IL-6 is a differentiation factor for M1 and WEHI-3B myeloid leukemic cells

IL-6 has multiple biologic activities in different cell systems including both the ability to support cell proliferation and to induce differentiation. We reported previously the isolation and functional expression of a mouse IL-6 (mIL-6) cDNA clone derived from bone marrow stromal cells. In this paper, we show that mIL-6 is a potent inducer of terminal macrophage differentiation for a mouse myeloid leukemic cell line, M1. Addition of mIL-6 to cultures of M1 cells rapidly inhibits their proliferation and induces phagocytic activity and morphologic changes characteristic of mature macrophages. These phenotypic changes are accompanied at the molecular level by a decrease in proto-oncogene c-myc mRNA accumulation and increases in Fc gamma R, proto-oncogenes c-fos and c-fms (CSF-1R) mRNA expression. Furthermore, IL-6 enhances the expression of Fc gamma R and c-fms in differentiation-responsive D+, but not unresponsive D- sublines of mouse myelomonocytic leukemic WEHI-3B cells. Together with our previous observation that IL-6 stimulates colony formation by normal myeloid progenitors, these results strongly suggest an important regulatory role for IL-6 in myeloid cell growth and differentiation.     

Proteomic analysis of macrophage differentiation. p46/52(Shc) Tyrosine phosphorylation is required for CSF-1-mediated macrophage differentiation

Macrophage colony stimulating factor (M-CSF or CSF-1) acts to regulate the development and function of cells of the macrophage lineage. Murine myeloid FDC-P1 cells transfected with the CSF-1 receptor (FD/WT) adopt a macrophage-like morphology when cultured in CSF-1. This process is abrogated in FDC-P1 cells transfected with the CSF-1 receptor with a tyrosine to phenyalanine substitution at position 807 (FD/807), suggesting that a molecular interaction critical to differentiation signaling is lost (Bourette, R. P., Myles, G. M., Carlberg, K., Chen, A. R., and Rohrschneider, L. R. (1995) Cell Growth Differ. 6, 631--645). A detailed examination of lysates of CSF-1-treated FD/807 cells by two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE) revealed a number of proteins whose degree of tyrosine phosphorylation was modulated by the Y807F mutation. Included in this category were three phosphorylated proteins that co-migrated with p46/52(Shc). Immunoprecipitation, Western blotting, and in vitro binding studies suggest that they are indeed p46/52(Shc). A key regulator of differentiation in a number of cell systems, ERK was observed to exhibit an activity that correlated with the relative degree of differentiation induced by CSF-1 in the two cell types. Transfection of cells with a non-tyrosine-phosphorylatable form of p46/52(Shc) prevented the normally observed CSF-1-mediated macrophage differentiation as determined by adoption of macrophage-like morphology and expression of the monocyte/macrophage lineage cell surface marker, Mac-1. These results are the first to suggest that p46/52(Shc) may play a role in CSF-1-induced macrophage differentiation. Additionally, a number of proteins were identified by two-dimensional SDS-PAGE whose degree of tyrosine phosphorylation is also modulated by the Y807F substitution. This group of molecules may contain novel signaling molecules important in macrophage differentiation.     

cAMP inhibits CSF-1-stimulated tyrosine phosphorylation but augments CSF-1R-mediated macrophage differentiation and ERK activation

Macrophage colony stimulating factor (M-CSF) or CSF-1 controls the development of the macrophage lineage through its receptor tyrosine kinase, c-Fms. cAMP has been shown to influence proliferation and differentiation in many cell types, including macrophages. In addition, modulation of cellular ERK activity often occurs when cAMP levels are raised. We have shown previously that agents that increase cellular cAMP inhibited CSF-1-dependent proliferation in murine bone marrow-derived macrophages (BMM) which was associated with an enhanced extracellular signal-regulated kinase (ERK) activity. We report here that increasing cAMP levels, by addition of either 8-bromo cAMP (8BrcAMP) or prostaglandin E(1) (PGE1), can induce macrophage differentiation in M1 myeloid cells engineered to express the CSF-1 receptor (M1/WT cells) and can potentiate CSF-1-induced differentiation in the same cells. The enhanced CSF-1-dependent differentiation induced by raising cAMP levels correlated with enhanced ERK activity. Thus, elevated cAMP can promote either CSF-1-induced differentiation or inhibit CSF-1-induced proliferation depending on the cellular context. The mitogen-activated protein kinase/extracellular signal-related protein kinase kinase (MEK) inhibitor, PD98059, inhibited both the cAMP- and the CSF-1R-dependent macrophage differentiation of M1/WT cells suggesting that ERK activity might be important for differentiation in the M1/WT cells. Surprisingly, addition of 8BrcAMP or PGE1 to either CSF-1-treated M1/WT or BMM cells suppressed the CSF-1R-dependent tyrosine phosphorylation of cellular substrates, including that of the CSF-1R itself. It appears that there are at least two CSF-1-dependent pathway(s), one MEK/ERK dependent pathway and another controlling the bulk of the tyrosine phosphorylation, and that cAMP can modulate signalling through both of these pathways.     

Stromal cell regulation of lymphoid and myeloid differentiation

In vitro microenvironmental influences seem to be critical for both B lymphocyte and myeloid differentiation. Studies on murine Dexter cultures and Whitlock-Witte lymphocyte cultures suggest the presence of two critical stromal regulatory cells: an alkaline-phosphatase-positive epithelioid cell and a macrophage. Further data suggest that these cells are capable of producing colony stimulating factor-1, granulocyte-macrophage CSF, a myeloid synergizing activity, and probably separate B cell growth factors. Isolation of a cell line from Dexter stroma was accomplished and this line produced CSF-1, GM-CSF, a pre-B cell and myeloid synergizing activity, and an activity acting on differentiated B cells. We speculate that the Dexter and Whitlock-Witte in vitro culture systems are regulated by factors produced by the two adherent cell types. A lineage nonspecific factor capable of inducing cells into the B lineage or synergizing with interleukin-3, GM-CSF, and CSF-1 is produced, which presumably acts on early stem cells. In addition, the cell line produces GM-CSF, CSF-1, and a factor acting on differentiated B cells. We speculate that in these culture systems, these "terminal differentiating hormones" regulate the final pathway of differentiation, whereas the pre-B-synergizing activity supports early stem cells that can then respond to the other differentiating hormones.     

Tuberculosis triggers a tissue-dependent program of differentiation and acquisition of effector functions by circulating monocytes

The origin and function of the different myeloid cell subsets that appear in the lung during pulmonary tuberculosis are unknown. Herein we show that adoptively transferred monocytes give rise to many of the macrophage and dendritic cell (DC) subsets that appear following aerosol infection with virulent Mycobacterium tuberculosis. Monocyte differentiation in infected peripheral tissue is surprisingly heterogeneous and results in the formation of five distinct myeloid subsets, including both classically activated macrophages, that produce inducible NO synthase via an IFN-gamma-dependent mechanism, and DC. In contrast, monocytes recruited to draining pulmonary lymph nodes are functionally different and acquire a mature DC phenotype. Thus, while monocytes are recruited to the lungs of uninfected mice, their differentiation and acquisition of myeloid effector functions are dramatically altered in the presence of inflammation and bacteria and are dependent on tissue localization. Therefore, our results support a model in which recruited monocytes are well poised to influence multiple aspects of host immunity to infections in the lungs. This report provides the first direct evidence for monocyte differentiation into both the macrophage and DC lineages in vivo following infection with a live human pathogen.     

Identification of tyrosine-phosphorylated colony-stimulating factor 1 (CSF-1) receptor and a 56-kilodalton protein phosphorylated in intact human cells in response to CSF-1

Colony-stimulating factor 1 (CSF-1) selectively supports the survival, proliferation, and maturation of hemopoietic cells of the monocyte/macrophage lineage. Although the cellular receptor for CSF-1, (the c-fms protein) is a protein-tyrosine kinase activated by the binding of CFS-1, the role of phosphorylation of cellular proteins in CSF-1 signal transduction is poorly understood. Therefore, we examined the CSF-1-stimulated phosphorylation of cellular proteins in human BeWo choriocarcinoma cell line (known to express the c-fms protein). BeWo cells were metabolically labeled with 32Pi, stimulated with recombinant human CSF-1, and extracted with detergent. Phosphotyrosyl proteins were isolated from detergent extracts by affinity chromatography on a highly specific antibody to phosphotyrosine. Rapid phosphorylation of 170-kd protein, followed closely by the phosphorylation of a 56-kd protein, was observed in response to CSF-1. The 170-kd phosphotyrosyl protein bound to wheat germ agglutinin and was secondarily immunoprecipitated with a specific anti-fms serum, consistent with its identity as the CSF-1 receptor. Although purified human macrophages that proliferate in culture in response to CSF-1 are not generally accessible, CSF-1 did stimulate the phosphorylation of a 56-kd protein in intact mononuclear leukocytes from human peripheral blood. Thus, the BeWo cell line may represent a good model for the study of CSF-1-stimulated cellular protein phosphorylation.     

Induction of macrophage colony-stimulating factor-dependent growth and differentiation after introduction of the murine c-fms gene into FDC-P1 cells.

A system has been established for analyzing the functions of the c-fms/macrophage colony-stimulating factor (M-CSF) receptor gene product in hematopoietic growth and differentiation. The murine c-fms gene was introduced into the factor-dependent murine hematopoietic cell line FDC-P1 by retroviral infection, and conversion to M-CSF-dependent growth was assayed in agar cultures. Expression of the c-fms gene in FDC-P1 cells, which normally do not express this gene, resulted in the conversion of resultant FD(c-fms) cells to M-CSF-dependent growth. Stimulation of FD(c-fms) cells by M-CSF led to the formation of colonies of altered morphology and produced reversible morphological changes suggestive of myeloid differentiation. M-CSF also induced expression of mature myeloid surface marker proteins in the FD(c-fms) cells. Neither multi-CSF nor granulocyte-macrophage CSF induced similar phenotypic changes but remained able to stimulate the proliferation of undifferentiated FD(c-fms) cells. These results indicate that the c-fms gene was expressed functionally in FDC-P1 cells and transmitted signals for growth. Also, the interaction of M-CSF with the c-fms gene product generated an additional signal for myeloid differentiation but did not irreversibly commit FD(c-fms) cells to terminal differentiation. This system can be used for molecular analysis of the growth- and differentiation-promoting activities of the c-fms proto-oncogene.     

C-FMS dependent HL-60 cell differentiation and regulation of RB gene expression

The dependence of induced myelomonocytic cell differentiation, and regulation of the RB tumor suppressor gene during this process, on the c-fms gene product, the CSF-1 lymphokine receptor, was determined in HL-60 promyelocytic leukemia cells. Adding a monoclonal antibody with specificity for the c-fms gene product to cells treated with various inducers of myelomonocytic or macrophage differentiation, including retinoic acid and 1,25-dihydroxy vitamin D3, inhibited the rate of differentiation. During the period of inducer treatment usually preceding onset of differentiation, longer periods of antibody exposure caused greater inhibition of differentiation. In a stable HL-60 transfectant overexpressing the CSF-1 receptor at the cell surface due to a constitutively driven c-fms trans gene, the rate of differentiation was enhanced compared to the wild type cell, consistent with a positive regulatory role for the CSF-1 receptor. The anti-fms antibody caused much less inhibition of differentiation in the transfectants than in wild type cells, consistent with a larger number of receptors causing reduced sensitivity. During the induced metabolic cascade leading to differentiation, the typical early down regulation of RB gene expression was inhibited by the antibody. The antibody itself caused an increase in RB expression per cell, which offset the decrease normally caused by differentiation inducers (1,25-dihydroxy vitamin D3 and retinoic acid). The changes in RB expression preceded changes in the RB protein to the hypophosphorylated state. Most of the RB protein in proliferating cells was phosphorylated and no significant accumulation of hypophosphorylated RB protein occurred until after onset of GO arrest. Thus the metabolic cascade leading to myelomonocytic differentiation of HL-60 cells appears to be driver by a function of the c-fms protein. Inhibiting that process by attacking this receptor impedes differentiation and also compromises the early down regulation of RB tumor suppressor gene expression which normally precedes differentiation. These findings provide additional support for a potential role for down regulating RB expression in promoting cell differentiation, and suggest the possibility that RB may be either a target or intermediate mediator of CSF-1 actions. © 1993 Wiley-Liss, Inc.     

The network of hemopoietic regulatory proteins in myeloid cell differentiation.

There are clones of myeloid leukemic cells that can be induced to undergo terminal cell differentiation to macrophages by normal hemopoietic regulatory proteins. Induction of differentiation in two different clones of myeloid leukemic cells with interleukin 6 (IL-6) or granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in induction of mRNA for the hemopoietic regulatory proteins IL-6, GM-CSF, interleukin 1 alpha and interleukin 1 beta, tumor necrosis factor, and transforming growth factor beta 1. In one of these clones, induction of differentiation with GM-CSF was also associated with induction of mRNA for macrophage colony-stimulating factor (M-CSF) but not for the receptor for M-CSF (c-fms), whereas in the other clone, induction of differentiation with IL-6 was associated with induction of mRNA for both c-fms and M-CSF. The clones also differed in their responsiveness to these regulators. There was no induction of mRNA for granulocyte colony-stimulating factor or interleukin 3 during differentiation of either clone. The results indicate that the genes for a nearly normal network of positive and negative hemopoietic regulatory proteins are induced during differentiation of these myeloid leukemic cells and that there are leukemic clones with specific defects in this network.     

Colony-stimulating factor-1 receptor (c-fms)

The macrophage colony-stimulating factor, CSF-1 (M-CSF), is a homodimeric glycoprotein required for the lineage-specific growth of cells of the mononuclear phagocyte series. Apart from its role in stimulating the proliferation of bone marrow-derived precursors of monocytes and macrophages, CSF-1 acts as a survival factor and primes mature macrophages to carry out differentiated functions. Each of the actions of CSF-1 are mediated through its binding to a single class of high-affinity receptors expressed on monocytes, macrophages, and their committed progenitors. The CSF-1 receptor (CSF-1R) is encoded by the c-fms proto-oncogene, and is one of a family of growth factor receptors that exhibits an intrinsic tyrosine-specific protein kinase activity. Transduction of c-fms sequences as a viral oncogene (v-fms) in the McDonough (SM) and HZ-5 strains of feline sarcoma virus has resulted in alterations in receptor coding sequences that affect its activity as a tyrosine kinase and provide persistent signals for cell growth in the absence of its ligand. The genetic alterations in the c-fms gene that unmask its latent transforming potential abrogate its lineage-specific activity and enable v-fms to transform a variety of cells that do not normally express CSF-1 receptors.