IL-6 as a corneal wound healing mediator in an in vitro scratch assay.

Corneal healing process under inflammatory conditions is not fully understood. We aimed at determining the effect of an inflammatory (presence of IL-6) or anti-inflammatory (presence of IL-10) environment and a mixture of both in the expression of IL-6 signaling pathway mediators, and on corneal wound healing in an in vitro scratch assay. For that purpose, human corneal epithelial cells were cultured until confluence. The effect of IL-6 (10 ng/ml), IL-10 (20 ng/ml) or IL-6 + IL-10 exposure on the expression of IL-6R, gp130, and STAT3 was determined by Western blotting and quantitative PCR, at different time points. The monolayer was mechanically wounded using a sterile 10 µl pipette tip. Wound healing rate in the presence or absence of these cytokines was measured immediately after cytokine exposure and after 4, 8, and 24 h. The effect of mitomycin C on wound healing rate, in control and IL-6-stimulated cells, was also evaluated. Detection of proliferative cells was performed with an EdU imaging kit. For the visualization of migrating cells, cold methanol-fixed cells were incubated with an α-actinin antibody. For the statistical analysis a two-factor design of experiment method was applied. Levene test was used to contrast equality of variances. If variances were equal, ANOVA was performed to test the equality of means. If variances were not equal, a Mood's median test was performed. We observed that IL-6 and IL-10 stimulation, and their combination, increased gp130 production at different time points. STAT3 production was increased in IL-6-stimulated cells, at 72 h. An increase in pSTAT3 production was found in IL-6- and IL-10-stimulated cells, that was sustained in time in IL-6 + IL-10 co-stimulated cultures. Scraped areas had an initial width of 570.57 ± 75.82 µm. In IL-6-exposed cells wound healing closure was faster than in control cells or IL-10-exposed cells. After 8 h, wound width in IL-10-exposed cells, was also significantly smaller than that of control cells. Cells exposed to IL-6 + IL-10 had the slowest wound healing rate, similar to control cells. Wounds were closed after 24 h regardless the experimental condition. Mitomycin C exposure increased the wound closure rate in every experimental condition. No significant differences in the percentage of proliferative cells at the edge of the scratch and in distant areas of the monolayer were found. At the edge of the scratch, some actin filaments of non-proliferative cells were directed through the cell-free area, independently of the stimulating condition. In conclusion, the presence of IL-10 and, most importantly, of IL-6, increased the wound healing rate in an in vitro corneal wound healing model. The combination of both cytokines did not have a synergistic action in wound healing. In our model, wound closure was the result of the combination of cell proliferation and cell migration.

CD44 and RHAMM hyaluronan receptors in human ocular surface inflammation.

BACKGROUND: CD44 and RHAMM hyaluronan (HA) receptors have been studied in several systemic diseases such as osteoarthritis and cancer. However, not too much is known about their role in ocular surface disorders. The purpose of this research was to determine if CD44 and RHAMM are implicated in human ocular surface inflammation. METHODS: Upper tarsal conjunctival epithelial samples from patients with active ocular surface inflammation (n = 17) and healthy controls (n = 14) were recovered by brush cytology. Patients were evaluated by an ophthalmologist and classified in different groups according to the etiology (immune atopic diseases or immune non-atopic diseases) and inflammation intensity (mild/moderate or severe). CD44, RHAMM, and p53 mRNAs were measured using real-time PCR. RESULTS: CD44, RHAMM, and p53 mRNAs were detected in all samples. In immune atopic diseases, higher levels of CD44 and RHAMM mRNAs were present, reaching a 300 % increase for RHAMM in severe inflammation (p < 0.001). In contrast, in immune non-atopic diseases, the HA receptors were downregulated. CD44 tended to decrease up to 30 % in severe patients (p = 0.06), and RHAMM decreased 40 % in severe inflammation (p = 0.021). CONCLUSIONS: RHAMM may be implicated in severe ocular surface inflammation affecting the upper tarsal conjunctiva.

A new human primary epithelial cell culture model to study conjunctival inflammation.

PURPOSE: To develop a complete and optimized method to expand in culture human conjunctival epithelial cells from cadaveric donor samples. METHODS: Epithelial cells were obtained from cadaveric conjunctival tissue (n = 47). Preplating and differential trypsinization were optimized to eliminate stromal contamination. Epithelial cells were grown with five different media: control, epithelial growth factor (EGF)-enriched, H2O2-supplemented, fibroblast-conditioned, and human serum media. Adhesion, proliferation, colony forming efficiency (CFE), and percentage of CK19(+) and Ki67(+) cells were determined with the five different media. Cells were characterized by immunofluorescence and/or Western blotting techniques for the expression of CK4, CK7, CK19, MUC5AC, vimentin, FSP-1, Ki67, E-cadherin, and zonula occludens (ZO)-1 markers. In addition, cells were treated with TNF-α and levels of secreted IL-6 were measured by enzyme-linked immunosorbent assay. RESULTS: Pure epithelial cell cultures were obtained. Human serum medium showed the best properties in proliferation and CFE, while maintaining epithelial phenotype. Cells with this medium were passaged up to five times, although they maintained all epithelial characteristics only through passage 3. Cultured cells expressed epithelial markers, but not stromal ones. The number of MUC5AC(+) cells increased throughout the passages, whereas Ki67(+) cell numbers decreased. Cells in culture maintained adherens and tight junctions, and responded to TNF-α treatment by releasing more IL-6, showing that they can be used for inflammation assays. CONCLUSIONS: We have developed a complete protocol to expand conjunctival epithelial cells from cadaveric tissue. This culture system responded to an inflammatory stimulus, so it could be used to develop a more complex in vitro model of inflammation.

Involvement of corneal epithelial cells in the Th17 response in an in vitro bacterial inflammation model.

PURPOSE: Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) are frequent causes of bacterial keratitis, an inflammatory process that can lead to vision loss. We used a human corneal epithelial (HCE) cell line to study the Th17 inflammatory pathway, including interleukin (IL-) 6, IL-17, and associated receptors, in response to stimulation by SA and PA culture supernatants. METHODS: Cells of the HCE cell line were exposed to either SA or PA supernatants in dilutions of 1:100 or 1:50, or to human recombinant IL-17A (20 ng/ml). Cell culture supernatants were collected at 6, 24, and 72 h, and protein and RNA were isolated. Expression of cytokine (IL-6, IL-17A), receptor (sIL-6R, IL-17RA), and mediator (soluble glycoprotein [sgp] 130, MIP3α) proteins and mRNAs were determined with enzyme-linked immunosorbent assay, immunohistochemistry, western blotting, and real-time, reverse-transcription quantitative PCR. In addition, IL-17RA was localized by transmission electron microscopy after immunogold labeling. RESULTS: Basal secretion of IL-6 and IL-17A by HCE cells occurred in a time-dependent manner. Expression of IL-6 was significantly enhanced by SA stimulation, but not by PA stimulation. IL-6 mRNA expression was higher in the control and SA-stimulated cells at 6 and 24 h, but not at 72 h. In the PA-stimulated cells, mRNA levels were significantly lower than the controls at 6 and 24 h. Expression of sIL-6R was not altered by SA or PA supernatants, but sgp130 expression was greater than controls at 6 h, less than controls at 24 h, and the same as controls at 72 h. HCE cells secreted IL-17A in a time-dependent manner that was not altered by stimulation; however, the IL-17A mRNA levels were lower than those of the controls at 6 h. With immunohistochemistry, IL-17RA was localized in perinuclear vesicles and in the cytosol and membranes of HCE cells. IL-17RA was also present in the epithelial cells from human ocular surface tissues. As quantified with western blotting, expression of IL-17RA was unchanged in HCE cells stimulated by SA or PA supernatants. CONCLUSIONS: HCE cells react to bacterial inflammation by enhancing the secretion of IL-6 and by regulating the proinflammatory response with differential secretion of sgp130. Under normal conditions, HCE cells and ocular surface tissues express IL-17RA. Additionally, HCE cells express IL-17RA after bacterial stimulation. All of these molecules are involved in the Th17 differentiation pathway, suggesting that corneal epithelial cells may act as indirect participants in the Th17 signaling pathway.

Structural and functional alteration of corneal epithelial barrier under inflammatory conditions.

PURPOSE: The aim of the study was to determine the effect of inflammatory conditions on the expression of tight junction (TJ) and adherens junction (AJ) proteins between human corneal epithelial cells and, consequently, on corneal epithelial barrier integrity. MATERIALS AND METHODS: Zonula occludens proteins ZO-1 and ZO-2, claudin-1 and -2 (CLDN-1 and CLDN-2), occludin (OCLN) as well as E-cadherin (E-cad) expression were analyzed in a human corneal epithelial cell line (HCE) at basal conditions and after stimulation with inflammatory cytokines (TNFα, TGFß, IL-10, IL-13, IL-17, IL-6), using real time RT-PCR, Western blotting and immunofluorescence. Actin cytoskeleton staining was performed after all stimulations. Transepithelial electrical resistance (TER) and fluorescein transepithelial permeability (TEP) were measured as barrier integrity functional assays. RESULTS: ZO-1, ZO-2, CLDN-1, CLDN-2, OCLN and E-cad were detected in HCE cell membranes at basal conditions. Cytokine stimulation resulted in significant changes in the expression of TJ and AJ proteins, both at mRNA and protein level, a remarkable change in their localization pattern, as well as a reorganization of actin cytoskeleton. Pro-inflammatory cytokines TNFα, TGFß, IL-13, IL-17 and IL-6 induced a structural and functional disruption of the epithelial barrier, while IL-10 showed a barrier protective effect. CONCLUSION: Simulated inflammatory conditions lead to an alteration of corneal barrier integrity by modulating TJ, and to a lesser extent also AJ, protein composition, at least In Vitro. The observed barrier protective effects of IL-10 support its well-known anti-inflammatory functions and highlight a potential therapeutic perspective.