Doxycycline inhibition of interleukin-1 in the corneal epithelium

PURPOSE. To evaluate the effect of doxycycline on the regulation of interleukin (IL)-1 expression and activity in human cultured corneal epithelium. METHODS. Human corneal limbal epithelium (HLE) was cultured from explants prepared from limbal rings of donor corneas. Primary cultured limbal epithelial cells were treated with either 10 μg/ml lipopolysaccharide (LPS), LPS with 10 μg/ml doxycycline, or LPS with 0.1 mg/ml methylprednisolone (MP) for 24 hours. The intracellular and supernatant protein amounts of IL-1α, the precursor and mature forms of IL-1β, IL-1 receptor antagonist (IL-1 RA), and the intracellular level of IL-1β-converting enzyme (ICE) were measured with enzyme-linked immunosorbent assays (ELISAs). Western blot analysis was performed to evaluate IL-1 RA protein. mRNA steady state amounts were determined by RNase protection assay (RPA) for IL-1α, IL-1β, IL-1 RA, and ICE. RESULTS. LPS increased the mRNA and protein amounts of intracellular and released IL-1α, mature IL-1β, and IL-1 RA. Doxycycline inhibited the LPS-induced IL-1β increased in the mRNA and protein amounts in the corneal epithelium and upregulated the expression of the anti-inflammatory IL-1 RA protein. In addition, doxycycline reduced the steady state level of the cellular ICE protein but did not affect the level of ICE transcripts. IL-1β secreted to the conditioned media of HLE was functionally active in inducing matrix metalloproteinase (MMP)-1 and MMP-3 in cultured corneal fibroblasts. Doxycycline significantly decreased IL-1β bioactivity in the supernatants from LPS-treated corneal epithelial cultures. These effects were comparable to those induced by the corticosteroid, MP. CONCLUSIONS. Doxycycline can suppress the steady state amounts of mRNA and protein of IL-β and decrease the bioactivity of this major inflammatory cytokine. These data may partially explain the clinically observed anti-inflammatory properties of doxycycline. The observation that doxycycline was equally potent as a corticosteroid, combined with the relative absence of adverse effects, makes it a potent drug for a wide spectrum of ocular surface inflammatory diseases.