CD44 Binds to Macrophage Mannose Receptor on Lymphatic Endothelium and Supports Lymphocyte Migration via Afferent LymphaticsNovelty and Significance
Rationale: Macrophage mannose receptor (MRC) is one of the few molecules known to be involved in lymphocyte trafficking via the lymphatic vessels. In endothelial cells of efferent lymphatics, it binds L-selectin on lymphocytes. In afferent lymphatics, MRC mediates trafficking of both normal and malignant L-selectin–negative cells to the draining lymph nodes.
Objective: This work was designed to search for additional lymphocyte ligands of MRC to elucidate how lymphocytes migrate into the draining lymph nodes.
Methods and Results: Using immunoprecipitation and binding studies with natural and recombinant proteins, we show that MRC and CD44 can interact with each other. Fine mapping revealed that the cysteine-rich domain of MRC binds to the chondroitin sulfate side chains of CD44. In vivo homing experiments with MRC- and CD44-deficient mice verified that MRC and CD44 function as a receptor-ligand pair in supporting lymphocyte migration via the afferent lymphatics into the draining lymph nodes.
Conclusions: These data identify a new counter-receptor for MRC and reveal CD44 as a new molecule involved in the poorly understood process of lymphocyte transit via the lymphatic vasculature.
Leukocyte migration from the periphery into the draining lymph nodes via the afferent lymphatics largely determines the magnitude of immune response within the node. Exit of lymphocytes from the lymph nodes via the lymphoid sinuses into efferent lymphatics also controls the recirculation potential of naive and activated lymphocytes. Furthermore, the lymphatic vasculature also presents an important route for cancer cells to metastasize.
Macrophage mannose receptor (MRC) is a scavenger receptor on macrophages and mediates the uptake of mannosylated glycoconjugates. It is also expressed on lymphatics and is involved in the migration of lymphocytes and melanoma cells into the draining lymph nodes.1 As a large protein consisting of a cysteine-rich (CR)/ricin R-type lectin domain, a fibronectin type II domain, and 8 C-type lectin domains, it has the potential to interact with diverse ligands.2 In vitro binding studies indicate that L-selectin functions as a leukocyte ligand for MRC at least in lymphoid sinuses.3,4 Because L-selectin–negative cells also migrate in an MRC-dependent manner via the afferent lymphatics,1 we wanted to identify additional leukocyte counter receptors for MRC.
On the basis of the protein structure and function, we hypothesized that CD44 might interact with MRC. CD44 is a proteoglycan having ≥40 isoforms as a result of the alternative splicing and abundant posttranslational modifications. It is involved in a multitude of functions, including apoptosis, adhesion to extracellular matrix, and lymphocyte homing from the blood into the lymphoid organs.5 The standard hematopoietic form of CD44 has glycosaminoglycan side chains, which are predominantly chondroitin-4 and -6-sulfates. In addition, the core protein incorporates sulfate directly to its other oligosaccharide components or amino acids.6,7 The best-characterized ligand for leukocyte CD44 is hyaluronan, present on the endothelial cell surface of blood vessels, and this interaction is important in the rolling phase of leukocyte extravasation. In addition, CD44 on mature leukocytes binds to endothelial selectins, and it can induce signals activating other adhesion molecules (eg, lymphocyte function-associated antigen-1).5,8
Full methods are described in the online-only Data Supplement.
The MRC knockout (MRC−/−) mice were a kind gift from M. Nussenzweig (Rockefeller University). The CD44 knockout (CD44−/−) mice9 and their wild-type (WT) controls were from Jackson Laboratories.
Frozen sections of mouse peripheral lymph nodes and ears were double stained for MRC and lymphatic endothelial hyaluronan receptor-1 (LYVE-1), MRC and F4/80, MRC and CD44, and plasmalemma vesicle-associated protein-1 and LYVE-1.
CD44 Chimera and MRC Constructs
The chimeric CD44 construct was a kind gift from D. Jackson (University of Oxford) and consisted of a full-length extracellular domain of the standard form of human CD44 fused to human Fc. The MRC constructs generated are presented in Figure 1.
Coprecipitation and Binding Studies
Binding of CD44 and MRC to affinity-purified human MRC and CD44 was tested by coprecipitation and detected with immunoblotting. The human material was used with the permission of the Ethical Committee of Turku University Hospital.
CD44-Fc and control chimera with and without chondroitinase treatment were incubated with different MRC transfectants. Bound chimera was detected with fluorescein isothiocyanate–conjugated anti-human IgG and analyzed using flow cytometry.
Binding of CD44 and control Namalwa transfectants to lymphatic endothelial cells in human lymph nodes was tested in ex vivo frozen section assays in the presence of anti-MRC or a control antibody. L-selectin was blocked with the function-blocking antibody Dreg56 during the assays.
Quantitative Polymerase Chain Reaction
Total RNA was extracted from peripheral lymph nodes, mesenteric lymph nodes, spleen, and liver of WT and MRC−/− mice (n=8). TaqMan Gene Expression Assays (Applied Biosystems) for mouse E-selectin, fibronectin-1, and Spp1 (osteopontin) were used in quantitative polymerase chain reaction, and the samples were run as triplicates. The expression values were normalized using mouse β-actin.
In Vivo Studies
Fluorescently labeled lymphocytes isolated from lymph nodes and spleens of CD44−/− and WT mice were injected subcutaneously into the footpads of MRC−/− and WT mice. After 12 hours, the popliteal lymph nodes were harvested and cell suspensions were analyzed using flow cytometry.
Data were analyzed with the Student t test (2 tailed).
CD44 and MRC Interact With Each Other
The CR domain of MRC recognizes chondroitin sulfate A and B and sulfated oligosaccharides of blood group Lewis a and Lewis x, as well as sulfated N-glycans of lutropin.10 Because CD44 has chondroitin sulfate side chains and sulfated carbohydrates linked to its core, we tested whether it could serve as a leukocyte counter receptor for MRC. We first affinity-purified CD44 from leukocyte lysates and ran lymphatic endothelial cells containing lymph node lysates through the columns. Immunoblotting analyses showed that MRC had specifically interacted with the immobilized CD44. Reciprocal experiments with immobilized natural MRC molecules confirmed the interaction with CD44. The band of CD44 appeared to be ≈180 kDa, suggesting it to be a certain splice variant or forms containing glycosaminoglycan side chains of CD44 (Figure 1A).
CR Domain of MRC Binds to Chondroitin Sulfate Side Chains of CD44
To characterize the binding in more detail, we produced recombinant CD44 as an Fc chimera. It showed a smeary appearance in the gel, typical for CD44 because of its abundant glycosylation. In addition, a small subpopulation of CD44 molecules were decorated by chondroitin sulfate side chains as demonstrated with 3 sharp bands showing up after chondroitinase ABC treatment (Figure 1B). We then tested the binding of the chimera to transfectants expressing different domains of MRC. The structure of the different domains is depicted in Figure 1C. Flow cytometric analyses showed that CD44 binds to the CR domain of MRC but not to the fibronectin type II domain or to the lectin domain (Figure 1D). Digestion of the chondroitin sulfate side chains of CD44-Fc with chondroitinase ABC abolished the binding to MRC (Figure 1E). Thus, MRC is a novel ligand for CD44, and the interaction takes place via binding of the glycosaminoglycan side chains of CD44 to the distal CR domain of MRC.
Expression Profiles of CD44 and MRC Are Compatible With the Ligand-Receptor Pair Concept
MRC is expressed both on LYVE-1+ lymphatic endothelial cells and on a subset of F4/80+ macrophages in the skin of WT mice (Figure 2A). In WT mice, CD44 is expressed mainly on hematopoietic cells in normal lymph nodes, whereas lymphatic endothelial cells are negative. MRC, however, is present on both afferent and efferent lymphatics and on many macrophages in the subcapsular sinus of the lymph node (Figure 2B). The MRC−/− and CD44−/− mice have apparently normal blood and lymphatic vasculature as detected by anti–plasmalemma vesicle-associated protein-1 and anti–LYVE-1, respectively (Figure 2C and Online Table I). Thus, the expression patterns of MRC on lymphatic endothelium and CD44 on lymphocytes are compatible with the possibility that the 2 molecules interact during lymphocyte migration through lymphatics. Furthermore, the expression of the previously reported CD44 counter receptors osteopontin, fibronectin, and E-selectin was not altered in MRC−/−mice compared with WT mice (Online Figure I), indicating that the absence of 1 ligand of CD44 (MRC) does not lead to compensatory changes in the expression of other ligands.
CD44-MRC Mediates Lymphocyte Trafficking via the Afferent Lymphatic Vessels In Vivo
The functional relevance of CD44-MRC interaction in supporting lymphocyte trafficking was then studied in adoptive cell transfer experiments. Fluorescently labeled lymphocytes from spleen and lymph nodes of WT and CD44−/− mice were injected into the footpads of WT and MRC−/− recipient mice, and their homing to the draining lymph nodes was measured. WT lymphocyte migration via the afferent lymphatics was impaired in MRC−/− mice, confirming our previous findings.1 CD44-negative lymphocytes also showed diminished capacity to home from the footpad into the draining lymph nodes in WT recipients (on average, 54% reduction; P<0.01). Furthermore, homing of CD44-negative lymphocytes through MRC−/− lymphatics was reduced to the similar extent (on average, 50% reduction; P<0.005). These functional assays show a new role for CD44 in lymphocyte migration via the lymphatics and indicate that MRC-CD44 is a receptor-ligand pair also in vivo (Figure 3A).
We further tested whether different lymphocyte populations are selectively dependent on CD44-MRC in their homing to draining lymph nodes. In general, T cells homed significantly better than B cells, and more L-selectin–negative cells homed than expected when their relative number in the input population was taken into account. However, their relative percentages were comparable in WT and MRC−/− mice (Online Figure II). Furthermore, because L-selectin can easily be shed from the surface of activated lymphocytes during the homing, we directly tested whether CD44-MRC interaction mediates lymphocyte binding to lymphatic endothelium independently of L-selectin using frozen section ex vivo assays. These experiments clearly showed that CD44 lymphoma transfectants bind better to lymphatic endothelial cells than their controls in the presence of a function-blocking antibody against L-selectin (P=0.03) and that blocking of MRC inhibits this CD44-mediated binding (P=0.03; Figure 3B).
In this work, we have identified a new ligand-receptor pair mediating lymphocyte trafficking from the periphery into the draining lymph nodes. Although the role of MRC in guiding lymphocyte migration within lymphatics was known earlier and the contribution of CD44 and hyaluronan interaction to leukocyte trafficking in the blood vessels is well recognized,1,5 CD44-MRC interaction with each other was not known. Furthermore, discovery of CD44 as a lymphocyte surface molecule supporting lymphocyte migration within the lymphatics helps to fill in the so-far incomplete understanding of the molecular mechanisms regulating cell trafficking within lymphatics.
Langerhans cell migration into the draining lymph nodes has been reported to be significantly reduced in CD44−/− mice.11 The reason is thought to be the lack of osteopontin-dependent chemotaxis because binding of osteopontin to variant exons of CD44 on Langerhans cells is important in this process.12,13 Furthermore, because both CD44 on leukocytes and LYVE-1 on lymphatic endothelium bind hyaluronan, bridging through hyaluronan has been speculated, but not shown, to contribute to leukocyte traffic within the lymphatics.14 Our current data now experimentally demonstrate for the first time a molecular mechanism by which CD44 mediates lymphocyte traffic in the lymphatic vasculature.
Besides normal leukocytes, most malignancies of hematopoietic origin express CD44, and it has also been demonstrated to be an important marker for cancer stem cells. Importantly, high expression of CD44 is associated with poor prognosis.15–17 As a result of multiple protumorigenic functions of CD44, its targeting in hematopoietic malignancies is actively explored in preclinical trials.16 From our findings, we speculate that binding of CD44 to MRC is perhaps involved in the metastatic spread of malignancies via the lymphatics, thus suggesting that both MRC and CD44 may be potential drug targets when metastatic process via the lymphatics needs to be prevented.
In conclusion, leukocyte trafficking within the lymphatics and blood vasculature is equally important for controlling the quality and quantity of the immune response. On the basis of our results, CD44 can be added to those few lymphocyte molecules known to mediate lymphocyte migration within the afferent lymphatics, and its potential as a drug target remains to be tested.
Sources of Funding
This work was supported by the Finnish Academy, Sigrid Juselius Foundation, Arvo and Inkeri Suominen Foundation, and Finnish Cancer Foundation (to S. Jalkanen).
In March 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.5 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.111.300476/-/DC1.
- CD44 knockout
- cysteine rich
- macrophage mannose receptor
- MRC knockout
- wild type
- Received November 7, 2012.
- Revision received April 18, 2013.
- Accepted April 19, 2013.
- © 2013 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Leukocyte trafficking via the lymphatic vessels is fundamental for proper functioning of the immune system.
Macrophage mannose receptor expressed on the lymphatic endothelial cells can bind to lymphocyte L-selectin to mediate lymphocyte migration into lymphatic vessels.
CD44 is a multifunctional adhesion molecule that mediates lymphocyte adhesion to extracellular matrix and vascular endothelium
What New Information Does This Article Contribute?
Lymphocyte CD44 is a novel counter receptor for macrophage mannose receptor.
CD44–macrophage mannose receptor interaction mediates lymphocyte migration via the afferent lymphatic vessels into the draining lymph nodes under physiological conditions.
Lymphocyte recirculation from the blood to tissues and then via the lymphatic vessels to lymphatic organs and back to blood is crucial for the generation of immune responses. Here, we show that binding of lymphocyte CD44 to macrophage mannose receptor, expressed on lymphatic endothelium, is involved in lymphocyte migration from the periphery to the draining lymph nodes. Identification of this novel receptor-ligand pair reinforces the multifunctionality of these 2 molecules. These findings add to our understanding of the molecular mechanisms guiding lymphocyte entrance into afferent lymphatic vessels and could provide new targets to regulate harmful cell trafficking in inflammatory diseases.