Elsevier

Experimental Eye Research

Volume 83, Issue 3, September 2006, Pages 526-535
Experimental Eye Research

Corticosteroid and doxycycline suppress MMP-9 and inflammatory cytokine expression, MAPK activation in the corneal epithelium in experimental dry eye

https://doi.org/10.1016/j.exer.2006.02.004Get rights and content

Abstract

We investigated the effects of corticosteroid and doxycycline on expression of matrix metalloproteinase (MMP)-9 and inflammatory cytokines and activation of mitogen-activated protein kinase (MAPK) signaling pathways, c-jun N-terminal kinases (JNK), extracellular-regulated kinases (ERK) and p38, in experimental murine dry eye. Experimental dry eye (EDE) was created in C57BL6 mice, with or without or topical treatment consisting of 1% methylprednisolone, 0.025% doxycycline or balanced salt solution four times per day. MMP-9 expression in the cornea epithelia was evaluated by laser scanning confocal microscopy. Gelatinase activity in the cornea was evaluated by in situ zymography and MMP-9 activity in tear washings was evaluated by gelatin zymography. Total and phosphorylated MAPKs (JNK1/2, ERK1/2, p38) were detected by Luminex immunobead assay. Levels of MMP-9, interleukin (IL)-1α, IL-1β and tumor necrosis factor (TNF)-α RNA transcripts were evaluated by real-time PCR. MMP-9 immunoreactivity was localized to the apical corneal epithelial cell membranes in normal control eyes. Desiccating stress significantly increased production of MMP-9 by the corneal epithelium and increased its activity in the corneal epithelium and tear fluid. Dryness also increased expression of IL-1α, IL-1β and TNF-α mRNA and stimulated phosphorylation of JNK1/2, ERK1/2 and p38 MAPKs in the corneal epithelium. Both methylprednisolone and doxycycline reduced expression and activity of MMP-9, decreased levels of inflammatory cytokines transcripts and reduced activation of MAPKs in the corneal epithelium in response to EDE. Desiccating stress stimulates expression of MMP-9, IL-1α, IL-1β and TNF-α mRNA , as well as activates MAPK signaling pathways in the corneal epithelium. Both corticosteroid and doxycycline suppressed this molecular stress response.

Introduction

Dry eye is a very common disease with a reported prevalence of 5.5–15% depending on the population studied, age and diagnostic criteria used in various epidemiological studies from around the world (Bjerrum, 1997, McCarty et al., 1998, Moss et al., 2000, Schaumberg et al., 2003, Schein et al., 1997). It is characterized by eye irritation symptoms, corneal surface irregularity, blurred and fluctuating vision and increased risk for corneal ulceration (de Paiva et al., 2003, Goto et al., 2002, Musch et al., 1983, Pflugfelder et al., 1998). Chronic dry eye has been demonstrated to cause inflammation, evidenced by an increase in pro-inflammatory cytokines and chemokines in the tear fluid, increased expression of immune activation and adhesion molecules (HLA-DR and intercellular adhesion molecule) by the conjunctival epithelium and an increased number of T lymphocytes in the conjunctiva (Baudouin et al., 2005, Jones et al., 1994, Pflugfelder et al., 1999, Rolando et al., 2005, Solomon et al., 2001, Stern et al., 2002).

A significant increase in the concentration and activity of matrix metalloproteinase 9 (MMP-9) has been reported in the tear fluid of human dry eye patients (Afonso et al., 1999, Sobrin et al., 2000, Solomon et al., 2001) as well as in the corneal epithelium and tear fluid of mice with experimental dry eye (EDE) (Pflugfelder et al., 2005). Hypersomolar stress and the elevated inflammatory cytokines present in dry eye (i.e. interleukin [IL]-1, tumor necrosis factor [TNF]-α and transforming growth factor [TGF]-β) have been found to increase expression of MMP-9 by the corneal epithelium (Li et al., 2002).

The desiccating and hyperosmolar stress in dry eye has been noted to activate mitogen-activated protein kinase (MAPK) intracellular signaling pathways in the corneal epithelium (Li et al., 2002, Luo et al., 2004, Luo et al., 2005). The MAPK cascades are well conserved signaling pathways that include three subtypes: c-jun N-terminal kinases (JNK), extracellular signal regulated kinases (ERK) and p38 MAPK. These kinase signaling pathways regulate the expression of cytokines, MMPs (MMP-9, -1, -3, and -13) and epithelial differentiation proteins (e.g. involucrin) that are involved in the pathogenesis of the ocular surface disease of dry eye (Adhikary et al., 2004, Li et al., 2002, Liacini et al., 2002, Mengshol et al., 2001, Reunanen et al., 2002, Woodgett et al., 1995, Zeigler et al., 1999).

Corticosteroids are global inhibitors of inflammation and have been reported to decrease the production of a number of inflammatory cytokines (IL-1, IL-6, IL-8, TNF-α, GM-CSF) and MMP-9 by the corneal epithelium (Dialilian, 2001). They have been successfully used to treat the corneal epithelial disease in dry eye (Marsh and Pflugfelder, 1999, Pflugfelder et al., 2004, Wachtel et al., 1999).

Doxycycline, a long-acting semisynthetic tetracycline, is well recognized for its therapeutic efficacy in treating MMP mediated ocular surface diseases, such as rosacea, recurrent epithelial erosion and sterile corneal ulceration (Akpek et al., 1997, Dursun et al., 2001, Seedor et al., 1987). Doxycycline has been found to inhibit MMP-9 activity in human corneal epithelial cells (Li et al., 2004), endothelial cells (Hanemaaijer et al., 1998), skin keratinocytes (Uitto et al., 1994) and prostate cancer cells (Lokeshwar et al., 1993, Lokeshwar, 1999).

The purpose of this study was to investigate the effects of two anti-inflammatory agents, with different mechanisms of action, the corticosteroid methylprednisolone and the tetracycline doxycycline on MMP-9 production and the activation of MAPKs in the corneal epithelium in response to experimental dry eye.

Section snippets

Mouse model of dry eye

This research protocol was approved by the Baylor College of Medicine Center for Comparative Medicine and it conformed to the standards in the Association for Research in Vision and Ophthalmology (ARVO) Statement for the use of animals in ophthalmic and vision research.

Experimental dry eye (EDE) was induced in C57BL6 mice, aged 6–8 weeks, by subcutaneous injection of 0.5 mg/0.2 ml scopolamine hydrobromide (Sigma–Aldrich, St. Louis, MO) in alternating hindquarters administered four times a day

Effects of methylprednisolone and doxycycline on corneal permeability in EDE

A hallmark of human dry eye is corneal epithelial disease, characterized by increased permeability to diagnostic dyes, such as sodium fluorescein. Corneal epithelial permeability to the high molecular weight fluorescent molecule OGD (70 kDa) was assessed in the UT and EDE controls and the three treatment groups (Fig. 1). Corneal uptake of OGD was significantly higher in EDE than UT controls, with areas of punctate and confluent dye staining (OGD score (mean ± standard error of mean [SEM] 0.73 ± 0.16

Discussion

We have previously reported that therapy of dry eye, with methylprednisolone and doxycycline was able to preserve the tight junction network and increase corneal smoothness (de Paiva et al., 2005). This study provides insight into the mechanism of action of these two anti-inflammatory agents in the treatment of the corneal epithelial disease. Our results showed that EDE significantly altered corneal barrier function and increased the production and activity of MMP-9, as measured by gelatin

Acknowledgements

Supported by NIH Grants EY11915 (S.C.P.), EY014553 (D.Q.L.), EY07001-29 (C.S.P.), National Eye Institute, an unrestricted grant from Research to Prevent Blindness, The Oshman Foundation, The William Stamps Farish Fund, and an unrestricted grant from Allergan. M.E.S. is an employee of Allergan.

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