Maturation rate of mouse neutrophilic granulocytes: Acceleration by retardation of proliferation, but no detectable influence from G-CSF or stromal cells

Our purpose was to examine the possible influence of stromal and humoral me diators on granulocytic maturation rates, Sorted immature murine progenitor (Lin(-)Sca-1(+)) cells were cultured in peritoneal diffusion chambers (DCs ) with or without a confluent layer of irradiated bone marrow stromal cells on one of the micropore membrane walls, In other experiments, 10 mu g/kg/d recombinant G-CSF (rhG-CSF) was administered continuously into DC host mic e through s.c. implanted osmotic minipumps, Operationally, maturation rate was assessed as the ratio between the number of polymorphonuclear cells (PM N) and proliferative granulocytes (PG) in short-term cultures, based on the differential cell counts, and supported by flow cytometric measurement of a granulocytic differentiation marker: and by the emergence time of PMN in the DCs, obtained by extrapolation. Also, increased maturation is associate d with increased cell density, as reflected by the positioning of the granu locytes during centrifugation in a discontinuous Percoll gradient. This met hod, as well as the conversion rate of H-3-thymidine labeled PG into the he avier non-PC maturational stages, were also used as indicators of maturatio n rate. After five, six, and seven days of culture in the peritoneal cavity , DC cells were harvested. Their proliferative status, based on measurement of incorporated bromodeoxyuridine, was determined, and their maturation sa tes were evaluated. Proliferation of immature granulocytic progenitor cells was apparently inhibited by direct contact with bone marrow stromal cells, and stimulated by G-CSF during the early stage of culturing. However, the subsequent maturation rate, which could be accelerated by increasing cultur e cellularity, thus decreasing PG proliferation rate, was not detectably in fluenced by either stromal cells or G-CSF.