Characterization of calcineurin in human neutrophils - Inhibitory effect of hydrogen peroxide on its enzyme activity and on NF-kappa B DNA binding

We describe here a specific calcineurin activity in neutrophil lysates, whi ch is dependent on Ca2+, inhibited by trifluoroperazine, and insensitive to okadaic acid. Immunoblotting experiments using a specific antiserum recogn ized both the A and B chains of calcineurin, Neutrophils treated with cyclo sporin A or FK 506 showed a dose-dependent inhibition of calcineurin activi ty. The effect of oxidant compounds on calcineurin activity was also invest igated. Neutrophils treated with hydrogen peroxide (H2O2), where catalase w as inhibited with aminotriazole, exhibited a specific inhibition of calcine urin activity. However, the addition of reducing agents to neutrophil extra cts partially reversed the inhibition caused by H2O2. A similar inhibitory effect of H2O2 on calcineurin activity was observed to occur in isolated ly mphocytes, This is the first demonstration that redox agents modulate calci neurin activity in a cellular system. In addition, electrophoretic mobility shift assays revealed that lipopolysaccharide-induced activation of NF-kap pa B in human neutrophils is inhibited by cell pretreatment with H2O2 in a dose-dependent manner. These data indicate that calcineurin activity regula tes the functional activity of lipopolysaccharide-induced NF-kappa B/Rel pr oteins in human neutrophils, These data indicate a role of peroxides in the modulation of calcineurin activity and that the H2O2-dependent NF-kappa B inactivation in neutrophils occurs in concert with inhibition of calcineuri n.