A3D8 induces monocytic differentiation of THP-1 and HL60 cells.

The data presented in panels A-E have been determined after addition of 2.5 μg/mL A3D8 at day 5 in the case of THP-1 cells (A,B) and at day 6 in the case of HL60 cells (C,D), except cytokine transcript synthesis, which has been analyzed at 24 hours (E). (A,C) Increased expression of lineage differentiation antigens. The expression of CD11b, CD14, and CD15 was measured as described in “Materials and methods” and in the legend to Figure 1A. (B,D) May-Grünwald Giemsa–stained cytosmears. (B) THP-1 cells: control THP-1 cells showed an immature phenotype: high nucleus-cytoplasmic ratio and numerous nucleoli. In contrast, A3D8-treated THP-1 cells showed a decreased nucleus-cytoplasmic ratio, chromatin condensation, and irregular cytoplasmic contours, all typical of mature monocytes. (D) HL60 cells: control cells showed an immature myeloblastic phenotype: high nucleus-cytoplasmic ratio and numerous nucleoli. A3D8-treated cells show a decrease in nucleus-cytoplasmic ratio, chromatin condensation, and irregular cytoplasmic contours typical of mature monocytes. Magnification: × 630. (E) Induction of synthesis of IL-1β, G-CSF, and IL-3 transcripts in HL60 cells. Total RNA from control and treated HL60 cells was prepared as described in “Materials and methods.” Using primers for glyceraldehyde phosphodehydrogenase, the amounts of cDNA were equilibrated to this internal standard. RT-PCR amplification products were separated by 1% agarose gel electrophoresis with 0.5 μg/mL ethidium bromide and were pictured under UV-light irradiation. Twenty-four hours after addition of A3D8, the expression of IL-1β, G-CSF, and IL-3 transcripts was detected. The specificity of the amplification was assessed by Southern blot hybridization with a specific oligonucleotide probe (data not shown). Pretreatment with genistein, a specific inhibitor of tyrosine kinases, abrogated this expression (data not shown).