(379c) Role of Fiber Length on the Inflammatory Response of Model Alveolar Macrophages

Asbestos fibers induce chronic lung inflammation and have been associated (WHO estimate) with 10⁵lung cancer deaths per year worldwide. In the lung, immune cells (macrophages) attempt to engulf inhaled foreign materials (like asbestos), secreting inflammatory molecules.  The inability of macrophages to effectively remove asbestos leads to chronic inflammation and disease.  Although the health effects of asbestos have been extensively investigated, this study examines the role of fiber length on phagocytosis and molecular inflammatory responses so as to characterize fiber toxicity at the cellular level.  A major challenge is obtaining fibers of the same diameter, d, but differing in length, L.  Glass fibers, with d ~1mm, were used as a model for asbestos.  Samples with different measured length distributions were prepared: aggressive crushing for short fibers (L < 15mm) and successive sedimentation for long fibers (L < 15mm).

The interactions of MH-S murine alveolar macrophages with the fibers were analyzed by time-lapse video microscopy, to qualitatively describe attempted phagocytosis in real time, and by flow cytometry, to quantitatively measure attachment and internalization of fibers.  Short fibers were observed to bind to macrophages, being internalized with little effort. Conversely, macrophages wrestled with long fibers over many hours, usually with unsuccessful internalization.  Short fibers were twice as likely to be internalized as the long fibers. Persistent macrophage activation, measured by secretion of pro-inflammatory biomolecules, is a critical indicator of fiber toxicity and a pathological hallmark. The production of the pro-inflammatory biomolecules tumor necrosis factor alpha (TNF-a) and cyclooxygenase-2 (COX-2), was quantified by ELISA after exposure of the macrophages to glass fibers of varying lengths. TNF-a was secreted in a dose-dependent manner for both short and long fibers. However, exposure to long fibers resulted in greater secretion of the cytokine compared to that of equivalently dosed short fibers. These results suggest that the persistence of long fibers due to incomplete internalization plays a critical role in persistent macrophage activation. This information can now be used to better understand molecular events occurring in vivo and inform future animal experiments.