Carmen García-Vázquez

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.

Genetically Engineered Elastin-Like Polymer as a Substratum to Culture Cells from the Ocular Surface

Purpose: To investigate epithelial cell adhesion and proliferation on a newly developed elastin-like polymer (ELP) that mimics the functional characteristics of extracellular matrices. Materials and Methods: A genetically engineered ELP with cell attachment sequences was adsorbed onto glass coverslips as 1, 2, or 3 molecular films. Conjunctival epithelial cells from a human cell line and human skin fibroblast cells (as controls) were plated onto coverslips with three different substrata: plain glass, Thermanox®, and ELP-coated. Cells (104) were plated after EDTA- or trypsin-based detachment. To test adhesion, epithelial and fibroblast cells were incubated for 4 hr, stained with hematoxylin, and counted. To study proliferation, Ki-67-positive epithelial cells were counted after 1, 3, and 5 days in culture. Immunostaining for conjunctival and adhesion markers was performed. Results: Epithelial cell, but not fibroblast, adhesion on ELP was significantly enhanced compared to that of control substrata. Epithelial cells detached with EDTA alone adhered significantly better than those detached with trypsin. By day 5, epithelial cell proliferation on ELP was significantly greater than that on plain glass. Epithelial cells grown on ELP expressed conjunctival and adhesion markers. Conclusions: The recombinant ELP resembling the ocular surface extracellular matrix was a suitable substratum to sustain epithelial cell attachment and growth. This type of polymer may be suitable for tissue engineering to restore vision by reconstructing the ocular surface.

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.

Gene Expression-Based Predictive Models of Graft Versus Host Disease-Associated Dry Eye.

PURPOSE To develop a predictive model based on inflammatory gene mRNA expression in conjunctival cells of graft versus host disease (GvHD)-associated dry eye (DE) patients, as well as to find meaningful correlations between gene signals and clinical signs. METHODS Twenty GvHD-DE patients and 14 healthy controls were recruited. Patients discontinued medications for 1 week before examination. Dry eye-related symptoms and signs were recorded, and conjunctival epithelial cells were collected by impression cytology after spending 20 minutes under standard conditions within a Controlled Environmental Research Laboratory. Gene expression of inflammatory molecules was determined by polymerase chain reaction, and the results were correlated with clinical signs. Shrinkage discriminant analysis, support vector machine, and k-nearest neighbor classifier methods were used to develop predictive models that were validated considering accuracy, calibration, and discriminant capability. RESULTS Out of the 84 genes analyzed, 34 showed significant differences in expression. IL-6, IL-9, CCL24, CCL18, IL-10, IFN-γ, and CCL2 were highly increased (>6-fold); 26 genes were moderately upregulated (2- to 6-fold), whereas EGFR was downregulated (2.63 fold) in GvHD-DE samples. A panel based on EGFR, IL-6, IL-9, and NAMPT had an area under the receiver operating characteristic curve of 0.994, a sensitivity of 100%, and a specificity of 92.9%. EGFR expression correlated negatively with ocular surface damage markers, while IL-6, IL-9, and NAMPT correlated positively with these tests. CONCLUSIONS EGFR, IL-6, IL-9, and NAMPT have the greatest potential as diagnostic biomarkers, with excellent sensitivity, specificity, and clinical relevance to the ocular surface status of GvHD.

Effect of TGF-β on ocular surface epithelial cells.

A role for transforming growth factor (TGF)-β in the pathogenesis of some ocular surface diseases has been proposed. We determined if secretion of TGF-β and expression of TGF-β 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-β. A human corneal epithelial (HCE) cell line and a conjunctival epithelial cell line (IOBA-NHC) were exposed to TGF-β1 and -β2 and to proinflammatory cytokines. TGF-β 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-β receptors and pSMAD2/SMAD2 were determined by Western blot and immunofluorescence assays. Secretion of TGF-β 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-β 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-β RI expression was down-regulated with IL-4 exposure, whereas TGF-β RII and TGF-β2 were upregulated by TNF-α in HCE cells. TGF-β 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-β treatment. TGF-β significantly up- and down-regulated secretion of several cytokines/chemokines by both cell lines and MMP by HCE cells. TGF-β2 and TGF-β3 were upregulated and TGF-β RIII mRNA was down-regulated in DE conjunctival epithelium. These results show that TGF-β plays an important role in directing local inflammatory responses in ocular surface epithelial cells.

RNA Collection From Human Conjunctival Epithelial Cells Obtained With a New Device for Impression Cytology

Purpose: To assess the ability of a new device (Eyeprim; Opia Technologies) designed for impression cytology (IC) to harvest RNA from conjunctival cells as compared with the conventional technique. Methods: Cell collection was performed in both eyes using both techniques (conventional and Eyeprim) in different eyes randomized to each technique to avoid bias. The collection order was also randomized. Subjective discomfort assessment was performed using the Symptom Assessment in Dry Eye questionnaire. RNA was quantified in a spectrophotometer. RNA yield and discomfort using each technique were evaluated. A P value ⩽0.05 was considered significant. Results: Twenty healthy subjects (8 men and 12 women) aged 24.7 ± 5.8 years were recruited. The mean corneal fluorescein staining scores were 0.10 ± 0.30 for both eyes (P = 1.0), and the mean phenol red thread tear scores were 28.6 ± 1.9 mm for the Eyeprim and 28.7 ± 2.5 mm for the conventional IC eye group (P = 0.64). No significant (P ≥ 0.45) differences were observed in the mean RNA yield between the Eyeprim and the conventional IC, neither in the total amount (0.32 ± 0.28 &mgr;g and 0.26 ± 0.28 &mgr;g, respectively) nor in the amount normalized to the membrane area (0.0046 ± 0.0040 &mgr;g/mm2 and 0.0040 ± 0.0043 &mgr;g/mm2, respectively). No significant differences were observed during (P ≥ 0.17) and after sample collection (P ≥ 0.36) in the frequency or intensity of discomfort (Symptom Assessment in Dry Eye scores). Conclusions: This pilot study shows that the Eyeprim provides similar RNA yield as the conventional harvesting conjunctival IC technique. It provides enough quantities of material useful for molecular analysis producing comparable levels of discomfort without using anesthesia.

Cytokine and chemokine tear levels in patients with uveitis

To determine whether the levels of cytokines and chemokines in tears differ in uveitis patients and healthy subjects.

Severity, therapeutic, and activity tear biomarkers in dry eye disease: An analysis from a phase III clinical trial

PURPOSE To evaluate the effect of 0.1%-fluorometholone (FML) on tear inflammatory molecule levels after 22-days treatment in dry eye disease (DED) patients exposed to an adverse controlled environment (ACE), identifying different biomarkers. METHODS Analysis of a double-masked randomized clinical trial. Forty-one DED patients received 4-drops daily of topical FML (FML-group) or polyvinyl-alcohol (PA-group) for 22 days. At day 21, patients were exposed to an ACE. Tear samples were collected at V1 (baseline), V2 (pre-ACE), V3 (post-2-h-ACE) and V4 (24-h post-ACE). Concentrations of 18 molecules (EGF, IFN-γ, TNF-α, IL-1β, IL-1RA, IL-2, IL-4, IL-6, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-17A, IP-10/CXCL10, MCP-1/CCL2, MIP-1α/CCL3, RANTES/CCL5 and MMP-9) were analyzed. Similarities among patients in molecule concentrations at V1 were evaluated. A linear-mixed effect model analyzed the influence of different variables on concentrations changes. RESULTS Multidimensional scaling (MDS) divided patients into two groups based on differences in EGF, IFN-γ, IL-8/CXCL8, RANTES/CCL5, and MMP-9 levels at V1. Groups had different clinical severities based on Schirmer test and conjunctival and corneal staining. IL-1RA, IL-2, and TNF-α were differentially affected by time, depending on treatment. Between V2-V3, there were significant changes in EGF, IL-1RA, IL-2, IL-8/CXCL8, IL-13, IP-10/CXCL10, TNF-α, and MMP-9. The strongest biomarker candidates were IFN-γ, RANTES/CCL5, and MMP-9 as DED severity biomarkers; IL-2 as DED therapeutic biomarker; and EGF as DED activity biomarker. CONCLUSIONS This clinical trial design using a controlled environment and the identified tear biomarkers could be useful to objectively select target patients, to define stress response, and to evaluate therapeutic endpoints in clinical trials.

Prehematopoietic Stem Cell Transplantation Tear Cytokines as Potential Susceptibility Biomarkers for Ocular Chronic Graft-Versus-Host Disease

Purpose To determine if cytokine tear levels before hematopoietic stem cell transplantation (HSCT) can help anticipate the occurrence of ocular chronic graft-versus-host disease (cGVHD). Methods In this pilot study, 25 patients undergoing HSCT were followed prospectively for ≤43 months. After ocular examinations, tears were collected before HSCT. Levels of 19 cytokines (epidermal growth factor [EGF], eotaxin 1/CCL11, fractalkine/CX3CL1, IL-1Ra, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8/CXCL8, IL-10, IL-12p70, IL-13, IL-17A, IP-10/CXCL10, IFN-γ, VEGF, TNF-α, and RANTES/CCL5) were measured by multiplex bead assay. A multistate model (MSM) based on four states (HSCT, systemic cGVHD, ocular cGVHD, and death) was developed to identify cytokines associated with each transition probability. Molecules included in the final multivariable model were selected by a supervised principal components analysis. Bootstrap resampling internally validated the final MSM. Model discriminatory ability was determined by time-dependent receiver operating characteristic curves and the corresponding area under the curve (AUC). Results The final model, based on fractalkine, IL-1Ra, and IL-6 tear levels, accurately influenced the transition between the four different states. The AUC for this model, based on a new variable built upon the combination of these three molecules, was 67% to 80% throughout follow-up and, thus, had good discriminatory ability. Conclusions In this prospective study, a model based on pre-HSCT tear levels of the inflammatory molecules fractalkine, IL-1Ra, and IL-6 had good prognostic ability for the development of ocular cGVHD after HSCT. These cytokines potentially could act as susceptibility biomarkers for the development of this disease after HSCT.