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Abstract
PURPOSE: A tripeptide chemoattractant(s) for neutrophils has been shown to release from alkali-degraded cornea. This study is designed to determine the source of the chemoattractant(s). METHODS: Whole corneas were degraded in 1 N NaOH for 10 minutes, 30 minutes, 1 hour, 4, 8, 24, and 48 hours to determine an optimal duration of alkali exposure for production of the chemoattractant. Whole cornea, cornea minus epithelium, cornea minus endothelium, and stroma alone were degraded in 1 N NaOH for 4 hours to determine the relative contribution of each corneal layer. In a separate experiment, epithelium alone, endothelium alone, cultured keratocytes alone, or bovine corneal collagen were treated separately in 1 N NaOH for 4 hours. Finally, human plasma, platelets, polymorphonuclear leukocytes (PMNL), and red blood cells were treated separately in 1 N NaOH for 4 hours to determine if a similar chemoattractant was released from alkali-treated noncorneal tissue. Neutralized suspensions of all samples were ultrafiltered and dialyzed. The chemotactic potential of each sample was determined in the polarization assay. RESULTS: Activation of the PMNL polarization response increased with the duration of exposure of corneal tissue to alkali, peaking at 6 hours. Release of the chemoattractant from alkali-degraded corneal tissue showed a significant decrease when the epithelium was removed from the stroma. All tissue layers showed a PMNL polarization response when treated with alkali. The response decreased from epithelium > endothelium > cultured keratocytes > collagen. Alkali degradation of human blood components, including plasma, showed significant polarization responses ranked in the following order: plasma > PMNL > tendon collagen > platelets = red blood cells. CONCLUSIONS: This study demonstrates that the tripeptide chemoattractant(s) is released from all layers of the cornea after alkali injury. The substance released from blood components is of similar size and biologic activity as that found in the cornea, but its exact molecular composition is yet to be determined. Timed response of alkali degradation determined the optimal conditions for generation of the chemoattractant(s) including clinically relevant time intervals.