Differential effects of volatile anesthetics on leukocyte integrin macrophage-1 antigen

Macrophage-1 antigen (Mac-1, M2) is a leukocyte adhesion molecule that plays a significant role in leukocyte crawling and phagocytosis, and is homologous to its sister protein leukocyte function-associated antigen-1 (LFA-1, L2). The authors have previously demonstrated that volatile anesthetics isoflurane and sevoflurane bound to and inhibited LFA-1. Here, the hypothesis tested was that isoflurane and sevoflurane would inhibit Mac-1. A binding assay of Mac-1 to its ligand inter-cellular adhesion molecule-1 (ICAM-1) using V-bottom plates was established. The effect of isoflurane and sevoflurane on Mac-1 was examined using this ICAM-1 binding assay and by probing exposure of activation-sensitive epitopes. The docking program Glide was used to predict anesthetic binding site(s) on Mac-1. The functional role of this predicted binding site was then assessed by introducing point mutations in this region. Lastly, the effect of anesthetic on activating mutants was evaluated. The results indicated that isoflurane inhibited binding of Mac-1 to ICAM-1, but sevoflurane did not. Isoflurane also attenuated the exposure of the activation-sensitive epitopes. The docking simulation predicted the isoflurane binding site to be at the cavity underneath the 7 helix of the ligand binding domain (the M I domain). Point mutants at this predicted binding site contained both activating and deactivating mutants, suggesting its functional significance. The binding of activating mutants M-Y267A 2-WT and M-L312A 2-WT to ICAM-1 was not affected by isoflurane, but binding of another activating mutant M-WT 2-L132A was inhibited supporting the binding of isoflurane to this cavity. The conclusion reached from these findings was that isoflurane inhibited Mac-1 binding to ICAM-1 by binding to the cavity underneath the 7 helix of the M I domain, but sevoflurane did not. Thus, because these common clinical volatile anesthetics demonstrated different effects on Mac-1, this implied their effects on the immune system might differ.