MODULATION OF MACROPHAGE COLONY STIMULATING FACTOR IN CULTURED HUMAN RETINAL-PIGMENT EPITHELIAL-CELLS

Steady-state mRNA expression and protein production of macrophage colony stimulating factor were measured in visually confluent monolayers of unstimulated cultured human retinal pigment epithelial cells and after cells were stimulated with recombinant cytokines. Using reverse transcription polymerase chain reaction, macrophage colony stimulating factor mRNA expression was detected in unstimulated cells obtained from each of four separate donors. In these cells, mRNA expression was accompanied by secretion of macrophage colony stimulating factor protein into cell-conditioned medium; 48 hr after cells were switched to fresh medium, the mean (±s.d.) quantity of macrophage colony stimulating factor, measured by enzyme-linked immunoassay, was 5·1±2·3 ng 10-6 cells. There was a dose- and time-dependent induction of macrophage colony stimulating factor mRNA after cells were exposed to recombinant human interleukin-1 and tumor necrosis factor α. Maximal mRNA induction was observed in cells exposed for 4 hr to interleukin-1β (5U ml-1) or for 4-8 hr to tumor necrosis factor α; under these conditions, macrophage colony stimulating factor mRNA was induced up to 23- and 46-fold after exposure to interleukin-1β and tumor necrosis factor α, respectively. Similarly, macrophage colony stimulating factor protein production was enhanced after cells were exposed to recombinant cytokines. Protein secretion increased 1·3-2·5-fold (P < 0·001) after exposure to interleukin-1β (5 U ml-1), and 1·2-1·6-fold after exposure to tumor necrosis factor α (P < 0.03). This cytokine mediated induction of macrophage colony stimulating factor secretion was preceded by an increase in cell-associated macrophage colony stimulating factor measured in cell lysates. The macrophage colony stimulating factor secreted by retinal pigment epithelial cells was biologically active, as determined by a bone marrow stimulation bioassay. The average number of macrophage specific colonies produced (±s.d.) was 190±42 10-5 bone marrow cells plated. In contrast, colonies were not produced with medium alone. The bioactivity was entirely due to macrophage colony stimulating factor activity, because colony growth was completely suppressed when the assay was conducted in the presence of neutralizing antibody to human macrophage colony stimulating factor. We conclude that human retinal pigment epithelial cells express macrophage colony stimulating factor mRNA, and synthesize and secrete biologically active protein under basal conditions; mRNA expression and protein production are modulated by the inflammatory cytokines interleukin-1β and tumor necrosis factor α. © 1992.