Normal Mouse Bone Marrow Research Articles

Partial purification and characterization of a growth factor for macrophage progenitor cells with high proliferative potential in mouse bone marrow

A population of macrophage progenitor cells, with high proliferative potential, has recently been demonstrated in postfluorouracil-treated and normal mouse bone marrow (BM) in vitro, when the newly discovered growth factor (synergistic activity, SA) is combined with a macrophage colony-stimulating factor (CSF) as a proliferative stimulus. SA, shown to be present in human spleen and placental conditioned media (HSCM and HPCM, respectively) have been studied and found to be unstable to trypsin digestion and to heating at 50 degrees C or above; stable between pH 4 and 9; nonadherent to Con-A-Sepharose; and to have an isoelectric point between pH 5 and 5.8 and a molecular weight of between 14,000 and 21,000 as indicated by gel filtration chromatography. SAs from both HSCM and HPCM have been purified 89- and 122-fold, respectively, by precipitation of extraneous proteins at pH 5 followed by chromatographing twice on Sephacryl S200. Neither of these partially purified SAs contain any CSF for mouse BM. These results indicate that the SAs from HSCM and HPCM may be closely related and that they are structurally different from CSFs derived from various murine sources that have been shown to be stable to proteolytic enzymes and heat.

Culture of macrophage cell lines from normal mouse bone marrow.

The use of semisolid medium for the culture and cloning of haematopoietic cells has helped our understanding of their proliferation and differentiation. It has been shown that mixed colonies of granulocytes and macrophages developed, in the presence of colony-stimulating factor (CSF), from their common precursor, granulocyte-macrophage colony-forming cells (GM-CFC). Attempts at recloning these colonies in semisolid medium suggested that granulocytes and macrophages were differentiated cells incapable of further proliferation. However, our studies on cultures of larger numbers of cells demonstrate that while this may be the case for granulocytes, macrophages seem to be capable of long-term proliferation.

Natural cell-mediated cytotoxicity in vitro and inhibition of tumor growth in vivo by murine lymphoid cells cultured with T cell growth factor (TCGF).

Lymphoid cells obtained from the spleen, thymus, bone marrow, peripheral blood, and peritoneal exudate of normal mice (BALB/c, BALB/c nude, C57BL/6, C3H) and from spleens of mice bearing a transplantable lung carcinoma or primary mammary carcinoma were expanded in culture for 1-9 months, with an increase in cell number of 10(5)- to 10(6)-fold per month, in crude or lectin-depleted medium containing T cell growth factor (TCGF). All these cultured lymphoid cell (CLC) lines exhibited strong cytotoxic activity in vitro (assessed by 51Cr-release assays) toward a variety of freshly harvested and cultured syngeneic, allogeneic, and xenogeneic tumor target cells, both lymphoid and solid (including metastatic growths) in origin. Extensive killing was observed against tumor targets that were resistant to lysis by natural killer (NK) cells as well as to NK-sensitive tumor lines. Low levels of cytotoxic reactivity were also demonstrated against fresh and cultured normal lymphoid cells. The CLC had some characteristics of NK cells but also expressed some typical T cell markers. In local Winn-type neutralization assays, CLC delayed or completely inhibited the growth of lymphomas and carcinomas in syngeneic and allogeneic recipients. In mice with metastatic growth of a second-generation transplant of mammary carcinoma, CLC were shown to have some therapeutic effect when administered IV 1 day after cyclophosphamide. No significant beneficial action of IV administered CLC was observed in the absence of chemotherapy in mice implanted with a lung carcinoma. The possibilities of employing TCGF-propagated cytotoxic effector cells in adoptive immunotherapy of human malignancies are discussed.

The simulation of bone marrow granulopoiesis of normal CBA mice in vitro and in vivo by serum obtained from leucophoretic rats.

The effect of leucophoretic serum (LS), obtained from rats with polyvinylpyrrolidone (PVP)-induced inflammation, on granulopoiesis in the bone marrow of normal CBA mice was studied. The following test systems were used: short term cultures (4 hr), diffusion chambers (8, 24, 48 and 72 hr) and in vivo assays (12, 24 and 48 hr). The results indicate that LS stimulates the proliferations of granulocytic cells by increasing the number of proliferative granulocytes in mitosis, as well as increasing the total number of proliferative granulocytes. LS did not appear to effect monocytes and other cell lines. It is concluded that a factor present in LS specifically stimulates the proliferation of granulocytic cells, both in vitro and in vivo.

Two Classes of Murine Granulocyte Progenitor Cells Expressed in Plasma Clot Diffusion Chamber Cultures

Two distinct classes of granulocyte progenitor cells present in normal mouse bone marrow are expressed sequentially in the vivo plasma clot diffusion chamber culture system. By several criteria, progenitor cells giving rise to granulocyte colonies on day 4 of culture (CFU-D4) are different from those giving rise to colonies on day 7 (CFU-D7). These differences include: cell cycle activity as measured by in vitro incubation with cytosine arabinoside, residual concentration in the bone marrow after in vivo treatment of donor mice with cytosine arabinoside or methotrexate, resistance to osmotic lysis, size as determined by velocity sedimentation, and the morphology of the granulocyte colonies to which these cells give rise. The CFU-D7 appears to represent an earlier progenitor cell than the CFU-D4 in the differentiation pathway of the granulocyte and is analagous in many respects to the BFU-E in the erythroid pathway.

Two classes of murine granulocyte progenitor cells expressed in plasma clot diffusion chamber cultures

Two distinct classes of granulocyte progenitor cells present in normal mouse bone marrow are expressed sequentially in the vivo plasma clot diffusion chamber culture system. By several criteria, progenitor cells giving rise to granulocyte colonies on day 4 of culture (CFU-D4) are different from those giving rise to colonies on day 7 (CFU-D7). These differences include: cell cycle activity as measured by in vitro incubation with cytosine arabinoside, residual concentration in the bone marrow after in vivo treatment of donor mice with cytosine arabinoside or methotrexate, resistance to osmotic lysis, size as determined by velocity sedimentation, and the morphology of the granulocyte colonies to which these cells give rise. The CFU-D7 appears to represent an earlier progenitor cell than the CFU-D4 in the differentiation pathway of the granulocyte and is analagous in many respects to the BFU-E in the erythroid pathway.

Granulopoiesis Associated With the C3HBA Tumor in Mice<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

Female C3H/HeJ mice were inoculated with syngeneic breast adenocarcinoma cells (C3HBA). A progressive neutrophilia developed as the tumors grew. A linear relationship was demonstrated between the tumor diameter and the extent of the neutrophilia. Local tumor excision caused a rapid fall (3 days) in the neutrophil count. Media conditioned with tumor cells, normal mouse kidney, and bone marrow of normal or tumor-bearing mice were prepared. Tumor cell-conditioned medium was found to have marked stimulating activity for granulocytic colony formation of mouse bone marrow cells. Sera from tumor-bearing mice also had colony-stimulating activity. This finding strongly suggested that the neutrophilia was caused by the release of a neutrophil-stimulating factor from the tumor.

Alkaline phosphatase in hematopoietic tumor cell lines of the mouse: high activity in cells of the B lymphoid lineage.

Alkaline phosphatase (EC 3.1.3.1) was assayed in a large number of cultured mouse tumor cell line using p-nitrophenylphosphate as the substrate. Of 19 lines of the B lymphoid lineage, including Abelson pre-B, B lymphoma, and plasma cell tumor lines, all but 1 had substantial activity averaging 407 nmol/min/mg protein (with a range from 5 to 900). Nine T lymphoid and 9 nonlymphoid hematopoietic lines examined had low activity of 0.7 to 4.2 nmol/min/mg protein. The enzyme was markedly enriched in plasma membrane preparations from the B lymphoid cells, but not in those from most T lymphoma cells. The activity of another plasma-membrane-bound enzyme, gamma-glutamyl transferase, did not vary systematically with the type of cell line but was exceptionally high in 1 T lymphoma line. Investigation of pH dependence and susceptibility to inhibition by L-phenylalanine and L-homoarginine indicated similarity of the alkaline phosphatase from B cell lines to the enzyme recoverable from normal mouse kidney, placenta, bone marrow, and lymphoid organs. The enzyme seems to provide a useful marker for tumor lines of the B lymphoid lineage and for their plasma membranes.

Characterization of a developmentally early macrophage progenitor found in normal mouse marrow.

In tissue culture high proliferation potential colony forming cells (HPP-CFC, Bradly & Hodgson, 1979) formed large colonies in which macrophages predominated. Granulocyte/macrophage colony forming cells (GM-CFC, Bradley & Metcalf, 1966) formed smaller colonies of granulocytes and macrophages. We compared HPP-CFC from normal adult mouse bone marrow with GM-CFC from the same source. We found that HPP-CFC differed from GM-CFC in the following ways: (1) HPP-CFC formed larger colonies containing more than 5 X 10(4) cells. (2) They required spleen conditioned medium (Metcalf & Johnson, 1978) as well as pregnant mouse uterine extract (PMUE, Bradley et al, 1971) as stimulants. (3) Only 9% of HPP-CFC were killed by the phase specific drug hydroxyurea. 36-55% of GM-CFC were killed by the same treatment. (4) The modal density of HPP-CFC was 1.070 g/cm3. That of GM-CFC was 1.080 g/cm3.

Continuous lines of basophil/mast cells derived from normal mouse bone marrow.

Nonadherent tissue culture cell lines were established from normal bone marrow of a variety of mouse strains. The lines possessed morphological and histochemical markers of the basophil/mast cell and contained committed stem cells for metachromatic cells. Their derivation from normal marrow and their lack of tumorigenicity despite long-term culture makes these cell lines potentially important for studies of the mechanisms of allergic reactions and inflammation as well as the differentiation pathways involving this subset of hematopoietic cells.

Cells in bone marrow and in T cell colonies grown from bone marrow can suppress generation of cytotoxic T lymphocytes directed against their self antigens.

Both normal mouse bone marrow and cells from T cell-containing colonies grown in vitro from normal bone marrow contain cells which can specifically suppress the development of cytotoxic T lymphocytes capable of recognizing alloantigens on the bone marrow or colony cells. Suppression, as assessed by reduction in cytotoxic activity, is produced by adding bone marrow or colony cells to mixed lymphocyte reactions between lymph node responder cells and irradiated histoincompatible spleen stimulator cells. The cytotoxic activity is reduced if the added bone marrow or colony cells are syngeneic or semisyngeneic to the stimulator cells but not if they are allogeneic. Suppression results from a reduction in the number of cytotoxic lymphocyte precursor cells activated in the cultures. The suppressor cells in bone marrow are radiation sensitive and Thy-1 negative; those in colonies grown from bone marrow are radiation resistant and Thy-1 positive.

2'-Deoxycytidine hydrochloride protection of mice against the lethal toxicity of cytosine arabinoside. Absence of protection of bone marrow lymphocytes.

Normal mice and mice with advanced leukemia were injected IP with cytosine arabinoside (araC) on a lethal treatment schedule. The simultaneous oral administration of deoxycytidine (CdR) in dose 2–3 times as high as those of araC prevented drug-related death of the animals. Although CdR protected myelo-and erythropoiesis in the bone marrow of normal mice treated with araC, it did not influence the suppression of bone marrow lymphopoiesis. However, in mice treated with araC+CdR, the peripheral blood levels of both lymphocytes and granulocytes were significantly higher than those in animals given araC only. AraC lowered the level of serum hemagglutinins in mice immunized with sheep red blood cells (SRBC), and prolonged skin allograft survival. CdR appeared to normalize only the cellular immune response. In mice with advanced L1210 lymphoblastic leukemia treated with araC+CdR, the effect of the metabolite was selective, i.e., CdR administration led to a complete abrogation of drug-induced lethality without interfering with the highly efficient therapeutic effect of araC. No antitumor effect of araC was shown in araC+CdR-treated mice with advanced granulocytic Graffi leukemia and erythroblastic Rauscher leukemia. We suppose that the combined effect of araC and CdR, araC being administered on a lethal treatment schedule, results in selective destruction of precursor cells with the emphasis on B lymphocytes.

Detection of Primitive Macrophage Progenitor Cells in Mouse Bone Marrow

Abstract A previously undetected population of macrophage progenitor cells with high proliferative potential (HPP-CFC; an average of 5 x 10(4) cells/colony) in nutrient agar cultures has been demonstrated in post- fluorouracil (FU) and fluorouracil plus endotoxin (FUEt) treated and normal mouse bone marrow, using a combination of colony-stimulating factors, pregnant uterus extract (PMUE) plus human spleen-conditioned medium (HUSPCM). Neither PMUE nor HUSPCM alone stimulated colony formation by the HPP-CFC. The incidences of HPP-CFC were 1 in 2380 nucleated cells in normal marrow, 1 in 380 for 10-day post-FU, and 1 in 118 in 8-day post-FUEt marrow cells. HPP-CFC were only depleted to 57% of normal at 2 days after FU treatment, whereas the cells responsive to PMUE alone (low proliferative potential, LPP-CFC) were depleted to 1.2%, indicating a marked difference in cycling status of the respective types of progenitor cells.

Detection of primitive macrophage progenitor cells in mouse bone marrow

A previously undetected population of macrophage progenitor cells with high proliferative potential (HPP-CFC; an average of 5 x 10(4) cells/colony) in nutrient agar cultures has been demonstrated in post-fluorouracil (FU) and fluorouracil plus endotoxin (FUEt) treated and normal mouse bone marrow, using a combination of colony-stimulating factors, pregnant uterus extract (PMUE) plus human spleen-conditioned medium (HUSPCM). Neither PMUE nor HUSPCM alone stimulated colony formation by the HPP-CFC. The incidences of HPP-CFC were 1 in 2380 nucleated cells in normal marrow, 1 in 380 for 10-day post-FU, and 1 in 118 in 8-day post-FUEt marrow cells. HPP-CFC were only depleted to 57% of normal at 2 days after FU treatment, whereas the cells responsive to PMUE alone (low proliferative potential, LPP-CFC) were depleted to 1.2%, indicating a marked difference in cycling status of the respective types of progenitor cells.

Regulation of normal differentiation in mouse and human myeloid leukemic cells by phorbol esters and the mechanism of tumor promotion

The control of cell multiplication and differentiation by tumor-promoting phorbol esters including 12-O-tetradecanoylphorbol-13-acetate (TPA) has been studied with different clones of mouse myeloid leukemic cells, a line of human myeloid leukemic cells, and normal mouse bone marrow myeloblasts. TPA induced normal cell differentiation in one of the mouse leukemic clones and this was mediated by induction of the protein inducer of differentiation to macrophages or granulocytes (MGI) in the cells that then differentiated. Other mouse clones were not induced to differentiate by TPA. In one of these clones, TPA induced cell susceptibility to externally added MGI. This effect was not due to a general induction of susceptibility to all compounds because TPA did not induce susceptibility to lypopolysaccharide or dexamethasone in this clone. In the human leukemic cell line, TPA also induced differentiation with the induction of MGI activity and enhanced susceptibility to added MGI. It is suggested that the clonal differences in induction of MGI activity and increased susceptibility to MGI may be associated with differences in receptors for TPA and the ability of TPA to modify receptors for MGI. Studies with normal bone marrow cells have indicated that TPA stimulated MGI activity and also increased susceptibility of normal myeloblasts to induction of multiplication by MGI. The ability of different phorbol esters to produce these effects on normal myeloblasts and myeloid leukemic cells paralleled their ability to act as tumor promoters. The results indicate that a tumor promoter such as TPA can induce the production of and increase cell susceptibility to a normal regulator of cell multiplication and differentiation. TPA has pleiotropic effects. It is suggested that, by these mechanisms, TPA may thus act as a tumor promoter by increasing cell multiplication in initiated cells, induce differentiation in some cells, or inhibit differentiation in other cells, depending on which molecules are being regulated in the TPA-treated cells.

Hematopoietic organs of mice after a single injection oxyhydrocortisone

Kinetics of the number and direction of CFU differentiating in mice after a single infusion of hydrocortisone in a dose of 5 mg per mouse was studied using the method of an exogenous cloning of the stem cells in the spleen and the bone marrow of the irradiated recipients. Against the background of the long-term involution of the thymicolymphoid apparatus a change of the number and direction of the CFU differentiating occurred. Along with this, CFU concentration in the spleen and bone marrow of mice remained constant. Under the hormone action CFU differentiating capacity in the spleen and bone marrow in the direction of erythro- and myelopoiesis sharply changes; CFU of the bone marrow behaves as CFU of the spleen, and CFU of the spleen behaves as CFU of the bone marrow of normal mice. It is suggested that the described effects of the CFU of the spleen and bone marrow of mice given hydrocortisone were due to the redistribution of T-lymphocytes and not to direct cytotoxic effect of hydrocortisone on the CFU population.

The proliferation of plasma cells from mouse bone marrow in vitro. III. Primary and secondary immune responses associated with thymic RNA.

In vitro primary and secondary immune responses involving the proliferation of IgG-forming plasma cells from cultures of normal or antigen-primed mouse bone marrow cells have been investigated in order to clarify the relationships between the bone marrow cells and thymic RNA derived from each animal. By an administration of soluble and insoluble antigens with thymic RNA to bone marrow cultures, 54 ± 13% of IgG-forming plasma cells were found to produce specific immunoglobulin following stimulation with antigen. Thymus independent antigen polyvinylpyrrolidone (PVP) induced the proliferation of anti-PVP specific antibody forming cells in the abcence of thymic RNA, although more of these cells were generated when PVP-primed thymic RNA was administered with PVP to normal bone marrow cultures. Other antigens such as keyhole limpet hemocyanin (KLH), flagellar antigen of Salmonella, apofferitin and SRBC were investigated using the same bone marrow culture system. These antigens induced various numbers of antibod...

Identification of T cell subpopulations binding phytohemagglutinin: functional characteristics.

AbstractLymphocytes binding 125I‐labeled phytohemagglutinin (125I‐PHA) were directly quantitated by autoradiography. The distribution of grain counts proved to be trimodal indicating the presence of three distinct subpopulations of PHA‐binding cells. Low intensity binders (median grain count of 8–12 grains/cell) predominated in spleen and lymph node, included B and T cells, and responded to the mitogen, lipopolysaccharide (LPS). High intensity binders (median grain count greater than 60 grains/cell) occurred at low frequency in every lymphoid organ, were T cells, and included mixed lymphocyte culture (MLC)‐reactive but not PHA‐reactive cells as judged by preferential sensitivity to suicide with 125I‐PHA. Medium intensity binders (median grain count of 40–45 grains/cell) predominated in the thymus, formed a subpopulation in the spleen and lymph nodes of normal mice, but were absent from bone marrow of normal mice and the spleen of nude mice. In addition, their numbers were markedly reduced following adult thymectomy and cortisone treatment indicating that they were most likely T cells of recent thymic origin.PHA‐reactive cells were considered to belong to the medium intensity PHA‐binding subpopulation. This conclusion was based on the following: first, medium intensity binders, like PHA‐reactive cells, were T cells; second, they were contained in the same subpopulation of T cells as PHA‐reactive cells. That is, they were of recent thymic origin (T1 cells) according to studies of tissue distribution, cortisone sensitivity and effect of adult thymectomy; third, “suicide” of the response to PHA as well as to allogeneic cells but not LPS was achieved by prior incubation of cells with high doses of soluble 125I‐PHA, whereas lower doses resulted only in abrogation of the MLC response. In other words, PHA‐reactive cells resided in the medium, rather than high intensity subpopulation of binding cells. By analogy with suicide of antigen‐binding cells, this observation implies that PHA binds preferentially to those cells responding to it, and confirms the validity of using mitogen activation as a model for antigen‐induced triggering. On the other hand, binding of PHA to a subpopulation of T cells casts doubt on the value of the PHA response as a measure of overall T Cell function. Support for this conclusion came from the failure of PHA suicide to abrogate effector T cell activities such as help and suppression.

The effect of helper virus on Abelson virus-induced transformation of lymphoid cells.

Abelson murine leukemia virus (A-MuLV)-transformed fibroblast nonproducer cells were used to prepare A-MuLV stocks containing a number of different helper viruses. The oncogenicity of the A-MuLV stocks was tested by animal inoculation and their ability to transform normal mouse bone marrow cells was measured in vitro. All of the A-MuLV stocks transformed fibroblast cells efficiently. However, only A-MuLV stocks prepared with helper viruses that are highly oncogenic were efficient in vivo and in vitro in hematopoietic cell transformation. In addition, inefficient helpers did not establish a stable infection in lymphoid nonproducer cells. Thus, helper virus has a more central role in lymphoid cell transformation than in fibroblast cell transformation.

Susceptibility of Hematopoietic Stem Cells (CFU-s) to 55 Fe Radiation Damage

The susceptibility of CFU-s to radiation damage from55 Fe was examined. In a preliminary experiment, irradiation emitted from ${}^{55}{\rm Fe}\text{-loaded}$ erythrocytes was found to have no effect on erythroid colony formation during 72 hr of incubation. This demonstrated the low biological radiation effect of extracellular55 Fe. Normal mouse bone marrow cell suspensions were then mixed with ${}^{55}{\rm Fe}\text{-loaded}$ bone marrow, ${}^{55}{\rm FeCl}{}_{2}$ , or $[{}^{55}{\rm Fe}]\text{desferrioxamine}$ . No effect on CFU-s was found after up to 1 hr of incubation. However, when normal bone marrow mixed with $[{}^{55}{\rm Fe}]\text{desferrioxamine}$ was frozen and stored at -196°C, measurable damage to CFU-s progressed with increased storage time. Normal marrow mixed with stable [ $[{}^{56}{\rm Fe}]\text{desferrioxamine}$ showed no loss of CFU-s over 13 storage weeks. In subsequ...