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Murine osteoclasts and B-lymphocates share a common progenitor cell whose abundance in bone marrow is regulated by estrogen

Osteoclasts develop from a hematopoietic precursor cell that can also differentiate into macrophages and granulocytes. In states of estrogen withdrawal, such as after ovariectomy (OVX), the number of osteoclast and lymphocyte precursor cells increases in bone marrow. We speculated that these events occur because estrogen withdrawal increases the abundance of common lympho-myeloid progenitor in the bone marrow. We analyzed B-lymphocyte lineage cells (B-cells)based on surface expression of B220 antigen. Mature B-cells were identified as expressing surface IgM. Immature B cells were designated by their expression of HSA (CD24), as either pro-B cells (HSA low) or pre-B cells (HSA high). Cells were stained with apropriate antibodies and purified as either immunomagnetic beads or FACS. The capacity of selected cells to form osteoclast-like cells (OCL) in culture was tested by treatment for 5-7 days with RANK-L ansd M-CSF (30 ng/ml both). We also evaluated expression of RANK, in cells by RT-PCR or by surface staining with a specific antibody. Both B220 posiutive (+) and B220 negative (-) cells were comparable in their ability to generate OCL after treatment with M-CSF and RANK-L. However virtually all the OCL progenitor activity within the B220+ cells was contained in the fraction with an immature phenotype (pre- and pro- B cells, 563+/- 34/well) as few OCL (4+/-2/well), were seen in cultures mature B cells (B220+, IgM+). OVX caused an approximately 50% increase in total B cells in bone marrow (p<.05), which was mostly results of a 2-fold increase in pre-B cells (p<.05). OVX also caused a 2-fold increase in the number of OCL that formed in cultures of B220+ bone marrow cells (p<.01). We next examined the expression of RANK in bone marroew cells, since this receptor is essential for OCL formation. Only 0.25% of the cells in bone marrow were RANK+ and essentially all of theese were mature B cells (B220+, IgM+). OVX did not alter the percentage of RANKL+ cells in marrow or the proportion of these that were IgM+. In addition essentially, all the B220* cells that formed OCL in 5-7 day culture with M-CSF and RANK-L were RANK-. This result implied that M-CSF stimulates RANK expression in OCL precursors. To confirm this, we cultured spleen cells without stromal cells and demonstrate a dramatic increase in RANK mRNA expression when M-CSF was present. These resulčts demonstrate that OVX increases the n umber of B220+ cells in bone marrow, that can form OCL. The OCL precursor, which is regulated by estrogen, appears to have the phenotype: B220+, IgM- and RANK-. The direct effect of estrogen withdrawal on these cells is likely an important event in the pathogenesis of the bone loss that occurs with OVX.

osteoclasts; B-lymphocytes; cell differentiation; cytokines

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