The precise control of neutrophil-mediated inflammation is critical for both host defense and the prevention of immunopathology. In vivo imaging studies in zebrafish, and more recently in mice, have made the novel observation that neutrophils leave a site of inflammation through a process called neutrophil reverse migration. The application of advanced imaging techniques to the genetically tractable, optically transparent zebrafish larvae was critical for these advances. Still, the mechanisms underlying neutrophil reverse migration and its effects on the resolution or priming of immune responses remain unclear. Here, we review the current knowledge of neutrophil reverse migration, its potential roles in host immunity, and the live imaging tools that make zebrafish a valuable model for increasing our knowledge of neutrophil behavior in vivo. 1. Introduction “Certain of the lower animals, transparent enough to be observed alive, clearly show in their midst a host of small cells with moving extensions. In these animals the smallest lesion brings an accumulation of these elements at the point of damage. In small transparent larvae, it can easily be shown that the moving cells, reunited at the damage point do often close over foreign bodies [1].” Ilya Mechnikov, one of the fathers of immunology, spoke these words at his Nobel Prize lecture in 1908. More than one hundred years after his seminal studies using transparent starfish larvae to illuminate a role for phagocytosis in immunity, we are again exploiting the power of transparent larvae for research on the immune system. Studies of neutrophils in both humans and mammalian model systems have brought great advances in our knowledge of their functions; however, zebrafish, a small tropical fish with transparent larvae, have demonstrated that direct observation of neutrophils in live animals can provide important insights that would have otherwise faced significant technical challenges using mice. Neutrophils are the most abundant leukocytes in both humans and zebrafish, and they are critical for defending the host against microbial infection [2]. In response to wounding, infection, or other inflammatory stimuli, neutrophils are rapidly recruited to perform their well-known effector functions: degranulation, phagocytosis, production of reactive oxygen species (ROS), secretion of proinflammatory cytokines, and extrusion of neutrophil extracellular traps (NETs) [3, 4]. These responses are acknowledged to kill and sequester microorganisms at their site of entry and promote the activation of the adaptive immune
G. J. Lieschke, A. C. Oates, M. O. Crowhurst, A. C. Ward, and J. E. Layton, “Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrafish,” Blood, vol. 98, no. 10, pp. 3087–3096, 2001.
J. Savill and C. Haslett, “Granulocyte clearance by apoptosis in the resolution ofinflammation,” Seminars in Cell Biology, vol. 6, no. 6, pp. 385–393, 1995.
D. L. Bratton and P. M. Henson, “Neutrophil clearance: when the party is over, clean-up begins,” Trends in Immunology, vol. 32, no. 8, pp. 350–357, 2011.
G. Cox, J. Crossley, and Z. Xing, “Macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary inflammation in vivo,” American Journal of Respiratory Cell and Molecular Biology, vol. 12, no. 2, pp. 232–237, 1995.
J. R. Mathias, B. J. Perrin, T. X. Liu, J. Kanki, A. T. Look, and A. Huttenlocher, “Resolution of inflammation by retrograde chemotaxis of neutrophils in transgenic zebrafish,” Journal of Leukocyte Biology, vol. 80, no. 6, pp. 1281–1288, 2006.
S. K. Yoo and A. Huttenlocher, “Spatiotemporal photolabeling of neutrophil trafficking during inflammation in live zebrafish,” Journal of Leukocyte Biology, vol. 21, no. 4, pp. 735–745, 2011.
A. Woodfin, M. B. Voisin, M. Beyrau et al., “The junctional adhesion molecule JAM-C regulates polarized transendothelial migration of neutrophils in vivo,” Nature Immunology, vol. 12, pp. 761–769, 2011.
S. A. Renshaw, C. A. Loynes, D. M. I. Trushell, S. Elworthy, P. W. Ingham, and M. K. B. Whyte, “A transgenic zebrafish model of neutrophilic inflammation,” Blood, vol. 108, no. 13, pp. 3976–3978, 2006.
P. Herbomel, B. Thisse, and C. Thisse, “Ontogeny and behaviour of early macrophages in the zebrafish embryo,” Development, vol. 126, no. 17, pp. 3735–3745, 1999.
C. Gray, C. A. Loynes, M. K. B. Whyte, D. C. Crossman, S. A. Renshaw, and T. J. A. Chico, “Simultaneous intravital imaging of macrophage and neutrophil behaviour during inflammation using a novel transgenic zebrafish,” Thrombosis and Haemostasis, vol. 105, no. 5, pp. 811–819, 2011.
F. Ellett, L. Pase, J. W. Hayman, A. Andrianopoulos, and G. J. Lieschke, “mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish,” Blood, vol. 117, no. 4, pp. e49–e56, 2011.
D. M. Langenau, A. A. Ferrando, D. Traver et al., “In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrafish,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 19, pp. 7369–7374, 2004.
N. Danilova and L. A. Steiner, “B cells develop in the zebrafish pancreas,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 21, pp. 13711–13716, 2002.
J. T. Dobson, J. Seibert, E. M. Teh et al., “Carboxypeptidase A5 identifies a novel mast cell lineage in the zebrafish providing new insight into mast cell fate determination,” Blood, vol. 112, no. 7, pp. 2969–2972, 2008.
K. M. Balla, G. Lugo-Villarino, J. M. Spitsbergen et al., “Eosinophils in the zebrafish: prospective isolation, characterization, and eosinophilia induction by helminth determinants,” Blood, vol. 116, no. 19, pp. 3944–3954, 2010.
E. Colucci-Guyon, J. Y. Tinevez, S. A. Renshaw, and P. Herbomel, “Strategies of professional phagocytes in vivo: unlike macrophages, neutrophils engulf only surface-associated microbes,” Journal of Cell Science, vol. 124, no. 18, pp. 3053–3059, 2011.
D. Le Guyader, M. J. Redd, E. Colucci-Guyon et al., “Origins and unconventional behavior of neutrophils in developing zebrafish,” Blood, vol. 111, no. 1, pp. 132–141, 2008.
Q. Deng, S. K. Yoo, P. J. Cavnar, J. M. Green, and A. Huttenlocher, “Dual roles for Rac2 in neutrophil motility and active retention in zebrafish hematopoietic tissue,” Developmental Cell, vol. 21, no. 4, pp. 735–745, 2011.
A. Cvejic, C. Hall, M. Bak-Maier et al., “Analysis of WASp function during the wound inflammatory response—live-imaging studies in zebrafish larvae,” Journal of Cell Science, vol. 121, no. 19, pp. 3196–3206, 2008.
K. B. Walters, J. M. Green, J. C. Surfus, S. K. Yoo, and A. Huttenlocher, “Live imaging of neutrophil motility in a zebrafish model of WHIM syndrome,” Blood, vol. 116, no. 15, pp. 2803–2811, 2010.
N. S. Trede, D. M. Langenau, D. Traver, A. T. Look, and L. I. Zon, “The use of zebrafish to understand immunity,” Immunity, vol. 20, no. 4, pp. 367–379, 2004.
S. A. Renshaw and N. S. Trede, “A model 450 million years in the making: zebrafish and vertebrate immunity,” Disease Models & Mechanisms, vol. 5, no. 1, pp. 38–47, 2012.
P. A. Morcos, “Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos,” Biochemical and Biophysical Research Communications, vol. 358, no. 2, pp. 521–527, 2007.
M. Dong, Y. F. Fu, T. T. Du et al., “Heritable and lineage-specific gene knockdown in Zebrafish embryo,” PLoS ONE, vol. 4, no. 7, Article ID e6125, 2009.
E. Wienholds, F. van Eeden, M. Kosters, J. Mudde, R. H. A. Plasterk, and E. Cuppen, “Efficient target-selected mutagenesis in zebrafish,” Genome Research, vol. 13, no. 12, pp. 2700–2707, 2003.
J. E. Foley, J. R. J. Yeh, M. L. Maeder et al., “Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by oligomerized pool ENgineering (OPEN),” PLoS ONE, vol. 4, no. 2, article e4348, 2009.
M. Christian, T. Cermak, E. L. Doyle et al., “Targeting DNA double-strand breaks with TAL effector nucleases,” Genetics, vol. 186, no. 2, pp. 756–761, 2010.
T. Li, S. Huang, W. Z. Jiang et al., “TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain,” Nucleic Acids Research, vol. 39, no. 1, pp. 359–372, 2011.
S. Hans, D. Freudenreich, M. Geffarth, J. Kaslin, A. Machate, and M. Brand, “Generation of a non-leaky heat shock-inducible Cre line for conditional Cre/lox strategies in zebrafish,” Developmental Dynamics, vol. 240, no. 1, pp. 108–115, 2011.
C. Mosimann, C. K. Kaufman, P. Li, E. K. Pugach, O. J. Tamplin, and L. I. Zon, “Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish,” Development, vol. 138, no. 1, pp. 169–177, 2011.
C. Mosimann and L. I. Zon, “Advanced zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments,” Methods in Cell Biology, vol. 104, pp. 173–194, 2011.
C. Hall, M. Flores, T. Storm, K. Crosier, and P. Crosier, “The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish,” BMC Developmental Biology, vol. 7, article 42, 2007.
A. H. Meijer, A. M. van der Sar, C. Cunha et al., “Identification and real-time imaging of a myc-expressing neutrophil population involved in inflammation and mycobacterial granuloma formation in zebrafish,” Developmental and Comparative Immunology, vol. 32, no. 1, pp. 36–49, 2008.
S. K. Yoo, Q. Deng, P. J. Cavnar, Y. I. Wu, K. M. Hahn, and A. Huttenlocher, “Differential regulation of protrusion and polarity by PI(3)K during neutrophil motility in live zebrafish,” Developmental Cell, vol. 18, no. 2, pp. 226–236, 2010.
Y. I. Wu, D. Frey, O. I. Lungu et al., “A genetically encoded photoactivatable Rac controls the motility of living cells,” Nature, vol. 461, no. 7260, pp. 104–108, 2009.
B. M. Burkel, G. Von Dassow, and W. M. Bement, “Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin,” Cell Motility and the Cytoskeleton, vol. 64, no. 11, pp. 822–832, 2007.
V. V. Belousov, A. F. Fradkov, K. A. Lukyanov et al., “Genetically encoded fluorescent indicator for intracellular hydrogen peroxide,” Nature Methods, vol. 3, no. 4, pp. 281–286, 2006.
P. Niethammer, C. Grabher, A. T. Look, and T. J. Mitchison, “A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish,” Nature, vol. 459, no. 7249, pp. 996–999, 2009.
S. K. Yoo, T. W. Starnes, Q. Deng, and A. Huttenlocher, “Lyn is a redox sensor that mediates leukocyte wound attraction in vivo,” Nature, vol. 480, no. 7375, pp. 109–112, 2011.
J. Hughes, R. J. Johnson, A. Mooney, C. Hugo, K. Gordon, and J. Savill, “Neutrophil fate in experimental glomerular capillary injury in the rat: emigration exceeds in situ clearance by apoptosis,” American Journal of Pathology, vol. 150, no. 1, pp. 223–234, 1997.
P. M. Elks, F. J. Van Eeden, G. Dixon et al., “Activation of hypoxia-inducible factor-1α (hif-1α) delays inflammation resolution by reducing neutrophil apoptosis and reverse migration in a zebrafish inflammation model,” Blood, vol. 118, no. 3, pp. 712–722, 2011.
S. B. Brown, C. S. Tucker, C. Ford, Y. Lee, D. R. Dunbar, and J. J. Mullins, “Class III antiarrhythmic methanesulfonanilides inhibit leukocyte recruitment in zebrafish,” Journal of Leukocyte Biology, vol. 82, no. 1, pp. 79–84, 2007.
N. G. Gurskaya, V. V. Verkhusha, A. S. Shcheglov et al., “Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light,” Nature Biotechnology, vol. 24, no. 4, pp. 461–465, 2006.
C. D. Buckley, E. A. Ross, H. M. McGettrick et al., “Identification of a phenotypically and functionally distinct population of long-lived neutrophils in a model of reverse endothelial migration,” Journal of Leukocyte Biology, vol. 79, no. 2, pp. 303–311, 2006.
B. A. Maletto, A. S. Ropolo, D. O. Alignani et al., “Presence of neutrophil-bearing antigen in lymphoid organs of immune mice,” Blood, vol. 108, no. 9, pp. 3094–3102, 2006.
D. V. Ostanin, E. Kurmaeva, K. Furr, et al., “Acquisition of antigen-presenting functions by neutrophils isolated from mice with chronic colitis,” The Journal of Immunology, vol. 188, no. 3, pp. 1491–1502, 2012.
J. Pillay, V. M. Kamp, E. van Hoffen, et al., “A subset of neutrophils in human systemic inflammation inhibits T cell responses through Mac-1,” The Journal of Clinical Investigation, vol. 122, no. 1, pp. 327–336, 2012.
I. Puga, M. Cols, C. M. Barra, et al., “B cell-helper neutrophils stimulate the diversification and production of immunoglobulin in the marginal zone of the spleen,” Nature Immunology, vol. 13, no. 2, pp. 170–180, 2012.
P. F. Bradfield, C. Scheiermann, S. Nourshargh et al., “JAM-C regulates unidirectional monocyte transendothelial migration in inflammation,” Blood, vol. 110, no. 7, pp. 2545–2555, 2007.
G. J. Randolph and M. B. Furie, “Mononuclear phagocytes egress from an in vitro model of the vascular wall by migrating across endothelium in the basal to apical direction: role of intercellular adhesion molecule 1 and the CD11/CD18 integrins,” Journal of Experimental Medicine, vol. 183, no. 2, pp. 451–462, 1996.
W. G. Tharp, R. Yadav, D. Irimia et al., “Neutrophil chemorepulsion in defined interleukin-8 gradients in vitro and in vivo,” Journal of Leukocyte Biology, vol. 79, no. 3, pp. 539–554, 2006.
M. C. Poznansky, I. T. Olszak, R. Foxall, R. H. Evans, A. D. Luster, and D. T. Scadden, “Active movement of T cells away from a chemokine,” Nature Medicine, vol. 6, no. 5, pp. 543–548, 2000.
P. Ogilvie, S. Paoletti, I. Clark-Lewis, and M. Uguccioni, “Eotaxin-3 is a natural antagonist for CCR2 and exerts a repulsive effect on human monocytes,” Blood, vol. 102, no. 3, pp. 789–794, 2003.
A. Huttenlocher and M. C. Poznansky, “Reverse leukocyte migration can be attractive or repulsive,” Trends in Cell Biology, vol. 18, no. 6, pp. 298–306, 2008.
B. Heit, S. Tavener, E. Raharjo, and P. Kubes, “An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients,” Journal of Cell Biology, vol. 159, no. 1, pp. 91–102, 2002.
S. S. G. Ferguson, “Evolving concepts in G protein-coupled receptor endocytosis: Tte role in receptor desensitization and signaling,” Pharmacological Reviews, vol. 53, no. 1, pp. 1–24, 2001.
P. A. Hernandez, R. J. Gorlin, J. N. Lukens et al., “Mutations in the chemokine receptor gene CXCR4 are associated with WHIM syndrome, a combined immunodeficiency disease,” Nature Genetics, vol. 34, no. 1, pp. 70–74, 2003.
K. Balabanian, B. Lagane, J. L. Pablos et al., “WHIM syndromes with different genetic anomalies are accounted for by impaired CXCR4 desensitization to CXCL12,” Blood, vol. 105, no. 6, pp. 2449–2457, 2005.
D. H. McDermott, Q. Liu, J. Ulrick, et al., “The CXCR4 antagonist plerixafor corrects panleukopenia in patients with WHIM syndrome,” Blood, vol. 118, no. 18, pp. 4957–4962, 2011.
D. C. Dale, A. A. Bolyard, M. L. Kelley, et al., “The CXCR4 antagonist plerixafor is a potential therapy for myelokathexis, WHIM syndrome,” Blood, vol. 118, no. 18, pp. 4963–4966, 2011.
J. Pillay, I. Den Braber, N. Vrisekoop et al., “In vivo labeling with 2H2O reveals a human neutrophil lifespan of 5.4 days,” Blood, vol. 116, no. 4, pp. 625–627, 2010.
K. W. Li, S. M. Turner, C. L. Emson, M. K. Hellerstein, and D. C. Dale, “Deuterium and neutrophil kinetics,” Blood, vol. 117, no. 22, pp. 6052–6053, 2011.
P. S. Tofts, T. Chevassut, M. Cutajar, N. G. Dowell, and A. M. Peters, “Doubts concerning the recently reported human neutrophil lifespan of 5.4 days,” Blood, vol. 117, no. 22, pp. 6050–6052, 2011.
G. Parsonage, A. Filer, M. Bik et al., “Prolonged, granulocyte-macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha,” Arthritis Research and Therapy, vol. 10, no. 2, article R47, 2008.
T. Tsukamoto, R. S. Chanthaphavong, and H. C. Pape, “Current theories on the pathophysiology of multiple organ failure after trauma,” Injury, vol. 41, no. 1, pp. 21–26, 2010.
E. Kardash, J. Bandemer, and E. Raz, “Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors,” Nature Protocols, vol. 6, no. 12, pp. 1835–1846, 2011.
H. Xu, E. Kardash, S. Chen, E. Raz, and F. Lin, “Gβγ signaling controls the polarization of zebrafish primordial germ cells by regulating Rac activity,” Development, vol. 139, no. 1, pp. 57–62, 2012.
M. E. Dodd, J. Hatzold, J. R. Mathias et al., “The ENTH domain protein Clint1 is required for epidermal homeostasis in zebrafish,” Development, vol. 136, no. 15, pp. 2591–2600, 2009.
J. R. Mathias, M. E. Dodd, K. B. Walters et al., “Live imaging of chronic inflammation caused by mutation of zebrafish Hai1,” Journal of Cell Science, vol. 120, no. 19, pp. 3372–3383, 2007.
