asbestos--crocidolite and arginyl-glycyl-aspartic-acid

Interstitial lung disease (ILD) produces disruption of alveolar walls with loss of functionality and scar tissue accumulation. Asbestosis is the ILD produced by the inhalation of asbestos fibers. This study attempts to elucidate the role of lung epithelial cells in the generation of asbestos-induced ILD. When exposed to crocidolite LA-4 cells had a decrease in viability and an increase in the release of lactate dehydrogenase (LDH) and 6-keto PGF(1alpha), a PGI(2) metabolite. PGI(2) release was mediated by cyclooxygenase-2 (COX-2) and vitronectin receptor (VNR). When LA-4 cells were treated with VNR inhibitors, either RGD (Arg-Gly-Asp) peptide or VNR blocking antibody, a statistically significant decrease in PGI(2) metabolite production was observed, but crocidolite-induced cytotoxicity was not prevented. These findings propose that crocidolite is coated by an RGD protein and binds VNR-inducing COX-2 expression and PGI(2) release. Moreover, when LA-4 cells were exposed to crocidolite in the presence of reduced serum culture media, PGI(2) production was prevented, and when bronchoalveolar lavage fluid (BALF) was added, PGI(2) production was rescued. Cytotoxicity did not occur, either in reduced serum culture media or when BALF was added. In conclusion, crocidolite requires the presence of an RGD protein coating the fibers to induce inflammation (PGI(2) production) and crocidolite alone cannot induce cytotoxicity in lung cells.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Asbestos, Crocidolite; Bronchoalveolar Lavage Fluid; Cell Line, Tumor; Cell Survival; Cyclooxygenase 2; Dose-Response Relationship, Drug; Epithelial Cells; Epoprostenol; Integrin alphaVbeta3; L-Lactate Dehydrogenase; Lung; Mice; Mice, Inbred BALB C; Oligopeptides; Signal Transduction

Phagocytosis of asbestos fibers may be a necessary step for asbestos-induced injury to mesothelial cells, but this has not been established because quantification of fiber uptake is difficult and ways to increase fiber phagocytosis without also increasing total dose were not available. We quantified phagocytosis by counting intracellular fibers after removing adherent fibers with trypsin; we selectively increased fiber phagocytosis by coating crocidolite asbestos fibers with the adhesive serum protein vitronectin (VN), which we have shown increases fiber uptake via integrins. We measured various aspects of asbestos-induced cytotoxicity: intracellular oxidation by the shift of fluorescence of cells loaded with an oxidative probe, DNA strand breakage by the alkaline unwinding ethidium bromide fluorometric assay, apoptosis by annexin V binding and by nuclear morphology, and cell-cycle progression. We found that, compared with control fibers or particles, asbestos increased intracellular oxidation, DNA strand breakage, and apoptosis. Selective increases in fiber uptake by VN-coating of the fibers further increased the oxidation, DNA strand breakage, and apoptosis, and induced a cell-cycle arrest in G2/M. Selective decreases in fiber uptake by cytochalasin or by integrin blockade with RGD peptides inhibited several of these measures of injury. We conclude that phagocytosis is important and perhaps necessary for asbestos-induced injury to mesothelial cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Asbestos, Crocidolite; Cells, Cultured; Cytochalasin B; DNA Damage; G2 Phase; Mitosis; Oligopeptides; Oxidative Stress; Phagocytosis; Pleura; Rabbits; Respiratory Mucosa; Serum Albumin, Bovine; Vitronectin