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It has been reported that vesnarinone, a new inotropic agent used in the treatment of cardiac failure, causes agranulocytosis as a side effect. To study the mechanisms by which this complication occurs, vesnarinone was introduced into a coculture system of HL60 and bone marrow (BM) stromal cells, in which HL60 cells were able to differentiate into mature granulocytes with no inducible exogenous factors added to the culture. When HL60 cells were cocultured with the human BM-derived stromal cell line LP101, HL60 cells were induced to differentiate into mature granulocytes, and expression of the mature granulocyte-macrophage surface antigen, CD11b was increased. Conditioned medium (CM) obtained from LP101 cells also showed the capacity to induce the maturation of HL60 cells, in a dose- and time-dependent manner. The differentiation of HL60 cells induced by CM was also determined by morphological analysis, expression of myeloperoxidase, and a nitroblue tetrazolium (NBT) reduction test. When HL60 cells were cocultured with LP101 in the presence of vesnarinone, the CD11b expression was greatly suppressed. CM obtained from vesnarinone-treated LP101 (ves-CM) lost the capacity to induce the differentiation of HL60 cells, at a concentration of 1 microg/mL of vesnarinone. Vesnarinone itself did not affect the proliferation of HL60 cells. Furthermore, the addition of vesnarinone or ves-CM to HL60 cultures incubated with CM did not alter the induction of CD11b expression, suggesting that vesnarinone has no effect on HL60 cells, but that it inhibits stromal cells from producing soluble factor(s) required for the differentiation of HL60 cells to mature granulocytes. All these findings indicate that vesnarinone causes the hematopoietic disorder agranulocytosis, via impairment of stromal function.

We have previously reported the establishment of a variety of human bone marrow stromal cell lines using a recombinant SV40-adenovirus vector. Using this vector, we obtained more clonal stromal cells. Here, we have characterized these cells and analyzed their capacity to support the proliferation and differentiation of hematopoietic cells. The stromal cells were cocultured with nonadherent human bone marrow cells used as hematopoietic cells. The total numbers of hematopoietic cells and CFU-GM in culture were counted every week. Two of the six stromal cell lines, AA101 and HAS303, supported the proliferation and differentiation of hematopoietic cells and CFU-GM for more than 9 weeks. Further, granulocytes, macrophages, and megakaryocytes were detected when cocultured with these cells. When hematopoietic cells were cocultured but separated from the two stromal cell lines by a 0.45-microns millipore membrane to prevent their attachment, almost all CFU-GM disappeared within 7 weeks. The supportive stromal cells produced GM-CSF and IL-6. However, other cell lines producing these humoral factors did not support hematopoietic cell proliferation for such a long time. These findings suggest that these established human bone marrow stromal cell lines will be useful, in that analysis of their supportive function in hematopoietic cell proliferation and differentiation through cell-to-cell interaction will shed some light on this area.