Acanthamoeba Cytopathic Protein Interacts with Phospholipids on Corneal Epithelial Cells and Induces Both Apoptosis and Pro-inflammatory Cytokines Through Cytosolic Phospholipase A2 (cPLA2) Activation

Abstract

Purpose: : Acanthamoeba keratitis is a severe and painful infection of the cornea caused by free-living amoebae. We have identified the interaction of Acanthamoeba castellanii with mannosylated protein on corneal epithelial cells that stimulates trophozoites to secrete a 133-kDa protease, which facilitates corneal invasion and induces apoptosis via the caspase-3 dependant pathway. The mechanism of MIP-133-induced apoptosis is unknown. The aim of this study is to determine if MIP-133 induces apoptosis and pro-inflammatory cytokines/chemokines in corneal epithelial cells via cytosolic phospholipase A₂ (cPLA₂) pathway.

Methods: : A. castellanii trophozoites were grown in peptone-yeast extract glucose with 100 mM methyl-α-D-mannopyranoside for 3-4 days. Supernatants were extracted at mid-log phase. MIP-133 protein was purified and characterized by fast protein liquid chromatography size exclusion and SDS-PAGE. Human corneal epithelial (HCE) and Chinese hamster corneal epithelial (HCORN) cells were incubated with or without MIP-133 at dosages of 7.5 μg/ml, 15 μg/ml and 50 μg/ml for 6 hr, 12 hr and 24 hr. The effect of cPLA₂ inhibitors on apoptosis, cPLA₂ and arachidonic acid release were tested in vitro. Inhibition of cPLA₂ involved pre-incubating the HCE and the HCORN cells for 1 hr with cPLA₂ inhibitors (10 μM of Methyl-arachidonyl fluorophosphonate or 20 μM Arachidonyl trifluoromethyl ketone) with or without 15 μg/ml MIP-133 for 24 hr. Induction of cPLA₂ at the mRNA and enzyme levels was examined by RT-PCR and by cPLA₂ assay kit respectively. Apoptosis of corneal epithelial cells was determined by caspase-3 assay. Expression of IL-8, IL-6, IL-1β and IFN-γ was examined by RT-PCR and ELISA.

Results: : MIP-133 induced significant arachidonic acid and cPLA₂ release from corneal cells while cPLA₂ inhibitors significantly reduced cPLA₂ and arachidonic acid release from these cells (P<0.05). cPLA₂ inhibitors significantly inhibited MIP-133-induced apoptosis in the corneal cells (P<0.05). MIP-133 induced significant (P<0.05) IL-8, IL-6, IL-1β and IFN-γ production in corneal epithelial cells.

Conclusions: : Results suggest that MIP-133 interacts with phospholipids on plasma membrane of HCE/HCORN cells; cPLA₂ is involved in apoptosis and arachidonic acid release from corneal epithelial cells induced by MIP-133. cPLA₂ inhibitors may be a therapeutic target in Acanthamoeba keratitis.

Keywords: Acanthamoeba • injection • amoeba