The present study was designed to test the hypothesis that inhaled MWCNT would cause greater lung injury and fibrosis in mice that had pre-existing allergic inflammation. We found that inhaled MWCNT caused no significant fibrosis in mice that did not receive ovalbumin challenge, nor did we observe any increase in total lung protein and LDH release into the BAL fluid from MWCNT-exposed mice. However, mice that received ovalbumin challenge before MWCNT inhalation exposure developed significant airway fibrosis as determined by quantitative morphometry. Some neutrophilic inflammation was observed in the lungs of mice that inhaled MWCNT, but this inflammation resolved by 2 weeks. Airway fibrosis in ovalbumin-challenged mice that inhaled MWCNT was accompanied by significant increases in two profibrogenic growth factors, PDGF-AA and TGF-β1. Ovalbumin alone induced only TGF-β1, whereas MWCNT alone only induced PDGF-AA. PDGF is the most potent fibroblast mitogen discovered to date and is important in expanding the lung population of fibroblasts (21
). TGF-β1 is the primary growth factor that stimulates fibroblasts to produce collagen, which defines fibrotic lesions (22
). Together, these two polypeptide growth factors orchestrate a fibrogenic response. While our study suggests potential roles for PDGF and TGF-β1, further work is needed to identify more definitive roles for these mediators in MWCNT-induced airway fibrosis using strategies such as RNAi, small molecule inhibitors, or neutralizing antibodies to knock-down or block the activity of these growth factors or their receptors in vivo
IL-13 is a well-known mediator of allergic asthma and was increased in the lavage fluid of ovalbumin-sensitized mice and also in ovalbumin-challenged mice that received MWCNT exposure. IL-13 has been reported to stimulate lung fibrosis by increasing TGF-β1 production and activating the release of active TGF-β1 from its latent complex (14
). Consistent with this report, we observed elevated TGF-β1 protein in the BAL fluid of ovalbumin-challenged mice. We did not observe a significant increase in PDGF-AA in the BAL fluid from ovalbumin-challenged mice, although we have previously reported that IL-13 stimulates the mitogenesis of human lung fibroblasts through a PDGF-AA autocrine loop (13
). We have also observed by immunohistochemistry that PDGF-AA and PDGF-CC are increased in the airway epithelium of mice after ovalbumin challenge (unpublished observation). It is possible that PDGF-AA in ovalbumin-challenged mice is not secreted by the airway epithelium in detectable amounts, although we clearly observed increased PDGF-AA in the BAL fluid of MWCNT-treated mice. IL-13, TGF-β1, and PDGF-AA are all elevated in human asthma, and play important roles in cell signaling pathways that mediate airway fibrosis (23
). MWCNT could have their most significant effect in potentiating the effects of allergen challenge by elevating PDGF.
IL-5 mRNA was increased only in mice that received both ovalbumin challenge and MWCNT inhalation. We did not measure IL-5 protein levels due to the limited amount of BAL fluid that was exhausted in ELISAs for IL-13, PDGF-AA, and TGF-β1. Previous studies have shown a modulatory role for IL-5 in promoting pulmonary fibrosis in different animal models (23
) and clinical studies indicate a similar effect in humans (25
). In the mouse ovalbumin model in particular, neutralization of IL-5 with monoclonal antibodies before multiple pulmonary challenges with ovalbumin decreases peribronchiolar fibrosis (26
). Based on these published reports, there is compelling evidence that IL-5 plays a role in airway fibrosis in asthma. It is therefore conceivable that IL-5, which is synergistically increased by the combination of ovalbumin and MWCNT, could play a significant role in mediating the effect of MWCNT toward potentiating airway fibrosis in allergic inflammation.
The calculated dose of MWCNT aerosol we used was an estimate of the dose used in earlier studies that employed the intratracheal instillation method for CNT delivery and which resulted in significant pulmonary fibrosis and granuloma formation (3
). Also, unlike instillation studies, the majority of MWCNT observed by light microscopy after inhalation were contained within alveolar macrophages, which is not surprising since macrophages avidly engulf inhaled particles and the (2-μm) size of the aggregated MWCNT aerosol was in the range for phagocytic uptake. Also, unlike intratracheal instillation studies (5
), histopathology of lungs in the present study showed no evidence of MWCNT agglomerates obstructing the conducting airways or the alveolar region. However, TEM showed that small aggregates of MWCNT or individual nanotubes were present on or beneath the epithelial cell surface throughout the lung.
The increased MCP-1 message expression from the lung tissue of MWCNT-treated mice could play a role in the recruitment of macrophages to sites of MWCNT deposition. The result that inhaled MWCNT agglomerates were almost completely localized in macrophages is consistent with a recent MWCNT inhalation study (10
). We previously reported that SWCNT delivered to the lungs of rats by intratracheal instillation stimulate a strong macrophage phagocytic response (6
). In that study, we described “carbon bridges,” which are bundles of SWCNT connecting two macrophages (6
). The frequency of carbon bridge formation induced by SWCNT was relatively low (< 5% of total macrophages), but provided a reliable biomarker of exposure. No such structures were found in the present study using MWCNT. This is perhaps because MWCNT are 30 to 50 nm in width, whereas SWCNT are only 1 to 3 nm in width; approximately the same width as actin filaments that compose the cytoskeleton. Effective sequestration of MWCNT agglomerates may explain why little inflammation or fibrosis was present by 14 days for unsensitized animals, since inflammation and fibrosis in response to MWCNT have previously been reported around sites where large agglomerates deposited and persisted after intratracheal instillation (9
). We observed many small aggregates and individual MWCNT by TEM after inhalation. Well-dispersed SWCNT have been reported to cause interstitial fibrosis in the lungs of mice when delivered by intratracheal instillation (4
). However, while our inhalation study resulted in well-dispersed MWCNT as determined by light microscopy and TEM evaluation, we did not observe a significant fibrogenic effect in the absence of pre-existing inflammation.
Ovalbumin sensitization by intraperitoneal injection followed by intranasal challenge creates a proinflammatory environment in the lung. Particulate pollutants have been reported to increase allergen-induced inflammation and lung function (17
). In particular, carbon nanoparticles can aggravate ovalbumin-induced airway inflammation and immunoglobulin production, which is more prominent with smaller particles (27
). In that study, the carbon nanoparticles caused airway inflammation but not fibrosis, which is consistent with our previous observation that carbon nanoparticles do not cause lung fibrotic reactions (6
). The present study is the first, to our knowledge, to show that a particulate pollutant of any kind can increase airway fibrosis in allergen-challenged mice. The fibrotic response that we observed with MWCNT and ovalbumin was confined to the bronchioles and did not extend into the interstitium, which differs from previous reports showing interstitial fibrosis at sites distant from the airways of unsensitized mice in response to SWCNT (4
). Although quantitative morphometry revealed a statistically significant increase in peribronchiolar fibrosis in sensitized animals that inhaled MWCNT, no significant increases in total lung collagen content were observed, which is consistent with our observation of a localized airway response. Moreover, the animals showed no signs of respiratory distress. However, we did not perform pulmonary function tests on mice as has been described previously (19
). Therefore, it is unknown whether the increased airway fibrosis in ovalbumin-challenged mice that inhaled MWCNT correlates with decreased lung function. This is an important issue that should be addressed in future investigations. It is also unknown whether the airway fibrotic lesions observed in mice that received a combination of allergen and MWCNT will eventually resolve or progress. Further work should include a longer time course evaluation to address this issue.
In the present study we used an aerosol concentration of approximately 100 mg/m3
MWCNT and a single exposure 6 hours in duration on a nose-only tower. This dose is admittedly high, but was specifically chosen to fall within the dosing range of MWCNT (0.5–20 mg/kg) delivered to mice by intratracheal instillation (7
) or to approximate a dose of SWCNT that we used previously administered to rats by intratracheal instillation. This type of comparison is the only way to directly assess the issue of whether inhalation exposure results in pathology similar to that of intratracheal instillation or pharyngeal aspiration. Moreover, the dose of MWCNT that we used in this study is roughly within the range of previous whole-body inhalation studies that were 0.2–2.7 mg/kg (10
) and 2–10.5 mg/kg (9
). We estimated an alveolar deposition dose of 12 mg/kg and a tracheobronchial dose of 4 mg/kg for an aerosol of average mass median aerodynamic diameter of approximately 700 nm. Although the airborne concentration of MWCNT in the present study is similar to those at the higher end of the dosing ranges in other MWCNT inhalation studies, it is 2,000-fold greater than the average, estimated concentration of 0.053 mg/m3
generated during the handling of SWCNT in occupational exposure scenarios (29
) and 20-fold greater than the 5 mg/m3
NIOSH occupational exposure standard for unregulated particulates (30
The MWCNT used in this study were purchased from a commercial source and characterization provided by the manufacturer and verified by an independent contract laboratory. TEM of the bulk material verified that these were nanotubes, not carbon nanofibers (31
), and that MWCNT were aggregated. TGA analysis from both sources were in agreement that bulk MWCNT were greater than 94% in purity. BET surface area was also consistent with an average of 109.29 m2
/g. The elemental composition of the bulk material was carbon > oxygen > nickel > lanthanum. It is important to note that Ni and La values measured by ICP-AES, in which the nanotubes are treated with strong acid, reflects dissolved surface metals. This measurement is probably more reasonable than EDX measurements for discerning bioavailable metals, since EDX also measures metals embedded within MWCNT. The metal concentrations as a percentage of the bulk material as determined by ICP-AES are low, at 0.34% (Ni) and 0.03% (La). The MWCNT aerosol was also characterized. TEM of the aerosol revealed that MWCNT were mostly agglomerates of approximately 2 μm in size with protruding nanotubes. Agglomeration is likely due to Van der Waals forces between MWCNT (1
) that were not disrupted by the Pluronic F-68 surfactant.
A caveat for the present study is that the high dose of MWCNT and the brief duration of exposure do not likely mimic occupational exposure scenarios, which will probably occur with airborne concentrations in the 0.05–5 mg/m3
range for up to 40 hours per week in duration over weeks to months to years. However, very high airborne concentrations and brief exposures are consistent with the use of inhalants in medicine, and MWCNT are proposed for use in drug delivery applications (33
). The result that no acute observable pulmonary hazard of airway fibrosis to normal lung tissue was observed is of importance, especially at the high dose that we used, since the data indicate that the NOAEL for inhaled MWCNT is probably greater than what may be achievable in “real life” occupational or therapeutic exposures. Our data do indicate a pulmonary hazard of increased airway fibrosis in inflamed lung tissue, but the degree of fibrosis was not sufficient to cause overt signs of pulmonary distress. This result supports our original hypothesis, which was that MWCNT cause greater lung injury and fibrosis under conditions of pre-existing allergic inflammation, indicating that individuals with asthma may be more susceptible to adverse effects from MWCNT than normal populations. This study should encourage future investigations into differences in dose–response-effect relationships in normal and asthmatic lung in response to inhaled CNT at airborne concentrations relevant to occupational and medical exposures.