Phospholipase A2 Activity in Normal and Staphylococcus aureus-Infected Rabbit Eyes

Abstract

Abstract: : Purpose: Phospholipase (PLA₂) provides an innate defense against ocular S. aureus infection. The purpose of this study is to quantify phospholipase activity in normal rabbit eyes and in eyes with S. aureus keratitis. Methods: PLA₂ was assayed by the killing of S. aureus at 33°C or by the release of arachidonic acid from S. aureus labeled with radioactive oleic acid. Myeloperoxidase (MPO) activity of the tear film, corneal homogenate, and aqueous humor were quantified using an o-dianisidine colorimetric assay. Rabbit corneas were injected intrastromally with 100 log phase CFU of S.aureus 8325-4. The tear film was sampled using swabs for MPO assays and with capillaries placed into the cul de sac (5-10 µl) for PLA₂ assays. Bacterial growth in the cornea was quantified as CFU per cornea (±SEM) by culturing corneal homogenates on trypticase soy agar plates. Results: The PLA₂-mediated killing of S. aureus by normal rabbit tears decreased by over 70% with age of the rabbits from 10 weeks to 28 weeks. In rabbits with S. aureus keratitis, the PLA₂ activity and MPO activity increased proportionally with time from 5 to 25 hours postinfection, as measured in the tear film, corneal homogenates, and aqueous humor. PLA₂ activity, as measured by counts per minute, increased six-fold in infected tears collected 25 hours postinfection as compared to normal tears, whereas a seven-fold increase was found in aqueous humor collected 25 hours postinfection. Infected eyes demonstrated a significant increase in MPO activity as compared to uninfected controls at 10 hours postinfection for the aqueous humor (P=0.02), at 16 hours postinfection for the tear film swabs (P=0.04), and at 25 hours postinfection for the corneal homogenate (P≤ 0.0001). Conclusion: The decrease in PLA₂ activity in the rabbit eye with age and its increase during the progression of infection are consistent with function of this molecule as an innate host defensive factor.

Keywords: 587 Staphylococcus • 449 keratitis • 469 microbial pathogenesis: experimental studies