Fig. 4

Blocking β2-integrins rather than α4-integrins inhibits shear resistant T cell arrest on EECM-BMEC-like cells expressing high cell surface levels of ICAM-1. Number of arrested human CD4⁺ Th1* cells on 16–24 h pro-inflammatory cytokine stimulated (7.6 IU/mL TNFα + 2 IU/mL IFNγ) EECM-BMEC-like cells derived from a healthy donor (A) or an MS patient (B) under physiological flow. Th1* cells were preincubated with different natalizumab constructs (1 μg/mL NTZ or 1 μg/mL bNTZ or 1 μg/mL mNTZ) and/or with 1 μg/mL of a function-blocking anti-β2-integrin antibody. An isotype control antibody was used as internal control (1 μg/mL Ctrl). Flow cytometry analysis for the cell-surface expression of ICAM-1 (C) and VCAM-1 (D) on BLEC, HBMEC and EECM-BMEC-like cells under non-stimulated (NS) and 16 h pro-inflammatory cytokine stimulated conditions (76 IU/mL TNFα + 20 IU/mL IFNγ) are shown. Bar graphs show geometric ΔMFI (MFI specific staining—MFI isotype). A–D Each figure shows the mean ± SEM of 3 independent experiments. Statistical analysis: one-way ANOVA followed by Tukey’s multiple-comparison test (p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = ****). Immunofluorescence staining of non-stimulated (NS) and 16 h pro-inflammatory cytokine stimulated (76 IU/mL TNFα + 20 IU/mL IFNγ) BLEC (E), HBMEC (F) and EECM-BMEC-like cells derived from a healthy donor (G) and a MS patient (H). E Immunostainings for ICAM-1 (green) and VCAM-1 (green) and ZO-1 (red) on BLEC are shown. F–H Immunostainings for ICAM-1 (red) and VCAM-1 (red) on HBMEC and EECM-BMEC-like cells are shown. E–H Nuclei were stained with DAPI (blue). Data are representative of 3 independent experiments. Scale bar = E 100 μm and F–H 50 μm