Steven Clasper

The extracellular matrix glycosaminoglycan hyaluronan (HA) is an abundant component of skin and mesenchymal tissues where it facilitates cell migration during wound healing, inflammation, and embryonic morphogenesis. Both during normal tissue homeostasis and particularly after tissue injury, HA is mobilized from these sites through lymphatic vessels to the lymph nodes where it is degraded before entering the circulation for rapid uptake by the liver. Currently, however, the identities of HA binding molecules which control this pathway are unknown. Here we describe the first such molecule, LYVE-1, which we have identified as a major receptor for HA on the lymph vessel wall. The deduced amino acid sequence of LYVE-1 predicts a 322-residue type I integral membrane polypeptide 41% similar to the CD44 HA receptor with a 212-residue extracellular domain containing a single Link module the prototypic HA binding domain of the Link protein superfamily. Like CD44, the LYVE-1 molecule binds both soluble and immobilized HA. However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels. Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels themselves.

The glycosaminoglycan hyaluronan is a key substrate for cell migration in tissues during inflammation, wound healing, and neoplasia. Unlike other matrix components, hyaluronan (HA) is turned over rapidly, yet most degradation occurs not locally but within distant lymph nodes, through mechanisms that are not yet understood. While it is not clear which receptors are involved in binding and uptake of hyaluronan within the lymphatics, one likely candidate is the lymphatic endothelial hyaluronan receptor LYVE-1 recently described in our laboratory (Banerji, S., Ni, J., Wang, S., Clasper, S., Su, J., Tammi, R., Jones, M., and Jackson, D.G. (1999)J. Cell Biol. 144, 789–801). Here we present evidence that LYVE-1 is involved in the uptake of hyaluronan by lymphatic endothelial cells using a new murine LYVE-1 orthologue identified from the EST data base. We show that mouse LYVE-1 both binds and internalizes hyaluronan in transfected 293T fibroblasts in vitro and demonstrate using immunoelectron microscopy that it is distributed equally among the luminal and abluminal surfaces of lymphatic vessels in vivo. In addition, we show by means of specific antisera that expression of mouse LYVE-1 remains restricted to the lymphatics in homozygous knockout mice lacking a functional gene for CD44, the closest homologue of LYVE-1 and the only other Link superfamily HA receptor known to date. Together these results suggest a role for LYVE-1 in the transport of HA from tissue to lymph and imply that further novel hyaluronan receptors must exist that can compensate for the loss of CD44 function. The glycosaminoglycan hyaluronan is a key substrate for cell migration in tissues during inflammation, wound healing, and neoplasia. Unlike other matrix components, hyaluronan (HA) is turned over rapidly, yet most degradation occurs not locally but within distant lymph nodes, through mechanisms that are not yet understood. While it is not clear which receptors are involved in binding and uptake of hyaluronan within the lymphatics, one likely candidate is the lymphatic endothelial hyaluronan receptor LYVE-1 recently described in our laboratory (Banerji, S., Ni, J., Wang, S., Clasper, S., Su, J., Tammi, R., Jones, M., and Jackson, D.G. (1999)J. Cell Biol. 144, 789–801). Here we present evidence that LYVE-1 is involved in the uptake of hyaluronan by lymphatic endothelial cells using a new murine LYVE-1 orthologue identified from the EST data base. We show that mouse LYVE-1 both binds and internalizes hyaluronan in transfected 293T fibroblasts in vitro and demonstrate using immunoelectron microscopy that it is distributed equally among the luminal and abluminal surfaces of lymphatic vessels in vivo. In addition, we show by means of specific antisera that expression of mouse LYVE-1 remains restricted to the lymphatics in homozygous knockout mice lacking a functional gene for CD44, the closest homologue of LYVE-1 and the only other Link superfamily HA receptor known to date. Together these results suggest a role for LYVE-1 in the transport of HA from tissue to lymph and imply that further novel hyaluronan receptors must exist that can compensate for the loss of CD44 function. hyaluronan lymphatic vessel endothelial HA receptor-1 fluorescein isothiocyanate polymerase chain reaction enzyme-linked immunosorbent assay phosphate-buffered saline HA receptor for endocytosis The extracellular matrix glycosaminoglycan hyaluronan (HA)1 is a large polymer ofN-acetyl-d-glucosamine andd-glucuronic acid (molecular mass 105-107 Da) which plays an important role in maintaining tissue integrity as well as facilitating the migration of cells during inflammation, wound repair, and embryonic development (1Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2086) Google Scholar,2Lee J.Y. Spicer A.P. Curr. Opin. Cell Biol. 2000; 12: 581-586Crossref PubMed Scopus (453) Google Scholar). By comparison with other macromolecules of the extracellular matrix, HA undergoes rapid turnover with a half-life of ∼24 h (1Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2086) Google Scholar). Intriguingly, most degradation occurs not locally, but within distant lymph nodes. During this process, tissue HA enters the afferent lymphatic vessels and is transported with the lymph fluid to the draining lymph nodes where ∼90% of the glycosaminoglycan is degraded by unknown mechanisms (3Fraser J.R. Kimpton W.G. Laurent T.C. Cahill R.N. Vakakis N. Biochem. J. 1988; 256: 153-158Crossref PubMed Scopus (161) Google Scholar, 4Fraser J.R. Laurent T.C. CIBA Found. Symp. 1989; 143: 41-53PubMed Google Scholar). The remaining 10–15% of the HA exits via the efferent lymphatics to the blood vasculature where it is rapidly endocytosed by the liver sinusoid endothelial HA receptor (5Yannariello-Brown J. McGary C.T. Weigel P.H. J. Cell. Biochem. 1992; 48: 73-80Crossref PubMed Scopus (17) Google Scholar), a 300-kDa heterotrimeric complex of α, β, and γ subunits that clears not only HA but also chondroitin and heparan sulfate from the circulation (6Zhou B. Oka J.A. Singh A. Weigel P.H. J. Biol. Chem. 1999; 274: 33831-33834Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). While it is clear that HA can rapidly permeate the lymphatics in skin and other tissues (7Brown T.J. Alcorn D. Fraser J.R. J. Invest. Dermatol. 1999; 113: 740-746Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), the mechanisms responsible for its transport across lymphatic endothelium, and the receptors involved in its uptake and transport within lymphatic vessels are all unknown. The majority of HA-binding proteins (8Toole B.P. Curr. Opin. Cell Biol. 1990; 2: 839-844Crossref PubMed Scopus (393) Google Scholar, 9Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1241Crossref PubMed Scopus (601) Google Scholar) identified to date belong to the Link protein superfamily, defined by the presence of a conserved HA-binding domain known as the Link module (10Neame P.J. Barry F.P. Experientia (Basel). 1993; 49: 393-402Crossref PubMed Scopus (110) Google Scholar, 11Day A.J. Biochem. Soc. Trans. 1999; 27: 115-121Crossref PubMed Scopus (76) Google Scholar). This is a unit of ∼100 amino acids that contains four conserved cysteine residues interspersed with tracts of both hydrophobic and charged residues. The three-dimensional structure of the Link module closely resembles that of the C-type lectin fold, comprising two β sheets flanked by two short α helices and stabilized by two disulfide linkages enclosing a central hydrophobic core (12Kohda D. Morton C.J. Parkar A.A. Hatanaka H. Inagaki F.M. Campbell I.D. Day A.J. Cell. 1996; 86: 767-775Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 13Brissett N.C. Perkins S.J. FEBS Lett. 1996; 388: 211-216Crossref PubMed Scopus (36) Google Scholar). Members of the Link superfamily include versican (14Zimmerman D.R. Ruoslahti E. EMBO J. 1989; 8: 2975-2981Crossref PubMed Scopus (502) Google Scholar), the cartilage structural proteins aggrecan and link protein (15Doege K.J. Sasaki M. Kimura T. Yamada Y. J. Biol. Chem. 1991; 266: 894-902Abstract Full Text PDF PubMed Google Scholar), the brain proteoglycans neurocan (16Rauch U. Karthikeyan L. Maurel P. Margolis R.U. Margolis R.K. J. Biol. Chem. 1992; 267: 19547-19563Abstract Full Text PDF Google Scholar) and brevican (17Yamada H. Watanabe K. Shimonaka M. Yamaguchi Y. J. Biol. Chem. 1994; 269: 10119-10126Abstract Full Text PDF PubMed Google Scholar), the inflammation-associated TSG-6 protein (18Wisniewski H.-G. Maier R. Lotz M. Lee S. Klampfer L. Lee T.H. Vilcek J. J. 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PubMed Scopus Google Scholar). the of CD44 in the by the that homozygous mice in the lymphatic R. J. N. T. A. A. J. A. C.J. PubMed Google Scholar, U. T. W. J. Immunol. 1999; Google Scholar). the that novel HA receptors must within the and and a for within the we identified a novel Link superfamily HA receptor LYVE-1 HA from a of the EST data and its expression to lymphatic endothelial cells within tissues S. J. S. J. R. M. D.G. J. Cell Biol. 1999; PubMed Scopus Google Scholar). that the LYVE-1 binds HA with a of and a role for the receptor in HA the luminal of lymphatic in an also S. L. M. A. D. R. S. J. R. D.G. L. K. EMBO J. PubMed Scopus Google Scholar, M. T. D.G. L. P. L. K. M. 7: PubMed Scopus Google Scholar, D.G. R. H. 7: PubMed Scopus Google Scholar, T. L. T. T. K.J. R. D.G. H. S. K. 7: PubMed Scopus Google Scholar, D.G. 7: Scholar, D.G. R. S. S. Immunol. Full Text Full Text PDF PubMed Scopus Google Scholar, D.G. R. J. S. Scholar) we a murine LYVE-1 orthologue and we its with that important with the CD44 Intriguingly, we that mouse LYVE-1 of HA and is both the luminal and abluminal of lymphatic endothelial The of these for the of LYVE-1 in are The cell 293T from the Cell The expression from The The for protein expression by to mouse from from and from from and from hyaluronan from and from from chondroitin and heparan sulfate from protein the extracellular domain of to the domain of by S. of knockout mice R. J. N. T. A. A. J. A. C.J. PubMed Google Scholar) the and from the for of by and the and by of for microscopy as described The amino acid of LYVE-1 to for murine within the mouse EST data using the via the The four and which a of to the The and and from mouse in a reaction using as In the of the and as in with of mouse D.G. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar) in a reaction and of In the of of the as in a reaction using the and The in are and the The both to its and amino acid of murine LYVE-1 and comparison with the and amino acid from the mouse LYVE-1 identified by the mouse EST data and from mouse The and and two for are an of the amino acid for mouse and CD44 with the and with residues in The the Link cysteine residues are and by are as the four conserved structural of the Link module the two conserved for and of CD44 Link a conserved cysteine within the for receptor and HA-binding in CD44 and a cysteine to LYVE-1 residues within the LYVE-1 Link module to in CD44 HA-binding are with In addition, a conserved of residues of the Link module in mouse and LYVE-1 and a in CD44 that an to the HA-binding domain are with a of mouse tissue of from and in with a the extracellular domain of mouse LYVE-1 with by to the in for and in for by in with by the The extracellular domain of mouse LYVE-1 the in from mouse D.G. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar) by of as described for LYVE-1 using the and by the and using of polymerase as in and the a the amino acids of mouse LYVE-1 to the of The both to expression and of the the transfected 293T cells using and in for to the of the by the of of the protein by a of protein with protein by the of of and the by HA by a of the of and B.P. Google Scholar) as described S. J. S. J. R. M. D.G. J. Cell Biol. 1999; PubMed Scopus Google Scholar). of HA with using the of and PubMed Scopus Google Scholar). of mouse LYVE-1 protein to HA in as described S. J. S. J. R. M. D.G. J. Cell Biol. 1999; PubMed Scopus Google Scholar). by with HA in and for h in and with mouse LYVE-1 protein in for h LYVE-1 S. J. S. J. R. M. D.G. J. Cell Biol. 1999; PubMed Scopus Google Scholar) and proteins as and with with protein with substrate in a with chondroitin chondroitin and heparan sulfate by the mouse LYVE-1 protein with the glycosaminoglycan for in The to and protein as described binding of LYVE-1 to with mouse LYVE-1 protein a protein in by and as described The with HA in a of HA as a for and using as as the binding of HA to cells these in HA and by in in with and a with 293T cells in transfected with mouse in LYVE-1 S. J. S. J. R. M. D.G. J. Cell Biol. 1999; PubMed Scopus Google Scholar) in using of with in the presence of a of HA for h cells with and by in with of cell in in HA and the other with for to cell by The of LYVE-1 the by cells and with LYVE-1 by In by using a of HA by cells as described for that cell LYVE-1 using a using a with of by with and to mouse LYVE-1 in and with in and to the of a with and by with mouse LYVE-1 protein in by two further in in by with mouse LYVE-1 protein protein as a to to the of the mice with of a of in as described in S. N. M. L. M. D. K. Cell 1999; PubMed Scopus Google the the mice by and which the surfaces of the and in the liver for and as described from mice homozygous knockout in and in to and by in and by in by in for and with a for to with LYVE-1 for with with for a further and with with tissues in using a and in to with LYVE-1 and with a of and in with and a of 293T these with LYVE-1 in for to in and with immunoelectron of mouse in and with LYVE-1 and with in in and in and in a We identified the LYVE-1 by the EST data for with the amino acid of the Link HA-binding domain of the CD44 Here we the LYVE-1 amino acid to the mouse orthologue by the mouse EST data with the The four and and that a of The which from mouse using contains a large of amino acids residues the with a and with a In the amino acid an hydrophobic to the extracellular domain four of which and are to the conserved disulfide that the link module two and and a likely to hydrophobic of the murine and LYVE-1 an the two of the extracellular the to the HA-binding Link unit are the are only of the in this are as are both the the Link that which is known to HA-binding in CD44, also important for LYVE-1 function. The of mouse and CD44 a of important the HA-binding of the two both within the Link module and in the the residues and in LYVE-1 Link to to the known CD44 HA-binding residues and D. A. J. Cell Biol. 1993; PubMed Scopus Google Scholar, J. B. Day A.J. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) are conserved in mouse LYVE-1 This further the that these amino acids HA in the LYVE-1 link a by results from not of the Link both mouse and LYVE-1 tracts of residues and conserved in a is also conserved in a LYVE-1 orthologue identified in a further of the EST data This functional in of the that a of residues in the domain of CD44 to to HA binding D. A. J. Cell Biol. 1993; PubMed Scopus Google Scholar). We also that an cysteine which is in CD44 is conserved in both mouse and LYVE-1 and This in important as this cysteine is to and to a that an disulfide to LYVE-1 In addition, the of mouse and LYVE-1 both a conserved cysteine and in the as in the CD44 that is in and HA binding D. J. 1996; PubMed Scopus Google Scholar, D. J. Immunol. Google Scholar). Together these the that CD44, a HA-binding domain that the link module J. B. Day A.J. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of the murine receptor we transfected 293T fibroblasts with in and binding of to the cell by the data in the majority of LYVE-1 but of the cells expression of the receptor in these by with mouse HA binding in we the extracellular domain of murine LYVE-1 as a protein and binding to in The results and the mouse binds both HA and HA in a binding to HA only by hyaluronan and not by the chondroitin chondroitin heparan sulfate results that the murine LYVE-1 receptor a for hyaluronan that is to that of the orthologue but is from the closely CD44 which binds both HA and chondroitin The of LYVE-1 to as a receptor for HA in where 293T cells with and the by from in these by both and of cells with to by the in 293T cells and The of of both cell and a within the of a of the HA by cells within this binding of by a of HA and uptake with 293T cells the of most likely LYVE-1 endocytosis of the by cells in these that to of cell LYVE-1 only of the The of the HA by microscopy which within the membrane these also to LYVE-1 cells with for and microscopy uptake of also in with 293T cells and data not results clear evidence that LYVE-1 both binding and of HA through an for the tissue of mouse LYVE-1 expression we in tissues by by the in to a only in and in not This with the orthologue where the is in of these tissues and is in S. J. S. J. R. M. D.G. J. Cell Biol. 1999; PubMed Scopus Google Scholar). results suggest are in the of LYVE-1 gene expression in LYVE-1 in and In to the LYVE-1 in murine tissues we a by with murine LYVE-1 The specific for the murine receptor as by an and to the LYVE-1 CD44 proteins to the LYVE-1 to the mouse orthologue not The of the LYVE-1 by of 293T cells transfected with LYVE-1 which the receptor in an the cell the binding of LYVE-1 protein to the presence of the domain not of a of mouse tissues large and and and using LYVE-1 of identified as lymphatics the of and the of a The vessels in tissues as the of large that an lymphatic and in by of the lymphatics from blood by with LYVE-1 and the endothelial which two of vessels and Together these results mouse its orthologue is restricted to lymphatic vessel of LYVE-1 in lymphatic but not from mice with to LYVE-1 and the endothelial to with as described are liver and In lymphatic vessels within are by a of In lymphatic vessels are the of large blood vessels HA in knockout mice suggest the that HA receptors in these the that LYVE-1 a role we its expression in tissues from and by means of The results equally of lymphatic vessels in both and mouse tissues with and in the of LYVE-1 in the of lymphatic vessels a of tissues the two mouse CD44 expression in the lymphatics of mice in tissues in the mice as by with the our not the of in LYVE-1 expression in of during the results that LYVE-1 is to compensate for the loss of CD44 expression and that as yet are likely to the that LYVE-1 as a receptor for the uptake of HA from the lymph as a receptor for HA in the tissues the lymphatics, we immunoelectron microscopy to LYVE-1 is to the luminal the of lymphatic endothelial Intriguingly, the expression of LYVE-1 both with and both of lymphatic endothelial cells as cells that of membrane and The of LYVE-1 in further the that the receptor is in transport of HA the vessel In this we described the and functional of the mouse lymphatic endothelial HA and that it both binding and of HA from the In addition, we that mouse to its is endothelial cells in lymphatic vessels and in tissues and in a lymphatic endothelial and using that the receptor is present both the luminal and abluminal endothelial We the of the HA receptors CD44 and LYVE-1 from and the of key conserved structural that in with CD44, contains an HA-binding domain that is to we evidence that the of LYVE-1 expression is not in homozygous knockout that as yet HA receptors are present within the In a S. 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Full Text Full Text PDF PubMed Scopus Google Scholar). to the LYVE-1 and are in The to a role for LYVE-1 in lymphatic HA The majority of HA turnover in tissues as the skin and is known to in draining lymph nodes. This is and that of the HA afferent lymphatics is degraded during through the nodes, the remaining rapidly by the liver and to the of (1Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2086) Google Scholar, J.R. Kimpton W.G. Laurent T.C. Cahill R.N. Vakakis N. Biochem. J. 1988; 256: 153-158Crossref PubMed Scopus (161) Google Scholar, 4Fraser J.R. Laurent T.C. CIBA Found. Symp. 1989; 143: 41-53PubMed Google Scholar). evidence to the HA receptor receptor for B. Weigel J.A. L. Weigel P.H. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) LYVE-1 as the receptor for in lymph to a endothelial HA receptor J. McGary C.T. Weigel P.H. J. Cell. Biochem. 1992; 48: 73-80Crossref PubMed Scopus (17) Google Scholar, B. Oka J.A. Singh A. 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The exit of antigen-presenting cells and lymphocytes from inflamed skin to afferent lymph is vital for the initiation and maintenance of dermal immune responses. How such an exit is achieved and how cells transmigrate the distinct endothelium of lymphatic vessels are unknown. We show that inflammatory cytokines trigger activation of dermal lymphatic endothelial cells (LECs), leading to expression of the key leukocyte adhesion receptors intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin, as well as a discrete panel of chemokines and other potential regulators of leukocyte transmigration. Furthermore, we show that both ICAM-1 and VCAM-1 are induced in the dermal lymphatic vessels of mice exposed to skin contact hypersensitivity where they mediate lymph node trafficking of dendritic cells (DCs) via afferent lymphatics. Lastly, we show that tumor necrosis factor alpha stimulates both DC adhesion and transmigration of dermal LEC monolayers in vitro and that the process is efficiently inhibited by ICAM-1 and VCAM-1 adhesion-blocking monoclonal antibodies. These results reveal a CAM-mediated mechanism for recruiting leukocytes to the lymph nodes in inflammation and highlight the process of lymphatic transmigration as a potential new target for antiinflammatory therapy.

The hyaluronan receptor LYVE-1 is selectively expressed in the endothelium of lymphatic capillaries, where it has been proposed to function in hyaluronan clearance and hyaluronan-mediated leukocyte adhesion. However, recent studies suggest that hyaluronan homeostasis is unperturbed in LYVE-1-/- mice and that lymphatic adhesion/transmigration may be largely mediated by ICAM-1 and VCAM-1 rather than LYVE-1. Here we have explored the possibility that LYVE-1 functions during inflammation and report that the receptor is down-regulated by pro-inflammatory cytokines. Using cultured primary lymphatic endothelial cells, we show that surface expression of LYVE-1 is rapidly and reversibly lost after exposure to tumor necrosis factor-α (TNFα) and TNFβ via internalization and degradation of the receptor in lysosomes, coupled with a shutdown in gene expression. Curiously, internalization does not result in significant uptake of hyaluronan, a process that is largely insensitive to the novel LYVE-1 adhesion blocking monoclonal antibody 3A, and proceeds almost equally in resting and inflammation-activated lymphatic endothelial cells. Finally, we show that TNF can induce down-modulation of LYVE-1 in ex vivo murine dermal tissue explants and present evidence that the process occurs in vivo, in the context of murine allergen-induced skin inflammation. These findings suggest that LYVE-1 can function independently of hyaluronan and have implications for the use of LYVE-1 as a histological marker for lymphangiogenesis in human pathology. The hyaluronan receptor LYVE-1 is selectively expressed in the endothelium of lymphatic capillaries, where it has been proposed to function in hyaluronan clearance and hyaluronan-mediated leukocyte adhesion. However, recent studies suggest that hyaluronan homeostasis is unperturbed in LYVE-1-/- mice and that lymphatic adhesion/transmigration may be largely mediated by ICAM-1 and VCAM-1 rather than LYVE-1. Here we have explored the possibility that LYVE-1 functions during inflammation and report that the receptor is down-regulated by pro-inflammatory cytokines. Using cultured primary lymphatic endothelial cells, we show that surface expression of LYVE-1 is rapidly and reversibly lost after exposure to tumor necrosis factor-α (TNFα) and TNFβ via internalization and degradation of the receptor in lysosomes, coupled with a shutdown in gene expression. Curiously, internalization does not result in significant uptake of hyaluronan, a process that is largely insensitive to the novel LYVE-1 adhesion blocking monoclonal antibody 3A, and proceeds almost equally in resting and inflammation-activated lymphatic endothelial cells. Finally, we show that TNF can induce down-modulation of LYVE-1 in ex vivo murine dermal tissue explants and present evidence that the process occurs in vivo, in the context of murine allergen-induced skin inflammation. These findings suggest that LYVE-1 can function independently of hyaluronan and have implications for the use of LYVE-1 as a histological marker for lymphangiogenesis in human pathology. LYVE-1, lymphatic vessel endothelial hyaluronan receptor-1, is a 322-residue type I transmembrane glycoprotein that was first identified through its homology with the inflammatory leukocyte homing receptor CD44 (1Banerji S. Ni J. Wang S.X. Clasper S. Su J. Tammi R. Jones M. Jackson D.G. J. Cell Biol. 1999; 144: 789-801Crossref PubMed Scopus (1303) Google Scholar, 2Aruffo A. Stamenkovic I. Melnick M. Underhill C.B. Seed B. Cell. 1990; 61: 1303-1313Abstract Full Text PDF PubMed Scopus (2160) Google Scholar). In common with CD44, the extracellular domain of LYVE-1 contains a single cartilage Link module (1Banerji S. Ni J. Wang S.X. Clasper S. Su J. Tammi R. Jones M. Jackson D.G. J. Cell Biol. 1999; 144: 789-801Crossref PubMed Scopus (1303) Google Scholar), the prototypic hyaluronan-binding domain conserved within all members of the Link superfamily (3Day A.J. Prestwich G.D. J. Biol. Chem. 2002; 277: 4585-4588Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). However, unlike CD44, which is widely expressed on cells of mesothelial, epithelial, and hematopoietic origin, LYVE-1 is almost entirely restricted to lymphatic endothelium, a property that makes it a powerful marker in studies of tumor lymphangiogenesis (4Jackson D.G. APMIS. 2004; 112: 526-538Crossref PubMed Scopus (173) Google Scholar). Although the precise role of LYVE-1 is currently unclear, its mutually exclusive pattern of expression with that of CD44 suggests a distinct physiological function, specific to the lymphatic vasculature. Hyaluronan (HA), 3The abbreviations used are: HA, hyaluronan; HARE, HA receptor for endocytosis; LEC, lymphatic endothelial cells; HDLEC, human dermal LEC; HMVEC, human dermal microvascular endothelial cells; Ab, antibody; mAb, monoclonal antibody; TNF, tumor necrosis factor; IL, interleukin; MIP3, macrophage-inflammatory protein 3; VEGF-C, vascular endothelial growth factor-C; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; DAPI, 4′,6-diamidino-2-phenylindole; MOPS, 4-morpholinepropanesulfonic acid; PDI, protein disulfide isomerase; β-COP, β-coat protein. the ligand common to both LYVE-1 and CD44, is a large, linear copolymer of d-glucuronic acid and N-acetyl-d-glucosamine (5Meyer K. Palmer J. J. Biol. Chem. 1934; 107: 629-634Abstract Full Text PDF Google Scholar) that is sequestered in tissues by matrix proteoglycans such as aggrecan, versican, and Link protein to form hygroscopic networks for CD44-mediated cell adhesion and migration (6Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1241Crossref PubMed Scopus (599) Google Scholar, 7Hardingham T.E. Fosang A.J. FASEB J. 1992; 6: 861-870Crossref PubMed Scopus (1012) Google Scholar, 8Toole B.P. Nat. Rev. Cancer. 2004; 4: 528-539Crossref PubMed Scopus (1678) Google Scholar). These HA networks are subject to constant turnover, initiated by proteolytic degradation and local uptake of glycosaminoglycan components in cells such as fibroblasts and macrophages. In addition, HA enters the lymphatics (9Fraser J.R. Kimpton W.G. Laurent T.C. Cahill R.N. Vakakis N. Biochem. J. 1988; 256: 153-158Crossref PubMed Scopus (159) Google Scholar, 10Fraser J.R. Laurent T.C. CIBA Found. Symp. 1989; 143: 41-53PubMed Google Scholar), where it is transported in afferent lymph for degradation within the draining lymph nodes, liver, and spleen sinusoids via the high affinity HA receptor for endocytosis (HARE, aka Stabilin II, FEEL II) (11Zhou B. Weigel J.A. Fauss L. Weigel P.H. J. Biol. Chem. 2000; 275: 37733-37741Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 12Tamura Y. Adachi Y. Osuga J. Ohashi K. Yahagi N. Sekiya M. Okazaki H. Tomita S. Lizuka Y. Shimano H. Nagai R. Kimura S. Tsujimoto M. Ishibashi S. J. Biol. Chem. 2003; 278: 12613-12617Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 13Weigel J.A. Raymond R.C. McGary C.T. Singh A. Weigel P.H. J. Biol. Chem. 2003; 278: 9808-9812Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 14Harris E.N. Kuyosseva S.V. Weigel J.A. Weigel P.H. J. Biol. Chem. 2007; 282: 2785-2797Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). This remote handling mechanism, which is responsible for catabolizing ∼30% of total body HA per day, is thought to be vital for protecting the tissues from prolonged exposure to short HA oligosaccharides with pro-angiogenic and pro-inflammatory properties (15Noble P.W. Matrix Biol. 2002; 21: 25-29Crossref PubMed Scopus (467) Google Scholar, 16West D.C. Kumar S. CIBA Found. Symp. 1989; 143: 187-201PubMed Google Scholar) that constitute danger signals for activating the immune system (17Termeer C.C. Hennies J. Voith U. Ahrens T. Weiss J.M. Prehm P. Simon J.C. J. Immunol. 2000; 165: 1863-1870Crossref PubMed Scopus (332) Google Scholar, 18Stern R. Asari A.A. Sugahara K.N. Int. J. Cell Biol. 2006; 85: 699-715Google Scholar). Previously, we hypothesized that LYVE-1 might participate in lymphatic HA metabolism, based on the capacity of the receptor to mediate specific, saturable binding and endocytosis of the glycosaminoglycan in transfected 293T human fibroblasts (19Prevo R. Banerji S. Ferguson D.J. Clasper S. Jackson D.G. J. Biol. Chem. 2001; 276: 19420-19430Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 20Jackson D.G. Trends Cardiovasc. Med. 2003; 13: 1-7Crossref PubMed Scopus (181) Google Scholar, 21Jackson D.G. Glycoforum. 2004; www.glycoforum.gr.jp/science/hyaluronan/HA28/HA28E.htmlGoogle Scholar). However, in studies on LYVE-1-/- knock-out mice, we found that loss of the receptor had no significant effect on either serum or tissue HA levels and no obvious consequences for HA-mediated adhesion/migration events such as exit of skin dendritic cells through afferent lymph (4Jackson D.G. APMIS. 2004; 112: 526-538Crossref PubMed Scopus (173) Google Scholar, 22Gale N.W. Prevo R. Espinosa-Fematt J. Ferguson D.J. Dominguez M.G. Yancopoulos G.D. Thurston G. Jackson D.G. Mol. Cell Biol. 2007; 27: 595-604Crossref PubMed Scopus (161) Google Scholar, 23Jackson D.G. Prevo R. Clasper S. Banerji S. Trends Immunol. 2001; 22: 317-321Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). A final anomaly is that we could not demonstrate binding of LYVE-1 to HA in normal lymphatic endothelial cells, most likely because of regulatory post-translational modifications to the receptor (24Nightingale T. Banerji S. Jackson D.G. Balazs E.A. Hascall V.C. Hyaluronan Structure, Metabolism, Biological Activities, Therpaeutic Applications. II. Matrix Biology Institute, Edgewater, NJ2005: 615-618Google Scholar). These various properties of LYVE-1 are reminiscent of the leukocyte HA receptor CD44, which is inactive by default in cells such as T lymphocytes and monocytes and binds HA only in response to inflammation or antigen receptor activation. This suggested to us that the function of LYVE-1, like that of CD44, might be more clearly inflammatory or specific In the present we have the of inflammation on LYVE-1 function both in and in than activating the we that pro-inflammatory such as and TNFβ induce internalization of LYVE-1 in lymphatic endothelial cells by degradation in we demonstrate that internalization does not LYVE-1 to or HA to that the process is not to HA These suggest that LYVE-1 does not function as receptor for HA in either or lymphatic endothelium and the of functions for protein. Cell human dermal lymphatic endothelial cells from human dermal microvascular endothelial cells by of as J.A. R. L. R. P. J. 2004; 165: Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, from human skin tissue by LYVE-1 as Clasper S. A. P. Jackson D.G. J. Med. 2006; PubMed Scopus Google Scholar). in both cultured in tissue that had been with cell and from and used the human growth growth growth growth VEGF-C, was used monoclonal antibody and the monoclonal and and human LYVE-1 as The was from protein and human LYVE-1 have been (19Prevo R. Banerji S. Ferguson D.J. Clasper S. Jackson D.G. J. Biol. Chem. 2001; 276: 19420-19430Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). The CD44 was from the for human and by of and was from was from was from the and from and and from and from and from with in and for with primary antibody by and with the and on a of and cells in in primary in serum and for by and with the in in and a tissue was in in PBS, in with and with the primary tissue was with for in the and in and on a 2000; for LYVE-1 cultured in either or with or TNFβ for by of the and by for LYVE-1 by in to with was used as a and protein was used as a in LYVE-1 was the and a antibody and for in a Cell of primary was by a that of to the of in was with of and to of endothelial cells cultured in for and of in was to the Cell and the in a of total from cultured was to and in with of either LYVE-1 or of by the and with by and high exposure to of total from after various in was and the by and to the LYVE-1 with either the LYVE-1 and or the on to and with either LYVE-1 or high of or of LYVE-1, cultured in in in PBS, and with and in serum and for with LYVE-1 and primary to either the marker or the marker by the of LYVE-1, in with LYVE-1 in the or of by in PBS, in and with cells with to either the marker or the marker by In both and the by in to in and on a 2000; of by in with CD44 to levels of CD44 expression in cultured (1Banerji S. Ni J. Wang S.X. Clasper S. Su J. Tammi R. Jones M. Jackson D.G. J. Cell Biol. 1999; 144: 789-801Crossref PubMed Scopus (1303) Google Scholar, Clasper S. A. P. Jackson D.G. J. Med. 2006; PubMed Scopus Google Scholar, D.J. B. G. Google and either the LYVE-1 or to the of and with or for a with for to HA (19Prevo R. Banerji S. Ferguson D.J. Clasper S. Jackson D.G. J. Biol. Chem. 2001; 276: 19420-19430Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar) by of by in with the and and with the of the and mice by of and the in on for and and and in and cultured in a in in the of murine mice by of in to the per The day, a of was to the the surface of the was in by of per the was with after and tissue was for as of LYVE-1 in the possibility that LYVE-1 during we first the of and growth on the levels of receptor in cultured primary such we used from primary human dermal microvascular endothelial cells The lymphatic endothelial of cells has been was by for the marker S. A. H. R. G. 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Thurston G. R. Wang J. Jackson D.G. T. Yancopoulos G.D. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google and had effect on receptor that loss of LYVE-1 was to be a of cell or of the response to that was both and as of LYVE-1 and with a in the of was with of surface receptor within of TNF as by The loss of LYVE-1 in response to TNF was by with the lymphatic endothelial marker the levels of which exposure to the and Finally, the of cell by was by than and by of and capacity to form in both of which by short with not of LYVE-1 by of LYVE-1 the of LYVE-1 we the of on LYVE-1 levels by of total The show that the and LYVE-1 (1Banerji S. Ni J. Wang S.X. Clasper S. Su J. Tammi R. Jones M. Jackson D.G. J. Cell Biol. 1999; 144: 789-801Crossref PubMed Scopus (1303) Google Scholar) are present only in cells and are lost within of of levels that the loss of LYVE-1 is within of exposure demonstrate that rapidly and reversibly LYVE-1 expression in lymphatic endothelial cells the of either or LYVE-1 and in the of LYVE-1 in HDLEC, we both and cells with LYVE-1 for by The that the receptor from a largely cell surface to a within of exposure and that only levels could be after a with degradation LYVE-1 was in However, had a to the most likely to protein in the the levels of LYVE-1 within the of than in cells as by that of the is mediated by surface of LYVE-1 in primary cells cultured for with either or to and with and either the marker or with and the of LYVE-1 is for cells with or the as A in the of to and with either or with and by a of the for LYVE-1 uptake and we the of receptor in and by of cells for by Although the of LYVE-1 in cells a with the and and β-COP, J. 1993; PubMed Scopus Google Scholar), most of was lost exposure to These findings are with shutdown of receptor by In a we the of LYVE-1 in by the receptor on the cell surface with LYVE-1 to with In we found a of the receptor with the marker and the marker in response to TNF In LYVE-1 with either marker in cells. These with that LYVE-1 is insensitive to the of during and G. suggest that LYVE-1 has a with Link superfamily such as in R. Banerji S. Ni J. Jackson D.G. J. Biol. 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