Monocyte stimulation by reactive oxygen species: role of superoxide and intracellular Ca2+.
OBJECTIVE
Production of reactive oxygen species has generally been linked to inflammatory processes. Whether the presence of superoxide and hydrogen peroxide affects mononuclear cell function was investigated with an in vitro model using isolated human mononuclear cells.


METHODS
Human mononuclear cells were isolated from buffy coat. Toxicity was measured with Trypan blue exclusion, secreted TNF-alpha was measured with an ELISA, reactive oxygen species within mononuclear cells were quantified with dichlorodihydrofluorescein diacetate fluorescence, intracellular TNF-alpha was measured with flow cytometry and fluorescence microscopy.


RESULTS
The enzymatic production of superoxide caused a dose-dependent increase in TNF-alpha synthesis, whereas hydrogen peroxide was ineffective. Flow cytometric measurements and immunofluorescence microscopy demonstrated that monocytes were the main TNF-alpha producing population. This proinflammatory reaction was further characterized by pharmacologically investigating Ca2+ signalling pathways. Superoxide stimulated TNF-alpha secretion was inhibited by intracellular Ca2+-buffers (MAPT-AM and BAPT-AM) or VOC operating antagonists (diltiazem and verapamil) and only to a small extent by pharmacological inhibitors of ligand-gated pathways (TMB-8 and SKF 96368).


CONCLUSIONS
We propose that superoxide activates human mononuclear cells in a Ca2+-dependent manner.
Volk
T
T
Department of Anaesthesiology and Intensive Therapy, University Hospital Charité, Berlin, Germany. thomas.volk@charite.de
Gerst
J
J
Faust-Belbe
G
G
Stroehmann
A
A
Kox
W J
WJ
D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism; D010599:Pharmacokinetics; D017382:Reactive Oxygen Species / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism
D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D002118:Calcium