October 2010
Volume 51, Issue 10
Free
Cornea  |   October 2010
Upregulation of Matrix Metalloproteinase Expression by Poly(I:C) in Corneal Fibroblasts: Role of NF-κB and Interleukin-1β
Author Affiliations & Notes
  • Kazuhiro Kimura
    From the Department of Ocular Pathophysiology, Yamaguchi University School of Medicine, and
  • Tomoko Orita
    the Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan.
  • Yukiko Kondo
    the Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan.
  • Hongyan Zhou
    the Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan.
  • Teruo Nishida
    the Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan.
  • Corresponding author: Kazuhiro Kimura, Department of Ocular Pathophysiology, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube City, Yamaguchi 755-8505, Japan; k.kimura@yamaguchi-u.ac.jp
Investigative Ophthalmology & Visual Science October 2010, Vol.51, 5012-5018. doi:10.1167/iovs.10-5167
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Kazuhiro Kimura, Tomoko Orita, Yukiko Kondo, Hongyan Zhou, Teruo Nishida; Upregulation of Matrix Metalloproteinase Expression by Poly(I:C) in Corneal Fibroblasts: Role of NF-κB and Interleukin-1β. Invest. Ophthalmol. Vis. Sci. 2010;51(10):5012-5018. doi: 10.1167/iovs.10-5167.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose.: To characterize the mechanism of corneal ulceration associated with viral keratitis, the authors investigated the effects of polyinosinic-polycytidylic acid [poly(I:C)], a synthetic analog of viral double-stranded RNA, on the expression of matrix metalloproteinases (MMPs) in human corneal fibroblasts.

Methods.: The expression of MMPs in human corneal fibroblasts cultured in the absence or presence of poly(I:C) was examined by immunoblot analysis, gelatin zymography, and quantitative reverse transcription–polymerase chain reaction analysis. The phosphorylation of the NF-κB inhibitor protein IκB-α was assessed by immunoblot analysis, and the concentration of interleukin (IL)-1β in culture supernatants was measured by enzyme-linked immunosorbent assay.

Results.: Poly(I:C) increased the expression of MMP-1 and MMP-3 in corneal fibroblasts at the mRNA and protein levels. It also induced the phosphorylation of IκB-α and the secretion of IL-1β in these cells. The poly(I:C)-induced expression of MMP-1 and MMP-3 was attenuated by a synthetic inhibitor of NF-κB signaling and by IL-1 receptor antagonist; the latter also inhibited poly(I:C)-induced phosphorylation of IκB-α.

Conclusions.: Poly(I:C) induces the expression of MMP-1 and MMP-3 in human corneal fibroblasts in a manner dependent on activation of the transcription factor NF-κB and on IL-1β secretion. These effects may play an important role in corneal ulceration associated with viral keratitis.

Ocular infection with viruses such as herpes simplex virus results in inflammation of the corneal stroma that can lead to corneal ulceration, perforation, and scarring. Viral stromal keratitis is a chronic immunopathologic disease induced by interactions between infiltrating immune cells, including polymorphonuclear leukocytes and T cells, and corneal resident cells, and it is a major cause of blindness. 1,2 Various factors, including cytokines, chemokines, and matrix metalloproteinases (MMPs), are produced by corneal resident cells in response to corneal infection 35 and may contribute to the destruction of the corneal stroma associated with viral keratitis. 
Keratocytes, which constitute the major cell type of the corneal stroma, produce the extracellular matrix of the stroma and maintain corneal transparency. Corneal fibroblasts (activated keratocytes) contribute to the modulation of local immune reactions through the secretion of cytokines and chemokines and the expression of adhesion molecules in response to stromal infection. 68 MMPs are zinc-dependent enzymes responsible for degradation of extracellular matrix proteins including collagen, fibronectin, and proteoglycans, and they are implicated in many physiological and pathologic processes including development, wound healing, cancer, and arthritis. 9,10 MMPs are secreted as proenzymes (proMMPs) and are activated by proteolytic cleavage. Collagenase (MMP-1) and stromelysin (MMP-3) are released at sites of corneal stromal wounds and contribute to remodeling of collagen fibrils. 11 The gelatinases MMP-2 and MMP-9 are also secreted by corneal stromal cells. 12,13 We previously showed that MMP-1, MMP-3, and MMP-9 are secreted by corneal fibroblasts. 14,15 Given that corneal ulceration results from collagen degradation in the stroma, MMPs are thought to play an important role in such corneal pathology. 16,17 Indeed, MMPs have been identified in ulcerated corneal tissue. 18  
The observation that corneal fibroblasts are able to produce MMPs suggests that these cells may be a major source of these enzymes in corneal ulceration. Little is known of the mechanism of corneal ulceration associated with viral infection, however. Polyinosinic-polycytidylic acid [poly(I:C)] is a synthetic analog of viral double-stranded RNA and, like such viral RNA, is recognized by Toll-like receptor 3 (TLR3). It has, therefore, been applied to study the effects of viral infection in several cell types. 16,17,19 We have now investigated the effects of poly(I:C) on MMP expression in human corneal fibroblasts. 
Methods
Materials
Eagle's minimum essential medium (MEM) and fetal bovine serum were obtained from Invitrogen-Gibco (Carlsbad, CA), and 24-well culture plates and 60-mm culture dishes were from Corning-Costar (Corning, NY). Poly(I:C) and the Trizol reagent were from Invivogen (San Diego, CA). Mouse monoclonal antibodies to MMP-1, MMP-2, MMP-3, or MMP-9 were obtained from Daiichi Fine Chemicals (Toyama, Japan), and rabbit polyclonal antibodies to phosphorylated forms of IκB-α were from Cell Signaling (Beverly, MA). Blocking antibodies to TLR3 (TLR3.7) were from Abcam (Cambridge, UK). Coomassie brilliant blue and gelatin were from Bio-Rad (Hercules, CA). A reverse transcription (RT) system was from Promega (Madison, WI). 
[5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide (IKK-2 inhibitor) was obtained from Calbiochem (La Jolla, CA), and interleukin-1 receptor antagonist (IL-1ra) was from R&D Systems (Minneapolis, MN). Protease inhibitor cocktail and antibodies to β-actin were obtained from Sigma (St. Louis, MO). Horseradish peroxidase–conjugated secondary antibodies, nitrocellulose membranes, and an enhanced chemiluminescence (ECL) kit were from GE Healthare (Uppsala, Sweden). 
Isolation and Culture of Human Corneal Fibroblasts
Human corneas were obtained for corneal transplantation surgery from NorthWest Lions Eye Bank (Seattle, WA). Corneal fibroblasts were prepared from the tissue remaining after surgery and were cultured as described previously. 6 The human tissue was used in strict accordance with the tenets of the Declaration of Helsinki. In brief, the endothelial layer of the remaining rim of the cornea 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 were used for experiments after four to eight passages. Cells were plated at a density approaching confluence (1 × 105 cells per well in 24-well plates; 3 × 105 cells per 60-mm dish) and were cultured in unsupplemented MEM for 24 hours before experiments. 
Immunoblot Analysis
Cells in 24-well culture plates were incubated with MEM containing various concentrations of poly(I:C) for 24 hours or the indicated times. For analysis of MMP secretion, culture supernatants were collected and subjected to SDS-polyacrylamide gel electrophoresis on a 10% gel. For analysis of IκB-α phosphorylation and β-actin, cells were lysed in 300 μL 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% SDS, 1 mM Na3VO4, and 1% protease inhibitor cocktail. The lysates were centrifuged at 15,000g for 10 minutes at 4°C, and the resultant supernatants were subjected to electrophoresis. Separated proteins were transferred to a nitrocellulose membrane, which was then exposed to blocking solution (20 mM Tris-HCl [pH 7.4], 5% dried skim milk, 0.1% Tween 20) before incubation for 16 hours at room temperature with primary antibodies at a 1:1000 dilution in blocking solution. After washing with a solution containing 20 mM Tris-HCl (pH 7.4) and 0.1% Tween 20, the membrane was incubated for 1 hour at room temperature with horseradish peroxidase–conjugated secondary antibodies at a 1:1000 dilution in the same solution, washed again, incubated with ECL reagents for 5 minutes, and then exposed to film. 
Blocking TLR3 with Antibodies to TLR3
Cells were incubated in the presence of the monoclonal antibodies (10 μg/mL) for 1 hour at 37°C before stimulation with poly(I:C) (1 μg/mL) for 24 hours; the secretion of MMP-1 and MMP-3 in culture supernatants was then measured with immunoblot analysis. 
ELISA for IL-1β
Cells in 24-well culture plates were incubated with MEM containing poly(I:C) for 24 hours, after which the concentration of interleukin-1β (IL-1β) in the culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA) with commercially available antibodies (R&D Systems). 
RT and Real-Time PCR Analysis
Cells in 60-mm culture dishes were incubated for 24 hours in MEM containing poly(I:C), after which total RNA was extracted from the cells with the use of the Trizol reagent and was subjected to RT. The resultant cDNA was subjected to real-time polymerase chain reaction (PCR) analysis with a thermocycler (LightCycler; Roche Molecular Biochemicals, Indianapolis, IN) and with primers specific for MMP-1, MMP-3, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as previously described. 20,21 Transcripts of the constitutively expressed gene for GAPDH served to normalize the amounts of MMP-1 and MMP-3 mRNAs in each sample. Real-time PCR data were analyzed with the thermocycler software (LightCycler version 3.1;Roche Molecular Biochemicals). 
Gelatin Zymography
Cells in 24-well plates were incubated with MEM containing poly(I:C) for 24 hours, after which the culture supernatants were collected and subjected to gelatin zymography as previously described. 22 In brief, culture supernatants were mixed with nonreducing SDS sample buffer (125 mM Tris-HCl [pH 6.8], 20% glycerol, 2% SDS, 0.002% bromophenol blue) and fractionated by SDS-polyacrylamide gel electrophoresis at 4°C on a 10% gel containing 0.1% gelatin. The gel was then washed with 2.5% Triton X-100 for 1 hour to promote the recovery of protease activity before incubation for 18 hours at 37°C in a reaction buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM CaCl2, and 1% Triton X-100. The gel was finally stained with Coomassie brilliant blue. 
Statistical Analysis
Quantitative data are presented as mean ± SE. Differences were analyzed by Dunnett's multiple comparison test or Student's unpaired t-test. P <0.05 was considered statistically significant. 
Results
Effect of Poly(I:C) on MMP-1 Expression in Human Corneal Fibroblasts
We first examined the effect of poly(I:C) on the expression of MMP-1 in human corneal fibroblasts. Immunoblot analysis of culture supernatants revealed that exposure of the cells to poly(I:C) (0–10 μg/mL) for 24 hours resulted in a concentration-dependent increase in the amount of proMMP-1, with this effect maximal at poly(I:C) concentrations of ≥0.3 μg/mL (Fig. 1A). This effect of poly(I:C) (1 μg/mL) was also time dependent and was first apparent at 6 hours (Fig. 1B). Quantitative RT-PCR analysis further revealed that exposure of human corneal fibroblasts to poly(I:C) for 24 hours resulted in a concentration-dependent increase in the amount of MMP-1 mRNA, with this effect significant at poly(I:C) concentrations of ≥1 μg/mL (Fig. 1C). 
Figure 1.
 
Effect of poly(I:C) on the expression of MMP-1 in human corneal fibroblasts. (A, B) Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C) (A) or for the indicated times with poly(I:C) at 1 μg/mL (B), after which the culture supernatants were collected and subjected to immunoblot analysis with antibodies to MMP-1. The position of the band corresponding to proMMP-1 is indicated. Data are representative of three independent experiments. (C) Cells were cultured as in (A), after which total RNA was isolated and subjected to RT and real-time PCR analysis of MMP-1 mRNA. Data were normalized by the abundance of GAPDH mRNA, are expressed in arbitrary units, and are mean ± SE from three separate experiments. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without poly(I:C).
Figure 1.
 
Effect of poly(I:C) on the expression of MMP-1 in human corneal fibroblasts. (A, B) Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C) (A) or for the indicated times with poly(I:C) at 1 μg/mL (B), after which the culture supernatants were collected and subjected to immunoblot analysis with antibodies to MMP-1. The position of the band corresponding to proMMP-1 is indicated. Data are representative of three independent experiments. (C) Cells were cultured as in (A), after which total RNA was isolated and subjected to RT and real-time PCR analysis of MMP-1 mRNA. Data were normalized by the abundance of GAPDH mRNA, are expressed in arbitrary units, and are mean ± SE from three separate experiments. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without poly(I:C).
Effect of Poly(I:C) on MMP-3 Expression in Human Corneal Fibroblasts
Immunoblot analysis showed that incubation of human corneal fibroblasts for 24 hours with poly(I:C) (0–10 μg/mL) resulted in a concentration-dependent increase in the amount of proMMP-3 in culture supernatants, with this effect maximal at poly(I:C) concentrations of ≥1 μg/mL (Fig. 2A). This effect of poly(I:C) (1 μg/mL) was also time dependent and was first apparent at 6 hours (Fig. 2B). RT and real-time PCR analysis revealed that exposure of the cells to poly(I:C) for 24 hours increased the amount of MMP-3 mRNA in a concentration-dependent manner, with this effect significant at poly(I:C) concentrations of ≥0.3 μg/mL (Fig. 2C). 
Figure 2.
 
Effect of poly(I:C) on the expression of MMP-3 in human corneal fibroblasts. (A, B) Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C) (A) or for the indicated times with poly(I:C) at 1 μg/mL (B), after which the culture supernatants were collected and subjected to immunoblot analysis with antibodies to MMP-3. The position of the band corresponding to proMMP-3 is indicated. Data are representative of three independent experiments. (C) Cells were cultured as in (A), after which total RNA was isolated and subjected to RT and real-time PCR analysis of MMP-3 mRNA. Data were normalized by the abundance of GAPDH mRNA, are expressed in arbitrary units, and are mean ± SE from three separate experiments. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without poly(I:C).
Figure 2.
 
Effect of poly(I:C) on the expression of MMP-3 in human corneal fibroblasts. (A, B) Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C) (A) or for the indicated times with poly(I:C) at 1 μg/mL (B), after which the culture supernatants were collected and subjected to immunoblot analysis with antibodies to MMP-3. The position of the band corresponding to proMMP-3 is indicated. Data are representative of three independent experiments. (C) Cells were cultured as in (A), after which total RNA was isolated and subjected to RT and real-time PCR analysis of MMP-3 mRNA. Data were normalized by the abundance of GAPDH mRNA, are expressed in arbitrary units, and are mean ± SE from three separate experiments. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without poly(I:C).
Effects of Poly(I:C) on the Expression of MMP-2 and MMP-9 in Human Corneal Fibroblasts
We next examined the effects of poly(I:C) on the expression of MMP-2 and MMP-9 in human corneal fibroblasts. Immunoblot analysis (Fig. 3A) and gelatin zymography (Fig. 3B) showed that poly(I:C) had no effect on the abundance or activity of proMMP-2 in culture supernatants. Similar analyses revealed that proMMP-9 protein and activity were virtually undetectable in the culture supernatants of cells incubated in the absence or presence of poly(I:C) (Fig. 3). 
Figure 3.
 
Effects of poly(I:C) on the expression of MMP-2 and MMP-9 in human corneal fibroblasts. Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C), after which the culture supernatants were collected and subjected either to immunoblot analysis with antibodies to MMP-2 and to MMP-9 (A) or to gelatin zymography (B). Positions of the bands corresponding to proMMP-2 and proMMP-9 are indicated. Data are representative of three independent experiments.
Figure 3.
 
Effects of poly(I:C) on the expression of MMP-2 and MMP-9 in human corneal fibroblasts. Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C), after which the culture supernatants were collected and subjected either to immunoblot analysis with antibodies to MMP-2 and to MMP-9 (A) or to gelatin zymography (B). Positions of the bands corresponding to proMMP-2 and proMMP-9 are indicated. Data are representative of three independent experiments.
Effect of Poly(I:C) on the Expression of MMP-1 and MMP-3 through TLR3 in Corneal Fibroblasts
We next examined the role of TLR3 about the expression of MMP-1 and MMP-3 induced by poly(I:C) on corneal fibroblasts by using blocking antibodies to TLR3. Immunoblot analysis revealed that blocking antibodies to TLR3 inhibited poly(I:C)-induced expression of MMP-1 and MMP-3, whereas control mouse IgG had no such effect (Fig. 4). 
Figure 4.
 
Effect of poly(I:C) on the effect of blocking antibodies to TLR3 on poly(I:C)-induced MMP-1 and MMP-3 expression in corneal fibroblasts. Cells were cultured for 1 hour in the absence or presence of blocking antibodies to TLR3 (TLR3Ab) or control mouse IgG at 10 μg/mL and then incubated for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL). Culture supernatants were then collected and subjected to immunoblot analysis. Data are representative of three independent experiments.
Figure 4.
 
Effect of poly(I:C) on the effect of blocking antibodies to TLR3 on poly(I:C)-induced MMP-1 and MMP-3 expression in corneal fibroblasts. Cells were cultured for 1 hour in the absence or presence of blocking antibodies to TLR3 (TLR3Ab) or control mouse IgG at 10 μg/mL and then incubated for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL). Culture supernatants were then collected and subjected to immunoblot analysis. Data are representative of three independent experiments.
Role of the NF-κB Signaling Pathway in Upregulation of MMP-1 and MMP-3 Expression by Poly(I:C)
We investigated whether the nuclear factor-κB (NF-κB) signaling pathway might contribute to the upregulation of MMP-1 and MMP-3 expression in human corneal fibroblasts by poly(I:C). Immunoblot analysis showed that poly(I:C) induced the phosphorylation of the NF-κB inhibitor protein IκB-α in the corneal fibroblasts (Fig. 5A). The effect of poly(I:C) on IκB-α phosphorylation was biphasic, with peaks at 2 and 12 hours. Immunoblot analysis also revealed that exposure of the cells to a synthetic inhibitor of IκB kinase-2 (IKK-2) for 1 hour before incubation with poly(I:C) (1 μg/mL) for 24 hours blocked the upregulation of proMMP-1 and MMP-3 in a concentration-dependent manner, with these effects maximal at inhibitor concentrations of ≥0.1 μM (Fig. 5B). The inhibitor also blocked the poly(I:C)-induced increase in the abundance of MMP-1 and MMP-3 mRNAs (Fig. 5C). 
Figure 5.
 
Role of NF-κB in the poly(I:C)-induced upregulation of MMP-1 and MMP-3 expression in human corneal fibroblasts. (A) Cells were incubated with 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-) and to β-actin. (B) Cells were incubated for 1 hour in the presence of the indicated concentrations of IKK-2 inhibitor and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which the culture supernatants were collected and subjected to immunoblot analysis with antibodies to MMP-1 and to MMP-3. (A, B) Data are representative of three independent experiments. (C) Cells were incubated for 1 hour in the absence or presence of the IKK-2 inhibitor (1 μM) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which total RNA was isolated and subjected to RT and real-time PCR analysis of MMP-1 and MMP-3 mRNAs. Data were normalized by the abundance of GAPDH mRNA, are expressed in arbitrary units, and are mean ± SE of three separate experiments. *P < 0.05 (Student's t-test).
Figure 5.
 
Role of NF-κB in the poly(I:C)-induced upregulation of MMP-1 and MMP-3 expression in human corneal fibroblasts. (A) Cells were incubated with 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-) and to β-actin. (B) Cells were incubated for 1 hour in the presence of the indicated concentrations of IKK-2 inhibitor and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which the culture supernatants were collected and subjected to immunoblot analysis with antibodies to MMP-1 and to MMP-3. (A, B) Data are representative of three independent experiments. (C) Cells were incubated for 1 hour in the absence or presence of the IKK-2 inhibitor (1 μM) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which total RNA was isolated and subjected to RT and real-time PCR analysis of MMP-1 and MMP-3 mRNAs. Data were normalized by the abundance of GAPDH mRNA, are expressed in arbitrary units, and are mean ± SE of three separate experiments. *P < 0.05 (Student's t-test).
Role of IL-1β in the Upregulation of MMP-1 and MMP-3 Expression by Poly(I:C)
To investigate the possible role of IL-1β in the poly(I:C)-induced expression of MMP-1 and MMP-3 in human corneal fibroblasts, we first examined the effect of poly(I:C) on IL-1β release from these cells. ELISA revealed that poly(I:C) induced a concentration-dependent increase in the release of IL-1β from corneal fibroblasts, with this effect significant at poly(I:C) concentrations of ≥0.3 μg/mL (Fig. 6). To determine whether the IL-1β released by the cells in response to poly(I:C) might be responsible for the upregulation of MMP-1 and MMP-3 expression by poly(I:C), we examined the effects of IL-1ra. Incubation of corneal fibroblasts with the IL-1 receptor antagonist (0.3 ng/mL) for 1 hour before exposure to poly(I:C) (1 μg/mL) for 24 hours inhibited the upregulation of MMP-1 and MMP-3 expression at both protein (Fig. 7A) and mRNA (Fig. 7B) levels. Finally, we found that IL-1ra did not inhibit the initial phosphorylation of IκB-α apparent at 1 to 2 hours after exposure to poly(I:C), but it did inhibit the second phase of IκB-α phosphorylation apparent at 12 hours (Fig. 8). 
Figure 6.
 
Effect of poly(I:C) on IL-1β release in human corneal fibroblasts. Cells were incubated with the indicated concentrations of poly(I:C) for 24 hours, after which the culture supernatants were collected and assayed for IL-1β by ELISA. Data are mean ± SE from three separate experiments. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without poly(I:C).
Figure 6.
 
Effect of poly(I:C) on IL-1β release in human corneal fibroblasts. Cells were incubated with the indicated concentrations of poly(I:C) for 24 hours, after which the culture supernatants were collected and assayed for IL-1β by ELISA. Data are mean ± SE from three separate experiments. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without poly(I:C).
Figure 7.
 
Effects of IL-1ra on the poly(I:C)-induced expression of MMP-1 and MMP-3 in human corneal fibroblasts. (A) Cells were incubated for 1 hour in the absence or presence of IL-1ra (0.3 ng/mL) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which the culture supernatants were collected and subjected to immunoblot analysis with antibodies to MMP-1 and to MMP-3. Data are representative of three independent experiments. (B) Cells were incubated for 1 hour in the absence or presence of IL-1ra (0.3 ng/mL) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which total RNA was isolated and subjected to RT and real-time PCR analysis of MMP-1 and MMP-3 mRNAs. Data were normalized by the abundance of GAPDH mRNA, are expressed in arbitrary units, and are mean ± SE from three separate experiments. *P < 0.05 (Student's t-test)
Figure 7.
 
Effects of IL-1ra on the poly(I:C)-induced expression of MMP-1 and MMP-3 in human corneal fibroblasts. (A) Cells were incubated for 1 hour in the absence or presence of IL-1ra (0.3 ng/mL) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which the culture supernatants were collected and subjected to immunoblot analysis with antibodies to MMP-1 and to MMP-3. Data are representative of three independent experiments. (B) Cells were incubated for 1 hour in the absence or presence of IL-1ra (0.3 ng/mL) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which total RNA was isolated and subjected to RT and real-time PCR analysis of MMP-1 and MMP-3 mRNAs. Data were normalized by the abundance of GAPDH mRNA, are expressed in arbitrary units, and are mean ± SE from three separate experiments. *P < 0.05 (Student's t-test)
Figure 8.
 
Effect of IL-1ra on poly(I:C)-induced IκB-α phosphorylation in human corneal fibroblasts. Cells were incubated in the absence or presence of IL-1ra (0.3 ng/mL) for 1 hour and then in the additional 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 IκB-α and to β-actin. Data are representative of three independent experiments.
Figure 8.
 
Effect of IL-1ra on poly(I:C)-induced IκB-α phosphorylation in human corneal fibroblasts. Cells were incubated in the absence or presence of IL-1ra (0.3 ng/mL) for 1 hour and then in the additional 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 IκB-α and to β-actin. Data are representative of three independent experiments.
Discussion
We have shown that poly(I:C), a synthetic analog of viral double-stranded RNA, increased the expression of MMP-1 and MMP-3 in cultured human corneal fibroblasts at both the mRNA and protein levels. In contrast, the expression of MMP-2 and MMP-9 in these cells was not affected by poly(I:C). Poly(I:C) also induced the phosphorylation of IκB-α in corneal fibroblasts, and the poly(I:C)-induced expression of MMP-1 and MMP-3 was attenuated by an IKK-2 inhibitor that blocks activation of the NF-κB signaling pathway. Finally, poly(I:C) induced the release of IL-1β from human corneal fibroblasts, and IL-1ra inhibited both the upregulation of MMP-1 and MMP-3 and a second phase of IκB-α phosphorylation induced by poly(I:C) in these cells. These results thus suggest that poly(I:C) upregulates the expression of MMP-1 and MMP-3 in human corneal fibroblasts in a manner dependent on NF-κB activation and, at least in part, on IL-1β secretion. 
Viral keratitis is caused by ocular infection with various viruses, including herpes simplex virus, herpes zoster virus, and adenovirus. The various clinical manifestations of viral keratitis include epithelial keratitis, necrotizing infectious stromal keratitis, stromal keratitis, and endothelitis. However, the pathophysiological mechanisms of these conditions remain poorly understood. Viral infection of the cornea can lead to corneal ulceration, which itself results from the destruction of collagen fibrils in the corneal stroma by proteolytic enzymes. MMPs are responsible in large part for the turnover and degradation of matrix components and are implicated in the pathogenesis of several diseases. 2325 The expression of collagenolytic and gelatinolytic MMPs is increased in bacterial corneal ulcers of mouse and rabbit. 26,27 Little is known about the pattern of MMP expression during viral keratitis, however. We have now shown that poly(I:C) increased the release of proMMP-1 and proMMP-3 from cultured human corneal fibroblasts. The activated corneal fibroblasts surrounding lesions of viral keratitis may contribute to the production of MMPs, given that cell density has been shown to affect the expression of these enzymes. 28 It is thus possible that corneal fibroblasts are the primary mediators of collagen degradation in corneal ulceration associated with viral keratitis. The degradation of extracellular matrix components at the corneal basement membrane, in addition to those in the corneal stroma, by MMPs produced by corneal fibroblasts may further contribute to the progression of corneal ulceration. 
Whereas the release of proMMP-1 and proMMP-3 from cultured human corneal fibroblasts was promoted by poly(I:C), the active (mature) forms of these MMPs were not detected. Immunoblot analysis also failed to detect the endogenous MMP inhibitors TIMP1 and TIMP2 in the culture supernatants of cells exposed to poly(I:C) (data not shown). We previously showed that plasminogen, the precursor of the fibrinolytic protease plasmin, is required for collagen degradation by cultured corneal fibroblasts. 14 Both plasmin and plasminogen activator are present in the cornea and tear fluid, 2931 and the plasminogen activator–plasmin system is implicated in the activation of MMPs. 32 These observations thus suggest that the plasminogen activator–plasmin system may be necessary for the activation of proMMPs secreted by corneal fibroblasts. 
The induction of cytokines, chemokines, and adhesion molecules in resident corneal cells in response to corneal infection or inflammation has been implicated in the pathogenesis of various corneal diseases. We have previously shown that corneal fibroblasts produce such proteins in response to stimulation with poly(I:C). 33 MMPs are secreted by various cell types in response to cytokines and growth factors. The proinflammatory cytokine IL-1β has also been shown to induce collagen degradation by rabbit corneal fibroblasts. 15 Moreover, IL-1β induces the synthesis of MMPs in corneal fibroblasts and other cell types. 3436 We have now shown that poly(I:C) induced not only the production of proMMP-1 and proMMP-3 but also the release of IL-1β by human corneal fibroblasts. Moreover, IL-1ra inhibited the upregulation of MMP-1 and MMP-3 expression induced in these cells by poly(I:C). These results suggest that the poly(I:C)-induced secretion of IL-1β by corneal fibroblasts may contribute to the upregulation of MMP expression induced by poly(I:C). IL-1β is produced not only by fibroblasts but also by infiltrated neutrophils. 3739 An additional supply of IL-1β from infiltrated neutrophils may thus modulate the synthesis of MMPs in corneal fibroblasts during viral corneal ulceration. We previously showed that neutrophils stimulate collagen degradation mediated by corneal fibroblasts. 15 Interactions of inflammatory cells with resident corneal fibroblasts thus likely play an important role in the inflammatory response of the stroma to viral infection. 
Epithelial cells also secrete cytokines and chemokines in response to viral or bacterial infection, resulting in upregulation of the expression of MMPs. 4042 The production of MMP-9 is induced in corneal epithelial cells by proinflammatory cytokines. 43 Prostaglandins also induce MMP expression in epithelial cells. 44 Poly(I:C) stimulates the production of cytokines and chemokines in corneal epithelial cells. 45,46 These various observations suggest that the corneal epithelium also contributes to the production of MMPs, either directly or indirectly, and that stromal-epithelial interactions play an important role in corneal ulceration. 
The induction of MMP expression by poly(I:C) has been demonstrated in other cell types, including synovial fibroblasts and lung epithelial cells. 47,48 Poly(I:C) was thus found to increase the expression of MMP-3 and MMP-13 in synovial fibroblasts. 48 We have now shown that poly(I:C) increased the expression of MMP-1 and MMP-3 but not of MMP-13 (data not shown) in human corneal fibroblasts. MMP-3 expression may thus be a common target of poly(I:C) in fibroblasts. Additional studies with physiological models may provide further insight into the role of viral double-stranded RNA, or poly(I:C), in corneal ulceration associated with the response of the corneal stroma to viral infection. 
Poly(I:C) activates several signaling pathways, including those mediated by mitogen-activated protein kinases (MAPKs) and NF-κB, in various cell types. 33,49,50 Indeed, the upregulation of cytokine and chemokine expression by poly(I:C) is mediated by such signaling pathways. 33,51,52 In the present study, poly(I:C) appeared to induce biphasic activation of the NF-κB signaling pathway in human corneal fibroblasts. The late phase (∼12 hours) of NF-κB activation appeared to be mediated by IL-1β secreted from the cells in response to poly(I:C) stimulation. The expression of MMP-1 and MMP-3 induced by poly(I:C) seemed to be dependent in part on this late phase of NF-κB activation as it was inhibited by IL-1ra. The inhibitory effect of IL-1ra on the induction of these MMPs, however, was less pronounced than that of the IKK-2 inhibitor. Whereas IL-1β activates the NF-κB signaling pathway, NF-κB also contributes to the expression of IL-1β, 53 giving rise to the operation of a positive feedback loop. 
In summary, we have shown that poly(I:C) induced the upregulation of MMP-1 and MMP-3 expression through activation of the NF-κB signaling pathway in human corneal fibroblasts. Poly(I:C) also induced the secretion of IL-1β from the cells, which in turn triggered further NF-κB activation and MMP expression. Our results therefore suggest that corneal fibroblasts may play an important role in collagen degradation during corneal ulceration associated with viral keratitis. 
Footnotes
 Supported in part by Grant 19791271 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Footnotes
 Disclosure: K. Kimura, None; T. Orita, None; Y. Kondo, None; H. Zhou, None; T. Nishida, None
The authors thank Yasumiko Akamatsu, Shizuka Murata, and Yukari Mizuno for technical assistance. 
References
Maertzdorf J Osterhaus AD Verjans GM . IL-17 expression in human herpetic stromal keratitis: modulatory effects on chemokine production by corneal fibroblasts. J Immunol. 2002;169:5897–5903. [CrossRef] [PubMed]
Duan R Remeijer L van Dun JM Osterhaus AD Verjans GM . Granulocyte macrophage colony-stimulating factor expression in human herpetic stromal keratitis: implications for the role of neutrophils in HSK. Invest Ophthalmol Vis Sci. 2007;48:277–284. [CrossRef] [PubMed]
Imanishi J . Expression of cytokines in bacterial and viral infections and their biochemical aspects. J Biochem. 2000;127:525–530. [CrossRef] [PubMed]
Lee S Zheng M Kim B Rouse BT . Role of matrix metalloproteinase-9 in angiogenesis caused by ocular infection with herpes simplex virus. J Clin Invest. 2002;110:1105–1111. [CrossRef] [PubMed]
Lundberg P Cantin E . A potential role for CXCR3 chemokines in the response to ocular HSV infection. Curr Eye Res. 2003;26:137–150. [CrossRef] [PubMed]
Kumagai N Fukuda K Ishimura Y Nishida T . Synergistic induction of eotaxin expression in human keratocytes by TNF-α and IL-4 or IL-13. Invest Ophthalmol Vis Sci. 2000;41:1448–1453. [PubMed]
Lu Y Liu Y Fukuda K Nakamura Y Kumagai N Nishida T . Inhibition by triptolide of chemokine, proinflammatory cytokine, and adhesion molecule expression induced by lipopolysaccharide in corneal fibroblasts. Invest Ophthalmol Vis Sci. 2006;47:3796–3800. [CrossRef] [PubMed]
Li YC Wu JJ Bi JJ Yang T Yang GH Wang BQ . [Influence of Wnt-1 recombinant adenovirus on differentiation of human epidermal stem cells.] Zhonghua Shao Shang Za Zhi. 2008;24:187–190. [PubMed]
Folgueras AR Pendas AM Sanchez LM Lopez-Otin C . Matrix metalloproteinases in cancer: from new functions to improved inhibition strategies. Int J Dev Biol. 2004;48:411–424. [CrossRef] [PubMed]
Gueders MM Foidart JM Noel A Cataldo DD . Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs in the respiratory tract: potential implications in asthma and other lung diseases. Eur J Pharmacol. 2006;533:133–144. [CrossRef] [PubMed]
Girard MT Matsubara M Kublin C Tessier MJ Cintron C Fini ME . Stromal fibroblasts synthesize collagenase and stromelysin during long-term tissue remodeling. J Cell Sci. 1993;104:1001–1011. [PubMed]
Fini ME Girard MT Matsubara M Bartlett JD . Unique regulation of the matrix metalloproteinase, gelatinase B. Invest Ophthalmol Vis Sci. 1995;36:622–633. [PubMed]
Sivak JM Fini ME . MMPs in the eye: emerging roles for matrix metalloproteinases in ocular physiology. Prog Retinal Eye Res. 2002;21:1–14. [CrossRef]
Hao JL Nagano T Nakamura M Kumagai N Mishima H Nishida T . Galardin inhibits collagen degradation by rabbit keratocytes by inhibiting the activation of pro-matrix metalloproteinases. Exp Eye Res. 1999;68:565–572. [CrossRef] [PubMed]
Li Q Fukuda K Lu Y . Enhancement by neutrophils of collagen degradation by corneal fibroblasts. J Leukoc Biol. 2003;74:412–419. [CrossRef] [PubMed]
Fini ME Girard MT Matsubara M . Collagenolytic/gelatinolytic enzymes in corneal wound healing. Acta Ophthalmol. 1992;202:26–33.
O'Brien TP Li QJ Sauerburger F Reviglio VE Rana T Ashraf MF . The role of matrix metalloproteinases in ulcerative keratolysis associated with perioperative diclofenac use. Ophthalmology. 2001;108:656–659. [CrossRef] [PubMed]
Fini ME Cook JR Mohan R . Proteolytic mechanisms in corneal ulceration and repair. Arch Dermatol Res. 1998;290(suppl):S12–S23. [CrossRef] [PubMed]
Tamesis RR Messmer EM Rice BA Dutt JE Foster CS . The role of natural killer cells in the development of herpes simplex virus type 1 induced stromal keratitis in mice. Eye. 1994;8:298–306. [CrossRef] [PubMed]
Leonardi A Cortivo R Fregona I Plebani M Secchi AG Abatangelo G . Effects of Th2 cytokines on expression of collagen, MMP-1, and TIMP-1 in conjunctival fibroblasts. Invest Ophthalmol Vis Sci. 2003;44:183–189. [CrossRef] [PubMed]
Fukuda K Fujitsu Y Kumagai N Nishida T . Inhibition of matrix metalloproteinase-3 synthesis in human conjunctival fibroblasts by interleukin-4 or interleukin-13. Invest Ophthalmol Vis Sci. 2006;47:2857–2864. [CrossRef] [PubMed]
Kumagai N Yamamoto K Fukuda K . Active matrix metalloproteinases in the tear fluid of individuals with vernal keratoconjunctivitis. J Allergy Clin Immunol. 2002;110:489–491. [CrossRef] [PubMed]
Deryugina EI Quigley JP . Matrix metalloproteinases and tumor metastasis. Cancer Metast Rev. 2006;25:9–34. [CrossRef]
Martin MD Matrisian LM . The other side of MMPs: protective roles in tumor progression. Cancer Metast Rev. 2007;26:717–724. [CrossRef]
Graham HK Horn M Trafford AW . Extracellular matrix profiles in the progression to heart failure: European Young Physiologists Symposium Keynote Lecture—Bratislava 2007. Acta Physiol (Oxf). 2008;194:3–21. [CrossRef] [PubMed]
Miyajima S Akaike T Matsumoto K . Matrix metalloproteinases induction by pseudomonal virulence factors and inflammatory cytokines in vitro. Microb Pathogen. 2001;31:271–281. [CrossRef]
Xue ML Wakefield D Willcox MD . Regulation of MMPs and TIMPs by IL-1β during corneal ulceration and infection. Invest Ophthalmol Vis Sci. 2003;44:2020–2025. [CrossRef] [PubMed]
Bachmeier BE Vene R Iancu CM . Transcriptional control of cell density dependent regulation of matrix metalloproteinase and TIMP expression in breast cancer cell lines. Thromb Haemost. 2005;93:761–769. [PubMed]
Berman M Leary R Gage J . Evidence for a role of the plasminogen activator–plasmin system in corneal ulceration. Invest Ophthalmol Vis Sci. 1980;19:1204–1221. [PubMed]
Thorig L Wijngaards G Van Haeringen NJ . Immunological characterization and possible origin of plasminogen activator in human tear fluid. Ophthalmic Res. 1983;15:268–276. [CrossRef] [PubMed]
Tervo T Salonen EM Vahen A . Elevation of tear fluid plasmin in corneal disease. Acta Ophthalmol. 1988;66:393–399. [CrossRef]
Lijnen HR . Extracellular proteolysis in the development and progression of atherosclerosis. Biochem Soc Trans. 2002;30:163–167. [CrossRef] [PubMed]
Liu Y Kimura K Yanai R Chikama T Nishida T . Cytokine, chemokine, and adhesion molecule expression mediated by MAPKs in human corneal fibroblasts exposed to poly(I:C). Invest Ophthalmol Vis Sci. 2008;49:3336–3344. [CrossRef] [PubMed]
Girard MT Matsubara M Fini ME . Transforming growth factor-β and interleukin-1 modulate metalloproteinase expression by corneal stromal cells. Invest Ophthalmol Vis Sci. 1991;32:2441–2454. [PubMed]
Sodin-Semrl S Taddeo B Tseng D Varga J Fiore S . Lipoxin A4 inhibits IL-1β-induced IL-6, IL-8, and matrix metalloproteinase-3 production in human synovial fibroblasts and enhances synthesis of tissue inhibitors of metalloproteinases. J Immunol. 2000;164:2660–2666. [CrossRef] [PubMed]
Han YP Downey S Garner WL . Interleukin-1α-induced proteolytic activation of metalloproteinase-9 by human skin. Surgery. 2005;138:932–939. [CrossRef] [PubMed]
Dularay B Westacott CI Elson CJ . IL-1 secreting cell assay and its application to cells from patients with rheumatoid arthritis. Br J Rheumatol. 1992;31:19–24. [CrossRef] [PubMed]
Re F Mengozzi M Muzio M Dinarello CA Mantovani A Colotta F . Expression of interleukin-1 receptor antagonist (IL-1ra) by human circulating polymorphonuclear cells. Eur J Immunol. 1993;23:570–573. [CrossRef] [PubMed]
Keller JF Carrouel F Colomb E . Toll-like receptor 2 activation by lipoteichoic acid induces differential production of pro-inflammatory cytokines in human odontoblasts, dental pulp fibroblasts and immature dendritic cells. Immunobiology. 2010;1:53–59. [CrossRef]
Lu J Chua HH Chen SY Chen JY Tsai CH . Regulation of matrix metalloproteinase-1 by Epstein-Barr virus proteins. Cancer Res. 2003;63:256–262. [PubMed]
Elkington PT Emerson JE Lopez-Pascua LD . Mycobacterium tuberculosis up-regulates matrix metalloproteinase-1 secretion from human airway epithelial cells via a p38 MAPK switch. J Immunol. 2005;175:5333–5340. [CrossRef] [PubMed]
Gursoy UK Kononen E Uitto VJ . Stimulation of epithelial cell matrix metalloproteinase (MMP-2, -9, -13) and interleukin-8 secretion by fusobacteria. Oral Microbiol Immunol. 2008;23:432–434. [CrossRef] [PubMed]
Li DQ Lokeshwar BL Solomon A Monroy D Ji Z Pflugfelder SC . Regulation of MMP-9 production by human corneal epithelial cells. Exp Eye Res. 2001;73:449–459. [CrossRef] [PubMed]
Pillinger MH Marjanovic N Kim SY . Matrix metalloproteinase secretion by gastric epithelial cells is regulated by E prostaglandins and MAPKs. J Biol Chem. 2005;280:9973–9979. [CrossRef] [PubMed]
Ueta M Hamuro J Kiyono H Kinoshita S . Triggering of TLR3 by polyI:C in human corneal epithelial cells to induce inflammatory cytokines. Biochem Biophys Res Commun. 2005;331:285–294. [CrossRef] [PubMed]
Kumar A Zhang J Yu FS . Toll-like receptor 3 agonist poly(I:C)-induced antiviral response in human corneal epithelial cells. Immunology. 2006;117:11–21. [CrossRef] [PubMed]
Ritter M Mennerich D Weith A Seither P . Characterization of Toll-like receptors in primary lung epithelial cells: strong impact of the TLR3 ligand poly(I:C) on the regulation of Toll-like receptors, adaptor proteins and inflammatory response. J Inflamm (Lond). 2005;2:16. [CrossRef] [PubMed]
Ospelt C Brentano F Rengel Y . Overexpression of toll-like receptors 3 and 4 in synovial tissue from patients with early rheumatoid arthritis: toll-like receptor expression in early and longstanding arthritis. Arthritis Rheum. 2008;58:3684–3692. [CrossRef] [PubMed]
Steer SA Moran JM Christmann BS Maggi LBJr Corbett JA . Role of MAPK in the regulation of double-stranded RNA- and encephalomyocarditis virus-induced cyclooxygenase-2 expression by macrophages. J Immunol. 2006;177:3413–3420. [CrossRef] [PubMed]
Zhao Y Rivieccio MA Lutz S Scemes E Brosnan CF . The TLR3 ligand polyI:C downregulates connexin 43 expression and function in astrocytes by a mechanism involving the NF-κB and PI3 kinase pathways. Glia. 2006;54:775–785. [CrossRef] [PubMed]
Maire M Parent R Morand AL . Characterization of the double-stranded RNA responses in human liver progenitor cells. Biochem Biophysical Res Commun. 2008;368:556–562. [CrossRef]
Berube J Bourdon C Yao Y Rousseau S . Distinct intracellular signaling pathways control the synthesis of IL-8 and RANTES in TLR1/TLR2, TLR3 or NOD1 activated human airway epithelial cells. Cell Signal. 2009;21:448–456. [CrossRef] [PubMed]
Son CG Shin JW Cho JH Cho CK Yun CH Han SH . Induction of murine interleukin-1β expression by water-soluble components from Hericium erinaceum . Acta Pharmacol Sinica. 2006;27:1058–1064. [CrossRef]
Figure 1.
 
Effect of poly(I:C) on the expression of MMP-1 in human corneal fibroblasts. (A, B) Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C) (A) or for the indicated times with poly(I:C) at 1 μg/mL (B), after which the culture supernatants were collected and subjected to immunoblot analysis with antibodies to MMP-1. The position of the band corresponding to proMMP-1 is indicated. Data are representative of three independent experiments. (C) Cells were cultured as in (A), after which total RNA was isolated and subjected to RT and real-time PCR analysis of MMP-1 mRNA. Data were normalized by the abundance of GAPDH mRNA, are expressed in arbitrary units, and are mean ± SE from three separate experiments. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without poly(I:C).
Figure 1.
 
Effect of poly(I:C) on the expression of MMP-1 in human corneal fibroblasts. (A, B) Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C) (A) or for the indicated times with poly(I:C) at 1 μg/mL (B), after which the culture supernatants were collected and subjected to immunoblot analysis with antibodies to MMP-1. The position of the band corresponding to proMMP-1 is indicated. Data are representative of three independent experiments. (C) Cells were cultured as in (A), after which total RNA was isolated and subjected to RT and real-time PCR analysis of MMP-1 mRNA. Data were normalized by the abundance of GAPDH mRNA, are expressed in arbitrary units, and are mean ± SE from three separate experiments. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without poly(I:C).
Figure 2.
 
Effect of poly(I:C) on the expression of MMP-3 in human corneal fibroblasts. (A, B) Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C) (A) or for the indicated times with poly(I:C) at 1 μg/mL (B), after which the culture supernatants were collected and subjected to immunoblot analysis with antibodies to MMP-3. The position of the band corresponding to proMMP-3 is indicated. Data are representative of three independent experiments. (C) Cells were cultured as in (A), after which total RNA was isolated and subjected to RT and real-time PCR analysis of MMP-3 mRNA. Data were normalized by the abundance of GAPDH mRNA, are expressed in arbitrary units, and are mean ± SE from three separate experiments. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without poly(I:C).
Figure 2.
 
Effect of poly(I:C) on the expression of MMP-3 in human corneal fibroblasts. (A, B) Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C) (A) or for the indicated times with poly(I:C) at 1 μg/mL (B), after which the culture supernatants were collected and subjected to immunoblot analysis with antibodies to MMP-3. The position of the band corresponding to proMMP-3 is indicated. Data are representative of three independent experiments. (C) Cells were cultured as in (A), after which total RNA was isolated and subjected to RT and real-time PCR analysis of MMP-3 mRNA. Data were normalized by the abundance of GAPDH mRNA, are expressed in arbitrary units, and are mean ± SE from three separate experiments. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without poly(I:C).
Figure 3.
 
Effects of poly(I:C) on the expression of MMP-2 and MMP-9 in human corneal fibroblasts. Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C), after which the culture supernatants were collected and subjected either to immunoblot analysis with antibodies to MMP-2 and to MMP-9 (A) or to gelatin zymography (B). Positions of the bands corresponding to proMMP-2 and proMMP-9 are indicated. Data are representative of three independent experiments.
Figure 3.
 
Effects of poly(I:C) on the expression of MMP-2 and MMP-9 in human corneal fibroblasts. Cells were incubated for 24 hours in the presence of the indicated concentrations of poly(I:C), after which the culture supernatants were collected and subjected either to immunoblot analysis with antibodies to MMP-2 and to MMP-9 (A) or to gelatin zymography (B). Positions of the bands corresponding to proMMP-2 and proMMP-9 are indicated. Data are representative of three independent experiments.
Figure 4.
 
Effect of poly(I:C) on the effect of blocking antibodies to TLR3 on poly(I:C)-induced MMP-1 and MMP-3 expression in corneal fibroblasts. Cells were cultured for 1 hour in the absence or presence of blocking antibodies to TLR3 (TLR3Ab) or control mouse IgG at 10 μg/mL and then incubated for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL). Culture supernatants were then collected and subjected to immunoblot analysis. Data are representative of three independent experiments.
Figure 4.
 
Effect of poly(I:C) on the effect of blocking antibodies to TLR3 on poly(I:C)-induced MMP-1 and MMP-3 expression in corneal fibroblasts. Cells were cultured for 1 hour in the absence or presence of blocking antibodies to TLR3 (TLR3Ab) or control mouse IgG at 10 μg/mL and then incubated for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL). Culture supernatants were then collected and subjected to immunoblot analysis. Data are representative of three independent experiments.
Figure 5.
 
Role of NF-κB in the poly(I:C)-induced upregulation of MMP-1 and MMP-3 expression in human corneal fibroblasts. (A) Cells were incubated with 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-) and to β-actin. (B) Cells were incubated for 1 hour in the presence of the indicated concentrations of IKK-2 inhibitor and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which the culture supernatants were collected and subjected to immunoblot analysis with antibodies to MMP-1 and to MMP-3. (A, B) Data are representative of three independent experiments. (C) Cells were incubated for 1 hour in the absence or presence of the IKK-2 inhibitor (1 μM) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which total RNA was isolated and subjected to RT and real-time PCR analysis of MMP-1 and MMP-3 mRNAs. Data were normalized by the abundance of GAPDH mRNA, are expressed in arbitrary units, and are mean ± SE of three separate experiments. *P < 0.05 (Student's t-test).
Figure 5.
 
Role of NF-κB in the poly(I:C)-induced upregulation of MMP-1 and MMP-3 expression in human corneal fibroblasts. (A) Cells were incubated with 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-) and to β-actin. (B) Cells were incubated for 1 hour in the presence of the indicated concentrations of IKK-2 inhibitor and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which the culture supernatants were collected and subjected to immunoblot analysis with antibodies to MMP-1 and to MMP-3. (A, B) Data are representative of three independent experiments. (C) Cells were incubated for 1 hour in the absence or presence of the IKK-2 inhibitor (1 μM) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which total RNA was isolated and subjected to RT and real-time PCR analysis of MMP-1 and MMP-3 mRNAs. Data were normalized by the abundance of GAPDH mRNA, are expressed in arbitrary units, and are mean ± SE of three separate experiments. *P < 0.05 (Student's t-test).
Figure 6.
 
Effect of poly(I:C) on IL-1β release in human corneal fibroblasts. Cells were incubated with the indicated concentrations of poly(I:C) for 24 hours, after which the culture supernatants were collected and assayed for IL-1β by ELISA. Data are mean ± SE from three separate experiments. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without poly(I:C).
Figure 6.
 
Effect of poly(I:C) on IL-1β release in human corneal fibroblasts. Cells were incubated with the indicated concentrations of poly(I:C) for 24 hours, after which the culture supernatants were collected and assayed for IL-1β by ELISA. Data are mean ± SE from three separate experiments. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without poly(I:C).
Figure 7.
 
Effects of IL-1ra on the poly(I:C)-induced expression of MMP-1 and MMP-3 in human corneal fibroblasts. (A) Cells were incubated for 1 hour in the absence or presence of IL-1ra (0.3 ng/mL) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which the culture supernatants were collected and subjected to immunoblot analysis with antibodies to MMP-1 and to MMP-3. Data are representative of three independent experiments. (B) Cells were incubated for 1 hour in the absence or presence of IL-1ra (0.3 ng/mL) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which total RNA was isolated and subjected to RT and real-time PCR analysis of MMP-1 and MMP-3 mRNAs. Data were normalized by the abundance of GAPDH mRNA, are expressed in arbitrary units, and are mean ± SE from three separate experiments. *P < 0.05 (Student's t-test)
Figure 7.
 
Effects of IL-1ra on the poly(I:C)-induced expression of MMP-1 and MMP-3 in human corneal fibroblasts. (A) Cells were incubated for 1 hour in the absence or presence of IL-1ra (0.3 ng/mL) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which the culture supernatants were collected and subjected to immunoblot analysis with antibodies to MMP-1 and to MMP-3. Data are representative of three independent experiments. (B) Cells were incubated for 1 hour in the absence or presence of IL-1ra (0.3 ng/mL) and then for 24 hours in the additional absence or presence of poly(I:C) (1 μg/mL), after which total RNA was isolated and subjected to RT and real-time PCR analysis of MMP-1 and MMP-3 mRNAs. Data were normalized by the abundance of GAPDH mRNA, are expressed in arbitrary units, and are mean ± SE from three separate experiments. *P < 0.05 (Student's t-test)
Figure 8.
 
Effect of IL-1ra on poly(I:C)-induced IκB-α phosphorylation in human corneal fibroblasts. Cells were incubated in the absence or presence of IL-1ra (0.3 ng/mL) for 1 hour and then in the additional 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 IκB-α and to β-actin. Data are representative of three independent experiments.
Figure 8.
 
Effect of IL-1ra on poly(I:C)-induced IκB-α phosphorylation in human corneal fibroblasts. Cells were incubated in the absence or presence of IL-1ra (0.3 ng/mL) for 1 hour and then in the additional 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 IκB-α and to β-actin. Data are representative of three independent experiments.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×