Regulation of Endothelial Intracellular Glutathione by ICAM‐1

Recent work from our lab has uncovered a role for ICAM‐1 in regulating endothelial glutathione levels. Here we provide further evidence for the involvement of this adhesion molecule in this process. Using cross‐linking antibodies, total GSH was measured after ICAM‐1 ligation on confluent endothelium. Next, confluent cells were treated overnight with 1, 10, or 100 ng/mL soluble ICAM‐1 (sICAM‐1) followed by total GSH measurements. Lastly, confluent endothelial cells were treated overnight with 10, 25, or 50 μM of a cell permeable peptide containing the transmembrane domain and cytoplasmic tail of ICAM‐1, and then GSH was measured. Cross linking ICAM‐1 on the endothelial surface, thus mimicking leukocyte adhesion, induced a significant increase in GSH levels at 16 hrs (135.02±15.32 nmol/mg protein vs 74.99±2.75 nmol mg/protein for untreated). Soluble ICAM‐1 at 1 ng/mL also significantly elevated endothelial GSH levels (88.52±9.76 nmol/mg protein for 1 ng/mL sICAM‐1 vs. 56.88±3.79 nmol/mg protein for untreated), and treatment with 25 and 50 μM of a cell permeable C‐terminal fragment of ICAM‐1 significantly increased GSH levels compared to the control peptide (36.14±1.07 and 39.65±1.27 nmol/mg protein for 25 and 50 μM ICAM‐1 peptide, respectively, vs. 19.82±1.15 and 24.39±0.63 nmol/mg protein for 25 and 50 μM control peptide, respectively). These data provide novel evidence that ICAM‐1 regulates endothelial cell redox status through modulation of pathways controlling intracellular glutathione.