Secretory Leukocyte Protease Inhibitor Binds to Annexin II, a Cofactor for Macrophage HIV-1 Infection

Ma, Ge; Greenwell-Wild, Teresa; Lei, Kejian; Jin, Wenwen; Swisher, Jennifer; Hardegen, Neil; Wild, Carl T.; Wahl, Sharon M.

Fluorescence Microscopy. 10 6 cells cultured in four-chamber glass slides (Nunc) were incubated with biotinylated rhSLPI from 5 s to 60 min at 37 Њ C, washed, and neutravidin-FITC (Jackson ImmunoResearch Laboratories) in PBS was added for 30 min at 4 Њ C. Slides were rinsed, fixed in 2% paraformaldehyde, incubated with propidium iodide (Sigma-Aldrich) for 5 min, rinsed, and mounted with SlowFade (Southern Biotechnology Associates, Inc.) for fluorescence microscopy. In additional experiments, monocytes were plated on glass coverslips in 24-well plates (2 ϫ 10 6 per well) and cultured as described above for 7-10 d. Before staining, coverslips were washed in PBS, fixed in 2% paraformaldehyde for 30 min, and then incubated in 100 mM glycine in PBS at room temperature for 20 min. Cells were rinsed in PBS, methanol permeabilized at Ϫ 20 Њ C for 5 min, and then blocked in 10% donkey serum for 30 min. 2 g/ml of primary antibody (mouse anti-annexin II; Transduction Labs) was added in 2% donkey serum in PBS, washed, and incubated for 1 h in 20 g/ml Alexa 647-conjugated donkey anti-mouse (Molecular Probes). Cells were washed twice in PBS, twice in dH 2 O, mounted using Fluormount G (Electron Microscopy Services), and visualized using the PerkinElmer UltraView LCI confocal system with a Nikon Eclipse microscope. For phagocytosis of apoptotic cells, Jurkat cells (200,000/ml) were treated with 0.2 M staurosporine at 37 Њ C for 7 h, washed, stained with 1 M carboxyfluorescein diacetate succinimidyl ester (Sigma-Aldrich) in PBS for 20 min, and washed before being added (4 ϫ 10 6 ) to macrophages on coverslips. After 10 min at 37 Њ C, the coverslips were vigorously washed, fixed, and stained for annexin II as described above.