Involvement of reactive oxygen species in TNF-α mediated activation of the transcription factor NF-κB in canine dermal fibroblasts

The cytokine tumor necrosis factor-alpha (TNF-α) plays a major role in inflammatory and immune-pathological reactions of the skin. With respect to a possible therapeutic modulation of TNF-α mediated activation of Nuclear Factor-kappa B (NF-κB) in canine cutaneous inflammation, we investigated the role of NF-κB and the involvement of reactive oxygen species (ROS) in the TNF-α signalling pathway in dermal fibroblasts of the dog. TNF-α treatment resulted in the activation of NF-κB as assessed by electrophoretic mobility shift assay (EMSA). Additionally, NF-κB translocation was induced with butylhydroperoxide and antimycin A, but not with hydrogen peroxide. TNF-α stimulated NF-κB activation was partially inhibited by preincubation with the antioxidants α-lipoic acid and butylated hydroxyanisol (BHA). No superoxide generation following TNF-α stimulation could be detected in the supernatant of canine fibroblasts with the superoxide dismutase-inhibitable cytochrome c reduction test. In contrast, production of TNF-α dependent intracellular hydrogen peroxide, the dismutation product of the superoxide radical, was demonstrated spectroscopically 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 indicating multiple hydrogen peroxide production sites. Peroxides, therefore, possibly play an important part in the redox-sensitive pathway of TNF-α dependent NF-κB activation in canine skin. An adjunctive therapy with appropriate antioxidants modulating NF-κB overactivation in cutaneous inflammation in the dog is promising.