Phenotypic characterization of aqueous humor-mediated suppression of natural killer cell activity

Purpose. To analyze aqueous humor-mediated suppression of natural killer cell activity in vitro. Methods. Murine spleen cells were incubated in 1,000 U/ml of IL-2 to generate activated natural killer (NK) cells. Aqueous humor (AH) was collected from freshly slaughtered rabbit eyes by paracentesis. Virally transformed corneal endothelial cells were used as target cells in a standard 4-hour chromium-release assay to measure NK cell activity. Cell viability was assessed by trypan blue exclusion and chromium release. FACS analysis was used to evaluate conjugate formation and expression of surface molecules. Cytoplasmic tyrosine phosphorylation was assessed by western blot analysis. Results. We have shown that rabbit AH inhibits murine NK cell-mediated cytolysis of corneal endothelial cells in vitro by 50-55%. AH did not impair the ability of NK cells to form conjugates with target cells nor did it modulate surface expression of signaling molecules (284, NK 1.1, and 5E6) that regulate NK cell activity. Incubation of NK cells with 50% AH profoundly downregulated tyrosine phosphorylation of cytoplasmic NK cell proteins known to play a crucial role in signaling and activation. Conclusions. A blockade in the signaling cascade resulting from inadequate cytoplasmic tyrosine phosphorylation could be responsible for the AH-mediated suppression of NK cell activity. Factors in AH may protect the MHC class I negative corneal endothelium from NK cell-mediated cytolysis but also allow choroidal melanomas to escape NK cell-mediated immune surveillance.