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Maud Gorbet, Doerte Luensmann, Sara Luck, Lyndon Jones; Response Of Tear Film Neutrophils To Different Stimuli. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5271.
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In the closed-eye environment (during sleep), changes in tear composition occur even in the absence of a contact lens. During sleep, an influx of neutrophils is seen, which typically returns to normal levels a few hours after waking. However, little is known about the phenotype of neutrophils after their extravasation (or diapedesis) from the blood vessels.This study was conducted to investigate the response of tear film neutrophils to inflammatory stimuli and this response was compared to the one of blood neutrophils.
Neutrophils were collected in phosphate buffer saline after sleep from healthy participants. Cells were resuspended in serum-containing-medium and concentrated. Collected cells were then incubated with fluorescently labeled antibodies against ICAM-1, Mac-1, CD66b (a degranulation membrane marker) and CD45 (pan leukocyte-marker) with or without phorbol myristate acetate (PMA) or Lipopolysaccharide (LPS). Reactive oxygen species were characterized using the fluorescent probe dichlorodihydro-fluorescein diacetate in the presence and absence of stimuli. Cell viability was assessed by trypan blue exclusion and PI. Neutrophils in blood as well as isolated neutrophils from blood were incubated in the same conditions. All samples were analysed by flow cytometry and at least 5000 events were analysed. Comparisons between fluorescent intensities of unstimulated (control) versus stimulated (PMA or LPS) were made.
After-sleep-collected neutrophils were alive as shown by trypan blue and PI exclusion. Tear film neutrophils were clearly identified by their side scatters and CD45 expression. Upon stimulation with LPS and PMA, tear-film neutrophils were found to upregulate ICAM-1 and produced reactive oxygen species. However, tear-film neutrophils appeared to be unable to upregulate the expression of CD66b and Mac-1 receptor in response to PMA or LPS stimulation. A similar lack of response to stimulus was observed for CD45. This lack of response to stimulus was significantly different from the response observed with blood neutrophils.
Our results indicate that while tear-film neutrophils are alive, they do not respond to inflammatory stimuli in the same manner as blood neutrophils. The process of extravasation appears to alter their phenotype. These phenomena have important implications on how we assess cell-material as well as cell-cell interactions in the closed-eye environment.
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