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ABSTRACTNeutrophils are thought to be involved in many infectious diseases and have been found in high numbers in the corneas of patients withAcanthamoebakeratitis. Using a Chinese hamster model of keratitis, conjunctival neutrophil migration was manipulated to determine the importance of neutrophils in this disease. Inhibition of neutrophil recruitment was achieved by subconjunctival injection with an antibody against macrophage inflammatory protein 2 (MIP-2), a powerful chemotactic factor for neutrophils which is secreted by the cornea. In other experiments, neutrophils were depleted by intraperitoneal injection of anti-Chinese hamster neutrophil antibody. The inhibition of neutrophils to the cornea resulted in an earlier onset and more severe infection compared to controls. Anti-MIP-2 antibody treatment produced an almost 35% reduction of myeloperoxidase activity in the cornea 6 days postinfection, while levels of endogenous MIP-2 secretion increased significantly. Recruitment of neutrophils into the cornea via intrastromal injections of recombinant MIP-2 generated an initially intense inflammation that resulted in the rapid resolution of the corneal infection. The profound exacerbation ofAcanthamoebakeratitis seen when neutrophil migration was inhibited, combined with the rapid clearing of the disease in the presence of increased neutrophils, strongly suggests that neutrophils play an important role in combating Acanthamoebainfections in the cornea.

Acanthamoebakeratitis is a rare, yet sight-threatening corneal infection. Ocular infection does not appear to induce protective immunity as repeated corneal infections occur in both humans and experimental animals. However, we have recently demonstrated that activation of the common mucosal immune system by oral immunization withAcanthamoebaantigens protects both Chinese hamsters and pigs against ocular infection with A. castellanii. Protection correlates closely with the appearance of anti- Acanthamoebaantibodies in the tears. To test the hypothesis that oral immunization induces specific protective IgA antibodies, two monoclonal IgA antibodies specific for Acanthamoebaantigens were generated. Both antibodies detected epitopes on the surface of fixed Acanthamoebatrophozoites. When delivered intraperitoneally, one monoclonal antibody (14E4) was detected in stool and tear samples. This clone also protected naive animals against ocular challenge with Acanthamoebatrophozoites (43% infection rate compared to a 91% infection rate in animals receiving control IgA). In vitro functional studies showed that neither antibody induced encystment or directly killedAcanthamoebatrophozoites. However, both monoclonal anti- AcanthamoebaIgA antibodies produced a three-fold inhibition in the adherence of trophozoites to corneal epithelial cells in vitro. These data show that monoclonal anti-AcanthamoebaIgA antibodies can protect against Acanthamoebakeratitis and suggest that this occurs by inhibiting adhesion of the parasite to the corneal epithelium.

ABSTRACTAcanthamoebakeratitis is a chronic inflammatory disease of the cornea which is highly resistant to many antimicrobial agents. The pathogenic mechanisms of this disease are poorly understood. However, it is believed that the initial phases in the pathogenesis of Acanthamoebakeratitis involve parasite binding and lysis of the corneal epithelium. These processes were examined in vitro, usingAcanthamoeba castellaniitrophozoites. Parasites readily adhered to Chinese hamster corneal epithelial cells in vitro; however, parasite binding was strongly inhibited by mannose but not by lactose. Although mannose prevented trophozoite binding, it did not affect cytolysis of corneal epithelial cells. Moreover, mannose treatment induced trophozoites to release cytolytic factors that lysed corneal epithelial cells in vitro. These factors were uniquely induced by mannose because supernatants collected from either untreated trophozoites or trophozoites treated with other sugars failed to lyse corneal cells. The soluble factors were size fractionated in centrifugal concentrators and found to be ≥100 kDa. Treatment of the supernatants with the serine protease inhibitor phenylmethylsulfonyl fluoride inhibited most, but not all, of the cytopathic activity. These data suggest that the binding ofAcanthamoebato mannosylated proteins on the corneal epithelium may exacerbate the pathogenic cascade by initiating the release of cytolytic factors.