INTRACELLULAR SURFACTANT REMOVAL FROM PHAGOCYTIZED MINERALS: Development of a Fluorescent Method Using a BODIPY-Labeled Phospholipid

Abstract

Lung surfactant serves as a protective coating when adsorbed on particle surfaces, so its removal or rate of removal in vivo may affect expression of mineral cytotoxicity. Removal of phospholipid surfactant components from the surface of mineral particles ingested by alveolar macrophages (AM) was measured using fluorescence microscopy. Dipalmitoylphosphatidylcholine with a fluorescent label (BODIPY) substituted for C1-C4 on the second acyl chain (DPPC*), was mixed with dioleoylphosphatidylcholine (DOPC) to coat respirable quartz and kaolin particles. Fluorescence from quartz or kaolin particles of 3-4, 5-6 and 8-9 µm size decreased in intensity with increasing ratios of DOPC/DPPC* for the same DOPC concentration of 0.4 mg/ml. There was a direct correlation between fluorescence and residual phospholipid surfactant remaining on particles using phospholipase A2 (PLA2) digestion in a cell-free system, indicating that the presence of the fluorophore on DPPC did not hinder enzymatic recognition. Lavaged primary AM obtained from male Fischer rats were challenged in vitro with DOPC/DPPC* (10:1 mol:mol) coated particles at 50 µg particles/10⁶ cells. In contrast to the biexponential response seen in cell-free experiments, the rate of fluorescence decay from ingested coated quartz or kaolin particles over 7 days was monoexponential, with the same t_1/2 (41 h) for each dust. This study suggests that the rate of phagolysosomal digestion and removal of the adsorbed surfactant is not a determinant of the different mineral-specific pathogenicities or toxicities of quartz and kaolin, although residual fluorescence remained on particles even after 7-8 days.