High-affinity binding of granulocyte-macrophage colony-stimulating factor to normal and leukemic human myeloid cells.

Purified natural and biosynthetic (recombinant) human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate colony formation by myeloid progenitor cells and enhance the function of mature neutrophils. Both of these actions occur at concentrations between 1 and 100 pM, with half-maximal stimulation at 10-20 pM. We have examined specific binding of 125I-labeled GM-CSF to responsive target cells in this range of concentrations. The results show a low number (50-250) of high-affinity (15-30 pM) binding sites on GM-CSF-responsive leukemic cells (KG-1, HL-60), as well as on peripheral blood neutrophils from normal donors. This high-affinity binding component was absent from unresponsive cell lines (KG-1a, K562). These results suggest that this binding site mediates the biological activities of GM-CSF on both proliferation and function of myeloid cells.     

Differentiation-inducing factor for a human leukemic cell line produced by colony-stimulating factor producing human lung cancer tissue

We have found that medium conditioned by a colony-stimulating factor producing tumor derived from a granulocytosis case with lung cancer contained a factor to differentiate a human promyelocytic leukemic cell line (HL-60) to macrophage-like cells that were butyrate esterase-positive and had phagocytosing activity and membrane Fc receptors. This differentiation-inducing factor was not active for a human myeloblastic cell line (KG-1), and was separated from a colony-stimulating factor by its molecular weight and isoelectric point. The conditioned medium did not contain a detectable amount of gamma interferon when tested by bioassay as well as by radioimmunoassay. This is the first report that a human lung cancer tissue produces not only a colony-stimulating factor, but also a differentiation-inducing factor. The conditioned medium is considered to be a good source of differentiation-inducing factor.     

Production of granulocyte colony-stimulating factor by a human melanoma cell line

We have isolated a human melanoma line (LD-1) from a patient with melanoma and unexplained leukocytosis. The LD-1 cells produced a colony-stimulating factor (CSF) which stimulated primarily granulocytic colonies in human and murine bone marrow cultures. Erythroid burst and mixed colony-stimulating activity was not detected. A single CSF species with a molecular weight of 21,000 was detected in LD-1-conditioned media by G-200 chromatography. Nude mice transplanted with LD-1 tumors developed granulocytosis and had increased blood CSF levels. Messenger RNA from LD-1 cells directed the synthesis of CSF by Xenopus oocytes. Northern blots of LD-1 RNA hybridized strongly with oligonucleotide probes based on the published sequences for human G-CSF, but not with a probe based on the human GM-CSF sequence. Northern blots hybridized with an oligonucleotide probe based on the CSF-1 sequence showed a high-molecular weight band; however, low-molecular weight CSF-1 mRNAs, which are present in the CSF-1-producing cell line MIA-PaCa-2, were not detected in the LD-1 mRNA. The CSF activity of LD-1 cells is best described as human granulocyte CSF.     

In vivo control of differentiation of myeloid leukemic cells by recombinant granulocyte-macrophage colony-stimulating factor and interleukin 3

The normal myeloid hematopoietic regulatory proteins include one class of proteins that induces viability and multiplication of normal myeloid precursor cells to form colonies (colony-stimulating factors [CSF] and interleukin 3 [IL-3], macrophage and granulocyte inducing proteins, type 7 [MGI-1]) and another class (called MGI-2) that induces differentiation of normal myeloid precursors without inducing cell multiplication. Different clones of myeloid leukemic cells can differ in their response to these regulatory proteins. One type of leukemic clone can be differentiated in vitro to mature cells by incubating with the growth-inducing proteins granulocyte-macrophage (GM) CSF or IL-3, and another type of clone can be differentiated in vitro to mature cells by the differentiation-inducing protein MGI-2. We have now studied the ability of different myeloid regulatory proteins to induce the in vivo differentiation of these different types of mouse myeloid leukemic clones in normal and cyclophosphamide-treated mice. The results show that in both types of mice (a) the in vitro GM-CSF- and IL-3-sensitive leukemic cells were induced to differentiate to mature cells in vivo in mice injected with pure recombinant GM-CSF and IL-3 but not with G-CSF, M-CSF, or MGI-2; (b) the in vitro MGI-2-sensitive leukemic cells differentiated in vivo by injection of MGI-2 and also, presumably indirectly, by GM-CSF and IL-3 but not by M-CSF or G-CSF; (c) in vivo induced differentiation of the leukemic cells was associated with a 20- to 60-fold decrease in the number of blast cells; and (d) all the injected myeloid regulatory proteins stimulated the normal myelopoietic system. Different normal myeloid regulatory proteins can thus induce in vivo terminal differentiation of leukemic cells, and it is suggested that these proteins can have a therapeutic potential for myeloid leukemia in addition to their therapeutic potential in stimulating normal hematopoiesis.     

Production of leukemic blast growth factor by a human bladder carcinoma cell line

Circulating blast cells from the peripheral blood of acute myeloblastic leukemia patients include a subpopulation capable of colony formation in the presence of phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). We describe the complete replacement in the blast assay of PHA-LCM by conditioned medium from a human bladder carcinoma cell line, HTB9. Both conditioned media contain a stimulator of blast cell growth that elutes as a single peak from a Sephadex G100 column with an apparent molecular weight of 30,000. It is shown that this leukemic blast growth factor is distinct from erythroid-potentiating activity (EPA) and possibly granulocyte macrophage colony-stimulating factor.     

Effect of interleukin 1, macrophage colony-stimulating factor, and factor increasing monocytopoiesis on the production of leukocytes in mice.

The present study was designed to establish that the factor increasing monocytopoiesis (FIM) is a unique factor that differs from interleukin 1 (IL-1) and macrophage colony-stimulating factor (M-CSF) in its effect on the production of granulocytes, lymphocytes, and monocytes in C3H/HeJ mice, which are unresponsive to lipopolysaccharide. [3H]thymidine, together with the cytokine under study, was used as a marker for newly formed cells. The number of each category of labeled leukocytes in the circulation was calculated from the number of leukocytes per microliter of blood, differential counts of the leukocytes, and their labeling indices, as determined by autoradiography. To compare the effect of the various cytokines on the production of leukocytes, the area under the curve (AUC) of the number of each category of leukocytes over a period of 96 h has been calculated. The results show that IL-1 causes, within 2 h, an increase in the number of circulating granulocytes, most probably by recruitment of these cells from the storage pool in the bone marrow and the marginating pool in the blood vessels. This is followed by an increased production of granulocytes; the production of monocytes and lymphocytes is not affected by IL-1. Administration of M-CSF had no significant effect on the production of granulocytes, lymphocytes, or monocytes in vivo. FIM specifically stimulated the production of monocytes and had no effect on the other cell lineages. Because FIM is synthesized and secreted by macrophages upon phagocytosis at the site of an inflammation, this study indicates that FIM is a monokine that acts as a long-range regulator to signal the bone marrow to increase monocyte production during an acute demand for more monocytes and (exudate) macrophages.     

Synergistic suppression of the clonogenicity of U937 leukemic cells by combinations of recombinant human interleukin 4 and granulocyte colony-stimulating factor.

The actions and interactions of purified recombinant human (rh) interleukin 4 (IL-4) and granulocyte colony-stimulating factor (G-CSF) on the clonogenicity of human leukemic cell line U937 were studied in vitro. Parameters analyzed were the suppression of stem cell generation using sequential clonal cultures, alterations of surface antigen expression, and morphological changes. IL-4 alone (10 U/ml) and G-CSF alone (1000 U/ml) only slightly reduced colony numbers (80% +/- 7% and 87% +/- 7% of control colonies, respectively). However, IL-4 interacted synergistically with G-CSF to further reduce the colony number (46% +/- 8% of control colonies) and suppress the self-renewal ability (clonogenicity) of U937 cells. This synergistic effect was not eliminated by cultures containing neutralizing concentrations of anti-granulocyte-macrophage colony-stimulating factor (anti-GM-CSF), anti-interleukin 6 (anti-IL-6), anti-interferon-alpha (anti-IFN-alpha), anti-IFN-gamma, anti-transforming growth factor-beta (anti-TGF-beta) serum, and anti-tumor necrosis factor-alpha (anti-TNF-alpha) serum. The coexistence of IL-4 and G-CSF was required for at least 48 h to reveal the synergistic action as assessed by preincubation and delayed addition experiments. Combinations of IL-4 and G-CSF showed a significant increase in CD11b expression on U937 cells. This action was not observed with HL60, K562, ML-1, or KG-1 leukemic cell lines, and IL-4 did not show any synergistic suppression of clonogenicity of U937 leukemic cells in combination with other cytokines tested in this study. These results suggest that IL-4 in combination with G-CSF may have some capacity to synergistically suppress human leukemic cells of specific types with loss of clonogenicity.     

Production of granulocyte-macrophage colony-stimulating factor by primary cultures of unstimulated rat microvascular endothelial cells

Small vessel (microvascular) endothelial cells are in close contact with hematopoietic progenitor cells in the bone marrow and therefore may have an important role in hematopoiesis. Although other studies have shown that endothelial cells produce various colony-stimulating factors (CSFs), these studies examined large vessel endothelial cells, which are different in many respects from microvascular endothelial cells and which do not contact cells in the bone marrow. We show in this study that primary cultures of unstimulated rat fat capillary endothelial cells grown in serum-free medium produce a substantial amount of granulocyte-macrophage CSF (GM-CSF). The medium conditioned by these cells stimulated proliferation of two different lines of GM- CSF-responsive cells--PT-18 mast cells and FDC-P1 cells--and supported the growth of cells of the granulocyte and macrophage lines in cultures of rat bone marrow cells. The factor responsible for this activity had physical properties consistent with those of GM-CSF, namely, a similar apparent mol wt by gel filtration, resistance to repeated freeze-thaws, resistance to boiling for ten minutes but not for 30 minutes, and resistance to heating to 56 degrees C for one hour. The factor causing target cell stimulation was not B cell-stimulating factor-1 (BSF-1, or IL 4), since it failed to stimulate a BSF-1-responsive cell line HT2- JH, and target cells (PT-18) did not respond appreciably to recombinant BSF-1. Northern blot analysis of mRNA from rat fat capillary endothelial cells showed high levels of expression of GM-CSF, confirming that this factor is produced by microvascular endothelial cells. This is the first report of CSF production by unstimulated microvascular endothelial cells, demonstrating that these ubiquitous cells are capable of producing sizable amounts of at least one growth factor for hematopoietic progenitor cells.     

Regulation of immunomodulatory functions by granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor in vivo

 The present study was designed to investigate in vivo immunomodulatory properties of hematopoietic growth factors. The influence on the activation of cytokine synthesis and on the expression of surface antigens associated with cellular activation of G-CSF or GM-CSF was investigated in cancer patients receiving these factors. One single dose of growth factor was administered to patients with bladder cancer (G-CSF group) or small cell lung cancer (GM-CSF group) before chemotherapy. After cytoreductive chemotherapy patients received supportive therapy with G-CSF or GM-CSF. Peripheral blood mononuclear cells and plasma samples were obtained for flow cytometry, Northern blot analysis, and assessment of cytokine protein levels after single-dose as well as after continous cytokine administration. Our results demonstrate differences in the induction of biological activities by GM-CSF and G-CSF in vivo which correlate well with in vitro findings. Among mature hematopoietic cells the effect of G-CSF is restricted to the granulocyte lineage. With GM-CSF moderate but unequivocal modulation of monocyte function was observed. On peripheral blood monocytes expression of MHC class-II molecules and CD44 was markedly stimulated. After one single dose of GM-CSF, plasma levels of sCD25 and IL-1RA were significantly induced (p<0.0001, p=0.032, respectively) and a trend to increased IL-8 levels was observed. The changes in plasma proteins were not correlated with shifts of mRNA expression for IL-8 and IL-1RA. T-cell activation was not observed with either cytokine. These results suggest that immunomodulatory features are differentially regulated by G-CSF and GM-CSF. The clinical relevance of a selective use of both hematopoietic growth factors in various disease settings remains to be determined. Received: 20 March 1996 / Accepted: 19 July 1996     

Regulation of human monocyte adherence by granulocyte-macrophage colony-stimulating factor.

Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to increase the adherence of purified peripheral blood monocytes to plastic surfaces and to monolayers of human umbilical vein endothelial cells. With plastic surfaces as a model 9-hr culture with GM-CSF was necessary for enhancement, and maximum levels were obtained after 24-hr stimulation. GM-CSF-stimulated adherence must require new RNA and protein synthesis because actinomycin D and cycloheximide abolished existing adherence and prevented further monocyte attachment. Interestingly, shorter incubations (1-2 hr) with cycloheximide increased adherence, suggesting a labile inhibitor. Formaldehyde fixation of monocytes but not of human vein endothelial cells abolished adherence, indicating the need for actively metabolizing monocytes. Thus, a hemopoietic growth factor, responsible for the proliferation and differentiation of monocytes, can also alter their adhesive characteristics. These observations may have important implications in pathological situations and in the in vivo use of GM-CSF.     

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.