Silke Hebel

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 (Zn2+), but this still awaits experimental verification. Zn2+ 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-α. Here we demonstrate that these effects are due to a physiological signaling system involving intracellular Zn2+ signals. An increase of the intracellular zinc ion concentration occurs upon stimulation of human leukocytes with Escherichia coli, LPS, Pam3CSK4, TNF-α, 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-κB, and abrogates the release of proinflammatory cytokines, including TNF-α. This function of Zn2+ 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 Ca2+.

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+).

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.

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 (Hg2+). Only EtHg and Hg2+ 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 Hg2+ 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.