Neutrophil and T-Cell Homeostasis in the Closed Eye

PURPOSE: This study sought to examine the changes and phenotype of the tear neutrophil and T-cell populations between early eyelid closure and after a full night of sleep. METHODS: Fourteen healthy participants were recruited and trained to wash the ocular surface with PBS for at-home self-collection of ocular surface and tear leukocytes following up to 1 hour of sleep and a full night of sleep (average 7 hours), on separate days. Cells were isolated, counted, and incubated with fluorescently labeled antibodies to identify neutrophils, monocytes, and T cells. For neutrophil analysis, samples were stimulated with lipopolysaccharide (LPS) or calcium ionophore (CaI) before antibody incubation. Flow cytometry was performed. RESULTS: Following up to 1 hour of sleep, numerous leukocytes were collected (2.6 × 10(5) ± 3.0 × 10(5) cells), although significantly (P < 0.005) more accumulated with 7 hours of sleep (9.9 × 10(5) ± 1.2× 10(6) cells). Neutrophils (65%), T cells (3%), and monocytes (1%) were identified as part of the closed eye leukocyte infiltration following 7 hours of sleep. Th17 cells represented 22% of the total CD4(+) population at the 7-hour time point. Neutrophil phenotype changed with increasing sleep, with a downregulation of membrane receptors CD16, CD11b, CD14, and CD15, indicating a loss in the phagocytic capability of neutrophils. CONCLUSIONS: Neutrophils begin accumulating in the closed eye conjunctival sac much earlier than previously demonstrated. The closed eye tears are also populated with T cells, including a subset of Th17 cells. The closed eye environment is more inflammatory than previously thought and is relevant to understanding ocular homeostasis.