Fig. 3. Effect of MCP-1 on actin cytoskeleton and intercellular TJs. Confluent CCR2^+/+ and CCR2^–/– mBMEC, were treated with recombinant mouse MCP-1 (100 nM) for the indicated time period (15 minutes and 2 hours) or served as normal controls (control). The cells were then fixed and stained with anti-occludin, ZO-1, ZO-2, claudin-5 antibodies or phalloidin Alexa 488 for F-actin. Untreated quiescent mBMEC (control, 7 days after initial plating) showed a typical polygonal shape, with actin filament distributed primarily in the cortical ring with a few stress fiber spanning the cells. They also had very specific continuous staining for occludin, ZO-1, ZO-2 and claudin-5 localized along the cell margins, possibly at the sites of cell-cell contact. In CCR2^+/+ cells, treatment with MCP-1 induced marked structural alterations in the distribution of actin filaments and TJ proteins in a time-dependent manner. In the absence of CCR2 receptors, the effect of MCP-1 on the actin cytoskeleton and TJ proteins were abrogated. Scale bar: 200 µm.

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