Characterization of Intraepithelial Lymphocyte Populations and Their Changes in Response to Experimental Desiccating Stress

Abstract

Purpose: : Intraepithelial lymphocytes (IEL) are a subset of lymphocytes that reside within the epithelium. The purpose of this study was to evaluate resident conjunctival IELs and the effects of desiccating ocular surface stress on these cells in C57BL/6 mice.

Methods: : Desiccating stress was induced in C57BL/6 mice by cholinergic blockade and exposure to a desiccating environment for 5 and 10 days. A separate group of non-stressed mice served as control. Eyes and adnexae were excised and prepared for either histology or frozen sections. Conjunctival goblet cell density was counted in periodic acid Schiff (PAS) stained sections. Immunohistochemistry was used to identify and count cells that were positive (+) for CD4, CD8α, integrin αEβ7 (also known as CD103, a marker of IEL), gamma-delta T cell (γΔ) and NK/NKT cells in conjunctival tissue. Immunohistochemistry results were confirmed using Flow Cytometry. Evaluation of conjunctival goblet cell density was also performed in non-stressed CD8α, CD103, γΔ knock-out (KO) mice.

Results: : IEL were observed in the goblet cell rich area of the conjunctiva, in close contact with the goblet cells and consisted of CD103+, γΔ+, NK/NKT+, CD8α+ and CD11c+ (25.89±2.10; 11.83±3.99; 6.17±0.43; 0.50±0.56; 3.8±2.0 mean±SD percentage of total lymphocytes, respectively). Among the IEL, CD103+γΔ+ and CD103+NK+ cells were the most abundant (11.15±4.87 and 0.63±0.31%, respectively). CD103+CD8α+ and CD103+CD11c+ cells were the least abundant (0.24±0.17 and 0.2±0.1%, respectively). Desiccating stress induced a significant decrease in the percentage of CD103+, γΔ+, CD8α+ and NK/NKT+ cells and an increase in CD4+ T cells. Filled goblet cell density correlated positively with the number of CD8α+ and CD103+ cells in the conjunctiva (R2=0.30 and R2=0.26, p<0.001 and p<0.01, respectively). Non-stressed CD8αKO, CD103KO and γΔKO had lower numbers of filled conjunctival goblet cells.

Conclusions: : The normal murine conjunctiva has a high number of IEL. The major IEL subsets are γΔ+ and NK/NKT+ cells. Desiccating stress decreases the IEL population, while decreasing the number of PAS+ goblet cells. Genetic deletion of certain IEL populations is associated with a decrease in goblet cells.

Keywords: stress response • inflammation • flow cytometry