J Korean Med Sci.  2015 Feb;30(2):145-150. 10.3346/jkms.2015.30.2.145.

Phenotypic and Functional Analysis of HL-60 Cells Used in Opsonophagocytic-Killing Assay for Streptococcus pneumoniae

• Kim KH
• Seoh JY
• Cho SJ

• KMID: 2129643
• DOI: http://doi.org/10.3346/jkms.2015.30.2.145

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 1alpha, 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.

Keyword

Opsonophagocytic-Killing Assay; Streptococcus pneumonia; HL-60; Differentiation

MeSH Terms

Antibodies, Bacterial/immunology
Antigens, CD11c/metabolism
Antigens, CD14/metabolism
Antigens, CD18/metabolism
Apoptosis/*immunology
Biological Assay
Cell Differentiation
Cell Line, Tumor
Cholecalciferol/pharmacology
Dimethylformamide/pharmacology
Flow Cytometry
HL-60 Cells
Humans
Phagocytosis/*immunology
Pneumococcal Vaccines/*immunology
Receptors, IgG/metabolism
Receptors, Immunologic/*biosynthesis
Respiratory Burst/immunology
Streptococcus pneumoniae/*immunology
Tretinoin/pharmacology
Antibodies, Bacterial
Antigens, CD11c
Antigens, CD14
Antigens, CD18
Cholecalciferol
Dimethylformamide
Pneumococcal Vaccines
Receptors, IgG
Receptors, Immunologic
Tretinoin