No abstract

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.

Purpose: Increased expression of transforming growth factor-b2 (TGF-b2) is reported in the conjunctiva of dry eye patients with no increase of anti-inflammatory activity of TGF-b2. Our aim was to compare the expression of molecules involved in TGF-b2 activation, thrombospondin-1 (TSP-1) and CD36, during murine and human conjunctival inflammation. Methods: Human conjunctival tissue from cadaveric donors, human conjunctival epithelial primary cells and fibroblasts, and murine conjunctivas were immunostained for TSP-1, CD36, or TGF-b2. Inflamed conjunctival tissues were obtained from C57BL/6 wild-type (WT) mice induced to develop experimental dry eye (EDE) with 10 days of desiccating conditions and scopolamine injections and TSP-1-deficient (TSP1 -/ -) mice, which spontaneously develop Sjögren's syndrome-associated conjunctival inflammation with age. Immunostaining intensities were compared using ImageJ software. Cultures of human conjunctival fibroblasts were stimulated with IL-1b and both secreted protein and message levels of TSP-1, CD36, and TGF-b2 were analyzed. Results: TSP-1 and CD36 were detectable in human and murine conjunctival tissues as well as primary conjunctival epithelial cells and fibroblasts. Increased conjunctival immunostaining of TGF-b2 and reduced CD36 were detected in EDE mice compared with WT mice. Interestingly, increased TGF-b2 and CD36 conjunctival immunostaining was detected in TSP1-/ -mice. The expression of TSP-1 and CD36 was downregulated in IL-1b-stimulated conjunctival fibroblasts at both the protein and message level, while active TGFb2 was undetected. Conclusions: The absence or reduced expression of either of the molecules involved in TGF-b2 activation supports proinflammatory conditions in the conjunctiva. Changes in TSP-1 and CD36 may serve as potential biomarkers of conjunctival inflammation.