Analysis of the secretion pattern of monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-beta 2 (TGF-beta2) by human retinal pigment epithelial cells

Abstract

Retinal pigment epithelial (RPE) cells, situated between the neurosensory retina and the vascularized choroid, form part of the blood-eye barrier and are important for homeostasis of the outer retina. These cells are able to produce a variety of cytokines which may play a role in the maintenance of the immunosuppressive milieu inside the eye and in intraocular inflammatory responses. In the present study, we investigated whether RPE cells secreted the anti-inflammatory cytokine TGF-beta2 and the proinflammatory cytokine MCP-1 in a polarized manner. Monolayers of human donor RPE cells were cultured on transwell filters. Secretion of TGF-beta2 and MCP-1 at either the apical or basal side of the RPE cell monolayers, that were not treated or stimulated with IL-1beta (200 U/ml), was analysed by ELISA. All three cell lines examined had a different TGF-beta2 secretion pattern. In two of the three donor RPE cell lines tested, TGF-beta2 secretion was polarized, but not in the same direction. TGF-beta2 secretion was not up-regulated by stimulation with IL-1beta. In contrast, IL-1beta strongly induced MCP-1 secretion preferentially into the basal compartment of all RPE monolayers tested. These data indicate that human RPE cells are able to secrete TGF-beta2 and MCP-1 in a polarized fashion. Our results suggest that MCP-1 can be secreted by RPE cells in the direction of choroidal vessels during inflammatory responses in the posterior part of the eye, which may limit damage to the neurosensory retina.

Fig. 1

TGF-β2 production by human donor retinal pigment epithelial (RPE) cell lines that were not treated or stimulated with IL-1β or tumour necrosis factor-alpha (TNF-α) for 48 h. RPE cells were seeded in 24-well plates and cultured until confluence in Iscove's modified Dulbecco's medium (IMDM) supplemented with 20% fetal calf serum (FCS). Before stimulation the RPE cells were cultured for 24 h in IMDM with 0.1% FCS. Cells were stimulated with IL-1β 200 U/ml or TNF-α 200 U/ml in 1 ml IMDM with 0.1% FCS for 48 h. The total TGF-β2 content (mature and latent) was determined by ELISA. Data are expressed as the mean of two or four wells ± s.e.m.

Fig. 2

Secretion of TGF-β2 by three donor retinal pigment epithelial (RPE) cell lines (372, 364, 605) into the basal compartment (segmented line) and into the apical compartment (continuous line) without stimulation (a) or after stimulation with IL-1β from the apical (b) or basal (c) side. RPE cells were seeded on transwell filters at a concentration of 1.6 × 10⁵ cells/cm² and cultured for at least 19 days in Iscove's modified Dulbecco's medium (IMDM) with 1% normal human serum (NHS) and had a transepithelial resistance (TER) > 30 Ω/cm². Filters were stimulated with 200 U/ml IL-1β in IMDM with 0.1% NHS. Data are expressed as absolute amounts of TGF-β2 per filter and are the mean of three or five filters ± s.e.m. *Statistically significant differences between upper and lower compartments.

Fig. 3

Secretion of MCP-1 by three donor retinal pigment epithelial (RPE) cell lines (372, 364, 605) into the basal compartment (segmented line) and into the apical compartment (continuous line) without stimulation (a) or after stimulation with IL-1β from the apical (b) or basal (c) side. RPE cells were seeded on transwell filters at a concentration of 1.6 × 10⁵ cells/cm² and cultured for at least 19 days in Iscove's modified Dulbecco's medium (IMDM) with 1% normal human serum (NHS) and had a transepithelial resistance (TER) > 30 Ω/cm². Filters were stimulated with 200 U/ml IL-1β in IMDM with 0.1% NHS. Data are expressed as absolute amounts of MCP-1 per filter and are the mean of three to five filters ± s.e.m. *Statistically significant differences between upper and lower compartments.