MWCNT have been reported to cause lung fibrosis in mice or rats (9
), yet little is known about the effect of pre-existing inflammation on the fibrogenic activity of MWCNT. This is an important issue, since the most susceptible individuals at greatest risk for environmental or occupational exposure to CNT would likely be those with pre-existing respiratory disease. In this study we investigated whether pre-existing lung inflammation caused by bacterial LPS (i.e., endotoxin), a ubiquitous environmental contaminant, would enhance interstitial lung fibrosis caused by MWCNT exposure. LPS pre-exposure enhanced MWCNT-induced lung fibrosis, increased MWCNT-induced lung injury as measured by LDH release and total protein levels, and synergistically elevated MWCNT-induced production of PDGF-AA, a central mediator of fibrosis (13
). Therefore, the data presented here support our hypothesis that pre-existing respiratory inflammation resulting from LPS pre-exposure exacerbates the lung fibrotic response to CNT.
LPS is a well-established stimulus of the lung inflammatory response and promotes acute inflammation characterized by the infiltration of circulating neutrophils into the lung, which play a critical role the development of LPS-induced airway disease in chronic exposures (19
). In the current study, we did not see an increase in PMNs in the BAL fluid of rats 48 hours after exposure to LPS alone, though there was a slight increase in the total cell numbers. This can be attributed to the short duration of the effects of a single exposure to LPS, which have been shown to resolve completely within 48 hours of exposure (25
). Many environmental and occupational lung diseases are caused by LPS that adheres to inhaled particles or fibers. For example, grain dust and cotton worker's lung disease are largely attributed to LPS (26
). Also, many ambient air pollution particles cause lung inflammation due to the presence of LPS, which contributes to a complex mixture of organic and inorganic components that comprise these particles (15
). Pristine CNT contain little to no LPS, since they are synthesized at very high temperatures. We were unable to detect LPS in our nanotube stock by a commercially available endotoxin assay, but the possibility exists that MWCNT could become contaminated with LPS after the manufacturing process depending upon storage and use conditions. This is because LPS is nearly ubiquitous in the environment and therefore is a common factor that could exacerbate respiratory challenges from other inhaled substances.
The mechanism whereby LPS exacerbates the lung fibrotic response to CNT is not yet known, but our findings suggest that amplification of PDGF-AA and its receptor could play a role in this interactive process. It is well-established that LPS mediates pro-inflammatory effects by binding and activating the transmembrane TLR4, which then signals intracellular signaling pathways that culminate in the production of cytokines such as TNF-α and IL-1, -6, and -8 (28
). In addition, LPS docking to the cell surface and coordinated binding to TLR4 is facilitated by the membrane CD14 receptor. We previously reported that LPS, acting through a CD14-dependent mechanism, increased the numbers of cell-surface PDGF receptors on rat lung fibroblasts (16
). Up-regulation of PDGF receptor levels increases the growth and chemotactic responses of fibroblasts to PDGF-AA secreted by macrophages and other lung cell types (13
). In the present study, we found that LPS exposure to the lungs of rats increased PDGF-AA protein levels in BAL fluid at 1 day after exposure, but not at 21 days after exposure. However, we detected PDGF-AA protein by immunohistochemistry at both 1 and 21 days. The reason for our inability to detect an increase in secreted PDGF-AA protein in BAL fluid at 21 days in mice treated with MWCNT and/or LPS is unclear. Since the PDGF-AA detected by immunohistochemistry most likely represents a membrane-tethered form and the PDGF-AA detected in BAL fluid by ELISA most likely represents PDGF-AA that has been cleaved from the cell membrane by proteolytic activity, it is conceivable that at 21 days the amount of the cleaved form was decreased to a level below our detection limits, reflecting a decrease in PDGF-AA activity at 21 days after exposure. We also showed that LPS increased PDGF-Rα mRNA levels in primary rat lung fibroblasts and NR8383 cells, a rat lung macrophage cell line. MWCNT exposure to the lungs of rats in vivo
or to cultured NR8383 cells in vitro
weakly stimulated PDGF-AA production, but this effect was synergistically amplified by LPS pre-exposure. Therefore, our data show that LPS could enhance MWCNT-induced lung fibrosis by amplifying MWCNT-induced PDGF-AA production in macrophages and epithelial cells and by increasing PDGF receptor levels on fibroblasts, which respond to macrophage- and epithelial-derived PDGF-AA.
Other investigations have shown that bacteria or bacterial-derived products modify the toxicity of CNT. For example, the pharyngeal aspiration of SWCNT into the lungs of mice followed by bacterial infection with Listeria monocytogenes
3 day later amplified lung inflammation and collagen formation, and decreased phagocytosis of bacteria by macrophages and bacterial clearance from the lungs of mice (29
). This study suggested that enhanced acute inflammation and pulmonary injury with delayed bacterial clearance after SWCNT exposure may lead to increased susceptibility to lung infection in exposed populations. In another study, mice that were exposed to SWCNT or MWCNT by intratracheal instillation at the same dose used in our present study (4 mg/kg) developed lung inflammation within 24 hours that was enhanced with LPS co-exposure (30
). Moreover, CNT tended to enhance expression of proinflammatory cytokines (TNF-α, IL-1β) in the lung and circulation in the presence of LPS, as well as in cultured mononuclear cells. These results suggested that LPS can facilitate CNT-induced systemic inflammation and possibly affect coagulation, at least in part, via the activation of mononuclear cells.
We recently reported that pre-existing allergic lung inflammation caused by ovalbumin challenge enhanced airway fibrosis in the lungs of C57BL6 mice exposed to MWCNT by inhalation (12
). That study also showed that inhaled MWCNT (the same source used in the present study) caused increased production of PDGF-AA in the lungs of mice but MWCNT alone did not cause airway fibrosis. Likewise, ovalbumin allergen challenge alone did not cause airway fibrosis but significantly increased the level of lung TGF-β1. However, the combination of ovalbumin allergen pre-exposure and MWCNT inhalation caused significant airway fibrosis and was accompanied by increases in both PDGF-AA and TGF-β1 (12
). These findings support the idea that both PDGF-AA (a stimulator of fibroblast replication) and TGF-β1 (the primary stimulator of fibroblast collagen synthesis) are required and sufficient for airway fibrosis. Moreover, since the mouse ovalbumin model is a well-established model of allergic airway disease, this study suggested that individuals with asthma might be at greater risk for the development of chronic airway disease if exposed to CNT.
The dose and administration methodology used to assess CNT toxicity and to determine the potential of CNT to cause disease are important considerations. Initial studies of CNT on rats and mice used intratracheal instillation or pharyngeal aspiration techniques to deliver a bolus of material to the lung (7
). The doses administered in these studies ranged from 1 to 4 mg/kg CNT. The dose of 4 mg/kg used in the present study is therefore consistent with these earlier studies. The concern with instillation or aspiration methodologies is that they do not produce deposition patterns similar to inhaled particles (12
). However, others have shown that inhalation exposure to SWCNT produce very similar fibrogenic effects to those seen after a pharyngeal exposure route (31
). While our methodology in the present studies achieved a well-dispersed dose of MWCNT in the lungs of rats, further study should address the effect LPS pre-exposure on inhaled MWCNT.
Our findings with MWCNT in the absence of LPS in the present study share some similarities and differences to a previous study in which we intratracheally instilled SWCNT into rats (11
), which provides a comparison of the pulmonary effects of MWCNT and SWCNT. In our previous study, rats exposed to SWCNT also developed interstitial fibrotic lesions in the same anatomic regions of the lung (terminal bronchiolar and alveolar regions) and had increased PDGF-AA (11
). The fibrotic lesions were relatively diffuse and localized to areas of CNT deposition, and therefore we did not detect a significant increase in total lung collagen content. For this reason, in the present study we did not measure total lung collagen by hydroxyproline or Sircol assay, but instead used a histopathologic scoring method. The trace metal content of MWCNT (nickel and lanthanum) differed from the trace metal content of SWCNT (cobalt and molybdenum). The lengths of both the SWCNT and MWCNT exceeded 10 μm; however, the MWCNT were 30 to 50 nm in width, whereas the SWCNT were 1 to 3 nm in width. Most importantly, SWCNT caused the formation of many unique bridge structures between alveolar macrophages in the lungs of rats (11
), whereas no such bridge structures were observed in the lungs of rats that were exposed to MWCNT.
The mechanism through which MWCNT or SWCNT increased PDGF-AA production is unknown. CNT have some features in common with asbestos fibers (e.g., high aspect ratio, residual metals), which are also known to increase PDGF by macrophages and fibroblasts (32
). However, metals in asbestos fibers (e.g., iron, magnesium) are naturally occurring, whereas CNT are manufactured using metal catalysts such as iron, nickel, and cobalt. Nickel was a catalyst present in the MWCNT used in the present study, and nickel has been reported to increase PDGF production by human macrophages (34
) and cause pulmonary fibrosis in experimental animals and in humans exposed occupationally (35
). Therefore, we speculate that the fibrogenic potential of MWCNT is due at least in part to residual nickel catalyst.
In our hands, SWCNT or MWCNT are weak inducers of TGF-β1 production in the lung. Our previous work with SWCNT showed no significant increase in lung TGF-β1 mRNA in rats exposed by pharyngeal aspiration (11
). In contrast, other work has shown that SWCNT increased TGF-β1 levels in the lungs of mice (10
). TGF-β1 levels in BAL fluid peaked at 7 days after exposure in these mice and then returned to near baseline levels by 28 days after exposure. Therefore, it is possible that in our studies with rats, which were evaluated either 1 or 21 days after exposure to SWCNT (11
) or MWCNT in the present study, we simply missed the window of TGF-β1 expression. We did observe low levels of TGF-β1 protein in the BAL fluid from rats exposed to LPS and MWCNT, which indicates that this important stimulator of collagen production is expressed under conditions that result in maximal fibrosis. A caveat is that we measured TGF-β1 by ELISA and such colorimetric assays are prone to some interference of protein binding and/or enzymatic activity by carbonaceous particles. While we were able to measure MWCNT-induced increases in the level of PDGF by ELISA, we cannot rule out some interference of nanoparticles in ELISA assays for measuring growth factor levels.
In summary, we report that pre-exposure to bacterial LPS enhances the fibrogenic effect of MWCNT delivered to the lungs of rats. Exacerbation of MWCNT-induced fibrosis by LPS is accompanied by enhanced production of PDGF-AA and its receptor in the lungs of rats, as well as in cultured rat lung macrophages and fibroblasts. Given the importance of the PDGF system in fibrotic diseases, our data indicate a possible mechanism of action whereby LPS increases MWCNT-induced fibrosis in two ways. First, LPS synergistically elevates macrophage production of PDGF-AA by MWCNT. Second, LPS up-regulates the receptor to which PDGF-AA binds on fibroblasts, thereby amplifying growth and chemotactic responses. In general, our data support the hypothesis that pre-existing inflammation exacerbates the fibrogenic response of the lungs to CNT.