Interleukin 1 and Tumor Necrosis Factor-α Stimulate the Production of Colony-Stimulating Factor 1 by Murine Astrocytes

Astrocytes have the ability to secrete colony-stimulating factor 1 (CSF-1), a growth factor known to stimulate the proliferation of brain macrophages. We have studied the effect of cytokines such as interleukin 1 (IL-1), tumor necrosis factor-alpha (TNF alpha), and interleukin 6 (IL-6) on the production of CSF-1 by cultured primary astrocytes and an astrocytic cell line derived from embryonic mouse brain. We observed that both TNF alpha and IL-1 increased CSF-1 mRNA and protein levels in the astrocytic cultures. In contrast, IL-6 was ineffective. The CSF-1 mRNA levels were strongly reduced by incubating immortalized astrocytic cells with staurosporine, a protein kinase C inhibitor, both in the absence and in the presence of cytokines. Conversely, 12-O-tetradecanoylphorbol 13-acetate, a protein kinase C activator, increased CSF-1 mRNA levels. These results suggest a mechanism whereby mononuclear phagocytes could favor their own recruitment in the CNS by producing cytokines.     

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.     

Vitamin D(3) and analogues modulate the expression of CSF-1 and its receptor in human dendritic cells

The active vitamin D(3)-metabolite 1,25(OH)(2)D(3) inhibits the interleukin 4/granulocyte-macrophage colony-stimulating factor (IL-4/GM-CSF)-induced differentiation of human monocytes into dendritic cells without altering survival. Colony-stimulating factor 1 (CSF-1) is an important survival factor for cells of the monocytic lineage. We therefore investigated whether the inhibitory activity of 1,25(OH)(2)D(3) is paralleled by a regulation of CSF-1 and its receptor. Purified human monocytes were cultured together with IL-4/GM-CSF in the presence of 1,25(OH)(2)D(3), its analogue tacalcitol, the low-affinity vitamin D receptor ligand 24,25(OH)(2)D(3), or the solvent ethanol for up to 5 days. Expression of CSF-1, CSF-1R, and GM-CSF mRNA was measured by RT-PCR. Protein secretion for CSF-1 was measured by ELISA, expression of CSF-1R by flow cytometry. The results showed that 1,25(OH)(2)D(3) and tacalcitol significantly up-regulated CSF-1 mRNA-expression and protein secretion in a dose-dependent manner. The effect of 1,25(OH)(2)D(3) occurred already after 1h of pre-treatment. In contrast, CSF-1R mRNA- and cell surface-expression was down-regulated simultaneously. The solvent ethanol and 24,25(OH)(2)D(3) were without effect. GM-CSF mRNA expression was not modulated in 1,25(OH)(2)D(3)-treated cells. These data point towards a distinct and specific regulation of CSF-1 and its receptor by 1,25(OH)(2)D(3) and its analogue tacalcitol in human monocytes which parallels the inhibition of differentiation into dendritic cells without altering survival.     

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation.

Production of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after stimulation by lipopolysaccharide (LPS) and phytohemagglutinin (PHA) was studied in 1/10 diluted whole blood (WB) culture and in peripheral blood mononuclear cell (PBMC) culture. Cytokines IL-1 beta, TNF-alpha and IL-6 are preferentially stimulated by LPS whereas IL-2, IFN-gamma and GM-CSF are stimulated by PHA. Combination of 5 micrograms/ml PHA and 25 micrograms/ml LPS gave the most reliable production of the six cytokines studied. IL-1 beta, TNF-alpha and IL-6 represent a homogeneous group of early-produced cytokines positively correlated among themselves and with the number of monocytes in the culture (LeuM3). Furthermore, IL-1 beta was negatively correlated with the number of T8 lymphocytes. IL-2, IFN-gamma and GM-CSF represent a group of late-produced cytokines. Kinetics and production levels of IL-6 and GM-CSF are similar in WB and PBMC cultures. In contrast, production levels of TNF-alpha and IFN-gamma are higher in WB than in PBMC whereas production levels of IL-6 and IL-2 are lower in WB than in PBMC. Individual variation in responses to PHA + LPS was always higher in PBMC cultures than in WB cultures. The capacity of cytokine production in relation to the number of mononuclear cells is higher in WB, or in PBMC having the same mononuclear cell concentration as WB, than in conventional cultures of concentrated PBMC (10(6)/ml). Because it mimics the natural environment, diluted WB culture may be the most appropriate milieu in which to study cytokine production in vitro.     

Contrasting effects of interferon gamma and interleukin 4 on responses of human vascular endothelial cells to tumour necrosis factor alpha.

Tumour necrosis factor alpha (TNF-alpha) and interleukin 4 (IL-4) selectively synergise in inducing expression of the mononuclear cell adhesion receptor VCAM-1 (vascular cell adhesion molecule-1) on human umbilical vein endothelial cells (HUVEC), which results in increased adhesiveness of HUVEC for T lymphocytes. This process may be crucial for adherence of circulating lymphocytes prior to their passage from the blood into inflammatory tissues. IL-4 also amplifies production of interleukin 6 (IL-6) and monocyte chemotactic protein-(MCP-1) from TNF-alpha-activated HUVEC. In the present study we demonstrate that IL-4 enhances production of granulocyte-macrophage colony-stimulating factor (GM-CSF) from TNF-alpha-stimulated HUVEC. Moreover, using cultured adult saphenous vein and umbilical artery endothelial cells, we show identical effects of IL-4 on TNF-alpha-induced responses to those observed with endothelial cells of foetal origin. Additionally, we report here that TNF-alpha and interferon gamma (IFN-gamma) synergise in the induction of both the lymphocyte adhesion receptor VCAM-1, and the TNF-alpha-inducible neutrophil adhesion receptor intercellular adhesion molecule-1, on all three endothelial cell types studied. In contrast, we found that GM-CSF secretion by endothelial cells treated with IFN-gamma plus TNF-alpha was markedly decreased when compared to the response induced by TNF-alpha alone. These results suggest that the combined actions of several cytokines, acting sequentially or in concert, may exert differential effects on activation and accumulation of circulating lymphocytes at sites of inflammation.     

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.     

Activation and distribution of inflammatory cells in the mouse uterus during the preimplantation period.

Northern blot analysis of mouse uterine RNA showed that IL-1 (alpha and beta), and TNF-alpha mRNA were abundant on day (D) 1 of pregnancy, reduced on D2, and remained basal throughout the remainder of the preimplantation period (D3 and D4). Elevated IL-1 beta and TNF-alpha mRNA levels on D1 were accompanied by increased levels of immunoreactive protein in uterine cytosol preparations as determined by ELISA. In situ hybridization detected IL-1 beta mRNA in cells located in the endometrial stroma and concentrated in subepithelial regions on D1. Immunocytochemical localization of IL-1 beta and TNF-alpha identified cells scattered throughout the endometrial stroma, but more concentrated in the subepithelial region on D1. On D3 and D4, cytokine-immunopositive cells decreased in number and became located predominantly at the endometrial-myometrial junction. Histochemical localization of peroxidase as a marker predominantly for eosinophils showed an abundance of these cells in the D1 uterus. The distribution of peroxidase-positive cells in the uterus followed the same temporal and spatial changes as cytokine-immunopositive cells during the preimplantation period. These data document the occurrence of an inflammatory response in the uterus on D1 of pregnancy, and demonstrate that as the preimplantation period progresses the distribution of inflammatory cells changes from the subepithelial region of the endometrial stroma to the periphery of the uterus at the endometrial-myometrial junction. Mechanisms regulating the uterine inflammatory response on D1 were investigated. Cytokine mRNA levels were not significantly elevated during the estrous cycle or after treatment of adult ovariectomized mice with estradiol-17 beta. In contrast, mating with vasectomized males resulted in an inflammatory response on D1 of pseudopregnancy similar to that on D1 of normal pregnancy, whereas mechanical stimulation of the uterine cervix failed to elicit such a response. These results strongly suggest a role for some factor(s) in the ejaculate, other than spermatozoa, in the initiation of a uterine inflammatory response after mating, but an effect of the act of mating cannot be excluded.     

Lithium chloride potentiates tumor necrosis factor-induced and interleukin 1-induced cytokine and cytokine receptor expression.

The antimalignant cell activity of tumor necrosis factor (TNF) in many cell types can be enhanced by lithium chloride (LiCl). This study shows the in vitro effect of LiCl on the TNF-induced or interleukin 1 (IL-1)-induced expression of IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, IL-2, and the IL-2 receptor-alpha (IL-2R alpha). The levels of IL-6 and GM-CSF in the medium of TNF-treated L929 fibrosarcoma cells were increased by cotreatment with LiCl. In contrast, enhancement of IL-6 production by dibutyryl cyclic AMP or cycloheximide was not affected by LiCl. The production of IL-6 and GM-CSF was not correlated with sensitivity to TNF-mediated cell killing. IL-1 by itself had no measurable effects on L929 cells. However, LiCl potentiated the IL-1-induced synthesis of IL-6, GM-CSF, IL-3, and IL-2 in PC60 murine T-cell hybridoma cells. TNF alone induced only GM-CSF production in these cells, but in the presence of LiCl, increased amounts of GM-CSF as well as small amounts of IL-2 and IL-6 could be detected. It is also shown that in these PC60 cells the expression of the IL-2R alpha was induced by TNF + LiCl treatment but not by TNF alone. IL-2R alpha expression was likewise considerably enhanced by IL-1 + LiCl treatment, as compared with treatment with IL-1 alone. The effects of LiCl on the TNF-induced and the IL-1-induced gene expression seem to be independent of the protein kinase A and C pathways. These results show that LiCl can modulate both TNF-mediated cytotoxicity and TNF-induced and IL-1-induced cytokine expression, suggesting that Li+ acts early in the TNF-signaling pathway, but at a step shared with the IL-1-signaling pathway.     

The effects of infection of thermal injury by Pseudomonas aeruginosa PAO1 on the murine cytokine response.

Pseudomonas aeruginosa infection, one of the major complications of burn wounds, may lead to sepsis and death. Using the Multi-Probe Template/RNase protection assay, we have compared the expression of different cytokine genes within the skin and livers of thermally injured mice infected with P. aeruginosa PAO1. Thermal injury alone enhanced or up-regulated certain cytokines, including macrophage colony-stimulating factor (M-CSF), interleukin 1 (IL-1)RI, IL-1 beta, macrophage inflammatory protein (MIP)-1 beta and MIP-2; while PAO1 challenge alone up-regulated tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) expression. The combination of thermal injury plus PAO1 infection enhanced the expression of several pro-inflammatory and haematopoietic cytokines [stem cell factor (SCF), leukocyte inhibitory factor (LIF), IL-6 and TNF-alpha]; induced the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF by 5 h and the expression of additional cytokines, including TGF-beta, TNF-beta, lymphotoxin beta (LT-beta), interferon gamma (IFN-gamma), and IFN-beta by 40 h post-burn/infection. While the most intense cytokine expression occurred in the skin, the majority of cytokines tested were also expressed in the liver by 40 h post-burn/infection. These results suggest that in P. aeruginosa infection of burn wounds: (1) up-regulation of the expression of different cytokines, locally and within the livers of burned mice, is an indication of P. aeruginosa -induced sepsis; and (2) IL-6 and G-CSF play an important role in the host response mechanism.     

Production of IL-10, TNF-alpha, IFN-gamma, TGF-beta1 by different populations of erythroid cells derived from human embryonal liver

It has previously been determined that erythroid cells of mice are capable of expressing such cytokines as interleukin (IL) 1 alpha and beta, IL-4, IL-6, interferon gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta (TGF-beta). It has been shown that glycophorin A(+) (GlA(+)) and antigen erythroblasts (AG-EB(+)) (both human erythroid cells of embryonic origin) are also capable of producing a series of cytokines such as IL-1 beta, IL-2, IL-4 and IL-6. The aim of this work was to study the capacity of erythroid cells from human embryonic liver to produce such cytokines as IFN-gamma, TGF-beta1, tumour necrosis factor alpha (TNF-alpha) and IL-10. The erythroid cells were isolated by means of antibodies specific to erythroblasts (GlA and AG-EB), as well as those from single erythroid colonies. The production level of some cytokines varies insignificantly under the action of erythropoietin (Epo) and quantitatively differs in GlA(+) and AG-EB(+) erythroid cells. Hence, the erythroid cells express IFN-gamma, TGF-beta1, TNF-alpha and IL-10. The erythroid cells could be involved through the production of these cytokines in the regulation of such processes as self-renewal, proliferation and differentiation of cells of other blood-forming sites.     

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.     

The effect of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on granulocyte macrophage-colony stimulating factor (GM-CSF) production by neuronal precursor cells

AIMS: To determine whether granulocyte macrophage-colony stimulating factor (GM-CSF) production by neuronal precursor (NT2) cells can be regulated by IL-1beta and TNF-alpha. BACKGROUND: We have previously demonstrated GM-CSF expression by neurons of the developing human brain, as well as by NT2 cells. IL-1beta and TNF-alpha upregulate GM-CSF production in glial cells, but GM-CSF regulation in neurons is as yet undefined. We hypothesized that IL-1beta and TNF-alpha would increase GM-CSF mRNA and protein production in NT2 cells. METHODS: The effect of IL-1beta and TNF-alpha on GM-CSF production was assessed by dose response (0 to 2,000 U/ml), and time course (0 to 48 hours incubation) experiments. GM-CSF mRNA and protein production were assessed by quantitative RT-PCR and by ELISA. The effect of these cytokines on cell turnover was determined by BrdU incorporation. RESULTS: IL-1beta increased GM-CSF mRNA and protein expression by NT2 cells. This effect was time and dose dependent, and the effective dose ranging from (20-200 U/ml). TNF-alpha increased GM-CSF mRNA expression to a lesser extent than did IL-1beta (maximal stimulation at 200 U/ml), and a minimal increase in net protein accumulation was noted. Neither cytokine increased NT2 cell turnover. CONCLUSIONS: IL-1beta and TNF-alpha both increase GM-CSF mRNA expression by NT2 cells, but only IL-1beta increases net GM-CSF protein accumulation.