A corneal epithelial inhibitor of stromal cell collagenase synthesis identified as TGF-beta 2.

PURPOSE To determine the molecular mechanisms whereby substances released by corneal epithelial cells act to inhibit the elaboration of collagenolytic activity by corneal stromal cells and to determine whether inhibitory activity might be mediated by interleukin-1 receptor antagonist (IL-1ra) or transforming growth factor (TGF)-beta. METHODS Conditioned media were generated from primary cultures of rabbit corneal epithelial cells, from passaged cultures of rabbit corneal stromal fibroblasts, and from cells of the rabbit corneal epithelial cell line, SIRC. Pure populations of stromal cells were isolated from rabbit cornea and used directly for bioassay of the conditioned media to detect substances that inhibit collagenase synthesis. The mink lung epithelial cell line, Mv1Lu, was used for bioassay of TGF-beta-like activity. The addition of specific neutralizing antisera to bioassays allowed an assessment of the contribution of each isoform to the net regulatory activity. Reverse transcription-polymerase chain reaction and Northern blot analysis were employed to detect the presence of IL-1ra or TGF-beta mRNA species in cells from cultures used to generate conditioned media. RESULTS Both stimulatory and inhibitory substances that regulate the synthesis of stromal cell collagenase are released by corneal epithelial cells in primary culture. In contrast, only stimulatory activity is produced by corneal fibroblasts or SIRCs. MRNAs for a TGF-beta isoform, TGF-beta 2, and IL-1ra were identified in epithelial cells. Stromal fibroblasts also expressed TGF-beta 2 mRNA, but no evidence was found for expression of TGF-beta 3 mRNA in any of the three cell types. TGF-beta 2 is released by epithelial cells in both active and latent forms. This cytokine mediates the major portion of the net inhibitory activity against stromal cell collagenase synthesis produced by corneal epithelial cells. CONCLUSIONS This study demonstrates the expression of TGF-beta 2 and IL-1ra by corneal epithelial cells in culture. It is the TGF-beta 2 that acts as the major inhibitor of collagenase synthesis by corneal stromal cells in culture. However, IL-1ra and TGF-beta 2 are likely to play important roles in epithelial-mesenchymal interactions regulating corneal development, homeostatic maintenance, and repair.