438 - MTOR limits endothelial-neutrophil interaction by suppressing ICAM-1 expression

Poster Number: 438
Publication Number: 438.430

Jin Whan Lim, University of Rochester School of Medicine and Dentistry, Rochester, NY, United States; Michelle Millar, University of Rochester, Rochester, NY, United States; Rauf A. Najar, University of Rochester School of Medicine and Dentistry, Rochester, NY, United States; Arshad Rahman, University of Rochester, Rochester, NY, United States

Background: Increased expression of intercellular adhesion molecule-1 (ICAM-1) leading to endothelial cell (EC)-neutrophil interaction is a critical component of inflammatory diseases such as acute lung injury.  Mechanistic Target of Rapamycin (MTOR) is a master regulator of cell growth and metabolism whose level is reduced in the inflamed lung.  However, it is unclear if a similar loss of MTOR in EC is linked to ICAM-1-mediated endothelial-neutrophil interaction. 

Objective: To examine if MTOR signaling is sufficient to limit EC-neutrophil interaction by suppressing ICAM-1 expression in EC.  To this end, we determined whether loss of MTOR in EC was sufficient to increase ICAM-1 expression and endothelial adhesivity toward neutrophils.

Design/Methods: Human Pulmonary Artery Endothelial Cells (HPAEC) were transfected with MTOR-specific siRNA (si-MTOR) to deplete MTOR. MTOR-depleted EC were analyzed by immunoblotting for ICAM-1 protein levels in the absence and presence of Y-27632, an inhibitor of Rho-associated kinase (ROCK). MTOR-depleted EC were also analyzed for cell surface expression of ICAM-1 by immunofluorescence.  The adhesive capacity of MTOR-depleted EC was assessed by neutrophil adhesion assay. In some adhesion assays, EC were briefly (~20 min) exposed to phorbol esters (PMA) or lipopolysaccharides (LPS) prior to incubation with neutrophils. The intent of these experiments was to test if brief PMA or LPS exposure (which is unlikely to cause new ICAM-1 protein synthesis) can increase the neutrophil-binding capacity of EC by inducing clustering of the existing ICAM-1. Data were analyzed by Student’s t-test or ANOVA. P < 0.05 was considered statistically significant.

Results: MTOR-depleted EC showed a dramatic increase in the protein and cell surface expression of ICAM-1 (Fig. 1A-C). Inhibiting ROCK caused a significant decrease in ICAM-1 protein levels in MTOR-depleted EC (Fig. 2). MTOR-depleted EC also displayed increased adhesivity toward naïve neutrophils (Fig. 3A&B). Exposure to PMA further increased the adhesivity of both control and MTOR-depleted EC toward neutrophils (Figs. 3C&D vs. 3A&B). LPS, a clinically relevant agonist, also increased EC adhesivity toward neutrophils (Fig. 3E). Conclusion(s): Loss of MTOR signaling in EC is sufficient to induce ICAM-1 expression via a ROCK-dependent mechanism and promote EC-neutrophil interaction, which may be further increased under inflammatory conditions, possibly via ICAM-1 clustering.  Thus, MTOR functions as an anti-inflammatory molecule in EC to limit neutrophil adhesion and migration across endothelial barrier
Figure 1. Depletion of MTOR in human pulmonary artery endothelial cells (HPAEC) induces ICAM-1 expression.HPAEC were transfected with Control or MTOR siRNA (si-Con or si-MTOR) for 48 h. (A&B) Cells were lysed and Western blot analysis was performed to determine (A) MTOR or (B) ICAM-1 levels. GAPDH levels were used to monitor loading. (C) Cells were fixed and stained with anti-ICAM-1 antibody (green) to mark cell surface ICAM-1 and DAPI (blue) to mark nuclei. surface ICAM-1 and DAPI (blue) to mark nuclei.
Figure 2. Inhibition of ROCK reduces ICAM-1 levels in MTOR-depleted EC.HPAEC were transfected with si-Con or si-MTOR. At 22 h after transfection, cells were treated with ROCK inhibitor (Y-27632; 10 uM). At 48 h after transfection, cells were lysed and analyzed by Western blot to measure ICAM-1 levels. Actin levels were used to monitor loading.