The Noninflammatory Phenotype of Neutrophils From the Closed-Eye Environment: A Flow Cytometry Analysis of Receptor Expression.

Abstract

In the closed-eye environment (during sleep), there is an influx of neutrophils into the tear film, and the phenotype of these cells has yet to be characterized. This study was conducted to investigate the response of tear-film neutrophils to inflammatory stimuli. Immediately upon awakening, cells from healthy participants (n = 12) were collected using a gentle eye-wash with PBS. Tear-film neutrophils were counted and cell viability was determined. Neutrophils were also isolated from blood by density-gradient centrifugation. Tear-film and blood-isolated neutrophils were stimulated with phorbol myristate acetate (PMA), lipopolysaccharide (LPS), or N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Changes in the expression of macrophage-1 antigen, intercellular adhesion molecule-1 (ICAM-1), CD66b (a degranulation membrane marker), C3aR (complement C3a receptor), CD45 (leukocyte common antigen) as well as reactive oxygen species (using dichlorodihydro-fluorescein diacetate) were characterized by flow cytometry. Hundreds of thousands of leukocytes were collected upon awakening. Tear-film neutrophils were alive as shown by trypan blue and propidium iodide (PI) exclusion. While tear-film neutrophils were able to mount an oxidative response, stimulation with LPS, PMA, or fMLP did not induce receptor upregulation. This lack of response to stimulus with tear-film neutrophils was significantly different from that of blood-isolated neutrophils. Incubation in the presence of tear film proteins did not affect the tear-film neutrophil response to stimuli. Our results indicate that while tear-film neutrophils are alive, they do not respond to inflammatory stimuli in the same manner as blood-isolated neutrophils. This refractory phenotype may be due to exposure to anti-inflammatory factors present in the tear film.