Fcr Receptor and Mac-1 Expression and Functional Differentiation of HL-60 Cells by All-trans Retinoic Acid

ABSTRACT

PURPOSE: During differentiation of HL-60 cells by all-trans retinoic acid(ATRA), we analyzed the expression of Fcr receptors and Mac-1 by molecules of equivalent soluble fluorochromes(MESF) and functional studies. METHODS: HL-60 cells were induced to differentiate by adding 1micrometer ATRA. On the 4th and 7th day after stimulation as well as before stimulation, the cells were analyzed for phenotypic and functional differentiation. Phenotypic analysis was performed by flow cytometry after staining the cells with PE-conjugated anti-human CD64(FcrRI), CD32(FcrRII), CD16(FcrRIII), CD11b, CD18. The measured fluorescent intensity was transformed into MESF. Phagocytic activity was measured by flow cytometry after incubation of the cells with fluorochrome-conjugated beads. Respiratory burst was measured by chemiluminescence assay. ADCC was measured by hemoglobin release assay. Opsonophagocytic activity was measured by fungicidal assay. Correlation between MESF of FcrR and Mac-1 and function of HL-60 was measured. RESULTS: Percent positive cells and MESF of CD11b and FcrRI increased on the 4th day and decreased on the 7th day. Percent positive cells of CD18 was 99% regardless of differentiation. But MESF of CD18 was increased on the 4th day and decreased on the 7th day. Percent positive cells of FcrRII were above 90% regardless of differentiation. MESF of FcrRII showed no significant change. FcrRIII expression was not induced. Phagocytic activity of HL-60 cells was increased twofold. Chemiluminescence of HL-60 cells was increased up to 60-fold on the 7th day. ADCC of HL-60 cells was incerased up to 2.5-fold on the 7th day. Opsonophagocytic activity increased twice on the 4th day. ADCC and opsonophagocytic activity correlates with the expression of CD11b/CD18 and FcrRII. CONCLUSION: Differentiation of HL-60 cells with ATRA induces several functional maturations until 7 days with expression of FcrR and Mac-1.