Zinc signals, i.e. changes in the free intracellular Zn(2+)concentration, are an integral component of signal transduction in several immune cells. The aim of the present study was to investigate if this is also the case in neutrophil granulocytes. One neutrophil function is NETosis, the release of a matrix composed of DNA, chromatin and granule proteins to capture extracellular bacteria within so-called neutrophil extracellular traps (NET). NETosis can be induced by the protein kinase C (PKC) activator 12-myristate 13-acetate (PMA). PMA treatment led to a zinc signal in neutrophil granulocytes. NETosis was inhibited when the zinc signal was sequestered by the membrane permeable high affinity chelator N,N,N',N',-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN). PKC-mediated NET formation depends on the production of reactive oxygen species (ROS) by NADPH oxidase. Inhibition of NADPH oxidase with diphenyleneiodonium chloride blocked ROS formation and NETosis, as well as the zinc signal. TPEN, however, had no effect on PKC activity and ROS production, indicating that Zn(2+) is not required for activation of PKC, but for signals downstream of ROS production. In conclusion, zinc signals are an essential component of the ROS-dependent signal transduction leading to NETosis.

Neutrophil granulocytes are the largest leukocyte population in the blood and major players in the innate immune response. Impaired neutrophil function has been reported in in vivo studies with zinc-deficient human subjects and experimental animals. Moreover, in vitro formation of neutrophil extracellular traps has been shown to depend on free intracellular Zn(2+). This study investigates the requirement of Zn(2+) for several other essential neutrophil functions, such as chemotaxis, phagocytosis, cytokine production, and degranulation. To exclude artifacts resulting from indirect effects of zinc deprivation, such as impaired hematopoietic development and influences of other immune cells, direct effects of zinc deprivation were tested in vitro using cells isolated from healthy human donors. Chelation of Zn(2+) by the membrane permeable chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) reduced granulocyte migration toward N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF) and IL-8, indicating a role of free intracellular Zn(2+) in chemotaxis. However, a direct action of Zn(2+) as a chemoattractant, as previously reported by others, was not observed. Similar to chemotaxis, phagocytosis, oxidative burst, and granule release were also impaired in TPEN-treated granulocytes. Moreover, Zn(2+) contributes to the regulatory role of neutrophil granulocytes in the inflammatory response by affecting the cytokine production by these cells. TPEN inhibited the lipopolysaccharide-induced secretion of chemotactic IL-8 and also anti-inflammatory IL-1ra. In conclusion, free intracellular Zn(2+) plays essential roles in multiple neutrophil functions, affecting extravasation to the site of the infection, uptake and killing of microorganisms, and inflammation.

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