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

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.

Functional Differentiation of HL-60 Cells by Dimethylsulfoxide and Phorbol 12-myristate 13-acetate

PURPOSE: During hematopoietic differentiation of HL-60 cells by DMSO and PMA, we demonstrated functional changes of HL-60 cells-phagocytic activity, respiratory burst, antibody-dependent cell-mediated cytotoxicity(ADCC) and opsonophagocytic activity. METHODS: HL-60 cells(ATCC CCL-240), cultured in RPMI 1640 and supplemented with 10% FBS, were induced to differentiate by adding 1.0% DMSO or 16nM PMA. On the 4th and 7th day after stimulation as well as before stimulation, the cells were analyzed for functional differentiation. 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 using Candida albicans. RESULTS: Phagocytic activity of HL-60 cells was not increased by differentiation with DMSO. But PMA induced increase of phagocytic activity on 7th day. Respiratory burst studied by chemiluminescence was increased up to 4-fold on 7th day by DMSO. PMA induced increase upto 3-fold on 4th day. ADCC was increased upto 3-fold on 4th day by DMSO, but PMA induced little increase in ADCC. Opsonophagocytic activity was increased upto 3-fold on 4th day by DMSO or PMA. On differentiation with DMSO, respiratory burst correlates with FcgammaRI and FcgammaRII. ADCC and opsonophagocytic activity correlate with CD11b/CD18. On differentiation with PMA, phagocytic activity correlates with FcgammaRII. Respiratory burst correlates with CD11b. ADCC and opsonophagocytic activity of HL-60 cells correlate with CD18. CONCLUSION: Treatment of HL-60 cells with DMSO or PMA induces functional maturation differently. Phagocytic activity, respiratory burst, ADCC and opsonophagocytic activity of HL-60 cells correlated with expresson of FcgammaR and Mac-1.