Differentiation of Mouse Bone Marrow Precursor Cells into Neutrophil Granulocytes by an Activity Separation from WEHI-3 Cell-Conditioned Medium

149

Colony-stimulating factors (CSFs) stimulate the differentiation of immature precursor cells to mature granulocytes and macrophages. Purified 1251-labeled murine L cell CSF has been used to develop a radioimmunoassay (RIA) that detects a subclass of CSFs that stimulates macrophage production. Murine CSF preparations that contain this subclass of CSF compete for all of the CSF binding sites on anti-L cell CSF antibody. With the exception of mouse serum, which can contain inhibitors of the bioassay, there is complete correspondence between activities determined by RIA and those determined by bioassay. The RIA is slightly more sensitive than the biosassay, detecting approximately 0.3 fmol of purified L cell CSF. It can also detect this subclass of CSF in chickens, rats, and humans. In the mouse, the subclass is distinguished from other CSFs by a murine cell bioassay dose-response curve in which 90% of the response occurs over a 10-fold (rather than a 100-fold) increase in concentration, by stimulating the formation of colonies containing a high proportion of mononuclear (rather than granulocytic) cells, and by certain physical characteristics.

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Colony-stimulating factor (CSF) radioimmunoassay: detection of a CSF subclass stimulating macrophage production.

Stanley

1979

149

“…Single-cell cloning studies indicated that many progenitor cells are bipotential and are able to form both granulocyte and macrophage progeny (2,3). In the presence of high regulator concentrations, even with GM-CSF purified to homogeneity, a high proportion of granulocytic colonies develops whereas in the presence of very low concentrations, only macrophage colonies develop (1,4,5). A relationship has also been observed between GM-CSF concentration and the rate of proliferation of developing colony cells (6).…”

mentioning

confidence: 99%

Clonal analysis of proliferation and differentiation of paired daughter cells: action of granulocyte-macrophage colony-stimulating factor on granulocyte-macrophage precursors.

Metcalf¹

1980

122

Mouse granulocyte-macrophage progenitor cells were stimulated to divide by the granulocyte-macrophage colony-stimulating factor (GM-CSF). The two daughter celis were separated; one daughter was transferred to medium containing a high concentration of GM-CSF, the other to medium containing a low concentration. Daughter cell-derived clones in the presence of 2500 units of GM-SF had average cell cycle times 3.5 + 2.5 (SEM) hr shorter than clones derived from the paired daughter cell stimulated by 50 units of GM-CSF. Final colony size achieved after stimulation by 50 units of GM-CSF was always smaller than that of colonies stimulated by 2500 units of GM-CSF. In 8 of 41 instances, colonies stimulated by 50 units of GM-CSF developed, or were composed only of, macrophage populations in contrast to the granulocytic composition of colonies derived from the paired daughter cell growing in the presence of 2500 units of GM-CSF. The regulator GM-CSF appears able to directly influence cell cycle times and the pathway of differentiation entered by many bipotential granulocyte-macrophage precursor cells.In the presence of adequate concentrations of the specific regulator, granulocyte-macrophage colony-stimulating factor (GM-CSF), the progenitors of granulocytes and macrophages are stimulated to proliferate in vitro to form colonies of differentiating granulocytes or macrophages or both (1). Single-cell cloning studies indicated that many progenitor cells are bipotential and are able to form both granulocyte and macrophage progeny (2, 3). In the presence of high regulator concentrations, even with GM-CSF purified to homogeneity, a high proportion of granulocytic colonies develops whereas in the presence of very low concentrations, only macrophage colonies develop (1,4,5). A relationship has also been observed between GM-CSF concentration and the rate of proliferation of developing colony cells (6). These observations suggested that GM-CSF is able to shorten cell cycle times of proliferating granulocyte-macrophage cells and to determine whether progeny cells exhibit granulocyte or macrophage differentiation.Interpretation of these data has been complicated by the demonstration of heterogeneity among granulocyte-macrophage progenitor cells (7,8). Subsets of precursors exist that appear to be preprogrammed to form only granulocytic progeny and to require high concentrations of GM-CSF for stimulation. Conversely, subsets of granulocyte-macrophage progenitors appear able to form only macrophage progeny and to respond to stimulation by low GM-CSF concentrations. It might be, therefore, that GM-CSF merely selectively activates differing subsets of preprogrammed progenitor cells without having any significant capacity to influence the number or type of progeny produced.The present experiments using paired daughter cells of in-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this...

“…The different CSA are known to differ in terms of the morphology of the colonies they elicit, but this does not correspond to the NZB abnormality. Most preparations of semipurified WEHI-3-M preferentially stimulate neutrophil colonies and LCCM elicits almost pure macrophage colonies (16,29). However, NZB precursors made at least normal numbers of granulocyte colonies in ES and failed to respond to batches of WEHI-3-M that were significantly contaminated with monocyte-macrophage stimulators.…”

mentioning

confidence: 99%

Abnormalities in clonable B lymphocytes and myeloid progenitors in autoimmune NZB mice.

Kincade¹,

Lee²,

Fernandes³

et al. 1979

Cloning procedures were used to study B lymphocytes and progenitors of granulocytes and macrophages in NZB mice. Numbers of B cells that were detected in sheep erythrocyte-containing semisolid cultures were only slightly elevated in NZB tissues, and these were normally sensitive to inhibition by anti-,u or anti-b antibodies or prostaglandin E.However, NZB mice rapidly developed large numbers of B cells that could be cloned in the presence of lipopolysaccharide, and these included unusual anti-,u resistant cells. Numbers of myeloid precursors in NZB bone marrow that were responsive to colony-stimulating activity in L-cell conditioned medium or endotoxin serum were at least normal, but at all ages granulocyte-macrophage precursors were poor responders in cultures stimulated by WEHI-3 cell conditioned medium. Almost no colonies were elicited in NZB cultures with a colony-stimulating activity moiety from WEHI-3 cells. Prostaglandin sensitivity of myeloid precursors from NZB and CBA mice was also different. Codominant genetic control of these abnormalities was suggested by their partial expression in F1 hybrid NZB X CBA and NZB X NZW mice. NZB mice expressed an unexpected IgD allotype allele.Abnormalities have been found in or attributed to T cells, thymic epithelium, B cells, macrophages, and stem cells in autoimmune NZB mice, and they produce high levels of xenotropic virus (1-6). The relevance of each of these abnormalities to the development of the hemolytic anemia in these mice is not clear, but genetic studies tend to minimize the role of viruses and certain hemopoietic abnormalities in the etiology of disease (5, 6). A possible temporal sequence is suggested by findings that B cells of NZB mice are polyclonally activated by 1 week of age, and autoantibodies with preference for suppressor T cells are detectable early in adult life (7-10). A primary B-cell abnormality or defects that are expressed at the B-cell level could thus predispose to subsequent aberrant T-cell function. On the other hand, production of antierythrocyte autoantibody may occur in the absence of significant numbers of T cells, and potential for autoimmunity is transferrable with fetal liver or bone marrow cells from NZB mice (3, 11). Some gene products influencing manifestation of autoimmunity might therefore be expressed in pre-B cells, B cells, or nonlymphoid cells capable of activating B cells. Limiting dilution cloning techniques are available for enumerating and characterizing several hemopoietic precursors as well as lymphocytes, and we propose that these can be of value for identifying intrinsic defects in various cell types and for following changes in these with progression of disease. Thus far, we have found that particular culture conditions reveal an exceptional category of B lymphocytes in NZB lymphoid tissues, which, unlike B cells from many other strains, proliferate in the presence of anti-ji antibody. In addition, a severe abnormality was detected in the ability of precursor cells of NZB mice to form myeloid colonie...