Zinc chelation decreases IFN-ß-induced STAT1 upregulation and iNOS expression in RAW 264.7 macrophages.

One consequence of lipopolysaccharide (LPS)-induced stimulation of macrophages is the release of Interferon (IFN)-ß, and subsequently the activation of the JAK-STAT1 pathway, resulting in the expression of inducible nitric oxide synthase (iNOS). Free intracellular zinc ions (Zn2+) have a profound impact as a second messenger in LPS-dependent gene expression. Previous work had indicated a Zn2+-dependent upregulation of STAT1 mRNA in response to LPS and IFN-ß, potentially affecting STAT1-dependent downstream signaling upon pre-incubation with these agents. The aim of the present study was to investigate the long-term influence of Zn2+ chelation on cellular STAT1 levels and their effect on protein levels and activity of iNOS. The LPS- and IFN-ß-mediated increase of STAT1 mRNA and protein levels was abrogated by chelation of Zn2+ with the membrane permeable chelator N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) in RAW 264.7 macrophages. After 48h pre-incubation together with IFN-ß, TPEN also led to reduced nitric monoxide formation in response to a second stimulation with LPS. Nonetheless, the latter was observed regardless of any pre-incubation with IFN-ß, suggesting that the effect of treatment with TPEN negatively affects iNOS induction independently from cellular STAT1 levels. In conclusion, long term Zn2+ chelation does affect STAT1 protein expression, but interferes with NO production by a different, yet unknown pathway not involving STAT1. However, as there are many additional STAT1-dependent genes, there might still be effects on targets other than iNOS.

Ethylmercury and Hg2+ induce the formation of neutrophil extracellular traps (NETs) by human neutrophil granulocytes.

Humans are exposed to different mercurial compounds from various sources, most frequently from dental fillings, preservatives in vaccines, or consumption of fish. Among other toxic effects, these substances interact with the immune system. In high doses, mercurials are immunosuppressive. However, lower doses of some mercurials stimulate the immune system, inducing different forms of autoimmunity, autoantibodies, and glomerulonephritis in rodents. Furthermore, some studies suggest a connection between mercury exposure and the occurrence of autoantibodies against nuclear components and granulocyte cytoplasmic proteins in humans. Still, the underlying mechanisms need to be clarified. The present study investigates the formation of neutrophil extracellular traps (NETs) in response to thimerosal and its metabolites ethyl mercury (EtHg), thiosalicylic acid, and mercuric ions (Hg(2+)). Only EtHg and Hg(2+) triggered NETosis. It was independent of PKC, ERK1/2, p38, and zinc signals and not affected by the NADPH oxidase inhibitor DPI. Instead, EtHg and Hg(2+) triggered NADPH oxidase-independent production of ROS, which are likely to be involved in mercurial-induced NET formation. This finding might help understanding the autoimmune potential of mercurial compounds. Some diseases, to which a connection with mercurials has been shown, such as Wegener's granulomatosis and systemic lupus erythematosus, are characterized by high prevalence of autoantibodies against neutrophil-specific auto-antigens. Externalization in the form of NETs may be a source for exposure to these self-antigens. In genetically susceptible individuals, this could be one step in the series of events leading to autoimmunity.

PTEN-inhibition by zinc ions augments interleukin-2-mediated Akt phosphorylation.

Free zinc ions (Zn(2+)) participate in several signaling pathways. The aim of the present study was to investigate a potential involvement of Zn(2+) in the PI3K/Akt pathway of interleukin (IL)-2 signaling in T-cells. The IL-2 receptor triggers three major pathways, ERK1/2, JAK/STAT5, and PI3K/Akt. We have previously shown that an IL-2-mediated release of lysosomal Zn(2+) into the cytoplasm activates ERK1/2, but not STAT5. In the present study, Akt phosphorylation in response to IL-2 was abrogated by the Zn(2+) chelator N,N,N',N'-tetrakis-2(pyridyl-methyl)ethylenediamine, and was induced by treatment with Zn(2+) and the ionophore pyrithione. The latter were ineffective in cells that were treated with siRNA against the phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a phosphatase that degrades the lipid second messenger PI(3,4,5)P3, which is produced by PI3K and leads to activation of Akt. Inhibition of recombinant PTEN by Zn(2+)in vitro yielded an IC50 of 0.59 nM. Considering a resting free cytoplasmic Zn(2+) level of 0.2 nM in the T-cell line CTLL-2, this seems ideally suited for dynamic regulation by cellular Zn(2+). Oxidation with H2O2 and supplementation with Zn(2+) led to similar changes in the CD spectrum of PTEN. Moreover, Zn(2+) partially prevented the oxidation of cysteines 71 and 124. Hence, we hypothesize that zinc signals affect the IL-2-dependent PI3K/Akt pathway by inhibiting the negative regulator PTEN through binding with a sub-nanomolar affinity to cysteine residues that are essential for its catalytic activity.

Application of Zinpyr-1 for the investigation of zinc signals in Escherichia coli.

Changes of the pico- to nanomolar concentration of free intracellular Zn(2+) are part of the signal transduction in mammalian cells. These zinc signals regulate the enzymatic activity of target proteins such as protein tyrosine phosphatases. For Escherichia coli, previous studies have reported diverging concentrations from femto- to picomolar, raising the question if Zn(2+) could also have a function in bacterial signaling. This manuscript explores the use of the low molecular weight fluorescent probe Zinpyr-1 in E. coli. The probe detects free Zn(2+) in these bacteria. Comparable to mammalian cells, other metal ions, especially Hg(2+) and Cd(2+), interfere with the detection of Zn(2+). Moreover, experiments in E. coli were particularly prone to artifacts based on cellular autofluorescence, necessitating corrections that are not required in mammalian cells. Based on measurements in lysates of E. coli and the mammalian cell line Jurkat, similar values between 0.1 and 0.2 nM free Zn(2+) were found. For E. coli, this corresponds to less than one free zinc ion per cell. Moreover, phosphatase inhibition by Zn(2+) was only observed in Jurkat, but not E. coli. This excludes a function for zinc signals as a regulator of bacterial phosphatases. Still, changes in the free Zn(2+) concentration were observed in response to elevated extracellular Zn(2+) and pH, or to addition of the detergent NP-40, suggesting that other processes could be controlled by the free intracellular Zn(2+) concentration.

Mercuric ions inhibit mitogen-activated protein kinase dephosphorylation by inducing reactive oxygen species.

Mercury intoxication profoundly affects the immune system, in particular, signal transduction of immune cells. However, the mechanism of the interaction of mercury with cellular signaling pathways, such as mitogen activated protein kinases (MAPK), remains elusive. Therefore, the objective of this study is to investigate three potential ways in which Hg(2+) ions could inhibit MAPK dephosphorylation in the human T-cell line Jurkat: (1) by direct binding to phosphatases; (2) by releasing cellular zinc (Zn(2+)); and (3) by inducing reactive oxygen species (ROS). Hg(2+) causes production of ROS, measured by dihydrorhodamine 123, and triggers ROS-mediated Zn(2+) release, detected with FluoZin-3. Yet, phosphatase-inhibition is not mediated by binding of Zn(2+) or Hg(2+). Rather, phosphatases are inactivated by at least two forms of thiol oxidation; initial inhibition is reversible with reducing agents such as Tris(2-carboxyethyl)phosphine. Prolonged inhibition leads to non-reversible phosphatase oxidation, presumably oxidizing the cysteine thiol to sulfinic- or sulfonic acid. Notably, phosphatases are a particularly sensitive target for Hg(2+)-induced oxidation, because phosphatase activity is inhibited at concentrations of Hg(2+) that have only minor impact on over all thiol oxidation. This phosphatase inhibition results in augmented, ROS-dependent MAPK phosphorylation. MAPK are important regulators of T-cell function, and MAPK-activation by inhibition of phosphatases seems to be one of the molecular mechanisms by which mercury affects the immune system.

Cadmium ions promote monocytic differentiation of human leukemia HL-60 cells treated with 1α,25-dihydroxyvitamin D3.

Cadmium exposure has multiple effects on the immune system. These can be stimulating, leading to improved clearance of infections, or inhibiting, increasing susceptibility toward infectious agents. One in vivo observation in cadmium-exposed individuals is increased monocyte numbers. Therefore, the objective of this study is to investigate the impact of cadmium on monocyte differentiation in the HL-60 model cell line. Administered alone, cadmium had no effect. However, cadmium amplified the expression of monocyte surface markers CD11b and CD14 when differentiation was induced by 1α,25-dihydroxyvitamin D3 (VD3). Furthermore, differentiation with VD3 in the presence of cadmium augmented key monocyte functions: the capacities to perform phagocytosis and generate an oxidative burst. One important signaling pathway required for monocyte differentiation involves extracellular signal-regulated kinase (ERK)1/2. Notably, cadmium induced ERK1/2 phosphorylation in HL-60 cells. Furthermore, U0126, which inhibits ERK1/2 phosphorylation by upstream MAPK/ERK kinases (MEK)1/2, reduced VD3-mediated differentiation and abrogated the effects of cadmium. In conclusion, cadmium can augment monocytic differentiation by activating ERK1/2 signaling, leading to increased generation of functional monocytes. These increased monocyte numbers could contribute to the impact of cadmium on the immune system owing to their role in the production of pro-inflammatory cytokines and activation of T-cells by antigen presentation.

Cadmium ions induce monocytic production of tumor necrosis factor-alpha by inhibiting mitogen activated protein kinase dephosphorylation.

Cadmium ions (Cd(2+)) are carcinogenic and have cytotoxic effects in a variety of organisms. In addition to its direct cytotoxicity, Cd(2+) acts as an immunomodulator at sub-toxic concentrations. Among other influences Cd(2+) can induce inflammation, but the molecular basis for this effect is not well investigated. In this manuscript, we analyze the impact of Cd(2+) on monocytes/macrophages, which are potent producers of pro-inflammatory cytokines, finding that Cd(2+) treatment induced tumor necrosis factor (TNF)-alpha secretion. Based on the observation that another group IIb metal, zinc (Zn(2+)), has a physiological role in these events, we investigated if Cd(2+) acts on the same molecular targets. Like Zn(2+), Cd(2+) inhibits phosphatases, and hereby dephosphorylation of mitogen activated protein kinases (MAPK). Consequently, treatment of cells with Cd(2+) resulted in stimulation of ERK 1/2 and p38 MAPK phosphorylation. Furthermore, Cd(2+)-induced release of TNF-alpha from primary human monocytes was blocked by inhibitors for ERK 1/2 (U0126) and p38 MAPK (SB202190), demonstrating that MAPKs are involved in the induction of TNF-alpha by Cd(2+).

Zinc ions cause the thimerosal-induced signal of fluorescent calcium probes in lymphocytes.

Most fluorescent probes for the investigation of calcium signaling also detect zinc ions. Consequently, changes in the intracellular zinc concentration could be mistaken for calcium signals. Thimerosal (TMS) is used as a calcium-mobilizing agent and we analyzed the contribution of zinc ions to the signal observed with fluorescent calcium probes after TMS stimulation. Our findings show that the fluorescent signal in lymphocytes is entirely due to zinc release. Experiments in the T lymphocyte cell line Jurkat and primary human lymphocytes show that TMS and its active metabolite, ethyl mercury, cause an increase in signal intensity with probes designed for the detection of either calcium or zinc ions. The TMS/ethyl mercury-induced signal of the calcium probes Fluo-4 and FURA-2 was completely absent when the zinc chelator TPEN [N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine] was added. In contrast, the signal caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was unaffected by TPEN. In light of these observations, zinc may also contribute to calcium signals caused by mercury-containing compounds other than TMS, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered.

Effects of long-term zinc supplementation and deprivation on gene expression in human THP-1 mononuclear cells.

Zinc is an essential trace element that is critical for cellular function and structural integrity. It has an important regulatory role in the immune system, in particular in monocytes. To identify the diverse cellular targets and mechanisms of action of zinc in this cell type, we used microarray technology to assess the effects of zinc supplementation and depletion on global gene expression. mRNA expression in the human monocytic cell line THP-1 was analyzed and compared in response to 40h supplementation with 50micromol/L zinc, or zinc deprivation by 2.5micromol/L of the membrane-permeant zinc chelator TPEN [N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine]. Analysis of microarrays consisting of approximately 19,000 unique oligonucleotides identified over 1400 genes, or approximately 7%, as zinc-sensitive. Notably, this yielded several sets of structurally or functionally related genes. Among those groups, which were mainly affected by zinc deprivation, were histones, S100 calcium and zinc binding proteins, and chemokines and their receptors. These groups of genes may mediate zinc-effects on chromatin regulation, zinc homeostasis, and chemotaxis, respectively. In addition, functional networks were analyzed, showing that the well known effect of zinc on pro-inflammatory cytokines is not limited to these genes; it acts on a number of functionally connected genes, as well. These results provide novel molecular targets and pathways that may aid in explaining the role of zinc in monocyte function.

Zinc signals are essential for lipopolysaccharide-induced signal transduction in monocytes.

Cytosolic alterations of calcium ion concentrations are an integral part of signal transduction. Similar functions have been hypothesized for other metal ions, in particular zinc (Zn(2+)), but this still awaits experimental verification. Zn(2+) is important for multiple cellular functions, especially in the immune system. Among other effects, it influences formation and secretion of pro-inflammatory cytokines, including TNF-alpha. Here we demonstrate that these effects are due to a physiological signaling system involving intracellular Zn(2+) signals. An increase of the intracellular zinc ion concentration occurs upon stimulation of human leukocytes with Escherichia coli, LPS, Pam(3)CSK(4), TNF-alpha, or insulin, predominantly in monocytes. Chelating this zinc signal with the membrane permeable zinc-specific chelator TPEN (N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine) completely blocks activation of LPS-induced signaling pathways involving p38 MAPK, ERK1/2, and NF-kappaB, and abrogates the release of proinflammatory cytokines, including TNF-alpha. This function of Zn(2+) is not limited to monocytes or even the immune system, but seems to be another generalized signaling system based on intracellular fluctuations of metal ion concentrations, acting parallel to Ca(2+).

Zinc-dependent suppression of TNF-alpha production is mediated by protein kinase A-induced inhibition of Raf-1, I kappa B kinase beta, and NF-kappa B.

Excessive and permanent cytokine production in response to bacterial LPS causes cell and tissue damage, and hence organ failure during sepsis. We have previously demonstrated that zinc treatment prevents LPS-induced TNF-alpha expression and production in human monocytes by inhibiting cyclic nucleotide phosphodiesterase (PDE) activity and expression, and subsequent elevation of the cyclic nucleotide cGMP. In the present study, we investigated the molecular mechanism by which cGMP signaling affects the LPS-induced signaling cascade to suppress TNF-alpha transcription and release from monocytes. Zinc-mediated cGMP elevation led to cross activation of protein kinase A. This zinc-induced protein kinase A activation inhibited Raf-1 activity by phosphorylation at serine 259, preventing activation of Raf-1 by phosphorylation of serine 338. By this mechanism, zinc suppressed LPS-induced activation of IkappaB kinase beta (IKKbeta) and NF-kappaB, and subsequent TNF-alpha production. Our study shows that PDE inhibition by zinc modulates the monocytic immune response by selectively intervening in the Raf-1/IKKbeta/NF-kappaB pathway, which may constitute a common mechanism for the anti-inflammatory action of PDE inhibitors.

Differential gene expression after zinc supplementation and deprivation in human leukocyte subsets.

An individual's zinc status has a significant impact on the immune system, and zinc deficiency, as well as supplementation, modulates immune function. To investigate the effects of zinc on different leukocyte subsets, we used microarray technology to analyze and compare the changes in mRNA expression in cell culture models of monocytes (THP-1), T cells (Jurkat), and B cells (Raji), in response to supplementation for 40 h with 50 microM zinc or 2.5 microM of the membrane-permeant zinc chelator TPEN [N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine], respectively. In each cell type, several hundred genes were identified to be zinc sensitive, but only a total of seven genes were commonly regulated in all three cell lines. The majority of those genes were involved in zinc homeostasis, and none in immune function. Nevertheless, further analysis revealed that zinc affects entire functional networks of genes that are related to proinflammatory cytokines and cellular survival. Although the zinc-regulated activities are similar throughout the gene networks, the specific genes that are affected vary significantly between different cell types, a situation that helps to elucidate the disparity of the effects that zinc has on different leukocyte populations.

Flow cytometric measurement of labile zinc in peripheral blood mononuclear cells.

Labile (i.e., free or loosely bound) zinc has the potential to modulate cellular function. Therefore, a flow cytometric assay for the measurement of labile zinc was developed to facilitate the investigation of the physiological roles of zinc. The zinc-sensitive fluorescent probe FluoZin-3 was used to quantify the amount of labile zinc in peripheral blood mononuclear cells isolated from human blood. Maximal fluorescence and autofluorescence of the probe were measured after the addition of zinc in the presence of the ionophore pyrithione, or the membrane-permeant chelator N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine, respectively. In this way, the intracellular concentrations of labile zinc in resting cells were estimated to be 0.17 nM in monocytes and 0.35 nM in lymphocytes. The method was successfully employed to monitor phorbol 12-myristate 13-acetate-induced zinc release, which occurred in monocytes but not lymphocytes, and the displacement of protein-bound zinc by the mercury-containing compounds HgCl(2) and thimerosal. Costaining with dyes that emit at higher wavelengths than FluoZin-3 allows multiparameter measurements. Two combinations with other dyes are shown: loading with propidium iodide to measure cellular viability and labeling with antibodies against the surface antigen CD4. This method allows measurement of the concentration of biologically active labile zinc in distinct cell populations.