We have previously reported that human promyelocytic leukemia HL-60 cells can be induced to differentiate into mature granulocytes when HL-60 co-cultivated with human bone marrow stromal LP101 cells. In the present study, we investigated which factors produced by LP101 cells induce HL-60 cells to differentiate into mature granulocytes. The expression of the cell surface antigen CD11b on HL-60 cells was increased after a 72-h culture with the conditioned medium (CM) obtained from LP101 cells. LP101 cells were observed to produce various cytokines, including TNF-α, GM-CSF and IL-6. The neutralizing antibodies against these cytokines partially suppressed the CM-induced differentiation of HL-60 cells. Recombinant TNF-α induced the differentiation of HL-60 cells, and GM-CSF and IL-6 additionally enhanced the effect of TNF-α. When the CM was divided into a low molecular weight (LMW) fraction and a high molecular weight (HMW) fraction by ultrafiltration, the LMW fraction synergistically enhanced the differentiation inducible activity of TNF-α. These results demonstrate that LP101 cells induce the differentiation of HL-60 cells by producing various cytokines including TNF-α, IL-6, and GM-CSF, and that unknown low molecular weight factors also participate.

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