Abstract
Purpose :
Hundreds of thousands of leukocytes (with approximately 60% of the population being polymorphonuclear neutrophils (PMNs)) can be collected from the ocular surface immediately on waking, while only a few are harvested during the daytime. To better understand their role in ocular surface homeostasis and inflammation, this study aimed to investigate potential factors contributing to the circadian infiltration of tear PMNs and changes in phenotype across different time points in a 24-hour cycle.
Methods :
Tear leukocytes were collected from 30 participants using a gentle eyewash after 2-hr and 7-hr of sleep at night, after 2-hr sleep during the day, and towards the end of the day (around 5 pm). Cell count and morphology were determined using a Moxi Z cell counter and May-Grunwald stain, respectively. Cells were stimulated by fMLP. Changes in the degranulation (lactoferrin, CD66b, CD63) and cell aging state (CD184) of PMNs were measured via flow cytometry. Neutrophil extracellular traps (NETs) were also identified by flow cytometry and microscopy following staining with myeloperoxidase, citrullinated histones, and CD15.
Results :
Significantly more cells were collected from the nighttime compared to the daytime (p<0.001). There was a positive correlation between IL-8 concentrations and PMN numbers, but not with C5a, suggesting that the recruitment of tear PMNs to the ocular surface may be driven mainly by IL-8. 2hr-sleep-derived tear PMNs were less degranulated than 7hr-sleep-derived tear PMNs (p<0.03) and possessed a larger functional activation potential in response to stimulus (p<0.03). Furthermore, 7hr-sleep-derived tear PMNs exhibited hyper-segmented nuclei and were prone to aggregation, when compared to 2hr-night-derived tear PMNs, suggesting an aged and activated phenotype. A significantly increased number of NETs were present in 7hr-night-derived tear samples (p<0.05).
Conclusions :
The diurnal-nocturnal recruitment pattern of tear PMNs may be driven by the increase in IL-8 in nighttime tears. Higher levels of degranulation and NETs indicate that tear PMNs become more activated on the ocular surface during prolonged eye closure at night. This PMN inflammatory response must then be balanced by other anti-inflammatory processes to prevent ocular surface damage.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.