Persistent Pleural Inflammation,fibrosis And Mesothelial Proliferation By Pulmonary Exposure Of Multi-walled Carbon Nanotubes In Rats

Background & Objective: Multi-walled carbon nanotubes(MWCNTs)are one of the most promising nanomaterials in the industry today.However,MWCNTs has a fibrous structure similar to asbestos,and exposure to MWCNTs may lead to pulmonary fibrosis,lung cancer,pleural plaques,malignant pleural mesothelioma and other asbestos-like diseases.With their increased use,biosafety of MWCNT becomes a health concern.While plenty of studies have shown that MWCNTs are able to cause lung inflammation,fibrosis and other diseases in rodents,a few researches are focusing pleural lesions induced by MWCNTs.In the present study,we aimed to find out if MWCNTs given to the rat lung by intratracheal spraying induce persistent pleural inflammation and lesions in both the visceral and parietal pleura,and analyze the relationship of pleural inflammatory reactions,especially the increased cytokines,with development of these pleural lesions.Method: 1.Ten week-old male Fisher344 rats were divided into eight groups;each group contained 6 rats.Suspensions of 2 kinds of MWCNTs and asbestos were administered to the rat lung by intratracheal spraying,250 ?g,2 times/week for 4 weeks.The rats were sacrificed at 1 day or at 3 months post the last spraying.Paraffin sections of the pleural tissues were prepared for HE staining,Azan-Mallory staining andimmunohistochemistry.2.Azan-Mallory staining was used to evaluate fibrosis in the pleura;Thickness of the visceral and parietal pleura was examined based on Azan-Mallory staining.3.Mesothelial cell proliferation was assessed by proliferating cell nuclear antigen(PCNA)immunohistochemistry.PCNA indices were calculated as the number of PCNA-positive mesothelial cells in total mesothelial cells of the visceral and parietal pleura.4.At sacrifice,pleural cavity lavage(PCL)was performed.Total inflammatory cells and differential leukocytes in the collected PLC fluids;the cell pellets were used for detection of MWCNT and crocidolite fibers with a polarized microscope;levels of cytokines in the supernatants of PCL were analyzed by Suspension Array.5.Cytokines found to be increased in the supernatants of PCL were used for in vitro cell proliferation assays.Western blotting and flow cytometry were used to examine cell proliferation signaling and analyze the cell cycle,respectively.Results: 1.MWCNTs and crocidolite administrated into the rat lung caused persistent pleural local thickening in both in the visceral and parietal pleura.The reason for the thickening was development of local fibrosis especially in or near severe inflammatory foci.2.PCNA immunohistochemistry indicated that treatment with MWCNTs and crocidolite induced mesothelial cell proliferation in the visceral and parietal pleura at 1day and at 3 months post the treatment.3.MWCNTs and crocidolite sprayed to the rat lung were transported into the pleural cavity,subsequently inducing inflammatory cell infiltration into the pleural cavity.The majority of the infiltrated cells were macrophages.Although the number of the fibers and the number of the infiltrated inflammatory cells were decreased with time,they were still significantly higher than those in the control group.4.Suspension Array analysis indicated that levels of IL-2,IL-18 and IP-10 in the supernatant of PCL increased in MWCNTs-or crocidolite-treated groups compared with those in the control groups.5.In vitro proliferation assays,addition of IL-2,IL-18 and IP-10 each alone did not promote the cell proliferation of human fibroblasts MRC-5 and mesothelial cells Met5 A up to 50 ng/ml,combination of the 3 cytokines synergistically stimulated MRC-5 and Met5 A proliferation in a dose-dependent manner.Upon stimulation with combination of the 3 cytokines,phosphorylation of Erk was enhanced as detected by Western blotting,and proportion of G2/M phase cells was increased as analyzed by flow cytometry cell cycle analysis.Conclusion: Like crocidolite,MWCNTs given to the rat lung lad to persistent pleural fibrosis and mesothelial cell proliferation in both the visceral and parietal pleura.MWCNTs and crocidolite fibers was found to be transported into the pleural cavity,subsequently inducing inflammatory responses in the pleural cavity.Increased production of cytokines IL-2,IL-18 and IP-10 synergistically promoted cell proliferation of fibroblasts and mesothelial cells.