Abstract
A large number of polymorphonuclear neutrophils (PMNs) invade the ocular surface during prolonged eye closure (sleep); these leukocytes are commonly referred as tear PMNs. PMNs contribute to homeostasis and possess an arsenal of inflammatory mediators to protect against pathogens and foreign materials. This study examined the ability of tear PMNs to generate reactive oxygen species (ROS), an essential killing mechanism for PMNs which can lead to oxidative stress and imbalance. Cells were collected after sleep from healthy participants using a gentle eye wash. ROS production in stimulated (phorbol-12-myristate-13-acetate (PMA), lipopolysaccharides (LPS) or N-Formylmethionyl-leucyl-phenylalanine (fMLP)) and unstimulated tear PMNs was measured using luminol-enhanced chemiluminescence for 60 min. A high level of constitutive/spontaneous ROS production was observed in tear PMNs in the absence of any stimulus. While tear PMNs were able to produce ROS in response to PMA, they failed to appropriately respond to LPS and fMLP, although fMLP-stimulated tear PMNs generated ROS extracellularly in the first three minutes. Higher ROS generation was observed in isolated tear PMNs which may be due to priming from the magnetic bead cell separation system. The differential responses of tear PMNs in ROS generation provide further evidence of their potential inflammatory roles in ocular complications involving oxidative stress.
Highlights
Polymorphonuclear neutrophils (PMNs), known as neutrophils, are potent innate immune cells
To gain a better understanding of the role and function of tear PMNs in ocular homeostasis, this study investigated their ability to produce reactive oxygen species (ROS) in response to fMLP and LPS, which act via G protein-coupled receptors (GPCRs) and toll like receptors (TLRs), respectively, and PMA, which directly activates protein kinase C (PKC)
Experiments were performed with tear PMNs that had been isolated from the eye wash collection using CD15+ positive selection with the MiniMACS column-based cell separation system (Miltenyi Biotec Inc., Auburn, CA, USA)
Summary
Polymorphonuclear neutrophils (PMNs), known as neutrophils, are potent innate immune cells. Unlike blood PMNs, tear PMNs collected after more than 6 hr of sleep (i.e., closed-eye conditions) have upregulated expression of Mac-1 (CD11b, a cell adhesion and activation marker) and CD66b (a degranulation marker) and exhibit a downregulation of L-selectin, a molecule reported to be shed upon activation or extravasation [4,6] These observations suggest that tear PMNs collected after sleep may have already been activated following their arrival on the ocular surface due to the presence of inflammatory cytokines and foreign particles such as bacteria [6]. The expression of cell membrane receptors associated with cell activation was assessed by flow cytometry following PMN isolation from the eye wash collection
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