Induction of Arachidonate 5‐Lipoxygenase and Arachidonate 5‐Lipoxygenase Activating Protein in Macrophages by Multi‐Walled Carbon Nanotubes

Abstract

Mammalian lungs respond to inhaled particulates including nanoparticles by eliciting inflammation. The initiation and resolution of inflammation are tightly controlled by potent proinflammatory and pro-resolving lipid mediators. Dysregulation of lipid mediator production and function contributes to failure of particle clearance and tissue repair, and chronic progression to fibrosis and cancer. Previous studies have shown that pulmonary exposure to multi-walled carbon nanotubes (MWCNTs, Mitsui-7) elicits acute inflammation accompanied by accumulation of M1 (classically activated) macrophages and elevated levels of proinflammatory lipid mediators in the lung and the bronco-alveolar lavage fluid. The current study aims to investigate mechanisms by which lipid mediators are regulated in J774.A1 murinemacrophages in culture, with focus on induction of arachidonate 5-lipoxygenase (ALOX5), which catalyzes the synthesis of proinflammatory lipid mediators, and ALOX5-activating protein (ALOX5AP), which activates ALOX5 by facilitating its translocation to membranes where its lipid substrates are abundantly available. Immunoblotting revealed that MWCNTs or tumor necrosis factor-α (TNF-α) induced ALOX5 by 3.5- and 4-fold, respectively, at day 1 post-exposure; MWCNTs also induced ALOX5AP by 1.5-fold at 1-day and by 2.5-fold at day 3 post-exposure. ELISA assays showed that MWCNTs induced TNF-a at 1- and 3- days post-exposure, which was comparable to induction by interferon-g (IFN-g) plus lipopolysaccharide (LPS), a classical inducer of M1 polarization. MWCNTs also induced interleukin-1b (IL-1b) protein at 1- and 3-days post-exposure, similarly to induction by TNF-a or IFN-g plus LPS. Therefore, MWCNTs alone can stimulate the production of TNF-a and IL-1b and induction of ALOX5 and ALOX5AP, which were also induced by an M1 inducer at a concentration for M1 polarization. We then examined the interaction between MWCNTs and M1 polarization for increased production of proinflammatory lipid mediators. Polarization of macrophages to an M1 phenotype by IFN-g plus LPS induced ALOX5 and ALOX5AP at day 1 through day 3 post-exposure as shown by immunoblotting. M1 polarization was accompanied by elevated levels of TNF-a and IL-1b proteins and increased production of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4), measured by ELISA, supporting that M1 macrophages are a major source of proinflammatory cytokines and proinflammatory lipid mediators. Treatment of M1 macrophages with MWCNTs at day 3 post-polarization markedly increased the production and secretion of TNF-a and IL-1b, and PGE2 and LTB4. These findings reveal that MWCNTs induce ALOX5 and ALOX5AP in macrophages, and strongly boost the production and secretion of proinflammatory lipid mediators from M1 macrophages. This study establishes a cellular model for analyzing the molecular pathways for induction of ALOX5 and ALOX5AP in macrophages by respirable particulates such as carbon nanotubes.