GM-CSF expression in the lung during Pneumocystis pneumonia.
GM-CSF expression in the lung during Pneumocystis pneumonia.
复制标题
肺孢子虫肺炎期间肺中 GM-CSF 的表达。
DOI：
10.1111/j.1550-7408.2006.00199.x
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Lee,Chao-Hung
中科院分区：
文献类型：
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作者：
Lasbury,MarkE;Durant,PamelaJ;Wang,Shao-Hung;Zhang,Chen;Liao,Chung-Ping;Tschang,Dennis;Lee,Chao-Hung
MATERIALS AND METHODSPneumocystis carinii organisms were isolated from infected rats and transtracheally inoculated into rats immunosuppressed with 1.8 mg/L dexamethasone in drinking water as previously described (Bartlett et al. 1988). When animals were moribund, or at the end of the study, they were anesthetized and sacrificed by cardiac exsanguination. Bronchoalveolar lavage fluids were obtained from rats as previously described (Lasbury et al. 2004). To reduce dilution of alveolar lining fluid, GM-CSF was analyzed in the first 5ml of saline instilled and recovered during the lavage process. Isolated BAL cells were suspended in lysis buffer, and soluble proteins were recovered after centrifugation. Bronchoalveolar lavage fluids were assayed immediately for GM-CSF levels using the Quantikine M Rat GM-CSF ELISA kit (R&D Systems, Minneapolis, MN). Fifty microliters of BAL fluids was combined with 50 μl assay diluent (supplied with kit) and incubated in wells of the assay plate coated with anti-rat GM-CSF antibody for 2 h at room temperature. The wells were washed with the kit-supplied wash solution and incubated for 2 h with 100 μl of peroxidase-linked polyclonal anti-GM-CSF antibody. The wash was repeated and 100 μl of substrate (hydrogen peroxide and tetramethylbenzidine) was incubated for 30min then stop solution (dilute HCl) was added, and the optical density was measured at 450 nm on a Vmax kinetic microplate reader (Molecular Devices, Sunnyvale, CA). The concentration was calculated by comparison with optical density readings of a recombinant GM-CSF standard curve. For immunoblotting, total soluble alveolar macrophage proteins were separated by gel electrophoresis on a 10% Bis-Tris gel (Invitrogen, Carlsbad, CA). After transferring proteins to PVDF membranes, the blot was blocked from non-specific binding using casein, and incubated with a chicken anti-GM-CSF antibody (Genway, San Diego, CA) followed by a rabbit anti-IgY peroxidase-conjugated secondary antibody (MP Biochemicals, Solon, OH). Integrated density was performed to identify sum pixel value (area x mean gray value) for the GM-CSF bands. The mean gray average was determined for the glyceraldehyde phosphate dehydrogeanse (GAPDH) internal controls, and the alveolar macrophage GM-CSF values were then normalized to GAPDH. To assess the role of calmodulin in GM-CSF production by rat macrophages, normal alveolar macrophages were isolated as described above and suspended in normal saline to a concentration of 2 x 106/ml. Five hundred liters of aliquots were transferred into wells of a 48-well plate. Triplicate wells were incubated for 30 min with 120 nM of the calmodulin inhibitor W-7 or an equal volume of saline as control.Compounds that stimulate GM-CSF production were examined for their effect on rat macrophages. Samples were incubated for 2 h with IFN-γ plus LPS (100 ng/ml of each), or 200 nM acetylcholine chloride; an equal volume of saline was added to controls. Soluble proteins from these alveolar macrophages were isolated and GM-CSF and GAPDH were assessed by immunoblot as described above.
