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First published online 28 October 2008
doi: 10.1242/jcs.030627

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Involvement of endothelial ephrin-B2 in adhesion and transmigration of EphB-receptor-expressing monocytes

¹ Joint Research Division Vascular Biology, Medical Faculty Mannheim, University of Heidelberg, and German Cancer Research Center (DKFZ-ZMBH Alliance), Heidelberg, Germany
² Institute of Neurology, Frankfurt University Medical School, Frankfurt, Germany
³ Group Microenvironment of Tumor Cell Invasion, German Cancer Research Center (DKFZ), Heidelberg, Germany
⁴ Institute of Physiology and Pathophysiology, Division of Cardiovascular Physiology, University of Heidelberg, Germany

View larger version (40K): [in this window] [in a new window]   Fig. 1. FACS analysis of EphBR expression in different hematopoietic cells. (A) FACS analysis of mouse bone-marrow-derived hematopoietic cells, peripheral blood leukocytes, splenocytes and macrophages (F4/80+) assessing EphBR expression by ephrin-B2-Fc binding (gray area) compared with Fc-control staining (white area). (B) FACS analysis of human monocytes (CD14+) and two monocytic cell lines (human U937, mouse J774) assessing EphBR expression by staining for ephrin-B2–Fc (gray area) compared with Fc control staining (white area). (C) FACS analysis of human monocytes (CD14+) that express EphB2 and EphB4 by using relevant antibodies (gray area) compared with IgG-control (white area).

View larger version (37K): [in this window] [in a new window]   Fig. 2. Expression of ephrin-B2 and C-ephrin-B2 in HUVECs and PAE cells. (A) Bright-field (BF) imaging and GFP live imaging of mock-transduced, ephrin-B2- and C-ephrin-B2-overexpressing HUVECs. Scale bar: 50 µm. (B) Ephrin-B2 and C-ephrin-B2 overexpression in porcine aortic ECs. (PAEC) detected by immunoprecipitation with EphB4-Fc followed by western blotting using anti-ephrin-B2 antibody. (C) MTT-proliferation assay of transduced HUVECs assayed over a period of 96 hours. (D) Confocal microscopy analysis of ephrin-B2 expressed in ECs validating its apical expression. Scale bar: 10 µm.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Preferential adhesion of EphBR-expressing monocytes to ephrin-B2-expressing ECs. (A,B) Adhesion of human U937 or mouse J774 cells to HUVEC monolayers. Adhesion was quantified after 30 minutes of incubation on a rocking platform. U937 and J774 cells adhered preferentially to ephrin-B2-overexpressing HUVECs, but not to C-ephrin-B2-overexpressing or mock-transduced HUVECs. (C) Adhesion of mouse J774 cells to HUVEC monolayers for 16 hours leading to saturation-level adhesion showing no difference to the three transduced monolayers. (D) Adhesion of J774 cells to HUVEC monolayers under flow conditions. Adhesion was quantified after 30 minutes of incubation under flow. J774 cells adhered preferentially to ephrin-B2-overexpressing HUVECs, but not to C-ephrin-B2-overexpressing or mock-transduced HUVECs. Values are expressed as the mean ± s.d. of one representative experiment of two independent experiments with similar results (**P<0.01).

View larger version (35K): [in this window] [in a new window]   Fig. 4. Single-cell force spectroscopy (SCFS) to study the adhesive strength of EphBR–ephrin-B2 interactions in a cellular context. (A) Model of SCFS. An individual J774 cell was attached to a cantilever and moved towards the surface of HUVECs that overexpressed ephrin-B2 or C-ephrin-B2, or towards mock-transduced HUVECs. The cantilever was then moved in the opposite direction to separate the cells following a cell-cell contact time of 1 minute. The adhesive force was calculated from the deflection of the cantilever and its spring constant. (B) J774 cell attached to the tip of the cantilever (left). Cantilever above the HUVEC layer (right). (C) Average maximum unbinding force and work needed to detach the J774 cell from ephrin-B2- or C-ephrin-B2-transduced HIVECs or from mock-transduced HUVECs. Statistical analysis using a one-way ANOVA followed by a multiple pairwise comparison (Holm-Sidak) revealed that there was no significant difference between either ephrin-B2 or C-ephrin-B2 whereas the difference between mock and both experimental groups was significant (*P<0.05). Average values were calculated from n=24-30 individual force-distance measurements. The average work required to separate the cells completely from each other was represented by the area under curve of the force plot. Scale bars: 50 µm.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Blocking of monocyte adhesion by using specific antibodies against EphBRs and ephrin-B2. (A) Pre-incubation of J774 cells with an anti-EphB2 or anti-EphB4 antibodies prior to seeding on HUVEC monolayers blocked the preferential adhesion to ephrin-B2-overexpressing HUVECs. Blocking with anti-EphB2 and anti-EphB4 antibodies further augmented the effect. (B) Pre-incubation of the HUVEC monolayer with anti-ephrin-B2 antibody prior to U937 cell seeding blocked the preferential adhesion to ephrin-B2-overexpressing HUVECs. Values are expressed as the mean ± s.d. of one out of three independent experiments with similar results (*P<0.05; **P<0.01).

View larger version (25K): [in this window] [in a new window]   Fig. 6. Augmented adhesion of EphB4-overexpressing monocytic cells to ephrin-B2-overexpressing ECs. (A) Western blot analysis of mock-transduced mouse J774 cells (J774), EphB4-GFP-overexpressing J774 cells (J774 EphB4) and C-EphB4–GFP-overexpressing J774 cells (J774 C-EphB4). All three cell populations expressed endogenous EphB4 as reflected by a faint, but detectable band at 130 kDa (middle arrow). Full-length EphB4-GFP (>130 kDa) and truncated C-EphB4–GFP (<130 kDa) are marked accordingly. Actin served as loading control. (B) Adhesion of mock-transduced, and EphB4- and C-EphB4-overexpressing J774 cells to ephrin-B2-overexpressing and mock-transduced HUVECs. Cells were incubated for 30 minutes on a rocking platform. Endogenous EphB4 expression facilitated the preferential adhesion to ephrin-B2-overexpressing HUVECs compared with mock-transduced HUVECs, confirming the findings obtained with J774 cells (Fig. 3). Overexpression of full-length EphB4 but not C-EphB4 in J774 cells further augmented the preferential adhesion of J774 cells to ephrin-B2-expressing HUVECs. Values are expressed as the mean ± s.d. of one out of three independent experiments with similar results (**P<0.01).

View larger version (51K): [in this window] [in a new window]   Fig. 7. Translocation of the EphBR–ephrinB complex to inter-endothelial junctions prior to internalization. HUVECs that overexpress full-length ephrin-B2 or C-ephrin-B2 were stimulated for various periods of time with EphB4-Fc after which monolayers were trypsinized, lyzed and processed for western blot analysis. (A) Ephrin-B2-overexpressing HUVECs presented ephrin-B2 on the cell surface. Trypsin treatment removed surface-expressed ephrin-B2 (lane 2 vs lane 1). Internalized ephrin-B2 was detectable within 15 minutes of EphB4-Fc stimulation. Maximal internalization of the EphB4–ephrin-B2 complex was detected after 30-60 minutes. Internalized ephrin-B2 was eventually degraded, as evidenced by the absence of ephrin-B2 after 4 hours. (B) By contrast, C-ephrin-B2, albeit being correctly expressed at the cell surface (B, lane 2 vs lane 1), was not endocytoced following EphB4-Fc stimulation; an unspecific band was detectable at the same size as C-ephrin-B2 (arrow in B). Yet, this band was also detectable in full-length ephrin-B2 overexpressing cells (arrow in A). (C) HUVECs that overexpress full-length ephrin-B2 or C-ephrin-B2 were stimulated with EphB4-Fc for various periods of time after which monolayers were fixed and stained for ephrin-B2. Unstimulated HUVECs overexpressed ephrin-B2 uniformly on their cell surface (0 min). Stimulation with EphB4-Fc for 20 minutes led to the translocation of the resulting EphB4–ephrin-B2 and EphB4–C-ephrin-B2 complex to inter-endothelial junctions (20 min). Complexes of full-length ephrin-B2 and EphB4-Fc were internalized within 60 minutes. By contrast, C-ephrin-B2 complexes with EphB4-Fc were retained at inter-endothelial junctions (60 min). Analysis by fluorescence microscopy. (D) HUVECs that overexpress full-length ephrin-B2 or C-ephrin-B2 were stimulated with EphB4-Fc for 30 minutes after which monolayer were fixed and stained for ephrin-B2 and VE-cadherin. Vascular integrity was not affected as evidenced by the unaltered junctional VE-cadherin expression. Ephrin-B2 did not colocalize with VE-cadherin; analysis by single scan confocal microscopy showing a single plain through the cell (notice the difference compared with images shown in C). Scale bars: 50 µm

View larger version (50K): [in this window] [in a new window]   Fig. 8. Preferential transmigration of EphBR-expressing monocytes through ephrin-B2-expressing EC monolayers. Mock-transduced cells as well as ephrin-B2- and C-ephrin-B2-overexpressing HUVEC were grown to confluence on gelatine-coated membrane filters. Confluence was monitored by calcein staining (top). J774 cells were allowed to transmigrate for 24 hours towards FCS. Quantification of transmigrated J774 cells revealed the preferential transmigration of J774 cells through ephrin-B2-overexpressing HUVEC monolayers but not through C-ephrin-B2-overexpressing or mock-transduced HUVEC monolayers (center). Representative images show PKH26-labeled transmigrated J774 cells on the lower side of the Transwell membrane (bottom). Values are expressed as the mean ± s.d. of one out of three independent experiments with similar results (*P<0.05). Scale bars: 500 µm.

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