cytokine, chemokines, and adhesion molecules. We also examined the effect of this agent on the migration of leukocytes stimulated by IL-1b-treated human corneal fibroblasts.

Materials and methods Materials Caffeic acid phenethyl ester (CAPE) was purchased from the Sigma Chemical Co. (St. Louis, MO). Eagle’s minimum essential medium (MEM), fetal bovine serum, and trypsin– EDTA were obtained from Invitrogen-Gibco (Carlsbad, CA),

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and 24-well culture plates and 60-mm culture dishes were from Corning-Costar (Corning, NY). The antibody to NF-kB was obtained from eBioscience (San Diego, CA). Antibodies against JNK, phospho (p)-JNK, p-ERK 1/2, p-p38, and p-IkBa were purchased from Cell Signaling (Danvers, MA). Antibodies against AKT, p-AKT, ERK, p38, and IkBa were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Nitrocellulose membranes and an enhanced chemiluminescence (ECL) kit were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). Isolation and culture of human corneal fibroblasts Human corneal fibroblasts were isolated from fresh human corneal stroma using a collagenase digestion protocol. The mean age of donor patients was 35.65 ± 12.55 years old, and all samples were obtained with informed consent. All protocols involving human cornea were approved by the Institutional Review Board at the Inje University and were consistent with the guidelines of the Declaration of Helsinki. A total of six peripheral corneas were obtained from braindead patients after keratoplasty. The corneal tissues were soaked in an antibiotic solution before being washed twice in 60 mm cell culture dishes containing 3 ml of DMEM culture media (Gibco, Carlsbad, CA). The corneal tissues were then placed in 10-mm cell-culture dishes with 0.2 ml DMEM culture media to prevent desiccation, cut into small pieces with two 21-G surgical blades, transferred to a 100-mm cell culture dish (Corning Life Sciences, Rochester, NY) with a 1-ml pipette, and digested with 100 U collagenase type IV (Sigma, St Louis, MO) for 1 h at 37  C, 5% CO2. The digested corneal tissue was then placed between glass slides and washed with 10 ml of DMEM to remove the collagenase. The isolated cells were cultured in serum-free DMEM for maintenance of the keratocytes in culture. Cells that had been passaged three times were used for RT-PCR and cells passaged five to eight times were used for the FACS analysis. In some instances, the cells were treated with recombinant human IFN-g (500 U/ml, R&D Systems, Minneapolis, MN) for 3 d. Cell line culture HL-60 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum. The HL-60 cells were induced to differentiate (dHL-60) with 1.75% (vol/vol) DMSO for 3–4 d to achieve the expression of the neutrophilic phenotype28. THP-1 cells, a human leukemia cell line of monocyte/macrophage lineage, were grown in RPMI-1640 medium with 10% fetal bovine serum. Determination of cell viability Cellular viability was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) method. Briefly, wells containing 1.5  l04 cells/ml were treated with CAPE. After incubation for 24 h, the cells were washed twice with PBS. CCK-8 was added to each well and incubated at 37  C for 1 h, followed by analysis at 450 nm using a microplate reader (Model EL800, BIO-TEK, Winooski, VT).

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The cells were pretreated with various concentrations of CAPE (10 or 20 mM in 0.2 % (vol/vol) DMSO) for 2 h before IL-1b stimulation. ELISA kits purchased from BioLegend (San Diego, CA) were used to measure IL-6 and MCP-1 levels, and a kit obtained from R&D Systems (Minneapolis, MN) was used to measure ICAM-1 levels. The absorbance at 450 nm was determined using a microplate reader (Model EL800, BIO-TEK, Winooski, VT).

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Western blot analysis The cells were washed three times with PBS and lysed with lysis buffer (Mammalian Cell-PE LB, G Biosciences, St. Louis, MO). Equal amounts of protein were separated on 10% SDS–polyacrylamide minigels and transferred to nitrocellulose membranes. After incubation with the appropriate primary antibody, the membranes were incubated for 1 h at room temperature with a secondary antibody conjugated to horseradish peroxidase. Following three washes in Trisbuffered saline Tween-20 (TBST), the immunoreactive bands were visualized using the ECL detection system. Electrophoretic mobility shift assay (EMSA) Nuclear extracts were prepared using NE-PER nuclear extraction reagent (Pierce, Rockford, IL). As a probe for the gel retardation assay, an oligonucleotide containing the immunoglobulin k-chain binding site (kB, 50 -GATCTCAGA GGGGACTTTCCGAGAGA-30 ) was synthesized. A nonradioactive method in which the 30 end of the probe was labeled with biotin was used in these experiments (Pierce, Rockford, IL). The binding reactions contained 5 mg of nuclear extract protein, buffer (10 mM Tris, pH 7.5, 50 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.05% Nonidet P-40, and 2.5% glycerol), 50 ng of poly (dI-dC), and 20 fM of the biotin-labeled DNA. The reactions were incubated for 20 min at room temperature in a final volume of 20 ml. The competition reactions were conducted by adding a 100-fold excess of unlabeled kB to the reaction mixture. The mixture was then separated by electrophoresis on a 5% polyacrylamide gel in 0.5 Tris-borate buffer and transferred to nylon membranes. The biotin-labeled DNA was detected using a LightShift chemiluminescent electrophoretic mobility shift assay kit (EMSA) (Pierce, Rockford, IL). Cell migration assay Migration of the dHL-60 and THP-1 cells was evaluated using a 24-well plate system at densities of 5  105 cells/ml and 5  106 cells/ml, respectively. The dHL-60 and THP-1 cells were added to the upper chambers of transwell cluster plates (24-well companion plate, Becton-Dickinson, Franklin Lakes, NJ) with 3 and 8 mm pore filters, respectively. IL-1b-induced human corneal fibroblasts were added to lower wells of the plates. The cells were allowed to migrate for 48 h at 37  C in 5% CO2 towards the lower chamber. The migrated cells from lower chamber were collected and then centrifuged at 400 g for 10 min. The number of cells that migrated to the lower wells was counted on a hemocytometer. Each experiment was performed in triplicate and repeated at least three times.

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Figure 2. The effect of CAPE on the viability of human corneal fibroblasts. The cells were treated with the indicated concentrations of CAPE (1–80 mM) for 2 h. The cell viability was assessed using the CCK8 assay, and the results are expressed as the percentage of surviving cells relative to control cells (DMSO). Each value indicates the mean ± S.D. and is representative of results obtained from three independent experiments. *p50.05, compared with the untreated control.

Statistical analysis The statistical analyses were conducted using Student’s t-test. The results are represented as the means ± S.D. of at least three separate experiments. p50.05 was considered to be statistically significant.

Results The effects of CAPE on the viability of human corneal fibroblasts Initially, we examined the viability of human corneal fibroblasts cells treated with CAPE (DMSO, 1, 10, 20, 40, and 80 mM) using the CCK-8 assay. There was no significant cytotoxicity to human corneal fibroblasts at doses up to 20 mM, but the cell viability was significantly reduced to 30% at the 40 mM dose of CAPE (Figure 2). Based on these results, a concentration of 10–20 mM was chosen for the subsequent experiments. No inhibitory effects on the expression of inflammatory mediators (IL-6 and MCP-1) by 1 mM of CAPE in IL-1b-induced human corneal fibroblasts were observed (data not shown). The effects of CAPE on the expression of IL-6, MCP-1, and ICAM-1 in IL-1b-induced human corneal fibroblasts As shown in Figure 3, the levels of IL-6, MCP-1, and ICAM-1 were considerably increased after stimulation of the human corneal fibroblasts with IL-1b. To evaluate the effect of CAPE on the protein expression of IL-6, MCP-1, and ICAM-1 in human corneal fibroblasts, we pretreated cells with CAPE (10 or 20 mM) before stimulation with IL-1b (0.1 ng/ml). The treatment with CAPE suppressed the IL-1binduced production of the IL-6, MCP-1, and ICAM-1 proteins. The effects of CAPE on the activation of mitogenactivated protein kinases and AKT signaling pathways in IL-1b-induced human corneal fibroblasts To elucidate the mechanisms underlying the effects of CAPE, we examined the activation of MAPKs and AKT using

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Figure 3. The effect of CAPE on the production of pro-inflammatory cytokines by IL-1b-stimulated human corneal fibroblasts. The levels of IL-6, MCP-1, and ICAM-1 proteins were determined by ELISA. The cells were pretreated with CAPE (10 or 20 mM) for 2 h prior to IL-1b stimulation for 24 h. Each bar represents the mean ± S.D. from three independent experiments. **p50.01, compared with IL-1b-stimulated values.

Western blot analysis. The stimulation of human corneal fibroblasts with IL-1b resulted in an increase in the phosphorylation of AKT and all three types of mitogenactivated protein kinases (MAPKs): JNK, p38, and ERK. As shown in Figure 4, pretreatment for 2 h with CAPE (20 mM) attenuated the phosphorylation of AKT induced by 30-min incubation with 0.1 ng/ml IL-1b, but it did not affect the phosphorylation of the MAPKs. The effects of CAPE on the activation of NF-kB in IL-1b-stimulated human corneal fibroblasts The production of pro-inflammatory cytokines, chemokines, and adhesion molecules are regulated by the transcription factor NF-kB. Therefore, to investigate the mechanism by which CAPE affects the expression IL-6, MCP-1, and ICAM1, we examined the effects of CAPE on NF-kB activation. Most inhibitors of NF-kB activation exert their effects via the suppression of IkBa phosphorylation and degradation.

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Figure 4. The effect of CAPE on IL-1b-induced phosphorylation of ERK-1/2, SAPK/JNK, p38 MAP kinase, and AKT in human corneal fibroblasts. The human corneal fibroblasts were treated with vehicle or the indicated concentrations of CAPE for 2 h prior to stimulation with IL-1b (0.1 ng/ml) for 30 min. Cell extracts were then prepared and subjected to Western blotting with antibodies specific for the phosphorylated forms of ERK-1/2, SAPK/JNK, p38, and AKT. The results presented are representative of three independent experiments. **p50.01, compared with IL-1b-stimulated values.

We found that CAPE inhibited the IL-1b-induced phosphorylation and degradation of IkBa, as well as the nuclear translocation of NF-kB p65 (Figure 5A). We next investigated the effect of CAPE on the DNA-binding activity of NF-kB using EMSA (Figure 5B). IL-1b treatment caused a significant increase in the DNA-binding activity of NF-kB, whereas treatment with CAPE markedly reduced the IL-1b-induced DNA-binding activity of NF-kB. The effects of CAPE on the migration of leukocytes towards IL-1b-stimulated human corneal fibroblasts To investigate whether CAPE affects the migration of dHL-60 and THP-1 cells, migration assays were performed using transwell cluster plates (Figure 6) with the two-compartment chambers separated by a polycarbonate membrane filter. The number of migrated dHL-60 and THP-1 cells co-cultured with IL-1b-treated human corneal fibroblasts were two and eight times greater than the vehicle-treated dHL-60 and THP-1 cells (4.67 ± 0.77 in control versus 9.85 ± 1.03 in IL-1b, and 11.37 ± 8.55 in control versus 88.17 ± 26.39 in IL-1b),

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Figure 5. The effect of CAPE on the phosphorylation/degradation of IkBa in the cytosol and the translocation and binding of NF-kB into the nuclei of IL-1b-stimulated human corneal fibroblasts. The human corneal fibroblasts were pre-treated with CAPE (10 or 20 mM) for 2 h and then stimulated with IL-1b for 1 h. (A) Cytosolic extracts were prepared as described in the Materials and methods section, and evaluated for IkBa and p-IkBa via Western blot analysis. Nuclear extracts were prepared as described in the Materials and methods section, and evaluated for NF-kB via Western blot analysis. (B) Nuclear extracts were prepared as described in the Materials and methods section and evaluated for NF-kB via EMSA.

respectively. However, the levels of infiltration of the dHL-60 and THP-1 cells were significantly attenuated by CAPE treatment when compared with the IL-1b-induced group (3.29 ± 0.20 and 4.85 ± 2.34, respectively, at 20 mM of CAPE).

Discussion Activated keratocytes, also known as corneal fibroblasts, contribute to the local inflammation associated with stromal infection of cornea by releasing various cytokines and chemokines and by expressing adhesion molecules8,12,29. These inflammatory mediators are expressed in response to Gram-negative bacterial infections, mediated by bacterially derived products such as lipopolysaccharide (LPS). The LPSmediated stimulation of the keratocytes leads to the recruitment of leukocytes into the corneal stroma and subsequent corneal ulceration and visual impairment. LPS induces the release of a number of pro-inflammatory cytokines, such as IL-1, IL-6, and TNF-a. IL-1b is a potent pro-inflammatory cytokine and is involved in the effector phase of inflammatory and immune responses. IL-1b is found in the epithelium,

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Figure 6. In vitro migration of cells from the differentiated human promyelocytic leukemia cell line (dHL-60) and human monocyte cell line (THP-1). The dHL-60 and THP-1 cells were added to the upper chambers of transwell cluster plates with 3 and 8 mm pore filters, respectively. IL-1b-induced human corneal fibroblasts were added to the lower wells of the plates. The inserts were incubated in the dark at 37  C. The numbers of cells that had migrated to the lower wells were counted using a hemocytometer. Each bar represents the mean ± S.D. from triplicates from three independent experiments. **p50.01, compared with IL-1b-stimulated values.

stroma, and endothelium of the cornea as well as in the tears of patients with ocular surface diseases30. The presence of the IL-1b protein has also been correlated with various models of ocular inflammation31. Neutralization of IL-1b activity can reduce the severity of corneal diseases32. In the present study, we have shown that CAPE inhibits IL-1b-induced IL-6, MCP-1, and ICAM-1 production by corneal fibroblasts and that these effects are associated with inhibition of the AKT and NF-kB signaling pathways. Our results thus suggest that CAPE is a potential candidate for the treatment of corneal inflammation. We first investigated the IL-1b-induced expression of IL-6, MCP-1, and ICAM-1 in human corneal fibroblasts. As shown in Figure 3, CAPE significantly inhibited the IL-1b-induced expression of IL-6, MCP-1, and ICAM-1 at concentrations of CAPE that were not cytotoxic to the corneal fibroblasts. Because CAPE inhibited the expression of the inflammatory mediators in IL-1b-stimulated human corneal fibroblasts cells, we further explored the mechanism of inhibitory action for CAPE. In a previous report, MAPKs were shown

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to be phosphorylated in IL-1b-stimulated human corneal fibroblasts33. Therefore, we examined whether the CAPE inhibits phosphorylation of the MAPKs. Interestingly, we found that CAPE did not affect the MAPK signaling pathways. Alternatively, it is known that IL-1b-induced activation of PI3K induces expression of MCP-134. The serine–threonine kinase AKT is one of the major downstream targets of PI3K. We therefore investigated the activation of the PI3K/AKT pathway by IL-1b in this study. As expected, when the human corneal fibroblast cells were treated with IL1b, AKT was phosphorylated. Our results show that CAPE inhibited the IL-1b-induced phosphorylation of AKT. Nuclear factor-kB is known to be a pleiotropic regulator of various genes involved in the production of many proinflammatory cytokines and enzymes related to the inflammatory process35. Due to its key role in several pathologic conditions, NF-kB is a major drug target in a variety of diseases. Our results revealed that IL-1b induced the activation of NF-kB. However, the IL-1b-induced activation of NF-kB p65 was completely abolished by pretreating the cells with CAPE. Furthermore, these effects of the CAPE were mediated by the inhibition of the phosphorylation and degradation of IkBa and subsequent inhibition of the translocation of NF-kB from the cytoplasm to the nucleus. Therefore, inhibition of the NFkB signaling pathways in human corneal fibroblasts by CAPE might result in the down-regulation of inflammatory mediators. Taken together, these results suggest that AKT and NFkB activation, but not MAPKs, are involved in the inhibitory effect of CAPE on IL-1b-induced IL-6, MCP-1, and ICAM-1 expression. One of the most important functions of IL-1b is its wellknown ability to induce neutrophil migration and adhesion to the endothelium36,37. After showing the inhibitory effects of CAPE on IL-1b-induced IL-6, MCP-1, and ICAM-1 expression, we expected that the CAPE would attenuate the migration of neutrophils and monocytes. IL-6, one of the cytokines expressed during bacteria and virus invasion, is involved in the recruitment of polymorphonuclear leukocytes (PMNs) to the site of inflammation by upregulating the expression ICAM-1, a key molecule involved in migration of neutrophils38–40. Once secreted, corneal-derived MCP may elicit its inflammatory effect by binding to specific receptors on neutrophils, lymphocytes, and monocytes, leading to leukocyte accumulation and activation at the site of inflammation41. Infection of the corneal stroma triggers immune and inflammatory responses that are mediated in part by interactions between corneal resident cells and infiltrating cells such as PMNs, T cells, and macrophages42–44. A functional validation was carried out by evaluating the inhibitory effect of CAPE on the migration of neutrophils and monocytes in vitro. The human promyelocytic HL-60 cell line, which can be induced to differentiate using agents such as dimethylsulfoxide (DMSO), is a valid model system to study neutrophil migration45. Additionally, THP-1 cells, a human acute monocytic leukemia cell line, have distinct monocytic markers and maintain these monocytic characteristics46. We therefore used dHL-60 and THP-1 cells to evaluate the antimigratory effects of CAPE. Figure 6 shows that treatment of dHL-60 and THP-1 cells with CAPE (20 mM) did indeed decrease migration by approximately 65 and 95%,

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respectively. In conclusion, CAPE treatment inhibited the expression of IL-6, MCP-1, and ICAM-1 in IL-1b-induced human corneal fibroblasts, and subsequently inhibited the migration of neutrophils and monocytes. This inhibition by CAPE may be the result of suppression of the phosphorylation and activation of AKT, as well as inhibition of the activation of NF-kB. Based on our results, these inhibitory effects of CAPE on inflammatory conditions should be considered as an effective therapeutic role for the treatment of corneal ulceration associated with bacterial keratitis.

Declaration of interest The authors report that they have no conflict of interest. This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare Affairs, Republic of Korea (A120006).

References 1. Foster CS. Immunologic disorders of the conjunctiva, cornea, and sclera. In: Albert DM, Jakobiec FA, eds. Principles and practice of ophthalmology. Philadelphia (PA): WB Saunders; 2000:803–828. 2. Dana MR, Qian Y, Hamrah P. Twenty-five year panorama of corneal immunology. Cornea 2000;19:625–643. 3. Cubitt CL, Tang Q, Monteiro CA, et al. IL-8 gene expression in cultures of human corneal epithelial cells and keratocytes. Invest Ophthalmol Vis Sci 1993;34:3199–3206. 4. Cubitt CL, Lausch RN, Oakes JE. Differences in interleukin-6 gene expression between cultured human corneal epithelial cells and fibroblasts. Invest Ophthalmol Vis Sci 1995;36:330–336. 5. Trans MT, Tellaetxe-Isusi M, Elner V, et al. Proinflammatory cytokines induce RANTES and MCP-1 synthesis in human corneal keratocytes but not in corneal epithelial cells. Beta-chemokine synthesis in corneal cells. Invest Ophthalmol Vis Sci 1996;37: 987–996. 6. Matsuda H, Smelser GK. Electron microscopy of corneal wound healing. Exp Eye Res 1973;16:427–442. 7. Jester JV, Petroll WM, Cavanagh HD. Corneal stromal wound healing in refractive surgery: the role of myofibroblasts. Prog Retin Eye Res 1999;18:311–356. 8. Fini ME. Keratocyte and fibroblast phenotypes in the repairing cornea. Prog Retin Eye Res 1999;18:529–551. 9. Daniels JT, Geerling G, Alexander RA, et al. Temporal and spatial expression of matrix metalloproteinases during wound healing of human corneal tissue. Exp Eye Res 2003;77:653–664. 10. Liu Y, Kimura K, Yanai R, et al. Cytokine, chemokine, and adhesion molecule expression mediated by MAPKs inhuman corneal fibroblasts exposed to poly(I:C). Invest Ophthalmol Vis Sci 2008;49:3336–3344. 11. Spandau UH, Toksoy A, Verhaart S, et al. High expression of chemokines Gro-a (CXCL-1), IL-8 (CXCL-8), and MCP-1 (CCL-2) in inflamed human corneas in vivo. Arch Ophthalmol 2003;121:825–831. 12. Kumagai N, Fukuda K, Fujitsu Y, et al. Lipopolysaccharideinduced expression of intercellular adhesionmolecule-1 and chemokines in cultured human corneal fibroblasts. Invest Ophthalmol Vis Sci 2005;46:114–120. 13. Arranz-Valsero I, Schulzem U, Contreras-Ruiz L, et al. Involvement of corneal epithelial cells in the Th17 response in an in vitro bacterial inflammation model. Mol Vis 2013;19:85–99. 14. Gallucci RM, Simeonova PP, Matheson JM, et al. Impaired cutaneouswound healing in interleukin-6-deficient and immunosuppressed mice. FASEB J 2000;14:2525–2531. 15. Gallucci RM, Sugawara T, Yucesoy B, et al. Interleukin-6 treatment augments cutaneous wound healing in immunosuppressed mice. J Interferon Cytokine Res 2001;21:603–609. 16. Tacke F, Randolph GJ. Migratory fate and differentiation of blood monocyte subsets. Immunobiology 2006;211:609–618. 17. Van Riper G, Siciliano S, Fischer PA, et al. Characterization and species distribution of high affinity GTP-coupled receptors for

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18. 19. 20. 21. 22. 23.

24. 25. 26. 27. 28. 29.

30.

31.

32.

human RANTES and monocyte chemoattractant protein 1. J Exp Med 1993;177:851–856. Carr MW, Roth SJ, Luther E, et al. Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant. Proc Natl Acad Sci USA 1994;91:3652–3656. Marlin SD, Springer TA. Purified intercellular adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte function-associated antigen 1 (LFA-1). Cell 1987;51:813–819. Liu Y, Shaw SK, Ma S, et al. Regulation of leukocyte transmigration: cell surface interactions and signaling events. J Immunol 2004;172:7–13. Fesen MR, Kohn KW, Leteurtre F, Pommier Y. Inhibitors of human immunodeficiency virus integrase. Proc Natl Acad Sci USA 1993; 90:2399–2403. Liao HF, Chen YY, Liu JJ, et al. Inhibitory effect of caffeic acid phenethyl ester on angiogenesis, tumor invasion, and metastasis. J Agric Food Chem 2003;51:7907–7912. Orban Z, Mitsiades N, Burke Jr TR, et al. Caffeic acid phenethyl ester induces leukocyte apoptosis, modulates nuclear factor-kappa B and suppresses acute inflammation. Neuroimmunomodulation 2000;7:99–105. Hepsen IF, Er H, Cekic O. Topically applied water extract of propolis of to suppress corneal neovascularization in rabbits. Ophtalmic Res 1999;31:426–431. Ilhan A, Iraz M, Gurel A, et al. Caffeic acid phenethyl ester exerts a neuroprotective effect on CNS against pentylenetetrazol-induced seizures in mice. Neurochem Res 2004;29:2287–2292. Hishikawa K, Nakaki T, Fujita T. Oral flavonoid supplementation attenuates atherosclerosis development in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 2005;25:442–446. Park EH, Kang JH. Suppressive effects of propolis in rat adjuvant arthritis. Arch Pharm Res 1999;22:554–558. Lehman N, Di Fulvio M, McCray N, et al. Phagocyte cell migration is mediated by phospholipases PLD1 and PLD2. Blood 2006;108: 3564–3572. Fukuda K, Kumagai N, Yamamoto K, et al. Potentiation of lipopolysaccharide-induced chemokine and adhesion molecule expression in corneal fibroblasts by soluble CD14 or LPS-binding protein. Invest Ophthalmol Vis Sci 2005;46:3095–3101. Weng J, Mohan RR, Li Q, Wilson SE. IL-1 upregulates keratinocyte growth factor and hepatocyte growth factor mRNA and protein production by cultured stromal fibroblast cells: interleukin-1b expression in the cornea. Cornea 1997;16:465–471. Thakur A, Xue M, Stapleton F, et al. Balance of pro- and antiinflammatory cytokines correlates with outcome of acute experimental Pseudomonas aeruginosa keratitis. Infect Immun 2002;70: 2187–2197. Rudner XL, Kernacki KA, Barrett RP, Hazlett LD. Prolonged elevation of IL-1 in Pseudomonas aeruginosa ocular infection

33. 34. 35.

36. 37. 38. 39. 40.

41. 42.

43. 44. 45.

46.

377

regulates macrophage-inflammatory protein-2 production, polymorphonuclear neutrophil persistence, and corneal perforation. J Immunol 2000;164:6576–6582. Zhou H, Kimura K, Orita T, et al. Inhibition by medroxyprogesterone acetate of interleukin-1b-induced collagen degradation by corneal fibroblasts. Invest Ophthalmol Vis Sci 2012;53:4213–4219. Bian ZM, Elner SG, Yoshida A, Elner VM. Differential involvement of phosphoinositide 3-kinase/Akt in human RPE MCP-1 and IL-8 expression. Invest Ophthalmol Vis Sci 2004;45:1887–1896. Jung WK, Lee DY, Park C, et al. Cilostazol is anti-inflammatory in BV2 microglial cells by inactivating nuclear factor-kappaB and inhibiting mitogen-activated protein kinases. Br J Pharmacol 2010; 159:1274–1285. Derevianko A, Graeber T, D’amico R, Simms HH. The role of neutrophil-derived oxidants as second messengers in interleukin1b-stimulated cells. Shock 1998;10:54–61. Grutkoski PS, D’amico R, Ayala A, Simms HH. IL-1b stimulation induces paracrine regulation of PMN function and apoptosis. Shock 1999;12:373–381. Fenton RR, Molesworth-Kenyon S, Oakes JE, Lausch RN. Linkage of IL-6 with neutrophil chemoattractant expression in virus-induced ocular inflammation. Invest Ophthalmol Vis Sci 2002;43:737–743. Cole N, Bao S, Willcox M, Husband AJ. Expression of interleukin6 in the cornea in response to infection with different strains of Pseudomonas aeruginosa. Infect Immun 1999;67:2497–2502. Youker K, Smith CW, Anderson DC, et al. Neutrophil adherence to isolated adult cardiac myocytes. Induction by cardiac lymph collected during ischemia and reperfusion. J Clin Invest 1992;89: 602–609. Bian ZM, Elner VM, Lukacs NW, et al. Glycated human serum albumin induces IL-8 and MCP-1 gene expression in human corneal keratocytes. Curr Eye Res 1998;17:65–72. Banerjee K, Biswas PS, Kim B, Lee S. Rouse BT. CXCR2/ mice show enhanced susceptibility to herpetic stromal keratitis: a role for IL-6-induced neovascularization. J Immunol 2004;172: 237–245. Kessler E, Mondino BJ, Brown SI. The corneal response to Pseudomonas aeruginosa: histopathological and enzymatic characterization. Invest Ophthalmol Vis Sci 1977;16:116–125. Vasanthi M, Prajna NV, Lalitha P, et al. A pilot study on the infiltrating cells and cytokine levels in the tear of fungal keratitis patients. Indian J Ophthalmol 2007;55:27–31. Hauert AB, Martinelli S, Marone C, Niggli V. Differentiated HL-60 cells are a valid model system for the analysis of human neutrophil migration and chemotaxis. Int J Biochem Cell Biol 2002;34: 838–854. Tsuchiya S, Yamabe M, Yamaguchi Y, et al. Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer 1980;26:171–176.

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Caffeic acid phenethyl ester inhibits the inflammatory effects of interleukin-1β in human corneal fibroblasts.

Expression of various inflammatory mediators in corneal fibroblasts contributes to corneal inflammation...
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