Dry eye disease and microbial keratitis: is there a connection?

Dry eye is a common ocular surface disease of multifactorial etiology characterized by elevated tear osmolality and inflammation leading to a disrupted ocular surface. The latter is a risk factor for ocular surface infection, yet overt infection is not commonly seen clinically in the typical dry eye patient. This suggests that important innate mechanisms operate to protect the dry eye from invading pathogens. This article reviews the current literature on epidemiology of ocular surface infection in dry eye patients and laboratory-based studies on innate immune mechanisms operating at the ocular surface and their alterations in human dry eye and animal models. The review highlights current understanding of innate immunity in dry eye and identifies gaps in our knowledge to help direct future studies to further unravel the complexities of dry eye disease and its sequelae.

Effect of TGF-ß on ocular surface epithelial cells.

A role for transforming growth factor (TGF)-ß in the pathogenesis of some ocular surface diseases has been proposed. We determined if secretion of TGF-ß and expression of TGF-ß receptors RI, RII, and RIII by human ocular surface epithelial cells were modified under inflammatory conditions. We also determined how these cells responded to TGF-ß. A human corneal epithelial (HCE) cell line and a conjunctival epithelial cell line (IOBA-NHC) were exposed to TGF-ß1 and -ß2 and to proinflammatory cytokines. TGF-ß receptor mRNAs were analyzed by real time reverse transcription polymerase chain reaction (RT-PCR) in both cell lines, and in conjunctival, limbal, and corneal epithelial cells from post-mortem human specimens. Expression of TGF-ß receptors and pSMAD2/SMAD2 were determined by Western blot and immunofluorescence assays. Secretion of TGF-ß isoforms, cytokine/chemokine, and metalloproteinases (MMPs) were analyzed in cell supernatants by immunobead-based assays. Secretory leukocyte proteinase inhibitor (SLPI) secretion was analyzed by enzyme-linked immunosorbent assay. TGF-ß isoform and receptor gene expression was determined by RT-PCR in conjunctival epithelium of dry eye (DE) patients and healthy subjects. Our results showed that TGF-ß RI expression was down-regulated with IL-4 exposure, whereas TGF-ß RII and TGF-ß2 were upregulated by TNF-α in HCE cells. TGF-ß RIII receptor expression was upregulated in IOBA-NHC cells by TNF-α and IFN-γ. SMAD2 phosphorylation occurred in HCE and IOBA-NHC cells after TGF-ß treatment. TGF-ß significantly up- and down-regulated secretion of several cytokines/chemokines by both cell lines and MMP by HCE cells. TGF-ß2 and TGF-ß3 were upregulated and TGF-ß RIII mRNA was down-regulated in DE conjunctival epithelium. These results show that TGF-ß plays an important role in directing local inflammatory responses in ocular surface epithelial cells.

Ocular mucin gene expression levels as biomarkers for the diagnosis of dry eye syndrome.

PURPOSE: To evaluate mRNA levels of the ocular mucins MUC1, MUC2, MUC4, MUC5AC, and MUC7 in conjunctival impression cytology samples from patients with moderate to severe dry eye syndrome (DES) compared with a population of healthy subjects; and to investigate the use of the levels of these mucin genes as biomarkers of DES and subsequently as a potential diagnostic test for DES. METHODS: This prospective study commenced in the year 2000 and ended in the year 2009. Thirty-eight patients with DES and 43 age- and sex-matched healthy subjects completed the initial part of the study. Investigations were repeated at a later stage in 16 healthy subjects and 30 patients with DES, which were used as external validation data. Conjunctival impression cytology was performed in all subjects to test gene expression of ocular mucin genes MUC1, MUC2, MUC4, MUC5AC, and MUC7. Statistical analysis was performed to determine whether there was a difference in the levels of mucin gene expression between the two groups of subjects. Sensitivity and specificity of mucin gene expression for the diagnosis of DES was calculated. RESULTS: Expressions of MUC1, MUC2, MUC4, and MUC5AC (P < 0.0001) were significantly lower in conjunctival epithelium of patients with DES compared with that in normal subjects. These results were replicated in the external control subject and patient groups. MUC1 expression levels demonstrated the greatest sensitivity (83.3%) and specificity (87.5%) among all genes tested. CONCLUSIONS: The data strongly suggest that the expression levels of MUC1 may be used as a diagnostic test in DES for investigational and selective clinical trials.

Advancements in anti-inflammatory therapy for dry eye syndrome.

PURPOSE: The goal of this literature review is to discuss recent discoveries in the pathophysiology of dry eye and the subsequent evolution of diagnostic and management techniques. The mechanisms of various anti-inflammatory treatments are reviewed, and the efficacy of common pharmacologic agents is assessed. Anti-inflammatory therapy is evaluated in terms of its primary indications, target population, and utility within a clinical setting. METHODS: The Medline PubMed database and the World Wide Web were searched for current information regarding dry eye prevalence, pathogenesis, diagnosis, and management. After an analysis of the literature, major concepts were integrated to generate an updated portrayal of the status of dry eye syndrome. RESULTS: Inflammation appears to play a key role in perpetuating and sustaining dry eye. Discoveries of inflammatory markers found within the corneal and conjunctival epithelium of dry eye patients have triggered recent advancements in therapy. Pharmacologic anti-inflammatory therapy for dry eye includes 2 major categories: corticosteroids and immunomodulatory agents. Fatty acid and androgen supplementation and oral antibiotics have also shown promise in dry eye therapy because of their anti-inflammatory effects. CONCLUSIONS: Anti-inflammatory pharmacologic agents have shown great success in patients with moderate to severe dry eye when compared with alternative treatment modalities. A deeper understanding of the link between inflammation and dry eye validates the utilization of anti-inflammatory therapy in everyday optometric practice.

Interleukin-1 receptor-1-deficient mice show attenuated production of ocular surface inflammatory cytokines in experimental dry eye.

PURPOSE: To compare inflammatory cytokine and defensin expression in response to experimental dry eye (EDE) in interleukin-1 receptor-1 (IL-1R1)-deficient (KO) mice with age-matched wild-type mice (WT). METHODS: EDE was induced by subcutaneous scopolamine injection, exposure to low humidity, and an air draft for 5 days in 4- to 6-week-old KO and WT mice. Expression of cytokines IL-1 alpha, IL-1 beta, tumor necrosis factor (TNF)-alpha, IL-6, and mouse beta-defensins (mBD)-1, mBD-2, and mBD-3 was evaluated by real-time polymerase chain reaction in scraped corneal epithelial cells and whole conjunctival tissues. A multiplex bead assay was performed to quantitate IL-1 alpha, IL-2, IL-4, IL-10, interferon (IFN)-gamma, and TNF-alpha levels in tear fluid, and an enzyme immunoassay was used to quantitate IL-1 beta levels in tear fluid. RESULTS: EDE significantly increased RNA transcripts for IL-1 alpha and beta in the conjunctiva and for TNF-alpha in the corneal epithelium of WT mice. Levels of IL-1 alpha, IL-1 beta, and IL-6 were significantly lower in the corneal epithelium and conjunctiva, and TNF-alpha was significantly lower in the cornea of KO mice after 5 days of EDE than WT mice. Tear fluid IL-1 alpha concentration increased above baseline on days 2-4 of EDE in WT and KO mice. A similar pattern was observed for tear TNF-alpha. Tear IL-1 beta increased throughout the 5 days of EDE in WT and KO mice. IFN-gamma, IL-2, IL-4, and IL-10 were undetectable in tear fluid of either strain before or after EDE. Corneal mBD-1 mRNA expression was unchanged and conjunctival mBD-1 transcripts decreased in WT and increased in KO mice with EDE. Untreated WT corneas, but not those of KO mice, expressed mBD-2 transcripts, whereas in the conjunctiva, mBD-2 increased in WT and decreased in KO mice with EDE. Corneal mBD-3 mRNA expression was undetected in WT mice, but increased after EDE in KO mice. Conjunctival mBD-3 transcripts were only detected in WT with EDE. CONCLUSIONS: These findings indicate that IL-1 signaling is responsible in part for the increased expression of inflammatory cytokines and the changes in mBDs by the ocular surface tissues in response to desiccating stress. These results show the important regulatory aspects of IL-1 on ocular surface epithelial inflammation.

In vitro activity of human beta-defensin 2 against Pseudomonas aeruginosa in the presence of tear fluid.

Pseudomonas aeruginosa causes vision-threatening keratitis and is difficult to treat due to emerging resistance. Human beta-defensin 2 (hBD-2) is an antimicrobial peptide expressed by ocular surface epithelia with broad-spectrum activity against various pathogens, including P. aeruginosa. The activity of hBD-2 against P. aeruginosa in the presence of human tears or NaCl was studied. In some experiments, tears were heat-inactivated, filtered, and separated into cationic/anionic fractions or mucin MUC5AC was removed by immunoprecipitation before use. Immunoprecipitation was performed to study the interaction between hBD-2 and MUC5AC. hBD-2 activity was reduced by 40 to 90% in the presence of 17.5 to 70% (vol/vol) tears. NaCl reduced hBD-2 activity, but at most it could account for only 36% of the inhibitory effect of tears. Heat inactivation and filtration attenuated the ability of tears to inhibit hBD-2 activity by 65 and 68%, respectively. Anionic tear fractions significantly reduced (86%) the activity of hBD-2, whereas only a 22% reduction was observed with the cationic fractions. In the absence of MUC5AC, the activity of hBD-2 was restored by 64%. Immunoprecipitation studies suggested that the loss of hBD-2 activity in tears is due to a direct binding interaction with MUC5AC. Our data showed that the antimicrobial activity of hBD-2 is sensitive to the presence of human tears and that this is partly due to the salt content and also the presence of MUC5AC. These data cast doubt on the effectiveness of hBD-2 as an antimicrobial peptide, and additional studies are required to conclusively elucidate its role in innate immunity at the ocular surface in vivo.

Effect of hyperosmolality on beta-defensin gene expression by human corneal epithelial cells.

PURPOSE: As human beta-defensins (hBD) are important antimicrobial peptides at epithelial surfaces, including the ocular surface, we tested the effect of hyperosmolar conditions on the expression of these peptides by human corneal epithelial cells (HCECs). METHODS: Simian virus 40-transformed HCECs (n = 5) or primary cultured HCECs (n = 5) were treated with serum-free media or serum-free hyperosmolar (400-500 mOsm/kg) media for 24 hours or serum-free 500 mOsm/kg media for 12 to 48 hours. The effect of hyperosmolality on interleukin-1beta (IL-1beta)-induced hBD-2 expression was also tested. IL-6 expression was studied as a marker of IL-1beta function. Expression of hBD-1, -2, and -3 and IL-6 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). The levels of active IL-1beta (culture supernatants and cell lysates) and pro-IL-1beta (cell lysates) were detected by enzyme-linked immunosorbent assay. RESULTS: HCECs constitutively expressed hBD-1 and -3 but not hBD-2. Hyperosmolar media had no effect on the basal expression of hBD-1 or -3 and did not induce the expression of hBD-2. Treatment with 500 mOsm/kg media for 24 hours decreased the ability of IL-1beta to upregulate hBD-2 and IL-6 expression. Active or pro-IL-1beta was not detected in any cell culture sample. CONCLUSION: Our results suggest that the hyperosmolar environment observed in diseases such as dry eye does not alter defensin expression. However, a hyperosmolar environment may influence cytokine function in ocular surface cells and thus affect their response to injury and inflammation.

Conjunctival cytokine expression in symptomatic moderate dry eye subjects.

PURPOSE: To compare ocular surface cytokine expression in healthy controls and subjects with moderate dry eye and to study the ability of interleukin (IL)-1beta to modulate cytokine expression in cultured human conjunctival epithelial cells (CECs). METHODS: Subjective (symptom questionnaire) and objective (tear osmolality, fluorescein tear break-up time [TBUT]) measures of dry eye were determined in five healthy controls and five subjects with moderate dry eye. Tear clearance rates were measured with a fluorophotometer. Enzyme immunoassay and a cytokine bead assay were used to quantify IL-1beta in tear fluid. RT-PCR was performed to detect expression of IL-1beta, IL-6, IL-8, growth-related oncogene (GRO)-beta, intercellular adhesion molecule (ICAM)-1, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and ephrin A5 in conjunctival impression cytology (CIC) samples and in CECs (IOBA-NHC cell line, n=3; primary cultured CEC, n=3) exposed to 10 ng/mL IL-1beta for 6 hours. RESULTS: Subjects with moderate dry eye had significantly higher symptom scores, higher tear osmolality, and shorter TBUT than healthy controls. Subjects with dry eye demonstrated slightly slower tear clearance (13.1% per minute) than healthy controls (15.4% per minute). Very low levels of IL-1beta protein were detected in the tear fluid of both groups. TRAIL was constitutively expressed in CIC samples, whereas IL-1beta, IL-6, and GRO-beta were absent. Weak expression of IL-8 (two healthy, four dry eye), ICAM-1 (four healthy, four dry eye), and ephrin A5 (one healthy, two dry eye) was observed. IL-1beta upregulated its own expression and that of IL-6, IL-8, GRO-beta, and ICAM-1 in cultured CECs but not that of ephrin A5 or TRAIL. CONCLUSIONS: The lack of major differences in ocular surface cytokine expression between the two groups of subjects implies other inflammatory pathways or etiologies are involved in moderate dry eye. Although IL-1beta modulated the expression of various cytokines in cultured CECs, its absence in tear fluid and CIC samples suggests that IL-1beta does not play a modulatory role in moderate dry eye.

The diagnosis and characteristics of moderate dry eye in non-contact lens wearers.

PURPOSE: To identify and characterize moderate dry eye in non-contact lens wearers with a new scoring system-based dry eye questionnaire and to determine which objective tests better differentiate patients with moderate dry eye from healthy patients. METHODS: Fifty-two healthy subjects (21 women and 31 men with a mean age of 27.8 +/- 9.2 years) and 37 subjects with moderate dry eye (33 women and 4 men with a mean age of 36.4 +/- 12.9 years) completed a 42-item dry eye questionnaire. Seventeen healthy subjects (11 women and 6 men with a mean age of 30.5 +/- 9.7 years) and 28 subjects with moderate dry eye (24 women and 4 men with a mean age of 38.50 +/- 3.8 years) underwent additional objective assessment of ocular surface health, tear osmolality, tear stability, and tear volume. RESULTS: Subjects with moderate dry eye scored significantly higher (49.8 +/- 20.3, P<0.0001) on the dry eye questionnaire than did normal subjects (11.7 +/- 10.3). Ocular irritation symptoms worsened with progression of time of day in both groups of subjects. Internal reliability (0.95 Cronbach alpha) was excellent, and concurrent validity (Spearman rho 0.507) was acceptable when compared to the McMonnies and Ho dry eye questionnaire. Significant differences in tear osmolality (P<0.00001), invasive tear breakup time (P<0.034), and corneal vital dye staining (P<0.0001) were detected between the two groups of subjects. A stepwise linear regression on objective clinical tests, however, did not account for 77% of the total variance in the questionnaire scores. CONCLUSIONS: A unique scoring system-based dry eye questionnaire was validated to separate non-contact lens wearers with moderate dry eye from healthy subjects. Objective tests of tear osmolality and stability and ocular surface integrity were better than other clinical measures at identifying differences between the two subject groups. The results strongly support the evidence that the diagnosis and treatment of moderate dry eye requires a detailed assessment of self-perceived symptoms and that objective clinical testing alone may be insufficient.

The effect of interleukin-1 on cytokine gene expression by human corneal epithelial cells.

The purpose of this study was to characterize the pattern of cytokine gene expression by human corneal epithelial cells (HCEC) in response to interleukin-1 (IL-1). Primary cultured HCEC (P-HCEC) or SV40 transformed HCEC (SV40-HCEC) were treated for 6 hr with serum-free growth-media alone or with recombinant human IL-1beta or IL-1alpha (10 ng ml(-1)). 33P labeled cDNA was generated from total RNA, then hybridized to a human cytokine expression array. An autoradiograph was generated for each experimental condition and results analysed semi-quantitatively. Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect mRNA for IL-8, growth related oncogene-beta (GRO-beta), intercellular adhesion molecule (ICAM)-1 and Ephrin A5. P-HCEC and SV40-HCEC demonstrated comparable cytokine profiles. For P-HCEC (n=2) the expression of 35 genes was upregulated or only detectable following IL-1beta treatment whereas the expression of nine genes was downregulated or undetectable after IL-1beta treatment. In SV40-HCEC (n=3), the expression of 48 genes was upregulated or only detectable following IL-1beta treatment and the expression of 10 genes was downregulated or undetectable after IL-1beta treatment. Some genes that demonstrated increased expression included cadherin-5, ICAM-1, GRO-alpha, GRO-beta, GRO-gamma, Activin A (bA subunit), tumor necrosis factor-alpha, IL-6, and IL-8. Genes that showed decreased expression included the chemokine receptor-CXCR-4, ciliary neurotrophic factor (CNTF), c-kit ligand, Ephrin A5, G-protein coupled receptor RDC-1 and FGF family FGFR2. Bayesian analysis of the SV40-HCEC data (n=3) revealed the expression of 15 genes that were significantly (p<0.05) differentially regulated. Within these 15 genes, the expression of chemokines (GRO-alpha, GRO-beta, IL-8), fibroblast growth factor 13 and the cytokine IL-6 were the most upregulated, while ephrin A5 and chemokine receptor-4 were the most downregulated. IL-1alpha treatment (n=1 P-HCEC; n=1 SV40-HCEC) produced results very similar to IL-1beta treatment. RT-PCR revealed differential regulation of IL-8, GRO-beta, ICAM-1 and ephrin A5 in accordance with gene array data. In conclusion, the data demonstrate that IL-1 treatment of HCEC differentially regulates the expression of other cytokine and related genes, thus adding to the body of evidence that IL-1 is a major mediator of ocular surface inflammatory reactions. Since the expression of a large number of genes can be studied simultaneously, gene array studies such as these offers the advantage of understanding global changes in response to a specific stimulus. Thus our study provides insight in to the ocular surface response in conditions of inflammation and corneal wound healing where the levels of IL-1 are known to be increased.

A comparison of tear volume (by tear meniscus height and phenol red thread test) and tear fluid osmolality measures in non-lens wearers and in contact lens wearers.

PURPOSE: Various measures are available to assess the tear film, yet little specific information is available on how they relate to each other. An exploratory study was undertaken to assess three measures and their relationship in non-contact lens wearers and in contact lens wearers. METHODS: Forty-three young subjects (mean age, 25.0 +/- 3.1 years; 19 men and 24 women) without overt ocular disease were recruited and categorized into four similarly sized groups based on contact lens wear (no lens wear, conventional daily wear hydrogels, silicone hydrogel lenses worn on a continuous basis, and gas-permeable contact lenses). Sets of measures, in random order and from both eyes whenever possible, were made using a phenol red thread (PRT) test over 15 seconds (open eye), biomicroscopy to assess tear meniscus height (TMH) from a perpendicular perspective using a 0.05-mm resolution graticule, and a borosilicate glass micropipette used to collect a 5-microL sample of tears for assessment of osmolality by vapor pressure measures. RESULTS: For the complete group of subjects, the TMH data averaged 0.22 +/- 0.07 mm; the average PRT wetting length was 18.0 +/- 6.1 mm; and the tear osmolality averaged 317 +/- 28 mOsm/kg. The intereye differences averaged 0.04 mm for TMH, 3.7 mm for PRT, and 15 mOsm/kg for tear osmolality. There were no detectable sex-related differences in the measures. Compared with the control group (average, 0.25 mm), the TMH data showed a trend to be lower in daily hydrogel (0.21 mm) and silicone hydrogel (0.20 mm) lens wearers, but not in gas-permeable lens wearers (0.24 mm). PRT data was bimodally distributed, with the control group showing slightly higher (average, 21.1 mm) wetting compared with hydrogel lens wearers (16.7 and 17.4 mm) and gas-permeable lens wearers (average, 17.3 mm). Hydrogel (319 mOsm/kg for both groups) and gas-permeable lens wearers (average, 324 mOsm/kg) had higher tear osmolality measures compared with the control group (average, 305 mOsm/kg). Although some of the differences approached statistical significance, any statistical differences were evident only after outliers were removed. However, on pooling all data, there was a statistically significant positive correlation between TMH and open-eye PRT measures (P < 0.001) and an indication of a negative correlation between open-eye PRT and tear osmolality measures. CONCLUSIONS: Even contemporary contact lens wear can have a small but measurable impact on the precorneal tear film osmolality or volume. The changes are internally consistent and, overall, support the idea that the PRT test provides a useful measure of tear meniscus volume.

The association of bulbar conjunctival folds with other clinical findings in normal and moderate dry eye subjects.

BACKGROUND: Conjunctival folds have been reported as incidental findings in routine biomicroscopic examination, especially related to patients with dry eye disease. We compared the number of lower temporal lid-parallel conjunctival folds (LIPCOF) in normal and moderate dry-eyed patients with specific biomicroscopic findings (corneal staining, limbal injection, and bulbar conjunctival injection) and tear osmolality. METHODS: Fourteen moderate dry eye subjects (12 female, 2 male; mean age, 43.4 years--range, 23 to 71 years) and eight normal subjects (3 female, 5 male; mean age, 37.25 years--range, 20 to 55 years) participated in this study. Bulbar conjunctival folds were quantified in the temporal aspect of each eye following fluorescein instillation. Tear osmolality was measured with a vapor pressure osmometer. RESULTS: There was a slight difference in the number of bulbar conjunctival folds between the moderate dry eye group and the normal controls (2.07 +/- 2.16 vs. 2.25 +/- 0.70, p = 0.676). Conjunctival folds were not correlated with any of the above parameters nor were they associated with the age of the subject. However, the study did reveal that tear osmolality (p = 0.005), corneal staining (p = 0.019), and conjunctival bulbar injection (p = 0.016) were significantly higher in the moderate dry eye group of subjects (Mann Whitney U Test). A significant correlation (Spearmans rho) was found between limbal injection (r2 = 0.535, p = 0.01) and comeal staining. Age was only found to be correlated with tear osmolality (r2 = 0.520, p = 0.013). CONCLUSIONS: Although there was a small difference in the number of conjunctival folds between normal and moderate dry eye subjects, they do not appear to be as discriminating as other variables measured in the study. Additionally, age does not affect the number of conjunctival folds.

Expression of human beta-defensins in conjunctival epithelium: relevance to dry eye disease.

PURPOSE: The goals of this study were to investigate whether beta-defensins are differentially expressed in the conjunctival epithelium of patients with moderate dry eye when compared with normal subjects and whether proinflammatory cytokines or bacteria can modulate the expression of human beta-defensins (hBDs)-1, -2, and -3 by conjunctival epithelial cells. METHODS: RNA extracted from conjunctival impression cytology specimens of eight normal subjects and nine patients with moderate dry eye was used in RT-PCR to detect mRNA for hBDs-1, -2, and -3. Two conjunctival epithelial cell lines and primary cultured conjunctival epithelial cells were treated with proinflammatory cytokines or heat-killed Pseudomonas aeruginosa. RT-PCR and immunoblot analysis were used to detect mRNA for hBD-1, -2, and -3 and protein secretion of hBD-2, respectively. RESULTS: hBD-2 message was detected in RNA samples of eight of nine patients with dry eye, but not in any of the normal subjects' samples, whereas hBD-1 and -3 were detected in all subjects tested. RT-PCR revealed an upregulation of hBD-2 but no difference in expression of hBD-1 and -3 in cultured conjunctival cells after a 24-hour treatment with 10 ng/mL interleukin (IL)-1beta, IL-1beta and tumor necrosis factor-alpha (10 ng/mL) or heat-killed Pseudomonas aeruginosa (1 million colony-forming units; n = 3). hBD-2 expression was upregulated from 4 hours of treatment with IL-1beta (at 10 ng/mL; (n = 2-3) and at a concentration of 0.1 ng/mL IL-1beta (24-hour treatment; n = 2-3). Immunoblots demonstrated protein secretion results corresponding to the RT-PCR data. CONCLUSIONS: hBD-2 was expressed only in the conjunctival epithelium of patients with moderate dry eye. Because cytokines such as IL-1beta and TNF-alpha induced the expression of hBD-2 by conjunctival epithelial cells and because increased proinflammatory cytokine activity is a feature of dry eye disease, it can be speculated that the hBD-2 upregulation observed in subjects with moderate dry eye is mediated by proinflammatory cytokine activity.