Effect of recombinant human alpha interferon (INF) and antilymphocyte globulin (ALG) on normal and aplastic anaemia haematopoietic progenitor cell growth.

The effect of recombinant alpha interferon (INF) and of antilymphocyte globulin (ALG) to the colony stimulating factor (CSF) production was examined with in vitro culture of the bone marrow of healthy and of aplastic anaemia (AA) persons. In healthy persons the supernatant of lymphocytes preincubated with PHA and ALG was found to show a stimulating effect to clonogenic properties of marrow progenitors, the mentioned effect being not in proportion to the concentration value. Similar properties are shown by interferon in these persons. In patients with aplastic anaemia, a considerable stimulating ALG effect to the granulocytic formation of colonies and a lesser stimulating effect of interferon were shown.     

Effects of human alpha-interferon on granulocyte-macrophage progenitor cells (CFU-GM) in vitro

Two preparations of human interferon (IFN)-alpha were assessed for their influence on granulocyte-macrophage progenitor cells (CFU-GM) in vitro. Both highly purified human IFN-alpha Ly and recombinant IFN-alpha 2a suppressed CFU-GM colony formation in a dose-dependent manner using low-density bone-marrow target cells. Suppression of CFU-GM colony formation was accompanied by an increase in clusters. However, depletion of monocytes, T lymphocytes and B lymphocytes from low-density bone-marrow cells resulted in insensitivity of progenitor cells to IFN-alpha. These results demonstrate that the effects of human IFN-alpha on myeloid progenitor cells (CFU-GM) are mediated by accessory cells within the bone marrow.     

Interferon induction in marrow-derived macrophages: regulation by L cell conditioned medium

Mouse bone marrow cells grown in medium enriched with L cell conditioned medium (LCM) as a source of colony stimulating factor (CSF) yield populations of adherent macrophages which are quite sensitive to induction of interferon (IFN) by viral and nonviral inducers. We examined the role of LCM in the sensitivity of marrow macrophage cultures to IFN induction. Removal of LCM from the cultures for as little as 3 hours markedly reduced the IFN titers induced by a double stranded ribopolynucleotide (poly I:C) or a lipopolysaccharide (LPS), while induction by Newcastle disease virus (NDV) was unaffected. Addition of anti-CSF serum to LCM medium also reduced IFN titers in response to polyI:C but had no effect on NDV induction. The inhibitory effect of anit-CSF indicates that the LCM requirement is at least partially related to the colony stimulating activity of the medium. We postulate that CSF regulates the initial interaction of macrophages with polyI:C or LPS rather than the synthesis and secretion of interferon by the phagocytes. Nearly complete restoration of IFN induction with polyI:C was obtained when LCM deprived cultures were reincubated with LCM medium previously conditioned by marrow cultures.     

Interferon-gamma inhibits proliferation, but not commitment, of murine granulocyte-macrophage progenitors.

We investigated the effects of interferon gamma (IFN-gamma) on the growth of murine hematopoietic progenitors. IFN-gamma inhibited granulocyte colony-stimulating factor (G-CSF)- and interleukin-3 (IL-3)-dependent colony growth by granulocyte-macrophage (GM) progenitors derived from the bone marrow cells of normal mice. However, the number of IL-3-dependent GM colonies formed by the bone marrow cells of 5-fluorouracil (5-FU)-treated mice was not influenced by the addition of IFN-gamma. Replating experiments suggested that IFN-gamma suppressed GM colony growth directly and that it exerted an inhibitory effect on the proliferation, but not on the commitment, of GM progenitors. In contrast, IFN-gamma failed to suppress colony growth by mast cell progenitors. Erythroid and megakaryocytic progenitors exhibited different responses to IFN-gamma depending on mouse strains. These results suggest that potent negative regulators are not always inhibitors of hematopoietic progenitors.     

Interferon is the suppressor of hematopoiesis generated by stimulated lymphocytes in vitro

Lymphocytes that inhibit hematopoiesis may have a pathogenic role in some forms of bone marrow failure, and lymphocyte-mediated suppression may also be important in the normal regulation of bone marrow function. We have investigated the mechanism of in vitro suppression of hematopoiesis by T cells by using the methylcellulose colony culture system. Total peripheral blood T cells and separated subpopulations of helper (OKT4+) and suppressor (OKT8+) cells that have been stimulated by exposure to lectin suppress autologous colony formation by bone marrow myeloid (CFU-C) and erythroid (BFU-E) progenitor cells. Medium conditioned by these cells is also inhibitory, indicating that the suppressor activity is a soluble factor. A strong correlation existed for the concentration of interferon and the degree of hematopoietic suppressor activity in these supernatants; both activities peaked at days 3 to 5 of incubation and had sharply declined by day 7. Interferon production was enhanced by exposure of lymphocytes to sheep red blood cells during the rosetting procedure. Specific antiserum and a monoclonal antibody directed against gamma-(immune) interferon abrogated the inhibitory activity for hematopoiesis produced by lectin-stimulated T cells; an antiserum to alpha-interferon was generally much less effective in neutralizing activity. We infer from these results that gamma-interferon is the mediator of hematopoietic suppression generated by lectin-treated T-cells.     

Negative regulation of human megakaryocytopoiesis by human platelet factor 4 (PF4) and connective tissue-activating peptide (CTAP-III)

We have previously reported that human platelet factor 4 (PF4) and beta-thromboglobulin inhibit human megakaryocyte (meg) colony formation in vitro. Here, we report further findings concerning the effect of PF4 as well as another platelet-derived factor: connective tissue-activating peptide (CTAP-III). Addition of these factors (2.5-10 micrograms/ml) into normal marrow cultures resulted in a significant decrease of meg colonies, especially the mixed-meg colonies, BFU-meg and large CFU-meg, suggesting that they inhibit both proliferation and maturation of meg progenitor cells, with a predominant effect on earlier progenitor cells. Comparison of the effects of the two factors showed that their major effects were similar, with some difference in inhibitory degree. These results indicate that both PF4 and CTAP-III are potent inhibitors of meg colony formation and involved in negative autocrine regulation of megakaryocytopoiesis.     

Interference of a human leukocyte interferon preparation with stimulatory activities in leukocyte-conditioned medium for human hematopoietic stem cells

Medium conditioned by leukocytes in the presence of phytohemagglutinin (PHA-LCM) promotes the growth of multilineage hemopoietic progenitors derived from human bone marrow. However, PHA-LCM prepared in the presence of a human leukocyte interferon preparation does not support mixed colony formation. Crude PHA-LCM preparations were characterized by gel filtration, affinity chromatography, and gel electrophoresis. The elution profile on Sephacryl S-300 of PHA-LCM prepared without interferon showed a distinct peak that stimulated the growth of pluripotent stem cells (CFU-gemm) and committed precursors (CFU-c, BFU-e). Gel filtration of PHA-LCM, prepared with 1000 U/ml of interferon, revealed a change in the elution profile. The eluted material demonstrated no growth-promoting activities. We conclude that the abolished stimulatory activity of PHA-LCM, prepared with human leukocyte interferon, might be due to a reduced production of stimulatory molecules, suggesting that interferon interferes with the molecular events required for colony formation of committed and noncommitted hemopoietic progenitors.     

Effects of recombinant human tumor necrosis factor (rhTNF) on normal human and mouse hemopoietic progenitor cells

We examined the effects of recombinant human tumor necrosis factor (rhTNF) on normal human and murine granulocyte-macrophage (CFU-gm) and erythroid (CFU-e, BFU-e) progenitor cells. We suppressed in vitro colony formation by human marrow CFU-gm, CFU-e and BFU-e or peripheral blood BFU-e by adding rhTNF to the culture in a dose-related manner. A half-maximal inhibition was observed with 1-10 ng/ml. Leukemic cell line K562 cells were found to be sensitive to rhTNF in the clonogenic colony assay. However, the clonal growth of murine marrow CFU-e and BFU-e colonies was less than 50% inhibited and CFU-gm growth was unaffected even at a concentration of 1,000 ng/ml. We observed slight to moderate inhibition after 24 h pulse exposure of both human and murine-committed progenitors to rhTNF prior to the culture. Intravenous injection of 1 mg/kg of rhTNF caused a marked decrease in marrow erythroid progenitors and consequently caused anemia in the mice. Our data indicate that rhTNF has a suppressive effect on normal human and murine hemopoietic colony formation in vitro and murine erythropoiesis in vivo.     

Inhibitory activity of interleukin 2-activated lymphocytes on human granulocyte precursors (CFU-g)

Interleukin 2-activated lymphocytes (lymphokine-activated killer [LAK] cells) cultured from 2 to 14 days were added to the cultures of granulocyte precursors (CFU-g). The LAK cells inhibited colony formation of granulocyte precursors; LAK cells cultured for five days showed the strongest inhibitory activity on colony formation. The presence of cell-to-cell interaction between LAK cells and bone marrow mononuclear cells (BMNC) in CFU-g assays emphasized the LAK cell-derived colony inhibitory activity (LAK-CIA), but cell-to-cell interaction was not always a requirement for LAK-CIA, since LAK cells were also found to inhibit colony formation without such interaction. This report shows that LAK cells can inhibit in vitro colony formation of granulocyte precursors. We therefore concluded that the observed CIA is caused by soluble factor(s) derived from LAK cells, and that E-rosette-forming cells are manifesting LAK-CIA.     

Expression of interferon consensus sequence binding protein (ICSBP) is downregulated in Bcr-Abl-induced murine chronic myelogenous leukemia-like disease, and forced coexpression of ICSBP inhibits Bcr-Abl-induced myeloproliferative disorder

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder resulting from the neoplastic transformation of a hematopoietic stem cell. The majority of cases of CML are associated with the (9;22) chromosome translocation that generates the bcr-abl chimeric gene. Alpha interferon (IFN-alpha) treatment induces hematological remission and prolongs life in 75% of CML patients in the chronic phase. It has been shown that mice deficient in interferon consensus sequence binding protein (ICSBP), a member of the interferon regulatory factor family, manifest a CML-like syndrome. We have shown that expression of Bcr-Abl in bone marrow (BM) cells from 5-fluorouracil (5-FU)-treated mice by retroviral transduction efficiently induces a myeloproliferative disease in mice resembling human CML. To directly test whether icsbp can function as a tumor suppressor gene, we examined the effect of ICSBP on Bcr-Abl-induced CML-like disease using this murine model for CML. We found that expression of the ICSBP protein was significantly decreased in Bcr-Abl-induced CML-like disease. Forced coexpression of ICSBP inhibited the Bcr-Abl-induced colony formation of BM cells from 5-FU-treated mice in vitro and Bcr-Abl-induced CML-like disease in vivo. Interestingly, coexpression of ICSBP and Bcr-Abl induced a transient B-lymphoproliferative disorder in the murine model of Bcr-Abl-induced CML-like disease. Overexpression of ICSBP consistently promotes rather than inhibits Bcr-Abl-induced B lymphoproliferation in a murine model where BM cells from non-5-FU-treated donors were used, indicating that ICSBP has a specific antitumor activity toward myeloid neoplasms. We also found that overexpression of ICSBP negatively regulated normal hematopoiesis. These data provide direct evidence that ICSBP can act as a tumor suppressor that regulates normal and neoplastic proliferation of hematopoietic cells.     

The nature of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated hemopoiesis, colony stimulating factor (CSF) requirement for colony formation, and the effect of TPA on [125I]CSF-1 binding to macrophages

The tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) was found to act both independently of and synergistically with the mononuclear phagocyte specific colony stimulating factor (CSF-1) to stimulate the formation of macrophage colonies in cultures of mouse bone marrow cells. In contrast, TPA did not synergize with other CSF subclasses that stimulate the formation of eosinophil, eosinophil-neutrophil, neutrophil, neutrophil-macrophage, and macrophage colonies, nor with either of the two factors required for megakaryocyte colony formation, megakaryocyte CSF, and megakaryocyte colony potentiator. In serum-free mouse bone marrow cell cultures TPA retained the ability to independently stimulate macrophage colony formation. However, TPA-stimulated colony formation was suboptimal and delayed in serum-free cultures that could support optimal colony formation in the presence of CSF-1. In addition, TPA did not directly compete with [125I]CSF-1 at 4 degrees C for its specific, high-affinity receptor on mouse peritoneal exudate macrophages. However, a 2-hour preincubation of the cells with TPA at 37 degrees caused almost complete loss of the receptor. Thus, TPA is able to mimic CSF-1 in its effects on CSF-1 responsive cells in some aspects (the spectrum of target cells, the morphology of resulting colonies, and the ability to down-regulate the CSF-1 receptor) but it is not able to mimic CSF-1 in other ways (TPA alone cannot stimulate the full CSF-1 response, TPA does not stimulate the most primitive CSF-1 responsive cells, and TPA does not bind to the CSF-1 receptor).     

兔再生肝提取物对骨髓细胞增殖,分化的影响

兔再生肝提取物（ＲＲＬＥ）中含一种对骨髓有明显影响的造血因子。通过给正常Ｂａｌｂ／ｃ小鼠体内注射ＲＲＬＥ,１天后骨髓有核细胞总数有意义减少,同时外周血和脾红髓内出现造血祖细胞,并伴贫血。取实验第２天脾细胞培养,ＣＦＵ—ｓ数明显升高,为混合性克隆形成单位。第４天后骨髓活跃增殖,产生以中性粒细胞系为主的大量骨髓细胞。血网织红细胞与白细胞值有意义升高,ＲＢＣ值于第９天恢复正常。上述结果提示ＲＲＬＥ中含有与其它造血因子作用不同的细胞因子。     

Established cell lines of mouse marrow adherent cells producing differentiation factor(s) for the granulocyte-macrophage lineage

Summary Two continuous cell lines derived from long-term cultures of AKR mouse bone marrow adherent cells were isolated. These cell lines release colony stimulating activity (CSA), a factor that induces in vitro differentiation of granulocyte-macrophage progenitor cells. The colony forming cells and cluster forming cells in mouse marrow responsive to CSA from cell line conditioned medium were compared with those responsive to CSA from mouse lung conditioned medium (MLCM). Colony forming cells were characterized by analysis of their density distribution after equilibrium centrifugation in density gradient. Cluster forming cells were characterized by analyzing the progeny of individual clusters after transfer to fresh semisolid culture medium containing MLCM. The results obtained indicate that the CSA from cell line conditioned medium closely compares with the CSA from MLCM in terms of the populations of colony and cluster forming cells stimulated. This work was supported by a research grant from the Institut National de la Santé et de la Recherche Médicale (CRL 802620), Paris, France.     

Cellular responsiveness to stimulation in vitro: increased responsiveness to colony stimulating factor of bone marrow colony-forming cells treated with surface-active agents and cyclic 3'5' AMP.

Addition of low concentrations (10 ng/ml) of saponin or Tween 80 to stimulated cultures of normal mouse bone marrow in agar increased the number of granulocyte-macrophage colonies which developed. Addition of cyclic AMP or dibutyryl cyclic AMP in low concentration (10(-8) to 10(-10) M) also enhanced colony numbers although concentrations above 10(-5) M were inhibitory. enhancement was found when marrow cells were pre-treated with these agents and cultured in their absence. The agents did not stimulate colony development in the absence of colony-stimulating factor and enhancement of colony number occurred only in cultures containing a concentration of colony-stimulating factor which was sub-optimal in terms of maximum colony development. There was no indication of increased colony-stimulating factor production by treated marrow cells under the experimental conditions used to show colony enhancement. It was concluded that the agents caused an increased responsiveness of colony-forming cells to colony-stimulating factor.     

Inhibition of human erythroid colony-forming units by interleukin-1 is mediated by gamma interferon.

IL-1 inhibits erythropoiesis in vivo and in vitro. This inhibition was studied by comparing the effect of recombinant human IL-1 (rhIL-1) on highly purified CFU-erythroid (E) generated from peripheral blood burst-forming units-erythroid (BFU-E) (mean purity 44.4%) with its effect on unpurified marrow CFU-E (mean purity 0.36%). Colony formation by marrow CFU-E was significantly inhibited by rhIL-1, while colony formation by highly purified CFU-E was not inhibited. However, purified CFU-E colonies were inhibited by rhIL-1 in the presence of autologous T-lymphocytes, and also by cell-free conditioned medium prepared from T-lymphocytes stimulated by rhIL-1. This inhibitory effect was ablated by neutralizing antibodies to gamma interferon (IFN), but not by antibodies to human IL-1, tumor necrosis factor, or beta IFN. Colony formation by highly purified CFU-E was also inhibited by recombinant human gamma IFN (rh gamma IFN). IL-1 and gamma IFN play significant roles in the pathogenesis of the anemia of chronic disease. These studies indicate that rhIL-1 inhibits CFU-E colony formation by an indirect mechanism involving T-lymphocytes and requiring gamma IFN and that gamma IFN itself is most probably the direct mediator of this effect.     

Enhanced expression of mRNA for nuclear factor kappaB1 (p50) in CD34+ cells of the bone marrow in rheumatoid arthritis

Bone marrow CD34+ cells from rheumatoid arthritis (RA) patients have abnormal capacities to respond to tumor necrosis factor (TNF)-alpha and to differentiate into fibroblast-like cells producing matrix metalloproteinase (MMP)-1. We explored the expression of mRNA for nuclear factor (NF)kappaB in RA bone marrow CD34+ cells to delineate the mechanism for their abnormal responses to TNF-alpha. CD34+ cells were purified from bone marrow samples obtained from 49 RA patients and 31 osteoarthritis (OA) patients during joint operations via aspiration from the iliac crest. The mRNAs for NFkappaB1 (p50), NFkappaB2 (p52) and RelA (p65) were examined by quantitative RT-PCR. The expression of NFkappaB1 mRNA in bone marrow CD34+ cells was significantly higher in RA than in OA, whereas there was no significant difference in the expression of mRNA for NFkappaB2 and RelA. The expression of NFkappaB1 mRNA was not correlated with serum C-reactive protein or with the treatment with methotrexate or oral steroid. Silencing of NFkappaB1 by small interfering RNA abrogated the capacity of RA bone marrow CD34+ cells to differentiate into fibroblast-like cells and to produce MMP-1 and vascular endothelial growth factor upon stimulation with stem cell factor, granulocyte-macrophage colony stimulating factor and TNF-alpha without influencing their viability and capacity to produce beta2-microglobulin. These results indicate that the enhanced expression of NFkappaB1 mRNA in bone marrow CD34+ cells plays a pivotal role in their abnormal responses to TNF-alpha and, thus, in the pathogenesis of RA.     

Proliferative effects of purified granulocyte colony-stimulating factor (G-CSF) on normal mouse hemopoietic cells

When granulocyte colony-stimulating factor (G-CSF), purified to homogeneity from mouse lung-conditioned medium, was added to agar cultures of mouse bone marrcw cells, it stimulated the formation of small numbers of granulocytic colonies. At high concentrations of G-CSF, a small proportion of macrophage and granulocyte-macrophage colonies also developed. G-CSF stimulated colony formation by highly enriched progenitor cell populations obtained by fractionation of mouse fetal liver cells using a fluorescence-activated cell sorter, indicating that G-CSF probably acts directly on target progenitor cells. Granulocytic colonies stimulated by G-CSF were small and uniform in size, and at 7 days of culture were composed of highly differentiated cells. Studies using clonal transfer and the delayed addition of other regulators showed that G-CSF could directly stimulate the initial proliferation of a large proportion of the granulocvte-macrophage progenitors in adult marrow and also the survival and/or proliferation of some multipotential, erythroid, and eosinophil progenitors in fetal liver. However, G-CSF was unable to sustain continued proliferation of these cells to result in colony formation. When G-CSF was mixed with purified granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), the combination stimulated the formation by adult marrow cells of more granulocyte-macrophage colonies than either stimulus alone and an overall size increase in all colonies. G-CSF behaves as a predominantly granulopoietic stimulating factor but has some capacity to stimulate the initial proliferation of the same wide range of progenitor cells as that stimulated by GM-CSF.     

Granulopoietic studies in acute lymphocytic leukemia of children.

Studies have been carried out on the levels of serum and urine colony stimulating activity (CSA) and peripheral blood and bone marrow colony forming cell numbers in children with acute lymphocytic leukemia (ALL) during various phases of their disease. These studies have suggested that serum and urine levels of colony stimulating factor are reduced during the inital or relapse phase of the disease compared to levels found during remission. It has also been found that the number of bone marrow colony forming cells is reduced in relapse or before treatment and elevated during remission while the number of peripheral blood colony forming cells is increased during relapse or before treatment and normal during remission. It has also been shown that mixing of serum or leukemic cells with normal human bone marrow cells inhibits colony formation.     

Inhibition of agar colony formation by partially purified granulocyte extracts (chalone)

Granulocytic extracts (GE) of different sources, presumably containing the granulocytic chalone, were prepared in different laboratories and purified to some extent. They specifically inhibited the formation of granulocyte and macrophage colonies in agar. The effect was however most pronounced on granulocyte and mixed granulocyte-macrophage colonies, and less on macrophage types. Addition of GE to bone marrow cells at the time of plating in agar, as well as short incubation of the cells together with GE prior to plating, inhibited subsequent colony formation. The inhibitory effect could easily be reversed by washing the cells with an excess of medium prior to plating during the first hour of preincubation, but not after five hours. Increasing the doses of colony stimulating activity (CSA) (at low doses of GE) released the inhibitory effect, but not at high doses of GE. The inhibitory effect of GE on colony formation was dose dependent down to almost 100% inhibition. No apparent cytotoxic effect of GE on bone marrow cells could be found and lymphoblastic cells were not inhibited. Extracts containing a specific inhibitor of erythropoiesis (EIF) stimulated myelopoietic colony formation in agar.