The production of hematopoietic growth factors by endothelial accessory cells

Monocytes are known to produce both hematopoietic growth factors and other factors, monokines, which do not directly stimulate hematopoiesis. Monokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF) may indirectly stimulate mesenchymal cells to produce hematopoietic growth factors. The identity of all the factors produced by monocytes or mesenchymal cells has not been established because of overlapping activities on biologic assay. The purpose of this study was to identify the individual growth factors produced by endothelial cells before and after stimulation with various monokines. We prepared conditioned media and extracted RNA from endothelial cells before and after stimulation with monokines. The results show that immortalized endothelial cells produce maximal detectable amounts of granulocyte-macrophage colony-stimulating factor (GM-CSF) constitutively. In contrast, GM-CSF production by primary endothelial cells requires induction with either IL-1 or TNF.     

Effects of endotoxin on proliferation of human hematopoietic cell precursors

In examining the effects of corticosteroids on hematopoiesis in vitro, we observed that results were highly dependent on the lot of commercial fetal calf serum (FCS) utilized. We hypothesized that this variability correlated with the picogram (pg) level of endotoxin contaminating the FCS. Randomly obtained commercial lots of FCS contained 0.39 to 187 pg/ml of lipopolysaccharide (LPS). Standard FCS concentrations in hematopoietic precursor proliferation assays (granulocyte-marcrophage colony forming units [CFU-GM]) resulted in final LPS levels as high as 40 pg/ml. LPS (2–5 pg/ml) added to essentially endotoxin-free cultures, induced human mononuclear cell release of interleukin (IL)-1, IL-6 and granulocyte colony stimulating factor (G-CSF). Lots of FCS induced the release of IL-1, IL-6, and G-CSF from human mononuclear cells and the release of these factors correlated with the level of contaminating LPS. Human bone marrow CFU-GM proliferation, in response to granulocyte-macrophage colony stimulating factor (GM-CSF), positively correlated with the level of LPS contaminating the FCS and the FCS-induced release of IL-6 from mononuclear cells. CFU-GM proliferation of human bone marrow cluster of differentiation (CD) 34+CD14-cells were not affected by the presence of endotoxin. These data suggest that LPS at 2–5 pg/ml may induce bone marrow accessory cell release of hematopoietic growth factors, thus altering proliferative response of hematopoietic precursors and confounding the study of exogenously added cytokines to culture systems. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interleukin-1 regulation of hematopoietic growth factor production by human stromal fibroblasts

The human stromal fibroblastoid cell strain designated ST-1 represents a normal population of cells capable of supporting hematopoiesis in vitro. These cells constitutively elaborate hematopoietic growth factor activity into the medium and the level of production of this activity dramatically increases following stimulation of the cells with IL-1. This enhanced production is due at least in part to increased expression of the genes for GM-CSF, G-CSF, and IL-6, but not IL-3. The IL-1 treatment had little effect on the expression of M-CSF, a factor made constitutively by the cells. These results are consistent with the model that hematopoiesis is regulated at least in part by constant short-range interactions of humoral factors produced by stromal cells both with other types of stromal cells and with the hematopoietic progenitors.     

Interleukin-4. A regulatory protein

Since its discovery in 1982, numerous biological activities of interleukin-4 (IL-4) have been described. Like other cytokines, IL-4 is highly pleiotropic, both with respect to the number of different target cells that are responsive to it and with respect to the number of different biological responses it elicits. Interleukin-4 was initially described as a costimulant for the proliferation of B lymphocytes stimulated with anti-IgM antibody. Synonyms for this cytokine are B cell growth factor-1 (BCGF-1) and B cell stimulatory factor-1 (BSF-1). After cloning of both the murine and human IL-4, the use of recombinant IL-4 enabled detailed studies of its biological functions. Many cell types, mainly of hematological origin, express receptors for IL-4. Accordingly, effects of IL-4 have been described on B lymphocytes, T lymphocytes, NK cells, mononuclear phagocytes, mast cells, fibroblasts and hematopoietic progenitor cells. Currently, there are three major areas in which IL-4 appears to play an important role: 1) regulation of B cell growth and of antibody isotype expression. In this context, a possible role for IL-4 in allergic reactions is of special interest. 2) Stimulation of T cell growth and the generation of cytotoxic T lymphocytes. In addition to the suppressive effects on the induction of non HLA-restricted cellular cytotoxicity by natural killer- (NK) and lymphokine-activated killer (LAK) cells, this suggests a role for IL-4 in the regulation of cellular immune responses. 3) Regulation of the growth and differentiation of hematopoietic bone marrow stem cells. IL-4 itself does not induce proliferation of hematological progenitor cells but it can modulate the growth-factor dependent proliferation of these cells. In this review the biological functions of IL-4, reported until present, are discussed.     

Killing of Escherichia coli by mononuclear phagocytes and neutrophils stimulated in vitro with beta-1,3-D-polyglucose derivatives.

Human monocytes, human peritoneal macrophages, mouse peritoneal macrophages and human peripheral neutrophils pretreated with beta-1,3-D-polyglucose derivatives showed pronounced bactericidal capacity to Escherichia coli compared to control cells. The increased bactericidal capacity was detectable in mononuclear phagocytes over a wide range of concentrations of bacteria. Granulocytes, however, showed bactericidal capacity only at low concentrations of bacteria. The pretreated mononuclear phagocytes released significant amounts of IL-1 and PGE2. However, there was no significant release of tumor necrosis factor (TNF). By incubating unstimulated cells with purified IL-1 and TNF, the bactericidal activity of neutrophils and mononuclear phagocytes was enhanced. Our data indicate that the inability of neutrophils stimulated with beta-1,3-D-polyglucose derivatives to kill large numbers of bacteria could be overcome by a combined treatment with purified IL-1 or TNF in addition to beta-1,3-D-polyglucose derivatives. By incubating unstimulated cells with medium from beta-1,3-D-polyglucose-treated human peritoneal macrophages, the bactericidal activity of the cells was enhanced to the same extent as cells pretreated with purified TNF and IL-1. Cells incubated with IL-1-depleted medium from beta-1,3-D-polyglucose-treated human peritoneal macrophages, showed reduced bactericidal activity compared to cells incubated with undepleted medium. These studies demonstrate that beta-1,3-D-polyglucose-treated mononuclear phagocytes and neutrophils show enhanced bactericidal activity. The enhanced activity is partly caused by stimulation of the cells with IL-1 released from mononuclear phagocytes and partly by other unknown effects of beta-1,3-D-polyglucose derivatives on both mononuclear phagocytes and neutrophils.     

Spred-1 negatively regulates interleukin-3-mediated ERK/mitogen-activated protein (MAP) kinase activation in hematopoietic cells

Sprouty/Spred family proteins have been identified as negative regulators of growth factor-induced ERK/mitogen-activated protein (MAP) kinase activation. However, it has not been clarified whether these proteins regulate cytokine-induced ERK activity. We found that Spred-1 is highly expressed in interleukin-3 (IL-3)-dependent hematopoietic cell lines and bone marrow-derived mast cells. To investigate the roles of Spred-1 in hematopoiesis, we expressed wild-type Spred-1 and a dominant negative form of Spred-1, DeltaC-Spred, in IL-3- and stem cell factor (SCF)-dependent cell lines as well as hematopoietic progenitor cells from mouse bone marrow by retrovirus gene transfer. In IL-3-dependent Ba/F3 cells expressing c-kit, forced expression of Spred-1 resulted in a reduced proliferation rate and ERK activation in response to not only SCF but also IL-3. In contrast, DeltaC-Spred augmented IL-3-induced cell proliferation and ERK activation. Wild-type Spred-1 inhibited colony formation of bone marrow cells in the presence of cytokines, whereas DeltaC-Spred-1 expression enhanced colony formation. Augmentation of ERK activation and proliferation in response to IL-3 was also observed in Spred-1-deficient bone marrow-derived mast cells. These data suggest that Spred-1 negatively regulates hematopoiesis by suppressing not only SCF-induced but also IL-3-induced ERK activation.     

Interleukin-1 beta (IL-1beta) induces tumor necrosis factor alpha (TNF-alpha) expression on mouse myeloid multipotent cell line 32D cl3 and inhibits their proliferation

Interleukin-1 alpha (IL-1alpha) and beta (IL-1beta) are well known factors that stimulate hematopoiesis, nevertheless there are reports that show that they can also inhibit this activity. While both IL-1alpha and IL-1beta induce the expression of hematopoietic cytokines, such as growth factors and their receptors on myeloid cells, helping thus to regulate hematopoiesis, it is not known if their inhibitory activity is also mediated through the induction of other specific cytokines. In this work we show that recombinant human IL-1beta (rhIL-1beta) inhibits the proliferation of a mouse IL-3-dependent myeloid multipotent cell line (32D cl3), without inducing its differentiation. We show that rhIL-1beta induces in 32D cl3 cells the expression of the tumor necrosis factor alpha (TNF-alpha) gene, a well known growth inhibitor, and that the rhIL-1beta growth inhibition property on 32D cl3 cells is partially due to this secreted TNF-alpha, hinting thus that the inhibition of hematopoiesis by IL-1 is mediated through other induced cytokines.     

NK cells regulate CD8+ T cell effector function in response to an intracellular pathogen

We studied the role of NK cells in regulating human CD8+ T cell effector function against mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Depletion of NK cells from PBMC of healthy tuberculin reactors reduced the frequency of M. tuberculosis-responsive CD8+IFN-gamma+ cells and decreased their capacity to lyse M. tuberculosis-infected monocytes. The frequency of CD8+ IFN-gamma+ cells was restored by soluble factors produced by activated NK cells and was dependent on IFN-gamma, IL-15, and IL-18. M. tuberculosis-activated NK cells produced IFN-gamma, activated NK cells stimulated infected monocytes to produce IL-15 and IL-18, and production of IL-15 and IL-18 were inhibited by anti-IFN-gamma. These findings suggest that NK cells maintain the frequency of M. tuberculosis-responsive CD8+IFN-gamma+ T cells by producing IFN-gamma, which elicits secretion of IL-15 and IL-18 by monocytes. These monokines in turn favor expansion of Tc1 CD8+ T cells. The capacity of NK cells to prime CD8+ T cells to lyse M. tuberculosis-infected target cells required cell-cell contact between NK cells and infected monocytes and depended on interactions between the CD40 ligand on NK cells and CD40 on infected monocytes. NK cells link the innate and the adaptive immune responses by optimizing the capacity of CD8+ T cells to produce IFN-gamma and to lyse infected cells, functions that are critical for protective immunity against M. tuberculosis and other intracellular pathogens.     

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.     

IL-9 inhibits oxidative burst and TNF-alpha release in lipopolysaccharide-stimulated human monocytes through TGF-beta

IL-9 is a Th2 cytokine that exerts pleiotropic activities on T cells, B cells, mast cells, hematopoietic progenitors, and lung epithelial cells, but no effect of this cytokine has been reported so far on mononuclear phagocytes. Human blood monocytes preincubated with IL-9 for 24 h before LPS or PMA stimulation exhibited a decreased oxidative burst, even in the presence of IFN-gamma. The inhibitory effect of IL-9 was specifically abolished by anti-hIL-9R mAb, and the presence of IL-9 receptors was demonstrated on human blood monocytes by FACS. IL-9 also down-regulated TNF-alpha and IL-10 release by LPS-stimulated monocytes. In addition, IL-9 strongly up-regulated the production of TGF-beta1 by LPS-stimulated monocytes. The suppressive effect of IL-9 on the respiratory burst and TNF-alpha production in LPS-stimulated monocytes was significantly inhibited by anti-TGF-beta1, but not by anti-IL-10Rbeta mAb. Furthermore, IL-9 inhibited LPS-induced activation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases in monocytes through a TGF-beta-mediated induction of protein phosphatase activity. In contrast, IL-4, which exerts a similar inhibitory effect on the oxidative burst and TNF-alpha release by monocytes, acts primarily through a down-regulation of LPS receptors. Thus, IL-9 deactivates LPS-stimulated blood mononuclear phagocytes, and the mechanism of inhibition involves the potentiation of TGF-beta1 production and extracellular signal-regulated kinase inhibition. These findings highlight a new target cell for IL-9 and may account for the beneficial activity of IL-9 in animal models of exaggerated inflammatory response.     

Multilineage, non-species specific hematopoietic growth factor(s) elaborated by a feline fibroblast cell line: enhancement by virus infection

In studies designed to determine the role of feline leukemia virus (FeLV) in the pathogenesis of marrow failure in the cat, we tested medium conditioned by uninfected and FeLV-infected feline embryonic fibroblasts (FEA) for its effect on hematopoietic colony growth in culture. As opposed to an inhibitory effect, we found that the conditioned medium (CM) from FEA or FEA/FeLV increased the in vitro growth of multiple hematopoietic progenitor cell types including erythroid burst-forming cells (BFU-E), granulocyte/macrophage colony-forming cells, megakaryocytic colony-forming cells, and mixed-cell colony-forming cells. Furthermore, CM enhanced the growth of progenitors in cultures of mouse or human marrow cells, as well as cat marrow cells. Stimulation of feline BFU-E was most marked with an increment in growth of 400% over control. The human burst promoting activity (BPA) of the CM was equivalent or better than other CM available in our laboratory. The evidence suggest that the growth-promoting activity is a constitutive product(s) released by FEA which was enhanced eightfold with virus infection. Studies with non-adherent and T-lymphocyte-depleted human marrow cells and human peripheral blood cells suggest that the growth factor(s) acts directly on progenitor cells and not through readily identified accessory cells. These findings are consistent with the concept that mesenchymal cells such as fibroblasts have the capacity to release hematopoietic growth factor(s) capable of acting on primitive hematopoietic progenitors. The results provide an example of how injury of such cells, through virus infection, may enhance growth factor(s) release and influence the hematopoietic microenvironment.     

Human mononuclear phagocyte activation antigens

Activation of mononuclear phagocytes causes changes in plasma membrane composition that include the expression of surface antigens and receptors. Monoclonal antibody technology has made it possible to identify and characterize newly expressed surface antigens. Among these "activation antigens" is a glycoprotein, Mo3, which (among hematopoietic cells) is selectively expressed by human mononuclear phagocytes that have been exposed to inflammatory factors in vitro and in vivo. Progress toward a functional and structural analysis of Mo3 is described.     

Basic fibroblast growth factor and epidermal growth factor downmodulate the growth of hematopoietic cells in long-term stromal cultures

The bone marrow microenvironment consists of stromal cells and extracellular matrix components which act in concert to regulate the growth and differentiation of hematopoietic stem cells. There is little understanding of the mechanisms which modulate the regulatory role of stromal cells. This study examined the hypothesis that mesenchymal growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) modulate stromal cell activities and thereby influence the course of hematopoiesis. Both bFGF and EGF were potent mitogens for marrow stroma. However, both factors proved to be inhibitory to hematopoiesis in primary log-term marrow cultures. Inhibition was also observed when hematopoietic cells and bFGF or EGF were added to subconfluent irradiated stromal layers, demonstrating that the decline of hematopoiesis was not due to overgrowth of the stromal layer. Loss of hematopoietic support in bFGF and EGF was dose-dependent. Removal of bFGF and EGF permitted stromal layers to regain their normal capacity to support hematopoiesis. In stroma-free long-term cultures, neither factor affected CFU-GM expansion. Basic FGF slightly enhanced granulocyte-macrophage colony forming unit (CFU-GM) cloning efficiency in short-term agarose culture. Basic FGF did not reduce the levels of interleukin-6 (IL-6), GM-CSF, or G-CSF released by steady state or IL-1-stimulated stroma. Similarly, the constitutive levels of steel factor (SF) mRNA and protein were not affected by bFGF. Basic FGF did not alter the level of TGF-β1 in stromal cultures. We conclude that bFGF and EGF can act as indirect negative modulators of hematopoietic growth in stromal cultures. The actual mediators of regulation, whether bound or soluble, remain to be identified. © 1995 Wiley-Liss, Inc.