Mast cells contribute to altered vascular reactivity and ischemia-reperfusion injury following cerium oxide nanoparticle instillation

Abstract

Cerium oxide (CeO₂) represents an important nanomaterial with wide ranging applications. However, little is known regarding how CeO₂ exposure may influence pulmonary or systemic inflammation. Furthermore, how mast cells would influence inflammatory responses to a nanoparticle exposure is unknown. We thus compared pulmonary and cardiovascular responses between C57BL/6 and B6.Cg-Kit(W-sh) mast cell deficient mice following CeO₂ nanoparticle instillation. C57BL/6 mice instilled with CeO₂ exhibited mild pulmonary inflammation. However, B6.Cg-Kit(W-sh) mice did not display a similar degree of inflammation following CeO₂ instillation. Moreover, C57BL/6 mice instilled with CeO₂ exhibited altered aortic vascular responses to adenosine and an increase in myocardial ischemia/reperfusion injury which was absent in B6.Cg-Kit(W-sh) mice. In vitro CeO₂ exposure resulted in increased production of PGD₂, TNF-α, IL-6 and osteopontin by cultured mast cells. These findings demonstrate that CeO₂ nanoparticles activate mast cells contributing to pulmonary inflammation, impairment of vascular relaxation and exacerbation of myocardial ischemia/reperfusion injury.

Figure 1

Characterization of CeO₂ nanoparticles. (A) Hydrodynamic size distribution of CeO₂ suspension obtained by two independent measurements, showing an average size of 90 ± 20 nm (shown in red). The standard deviations are shown in green. (B) Zeta potential of CeO₂ suspension, indicating an average value of −52.7 mV. (C) TEM image of dehydrated CeO₂ suspension. A solution of 1 μg/μl CeO₂ was used for characterization.

Figure 2

Raman spectroscopy of CeO₂ nanoparticles in mouse lung tissue. (A) An optical image of a CeO₂-instilled C57BL/6 mouse lung section showing black colored CeO₂ at the surface. (B) The corresponding Raman image of the mouse lung section shown in (A). The red colored region is a map of the characteristic Raman peak (shown in the inset ~ 464 cm⁻¹) for CeO₂ nanoparticles. (C) Optical image of CeO₂ instilled mouse lung section showing CeO₂ embedded at ~ 4 μm depth. (D) The corresponding Raman map of the 464 cm⁻¹ peak for the section shown in (C).

Figure 3

Expression of inflammatory mediators in lung tissue and BALF in C57BL/6 and B6.Cg-Kit^W-sh mice 24 hours after instillation of 100 μg CeO₂. (A) IL-6, (B) MIP-1α, (C) IL-10 protein levels measured in lung tissue 24 h following instillation of CeO₂ in C57BL/6 and B6.Cg-Kit^W-sh mice. (D) Osteopontin protein levels measured in BALF of saline or CeO₂ instilled C57BL/6 and B6.Cg-Kit^W-sh mice. n = 7–9 mice/group. ^★p ≤ 0.05.

Figure 4

Effect of CeO₂ instillation on the contraction and relaxation of isolated thoracic aorta from C57BL/6 or B6.Cg-Kit^W-sh mice. (A) Adenosine concentration response profiles; (B) norepinephrine concentration response profiles and (C) acetylcholine concentration response profiles. Aortic rings were prestimulated with 1 μM phenylephrine. n = 6–10 mice/group. ^★p ≤ 0.05 versus C57BL/6.

Figure 5

Comparison of the adenosine dose-response of thoracic aortic ring segments from C57BL/6 mice exposed to 10, 30 and 100 μg of CeO₂ and B6.Cg-Kit^W-sh mice exposed to 100 μg of CeO₂. n = 6–10 mice/group. ^★p ≤ 0.05 as compared to saline treated mice.

Figure 6

Comparison of the size of myocardial infarction in C57BL/6 exposed to 10, 30 and 100 μg of CeO₂ and B6.Cg-Kit^W-sh mice exposed to 100 μg of CeO₂. Twenty-four hours after instillation with either saline or CeO₂, the LAD was ligated for 20 min and then reperfused for 2 h. n = 3–4 mice/group. ^★p ≤ 0.05 ^★★★ p ≤ 0.001.

Figure 7

Expression of osteopontin, TNF-α and TGF-β in non-infarcted heart tissue following CeO₂ instillation. (A) mRNA levels of osteopontin, TNF-α and TGF-β in non-infarcted heart tissue following CeO₂ instillation in C57BL/6 or B6.Cg-Kit^W-sh mice. (B) Osteopontin and (C) TGF-β protein levels in non-infarcted heart tissue following CeO₂ instillation in C57BL/6 or B6.Cg-Kit^W-sh mice. n = 3–4 mice/group. ^★p ≤ 0.05.

Figure 8

Effect of CeO₂ on in vitro BMMC activation. (A) Fold change in mRNA expression of IL-6, IL-13, osteopontin (Spp1), TNF-α and TGF-β 2 hours following CeO₂ treatment of BMMCs as compared to untreated cells. (B) Osteopontin protein levels measured in the supernatant of BMMCs exposed to CeO₂ for 24 h as measured by ELISA. (C) PGD₂ levels measured in supernatant of BMMCs exposed to CeO₂ for 1 h as measured by EIA. n = 3–5 independent experiments. ^★p ≤ 0.05.