Lymphohematopoietic Progenitors Research Articles

Dual stimulatory and inhibitory effect of NK cell stimulatory factor/IL-12 on human hematopoiesis.

NK cell stimulatory factor, or IL-12 (NKSF/IL-12), is a heterodimeric cytokine produced by monocyte-macrophages, B cells, and possibly other accessory cell types. Although the major biologic effects of NKSF/IL-12 have been demonstrated on mature T and NK cells, in which it induces cytokine secretion, increased cytotoxicity, and proliferation, recent evidence in the murine system has suggested that NKSF/IL-12 may play a role in the differentiation of early lymphohematopoietic progenitor cells and thymocytes. In this paper, we have analyzed the effect of human rNKSF/IL-12 on the formation of colonies by highly enriched hematopoietic progenitor cells from human peripheral blood and bone marrow. At concentrations between 1 and 10 ng/ml, NKSF/IL-12 synergizes with a combination of steel factor and IL-3 to induce formation of mixed, erythroid, and myeloid colonies. Therefore, human NKSF/IL-12, like murine NKSF/IL-12, seems to belong to a small group of early acting cytokines, including IL-6, granulocyte-CSF, leukemia-inhibitory factor, and IL-11, which are able to synergize with steel factor and IL-3 to induce proliferation and differentiation of very early hematopoietic progenitor cells. However, in the presence of enriched preparations of NK cells cultured together with the progenitor cells, NKSF/IL-12 inhibits formation of hematopoietic colonies supported by IL-3 and granulocyte-macrophage CSF, by inducing production of IFN-gamma and TNF-alpha, two cytokines with synergistic inhibitory effects on hematopoietic colony formation. Because cell types that are able to produce NKSF/IL-12 are present in normal bone marrow and NKSF/IL-12 production in vivo and can be stimulated during bacterial or parasitic infection, it is possible that the direct stimulatory effect of NKSF/IL-12 on hematopoietic progenitor cells and the indirect inhibitory effect mediated by secondary cytokine production by lymphoid cells may play a role in the regulation of physiologic hematopoiesis and in its alterations during infection.

Synergistic Interaction Between Interleukin-12 and Steel Factor in Support of Proliferation of Murine Lymphohematopoietic Progenitors in Culture

We have investigated the effects of interleukin (IL)-12 (natural killer cell stimulatory factor/cytotoxic lymphocyte maturation factor) on the proliferation of murine myeloid and lymphohematopoietic progenitors in methylcellulose culture. In the presence of erythropoietin (Ep), IL-12 alone failed to support colony formation by mononuclear and enriched marrow cells of normal mice. Steel factor (SF) alone supported primarily formation of granulocyte/macrophage (GM) colony formation. However, the combination of the two cytokines yielded a significant number of multilineage colonies. When tested on marrow cells from 5-fluorouracil (5-FU)-treated mice, the combination of IL-12 and SF, but not the single factors, was effective in support of formation of various types of colonies. Approximately 25% of these colonies yielded pre-B-cell colonies when replated in secondary culture containing SF and IL-7, indicating that IL-12 can interact with SF in supporting the development of primitive lymphohematopoietic progenitors. These results demonstrate that IL-12, a cytokine believed to be involved in the development of cell-mediated immune responses, has a wider range of activity, including committed myeloid and multipotent lymphohematopoietic progenitors.

Synergistic interaction between interleukin-12 and steel factor in support of proliferation of murine lymphohematopoietic progenitors in culture

We have investigated the effects of interleukin (IL)-12 (natural killer cell stimulatory factor/cytotoxic lymphocyte maturation factor) on the proliferation of murine myeloid and lymphohematopoietic progenitors in methylcellulose culture. In the presence of erythropoietin (Ep), IL-12 alone failed to support colony formation by mononuclear and enriched marrow cells of normal mice. Steel factor (SF) alone supported primarily formation of granulocyte/macrophage (GM) colony formation. However, the combination of the two cytokines yielded a significant number of multilineage colonies. When tested on marrow cells from 5- fluorouracil (5-FU)-treated mice, the combination of IL-12 and SF, but not the single factors, was effective in support of formation of various types of colonies. Approximately 25% of these colonies yielded pre-B-cell colonies when replated in secondary culture containing SF and IL-7, indicating that IL-12 can interact with SF in supporting the development of primitive lymphohematopoietic progenitors. These results demonstrate that IL-12, a cytokine believed to be involved in the development of cell-mediated immune responses, has a wider range of activity, including committed myeloid and multipotent lymphohematopoietic progenitors.

Impaired proliferation and differentiation of myelodysplastic CD34+ cells.

The myelodysplastic syndromes (MDS) are a heterogeneous group of disorders of hematopoiesis entailing hyperproliferative and ineffective hematopoiesis resulting in refractory cytopenia(s), and increased risk of transformation into acute myeloblastic leukemia (AML). The widely used classification defined by the French-American-British group (FAB) recognizes 5 cytological subtypes of different prognosis, based essentially on the presence and the frequency of marrow blasts. The percentage of marrow blasts does not exceed 30%, hence, direct investigations of biological and biochemical events of MDS blast cells have been hampered. The CD34 antigen is currently unique in its narrow specificity of expression on human lymphohematopoietic progenitor cells. This cell membrane phosphoglycoprotein has been used for immunologic blast cell purification, notwithstanding the frequency of marrow blasts, and has provided a set of tools for investigations of MDS i.e. a direct comparison of the nature of blast cells in each of the MDS subtypes, using immunologic, biologic, biochemical and molecular biological methodology. A combination of serum-free medium and a purification method for blast cells provided evidence that the progenitor cell growth abnormalities in these disorders involve a defect in the capacity of progenitor cells to respond to stimulation with growth factor(s), and has presented direct evidence for the manner in which myelodysplastic CD34+ cells are impaired.

Growth factor requirement for survival in cell-cycle dormancy of primitive murine lymphohematopoietic progenitors

We used enriched marrow cells from mice administered three doses of 150 mg/kg 5-fluorouracil (5-FU) 1, 3 and 7 days before they were killed to study the effects of different growth factors on the survival of primitive, cell-cycle dormant progenitors in culture. This cell population yielded substantially fewer colonies in response to single growth factors than corresponding preparations from day 2 post-5-FU bone marrow samples, and the majority of progenitors were multipotential in nature. These observations were consistent with the prediction that multiple cycles of 5-FU treatment would further enrich for primitive cells. With this cell population, we found that among all the factors tested, interleukin-3 (IL-3) and steel factor (SF) as single factors are the most effective in supporting survival of dormant primitive progenitors. Interleukin-6 (IL-6), granulocyte colony- stimulating factor (G-CSF), interleukin-11 (IL-11), interleukin-4 (IL- 4), interleukin-1 alpha (IL-1 alpha), and tumor necrosis factor-alpha (TNF-alpha) also supported survival of a few progenitors, but much less effectively than either IL-3 or SF. The hematopoietic progenitors that survived for 1 week in liquid culture supplemented with either IL-3 or SF retained the capability to develop pre-B-cell colonies in secondary culture. Our results demonstrate that survival of dormant murine lymphohematopoietic cells in culture is dependent on the presence of specific growth factors, and that this growth factor requirement can be satisfied well by SF or IL-3.

Growth Factor Requirement for Survival in Cell-Cycle Dormancy of Primitive Murine Lymphohematopoietic Progenitors

We used enriched marrow cells from mice administered three doses of 150 mg/kg 5-fluorouracil (5-FU) 1, 3 and 7 days before they were killed to study the effects of different growth factors on the survival of primitive, cell-cycle dormant progenitors in culture. This cell population yielded substantially fewer colonies in response to single growth factors than corresponding preparations from day 2 post-5-FU bone marrow samples, and the majority of progenitors were multipotential in nature. These observations were consistent with the prediction that multiple cycles of 5-FU treatment would further enrich for primitive cells. With this cell population, we found that among all the factors tested, interleukin-3 (IL-3) and steel factor (SF) as single factors are the most effective in supporting survival of dormant primitive progenitors. Interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), interleukin-11 (IL-11), interleukin-4 (IL-4), interleukin-1 alpha (IL-1 alpha), and tumor necrosis factor-alpha (TNF-alpha) also supported survival of a few progenitors, but much less effectively than either IL-3 or SF. The hematopoietic progenitors that survived for 1 week in liquid culture supplemented with either IL-3 or SF retained the capability to develop pre-B-cell colonies in secondary culture. Our results demonstrate that survival of dormant murine lymphohematopoietic cells in culture is dependent on the presence of specific growth factors, and that this growth factor requirement can be satisfied well by SF or IL-3.

Antigenic analysis of hematopoiesis: a review.

The ability to immunophenotype the heterogeneous populations of cells within bone marrow has given investigators the tools to identify, enumerate, and isolate cells of any lymphohematopoietic lineage. These cells can, in turn, be studied in morphologic, growth, biochemical, and molecular biological assays, and used for clinical transplantation, with or without in vitro expansion. Although the map of normal marrow lymphohematopoiesis has been largely determined, there is still some controversy regarding the identity and maturation pathways of lymphohematopoietic progenitors and stem cells. The currently available information on this area is reviewed and interpreted in this article.

Activated protein kinase C directly phosphorylates the CD34 antigen on hematopoietic cells.

The CD34 antigen is a human leukocyte membrane protein expressed specifically by lymphohematopoietic progenitor cells. We found that CD34 is a phosphoprotein and therefore examined the regulation of its phosphorylation. Activation of protein kinase C (PKC) enhanced CD34 phosphorylation. The PKC activators, 12-O-tetradecanoylphorbol-13-acetate and bryostatin-1, induced rapid, stoichiometric hyperphosphorylation of CD34 protein in cells, resulting in a 5-fold increase in CD34 phosphorylation. In vitro kinase studies revealed that purified PKC could directly phosphorylate purified CD34. Only serine phosphorylation was detected in the CD34 molecule. Two-dimensional phosphopeptide mapping experiments indicated that PKC induces the phosphorylation of identical serine residue(s) in vitro and in vivo (in KG1 cells). These are newly phosphorylated serine residue(s), which are not detectably phosphorylated in CD34 from exponentially growing KG1 cells. These data indicate that the developmental stage-specific molecule, CD34, is a phosphorylation target for activated PKC. Furthermore, these findings raise the possibility that PKC activation and phosphorylation of the CD34 molecule may play a role in signal transduction during early lymphohematopoiesis.

Establishment of a leukemic cell line MT-ALL with multilineage differentiation potential.

Multiple steps are involved in hematopoietic differentiation and maturation from multipotent progenitor cells to terminally differentiated cells of each lineage. The availability of recombinant growth factors and of various clonogenic precursor cell assays has provided useful information about hematopoietic precursors, notably of the myeloid lineage [1–3]. However, the direct evaluation of primitive human lymphohematopoietic progenitor cells has been hampered by their low frequency in normal donors and the difficulties involved in expanding them in in vitro culture [4, 5]. Therefore, little is known about the events and mechanisms that result in the irreversible commitment of multipotent uncommitted progenitors to a specific cell lineage. Leukemias have been traditionally used as a model for lymphohematopoietic development and have been studied extensively for the expression of differentiation antigens [6, 7] and recently for rearrangements of genes of the immunoglobulin supergene family [8, 9]. They are considered to represent clonal expansions of lymphoid and myeloid progenitor cells and therefore provide an opportunity to study homogeneous populations of lymphohematopoietic progenitor cells. However, leukemic cell lines with the potential for multilineage differentiation that would allow the study of early events of lymphohematopoietic development are very rare [10, 11]. We report a novel acute lymphoblastic leukemia (ALL), MT-ALL, with a predominantly mature CD3+ TCR α/β+ phenotype, which in response to various growth factors displays the potential for multilineage differentiation in vitro. This leukemia may be instrumental in elucidating mechanisms involved in early lymphohematopoietic development.

Effect of Continuous, Whole-Body Gamma Irradiation upon Canine Lymphohematopoietic (CFU-GM, CFU-L) Progenitors and a Possible Hematopoietic Regulatory Population

Clonogenic assays for granulocytes-macrophages (CFU-GM) in bone marrow and for T lymphocytes (CFU-L) in peripheral blood were performed on dogs continuously exposed to 60Co irradiation (0.02, 0.04, or 0.11 Gy/day). When decreased numbers of CFU-GM were observed they correlated well with the clinical status of the dogs but were not generally associated with increasing cumulative doses of absorbed irradiation. In clinically normal, irradiated animals, decreased CFU-GM values and myeloid-erythroid ratios were observed, suggesting that chronic irradiation may affect the granulocytic series well before decreased peripheral blood values are seen. In hypocellular dogs the number of CFU-GM were significantly decreased compared to values obtained from control or clinically normal irradiated dogs, while virtually no CFU-GM were observed in the leukemic dogs. Only the CFU-GM values of the hypocellular group showed an association, e.g., a suggestion of an abortive regenerative effort, with increasing absorbed dose. Proliferative capacity of T lymphocytes (CFU-L) was not affected by either increasing absorbed irradiation or the presence of leukemia. D0 values were determined on marrow fibroblastic cells to ascertain whether a radioresistant subpopulation of stromal elements would result from continuous in vivo irradiation. No correlation was found between absorbed dose and increased D0 values. However, seven of eight dogs which developed acute nonlymphocytic leukemia displayed marrow fibroblastic cells with elevated D0 values. These radioresistant marrow fibroblastic cells were assayed for their ability to support normal granulopoiesis and found to be not significantly different from control fibroblasts.