Involvement of reactive oxygen species in TNF-alpha mediated activation of the transcription factor NF-kappaB 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 Nuclear Factor-kappa B (NF-kappaB) in canine cutaneous inflammation, we investigated the role of NF-kappaB and the involvement of reactive oxygen species (ROS) in the TNF-alpha signalling pathway in dermal fibroblasts of the dog. TNF-alpha treatment resulted in the activation of NF-kappaB as assessed by electrophoretic mobility shift assay (EMSA). Additionally, NF-kappaB translocation was induced with butylhydroperoxide and antimycin A, but not with hydrogen peroxide. TNF-alpha stimulated NF-kappaB activation was partially inhibited by preincubation with the antioxidants alpha-lipoic acid and butylated hydroxyanisol (BHA). No superoxide generation following TNF-alpha stimulation could be detected in the supernatant of canine fibroblasts with the superoxide dismutase-inhibitable cytochrome c reduction test. In contrast, production of TNF-alpha 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-alpha dependent NF-kappaB activation in canine skin. An adjunctive therapy with appropriate antioxidants modulating NF-kappaB overactivation in cutaneous inflammation in the dog is promising.
Köhler
H B
HB
Klinik für kleine Haustiere, Tierärztliche Hochschule Hannover, Germany. hkoehler@stud.tiho-hannover.de
Knop
J
J
Martin
M
M
de Bruin
A
A
Huchzermeyer
B
B
Lehmann
H
H
Kietzmann
M
M
Meier
B
B
Nolte
I
I
D000818:Animals; D002563:Cerium / Q000737:chemistry; D003574:Cytochrome c Group / Q000032:analysis; D015232:Dinoprostone / Q000096:biosynthesis; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D005347:Fibroblasts / Q000276:immunology*; D006861:Hydrogen Peroxide / Q000032:analysis; D008854:Microscopy, Electron / Q000662:veterinary; D016328:NF-kappa B / Q000276:immunology*; D017382:Reactive Oxygen Species / Q000276:immunology*; D011994:Recombinant Proteins; D015398:Signal Transduction / Q000276:immunology; D012867:Skin / Q000276:immunology*; D013482:Superoxide Dismutase / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
D003574:Cytochrome c Group; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D002563:Cerium; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D015232:Dinoprostone