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Neutrophil Extravasation Cascade: What Can We Learn from Two-Photon Intravital Imaging?

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Neutrophil Extravasation Cascade: What Can We Learn from Two-Photon Intravital Imaging? REVIEW ARTICLE https://doi.org/10.4110/in.2016.16.6.317 pISSN 1598-2629 · eISSN 2092-6685 Neutrophil Extravasation Cascade: What Can We Learn from Two-photon Intravital Imaging? Sang A Park and Young-Min Hyun* Department of Anatomy, Yonsei University College of Medicine, Seoul 03722, Korea Immune cells (leukocytes or white blood cells) move actively and sensitively based on body conditions. Despite their important role as protectors inside the body, it is difficult to directly observe the spatiotemporal momentum of leukocytes. With advances in imaging technology, the introduction of two-photon microscopy has enabled researchers to look deeper inside tissues in a three-dimensional manner. In observations of immune cell movement along the blood vessel, vascular permeability and innate immune cell movements remain unclear. Here, we describe the neutrophil extravasation cascade, which were observed using a two-photon intravital imaging technique. We also provide evidence for novel mechanisms such as neutrophil body extension and microparticle formation as well as their biological roles during migration. [Immune Network 2016;16(6):317-321] Keywords: Neutrophil extravasation, Microparticle formation, Two-photon microscopy, Intravital imaging TWO-PHOTON MICROSCOPY the focal point (1) and to capture images for much longer periods of time compared to single-photon confocal Principle of two-photon microscopy microscopy. In single-photon confocal microscopy, a photon is excited and then emitted for visualization of the fluorescent cell Intravital imaging using two-photon microscope or tissue. In the process, fluorescence is emitted along Confocal fluorescent microscopy has been widely used the whole path of a laser beam focused on a sample of over the past several decades to observe the morphology fluorescent dye, forming a long trail of light (1). Two- and motility of cells and tissues. Many valuable dis- photon microscopy, however, doubles the energy of two coveries such as imaging of protein interactions and the near-infrared photons that are absorbed simultaneously analysis of gene expression in living animals (4) have (2). Indeed, the light ranges from 700 to 1000 nm (3). been made using single-photon confocal microscopy. As two-photon microscopy uses photons with longer Also, several research groups introduced novel pheno- wavelengths and less energy than single-photon confocal mena of immune response from intravital imaging microscopy, this results in deep penetration of the photon using single-photon confocal microscopy (5,6). Yet, this into the tissue with less tissue damage. This mechanism microscopic system contains three major limitations: also enables researchers to visualize only the focal point inability to observe ‘deep’ into biological tissues, damage of a laser beam within a range of 1 mm above and below to nearby tissues from photobleaching, and loss of image Received on July 22, 2016. Revised on September 19, 2016. Accepted on September 29, 2016. This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons. org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. * Corresponding Author. Young-Min Hyun, Department of Anatomy, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea. Tel: 82-2-2228-1655; Fax: 82-2-365-0700; E-mail: [email protected] http://immunenetwork.org 317 Neutrophil Extravasation contrast (2). Thus, single-photon confocal microscopy to obtain spatiotemporal information such as the motility is more widely used for in vitro or ex vivo imaging of and morphology of target cells and tissues is only possible target tissues for one time point or short periods rather using intravital imaging, which reveals the dynamics of than in vivo or time lapse manner. In addition, this living targets of intact organisms (7). Leukocyte activity imaging system is frequently limited to two-dimensional is one of the most dynamic phenomena in live animals. imaging or very thin three-dimensional imaging (3). Two- Therefore, many researchers use the features of two- photon confocal microscopy can be used to compensate photon microscope to determine the mechanism and the limitations of conventional single-photon confocal pathology of leukocytes in the immune response. microscopy and enables researchers to investigate deeper regions of the tissues for as long as several hours, particularly in live animals (7). Confocal microscopy NEUTROPHIL MIGRATION IN PERIPHERAL VESSELS typically suggests the use of single-photon confocal microscopy; “two-photon microscopy” is widely used to When does neutrophil migrate? describe two-photon confocal microscopy. When tissue is physically damaged or foreign substances such as bacteria and virus invade the body of live Two-photon intravital imaging is useful for understanding specimens, the immune system is activated in order to leukocyte migration reduce the damage and maintain the homeostasis in the Since two-photon microscopic imaging shows a lower body. The first stage of this system is innate immunity, probability of causing phototoxicity, it is considered which is non-specific to pathogens and responds rapidly. an essential tool in intravital imaging for long periods Several leukocyte populations are key players in this from several hours through a few days (8). In addition, innate response. Among the various leukocytes, neutrophil researchers can analyze actual morphology and motility identification at the site of infection is considered a of live specimens in the deep area of the tissues because hallmark of inflammation (9), as it is the first to arrive to of the ability of two-photon microscopy to penetrate the affected location (10). In the bone marrow of humans, deep inside the tissue up to 1 millimeter. The capability nearly 1011 neutrophils are generated and emitted into Figure 1. Schema of neutrophil extravasation cascade. In the process of intravascular migration, neutrophil tethers itself to the endothelial cells via (1) rolling, (2) adhesion and (3) crawling. Thereafter neutrophil is (4) firmly adhered to the luminal surface of the vessel. After approaching to the proper site of extravasation, (5) leukocyte transmigrate through the endothelial cells, pericyte and basement membrane. In this process of extravasation, neutrophil undergoes (a) intrusion, (b) perivascular embedment & crawling, (c) protrusion, and then finally (d) uropod elongation & microparticle formation. Microparticles (red dot) are formed in this stage, and usually embedded between endothelial cells and pericytes. When extravasation is over, (6) leukocyte starts interstitial migration. 318 IMMUNE NETWORK Vol. 16, No. 6: 317-321, December, 2016 Neutrophil Extravasation the blood circulation each day (10). Neutrophils must below. Extravasation is the actual step to overcome penetrate the blood vessels in order to migrate damage the vessel wall during neutrophil migration cascade. to tissue under inflammatory conditions. The neutrophil It is known that neutrophils undergo transendothelial migration cascade is composed of intravascular migration, migration in two different ways. One is the paracellular extravasation, and interstitial migration. route which passes between the endothelial cell junctions while the other is the transcellular route which travels Neutrophil migration cascade directly through the endothelial cell body (10). It was The blood vessel wall is composed of the endothelial reported that majority of neutrophil transendothelial cell layer, endothelial basement membrane, and pericytes migration occurs via the paracellular route (17). (Fig. 1). Therefore, neutrophils must enter between Extravasation process in detail: First, neutrophils that these arrangements, which may result in morphological complete the crawling process intrude under the endo- modification. Pericytes are located at the abluminal thelial cell layer. Next, some neutrophils are embedded side of microvessels (11). These cells are known to be in the basement membrane area, while others migrate involved in controlling capillary permeability (12). The within the endothelial basement membrane, known as endothelial basement membrane is composed of laminins perivascular crawling. Finally, most neutrophils overcome and collagen IV, which serve as ligands for adhesion the endothelial basement membrane through a specific molecules on migrating leukocytes (13,14). Thus, endo- location at which a small gap is formed between pericytes; thelial cells also interconnect macromolecules, supporting this phenomenon is known as perivascular crawling and structures, and signaling properties of the vessel (10). extension of ventral membrane protrusions. Neutrophils Intravascular migration: Intravascular migration refers typically protrude through physically permissive sites to all processes inside blood vessels, including rolling, rather than altering basement membrane formation. As adhesion, crawling, and firm adhesion of leukocytes to neutrophils project throughout a small space, a uropod is the endothelial cell layer (Fig. 1). formed and elongated. Recently, we found that the small A complex cascade of intravascular migration is initiated end-tip of the uropod remains tied along the endothelial by selectin-mediated “tethering and rolling” of leukocytes basement membrane region during tail retraction, forming to the luminal side of
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