Immune response in the conjunctival epithelium of patients with dry eye

The aim of our project was to test the hypothesis that patients with dry eye have a significant degree of inflammation and lymphocyte infiltration in conjunctival epithelium by using flow cytometry analysis of cells stored in cell culture medium. Impression cytology specimens were collected in 15 normal subjects and 15 dry eye patients. Samples collected from the right eye were placed in Phosphate Buffered Saline containing 0.05% paraformaldehyde (PFA), and samples from the left eye in cell culture medium containing 10% foetal calf serum (FCS). Phenotypic analysis was performed on cells derived from 21 dry eye patients and 16 healthy controls. The cells collected in FCS were stained for the expression of CK19, CD3, CD4, CD8, CD56, CD19, CD20, CD14 and HLA-DR, and analyzed by flow cytometry.FCS samples contained a statistically increased number of cells when compared to PFA samples. No statistically significant differences were present in the number of CD45 + CK19- cells, CD3+ and CD4 + T cells, B and NK cells in dry eye patients compared to healthy controls. In the dry eye group there was a significant difference in the CD4/CD8 ratio respect to what observed in normal subjects, and an increased number of CD14+ cells. HLA-DR expression was increased only in CK19+ conjunctival epithelial cells of dry eye patients.This study indicates that immune cells isolated from the superficial layer of the conjunctiva may play a pivotal role in the pathogenesis of dry eye, and that a new method of preservation of impression cytology samples can enhance flow cytometry analysis of epithelial and immune cells of the conjunctiva.