U937 and THP-1 cells do not release LTB4, LTC4, or LTD4 in response to A23187

U937 and THP-1 cells possess some characteristics of human mononuclear phagocytes, cells which synthesize and release LTB4, LTC4, and LTD4. Incubation of these cells with recombinant human interferon-gamma (IFN-gamma) or Phorbol Myristate Acetate (PMA) induces a more differentiated cell state. We hypothesized that U937 and THP-1 cells would release LTB4, LTC4, and LTD4 in response to stimulation with the non-physiologic agonist, calcium ionophore A23187 and that preincubation with IFN-gamma or PMA might alter leukotriene release by these cells. We cultured both cell lines for 48 hours in the presence and absence of IFN-gamma (1000 units/ml) and for 120 hours in the presence and absence of PMA (160 nM) and then challenged them with A23187 (5uM) for 30 minutes at 37 degrees C. The supernatants were deproteinated and assayed by RIA for LTB4 and LTC4 and by RP-HPLC for LTB4, LTC4, and LTD4. Neither U937 nor THP-1 cells released quantities of leukotrienes detectable by RIA, less than 0.3ng/5 X 10(6) cells. Peripheral blood mononuclear phagocytes from normal volunteers, cultured and challenged in vitro at under identical conditions, released 11.3 +/- 2.9 ng LTB4 and 2.0 +/- 1.5 ng LTC4/10(6) viable monocytes. The lack of leukotriene production by U937 and THP-1 cells was not altered by preincubation for 48 hours with IFN-gamma (n = 3) nor by preincubation with PMA for 120 hours (n = 3). We conclude 1) U937 and THP-1 cells do not appear to be appropriate in vitro models for the examination of leukotriene release from normal mononuclear phagocytes. 2) Pre-incubation of U937 and THP-1 cells with IFN-gamma or PMA under the conditions tested, does not induce the ability of these cell lines to release leukotrienes.     

The vitamin D receptor-mediated activation of phosphatidylinositol 3-kinase (PI3Kalpha) plays a role in the 1alpha,25-dihydroxyvitamin D3-stimulated increase in steroid sulphatase activity in myeloid leukaemic cell lines

In this article we show that 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) stimulates the activity of the class IA phosphatidylinositol 3-kinase PI3Kalpha and its downstream target Akt in HL60, U937 and THP-1 myeloid leukaemic cell lines. Furthermore, we show that the classical nuclear vitamin D receptor (VDR(nuc)) is involved in this activation of the PI3K/Akt signalling in these cell lines. We have previously shown that the activity of steroid sulphatase is stimulated in HL60, U937 and THP-1 myeloid leukaemic cell lines by 1alpha,25(OH)(2)D(3) (Hughes et al., [2001] Biochem J 355:361-371; Hughes et al., [2005] J Cell Biochem 94:1175-1189; Hughes and Brown [2006] J Cell Biochem 98:590-617). In this article we show that the 1alpha,25(OH)(2)D(3)-stimulated increase in signalling via the PI3K/Akt pathway plays a role in the increase in steroid sulphatase activity in the HL60 U937 and THP-1 cell lines. We used a variety of pharmacological and biochemical approaches to show that activation of PI3Kalpha mediates the 1alpha,25(OH)(2)D(3)-stimulated increase in steroid sulphatase activity in myeloid leukaemic cells. We also show that the PI3K/Akt dependent activation of NF-kappaB plays a role in the 1alpha,25(OH)(2)D(3)-stimulated increase in steroid sulphatase activity in myeloid leukaemic cells.     

IFN-gamma and transforming growth factor-beta 1 differently regulate fibronectin and laminin receptors of human differentiating monocytic cells.

The regulation of extracellular matrix (ECM) protein receptor expression was followed in the human promonocytic cell line U937 before and after stimulation either with PMA or various cytokines implicated in monocytopoiesis. On undifferentiated U937 cells, alpha-chains of very late Ag (VLA)-4, VLA-5, and VLA-6 were constitutively expressed whereas alpha-chains of VLA-2 (alpha 2) and vitronectin receptor (alpha V) were not. Maturation of U937 cells with PMA resulted in a marked decrease in alpha 4 expression (25% of control by day 5), and a small but significant increase in the expression of alpha 2 and alpha v over 4 days of stimulation. Unstimulated U937 cells attached to fibronectin (FN) but not to laminin (LM), collagens I/IV-coated surfaces. After PMA stimulation, U937 cells exhibited enhanced adherence on FN and expressed the ability to adhere to LM. PMA stimulation also promoted U937 spreading both on FN and LM. Adhesion on FN all along the maturation pathway was specifically and totally inhibited by anti-alpha 5 mAb but not by anti-alpha 4 mAb. Anti-beta 1, anti-alpha 6, anti-alpha 2, and anti-alpha v mAb, as well as Tyr-Ile-Gly-Ser-Arg and Arg-Gly-Asp synthetic peptides from LM, had no effect on adhesion of PMA-stimulated cells on LM, implying that U937 cell adherence to LM is mediated through hitherto distinct receptors. In the presence of rIFN-gamma, differentiating U937 cells did not adhere to LM and lost the capacity to bind to FN. Loss of adhesion to FN was correlated with the concomitant decrease in the expression of alpha 4 and alpha 5 integrin subunits. In contrast, TGF-beta 1 mimicked most of the effects of PMA by enhancing the attachment of maturating U937 cells on FN through alpha 5 receptors and by promoting adherence to LM. TGF-beta 1 stimulation also promoted U937 cell spreading on both FN- and LM-coated surfaces. The data suggest that inflammatory cytokines such as IFN-gamma and TGF-beta 1 may be critically important in the homing of monocytic cells at sites of inflammation by modulating cell-surface expression of ECM receptors.     

1,25-Dihydroxyvitamin D3 suppresses the expression of the VCAM-1 receptor, VLA-4 in human leukemic HL-60 cells

Very late antigen-4 (VLA-4) is the complex with alpha4 and beta1 integrins, which is the receptors to fibronectin and VCAM-1. We evaluate the effect of 1,25(OH)2D3 on the expression of VLA-4 in human leukemic HL-60, U937 cells and human melanoma A375 cells. Flow cytometric analysis demonstrate that the expression of alpha4 integrin is negatively regulated in the cell lines we studied. The expression of beta1 integrin is also decreased in HL-60 and U937 cells. The mRNA expression of alpha4 integrin is significantly decreased by the treatment with 1,25(OH)2D3, whereas 1,25(OH)2D3 does not alter the expression of beta1 mRNA. The adhesion assay demonstrate that the number of adherent cells treated with 1, 25(OH)2D3 is significantly lower than that untreated on VCAM-1-coated wells. Because VCAM-1 is highly expressed in the endothelial cells, it is possible that 1,25(OH)2D3 prevents the attachment of the cells from the endothelial cells in vivo.     

And THP-1 cells do not release LTB4, LTC4, or LTD4 in response to A23187

U937 and THP-1 cells possess some characteristics of human mononuclear phagocytes, cells which synthesize and release LTB₄, LTC₄, and LTD₄. Incubation of these cells with recombinant human interferongamma (IFN-gamma) or Phorbol Myristate Acetate (PMA) induces a more differentiated cell state. We hypothesized that U937 and THP-1 cells would release LTB₄, LTC₄, and LTD₄ in response to stimulation with the non-physiologic agonist, calcium ionophore A23187 and that preincubation with IFN-gamma or PMA might alter leukotriene release by thes cells. We cultured both cell lines for 48 hours in the presence and absence of IFN-gamma (10000 units/ml)n and for 120 hours in the presence and absence of PMA (160 nM) and then challenged them with A23187 (5uM) for 30 minutes at 37°C. The supernatants were deproteinated and assayed by RIA for LTB₄ and LTC₄ and by RP-HPLC for LTB₄, LTC₄, and LTD₄. Neither U937 nor THP-1 cells released quantities of leukotrienes detectable by RIA, <0.3ng/5 × 10⁶ cells. Peripheral blood mononuclear phagocytes from normal volumteers, cultured and challenged in vitro at under identical conditions, released 11.3 ± 2.9 ng LTB₄ and 2.0 ± 1.5 ng LTC₄/10⁶ viable monocytes. The lack of leukotriene production by U937 and THP-1 cells was not altered by preincubation for 48 hours with IFN-gamma (n=3) nor by preincubation with PMA for 120 hours (n=3). We conclude 1) U937 and THP-1 cells do not appear to be appropriate in vitro models for the examination of leukotriene release from normal mononuclear phagocytes. 2) Pre-incubation of U937 and THP-1 cells with IFN-gamma or PMA under the conditions tested, does not induce the ability of these cell lines to release leukotrienes.     

Stimulation of arachidonic acid metabolism in primary cultures of osteoblast-like cells by hormones and drugs

The effects of parathyroid hormone (PTH), dihydroxycholecalciferol (1,25-(OH)2 D3), thrombin, epidermal growth factor (EGF) and 12-o-tetradecanoylphorbol-13-acetate (PMA) on the biosynthesis and release of arachidonic acid metabolites were studied in primary cultures of osteoblast-like cells isolated from 18-day-old chick embryo calvaria. Cells were labelled with (14C)-arachidonic acid for 30 h. The radioactive eicosanoids were extracted from the cell culture media after a further 30 h stimulation period and analysed on a PRP-1 column by HPLC. The radioactive products were characterized by co-elution of (3H) standard prostanoids. Osteoblasts showed a basal release of the prostanoids 6-keto-PGF1 alpha, TXB2, PGF2 alpha, PGE2, PGD2 and PGB2, the latter being the most abundant one. Indomethacin (10(-5) M) effectively inhibited the basal release, but not that of an as yet unidentified compound. The release of prostanoids was stimulated by PTH (2 U/ml), thrombin (0.4 NIH/ml), EGF (50 ng/ml) and PMA (25 ng/ml), the latter being by far the most potent one. 1,25-(OH)2D3 was found to slightly inhibit the prostanoid release. These results indicate: (1) primary cultures of osteoblasts synthesize several prostaglandins, thromboxane B2 and one unidentified product. (2) the action on bone of PTH and the various drugs tested may be, at least partly, mediated by an increased prostaglandin production by osteoblasts. Clearly this does not apply to 1,25-(OH)2D3.     

Differential regulation of interleukin-6, macrophage inflammatory protein-1, and JE/MCP-1 cytokine expression in macrophage cell lines.

Activated macrophages produce a number of proinflammatory cytokines including IL-6, JE, MIP-1 alpha and MIP-1 beta. The induction requirements for production of either IL-6 or the MIP-1 related inflammatory proteins (MIP-1 alpha, MIP-1 beta, and JE) have been analyzed independently using fibroblasts, monocytes, or endothelial cells. However, little is known about the regulation of these cytokines in macrophages. Since activated macrophages produce prostaglandins (PGE2) which may participate in the autoregulation of cytokine production by stimulation of adenylate cyclase and the induction of cAMP-dependent signal pathways, we determined the effects of PGE on the production of IL-6 and MIP-1-related proteins. Murine macrophage cell lines were incubated with PGE1, PGE2, cholera toxin, or dibutyryl cAMP in the presence of absence suboptimal doses of LPS. Pharmacologic agents alone did not induce IL-6 production but incubation of macrophages with combinations of adenylate cyclase stimulators and LPS or dcAMP and LPS led to the dose-dependent enhancement of IL-6 secretion and mRNA expression. In contrast, PGE1 inhibits LPS-induced JE, MIP-1 alpha, and MIP-1 beta mRNA expression and this inhibition is partially dependent on a cAMP-mediated pathway of signal transduction. In previous work we demonstrated that IFN-gamma and PMA do not stimulate the production of IL-6 by macrophages. Here we show that incubation of macrophages with either IFN-gamma or PMA induces the expression of JE, MIP-1 alpha and MIP-1 beta mRNA expression. JE mRNA expression is much more responsive to the stimulatory effects of IFN-gamma than are the MIP-1 genes. Finally, PGE inhibits PMA and IFN-gamma-induced JE and MIP-1-related mRNA expression.     

Novel vitamin D3 derivatives, 26-homo-delta 22-dehydro-1 alpha,25(S)-dihydroxyvitamin D3 and 26-homo-delta 22-dehydro-1 alpha,25(R)-dihydroxyvitamin D3: preferential activity in c-myc mRNA production and in induction of phenotypic differentiation of HL-60 cells

Novel vitamin D3 derivatives, 26-homo-delta 22-1 alpha,25(S)-dihydroxyvitamin D3 and 26-homo-delta 22-1 alpha,25(R)-dihydroxyvitamin D3 were compared with the native hormone, 1,25-dihydroxyvitamin D3, and with other vitamin D3 derivatives, in inhibition of cell growth, induction of phenotypic differentiation, and c-myc mRNA reduction of HL-60 cells. The degree of inhibition in cell growth caused by 26-homo-delta 22-1 alpha,25(S)-(OH)2D3 was the greatest followed by 26-homo-delta 22-1 alpha,25(R)-(OH)2D3. The ability to reduce NBT was parallel to that to inhibit cellular proliferation. 26-homo-delta 22-1 alpha,25(S)-(OH)2D3, 26-homo-delta 22-1 alpha,25(R)-(OH)2D3, 24-homo-24-F2-1 alpha,25-(OH)2D3, and 1 alpha,24(R)-(OH)2-26-Cl-D3 were more active than 1 alpha,25-(OH)2D3 in the induction of OK-M1+ and OK-Mo-2+ HL-60 cells. Using two color flow cytometric analysis, the percentages of OK-M5(+)- and OK-DR(+)-HL-60 cells were 33% in the treatment with 26-homo-delta 22-1 alpha,25(S)-(OH)2D3 plus interferon-gamma (IFN-gamma) but 14% in the treatment with 1 alpha,25-(OH)2D3 plus IFN-gamma. 26-Homo-delta 22-1 alpha,25(S)-(OH)2D3 has an inhibitory effect on c-myc reduction in treated HL-60 cells. These results suggest that the novel vitamin D3 derivatives, 26-homo-delta 22-1 alpha,25(S)-(OH)2D3 and 26-homo-delta 22-1 alpha,25(R)-(OH)2D3, have preferential activity in inducing phenotypic differentiation and in inducing cell proliferation related c-myc mRNA activity.     

Nuclear receptors are involved in the enhanced IL-6 production by melatonin in U937 cells

This report shows that melatonin enhances IL-6 production by U937 cells via a nuclear receptor-mediated mechanism. Resting U937 cells only express membrane (mt1) melatonin receptors. In these cells, melatonin did not modify basal production of IL-6 or when activated by PMA plus lipopolysaccharide, a treatment that downregulates the expression of mt1 receptor. However, in U937 cells activated with IFN-gamma, which induces the expression of the ROR alpha 1 and ROR alpha 2 nuclear receptors and represses the expression of the mt1 receptor, melatonin can activate IL-6 production. These results show that the expression of nuclear melatonin receptor but not membrane receptors is sufficient for melatonin to activate cytokine production in human lymphocytic and monocytic cell lines.     

Membrane-associated interleukin 1 activity on human U937 tumor cells: stimulation of PGE2 production by human chondrosarcoma cells

Membrane-associated interleukin 1 (IL 1) activity was induced on the human macrophage tumor cell line, U937, by pretreatment with phorbol myristic acid (PMA). Incubation of PMA-treated, paraformaldehyde-fixed U937 cells with the murine cell line D10.G4.1 in the presence of concanavalin A caused an increase in DNA synthesis as measured by the uptake of tritiated thymidine. Paraformaldehyde-fixed U937, not pretreated with PMA, showed little or no activity. A rabbit polyclonal antibody directed against human IL 1 neutralized all membrane-associated IL 1-like activity, as measured by the inhibition of D10.G4.1 cell proliferation. PMA-treated U937 caused a pronounced enhancement of PGE2 production from a human chondrosarcoma cell line, SW-1353. Membrane-associated IL 1 induced a more potent PGE2 response than did a maximal concentration of soluble IL 1. Rabbit antihuman IL 1 neutralized membrane-bound IL 1 induction of PGE2. The data presented here raise the possibility that membrane-bound IL 1 may play a primary role in the pathophysiology of the inflammatory disease process.     

Synthesis of active metabolite(s) from 1 alpha-hydroxyvitamin D3 by human monocytic leukemia cells.

Synthesis of the biologically active metabolite(s) from 1 alpha-hydroxyvitamin D3 (1 alpha(OH)D3) was examined in various types of human leukemia cell lines. Untreated monocytoid leukemia cells (U937 and HEL/S) metabolized 1 alpha (OH)D3 to the active metabolite(s), possibly 1 alpha, 24- and/or 1 alpha, 25-dihydroxyvitamin D3, and these cells were efficiently induced to differentiate by treatment with 1 alpha (OH)D3. However, the other types of leukemia cells did not efficiently metabolize it and were not induced to differentiate by 1 alpha (OH)D3. The possible therapeutic advantage of 1 alpha (OH)D3 in the treatment of monocytic leukemia is discussed.     

Phagocytic cells metabolize 25-hydroxyvitamin D3 to 10-oxo-19-nor-25-hydroxyvitamin D3 and a new metabolite, 8 alpha,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one

The metabolism of 25-hydroxyvitamin D3 [25(OH)D3] was examined in several phagocytic cells including alveolar macrophages and myeloid leukemia cells (M1, HL-60 and U937). Phagocytic cells converted 25(OH)D3 to 10-oxo-19-nor-25-hydroxyvitamin D3 and a new metabolite. The former metabolite was dominant in shorter incubation periods (1 h), whereas the latter dominated over longer incubation periods (24 h). The new metabolite was produced from 25(OH)D3 directly but not through 10-oxo-19-nor-25-hydroxyvitamin D3. The new metabolite was unequivocally identified as 8 alpha,25-dihydroxy-9-10-seco-4,6,10(19)-cholestatrien-3-one. These results suggest that phagocytic cells somehow promote oxidation of the triene part of vitamin D compounds.     

Regulation of the expression of leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) on a human eosinophilic leukemia cell line EoL-3.

The effects of several cytokines and phorbol myristate acetate (PMA) on LFA-1 and ICAM-1 expression on a human eosinophilic leukemia cell line, EoL-3, were investigated and compared with those of a human monocytic leukemia cell line, U937. EoL-3 cells expressed large amounts of LFA-1 and small amounts of ICAM-1, and their expression was regulated similarly in EoL-3 cells and U937 cells. Interferon-gamma (IFN-gamma) enhanced ICAM-1 expression but not LFA-1 expression, and PMA augmented both LFA-1 and ICAM-1 expression. IFN-gamma and PMA showed an additive effect on ICAM-1 expression. These results collectively suggest that expression of LFA-1 and ICAM-1 is regulated differently and that IFN-gamma and PMA regulate the expression through different mechanisms. PMA but not IFN-gamma induced homotypic adhesion of EoL-3 and U937 cells, suggesting that PMA but not IFN-gamma activated the adhesive function of these cells. Staurosporin, an inhibitor of protein kinases (PKs), partly suppressed IFN-gamma- and PMA-augmented expression of ICAM-1 on EoL-3 and U937 cells, but did not affect PMA-augmented LFA-1 expression, suggesting that staurosporin-sensitive PKs are involved in IFN-gamma- and PMA-augmented ICAM-1 expression but not in PMA-augmented LFA-1 expression. The role of protein kinase C (PK-C) in these mechanisms was not revealed because a PK-C inhibitor, H-7, did not show any definitive effect on IFN-gamma- and PMA-induced expression of LFA-1 and ICAM-1. Moreover, cyclic AMP (cAMP)- and cGMP-dependent pathways were not shown to be involved in the augmentation of the expression of these molecules.     

LPS-stimulated release of prostaglandin E and thromboxane B2 from the U937 cell line

Human monocytes are known to metabolize arachidonic acid (AA) and to release prostaglandins upon stimulation. Previous data indicate that in vitro maturation and differentiation of monocytes result in alteration of this property with greatly diminished response to stimulators of release of prostaglandin E (PGE) and thromboxane B2 (TxB2) occurring after cells have been cultured. To further study the effects of differentiation on human monocyte AA metabolism, a model system was established based upon the human histiocytic cell line U937. Among tested stimulants, which included opsonized zymosan, complement fragment C3b, phorbol myristate acetate (PMA), calcium ionophore A23187, and concanavalin A, it was found that Escherichia coli lipopolysaccharide (LPS) was unique in that it stimulated increased release of TxB2 from U937 cells. The effect of the phorbol ester PMA, a compound commonly used to induce differentiation of U937, on the ability of U937 to respond to LPS was examined. Following 48 hr of treatment with PMA, U937 became capable of releasing both PGE and TxB2 in response to small doses of LPS. As previously observed for human monocytes, the release of PGE was delayed for several hours following stimulation and failed to reach maximal cumulative levels in culture until 24-48 hr following stimulation. In contrast to human monocytes, PMA-induced U937 were capable of maintaining their responsiveness to LPS for several days. Thus, the U937 cell line provides a useful model for study of the effects of differentiation of human mononuclear phagocytes on their ability to metabolize AA, and for the effects of LPS on histiocytic tumor cell prostaglandin release.     

Establishment of murine macrophage-like mutant and hybrid cell lines: comparative analysis of the differentiation induced by 1 alpha,25-dihydroxyvitamin D3 and recombinant murine interferon-gamma

The purpose of this study is to establish the monocyte/macrophage-like cell lines which are sensitive to potent systemic and local factors, 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2 VD3) and interferon-gamma (IFN-gamma). We established two variant mouse macrophage-like cell lines, whose responses to 1 alpha,25(OH)2 VD3 and IFN-gamma differed from one another. The AH-sensitive mutant cell line (G3) was induced by allowing P388D1 tolerant to 8-azaguanine. G3 mutant cells were then fused with the 1 alpha,25(OH)2 VD3-stimulated bone marrow cells isolated from DBA/2 mice. After AH selection the hybrid cell line (XC) was established. The G3 mutant cell line and the XC hybrid cell line had macrophage-like characteristics, such as surface antigens, Fc receptor, C3 receptor, and lysosomal enzymes. The treatment of G3 mutant cells with 1 alpha,25(OH)2 VD3 inhibited cell proliferation with morphological changes, and increased acid phosphatase activity, phagocytic activity, and F4/80 antigen expression on the cell surface. In contrast, IFN-gamma inhibited cell proliferation without effect on acid phosphatase activity and phagocytic activity but increased F4/80 antigen expression. In XC hybrid cells, on the other hand, IFN-gamma, but not 1 alpha,25(OH)2 VD3, inhibited cell proliferation with morphological changes but increased phagocytic activity and F4/80 antigen expression. In addition, IFN-gamma, but not 1 alpha,25(OH)2 VD3, dose-dependently increased multinucleated cell formation of both cells. These findings suggest that the G3 mutant cell line with macrophage-like characteristics is 1 alpha,25(OH)2 VD3- and IFN-gamma-sensitive, and that the XC hybrid cell line is, despite its macrophage-like characteristics, only IFN-gamma-sensitive. Therefore, these newly established cell lines will provide useful systems in studying the differentiation of monocyte/macrophage lineage.     

Biological activity of 24,24-difluoro-1 alpha, 25-dihydroxyvitamin D3 and 1 alpha, 25-dihydroxyvitamin D3-26,23-lactone in inducing differentiation of human myeloid leukemia cells

Vitamin D compounds added to the culture medium induce differentiation of human myeloid leukemia cells (HL-60 cells) by binding to a specific cytosol receptor protein. This system provides a biologically relevant and technically simple assay to examine the relationship between molecular structure and biological activity of vitamin D compounds. Using this culture system, the biological activity of 24,24-F2-1 alpha,25(OH)2D3 and 1 alpha,25(OH)2D3-26,23-lactone was assayed. 24,24-F2-1 alpha,25(OH)2D3 was four to seven times more potent than 1 alpha,25(OH)2D3 in inducing phagocytosis and C3 rosette formation of HL-60 cells, though both compounds bound equally well to the cytosol receptor, suggesting that the defuorination at the 24-carbon position may stimulate membrane permeability of the compound. 1 alpha,25(OH)2D3-26,23-lactone, on the other hand, was only 1/200th as active as 1 alpha,25(OH)2D3. The binding affinity of the lactone for the cytosol receptor was identical with that of 1 alpha (OH)D3, suggesting that the lactone formation between the 26 and 23 positions masks the function of the 25-hydroxyl group. The binding affinity of vitamin D3 derivatives to the specific cytosol receptor of HL-60 cells was well correlated with that of intestinal cytosol protein specifically bound to 1 alpha,25(OH)2D3.     

Time dependent production of cytokines and eicosanoids by human monocytic leukaemia U937 cells; effects of glucocorticosteroids

In the present study the human monoblast cell line U937 has been used as a model to study the function of human mononuclear phagocytes in asthma. The kinetics of the production of eicosanoids and cytokines, which are thought to play a role in the pathogenesis of asthma, were studied. In addition, the effects of glucocorticosteroids were investigated, as these drugs are of great importance for the treatment of asthmatic patients. After stimulation with phorbol-12 myristate acetate (PMA) for 24 h, U937 cells were cultured in the absence or presence of lipopolysaccharide (LPS: 1 and 5 microg ml(-1)) and glucocorticosteroids (budesonide, fluticasone propionate and prednisolone: 10(-11), 10(-9) and 10(-7) M) for 96 h. The production of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) gradually increased in time after stimulation with LPS, whereas the transient production of tumor necrosis factor alpha (TNF-alpha) reached its maximum between 6 and 12 h. Interferon-gamma (IFN-gamma), interleukin-10 (IL-10) and leukotriene B4 (LTB4) were not detectable. All three glucocorticosteroids (budesonide, fluticasone propionate and prednisolone) completely inhibited the production of both eicosanoids and cytokines. The production of eicosanoids was more sensitive to these glucocorticoids than the production of cytokines. The observed differences in the kinetics of the production of eicosanoids and cytokines stress the importance of time course experiments in studies on the effect of drugs on mononuclear cells.     

Involvement of prostaglandin E2 in the inhibition of osteocalcin synthesis by human osteoblast-like cells in response to cytokines and systemic hormones

The stimulation of the production of osteocalcin by human osteoblast-like cells in response to 1,25(OH)2D3 is antagonized by several agents that induce the synthesis of prostaglandin E2 (PGE2) including interleukin 1 (IL-1), tumour necrosis factor (TNF) and parathyroid hormone (PTH). The mechanism whereby these agents inhibit the synthesis of osteocalcin is not known. In this report we show that exogenous PGE2 inhibits this stimulatory action of 1,25(OH)2D3 on human osteoblast-like cells in a dose-dependent manner, suggesting that PGE2 may contribute to the inhibition of osteocalcin synthesis in response to these agents. Assessment of the inhibitory role of endogenous PGE2 synthesis in the action of rhIL-1 alpha, rhIL-1 beta and rhTNF alpha on the production of osteocalcin demonstrated that the inhibition by these agents could be partially overcome by the addition of indomethacin, an inhibitor of PGE2 synthesis. In contrast, the inhibitory action observed with bPTH (1-84) was unaffected by indomethacin. These observations indicate that endogenous PGE2 synthesis mediates, in part, some of the inhibitory actions of the cytokines on the induction of osteocalcin synthesis in response to 1,25(OH)2D3, but not of PTH. Since the antagonism of the synthesis of osteocalcin by rhIL-1 alpha, rhIL-1 beta and rhTNF alpha was not completely abolished following the inhibition of PGE2 synthesis this would indicate that additional PGE2-independent mechanisms also account for the action of these cytokines on osteocalcin production. The nature of these mechanisms is currently not known.