Inflammation-induced Uptake and Degradation of the Lymphatic Endothelial Hyaluronan Receptor LYVE-1

Abstract

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. K. W. K. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), the lymphatic G. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and the marker in with and by the in A and the expressed high levels of of in to LYVE-1 for LYVE-1 and and a of receptor from studies Clasper S. A. P. Jackson D.G. J. Med. 2006; PubMed Scopus Google and G. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of or as by expression and was the of the on a lymphatic had been of LYVE-1 down-modulation in cultured A and cultured either in of for or a of a of and with and for of LYVE-1 expression was by cells either in the of or in for in both by and for the are for in surface expression of LYVE-1 and was on cells cultured with or by after with and and with the and with of surface expression in and cells are in with the pro-inflammatory and in loss of LYVE-1 and and not In exposure to growth the and or the growth M. A. M. H. M. K. 276: PubMed Scopus Google Scholar), N.W. 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. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) and that the of through of endocytosis and of LYVE-1 the for HA the LYVE-1 in to be as cultured to binding of to the cell surface the in vivo evidence that lymphatic tissue HA for degradation in lymph nodes, we the possibility that internalization of LYVE-1 might as a for the receptor in to and hyaluronan for we uptake of in primary by and the of LYVE-1 in the process by of a monoclonal antibody that HA binding to the The of novel is by its capacity to binding to 293T cells, which in to the receptor in form of uptake by primary that cells in significant uptake of HA However, the to which TNF the process was in uptake after the of on HA uptake only the for clearance of the receptor and in in in which the of LYVE-1 internalization is not In show that LYVE-1 internalization does not to the process of HA internalization in of LYVE-1 to be a for HA in such of LYVE-1 internalization on uptake of by for with the blocking and either the blocking or the by the of and and of after the of LYVE-1 in ex of LYVE-1 by in primary HDLEC, we a process occurs within the endothelium of lymphatic ex we dermal explants and the expression of LYVE-1 within the of lymphatic with and both and after with by the in lymphatic in normal LYVE-1 with both and present in tissue had lost all LYVE-1 expression. we for evidence of LYVE-1 in vivo, in the context of the skin response in and with is to a inflammatory response that lymph of dendritic cells via afferent dermal lymphatics S. 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Med. 2001; PubMed Scopus Google Scholar) the findings with tissue explants and the that loss of LYVE-1 is process rather than the result of vessel necrosis or Finally, to the of we of from the and after and to for LYVE-1 and by of vessel The and show that the of lymphatic LYVE-1 by almost and after and that by only of had LYVE-1 expression. such with no skin not The that LYVE-1 is down-regulated in inflammation has consequences for the function of the In recent studies knock-out mice, we found that expression of LYVE-1 in lymphatic endothelium to be for both leukocyte and hyaluronan normal physiological This us to that the function of the receptor might only be of such as immune signals in leukocyte and matrix The internalization and degradation of the receptor that we in response to inflammation was it is that LYVE-1 could function in or HA such LYVE-1 could inflammatory leukocyte migration by on cells H. A. J. Immunol. 2002; PubMed Scopus Google Scholar, A. 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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), as is not expressed in could of CD44 present in cultured uptake was insensitive to the CD44 be to the of afferent lymphatic endothelium to HA both in of findings and in of recent that Espinosa-Fematt and G. The of LYVE-1 by TNF with the of the on CD44, which via and S. K. Kumar A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), coupled with of HA via A. G. P. 282: PubMed Scopus Google Scholar) and protein of a Cell Immunol. 1999; PubMed Scopus Google Scholar, K. M. Kumar A. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). Curiously, LYVE-1 to that the can LYVE-1 down-modulation in by to the for LYVE-1, in to that of CD44, is not coupled to activation. the physiological that to of LYVE-1 and the that the of the receptor a it is that the that HA binding in LYVE-1 could function in like to CD44 that the homing and R.C. R. U. S. A. 2000; PubMed Scopus Google Scholar, S. R.C. R. J. Cell Biol. 2001; PubMed Scopus Google Scholar, S. R. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). are currently and by for with LYVE-1 and The studies in report have the of LYVE-1 in a of and we have to the to a that has been to lymphangiogenesis J. 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