The Differential Reactive Oxygen Species Production of Tear Neutrophils in Response to Various Stimuli In Vitro.

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.

Tear-Derived Exosome Proteins Are Increased in Patients with Thyroid Eye Disease.

Exosomes contain proteins, lipids, RNA, and DNA that mediate intercellular signaling. Exosomes can contribute to the pathological processes of various diseases, although their roles in ocular diseases are unclear. We aimed to isolate exosomes from tear fluids (TF) of patients with Thyroid eye disease (TED) and analyze the exosomal proteins. TFs were collected from eight patients with TED and eight control subjects. The number of TF exosomes were measured using nanoparticle-tracking analysis. The expression of specific proteins in the purified exosome pellets were analyzed using a Proteome Profiler Array Kit. Cultured normal orbital fibroblasts were incubated with TF exosomes from patients with TED and control subjects, and changes in inflammatory cytokine levels were compared. TF exosomes from TED patients showed more exosomes than the control subjects. The expression levels of exosomal proteins vitamin D-binding (VDB) protein, C-reactive protein (CRP), chitinase 3-like 1 (CHI3L1), matrix metalloproteinase-9 (MMP-9), and vascular adhesion molecule-1 (VCAM-1) were significantly increased in patients with TED, compared to those of controls. Orbital fibroblasts exposed to TF exosomes from patients with TED showed significantly higher levels of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) production than those treated with control TF exosomes. Specific proteins showed higher expression in exosomes from TED patients, implying that they may play keys roles in TED pathogenesis.

[Sjögren's syndrome]. / Sjögren-Syndrom.

Sjögren's syndrome is an autoimmune disease characterized by lymphocytic infiltration of the tear and salivary glands leading to dryness of the mouth and eyes. The awareness that extraglandular manifestations, such as polyneuropathy, arthritis or recurrent airway infections may indicate Sjögren's syndrome is important. In the diagnostic procedure, the tear and saliva production and antibodies against Sjögren's syndrome A (SS-A) and SS-B should be measured. A salivary gland biopsy should be performed when the diagnosis is not still clear. The therapy of oral and ocular dryness is mainly symptomatic whereas the treatment of extraglandular manifestations is based on experience with treatment of these manifestations in systemic lupus erythematosus (SLE).

High Level of Inflammatory Cytokines in the Tears: A Bridge of Patients with Concomitant Exotropia and Dry Eye.

Concomitant exotropia have obvious symptoms of eye discomfort in adults, and the presence of ocular surface inflammation in patients may be important mediators between concomitant exotropia and dry eye. Oculus Keratograph eye comprehensive analyzer was performed to detect noninvasive tear break time, noninvasive tear height, and eye red index, while the ocular surface disease index and schirmer I testing were made. The levels of IL-6, IL-10, IL-17A, IL-12P70, INF-γ, and TNF-α were detected in tears in patients with concomitant exotropia and healthy controls matched by age and gender through the Simoa technology. IL-6 was significantly higher in patients with concomitant exotropia (4.683 ± 1.329) pg/mL than that in normal group (1.455 ± 0.391) pg/mL, p = 0.0304. TNF-α was also significantly higher in patients (0.2095 ± 0.0703) pg/mL than normal group (0.0513 ± 0.0149) pg/mL, p = 0.0397. The levels of inflammatory factors in strabismic patients vs. normal controls were as follows: IL-17A (0.1551 pg/mL︰0.0793 pg/mL), IL-10 (0.3358 pg/mL︰0.0513 pg/mL), IL-12p70 (0.0253 pg/mL︰0.0099 pg/mL), and INF-γ (0.0284 pg/mL︰0.009 pg/mL) were detected, and the median of them in concomitant strabismus was 1.96-6.55-fold as much as the control group. High levels of inflammatory cytokines in tears of patients with concomitant exotropia, which may be a potentially factor promoted the occurrence of dry eye in the patients with concomitant exotropia.

Corneal sub-basal nerve plexus assessment and its association with phenotypic features and lymphocyte subsets in Sjögren's Syndrome.

PURPOSE: To assess and compare corneal sub-basal nerve plexus morphology with circulating lymphocyte subsets, immunologic status and disease activity in Sjögren syndrome (SjS) patients. METHODS: Fifty-five SjS patients, 63 Sicca patients (not fulfilling SjS criteria), 18 rheumatoid arthritis (RA) patients and 20 healthy controls (HC) were included. Systemic disease activity in SjS was assessed with the ESSDAI score. Lymphocyte subpopulations were studied with flow cytometry. Corneal confocal microscopy and ImageJ software were used to characterize corneal sub-basal nerve plexus in terms of nerve density (CNFD), length (CNFL) and tortuosity (CNFT). Conventional dry eye tests were also performed. RESULTS: CNFL and CNFD were lower in SjS, Sicca and RA groups, compared to HC (p < 0.001 for both SjS and Sicca); CNFL p = 0.005, CNFD p = 0.018 in RA). CNFT was higher in SjS, followed by Sicca, RA and HC. A negative correlation was found between ESSDAI score and CNFL (r=-0.735, p = 0.012). CNFL correlated negatively with IL21+ CD8+ T cells (r=-0.279, p = 0.039) and a positively with total memory (r = 0.299, p = 0.027), unswitched memory (r = 0.281, p = 0.038) and CD24Hi CD27+ (r = 0.278, p = 0.040) B cells. CNFD showed a tendency to significance in its negative correlation with ESSDAI (r=-0.592, p = 0.071) and in its positive correlation with switched memory B cells (r = 0.644, p = 0.068). CONCLUSIONS: This is the first study aiming to correlate ocular findings with lymphocyte subsets in SjS. The associations founded between CNFL and CNFD and disease activity, IL21+ follicular T cells and some B-cell subsets suggest that corneal nerve damage may parallel systemic disease activity and inflammatory cells' dynamics.

Exfoliating goblet cell hyperplasia mimicking pterygium: a clinicopathological case report.

We present an interventional case report of exfoliating goblet cell hyperplasia mimicking pterygium. A 44-year-old male with bilateral lesions of the nasal bulbar conjunctiva extending over the cornea, consistent with pterygia, underwent surgical excision of the lesion in his left eye. Histopathologic examination revealed an exophytic lesion made up of proliferated goblet cells with benign cytologic features. Some of the goblet cells were atrophic and seemed to be desquamating from the lesion surface. Cytologic examination of a tear specimen collected from the right eye revealed the presence of exfoliated goblet cells admixed with mucin material. The lesion had not recurred three months after surgery. Exfoliating goblet cell hyperplasia, a condition not previously reported on the ocular surface, may mimic pterygium. Histopathologic examination is required to establish the diagnosis.

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

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.

Tear film analysis and evaluation of optical quality: A review of the literature.

Dry eye is a complex multifactorial disease of the ocular surface and tears. It is associated with ocular surface symptoms and is one of the most common causes for ophthalmologic consultation. Despite their frequent use in clinical practice, the usual tests to evaluate dry eye and ocular surface disease-history of symptoms, tear break-up time (TBUT), Meibomian gland evaluation, corneal fluorescein staining, Schirmer test-have shown low reproducibility and reliability. In addition, subjective symptoms are often weakly or poorly correlated with objective signs. Since the tear film is the first system through which light must pass, the optical quality of the eye is highly dependent on the homogeneity of the tear film. Various investigative methods have been developed to evaluate both the structural and functional quality of the tear film, such as corneal topography, interferometry, tear meniscus measurement, evaporation rate, tear osmolarity and even aberrometry. Some are easily accessible to clinicians, while others remain in the field of clinical research. All of these tests provide a better understanding of the pathophysiology of the tear film. This review hopes to provide an overview of the existing tests and their role in evaluating the significance of the tear film in visual function.

Trypsin inhibitory capacity in vernal keratoconjunctivitis.

PURPOSE: To study tear trypsin inhibitory capacity (T-TIC), serum trypsin inhibitory capacity (S-TIC) and their relationship to matrix metalloproteinases (MMP)-1 and -9 in patients with vernal keratoconjunctivitis (VKC). METHODS: In the first phase of the study, inactivation of alpha-1 antitrypsin (AAT) by MMP-1 and -9 was investigated in vitro. Subsequently, tear samples were collected after clinical evaluation from 14 patients with active VKC and 15 normal control subjects. Tear cytology was performed on all samples. Levels of T-TIC and S-TIC were determined by spectrophotometry, whereas levels of tear pro-MMP-1, pro-MMP-9, and active MMP-1 and -9 were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: MMP-1 and -9 inactivated AAT in vitro. S-TIC was significantly higher (P < 0.0001), and T-TIC (P < 0.0001) significantly lower in VKC samples. Tear levels of pro-MMP-1 and pro-MMP-9, and the activity of MMP-1 and -9 was significantly greater in patients with VKC than in healthy subjects (P < 0.0001). There was no significant correlation between T-TIC, MMP-1, and MMP-9 activity. In addition, T-TIC was not correlated to the total clinical score or to single clinical sign scores. CONCLUSIONS: In this study, tear trypsin inhibitory capacity was shown to be reduced and tear MMP-1 and -9 activity increased in patients with VKC. Although T-TIC did not correlate with VKC severity, a local reduced inhibitory capacity of ATT may facilitate or prolong conjunctival inflammation in VKC.

Non-contact confocal microscopy of the tear film in unoperated eyes.

PURPOSE: The NIDEK ConfoScan4 (CS4) is a digital scanning slit confocal microscope. The corneal structure and tear film can be viewed, magnified, measured, and photographed at magnifications up to 500x, in vivo, in a noninvasive manner. The objective of this study was to evaluate and illustrate various conditions related to dry eye using the CS4 confocal microscope with the 20x noncontact lens. METHODS: The CS4 was used to evaluate the natural tear film in 58 eyes of 29 patients with normal examinations, allergic conjunctivitis, nonspecific conjunctivitis, and dry eyes. In a subset of this patient population, subjective and objective findings were used to classify mild, moderate, and severe dry eye disease states. The usefulness of confocal microscopy as an objective tool to diagnose and manage different tear film-related ocular disease was also evaluated. RESULTS: The differences in tear film composition were visible using confocal microscopy. Photographs demonstrate confocal noncontact 20x microscopy as a diagnostic tool. CONCLUSIONS: Noncontact confocal microscopy is a valuable tool in the diagnosis and treatment of dry eye syndrome and other ocular states such as allergic and nonspecific conjunctivitis. It provides a simple and effective way to observe, classify, and treat the tear film. As investigators visualize and learn more, understanding of this structure will continue to improve.

A pilot study on the infiltrating cells and cytokine levels in the tear of fungal keratitis patients.

AIM: To determine the cellular profile and cytokine levels in the tear fluid of fungal keratitis patients. MATERIALS AND METHODS: Tear samples were collected from six fungal keratitis patients (Group I) from active stages of the disease up to resolution. Tears collected from the following served as controls: uninfected fellow eye (Group II A) of Group I, patients undergoing cataract surgery (Group II B) and acute conjunctivitis (Group II C). The cellular profile was evaluated. Interleukines (IL-6, IL-8 and IL-1beta) were estimated using sandwich enzyme immunoassay. Statistical analysis was carried out using nonparametric two-sample median test. RESULTS: Polymorphonuclear leukocytes (PMN) were the predominant infiltrating cells in Group I. During the initial stages of fungal infection, levels of IL-6 and IL-8 in the tear samples were found to be significantly increased when compared with Group II A (P=0.019 for IL-6, P<0.001 for IL-8). This was also true for IL -8 (P=0.008) levels in Group I and Group II B). While IL-6 levels decreased significantly towards healing, IL-8 remained slightly elevated even after healing. These cytokines were at the base level in Group II A. Lymphocytes and PMN were present in equal proportions in Group II C, which showed elevated levels of cytokines but not to the extent of Group I. CONCLUSION: This horizontal study indicates that understanding the nature of the inflammatory response in the tears of fungal keratitis patients is of considerable interest and warrants further investigations.

[Morphometric analysis of the structure of tear and aqueous humor in senile cataract].

The parameters of the wedge dehydrated samples of aqueous humor and tear taken from patients with senile cataract were compared by a morphometric assay. Computer-aided processing of the images of the test biological fluids was made on a Morphotest-1 hardware-software complex. The study has provided evidence for the uniformity of distribution of major structural components in the dehydrated samples of tear and aqueous humor in each specific examinee. This fact opens up possibilities for studying the signs of different intraocular pathological conditions from the morphological pattern of tear.

Effect of overnight orthokeratology on conjunctival goblet cells.

OBJECTIVE: To evaluate the differences between goblet cell density (GCD) and symptomatology after one month of orthokeratology lens wear. METHODS: A pilot, short-term study was conducted. Twenty-two subjects (29.7±7.0 years old) participated voluntarily in the study. Subjects were divided into two groups: habitual silicone hydrogel contact lens wearers (SiHCLW) and new contact lens wearers (NCLW). Schirmer test, tear break up time (TBUT), Ocular Surface Disease Index (OSDI) questionnaire and conjunctival impression cytology. GCD, mucin cloud height (MCH) and cell layer thickness (CLT) were measured. All measurements were performed before orthokeratology fitting and one month after fitting to assess the evolution of the changes throughout this time. RESULTS: No differences in tear volume and TBUT between groups were found (p>0.05). However, the OSDI score was statistically better after one month of orthokeratology lens wear than the baseline for the SiHCLW group (p=0.03). Regarding the goblet cell analysis, no differences were found in CLT and MCH from the baseline visit to the one month visit for the SiHCLW compared with NCLW groups (p>0.05). At baseline, the GCD in the SiHCLW group were statistically lower than NCLW group (p<0.001). There was a significant increase in GCD after orthokeratology fitting from 121±140cell/mm(2) to 254±130cell/mm(2) (p<0.001) in the SiHCLW group. CONCLUSION: Orthokeratology improves the dry eye subject symptoms and GCD after one month of wearing in SiHCLW. These results suggest that orthokeratology could be considered a good alternative for silicone hydrogel contact lens discomfort and dryness.

Interobserver variability of an open-source software for tear meniscus height measurement.

PURPOSE: Different values of the lower tear meniscus height (TMH) can be obtained depending on the method and technique of measurement employed. The aim of this study was to analyse the interobserver variability of a method for measuring TMH by using an open source software. MATERIAL AND METHODS: On a group of 176 subjects, two videos of the central lower tear meniscus, first under slit-lamp illumination and ten minutes later under Tearscope illumination, were generated by a digital camera attached to a slit-lamp. Images were extracted from each video by a masked observer. Two further observers measured in a masked and randomized order the TMH in each illumination method by using an open source software based on Java (NIH ImageJ). TMH was measured from the lower lid to the upper limit of the tear meniscus for both slit-lamp (TMH-SL) and Tearscope (TMH-Tc) illumination methods. Subsequently, in different order, observers assigned a four-grading and a healthy/abnormal subjective classification to each central meniscus. RESULTS: No significant differences were found between the TMH measurements obtained by both investigators in slit-lamp or Tearscope image datasets (t-test; both p≥0.136). When comparing TMH measurements stratified by grade, only interobserver significant differences were observed for grades 3 and 4 with silt-lamp (t-test; both p≤0.009). Significant differences on TMH results between subjective subgroups were observed for both illumination techniques (ANOVA, all p≤0.045). CONCLUSION: This study showed a useful tool to objectively measure TMH by photography.

Evaluating tear film stability in the human eye with high-speed videokeratoscopy.

High-speed videokeratoscopy (HSV) is an emerging technology that has the potential to acquire information on the dynamics of corneal topography and tear-film behavior. We show that the surface regularity and asymmetry indices, which are traditionally used for characterizing the stability of precorneal tear film, have limitations in the context of HSV because they are highly sensitive to natural ocular microfluctuations. To overcome this problem, we propose a new microfluctuation-independent surface indicator. It is based on the root-mean-square of the error of the parametric model fit to the surface. Further, we develop techniques for estimating the tear film build-up and break-up times. The tear film build-up time estimator is based on the proposed RMS fit surface indicator while the tear film break-up time estimator is derived directly from a set of consecutive HSV digital images, without the need for estimating the resulting corneal surface.

Goblet cell density association with tear function and ocular surface physiology.

PURPOSE: To determine the relationship of goblet cell density (GCD) with tear function and ocular surface physiology. METHODS: This was a cross-sectional study conducted in 35 asymptomatic subjects with mean age 23.8±3.6 years. Tear film assessment, conjunctiva and cornea examination were done in each subject. Conjunctival impression cytology was performed by applying Nitrocellulose Millipore MF™-Membrane filter over the superior bulbar conjunctiva. The filter paper was than fixed with 96% ethanol and stained with Periodic Acid Schiff, Hematoxylin and Eosin. GCD was determined by optical microscopy. Relation between GCD and Schirmer score, tear break-up time (TBUT), bulbar redness, limbal redness and corneal staining was determined. RESULTS: The mean GCD was 151±122 cells/mm(2). GCD was found higher in eyes with higher Schirmer score but it was not significant (p=0.75). There was a significant relationship of GCD with TBUT (p=0.042). GCD was not correlated with bulbar redness (p=0.126), and limbal redness (p=0.054) as well as corneal staining (p=0.079). No relationship of GCD with age and gender of the subjects (p>0.05) was observed. CONCLUSION: GCD was found correlated with TBUT but no significant correlation was found with the aqueous portion of the tear, limbal as well as bulbar redness and corneal staining.

Non invasive assessment of the human tear film dynamics.

Dry eye disease, or keratoconjunctivitis sicca, is a multifactorial syndrome with altered tear film homeostasis leading to ocular irritations. These alterations cause discomfort and stress for the patient, but only a few objective parameters allow for proper differential diagnosis into different subtypes of this condition. The mostly invasively performed standard assessment procedures for tear film diagnosis are manifold, but often correlate quite poorly with the subjectively reported symptoms. Due to the inherent limitations, e.g. the subjectivity of the commonly performed invasive tests, a number of devices have been developed to assess the human tear film non-invasively. Since the production, delivery, distribution and drainage of the tear film is a dynamic process, we have focused our review on non-invasive methods which are capable of continuous or repetitive observations of the tear film during an inter-blink interval. These dynamic methods include (1) Interferometry, (2) Pattern Projection, (3) Aberrometry, (4) Thermography; and (5) Evaporimetry. These techniques are discussed with respect to their diagnostic value, both for screening and differential diagnostic of Dry Eye Disease. Many of the parameters obtained from these tests have been shown to have the potential to reliably discriminate patients from healthy subjects, especially when the tests are performed automatically and objectively. The differentiation into subtypes based solely on a single, dynamic parameter may not be feasible, but the combination of non-invasively performed procedures may provide good discrimination results.

Repeatability of tear meniscus evaluation using spectral-domain Cirrus® HD-OCT and time-domain Visante® OCT.

PURPOSE: To investigate the intra-rater, inter-rater and inter-device repeatability of a spectral-domain OCT (Cirrus) and a time-domain OCT (Visante) for tear meniscus height (TMH) and area (TMA) measurements. METHODS: 20 participants with no known eye disease were recruited. Both eyes of participants were imaged with both OCTs under the similar conditions. The inferior tear meniscus was imaged at 6 o'clock position whereas the superior meniscus was imaged at 12 o'clock position. Data from the right eyes was analyzed. Two raters independently measured TMH and TMA using the OCT images, and one rater repeated the measurements. Intra-rater, inter-rater and inter-device repeatability of measurements were assessed using Bland-Altman plots and pooled standard deviation. RESULTS: For intra-rater repeatability, TMH and TMA measurements were more repeatable in Cirrus than Visante (95% limits of agreement (LOA): TMH (µm), -22 to 66 (Cirrus), -125 to 45 (Visante); TMA (µm(2)), -1632 to 5331 (Cirrus), -38,050 to 21,874 (Visante)). For inter-rater agreement, TMH and TMA were also more repeatable in Cirrus than Visante (95%LOA: TMH (µm), -29 to 107 (Cirrus), -215 to 252 (Visante); TMA (µm(2)), -6650 to 9567 (Cirrus), -33,119 to 39,272 (Visante)). Inter-device agreement was poor (95%LOA: TMH (µm), -158 to 150; TMA (µm(2)), -32,903 to 14,076). There was no significant difference in inferior TMH between Cirrus and Visante (p>0.05). Inferior TMA was significantly lower in Cirrus by a mean difference of 10,223µm(2) (95% confidence interval, 5479, 14,966) (p=0.0002). CONCLUSION: Spectral-domain OCT is superior to time-domain OCT for intra-rater and inter-rater repeatability of TMH and TMA measurements.

The Noninflammatory Phenotype of Neutrophils From the Closed-Eye Environment: A Flow Cytometry Analysis of Receptor Expression.

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.