doi:10.1016/j.ultrasmedbio.2005.11.010 ● Original Contribution ULTRASOUND EXPOSURE CAN INCREASE THE MEMBRANE PERMEABILITY OF HUMAN NEUTROPHIL GRANULOCYTES CONTAINING MICROBUBBLES WITHOUT CAUSING COMPLETE CELL DESTRUCTION GRIGORIOS KOROSOGLOU,* STEFAN E. HARDT,* RAFFI BEKEREDJIAN,* JUERGEN JENNE, † MATHIAS KONSTANTIN,* MARCO HAGENMUELLER,* HUGO A. KATUS,* and HELMUT KUECHERER* *Department of Cardiology, University of Heidelberg, Heidelberg, Germany; and † German Cancer Research Center (DKFZ), Heidelberg, Germany (Received 20 July 2005, revised 7 November 2005, in final form 17 November 2005) Abstract—Activated polymorphonuclear neutrophil (PMN) granulocytes can bind and subsequently phagocytose microbubbles used as ultrasound (US) contrast agents. The purpose of the present study was to assess insonation effects on cell membrane integrity and metabolic activity of activated PMN. Furthermore, we investigated whether or not there is an acoustic threshold at which insonation of PMN results in increase of membrane permeability without causing complete cell destruction. PMN isolated from healthy volunteers were activated with phorbol myristate acetate (PMA) for 15 min to allow phagocytosis of albumin and lipid microbubbles and were subsequently exposed to US with a mechanical index between 0.15 and 1.8. Apoptosis, loss of membrane integrity and formation of cell fragments were evaluated by measurement of lactate dehydrogenase leakage and by double staining with annexin V and propidium iodide, using flow cytometry. Neutrophil superoxide anion generation was measured photometrically. Insonation of activated PMN in the presence of microbubbles amplified apoptosis and lactate dehydrogenase leakage and induced loss of membrane integrity and complete cell destruction with increasing acoustic pressures. The bioeffects observed by insonation with high mechanical indices (1.0 to 1.8), and particularly the formation of cell fragments, were significantly more pronounced in the presence of albumin microbubbles. Insonation in the presence of lipid microbubbles increased cell membrane permeability, but caused significantly less cell destruction and left the metabolic activity of activated PMN uninfluenced. Thus, both albumin and lipid microbubbles induce apoptosis and membrane injury during insonation of activated PMN. However, insonation in the presence of lipid microbubbles seems to influence cell viability to a smaller extent. This could be of advantage in the setting of US-guided local drug delivery. In this setting, increase of membrane permeability may allow bioactive substances to enter into cells, which survive the US treatment, and specifically modify their function. (E-mail: Grigorios_Korosoglou@med.uni-heidelberg.de) © 2006 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound microbubbles, Neutrophil granulocytes, Phagocytosis. INTRODUCTION Second-generation contrast agents are microbubbles composed of an albumin or lipid shell and filled with inert gas. To assess microvascular perfusion (Wei et al. 1998; Korosoglou et al. 2004b, 2004c, 2005, 2006), microbubbles are designed to possess rheological prop- erties similar to those of red blood cells and to transit through the microcirculation unimpeded (Keller et al. 1989; Jayaweera et al. 1994). Contrast agents have been found to be safe for clinical use within the US Food and Drug Administration approval process and severe ad- verse effects have not been reported in human beings so far (Moore et al. 1986; Geny et al. 1997; Ten Cate et al. 1993). However, imaging with high acoustic pressures and high doses of contrast agents has been reported to induce premature ventricular contractions in human beings (van Der Wouw et al. 2000). In the experimental setting, insonation of tissue containing microbubbles caused pe- Address correspondence to: Dr. Grigorios Korosoglou, M.D., Department of Cardiology, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg 69120 Germany. E-mail: Grigorios_ Korosoglou@med.uni-heidelberg.de Ultrasound in Med. & Biol., Vol. 32, No. 2, pp. 297–303, 2006 Copyright © 2006 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/06/$–see front matter 297