Hematopoietic regulatory factors produced in long-term murine bone marrow cultures and the effect of in vitro irradiation

Abstract

The nature of hematopoietic regulatory factors elaborated by the adherent (stromal) cells of long-term murine bone marrow cultures and the effect of in vitro stromal irradiation (XRT) on the production of these factors was investigated. Using an in situ stromal assay employing a double layer of semisolid agar, it was possible to demonstrate stromal elaboration of at least two colony-stimulating activities, ie, granulocyte/macrophage colony-stimulating activity (G/M- CSA) and megakaryocyte colony-stimulating activity (Meg-CSA). Exposure of the stroma to XRT resulted in dose-dependent elevations of both activities that correlated inversely with total myeloid cell mass as determined by concurrent reductions in total supernatant cell recoveries from irradiated cultures. Mixture experiments that combined control and irradiated stroma revealed that the hematopoietically active control stroma could block detection of XRT-related G/M-CSA elevations. These data implicate a local negative feedback mechanism in the regulation of hematopoiesis. Antiserum directed against purified L cell colony-stimulating factor (CSF) reduced granulocyte/macrophage colony formation in the target layer but did not effect the increased Meg-CSA. While a radioimmunoassay for L-cell type CSF was unable to detect significant differences in concentrated media from control and irradiated cultures, bioassays of these media revealed XRT-related G/M- CSA elevations. These results indicate that the G/M-CSA elaborated in these cultures is immunologically distinct from the Meg-CSA produced, and although distinct from L cell CSF, the G/M-CSA is crossreactive with the L cell CSF antiserum. Morphologic, histochemical, and factor VII antigen immunofluorescent studies were performed on the stromal cell population responsible for production of these stimulatory activities. In addition to “fat” cells, the stromal cells remaining after XRT were composed of two predominant cell populations. These included a major population of acid phosphatase and nonspecific esterase-positive macrophage-like cells and a minor population of factor VII antigen negative epithelioid cells.

Effects of Myelotoxic Agents on Cytokine Production in Murine Long‐Term Bone Marrow Cultures

“…This may occur as a consequence of reduced numbers of myeloid elements or altered stromal cells leading to lower CSA or IL-6 requirements. The phenomenon of growth factor utilization in hematopoietically active cultures compared with irradiated cultures has been demonstrated for GM-CSA and CSA [9,11].…”

“…Cytokines play a central regulatory role in hematopoiesis [1][2][3][4][5]. In murine long-term bone marrow cultures (LTBMC) the production of colony-stimulating activity (CSA), macrophage-colony stimulating factor (M-CSF), GM-CSF, G-CSF, interleukin 6 (IL-6), stem cell factor (c-kit ligand), and IL-3 by stromal cells has been reported [4,[6][7][8][9][10][11][12]. However, few studies have examined the kinetics of cytokine production in this culture system [8,[11][12][13].…”

Isolation of a human stromal cell strain secreting hemopoietic growth factors

“…The stromal layer consists of an extracellular matrix and several types of cells, including fibroblasts, "blanket cells," adipocytes, macrophages, and endothelial cells (Allen and Dexter, 1984). Though early studies using the long-term liquid culture systems failed to detect biologically active GM-CSA in the conditioned medium from these cultures, it has been detected in more recent experiments (Heard et al, 1982;Shadduck et al, 1983;Gualtieri et al, 1984). This suggests that hemopoietic growth regulators are produced locally by stromal cells and bind immediately to receptors on the nearby devel-0 + ST-1 Monolayer -0 - oping progenitors and precursors.…”

“…While only limited direct evidence exists for either or the first two mechanisms, the in vitro dependence of hemopoietic colony growth on exogenous colony-stimulating activities (CSA) suggests that accessory cells capable of secreting hemopoietic growth factors may be involved in the regulation of hemopoiesis in vivo (Bradley and Metcalf, 1966;Pike and Robinson, 1970). Although early studies failed to detect production of either granulocyte-macrophage colony-stimulating activity (GM-CSA) or erythroid burst-promoting activity (BPA) by stromal cells, recent studies clearly show that stroma of bone marrow long-term culture can produce CSA (Heard et al, 1982;Shadduck et al, 1983;Gualtieri et al, 1984). While detailed analysis of the cellular and molecular basis of stromal cell-progenitor cell interaction requires the isolation of the specific stromal cell types involved, no homogeneous human stromal cell preparation has yet been described.…”

Persistent production of colony‐stimulating factor (CSF‐1) by cloned bone marrow stromal cell line D2XRII after X‐irradiation

“…Irradiation of intact, hematopoietically active whole bone marrow cultures to 900 rad produces a n increase in the levels of detectable CSF-1 (Gualtieri et al, 1984). In contract, irradiation of purified bone marrow stromal cell cultures has been shown to decrease the levels of detectable CSF-1 (Greenberger et al, 1984a).…”