UDP -G activates oxidative stress and respiratory burst in human polymorphonuclear cells.

REGUEIRO, M; ACOSTA, JM; REPETTO, MG; LAZAROWSKI, A; MERELLI, A

Buenos Aires

Congreso; IV International Congress in Translational Medicine.; 2018

Maestría Internacional en Ciencias Biomédicas (IMBS). Universidad de Freiburg (Alemania) y Universidad de Buenos Aires

Defects in the intracellular phagocyte respiratory burst cause in affected patients recurrent infections, immunodeficiency, chronic and severe diseases, often with sepsis and hypoxia. The aim of this study is to standardize the methodological conditions to evaluate the respiratory burst and oxidative stress in human blood neutrophils (PMN) after in vitro stimulation. Methodology: Phagocyte respiratory burst was evaluated by measuring oxygen consumption with an oxygen electrode type Clark, and cells chemiluminescence with a scintillation photon counter, after PMN activation with lipoplysacarid (LPS, ), phorbol myristate acetate (PMA) or uracile diphosphate glucose (UDP-G). Chemiluminescence and oxygen consumption were measured after 15 min of exposure to different stimulus. Results: An enhancement in prooxidant species production takes place over a broader time-window 3-7 in a cell dependent manner. Interestingly, GSH does not become oxidized during the first 7 hours of exposure. In accordance, addition of N-acetylcysteine (NAC) does not prevent cell death. However, addition of GSH as well as its oxidized form GSSG to the medium fully prevents the toxicity of the metal. GSH depletion by buthionine-sulfoximine (BSO) dramatically enhances cell death upon Cu exposure which cannot be rescued by replenishment of soluble ?SH groups by means of NAC on top of GSH depleted cells indicating that GSH is likely to be acting as a protective molecule, independently of its ?SH group. Cu exposure leads to a ubiquitous phosphorylation of JNK and may lead to caspase 3 cleavage and activation in a cell dependent manner. Although, even when active, caspase 3 plays a secondary role in cell death since its inhibition by ZVAD has a minor effect on the survival of the cell. Finally, the gene expression profile shows a largely increased expression of heat shock proteins, ER stress response related genes, autophagy and genes related with proteasome degradation processes such as ubiquitin. In accordance, a large number of genes under control of transcription factors involved in protein folding show a consistent enhanced expression. Discussion: Cu toxicity is likely related to the high reactivity of the metal. However, the molecular targets once the metal enters the cell are uncertain. The capacity of the metal to undergo redox cycling while giving rise to the highly reactive hydroxyl radical by Fenton chemistry makes the redox hypothesis a tempting explanation to the toxicity of the metal. However, according to these results, the manipulation of the free ?SH pool by exogenous addition of NAC had no effect on cell death. Moreover, the fact that GSSG as well as GSH addition result in a protective effect suggests the capacity of GSH to act independently of its ?SH group, and redox active part of the molecule. Interestingly, the gene expression profile suggests that Cu ions interact with proteins, thus leading to improper folding and heat shock response followed by enhanced proteasome degradation and autophagy. Conclusion: Cu induced cell death is delayed by intracellular GSH which acts independently of the ?SH. The metal overload may trigger caspase 3 activation. However, apoptosis is only secondary to a more critical caspase independent event.