Purpose: The goal of the present study was to investigate the role of platelet activating factor (PAF) and PAF receptor (PAF-R) in the recruitment of eosinophils into the conjunctiva in the course of PAF induced conjunctivitis. Eosinophils are important players in the immediate hypersensitivity reactions and in allergic conjunctivitis. PAF-R is expressed in many ocular tissues including conjunctival cells. Although it is known that PAF is one of the most potent chemotactic agents for the recruitment of eosinophils, factors responsible for it in conjunctivitis are not clear. Colocalization analysis has been employed to quantify the degree of colocalization of major basic protein (MBP) and PAF-R antigens in the course of PAF induced conjunctivitis.

Methods: A 1% solution of PAF was applied in eye drops to male Brown Norway rats. Eyes were harvested with intact conjunctivas at different time points and examined using histology, immunohistochemistry, confocal immunofluorescence microscopy, and reverse transcription-polymerase chain reaction. PAF-R and MBP (a marker of eosinophils) antibodies have been used for immunohistochemical studies. Quantitative analysis of the colocalization of PAF-R and major basic protein (MBP) antigens was performed.

Results: Instillation of PAF caused a time dependent recruitment of eosinophils. Eosinophils revealed PAF-R in the intact state. An influx of eosinophils into the conjunctiva was caused by the interaction of PAF with PAF-R and, possibly, with MBP antigen. PAF appeared to enhance the expression of PAF-R by eosinophils and to act toward the PAF-R directly, without chemokine mediation.

Conclusions: Quantitative colocalization analysis helped to determine that the recruitment of eosinophils in PAF induced conjunctivitis is accomplished via direct action of PAF toward the PAF-R. It also ensured an objective evaluation of the changes of the degree of colocalization of MBP and PAF-R antigens and the degree of PAF-R expression in dynamics, the findings not otherwise obtainable using qualitative approaches alone.