Fig. 1.

Env-ms specifically induces over-expression of ICAM-1 on HCMEC/D3 cells but not Env-syn. (A) Indirect immunostaining of ICAM-1 (red) on untreated HCMEC/D3 cells or after overnight treatment with Env-ms at 2 µg ml⁻¹. Nuclei are stained in blue. Scale bar: 20 µm. (B and C). Flow cytometry analysis of ICAM-1 expression after overnight treatment of HCMEC/D3 cells (B) with TNF-α (100U ml⁻¹) or Env-ms (2 µg ml⁻¹) (NT: not treated) and (C) with TNF-α (100U ml⁻¹), Env-ms or Env-syn. Values are means ± SE of fluorescence intensity in two independent experiments each performed in duplicate. Negative control: an irrelevant antibody of the same isotype (IgG1) as the monoclonal antibody specific for ICAM-1. (D) Inhibition using monoclonal antibodies. HCMEC/D3 cells were stimulated overnight with Env-ms (2 µg ml⁻¹) pre-incubated for 40min at 4°C with mAbs (30 µg ml⁻¹) specific for Env-ms (GN-mAb03) or gag (GN-mAb12) proteins. ICAM-1 expression was then measured by flow cytometry. Results represent the mean of the fold of increase (ratio between mean of fluorescence of the activated cells and untreated cells) ± SE of three independent experiments. (E) PB inhibition assay: HCMEC/D3 cells were treated overnight with Env-ms (2 µg ml⁻¹) or LPS (0.1 µg ml⁻¹), pre-incubated or not for 30min at 37°C with PB (25 µg ml⁻¹). ICAM-1 expression was measured by flow cytometry (see above). Results represent the mean ± SE of two independent experiments. (F) Env-ms stimulates the adhesion between immune cells and the endothelial monolayer. Results represent means of fold of increase compared to untreated condition ± SE of two independent experiments each performed in triplicate. (G) Env-ms (2 µg ml⁻¹) stimulates the transmigration of HL-60 cells through the endothelial monolayer. TNF (100U ml⁻¹) was used as a positive control for transmigration. Results represent means of fold of increase compared to untreated condition ± SE of three independent experiments each performed in duplicate. *P ≤ 0.05; **P ≤ 0.01.