Eosinophil Adhesion to Conjunctival Epithelial Cells and Eosinophil Degranulation are Upregulated By Anti-IgE Activated Conjunctival Mast Cells in vitro

Abstract

Abstract: : Purpose: Evidence of eosinophil derived mediators has been associated with chronic ocular allergic inflammation, such as vernal keratoconjunctivitis, yet the mechanism for eosinophil degranulation once it reaches the ocular surface is unknown. Incubation of conjunctival epithelial cells with supernates from anti-IgE stimulated conjunctival mast cells upregulates ICAM-1 via a mechanism specifically involving TNFα; (Cook et al., Annals Allergy Immunol, 2001). The purpose of this study was to determine whether this upregulation of ICAM-1 was biologically relevant by examining the effect on eosinophil adhesion in vitro and to further investigate whether supernates from activated mast cells can stimulate eosinophil degranulation as measured by release of eosinophil derived neurotoxin (EDN). Methods: Conjunctival mast cells and epithelial cells were acquired by enzymatic digestion of cadaveric conjunctival tissues and Percoll gradient separation. Eosinophils were obtained from peripheral blood and purified using negative magnetic bead selection. In some experiments, mast cells were pre-incubated with the mast cell stabilizer/antihistamine, olopatadine, prior to anti-IgE challenge. For adhesion, conjunctival epithelial cells were pre-incubated (24 hrs) with anti-IgE stimulated conjunctival mast cell supernates. Results: Adhesion of IL-5 primed eosinophils (expressed as percent of total eosinophils minus spontaneous adhesion) was increased by anti-IgE stimulated mast cell supernates over unstimulated controls (21.5 ± 5.9% and 1.37 ± 1.32% respectively, p <0.05). Anti-IgE stimulated mast cell supernates increased release of EDN over unstimulated supernate controls. Concentrations of EDN released were 61.9 and 24.9 ng/106 cells (minus spontaneous release) for anti-IgE and unstimulated supernates respectively (20% and 8% of positive control release). Pre-incubation of mast cells with olopatadine decreased mast cell induced EDN release resulting in concentrations of 31.3 and 3.3 ng/106 for anti-IgE and unstimulated supernates respectively (10% and 1% of positive control release). Conclusion: These results demonstrate that mast cell mediators may be directly involved in initiating and maintaining eosinophil mediated events leading to ocular surface damage in allergic inflammation.

Keywords: 365 conjunctiva • 437 inflammation • 380 cytokines/chemokines