Thiol antioxidants block the activation of antigen-presenting cells by contact sensitizers

Strong contact sensitizers are able to induce signal transduction mechanisms such as tyrosine phosphory-lation and activation of MAP kinases in antigen-presenting cells. We studied the capacity of different antioxidants (ascorbic acid, alpha-tocopherol, pyrrolidine dithiocarbamate, N-acetylcysteine, and glutathione) to block the increase in tyrosine phosphorylation in human monocytes seen after stimulation with strong contact sensitizers. Human peripheral blood mononuclear cells were stimulated with 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone in the presence or absence of these antioxidants. The total amount of membrane-associated phosphotyrosine in CD14(+) cells was quantified using flow cytometric techniques. Complete inhibition of tyrosine phosphorylation was noticed when cells were stimulated in the presence of N-acetylcysteine or glutathione. Using N-acetylcysteine as inhibitor similar results were obtained for cells stimulated with formaldehyde, thimerosal methyldibromoglutaronitrile, diphenylcyclopropenone, p-phenylenediamine, toluene-2,5-diamine, and 2,4-dinitrofluorobenzene. By use of a trinitrophenyl-specific monoclonal antibody it was shown that N-acetylcysteine as well as cysteine prevents the binding of 2,4,6-trinitrochlorobenzene to proteins in monocytes and monocyte-derived mature dendritic cells. Furthermore, the capacity of N-acetylcysteine to block the activation of p38 and ERK1/2 MAP kinases by 2,4,6-trinitrochlorobenzene was demonstrated. The radical scavengers ascorbic acid and alpha-tocopherol as well as the nuclear factor kappaB inhibitor pyrrolidine dithiocarbamate failed to prevent the increase in tyrosine phosphorylation. Our data present evidence that reactive oxygen species as well as transcription factor nuclear factor kappaB seem to be unimportant for the induction of tyrosine phosphorylation by contact sensitizers. On the other hand, protection of thiol groups using compounds with free sulfhydryl groups is very effective to block this process. This finding may have implications for prevention of occupational sensitization to strong contact allergens.