Effect of cytokines and growth factors on the macrophage colony-stimulating factor secretion by human bone marrow stromal cells

We have investigated the effect of growth factors, inflammatory and anti-inflammatory cytokines on the macrophage colony-stimulating factor (M-CSF) secretion by cultured human bone marrow stromal cells. Their production of M-CSF cultured in serum-free medium is enhanced in a time-dependent manner in response to tumour necrosis factor (TNF-)alpha and interleukin (IL-)4 but not to IL-1, IL-3, IL-6, IL-7, IL-10, SCF, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, bFGF and transforming growth factor (TGF-)beta. The co-addition of IL-4 and TNF-alpha has a greater than additive effect on the secretion of M-CSF suggesting that they act synergistically. The anti-inflammatory molecules IL-10 and TGF-beta have no effect on the TNF-alpha-induced M-CSF synthesis by marrow stromal cells. In conclusion TNF-alpha and IL-4 are potent stimulators of the M-CSF synthesis by human bone marrow stromal cells, a result of importance regarding the role of M-CSF in the proliferation/differentiation of mononuclear-phagocytic cells and the role of marrow stromal cells as regulators of marrow haematopoiesis.     

Production of granulocyte colony-stimulating factor and granulocyte/macrophage-colony-stimulating factor after interleukin-1 stimulation of marrow stromal cell cultures from normal or aplastic anemia donors.

We have studied stromal cell function in naive or interleukin-1 (IL-1)-stimulated (100 pg/ml) long-term marrow cultures (LTC) from 12 normal donors and 21 patients with severe aplastic anemia (AA). Conditioned media (CM) from normal LTC contained levels of erythroid burst-promoting activity (BPA) and granulocyte/macrophage (GM) colony-stimulating activity (CSA) comparable to those previously described (Migliaccio et al., [1990] Blood, 75:305-312). The addition of IL-1 to these cultures increased the level of CSA and, specifically, of granulocyte colony-stimulating factor (G-CSF) released. Anti-GM-CSF antibody neutralized BPA and CSA in normal naive LTC CM but only the CSA in the CM from IL-1-stimulated LTC. Since the concentrations of GM-CSF, as detected with a specific immunoassay, did not increase after IL-1 treatment, these data suggest that IL-1-stimulated cultures contain an unidentified growth factor having BPA. CM from AA stromal cells contained levels of CSA comparable to those observed in normal stromal cell CM but had significantly lower levels of BPA. Neither anti-GM-CSF nor anti-IL-3 antibodies neutralized the BPA in AA stromal cell CM. This activity may be related to that found in the CM of IL-1-treated normal stromal cells. In nearly 50% of stromal cell cultures of AA patients, addition of IL-1 failed to increase the BPA, CSA, or G-CSF. The presence of an inhibitor in naive or IL-1-treated AA stromal cell CM was excluded by adding the CM to IL-3-stimulated cultures. These findings suggest that G-CSF and GM-CSF genes are differentially regulated in the marrow microenvironment. Furthermore, a marrow microenvironment, deficient in BPA production and, in some cases, unresponsive to IL-1 could contribute to marrow failure in some patients with AA.     

Synergism among interleukin 1, interleukin 3, and interleukin 5 in the production of eosinophils from primitive hemopoietic stem cells

The in vitro production of eosinophils from committed progenitor cells is influenced by interleukin (IL)-5 (eosinophil differentiation factor) and to a lesser extent by IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). In primary suspension cultures of marrow cells taken from eosinophilic mice, IL-3 induced a modest stimulation of eosinophil production compared to IL-5. In contrast, IL-3 was sevenfold more effective than IL-5 in generating eosinophil progenitors (eosinophil colony-forming units (CFU-eo] from more primitive precursors present in the marrow of normal mice. Pre-incubation of marrow cells in suspension culture with IL-3, but not IL-5, increased the recovery of myeloid precursors responsive to G-CSF, GM-CSF, CSF-1, or IL-3 two- to fourfold while eosinophil progenitor cells responsive to IL-5 were increased by more than 70-fold. Similarly, pre-incubation of bone marrow cells under clonal conditions with IL-3, but not IL-5, resulted in a more than 50 fold increase in CFU-eo responsive to IL-5 over input values. Bone marrow from mice pre-treated with 5-fluorouracil is greatly depleted of progenitor cells directly responsive to IL-3 or IL-5. IL-1 which synergistically interacts with various CSF species to confer a clonogenic response by primitive stem cells present in 5-fluorouracil-treated marrow also failed to stimulate eosinophil production. A marked synergism was observed when IL-1 and IL-3 were combined in the suspension pre-culture phase with a more than sixfold recovery of CFU-eo than induced by either factor alone. Furthermore, pre-culture of 5-fluorouracil-treated marrow cells with a combination of IL-1 and IL-3 resulted in a more than 260-fold increase of CFU-eo over input numbers. These data suggest that the concatenate action of IL-1, IL-3, and IL-5 is an absolute requirement for the in vitro generation of eosinophils from primitive hemopoietic stem cells.     

Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells

Mesenchymal stem cells (MSCs) are a population of pluripotent cells within the bone marrow microenvironment defined by their ability to differentiate into cells of the osteogenic, chondrogenic, tendonogenic, adipogenic, and myogenic lineages. We have developed methodologies to isolate and culture-expand MSCs from human bone marrow, and in this study, we examined the MSC's role as a stromal cell precursor capable of supporting hematopoietic differentiation in vitro. We examined the morphology, phenotype, and in vitro function of cultures of MSCs and traditional marrow-derived stromal cells (MDSCs) from the same marrow sample. MSCs are morphologically distinct from MDSC cultures, and flow cytometric analyses show that MSCs are a homogeneous cell population devoid of hematopoietic cells. RT-PCR analysis of cytokine and growth factor mRNA in MSCs and MDSCs revealed a very similar pattern of mRNAs including IL-6, -7, -8, -11, -12, -14, and -15, M-CSF, Flt-3 ligand, and SCF. Steady-state levels of IL-11 and IL-12 mRNA were found to be greater in MSCs. Addition of IL-1α induced steady-state levels of G-CSF and GM-CSF mRNA in both cell preparations. In contrast, IL-1α induced IL-1α and LIF mRNA levels only in MSCs, further emphasizing phenotypic differences between MSCs and MDSCs. In long-term bone marrow culture (LTBMC), MSCs maintained the hematopoietic differentiation of CD34⁺ hematopoietic progenitor cells. Together, these data suggest that MSCs represent an important cellular component of the bone marrow microenvironment. J. Cell. Physiol. 176:57–66, 1998. © 1998 Wiley-Liss, Inc.     

Cytokine expression by human marrow-derived mesenchymal progenitor cells in vitro: Effects of dexamethasone and IL-1α

We previously reported the purification, culture-expansion, and osteogenic differentiation potential of mesenchymal progenitor cells (MPCs) derived from human bone marrow. As a first step to establishing the phenotypic characteristics of MPCs, we reported on the identification of unique cell surface proteins which were detected with monoclonal antibodies. In this study, the phenotypic characterization of human marrow-derived MPCs is further established through the identification of a cytokine expression profile under standardized growth medium conditions and in the presence of regulators of the osteogenic and stromal cell lineages, dexamethasone and interleukin-1α (IL-1α), respectively. Constitutively expressed cytokines in this growth phase include G-CSF, SCF, LIF, M-CSF, IL-6, and IL-11, while GM-CSF, IL-3, TGF-β2, and OSM were not detected in the growth medium. Exposure of cells in growth medium to dexamethasone resulted in a decrease in the expression of LIF, IL-6, and IL-11. These cytokines have been reported to exert influence on the differentiation of cells derived from the bone marrow stroma through target cell receptors that utilize gp130-associated signal transduction pathways. Dexamethasone had no effect on the other cytokines expressed under growth medium conditions and was not observed to increase the expression of any of the cytokines measured in this study. In contrast, IL-1α increased the expression of G-CSF, M-CSF, LIF, IL-6, and IL-11 and induced the expression of GM-CSF. IL-1α had no effect on SCF expression and was not observed to decrease the production of any of the cytokines assayed. These data indicate that MPCs exhibit a distinct cytokine expression profile. We interpret this cytokine profile to suggest that MPCs serve specific supportive functions in the microenvironment of bone marrow. MPCs provide inductive and regulatory information which are consistent with the ability to support hematopoiesis, and also supply autocrine, paracrine, and juxtacrine factors that influence the cells of the marrow microenvironment itself. In addition, the cytokine profiles expressed by MPCs, in response to dexamethasone and IL-1α, identify specific cytokines whose levels of expression change as MPCs differentiate or modulate their phenotype during osteogenic or stromagenic lineage entrance/progression. © 1996 Wiley-Liss, Inc.     

Endogenous production of tumor necrosis factor by primary cultures of murine calvarial cells: influence on IL-6 production and osteoclast development

We have previously shown that tumor necrosis factor (TNF) and interleukin-1 (IL-1) acted synergistically to stimulate the production of IL-6 by bone marrow stromal and osteoblastic cells; and that an antibody to IL-6 inhibited TNF-induced osteoclast development in murine calvarial cell cultures. Prompted by this evidence, we have now examined whether TNF and/or IL-1 are produced by murine calvarial cells, and whether these cytokines are involved in IL-6 production and osteoclast formation. When cultured under basal conditions, calvarial cells produced TNF and IL-6, and were able to form bone resorbing osteoclasts. A neutralizing antibody against TNF suppressed both basal IL-6 production and the formation of bone resorbing osteoclasts. The anti-TNF antibody also inhibited IL-6 production in response to exogenous IL-1 or parathyroid hormone (PTH). In contrast, a neutralizing anti-IL-1 receptor antibody had no effect on basal, TNF- or PTH-stimulated IL-6 production. These findings suggest that TNF, but not IL-1, is produced by murine bone cells and that endogenous TNF induces the IL-6 production, osteoclast formation, and bone resorption exhibited by these cultures under basal conditions. Furthermore, bone cell-derived TNF amplifies the stimulatory effect of exogenous IL-1 or PTH on IL-6 production by calvarial cells.     

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.     

Human articular cartilage and chondrocytes produce hemopoietic colony-stimulating factors in culture in response to IL-1.

The hemopoietic CSF, granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF), are cytokines that mediate the clonal proliferation and differentiation of progenitor cells into mature macrophages and/or granulocytes. We have employed an all-human cell culture system, specific ELISA for GM-CSF and G-CSF, and Northern analysis to investigate whether chondrocytes are a potential source of CSF in rheumatoid disease. We report that human rIL-1 stimulated in a dose-dependent manner the production of GM-CSF and G-CSF by human articular cartilage and chondrocyte monolayers in organ and cell culture, respectively. Increased levels of the CSF Ag were detected after 2 to 8 h stimulation with IL-1, and the optimum dose of IL-1 was 10 to 100 U/ml (0.06 to 0.6 nM IL-1 alpha; 0.02 to 0.2 nM IL-1 beta); neither CSF was detectable in nonstimulated cultures nor in IL-1-stimulated cultures treated with actinomycin D or cycloheximide, indicating the requirement for de novo RNA and protein synthesis. The IL-1-mediated increase in GM-CSF could also be inhibited by the corticosteroid, dexamethasone, but not by the cyclo-oxygenase inhibitor, indomethacin. Although having little effect when tested alone, TNF-alpha and lymphotoxin (TNF-beta) could synergize with IL-1 for the production of GM-CSF. Basic fibroblast growth factor, platelet-derived growth factor, and IFN-alpha and IFN-gamma each had no effect on GM-CSF levels. Results obtained by Northern analysis of chondrocyte total RNA reflected those found for the CSF Ag, namely that CSF mRNA levels were elevated in response to IL-1, but not TNF, and that there was synergy between these two cytokines. We propose that chondrocyte CSF production in response to IL-1, and the concurrent destruction of cartilage by IL-1, could provide a mechanism for the chronic nature of rheumatoid disease.     

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.     

Effect of cytokines on Japanese encephalitis virus production by human monocytes

Japanese encephalitis (JE) virus was shown to grow in in vitro cultures of human monocytes. Interferon (IFN)-alpha and IFN-gamma inhibited JE virus production by the infected monocytes in the absence of anti-JE virus antibody, but interleukin (IL)-1 alpha, IL-2, IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-CSF (G-CSF), and tumor necrosis factor (TNF)-alpha did not show a significant inhibition. Antibody against JE virus increased the JE virus production by the infected monocytes probably by enhanced uptake of virus-antibody complexes via Fc receptors. IFN-gamma and GM-CSF increased JE virus production by monocytes in the presence of anti-JE virus antibody, whereas IFN-alpha inhibited JE virus production even in the presence of the antibody. The other 5 cytokines (IL-1 alpha, IL-2, IL-3, G-CSF, and TNF-alpha) did not show a significant effect on JE virus production by monocytes in the presence or absence of the antibody.     

Induction of hepatocyte growth factor/scatter factor by interferon-γ in human leukemia cells

Induction of hepatocyte growth factor/scatter factor (HGF/SF) may be one of the critical steps in organ regeneration, wound healing, and embryogenesis. We previously reported the production of HGF/SF from various human leukemia cell lines and a high level of the growth factor in blood and bone marrow plasma from patients with various types of leukemia. We determined here the effects of hematopoietic cytokines on HGF/SF production in human leukemia cell lines, KG-1, a myeloid cell line, and RPMI-8226, a B cell line. Interferon (IFN)-γ remarkably stimulated HGF/SF production in both cell lines at concentrations of more than 0.1 or 1 IU/ml. IFN-α and IFN-β were as effective as IFN-γ in RPMI-8226 cells, but less than IFN-γ in KG-1 cells. HGF/SF gene expression in KG-1 cells was also up-regulated by IFN-γ. Granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-5 and IL-6 had no effect on HGF/SF production in the 2 leukemia cell lines. We also determined the effects of HGF/SF inducers known for human fibroblasts on the growth factor production in leukemia cells. Out of phorbol 12-myristate 13-acetate (PMA), cholera toxin, IL-1β, and tumor necrosis factor (TNF)-α, the former three were as effective as IFN-γ in KG-1 cells, but only TNF-α stimulated HGF/SF production in RPMI-8226 cells, whose effect was less than those of IFN-α, IFN-β, and IFN-γ. The effect of IFN-γ in KG-1 cells was synergistic with that of PMA. In contrast with the effect in leukemia cells, HGF/SF induction by IFN-γ in human skin fibroblasts was much less than that by PMA or cholera toxin. These results indicated that IFN-γ is a potent inducer of HGF/SF in human leukemia cells. This finding suggests the presence of a homeostatic control mechanism in liver regeneration and repair: hepatic injury, DNA synthesis inhibition, or apoptosis caused by IFN-γ is subsequently overcome by cytokine-induced HGF/SF, a potent promoter of liver DNA synthesis. J. Cell. Physiol. 174:107–114, 1998. © 1998 Wiley-Liss, Inc.     

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.     

The differential inhibition of hemopoietic growth factor activity by cytotoxins and interferon-gamma

The effects of recombinant human tumor necrosis factor (TNF), lymphotoxin (LT), and interferon-gamma (IFN-gamma) on the growth of human hemopoietic progenitor cells in clonal culture have been examined. Colony growth was induced by using granulocyte colony-stimulating factor (G-CSF), or granulocyte-macrophage colony-stimulating factor (GM-CSF). A suppressive effect of TNF, LT, and IFN-gamma on the development of granulocyte, macrophage, and mixed granulocyte/macrophage colonies was shown. Suppression of colonies formed after stimulation with G-CSF was greater than that observed after stimulation with GM-CSF. In the presence of a monoclonal antibody to TNF, or polyclonal antibodies to either LT or IFN-gamma, the inhibitory effect of the molecule to which the antibody was directed was abrogated. These findings suggest that progenitor cells responsive to G-CSF or GM-CSF have different sensitivities to the effects of TNF, LT, and IFN-gamma. Defining the interactions of growth factors and inhibitors should increase understanding of mechanisms underlying diseases associated with suppression of normal hemopoiesis, and in predicting the effects in vivo of these bioregulatory molecules in clinical medicine.     

Effect of cytokines on the proliferation/differentiation of stroma-initiating cells

A culture system that identifies the precursor of murine bone marrow fibroblastic stromal cells (stroma-initiating cells, SIC) has been developed. In this system, mature fibroblasts are depleted by adherence to plastic dishes and the nonadherent cells are seeded at a low density, which results in the formation of colonies composed of fibroblastic cells. Macrophage colony-stimulating factor (M-CSF) has been shown to accelerate the colony formation in the system. In this study, we examined the stroma-inducing activity of a number of cytokines. Neither granulocyte-CSF, stem cell factor, interleukin (IL)-1, IL-6, transforming growth factor, epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, nor fibroblast growth factor showed the activity. Similarly, tumor necrosis factor (TNF) did not show any stroma-inducing activity, but the factor inhibited the stromal colony formation induced by M-CSF. In this study, we found that granulocyte/macrophage-CSF (GM-CSF) and IL-3, as well as M-CSF had the stroma-inducing activity. Neither an additive nor synergistic effect was observed when the three factors were assayed in various combinations. The stroma-inducing activity of M-CSF, GM-CSF and IL-3 was observed even if lineage-negative bone marrow cells were used as target cells, suggesting that mature hematopoietic cells such as macrophages and granulocytes were not involved in the induction of stromal colony formation by these factors. Our results raise the possibility that GM-CSF and IL-3 as well as M-CSF stimulate the proliferation or differentiation of the precursor of bone marrow fibroblastic stromal cells.     

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.     

IL-4 and TNF-α-MEDIATED PROLIFERATION OF THE HUMAN MEGAKARYOCYTIC LINE M-O7E IS REGULATED BY INDUCED AUTOCRINE PRODUCTION OF GM-CSF

In this study, the authors examined the effects of recombinant human interleukin 4 (rhIL-4) and recombinant human tumour necrosis factor alpha (rhTNF-α) alone or in combination on proliferation of the human cytokine dependent myeloid cell line, M-O7e. While rhIL-4 or rhTNF-α alone induced only a weak proliferative response, a synergistic proliferative signal was clearly evident on stimulation of cells with a combination of both cytokines. The stimulatory effect of rhTNF-α is mediated predominantly by the 55-kDa TNF receptor because the agonistic monoclonal antibody Htr-9 and the Trp₃₂Thr₈₆TNF-α mutant protein specific for this receptor type produced similar results to rhTNF-α. In contrast, the Asn₁₄₃Arg₁₄₅TNF-α mutant protein specific for the 75-kDa TNF receptor produced only minimal proliferation of M-O7e cells. Using RT-PCR, we found that rhTNF-α rapidly and strongly induced granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA production, while rhIL-4 was a slow and less efficient inducer of GM-CSF mRNA. However, there was little evidence of the TNF-α/IL-4 combination acting synergistically on GM-CSF mRNA production as the levels of GM-CSF mRNA increased only marginally compared with IL-4 or TNF-α alone. Thus, the observed synergistic effect of TNF-α/IL-4 costimulation of M-O7e cells seems to be mediated via induction of GM-CSF secretion rather than an enhanced production of GM-CSF mRNA. Higher levels of GM-CSF were detectable in supernatants of cells treated with both rhIL-4 and rhTNF-α than in cells stimulated with either cytokine alone. Furthermore, addition of a neutralising antibody against GM-CSF abrogated the observed synergistic effect of rhIL?4 and rhTNF-α treatment, indicating that the rhIL-4/THF-α combination acts to significantly increase GM-CSF release which then acts in an autocrine manner to enhance the proliferation of M-O7e cells.     

Tumour necrosis factor-alpha (TNFalpha) stimulates the growth of human bone marrow stromal cells

This study reports that TNF-alpha is a potent mitogen for human bone marrow sternal cells in vitro (assessed by [(3)H]-thymidine incorporation into DNA and cell counts). In contrast, cytokines such as IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-6, LIF, SCF, M-CSF, G-CSF and GM-CSF had no effect. The effect of TNF-alpha on the growth of human bone marrow stromal cells could be of importance during inflammatory processes which take place in the marrow, for example marrow fibrosis.     

Interleukin 1 stimulates human endothelial cells to produce granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor

Endothelial cells are a potent source of hematopoietic growth factors when stimulated by soluble products of monocytes. Interleukin 1 (IL 1) is released by activated monocytes and is a mediator of the inflammatory response. We determined whether purified recombinant human IL 1 could stimulate cultured human umbilical vein endothelial cells to release hematopoietic growth factors. As little as 1 U/ml of IL 1 stimulated growth factor production by the endothelial cells, and increasing amounts of IL 1 enhanced growth factor production in a dose-dependent manner. Growth factor production increased within 2 to 4 hr and remained elevated for more than 48 hr. To investigate the molecular basis for these findings, oligonucleotide probes for granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and multi-CSF were hybridized to poly(A)-containing RNA prepared from unstimulated and IL 1-stimulated endothelial cells. Significant levels of GM-CSF and G-CSF, but not M-CSF or multi-CSF, mRNA were detected in the IL 1-stimulated endothelial cells. Biological assays performed on the IL 1-stimulated endothelial cell-conditioned medium confirmed the presence of both GM- and G-CSF. These results demonstrate that human recombinant IL 1 can stimulate endothelial cells to release GM-CSF and G-CSF, and provide a mechanism by which IL 1 could modulate both granulocyte production and function during the course of an inflammatory response.     

Synergistic effects of purified recombinant human and murine B cell growth factor-1/IL-4 on colony formation in vitro by hematopoietic progenitor cells. Multiple actions

Purified recombinant human B cell growth factor-1/IL-4 was evaluated, alone and in combination, with purified preparations of recombinant human (rhu) CSF or erythropoietin (Epo) for effects on colony formation by human bone marrow CFU-GM progenitor cells (GM) and burst forming unit-E progenitor cells. rhu IL-4 synergized with rhu G-CSF to enhance granulocyte colony formation, but had no effect on CFU-GM colony formation stimulated by rhu GM-CSF, rhu IL-3, or rhu CSF-1. Rhu IL-4 synergized with Epo to enhance BFU-E colony formation equal to that of Epo plus either rhu IL-3, rhu GM-CSF, or rhu G-CSF. Removal of adherent cells and T lymphocytes did not influence the synergistic activities of rhu IL-4. Rmu IL-4, synergized with rhu G-CSF, but not with rmu GM-CSF, rmu IL-3, or natural mu CSF-1, to enhance CFU-GM (mainly granulocyte) colony numbers by a greater than 90% pure preparation of murine CFU-GM. Also, rhu IL-4 at low concentrations enhanced release of CSF and at higher concentrations the release also of suppressor molecules from human monocytes and PHA-stimulated human T lymphocytes. Use of specific CSF antibodies suggested that rhu IL-4 was enhancing the release of G-CSF and CSF-1 from monocytes and the release of GM-CSF and possibly G-CSF from PHA-stimulated T lymphocytes. Use of antibodies for TNF-alpha, IFN-gamma, or TNF-beta as well as measurement of TNF and IFN titers suggested that the suppressor molecule(s) released from monocytes were acting with TNF-alpha and those released from PHA-stimulated T lymphocytes were acting with IFN-gamma. These results implicate B cell growth factor-1/IL-4 as a synergistic activity for hematopoietic progenitors and suggest that the actions can be on both progenitor and accessory cells.