Amalia Enríquez-de-Salamanca

Cytokine and chemokine levels in tears from healthy subjects

Acta Ophthalmol. 2010: 88: e250–e258

Cytokine responses by conjunctival epithelial cells: An in vitro model of ocular inflammation

OBJECTIVES We examined the differential secretion of cytokines by a conjunctival epithelial cell line in response to proinflammatory cytokines to identify the potential contributions during ocular surface inflammation. METHODS A conjunctival epithelial cell line was exposed to IFN-gamma, TNF-alpha, IL-4, or IL-13, and cytokine production was determined in supernatants at different times after exposure. Cell apoptosis was measured by flow cytometry. RESULTS TNF-alpha induced the greatest effect on cytokine secretion, which was time-dependent. TNF-alpha-stimulated secretion of IL-12p40 was significantly increased by 30 min; GM-CSF, MCP-1, IL-6, IL-7, IL-8, and RANTES were significantly increased by 2 h, and IFN-gamma and IL-1alpha by 24 h. After 48 h, TNF-alpha also induced a significant increase in IL-1beta, IL-3, and IP-10 secretion. IFN-gamma significantly enhanced IP-10 and RANTES secretion after 48 h of exposure. Following IL-4 treatment there was a significant increase in eotaxin-1 after 24h, and IL-12p40 and IL-3 after 48 h. IL-13 significantly increased the secretion of eotaxin-1 after 24 h, and IL-8 after 48 h. CONCLUSION Our results suggest that conjunctival epithelial cells are an important source of cytokines and chemokines that are regulated by proinflammatory cytokines and may play an important role in ocular surface inflammation.

Dry Eye Disease as an Inflammatory Disorder

Purpose: Dry eye disease (DED) is a prevalent inflammatory disorder of the lacrimal functional unit of multifactorial origin leading to chronic ocular surface disease, impaired quality of vision, and a wide range of complications, eventually causing a reduction in quality of life. It still is a frustrating disease because of the present scarcity of therapies that can reverse, or at least stop, its progression. Methods: A comprehensive literature survey of English-written scientific publications on the role of inflammation in DED. Results: New investigations have demonstrated that a chronic inflammatory response plays a key role in the pathogenesis of human DED. Additionally, correlations between inflammatory molecules and clinical data suggest that inflammation can be responsible for some of the clinical symptoms and signs. Conclusions: Research efforts to clarify its pathophysiology are leading to a better understanding of DED, demonstrating that inflammation, in addition to many other factors, plays a relevant role.

Dry eye exacerbation in patients exposed to desiccating stress under controlled environmental conditions.

PURPOSE To determine if controlled environmental conditions can induce acute exacerbations of signs and symptoms in dry eye and asymptomatic subjects. DESIGN Prospective cross-sectional study. METHODS Nineteen patients with dry eye and 20 asymptomatic controls were exposed to controlled low humidity (5% relative humidity, desiccating environment) for 2 hours in our Controlled Environmental Research Laboratory at the University of Valladolid. The patients completed the Single-Item Score Dry Eye Questionnaire and the following diagnostic tests were performed before and after exposure: tear osmolarity, phenol red thread test, conjunctival hyperemia, fluorescein tear film break-up time, Schirmer test, and ocular surface vital staining. Sixteen molecules in the tears samples were analyzed by multiplex bead analysis. RESULTS After exposure, the patients and controls had a significant (P ≤ .003) increase in corneal staining (from 0.68 ± 0.15 to 1.16 ± 0.14 and from 0.50 ± 0.15 to 1.30 ± 0.19, respectively), significantly decreased (P ≤ .01) fluorescein tear film break-up time values (from 2.78 ± 0.56 seconds to 1.94 ± 0.24 seconds and from 2.81 ± 0.24 seconds to 2.13 ± 0.19 seconds, respectively), and significantly increased (P ≤ .03) matrix metalproteinase 9 tear levels (from 10 054.4 ± 7326.6 pg/mL to 25 744.5 ± 13 212.4 pg/mL and from 10 620.5 ± 4494.3 pg/mL to 16 398.7 ± 5538.3 pg/mL, respectively). In the control group, the epidermal growth factor tear levels decreased significantly (P = .007; from 1872.1 ± 340.7 pg/mL to 1107.1 ± 173.6 pg/mL), and interleukin 6 levels increased significantly (P < .001; from 29.6 ± 5.8 pg/mL to 54.3 ± 8.3 pg/mL) after exposure. CONCLUSIONS Adult patients with mild-to-moderate dry eye and asymptomatic subjects of similar ages can experience acute exacerbation in an environmental chamber that resembles the sudden worsening that patients with dry eye experience daily.

Influence of a Controlled Environment Simulating an In-Flight Airplane Cabin on Dry Eye Disease

PURPOSE To evaluate symptoms, signs, and the levels of 16 tears inflammatory mediators of dry eye (DE) patients exposed to an environment simulating an in-flight air cabin in an environmental chamber. METHODS Twenty DE patients were exposed to controlled environment simulating an in-flight airplane cabin (simulated in-flight condition [SIC]) of 23°C, 5% relative humidity, localized air flow, and 750 millibars (mb) of barometric pressure. As controls, 15 DE patients were subjected to a simulated standard condition (SSC) of 23°C, 45% relative humidity, and 930 mb. A DE symptoms questionnaire, diagnostic tests, and determination of 16 tear molecules by multiplex bead array were performed before and 2 hours after exposure. RESULTS After SIC exposure, DE patients became more symptomatic, suffered a significant (P ≤ 0.05) decrease in tear stability (tear break up time) (from 2.18 ± 0.28 to 1.53 ± 0.20), and tear volume (phenol red thread test), and a significant (P ≤ 0.05) increase in corneal staining, both globally (0.50 ± 0.14 before and 1.25 ± 0.19 after) and in each area (Baylor scale). After SSC, DE patients only showed a mild, but significant (P ≤ 0.05), increase in central and inferior corneal staining. Consistently, tear levels of IL-6 and matrix metalloproteinase (MMP)-9 significantly increased and tear epidermal growth factor (EGF) significantly decreased (P ≤ 0.05) only after SIC. CONCLUSIONS The controlled adverse environment conditions in this environmental chamber can simulate the conditions in which DE patients might be exposed during flight. As this clearly impaired their lacrimal functional unit, it would be advisable that DE patients use therapeutic strategies capable of ameliorating these adverse episodes.

Tear fluid biomarkers in ocular and systemic disease: potential use for predictive, preventive and personalised medicine

In the field of predictive, preventive and personalised medicine, researchers are keen to identify novel and reliable ways to predict and diagnose disease, as well as to monitor patient response to therapeutic agents. In the last decade alone, the sensitivity of profiling technologies has undergone huge improvements in detection sensitivity, thus allowing quantification of minute samples, for example body fluids that were previously difficult to assay. As a consequence, there has been a huge increase in tear fluid investigation, predominantly in the field of ocular surface disease. As tears are a more accessible and less complex body fluid (than serum or plasma) and sampling is much less invasive, research is starting to focus on how disease processes affect the proteomic, lipidomic and metabolomic composition of the tear film. By determining compositional changes to tear profiles, crucial pathways in disease progression may be identified, allowing for more predictive and personalised therapy of the individual.This article will provide an overview of the various putative tear fluid biomarkers that have been identified to date, ranging from ocular surface disease and retinopathies to cancer and multiple sclerosis. Putative tear fluid biomarkers of ocular disorders, as well as the more recent field of systemic disease biomarkers, will be shown.

Clinical and Molecular Inflammatory Response in Sjögren Syndrome-Associated Dry Eye Patients Under Desiccating Stress.

PURPOSE To evaluate the response of the lacrimal function unit in Sjögren syndrome (SS)-associated dry eye patients exposed to 2 simulated daily life environmental conditions. DESIGN Prospective crossover pilot study. METHODS Fourteen female SS dry eye patients were exposed for 2 hours to a controlled normal condition (23 C, 45% relative humidity, and air flow 0.10 m/s) and a controlled adverse condition that simulates desiccating stress (23 C, 5% relative humidity, and air flow 0.10 m/s). The following dry eye tests were performed before and after the exposure: tear osmolarity, phenol red thread test, conjunctival hyperemia, fluorescein tear break-up time, corneal fluorescein staining, conjunctival lissamine green staining, and Schirmer test. Levels of 16 molecules were analyzed in tears by multiplex immunobead analysis. RESULTS Clinical evaluation showed lacrimal functional unit impairment after the desiccating stress: significantly increased tear osmolarity (315.7 ± 3.0 vs 327.7 ± 5.1 mOsm/L, P = .03), conjunctival hyperemia (1.3 ± 0.1 vs 1.6 ± 0.1, P = .05), and corneal staining in temporal (3.5 ± 0.5 vs 4.7 ± 0.4, P = .01) and nasal (3.6 ± 0.5 vs 4.5 ± 0.5, P = .04) areas. Tear concentrations increased for interleukin-1 receptor antagonist (16 557.1 ± 4047.8 vs 31 895.3 ± 5916.5 pg/mL, P = .01), interleukin-6 (63.8 ± 20.2 vs 111.5 ± 29.6 pg/mL, P = .02), interleukin-8 (2196.1 ± 737.9 vs 3753.2 ± 1106.0 pg/mL, P = .03), and matrix metalloproteinase-9 (101 515.6 ± 37 088.4 vs 145 867.1 ± 41 651.5 pg/mL, P = .03). After the simulated normal condition, only a significant increase in nasal corneal staining (2.9 ± 0.5 vs 3.6 ± 0.5, P = .03) was observed. CONCLUSIONS Even a short exposure to a desiccating environment can produce a significant deterioration of the lacrimal function unit in female SS dry eye patients. The often unnoticed exposure to these conditions during daily life may increase inflammatory activity rapidly, triggering an ocular surface deterioration.

Molecular and cellular biomarkers in dry eye disease and ocular allergy.

Purpose of reviewTo summarize recent studies in biomarkers in dry eye disease (DED) and ocular allergy in the last 18 months. Recent findingsNew significant correlations with clinical symptoms and signs have been described for some tear molecules (proteins, cytokines/chemokines, metalloproteinases, mucins and lipids). Correlation with disease severity has been shown for some of them. New proposed DED biomarkers include some proteins, such as S100A8, S100A9, lipocalin-1, secretory phospholipase A2 and some cytokines and chemokines; mucin-1 mRNA conjunctival expression levels are shown to be a very sensitive and specific DED diagnosis biomarker; metalloproteinase-9 tear level is confirmed as a good DED biomarker; cell parameters such as conjunctival cell viability and proliferative capacity have been also proposed as DED biomarkers. In ocular allergy, protein activated receptor-2, heat shock protein-70, eosinophil cationic protein and hemopexin have been pointed as potential biomarkers. Characterization of neuromediators tear levels confirmed the involvement of these molecules in both DED and ocular allergy. SummaryBiomarkers are essential to monitor health status. Several studies have proposed different molecules and/or cellular parameters as potential biomarkers for DED and ocular allergy. The knowledge presented in these studies will further add support to the discovery and development of new drugs and the establishment of personalized medicine.

Intra- and inter-day variation of cytokines and chemokines in tears of healthy subjects

Tear levels of certain cytokines/chemokines can potentially serve as biomarkers for dry eye and other ocular surface diseases if they remain stable from day-to-day in healthy eyes. The aim of this study was to determine the normal intra- and inter-day variation of selected tear cytokines/chemokines. Tear samples from 24 young, healthy adults were collected 11:00 AM-1:00 PM (mid-day) and 5:00-7:00 PM (evening) on three non-consecutive days. Concentrations of 18 cytokines/chemokines (EGF, eotaxin, CX3CL1/fractalkine, GM-CSF, IFN-γ, IL-10, IL-1β, IL-13, IL-17A, IL-1RA, IL-5, IL-6, CXCL8/IL-8, IL-9, CXCL10/IP-10, CCL5/RANTES, TNF-α, and VEGF) were measured by multiplex bead analysis. Ocular surface disease was ruled out by clinical tests. A random-effects ANOVA model was used to evaluate intra- and inter-day effects on cytokine/chemokine levels. Repeatability of intra-subject inter-day measurements was assayed by coefficient of variation. Ten out of the 18 molecules had detectable tear levels in >50% of the subjects. Of those, only IL-10 and IL-1β levels had significant inter-day variations. EGF, CX3CL1/fractalkine, CXCL10/IP-10, and VEGF were consistently higher in the evening compared to the mid-day measurements. EGF, CXCL10/IP-10, VEGF and CXCL8/IL-8had good intra-subject reproducibility. In conclusion, tear cytokines/chemokines can be measured reproducibly over time, with most not having significant inter-day variability. Some varied significantly depending upon the time of tear collection, and these variations should be taken into account when comparisons are made. The good intra-subject reproducibility for EGF, CXCL10/IP-10, CXCL8/IL-8, and VEGF indicates that these molecules could potentially serve as biomarkers of ocular surface disease.

Biomarkers in Ocular Chronic Graft Versus Host Disease: Tear Cytokine- and Chemokine-Based Predictive Model

PURPOSE To develop a tear molecule level-based predictive model based on a panel of tear cytokines and their correlation with clinical features in ocular chronic graft versus host disease (cGVHD). METHODS Twenty-two ocular cGVHD patients and 21 healthy subjects were evaluated in a controlled environmental research laboratory (CERLab). Clinical parameters were recorded, and tears were collected. Levels of 15 molecules (epidermal growth factor [EGF], IL receptor antagonist [IL-1Ra], IL-1β, IL-2, IL-6, IL-8/CXCL8, IL-10, IL-12p70, IL-17A, interferon inducible protein [IP]-10/CXCL10, IFN-γ, VEGF, TNF-α, eotaxin 1, and regulated on activation normal T cell expressed and secreted [RANTES]) were measured by multiplex-bead assay and correlated with clinical parameters. Logistic regression was used to develop a predictive model. Leave-one-out cross-validation was applied. Classification capacity was evaluated in a cohort of individuals with dry eye (DE) of other etiologies different from GVHD. RESULTS Epidermal growth factor and IP-10/CXCL10 levels were significantly decreased in ocular cGVHD, positively correlating with tear production and stability and negatively correlating with symptoms, hyperemia, and vital staining. Interleukin-1Ra, IL-8/CXCL8, and IL-10 were significantly increased in ocular cGVHD, and the first two correlated positively with symptoms, hyperemia, and ocular surface integrity while negatively correlating with tear production and stability. Predictive models were generated, and the best panel was based on IL-8/CXCL8 and IP-10/CXCL10 tear levels along with age and sex, with an area under the receiving operating curve of 0.9004, sensitivity of 86.36%, and specificity of 95.24%. CONCLUSIONS A predictive model based on tear levels of IL-8/CXCL8 and IP-10/CXCL10 resulted in optimal sensitivity and specificity. These results add further knowledge to the search for potential biomarkers in this devastating ocular inflammatory disease.