Figure 1. Human brain endothelial cells internalize myelin particles.

Confluent monolayers of brain endothelial cells (BECs) were stimulated with 5 ng/ml TNFα for 24 hr. (A) Expression of MHC-I, MHC-II, CD40, PD-L1 and VCAM−1 was determined by flow cytometry. Histograms depict expression of indicated markers in resting (grey solid line) and activated (black solid line) BECs. Dashed lines indicate isotype controls. (B) The MFI of expression of the indicated markers is shown. Data are presented as the mean ± SD of duplicate values (n = 5 independent experiments). *p<0.05, **p<0.01, ***p<0.001 (Student t-test). (C–E) Fluorescent labeled human myelin was added to resting or activated BECs for 4 hr or 24 hr and uptake was analyzed by (C) flow cytometry or (D–E) imaging flow cytometry. (C) Representative facs plots of myelin uptake by BECs, numbers in plots indicate the MFI of myelin-positive cells. The percentage of myelin-positive resting and activated BECs at 4 and 24 hr after loading with antigen is shown in a graph. (D–E) Myelin-positive BECs internalized between 1–3 particles/cell. On average, BECs acquired 2–3 myelin particles/cell. Activation of BECs did not affect the number of internalized particles. The average number of internalized myelin particles per cell is shown in a bar graph. Data presented are the means of triplicate values ± SEM of at least three independent experiments. *p<0.05, **p<0.01, ***p<0.001 (Student t-test).

DOI: http://dx.doi.org/10.7554/eLife.13149.003

Figure 1—figure supplement 1. Human brain endothelial cells increase MHC and costimulatory molecule expression upon activation by IFN-γ.

Confluent monolayers of brain endothelial cells (BECs) were stimulated with 10 ng/ml IFN-γ for 24 hr. Expression of MHC-I, MHC-II, CD40 and VCAM−1 was determined by flow cytometry. Histograms depict expression of indicated markers in resting (orange line) and activated (green line) BECs. Red and blue lines indicate isotype controls.

DOI: http://dx.doi.org/10.7554/eLife.13149.004

Figure 1—figure supplement 2. Brain endothelial cells internalize myelin particles.

BECs were loaded with fluorescently labeled human myelin and internalization of myelin particles was assessed by imaging flow cytometry. To determine internalization scores, a mask was designed based on the surface of BECs in the brightfield image. This mask was then eroded to exclude the cell membrane. The resulting mask was applied to the fluorescence channel. The internalization score, interpreted as a ratio of the intensity of the intracellular space versus the intensity of the whole cell, was calculated on this mask using the internalization feature of the Ideas v6.0 software (AMNIS Merck Millipore). Cells that have internalized antigens have positive scores, as depicted here for BECs.

DOI: http://dx.doi.org/10.7554/eLife.13149.005