Levels Of Colony-stimulating Activity Research Articles

Human lung cancer cell line (KSNY) producing colony-stimulating activity which affects both human and mouse marrow cells.

A cell line (KSNY) in vitro, which produces colony-stimulating activity (CSA) for human and mouse marrow cells, has been established. A biopsy was performed on the tumor mass of a lung cancer patient who had developed extensive leucocytosis. A piece of the tumor was transplanted to a nude mouse. The seconarily transplanted mice, in turn, developed extensive leucocytosis. The mouse tumor was then removed and placed in culture bottles. To date, the KSNY cells have been maintained in vitro continuously for 15 months. By the use of a methylcellulose bone-marrow colony-formation technique, a high level of CSA in the supernatant of the tumor cell culture was recognized. Doubling time of the cell line is 46 hr. The modal chromosome number is 52, ranging from 45 to 106.

Age-related changes in myelopoietic response to lipopolysaccharide in senescence-accelerated (SAM) mice

The effects of in vivo lipopolysaccharide (LPS) administration on myelopoiesis were examined in senescence-accelerated (SAM) mice. Young mice injected with LPS exhibited: (a) increased femoral proliferative pool size; (b) transient reduction in femoral non-proliferative pool size and number of femoral colony forming unit-granulocyte macrophages (CFU-GMs); (c) marked increase in splenic CFU-GMs; and (d) transient increase in S-phase of femoral CFU-GMs. The responses of old mice after LPS administration differed from those of young mice in the following points: (a) no recovery of the femoral non-proliferative pool or femoral CFU-GMs, (b) less significant augmentation of the femoral proliferative pool and splenic CFU-GMs, and (c) prolonged reduction in S-phase of femoral CFU-GM. Injection of LPS into mice resulted in a hyperproduction of colony-stimulating activity (CSA) in bone followed by production of colony-inhibitory activity (CIA) in young mice and in contrast, an excessive CIA secretion from bone without an increase in CSA levels in old mice. These imbalances in the regulatory factors derived from non-hemopoietic cells in the bones may lead to an inappropriate response of myelopoiesis in aged SAM mice after LPS administration, which may play a key role in infections.

Petiveria Alliacea I. Extract Protects Mice Against Listeria Monocytogenes Infection-Effects on Bone Marrow Progenitor Cells

In this study we have investigated the effects of Petiveria alliacea on the hematopoietic response of mice infected with Listeria monocytogenes. Our results demonstrate a protective effect of the crude extract of P. alliacea since the survival of the treated/infected was higher than that in the infected group. Moreover, the number of granulocyte/macrophage colonies (CFU-GM) and the serum colony stimulating activity levels were increased in the treated/infected mice in relation to the infected group. These results suggest an immunomodulation of Petiveria alliacea extract on hematopoiesis, which may be responsible, at least in part, for the increased resistance of mice to Listeria monocytogenes infection.

Modulation of growth supportive and oncogenic properties of murine leukemia cells

A leukemoid reaction occurs after inoculation of L1210 leukemic cells into recipient mice and the degree of granulocytosis is correlated with tumor progression. It was found that the sera of leukemic mice contained elevated levels of colony stimulating activity (CSA) when compared with normal mouse sera. Media conditioned by L-1210 cells in vitro (L-1210-CM) contained CSA which stimulated normal bone marrow myeloid colony growth and an auto-stimulatory activity (ASA) which stimulated L-1210 cell proliferation. We studied the effects of trans-retinoic acid (RA) and 1,25 dihydroxyvitamin D3 (VD3) on the production of growth substances by L1210 cells. When L1210-CM was prepared in the presence of RA and VD3, the CSA and ASA were markedly inhibited. A combination of the two agents was more effective than either agent. Mice inoculated with 1 × 10 5 L1210 suspension culture cells treated with either agent or both combined survived significantly longer than controls. Mice inoculated with L1210 cells treated with the two agents combined survived longest. By using antibodies, preliminary analysis of growth substances generated in L1210-CM showed that it contains primarily GM-CSF and M-CSF-like activities which were distinct from ASA. Combination antibody titer assays revealed that ASA was not significantly inhibited with anti-GM-CSF and anti-M-CSF antibodies, while CSA was inhibited by between 61 and 84%. We conclude that RA and VD3 synergistically inhibit the release of growth-enhancing substances by L1210 cells which may reduce the growth advantage of leukemic cells and the resulting leukocytosis in lymphocytic leukemia.

Accelerated Hematopoietic Recovery and Protective Effect of the Cyclooxygenase Inhibitor Indomethacin in Bacterial Infection of Neutropenic Mice

The effects of indomethacin administration on Pseudomonas aeruginosa infection were investigated in neutropenic mice. Cyclophosphamide-treated mice received the drug at 2.5 to 12 mg/kg according to different regimens, to be challenged with a lethal intraperitoneal inoculum of P. aeruginosa 5 days after myelosuppression. A single exposure of the neutropenic mice to 7 mg/kg indomethacin during the first 6 to 48 hr after myelosuppression was found to optimally restore the animals' antibacterial resistance, both in terms of survival of infected mice and clearance of the organisms from the peritoneal cavity. However, when administered 24 hr before challenge, the same drug dosage had no effect in enhancing survival. Cure was associated with accelerated hematopoietic recovery, as revealed by peripheral blood leukocyte counts, spleen weight and cellularity, cellular response to infection in the peritoneal cavity, and enumeration in vitro of bone marrow and splenic granulocyte-macrophage colony-forming cells. Following indomethacin administration, a rapid burst in the levels of colony-stimulating activity was detected in the bloodstream, and exposure of splenic macrophages or marrow cells to indomethacin in vitro was found to result in enhanced expression of transcripts specific for granulocyte-macrophage colony-stimulating factor. These data support the notion that the administration of cyclooxygenase inhibitors may be useful in promoting hematopoiesis and reducing the risk of opportunistic infections in myelosuppressed hosts.

Enhancement of Granulocytepoiesis by Freund's Complete Adjuvant in Carp.

To study the regulation mechanisms of carp granulopoiesis, we injected Freund's complete adjuvant (FCA) into carp and measured the effect of FCA on (i) colony-stimulating activity (CSA) in serum, (ii) colony-forming cell response in hematopoietic tissues by using the semi-solid agar culture technique. Inlection of FCA caused a high level of serum CSA, which began to reise 1 day after injection. Peak activity occurred after 6 days, at which time CSA was 2.5-10 times higher than that of the pre-injection level. FCA-injection also increased the number of colony-forming cells in hematopoietic tissudes. When the hematopoietic cells obtained from non-injected carp were cultured, little or no colony formation was observed. But when the hematopoietic cells from FCA-injected carp were cultured, more than 40 colonies were formed. The colonies were mostly composed of basophilic granulocytes. A few neutrophilic granulocytes were also observed in the same colonies.

Production of Colony-Stimulating Activity by Circulating Leukocytes from Patients with Chronic Myeloid Leukemia at Various Phases of the Disease

The production of colony-stimulating activity (CSA) by peripheral blood leukocytes (PBL) from 107 patients with chronic myeloid leukemia (CML), 58 at diagnosis, 34 during hematological remission, 33 in relapse from hematological control, 39 in accelerated phase, and 28 in blast phase, was measured in the double-layer agar culture system using normal nonadherent bone marrow cells as the source of CFU-GM. The median CSA levels in various stages decreased to 28-60% of control, whereas in hematological remission, there was a normalization of CSA. Five fractionation experiments using monocytes as feeder layers did not show CSA production in untreated CML. The leukemic PBL of 6 patients in blast crisis had no apparently inhibitory effect on colony formation stimulated by normal PBL. There was no correlation between the CSA of PBL and the number of CFU-GM in their bone marrow or peripheral blood at different disease states. Likewise, we failed to find a relationship between the PBL CSA levels and the total WBC counts or the differential counts in the peripheral blood as well as in the feeder layers. Our study indicated that circulating leukocytes from patients with CML at various phases except in hematological remission produce less CSA, which is not attributed to a low number of monocytes or the inhibitory effects of leukemic cells.

Modulation of circulating colony-stimulating activity in mice: Combined effects of IL-1 and bacterial or indomethacin treatment

We have investigated the effects of interleukin 1 (IL-1) administration on circulating levels of colony-stimulating activity (CSA) in intact or neutropenic mice. Intact or cyclophosphamide-treated mice received human rIL-1β according to different regimens, and their sera were assayed for CSA at 4, 24 or 48 h. The results indicated that (1) cyclophosphamide alone significantly increased the level of circulating CSA, (2) administration of IL-1 to intact or neutropenic mice resulted in a biphasic pattern of CSA response, an early burst at 4 h being followed at 24–48 h by a significant decrease. In nongranulocytopenic mice, the combined treatment with IL-1 and bacterial cells also resulted in a biphasic pattern of CSA response. However, when IL-1 was administered in concurrence with the cyclo-oxygenase inhibitor indomethacin, sustained CSA levels could be observed for a prolonged period of time. These data expand upon our previous observations on modulation of CSA by IL-1 in granulocytopenic mice, and further support the concept that IL-1 may have both positive and negative effects on the expression of circulating CSA.

Enhancement of hematopoietic response of mice by intraperitoneal administration of a .BETA.-glucan, SSG, obtained from Sclerotinia sclerotiorum.

Effects of intraperitoneal (i.p.) administration of SSG, a (1----3)-beta-D-glucan obtained from the culture filtrate of Sclerotinia sclerotiorum IFO 9395, on hematopoietic responses of mice were investigated. Numbers of spleen and peripheral leukocytes increased 7 d after administration of SSG (250 micrograms/mouse). The percentages of polymorphonuclear leukocytes (PMN) in both spleen and peripheral blood increased markedly. The numbers of macrophage progenitor cells also increased in both spleens and femurs of the mice administered SSG. Furthermore, levels of colony-stimulating activity (CSA) in sera were elevated, and two peaks were observed (at 6 h and on day 7 after administration). Elevated CSAs were also observed in the cultures of peritoneal cells taken from mice 3 or 6 h after administration of SSG. These results may suggest that i.p. administration of SSG into mice enhanced the production of colony-stimulating factors (CSFs), and then increased the numbers of both spleen and peripheral blood leukocytes.

Autocrine growth mechanisms of the progenitors of blast cells in acute myeloblastic leukemia

Autocrine growth mechanisms of leukemic blast progenitors in acute myeloblastic leukemia (AML) were investigated. Colony formation of leukemic blast progenitors was observed in 14 of 14 patients tested when purified blast cell fraction depleted of both T cells and monocytes was plated in methylcellulose without any colony-stimulating factor (CSF). However, there existed a minimal cell density required to initiate blast progenitor growth with marked patient-to-patient variation. To clarify the role of cell density on the spontaneous growth of blast progenitors, we tested whether leukemic cells produced and secreted some stimulatory humoral factor(s). Production of colony- stimulating activity (CSA) by blast cells was observed in 17 of 18 patients tested. Following further depletion of monocytes, the CSA levels decreased markedly in 14 patients, indicating that blast cells with monocytoid differentiation were responsible for CSA production. We also confirmed granulocyte colony-stimulating factor (G-CSF) and/or granulocyte macrophage-colony-stimulating factor (GM-CSF) production by leukemic blasts using specific immunologic assays. When leukemic cells were divided into nonadherent nonphagocytic cell fraction and adherent cell fraction, only nonadherent nonphagocytic cells showed clonogenecity and adherent blast cells lacked the colony-forming capacity. The results indicate that there are at least two blast cell subpopulations in AML: one is proliferating subpopulation with self- renewal capacity and the other is supporting subpopulation with functions such as CSF production. The quite intimate relationship between these two blast cell subpopulations in AML may play an important role on the growth of leukemic blast progenitors in vitro.

Autocrine Growth Mechanisms of the Progenitors of Blast Cells in Acute Myeloblastic Leukemia

AbstractAutocrine growth mechanisms of leukemic blast progenitors in acute myeloblastic leukemia (AML) were investigated. Colony formation of leukemic blast progenitors was observed in 14 of 14 patients tested when purified blast cell fraction depleted of both T cells and monocytes was plated in methylcellulose without any colony-stimulating factor (CSF). However, there existed a minimal cell density required to initiate blast progenitor growth with marked patient-to-patient variation. To clarify the role of cell density on the spontaneous growth of blast progenitors, we tested whether leukemic cells produced and secreted some stimulatory humoral factor(s). Production of colonystimulating activity (CSA) by blast cells was observed in 17 of 18 patients tested. Following further depletion of monocytes, the CSA levels decreased markedly in 14 patients, indicating that blast cells with monocytoid differentiation were responsible for CSA production. We also confirmed granulocyte colony-stimulating factor (G-CSF) and/or granulocyte macrophage-colony-stimulating factor (GM- CSF) production by leukemic blasts using specific immunologic assays. When leukemic cells were divided into nonadherent nonphagocytic cell fraction and adherent cell fraction, only nonadherent nonphagocytic cells showed clonogenecity and adherent blast cells lacked the colonyforming capacity. The results indicate that there are at least two blast cell subpopulations in AML: one is proliferating subpopulation with self-renewal capacity and the other is supporting subpopulation with functions such as CSF production. The quite intimate relationship between these two blast cell subpopulations in AML may play an important role on the growth of leukemic blast progenitors in vitro.

Interleukin 1-dependent paracrine granulopoiesis in chronic granulocytic leukemia of the juvenile type.

Marrow and peripheral blood cells from nine children with juvenile chronic granulocytic leukemia (JCGL) demonstrated intense (94 +/- 16% maximum) spontaneous granulocyte/macrophage colony growth but cells from five children with the adult variety of CGL did not. This unusual pattern of colony growth depended upon a stimulatory protein(s) produced by mononuclear phagocytes. No GM-CSA activity was found in any chromatofocused fraction of JCGL monocyte-conditioned media but an activity that induced GM-CSA in umbilical vein endothelial cells was detected at pI 6.9-7.2. Moreover, the CSA-inducing monokine was neutralized by an anti-IL-1 antibody in vitro and, in the one case so tested, the same antibody also inhibited "spontaneous" colony growth. Therefore granulocyte/macrophage colony growth in JCGL is characteristically abnormal and distinguishes JCGL from the adult form of the disease. This abnormality depends upon the production, by mononuclear phagocytes, of IL-1 which, in turn, stimulates the release of high levels of colony stimulating activity by other cells. The high proliferative activity of CFU-GM we found in JCGL patients, and the high levels of GM-CSA found in their serum are compatible with the view that the in vitro abnormality reflects a similar abnormality in vivo.

Proliferation of myeloid progenitor cells in human long-term bone marrow cultures is stimulated by interleukin-1 beta

To study the effect of interleukin-1 (IL-1) beta on the proliferation of hematopoietic progenitor cells (HPC) in long-term bone marrow cultures (LTBMC), stromal cell layers were established from normal human bone marrow. Autologous cryopreserved mononuclear phagocyte- and T-lymphocyte-depleted bone marrow cells were reinoculated on the stromal layers in fresh culture medium, with or without the addition of human IL-1 beta (30 U/mL). Once a week, half of the culture supernatant was replaced with fresh culture medium with or without IL-1, and all nonadherent cells were returned to the flasks. At weekly intervals during a period of 5 weeks, one culture was sacrificed to determine the total number of cells and hematopoietic progenitor cells, present in the adherent and the nonadherent cell fractions. In IL-1-stimulated cultures, the number of cells recovered during a period of 5 weeks exceeded the number of cells in unstimulated control cultures by 1.5 times. This difference was attributed to a twofold increase in the number of adherent cells. The number of HPC recovered from IL-1-stimulated cultures was not different from that recovered from controls. The levels of colony-stimulating activity (CSA) in supernatants from IL-1-stimulated cultures were significantly higher than those in supernatants from control cultures. These results indicate that IL-1 enhances the recovery of cells in LTBMC by stimulating the proliferation of HPC with the concurrent release of CSA from stromal cells, without diminishing the number of HPC.

Correlation between production of colony-stimulating activity (CSA) and adipose conversion in a murine marrow-derived preadipocyte line (H-1/A).

The correlation between adipose conversion of cloned H-1 cells (H-1/A) and their production of colony-stimulating activity (CSA) was examined. The production of CSA from H-1/A cells declined after adipose conversion, although H-1/A cells are active producers of CSA during their fibrocytic stage. The addition of 2 X 10(-5) M 5-bromo-2'-deoxyuridine to the cultures almost completely inhibited adipose conversion and there was no reduction of CSA levels after 9 days of culture. On the other hand, the addition of 10(-6) M hydrocortisone sodium succinate to the culture markedly enhanced adipose conversion, and a greater reduction in the CSA level was observed in the supernatants than in the control cultures after 12 days of culture. Indomethacin had no effect on the production of CSA or on adipose conversion. Furthermore, there were no significant differences between the CSA levels of nondialyzed supernatants and dialyzed supernatants from the control cultures during the entire course of the experiment. Supernatants during the adipocyte stage of H-1/A cultures did not inhibit the CSA derived from the fibrocytic stage. There were no differences in colonies in agar cultures stimulated by supernatants derived from cultures that had undergone either of the above treatments. These results suggest that the reduction of CSA is not due to the production of inhibitors, but that the production of CSA declines after adipose conversion of H-1/A cells. Preadipocytes in bone marrow therefore appear to contribute to granulopoiesis during the fibrocytic stage and are hematopoietically inactive when they convert to adipocytes.

Superoxide anion generation by polymorphonuclear leukocytes enhanced in a patient with colony-stimulating activity-producing lung cancer

We examined superoxide (O 2 −)-generating activity of polymorphonuclear leukocytes (PMN) from a patient with lung cancer in whom there was a marked granulocytosis. There was a high level of colony-stimulating activity (CSA) in both the serum and the supernatant of the culture of the primary tumor. The PMN from the patient produced almost three times more O 2 − than did the PMN from healthy donors and other patients with lung cancers but with no granulocytosis. The binding of wheat germ agglutinin (WGA), used as a stimulus for O 2 − generation, to the surface membrane of cells and the protein content in the cells were about double that noted in the case of healthy donors. Following total extirpation of the tumor, the level of CSA in the serum decreased, as did activity of PMN for O 2 − generation, and the binding of WGA to the cells reverted to normal levels.

マウス骨髄細胞の長期培養により形成された吸着細胞層によるコロニー刺激因子の産生

Conditioned medium prepared from adherent cells in long-term culture of mouse bone marrow stimulated in vitro colony formation by mouse bone marrow cells. Colonystimulating activity (CSA) was detected in the medium from 12-week-old cultures, and increased weekly to reach a plateu level 25 weeks after initiation of cultures. The CSA level was maintained over a 40-week period. The cultures were not recharged with fresh bone marrow cells throughout the experiment, and then no non-adherent cell production was observed in them. Two types of colony-stimulating factor were separated by gel filtration on Ultro-gel AcA 34 column. The larger molecular weight component that was heat-stable and trypsinresistant stimulated macrophage colony formation. The smaller molecular weight component that was heat-labile and trypsin-sensitive stimulated granulocyte colony formation.

Spontaneous production of colony-stimulating activity by splenic mac-1 antigen-positive cells from autoimmune motheaten mice

Cultured splenocytes from 3-wk-old autoimmune motheaten mice (me/me) spontaneously produced colony-stimulating activity (CSA), which stimulated the formation of bone marrow myeloid colonies. The production of CSA was not dependent on the presence of serum; this activity was not produced by spleen cells from their phenotypically normal littermates (+/-) or from other normal mouse strains. The peak level of CSA occurred early during the culture period, and within 48 hr the activity was markedly diminished. Cell fractionation studies demonstrated that cells expressing Mac-1 antigen produce CSA and are most likely to be mononuclear phagocytes. The unusual proliferative capacity in vitro of splenic mononuclear phagocytes from motheaten mice probably results from the spontaneous production of CSA by Mac-1 antigen-positive cells. Defective regulation of the production of monokines may contribute to the severity of the immunologic disease of these mutant mice.

Sequential effects of lithium on haematopoiesis.

The administration of lithium salts to haematologically normal subjects is associated with increased blood neutrophil concentrations and marrow neutrophil production and with enhanced release of colony stimulating activity (CSA) required for growth of granulocyte-macrophage progenitor cells (CFU-GM) in vitro. To examine the haematopoietic changes associated with lithium salts, mice were given LiCl daily. Blood neutrophils and serum CSA levels increased as did blood platelet concentrations. These increments were preceded by expansion of marrow neutrophil production and increased concentrations of CFU-GM as well as progenitor cells for megakaryocytes (CFU-M) and erythrocytes (BFU-E and CFU-E). An earlier and sustained increase of transplantable pluripotential stem cells (CFU-S) was detected beginning at day 2 of lithium administration. The sustained increase of CFU-S with lithium was not associated with detectable changes of endogenous stem cells (E-CFU) suggesting that a portion of the stem cell pool is resistant to the proliferative effects of lithium. These studies indicate that lithium acts initially to directly or indirectly increase marrow CFU-S with later increments of progenitor cells. The more sustained increase of blood neutrophils with lithium administration may be the result of subsequent increments of CSA resulting in enhanced marrow granulocyte production.

Colony promoting activity (CPA) produced in long-term culture of murine bone marrow cells

The differences between colony promoting activity(CPA) and colony stimulating activity(CSA) in the culture media of murine long-term bone marrow cultures(LTBMC) were demonstrated and the role of adherent cells and nonadherent cells in the production of CPA was studied in this culture system. Supernatant harvested from intact continuous marrow cultures showed high CPA but contained no CSA. Assayable CSA was detected in concentrated supernatant. However, there was no significant relationship between levels of CPA and CSA in the supernatant. When adherent cells and nonadherent cells from LTBMC were separately cultured, CPA was detected in the conditioned medium of adherent cells but not in that of nonadherent cells. The CPA level in LTBMC was related inversely to the number of nonadherent cells and addition of nonadherent cells to adherent cell cultures reduced the level of CPA. Conditioned medium of nonadherent cells showed no inhibitory activity to CPA. These results indicate that CPA is produced by bone marrow adherent cells and that it may be consumed by myeloid progenitor cells in nonadherent cells.

Spontaneous production of colony-stimulating activity by splenic Mac-1 antigen-positive cells from autoimmune motheaten mice.

Cultured splenocytes from 3-wk-old autoimmune motheaten mice (me/me) spontaneously produced colony-stimulating activity (CSA), which stimulated the formation of bone marrow myeloid colonies. The production of CSA was not dependent on the presence of serum; this activity was not produced by spleen cells from their phenotypically normal littermates (+/-) or from other normal mouse strains. The peak level of CSA occurred early during the culture period, and within 48 hr the activity was markedly diminished. Cell fractionation studies demonstrated that cells expressing Mac-1 antigen produce CSA and are most likely to be mononuclear phagocytes. The unusual proliferative capacity in vitro of splenic mononuclear phagocytes from motheaten mice probably results from the spontaneous production of CSA by Mac-1 antigen-positive cells. Defective regulation of the production of monokines may contribute to the severity of the immunologic disease of these mutant mice.