Inflammatory Mediators in Alkali-Burned Corneas: Preliminary Characterization

Abstract

Leukocyte chemotactic factors (LCF) are important inflammatory mediators which activate and recruit leukocytes from the circulation into sites of tissue damage. These factors were recently detected in the tear fluid of inflamed eyes induced by alkali burn. It remains unclear, however, whether the detected LCF are released from injured corneal tissues or leaked from the circulation. Using a corneal cup model developed in our laboratory, we began examining the capability of corneal tissues to produce LCF in response to alkali injury. We also evaluated the influence of citric acid on the production of LCF from corneas preinjured by the alkali sodium hydroxide (NaOH). For these studies, the epithelial surfaces of corneas isolated from bovine and human eyes were exposed to IN NaOH for 35 seconds at room temperature. The NaOH was then removed and the epithelial surfaces washed once with buffer and incubated with culture medium for 1, 2, 4, and 6 hours at 37°C/5% CO₂ atmosphere. Our results showed that (1) NaOH induced corneal epithelial cell injury ranging from cell discoloration and moderate damage of the upper half of the epithelium (1–4 hrs) to total destruction of the epithelium (6 hrs); (2) NaOH-injured corneas (2 hr incubation post injury) produced singificant levels of chemotactic activity (via checkerboard analysis) specific for neutrophils (115% maximum chemotactic response [MCR]) and mononuclear cells (94% MCR); (3) preliminary characterization of these factors revealed that they are protease and heat sensitive, extractable by organic solvents, and possess molecular weight values greater than 100,000 daltons; and (4) incubation of NaOH-pretreated corneas with 0.01% citric acid for 2 hours markedly inhibited the production of LCF for both neutrophils (98% inhibition) and mono-nuclear cells (91% inhibition). Results of these studies indicate that alkali-burned bovine and human corneas generate leukocyte chemoattractants which differ in their biochemical characteristics from previously known low molecular weight chemotactic factors such as C5a, interleukin-1, or leukotriene B₄.