Phenotypic and functional analysis of HL-60 cells used in opsonophagocytic-killing assay for Streptococcus pneumoniae

Abstract

Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1α, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.

Keywords: Differentiation; HL-60; Opsonophagocytic-Killing Assay; Streptococcus pneumonia.

Fig. 1

Analysis of apoptotic cell death during HL-60 differentiation. HL-60 cells were incubated with or without DMF (0.8%), ATRA (1 µM), or VitD₃ (20 µg/mL) for the indicated time period. Cells were stained with 20 µg/mL of 7-AAD and percentage of apoptotic cell populations were determined by analyzing fluorescence intensity of moderately stained cells with flow cytometry. ^*Indicates P < 0.05 when the values were compared with that of the day 0. DMF, dimethylformamide; ATRA, all-trans retinoic acid; VitD₃, vitamin D₃; 7-ADD, 7-aminoactinomycin D.

Fig. 2

Analysis of respiratory burst during HL-60 differentiation. HL-60 cells were incubated with or without DMF (0.8%), ATRA (1 µM), or VitD₃ (20 µg/mL) for the indicated time period. The HL-60 cells were incubated with 50 mM luminal and 500 nM PMA for 2 hr. Chemiluminescence intensity was examined with a luminometer. One of the three similar results is shown. DMF, dimethylformamide; ATRA, all-trans retinoic acid; VitD₃, vitamin D₃; 7-ADD, 7-aminoactinomycin D.

Fig. 3

Opsonophagocytic-killing assay activity of HL-60 cells during differentiation. HL-60 cells were incubated with or without DMF (0.8%), ATRA (1 µM), or VitD₃ (20 µg/mL) for the indicated time period. The HL-60 cells were applied to a conventional pneumococcal OPKA against serotype 19F as described in the Materials and Methods. DMF, dimethylformamide; ATRA, all-trans retinoic acid; VitD₃, vitamin D₃; 7-ADD, 7-aminoactinomycin D.