Investigation of the response of tear-film neutrophils to interleukin 8 and their sensitivity to centrifugation, fixation, and incubation

Abstract

During eye closure, a large number of neutrophils (polymorphonuclear neutrophils, PMNs) invade the ocular surface and are often referred to as tear-film PMNs. While immunophenotyping experiments have been performed on tear-film PMNs, the impact of commonly used experimental procedures on their phenotype as well as their response to interleukin-8 (IL-8), a physiological inflammatory mediator, have not yet been investigated. A gentle eye wash method was used to collect cells at home. In the morning upon awaking, participants washed their eyes with sterile phosphate buffer saline (PBS) and collected the runoff into a sterile polypropylene tube. The cell collection was then delivered to the lab within two hours. The effects of centrifugation, incubation and fixation with paraformaldehyde (PFA) before (pre-fixed staining) or after (post-fixed staining) incubation with antibodies were characterized. Tear-film PMNs as well as blood PMNs (used for comparison) were also stimulated with IL-8. To assess the reproducibility of cell collection and variability in receptor expression over time, participants were also asked to collect cells three times over a period of a month. The change in expression of surface receptors, CD11b, CD16, CD55, CD66b, important inflammatory and activation markers, and CD45 (PAN leukocyte marker) was assessed by flow cytometry. Fixing tear-film PMNs prior to the staining with antibodies resulted in a significant (fivefold or more) reduction in the expression of CD11b, CD16 and CD45 when compared to unfixed samples, while CD16 was the only receptor to undergo significant downregulation upon post-staining fixation. Furthermore, additional centrifugation step prior to antibody incubation as well as long (4 h) incubation at 37 °C resulted in significant reductions in expression of CD11b, CD16 and CD55 when compared to control samples. As opposed to blood PMNs, stimulating tear-film PMNs with IL-8 did not induce any significant changes in expression of CD11b, CD16, CD55 and CD66b. When working with collected tear-film PMNs, our results suggest that any additional centrifugation and incubation step should be avoided, or at least limited, and post fixation staining is recommended in order to preserve cell phenotype and cell integrity of tear film PMNs. Our study also adds further information on the reproducibility of the gentle eye wash as well as the inability of tear-film PMNs to modulate their surface receptors upon stimulation with IL-8. The latter may be due to prior exposure to IL-8, activation in the closed-eye environment, or a reduced ability to respond to inflammatory stimulus. Further mechanistic studies will be needed to gain a better understanding of the tear-film neutrophil phenotype.

Figure 1

Effect of paraformaldehyde (PFA) on the expression of cell membrane receptors on tear-film PMNs. Cells were either fixed with 2% PFA at 4 °C for 15 min prior to staining with antibodies (pre-fixed staining) or stained with antibodies and then fixed with 2% PFA (post-fixed staining). Control samples were stained with antibodies and immediately analyzed by flow cytometry (non-fixed staining). All incubation with antibodies were performed at RT, in the dark, for 20 min. Fluorescence intensities for CD11b, CD16, CD45, CD66b, and CD55 were recorded as the arbitrary fluorescent units by flow cytometry and are expressed as a ratio of expression level on fixed samples compared to the control (non-fixed staining samples). The dotted line represents a ratio of 1 (no difference between fixed and control samples). Mean ± SD, n = 9, *significantly different from non-fixed samples, p < 0.01.

Figure 2

Effect of exposure to 37 °C for 4 h on the expression of cell membrane receptors on tear-film PMNs. Following collection, tear film PMNs were either processed for flow cytometry (antibody staining followed by PFA fixing) or incubated for 4 h at 37 °C, 5% CO₂. Following 4 h incubation, cells were stained with antibodies and fixed with PFA. Fluorescence intensities for CD11b, CD16, CD45, CD66b, and CD55 were recorded as arbitrary fluorescent units by flow cytometry and are expressed as the ratio of expression level on incubated samples compared to the control (non-incubated samples, 0 h sample). The dotted line represents a ratio of 1 (no difference between fixed and control samples). Mean ± SD, n = 8, *significantly different from non-incubated samples, p ≤ 0.04.

Figure 3

Effect of centrifugation on the expression of cell membrane receptors on tear-film PMNs. Following collection and concentration, tear film PMNs were either subjected to another centrifugation step or incubated for 4 h at 37 °C–5%CO₂ and then centrifuged. Samples were processed for flow cytometry (antibody staining followed by PFA fixing). Fluorescence intensities for CD11b, CD16, CD45, CD66b, and CD55 were recorded as arbitrary fluorescent units by flow cytometry and are expressed as percentage of expression level on centrifuged samples compared to controls (non-centrifuged samples). The dotted line represents a ratio of 1 (no difference between centrifuged and non-centrifuged samples). Mean ± SD, n = 8–10, *significantly different from control, p ≤ 0.03.

Figure 4

Variations in the level of expression of membrane receptors on tear-film PMNs collected over 3 collection days (MFI: Mean Fluorescent Intensity). (a) CD11b; (b) CD16; (c) CD45; (d) CD55; (e) CD66b. n = 12. Twelve participants collected cells on day 1, day 2 and day 29 (one participant did not have enough cells on day 29). Note that (c) shows duplicates as CD45 expression on PMNs is measured used in 2 combination of antibodies (CD11/CD16/CD45 and CD66b/CD55/CD45).

Figure 5

The expressions of surface receptors of blood PMNs and tear-film PMNs following stimulation with IL-8. PMNs were stimulated with IL-8 (10 ng/mL), and their expression of surface receptor of CD11b, CD16, CD45, CD55 and CD66b was assessed through flow cytometry. Data is expressed as the ratio between stimulated (IL-8) and unstimulated samples. The dotted line represents the unstimulated value. Results are presented as means ± standard deviations from 13 participants collecting tear-film PMNs several times (n = 30) and 3 participants donating blood at least once (n = 4). *Significantly different from blood PMNs (p < 0.03).