Hyaluronan and Its Receptors: Key Mediators of Immune Cell Entry and Trafficking in the Lymphatic System

Abstract

Entry to the afferent lymphatics marks the first committed step for immune cell migration from tissues to draining lymph nodes both for the generation of immune responses and for timely resolution of tissue inflammation. This critical process occurs primarily at specialised discontinuous junctions in initial lymphatic capillaries, directed by chemokines released from lymphatic endothelium and orchestrated by adhesion between lymphatic receptors and their immune cell ligands. Prominent amongst the latter is the large glycosaminoglycan hyaluronan (HA) that can form a bulky glycocalyx on the surface of certain tissue-migrating leucocytes and whose engagement with its key lymphatic receptor LYVE-1 mediates docking and entry of dendritic cells to afferent lymphatics. Here we outline the latest insights into the molecular mechanisms by which the HA glycocalyx together with LYVE-1 and the related leucocyte receptor CD44 co-operate in immune cell entry, and how the process is facilitated by the unusual character of LYVE-1 • HA-binding interactions. In addition, we describe how pro-inflammatory breakdown products of HA may also contribute to lymphatic entry by transducing signals through LYVE-1 for lymphangiogenesis and increased junctional permeability. Lastly, we outline some future perspectives and highlight the LYVE-1 • HA axis as a potential target for immunotherapy.

Keywords: CD44; LYVE-1; T cell; dendritic cell; glycocalyx; hyaluronan; immune cell; lymphatic endothelium; macrophage; trafficking.

Figure 1

Structure and properties of HA. Top left and right panels, respectively, show the basic disaccharide repeat unit of long unbranched HA chains (represented in chair conformation) and the 3D volume occupied by a typical HMW HA chain (example shown is a 5000 kDa polymer) relative to average-sized globular proteins and larger globular/fibrous macromolecules depicted in 2D. Bottom left panel shows an illustration of a large noncovalent HMW HA complex containing the chondrotin sulphate proteoglycans aggrecan, versican and Link protein typical of those found in extracellular/pericellular matrices surrounding, e.g., chondrocytes in connective tissue anchored by CD44, and an inflammation-induced IαI heavy chain covalent HA complex (a.k.a. SHAP, serum HA associated protein) containing the cross-linking tetramer pentraxin. Formation of SHAP is catalysed by the small HA-binding protein TSG-6, which forms transient covalent intermediates with HA (not shown), but TSG-6 can also form cross-linked noncovalent complexes that have enhanced binding avidity for LYVE-1 and CD44 [49,50] (see text for details). Bottom right panel shows the different consequences of HA glycocalyx formation on cell behaviour including adhesion to neighbouring cells expressing appropriate HA receptors, repulsion by neighbouring cells with similar HA glycocalyces and camouflage of surface receptors and adhesion molecules depicted as red boxes, beneath the canopy formed by the HA glycocalyx.

Figure 2

Functionally specialised junctions between endothelial cells of initial lymphatic vessels. (A–C), Blind-ended initial capillaries of whole mount mouse ear dermis, immunolabeled with anti-LYVE-1/AF647-conjugated anti-rabbit antibody (blue) and anti-VE-cadherin/AF568-conjugated anti-rat antibody (red) shown at low (100×, panel A) and high magnification (630×, panel B), with further digital zoom (panel C), to illustrate the arrangement of LYVE-1 along endothelial flaps pinned by VE-cadherin in button-like junctions where immune cells transmigrate (see text for details). The same images are re-drawn as illustration models in panels (D–F).

Figure 3

The HA glycocalyx on the surface of DCs and its involvement in docking to lymphatic endothelium via LYVE-1 transmigratory cups. Panel (A) shows the HA glycocalyx on the surface of an LPS-matured mouse bmDC, detected with bVG1/streptavidin-AF647, dual stained for CD44/AF488 and counterstained for nuclei with DAPI, imaged by confocal microscopy with Airyscan detection and digital zoom (right). Panel (B) illustrates the docking of a mouse bmDC to the surface of a LEC through formation of a LYVE-1 transmigratory cup that encircles the cell and engages with the HA glycocalyx. Shown are confocal microscopy images with orthogonal views of mouse LEC monolayers immunostained with anti-LYVE-1/AF548 (red), recorded 3 h after the addition of LPS-matured fluorescent bmDCs (green) dual immunostained for anti-CD44/AF594 (blue), and bVG1/streptavidin-AF647 (yellow), and counterstained for nuclei with DAPI (grey). Adapted from Johnson et al. 2021 [114] under Creative Commons BY 4.0.

Figure 4

Dynamics of the DC HA glycocalyx and its influence on engagement of underlying adhesion receptors with lymphatic endothelium during vessel entry. (A) Initial contact with lymphatic endothelium triggers polarisation of migrating DCs and redistribution of CD44 to the adhesive uropod as observed by confocal microscopy [114] and as depicted in the illustration shown. Bar = 10 µm. (B) The estimated length of HMW HA chains in the glycocalyx (≤1 µm) extends beyond that of other key underlying adhesion receptors such as leucocyte integrins and their ligands on the DC surface, likely positioning the LYVE-1 • HA axis for making the first adhesive contacts between DC and endothelium during the process of transmigration. (C) Speculative organisation of DC • endothelial contacts during transmigration and the possible influence of the HA glycocalyx on underlying β2 integrin accessibility and adhesive function. The illustration depicts the possible imposition of focal clustering of DC integrins by the bulky HA chains, enabling their conformational activation and subsequent contribution to endothelial adhesion via ICAM-1 during diapedesis [128]. How and when integrins become accessible beneath the glycocalyx is, however, still unclear. The confocal image in panel A is from Johnson et al. 2021, [114] and the cartoon in panel B is adapted from Barclay et al. 1997, [129] with permission from the publisher (Elsevier).

Figure 5

Characteristics of LYVE-1 that regulate its interactions with HA in lymphatic endothelium. (A) Endogenous LYVE-1 forms homodimers on the endothelial surface via an unpaired cysteine residue C201 in a process that is critical for HA-binding in vivo and which increases binding affinity some 15-fold as assessed by Biacore analysis [147]. The ratio of homodimer to monomer is subject to regulation by the local redox environment. (B) Engagement with HA also requires clustering of LYVE-1 on the endothelial surface, induced by interaction with cross-linked macromolecular HA • protein complexes that have heightened binding avidity (see also Figure 1), and local disassembly of the sub-membrane actin meshwork that normally constrains LYVE-1 lateral mobility in the plasma membrane [49,150]. The figure is modified from Jackson (2019) [23] with permission from the publisher (Elsevier).

Figure 6

3D structure of the LYVE-1 HA-binding Link domain. (A) Structure-based models of the HA-binding domain in human LYVE-1 based on the high-resolution crystal structure of its closest homologue CD44 [152] below. Illustrations depict membrane-proximal domains in each case decorated with O-linked sugars (red lines). The yellow circle in LYVE-1 represents the unpaired cysteine residue C201 involved in homodimer formation (see Figure 5). (B) Alignment of the human LYVE-1 and CD44 amino acid sequences highlighting the regions encoding the conserved disulphide-bridged α/β fold of the consensus Link domain (mauve) and the β stranded N- and C-terminal extensions peculiar to CD44 (orange) stabilised by the additional disulphides C1-C6 and C2-C5 predicted to be present also in LYVE-1 from secondary structure program analysis using JPRED and JUFO. Notably in LYVE-1, the β8 and β9 strands of the C-terminal extension are substituted by serine and threonine-rich tracts that predict formation of an elongated, O-glycosylated membrane-proximal stalk [146]. Asterisks mark amino acid motifs (NXS/T) for N-glycosylation. Sites for LYVE-1 ectodomain cleavage (scissors symbol) by MMPs and ADAMs are boxed in red. The figure is modified from Jackson (2019) [23] with permission from the publisher (Elsevier).