Microbubble Persistence in the Microcirculation During Ischemia/Reperfusion and Inflammation Is Caused by Integrin- and Complement-Mediated Adherence to Activated Leukocytes

Abstract

Background —Albumin microbubbles that are used for contrast echocardiography persist within the myocardial microcirculation after ischemia/reperfusion (I-R). The mechanism responsible for this phenomenon is unknown. Methods and Results —Intravital microscopy of the microcirculation of exteriorized cremaster muscle was performed in 12 wild-type mice during intravenous injections of fluorescein-labeled microbubbles composed of albumin, anionic lipids, or cationic lipids. Injections were performed at baseline and after 30 to 90 minutes of I-R in 8 mice and 2 hours after intrascrotal tumor necrosis factor-α (TNF-α) in 4 mice. Microbubble adherence at baseline was uncommon (<2/50 high-power fields). After I-R, adherence increased ( P <0.05) to 9±5 and 5±4 per 50 high-power fields for albumin and anionic lipid microbubbles, respectively, due to their attachment to leukocytes adherent to the venular endothelium. TNF-α produced even greater microbubble binding, regardless of the microbubble shell composition. The degree of microbubble attachment correlated ( r =0.84 to 0.91) with the number of adhered leukocytes. Flow cytometry revealed that microbubbles preferentially attached to activated leukocytes. Albumin microbubble attachment was inhibited by blocking the leukocyte β 2 -integrin Mac-1, whereas lipid microbubble binding was inhibited when incubations were performed in complement-depleted or heat-inactivated serum rather than control serum. Conclusions —Microvascular attachment of albumin and lipid microbubbles in the setting of I-R and TNF-α–induced inflammation is due to their β 2 -integrin– and complement-mediated binding to activated leukocytes adherent to the venular wall. Thus, microbubble persistence on contrast ultrasonography may be useful for the detection and monitoring of leukocyte adhesion in inflammatory diseases.