Abstract
Purpose: :
The purpose of this study was to determine whether activating the conjunctival macrophage would affect the course of the disease in the Chinese hamster model of Acanthamoeba keratitis.
Methods: :
Chinese hamster spleen cells were stimulated with concanavalin A (Con A) and interferon gamma (IFN–γ) containing supernatants were collected 24 hours later. The IFN–γ containing supernatants were loaded into liposomes, which were administered to peritoneal macrophages in vitro. Macrophage activation was assessed by testing for the production of nitric oxide (NO) using Griess reagents. Conjunctival macrophages were activated in situ by subconjunctival injection of liposomes containing Con A activated spleen cell culture supernatants. Control liposomes were loaded with bovine serum albumin (BSA).
Results: :
Macrophages exposed to supernatants from Con A stimulated spleen cells produced 4 fold higher amounts of NO than unstimulated macrophages. Activation of macrophages via subconjunctival injection of liposomes containing supernatants from Con A stimulated spleen cell cultures resulted in rapid resolution of the corneal infection. Approximately 80% of animals treated with BSA–containing liposomes still demonstrated evidence of corneal disease at day 14 compared to a 10% incidence of infection in the Con A treated group. Moreover, at all time points examined, the clinical appearance of the keratitis in animals treated with liposomes containing Con A supernatants was significantly reduced compared to the group treated with liposomes containing BSA (P<0.05). Macrophages stimulated with IFN–γ containing supernatants killed significant numbers of the trophozoites (P<0.05). Killing was inhibited by cytochalasin D but not by L–NIL, which is a selective inhibitor of inducible NO synthase (INOS).
Conclusions: :
Activated macrophages kill Acanthamoeba trophozoites by contact–dependent mechanisms. In vivo activation of macrophages has a profound effect on the incidence, severity and chronicity of keratitis in Chinese hamsters. These results strongly suggest that macrophages play an important role in controlling corneal infection with Acanthamoeba trophozoites by acting as a first line of defense, and T cell–related activation of macrophages further enhances macrophage–mediated elimination of trophozoites.
Keywords: Acanthamoeba • amoeba • cornea: basic science