TUMOR NECROSIS FACTOR ALPHA AND INTERFERON GAMMA COOPERATIVELY INDUCE OXIDATIVE STRESS AND MOTONEURON DEATH IN RAT SPINAL CORD EMBRYONIC EXPLANTS

The accumulation of reactive microglia in the degenerating areas of amyotrophic lateral sclerosis (ALS) tissue is a key cellular event creating a chronic inflammatory environment that results in motoneuron death. We have developed a new culture system that consists in rat spinal cord embryonic explants in which motoneurons migrate outside the explant, growing as a monolayer in the presence of glial cells. The proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) have been proposed to be involved in ALS-linked microglial activation. In our explants, the combined exposure to these cytokines resulted in an increased expression of the pro-oxidative enzymes inducible nitric oxide synthase (MOS), the catalytic subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, gp91(phox) and cyclooxygenase-2 (COX-2), as compared to each cytokine alone. This effect was related to their cooperation in the activation of the transcription factor nuclear factor kappa B (NF-kappa B). TNF-alpha and IFN-gamma also cooperated to promote protein oxidation and nitration, thus increasing the percentage of motoneurons immunoreactive for nitrotyrosine. Apoptotic motoneuron death, measured through annexin V-Cy3 and active caspase-3 immunoreactivities, was also found cooperatively induced by TNF-alpha and IFN-gamma. Interestingly, these cytokines did not affect the viability of purified spinal cord motoneurons in the absence of glial cells. It is proposed that the proinflammatory cytokines TNF-alpha and IFN-gamma have cooperative/complementary roles in inflammation-induced motoneuron death. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.