FIGURE 2.

A, mBMEC monolayers were treated with CCL2 (100 ng/ml) in serum-free medium for the indicated periods (0–120 min). TEER was evaluated during this time period by ECIS assay. In a separate set of experiments after 60 min of CCL2 treatment (100 ng/ml), the CCL2 was removed by replacing media with fresh DMEM, and the cells were observed for an additional 60 min (+recovery). Each TEER tracing represents an average of three replicate wells and from three independent experiments. Impedance values were normalized by dividing by the impedance measured just prior to the addition of CCL2. B, the value of TEER of brain endothelial barrier in the presence or absence of CCL2 and 5 μg/ml cycloheximide (CHX) or 1 μm lactacystein (LC) as well as during the recovery time. Both inhibitors, CHX and LC, did not disturb the brain endothelial barrier opening or recovery. Data represent an average ± S.D. for n = 5 independent experiments. C, time-lapse confocal microscopy. bEnd.3 monolayers, expressing either GFP-claudin-5 or GFP-occludin, were exposed to CCL2 (100 ng/ml). Images were obtained every 5 min over a time period of 0–120 min. After 60 min of treatment, CCL2 was removed from cells by replacing media with fresh DMEM, and the cells were observed for an additional 60 min. The representative images show some critical time points during CCL2-induced alterations in brain endothelial barrier permeability and during recovery. There is a fragmented pattern of occludin and claudin-5 staining at the cell-cell border of endothelial cells during CCL2 exposure, but this returns to a “control” pattern of staining along the cell borders after CCL2 removal. *, the location shown in magnification in the lower images; arrows indicate alterations in the localization GFP-claudin-5 and GFP-occludin in the presence of CCL2. Scale bar, 20 μm. D, biochemical changes in occludin and claudin-5 after CCL2 treatment. There was a redistribution of these proteins first from the membrane fraction (MF, Triton X-100-insoluble) to cytosolic (CF, Triton X-100-soluble) and actin cytoskeletal (ACF, Triton X-100-insoluble) fractions during CCL2 exposure and back to membrane fraction after removing CCL2 stimulus. To demonstrate the purity of the different fractions, cytochrome P-450 reductase, calpain, and vimentin were used as specific markers for membrane, cytosolic, and actin cytoskeletal fractions, respectively. While affecting fractional distribution of claudin-5 and occludin, CCL2 and its removal did not affect the total protein content of either protein. Blots represent one of three successful experiments.