Ocular fibroblast diversity: implications for inflammation and ocular wound healing

Abstract

Purpose: Various ocular and orbital tissues differ in their manifestations of inflammation, although the reasons for this are unclear. Such differences may be due to behaviors exhibited by resident cell types, including fibroblasts. Fibroblasts mediate immune function and produce inflammatory mediators. Chronic stimulation of ocular fibroblasts can lead to prolonged inflammation and, in turn, to impaired vision and blindness. Interleukin (IL)-1β, which is produced by various cells during inflammation, is a potent activator of fibroblasts and inducer of the expression of inflammatory mediators. The hypothesis for this study was that that human fibroblasts derived from distinct ocular tissues differ in their responses to IL-1β and that variations in the IL-1 signaling pathway account for these differences.

Methods: Human fibroblasts were isolated from the lacrimal gland, cornea, and Tenon's capsule and treated with IL-1β in vitro. Cytokine and prostaglandin (PG)E(2) production were measured by ELISA and EIA. Cyclooxygenase (Cox)-2 expression was detected by Western blot. Components of the IL-1 signaling pathway were detected by flow cytometry, ELISA, Western blot, and immunofluorescence.

Results: Cytokine and PGE(2) production and Cox-2 expression were greatest in corneal fibroblasts. VEGF production was greatest in Tenon's capsule fibroblasts. Variations in IL-1 receptor and receptor antagonist expression, IκBα degradation and p65 nuclear translocation, however, did not account for these differences, but overexpression of the NF-κB member RelB dampened Cox-2 expression in all three fibroblast types.

Conclusions: The results highlight the inherent differences between ocular fibroblast strains and provide crucial insight into novel, tissue-specific treatments for ocular inflammation and disease, such as RelB overexpression.

Figure 1.

IL-1β increased proinflammatory cytokine and VEGF production in ocular fibroblasts. Fibroblasts isolated from the lacrimal gland, cornea and Tenon's capsule were treated with IL-1β at 10 ng/mL for 24 hours, after which IL-6 (A), IL-8 (B), MCP-1 (C), and VEGF (D) were detected in the supernatants by ELISA. Production of IL-6, IL-8, and MCP-1 was increased most dramatically in corneal fibroblasts, whereas VEGF production was increased most noticeably in Tenon's capsule fibroblasts. *P < 0.05, IL-1β-treated versus vehicle-treated fibroblasts. †P < 0.05, IL-1β-treated lacrimal gland or Tenon's capsule fibroblasts versus IL-1β-treated corneal fibroblasts. ‡P < 0.05, IL-1β-treated lacrimal gland or corneal fibroblasts versus IL-1β-treated Tenon's capsule fibroblasts (n = 3 per group).

Figure 2.

Enumeration of the frequency of IL-6 and -8 production by individual fibroblasts. Lacrimal gland, cornea, and Tenon's capsule fibroblasts were treated with IL-1β (10 ng/mL) for 6 hours, and IL-6 and -8 production was measured with a plate reader. (A) Representative images of IL-6 and -8 results. (B, C) Quantitation of IL-6 and -8 spot intensity. (D, E) IL-6 and -8 spot frequency expressed as the number of spots per 100 cells. *P < 0.05, IL-1β-treated versus vehicle-treated fibroblasts. †P < 0. 05, IL-1β-treated lacrimal gland or Tenon's capsule fibroblasts versus IL-1β-treated corneal fibroblasts (n = 3 per group).

Figure 3.

IL-1β-induced Cox-2 expression and PGE₂ production were greater in corneal than in lacrimal gland and Tenon's capsule fibroblasts. Fibroblasts isolated from the lacrimal gland, cornea, and Tenon's capsule were treated with IL-1β at 10 ng/mL for 24 hours, and Cox-2 expression was detected in whole cell lysates by Western blot (A). Results from fibroblasts from two different subjects are shown. GAPDH is included as a housekeeping protein. PGE₂ was detected in the supernatants of these cultures by EIA (B). *P < 0.05 IL-1β-treated versus vehicle-treated fibroblasts (n = 3 per group).

Figure 4.

IL-1β-treated ocular fibroblasts did not exhibit differences in IL-1 receptor expression and IL-1 receptor antagonist production. Fibroblasts were treated with IL-1β for 24 hours at 10 ng/mL, after which cell surface expression of IL-1RI and -II were measured by flow cytometry (A, B), and IL-1RA production was measured in the supernatants by ELISA (C). IL-1RI and -II expression were higher in lacrimal gland fibroblasts, whereas IL-1RA production was notably higher in Tenon's capsule fibroblasts. Flow cytometry measurements are presented as a percentage of the maximum. *P < 0.05 IL-1β-treated versus vehicle-treated fibroblasts (n = 3 per group).

Figure 5.

IL-1β-treated ocular fibroblasts did not exhibit differences in p65 nuclear translocation and IκBα degradation. Fibroblasts were treated with IL-1β (10 ng/mL) for either 20 minutes or 6 hours, after which p65 nuclear translocation was detected by immunofluorescent microscopy (A; 20 minutes only), and IκBα expression was detected by Western blot (B). p65 nuclear localization and IκBα expression or phosphorylation did not vary dramatically among the fibroblast types.

Figure 6.

Overexpression of the NF-κB family member RelB dampened IL-1β-induced Cox-2 expression. Fibroblasts were infected with either a control or RelB recombinant adenovirus at 10,000 MOI for 24 hours, after which they were exposed to IL-1β at 10 ng/mL for 24 hours. Cox-2 and RelB expression were detected in whole cell lysates by Western blot. IL-1β-induced Cox-2 expression was reduced in all three types of ocular fibroblasts after RelB overexpression (red boxes). Basal Cox-2 expression was also decreased after RelB overexpression in corneal fibroblasts. All samples from each blot were transposed from different locations on the same gel, but are shown separated by white space.