Role of Hydrogen Peroxide in Mycoplasma pneumoniae-induced Mast Cell IL-4 Production

RATIONALE: We have previously described activation of mast cells for cytokine production by Mycoplasma pneumoniae, which is known to produce large amounts of H2O2 during normal metabolism. H2O2 is now recognized to function as a signaling molecule, augmenting tyrosine phosphorylation by oxidizing an essential cysteine within the active site of protein tyrosine phosphatases. We hypothesized that adherent M. pneumoniae activates mast cells through H2O2 produced at the mast cell membrane contact point.METHODS: RBL 2H3 mast cells were co-cultured for 4-6 hours with M. pneumoniae strain M129 (bacteria to cell ratio 100:1) in 24 well flat bottom culture plates in RPMI 1640 with 10% fetal bovine serum without antibiotics with 10 mM HEPES (pH 7.0). Culture supernatants were filtered and stored at -80°C until assayed for IL-4 protein by ELISA.RESULTS: Augmentation of cellular antioxidant defenses with exogenous reduced glutathione (GSH) and depletion of extracellular H2O2 with the addition of catalase to the medium results in a dose-dependent inhibition of M. pneumoniae-induced mast cell IL-4 production of up to 50-60%. Further, addition of horseradish peroxidase (HRP), which utilizes H2O2 to oxidize a variety of cellular substrates, results in about 50% augmentation of mast cell IL-4 production in the presence of M. pneumoniae.CONCLUSION: Our previous studies together with the current results support a model in which the binding of the organism to the surface of eukaryotic cells produces cellular activation by activating sialated surface receptors including FcεRI while simultaneously augmenting the effect of receptor activation through production of H2O2 at the membrane-membrane junction. RATIONALE: We have previously described activation of mast cells for cytokine production by Mycoplasma pneumoniae, which is known to produce large amounts of H2O2 during normal metabolism. H2O2 is now recognized to function as a signaling molecule, augmenting tyrosine phosphorylation by oxidizing an essential cysteine within the active site of protein tyrosine phosphatases. We hypothesized that adherent M. pneumoniae activates mast cells through H2O2 produced at the mast cell membrane contact point. METHODS: RBL 2H3 mast cells were co-cultured for 4-6 hours with M. pneumoniae strain M129 (bacteria to cell ratio 100:1) in 24 well flat bottom culture plates in RPMI 1640 with 10% fetal bovine serum without antibiotics with 10 mM HEPES (pH 7.0). Culture supernatants were filtered and stored at -80°C until assayed for IL-4 protein by ELISA. RESULTS: Augmentation of cellular antioxidant defenses with exogenous reduced glutathione (GSH) and depletion of extracellular H2O2 with the addition of catalase to the medium results in a dose-dependent inhibition of M. pneumoniae-induced mast cell IL-4 production of up to 50-60%. Further, addition of horseradish peroxidase (HRP), which utilizes H2O2 to oxidize a variety of cellular substrates, results in about 50% augmentation of mast cell IL-4 production in the presence of M. pneumoniae. CONCLUSION: Our previous studies together with the current results support a model in which the binding of the organism to the surface of eukaryotic cells produces cellular activation by activating sialated surface receptors including FcεRI while simultaneously augmenting the effect of receptor activation through production of H2O2 at the membrane-membrane junction.