Carbon nanotube instillation in mice causes sustained pulmonary inflammation and apoptosis of alveolar macrophages

Introduction: Inhalation of nanoparticles has been associated with acute and chronic pulmonary inflammation. The underlying pathways however are mostly unknown. Due to their phagocytic activity, alveolar macrophages (AM) are considered to play an important role in triggering the toxicological response to inhaled particles.

Results: Here, we show that a single exposure of mice to fibrous carbon nanotubes (CNT) but not isometric carbon nanoparticles (CNP) induces sustained lung inflammation and cell death of AM until day 90 post instillation. Although lavage analysis revealed increasing CCL2 levels and macrophage numbers for CNT but not CNP instillation, free, not phagocytosed particle agglomerates where detected for CNT only. Immunohistochemical detection of different apoptotic mediators – the general effector caspase-3, as well as the extrinsic Fas-receptor and initiator caspase-8 and the intrinsic caspase-9– on lavaged cells revealed a significant expression merely in AM of CNT exposed mice. Signal intensity per cell increased for both caspase-dependent pathways over time: initiator caspase-9 and effector caspase-3 show the highest expression after 90 days while Fas expression peaked on day 14. Intriguingly, cell death related protein expression was abundant only for CNT-laden AM (macrophages colocalizing with CNT agglomerates), although particle laden AM where detected till day 90 after CNT and CNP exposure. Similar results were observed in lung sections.

Discussion: Our data suggest a causal relation of CNT induced apoptotic macrophage cell death and non-resolving, persistent lung inflammation, initiated by phagocytotic uptake of particle agglomerates and followed by macrophage cell death and sustained lung inflammation. Since apoptosis is considered a programmed process of autonomous cellular dismantling that actively inhibits inflammation, further analysis shall focus on the occurrence of necrosis or pyroptosis upon CNT phagocytosis, and their impact to trigger chronic lung inflammation.