FIGURE 8.

Mast cell-derived PGD₂ activates orbital fibroblasts to produce HA. A, contact co-culture. Confluent orbital fibroblasts were seeded with HMC-1 cells at a cell ratio of 1:1 for 4 h. The mast cells were then removed, the fibroblasts were washed, and fresh medium was added for another 18 h. The medium was collected for HA ELISA. Co-culture of orbital fibroblasts with HMC-1 cells significantly increased HA synthesis (**, p < 0.001). B, transwell co-culture. HMC-1 cells and confluent fibroblasts were co-cultured in a transwell system, where the fibroblasts and HMC-1 cells were separated by a 0.4-μm membrane; the HMC-1/orbital fibroblast (OF) ratio was 5:1, 10:1, or 20:1. The conditioned medium from both chambers was collected for HA ELISA. There was a significant difference in HA levels between HMC-1 (upper chamber, open bars) and orbital fibroblasts (lower chamber, black bars) (#, p < 0.05; ###, p < 0.001). Co-culture of orbital fibroblasts with HMC-1 cells significantly increased HA production only by the fibroblasts (lower chamber) (***, p < 0.001 compared with no HMC-1 cells). C, inhibition of PGD₂ secretion by HMC-1 cells prevents HA production by orbital fibroblasts. HMC-1 cells were treated with the H-PGDS inhibitor HQL-79 prior to co-culturing (ratio 20:1) with orbital fibroblasts. There was a significant increase in HA synthesis when fibroblasts were cultured with HMC-1 cells (**, p < 0.01 compared with no HMC). This increase in HA was attenuated when PGD₂ production in mast cells was prevented by HQL-79 (##, p < 0.001, HQL-79 compared with vehicle).