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The effects of colony-stimulating factor 1 (CSF-1), the primary regulator of mononuclear phagocyte production, are thought to be mediated by the CSF-1 receptor (CSF-1R), encoded by the c-fms proto-oncogene. To investigate the in vivo specificity of CSF-1 for the CSF-1R, the mouse Csf1r gene was inactivated. The phenotype of Csf1(-)/Csf1r(-) mice closely resembled the phenotype of CSF-1-nullizygous (Csf1(op)/Csf1(op)) mice, including the osteopetrotic, hematopoietic, tissue macrophage, and reproductive phenotypes. Compared with their wild-type littermates, splenic erythroid burst-forming unit and high-proliferative potential colony-forming cell levels in both Csf1(op)/Csf1(op) and Csf1(-)/Csf1r(-) mice were significantly elevated, consistent with a negative regulatory role of CSF-1 in erythropoiesis and the maintenance of primitive hematopoietic progenitor cells. The circulating CSF-1 concentration in Csf1r(-)/Csf1r(-) mice was elevated 20-fold, in agreement with the previously reported clearance of circulating CSF-1 by CSF-1R-mediated endocytosis and intracellular destruction. Despite their overall similarity, several phenotypic characteristics of the Csf1r(-)/Csf1r(-) mice were more severe than those of the Csf1(op)/Csf1(op) mice. The results indicate that all of the effects of CSF-1 are mediated via the CSF-1R, but that subtle effects of the CSF-1R could result from its CSF-1-independent activation.

We have shown that pertussis toxin (PTx) modulates the effect of tumor necrosis factor-alpha (TNF-alpha) in inducing monocytic differentiation of WEHI-3B (JCS) myeloid leukemic cells in vitro. PTx (0.1-2 ng/ml) alone was not cytotoxic and did not induce morphological changes in JCS cells. In the presence of a suboptimal concentration of TNF-alpha (25 U/ml), however, PTx (1 ng/ml) acted synergistically in inhibiting proliferation and in inducing monocytic differentiation of the JCS cells. Expression of the macrophage differentiation marker (Mac-1) on JCS cells was increased by the combination of PTx and TNF-alpha, and phagocytic activity of the cells was also enhanced. Moreover, JCS cells treated with PTx and TNF-alpha had reduced tumorigenic capacity in vivo. The data suggest that a PTx-sensitive G protein may be involved in regulating the TNF-alpha-induced monocytic differentiation of the myeloid leukemic JCS cells and that combination of PTx and TNF-alpha may be useful in the treatment of some forms of myelomonocytic leukemia.

Murine hematopoietic cells can be transformed in vitro by recombinant retroviruses that express the myb oncogene, and hematopoietic growth factor (HGF)-dependent myeloid cell lines can be derived from these transformed primary cells. In this study, the differentiation state and responses of myb-transformed hematopoietic cells (MTHCs) have been investigated. We find that MTHCs exhibit properties of early myeloid progenitors including synergistic responses to combinations of HGFs and expression of certain surface markers. As reported previously, MTHCs respond well to granulocyte-macrophage colony-stimulating factor (GM-CSF) but can also respond to interleukin-3 (IL-3); the response to the latter factor depends on the mouse strain from which the cells are derived. Although these single factors stimulate MTHCs, combinations of these factors with colony-stimulating factor-1 (CSF-1 or M-CSF) or Steel factor (SLF or SCF) act synergistically to promote colony formation. The surface markers expressed by MTHCs include both granulocyte-macrophage lineage specific antigens Gr-1, 7/4, F4/80, and Mac-1, as well as two antigens found on early progenitors and stem cells--Thy-1 and Sca-1 (Ly6E). Expression of the latter markers is often heterogeneous and can be modulated by the growth factors to which the cells are exposed. Finally, we show that monocytic differentiation of MTHCs can be induced by exposure to tumor necrosis factor (TNF alpha). Taken together, these results suggest that MTHCs will be a useful model for studying HGF/cytokine responses in both proliferation and differentiation.

We have isolated a subclone (JCS) of the WEHI 3B myelomonocytic leukemia, which acquires the characteristics of mature macrophage lineage cells in the presence of PMA or noncytotoxic concentrations of TNF-alpha (600-1200 U/ml). JCS cells were compared with D+ and D- subclones of WEHI 3B. Unlike D+ cells, JCS cells did not produce differentiated granulocyte-macrophage colonies in the presence of postendotoxin serum or recombinant G-CSF. Stimulation with PMA or TNF-alpha reduced proliferation of JCS cells. TNF-alpha decreased the level of cell surface J11D antigen with concurrent increased expression of Mac-1 and FcR antigens and phagocytic activity. These TNF-alpha-mediated effects were enhanced by addition of IFN-gamma to the cultures. Furthermore, differentiation-inducing activity of PMA could be prevented using neutralizing anti-TNF-alpha antibodies. The results indicate that exogenous TNF-alpha can act as a differentiative agent for JCS cells and that endogenous TNF-alpha is the active substance when PMA is used to stimulate macrophage differentiation of JCS cells.

A set of primers (MF43, MF44 and MF45) were designed and used in the polymerase chain reaction to distinguish between the expression of mouse IL-3 receptor and mouse IL-3 receptor-like mRNAs. Primers MF43 and MF45 were specific for IL-3 receptor mRNA while the primers MF44 and MF45 were specific for IL-3 receptor-like mRNA. Primers MF44 and MF45 could not amplify IL-3 receptor cDNA even at an annealing temperature of 46 degrees C which is 20 degrees C below the melting temperature of the primers, or at high template concentrations (up to 100 ng cDNA). The optimal range of Mg2+ concentrations for the two pairs of primers MF43, MF45 and MF44, MF45, were essentially the same and this permits comparisons of the expression level of these two mRNAs under identical PCR conditions. Both the IL-3 receptor and IL-3 receptor-like mRNAs could be detected in normal bone marrow cells and IL-3-dependent cell lines (FDC-P1 and 32D cl-23), as well as in the IL-3 independent cell lines P388D1 and WEHI-3B, the latter being a constitutive producer of IL-3. In contrast, neither species of mRNA was detected in the T lymphoma cell line (EL-4). The ratio of IL-3 receptor-like mRNA to IL-3 receptor mRNA was usually greater than 1, except in 32D cl-23 cells where it was 0.66.

Four myeloid cell lines (M1, WEHI-3B D+, FDC-P1, and 32D) were screened for the presence of J11d antigen. One of these cell lines, the myeloid leukemia M1, was found to express a high level of J11d antigen on the cell surface. Recombinant mouse leukemic inhibitory factor (rm-LIF), recombinant human LIF (rh-LIF), and steroids (hydrocortisone and dexamethasone) could induce M1 cells to undergo monocytic differentiation. The level of J11d antigen was greatly reduced after treatment of the cells with LIF or steroids. Western blotting revealed that the apparent molecular weight of the J11d antigen on M1 cells was 45-48 kDa. Furthermore, the level of J11d mRNA was also reduced during LIF-induced differentiation of M1 cells.

In the present study, we show that recombinant human interleukin-1 beta (rhIL-1 beta), which has no effect on the proliferation of human progenitor cells, has synergistic effects on the expansion of human progenitor cells induced by rhIL-3 in liquid cultures. The synergistic effects of rhIL-1 beta with rhIL-3 were observed in liquid cultures using not only fresh bone marrow mononuclear cells, but also selected populations of nonadherent cells, non-T nonadherent cells, and CD34-positive cells. Anti-granulocyte-macrophage colony-stimulating factor (anti-GM-CSF) antibody partially blocked the synergistic effects of rhIL-1 beta on the proliferation of colony-forming unit (CFU)-GM burst-forming unit-erythroid (BFU-E), and CFU-Mix in liquid cultures in the presence of rhIL-1 beta plus rhIL-3, suggesting that the synergistic effects of rhIL-1 beta plus rhIL-3 are explained in part by the secondary production of GM-CSF. Limiting dilution assays and liquid culture assays using CD34-positive cells indicate that rhIL-1 beta directly increases the numbers of colony-forming cells in liquid cultures. These results suggest that rhIL-1 beta has unique direct and indirect effects on the expansion of hematopoietic progenitor cells in liquid cultures.

Interleukin-3 (IL-3) (multipotential colony-stimulating factor [multi-CSF] ) is an important regulator of hemopoiesis. We recently isolated a cDNA clone of this gene and describe in this manuscript the biologic properties of the expressed gene product. An SV40 expression vector carrying cDNA encoding murine interleukin-3 was constructed so that expression of the IL-3 gene was placed under the control of the SV40 early promoter. When the expression vector was transfected to COS-1 monkey cells, IL-3 activity was secreted into the medium, reaching maximal levels 72 hours after transfection. The IL-3 produced by the COS-1 cells was partially purified using diethylaminoethyl Sephacel and phenyl-Sepharose, and its chromatographic properties were the same as IL-3 produced by the WEHI-3 cell line. The biologic activities of the "expressed" IL-3 include the "induction" of 20-alpha-hydroxysteroid dehydrogenase (20-alpha-SDH) in splenic lymphocytes from nu/nu mice, proliferative activity for 32D cl-23 and FDC-P1 cell lines, and colony-stimulating activity for granulocyte-macrophage, eosinophil, megakaryocyte, natural killer-like, erythroid, and multipotential colony-forming cells from murine fetal liver and adult bone marrow.

trans-4-Hydroxy-2-nonenal (HNE) is an aldehyde end-product of lipid peroxidation in biological systems which is capable of producing a range of powerful biological effects. We wish to describe a sensitive and selective strategy for the determination of HNE in biological samples. The method is based on the formation of the O-pentafluorobenzyl (O-PFB) oxime derivatives of HNE and its deuterated internal standard which, after sample clean-up by solid-phase extraction and purification by high-performance liquid chromatography (HPLC), were derivatised further to trimethylsilyl ethers. Subsequent capillary column gas chromatography-negative-ion chemical ionisation mass spectrometry (GC-NICIMS) using selected-ion monitoring allowed quantitation in the low ng/ml range. The use of an internal standard and the O-PFB oxime derivatives circumvented the problems encountered previously by other workers because of the volatility and instability of HNE. The syn-isomer of HNE O-PFB oxime followed the anti-isomer on the HPLC and GC columns used, giving a distinctive pair of peaks of characteristic relative proportion. Moreover, the NICI mass spectra of the geometrical isomers were significantly different, providing further evidence to validate the identity of any endogenous HNE recovered. The method was used to identify and quantify HNE in platelets, monocytes, plasma and oxidised low-density lipoprotein.

In order to facilitate the identification of human haemopoietic growth factors (HGFs), in complex conditioned media from limited cell sources, we have developed a method for the rapid assay of HGFs using bone marrow stem cells (BMSCs) obtained by Percoll density gradient enrichment of non-adherent cells from long-term human bone marrow culture. These BMSCs provide the basis for a 72 h assay where proliferation is triggered by the presence of GM-CSF, G-CSF and IL-3. The assay is up to 1000 times more sensitive than the conventional colony assay in detecting the presence of HGFs. We also describe methods for the rapid separation and identification of HGFs using fast protein liquid chromatography (FPLC) with phenyl-Superose and Mono Q columns. This enables individual HGFs present in complex conditioned media to be rapidly identified from the elution profile of HGF activity determined in the BMSC assay.

The cell lines FDC-Pl and 32D cl-23 have previously been used as unique indicators for the growth-promoting activity of interleukin-3. We show that FDC-Pl cells respond to granulocyte/macrophage colony-stimulating factor (GM-CSF, CSF-2) as well as to interleukin-3. In keeping with this finding, FDC-Pl cells express the macrophage-specific marker, F4/80. FDC-Pl cells do not, however, respond to macrophage CSF (M-CSF, CSF-1). In contrast, 32D cl-23 cells do not respond to GM-CSF and lack F4/80. Instead, 32D cl-23 cells respond to an as yet undefined factor in conditioned medium (CM) from the primate T cell line, MLA-144, and CM from mitogen-stimulated human lymphocytes (HLCM). 32D cl-23 cells are Lyt-1+. Both FDC-Pl and 32D cl-23 cells consume interleukin-3, but only FDC Pl cells consume GM-CSF. Similarly, 32D cl-23, but not FDC-Pl, cells consume 32D cl-23 growth factor from MLA-144 CM and HLCM. Interleukin-3-dependent cell lines must therefore concurrently express different functional cell surface receptors for a variety of biochemically distinct growth factors.

Spleen cells from mice immunized with vaccinia or influenza viruses were mixed with spleen cells from mice immunized by dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH) and transferred into irradiated syngeneic recipient mice which were subsequently restimulated with trinitrophyenylates (TNP) or unmodified viruses. One week later, spleens were removed and assayed for indirect anti-DNP plaque-forming cells (PFC). When spleen cells from both influenza and vaccinia primed mice were restimulated with the haptenated form of the virus used for the initial immunization there was an enhanced PFC response compared to that seen with spleen cells from unprimed mice.

Human bone marrow cells cultured for 21 days in the presence of recombinant human interleukin-3 (IL-3) produced up to 28 times more colony-forming cells (CFC) than could be obtained from cultures stimulated with granulocyte colony stimulating factor (G-CSF) or granulocyte-macrophage CSF (GM-CSF). IL-3-cultured cells retained a multipotent response to IL-3 in colony assays but were restricted to formation of granulocyte colonies in G-CSF and granulocyte or macrophage colonies in GM-CSF. Culture of bone marrow cells in IL-3 also led to accumulation of large numbers of eosinophils and basophils. These data contrast with the effects of G-CSF, GM-CSF, and IL-3 in seven-day cultures. Here both GM-CSF and IL-3 amplified total CFC that had similar multipotential colony-forming capability in either factor. G-CSF, on the other hand, depleted IL-3-responsive colony-forming cells dramatically, apparently by causing these cells to mature into granulocytes. The data suggest that a large proportion of IL-3-responsive cells in human bone marrow express receptors for G-CSF and can respond to this factor, the majority becoming neutrophils. Furthermore, the CFC maintained for 21 days in IL-3 may be a functionally distinct population from that produced after seven days culture of bone marrow cells in either IL-3 or GM-CSF.

Adherent monolayer cultures of human blood monocytes, peritoneal macrophages, bone marrow macrophages, and colonic mucosa macrophages were examined for their ability to produce and secrete minactivin, a specific inactivator of urokinase-type plasminogen activator. All except colonic mucosa macrophages produced and secreted appreciable amounts of minactivin, but only blood monocytes were stimulated by muramyl dipeptide (adjuvant peptide) to increase production. The minactivin from each of these populations could be shown to preferentially inhibit urokinase-type plasminogen activator and not trypsin, plasmin, or "tissue"-type plasminogen activator (HPA66). A plasminogen-activating enzyme present in monocyte cultures appeared unaffected by the presence of minactivin and could be shown to be regulated independently by dexamethasone.