November 2010
Volume 51, Issue 11
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Cornea  |   November 2010
Poly(I:C)-Induced Adhesion Molecule Expression Mediated by NF-κB and Phosphoinositide 3-Kinase–Akt Signaling Pathways in Human Corneal Fibroblasts
Tomoko Orita; Kazuhiro Kimura; Hong-Yan Zhou; Teruo Nishida
Author Affiliations & Notes
  • Tomoko Orita
    From the Departments of Ophthalmology and
  • Kazuhiro Kimura
    Ocular Pathophysiology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan.
  • Hong-Yan Zhou
    From the Departments of Ophthalmology and
  • Teruo Nishida
    From the Departments of Ophthalmology and
    Ocular Pathophysiology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan.
  • Corresponding author: Kazuhiro Kimura, Department of Ocular Pathophysiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube City, Yamaguchi 755-8505, Japan; [email protected]. 
Investigative Ophthalmology & Visual Science November 2010, Vol.51, 5556-5560. doi:https://doi.org/10.1167/iovs.09-4909
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      Tomoko Orita, Kazuhiro Kimura, Hong-Yan Zhou, Teruo Nishida; Poly(I:C)-Induced Adhesion Molecule Expression Mediated by NF-κB and Phosphoinositide 3-Kinase–Akt Signaling Pathways in Human Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2010;51(11):5556-5560. https://doi.org/10.1167/iovs.09-4909.

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Abstract

Purpose.: Viral infection at the ocular surface can lead to the chronic condition of viral stromal keratitis. Polyinosinic-polycytidylic acid [poly(I:C)], an analog of viral double-stranded RNA, induces the expression of adhesion molecules in cultured corneal fibroblasts. The authors investigated the roles of nuclear factor (NF)-κB and phosphoinositide 3-kinase (PI3K)-Akt signaling pathways in the poly(I:C)-induced expression of adhesion molecules in corneal fibroblasts.

Methods.: Human corneal fibroblasts were cultured with poly(I:C) in the absence or presence of IκB kinase 2 (IKK2) inhibitor (an inhibitor of NF-κB activation) or the PI3K inhibitor LY294002. The expression of vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, ICAM-2, and E-selectin, as well as the phosphorylation of the NF-κB inhibitory protein IκB-α and Akt, were examined by immunoblot analysis. The subcellular localization of adhesion molecules was determined by immunofluorescence analysis.

Results.: Poly(I:C) increased the expression of VCAM-1 and ICAM-1 but not that of ICAM-2 or E-selectin in corneal fibroblasts. Poly(I:C) also induced the phosphorylation of IκB-α and Akt. The poly(I:C)-induced expression of VCAM-1 and ICAM-1 was attenuated by both IKK2 inhibitor and LY294002.

Conclusions.: The NF-κB and PI3K-Akt signaling pathways mediate the poly(I:C)-induced upregulation of VCAM-1 and ICAM-1 in corneal fibroblasts, with PI3K acting upstream of NF-κB activation. These pathways thus likely modulate local immune and inflammatory responses to viral infection in the corneal stroma.

Virus infection at the ocular surface initially affects the corneal epithelium but eventually leads to disorders of the corneal stroma, including inflammation, ulceration, and fibrosis. 1 Viral stromal keratitis is a chronic inflammatory condition mediated by both resident cells of the corneal stroma and infiltrated cells, including polymorphonuclear leukocytes, T lymphocytes, and macrophages. 2,3 The stromal edema and cellular infiltration associated with viral infection of the corneal stroma can lead to corneal opacity. 4,5  
Keratocytes are the major resident cells of the corneal stroma and play a key role in the maintenance of corneal transparency. Activated keratocytes (also known as corneal fibroblasts) contribute to local inflammatory and immune responses to viral infection through the expression of adhesion molecules and the secretion of regulatory factors such as cytokines and chemokines. 6 –8 Adhesion molecules whose expression is associated with inflammation include intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, ICAM-2, and E-selectin. 9,10 The expression of ICAM-1 or VCAM-1 in corneal fibroblasts is induced by polyinosinic-polycytidylic acid [poly(I:C)], 8 a synthetic analog of viral double-stranded RNA, as well as by bacterial lipopolysaccharide. 11 These adhesion molecules, together with secreted cytokines and chemokines, promote the infiltration of leukocytes into the corneal stroma and thereby contribute to stromal disorders associated with viral keratitis. 
We have previously examined the effects of poly(I:C) on cultured human corneal fibroblasts to study the effects of viral infection on the corneal stroma. Poly(I:C) induced the activation of nuclear factor (NF)-κB in corneal fibroblasts and increased the expression of adhesion molecules in a manner dependent in part on mitogen-activated protein kinase (MAPK) signaling. 8 Signaling pathways mediated by NF-κB and by phosphoinositide 3-kinase (PI3K) and the protein kinase Akt also play important roles in the innate immune system. We have now examined the possible contributions of these two signaling pathways in corneal fibroblasts to corneal stromal keratitis associated with viral infection. 
Materials and Methods
Eagle's minimum essential medium (MEM), fetal bovine serum, and trypsin-EDTA were obtained from Invitrogen-Gibco (Carlsbad, CA); 24-well culture plates and 60-mm culture dishes were from Corning-Costar (Corning, NY); poly(I:C) was from InvivoGen (San Diego, CA); and IKK2 inhibitor was from Calbiochem (La Jolla, CA). Protease inhibitor cocktail was from Sigma-Aldrich (St. Louis, MO). Antibodies to VCAM-1, to ICAM-1, and to ICAM-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies to Akt, phosphorylated Akt, phosphorylated IκB-α, and LY294002 were from Cell Signaling (Beverly, MA); and antibodies to E-selectin were from R&D Systems (Minneapolis, MN). Nitrocellulose membranes and an enhanced chemiluminescence (ECL) kit were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden), and AlexaFluor 488–labeled goat antibodies to mouse immunoglobulin G and 4′,6-diamidino-2-phenylindole (DAPI) were from Molecular Probes (Eugene, OR). All media and reagents for cell culture were endotoxin minimized. 
Isolation and Culture of Human Corneal Fibroblasts
Human corneal fibroblasts were isolated and cultured as described previously. 6 Human corneas were obtained for corneal transplantation surgery from North West Lions Eye Bank (Seattle, WA) and were used in strict accordance with the tenets of the Declaration of Helsinki. The endothelial layer of the rim of the cornea remaining after transplantation surgery was removed mechanically, and the tissue was then incubated with dispase (2 mg/mL in MEM) for 1 hour at 37°C. After mechanical removal of the epithelial sheet, the tissue was treated with collagenase (2 mg/mL in MEM) at 37°C until a single-cell suspension of corneal fibroblasts was obtained. The isolated cells were maintained under a humidified atmosphere of 5% CO2 at 37°C in MEM supplemented with 10% fetal bovine serum, and they were used for experiments after four to seven passages. Cells were negative for the expression of α-smooth muscle actin (a marker of myofibroblasts), as described previously, 12 and they were positive for the expression of Toll-like receptor 3 (the receptor for viral double-stranded RNA), also as described previously. 8  
Immunoblot Analysis
Corneal fibroblasts were cultured in 60-mm dishes until they achieved confluence, after which the culture medium was replaced with serum-free MEM for 24 hours before exposure of the cells to poly(I:C) in MEM. The cells were then lysed in 300 μL of a solution containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 5 mM NaF, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM Na3VO4, and 1% protease inhibitor cocktail. Cell lysates (10 μg protein) were subjected to SDS-polyacrylamide gel electrophoresis on a 10% gel, the separated proteins were transferred electrophoretically to a nitrocellulose membrane, and the membrane was incubated with primary antibodies. Immune complexes were detected with the use of secondary antibodies and ECL reagents. 
Immunofluorescence Staining
For immunostaining of VCAM-1, ICAM-1, ICAM-2, or E-selectin, corneal fibroblasts were cultured in 24-well plates until they achieved confluence. The medium was then changed to serum-free MEM, and the cells were cultured for an additional 24 hours before exposure to poly(I:C) in MEM. The cells were washed twice with phosphate-buffered saline (PBS), fixed for 15 minutes at room temperature with PBS containing 1% paraformaldehyde, washed three times with PBS containing 1% bovine serum albumin (BSA), and incubated for 1 hour at room temperature with PBS-BSA containing mouse monoclonal antibodies to VCAM-1, ICAM-1, ICAM-2, or E-selectin. The cells were then washed three times with PBS-BSA before incubation for 1 hour at room temperature with DAPI and AlexaFluor 488–conjugated goat antibodies to mouse immunoglobulin G. The cells were finally washed with PBS, mounted with the use of mounting medium (Vectashield; Vector Laboratories, Burlingame, CA), and observed with a fluorescence microscope (Axioskop 50; Carl Zeiss, Oberkochen, Germany). 
Results
Effects of Poly(I:C) on VCAM-1, ICAM-1, ICAM-2, and E-selectin Expression in Corneal Fibroblasts
We first examined the effects of poly(I:C) on the expression of adhesion molecules in human corneal fibroblasts. Immunoblot analysis revealed that incubation of the cells with poly(I:C) at concentrations of 0 to 30 μg/mL for 24 hours resulted in a concentration-dependent increase in the abundance of VCAM-1 and ICAM-1 (Fig. 1A). The effects of poly(I:C) on VCAM-1 and ICAM-1 expression appeared maximal at concentrations of 1 to 3 μg/mL. In contrast, poly(I:C) had no effect on the amounts of ICAM-2 or E-selectin in corneal fibroblasts (Fig. 1A). Immunofluorescence analysis revealed that immunoreactivity for VCAM-1 or ICAM-1 was prominent at the cell margins, indicative of surface expression of these adhesion molecules and that the amount of such immunoreactivity was markedly increased by exposure of the cells to poly(I:C) at 1 μg/mL (Fig. 1B). Immunofluorescence staining for ICAM-2 or E-selectin appeared diffuse and was not affected by poly(I:C) (Fig. 1B). 
Figure 1.
Effects of poly(I:C) on the expression of VCAM-1, ICAM-1, ICAM-2, and E-selectin in human corneal fibroblasts. (A) Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1, ICAM-1, ICAM-2, E-selectin, or β-actin (loading control). (B) Cells were incubated for 24 hours in the absence (a–d) or presence (e–h) of poly(I:C) (1 μg/mL) and were then subjected to immunofluorescence staining with antibodies to VCAM-1 (a, e), ICAM-1 (b, f), ICAM-2 (c, g), or E-selectin (d, h) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are representative of three independent experiments.
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Effects of poly(I:C) on the expression of VCAM-1, ICAM-1, ICAM-2, and E-selectin in human corneal fibroblasts. (A) Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1, ICAM-1, ICAM-2, E-selectin, or β-actin (loading control). (B) Cells were incubated for 24 hours in the absence (a–d) or presence (e–h) of poly(I:C) (1 μg/mL) and were then subjected to immunofluorescence staining with antibodies to VCAM-1 (a, e), ICAM-1 (b, f), ICAM-2 (c, g), or E-selectin (d, h) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are representative of three independent experiments.
Figure 1.
 
Effects of poly(I:C) on the expression of VCAM-1, ICAM-1, ICAM-2, and E-selectin in human corneal fibroblasts. (A) Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1, ICAM-1, ICAM-2, E-selectin, or β-actin (loading control). (B) Cells were incubated for 24 hours in the absence (a–d) or presence (e–h) of poly(I:C) (1 μg/mL) and were then subjected to immunofluorescence staining with antibodies to VCAM-1 (a, e), ICAM-1 (b, f), ICAM-2 (c, g), or E-selectin (d, h) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are representative of three independent experiments.
Effects of poly(I:C) on the expression of VCAM-1, ICAM-1, ICAM-2, and E-selectin in human corneal fibroblasts. (A) Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1, ICAM-1, ICAM-2, E-selectin, or β-actin (loading control). (B) Cells were incubated for 24 hours in the absence (a–d) or presence (e–h) of poly(I:C) (1 μg/mL) and were then subjected to immunofluorescence staining with antibodies to VCAM-1 (a, e), ICAM-1 (b, f), ICAM-2 (c, g), or E-selectin (d, h) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are representative of three independent experiments.
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Effects of NF-κB Inhibition on VCAM-1 and ICAM-1 Expression Induced by Poly(I:C) in Corneal Fibroblasts
We investigated the effects of NF-κB inhibition on poly(I:C)-induced expression of adhesion molecules in corneal fibroblasts. We first confirmed that IκB kinase 2 (IKK2) inhibitor, which blocks NF-κB activation, indeed prevented the poly(I:C)-induced translocation of the p65 subunit of NF-κB from the cytosol to the nucleus in these cells (data not shown). Immunoblot analysis revealed that the upregulation of VCAM-1 and ICAM-1 induced by poly(I:C) (1 μg/mL) was attenuated by IKK2 inhibitor in a concentration-dependent manner, with inhibition being complete at an inhibitor concentration of 3 μM (Fig. 2A). Immunofluorescence analysis also showed that IKK2 inhibitor (1 μM) largely prevented the poly(I:C)-induced increase in the surface expression of VCAM-1 and ICAM-1 (Fig. 2B). 
Figure 2.
Effects of IKK2 inhibitor on the poly(I:C)-induced expression of adhesion molecules in corneal fibroblasts. (A) Cells were incubated first for 1 hour in the presence of the indicated concentrations of IKK2 inhibitor and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1 or ICAM-1. (B) Cells were incubated for 1 hour in the absence or presence of IKK2 inhibitor (1 μM) and for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL). Cells were then subjected to immunofluorescence analysis with antibodies to VCAM-1 (a–c) or ICAM-1 (d–f) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are representative of three independent experiments.
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Effects of IKK2 inhibitor on the poly(I:C)-induced expression of adhesion molecules in corneal fibroblasts. (A) Cells were incubated first for 1 hour in the presence of the indicated concentrations of IKK2 inhibitor and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1 or ICAM-1. (B) Cells were incubated for 1 hour in the absence or presence of IKK2 inhibitor (1 μM) and for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL). Cells were then subjected to immunofluorescence analysis with antibodies to VCAM-1 (a–c) or ICAM-1 (d–f) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are representative of three independent experiments.
Figure 2.
 
Effects of IKK2 inhibitor on the poly(I:C)-induced expression of adhesion molecules in corneal fibroblasts. (A) Cells were incubated first for 1 hour in the presence of the indicated concentrations of IKK2 inhibitor and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1 or ICAM-1. (B) Cells were incubated for 1 hour in the absence or presence of IKK2 inhibitor (1 μM) and for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL). Cells were then subjected to immunofluorescence analysis with antibodies to VCAM-1 (a–c) or ICAM-1 (d–f) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are representative of three independent experiments.
Effects of IKK2 inhibitor on the poly(I:C)-induced expression of adhesion molecules in corneal fibroblasts. (A) Cells were incubated first for 1 hour in the presence of the indicated concentrations of IKK2 inhibitor and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1 or ICAM-1. (B) Cells were incubated for 1 hour in the absence or presence of IKK2 inhibitor (1 μM) and for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL). Cells were then subjected to immunofluorescence analysis with antibodies to VCAM-1 (a–c) or ICAM-1 (d–f) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are representative of three independent experiments.
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Activation of Akt by Poly(I:C) in Corneal Fibroblasts
We next examined the effect of poly(I:C) on the activation status of Akt in corneal fibroblasts. Immunoblot analysis revealed that poly(I:C) (1 μg/mL) induced a time-dependent increase in the phosphorylation (activation) of Akt that was first apparent at 30 minutes, maximal at 30 to 60 minutes, and no longer apparent at 90 minutes (Fig. 3). 
Figure 3.
Effect of poly(I:C) on the activation status of Akt in corneal fibroblasts. Cells were incubated in the absence or presence of poly(I:C) (1 μg/mL) for the indicated times, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to phosphorylated (p-) or total forms of Akt. Data are representative of three independent experiments.
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Effect of poly(I:C) on the activation status of Akt in corneal fibroblasts. Cells were incubated in the absence or presence of poly(I:C) (1 μg/mL) for the indicated times, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to phosphorylated (p-) or total forms of Akt. Data are representative of three independent experiments.
Figure 3.
 
Effect of poly(I:C) on the activation status of Akt in corneal fibroblasts. Cells were incubated in the absence or presence of poly(I:C) (1 μg/mL) for the indicated times, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to phosphorylated (p-) or total forms of Akt. Data are representative of three independent experiments.
Effect of poly(I:C) on the activation status of Akt in corneal fibroblasts. Cells were incubated in the absence or presence of poly(I:C) (1 μg/mL) for the indicated times, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to phosphorylated (p-) or total forms of Akt. Data are representative of three independent experiments.
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Effects of PI3K Inhibitor on Poly(I:C)-Induced Adhesion Molecule Expression and NF-κB Activation in Corneal Fibroblasts
We first examined the effects of the PI3K inhibitor on poly(I:C)-induced expression of adhesion molecules in corneal fibroblasts. Immunoblot analysis showed that LY294002 inhibited the poly(I:C)-induced expression of VCAM-1 and ICAM-1 in a concentration-dependent manner (Fig. 4A). Immunofluorescence analysis confirmed that LY294002 (10 μM) inhibited the upregulation of VCAM-1 and ICAM-1 by poly(I:C) (Fig. 4B). We then investigated the effect of PI3K inhibitor LY294002 on poly(I:C)-induced NF-κB activation in corneal fibroblasts. Immunoblot analysis revealed that LY294002 (0–50 μM) inhibited the poly(I:C)-induced phosphorylation of the endogenous NF-κB inhibitory protein IκB-α in a concentration-dependent manner (Fig. 5). 
Figure 4.
Effects of a PI3K inhibitor on adhesion molecule expression in corneal fibroblasts. (A) Cells were incubated first for 30 minutes in the presence of the indicated concentrations of LY294002 and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1 or ICAM-1. (B) Cells were incubated first for 30 minutes in the absence or presence of LY294002 (10 μM) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which they were subjected to immunofluorescence analysis with antibodies to VCAM-1 (a–c) or ICAM-1 (d–f) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. Data are representative of three independent experiments.
View OriginalDownload Slide
 
Effects of a PI3K inhibitor on adhesion molecule expression in corneal fibroblasts. (A) Cells were incubated first for 30 minutes in the presence of the indicated concentrations of LY294002 and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1 or ICAM-1. (B) Cells were incubated first for 30 minutes in the absence or presence of LY294002 (10 μM) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which they were subjected to immunofluorescence analysis with antibodies to VCAM-1 (a–c) or ICAM-1 (d–f) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. Data are representative of three independent experiments.
Figure 4.
 
Effects of a PI3K inhibitor on adhesion molecule expression in corneal fibroblasts. (A) Cells were incubated first for 30 minutes in the presence of the indicated concentrations of LY294002 and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1 or ICAM-1. (B) Cells were incubated first for 30 minutes in the absence or presence of LY294002 (10 μM) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which they were subjected to immunofluorescence analysis with antibodies to VCAM-1 (a–c) or ICAM-1 (d–f) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. Data are representative of three independent experiments.
Effects of a PI3K inhibitor on adhesion molecule expression in corneal fibroblasts. (A) Cells were incubated first for 30 minutes in the presence of the indicated concentrations of LY294002 and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1 or ICAM-1. (B) Cells were incubated first for 30 minutes in the absence or presence of LY294002 (10 μM) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which they were subjected to immunofluorescence analysis with antibodies to VCAM-1 (a–c) or ICAM-1 (d–f) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. Data are representative of three independent experiments.
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Figure 5.
Effects of a PI3K inhibitor on poly(I:C)-induced NF-κB activation in corneal fibroblasts. Cells were incubated first for 30 minutes in the presence of the indicated concentrations of LY294002 and then for 90 minutes in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to the phosphorylated form (p-) of IκB-α. All data are representative of three independent experiments.
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Effects of a PI3K inhibitor on poly(I:C)-induced NF-κB activation in corneal fibroblasts. Cells were incubated first for 30 minutes in the presence of the indicated concentrations of LY294002 and then for 90 minutes in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to the phosphorylated form (p-) of IκB-α. All data are representative of three independent experiments.
Figure 5.
 
Effects of a PI3K inhibitor on poly(I:C)-induced NF-κB activation in corneal fibroblasts. Cells were incubated first for 30 minutes in the presence of the indicated concentrations of LY294002 and then for 90 minutes in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to the phosphorylated form (p-) of IκB-α. All data are representative of three independent experiments.
Effects of a PI3K inhibitor on poly(I:C)-induced NF-κB activation in corneal fibroblasts. Cells were incubated first for 30 minutes in the presence of the indicated concentrations of LY294002 and then for 90 minutes in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to the phosphorylated form (p-) of IκB-α. All data are representative of three independent experiments.
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Discussion
We have shown that poly(I:C), a synthetic analog of viral double-stranded RNA, induced the expression of VCAM-1 and ICAM-1 in human corneal fibroblasts in a concentration-dependent manner, as revealed by both immunoblot and immunofluorescence analyses. In contrast, the expression of neither ICAM-2 nor E-selectin was affected by poly(I:C) in these cells. The expression of VCAM-1 and ICAM-1 induced by poly(I:C) was attenuated by IKK2 inhibitor and by the PI3K inhibitor LY294002 in concentration-dependent manners. Finally, the PI3K inhibitor attenuated the phosphorylation of IκB-α elicited by poly(I:C), and poly(I:C) induced the activation of Akt in corneal fibroblasts. These results thus suggest that poly(I:C) upregulates the expression of the adhesion molecules VCAM-1 and ICAM-1 in corneal fibroblasts in a manner dependent on both the NF-κB and the PI3K-Akt signaling pathways. 
The expression of VCAM-1 and ICAM-1 is increased in various cell types in response to inflammation or infection. 9,13 The expression of these molecules in such cells contributes to the adhesion of leukocytes and plays an important role in modulation of inflammation. 10 Lipopolysaccharide, a glycolipid component of the outer membrane of Gram-negative bacteria, induces the expression of ICAM-1 in corneal fibroblasts. 11 Moreover, interleukin-4 and tumor necrosis factor-α each upregulates the expression of ICAM-1 and VCAM-1 in corneal fibroblasts and thereby promotes the adhesion of eosinophils to these cells. 14 Our observation that poly(I:C) induces the expression of VCAM-1 and ICAM-1 in corneal fibroblasts suggests that corneal fibroblasts may contribute to the infiltration of leukocytes into the corneal stroma associated with viral infection. 
Toll-like receptors recognize structurally conserved features of pathogens, including bacterial lipopolysaccharide and flagellin as well as viral double-stranded RNA, and they thereby play an important role in the innate immune response to infection. 15 Interaction of these receptors with their cognate ligands results in the activation of various signaling pathways, including those mediated by MAPKs, PI3K-Akt, and NF-κB, in various cell types. 16,17 We have previously shown that the poly(I:C)-induced expression of VCAM-1 and ICAM-1 in corneal fibroblasts is dependent only in part on MAPK signaling, with the upregulation of ICAM-1 attenuated only by an inhibitor of c-Jun NH2-terminal kinase (JNK) and that of VCAM-1 not affected by inhibitors of extracellular signal–regulated kinase (ERK), p38 MAPK, or JNK signaling. 8 We have also previously shown that lipopolysaccharide induces the expression of ICAM-1 and the phosphorylation of IκB-α in corneal fibroblasts. 18 We previously showed that poly(I:C) activates the NF-κB signaling pathway in corneal fibroblasts, 8 and in the present study we demonstrate that the upregulation of VCAM-1 and ICAM-1 expression by poly(I:C) in these cells was attenuated by IKK2 inhibitor. These results thus suggest that NF-κB signaling may be a common pathway by which pathogens induce the expression of adhesion molecules in human corneal fibroblasts. 
We have now shown that the poly(I:C)-induced expression of VCAM-1 and ICAM-1 in corneal fibroblasts was attenuated by the PI3K inhibitor LY294002, suggesting that the PI3K-Akt signaling pathway also contributes to local inflammation in the corneal stroma associated with viral infection. Moreover, we found that poly(I:C) increased the phosphorylation of Akt and that the phosphorylation of IκB-α induced by poly(I:C) was also inhibited by LY294002. These results thus suggest that PI3K acts upstream of the NF-κB signaling pathway in the regulation of VCAM-1 and ICAM-1 expression by poly(I:C). 
In summary, we have shown that poly(I:C) upregulates the expression of VCAM-1 and ICAM-1 in human corneal fibroblasts in a manner dependent on activation of the PI3K-Akt and NF-κB signaling pathways. Our results therefore suggest that PI3K-Akt and NF-κB signaling in corneal fibroblasts may play an important role in the modulation of local immune and inflammatory responses associated with viral infection in the corneal stroma. 
Footnotes
 Supported in part by Grant 21791687 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Footnotes
 Disclosure: T. Orita, None; K. Kimura, None; H.-Y. Zhou, None; T. Nishida, None
The authors thank Yasumiko Akamatsu and the staff of Yamaguchi University Center for Gene Research for technical assistance. 
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Figure 1.
Effects of poly(I:C) on the expression of VCAM-1, ICAM-1, ICAM-2, and E-selectin in human corneal fibroblasts. (A) Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1, ICAM-1, ICAM-2, E-selectin, or β-actin (loading control). (B) Cells were incubated for 24 hours in the absence (a–d) or presence (e–h) of poly(I:C) (1 μg/mL) and were then subjected to immunofluorescence staining with antibodies to VCAM-1 (a, e), ICAM-1 (b, f), ICAM-2 (c, g), or E-selectin (d, h) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are representative of three independent experiments.
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Effects of poly(I:C) on the expression of VCAM-1, ICAM-1, ICAM-2, and E-selectin in human corneal fibroblasts. (A) Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1, ICAM-1, ICAM-2, E-selectin, or β-actin (loading control). (B) Cells were incubated for 24 hours in the absence (a–d) or presence (e–h) of poly(I:C) (1 μg/mL) and were then subjected to immunofluorescence staining with antibodies to VCAM-1 (a, e), ICAM-1 (b, f), ICAM-2 (c, g), or E-selectin (d, h) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are representative of three independent experiments.
Figure 1.
 
Effects of poly(I:C) on the expression of VCAM-1, ICAM-1, ICAM-2, and E-selectin in human corneal fibroblasts. (A) Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1, ICAM-1, ICAM-2, E-selectin, or β-actin (loading control). (B) Cells were incubated for 24 hours in the absence (a–d) or presence (e–h) of poly(I:C) (1 μg/mL) and were then subjected to immunofluorescence staining with antibodies to VCAM-1 (a, e), ICAM-1 (b, f), ICAM-2 (c, g), or E-selectin (d, h) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are representative of three independent experiments.
Effects of poly(I:C) on the expression of VCAM-1, ICAM-1, ICAM-2, and E-selectin in human corneal fibroblasts. (A) Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1, ICAM-1, ICAM-2, E-selectin, or β-actin (loading control). (B) Cells were incubated for 24 hours in the absence (a–d) or presence (e–h) of poly(I:C) (1 μg/mL) and were then subjected to immunofluorescence staining with antibodies to VCAM-1 (a, e), ICAM-1 (b, f), ICAM-2 (c, g), or E-selectin (d, h) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are representative of three independent experiments.
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Figure 2.
Effects of IKK2 inhibitor on the poly(I:C)-induced expression of adhesion molecules in corneal fibroblasts. (A) Cells were incubated first for 1 hour in the presence of the indicated concentrations of IKK2 inhibitor and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1 or ICAM-1. (B) Cells were incubated for 1 hour in the absence or presence of IKK2 inhibitor (1 μM) and for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL). Cells were then subjected to immunofluorescence analysis with antibodies to VCAM-1 (a–c) or ICAM-1 (d–f) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are representative of three independent experiments.
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Effects of IKK2 inhibitor on the poly(I:C)-induced expression of adhesion molecules in corneal fibroblasts. (A) Cells were incubated first for 1 hour in the presence of the indicated concentrations of IKK2 inhibitor and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1 or ICAM-1. (B) Cells were incubated for 1 hour in the absence or presence of IKK2 inhibitor (1 μM) and for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL). Cells were then subjected to immunofluorescence analysis with antibodies to VCAM-1 (a–c) or ICAM-1 (d–f) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are representative of three independent experiments.
Figure 2.
 
Effects of IKK2 inhibitor on the poly(I:C)-induced expression of adhesion molecules in corneal fibroblasts. (A) Cells were incubated first for 1 hour in the presence of the indicated concentrations of IKK2 inhibitor and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1 or ICAM-1. (B) Cells were incubated for 1 hour in the absence or presence of IKK2 inhibitor (1 μM) and for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL). Cells were then subjected to immunofluorescence analysis with antibodies to VCAM-1 (a–c) or ICAM-1 (d–f) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are representative of three independent experiments.
Effects of IKK2 inhibitor on the poly(I:C)-induced expression of adhesion molecules in corneal fibroblasts. (A) Cells were incubated first for 1 hour in the presence of the indicated concentrations of IKK2 inhibitor and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1 or ICAM-1. (B) Cells were incubated for 1 hour in the absence or presence of IKK2 inhibitor (1 μM) and for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL). Cells were then subjected to immunofluorescence analysis with antibodies to VCAM-1 (a–c) or ICAM-1 (d–f) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are representative of three independent experiments.
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Figure 3.
Effect of poly(I:C) on the activation status of Akt in corneal fibroblasts. Cells were incubated in the absence or presence of poly(I:C) (1 μg/mL) for the indicated times, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to phosphorylated (p-) or total forms of Akt. Data are representative of three independent experiments.
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Effect of poly(I:C) on the activation status of Akt in corneal fibroblasts. Cells were incubated in the absence or presence of poly(I:C) (1 μg/mL) for the indicated times, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to phosphorylated (p-) or total forms of Akt. Data are representative of three independent experiments.
Figure 3.
 
Effect of poly(I:C) on the activation status of Akt in corneal fibroblasts. Cells were incubated in the absence or presence of poly(I:C) (1 μg/mL) for the indicated times, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to phosphorylated (p-) or total forms of Akt. Data are representative of three independent experiments.
Effect of poly(I:C) on the activation status of Akt in corneal fibroblasts. Cells were incubated in the absence or presence of poly(I:C) (1 μg/mL) for the indicated times, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to phosphorylated (p-) or total forms of Akt. Data are representative of three independent experiments.
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Figure 4.
Effects of a PI3K inhibitor on adhesion molecule expression in corneal fibroblasts. (A) Cells were incubated first for 30 minutes in the presence of the indicated concentrations of LY294002 and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1 or ICAM-1. (B) Cells were incubated first for 30 minutes in the absence or presence of LY294002 (10 μM) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which they were subjected to immunofluorescence analysis with antibodies to VCAM-1 (a–c) or ICAM-1 (d–f) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. Data are representative of three independent experiments.
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Effects of a PI3K inhibitor on adhesion molecule expression in corneal fibroblasts. (A) Cells were incubated first for 30 minutes in the presence of the indicated concentrations of LY294002 and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1 or ICAM-1. (B) Cells were incubated first for 30 minutes in the absence or presence of LY294002 (10 μM) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which they were subjected to immunofluorescence analysis with antibodies to VCAM-1 (a–c) or ICAM-1 (d–f) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. Data are representative of three independent experiments.
Figure 4.
 
Effects of a PI3K inhibitor on adhesion molecule expression in corneal fibroblasts. (A) Cells were incubated first for 30 minutes in the presence of the indicated concentrations of LY294002 and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1 or ICAM-1. (B) Cells were incubated first for 30 minutes in the absence or presence of LY294002 (10 μM) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which they were subjected to immunofluorescence analysis with antibodies to VCAM-1 (a–c) or ICAM-1 (d–f) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. Data are representative of three independent experiments.
Effects of a PI3K inhibitor on adhesion molecule expression in corneal fibroblasts. (A) Cells were incubated first for 30 minutes in the presence of the indicated concentrations of LY294002 and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1 or ICAM-1. (B) Cells were incubated first for 30 minutes in the absence or presence of LY294002 (10 μM) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which they were subjected to immunofluorescence analysis with antibodies to VCAM-1 (a–c) or ICAM-1 (d–f) and with AlexaFluor 488–conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. Data are representative of three independent experiments.
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Figure 5.
Effects of a PI3K inhibitor on poly(I:C)-induced NF-κB activation in corneal fibroblasts. Cells were incubated first for 30 minutes in the presence of the indicated concentrations of LY294002 and then for 90 minutes in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to the phosphorylated form (p-) of IκB-α. All data are representative of three independent experiments.
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Effects of a PI3K inhibitor on poly(I:C)-induced NF-κB activation in corneal fibroblasts. Cells were incubated first for 30 minutes in the presence of the indicated concentrations of LY294002 and then for 90 minutes in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to the phosphorylated form (p-) of IκB-α. All data are representative of three independent experiments.
Figure 5.
 
Effects of a PI3K inhibitor on poly(I:C)-induced NF-κB activation in corneal fibroblasts. Cells were incubated first for 30 minutes in the presence of the indicated concentrations of LY294002 and then for 90 minutes in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to the phosphorylated form (p-) of IκB-α. All data are representative of three independent experiments.
Effects of a PI3K inhibitor on poly(I:C)-induced NF-κB activation in corneal fibroblasts. Cells were incubated first for 30 minutes in the presence of the indicated concentrations of LY294002 and then for 90 minutes in the additional absence or presence of poly(I:C) (1 μg/mL), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to the phosphorylated form (p-) of IκB-α. All data are representative of three independent experiments.
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