Hypoxia Potentiates LPS-Mediated Cytotoxicity of BV2 Microglial Cells In Vitro by Synergistic Effects on Glial Cytokine and Nitric Oxide System

Abstract

Background Microglial activation due to a variety of stimuli induces secretion of neurotoxic substances including inflammatory cytokines and nitric oxide (NO). Clinical studies indicate a cross-link between inflammatory and hypoxia-regulated pathways suggesting that bacterial infections markedly sensitize the immature brain to hypoxic injury.

Methods The impact of inflammation and hypoxia on interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α), and NO secretion and microglia-induced cytotoxicity was investigated exposing BV2 cells to lipopolysaccharides (LPS) and hypoxia (1% O₂). Cytotoxicity, NO, and cytokine release was quantified by MTS and Griess assays and by enzyme-linked immunosorbent assays, respectively.

Results LPS exposure of BV2 cells induced a significant, persistent production of NO, IL-1β, IL-6, and TNF-α. Even after LPS removal, ongoing NO and cytokine secretion was observed. Hypoxia mediated exclusively a significant, short-term IL-1β increase, but enhanced LPS-induced cytokine and NO secretion significantly. In addition, LPS-induced supernatants exhibited a stronger cytotoxic effect in glial and neuronal cells than LPS exposition (p < 0.001). Hypoxia potentiated LPS-induced cytotoxicity.

Conclusion Present data prove that LPS-induced soluble factors rather than LPS exposure mediate microglial toxicity under conditions of hypoxia in vitro. Apart from potential protective effects of the hypoxia-inducible transcription factor (HIF)-1α system, activation of proinflammatory pathways may markedly sensitize microglial cells to promote hypoxia-induced injuries of the developing brain.

Keywords

microglia - inflammation - cytokines - hypoxia - HIF-1α - neurotoxicity

^* These authors contributed equally to the work.

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