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Immunology and Microbiology  |   July 2015
The Noninflammatory Phenotype of Neutrophils From the Closed-Eye Environment: A Flow Cytometry Analysis of Receptor Expression
Author Affiliations & Notes
  • Maud Gorbet
    Systems Design Engineering University of Waterloo, Waterloo, Ontario, Canada
    School of Optometry and Vision Science, University of Waterloo, Waterloo, Ontario, Canada
  • Cameron Postnikoff
    Systems Design Engineering University of Waterloo, Waterloo, Ontario, Canada
  • Sara Williams
    Systems Design Engineering University of Waterloo, Waterloo, Ontario, Canada
  • Correspondence: Maud Gorbet, Department of Systems Design Engineering, University of Waterloo, 200 University Avenue West, Waterloo, ON, N2L 3G1, Canada; mgorbet@uwaterloo.ca
Investigative Ophthalmology & Visual Science July 2015, Vol.56, 4582-4591. doi:10.1167/iovs.14-15750
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      Maud Gorbet, Cameron Postnikoff, Sara Williams; The Noninflammatory Phenotype of Neutrophils From the Closed-Eye Environment: A Flow Cytometry Analysis of Receptor Expression. Invest. Ophthalmol. Vis. Sci. 2015;56(8):4582-4591. doi: 10.1167/iovs.14-15750.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Neutrophils represent 40% to 60% of the leukocyte population (3–5 × 106 neutrophils/mL) and, as cells of the innate immune system, are our first line of defense and play a critical role in inflammation and preventing infection.1 While neutrophils circulate in blood, they are also found in tissue following extravasation in response to an inflammatory stimulus. Chemotactic molecules, such as complement activation products and cytokines, have been recognized to play a role in extravasation. In response to inflammatory stimulus (complement activation proteins, cytokines, chemokines, lipid mediators) and infection, neutrophils will undergo activation and release potent mediators such as inflammatory cytokines, metalloproteinases (which can degrade the extracellular matrix), lipoxygenase, and cyclooxygenase enzymes (which can further generate both pro- or anti-inflammatory lipid mediators) and oxidative products.1,2 
The anterior segment of the eye represents a unique environment due to its immune privilege. This environment is meant to protect the eye from the damage that inflammatory cells may cause upon activation.24 The presence of neutrophils in the anterior eye is thus highly regulated. In inflammatory diseases such dry eye5 and allergy,6,7 neutrophils have been observed in the precorneal tear film and their relevance and contribution to disease is currently being investigated. Any wound to the cornea, such as the ones induced by chemical burns or puncture, also leads to the invasion of the cornea by neutrophils. During wound healing, neutrophils migrate into the tear film by leakage through the blood vessels and research has identified lumican as a key player in neutrophil extravasation to the cornea.8,9 The presence of neutrophils contributes to the resolution of inflammation and ocular wound closure,2,10,11 and Wang et al.12 recently demonstrated that lipid mediators can control the state of neutrophil activation inflammatory resolution in the mouse cornea. 
However, in the absence of any wound or infection, the closed-eye environment represents a unique condition in the eye where a significant influx of neutrophils regularly takes place: every day, during sleep, changes in tear composition with increases in complement activity, metalloproteinases and cytokines correlate with the recruitment of leukocytes to the ocular surface.1315 Wilson et al.14 first identified the presence of leukocytes in ocular cell collection upon awakening. Tan et al.15 further reported that neutrophil recruitment, during sleep, appears to be intensified between 3 and 5 hours, directly following an increase in complement activation. 
Ocular neutrophil recruitment by corneal infection, wounding or inflammation due to ocular diseases has seen extensive investigation in rabbit1618 and mouse11,12,19,20 models. However, these inflammatory conditions are significantly different from the closed-eye environment, and thus mechanisms of cell migration/recruitment and activation likely differ altogether. Furthermore, it is difficult to draw conclusions about the phenotype of closed-eye neutrophils in animal models given that these animals do not close their eyes. 
This current study presents a simple and rapid method to collect tear-film neutrophils and was conducted to characterize the phenotype of closed-eye tear-film neutrophils. Their response to inflammatory stimuli was investigated and compared with that of blood-isolated neutrophils, as blood-isolated neutrophils have been commonly used to characterize interactions with ophthalmic biomaterials and/or ocular bacteria.21,22 Cell viability was assessed following cell collection and flow cytometry was used to characterize cell activation through the expression of selected cell membrane receptors and oxidative products.23 
Materials and Methods
Reagents and Monoclonal Antibodies
Lipopolysaccharide (LPS) from Escherichia coli serotype 0111:B4, phorbol-12-myristate-13-acetate (PMA), N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), endotoxin-free water, EDTA, dichlorodihydro-fluorescein diacetate (DCF), Giemsa stain, and paraformaldehyde were purchased from Sigma-Aldrich Co. (Oakville, ON, Canada). Phosphate-buffered saline (pH 7.4) was acquired through Lonza (Allendale, NJ, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and Hoechst were purchased from Life Technologies (Burlington, ON, Canada). All other chemicals were of analytical reagent grade. 
Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies against human CD11b, CD16, and CD66b, R-phycoerythrin (PE)-conjugated monoclonal antibodies against human C3aR, CD15, and CD54 and R-phycoerythrin-cytochrome 5 (PE-Cy5)-conjugated monoclonal antibody against CD45 were purchased from Becton Dickinson (San Diego, CA, USA). Fluorescein isothiocyanate–conjugated annexin V was also from BD. 
Tear-Film Cell Collection
Neutrophils were collected after sleep from healthy participants. This study was conducted in accordance with the tenets of the Declaration of Helsinki and received ethics clearance from the University of Waterloo Human Research Ethics Committee (Waterloo, ON, Canada). Twelve healthy study participants, six female and six male, within the age range of 21 to 44 years, were involved. All participants were trained to self-collect their tear-film neutrophils using a polypropylene pipette containing sterile PBS. After 8 hours of sleep, participants were instructed to gently wash their eyes with approximately 5 mL PBS for each eye; the eye wash was collected in one sterile polypropylene tube (pooled sample). The collected samples were brought to the laboratory within 90 minutes of collection and processed immediately. Serum-containing medium (DMEM with 10% FBS, to be referred to as DMEM/FBS) was then added and the cell collection was centrifuged at 250g and cells were resuspended in a small volume of DMEM/FBS. In some experiments, cells were resuspended in artificial tear solution instead (ATS; see Table 1 for ATS composition). 
Table 1
 
Composition of Artificial Tear Solution53
Table 1
 
Composition of Artificial Tear Solution53
Peripheral Blood Neutrophil Isolation
Blood was drawn from the same healthy participants who were free of medication for at least 48 hours. Low molecular weight heparin (10 U/mL) was used as an anticoagulant. Platelet-rich plasma was first removed by centrifugation at 100g. Blood neutrophils were then isolated using the density gradient PolymorphPrep, washed three times in EDTA and serum-containing media, and were ultimately resuspended in DMEM/FBS. 
For the experiments performed to assess the effect of tear proteins on cell activation, the last wash was performed in an excess of PBS to remove the serum proteins present in the resuspended blood-isolated neutrophils. After centrifugation, supernatant was aspirated and cells were resuspended in either ATS, DMEM/FBS, PBS, or PBS+Lactoferrin (LF). 
Leukocyte Viability
For blood-isolated leukocytes and tear-film cell collection, viability, and cell count were determined using a hemocytometer following 1:1 dilution of a small aliquot in Trypan Blue. The fluorescent dye Hoechst was also used. Tear film cell collection was incubated with Hoechst for 30 minutes at 37°C prior examination by fluorescence microscopy. 
Viability of tear-film neutrophils and apoptosis were further characterized by flow cytometry using propidium iodide (PI) and the fluorescently-labeled pan-caspase inhibitor (FITC-VAD-FMK; Calbiochem, San Diego, CA, USA). Tear-film neutrophils were incubated with FITC-VAD-FMK for 1 hour at 37°C. Samples were washed and resuspended in wash buffer before immediate analysis by flow cytometry. 
Cell Stimulation
Cells were allowed to “equilibrate” in medium for 30 minutes prior to incubation with stimulus. Three stimuli, that are recognized to induce an inflammatory response in leukocytes, were used: PMA (2 μM, final concentration), a protein kinase C activator; LPS (2 μg/mL, final concentration), also known as bacterial endotoxin; and fMLP (17 nM, final concentration), which induces a G-protein coupled receptor mediated physiological activation. Samples were divided in aliquots (Rest, i.e., unstimulated; fMLP-stimulated; LPS-stimulated, and PMA-stimulated). Stimulation was performed at 37°C for 15 minutes (fMLP-stimulated samples) and 30 minutes (LPS-stimulated samples). Phorbol myristate acetate–stimulated samples were incubated for 20 minutes at room temperature. 
Expression of Membrane Receptors on Neutrophils and Oxidative Response
After incubation with stimulus, 30 μL cell suspension was transferred into tubes containing fluorescently labeled antibodies against C3aR, CD11b, CD16, CD45, CD54, CD66b (Table 2), and incubated for another 30 minutes at room temperature in the dark. At the end of the incubation, samples were diluted and fixed with paraformaldehyde (1% final concentration). All samples were analyzed on the flow cytometer within 5 days. 
Table 2
 
Antibodies and Stains Used in Flow Cytometry to Characterize Cell Activation and the Phenotype of Tear-Film Neutrophils
Table 2
 
Antibodies and Stains Used in Flow Cytometry to Characterize Cell Activation and the Phenotype of Tear-Film Neutrophils
The formation of reactive oxygen species in stimulated and unstimulated samples was characterized using the fluorescent probe dichlorodihydro-fluorescein diacetate (DCFH-DA). Dichlorodihydro-fluorescein diacetate, a small nonpolar, nonfluorescent molecule, diffuses into the cells and is oxidized by H2O2 to the fluorescent molecule DCF.24 There is also evidence that nitric oxide may be able to convert DCFH to the fluorescent probe DCF,25 and thus DCF may assess more than just the production of reactive oxygen species. Dichlorodihydro-fluorescein was added to samples (48 μM, final concentration). After 30-minute incubation at 37°C, samples were diluted and immediately analyzed on the flow cytometer. 
Flow Cytometry
All samples were acquired on a Becton Dickinson FACSCalibur flow cytometer (Mountain View, CA, USA) using CELLQuest Software (Becton Dickinson, Mountain View, CA, USA). At least 5000 events were acquired. Appropriate isotype controls were used with each experiment. Data analysis was performed using CELLQuest post data acquisition. 
When reporting all results, the following nomenclature applies: neutrophils collected after sleep are referred to as “tear-film neutrophils,” neutrophils isolated from blood as “blood-isolated neutrophils.” To allow for comparisons between the different types of neutrophils, the mean fluorescence intensity in arbitrary units (MFI) as well as the ratio of the fluorescent intensities of rest (unstimulated sample) versus stimulated are presented. 
Statistical Analysis
All results are reported as means ± SD. To evaluate the significance of the differences in the ratio of cell activation, an ANOVA was performed followed by multiple pairwise comparisons using the Tukey test. Analysis was performed using Statistical Analysis Software (SAS; Cary, NC, USA) and a P value of less than 0.05 was required for statistical significance. 
Results
Characterization of Tear-Film Neutrophils, Cell Count, and Viability
After receiving the closed-eye cell collections, samples were processed immediately. A viable leukocyte cell count was performed using trypan blue exclusion and viability was determined to be 98%. Corneal epithelial cells, as identified by their cobblestone shape, were also observed during cell count but were not counted; in most cases, corneal epithelial cells were found to be dead (stained with trypan blue; Fig. 1). Based on the trypan blue count, total cell collection led to a leukocyte count of 345,000 ± 218,000 (ranging from 58,000 to 1 million leukocytes). 
Figure 1
 
Ocular cell collection stained with trypan blue. Cells were collected immediately upon awakening. Many viable leukocytes and some dead epithelial cells (blue-stained cells) were present.
Figure 1
 
Ocular cell collection stained with trypan blue. Cells were collected immediately upon awakening. Many viable leukocytes and some dead epithelial cells (blue-stained cells) were present.
Hoechst staining further confirmed the viability of tear-film neutrophils and significant presence of cells with the trilobed nucleus, which is a hallmark of neutrophils (Fig. 2). A Giemsa stain of a closed-eye cell collection is shown in Figure 3, which highlights the presence of neutrophils in the cell collection. Tear-film neutrophils were found to have a smaller diameter than blood-isolated neutrophils (P < 0.03), of 9.0 ± 0.3 μm and 9.5 ± 0.3 μm respectively, as measured by the Moxi Z automated cell counter (ORFLO; Hailey, ID, USA). 
Figure 2
 
Ocular cell collection stained with Hoechst. Cells were collected immediately upon awakening, stained with Hoechst, and observed under an epifluorescence microscope. The tear-film neutrophils show the typical multilobed nucleus.
Figure 2
 
Ocular cell collection stained with Hoechst. Cells were collected immediately upon awakening, stained with Hoechst, and observed under an epifluorescence microscope. The tear-film neutrophils show the typical multilobed nucleus.
Figure 3
 
Giemsa stain of tear-film neutrophils.
Figure 3
 
Giemsa stain of tear-film neutrophils.
The leukocyte population in the closed-eye collection samples was identified using the pan-leukocyte marker CD45. As shown in Figure 4, regions were created around the neutrophil population (R1 and R2) using light scatter characteristics (R1, based on cell size and granularity, Fig. 4a) and CD45 fluorescent values (R2, based on granularity and expression of CD45, Fig. 4b) were obtained by gating on the neutrophil population only (cells in R1 and R2). The neutrophil population in the closed-eye cell collection was easily identifiable by its scatter characteristics (Fig. 4a); the presence of lymphocytes and monocytes in the cell collection could also be identified by flow cytometry. In all the collection performed, the neutrophil population represented 58 ± 18% of the total ocular cell collection. To further confirm that the double-gating strategy (R1 and R2) identified the neutrophil population, the expression of CD16 (a marker for neutrophils) was also assessed. As shown in Figure 4c, 97% of the double-gated cell population expressed CD16. The neutrophil phenotype (identified by the R1 and R2) region was also confirmed by positive expression of CD15 (an antigen also found on neutrophils) and a lack of expression of CD14 (an antigen mainly present on monocytes; data not shown). Consistent with the process of extravasation and the interaction of L-selectin with endothelial cells lining the blood vessels,26,27 tear-film neutrophils appeared to have shed L-selectin: L-selectin expression on tear-film neutrophils was close to background levels (Fig. 5). 
Figure 4
 
Identification of the neutrophil population in the ocular cell collection using flow cytometry analysis. The neutrophil population was identified by double-gating: a region R1 in the side scatter dot plot to select neutrophils based on cell size and granularity (a); side scatter (SSC-H) versus CD45 fluorescence (FL3-H) dot plots to identify CD45 positive cells (PAN leukocyte marker) based on their granularity (b); expression of CD16 (neutrophil marker) on the double-gated (R1 and R2) population (c). These plots are representative of all experiments performed in this study (n > 12).
Figure 4
 
Identification of the neutrophil population in the ocular cell collection using flow cytometry analysis. The neutrophil population was identified by double-gating: a region R1 in the side scatter dot plot to select neutrophils based on cell size and granularity (a); side scatter (SSC-H) versus CD45 fluorescence (FL3-H) dot plots to identify CD45 positive cells (PAN leukocyte marker) based on their granularity (b); expression of CD16 (neutrophil marker) on the double-gated (R1 and R2) population (c). These plots are representative of all experiments performed in this study (n > 12).
Figure 5
 
Fluorescent histogram for L-selectin expression on tear-film and blood-isolated neutrophils. Black-filled histogram: tear-film neutrophils; black-line histogram: blood-isolated neutrophils. Shedding of L-selectin as shown by the significantly reduced expression of L-selectin is evident on tear-film neutrophils.
Figure 5
 
Fluorescent histogram for L-selectin expression on tear-film and blood-isolated neutrophils. Black-filled histogram: tear-film neutrophils; black-line histogram: blood-isolated neutrophils. Shedding of L-selectin as shown by the significantly reduced expression of L-selectin is evident on tear-film neutrophils.
Flow cytometry was also used to further assess the viability of tear-film neutrophils (PI staining) and the presence of apoptotic cells was determined using FITC-FAD-FMK, a caspase inhibitor that binds to activated caspases in cells. Few leukocytes were observed to stain with PI and caspase, as illustrated in Figure 6, which further confirmed that the collected tear-film neutrophils were alive and did not display signs of apoptosis. Tear-film neutrophils also did not stain with Annexin V, another marker for apoptosis (data not shown). 
Figure 6
 
Flow cytometry analysis of caspase activity in tear-film neutrophils (a) and BAK-treated tear-film neutrophils (b). BAK was used as a positive control to induce apoptosis and necrosis. Minimal caspase activity (less than 2%) was observed in tear-film neutrophils. A representative experiment (n = 10) is depicted. BAK, benzalkonium chloride.
Figure 6
 
Flow cytometry analysis of caspase activity in tear-film neutrophils (a) and BAK-treated tear-film neutrophils (b). BAK was used as a positive control to induce apoptosis and necrosis. Minimal caspase activity (less than 2%) was observed in tear-film neutrophils. A representative experiment (n = 10) is depicted. BAK, benzalkonium chloride.
Tear-Film Neutrophil Response to Stimulus In Vitro
All results that are presented refer to the double-gated cell population that specifically identify neutrophils. 
Membrane Receptor Expression.
To characterize tear-film neutrophils, their response to various inflammatory stimuli was determined and then compared with blood-isolated neutrophils. The response is expressed as the ratio between rest (unstimulated) versus stimulated samples to allow for comparisons between different sources of neutrophils and accounts for difference in baseline level of receptor expression. As illustrated in Figure 7a, in response to LPS, PMA, and fMLP stimulation, all ratios remained at approximately 1.0 indicating that the tear-film neutrophils were unable to upregulate their expression of Mac-1, ICAM-1, CD66b, or CD45. This response to stimulation (or lack thereof) was significantly different from that of blood-isolated neutrophils, where a significant upregulation of receptors was observed (Fig. 7b). Blood-isolated neutrophils responded to stimulation in the expected manner; PMA, LPS, and fMLP have all been reported to upregulate Mac-1, CD54, and CD66b in blood neutrophils.23,28 We also observed high ratio of activation with Mac-1, ICAM-1, CD66b, and CD45 in nonisolated blood neutrophils (Table 3). The lack of change in receptor expression on tear-film neutrophils is further illustrated in Figure 8 with the fluorescent histograms of expression before and after LPS-stimulation. It is interesting to note that a double peak is observed for the CD66b expression on the unstimulated tear-film neutrophils, which suggests that tear film neutrophils may have undergone degranulation in the closed-eye environment. For C3aR, in the case of a 30 minute-stimulation with PMA at 37°C, C3aR is generally internalized (i.e., a downregulation of C3aR expression is commonly observed).28 However, in our PMA-stimulation condition (20 minutes at 24°C), no significant C3aR internalization occurred. 
Figure 7
 
Receptor upregulation of tear-film neutrophils collected after sleep (a) and blood-isolated neutrophils (b) following stimulation with LPS, PMA, and fMLP in DMEM/FBS. Fluorescence intensities were recorded by flow cytometry and are expressed as a ratio between stimulated and unstimulated samples. n = 12, mean ± SD. *Significantly different from activation ratio of blood isolated neutrophils (P < 0.001).
Figure 7
 
Receptor upregulation of tear-film neutrophils collected after sleep (a) and blood-isolated neutrophils (b) following stimulation with LPS, PMA, and fMLP in DMEM/FBS. Fluorescence intensities were recorded by flow cytometry and are expressed as a ratio between stimulated and unstimulated samples. n = 12, mean ± SD. *Significantly different from activation ratio of blood isolated neutrophils (P < 0.001).
Table 3
 
Receptor Upregulation of Neutrophils in Whole Blood After Stimulation With LPS and PMA
Table 3
 
Receptor Upregulation of Neutrophils in Whole Blood After Stimulation With LPS and PMA
Figure 8
 
Overlay of fluorescent histograms for Mac-1, ICAM-1, CD54, and CD45 expression on tear-film and blood-isolated neutrophils. Black-filled histogram: unstimulated neutrophils; gray-line histogram: LPS-stimulated neutrophils. The lack of upregulation of receptor expression following LPS-stimulation is seen by the absence of a shift in fluorescence intensity. Histograms are representative of all experiments performed.
Figure 8
 
Overlay of fluorescent histograms for Mac-1, ICAM-1, CD54, and CD45 expression on tear-film and blood-isolated neutrophils. Black-filled histogram: unstimulated neutrophils; gray-line histogram: LPS-stimulated neutrophils. The lack of upregulation of receptor expression following LPS-stimulation is seen by the absence of a shift in fluorescence intensity. Histograms are representative of all experiments performed.
A difference in the baseline level of receptor expression was also noted: the mean fluorescence values of membrane receptor expression are presented in Table 4. Significantly higher levels (P < 0.035) of expression of ICAM-1, CD45, and CD66b were observed on the unstimulated tear-film neutrophils when compared with blood-isolated neutrophils. 
Table 4
 
Expression of Membrane Receptor on Unstimulated Tear-Film and Blood-Isolated Neutrophils (Resting Level) Resuspended in DMEM/FBS
Table 4
 
Expression of Membrane Receptor on Unstimulated Tear-Film and Blood-Isolated Neutrophils (Resting Level) Resuspended in DMEM/FBS
Oxidative Response.
Dichlorodihydrofluorescein diacetate was used to assess oxidative products in neutrophils following stimulation. Significant variations in the generation of oxidative products were found, which resulted in a high SD for both tear-film and blood-isolated neutrophils, an observation that has been shared by other researchers.29 As shown in Table 5, tear-film neutrophils appear to be unable to mount a significant oxidative response following stimulus. The baseline level of oxidative products was also significantly higher with tear-film neutrophils when compared with blood-isolated neutrophils (Table 5; P < 0. 001). 
Table 5
 
Oxidative Burst Response, as Measured by DCF-HA, by Tear-Film and Blood-Isolated Neutrophils Following fMLP, LPS, and PMA Stimulation in DMEM/FBS
Table 5
 
Oxidative Burst Response, as Measured by DCF-HA, by Tear-Film and Blood-Isolated Neutrophils Following fMLP, LPS, and PMA Stimulation in DMEM/FBS
The Effect of an Artificial Tear Solution on Neutrophil Response to Stimuli.
To determine if the response of tear-film neutrophils to different stimuli may be dependent on the presence of tear film proteins, activation was performed in ATS containing appropriate concentrations of proteins known to be present in the tear film, namely lactoferrin, lysozyme, mucin, IgG, and albumin (see Table 1 for protein concentration).15 Stimulating tear-film neutrophils in ATS resulted in a similar response, or lack thereof, as previously observed in DMEM/FBS (Table 6). In ATS, blood-isolated neutrophils were still able to mount an inflammatory response as shown by an activation ratio above 1.0 for all markers (Table 6). When compared with ratios observed with DMEM/FBS, a reduction in the ability of blood-isolated neutrophils to upregulate the expression of Mac-1 and CD66b in response to LPS stimulation was identified (P < 0.045 for CD66b). Isolated blood neutrophils were able to mount a normal response to fMLP and PMA stimulation in the presence of ATS. Interestingly, the baseline levels of Mac-1 and CD66b expression were reduced in ATS when compared with the levels observed in DMEM/FBS (see Table 4), with 45 ± 22 (P < 0.03) and 32 ± 16 for Mac-1 and CD66b expression, respectively. 
Table 6
 
Response of Tear-Film and Blood-Isolated Neutrophils to LPS, fMLP, and PMA Stimulation in Artificial Tear Solution
Table 6
 
Response of Tear-Film and Blood-Isolated Neutrophils to LPS, fMLP, and PMA Stimulation in Artificial Tear Solution
As DMEM/FBS is a complex salt and protein solution, to allow for better comparisons with stimulation in ATS, a series of stimulation experiments with physiological stimuli (LPS and fMLP) was performed using blood-isolated neutrophils in PBS supplemented with various components (ATS is a PBS solution supplemented with tear film proteins as shown in Table 1). There was no significant difference between the level of response to stimulation between DMEM/FBS and PBS, suggesting that the lower response to stimulation observed with ATS was due to the presence of the tear film proteins. Indeed, a significant difference in upregulation of Mac-1 and CD66b was observed in the response of blood-isolated neutrophils to stimulation in ATS versus PBS (Tables 6, 7). Adding lactoferrin (1.80 mg/mL) to PBS resulted in a reduction of the upregulation of all the activation markers tested (albeit the difference did not reach statistical significance P > 0.1); upregulation of CD66b was also still higher in PBS+LF when compared with stimulation in ATS (Tables 6, 7). It is worth noting that a reduction in the baseline level of expression of CD66b was observed in the presence of lactoferrin, with 31 ± 11 and 20 ± 6 for PBS and PBS+LF, respectively (P = 0.08). While this highlights that lactoferrin has an anti-inflammatory role, these results also suggest that other components of the ATS play a significant role in the reduced the inflammatory response observed with blood-isolated neutrophils stimulated in ATS. 
Table 7
 
Effect of Stimulating Blood-Isolated Neutrophils in PBS and PBS Supplemented With Lactoferrin (1.80 mg/mL)
Table 7
 
Effect of Stimulating Blood-Isolated Neutrophils in PBS and PBS Supplemented With Lactoferrin (1.80 mg/mL)
Discussion
While it has been recognized that neutrophils invade the ocular surface during closed-eye conditions,14,30,31 there has been no study aimed at characterizing the phenotype of these cells. In a previous lens wear study by Wilson et al.,14 between 630 and 22,000 leukocytes were collected in one eye (median for cell collection was 6500 leukocytes) following 8 hours of sleep. Tan et al.31 also performed an ocular wash after sleep and reported 1529 ± 18 and 6583 ± 2354 leukocytes after 5 and 8 hours of sleep, respectively. Using our collection protocol, we were able to collect a significantly higher numbers of leukocytes; difference in materials used for the collection (polypropylene tubes, phosphate buffer),the centrifugation protocol and training of the study participants are likely responsible for the higher yield obtained in our study. Based on the numbers of neutrophils collected and the volume of tears, the concentration of inflammatory cells at the ocular surface during closed-eye conditions may be approximated to be 3 × 107 cells/mL, which is at least five times as concentrated as neutrophils in the blood. For such a high concentration of inflammatory cells to be present on a nightly basis, a balance needs to exist between protection of the eye against pathogens and preservation of the integrity of the corneal epithelium. 
While refractory behavior, whereby neutrophils respond distinctively from blood-isolated neutrophils, has been observed previously in neutrophils extravasated to the lungs32 and inflamed tissues,33,34 our work is the first report of this observation with neutrophils from the ocular surface. A recent study by Baines et al.35 reported significant differences in level of cytokine synthesis in resting sputum neutrophils from asthmatics versus healthy participants as well as following response to LPS stimulation. Lakschevitz et al.36 also reported different cytokine and receptor expression profiles for oral neutrophils compared with circulating neutrophils. Extravasated neutrophils have been shown to have higher levels of CD66b34 and β-catenin, a receptor important in epithelial repair.37 Assuming neutrophils collected upon awakening extravasated from ocular blood vessels, the complex process of activation, adhesion to the endothelium, and ultimately endothelial transmigration may have contributed to the phenotype of tear-film neutrophils.26 
Changing the medium of incubation for tear-film neutrophils from DMEM/FBS to ATS did not result in any changes in the response to inflammatory stimuli, suggesting that the impaired response to stimulus was not due to the absence of a specific factor. On the other hand, significant changes were observed in the response of blood-isolated neutrophils where a reduction in CD66b upregulation was observed when stimulation took place in ATS or in PBS+lactoferrin, suggesting that interactions with specific proteins in the tear film may contribute to inhibit degranulation (CD66b is a marker of degranulation). The concept of anti-inflammatory properties of tear film proteins is not new.38,39 Among the many proteins of the tear film, numerous studies on lactoferrin have been published as it is present in both blood and tears and is also a secretory product of neutrophils. Lactoferrin is known for its antimicrobial properties40 but is also recognized to have anti-inflammatory properties with leukocytes.41,42 The role of lactoferrin in production of oxidative species appears to be more complex and dependent on experimental conditions. Gahr et al.43 have shown that the presence of lactoferrin at 500 μg/mL induced greater motility as well as release of superoxide in neutrophils. However, lactoferrin at 50 μg/mL inhibited LPS-induced production of reactive oxygen species.44 Our results with ATS, thus support previous finding that lactoferrin at 1800 μg/mL can reduce leukocyte response to inflammatory stimulus and that this inhibition is stimulus dependent. It is therefore possible that the anti-inflammatory phenotype of tear-film neutrophils may be the result of prolonged exposure to lactoferrin and other anti-inflammatory compounds in the tear film. There is significant evidence that exposure to cytokines will result in phenotypic and functional changes in neutrophils in injured tissues; this mechanism is believed to relate to innate and adaptive immunity and further highlights the heterogeneity in the neutrophil population.45 
While the tear film proteins such as lactoferrin and lysozyme were present in the ATS, the ATS did not include complement proteins and cytokines such as IL-8 and -6 that are known to be present during closed-eye conditions (lactoferrin and lysozyme concentrations are similar in both conditions46). A recent pilot study has shown that in ATS containing human serum (and hence complement products), tear-film neutrophils are still unable to upregulate markers of activation such as Mac-1 and CD66b in the presence of bacteria (Gorbet M, et al. IOVS 2015;56:ARVO E-Abstract 4048), and thus the observed unresponsiveness to PMA, fMLP, and LPS is unlikely to be due to the absence of specific proteins in the medium of incubation. 
The noninflammatory phenotype observed in the tear-film neutrophils may seem in contradiction to the fact that ocular neutrophils are also exposed to high levels of inflammatory mediators such as IL-8 and C3a. Exposure to high levels of C3a may also lead the refractory or noninflammatory phenotype of neutrophils in tears. Such an effect has been previously described with cytokines47 and C5a, which, similarly to C3a, is a potent chemoattractant and inflammatory mediator in neutrophils under normal conditions. Impaired neutrophil function in sepsis has been linked to prolonged exposure to C5a48 and internalization of the C3aR has also been observed as a negative control mechanism.28 
It is also possible that tear-film neutrophils have a distinct phenotype that is not induced by the closed-eye environment but is “inherited.” Several recent studies suggest that unique population of leukocytes exists in blood33,49 and in murine lacrimal glands (Gronert K, et al. IOVS 2014;55:ARVO E-Abstract 1855 and Gao Y, et al. IOVS 2014;55:ARVO E-Abstract 1875). Further investigations are required to determine the origin of the tear-film neutrophil phenotype, to understand how components of the tear film affect the neutrophil phenotype, and to investigate the potential of tear-film neutrophils to contribute to a pro- or anti-inflammatory response. 
Conclusions
The current study highlights how neutrophils collected from the ocular surface following closed-eye conditions are alive and not apoptotic, but in a state where various inflammatory stimuli induce a limited cellular response, as measured by receptor expression and oxidative products. The response of tear-film neutrophils to physiological (fMLP, LPS) and chemical (PMA) stimuli is in contrast to that of blood-isolated neutrophils. The presence of tear-film proteins did not change the tear-film neutrophil response to stimulus, but did somewhat affect receptor expression and stimulus-induced response in blood-isolated neutrophils. This suggests that the noninflammatory or refractory tear-film neutrophils phenotype may be induced in part by exposure to tear-film proteins. Due to their short life-span and limited DNA material, neutrophils have often been considered terminally differentiated with the assumption that minimal changes in gene expression and phenotype occur once they leave the bone marrow. Our study adds to the mounting evidence from the peritoneum,50 lung,51 mouth,36 nose,52 and ocular surface49 that such concepts are ill-defined and blood-isolated neutrophils may not always represent an appropriate model to study mechanism of inflammation in extravasated neutrophils. Further investigations are required to fully phenotype the tear-film neutrophils from the closed-eye environment and determine the mechanisms leading to the observed refractory phenotype. 
Acknowledgments
The authors thank Miriam Heynen, our phlebotomist, and Elena Kreinin for helping with experiments, as well as Doerte Luensmann and Karsten Gronert for fruitful discussion related to inflammation and the ocular surface. 
Supported by grants from 20/20: the Natural Sciences and Engineering Research Council (NSERC) Ophthalmic Materials Network (Hamilton, ON, Canada) as well as NSERC (Ottawa, ON, Canada). 
Disclosure: M. Gorbet, None; C. Postnikoff, None; S. Williams, None 
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Figure 1
 
Ocular cell collection stained with trypan blue. Cells were collected immediately upon awakening. Many viable leukocytes and some dead epithelial cells (blue-stained cells) were present.
Figure 1
 
Ocular cell collection stained with trypan blue. Cells were collected immediately upon awakening. Many viable leukocytes and some dead epithelial cells (blue-stained cells) were present.
Figure 2
 
Ocular cell collection stained with Hoechst. Cells were collected immediately upon awakening, stained with Hoechst, and observed under an epifluorescence microscope. The tear-film neutrophils show the typical multilobed nucleus.
Figure 2
 
Ocular cell collection stained with Hoechst. Cells were collected immediately upon awakening, stained with Hoechst, and observed under an epifluorescence microscope. The tear-film neutrophils show the typical multilobed nucleus.
Figure 3
 
Giemsa stain of tear-film neutrophils.
Figure 3
 
Giemsa stain of tear-film neutrophils.
Figure 4
 
Identification of the neutrophil population in the ocular cell collection using flow cytometry analysis. The neutrophil population was identified by double-gating: a region R1 in the side scatter dot plot to select neutrophils based on cell size and granularity (a); side scatter (SSC-H) versus CD45 fluorescence (FL3-H) dot plots to identify CD45 positive cells (PAN leukocyte marker) based on their granularity (b); expression of CD16 (neutrophil marker) on the double-gated (R1 and R2) population (c). These plots are representative of all experiments performed in this study (n > 12).
Figure 4
 
Identification of the neutrophil population in the ocular cell collection using flow cytometry analysis. The neutrophil population was identified by double-gating: a region R1 in the side scatter dot plot to select neutrophils based on cell size and granularity (a); side scatter (SSC-H) versus CD45 fluorescence (FL3-H) dot plots to identify CD45 positive cells (PAN leukocyte marker) based on their granularity (b); expression of CD16 (neutrophil marker) on the double-gated (R1 and R2) population (c). These plots are representative of all experiments performed in this study (n > 12).
Figure 5
 
Fluorescent histogram for L-selectin expression on tear-film and blood-isolated neutrophils. Black-filled histogram: tear-film neutrophils; black-line histogram: blood-isolated neutrophils. Shedding of L-selectin as shown by the significantly reduced expression of L-selectin is evident on tear-film neutrophils.
Figure 5
 
Fluorescent histogram for L-selectin expression on tear-film and blood-isolated neutrophils. Black-filled histogram: tear-film neutrophils; black-line histogram: blood-isolated neutrophils. Shedding of L-selectin as shown by the significantly reduced expression of L-selectin is evident on tear-film neutrophils.
Figure 6
 
Flow cytometry analysis of caspase activity in tear-film neutrophils (a) and BAK-treated tear-film neutrophils (b). BAK was used as a positive control to induce apoptosis and necrosis. Minimal caspase activity (less than 2%) was observed in tear-film neutrophils. A representative experiment (n = 10) is depicted. BAK, benzalkonium chloride.
Figure 6
 
Flow cytometry analysis of caspase activity in tear-film neutrophils (a) and BAK-treated tear-film neutrophils (b). BAK was used as a positive control to induce apoptosis and necrosis. Minimal caspase activity (less than 2%) was observed in tear-film neutrophils. A representative experiment (n = 10) is depicted. BAK, benzalkonium chloride.
Figure 7
 
Receptor upregulation of tear-film neutrophils collected after sleep (a) and blood-isolated neutrophils (b) following stimulation with LPS, PMA, and fMLP in DMEM/FBS. Fluorescence intensities were recorded by flow cytometry and are expressed as a ratio between stimulated and unstimulated samples. n = 12, mean ± SD. *Significantly different from activation ratio of blood isolated neutrophils (P < 0.001).
Figure 7
 
Receptor upregulation of tear-film neutrophils collected after sleep (a) and blood-isolated neutrophils (b) following stimulation with LPS, PMA, and fMLP in DMEM/FBS. Fluorescence intensities were recorded by flow cytometry and are expressed as a ratio between stimulated and unstimulated samples. n = 12, mean ± SD. *Significantly different from activation ratio of blood isolated neutrophils (P < 0.001).
Figure 8
 
Overlay of fluorescent histograms for Mac-1, ICAM-1, CD54, and CD45 expression on tear-film and blood-isolated neutrophils. Black-filled histogram: unstimulated neutrophils; gray-line histogram: LPS-stimulated neutrophils. The lack of upregulation of receptor expression following LPS-stimulation is seen by the absence of a shift in fluorescence intensity. Histograms are representative of all experiments performed.
Figure 8
 
Overlay of fluorescent histograms for Mac-1, ICAM-1, CD54, and CD45 expression on tear-film and blood-isolated neutrophils. Black-filled histogram: unstimulated neutrophils; gray-line histogram: LPS-stimulated neutrophils. The lack of upregulation of receptor expression following LPS-stimulation is seen by the absence of a shift in fluorescence intensity. Histograms are representative of all experiments performed.
Table 1
 
Composition of Artificial Tear Solution53
Table 1
 
Composition of Artificial Tear Solution53
Table 2
 
Antibodies and Stains Used in Flow Cytometry to Characterize Cell Activation and the Phenotype of Tear-Film Neutrophils
Table 2
 
Antibodies and Stains Used in Flow Cytometry to Characterize Cell Activation and the Phenotype of Tear-Film Neutrophils
Table 3
 
Receptor Upregulation of Neutrophils in Whole Blood After Stimulation With LPS and PMA
Table 3
 
Receptor Upregulation of Neutrophils in Whole Blood After Stimulation With LPS and PMA
Table 4
 
Expression of Membrane Receptor on Unstimulated Tear-Film and Blood-Isolated Neutrophils (Resting Level) Resuspended in DMEM/FBS
Table 4
 
Expression of Membrane Receptor on Unstimulated Tear-Film and Blood-Isolated Neutrophils (Resting Level) Resuspended in DMEM/FBS
Table 5
 
Oxidative Burst Response, as Measured by DCF-HA, by Tear-Film and Blood-Isolated Neutrophils Following fMLP, LPS, and PMA Stimulation in DMEM/FBS
Table 5
 
Oxidative Burst Response, as Measured by DCF-HA, by Tear-Film and Blood-Isolated Neutrophils Following fMLP, LPS, and PMA Stimulation in DMEM/FBS
Table 6
 
Response of Tear-Film and Blood-Isolated Neutrophils to LPS, fMLP, and PMA Stimulation in Artificial Tear Solution
Table 6
 
Response of Tear-Film and Blood-Isolated Neutrophils to LPS, fMLP, and PMA Stimulation in Artificial Tear Solution
Table 7
 
Effect of Stimulating Blood-Isolated Neutrophils in PBS and PBS Supplemented With Lactoferrin (1.80 mg/mL)
Table 7
 
Effect of Stimulating Blood-Isolated Neutrophils in PBS and PBS Supplemented With Lactoferrin (1.80 mg/mL)
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