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Figure 7 | Journal of Neuroinflammation

Figure 7

From: Sphingosine 1-phosphate receptor 5 mediates the immune quiescence of the human brain endothelial barrier

Figure 7

S1P 5 knockdown increases monocyte transmigration and adhesion. (a) Adhesion of primary human monocytes to hCMEC/D3, mock, and S1P5 cells was assayed in time. A significant increase in monocyte adhesion to S1P5 knockdown cells was already observed after 15 minutes (non-targeting control: 112.5% ± 3.4; S1P5 knockdown 159.0% ± 3.0 at 60 minutes, n = 6). (b) Monocyte migration across confluent monolayers of hCMEC/D3, mock, and S1P5 knockdown cells was assayed by time-lapse microscopy. A strong increase in monocyte migration of 193% was observed across S1P5 knockdown cells compared to mock cells (non-targeting control: 145.5% ± 2.6; S1P5 knockdown: 338.1% ± 34.2, n = 3). (c) VCAM-1 and ICAM-1 gene expression levels in mock and S1P5 knockdown cells were studied by qPCR. VCAM-1 gene expression increased 204% (non-targeting control: 100.0% ± 0.5; S1P5 knockdown 304.4 ± 18.8, n = 3) and ICAM-1 gene expression increased 80% (non-targeting control: 100.0% ± 10.6; S1P5 knockdown 179.6% ± 10.8, n = 3) in S1P5 knockdown cells compared to mock cells. (d) FACS analysis of ICAM-1 and VCAM-1 expression by mock cells and S1P5 knockdown cells demonstrated a 76% (non-targeting control: 100.0% ± 6.6; S1P5 knockdown: 175.8% ± 13.1, n = 3) increase in ICAM-1 protein expression and a 23% (non-targeting control: 100.0% ± 3.1; S1P5 knockdown: 122.8% ± 2.7, n = 3) increase in VCAM-1 protein expression by S1P5 knockdown cells. Statistical significance (t-test) is indicated with asterisks: *P < 0.05, **P < 0.005 and ***P < 0.001.

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