Involvement of reactive oxygen species in TNF-alpha mediated activation ofthe transcription factor NF-kappa B in canine dermal fibroblasts

The cytokine tumor necrosis factor-alpha (TNF-alpha) plays a major role in inflammatory and immune-pathological reactions of the skin. With respect to a possible therapeutic modulation of TNF-alpha mediated activation of Nucl ear Factor-kappa B (NF-kappa B) in canine cutaneous inflammation, we invest igated the role of NF-kappa B and the involvement of reactive oxygen specie s (ROS) in the TNF-alpha signalling pathway in dermal fibroblasts of the do g. TNF-alpha treatment resulted in the activation of NF-kappa B as assessed by electrophoretic mobility shift assay (EMSA). Additionally, NF-kappa B t ranslocation was induced with butylhydroperoxide and antimycin A, but not w ith hydrogen peroxide. TNF-alpha stimulated NF-kappa B activation was parti ally inhibited by preincubation with the antioxidants alpha-lipoic acid and butylated hydroxyanisol (BHA). No superoxide generation following TNF-alph a stimulation could be detected in the supernatant of canine fibroblasts wi th the superoxide dismutase-inhibitable cytochrome c reduction test. In con trast, production of TNF-alpha dependent intracellular hydrogen peroxide, t he dismutation product of the superoxide radical, was demonstrated spectros copically by Formation of electron dense cerium-hydroperoxide precipitates. With electron energy loss spectroscopy (EELS) significant cerium deposits were detected in the mitochondria, the endoplasmatic reticulum, the cytosol and to a lesser extent on the plasma membrane of canine fibroblasts indica ting multiple hydrogen peroxide production sites. Peroxides, therefore, pos sibly play an important part in the redox-sensitive pathway of TNF-alpha de pendent NF-kappa B activation in canine skin. An adjunctive therapy with ap propriate antioxidants modulating NF-kappa B overactivation in cutaneous in flammation in the dog is promising. (C) 1999 Elsevier Science B.V. All righ ts reserved.