Fig 1: CD31 interactions promote the recovery of endothelial integrity following endothelial contraction induced by MHC molecule triggering.a–d Following MHC or ICAM-1 and/or CD31 antibody-mediated co-ligation for 30 min, EC were fixed and stained with rhodamine-phalloidin. Images taken on EC monolayers seeded at identical density are shown in (a, b). The average F-actin intensity per cell of three independent experiments is shown in (c, d). Scale bar, 20 µm. (n = 3 biologically independent samples, N = 3 independent experiments, data are mean ± SD). One-way Anova with Tuckey post-hoc test. MHC vs all ****p < 0.0001, MHC + CD31 vs Isc ****p < 0.0001, MHC + CD31 vs all ****p < 0.0001. e Western blot (WB) analysis of Erk activation by WT and cd31-/- EC 30 min after MHC stimulation. The bar graph shows relative protein expression ± SEM. N = 3 independent experiments (data are mean ± SD). One-way Anova with Tuckey post-hoc test. cd31-/- MHC vs cd31-/- IsC ***p = 0.0002, cd31-/- MHC vs all ****p < 0.0001. f Western blot (WB) analysis of RhoA activation by WT and cd31-/- EC 30 min after MHC stimulation. The bar graph shows relative protein expression ± SEM. N = 3 independent experiments (data are mean ± SD). One-way Anova with Tuckey post-hoc test. cd31-/- 15' vs all ***p = 0.0003, cd31-/- 30' vs all ****p < 0.0001. g Immunoprecipitation of CD31 molecules from WT EC exposed to MHC/ICAM-1 stimulation for 30 min followed by immunoblotting with an anti-phosphotyrosine antibody and an anti-SHP2 antibody. The bar graph shows relative protein expression ± SEM. N = 3 independent experiments. One-way Anova with Tuckey post-hoc test. pTyr20 MHC vs all ****p < 0.0001, SHP2 MHC vs all ****p < 0.0001, pTyr20 ICAM-1 vs all ****p < 0.0001. h WT or cd31-/- mice (n = 6 mice, N = 2 independent experiments) received anti-MHC and secondary cross-linking antibody (0.67 µg and 0.33 µg/kg body weight, respectively) in saline solution i.v. After 3 or 6 h, 100 µL of 2% Evans blue in saline solution was injected. Dye was allowed to circulate for 45 min before organs dye content was assessed spectrophotometrically and normalized to plasma levels (data are mean ± SEM). One-way Anova with Tuckey post-hoc test. Lung cd31-/- vs WT *p = 0.0352, heart cd31-/- vs WT **p = 0.0045, kidney cd31-/- vs WT ***p = 0.0009, liver cd31-/- vs WT **p = 0.0071, spleen cd31-/- vs WT **p = 0.002. i H-2B-specific alloreactive T-cells were obtained by stimulation of Balb/C mice-derived splenocytes (H2-D) with sublethally irradiated WT splenocytes in the presence of 20 U/ml IL-2. T-cells were harvested 72 h after stimulation and seeded (105/well) onto confluent WT or cd31-/- EC monolayers grown on 0.2 µm-pore transwells and previously treated with IFN-? for 48 h to upregulate MHC expression. TEER was measured as described in the Methods section. n = 3 biologically independent samples, N = 2 independent experiments. (data are mean ± SD) One-way Anova with Tuckey post-hoc test. 3 h WT vs cd31-/- ****p < 0.0001, 6 h WT vs cd31-/- ****p < 0.0001, 24 h WT vs cd31-/- ***p = 0.0005. j WT or cd31-/- mice (n = 6 mice, N = 2 independent experiments) were treated either with saline solution or with IFN-? i.p. (70,000 U/mouse) i.p. After 48 h, some mice received an i.v. injection of the SEA superantigen (60 ng/mouse). After a further 4 h, 100 µL of 2% Evans blue in saline solution was injected. Dye was allowed to circulate for 45 min before organs dye content was assessed spectrophotometrically and normalized to plasma levels (data are mean ± SEM). One-way Anova with Tuckey post-hoc test. lung cd31-/- SEA vs WT SEA ***p = 0.0006, lung cd31-/- SEA vs cd31-/- PBS **p = 0.002, liver cd31-/- SEA vs WT SEA **p = 0.00124, liver cd31-/- SEA vs cd31-/- PBS ** = 0.004, heart cd31-/- SEA vs WT SEA ***p = 0.0007, heart cd31-/- SEA vs cd31-/- PBS **p = 0.0088, spleen cd31-/- SEA vs WT SEA ****p < 0.0001, spleen cd31-/- SEA vs cd31-/- ***p = 0.0002, kidney spleen cd31-/- SEA vs WT SEA ****p < 0.0001, kidney cd31-/- SEA vs cd31-/- ***p = 0.0008.