The increased expression and activity of COX-2 and iNOS in macrophages are two hallmarks of inflammatory and immune responses to a variety of stimuli, including LPS, metals, and oxidative stress. MWCNTs delivered to the lungs of mice by inhalation or oropharyngeal aspiration, or to rats by intratracheal instillation, are avidly engulfed by alveolar macrophages and MWCNT-containing macrophages are associated with progressive inflammatory and fibrotic lesions in the lung alveolar region, airways, or pleura of these animals
]. In this study, we found that MWCNTs increased the expression of COX-2 and iNOS, and the induction of these two enzymes correlated with increased production of PGE2
and NO, respectively. Therefore, the induction of COX-2 and iNOS in RAW264.7 macrophages in vitro
observed in the present study suggest that these enzymes and their products could play a role in the lung’s inflammatory or fibrogenic response to MWCNTs.
We further investigated upstream signaling that might mediate the induction of COX-2 and iNOS in RAW264.7 macrophages and found that MWCNTs increased the expression of COX-2 via an ERK1,2-dependent mechanism as demonstrated by blocking ERK activation with the MEK inhibitor U0126. While COX-2 expression was blocked by U0126, there was no discernable effect of U0126 on MWCNT-induced iNOS levels. MAPK signaling has been reported to regulate LPS-induced COX-2 expression in RAW264.7 cells . However, LPS-induced COX-2 expression was partially blocked by inhibitors of ERK1,2 or p38 MAP kinase and combined blockade of these two kinases was required to completely inhibit COX-2 expression
]. In the present study we demonstrated that COX-2 induction in RAW264.7 macrophages by LPS, V2
, NiNPs, or MWCNTs was significantly inhibited by treatment with U0126, indicating that diverse organic and inorganic stimuli are able to induce COX-2 via ERK1,2-dependent signaling. In addition, we did not observe increased JNK or p38 MAP activation in RAW264.7 cells following MWCNT treatment (data not shown). Taken together, these findings suggest that ERK1,2 is the major pathway for MWCNT induction of COX-2 expression in these cells. However, a caveat of our data is that ERK was phosphorylated by relatively low concentrations of MWCNT compared to COX-2 induction (Figures
&). These findings suggest that ERK phosphorylation is required but perhaps not sufficient to induce COX-2 at low MWCNT doses in RAW264.7 cells. Possibly at low MWCNT doses other intracellular signaling intermediates could play contributory roles in COX-2 induction. For example, NFκB and C/EBPbeta have been reported to mediate air pollution particulate matter-induced COX-2 expression in human bronchial epithelial cells
The biological effects of MWCNTs could be due to multiple factors, including aspect (length to width) ratio, surface properties, aggregation or dispersion, and residual metal catalysts. For example, the purification of MWCNTs to remove residual metal catalysts used in the manufacturing process reduces the toxicity and pro-fibrogenic activity of MWCNTs
]. Our results show that NiNPs are a potent inducer of COX-2. This suggests that at least part of the bioactivity of the MWCNTs used in our study could be due to residual Ni from the manufacturing process. While relatively high concentrations of Ni clearly induced COX-2 (Figure
A), removal of ~60% of Ni from MWCNT (4.49% Ni in AP-MWCNT reduced to 1.8% Ni in PD-MWCNT) did not have a significant effect on MWCNTs ability to induce COX-2 induction by MWCNT (Figure
B). Other groups have shown that the high aspect ratio (i.e., length) of MWCNTs, as well as other nanomaterials such as nickel nanowires, is perhaps the most important factor in determining macrophage activation, clearance, and ultimately disease outcome
]. Given the data presented in Figure
B we speculate that other factors in addition to Ni (e.g., nanotube length) are important to COX-2 expression in macrophages. However, as acid purification did not remove all residual nickel and even purified samples are not completely metal-free, Ni may still have a role in the induction of COX-2 in our studies. Furthermore, the metal catalysts present in MWCNT may not be bioavailable
]. For example, the Ni present in MWCNTs appears to be encapsulated by carbon as observed by TEM (unpublished observation). Therefore, the relative contribution of Ni, nanotube length, and perhaps other factors, to COX-2 induction requires further study.
It is unknown whether ROS generation is involved in MWCNT induction of COX-2. MWCNTs have been reported to increase ROS production in lung cells in vitro
]. It has also been shown that particulate matter-induced ROS generation is primarily of mitochondrial origin and results in increased COX-2 expression and IL-6 release by cultured bronchial epithelial cells
]. In addition, the organic diesel exhaust constituent 1,2-napthoquinone caused mitochondrial production of H2
and increased levels of COX-2 and IL-8, both of which were diminished by the over-expression of catalase, which degrades H2
]. We previously reported that vanadium pentoxide-induced H2
production in human lung fibroblasts occurs via NADPH oxidases
]. Furthermore, p-ERK1,2, which was shown to mediate MWCNT-induced COX-2 in the present study, is also strongly activated by H2
in lung myofibroblasts
]. While it is possible that MWCNTs induce ERK1,2-dependent COX-2 expression via ROS generation, the origin of ROS generation is complex and elucidation of ROS involvement in MWCNTs activity will require further study.
Others have reported that iNOS or reactive nitrogen species (RNS) generated by iNOS influence COX-2 activity. For example, iNOS activates COX-2 in LPS-stimulated RAW264.7 cells through generation of NO
]. Furthermore, iNOS inhibitors have been reported to reduce PG production in carrageenan-induced inflammation in rats
]. Based on these studies, cross-talk between iNOS and COX pathways has been proposed as an important contributing mechanism for inflammatory diseases
]. However, in the present study the inhibition of iNOS with L-NAME did not significantly reduce MWCNT-induced COX-2 levels in RAW264.7 cells.
Both protective and pathogenic roles for COX-2 and its metabolites have been proposed. For example, PGE2
generated by COX-2 inhibits fibroblast and epithelial cell growth and reduces platelet-derived growth factor receptor expression in rat lung fibroblasts in vitro
]. Furthermore, COX-2 knock-out mice display more inflammation and fibrosis in response to metals or allergens
]. Our unpublished observations also show that COX-2 knock-out mice have exaggerated airway inflammation and production of IL-13 after combined exposure to ovalbumin and MWCNTs. Collectively these transgenic mouse models suggest that COX-2 is protective in lung inflammation and fibrogenesis. It is also noteworthy that patients with idiopathic pulmonary fibrosis have reduced levels of COX-2
]. Despite the evidence that COX-2 is protective in lung inflammatory and fibrotic diseases, there is also evidence that COX-2 and its metabolites have detrimental roles in mediating the pathogenesis of other diseases, particularly in arthritis and cancer
]. Therefore, the significance of COX-2 in the pathogenesis of MWCNT-induced lung disease is unclear at present.
Both beneficial and potentially detrimental effects have also been ascribed to NO. It is well established that NO exerts a beneficial role through its action as a vasodilator and exogenous NO has been proposed to have therapeutic value for the treatment of asthma
]. On the other hand, NO could have potential deleterious effect as it forms the highly toxic peroxynitrite (ONOO-
) in the presence of H2
and acts as a potent signaling intermediate; causing tyrosine nitration and the activation of the EGF receptor and MAPK signaling pathways
]. The generation of reactive nitrogen species derived from NO metabolism plays important roles in particulate-induced lung disease
in particular has been implicated in the pathogenesis of lung and pleural disease associated with asbestos fibers
]. Because MWCNTs have been compared to asbestos fibers with respect to their pathogenicity, it will be important to further elucidate whether MWCNTs are capable of generating RNS such as ONOO-
. Our data show that MWCNTs increase NO and others have reported that MWCNTs increase ROS in lung cells
]. Therefore, if NO and ROS are generated simultaneously then it is likely that ONOO-
will be formed in the lungs of rodents or humans exposed to MWCNTs.
Whether or not COX-2 and its metabolites, or iNOS-generated NO, are beneficial or detrimental following MWCNTs exposure remains to be elucidated. COX-2 deletion in mice results in susceptibility to metal-induced lung fibrosis
] or allergen-induced lung inflammation
], and the severity of lung inflammation in COX-2-deficient mice is due to reduced PGE2
]. MWCNTs also induce fibrogenesis in the lungs of exposed mice and rats
] and impair lung function
]. Therefore, it would be important to determine whether MWCNT-induced inflammation and fibrosis are altered in the lungs of COX-2 deficient mice.
Modifications to alter the surface properties of MWCNTs could also alter biological activity. It has been recently shown that modification of MWCNTs by addition of carboxyl groups, as well as the dispersal state of MWCNTs affect the fibrogenic cellular responses that correlate with the extent of fibrosis in mice
]. Furthermore, modifications of other types of nanoparticles, such as silica, by the addition of carboxyl or amine groups, changes the surface properties of these nanoparticles to alter intracellular localization and cytotoxicity in macrophages
]. Therefore, COX-2 or iNOS induction in cultured cells could be indicative of the inflammatory or fibrogenic activity of an increasing diversity of CNTs or other engineered nanoparticles. Such information should allow us to better predict relative toxicity to humans and this information should aid in the design of safer nanomaterials.