PMA-differentiated U937 Cells Research Articles

Supercritical carbon dioxide Cannabis sativa L. and Humulus lupulus extracts and their influence on human macrophages inflammatory state*

Herbal extracts are promising immunomodulating compounds. Their standardization may improve clinical outcome in various conditions related to inflammatory state. The aim of this study was to assess the utility of Cannabis sativa L. and Humulus lupulus extracts obtained by supercritical carbon dioxide (scCO2) in the reduction of pro-inflammatory cytokines release after LPS stimulation in the in vitro model. After scCO2 extraction, the cytotoxic potential of the obtained compounds was determined. The highest non-cytotoxic concentrations were selected for further inflammatory testing. PMA-differentiated U937 cells were used as an LPS induced model of the inflammation to assess the extracts potential to decrease the level of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. Either individually tested or in combination scCO2 extracts markedly reduced the level of released pro-inflammatory cytokines in comparison to LPS stimulated positive control. Our results show that the usage of standardized Cannabis sativa L. and Humulus lupulus extracts might be beneficial in reducing the inflammatory state. Application of the mixed extracts not only reduces the need for a high concentration of pure compounds, but also broadens the possible therapeutic effect. Moreover, scCO2 extraction may serve as the efficient method of obtaining functional anti-inflammatory extracts from either hop cones or cannabis.

1,25-Dihydroxyvitamin D3 induces human myeloid cell differentiation via the mTOR signaling pathway

1,25-Dihydroxyvitamin D3 or 1,25(OH)2D3 is known to play an important role in the differentiation of human myeloid cells. However, the molecular mechanism underlying the 1,25(OH)2D3-mediated differentiation of human myeloid cells is incompletely understood. Here, we report that 1,25(OH)2D3 induces differentiation of human myeloid cell lines such as U937 and THP-1 cells via the mammalian target of rapamycin (mTOR) signaling pathway. Both the expression of the differentiation marker CD14 and activation of the mTOR signaling pathway were induced by 1,25(OH)2D3 in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 and THP-1 cells. The 1,25(OH)2D3-induced expression of CD14 in PMA-differentiated U937 and THP-1 cells was prevented by mTOR inhibitors, PP242 and Torin1. The 1,25(OH)2D3-induced morphological changes as characteristics of differentiated myeloid cells were also reversed after PP242 and Torin1 treatment. Silencing of either regulatory-associated protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) in PMA-differentiated THP-1 cells with small-interfering RNA resulted in the inhibition of CD14 expression and morphological changes induced by 1,25(OH)2D3, indicating that both mTORC1 and mTORC2 were important for the differentiation of myeloid THP-1 cells. Previous studies have shown that phosphatidic acid (PA) maintains the stability of the mTOR complex. Here we found that the attenuation of PA production with 1-butanol or a PLD inhibitor prevented the 1,25(OH)2D3-induced upregulation of CD14. Taken together, our results show that 1,25(OH)2D3 enhances the differentiation of human myeloid cells through the mTOR signaling pathway.

Engulfment, persistence and fate of Bdellovibrio bacteriovorus predators inside human phagocytic cells informs their future therapeutic potential

In assessing the potential of predatory bacteria, such as Bdellovibrio bacteriovorus, to become live therapeutic agents against bacterial infections, it is crucial to understand and quantify Bdellovibrio host cell interactions at a molecular level. Here, we quantify the interactions of live B. bacteriovorus with human phagocytic cells, determining the uptake mechanisms, persistence, associated cytokine responses and intracellular trafficking of the non-growing B. bacteriovorus in PMA-differentiated U937 cells. B. bacteriovorus are engulfed by U937 cells and persist for 24 h without affecting host cell viability and can be observed microscopically and recovered and cultured post-uptake. The uptake of predators is passive and depends on the dynamics of the host cell cytoskeleton; the engulfed predators are eventually trafficked through the phagolysosomal pathway of degradation. We have also studied the prevalence of B. bacteriovorus specific antibodies in the general human population. Together, these results quantify a period of viable persistence and the ultimate fate of B. bacteriovorus inside phagocytic cells. They provide new knowledge on predator availability inside hosts, plus potential longevity and therefore potential efficacy as a treatment in humans and open up future fields of work testing if predators can prey on host-engulfed pathogenic bacteria.

Dietary Glycation Products Regulate Immune Homeostasis: Early Glycation Products Promote Prostate Cancer Cell Proliferation through Modulating Macrophages.

Well-controlled glycation (generally limited to the early stages) has been proposed as a strategy to improve the physiochemical properties of dietary proteins, but the functional studies of glycation products are mostly on advanced glycation end-products (AGEs) rather than early glycation products (EGPs). Since cytokines are important modulators of various biological processes, this study aims to determine whether EGPs and AGEs affected immune homeostasis differentially and do so through modulating macrophage-derived factors. Two systems (glycine-glucose and whey protein isolate (WPI)-glucose) are established to generate glycation products. They are applied to human macrophages (PMA-differentiated U937 cells), and cell viability and cytokine production are measured. Furthermore, EGPs, AGEs, and their conditioned medium (CM) from macrophages are applied to human prostate cancer (PCa) cells with different etiology (LNCaP and PC-3) and murine PCa cells (TRAMP-C2) to determine their direct and indirect effects on PCa cell proliferation. EGPs enhance the production of immunosuppressive cytokines, and this enhancement is associated with increased PCa cell proliferation. In contrast, AGEs inhibit macrophages to secret cytokines, but increase PCa cell proliferation directly. Our data suggest that EGPs promote the prostate tumor proliferation indirectly through modulating macrophages, while AGEs have a direct effect.

Effect of Bee Venom and Its Fractions on the Release of Pro-Inflammatory Cytokines in PMA-Differentiated U937 Cells Co-Stimulated with LPS.

The venom of Apis mellifera (honey bee) has been reported to play a role in immunotherapy, but existing evidence to support its immuno-modulatory claims is insufficient. Four fractions from whole bee venom (BV) were separated using medium pressure liquid chromatography. Their ability to induce the production of cytokines TNFα, IL-1β and IL-6 in phorbol-12-myristate-13-acetate (PMA)-treated U937 cells was assessed. The levels of the three cytokines produced by stimulation with the four fractions and crude BV without LPS were not significantly different from negative control values. However, co-stimulation of the cells with LPS and Fraction 4 (F-4) induced a 1.6-fold increase in TNF-α level (p < 0.05) compared to LPS alone. Likewise, LPS-induced IL-1β production was significantly synergised in the presence of F-1 (nine-fold), F-2 (six-fold), F-3 (four-fold) and F-4 (two-fold) fractions, but was only slightly enhanced with crude BV (1.5-fold) relative to LPS. Furthermore, the LPS-stimulated production of IL-6 was not significantly increased in cells co-treated with F-2 and F-3, but the organic fraction (F-4) showed an inhibitory effect (p < 0.05) on IL-6 production. The latter was elucidated by NMR spectroscopy and found to contain(Z)-9-eicosen-1-ol. The effects observed with the purified BV fractions were more marked than those obtained with the crude sample.

Regulation and trafficking of the HLA-E molecules during monocyte-macrophage differentiation.

HLA-E is a nonclassical HLA-class I molecule whose best known role is to protect from the natural killer cells. More recently, an additional function more similar to that of classical HLA-class I molecules, i.e., antigen presentation to T cells, is emerging. However, much remains to be explored about the intracellular trafficking of the HLA-E molecules. With the use of 3 different cellular contexts, 2 monocytic cell lines, U937 and THP1, and peripheral blood monocytes, we show here a remarkable increase of HLA-E during monocyte-macrophage differentiation. This goes independently from the classical HLA-class I, the main source of HLA-E-specific peptides, which is found strongly up-regulated upon differentiation of peripheral blood monocytes but not at all in the case of U937 and THP1 cell lines. Although in all cases, there was a moderate increase of HLA-E expressed in the cell surface, lysis by natural killer cells is comparably restored by an anti-NKG2A antibody in untreated as well as in PMA-differentiated U937 cells. Instead, the great majority of the HLA-E is retained in the vesicles of the autophagy-lysosome network, where they colocalize with the microtubule-associated protein light chain 3, as well as with the lysosomal-associated membrane protein 1. We conclude that differently from the classical HLA-class I molecules, the primary destination of the newly synthesized HLA-E molecules in macrophages is, rather than the cell membrane, the intracellular autophagy-lysosomal vesicles where they are stored and where they can encounter the exogenous antigens.

1α,25-Dihydroxyvitamin D3 upregulates HIF-1 and TREM-1 via mTOR signaling

Triggering receptor expressed on myeloid cells-1 (TREM-1) is induced by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in human monocytes/macrophages and epithelial cells. However, little information is available regarding the mechanism of 1,25(OH)2D3-induced TREM-1 expression in human monocytes/macrophages. In this study, 1,25(OH)2D3 was shown to strongly upregulate hypoxia-inducible transcription factor (HIF) in PMA-differentiated U937 cells. However, HIF was not mainly involved in 1,25(OH)2D3-induced TREM-1 expression. Instead, 1,25(OH)2D3-induced expression of TREM-1 was inhibited by rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway, indicating the involvement of mTOR. Induction of HIF proteins by 1,25(OH)2D3 was also inhibited by rapamycin. In addition, 1,25(OH)2D3 induced the phosphorylation of p70S6 kinase, a target of mTOR complex 1 (mTORC1). Our results suggest that 1,25(OH)2D3 induces the expression of TREM-1 through the mTOR signaling pathway in human macrophages.

Pseudomonas pyocyanin stimulates IL-8 expression through MAPK and NF-κB pathways in differentiated U937 cells

BackgroundPyocyanin (PCN), an extracellular product of Pseudomonas aeruginosa and a blue redox active secondary metabolite, plays an important role in invasive pulmonary infection. However, the detailed inflammatory response triggered by PCN infection in inflammatory cells (particularly macrophages), if present, remains to be clarified. To investigate the effects of PCN on macrophages, the ability of PCN to induce inflammation reaction and the signaling pathway for IL-8 release in PCN-induced differentiated U937 cells were examined.ResultsIt was found that PCN increased IL-8 release and mRNA expression in Phorbol 12-myristate 13-acetate (PMA) differentiated U937 cells in both a concentration- and time-dependent manner by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). P38 and ERK MAPKs were activated after 10 min of induction with PCN and their levels returned to baselines after 30 min by Western blotting. It was also found that within 10 min of PCN incubation, the level of p-I-κBα in the cytosol was increased, which returned to baseline level after 60 min. Meanwhile, the level of p-p65 was increased in the nuclear extract and cytosol, and maintained high in total cell lysates. The results were further confirmed by the observation that p38, ERK1/2 and NF-κB inhibitors inhibited PCN-induced NF-κB activation and attenuated PCN-induced IL-8 expression in U937 cells as a function of their concentrations. Moreover, it was shown that PCN induced oxidative stress in U937 cells and N-acetyl cysteine, an antioxidant, was able to inhibit PCN-induced IL-8 protein expression.ConclusionsIt is concluded that PCN induces IL-8 secretion and mRNA expression in PMA-differentiated U937 cells in a concentration- and time- dependent manner. Furthermore, p38 and ERK MAPKs and NF-κΒ signaling pathways may be involved in the expression of IL-8 in PCN-incubated PMA-differentiated U937 cells.

Pyocyanin from Pseudomonas induces IL-8 production through the PKC and NF-κB pathways in U937 cells

Pyocyanin, an extracellular product of Pseudomonas aeruginosa (P. aeruginosa), is important in invasive pulmonary infection. Pseudomonas infections are characterized by a marked influx of polymorphonuclear cells (neutrophils). An increased release of interleukin-8 (IL-8), a potent neutrophil chemoattractant, in response to pyocyanin may contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage observed in Pseudomonas-associated lung diseases. In order to explore the pathogenesis of pyocyanin on macrophages, we treated phorbol-12-myristate-13-acetate (PMA)-differentiated U937 cells with pyocyanin in vitro and explored the expression of IL-8 using reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). In the present study, we also investigated the activation of protein kinase C (PKC) and nuclear factor (NF)-κB in PMA-differentiated U937 cells by western blotting and immunohistochemical methods. It was found that pyocyanin increased IL-8 release and mRNA expression in differentiated U937 cells in a concentration- and time-dependent manner. Calphostin C (Cal C), a protein kinase C (PKC) inhibitor, and pyrrolidine dithiocarbamate (PDTC) , an NF-κB inhibitor, blocked IL-8 expression in a concentration-dependent manner in pyocyanin-induced U937 cells. We concluded that pyocyanin promotes IL-8 secretion and mRNA expression in a concentration- and time-dependent manner and furthermore, that the PKC and NF-κΒ signaling pathways may be involved in the expression of IL-8 in pyocyanin-infected PMA-differentiated U937 cells.

A Novel Macrolide Solithromycin Exerts Superior Anti-inflammatory Effect via NF-κB Inhibition

Macrolides are reported to reduce exacerbation of chronic inflammatory respiratory disease, such as chronic obstructive pulmonary disease (COPD), and also show anti-inflammatory effects in vitro and in vivo. However the anti-inflammatory efficacies of current macrolides are relatively weak. Here we found that a novel macrolide/fluoroketolide solithromycin (CEM-101) showed superior anti-inflammatory effects to macrolides in current clinical use. The effects of solithromycin (SOL) on lipopolysaccharide-induced TNFα (tumor necrosis factor α) and/or CXCL8 (C-X-C motif chemokine ligand 8; interleukin-8) release, phorbol 12-myristate 13-acetate-induced MMP9 (matrix metalloproteinase 9) activity and NF-κB (nuclear factor-κB) activity under conditions of oxidative stress have been evaluated and compared with the effects of erythromycin, clarithromycin, azithromycin, and telithromycin in macrophage-like PMA-differentiated U937 cells and peripheral blood mononuclear cells (PBMC) obtained from COPD patients. We also examined effect of SOL on cigarette smoke-induced airway inflammation in mice. SOL exerted superior inhibitory effects on TNFα/CXCL8 production and MMP9 activity in monocytic U937 cells. In addition, SOL suppressed TNFα release and MMP9 activity in PBMC from COPD patients at 10 µM, which is 10 times more potent than the other macrolides tested. Activated NF-κB by oxidative stress was completely reversed by SOL. SOL also inhibited cigarette smoke-induced neutrophilia and pro-MMP9 production in vivo, although erythromycin did not inhibit them. Thus, SOL showed better anti-inflammatory profiles compared with macrolides currently used in the clinic and may be a promising anti-inflammatory and antimicrobial macrolide for the treatment of COPD in future.

Upregulation of interleukin-1β production by 1,25-Dihydroxyvitamin D3 in activated human macrophages

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) plays important roles in the immune system. In contrast to its well known function in the adaptive immune system, much less is known about the immunoregulatory effects of 1,25(OH)(2)D(3) in the innate immune system, especially on activated human macrophages. Here we found that 1,25(OH)(2)D(3) strongly stimulated the production of interleukin-1β (IL-1β) in PMA-differentiated U937 cells and human monocyte-derived macrophages treated with lipopolysaccharide (LPS) or PMA. In this study, Erk1/2 appeared to mediate 1,25(OH)(2)D(3)-induced expression of IL-1β. Parallel to the increased production of IL-1β, 1,25(OH)(2)D(3) increased the expression and phosphorylation of the CCAAT enhancer-binding protein β (C/EBPβ), which is one of the key transcriptional regulatory factors for IL-1β transcription. These results suggest that 1,25(OH)(2)D(3) may function as a proinflammatory molecule in inflammatory macrophages.

Human monocyte differentiation stage affects response to arachidonic acid

AA-induced cell death mechanisms acting on human monocytes and monocyte-derived macrophages (MDM), U937 promonocytes and PMA-differentiated U937 cells were studied. Arachidonic acid induced apoptosis and necrosis in monocytes and U937 cells but only apoptosis in MDM and U937D cells. AA increased both types of death in Mycobacterium tuberculosis-infected cells and increased the percentage of TNFα+ cells and reduced IL-10+ cells. Experiments blocking these cytokines indicated that AA-mediated death was TNFα- and IL-10-independent. The differences in AA-mediated cell death could be explained by high ROS, calpain and sPLA-2 production and activity in monocytes. Blocking sPLA-2 in monocytes and treatment with antioxidants favored M. tuberculosis control whereas AA enhanced M. tuberculosis growth in MDM. Such evidence suggested that AA-modulated effector mechanisms depend on mononuclear phagocytes’ differentiation stage.

Montelukast inhibits tumour necrosis factor‐α‐mediated interleukin‐8 expression through inhibition of nuclear factor‐κB p65‐associated histone acetyltransferase activity

Montelukast is a potent cysteinyl leukotriene-1 receptor antagonist possessing some anti-inflammatory effects although the molecular mechanism of these anti-inflammatory effects is unknown. In this study, we aimed to investigate the effect of montelukast on nuclear factor (NF)-kappaB-associated histone acetylation activity in phorbol myristate acetate (PMA)-differentiated U937 cells. We examined the inhibitory effects of montelukast on TNF-alpha-induced IL-8 production in PMA-differentiated U-937 cells. U-937 cells were exposed to PMA (50 ng/mL) for 48 h to allow differentiation to macrophages. Macrophages were then exposed to TNF-alpha (10 ng/mL) in the presence or absence of montelukast (0.01-10 microm) for 24 h. After this time, the concentration of IL-8 in the culture supernatant was measured by sandwich-type ELISA kit. The effect of signalling pathways on TNF-alpha-induced IL-8 release was examined pharmacologically using selective NF-kappaB/IKK2 (AS602868, 3 microm), (PD98059, 10 microm) and p38 mitogen activated protein kinase (MAPK) (SB203580, 1 microm) inhibitors. NF-kappaB DNA binding activity was measured by a DNA-binding ELISA-based assay. NF-kappaB-p65-associated histone acetyltransferase (HAT) activity was measured by immunoprecipitation linked to commercial fluorescent HAT. TNF-alpha-induced IL-8 release was suppressed by an NF-kappaB inhibitor but not by MEK or p38 MAPK inhibitors. Montelukast induced a concentration-dependent inhibition of TNF-alpha-induced IL-8 release and mRNA expression that reached a plateau at 0.1 microm without affecting cell viability. Montelukast did not affect NF-kappaB p65 activation as measured by DNA binding but suppressed NF-kappaB p65-associated HAT activity. Montelukast inhibits TNF-alpha-stimulated IL-8 expression through changes in NF-kappaB p65-associated HAT activity. Drugs targeting these enzymes may enhance the anti-inflammatory actions of montelukast.

Regulation of 5-hydroxyeicosanoid dehydrogenase activity in monocytic cells

The 5-lipoxygenase product 5-oxo-ETE (5-oxo-eicosatetraenoic acid) is a highly potent granulocyte chemoattractant that is synthesized from 5-HETE (5-hydroxyeicosatetraenoic acid) by 5-HEDH (5-hydroxyeicosanoid dehydrogenase). In the present study, we found that 5-HEDH activity is induced in U937 monocytic cells by differentiation towards macrophages with PMA and in HL-60 myeloblastic cells by 1,25-dihydroxy-vitamin D3. We used PMA-differentiated U937 cells to investigate further the regulation of 5-HEDH. This enzyme exhibits approx. 10000-fold selectivity for NADP+ over NAD+ as a cofactor for the oxidation of 5-HETE, which is maximal at pH 10.2. In contrast, the reverse reaction (5-oxo-ETE-->5-HETE) is NADPH-dependent and is maximal at pH 6. Although the K(m) for the forward reaction (670 nM) is about twice that for the reverse reaction at neutral pH, the V(max) is approx 8-fold higher. The oxidation of 5-HETE to 5-oxo-ETE is supported by very low concentrations of NADP(+) (K(m) 139 nM), inhibited by NADPH (K(i) 224 nM) and is consistent with a ping-pong mechanism. The amount of 5-oxo-ETE synthesized by 5-HEDH depends on the ratio of NADP+ to NADPH. Exposure of U937 cells to oxidative stress (t-butyl hydroperoxide) increased the ratio of NADP+ to NADPH from approx. 0.08 in resting cells to approx. 3, and this was accompanied by a dramatic increase in 5-HETE oxidation to 5-oxo-ETE. We conclude that differentiation of monocytic cells towards macrophages results in enhanced 5-oxo-ETE synthesis and that the ability of cells to synthesize 5-oxo-ETE is tightly regulated by the ratio of intracellular NADP+ to NADPH.

Nitric Oxide Down-regulates Polo-like Kinase 1 through a Proximal Promoter Cell Cycle Gene Homology Region

Polo-like kinase 1 (PLK1) is an evolutionarily conserved serine/threonine kinase essential for cell mitosis. As a master cell cycle regulator, p21/Waf1 plays a critical role in cell cycle progression. Nitric oxide (NO.) has been shown to down-regulate PLK1 and up-regulate p21/Waf1 independent of cGMP. Here, the respective roles of p38 MAPK and p21/Waf1 in NO.-mediated PLK1 repression were investigated using differentiated U937 cells that lack soluble guanylate cyclase. NO. was shown to down-regulate both PLK1 mRNA and protein. Nuclear run-on assays and mRNA stability studies demonstrated that the effect of NO. on PLK1 expression was associated with decreased transcription without changes in transcript stability. SB202190, a p38 MAPK inhibitor, prevented transcriptional repression of PLK1 by NO.. Transfection with dominant-negative p38 MAPK mutant eliminated the NO. effect on both p21/Waf1 and PLK1 gene expression. Knockdown of p21/Waf1 with siRNA also substantially reduced the regulatory effect of NO. on PLK1. Reporter gene experiments showed that NO. decreased activity of the PLK1 proximal promoter, an effect that was blocked by p38 MAPK inhibitor. Deletion or mutation of the CDE/CHR promoter site, an element regulated by p21/Waf1, increased base-line promoter activity and abolished NO. repression of the PLK1 promoter. Likewise, electrophoretic mobility shift assays with CDE/CHR probe revealed a NO.-mediated change in protein-probe complex formation. Competition with various unlabeled CDE/CHR mutant sequences showed that NO. increased nuclear protein binding to intact CHR. These results demonstrate that a NO.-p38 MAPK-p21/Waf1 signal transduction pathway represses PLK1 through a canonical CDE/CHR promoter element.

The Effects of Indirubin-3′-Monoxime, A Novel AHR Ligand, on Stress and Toxicity-Related Gene/Protein Expression in Human U937 Cells Undergoing Differentiation and Activation

Indirubin-3-monoxime is an aryl hydrocarbon receptor (AhR)-binding, idole-derived compound known to be a potent inducer of CYP1A1, at least in hepatic cell lines. To date, very little is known about the effects of indirubin-3-monoxime on cells relevant to the immune system. We treated human U937 histiocytic lymphoma cells with indirubin-3′-monoxime in four different regimens, representing various stages of PMA-induced differentiation and further activation with LPS. Gene expression profiles were monitored using commercial macroarrays for 96 select stress and toxicity genes. The four treatment regimens were (A) the monocyte model: U937 cells were treated with 1 μM indirubin-3′-monoxime or carrier control for 24 hrs; (B) the differentiated model: PMA-differentiated U937 cells were treated with 1 μM indirubin-3′-monoxime or carrier control for 24 hrs; (C) the differentiation process model: U937 cells were treated for 24 hours with 1 μM indirubin-3′-monoxime or carrier control, followed by PMA-induced differentiation over a 24-hr period; and (D) the LPS-activated model: PMA-differentiated and LPS-activated U937 cells were treated with 1 μM indirubin-3′-monoxime or carrier control for 24 hrs. Indirubin-3′-monoxime treatment caused a 12.7-, 2.1-, 3.2-, and 4.5-fold increases in CYP1A1 in regimes A, B, C, and D, respectively. Indirubin-3′-monoxime treatment also enhanced cyclo-oxygenase 2 expression in PMA-differentiated cells, but reduced the expression of macrophage inflammatory protein, catalase, heme oxygenase-1, glutathione peroxidase, and manganese superoxide dismutase; a scenario consistent with oxidative stress. Macroarray validity was confirmed by RT-PCR using CYP1A1, COX-2, MIP-β, and GADPH as target genes, and at the protein level for CYP1A1, COX-2, and β-actin. To our knowledge, this is the first study to examine the effects of indirubin-3-monoxime in a human cell line relevant to the immune system. Overall, our study suggests that indirubin-3′-monoxime is either a classical AhR ligand, or that it modulates endogenous AhR-regulated activity.

Monocytes stimulate COX-2 expression in pancreatic cancer cells

COX-2 is an enzyme that modulates human pancreatic cancer (PaCa) cell survival, proliferation, and tumor metastasis. 60–80% of human PaCa express COX-2. However, the regulation of COX-2 expression in PaCa cells is not clear. We hypothesize that inflammatory cell infiltrate—namely monocytes/macrophages found commonly in PaCa—regulates the expression and activity of COX-2. The human histiocytic lymphoma cell line U937 was terminally differentiated with phorbol ester (PMA) and served as a human monocyte/macrophage model. IL-1β levels in the culture medium were measured by ELISA. PaCa cell lines (BxPC-3, Capan-2, HPAF-II) were stimulated with U937 conditioned culture medium (CM). COX-2 protein expression was determined by immunoblotting and prostaglandin E2 (PGE2) production by ELISA. To determine, whether the effect of U937 CM was mediated by IL-1β, we used an IL-1 receptor antagonist (IL-1RA). The effect of recombinant IL-1β on COX-2 expression, PGE2 production, and ERK1/ERK2 phosphorylation in human PaCa cells was analyzed. Since PMA-differentiated U937 cells secreted low levels of IL-1β (15 ± 2 pg/ml), we stimulated differentiated U937 cells with LPS markedly increasing IL-1β secretion (1670 ± 145 pg/ml). CM of U937 cells increased COX-2 protein expression after 24 hours 5.6-fold, 7.7-fold, and 5.3-fold in the BxPC-3, Capan-2, and HPAF-II cells, respectively. Compared to control, U937 CM increased PGE2 production in BxPC-3 (202 ± 8 vs. 516 ± 24 pg/ml, p < 0.01), Capan-2 (19 ± 1 vs. 39 ± 1 pg/ml, p < 0.01), and HPAF-II cells (173 ± 10 vs. 429±33 pg/ml, p<0.01). The effect of U937 CM on COX-2 expression and PGE2 production was only partially inhibited by IL-1RA. Recombinant IL-1β dose- and time dependently increased COX-2 expression, PGE2 production, and ERK1/ERK2 activation in BxPC-3, Capan-2, and HPAF-II cells, an effect which was inhibited by IL-1RA. IL-1β secreted by monocytes stimulates expression of COX-2 in human pancreatic cancer cells. Other cytokines secreted from U937 cells may also activate COX-2 expression. Studies combining immunomodulation with COX-2 inhibition for PaCa treatment may yield significant results.

Estrogen regulates cytokine production and apoptosis in PMA-differentiated, macrophage-like U937 cells.

We have investigated the effects of sex steroids, estradiol (E2), and testosterone (T) on the synthesis of tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) in phorbol-myristate-acetate (PMA)-differentiated human monoblastic U937 cells. The ability of both hormones to modulate the viability and programmed cell death of macrophage-like PMA-differentiated U937 cells was also inspected. E2 increased TNF-alpha synthesis, whereas T had no effect on the production of this cytokine. The combination of E2 and its antagonist tamoxifen or ICI-182,789 completely abolished the induction of TNF-alpha, while combination of T and its antagonist Casodex (CSDX) did not significantly affect TNF-alpha production by U937 cells. Exposure of cells to E2 resulted in a dose-dependent decrease of IL-10 synthesis, while again T did not show any detectable effect. In addition, E2 induced a significant increase of apoptosis in macrophage-like U937 cells and this increase was inhibited by the simultaneous addition of either tamoxifen or ICI-182. In contrast, T alone or in combination with CSDX did not modify apoptotic rates of U937 cells. This evidence, taken together, suggests that estrogens, but not androgens, exert a pro-inflammatory action through the modulation of TNF-alpha and IL-10, and regulate the immune effector cells by the induction of programmed cell death.

Non Fc receptor-mediated infection of human macrophages by dengue virus serotype 2.

Four human monocyte-derived macrophage membrane proteins, with apparent molecular masses of 27, 45, 67 and 87 kDa, were identified as possible receptors for dengue virus serotype 2 (DEN-2) (Mexican isolate 200787/1983), based on affinity chromatography, immunofluorescence, virus overlay protein-binding assays and Western blotting. Additionally, mouse polyclonal antibodies raised against each of the four proteins were capable of partially inhibiting in vitro DEN-2 infection of monocyte-macrophages, thus supporting the notion of a role for such proteins as DEN-2 receptors. Parallel studies were carried out using the human promonocytic U-937 cell line, both as undifferentiated cells and as monocyte-like phorbol myristate acetate (PMA)-differentiated cells, as target cells. Whereas interaction between DEN-2 and undifferentiated U-937 cells was almost negligible, PMA-differentiated U-937 cells were shown to harbour putative receptors (with molecular masses of 45 and 67 kDa) for DEN-2, similar to those found in human monocyte-derived macrophages. To our knowledge, this is the first report that describes putative receptors for DEN-2 in primary cultures of human macrophages.

Signaling by eNOS through a superoxide-dependent p42/44 mitogen-activated protein kinase pathway

Expression of endothelial nitric oxide synthase (eNOS) in transfected U-937 cells upregulates phorbol 12-myristate 13-acetate (PMA)-induced tumor necrosis factor-alpha (TNF-alpha) production through a superoxide (O(2)(-))-dependent mechanism. Because mitogen-activated protein kinases (MAPK) have been shown to participate in both reactive oxygen species signaling and TNF-alpha regulation, their possible role in eNOS-derived O(2)(-) signal transduction was examined. A redox-cycling agent, phenazine methosulfate, was found to both upregulate TNF-alpha (5.8 +/- 1.0 fold; P = 0.01) and increase the phosphorylation state of p42/44 MAPK (3.1 +/- 0.2 fold; P = 0.01) in PMA-differentiated U-937 cells. Although S-nitroso-N-acetylpenicillamine, a nitric oxide (NO) donor, also increased TNF-alpha production, NO exposure led to phosphorylation of p38 MAPK, not p42/44 MAPK. Upregulation of TNF-alpha production by eNOS transfection was associated with increases in activated p42/44 MAPK (P = 0.001), whereas levels of phosphorylated p38 MAPK were unaffected. Furthermore, cotransfection with Cu/Zn superoxide dismutase, which blocks TNF-alpha upregulation by eNOS, also abolished the effects on p42/44 MAPK. Expression of Gln(361)eNOS, a mutant that produces O(2)(-) but not NO, still resulted in p42/44 MAPK phosphorylation. In contrast, two NADPH binding site deletion mutants of eNOS that lack oxidase activity had no effect on p42/44 MAPK. Finally, PD-98059, a p42/44 MAPK pathway inhibitor, blocked TNF-alpha upregulation by eNOS (P = 0.02). Thus O(2)(-) produced by eNOS increases TNF-alpha production via a mechanism that involves p42/44 MAPK activation.