IgE-anti-IgE-induced prostaglandin D2 release from cultured human mast cells

To elucidate the characterization of cultured human mast cells, the generation of prostaglandin D2 (PGD2) upon stimulation with IgE-anti IgE challenge was determined by using gas chromatography/mass spectrometry/selected ion monitoring (GC/MS/SIM) assay. Mononuclear cells obtained from human umbilical cord blood were cultured in the presence of Steel Factor, IL-6, and PGE2. After 8 weeks of culture, approximately 90 to 95% of cultured cells became tryptase-positive and included basophilic granules. At 12 weeks of culture, cells were harvested and incubated with 1 microgram/ml of human IgE for 1 hr and then challenged with 10 micrograms/ml of anti-IgE. The level of PGD2 release into the supernatant was measured by GC/MS/SIM with the stable isotope dilution method. Cyclooxygenase inhibitors affected the PGD2 release from the cell by IgE-anti IgE challenge incubation. Indomethacin inhibited 98% of PGD2 release. Acetylsalicylic acid (aspirin) and NS-398 (potent COX-2 inhibitor) also inhibited PGD2 release by 85 and 45% respectively.