First published online 24 October 2006
doi: 10.1242/jcs.03222
Journal of Cell Science 119, 4634-4643 (2006)
Published by The Company of Biologists 2006
Junctional expression of the prion protein PrPC by brain endothelial cells: a role in trans-endothelial migration of human monocytes
Pedro Viegas1,2,3,4,
Nathalie Chaverot1,2,3,4,
Hervé Enslen5,6,7,
Nicolas Perrière1,2,3,4,
Pierre-Olivier Couraud1,2,3,4 and
Sylvie Cazaubon1,2,3,4,*
1 Institut Cochin, Département Biologie Cellulaire, Paris 75014, France
2 Inserm, U567, Paris 75014, France
3 CNRS, UMR 8104, Paris 75014, France
4 Université Paris 5, Faculté de Médecine René Descartes, UM 3, Paris 75014, France
5 Institut du Fer à Moulin, Paris 75005, France
6 Inserm, U536, Paris 75005, France
7 Université Pierre et Marie Curie (UPMC-Paris 6), Paris 75005, France
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Fig. 1. Expression and localization of PrPC in brain blood vessels. Transmission and immunofluorescence labeling of PrPC and ZO-1 of brain sections from wild-type (WT) and PrPC-deficient knockout (KO) mice. Sections of striatum were incubated with anti-PrPC monoclonal antibodies (SAF32 plus SAF61, both at 1 µg/ml) and anti-ZO-1 polyclonal antibodies (5 µg/ml) for 16 hours at 4°C, then with Alexa Fluor 488-conjugated anti-mouse antibodies and Cy3-conjugated anti-rabbit antibodies for 45 minutes at 25°C. Images were collected with a confocal fluorescence microscope. Arrows in the left panel indicate blood vessels; insets show microvessels (chosen from a different field). Bars, 40 µm or 10 µm (inset).
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Fig. 2. Expression of PrPC at the cell-cell contacts of primary brain endothelial cells. Immunofluorescence labeling of PrPC, PECAM-1 and ZO-1 in freshly isolated brain microvessels. Cells were incubated with anti-PrPC (SAF32, 2 µg/ml), anti-PECAM-1 (3A12, 2 µg/ml) or anti-ZO-1 (1 µg/ml) antibodies for 16 hours at 4°C, then with Cy2-conjugated anti-mouse antibodies or Cy3-conjugated anti-rabbit antibodies for 45 minutes at 25°C. Images were collected with a confocal fluorescence microscope. Bars, 20 µm (A, xy), 10 µm (A, z), 10 µm (B).
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Fig. 3. Expression of PrPC in brain endothelial cell lines. (A) Immunofluorescence labeling of PrPC in rat (RBE4) and human (hCMEC/D3) brain endothelial cell lines. Cells were incubated with anti-PrPC (SAF32, 2 µg/ml) monoclonal antibodies for 16 hours at 4°C, then with Cy2-conjugated anti-mouse antibodies for 45 minutes at 25°C. Images were collected with a confocal fluorescence microscope. Bars, 20 µm. (B) RT-PCR detection of PrPC transcripts in rat (RBE4) and human (hCMEC/D3) brain endothelial cells; primary rat astrocytes (Astro) and the human U373 astrocytoma line (U373) were used as positive controls, respectively. Rat HPRT or human GAPDH were used for standardization. The size of the transcripts (rat: 244 bp; human: 243 bp) was as expected. Control lanes show no amplified fragments in the absence of cDNA.
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Fig. 4. Co-localization of PrPC and PECAM-1 in raft/caveolae-like microdomains in brain endothelial cells. (A) RBE4 cells were harvested by scraping and submitted to subcellular fractionation in the presence of sodium carbonate as described in the Materials and Methods. Fractions (1-10) were collected from the top of the gradient and proteins in each fraction were concentrated by precipitation with 10% trichloroacetic acid. Equal volumes (15 µl) of concentrated fractions were analyzed by SDS-PAGE and processed for immunoblot analyses. Membranes were incubated with antibodies to caveolin-1 (0.5 µg/ml), PrPC (SAF32, 1 µg/ml), PECAM-1 (M20, 1 µg/ml) or ß-catenin (1 µg/ml). PrPC and PECAM-1 are detected in fraction 4 containing raft/caveolae-like microdomains; PrPC appears as a typical smear, as seen in the whole cell lysate (panel B). Other forms of PrPC were detected in fraction 10, which contains the majority of cellular proteins, including ß-catenin; these forms of PrPC probably correspond to intermediate glycosylation forms. (B) Whole RBE4 cell lysate was treated (+) or not (–) with PNGase F and processed for western blot analyses using SAF32 anti-PrPC antibodies (1 µg/ml). Following PNGase F treatment (+) a single band of approximately 25 kDa is detected, corresponding to unglycosylated PrPC. The results are representative of three independent experiments.
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Fig. 5. Translocation of PrPC to intercellular junctions is induced by cell-cell contacts. Immunofluorescence labeling of PrPC and PECAM-1 in a pre-confluent culture of RBE4 cells. Nonpermeabilized cells were incubated with anti-PrPC (SAF32) (A) or anti-PECAM-1 (3A12) antibodies (B) at 2 µg/ml for 16 hours at 4°C, then with Cy2-conjugated anti-mouse antibodies for 45 minutes at 25°C. Images were collected with a confocal fluorescence microscope. Arrows indicate PrPC (A) or PECAM-1 (B) accumulation at cell-cell contacts. Bars, 20 µm.
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Fig. 6. Translocation of PrPC to intercellular junctions is dependent on PrPC expression by adjacent cells. Double immunofluorescence labeling of PrPC and ß-catenin in a mixed primary culture of wild-type (WT) and PrPC-deficient knockout (KO) mouse brain endothelial cells. Cells were permeabilized with 0.05% saponin for 1 hour before incubation with anti-PrPC (SAF32, 2 µg/ml) monoclonal antibody plus anti-ß-catenin rabbit polyclonal antibodies (1 µg/ml) for 16 hours at 4°C. ß-catenin and PrPC were revealed using Cy2-conjugated anti-rabbit antibodies (A) and Cy3-conjugated anti-mouse antibodies (B), respectively. Images were collected with a confocal fluorescence microscope. Arrows indicate a junction between two WT cells showing a positive staining for ß-catenin (A) and PrPC (B), arrowheads indicate a junction between one WT cell and one KO cell showing a positive staining for ß-catenin (A) but no staining for PrPC (B). A merged image of the same field is presented in the right hand panel (merged), with PrPC staining in red and ß-catenin staining in green: co-localization (yellow) of PrPC and ß-catenin is observed only at junctions between two adjacent WT cells. Bar, 20 µm.
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Fig. 7. Expression of PrPC, PECAM-1 and VLA-4 by U937 human monocytic cells. U937 cells were incubated with anti-PrPC (SAF32), anti-PECAM-1 (HEC-7) or anti-VLA-4 antibodies (2 µg/ml) for 10 minutes at 25°C, then with FITC-conjugated anti-mouse antibodies. Samples were analyzed by flow cytometry and at least 5000 cells were counted. Black areas represent staining profiles with the indicated antibodies, gray areas represent staining profiles with first antibodies omitted as negative control.
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Fig. 9. Transmigration of U937 monocytic cells through human brain endothelial cells is prevented by anti-PrPC antibodies. (A) hCMEC/D3 cells were grown to confluence on Transwell filters with 3 µm pore size and activated (+) or not (–) with TNF- (200 U/ml) and IFN- (200 U/ml) for 24 hours. CMFDA-labeled U937 monocytic cells were then added and allowed to transmigrate for 16-18 hours in the presence (+) or absence (–) of SDF-1 (100 ng/ml) in basal compartments. Transmigrated U937 cells were counted by FACS analysis of basal compartments. Results are presented as percentages of transmigrated cells. Average values (± s.e.m.) from one representative experiment out of three independent experiments performed in triplicate are presented. (B) The transmigration assay was performed as above in the presence of SDF-1 (100 ng/ml), following separate incubation of activated hCMEC/D3 cells and U937 monocytic cells with the indicated antibodies at 20 µg/ml for 1 hour. Antibodies tested were specific for: the irrelevant membrane protein CD71; PECAM-1 (HEC-7 antibody), known to support monocyte transmigration; or PrPC (SAF32, SAF34, SAF61, 6H4 antibodies). Average values (± s.e.m.) are presented of triplicates from three to five independent experiments with 100% being the number of transmigrated U937 cells following incubation with anti-CD71 irrelevant antibodies (*P<0.01 versus cells pre-treated with anti-CD71 antibody). (C) Migration of CMFDA-labeled U937 monocytic cells was performed through endothelial-cell-free filters coated with fibronectin and collagen, for 16-18 hours in the presence of SDF-1 (100 ng/ml) in basal compartments. Cells were preincubated with the indicated antibodies at 20 µg/ml for 1 hour before the migration assay, as above, with anti-CD71 or anti-PrPC (SAF34, SAF32) antibodies. Results are presented as percentages (%) of transmigrated cells with 100% being the number of transmigrated U937 cells following incubation with anti-CD71 irrelevant antibodies. Average values (± s.e.m.) from one representative experiment out of three independent experiments performed in triplicate are presented. Numbers of migrated U937 cells following incubation with SAF32 or SAF34 anti-PrPC antibodies were not statistically different from control values (migrated U937 cells following incubation with anti-CD71 antibodies), indicating that pre-incubation with the indicated antibodies did not differentially affect the migration capacity of U937 cells.
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Fig. 11. PrPC expressed by monocytes and endothelial cells is involved in transmigration of freshly isolated monocytes. Freshly isolated monocytes were labeled as above and separately incubated (monocytes) with anti-CD71, anti-PECAM-1 or anti-PrPC antibodies at 20 µg/ml for 1 hour, as indicated. Similarly, hCMEC/D3 endothelial cells, grown and activated as above, were separately incubated in the same conditions (hCMEC/D3). In parallel, both monocytes and hCMEC/D3 endothelial cells (monocytes + hCMEC/D3) were separately incubated in the same conditions. Monocytes were then added to the apical compartments of Transwell filters and allowed to transmigrate for 3 hours in the presence of MCP-1 (25 ng/ml) in basal compartments, as above. Average values (± s.e.m.) from one representative experiment out of three independent experiments are presented. The number of migrated cells following incubation with CD71 antibodies was taken as 100% in each condition (*P<0.01 versus cells pre-treated with anti-CD71 antibody).
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© The Company of Biologists Ltd 2006
