Dendritic cell entry to lymphatic capillaries is orchestrated by CD44 and the hyaluronan glycocalyx.

DCs play a vital role in immunity by conveying antigens from peripheral tissues to draining lymph nodes, through afferent lymphatic vessels. Critical to the process is initial docking to the lymphatic endothelial receptor LYVE-1 via its ligand hyaluronan on the DC surface. How this relatively weak binding polymer is configured for specific adhesion to LYVE-1, however, is unknown. Here, we show that hyaluronan is anchored and spatially organized into a 400-500 nm dense glycocalyx by the leukocyte receptor CD44. Using gene knockout and by modulating CD44-hyaluronan interactions with monoclonal antibodies in vitro and in a mouse model of oxazolone-induced skin inflammation, we demonstrate that CD44 is required for DC adhesion and transmigration across lymphatic endothelium. In addition, we present evidence that CD44 can dynamically control the density of the hyaluronan glycocalyx, regulating the efficiency of DC trafficking to lymph nodes. Our findings define a previously unrecognized role for CD44 in lymphatic trafficking and highlight the importance of the CD44:HA:LYVE-1 axis in its regulation.

Extracellular bacterial lymphatic metastasis drives Streptococcus pyogenes systemic infection.

Unassisted metastasis through the lymphatic system is a mechanism of dissemination thus far ascribed only to cancer cells. Here, we report that Streptococcus pyogenes also hijack lymphatic vessels to escape a local infection site, transiting through sequential lymph nodes and efferent lymphatic vessels to enter the bloodstream. Contrasting with previously reported mechanisms of intracellular pathogen carriage by phagocytes, we show S. pyogenes remain extracellular during transit, first in afferent and then efferent lymphatics that carry the bacteria through successive draining lymph nodes. We identify streptococcal virulence mechanisms important for bacterial lymphatic dissemination and show that metastatic streptococci within infected lymph nodes resist and subvert clearance by phagocytes, enabling replication that can seed intense bloodstream infection. The findings establish the lymphatic system as both a survival niche and conduit to the bloodstream for S. pyogenes, explaining the phenomenon of occult bacteraemia. This work provides new perspectives in streptococcal pathogenesis with implications for immunity.

The cortical actin network regulates avidity-dependent binding of hyaluronan by the lymphatic vessel endothelial receptor LYVE-1.

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) mediates the docking and entry of dendritic cells to lymphatic vessels through selective adhesion to its ligand hyaluronan in the leukocyte surface glycocalyx. To bind hyaluronan efficiently, LYVE-1 must undergo surface clustering, a process that is induced efficiently by the large cross-linked assemblages of glycosaminoglycan present within leukocyte pericellular matrices but is induced poorly by the shorter polymer alone. These properties suggested that LYVE-1 may have limited mobility in the endothelial plasma membrane, but no biophysical investigation of these parameters has been carried out to date. Here, using super-resolution fluorescence microscopy and spectroscopy combined with biochemical analyses of the receptor in primary lymphatic endothelial cells, we provide the first evidence that LYVE-1 dynamics are indeed restricted by the submembranous actin network. We show that actin disruption not only increases LYVE-1 lateral diffusion but also enhances hyaluronan-binding activity. However, unlike the related leukocyte HA receptor CD44, which uses ERM and ankyrin motifs within its cytoplasmic tail to bind actin, LYVE-1 displays little if any direct interaction with actin, as determined by co-immunoprecipitation. Instead, as shown by super-resolution stimulated emission depletion microscopy in combination with fluorescence correlation spectroscopy, LYVE-1 diffusion is restricted by transient entrapment within submembranous actin corrals. These results point to an actin-mediated constraint on LYVE-1 clustering in lymphatic endothelium that tunes the receptor for selective engagement with hyaluronan assemblages in the glycocalyx that are large enough to cross-bridge the corral-bound LYVE-1 molecules and thereby facilitate leukocyte adhesion and transmigration.

Dendritic cells enter lymph vessels by hyaluronan-mediated docking to the endothelial receptor LYVE-1.

Trafficking of tissue dendritic cells (DCs) via lymph is critical for the generation of cellular immune responses in draining lymph nodes (LNs). In the current study we found that DCs docked to the basolateral surface of lymphatic vessels and transited to the lumen through hyaluronan-mediated interactions with the lymph-specific endothelial receptor LYVE-1, in dynamic transmigratory-cup-like structures. Furthermore, we show that targeted deletion of the gene Lyve1, antibody blockade or depletion of the DC hyaluronan coat not only delayed lymphatic trafficking of dermal DCs but also blunted their capacity to prime CD8+ T cell responses in skin-draining LNs. Our findings uncovered a previously unknown function for LYVE-1 and show that transit through the lymphatic network is initiated by the recognition of leukocyte-derived hyaluronan.

Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium.

The lymphatic vessel endothelial receptor LYVE-1 is implicated in the uptake of hyaluronan (HA) and trafficking of leukocytes to draining lymph nodes. Yet LYVE-1 has only weak affinity for hyaluronan and depends on receptor clustering and higher order ligand organization for durable binding in lymphatic endothelium. An unusual feature of LYVE-1 not found in other HA receptors is the potential to form disulfide-linked homodimers. However, their influence on function has not been investigated. Here we show LYVE-1 homodimers are the predominant configuration in lymphatic endothelium in vitro and in vivo, and formation solely requires the unpaired cysteine residue Cys-201 within the membrane-proximal domain, yielding a 15-fold higher HA binding affinity and an ∼67-fold slower off-rate than the monomer. Moreover, we show non-dimerizing LYVE-1 mutants fail to bind HA even when expressed at high densities in lymphatic endothelial cells or artificially cross-linked with antibody. Consistent with these findings, small angle X-ray scattering (SAXS) indicates the Cys-201 interchain disulfide forms a hinge that maintains the homodimer in an "open scissors" conformation, likely allowing arrangement of the two HA binding domains for mutual engagement with ligand. Finally, we demonstrate the Cys-201 interchain disulfide is highly labile, and selective reduction with TCEP-HCl disrupts LYVE-1 homodimers, ablating HA binding. These findings reveal binding is dependent not just on clustering but also on the biochemical properties of LYVE-1 homodimers. They also mark LYVE-1 as the first Link protein superfamily member requiring covalent homodimerization for function and suggest the interchain disulfide acts as a redox switch in vivo.

A single molecule assay to probe monovalent and multivalent bonds between hyaluronan and its key leukocyte receptor CD44 under force.

Glycosaminoglycans (GAGs), a category of linear, anionic polysaccharides, are ubiquitous in the extracellular space, and important extrinsic regulators of cell function. Despite the recognized significance of mechanical stimuli in cellular communication, however, only few single molecule methods are currently available to study how monovalent and multivalent GAG·protein bonds respond to directed mechanical forces. Here, we have devised such a method, by combining purpose-designed surfaces that afford immobilization of GAGs and receptors at controlled nanoscale organizations with single molecule force spectroscopy (SMFS). We apply the method to study the interaction of the GAG polymer hyaluronan (HA) with CD44, its receptor in vascular endothelium. Individual bonds between HA and CD44 are remarkably resistant to rupture under force in comparison to their low binding affinity. Multiple bonds along a single HA chain rupture sequentially and independently under load. We also demonstrate how strong non-covalent bonds, which are versatile for controlled protein and GAG immobilization, can be effectively used as molecular anchors in SMFS. We thus establish a versatile method for analyzing the nanomechanics of GAG·protein interactions at the level of single GAG chains, which provides new molecular-level insight into the role of mechanical forces in the assembly and function of GAG-rich extracellular matrices.

Critical importance of appropriate fixation conditions for faithful imaging of receptor microclusters.

Receptor clustering is known to trigger signalling events that contribute to critical changes in cellular functions. Faithful imaging of such clusters by means of fluorescence microscopy relies on the application of adequate cell fixation methods prior to immunolabelling in order to avoid artefactual redistribution by the antibodies themselves. Previous work has highlighted the inadequacy of fixation with paraformaldehyde (PFA) alone for efficient immobilisation of membrane-associated molecules, and the advantages of fixation with PFA in combination with glutaraldehyde (GA). Using fluorescence microscopy, we here highlight how inadequate fixation can lead to the formation of artefactual clustering of receptors in lymphatic endothelial cells, focussing on the transmembrane hyaluronan receptors LYVE-1 and CD44, and the homotypic adhesion molecule CD31, each of which displays their native diffuse surface distribution pattern only when visualised with the right fixation techniques, i.e. PFA/GA in combination. Fluorescence recovery after photobleaching (FRAP) confirms that the artefactual receptor clusters are indeed introduced by residual mobility. In contrast, we observed full immobilisation of membrane proteins in cells that were fixed and then subsequently permeabilised, irrespective of whether the fixative was PFA or PFA/GA in combination. Our study underlines the importance of choosing appropriate sample preparation protocols for preserving authentic receptor organisation in advanced fluorescence microscopy.

Micromechanical Analysis of the Hyaluronan-Rich Matrix Surrounding the Oocyte Reveals a Uniquely Soft and Elastic Composition.

The cumulus cell-oocyte complex (COC) matrix is an extended coat that forms around the oocyte a few hours before ovulation and plays vital roles in oocyte biology. Here, we analyzed the micromechanical response of mouse COC matrix by colloidal-probe atomic force microscopy. We found that the COC matrix is elastic insofar as it does not flow and its original shape is restored after force release. At the same time, the COC matrix is extremely soft. Specifically, the most compliant parts of in vivo and in vitro expanded COC matrices yielded Young's modulus values of 0.5 ± 0.1 Pa and 1.6 ± 0.3 Pa, respectively, suggesting both high porosity and a large mesh size (≥100 nm). In addition, the elastic modulus increased progressively with indentation. Furthermore, using optical microscopy to correlate these mechanical properties with ultrastructure, we discovered that the COC is surrounded by a thick matrix shell that is essentially devoid of cumulus cells and is enhanced upon COC expansion in vivo. We propose that the pronounced nonlinear elastic behavior of the COC matrix is a consequence of structural heterogeneity and serves important functions in biological processes such as oocyte transport in the oviduct and sperm penetration.

Binding of Hyaluronan to the Native Lymphatic Vessel Endothelial Receptor LYVE-1 Is Critically Dependent on Receptor Clustering and Hyaluronan Organization.

The lymphatic endothelial receptor LYVE-1 has been implicated in both uptake of hyaluronan (HA) from tissue matrix and in facilitating transit of leukocytes and tumor cells through lymphatic vessels based largely onin vitrostudies with recombinant receptor in transfected fibroblasts. Curiously, however, LYVE-1 in lymphatic endothelium displays little if any binding to HAin vitro, and this has led to the conclusion that the native receptor is functionally silenced, a feature that is difficult to reconcile with its proposedin vivofunctions. Nonetheless, as we reported recently, LYVE-1 can function as a receptor for HA-encapsulated Group A streptococci and mediate lymphatic dissemination in mice. Here we resolve these paradoxical findings and show that the capacity of LYVE-1 to bind HA is strictly dependent on avidity, demanding appropriate receptor self-association and/or HA multimerization. In particular, we demonstrate the prerequisite of a critical LYVE-1 threshold density and show that HA binding may be elicited in lymphatic endothelium by surface clustering with divalent LYVE-1 mAbs. In addition, we show that cross-linking of biotinylated HA in streptavidin multimers or supramolecular complexes with the inflammation-induced protein TSG-6 enables binding even in the absence of LYVE-1 cross-linking. Finally, we show that endogenous HA on the surface of macrophages can engage LYVE-1, facilitating their adhesion and transit across lymphatic endothelium. These results reveal LYVE-1 as a low affinity receptor tuned to discriminate between different HA configurations through avidity and establish a new mechanistic basis for the functions ascribed to LYVE-1 in matrix HA binding and leukocyte traffickingin vivo.

Rapid Lymphatic Dissemination of Encapsulated Group A Streptococci via Lymphatic Vessel Endothelial Receptor-1 Interaction.

The host lymphatic network represents an important conduit for pathogen dissemination. Indeed, the lethal human pathogen group A streptococcus has a predilection to induce pathology in the lymphatic system and draining lymph nodes, however the underlying basis and subsequent consequences for disease outcome are currently unknown. Here we report that the hyaluronan capsule of group A streptococci is a crucial virulence determinant for lymphatic tropism in vivo, and further, we identify the lymphatic vessel endothelial receptor-1 as the critical host receptor for capsular hyaluronan in the lymphatic system. Interference with this interaction in vivo impeded bacterial dissemination to local draining lymph nodes and, in the case of a hyper-encapsulated M18 strain, redirected streptococcal entry into the blood circulation, suggesting a pivotal role in the manifestation of streptococcal infections. Our results reveal a novel function for bacterial capsular polysaccharide in directing lymphatic tropism, with potential implications for disease pathology.

Analysis of CD44-hyaluronan interactions in an artificial membrane system: insights into the distinct binding properties of high and low molecular weight hyaluronan.

CD44 is a major cell surface receptor for the large polydisperse glycosaminoglycan hyaluronan (HA). Binding of the long and flexible HA chains is thought to be stabilized by the multivalent nature of the sugar molecule. In addition, high and low molecular weight forms of HA provoke distinct proinflammatory and anti-inflammatory effects upon binding to CD44 and can deliver either proliferative or antiproliferative signals in appropriate cell types. Despite the importance of such interactions, however, neither the stoichiometry of multivalent HA binding at the cell surface nor the molecular basis for functional distinction between different HA size categories is understood. Here we report on the design of a supported lipid bilayer system that permits quantitative analysis of multivalent binding through presentation of CD44 in a stable, natively oriented manner and at controlled density. Using this system in combination with biophysical techniques, we show that the amount of HA binding to bilayers that are densely coated with CD44 increases as a function of HA size, with half-maximal saturation at ∼30 kDa. Moreover, reversible binding was confined to the smaller HA species (molecular weight of ≤10 kDa), whereas the interaction was essentially irreversible with larger polymers. The amount of bound HA decreased with decreasing receptor surface density, but the stability of binding was not affected. From a physico-chemical perspective, the binding properties of HA share many similarities with the typical behavior of a flexible polymer as it adsorbs onto a homogeneously attractive surface. These findings provide new insight into the multivalent nature of CD44-HA interactions and suggest a molecular basis for the distinct biological properties of different size fractions of hyaluronan.

Distinctive properties of the hyaluronan-binding domain in the lymphatic endothelial receptor Lyve-1 and their implications for receptor function.

The lymphatic endothelial hyaluronan (HA) receptor Lyve-1 is a member of the Link protein superfamily most similar to the leukocyte HA receptor CD44. However, the structure of Lyve-1 and the nature of its interaction with ligand are obscure. Here we present new evidence that Lyve-1 is functionally distinct from CD44. Using truncation mutagenesis we confirm that Lyve-1 in common with CD44 contains an extended HA-binding unit, comprising elements flanking the N and C termini of the consensus lectin-like Link module, bridged by a third conserved disulfide linkage that is critical for HA binding. In addition, we identify six essential residues Tyr-87, Ile-97, Arg-99, Asn-103, Lys-105, and Lys-108 that define a compact HA-binding surface on Lyve-1, encompassing the epitope for an adhesion-blocking monoclonal antibody 3A, in an analogous position to the HA-binding surface in CD44. The overtly electrostatic character of HA binding in Lyve-1 and its sensitivity to ionic strength (IC(50) of 150 mm NaCl) contrast markedly with CD44 (IC(50) > 2 m NaCl) in which HA binding is mediated by hydrogen bonding and hydrophobic interactions. In addition, unlike the extended Link module in CD44, which binds HA efficiently when expressed as a soluble monomer (K(d) = 65.7 mum), that of Lyve-1 requires artificial dimerization, although the full ectodomain is active as a monomer (K(d) = 35.6 mum). Finally, full-length Lyve-1 did not form stable dimers in binding-competent 293T transfectants when assessed using bioluminescent resonance energy transfer. These results reveal that elements additional to the extended Link module are required to stabilize HA binding in Lyve-1 and indicate important structural and functional differences with CD44.

A mechanism of sialylation functionally silences the hyaluronan receptor LYVE-1 in lymphatic endothelium.

The major lymphatic endothelial hyaluronan receptor LYVE-1, a Link superfamily glycoprotein similar to the hyaluronan-binding/inflammatory leukocyte homing receptor CD44, was initially implicated in hyaluronan (HA)-mediated cell adhesion and lymph-borne hyaluronan metabolism. However, the apparently normal phenotype of Lyve-1 knock-out mice and the recent demonstration that the receptor undergoes cytokine-induced endocytosis independent of HA uptake have cast doubt on such functions. Here we present new data that reconcile these anomalies by showing that LYVE-1 is functionally "silenced" in a cell-specific fashion by autoinhibitory glycosylation. We demonstrate that LYVE-1 transfected in HEK 293T fibroblasts and Jurkat T cells is competent to bind HA, whereas the endogenous receptor in cultured lymphatic endothelial cells or the receptor transfected in Chinese hamster ovary and HeLa cells is not. Moreover, through a combination of mutagenesis and functional analysis in HEK 293T fibroblasts and glycosylation-defective Chinese hamster ovary cell lines, we reveal that the inhibitory mechanism is reversible and is exerted by terminal sialylation, most likely through alpha2-3 or alpha2-6 linkage to O-glycans. Finally, we provide evidence that the mechanism operates in vivo by showing that native LYVE-1 in primary lymphatic endothelial cells is extensively sialylated and that HA binding can be reactivated by neuraminidase treatment of the soluble ectodomain. These results reveal unexpected complexity in the regulation of LYVE-1 function and raise the possibility that this receptor, like CD44, may become active after appropriate unmasking in vivo.

Structures of the Cd44-hyaluronan complex provide insight into a fundamental carbohydrate-protein interaction.

Regulation of transient interactions between cells and the ubiquitous matrix glycosaminoglycan hyaluronan is crucial to such fundamental processes as embryonic development and leukocyte homing. Cd44, the primary cell surface receptor for hyaluronan, binds ligand via a lectin-like fold termed the Link module, but only after appropriate functional activation. The molecular details of the Cd44-hyaluronan interaction and hence the structural basis for this activation are unknown. Here we present the first crystal structure of Cd44 complexed with hyaluronan. This reveals that the interaction with hyaluronan is dominated by shape and hydrogen-bonding complementarity and identifies two conformational forms of the receptor that differ in orientation of a crucial hyaluronan-binding residue (Arg45, equivalent to Arg41 in human CD44). Measurements by NMR indicate that the conformational transition can be induced by hyaluronan binding, providing further insight into possible mechanisms for regulation of Cd44.

Rapid plasma membrane-endosomal trafficking of the lymph node sinus and high endothelial venule scavenger receptor/homing receptor stabilin-1 (FEEL-1/CLEVER-1).

The sinusoidal endothelia of liver, spleen, and lymph node are major sites for uptake and recycling of waste macromolecules through promiscuous binding to a disparate family of scavenger receptors. Among the most complex is stabilin-1, a large multidomain protein containing tandem fasciclin domains, epidermal growth factor-like repeats, and a C-type lectin-like hyaluronan-binding Link module, which functions as an endocytic receptor for acetylated low density lipoprotein and advanced glycation end products. Intriguingly, stabilin-1 has also been reported to mediate both homing of leukocytes across lymph node high endothelial venules and adhesion of metastatic tumor cells to peritumoral lymphatic vessels. Currently, however, it is not clear how stabilin-1 mediates these distinct functions. To address the issue, we have investigated the tissue and subcellular localization of stabilin-1 in detail and assessed the functional status of its Link module. We show that stabilin-1 is almost entirely intracellular in lymph node high endothelial venules, lymphatic sinus endothelium, and cultured endothelial cells but that a finite population, detectable only by fluorescent antibody or fluorescein-labeled (Fl)-acetylated low density lipoprotein uptake, cycles rapidly between the plasma membrane and EEA-1+ve (early endosome antigen 1) early endosomes. In addition, we show using full-length stabilin-1 cDNA and a stabilin-1/CD44 chimera in HeLa cells that intracellular targeting is influenced by the transmembrane domain/cytoplasmic tail, which contains a putative dileucine (DXXLL) Golgi to endosomal sorting signal. Finally, we provide evidence that the stabilin-1 Link domain binds neither hyaluronan nor other glycosaminoglycans. These properties support a role for stabilin-1 as a rapidly recycling scavenger receptor and argue against a role in cell adhesion or lymphocyte homing.

Structure of the regulatory hyaluronan binding domain in the inflammatory leukocyte homing receptor CD44.

Adhesive interactions involving CD44, the cell surface receptor for hyaluronan, underlie fundamental processes such as inflammatory leukocyte homing and tumor metastasis. Regulation of such events is critical and appears to be effected by changes in CD44 N-glycosylation that switch the receptor "on" or "off" under appropriate circumstances. How altered glycosylation influences binding of hyaluronan to the lectin-like Link module in CD44 is unclear, although evidence suggests additional flanking sequences peculiar to CD44 may be involved. Here we show using X-ray crystallography and NMR spectroscopy that these sequences form a lobular extension to the Link module, creating an enlarged HA binding domain and a formerly unidentified protein fold. Moreover, the disposition of key N-glycosylation sites reveals how specific sugar chains could alter both the affinity and avidity of CD44 HA binding. Our results provide the necessary structural framework for understanding the diverse functions of CD44 and developing novel therapeutic strategies.

Absence of lymphangiogenesis and intratumoural lymph vessels in human metastatic breast cancer.

Early metastasis to lymph nodes is a frequent complication in human breast cancer. However, the extent to which this depends on lymphangiogenesis or on invasion of existing lymph vessels remains controversial. Although proliferating intratumoural lymphatics that promote nodal metastasis have been demonstrated in experimental breast tumours overexpressing VEGF-C, it has yet to be determined whether the same phenomena occur in spontaneous human breast cancers. To address this important issue, the present study investigated the lymphatics in primary human breast carcinoma (75 cases of invasive ductal and lobular breast cancer) by quantitative immunohistochemical staining for the lymphatic endothelial hyaluronan receptor LYVE-1, the blood vascular marker CD34, and the nuclear proliferation marker pKi67. None of the breast carcinomas was found to contain dividing lymph vessels, even in areas of active haemangiogenesis. Furthermore, the majority of non-dividing lymph vessels were confined to the tumour periphery where their incidence was low and unrelated to tumour size, grade or nodal status; rather, their density was inversely correlated with tumour aggressiveness as assessed by macrophage density (p = 0.009), and blood microvessel density (p = 0.05, Spearman Rank), as well as with distance from the tumour edge. Finally, a proportion of the peritumoural lymphatics contained tumour emboli associated with hyaluronan, indicating a possible role for LYVE-1/hyaluronan interactions in lymphatic invasion or metastasis. These results suggest that naturally occurring breast carcinomas invade and destroy lymph vessels rather than promoting their proliferation; that breast tumour lymphangiogenesis may not always occur at physiological VEGF-C levels; and that nodal metastasis can proceed via pre-existing lymphatics.

Intratumoral lymphangiogenesis and lymph node metastasis in head and neck cancer.

How tumors access and spread via the lymphatics is not understood. Although it is clear that dissemination via the blood system involves hemangiogenesis, it is uncertain whether tumors also induce lymphangiogenesis or simply invade existing peritumoral vessels. To address the issue we quantitated tumor lymph vessels in archival specimens of head and neck cancer by immunostaining for the recently described lymphatic endothelial marker LYVE-1, the vascular endothelial marker CD34, and the pKi67 proliferation marker, correlating lymph vessel density and proliferation index with clinical and pathological variables. Discrete "hotspots" of intratumoral small proliferating lymphatics were observed in all carcinomas, and a high intratumoral lymph vessel density was associated with neck node metastases (n = 23; P = 0.027) and an infiltrating margin of tumor invasion (P = 0.046) in the oropharyngeal subgroup. Quantitation of the lymphangiogenic growth factor vascular endothelial growth factor C by real-time PCR and immunohistochemistry revealed higher levels of mRNA in tumor tissue than in normal samples (n = 8; P = 0.017), but no obvious correlation with intratumoral lymphatics. Our results provide new evidence that proliferating lymphatics can occur in human cancers and may in some cases contribute to lymph node metastasis.