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.

Training Opportunities

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.

References cited

  1. 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.
  2. Jackson  DG.  2009.  Immunological   functions  of  hyaluronan  and  its  receptors  in  the lymphatics. Immunol. Rev., 230 (1), pp. 216-31.
  3. 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.
  4. 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.
  5. Johnson LA, Jackson DG. 2014. Control of dendritic cell trafficking in lymphatics by chemokines. Angiogenesis, 17 (2), pp. 335-45.
  6. 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.
  7. 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.
  8. 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.
  9. 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.
  10. 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.
  11. 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.

For further information, please contact:

Prof David Jackson David.jackson@imm.ox.ac.uk

Jackson Figure 1-1
Fig. 1. Cartoon showing the main stages during the migration and entry of dendritic cells from the tissues to initial lymphatic vessels, enroute to draining lymph nodes for the generation of immune responses. The main candidates involved are listed to the right. 
 
Jackson Figure 1-2
Fig. 2. Confocal images and diagrammatic cartoon showing the distinctive overlapping junctions of initial lymphatic capillaries in tissues such as skin, and the button-like portals lined with LYVE-1 that represent sites for dendritic cell entry.Jackson Figure 1-3
Fig 3. Hypothetical model showing the proposed mechanism by which interaction of hyaluronan coated dendritic cells with LYVE-1 in the lymphatic endothelium leads to changes in junctional integrity that admit entry to the vessel lumen.