Multi-walled carbon nanotubes induce COX-2 and iNOS expression via MAP Kinase-dependent and -independent mechanisms in mouse RAW264.7 macrophages

Particle and Fibre Toxicology (Impact Factor: 7.11). 05/2012; 9(1):14. DOI: 10.1186/1743-8977-9-14
Source: PubMed


Carbon nanotubes (CNTs) are engineered graphene cylinders with numerous applications in engineering, electronics and medicine. However, CNTs cause inflammation and fibrosis in the rodent lung, suggesting a potential human health risk. We hypothesized that multi-walled CNTs (MWCNTs) induce two key inflammatory enzymes in macrophages, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), through activation of extracellular signal-regulated kinases (ERK1,2).

RAW264.7 macrophages were exposed to MWCNTs or carbon black nanoparticles (CBNPs) over a range of doses and time course. Uptake and subcellular localization of MWCNTs was visualized by transmission electron microscopy (TEM). Protein levels of COX-2, iNOS, and ERK1,2 (total ERK and phosphorylated ERK) were measured by Western blot analysis. Prostaglandin-E2 (PGE2) and nitric oxide (NO) levels in cell supernatants were measured by ELISA and Greiss assay, respectively.

MWCNTs, but not CBNPs, induced COX-2 and iNOS in a time- and dose-dependent manner. COX-2 and iNOS induction by MWCNTs correlated with increased PGE2 and NO production, respectively. MWCNTs caused ERK1,2 activation and inhibition of ERK1,2 (U0126) blocked MWCNT induction of COX-2 and PGE2 production, but did not reduce the induction of iNOS. Inhibition of iNOS (L-NAME) did not affect ERK1,2 activation, nor did L-NAME significantly decrease COX-2 induction by MWCNT. Nickel nanoparticles (NiNPs), which are present in MWCNTs as a residual catalyst, also induced COX-2 via ERK-1,2. However, a comparison of COX-2 induction by MWCNTs containing 4.5 and 1.8% Ni did not show a significant difference in ability to induce COX-2, indicating that characteristics of MWCNTs in addition to Ni content contribute to COX-2 induction.

This study identifies COX-2 and subsequent PGE2 production, along with iNOS induction and NO production, as inflammatory mediators involved in the macrophage response to MWCNTs. Furthermore, our work demonstrates that COX-2 induction by MWCNTs in RAW264.7 macrophages is ERK1,2-dependent, while iNOS induction by MWCNTs is ERK1,2-independent. Our data also suggest contributory physicochemical factors other than residual Ni catalyst play a role in COX-2 induction to MWCNT.

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    • "For instance, it has been shown that exposures to MWCNTs in primary peritoneal or monocyte-derived macrophages were associated with increased TNF production (Clift et al. 2014; Gasser et al. 2012; He et al. 2011; Liu et al. 2012; Muller et al. 2005), whereas exposure to CNTs did not increase the TNF secretion in RAW 264.7 cells (Di Giorgio et al. 2011). However, secretions of other inflammatory proteins (IL1β, IL6, IL10, CCL2 and prostaglandin E2) from CNT-exposed RAW 264.7 cells have been documented (He et al. 2011; Lee et al. 2012). CNTs did not increase the release of IL8 from A549 cells, or TNF from rat alveolar (NR8383) macrophages after 24-h exposure (Pulskamp et al. 2007). "
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    • "Transmission electron microscopy was conducted by Bonner et al., on the murine RAW 264.7 cells for the assessment of cellular uptake and sublocalization of MWCNTs. TEM results showed that the RAW 264.7 macrophages successfully engulfed the MWCNTs [96]. Similarly, Sitharaman et al. reported the efficacy of europium (Eu) catalyzed SWCNTs (Eu-SWCNTs), as visible nanoprobes for cellular imaging after observing the internalization of Eu-SWCNTs in the breast cancer cells (SK-BR3 and MCF-7) via cellular endocyte formation as imaged by confocal fluorescence microscopy and TEM [97]. "
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    • "In vitro toxic effects of CNTs have also been investigated in a variety of cultured cells and several adverse responses such as cellular and DNA damage, and modulation of cell signaling have been reported following treatment with MWCNTs (Cavallo et al., 2012; Lee et al., 2012). The adverse human health effects of active and passive smoking are widely known and include increased risk for a variety of respiratory diseases including lung cancer (Hecht, 1999). "
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