Pre-B cells in mouse bone marrow: immunofluorescence stathmokinetic studies of the proliferation of cytoplasmic mu-chain-bearing cells in normal mice

Abstract

By using a technique that combines metaphase arrest with immunofluorescence labeling, the proliferation of specifically identified pre-B cells in mouse bone marrow has been analyzed under physiological conditions in vivo. Pre-B cells bearing cytoplasmic mu-chains and no surface mu-chains constituted 12% of marrow nucleated cells, or 27 X 10(5) cells/femur, whereas surface mu-bearing B lymphocytes totaled 33 X 10(5) cells/femur. Pre-B cells measured 7 to 14 micron in diameter, the small number seen in metaphase (1 to 2%) being large cells (greater than 10 microns). After vincristine injection, the metaphase incidence (Imet) of pre-B cells increased with cell size; a broad-dose range of vincristine gave similar Imet values. Mitoses were arrested for 4 hr with no apparent cell death. Linear regression analysis of the increase in Imet of pre-B cells 2 to 4 hr after vincristine revealed a rate of entry into mitosis of 6.3%/hr, relative to all pre-B cells (average compartment turnover time, 16 hr), and 15.3%/hr for the large proliferating pre-B cell subset. This represented a pre-B cell production of 1.3 X 10(5) cells/femoral shaft/hr or 0.5 X 10(8) cells/whole bone marrow organ/day, emphasizing the magnitude of B lymphocyte genesis in normal bone marrow. Combined with reported renewal rates for small pre-B cells and small B lymphocytes, these values form a kinetic model of B lymphocyte development. The results reveal an apparent overproduction of large pre-B cells, consistent with a speculative post-mitotic loss of some immature primary B lymphocytes.