Hyaluronan Anchoring and Regulation on the Surface of Vascular Endothelial Cells Is Mediated through the Functionally Active Form of CD44

CD44 on lymphocytes binding to its carbohydrate ligand hyaluronan can mediate primary adhesion (rolling interactions) of lymphocytes on vascular endothelial cells. This adhesion pathway is utilized in the extravasation of activated T cells from the blood into sites of inflammation and therefore influences patterns of lymphocyte homing and inflammation. Hyaluronan is a glycosaminoglycan found in the extracellular matrix and is involved in a number of biological processes. We have shown that the expression of hyaluronan on the surface of endothelial cells is inducible by proinflammatory cytokines. However, the manner through which hyaluronan is anchored to the endothelial cell surface so that it can resist shear forces and the mechanism of the regulation of the level of hyaluronan on the cell surface has not been investigated. In order to characterize potential hyaluronan receptors on endothelial cells, we performed analyses of cell surface staining by flow cytometry on intact endothelial cells and ligand blotting assays using membrane fractions. Hyaluronan binding activity was detected as a major species corresponding to the size of CD44, and this was confirmed to be the same by Western blotting and immunoprecipitation. Moreover, alterations in the surface level of hyaluronan after tumor necrosis factor-α stimulation is regulated primarily by changes in the cell surface levels of the hyaluronan-binding form of CD44. In laminar flow assays, lymphoid cells specifically roll on hyaluronan anchored by purified CD44 coated on glass tubes, indicating that the avidity of the endothelial CD44/hyaluronan interaction is sufficient to support rolling adhesions under conditions mimicking physiologic shear forces. Together these studies show that CD44 serves to anchor hyaluronan on endothelial cell surfaces, that activation of CD44 is a major regulator of endothelial surface hyaluronan expression, and that the non-covalent interaction between CD44 and hyaluronan is sufficient to provide resistance to shear under physiologic conditions and thereby support the initial steps of lymphocyte extravasation. CD44 on lymphocytes binding to its carbohydrate ligand hyaluronan can mediate primary adhesion (rolling interactions) of lymphocytes on vascular endothelial cells. This adhesion pathway is utilized in the extravasation of activated T cells from the blood into sites of inflammation and therefore influences patterns of lymphocyte homing and inflammation. Hyaluronan is a glycosaminoglycan found in the extracellular matrix and is involved in a number of biological processes. We have shown that the expression of hyaluronan on the surface of endothelial cells is inducible by proinflammatory cytokines. However, the manner through which hyaluronan is anchored to the endothelial cell surface so that it can resist shear forces and the mechanism of the regulation of the level of hyaluronan on the cell surface has not been investigated. In order to characterize potential hyaluronan receptors on endothelial cells, we performed analyses of cell surface staining by flow cytometry on intact endothelial cells and ligand blotting assays using membrane fractions. Hyaluronan binding activity was detected as a major species corresponding to the size of CD44, and this was confirmed to be the same by Western blotting and immunoprecipitation. Moreover, alterations in the surface level of hyaluronan after tumor necrosis factor-α stimulation is regulated primarily by changes in the cell surface levels of the hyaluronan-binding form of CD44. In laminar flow assays, lymphoid cells specifically roll on hyaluronan anchored by purified CD44 coated on glass tubes, indicating that the avidity of the endothelial CD44/hyaluronan interaction is sufficient to support rolling adhesions under conditions mimicking physiologic shear forces. Together these studies show that CD44 serves to anchor hyaluronan on endothelial cell surfaces, that activation of CD44 is a major regulator of endothelial surface hyaluronan expression, and that the non-covalent interaction between CD44 and hyaluronan is sufficient to provide resistance to shear under physiologic conditions and thereby support the initial steps of lymphocyte extravasation. hyaluronan soluble HA bovine proteoglycan endothelial cell fluoresceinated HA human dermal microvascular endothelial cells reverse transcription-polymerase chain reaction soluble CD44-immunoglobulin fusion protein tumor necrosis factor-α bovine serum albumin fetal calf serum Dulbecco's modified Eagle's medium phosphate-buffered saline fluorescence-activated cell sorter wall shear stress SV40 virus-transformed endothelial cells major histocompatibility complex Hyaluronan (HA)1 is a high molecular weight nonsulfated linear glycosaminoglycan comprised of a range of repeating disaccharide subunits, β1,3-N-acetyl-d-glucosamine in linkage to β1,4-d-glucuronic acid, and ranging in molecular mass up to 1 × 107 Da. As a prominent ubiquitously expressed extracellular matrix component, HA can be produced by a wide variety of cell types and tissues and is most prevalent in soft connective tissues. HA has been suggested to play a key role in several biological processes including embryonic development (1.Toole B.P. J. Intern. Med. 1997; 242: 35-40Crossref PubMed Scopus (282) Google Scholar), extracellular matrix organization and turnover (2.Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2086) Google Scholar, 3.Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1241Crossref PubMed Scopus (601) Google Scholar, 4.Nishida Y. Knudson C.B. Nietfeld J.J. Margulis A. Knudson W. J. Biol. Chem. 1999; 274: 21893-21899Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), wound healing (5.King S.R. Hickerson W.L. Proctor K.G. Surgery. 1991; 109: 76-84PubMed Google Scholar, 6.Oksala O. Salo T. Tammi R. Hakkinen L. Jalkanen M. Inki P. Larjava H. J. Histochem. Cytochem. 1995; 43: 125-135Crossref PubMed Scopus (230) Google Scholar), tumor growth (7.Bartolazzi A. Peach R. Aruffo A. Stamenkovic I. J. Exp. Med. 1994; 180: 53-66Crossref PubMed Scopus (322) Google Scholar), and angiogenesis (8.West D.C. Hampson I.N. Arnold F. Kumar S. Science. 1985; 228: 1324-1326Crossref PubMed Scopus (979) Google Scholar). It is generally thought to exert many of these functions by establishing a provisional matrix for supporting cellular migration and adherence (9.Laurent T.C. Fraser J.R. CIBA Found. Symp. 1986; 124: 9-29PubMed Google Scholar). Moreover, studies have demonstrated that HA may function as a cellular signaling molecule under certain circumstances (10.Bourguignon L.Y. Lokeshwar V.B. Chen X. Kerrick W.G. J. Immunol. 1993; 151: 6634-6644PubMed Google Scholar, 11.Hall C.L. Wang C. Lange L.A. Turley E.A. J. Cell Biol. 1994; 126: 575-588Crossref PubMed Scopus (225) Google Scholar) and can deliver signals leading to or regulating cellular proliferation (2.Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2086) Google Scholar, 12.Papakonstantinou E. Karakiulakis G. Roth M. Block L.H. Proc. Natl. Acad. Sci. U. S.A. 1995; 92: 9881-9885Crossref PubMed Scopus (78) Google Scholar, 13.Evanko S.P. Angello J.C. Wight T.N. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1004-1013Crossref PubMed Scopus (421) Google Scholar, 14.Brecht M. Mayer U. Schlosser E. Prehm P. Biochem. J. 1986; 239: 445-450Crossref PubMed Scopus (281) Google Scholar). Although a member of the family of glycosaminoglycans, HA is distinct from other members in that it is not found covalently linked to a peptide core (2.Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2086) Google Scholar). This is also in marked contrast to other carbohydrate ligands participating in extravasation, such as selectin ligands, which are generally glycoproteins anchored by transmembrane domains (15.Kansas G.S. Blood. 1996; 88: 3259-3287Crossref PubMed Google Scholar). In further contradistinction to other glycosaminoglycans and proteoglycans, HA is unique in its mode of synthesis. Rather than being confined to the Golgi apparatus or post-Golgi compartment, the assembly of HA by HA synthase is located at the plasma membrane where HA is synthesized and polymers are directly extruded into the extracellular space (16.Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar, 17.Prehm P. Biochem. J. 1984; 220: 597-600Crossref PubMed Scopus (293) Google Scholar, 18.Philipson L.H. Schwartz N.B. J. Biol. Chem. 1984; 259: 5017-5023Abstract Full Text PDF PubMed Google Scholar). Although its extracellular organization and disposition in a number of solid tissue microenvironments has been examined, HA on endothelium at the interface with the bloodstream has not been extensively studied. The recognition of endothelial cells by leukocytes and their subsequent extravasation through the blood vessel wall are based on a multistep pathway of sequential receptor engagement, in which a variety of molecular ligands participate (19.Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6414) Google Scholar, 20.Butcher E.C. Picker L.J. Science. 1996; 272: 60-66Crossref PubMed Scopus (2519) Google Scholar, 21.Butcher E.C. Cell. 1991; 67: 1033-1036Abstract Full Text PDF PubMed Scopus (2530) Google Scholar). We have described a novel primary (rolling) interaction between T cells and endothelial cells that is similar under laminar flow conditions to that mediated by selectins and also has as its basis a distinct protein-carbohydrate ligand interaction, namely that between the cartilage link protein family member CD44, a broadly distributed complex multifunctional family of cell surface glycoproteins (22.Lesley J. Hyman R. Kincade P.W. Adv. Immunol. 1993; 54: 271-335Crossref PubMed Scopus (1032) Google Scholar), and its principal ligand HA (23.DeGrendele H.C. Estess P. Picker L.J. Siegelman M.H. J. Exp. Med. 1996; 183: 1119-1130Crossref PubMed Scopus (364) Google Scholar). One well known consequence of antigen stimulation on T cells is increased surface levels of CD44 (22.Lesley J. Hyman R. Kincade P.W. Adv. Immunol. 1993; 54: 271-335Crossref PubMed Scopus (1032) Google Scholar, 24.McHeyzer-Williams M.G. Davis M.M. Science. 1995; 268: 106-111Crossref PubMed Scopus (405) Google Scholar, 25.DeGrendele H.C. Estess P. Siegelman M.H. J. Immunol. 1997; 159: 2549-2553PubMed Google Scholar). However, elevated levels of CD44 do not necessarily correlate with increased HA binding, and thus the ability of CD44 to bind HA is not constitutive; rather, CD44 requires some form of structural alteration to engage this ligand (22.Lesley J. Hyman R. Kincade P.W. Adv. Immunol. 1993; 54: 271-335Crossref PubMed Scopus (1032) Google Scholar,26.Katoh S. Zheng Z. Oritani K. Shimozato T. Kincade P.W. J. Exp. Med. 1995; 182: 419-429Crossref PubMed Scopus (230) Google Scholar, 27.Lesley J. English N. Perschl A. Gregoroff J. Hyman R. J. Exp. Med. 1995; 182: 431-437Crossref PubMed Scopus (177) Google Scholar). Although the mechanism by which CD44 is altered to bind HA remains to be completely elucidated, evidence has accumulated that T cell stimulation of normal lymphocytes in vitro or in vivo via signaling through the T cell receptor induces the activated form of CD44 and attendant rolling interactions on HA substrate (22.Lesley J. Hyman R. Kincade P.W. Adv. Immunol. 1993; 54: 271-335Crossref PubMed Scopus (1032) Google Scholar, 25.DeGrendele H.C. Estess P. Siegelman M.H. J. Immunol. 1997; 159: 2549-2553PubMed Google Scholar, 28.DeGrendele H.D. Estess P. Siegelman M.H. Science. 1997; 278: 672-675Crossref PubMed Scopus (481) Google Scholar). These observations have established the HA-binding form of CD44 as an early activation marker on T cells after T cell receptor stimulation and support a role for this interaction during the course of an immune response. In particular, CD44/HA interactions have been shown to be required for extravasation of superantigen-stimulated T cells into an inflamed site in a mouse model (28.DeGrendele H.D. Estess P. Siegelman M.H. Science. 1997; 278: 672-675Crossref PubMed Scopus (481) Google Scholar). CD44 has been prominently associated with human arthritis and with a model of collagen-induced murine arthritis (29.Haynes B.F. Hale L.P. Patton K.L. Martin M.E. McCallum R.M. Arthritis & Rheum. 1991; 34: 1434-1443Crossref PubMed Scopus (193) Google Scholar, 30.Estess P. DeGrendele H.C. Pascual V. Siegelman M.H. J. Clin. Invest. 1998; 102: 1173-1182Crossref PubMed Scopus (97) Google Scholar, 31.Mikecz K. Brennan F.R. Kim J.H. Glant T.T. Nat. Med. 1995; 1: 558-563Crossref PubMed Scopus (259) Google Scholar, 32.Verdrengh M. Holmdahl R. Tarkowski A. Scand. J. Immunol. 1995; 42: 353-358Crossref PubMed Scopus (70) Google Scholar), and more recently with a murine model of multiple sclerosis (33.Brocke S. Piercy C. Steinman L. Weissman I.L. Veromaa T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6896-6901Crossref PubMed Scopus (263) Google Scholar). CD44 interactions with HA are also thought to be important in allogeneic graft rejection (34.Knoflach A. Azuma H. Magee C. Denton M. Murphy B. Iyengar A. Buelow R. Sayegh M.H. J. Am. Soc. Nephrol. 1999; 10: 1059-1066Crossref PubMed Google Scholar, 35.Knoflach A. Magee C. Denton M.D. Kim K.S. Buelow R. Hancock W.W. Sayegh M.H. Transplantation. 1999; 67: 909-914Crossref PubMed Scopus (21) Google Scholar). Based on our observations, we have postulated that CD44 on lymphocytes interacts with HA on endothelium and participates in the well known preferential homing of activated lymphocytes to tertiary sites of inflammation. In support of this model, circulating lymphocytes bearing activated CD44 have been shown to be elevated during autoimmune exacerbations in both arthritis and systemic lupus erythematosus in humans (30.Estess P. DeGrendele H.C. Pascual V. Siegelman M.H. J. Clin. Invest. 1998; 102: 1173-1182Crossref PubMed Scopus (97) Google Scholar). A clear implication of this model is that regulation of HA on vascular endothelium in response to local pathophysiologic conditions should create a receptive site for leukocyte recruitment and therefore represents an important control point for extravasation. It has been reported that hyaluronan is found both in vitro and in vivo on endothelial cells (23.DeGrendele H.C. Estess P. Picker L.J. Siegelman M.H. J. Exp. Med. 1996; 183: 1119-1130Crossref PubMed Scopus (364) Google Scholar, 36.Bennett K.L. Modrell B. Greenfield B. Bartolazzi A. Stamenkovic I. Peach R. Jackson D.G. Spring F. Aruffo A. J. Cell Biol. 1995; 131: 1623-1633Crossref PubMed Scopus (145) Google Scholar, 37.Harder R. Uhlig H. Kashan A. Schutt B. Duijvestijn A. Butcher E.C. Thiele H.G. Hamann A. Exp. Cell Res. 1991; 197: 259-267Crossref PubMed Scopus (34) Google Scholar, 38.Green S.J. Tarone G. Underhill C.B. J. Cell Sci. 1988; 90: 145-156Crossref PubMed Google Scholar, 39.Ausprunk D.H. Am. J. Anat. 1986; 177: 313-331Crossref PubMed Scopus (50) Google Scholar, 40.Aruffo A. Stamenkovic I. Melnick M. Underhill C.B. Seed B. Cell. 1990; 61: 1303-1313Abstract Full Text PDF PubMed Scopus (2169) Google Scholar, 41.Tammi R. Paukkonen K. Wang C. Horsmanheimo M. Tammi M. Arch. Dermatol. Res. 1994; 286: 21-29Crossref PubMed Scopus (38) Google Scholar). In addressing the potential regulation of HA on endothelial cells, we previously demonstrated that its expression on cultured endothelial cell lines and primary endothelial cultures is inducible by the proinflammatory cytokines TNFα, interleukin-1β, interleukin-15, and bacterial lipopolysaccharide (42.Mohamadzadeh M. DeGrendele H.C. Estess P. Siegelman M.H. J. Clin. Invest. 1998; 101: 97-108Crossref PubMed Scopus (273) Google Scholar, 43.Estess P.E. Nandi A. Siegelman M.H. J. Exp. Med. 1999; 190: 9-19Crossref PubMed Scopus (66) Google Scholar). In addition, this inducibility appeared strikingly restricted to endothelial cells derived from microvascular but not large vessel sources, consistent with a role in the vessels where the majority of leukocyte trafficking occurs. The elevated HA levels induced by cytokines further resulted in increased adhesive interactions in both non-static shear and laminar flow adhesion assays. However, the changes in surface HA levels did not appear dependent either on described HA synthetic or degradative enzymes, suggesting other mechanisms for such regulation. These data added to the selectin and immunoglobulin gene families a new inducible endothelial adhesive molecule, hyaluronan, and helped to further our understanding of the potential physiologic roles of the CD44/HA interaction. However, the manner by which HA is anchored to the endothelial surface and the requirements for cell surface retention under conditions of physiologic shear forces has remained unclear. A number of proteins have been characterized that can bind HA and anchor it in the tissues and on cell surfaces. The proteoglycans aggrecan and link protein are major binders of HA in cartilage and soft tissues (44.Hardingham T.E. Fosang A.J. FASEB J. 1992; 6: 861-870Crossref PubMed Scopus (1013) Google Scholar). A specialized receptor for HA internalization is found on liver sinusoidal endothelial cells (45.Eriksson S. Fraser J.R. Laurent T.C. Pertoft H. Smedsrod B. Exp. Cell Res. 1983; 144: 223-228Crossref PubMed Scopus (261) Google Scholar, 46.Raja R.H. McGary C.T. Weigel P.H. J. Biol. Chem. 1988; 263: 16661-16668Abstract Full Text PDF PubMed Google Scholar). Other cell surface HA receptors include the widely distributed CD44 molecule (22.Lesley J. Hyman R. Kincade P.W. Adv. Immunol. 1993; 54: 271-335Crossref PubMed Scopus (1032) Google Scholar), and an unrelated protein, receptor for hyaluronan-mediated motility (RHAMM) (47.Hall C.L. Turley E.A. J. Neurooncol. 1995; 26: 221-229Crossref PubMed Scopus (78) Google Scholar). In these studies we show that CD44 is the predominant cell surface molecule on endothelial surfaces responsible for HA binding and that it is the regulation of the activated form of CD44 that largely determines the level of surface HA expression. Furthermore, using isolated CD44 bound to HA, we establish that this interaction is sufficient to resist hemodynamic drag forces and support rolling adhesions of lymphocytes under physiologic laminar flow conditions. Rooster comb HA was purchased from Sigma. Streptococcal hyaluronidase was purchased from ICN (Irvine, CA). Fluorescein-conjugated HA (Fl-HA) was prepared as described (48.de Belder A.N. Wik K.O. Carbohydr. Res. 1975; 44: 251-257Crossref PubMed Scopus (174) Google Scholar). Biotinylated bovine proteoglycan (bPG) from nasal cartilage was kindly provided by C. Underhill (Georgetown University School of Medicine, Washington, D. C.). Trypsin-EDTA was purchased from Life Technologies, Inc.; recombinant murine TNFα (5 × 107 units/mg) was purchased from Genzyme, Inc. (Cambridge, MA); and recombinant human TNFα (1 × 107 units/mg) was obtained from Fisher. The rat anti-mouse CD44 antibody producing cell lines KM81 (HA-blocking, IgG2a, κ) and KM703 (non-HA-blocking, IgG2a, κ) (49.Miyake K. Medina K.L. Hayashi S. Ono S. Hamaoka T. Kincade P.W. J. Exp. Med. 1990; 171: 477-488Crossref PubMed Scopus (534) Google Scholar) were obtained from the American Type Culture Collection (Manassas, VA). HA-blocking mouse anti-human CD44 (clone 515, IgG1, κ) was kindly provided by Dr. G. Kansas (Northwestern University Medical School, Chicago) (50.Cannistra S.A. Kansas G.S. Niloff J. DeFranzo B. Kim Y. Ottensmeier C. Cancer Res. 1993; 53: 3830-3838PubMed Google Scholar, 51.Kansas G.S. Wood G.S. Dailey M.O. J. Immunol. 1989; 142: 3050-3057PubMed Google Scholar), and non-HA-blocking mouse anti-human CD44 Hermes-3 (IgG2a, κ) (52.Jalkanen S. Bargatze R.F. de los Toyos J. Butcher E.C. J. Cell Biol. 1987; 105: 983-990Crossref PubMed Scopus (479) Google Scholar) was kindly provided by Dr. L. Picker (University of Texas Southwestern Medical Center, Dallas). All anti-CD44 antibodies bind to epitopes in the invariant portions of CD44. Rat anti-mouse MHC class I (pan-anti-H-2, clone M1/42), was provided by Dr. K. Fisher-Lindahl (53.Kennett R. Monoclonal Antibodies. Plenum Publishing Corp., New York1980: 423Google Scholar). Soluble human CD44-immunoglobulin fusion construct (26.Katoh S. Zheng Z. Oritani K. Shimozato T. Kincade P.W. J. Exp. Med. 1995; 182: 419-429Crossref PubMed Scopus (230) Google Scholar) was expressed as a stable transfectant in BW5147 cells. Antibodies were purified from tissue culture supernatants by protein A-Sepharose column chromatography. Phycoerythrin and biotin-conjugated IM7, a rat anti-mouse CD44 that cross-reacts with human and that does not displace bound HA (IgG2b, κ) (55.Trowbridge I.S. Lesley J. Schulte R. Hyman R. Trotter J. Immunogenetics. 1982; 15: 299-312Crossref PubMed Scopus (319) Google Scholar), and phycoerythrin-labeled streptavidin were purchased from PharMingen (San Diego, CA). Anti-fluorescein antibody was obtained from Sigma. Fluorescein isothiocyanate-conjugated goat anti-rat immunoglobulin was obtained from Caltag (Burlingame, CA). BW5147 murine T cells were maintained in RPMI 1640 containing 10% FCS, 100 mm sodium pyruvate, 200 mm l-glutamine, and 50 μmβ-mercaptoethanol. The murine lymph node endothelial cell line SVEC4-10 (56.O'Connell K.A. Edidin M. J. Immunol. 1990; 144: 521-525PubMed Google Scholar) was grown and maintained in DMEM containing 10% FCS and 200 mm l-glutamine. For TNFα stimulation, cell monolayers were grown to 60–80% confluence, washed with DMEM, and stimulated with TNFα (10 ng/ml) for 4 h or as indicated in time course analyses. Single cell suspensions were made by incubation of monolayers in Versene (Life Technologies, Inc.) at 37 °C for 10 min for staining or immunoprecipitation. For Fl-HA binding experiments, the cells were additionally treated with 20 units/ml hyaluronidase for 1 h at 37 °C. Where indicated, trypsin was used at a final concentration of 0.5 mg/ml for the indicated times at 37 °C. Trypsin was inactivated by the addition of 10% fetal bovine serum. Primary cultures of human dermal microvascular endothelium (HDMEC) were obtained from the Skin Center at Emory University (S. W. Caughman, Atlanta, GA) and maintained in Iscove's modified Dulbecco's medium supplemented with 20% human serum, 2.5 μg/ml cAMP, 10 ng/ml epidermal growth factor, and 5 ng/ml hydrocortisone. After reaching initial confluence, cells were passaged and used directly or after one additional passage to fresh plates. Cells were stimulated with human TNFα at 10 ng/ml for 4 h. 5 × 105 cells were stained with Fl-HA, anti-CD44-PE (IM7), anti-H-2, or bPG-biotin in 100 μl of PBS, 5% FCS for 30 min on ice and then washed with 1 ml of PBS containing 5% FCS. Cells were incubated with Fl-HA for 10 min prior to addition of IM7-PE. Fluorochrome-labeled streptavidin or secondary antibody was added for 20 min as indicated, and cells were again washed before analysis. For blocking of Fl-HA staining, unlabeled blocking anti-CD44 antibody (KM81 or 515) was incubated with cells for 10 min prior to staining with Fl-HA. Data were collected using FACScanTM analytical instrument (Becton Dickinson, San Jose, CA) and analyzed using CellQuestTM software. SVEC4-10 cells were grown in 80-mm tissue culture dishes to 60–80% confluence in DMEM containing 10% FCS. The monolayer was washed with DMEM and stimulated with TNFα (10 ng/ml) for 4 h. The monolayer was again washed with DMEM, treated with 20 units/ml hyaluronidase for 1 h at 37 °C, washed with chilled PBS, and cells scraped off the dish in lysis buffer (50 mm Tris, pH 8.0, 100 mm NaCl, 5 mmEDTA, 1 mm phenylmethylsulfonyl fluoride). BW5147 cells were grown in suspension, washed twice with chilled PBS, and resuspended in lysis buffer. The cell lysates were sonicated briefly, and membranes were prepared by centrifugation at 100,000 ×g (57.Visweswariah S.S. Ramachandran V. Ramamohan S. Das G. Ramachandran J. Eur. J. Biochem. 1994; 219: 727-736Crossref PubMed Scopus (32) Google Scholar). Pellets were resuspended in non-reducing sample buffer and resolved on a 10% polyacrylamide gel containing 0.1% SDS, after which proteins were transferred to nitrocellulose (Amersham Pharmacia Biotech). Ligand blotting was performed based on described methods (58.Yannariello-Brown J. Zhou B. Ritchie D. Oka J.A. Weigel P.H. Biochem. Biophys. Res. Commun. 1996; 218: 314-319Crossref PubMed Scopus (20) Google Scholar). Membranes were incubated with Fl-HA/anti-fluorescein-biotin or IM-7-biotin. Fl-HA was used 1:100 diluted in a 50% Blocking solution (ECL kit, Roche Molecular Biochemicals) and was incubated 4 h at ambient temperature. Blots were washed with PBS, 0.1% Tween 20, 100 mm NaCl for 1 h three times. Biotinylated anti-fluorescein or anti-CD44 antibody was used at 1:1000 dilution and incubated for 2 h at room temperature and washed as above. Blots were developed with a chemiluminescence kit using streptavidin-POD according to manufacturer's instructions (Roche Molecular Biochemicals). SVEC4-10 membrane lysates were prepared as described above for Western blots. The pellet was resuspended in immunoprecipitation buffer (50 mm Tris, pH 8.0, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 1% Nonidet P-40). Fl-HA/anti-fluorescein-biotin or IM7-biotin was added to the above mixture and incubated at 4 °C for 2 h on a rocking platform. Complexes were precipitated using streptavidin-Sepharose (Sigma). Bound material was eluted by lowering the pH to 3.0 with 100 mm glycine HCl, neutralized with 100 mm Tris-HCl, pH 8.0, and analyzed by SDS-PAGE on a 10% polyacrylamide gel under reducing conditions. Following transfer to nitrocellulose, precipitated material was probed with IM7-biotin and developed with chemiluminescence using streptavidin-conjugated peroxidase. SVEC4-10 cells were grown to 60–80% confluence and then stimulated with 10 ng/ml TNFα for 4 h. Total RNA was prepared according to manufacturer's instructions using Trizol reagent (Life Technologies, Inc.). The reverse transcriptase reaction and PCR amplification were performed as described (59.Saiki R.K. Bugawan T.L. Horn G.T. Mullis K.B. Erlich H.A. Nature. 1986; 324: 163-166Crossref PubMed Scopus (1458) Google Scholar). CD44 forward primer sequence was 5′-GCACACCTACCTTCCTAC-3′ and reverse primer sequence was 5′-CTGGAATCTGAGGTCTCCTC-3′. β-Actin primers were used as control. PCR product was analyzed by agarose gel electrophoresis. Glass capillary tubes (1.41 mm inner diameter; Drummond Scientific, Broomall, PA) were coated with 1% BSA, soluble recombinant human CD44-Ig (350 μg/ml), control human IgG (350 μg/ml), soluble HA (1 mg/ml), or protein followed by sHA. To coat tubes with two substrates, tubes were first incubated with BSA, sCD44-Ig, or human IgG for 16 h at 4 °C and then further incubated with 1 mg/ml HA solution for 3 h at room temperature. sHA binding to sCD44-Ig-coated tubes was blocked by preincubation with HA-blocking anti-CD44 antibody prior to the addition of sHA to the tubes. All tubes were additionally blocked with 1% BSA/PBS for 1 h at room temperature. BW5147 murine T cells were washed and resuspended in RPMI 1640 medium at a concentration of 2 × 106/ml. The medium and the flow chamber were equilibrated to 37 °C. Medium containing BW5147 cells was continuously pulled through the capillary tube by means of a Harvard syringe pump at flow rates of between 0.5 and 5 ml/min, corresponding to shear stresses of 0.3–3.0 dynes/cm2 (60.Berg E.L. Robinson M.K. Warnock R.A. Butcher E.C. J. Cell Biol. 1991; 114: 343-349Crossref PubMed Scopus (271) Google Scholar). Interaction of the BW5147 cells with the wall of the capillary tubes (rolling) was monitored by the use of a Nikon Diaphot-TMD inverted phase contrast microscope connected to a video camera and recorder. Rolling numbers were determined by counting the number of cells/min crossing a fixed position perpendicular to the flow of cells on the video screen. Cell surface HA is often found non-covalently associated with membrane proteins, of which CD44 would be a prime candidate (22.Lesley J. Hyman R. Kincade P.W. Adv. Immunol. 1993; 54: 271-335Crossref PubMed Scopus (1032) Google Scholar). To characterize the nature of the association of HA with the endothelial surface, the peripheral lymph node-derived microvascular EC line SVEC4-10 was used after TNFα activation to induce high HA expression as described (42.Mohamadzadeh M. DeGrendele H.C. Estess P. Siegelman M.H. J. Clin. Invest. 1998; 101: 97-108Crossref PubMed Scopus (273) Google Scholar). Cells were then stained with the HA-binding proteoglycan bPG to assess HA surface levels. To measure other surface markers, cells were stained simultaneously with anti-CD44 or anti-H-2 and then subjected to flow cytometric analysis before and after various intervals of treatment with trypsin. The ability of bPG to bind to this cell line was completely trypsin-sensitive (Fig.1). Diminution of staining was evident within 5 min of trypsin treatment, and staining was 50% reduced by 10 min and completely ablated by 30 min. Staining with anti-CD44 IM7-PE diminished in parallel with bPG staining, correlating the trypsin sensitivity of HA expression with that of CD44, suggestive that this may be the HA-anchoring protein. In contrast, staining with anti-H-2 antibody was unaffected by trypsin treatment, consistent with the trypsin insensitivity of the extracellular portion of mouse class I MHC molecules (61.Bernard O. Scheid M.P. Ripoche M.A. Bennett D. J. Exp. Med. 1978; 148: 580-591Crossref PubMed Scopus (42) Google Scholar). Thus, HA is attached to endothelial surfaces through a trypsin-sensitive moiety that correlates with the cell surface levels of CD44. To establish further the nature of the EC surface HA-binding protein, we used a strategy of removing surface HA by digestion wi