Airborne agricultural particulate matter induces inflammatory cytokine secretion by respiratory epithelial cells: Mechanisms of regulation by eicosanoid lipid signal mediators.

The purpose of this study was to elucidate the mechanism of the airborne poultry dust (particulate matter, PM)-induced respiratory tract inflammation, a common symptom in agricultural respiratory diseases. The study was based on the hypothesis that poultry PM would induce the release of inflammatory cytokine interleukin-8 (IL-8) by respiratory epithelial cells under the upstream regulation by cytosolic phospholipase A2 (cPLA2) activation and subsequent formation of cyclooxygenase (COX)- and lipoxygenase (LOX)-catalyzed arachidonic acid (AA) metabolites (eicosanoids). Human lung epithelial cells (A549) in culture were treated with the poultry PM (0.1-1.0 mg) for different lengths of time, following which PLA2 activity, release of eicosanoids and secretion of IL-8 in cells were determined. Poultry PM (1.0 mg/ml) caused a significant activation of PLA2 in a time-dependent manner (15-60 min), which was significantly attenuated by the calcium-chelating agents, cPLA2-specific inhibitor (AACOCF3) and antioxidant (vitamin C) in A549 cells. Poultry PM also significantly induced the release of COX- and LOX-catalyzed eicosanoids (prostaglandins, thromboxane A2 and leukotrienes B4 and C4) and upstream activation of AA LOX in the cells. Poultry PM also significantly induced release of IL-8 by the cells in a dose- and time-dependent manner, which was significantly attenuated by the calcium chelating agents, antioxidants and COX- and LOX-specific inhibitors. The current study for the first time revealed that the poultry PM-induced IL-8 release from the respiratory epithelial cells was regulated upstream by reactive oxygen species, cPLA2-, COX- and LOX-derived eicosanoid lipid signal mediators.

Choice of cyclodextrin for cellular cholesterol depletion for vascular endothelial cell lipid raft studies: Cell membrane alterations, cytoskeletal reorganization and cytotoxicity.

The use of cyclodextrins as tools to establish the role of cholesterol rafts in cellular functions has become a widely accepted procedure. However, the adverse effects of cyclodextrins as the cholesterol-depleting agents on cellular structure and functions are not reported in detail. Therefore, in the current study, we investigated the membrane-perturbing actions and cytotoxicity of the two widely used cellular cholesterol-depleting cyclodextrins methyl-b-cyclodextrin (MbCD) and hydroxypropyl-b-cyclodextrin (HPCD) in our well-established bovine pulmonary artery endothelial cell (BPAEC) in vitro model system. BPAECs treated with different concentrations of MβCD and HPCD (2% and 5%, wt/vol.) for 15-180 min showed significant loss of membrane cholesterol, cytotoxicity, cell morphology alterations, actin cytoskeletal reorganization, alterations in cellular proteins and membrane fatty acid composition, and decrease in trans-endothelial electrical resistance (TER). MbCD induced a marked loss of cellular proteins, as compared to that caused by HPCD under identical conditions. More noticeably, MbCD caused a drastic loss of membrane lipid fatty acids in BPAECs, as compared to HPCD which failed to cause such alteration. Removal of cholesterol by cyclodextrin (especially MβCD) treatment apparently caused loss of fluidity of the cell membrane and leakage of vital cellular molecules including proteins and fatty acids, and thus caused cytotoxicity and loss of cell morphology in BPAECs. Replenishment of cells with cholesterol following its depletion by MbCD treatment significantly attenuated the depletion of cellular cholesterol, cytotoxicity and morphological alterations in BPAECs, indicating the importance of membrane cholesterol in vascular EC integrity. Also, the current study offered a safer method of cholesterol removal from membranes and lipid rafts by HPCD, suggesting its use in studies to investigate the role of lipid raft-associated cholesterol in cellular functions.