NF-kappaB dependent and independent mechanisms of quartz-induced proinflammatory activation of lung epithelial cells.

Damien Van Berlo,Ad M Knaapen,Frederik-Jan Van Schooten,Roel Pf Schins,Catrin Albrecht

doi:10.1186/1743-8977-7-13

Abstract

In the initiation and progression of pulmonary inflammation, macrophages have classically been considered as a crucial cell type. However, evidence for the role of epithelial type II cells in pulmonary inflammation has been accumulating. In the current study, a combined in vivo and in vitro approach has been employed to investigate the mechanisms of quartz-induced proinflammatory activation of lung epithelial cells. In vivo, enhanced expression of the inflammation- and oxidative stress-related genes HO-1 and iNOS was found on the mRNA level in rat lungs after instillation with DQ12 respirable quartz. Activation of the classical NF-κB pathway in macrophages and type II pneumocytes was indicated by enhanced immunostaining of phospho-IκBα in these specific lung cell types. In vitro, the direct, particle-mediated effect on proinflammatory signalling in a rat lung epithelial (RLE) cell line was compared to the indirect, macrophage product-mediated effect. Treatment with quartz particles induced HO-1 and COX-2 mRNA expression in RLE cells in an NF-κB independent manner. Supernatant from quartz-treated macrophages rapidly activated the NF-κB signalling pathway in RLE cells and markedly induced iNOS mRNA expression up to 2000-fold compared to non-treated control cells. Neutralisation of TNFα and IL-1β in macrophage supernatant did not reduce its ability to elicit NF-κB activation of RLE cells. In addition the effect was not modified by depletion or supplementation of intracellular glutathione.The results from the current work suggest that although both oxidative stress and NF-κB are likely involved in the inflammatory effects of toxic respirable particles, these phenomena can operate independently on the cellular level. This might have consequences for in vitro particle hazard testing, since by focusing on NF-κB signalling one might neglect alternative inflammatory pathways.

Highlights

The highly abundant mineral quartz is present in most rocks and minerals to some extent
Nuclear Factor kappa-B (NF-κB) is activated in vivo in rats after quartz exposure To determine the effects of respirable quartz exposure on in vivo activation of the classical NF-κB pathway, lung tissue sections of quartz-exposed rats were analyzed for Ser32/36-phosphorylated Inhibitor of κB alpha (IκBα) by immunohistochemistry
The qRT-PCR data obtained in this study indicate that inducible Nitric Oxide Synthase (iNOS), which was shown to be upregulated in vivo in the rat lung after quartz exposure, can be highly induced in rat lung epithelial cells by products of quartz-treated macrophages, while to a lesser degree and via more gradual kinetics by quartz

Summary

Introduction

The highly abundant mineral quartz is present in most rocks and minerals to some extent. The transcription factor Nuclear Factor-kappa B (NFκB) is pivotal in mediating inflammatory processes in general and is considered as the central regulator activating cells in response to silica [6]. In the classical NF-κB activation pathway, the inhibitor protein IκBα is phosphorylated at serines 32 and 36 by the IKK enzyme complex, ubiquitinated at lysines 21 and 22, and subsequently degraded by the 26S proteasome. This unmasks the nuclear localization signal of NF-κB and liberates it from the nuclear export signal of IκBα, allowing NF-κB to migrate into the nucleus, where it binds to the DNA, and activates the transcription of many pro-inflammatory genes [7]. Activation of NF-κB by silica has been shown in vivo [20,21,22], with activation localized to alveolar macrophages and pulmonary epithelial cells in particular [21,22]

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