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. 2010 May 19:16:862-73.

Tear cytokine and chemokine analysis and clinical correlations in evaporative-type dry eye disease

Amalia Enríquez-de-Salamanca  1 , Evangelina Castellanos, Michael E Stern, Itziar Fernández, Ester Carreño, Carmen García-Vázquez, Jose M Herreras, Margarita Calonge
Affiliations

Tear cytokine and chemokine analysis and clinical correlations in evaporative-type dry eye disease

Amalia Enríquez-de-Salamanca et al. Mol Vis. .

Abstract

Purpose: Inflammatory molecules have been demonstrated in the tear film of patients with severe dry eye disease (DED). However, little attention has been paid to the most frequent moderate forms of DED. This study analyzes tear cytokine levels and their clinical correlations in patients with moderate evaporative-type DED due to meibomian gland disease (MGD).

Methods: Twenty three evaporative-type DED patients (46 eyes) of mild-to-moderate intensity and nine healthy subjects (18 eyes) were recruited. Two symptom questionnaires were self-answered and multiple DED-related clinical tests were performed. Unstimulated tears from each eye were isolated and were not pooled. Levels of 15 cytokines and chemokines were measured by multiplex bead analysis, compared with control levels, and correlated with clinical tests.

Results: Fourteen out of the 15 molecules were reliably detected in 1 microl of unstimulated tears from DED patients. Epidermal growth factor (EGF), fractalkine/CX3CL1, interleukin (IL) 1-receptor antagonist (Ra), IL-8/CXCL8, interferon inducible protein (IP)-10/CXCL10, and vascular endothelial growth factor (VEGF) were found in 94%-100% of samples; IL-6 in 65% (significantly more detected in older patients); IL-1beta, interferon gamma (IFN-gamma), and IL-10 in 30%-48%; IL-17 in 13%; granulocyte macrophage colony stimulating factor (GM-CSF), IL-13, and tumor necrosis factor alpha (TNF-alpha) in 2%-9%; and IL-5 was never detected. EGF, fractalkine/CX3CL1, IL-1Ra, IP-10/CXCL10, and VEGF levels were significantly increased compared to normal controls. Pain was correlated with IL-6 and IL-8/CXCL8. Tear break-up time correlated inversely with IL1-Ra. Schirmer test and tear lysozyme levels negatively correlated with IL-1Ra, IL-8/CXCL8, fracktalkine/CX3CL1, IL-6, IP-10/CXCL10, and VEGF had the same tendency. Conjunctival staining correlated negatively with EGF and positively with IL-6.

Conclusions: In this sample of moderate evaporative-type DED patients, five inflammatory molecules were elevated. Fracktalkine was demonstrated to be present and elevated in tears in human DED. IL-1Ra, IL-6, IL-8/CXCL8, and EGF levels correlated with pain and with clinical parameters measuring tear stability, tear production or ocular surface integrity. These results suggest that inflammation plays a role not only in severe DED but also in moderate evaporative DED.

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Figures

Figure 1
Figure 1
Cytokines and chemokines in tears of DED patients. Fourteen out of the 15 molecules analyzed were detected. EGF, Fractalkine/CX3CL1, IL-1Ra, IP-10/CXCL10, VEGF, and IL-8 were the more detected molecules in evaporative-type DED tear samples and were also the ones with the highest concentration. Data are presented on a logarithmic scale and bars represent values for OS and OD together. Percents of patients having detectable amounts are shown at the top of each bar. EGF, epidermal growth factor; IFN-γ, interferon gamma; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; IL-1Ra, interleukin 1 receptor antagonist; TNFα, tumor necrosis factor alpha; IP-10, interferon inducible protein 10; VEGF, vascular endothelial growth factor.
Figure 2
Figure 2
Comparison of tear cytokine/chemokine levels in DED patients with control subjects. EGF, Fractalkine/CXC3L1, IL-1Ra, IP-10/CXCL10, and VEGF levels were significantly increased in evaporative-type DED tear samples compared to controls. Values are presented on a logarithmic scale. EGF, epidermal growth factor; IL, interleukin; IL-1Ra, interleukin 1 receptor antagonist; IP-10, interferon inducible protein 10; VEGF, vascular endothelial growth factor. OD, right eye; OS, left eye. *p
Figure 3
Figure 3
Fractalkine/CX3CL1 and IL-8/CXCL8 levels correlation with age in DED patients. Fractalkine/CX3CL1 and IL-8/CXCL8 levels correlated positively with age in evaporative-type DED tear samples. Spearman ranked correlation test was used and the strength of correlation between variables was determined by the Spearman's rho correlation coefficient. Dotted lines indicate the 95% confidence bands. IL, interleukin; OD, right eye; OS, left eye.
Figure 4
Figure 4
Correlation studies with SODQ pain score. IL-6 and IL-8/CXCL8 were significantly increased for pain score 1 compared to pain scores 0 and 2 (OS *p=0.003 for IL-6; *p=0.027 for IL-8/CXCL8; OD p-values were not significant but followed the same trend as OS). IL-1Ra levels increased with pain in both eyes but did not reach statistical significance. IL, interleukin; OD, right eye; OS, left eye.
Figure 5
Figure 5
Correlation of OD and OS tear cytokine/chemokine levels with corresponding OD and OS clinical measurements of tear production and ocular surface integrity in DED patients. IL-1Ra, IL-8/CXCL8, and Fractalkine/CX3CL1 levels correlated negatively with tear production tests, and IL-6 levels correlated positively with rose Bengal staining test (measuring ocular surface integrity). A: Schirmer test, B: lysozyme levels, C: BUT, and D: Rose Bengal staining. Cytokine/chemokine values are expressed in pg/ml in a 100× (IL-1Ra, IL-8 and fractalkine) or 10× (IL-6) scale. Spearman's rho correlation coefficient was determined. Dotted lines indicate the 95% confidence bands. IL, interleukin; IL-1Ra, interleukin 1 receptor antagonist; OD, right eye; OS, left eye.
Figure 6
Figure 6
Correlation of OD and OS tear EGF with corresponding OD and OS clinical measurements of tear production and ocular surface integrity in DED patients. EGF levels correlated negatively with rose Bengal and fluorescein staining tests (measuring ocular surface integrity) and positively with Lysozyme and Schirmer tests (measuring tear production). EGF values are expressed in pg/ml in a 100× scale. Spearman ranked correlation test was used and the strength of correlation between variables was determined by the Spearman's rho correlation coefficient. EGF, epidermal growth factor; OD, right eye; OS, left eye.
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