Figure 4.
Immunofluorescence and Western blot analysis of BBB cocultures after HIV-infected PBMC transmigration in response to CCL2 indicates that TJP expression in BBB cells is reduced. After transmigration (24 h) of uninfected (Control) or HIV-infected PBMC in response to CCL2, cocultures were immunostained with occludin, claudin-1, and ZO-1 antibodies and examined by confocal microscopy. After transmigration of uninfected cells, surface scanning of our coculture model showed a characteristic distribution of occludin (A) and claudin-1 (D) in cell-to-cell apposition membranes, whereas ZO-1 immunoreactivity (G) exhibited a diffuse pattern of expression. This is attributable to the reactivity of astrocyte foot processes, positive for TJP, that protrude into the EC layers. After HIVADA-infected (similar results were obtained for HIVJR-CSF) or HIV92UG021-infected PBMC transmigration, the expression of occludin (B, C), claudin-1 (E, F), and ZO-1 (H, I) was greatly reduced. The inset in H represents the staining for VWF (red) and GFAP (green) to demonstrate astrocyte end foot processes on the endothelial monolayer. Scale bar, 50 μm. These results are representative of 12 separate experiments. Protein lysates of BBB cells were prepared from cocultures by scraping off the cells from 16 inserts after transmigration for 24 h of uninfected (Con) and HIV-infected PBMCs in the absence (−) or presence of CCL2 (M). Lysates were analyzed by Western blotting for occludin, claudin-1, and ZO-1. Occludin, claudin-1, and ZO-1 expression in cocultures was reduced only after HIVADA-, HIVJR-CSF-, and HIV92UG021-infected PBMC transmigration in the presence of CCL2 (J). EC protein lysate (S) was used as a positive control to identify the electrophoretic mobility of TJP reactive bands. Blots were stripped and incubated with a histone-1 antibody to document equivalent protein loading. These results are representative of five separate experiments.