Presence Of Colony-stimulating Activity Research Articles

Differences in radiation response between cells in S-phase and non-S-phase cells of the granulocyte/macrophage progenitor (GM-CFC) compartment

Studies were performed to investigate the radiation response of granulocyte/macrophage progenitor cells from canine bone marrow in different proliferative states, and in which way it will change if the S-phase cells are eliminated from the irradiated populations. To obtain progenitor cells of different proliferative states, bone marrow cell suspensions were kept in liquid cultures for 1 or 3 days in the presence of colony stimulating activity. Radiation dose response curves were determined (a) for the total population of progenitor cells under normal conditions (fraction of cells in S-phase 35%), (b) in a state of rapid cycling (fraction of S-phase cells 53% to 57%), and (c) after sterilization of S-phase cells by pretreatment with 3H-thymidine. The rapidly proliferating progenitor cells showed a strong decrease in their radiosensitivity (D 0 = 0.84 Gy) within the first day in suspension culture when compared to the normal population (D 0 = 0.50 Gy). The cell populations from which the S-phase cells had been eliminated were found more sensitive than the respective total populations (D0 values in the range from 0.44 Gy to 0.50 Gy). The D 0 values for the S-phase cells were between 0.57 Gy and 1.13 Gy depending on the proliferative state of the cell populations. These data indicate that granulocyte/macrophage progenitor cells during progression through the S-phase become less radiosensitive than they are in other phases of the cell cycle.

Synergistic action of recombinant-derived murine interleukin-1 on the augmentation of colony stimulating activity on murine granulocyte-macrophage hematopoietic stem cells in vitro.

This communication reports studies that were designed to investigate the role of recombinant-derived murine interleukin-1 on granulocyte macrophage (CFU-GM) progenitor stem cells in vitro. Interleukin-1 (IL-1, 5-25 units/ml culture) in a dose-dependent fashion, stimulated CFU-GM with a maximum effect at 25 units (147.3 +/- 6.4 colonies/10(5) nonadherent-derived marrow cells). At a higher concentration (50 units/ml) this stimulation was significantly reduced. IL-1, in combination with various types of conditioned media: pokeweed mitogen stimulated spleen cell, mouse lung and WEHI-3 cell, as a source of colony-stimulating activity (CSA), produced more CFU-GM when compared to controls (conditioned media alone). Furthermore, when assayed for their esterase activity, IL-1 increased both types of nonspecific and specific esterase content in CFU-GM colonies. Also, the actual ratio between neutrophilic and monocyte/macrophage colonies was reduced when compared to cultures stimulated in the presence of CSA, indicating that IL-1 increased myeloid differentiation. In the presence of indomethacin, an effective inhibitor of prostaglandin synthesis (1 microgram/ml), greater numbers of CFU-GM were present with IL-1 and/or CSA than without indomethacin. Cultures with anti-CSA demonstrated a reduced number of CFU-GM when plated in the presence of CSA but not with IL-1, demonstrating the specificity of IL-1 to stimulate CFU-GM in the presence of anti-CSA antibody. These results indicate a role for IL-1 in modulating granulopoiesis in vitro.

Release of Granulocyte-Specific Colony-Stimulating Activity by Human Bone Marrow Exposed to Phorbol Esters

AbstractGranulocyte-macrophage colony growth depends on the presence of colony-stimulating activity (CSA). Phorbol esters induce concentration-dependent colony formation in the absence of exogenous CSA. We questioned whether phorbol esters mimicked the action of CSA by directly stimulating colony growth, or whether phorbol esters acted indirectly by inducing marrow cells to release CSA. First, after incubating human bone marrow cells with phorbol 12,13-dibutyrate (PDP) for 3 days, we separated PDB from the protein peak of the conditioned medium by Sephadex G-10 gel filtration and tested this peak for the presence of CSA. When diluted 1:10 in the agar colony assay, this material induced 133 ± 15 colonies/105 bone marrow cells. Second, to determine whether bone marrow cells required the continued presence of PDB in order to release CSA, PDB was removed from bone marrow cells by washing, and these cells were reincubated in fresh medium in the absence of PDB. CSA was found in the medium of these cultures; its release was maximal after preincubation of bone marrow cells with 5 × 10−8M PDB for 3 days, followed by incubation for 3 days in the absence of PDB. This CSA stimulated granulopoiesis out of proportion to monocytopoiesis, with 85% ± 17% of the colonies being granulocytic (as indicated by histochemical staining for chloroacetate esterase), and 12% ± 3% being monocytic (as indicated by nonspecific esterase). Inhibitors of monocyte colony formation, including PGE1, were not present in the medium that contained this CSA. These studies demonstrate that normal human bone marrow cells exposed to PDB release CSA and that this CSA selectively stimulates granulopoiesis in vitro.

An In Vitro Model of Hematopoietic Injury In Chronic Hypoplastic Anemia

Mice treated with high-dose busulfan develop a 'latent' form of bone marrow failure characterized by near-normal peripheral blood counts and marrow cellularity, but marked reductions in marrow pluripotent stem cells (CFUs) and myeloid progenitor cells (CFUc). Spleen cell suspensions from control and 'latent' mice were placed in liquid culture in the presence of colony-stimulating activity. Cells were harvested at intervals up to 14 days and sub-cultured in agar to assay for CFUc. Baseline splenic CFUc did not differ significantly between control and 'latent' mice. Splenic CFUc from control mice increased 50-fold and reached a peak at day 10 in liquid culture. In contrast, splenic CFUc from 'latent' mice increased only 7-fold and reached a peak at day 3. Our results indicate that although splenic CFUc are present in normal numbers in 'latent' mice, their proliferative capacity is markedly reduced, either as the result of defective CFUc self-renewal or defective feed-in from CFUs or both.

Colony-Stimulating and Colony-Forming Cells in Peripheral Blood as Prognostic Markers in Acute Myelogenous Leukemia

Colony-forming cells (CFC) and colony-stimulating activity (CSA) in peripheral blood cells have been studied before and repeatedly during treatment of 32 patients with acute myelogenous leukemia. WBC obtained after Isopaque-dextran separation were cultured in vitro by a double-layer agar technique. Before treatment 19 patients out of 32 had CSA and all had CFC; both CSA and CFC were found in 19 patients. In follow-up studies during treatment, CSA was mainly unaffected during the leukopenic phase, while CFC were suppressed. No CFC were found at WBC counts below 1,000/mm3. This seems to imply that CFC are more sensitive to chemotherapy than colony-stimulating cells. 14 patients entered remission; all of them had CSA. 16 out of the 18 nonresponders lacked on or both types of cells. The presence of CSA and CFC in peripheral blood therefore appears to be a sign of favorable prognosis, while the absence of CSA and/or CFC implies lack of response to treatment. The conclusion from this study is that the presence of both CFC and CSA in peripheral WBC is a sign of a good prognosis. Absence of CFC and/or CSA in the initial sample indicates that the patient is unlikely to respond to conventional therapy with cytotoxic drugs. The presence or absence of CSA alone has a higher prognostic significance than the presence or absence of CFC.

Immunochemical quantification of in vitro neutrophilic granulocyte differentiation

IN the presence of colony stimulating activity (CSA)1–5, both normal and neoplastic cells of the neutrophilic granulocyte (neutrophil) and monocyte–macrophage cell series proliferate and differentiate in semi-solid and liquid cultures6–9. However, as conventionally performed, the clonal assay for CSA depends on cellular proliferation, and does not provide quantitative information on the role of CSA in differentiation. Lactoferrin (LF) is a specific protein marker for the neutrophil cell series10–12, and among bone marrow cells is found exclusively in the specific granules of the maturing neutrophil13,14. These granules first appear at the myelocyte stage of cytodifferentiation15. Thus, synthesis of LF is a biochemical event which may be used to quantify a specific state of neutrophil differentiation. We previously reported the purification of murine LF16, and its quantification by radioimmunoassay17, and also the development of a slide chamber method for culturing bone marrow cells in liquid medium18. We now report that in similar cultures, the net synthesis of LF is correlated in a dose-dependent manner with CSA concentration. To our knowledge, this is the first demonstration of a quantitative relationship at both the biochemical and cellular levels between CSA and a specific marker of neutrophilic granulocyte differentiation19.

Slide chamber culture system for the in vitro study of humoral regulation of granulocyte and monocyte-macrophage proliferation and differentiation

A liquid culture system employing slide chambers was developed to facilitate the study of proliferation and differentiation of mouse neutrophilic granulocytes and cells of the monocyte-macrophage series. Cultures were initiated with 1–2.5 X 10(4) light density marrow cells which had been fractionated on Ficoll-Hypaque. In the presence of colony-stimulating activity (CSAHU), two types of clusters were observed. One was tight, spheroidal, and composed of neutrophilic granulocytes, while the other was a loose grouping of flattened cells of the monocyte-macrophage series. The slide chamber culture is adaptable to microscopic assay techniques (e.g., cluster size, autoradiography, immunofluorescence) as well as quantitative biochemical methods (e.g., rate of 3H-thymidine incorporation, DNA quantitation). We have demonstrated both a shortening of the generation time of granulocytes in tight clusters and increasing rates of 3H- thymidine incorporation into DNA, with a corresponding increase in total culture DNA as a function of CSAHU concentration. Granulocytic differentiation in the tight spheroidal clusters has been demonstrated by histochemical stains and immunofluorescence of an antibody to a marker protein specific for the secondary granule of the neutrophil (lactoferrin).

Slide Chamber Culture System for the In Vitro Study of Humoral Regulation of Granulocyte and Monocyte-Macrophage Proliferation and Differentiation

A liquid culture system employing slide chambers was developed to facilitate the study of proliferation and differentiation of mouse neutrophilic granulocytes and cells of the monocyte-macrophage series. Cultures were initiated with 1-2.5 X 10(4) light density marrow cells which had been fractionated on Ficoll-Hypaque. In the presence of colony-stimulating activity (CSAHU), two types of clusters were observed. One was tight, spheroidal, and composed of neutrophilic granulocytes, while the other was a loose grouping of flattened cells of the monocyte-macrophage series. The slide chamber culture is adaptable to microscopic assay techniques (e.g., cluster size, autoradiography, immunofluorescence) as well as quantitative biochemical methods (e.g., rate of 3H-thymidine incorporation, DNA quantitation). We have demonstrated both a shortening of the generation time of granulocytes in tight clusters and increasing rates of 3H-thymidine incorporation into DNA, with a corresponding increase in total culture DNA as a function of CSAHU concentration. Granulocytic differentiation in the tight spheroidal clusters has been demonstrated by histochemical stains and immunofluorescence of an antibody to a marker protein specific for the secondary granule of the neutrophil (lactoferrin).

CGMP stimulation of stem cell proliferation

The process by which resting hemopoietic stem cells become activated is poorly understood, but it has been suggested that cyclic nucleotide levels in the cell may play an important role. In the present study, the effect of various, nucleotides and stimulators of nucleotide synthesis upon the formation of in vitro granulocyte colonies has been examined. It was found that physiologic concentrations of 3′, 5′-cyclic guanosine monophosphate enhanced the formation of granulocyte- macrophage colonies in the presence of colony-stimulating activity. The cells sensitive to cGMP activation were resistant to thymidine suicide and could not be activated by colony-stimulating activity alone. Therefore it was suggested that the cGMP sensitive stem cell was an ordinarily resting stem cell which was triggered into a proliferative state by cGMP.

CGMP Stimulation of Stem Cell Proliferation

The process by which resting hemopoietic stem cells become activated is poorly understood, but it has been suggested that cyclic nucleotide levels in the cell may play an important role. In the present study, the effect of various, nucleotides and stimulators of nucleotide synthesis upon the formation of in vitro granulocyte colonies has been examined. It was found that physiologic concentrations of 3', 5'-cyclic guanosine monophosphate enhanced the formation of granulocyte-macrophage colonies in the presence of colony-stimulating activity. The cells sensitive to cGMP activation were resistant to thymidine suicide and could not be activated by colony-stimulating activity alone. Therefore it was suggested that the cGMP sensitive stem cell was an ordinarily resting stem cell which was triggered into a proliferative state by cGMP.

Prognostic Value of Colony‐stimulating and Colony‐forming cells in Peripheral Blood in Acute Non‐lymphoblastic Leukemia

Colony-forming cells (CFC) and colony-stimulating activity (CSA) in peripheral blood cells have been studied before and repeatedly during treatment of 30 patients with acute non-lymphoblastic leukemia. WBC obtained after Isopaque-dextran separation were cultured in vitro by a double-layer agar technique. Before treatment 16 patients out of 30 had CSA and 22 out of 29 had CFC; both CSA and CFC were found in 15 patients. In follow-up studies during treatment, CSA was mainly unaffected during the leukopenic phase, while CFC were suppressed. No CFC were found at WBC counts below 900/mm3. This seems to imply that CFC are more sensitive to cytotoxic agents than colony-stimulating cells. Twelve patients entered remission; all of them had CSA and all the 11 who were investigated for CFC had CFC before treatment. Fourteen out of 18 non-responders lacked one or both types of cells. The presence of CSA and CFC in peripheral blood therefore appears to be a sign of favorable prognosis, while the absence of CSA and/or CFC implies lack of response to treatment.