Increased production of transforming growth factor-beta 2 from cultured human retinal pigment epithelial cells by photocoagulation

Abstract

Purpose: To examine whether laser photocoagulation increased production of growth factors and cytokines, especially transforming growth factor-beta 2 (TGF-beta 2), by cultured human retinal pigment epithelial (RPE) cells.

Methods: Dot blot analysis was performed to identify the changes using the conditioned medium of cultured human RPE cells with or without laser photocoagulation. The following growth factors and cytokines were examined: TGF-beta 1, TGF-beta 2, platelet-derived growth factor-BB, epidermal growth factor, interleukin-1 beta, tumor necrosis factor-alpha, and basic fibroblast growth factor. Immunostaining of the photocoagulated RPE cells was also performed by using the antibodies. Growth inhibition assay using mink lung epithelial cells (CCL-64) was employed both to quantitate active and latent forms of TGF-beta 2 secreted in the conditioned media and to determine the type of TGF-beta by using a neutralizing antibody.

Results: In dot blot analysis, among the growth factors and cytokines examined, production of TGF-beta 2 was changed most markedly by laser photocoagulation. This result agreed with the immunocytochemical staining pattern. Photocoagulated RPE-conditioned medium inhibited [3H]-thymidine incorporation of CCL-64. The inhibitory activity was blocked by a neutralizing antibody to TGF-beta 2. Production of active TGF-beta 2 was increased from 1.0 pg/cm2 +/- 0.6 pg/cm2 per 24 hours to 11.0 pg/cm2 +/- 1.0 pg/cm2 per 24 hours by laser photocoagulation, and that of total TGF-beta 2 was increased from 11 pg/cm2 +/- 1.0 pg/cm2 per 24 hours to 100 pg/cm2 +/- 20.1 pg/cm2 per 24 hours.

Conclusions: Laser photocoagulation of cultured RPE cells increases their production of TGF-beta 2. It thus seems possible that the TGF-beta 2 produced by the RPE cells plays an important role in the processes that occur after laser photocoagulation.