TNF-α dependent NF-κB activation in cultured canine keratinocytes is partly mediated by reactive oxygen species

The cytokine TNF-alpha plays a major role in inflammatory and immunological reactions of canine skin. With respect to a possible therapeutic modulation, we investigated the role of the transcription factor NF-kappaB and the involvement of reactive oxygen species (ROS) in the TNF-alpha signalling pathway in cultured canine keratinocytes. TNF-alpha treatment resulted in activation of NF-kappaB which was partially inhibited by the antioxidant alpha -lipoic acid. Using the cytochrome c reduction test no superoxide production could be detected in the supernatant of TNF-alpha stimulated keratinocytes. However, TNF-alpha dependent intracellular hydrogen peroxide production was demonstrated spectroscopically. With electron energy loss spectroscopy (EELS) significant hydrogen peroxide formation was detected in the mitochondria, the endoplasmic reticulum, the cytosol and partially on the plasma membrane of the keratinocytes. Hence, ROS possibly play an important role in the TNF-alpha signalling pathway leading to NF-kappaB activation in canine skin. An adjunctive therapy with natural potent antioxidants modulating NF-kappaB overactivation in canine cutaneous inflammation may be of therapeutic benefit.