Source Of Colony-stimulating Factor Research Articles

Effect of alcoholic extract of Ayurvedic herb Tinospora cordifolia on the proliferation and myeloid differentiation of bone marrow precursor cells in a tumor-bearing host

The present study investigates the effect of in vivo administration of alcoholic extract of Tinospora cordifolia whole plant (ALTC) on the proliferation and myeloid differentiation of bone marrow hematopoietic precursor cells in mice bearing a transplantable T cell lymphoma of spontaneous origin designated as Dalton's lymphoma (DL). BMC obtained from ALTC administered DL-bearing mice showed an enhanced BMC proliferation and colony forming ability in vitro in response to L929 conditioned medium as a source of colony stimulating factor (CSF). The number of granulocyte–macrophages colony (CFU-GM) was predominantly higher in the cultures of BMC obtained from ALTC administered mice as compared to mice injected with PBS alone. An increase in the count of bone marrow derived macrophages (BMDM) from ALTC administered mice was also observed along with an increase in the count of tumor associated macrophages. The BMDM obtained from ALTC administered mice showed an enhanced response to signal of LPS for activation to produce IL-1 and TNF. This study indicates that the T. cordifolia can influence the myeloid differentiation of bone marrow progenitor cells and the recruitment of macrophages in response to tumor growth in situ.

The development and magnitude of thermotolerance during chronic hyperthermia in murine bone marrow granulocyte-macrophage progenitors: I.

Murine bone marrow granulocyte-macrophage progenitors (CFU-GM) are capable of developing thermotolerance during exposure to temperatures < 42.5 degrees C. Bone marrow from the tibia and femora was heated to 40-42 degrees C (i.e. chronic hyperthermia), and challenged immediately with 15 min at 44 degrees C at regular intervals during treatment (step-up heating). CFU-GM were heated and cultured in McCoy's 5A medium + 15% FBS (fetal bovine serum) and lung-conditioned medium (source of colony stimulating factor) in semisolid agar. The kinetics of thermotolerance development and decay, and the magnitude of the thermotolerance during chronic heating with temperatures of 40-41.5 degrees C were similar. Survival increased rapidly to a maxima by approximately 120 min of hyperthermia (temperatures of 40-41.5 degrees C) and thereafter decreased with a slope similar to the controls. Normalization for cell killing by chronic hyperthermia that occurred during "step-up' heating permitted analysis of thermotolerance in the surviving cells. The surviving fraction after 15 min at 44 degrees C, during incubation at 40, 41 and 41.5 degrees C increased from 0.13 to maxima of 0.56 +/- 0.04, 0.71 +/- 0.03 and 0.82 +/- 0.03 respectively, by 150 min and did not decrease for up to 480 min during chronic hyperthermia. The surviving fraction after 15 min at 44 degrees C during incubation at 42 degrees C increased more slowly than during incubations at 40-41.5 degrees C. The survival of thermotolerant cells after exposure to 15 min at 44 degrees C during 42 degrees C chronic hyperthermia was maximal at 0.87 +/- 0.08 by 120 min and then decreased after approximately 150 min of exposure to 42 degrees C. The thermotolerance ratios (TTR's) were 4.0, 5.4, 6.7 and 6.9 for temperatures of 40, 41, 41.5 and 42 degrees C respectively. The results suggest that chronic hyperthermia temperatures (i.e. 40-42 degrees C) induce rapid thermotolerance development in CFU-GM during the thermal exposure and protect this normal marrow progenitor during whole body hyperthermia or ex vivo purging of leukaemic cells.

Intrinsic thermal response, thermotolerance development and stepdown heating in murine bone marrow progenitor cells.

Thermal response, thermotolerance development and stepdown heating (SDH) in the murine bone marrow granulocyte-macrophage (CFU-GM) progenitors were determined in vitro. Marrow was removed from femora and tibia, heated in McCoy's 5A medium plus 15% FBS and cultured in soft agar in the presence of three different sources of colony stimulating factor. D0's (+/- SE) for survival curves of CFU-GM heated in vitro were 147 +/- 13, 71 +/- 9, 37 +/- 2, 19 +/- 0.7, 11 +/- 1, and 4.3 +/- 0.3 min, for temperatures of 41.8, 42, 42.3, 42.5, 43 and 44 degrees C, respectively. Arrhenius analysis showed inactivation enthalpies of 812 +/- 9 KJoules/mole (193 +/- 2 Kcal/mole) above, and 2142 +/- 157 KJoules/mole (509 +/- 37 Kcal/mole) below, an inflection at 42.5 degrees C. Thermotolerance development was evident during prolonged hyperthermia exposure at temperatures below 42.5 degrees C (chronic hyperthermia) as a change in the slope of the survival curves after approximately 110 min of heating. Thermotolerance development at 37 degrees C after exposure to temperatures of 43 degrees C or greater (acute hyperthermia) was assessed by fractionated heat treatments consisting of an initial heat treatment (15 min at 44 degrees C) followed by incubation at 37 degrees C and challenge with 15 min or 25 min at 44 degrees C. Maximum thermotolerance occurred after 210 and 330 min at 37 degrees C, respectively. The half-time for maximum thermotolerance development was 36 min. Depending on the amount of heat damage and the maximum amount of thermotolerance development, the decay of thermotolerance was complete after approximately 48-72 h at 37 degrees C. An exposure of 10 min at 44 degrees C before incubation at 40 or 41 degrees C (stepdown heating) reduced the slope of the 40 or 41 degrees C survival curves by inhibiting thermotolerance development that would have otherwise occurred. D0's were 100 +/- 19 and 45 +/- 5 min for 40 and 41 degrees C incubation preceded by 10 min at 44 degrees C, respectively. These studies indicate that whole-body or regional hyperthermia protocols designed either to treat solid tumours or to purge leukemic stem cells from marrow ex vivo should avoid inadvertent temperature elevations to large volumes of marrow. Although, marrow progenitors are capable of thermotolerance development during exposure to temperatures up to 42.3 degrees C, results suggest that conditions of stepdown heating may prevent thermotolerance development.

The characteristics of macrophage-like cell lines derived from normal sheep spleens

Macrophage-like cell lines were derived from sheep spleens using conditioned medium from L-929 mouse cells as a source of colony stimulating factor. In seven out of ten attempts colonies of macrophage-like cells appeared after 2-3 weeks of culture. The cells were established in culture as cell lines, and survived 120 passages. They were strongly (+ +) positive for non-specific esterase but negative for peroxidase and produced detectable but small amounts of lysozyme (0.21-1.76 micrograms/10(6) cells). Latex particles were actively phagocytosed. Bacteria (Staphylococcus albus, Staphylococcus aureus) attached to the cell surface and were internalized in the presence of specific antibody. Expression of receptors for immunoglobulin and complement varied somewhat between the different cell lines: the proportion of receptor-bearing cells ranged between 9 and 26% FC-receptors, and 10 and 38% for C-receptors. The cell lines displayed a peculiar karyotype as well as protein profile that were different from normal sheep but similar between the different cell lines. Culture supernatants of the cell lines contained a colony stimulating activity which was used to establish further cell lines. They also spontaneously produced an interleukin-1-like activity that had no effect on baseline proliferation of sheep lymphocytes but enhanced their response to PHA (1.7-fold) particularly in conjunction with sheep IL-2 (4-fold). Prostaglandin E2 was produced in a growth-cycle dependent manner: the peak production occurred on the second day (77-140 pg/ml) at 2 x 10(5) cells and declined to 33-50 pg/ml on the eighth day when cell numbers had increased to 2-3 x 10(6). These easily cultured cell lines derived from normal tissue without the introduction of viral DNA should provide a useful source of material for studies of macrophage function in sheep.

Clonogenic methods in vitro for the enumeration of granulocyte-macrophage progenitor cells (CFU-GM) in human bone marrow and mouse bone marrow and spleen

Protocols and techniques are described for the in vitro enumeration of granulocyte and macrophage progenitors found in human bone marrow and mouse bone marrow and spleen. Study of the colony forming unitgranulocyte-macrophage (CFU-GM) in human bone marrow is usually first accomplished by density separation of whole bone marrow “buffy coats” through Ficoll-Paque into low- and high-density fractions. After collection of the low-density fraction (containing most or all of the CFU-GM), cells are washed and suspended, at a known cell concentration, in a mixture of culture medium, fetal bovine serum (or serum-supplements), agar or agarose, with a source of colony stimulating factor(s). Cultures are allowed to solidify and are then placed in a humidified 37°C incubator at 5% CO2 in lowered (5%) O2 tension for 7 to 14 d. Colonies (>40 cells/ aggregate) and clusters (3 to 40 cells/ aggregate) are then scored. Murine CFU-GM are cultured and characterized in a similar manner except tissues are separated into a single cell suspension without a density separation, and the culture time is reduced to 5 to 7 d. Colonies and clusters are scored as previously described. Purification of CFU-GM can be performed and these cells cultured as above.

The effects of oxidizing species derived from molecular oxygen on the proliferation in vitro of human granulocyte-macrophage progenitor cells.

In order to better understand the enhancing effects of lowered oxygen (O2) tension on the growth in vitro of granulocyte-macrophage progenitor cells (CFU-GM), the effects of oxidizing species derived from molecular O2 were assessed on CFU-GM. Low density or nonadherent low density normal human bone marrow cells were plated at ambient (20%) or lowered (5%) O2 tension in the presence of a source of colony stimulating factors, and in the absence or presence of superoxide dismutase, catalase, glucose oxidase or horseradish peroxidase, alone or in various combinations. Enhanced colony and cluster formation of CFU-GM was noted when low density cells were grown at 5% O2, or when cells were grown at 20% O2 in the presence of superoxide dismutase or glucose oxidase. Both of these enzymes are capable of generating hydrogen peroxide (H2O2), although by different mechanisms. Low concentrations of glucose oxidase resulted in increased formation of colonies and clusters, but higher concentrations of glucose oxidase were inhibitory. Catalase, which converts H2O2 to H2O, had no effect by itself on cells growing at 20% O2, but it eliminated the superoxide dismutase and glucose oxidase enhancing effects. Catalase decreased colony formation of cells grown at 5% O2. Removal of adherent cells ablated the growth-enhancing effects noted at lowered (5%) O2 tension and also the superoxide dismutase and catalase effects at 20% or 5% O2. Horseradish peroxidase, which converts H2O2 to a more toxic oxidant, hypochlorite, had a suppressive effect on colony and cluster numbers and at 20% O2 converted the glucose oxidase effects from stimulatory to inhibitory. The results suggest that adherent cells and low concentrations of H2O2 may mediate growth-enhancing effects of CFU-GM seen at lowered (5%) O2 tension.

HSV-1 DNA sequence determining intraperitoneal pathogenicity in mice is required for transcription of viral immediate-early genes in macrophages.

The relationship between intraperitoneal (ip) pathogenicity in vivo of herpes simplex virus type 1 (HSV-1) and infection of macrophages (m phi) in vitro was studied. The apathogenic HSV-1 strain HFEM disappeared from the peritoneum of infected mice following ip inoculation, while the pathogenic F strain persisted in the peritoneum and penetrated the mouse nervous system, and eventually the mice died, showing severe neurological signs. When peritoneal m phi were infected in vitro, a direct correlation with pathogenicity in vivo was found with several HSV-1 strains and recombinants. HSV-1 strains (F, KOS, R-M1C1) which were pathogenic for mice by the ip route, induced cytopathic effect (CPE) in m phi in vitro. Strain F transcribed viral immediate-early genes and synthesized viral DNA in m phi that were treated with L-cell conditioned medium (as a source of colony-stimulating factor) prior to infection. Apathogenic HSV-1 strains (HFEM, R-15, R-19) did not cause CPE in m phi. The HFEM strain was already blocked in the transcription of viral alpha genes in the infected m phi, but replicated well in control BSC-1 cells. An intratypic recombinant (R-M1C1), produced by cotransfection of HFEM DNA with a cloned Mlul-Mlul DNA fragment (coordinates 0.7615-0.796) from HSV-1 strain F, that was shown [Becker et al. (1986). Virology 149, 255-259] to have regained partial ip virulence for mice, now transcribed alpha genes, synthesized viral DNA, and induced CPE in m phi. It appears that the viral DNA fragment responsible for ip virulence is involved in tissue-specific recognition of virus by infected m phi, a function necessary for transcription of viral alpha genes.

In Vitro Action of Tumor Necrosis Factor on Myeloid Leukemia Cells

We investigated the in vitro action of recombinant tumor necrosis factor alpha (TNF) on the clonal growth of normal and malignant myeloid cells. Clonogenic cells from six of nine myeloid leukemia cell lines were very sensitive to the effects of TNF with 50% of the colonies inhibited (ED50) by concentrations of TNF that ranged between 6 and 150 U/mL. A decrease in DNA, RNA, and protein synthesis and in cloning efficiency occurred within three hours of exposure of HL-60 promyelocytes to TNF. The TNF in combination with recombinant interferons produced an additive or synergistic inhibition of colony formation of HL-60 and THP-1 myelomonoblasts. Normal human CFU-GM are sensitive to TNF (ED50 between 100 and 50,000 U/mL), but their sensitivity to TNF depends on the source of colony stimulating factor (CSF) with T lymphocyte derived GM-CSF (recombinant or natural) partially protecting the CFU-GM from the suppression exerted by TNF (and interferons). In eight of 15 cases the clonogenic myeloid leukemia cells from patients with acute or chronic myeloid leukemia were more sensitive than normal CFU-GM using GM-CSF as a source of colony stimulating activity. Further studies showed that the action of TNF on myeloid leukemia cells probably can be only partially explained by differentiation. Our finding of a possible selective cytotoxicity to leukemic clonogenic cells by TNF suggests that TNF may have value in the therapy of some patients with myeloid leukemia.

In vitro action of tumor necrosis factor on myeloid leukemia cells

We investigated the in vitro action of recombinant tumor necrosis factor alpha (TNF) on the clonal growth of normal and malignant myeloid cells. Clonogenic cells from six of nine myeloid leukemia cell lines were very sensitive to the effects of TNF with 50% of the colonies inhibited (ED50) by concentrations of TNF that ranged between 6 and 150 U/mL. A decrease in DNA, RNA, and protein synthesis and in cloning efficiency occurred within three hours of exposure of HL-60 promyelocytes to TNF. The TNF in combination with recombinant interferons produced an additive or synergistic inhibition of colony formation of HL-60 and THP-1 myelomonoblasts. Normal human CFU-GM are sensitive to TNF (ED50 between 100 and 50,000 U/mL), but their sensitivity to TNF depends on the source of colony stimulating factor (CSF) with T lymphocyte derived GM-CSF (recombinant or natural) partially protecting the CFU-GM from the suppression exerted by TNF (and interferons). In eight of 15 cases the clonogenic myeloid leukemia cells from patients with acute or chronic myeloid leukemia were more sensitive than normal CFU-GM using GM-CSF as a source of colony stimulating activity. Further studies showed that the action of TNF on myeloid leukemia cells probably can be only partially explained by differentiation. Our finding of a possible selective cytotoxicity to leukemic clonogenic cells by TNF suggests that TNF may have value in the therapy of some patients with myeloid leukemia.

Functional defects of culture-grown bone marrow-derived macrophages from autoimmune MRL/MpJ-lpr/lpr mice.

To investigate the primary defects and development of macrophages in MRL/MpJ-/pr/lpr (MRL/l) mice, we used a pure population of macrophages derived from bone marrow precursor cells cultured in the presence of L-cell conditioned medium (LCM) as a source of colony stimulating factor. Bone marrow-derived macrophages (BMM phi) from MRL/l mice had lower antigen presenting activity as detected by the induction of antigen-specific T cell proliferation, than age- and sex-matched control mice (CBA/J). Cell surface antigens (Ia and Mac-1) were determined quantitatively by a cell sorter as markers of macrophage differentiation. The BMM phi from MRL/l contained a much smaller number of Ia antigen-positive macrophages than those from normal mice. Treatment of BMM phi with an Ia-inducing of factor (IFN-gamma) markedly increased the expression of Ia antigens. This increase was significantly greater in BMM phi from MRL/l mice than in BMM phi from control mice. Expression of Mac-1 antigen was not different in BMM phi from the two strains. The Fc-mediated phagocytosis of IgG-coated sheep red blood cells was decreased in BMM phi from MRL/l mice compared with those from control mice. The function of nonspecific phagocytosis as measured by latex-bead incorporation was also impaired in MRL/l mice. The functional defects of MRL/l BMM phi found in these experiments are not secondary defects acquired under the influence of environmental signals during development, but are derived from the primary abnormalities which already exist in myeloid stem cells.

A characteristic CFU-GM growth pattern in acute promyelocytic leukemia

Bone marrow and/or peripheral blood cells from 12 patients with acute promyelocytic leukemia (APL) were cultured in soft agar or methylcellulose in the presence of 15% human placental conditioned medium as a source of colony stimulating factor. Buffy coat cells, taken from ten patients when APL was diagnosed, produced a growth pattern in soft agar that was characterized by a high incidence of small, relatively uniform clusters of promyelocytes, which, when stained in situ, reacted strongly with myeloperoxidase, Sudan black B and chloroacetate esterase. In six cases, the leukemic origin of the cluster forming cells was demonstrated by the presence of the t(15;17) in these cells. During periods of complete remission the small clusters were replaced by larger and more diverse aggregates which had a normal karyotype. At relapse the small cluster growth pattern returned. The growth pattern of small clusters is more commonly associated with APL than with other types of acute myeloid leukemia.

Differentiation of rat bone marrow cells into macrophages under the influence of mouse L929 cell supernatant.

Bone marrow cells (BMC) flushed from femora of Lewis rats were cultured in Dulbecco's modification of Eagle's medium supplemented with mouse L929 cell supernatant as a source of colony-stimulating factor (CSF). Differentiation of macrophage progenitor cells into macrophages (M phi) and expression of various markers were kinetically assessed. The proportion of M phi increases from approximately 4% in freshly isolated BMC to 100% after 7-8 days of cell culture. These cells, termed bone marrow cell-derived macrophages (BMDM phi), adhere to and spread on plastic surface; exhibit M phi morphology; stain intensely for nonspecific esterase; are able to phagocytose latex particles, IgG-sensitized erythrocytes, and C3-coated red cells; and express receptors for IgG and C3. A subpopulation of BMDM phi expresses MHC class II antigens as demonstrated by immunofluorescence using MRC OX6 and MRC OX17 monoclonal antibodies which recognize antigens coded in the I-A or I-E subregion of the MHC, respectively. Collectively, our results show that supernatant from mouse L929 cells supports and is continuously required for proliferation and differentiation of rat BMC into typical M phi, and suggest that mouse CSF cross-reacts with the putative receptor on rat M phi.

The effect of bone marrow depletion on prostaglandin E-producing suppressor macrophages in mouse spleen.

The i.p. injection of Corynebacterium parvum (CP) into CBA/J mice effected increases in macrophage colony-forming cells (M-CFC) when spleen cells were cultured with L cell culture filtrate as a source of colony-stimulating factor. Significant increases in phagocytic macrophages (M phi) with Fc receptors for IgG2a and IgG2b immune complexes were additionally noted among the spleen cells in these mice. These M phi effectively inhibited Con A-induced lymphocyte proliferation, probably reflecting a 10-fold increase above normal controls in prostaglandin E to 47 ng/3 X 10(6) spleen cells/ml. To determine whether the suppressor M phi are immediate derivatives of splenic M-CFC, we tried to induce suppressor M phi by the injection of CP into mice depleted of bone marrow M-CFC by the earlier administration of the bone-seeking isotope, 89Sr. This procedure reduced M-CFC in the bone marrow to less than 1% of normal for more than 30 days. Monocytes in the blood fell to 5% of normal by day 10 and were 30% on day 30. Levels of resident peritoneal M phi showed relatively little change in this period. By contrast, splenic M-CFC increased to 20-fold higher than the "cold" 88Sr controls. CP-induced suppressor M phi activity, however, was sharply reduced in 89Sr marrow-depleted mice on day 10, despite the striking increase in M-CFC. There was a threefold increase in the number of phagocytic M phi binding IgG2a immune complexes, with no significant increase in IgG2b binding M phi. The kinetics of recovery of suppressor M phi activity showed that on days 20, 30, and 50 after 89Sr injection the activities reached 20%, 30%, and 70% of the "cold" control, respectively, and correlated with the recovery of significant levels of M-CFC in the bone marrow. Taken together, these observations suggest that splenic M-CFC are not an immediate source of PGE-suppressor M phi in vivo. It appears more likely that the CP-inducible suppressor M phi, in particular, originate from radiosensitive bone marrow cells or require for differentiation a microenvironment provided by bone marrow cells. The data also suggest that the expression of the Fc gamma 2b receptor and of suppressor activity by CP-induced splenic M phi are related phenomena.

Modulation of in vitro eosinophil progenitors by hydrocortisone: role of accessory cells and interleukins

AbstractThe growth of human eosinophil progenitors (CFU-Eo) and the modulation of growth by hydrocortisone were studied as functions of the presence of lymphocytes and monocytes in marrow cells under study; and the source of colony-stimulating factors, specifically, media conditioned by macrophage-like cell line, GCT; phytohemagglutinin-stimulated mononuclear cells (PHA-LCM); or the T cell line, MO. CFU-Eo growth was greatest in marrow containing accessory cells as compared to marrow depleted of accessory cells; and in marrow treated with phytohemagglutinin-stimulated leukocyte conditioned media (PHA-LCM) or MO (T cell line)-conditioned medium (MO-CM) as compared with GCT cell- conditioned medium (GCT-CM). Hydrocortisone reproducibly inhibited eosinophil progenitor growth in unfractionated marrow stimulated by GCT- CM. This effect was abrogated by admixing irradiated mononuclear cells or T lymphocytes with the target marrow or by adding interleukin 1 or interleukin 2 (IL-1, IL-2). Inhibition by hydrocortisone did not occur when monocyte and T lymphocyte depleted marrow was studied. Unlike GCT- CM, MO-CM and PHA-LCM stimulated equal proportions of eosinophil progenitors in nondepleted and accessory cell-depleted marrow and demonstrated less hydrocortisone inhibition. However, both GCT-CM and PHA-LCM produced in the presence of hydrocortisone stimulated significantly fewer CFU-Eos in both unfractionated and accessory cell- depleted marrow target populations. These results indicate that the growth of CFU-Eo and inhibition of growth by hydrocortisone is a direct function of a monocyte-T cell interaction and probably is mediated through effects on the production/release of eosinophil colony stimulating factor (Eo-CSF).

Modulation of in vitro eosinophil progenitors by hydrocortisone: role of accessory cells and interleukins

The growth of human eosinophil progenitors (CFU-Eo) and the modulation of growth by hydrocortisone were studied as functions of the presence of lymphocytes and monocytes in marrow cells under study; and the source of colony-stimulating factors, specifically, media conditioned by macrophage-like cell line, GCT; phytohemagglutinin-stimulated mononuclear cells (PHA-LCM); or the T cell line, MO. CFU-Eo growth was greatest in marrow containing accessory cells as compared to marrow depleted of accessory cells; and in marrow treated with phytohemagglutinin-stimulated leukocyte conditioned media (PHA-LCM) or MO (T cell line)-conditioned medium (MO-CM) as compared with GCT cell- conditioned medium (GCT-CM). Hydrocortisone reproducibly inhibited eosinophil progenitor growth in unfractionated marrow stimulated by GCT- CM. This effect was abrogated by admixing irradiated mononuclear cells or T lymphocytes with the target marrow or by adding interleukin 1 or interleukin 2 (IL-1, IL-2). Inhibition by hydrocortisone did not occur when monocyte and T lymphocyte depleted marrow was studied. Unlike GCT- CM, MO-CM and PHA-LCM stimulated equal proportions of eosinophil progenitors in nondepleted and accessory cell-depleted marrow and demonstrated less hydrocortisone inhibition. However, both GCT-CM and PHA-LCM produced in the presence of hydrocortisone stimulated significantly fewer CFU-Eos in both unfractionated and accessory cell- depleted marrow target populations. These results indicate that the growth of CFU-Eo and inhibition of growth by hydrocortisone is a direct function of a monocyte-T cell interaction and probably is mediated through effects on the production/release of eosinophil colony stimulating factor (Eo-CSF).

Serum-free conditions for the growth of avian granulocyte and monocyte clones and primary leukemic cells induced by AMV, and the apparent conversion of granulocytic progenitors into monocytic cells by a factor in chicken serum.

The supportive activity of chicken serum for the soft-agar growth of chicken granulocyte and monocyte clones could be replaced with defined ingredients [bovine serum albumin (BSA), conalbumin, selenium, linoleic acid and for routine work, a liquid soy lecithin preparation (59% phospholipids, 39% linoleic acid, and less than 2% of inositol and choline)]. The lecithin preparation could be replaced with L-alpha-phosphatidylcholine. The source of colony-stimulating factor was medium conditioned by fibroblasts cultured under protein-free conditions. AMV-induced leukemic cells could be cloned under identical conditions. In the presence of both chicken serum (10%) and the replacement ingredients, most of the proliferative clones produced were monocytic (84%). In the presence of serum alone, all of them were monocytic. Under serum-free conditions, all the clones produced were granulocytic when a day-3 conditioned medium (CM) was used; monocytic ones were also present when a day-6 CM was used. When the serum was serially diluted in the presence of the replacement ingredients, the number of proliferative monocytic clones progressively decreased while the number of proliferative granulocytic clones progressively increased and the kinetics of each were essentially the same, only opposite in direction. Moreover, the total number of proliferative clones did not change more than 33% at any dilution (or in the absence of serum). We postulate the existence in the chick system of a serum macrophage differentiation factor (M-DF) which converts early granulocytic progenitors (or exclusively diverts a common progenitor) into monocytic cells.

Development of macrophage and granulocyte colonies from human peripheral blood

The presence of macrophage and granulocyte progenitor cells in the human peripheral blood enabled the establishment of colonies from this accessible tissue and obviated the need for bone marrow to achieve this task. We have developed a method of obtaining reproducible growth of macrophage/granulocyte colonies from human peripheral blood. Colonies of macrophages and granulocytes were obtained by plating peripheral blood mononuclear cells (PBM) in methylcellulose containing medium in the presence of medium conditioned by non-stimulated PBM (CM). At early stages of colony growth both macrophage and granulocyte colonies were detected while following 20–25 days in culture all colonies tested revealed monocyte/macrophage morphology. To obtain higher numbers of colonies, we tested different cell sources, different CM preparations and the effect of steroid hormones on colony development. We found that the mononuclear cells obtained from cord blood (CB) or from some patients with inflammatory bowel disease yielded much higher numbers of colonies than PBM from normal individuals. Colony development from these two sources did not depend on an external source of colony stimulating factor (CSF) but was augmented as a result of CSF supplementation. CM obtained from CB mononuclear cells as well as supernatants from some human monoblastic cell lines were similar in their CSF activity to CM from normal PBM and made possible the development of macrophage/granulocyte colonies. Higher numbers of colonies were induced by including physiological concentrations of estradiol in the culture medium, in the absence of external sources of CSF. The system described above enabled the analysis of cloned macrophages and their circulating progenitor cells as well as the assay of different preparations of CSF.

Inhibitory activity in mouse lung-conditioned medium studied in the agar assay for bone marrow colony-forming cells: removal by vitamin C.

The growth of granulocyte/macrophage colony-forming cells (CFC) from bone marrow, with mouse lung-conditioned medium (LCM) as source of colony-stimulating factor, was tested in five strains of mice. Maximum stimulation of growth was usually obtained at a LCM dilution of 1:5 (final concentration, 1.8% vol/vol). Higher concentrations nearly always caused inhibition. The inhibitory activity was resistant to heating (56 degrees C, 30 min) and was not removed by dialysis, but could be extracted with chloroform, suggesting a lipophilic inhibitor of high molecular weight. On gel filtration (Sephadex G50) the stimulatory activity eluted in the first fraction, whereas the inhibitory activity could not be recovered. Ascorbic acid reduced or prevented the inhibition (optimum concentration, 0.45 mmol/l), and provides a convenient means of stabilizing the CFC agar assay system. In physiological conditions the vitamin might influence the responsiveness of CFC to growth-regulating factors.

In vitro differentiation of myeloid progenitor cells in patients with eosinophilia

Colony-forming cells of the granulocytic/macrophage ( CFCGM ) and eosinophilic ( CFCEo ) series were grown from bone marrow and/or peripheral blood of 20 patients with persistent eosinophilia mainly due to helminthic diseases and of 17 patients without eosinophilia. The semi-solid culture technique of haemopoietic cells was applied using different sources of colony-stimulating factor (CSF). After 14 d of incubation the number of CFCs as well as their differentiation into the CFCGM and CFCEo was evaluated. The total median CFC count in the bone marrow differed significantly between the patient and the control group. No difference of the total colony count was observed when culturing peripheral-blood lymphocytes of patients or controls. 47% Luxol-fast-blue positive colonies were identified in the peripheral blood cultures from patients with eosinophilia as compared with 56% from controls using human placenta-conditioned medium as source of CSF. In contrast, bone-marrow cultures showed a significantly lower proportion of CFCEo (14% v. 18% resp.). No difference could be observed with regard to the source of stimulus. Thus, the discrepancy of the peripheral-blood and bone-marrow eosinophilia and of the in vitro culture findings may indicate that the commitment of granulocytic progenitor cells into the eosinophilic series is unaffected by a defined stress, such as helminthic diseases or is not measurable in the culture dish.

Inhibition of CFU-NM and CFU-EOS by mature granulocytes

Approximately half of the colony-forming units-culture (CFU-C) from normal peripheral blood are eosinophilic. The purpose of our study was to determine: (1) whether progenitor cells committed to eosinophil or neutrophil maturation would be differentially affected by feedback inhibition, and (2) whether mature eosinophils added to the feeder layers of the culture would inhibit the proliferation of CFU-C in a manner similar to that described for neutrophils. Concentrated eosinophils and neutrophils, obtained by separation on a metrizamide gradient, were added to feeder layers containing either 10(6) autologous whole mononuclear cells (WMNC) or 0.1 ml of leukocyte conditioned media (LCM). The average number of colonies was 123/10(6) nonadherent cells (NAC) cultured. When neutrophils or eosinophils were added to the WMNC feeder layer, the percent inhibition of growth was 40.2% +/- 1.6% (mean +/- SEM) and 42.3% +/- 5.4%, respectively, but the ratio of neutrophil to eosinophil colonies remained constant. No effect was seen when neutrophils or eosinophils were added to an LCM feeder layer. Thus, it appears that the differential control of neutrophil versus eosinophil production in vitro is not regulated through feedback inhibition by mature granulocytes. In addition, these studies suggest that eosinophils, as well as neutrophils, cause inhibition of CFU-C growth when intact cells are the source of colony-stimulating factor (CSF).