Mechanisms underlying the trafficking of leucocytes from tissues via the lymphatics in immunity and inflammation
Supervisor: Prof David Jackson
Fundamental to generating a host immune response to infection is the mobilisation of antigen loaded dendritic cells (DCs) in the affected tissues and their migration via afferent lymph to draining nodes where they prime and activate T cells in the paracortex (reviewed in Jackson 2009, "Lymphatic regulation of cell trafficking" http://www.omicsonline.org/open-access/lymphatic-regulation-of- cellular-trafficking-2155-9899-5-258.php?aid=31116) 10.4172/2161-0681.1000241 ). The trafficking of DCs through afferent lymphatic vessels is vital to such immunity, and involves a series of co-ordinated steps driven by chemokines, including CCL21 and CX3CL1 secreted from initial lymphatic capillaries (Johnson and Jackson 2010, 2013; reviewed in Johnson et al. 2014) and supported by a plethora of adhesion receptors (see Fig 1) whose expression is frequently upregulated during inflammation (Johnson et al 2006). Besides DCs, other leucocyte populations such as macrophages and neutrophils also migrate through lymphatics, and such migration is important for their clearance from the tissues during the resolution of inflammation. The precise choreography of directional guidance, endothelial adhesion and transmigration, and downstream trafficking to lymph nodes are however unclear.
Most leucocytes are thought to access lymphatic capillaries in the tissues at specialised overlapping junctions, distinct from the tight junctions of blood capillaries. Here, the flap-like edges of oakleaf shaped endothelial cells interdigitate to form button-like portals, whose tips are decorated with the lymphatic vessel specific hyaluronan receptor LYVE-1 (Banerji et al, 1999; Baluk et al 2007 and reviewed in Jackson 2014), and whose sides are pinned by adherens and tight junction receptors VE- cadherin, claudins and JAMs (Fig 2). The implication is that engagement of migrating cells with these portals leads to loosening of the junctions and entry to the vessel lumen (Fig 3).
Recent work in my laboratory using a combination of in vitro and in vivo approaches has yielded compelling evidence that DCs initially adhere to lymphatic endothelium via LYVE-1, and that the interaction involves reversible avidity-dependent binding to hyaluronan arranged appropriately within the DC surface glycocalyx ((Lawrance et al 2016; Johnson et al 2017), thus enabling the migrating cells to dock via transmigratory cups and transit to the vessel lumen. An analogous mechanism appears to mediate systemic spread of virulent strains of Group A streptococci, the agents of bacterial tonsillitis and necrotizing fasciitis, whose dense hyaluronan capsule mediates both lymphatic tropism and protection against host phagocytosis (Lynskey et al. 2015). Ongoing work suggests that monocyte/macrophages may also utilize LYVE-1 to navigate the lymphatics, during the course of tissue inflammation. How LYVE-1 selectively binds.
The project on offer will provide an exciting opportunity to explore the in vivo anatomy of LYVE-1 mediated leucocyte interactions during leucocyte trafficking in more detail, using intravital video microscopic imaging as well as in vitro analytical approaches. In particular it will ask - How does the LYVE-1:HA adhesion axis integrate with chemotaxis for vessel entry ? Does LYVE-1 mediated entry involve concerted interactions with other receptors such as ICAMs that are upregulated in inflamed lymphatics ? Are LYVE-1:HA interactions involved primarily in leucocyte docking or do they also assist in DC crawling within the vessel lumen ? Do they also mediate subsequent interactions with the subcapsular cortical or medullary sinuses in downstream lymph nodes ? Does the function of LYVE-1 integrate with that of the leucocyte HA receptor CD44 and do they both contribute to the recruitment of leucocytes during the initiation and resolution of tissue inflammation?
The successful candidate will have the benefit of supervision both from the PI, and from a dedicated senior research scientist, as well as close Unit support and international collaborations with other leading scientists in the field. We anticipate the new insights gained in these preclinical studies to seed future development of novel therapeutic strategies for inflammatory diseases.
The work will involve the use of animal models of inflammation, ex vivo tissue explants and in vitro culture models to visualize, characterise, manipulate and quantify leucocyte trafficking, hyaluronan glycocalyx/complex formation and tissue vasculature by confocal and intravital imaging. Experiments will take advantage of constitutive and conditional LYVE-1-/- mice, LYVE-1 function blocking mAbs and appropriate fluorescent reporter mice that will be available in the host laboratory.
The successful candidate will receive training in flow cytometry, confocal/intravital microscopy, endothelial cell culture and characterization, ex vivo/in vitro transmigration assays, in vivo studies in animal inflammatory disease models, immunoassays, molecular biology techniques and protein analysis.The host Institute, and the MRC Human Immunology Unit have world class, cutting edge facilities for microscopic imaging, including super-resolution (STED) microscopy and a dedicated suite for intravital imaging.
- Banerji S, Ni J, Wang SX, Clasper S, Su J, Tammi R, Jones M, Jackson DG. 1999. LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J. Cell Biol., 144 (4), pp. 789-801.
- Jackson DG. 2009. Immunological functions of hyaluronan and its receptors in the lymphatics. Immunol. Rev., 230 (1), pp. 216-31.
- Johnson LA, Jackson DG. 2010. Inflammation-induced secretion of CCL21 in lymphatic endothelium is a key regulator of integrin-mediated dendritic cell transmigration. Int. Immunol., 22 (10), pp. 839-49.
- Johnson LA, Jackson DG. 2013. The chemokine CX3CL1 promotes trafficking of dendritic cells through inflamed lymphatics. J. Cell. Sci., 126 (Pt 22), pp. 5259-70.
- Johnson LA, Jackson DG. 2014. Control of dendritic cell trafficking in lymphatics by chemokines. Angiogenesis, 17 (2), pp. 335-45.
- Johnson LA, Clasper S, Holt AP, Lalor PF, Baban D, Jackson DG. 2006. An inflammation-induced mechanism for leukocyte transmigration across lymphatic vessel endothelium. J. Exp. Med., 203 (12), pp. 2763-77.
- Lawrance W, Banerji S, Day AJ, Bhattacharjee S, Jackson DG. 2016. Binding of Hyaluronan to the Native Lymphatic Vessel Endothelial Receptor LYVE-1 Is Critically Dependent on Receptor Clustering and Hyaluronan Organization. J. Biol. Chem., 291 (15), pp. 8014-30.
- Baluk P, Fuxe J, Hashizume H, Romano T, Lashnits E, Butz S, Vestweber D, Corada M, Molendini C, Dejana E, McDonald DM. 2007. Functionally specialized junctions between endothelial cells of lymphatic vessels. J. Exp. Med., 204 (10), pp. 2349-62.
- Tal O, Lim HY, Gurevich I, Milo I, Shipony Z, Ng LG, Angeli V, Shakhar G. 2011. DC mobilization from the skin requires docking to immobilized CCL21 on lymphatic endothelium and intralymphatic crawling. J. Exp. Med., 208 (10), pp. 2141-53.
- Lynskey NN, Banerji S, Johnson LA, Holder KA, Reglinski M, Wing PA, Rigby D, Jackson DG, Sriskandan S. 2015. Rapid Lymphatic Dissemination of Encapsulated Group A Streptococci via Lymphatic Vessel Endothelial Receptor-1 Interaction. PLoS Pathog., 11 (9), pp. e1005137.
- McDonald B, McAvoy EF, Lam F, Gill V, de la Motte C, Savani RC, Kubes P. 2008. Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids. J. Exp. Med., 205 (4), pp. 915-27.
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Prof David Jackson David.firstname.lastname@example.org