Characterization of a Hyaluronan Receptor on Rat Sinusoidal LiverEndothelial Cells and Its Functional Relationship

to Scavenger Receptors

PETER A. G. MCCOURT,1,3 BÅRD H. SMEDSRØD,1 JUKKA MELKKO,2 AND STAFFAN JOHANSSON3

Hyaluronan is a widely distributed extracellular compo-nent of connective tissue with several mechanical and cellbiological functions. The serum level of hyaluronan iselevated in rheumatic and liver diseases and in certainmalignancies. The major route of hyaluronan clearancefrom the blood is via the liver, taken up predominantly bysinusoidal liver endothelial cells. We have purified a novelhyaluronan binding protein from liver that also has anaffinity for the N-terminal propeptide of type I procollagen,a physiological scavenger receptor ligand. A polyclonalantibody raised against the protein was found to inhibit thebinding and degradation of hyaluronan as well as twoscavenger receptor ligands by cultured sinusoidal liverendothelial cells. Immunostaining of nonpermeabilizedliver cells and liver sections showed that the antibodyspecifically stains the surface of sinusoidal liver endothelialcells. After pretreatment with monensin to block the recircu-lation of endocytic receptors, the immunostaining wasspecifically associated with early endosomes of these cells.Thus, this rat sinusoidal liver endothelial cell hyaluronanreceptor shares functional properties with the scavengerreceptor family, a group of proteins shown to play a key rolein the uptake of atherogenic lipids and other waste productsfrom the tissues. (HEPATOLOGY 1999;30:1276-1286.)

Hyaluronan (hyaluronic acid; HA) is a widely distributednonsulfated polysaccharide with many biological functionssuch as space filling and lubrication as well as other more

specific effects on cell functions.1 Its simple chemical struc-ture of repeating disaccharide units of N-acetyl-D-glu-cosamine and D-glucuronic acid linked by b(1-4) and b(1-3)glycosidic bonds, respectively, belies the range of uniqueviscoelastic and physiological properties that this anionicpolysaccharide has. Such qualities are probably conferredboth by its high molecular mass and its secondary and tertiarystructures that include 2-fold helices and extensive branchednetworks2 and possibly sheets and tubular structures.3 Theformation of these secondary and tertiary structures is likelyfacilitated by both hydrophobic and hydrophilic interactionsbetween HA monomers.3

Accumulation of HA in the extracellular matrix is an earlyevent in tissue repair.4 The appearance and removal of HA atspecific locations are precisely controlled during embryogen-esis.4 In certain disease states such as rheumatoid arthritis,osteoarthritis, liver cirrhosis, Werner syndrome, renal failure,psoriasis, and various malignancies (e.g., Wilms’ tumor), theserum level of HA is elevated.5 These increased levels arecaused either by overproduction of HA or by impairedclearance from the blood. In the case of Wilms’ tumor, theoverproduction of HA is so great that it causes the blood tobecome overly viscous6,7 as well as causing defects in bloodclotting.8 This last example shows the consequences ofexcessive levels of HA in circulation and therefore theimportance of its removal.

The disposal of HA is, in the healthy individual, almostentirely brought about by catabolism rather than excretionand occurs at 3 main levels: the first is local degradation inthe tissues where HA is synthesized, the second is in thelymph nodes after displacement from the tissues, and thethird is in the blood of HA that is not metabolized in thelymph nodes.9 The major part of the blood-borne HA isremoved by the liver,10-12 with the remainder removed by thespleen,10 kidneys,13 and bone marrow.11 In the liver, thesinusoidal liver endothelial cells (LECs) are the most impor-tant sites of hepatic extraction of HA, with the Kupffer cellsplaying a relatively minor role in HA clearance under normalconditions.14-17

The removal of HA from the blood is very efficient,resulting in a half-life of less than 1 minute (in rats)18 and isfacilitated by endocytic cell surface receptors on LECs, whichalso recognize chondroitin sulfate (CS).15,16 Formaldehyde-treated albumin (FSA), a scavenger receptor (SR) ligand,blocks the uptake of CS,19 suggesting that the HA/CS receptoris functionally related to the family of SRs. The affinity of thereceptor(s) for HA is dependent on the size of the latter: forexample, the Kd values for HA octasaccharides and HA of Mr

Abbreviations: HA, hyaluronan; LEC, sinusoidal liver endothelial cell; CS, chondroi-tin sulfate; FSA, formaldehyde-treated bovine serum albumin; SR, scavenger receptor;PBS, phosphate-buffered saline; EDA, ethylenediamine; PINP, N-terminal propeptide ofprocollagen type I; PI, protease inhibitors; EDTA, ethylenediaminetetraacetic acid;SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HABP, HA-binding protein; IgG, immunoglobulin G; TBS, Tris-buffered saline; LDL, low-densitylipoprotein.

From the 1Department of Experimental Pathology, University of Tromsø, Tromsø,Norway; 2Department of Pathology, University of Oulu, Oulu, Finland; and 3Depart-ment of Medical Biochemistry and Microbiology, University of Uppsala, Uppsala,Sweden

Received February 22, 1999; accepted August 9, 1999.Supported by grants from Polysackaridforskning i Uppsala AB, the Swedish Medical

Research Council (grant 7147), the Konung Gustav V:s 80-årsfond, the NorwegianResearch Council (grant 312.92/017), and the Norwegian Cancer Foundation (grant88.100).

Address reprint requests to: Peter McCourt, Ph.D., Department of ExperimentalPathology, University of Tromsø, MH Building, N-9037 Tromsø, Norway. E-mail:petermcc@fagmed.uit.no; fax: (47) 77 64 54 00.

Copyright r 1999 by the American Association for the Study of Liver Diseases.0270-9139/99/3005-0023$3.00/0

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6.4 3 106 are 1.4 µmol/L and 9 pmol/L, respectively.15 Usingphotoaffinity labeling and ligand blotting of reduced LECmembranes, 3 species of 166, 175, and 300-400 kd have beenproposed as potential LEC HA receptors.20-23 The identity andfunction of these proteins remain to be established.

In the present report we describe studies that have led tothe purification and characterization of an HA receptor onLEC using HA affinity chromatography, immunohistochemis-try, and antibody-ligand inhibition studies.

MATERIALS AND METHODS

Chemicals and Reagents. Collagenase (Grade V), hyaluronidase(bovine testes type IV-S), lactoperoxidase, glucose oxidase (typeII-S), leupeptin, pepstatin A, phenylmethyl sulfonyl fluoride, benza-midine, ethylene diamine, N-ethylmaleimide, N-acetylglucosamine,a-methyl mannoside, mannan, bovine serum albumin, and TritonX-100 (molecular biology grade) were obtained from Sigma Chemi-cal Co. (St. Louis, MO). Aprotinin was obtained from Bayer(Leverkusen, Germany). 125I was obtained from Nordion Inc,Kanata, Canada. The monoclonal antibody to the mouse macro-phage scavenger receptor, 2F8, was obtained from Serotec (Oxford,UK). Anti-rabbit–Cy2 and –Cy3 were purchased from JacksonImmunoResearch Laboratories (West Grove, PA). Fluoromount Gwas purchased from Southern Biotechnology Associates (Birming-ham, AL). High-range prestained molecular mass standards wereobtained from Bio-Rad Laboratories (Hercules, CA). 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide was obtained from FlukaChemika-BioChemika (Buchs, Switzerland). CHAPS was obtainedfrom CalBiochem (La Jolla, CA). Wheat germ agglutinin wasobtained from the Separation Center, Biomedical Center (Uppsala,Sweden). Enhanced chemiluminescence kit, Cyanogen bromide–activated Sepharose 4B, PD 10 columns, and Protein A Hi Trapcolumns were obtained from Amersham Pharmacia Biotech (Uppsala,Sweden). HA (approximately 900 kd) was a kind gift from Dr. OveWik, Pharmacia (Uppsala, Sweden), and Healon (approximately3.5 3 103 kd HA) was a kind gift of Pharmacia and Upjohn(Uppsala, Sweden). Metabolically labeled 3H-HA (4.2 3 105 dpm/µg, Mr 3.85 3 106)(10) was a kind gift of Professor Robert Fraser(Department of Biochemistry, Monash University, Clayton, Austra-lia). Collagen (Vitrogen 100) was from Collagen Biomaterials,Collagen Corporation (Palo Alto, CA). Nidogen was a kind gift ofDr. Ulrike Mayer (Max-Planck-Institute for Biochemistry, Martins-ried bei Munchen, Germany). Mouse liver nonparenchymal cellswere a kind gift of Ms. Beatriz Arteta (University of Tromsø, Tromsø,Norway).

Animals. All rats (Sprague-Dawley, male) received humane careaccording to the guidelines of the National Institutes of Health(Bethesda, MD).

Preparation of HA Oligosaccharides for Coupling to Sepharose and forElution of Bound Proteins. HA oligosaccharides were prepared bydigestion of high Mr HA (210 mg) in 150 mmol/L NaCl, 100 mmol/LNaCH3COO, pH 5.0 (100 mL) with bovine testes hyaluronidase typeIV-S (8,000 U/g of HA) for 4 hours at room temperature. Thedigested material was placed in boiling water for 45 minutes toinactivate the enzyme, and then dialyzed (1.2 3 104 molecularweight cut off) against water (1.6 3 104 volumes) before beinglyophilized to dryness. The resulting oligosaccharides were dis-solved in phosphate-buffered saline (PBS) (pH 7.4) (137 mmol/LNaCl, 2.7 mmol/L KCl, 6.5 mmol/L Na2HPO4, and 1.5 mmol/LKH2PO4) and chromatographed on a calibrated S-300 size exclusioncolumn. HA fractions corresponding to 25 to 40 kd were pooled anddialyzed against water as before and then lyophilized to dryness. Theresulting lyophilisate was dissolved either in water for coupling toaminoethyl-Sepharose or PBS for elution studies.

Preparation of HA-Ethylenediamine Sepharose 4B and Control-Ethylenedi-amine-Sepharose 4B. HA-ethylenediamine (HA-EDA) Sepharose 4Band control-EDA (1-amino-2-acetoamidoethane)-Sepharose 4B wereprepared as previously described.24

Preparation of N-Terminal Propeptide of Type I Procollagen-Sepharose andControl-Sepharose. N-terminal propeptide of type I procollagen (PINP)was prepared from the culture medium of human osteosarcomaMG-63 cells (American Type Culture Collection, Rockville, MD) asdescribed previously.25 One milligram of PINP was coupled tocyanogen bromide-activated Sepharose 4B (3 mL) according to themanufacturer’s instructions. Residual active groups on the gel wereblocked by the addition of 2 mol/L Tris (pH 8.8) and mixingend-over-end for 2 hours at room temperature.

Control-Sepharose was prepared by the addition of 2 mol/L Tris(pH 8.8) directly to reswollen cyanogen bromide-activated Sepha-rose 4B and mixing end-over-end for 2 hours at room temperature.

Isolation of Rat Hepatocytes and LECs. Single-cell suspensions wereprepared from the livers of rats by collagenase perfusion accordingto Obrink.26 Hepatocytes and LECs were isolated after Percollgradient centrifugation and selective adherence according to Smeds-rød and Pertoft.27 LECs were either solubilized directly in PBScontaining 1% (vol/vol) Triton X-100 and protease inhibitors (PI)(10 mmol/L benzamidine, 50 KIE/mL Aprotinin, 0.2 mmol/Lphenylmethyl sulfonyl fluoride, 1 mg/mL leupeptin, 0.5 mmol/Lethylenediaminetetraacetic acid [EDTA], and 1 µg/mL pepstatin A)or cultured overnight at 37°C in RPMI 1640 medium on fibronectin-coated plates and coverslips for surface labeling experiments (de-scribed later), antibody inhibition studies, and immunohistochemis-try. Hepatocytes were cultured on fibronectin-coated coverslips onlyin the presence of 10% calf serum.

Surface Labeling of Rat LECs. Overnight monolayer cultures ofapproximately 10 3 106 LECs on 60 cm2 plates were surface labeledwith 125I, using the lactoperoxidase method28 as previously de-scribed29 with minor modifications. Briefly, LECs were washed 3times with ice-cold PBS and then incubated with PBS (1 mL)containing 5 mmol/L b-D-glucose, 125I (0.5 mCi/mL), lactoperoxi-dase (30 µg/mL), and glucose oxidase (2.4 µg/mL) for 15 minutes,with gentle rocking. The cells were then washed 6 times with PBSand solubilized in PBS containing 1% (vol/vol) Triton X-100 (1 mL)and PI for 1 hour at 4°C on a rocking platform.

Affinity Chromatography of Surface-Labeled LEC Extracts on HA-EDA–Sepharose and PINP-Sepharose. Columns (17 mm diameter) werepacked with 3 mL each of HA-Sepharose, EDA-Sepharose, control-EDA–Sepharose, PINP-Sepharose, and control-Sepharose and equili-brated with PBS containing 0.1% (vol/vol) Triton X-100 plus PI. Todetermine which cell surface proteins bound to the above resins,Triton X-100 extracts (of approximately 3 3 106 125I-surface labeledLEC) were diluted to 0.1% (vol/vol) Triton X-100 with PBScontaining PI (3.0 mL) and applied to the columns at 0.6 mL every 5minutes. The flow-throughs from the HA-EDA– and PINP-Sepharose were applied directly to fresh columns (3 mL) of PINP-and HA-EDA–Sepharose, respectively. The columns were washedwith equilibration buffer (15 mL) and bound proteins eluted eitherwith a 500-µL aliquot of 10 mg/mL HA oligosaccharides followed byequilibration buffer (500 µL every 10 minutes) (HA-EDA– andcontrol-EDA–Sepharose columns), or with 0.1 mol/L NaCH3COOpH 3.5 (500 µL every 10 minutes) (PINP- and control-Sepharose);500-µL fractions were collected. All chromatographic steps wereperformed at 6°C. Fractions containing the most radioactivity wereanalyzed by sodium dodecyl sulfate-polyacrylamide gel electropho-resis (SDS-PAGE) (7.5% acrylamide) and autoradiography.

Large-Scale Purification of LEC HA Binding Proteins. All steps wereperformed at 6°C unless otherwise stated. Purification was achievedusing both whole rat liver and LECs as a source for LEC HA bindingproteins (LEC HABPs). For studies with whole liver, typically 10snap-frozen rat livers were homogenized on ice in PBS (250 mL)containing 2% Triton X-100, 10 mmol/L EDTA, 1 mmol/L N-ethylmaleimide, and PI (buffer A). The homogenate was centrifugedat 25,000g for 1 hour and the supernatant filtered through glasswool. At this stage, we found that freezing the above supernatant at220°C for at least 1 week (freezing overnight was found to beinsufficient) resulted in the precipitation of solid material (approxi-mately 20% of the supernatant volume), which could be easily

HEPATOLOGY Vol. 30, No. 5, 1999 MCCOURT ET AL. 1277

pelleted with centrifugation at 5,000g for 20 minutes. This resultedin an almost clear supernatant that was applied at a flow rate of 7cm/h to a wheat germ agglutinin–Sepharose column (50 mL bedvolume, 250 mg wheat germ agglutinin) that had been equilibratedwith buffer A. Omission of the week-long freezing step resulted inclogging of the wheat germ agglutinin column and markedlyreduced yields of protein. The column was then washed with 7volumes of PBS containing 0.1% Triton X-100, 1 mmol/L N-ethylmaleimide, and PI (buffer B) and eluted with the same buffercontaining 350 mmol/L N-acetylglucosamine (350 mL).

The eluted fractions were pooled and concentrated to 10 to 15 mLon an Amicon YM 30 membrane and then applied at a flow rate of 10cm/h to an S-300 column (500-mL bed volume, 2.5 cm diameter)that had been equilibrated with buffer B. Proteins were eluted at thesame flow rate with PBS containing 0.1% Triton X-100 and 10-mLfractions collected. Samples of fractions 17 to 45 were subjected toSDS-PAGE and silver stain analysis, and those fractions enriched inproteins $80 kd (typically fractions 18-30) were pooled for the nextstep.

The pooled fractions were then applied at a flow rate of 5 cm/h tocontrol-EDA–Sepharose (5 mL) and HA-EDA–Sepharose (5 mL)columns connected in series and in that order that had beenequilibrated with buffer B. The column series was washed at 10 cm/hwith 30 mL buffer B, and then the HA-EDA–Sepharose column wasdetached and washed with 20 mL of PBS containing 0.49% CHAPS,1 mmol/L N-ethylmaleimide and PI minus EDTA (buffer C). Boundproteins were eluted with a pulse of HA oligosaccharides (10 mg in 1mL buffer C) followed by buffer C, applied at 5 cm/h; 1-mL fractionswere collected and analyzed with SDS-PAGE and silver staining.

Fractions from 6 to 8 purifications (i.e., from 60-80 rat livers)were then pooled and applied at 5 cm/h to a lentil lectin-Sepharosecolumn (1 mL). The column was washed with buffer C (3 mL), andbound proteins were eluted with 350-mmol/L a-methyl mannosidein the same buffer (100 mL).

The eluate was then concentrated in a Millipore Biomax Ultra-free-30 centrifugal concentrator, and fractionated on a Superose 6column in buffer C. Fractions eluted in the void volume weresubjected to preparative SDS-PAGE (6% acrylamide) under nonre-ducing conditions, and Coomassie stained bands at 180 andapproximately 270 kd were excised, washed 4 times for 30 minutesin PBS, and then stored at 220°C.

For studies with LECs, fresh Percoll-purified nonparenchymalcells (consisting of approximately 85% LECs) from 10 rat livers weresolubilized in PBS (20 mL) containing 1% Triton X-100 and PI, for 1hour at 6°C. The solubilized extract was centrifuged at 10,000g for10 minutes, and the supernatant was diluted to 0.1% (vol/vol) TritonX-100 with PBS (containing PI) (180 mL) and applied to thecontrol-EDA–Sepharose/HA-EDA–Sepharose column series as de-scribed previously. Fractions eluted from the HA column werechromatographed on lentil lectin as described previously, and theeluted fractions were concentrated before freezing at 270°C.

Amino Acid Sequence Analysis of Peptides Isolated From LEC HA BindingProtein. Preparative SDS-PAGE was performed as described previ-ously using LEC HABP purified from 60 to 80 rat livers. Theapproximately 270-kd protein band was visualized with CoomassieBrilliant Blue, excised, and digested ‘‘in-gel’’ with Lys-C (lysylendopeptidase) according to the method of Rosenfeld et al.30 Theliberated peptides were then purified by reverse phase high perfor-mance liquid chromatography on a µRPC SC2.1/10 C2/18 column(Pharmacia) on a SMART chromatograph with automatic peakcollection. Peptides were eluted with a gradient from 0% to 50%acetonitrile in water and 0.05% trifluoroacetic acid over 75 minutesand then sequenced.

Preparation of Antibodies to Purified LEC HA Binding Protein. A gelpiece containing the approximately 270-kd LEC HABP (approxi-mately 100 µg protein) was homogenized in PBS (3 mL), dispensedinto 0.3-mL aliquots and stored at 220°C. A New Zealand Whiterabbit was immunized first with one homogenized gel aliquot inFreund’s complete adjuvant (0.3 mL) and subsequently with an

aliquot of the same material in Freund’s incomplete adjuvant every 2weeks (3 immunizations) and thereafter once every 6 weeks. Serumwas taken from the rabbit every 2 weeks after the fourth immuniza-tion.

Purification of Immunoglobulin G From Rabbit Serum. Control andimmune rabbit serum (3 mL) was applied to Protein A HiTrap (1mL) columns at a rate of 1 mL/min. The columns were washed with50 mmol/L sodium phosphate (pH 7.0) and the bound immunoglobu-lin G (IgG) eluted with 100 mmol/L glycine pH 3.0. Fractionscontaining the most IgG were then applied to PD 10 columns andeluted in PBS before sterile filtration and storage at 6°C.

Immunoblotting. After SDS-PAGE, proteins were transferred tonitrocellulose,31 blocked with skim milk (10% in PBS), and probedwith rabbit immune serum against LEC HABP at 1:200 dilution (4hours at 25°C), followed by donkey anti-rabbit horseradish peroxi-dase conjugate at 1:5000 dilution (1 hour at 25°C). After visualiza-tion of immunoreactive bands with the enhanced chemilumines-cence kit (according to the manufacturer’s instructions), theimmunoblots were stripped with 100 mmol/L 2-mercaptoethanol,2% SDS, 62.5 mmol/L Tris-HCl, pH 6.7, at 60°C for 30 minutes. Thestripped immunoblots were blocked with 1% bovine serum albuminin Tris-buffered saline (150 mmol/L NaCl, 20 mmol/L Tris, pH 7.6)(TBS) and probed with the anti-mouse macrophage scavengerreceptor monoclonal antibody 2F832 (5 µg/mL) (2 hours at 25°C).The secondary antibody (rabbit anti-rat) was used at 1:2,000dilution (1 hour at 25°C) and the tertiary antibody (donkeyanti-rabbit horseradish peroxidase) was used at 1:10,000 dilution (1hour at 25°C). Immunoreactive protein bands were visualized asdescribed earlier.

Immunostaining of LECs, Hepatocytes, and Mouse Liver Sections. LECsand hepatocytes grown on fibronectin-coated coverslips (14 mm)were fixed in PBS containing 2% paraformaldehyde for 10 minutes atroom temperature, washed 3 times with PBS, and stored in PBScontaining 0.02 % NaN3 at 4°C. Fixed cells were blocked with 10%goat serum in PBS (blocking buffer) for 1 hour at room temperature.Preimmune and immune serum at a dilution of 1:200 in blockingbuffer was applied to the fixed cells for 1 hour at room temperature.The coverslips were washed 3 times for 10 minutes with PBScontaining 0.05% Tween-20 (wash buffer), and then the goatanti-rabbit-Cy3 at a dilution of 1:400 in blocking buffer was appliedfor 1 hour at room temperature in darkness. The coverslips werewashed 3 times for 5 minutes with wash buffer, dipped once inwater, touched against blotting paper to remove excess fluid, andthen mounted with Fluoromount G on a microscope slide. The cellspecimens were then examined under a conventional fluorescencemicroscope.

For immunostaining of tissue sections, paraffin mouse liversections33 were soaked in xylene (10 minutes), 100% ethanol (3minutes), 96% ethanol (3 minutes), 70% ethanol (3 minutes), andwater (10 minutes) at room temperature and in that order. Thesections were then treated with trypsin (Dibco, 1 mg/mL) in TBScontaining CaCl2 (6 mmol/L) for 10 minutes at 37°C, before beingplaced in TBS for 10 minutes at 4°C. The sections were then washedwith TBS, blocked with goat serum, and immunostained withimmune serum as described earlier, except that the primary anti-body was incubated on the section overnight at 4°C, and goatanti-rabbit-Cy2 was used as the secondary antibody.

Preparation of Denatured Collagen and FSA. Denatured (gelatinized)collagen was generated by heating at 60°C for 30 minutes. Formalde-hyde treatment of bovine serum albumin was performed as de-scribed.34

Ligand Labeling Techniques. FSA, nidogen, PINP, mannan, anddenatured collagen were labeled with 125I or TRITC (collagen only)as previously described.35

HA Binding Protein/Denatured Collagen Colocalization Studies. Theeffect of monensin (an ionophore that blocks endocytic vesiclefusion and receptor recirculation36) on the distribution of thepolyclonal antibody staining in cultured LECs was tested. Freshlyprepared monolayer cultures of LECs on fibronectin-coated cover-

1278 MCCOURT ET AL. HEPATOLOGY November 1999

slips (14 mm) were incubated in the presence of monensin (1µmol/L) for 15 minutes at 37°C. A marker of LEC-specific endocyto-sis,35 TRITC-labeled denatured collagen (T-COL), was added to aconcentration of 0.1 mg/mL for 30 minutes at 37°C. Excessunbound T-COL was washed away with PBS, and then the cells werefixed, treated with PBS/0.05% Tween-20, immunostained, andmounted as described earlier, with the exception that the secondaryantibody used was goat anti-rabbit fluorescein isothiocyanate at adilution of 1:20.

Antibody Inhibition Studies. Cultures of LECs (0.5 3 106 cells) wereestablished in 2-cm2 wells and maintained in RPMI 1640. Afterwashing, the cultures were incubated at 37°C for 30 minutes in 200µL medium containing IgG from control and immune serum atindicated concentrations. Radiolabeled ligands were added in 20 µLand the cultures returned to 37°C for a 1-hour incubation tomeasure endocytosis. For binding studies, the cultures were placedat 4°C before the radiolabeled ligand (20 µL) was added for 2 hoursat the same temperature. 3H-HA was incubated with the cells at 0.2µg/mL (approximately 20,000 cpm per well), and subsequently theincubation medium was decanted and the cells were washed withPBS (500 µL). The medium and the wash were pooled in the sametube. For experiments at 4°C, the pool was mixed directly withscintillation fluid (5 mL). For endocytosis (uptake)-inhibitionexperiments (at 37°C), the pool was chromatographed on PD10columns; 1-mL fractions were collected and mixed with scintillationfluid (2 mL). The cell layer, containing bound 3H-HA, was solubi-lized in 1% SDS (500 µL) and transferred to a scintillation tube. Thewell was washed with a further aliquot of 1% SDS, which was pooledwith the cell-bound material and scintillation fluid (5 mL), added.The radioactivity was then analyzed in a beta counter. For binding-inhibition studies with denatured collagen and PINP, these ligandswere incubated with the cells at 4°C for 2 hours and then treated asfor the HA binding studies described earlier. For endocytosis-inhibition studies with denatured collagen, mannan, PINP, FSA, andnidogen, these ligands were incubated with the cells at 37°C for 1hour and the cells and media treated as described in Hellevik et al.35

RESULTS

Affinity Chromatography of Iodinated LECs. Potential LECHABPs had previously been identified as consisting of approxi-mately 200- and 400-kd proteins,24 using HA-EDA–Sepha-rose affinity chromatography. The affinity of these proteinsfor another molecule specifically cleared from the blood byLECs, namely PINP, was tested. Shown on the SDS-PAGEautoradiographs in Fig. 1 is material eluted from HA-EDA–Sepharose (Fig. 1A, lanes 1 and 2) and PINP-Sepharose (Fig.1A, lanes 3 and 4) after 125I-surface labeled LECs were appliedto each column. A similar band pattern was obtained fromboth columns: the unreduced proteins migrated as 200- andapproximately 350-kd bands, whereas 3 components of 210,approximately 300, and approximately 350 kd were seen afterreduction. When the nonbinding (flow-through) fractionsfrom the HA column were applied to the PINP column (Fig.1B, lanes 1 and 2) and vice versa (Fig. 1B, lanes 3 and 4), no(or very little) protein could subsequently be eluted from thelatter column. This indicated that the LEC HABPs also had anaffinity for PINP ligands. There were no visible bands elutedfrom the control-EDA–Sepharose (Fig. 1C, lane 1); a faintband (170 kd) was eluted from the control-Sepharose (Fig.1C, lane 2). It should be noted that the molecular masses ofthe 300- and 350-kd species are tentative estimates, as therewere no available standards with a molecular mass greaterthan 205 kd.

Purification of LEC HA Binding Proteins. Attempts were madeto purify the above HABPs using LEC and liver as starting

FIG. 1. SDS-PAGE (7.5% gel) autoradiographs of 125I surface-labeled LECextracts chromatographed on HA-EDA–Sepharose and PINP-Sepharose (Aand B) and their respective control resins (C). (A) Lanes 1 and 2 showmaterial (reduced and nonreduced, respectively) eluted from HA-EDA–Sepharose using free HA oligosaccharides, and lanes 3 and 4 show material(reduced and nonreduced, respectively) eluted from PINP-Sepharose usinglow pH. (B) Lanes 1 and 2 show material (reduced and nonreduced,respectively) eluted from HA-EDA–Sepharose using free HA oligosaccha-rides, after the applied LEC extract had been precleared on PINP-Sepharose,and lanes 3 and 4 show material (reduced and nonreduced, respectively)eluted from PINP-Sepharose using low pH, after the applied LEC extract hadbeen precleared on HA-EDA–Sepharose. (C) Lanes 1 and 2 show material(nonreduced) eluted from control-EDA–Sepharose using free HA oligosaccha-rides and from control-Sepharose with low pH. (c), Molecular weightstandards; (arrowhead), radio-labeled protein bands in kd.

HEPATOLOGY Vol. 30, No. 5, 1999 MCCOURT ET AL. 1279

material. Using detergent solubilized LEC extracts from 10rats as starting material, approximately 4 µg of both a 170- to180-kd doublet and an approximately 270-kd species waspurified; shown in Fig. 2A is a silver-stain of this materialafter SDS-PAGE (6% gel). Again, the molecular mass of theapproximately 270-kd species is an estimate, based onstandards with a molecular mass less than 213 kd. When thismaterial was run on 7.5% SDS-PAGE, the apparent molecularmasses of both the upper and lower bands shifted upward; thelower band to 200 kd and the upper band to (approximately)350 kd (not shown).

At this point a procedure for the purification of the LECHABPs from whole rat liver was developed, because theisolation of LECs is an expensive and laborious method, andthe yield of purified protein was relatively low. The procedureusing whole liver has resulted in the purification of the 170-to 180-kd doublet and approximately 270-kd species, withtypical yields of 0.5 to 1.0 µg of each species per liver. Shownin Fig. 2B is a silver-stain of this material after SDS-PAGE (6%gel). As with material purified from LEC described earlier,there was an apparent upward shift of the molecular massesof both the upper and lower bands when analyzed on 7.5%SDS-PAGE. The band at 150 kd (indicated with an arrow) is acontaminant that also has affinity for control-EDA–Sepha-rose. The upper approximately 270-kd LEC HABP waspurified in sufficient amounts for antibody production andamino acid sequencing. Peptides with the sequences KSFWL-SRNNW, KADILDYLLSPEGS, KGVIHGLEK, and KNTVG-QILDEGGPY were obtained. When attempting sequencealignments of these peptides on the NCBI BLAST database, no

significant homologies to any protein were found, suggestingthat the approximately 270-kd species is a novel protein.

Immunoblotting and Immunohistochemistry With the Anti–270-kdLEC HABP Antibody and the Anti-Mouse Macrophage ScavengerReceptor Monoclonal 2F8. A rabbit polyclonal antibody usingthe approximately 270-kd protein as immunogen was pro-duced. This antibody was found to recognize the 170- to180-kd and the approximately 270-kd species in immuno-blots of LEC extracts (Fig. 3A) and partially purified LECHABP from liver (Fig. 3B) providing evidence that the upperband and the lower doublet are related proteins.

To investigate the relationship of the LEC HABP to themacrophage scavenger receptor type A (which is expressedon LECs), immunoblots of rat LEC extracts and mouse livernonparenchymal cell extracts were probed with the 2F8monoclonal. The immunoblots were first probed with theanti–LEC HABP antibody, stripped, and then probed with2F8. The anti–LEC HABP antibody recognized 170- to180-kd and approximately 270-kd species in the rat (Fig. 3A)and mouse (Fig. 4A) cell extracts. The 2F8 rat monoclonaldid not recognize any bands in rat LEC extracts (not shown),but did recognize an approximately 230-kd band in mouseliver nonparenchymal cell extracts (Fig. 4B).

The anti–LEC HABP antibody specifically immunostainsnonpermeabilized cultured LECs (Fig. 5A) but does not staincultured hepatocytes (not shown); the background stainingof preimmune serum is minimal (Fig. 5B). General stainingover the entire surface of the LECs was evident at this (Fig.5A) and at a magnification 31,000 (not shown), with higherintensity about the nucleus, and lower staining at the (flatter)cell extremities. Particularly intense staining about the nucleuswas noted in some LECs, but no other regions displayed any

FIG. 2. SDS-PAGE (6% gel)-silver stain of HABP purified from rat LECs(A) and whole rat liver (B). (c), Molecular weight standards; (arrowhead),silver-stained protein bands; (open arrow), contaminant in kd.

FIG. 3. Immunoblots of rat LEC lysate (A) and partially purified LECHABP from whole rat liver (B) probed with the anti-LEC HABP polyclonalantibody. (c), Molecular weight standards; (arrowhead), immunoreactiveprotein bands in kd.

1280 MCCOURT ET AL. HEPATOLOGY November 1999

notable pattern of intense staining. The antibody was alsotested for its ability to stain liver sections. The antibody stainsthe sinusoidal endothelium (indicated with arrows) but notthe large vessel endothelium in mouse liver sections (Fig. 6).

The effect of monensin (an ionophore which blocksendocytic vesicle fusion and receptor recycling36) on thedistribution of the anti-HABP staining in cultured LECs wasalso tested. In the monensin-treated cells, the staining wasredistributed to intracellular ring structures (Fig. 7A) insteadof the predominant general surface location in untreated cells(Fig. 5A). TRITC-collagen, which is endocytosed by LECs viaa separate receptor,35 colocalized to the same ring structuresin the monensin-treated LECs (Fig. 7B). This shows that theanti-HABP staining colocalizes to endocytic vesicles, suggest-ing that it is an endocytic protein.

Antibody/Ligand Inhibition Studies. The ability of the immuneIgG at various concentrations to inhibit the binding of highmolecular weight 3H-HA and other molecules taken up byLECs was tested at 4°C, using nonimmune IgG as a control;the results are shown in Table 1. In all experiments, theimmune IgG inhibited 3H-HA binding to LECs to a greaterdegree than the nonimmune IgG at IgG concentrations $0.5mg/mL; however, nonimmune IgG had some inhibitory effecton binding at all IgG concentrations. Shown also in Table 1 isthe binding by LECs at 4°C of a labeled SR ligand, 125I-PINP,and labeled denatured collagen (125I-collagen) in the presenceof nonimmune and immune IgG. The immune IgG reducedthe capacity of LECs to bind 125I-PINP by 35% but had noeffect on the binding of 125I-collagen.

The ability of immune IgG to inhibit the LEC endocytosisof high molecular weight 3H-HA at 37°C was tested. Shown inFig. 8 is the total endocytosed (Fig. 8A), cell associated (Fig.8B), and degraded (Fig. 8C) 3H-HA. LECs incubated withimmune IgG had a markedly reduced capacity to bind anddegrade 3H-HA, resulting in the reduction of endocytosis to61%, 37%, and 16% of control levels, in the presence of 0.2,0.5, and 1.0 mg/mL IgG, respectively. Nonimmune IgG hadessentially no effect on the endocytosis of 3H-HA.

The effect of immune IgG on the LEC endocytosis of 3labeled SR ligands, namely 125I-FSA, 125I-PINP and 125I-nidogen, and 125I-collagen at 37°C was tested. Shown in Fig. 9is the total endocytosed (Fig. 9A), cell associated (Fig. 9B),and degraded (Fig. 9C) 125I-FSA, 125I-PINP, and 125I-collagen.The ability of LECs to bind and degrade 125I-FSA was alsoreduced (to 61% and 20% of control levels, respectively) bythe presence of immune IgG, resulting in the reduction ofendocytosis to 33% of control levels. Nonimmune IgGinhibited endocytosis slightly, to 93% of control levels. Theability of LECs to degrade 125I-PINP was reduced by immuneIgG to 27% of control levels, whereas the cell-associatedfraction was unaffected, resulting in the reduction of endocy-tosis to 77% of control levels. Nonimmune IgG had noinhibitory effects on binding, degradation, and endocytosis of125I-PINP. The endocytosis of nidogen was not inhibited at allby immune IgG (data not shown). The binding, degradation,and endocytosis of 125I-collagen were not inhibited by im-mune IgG or nonimmune IgG.

The effect of immune IgG on the endocytosis of a ligandtaken up by the mannose receptor, namely mannan, wastested. The endocytosis of 125I-mannan was inhibited by 20%with immune IgG, whereas nonimmune IgG had no effect(not shown).

FIG. 4. Immunoblots of mouse liver nonparenchymal cell lysate probedwith the anti–LEC HABP polyclonal antibody (A) and the anti-mousemacrophage scavenger receptor monoclonal antibody 2F8 (B). (c), Molecu-lar weight standards; (arrowhead), immunoreactive protein bands in kd.

FIG. 5. Nonpermeabilized cultured rat LECs immunostained with anti–LEC HABP polyclonal antibody (1:200) (A) or preimmune serum (1:200)(B). Secondary antibody was goat anti-rabbit-Cy3 (1:400). Bar indicates40 µm.

HEPATOLOGY Vol. 30, No. 5, 1999 MCCOURT ET AL. 1281

DISCUSSION

It has previously been reported that rat intercellularadhesion molecule 1 could serve as an HA receptor onLECs.37 However, this finding is most likely an artifact,because of complications caused by the use of HA attached toSepharose via a 6-carbon diaminohexane linker.37,38 Subse-quent studies showed that this linker bound many proteinsnonspecifically, including intercellular adhesion molecule 1,irrespective of whether HA was coupled or not.24 However,the use of HA attached via a shorter 2-carbon EDA linker toSepharose avoided the problems of nonspecific binding, andfacilitated the purification of 2 proteins of 200 and (approxi-mately) 400 kd from Triton X-100 extracts of 125I-surface–labeled LECs.24 The molecular masses and the double bandpattern of these polypeptides are similar to that reported forLEC HABPs by others.20-23

The purification of the LEC HABP in amounts sufficient forantibody production and amino acid sequencing was neces-sary to show its HA receptor function and to resolve thequestion of its multiple affinities and its relationship (if any)to other proteins. The protocol outlined in the methodssection uses whole liver as starting material, obviating theneed for pure LECs, which are difficult and costly to produce.Furthermore, the protocol ensures the purification of HABPs,through the inclusion of the penultimate specific affinity step,namely the elution with HA of protein bound to HA-EDA–Sepharose. The procedure yielded submilligram amounts ofproteins of 170 to 180 kd and approximately 270 kd, whichhave a molecular mass rather less than those HABPs (200 andapproximately 350 kd) identified on surface-labeled LECs.This apparent inconsistency can be explained by the fact thatthe SDS-PAGE analysis was performed at different acrylamideconcentrations, resulting in an apparent change in molecularmass (relative to the standards). We found that increasing theacrylamide concentration increased the apparent molecularmass of material purified from both LECs and whole liver.This unusual behavior is similar to that described for LECHABPs by others23 suggesting that the same proteins havebeen detected by independent techniques. That the 170- to

FIG. 6. Mouse liver sections immunostained with anti–LEC HABPpolyclonal antibody (1:200). Secondary antibody was goat anti-rabbit-Cy2.Arrows indicate specifically stained sinusoidal cells. Bar indicates 20 µm. LV,large vessel.

FIG. 7. Location of LEC HABP (A) and denatured collagen (B) inmonensin (1 µmol/L) treated cultured LECs (bar indicates 20 µm). The HAreceptor (green fluorescence) was immunostained with anti–LEC HA receptorantibody (1:200) and anti rabbit IgG–fluorescein isothiocyanate (1:40). Thelocation of TRITC-labeled denatured collagen is indicated by red fluorescence.

TABLE 1. Antibody Inhibition of Ligand Binding to LECs

LigandIgG Concentration

(mg/mL)

% Control Binding

Anti–HABP IgG Nonimmune IgG

3H-HA 0.20 84 (n 5 4) 91 (n 5 3)SD 5 10 SD 5 4

0.50 59 (n 5 3) 86 (n 5 3)SD 5 10 SD 5 10

1.00 52 (n 5 4) 82 (n 5 3)SD 5 4 SD 5 1

125I-PINP 0.20 57 (n 5 3) 92 (n 5 3)SD 5 5 SD 5 3

125I-collagen 0.50 106 (n 5 1) 96 (n 5 1)

NOTE. LECs were incubated in the presence or absence of indicated IgGfor 30 minutes at 37°C. The cultures were transferred to 4°C and incubatedwith the indicated ligands for 2 hours to determine binding. 100% binding isdefined as ligand binding in the absence of added IgG, and corresponds to6.1%, 12.3%, and 14.4% of applied radioactivity for 3H-HA, 125I-PINP, and125I-collagen, respectively. n 5 The number of independent experiments.

1282 MCCOURT ET AL. HEPATOLOGY November 1999

180-kd doublet in Fig. 2 is not evident as a doublet in theautoradiographs at 200/<210 kd in Fig. 1 is also probablybecause of the higher percentage gel used, which would notallow sufficient separation of these 2 proteins.

FIG. 8. Inhibition of the endocytosis (A), cell binding (B), and degrada-tion (C) of 3H-HA by LECs at 37°C in the presence of various concentrationsof anti–LEC HA receptor IgG (open bars) or nonimmune IgG (solid bars).Endocytosis is the sum of cell-bound and degraded 3H-HA. Results, given aspercent of control, are the mean of 2 experiments. 100% corresponds to 25%,16%, and 9% of added radioactivity for endocytosed, cell-associated, anddegraded 3H-HA, respectively. Variation was less than 15%.

FIG. 9. Inhibition of the endocytosis (A), cell binding (B), and degrada-tion (C) of 125I-FSA, 125I-PINP, and 125I-collagen by LECs at 37°C in thepresence of anti–LEC HA receptor IgG (open bars) or nonimmune IgG (solidbars). IgG concentration in the 125I-FSA and 125I-PINP experiments was 0.2mg/mL; in 125I-collagen experiments the concentration was 0.5 mg/mL.Endocytosis is the sum of cell-bound and degraded ligand. Results, given aspercent of control, are the mean of 3 experiments (125I-FSA and 125I-PINP;standard deviation indicated) or 2 experiments (125I-collagen; variation , 10%). For the (i) 125I-FSA, (ii) 125I-PINP, and (iii) 125I-collagen data, 100%corresponds to (i) 26%, 9%, and 17%; (ii) 38%, 18%, and 20%; and (iii) 46%,32%, and 14% of added radioactivity for total endocytosed, cell associated,and degraded ligand, respectively.

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An alternative method for the purification of LEC HABPshas been reported by Yannariello-Brown et al.,23 using Sepha-cryl-400 gel filtration of a detergent (CHAPS) solubilizedmixture of LECs and Kupffer cell membranes, and monitor-ing HA binding activity of eluted fractions using 125I-HA in adot-blot assay. These investigators proposed that a 175-kdband, enriched in the peak HA binding fractions, is theligand-binding subunit of the LEC HA receptor. A questionthat arises is that major bands at both 300 and 175 kd wereshown by these investigators in ligand blots of LEC mem-branes, yet only minor amounts of an approximately 300-kdband (relative to the 175-kd band) were enriched in the aboveprocedure. It is possible that the Coomassie-stained 175-kdband may be an unrelated protein, with smaller amounts ofthe bona fide receptor migrating at the same or similar Mr. Inthis case, the 125I-HA ligand-blotting of these fractions wouldnot determine if such a contaminant was present. Thefunction of this 175-kd protein remains to be determined, aswell as its relationship to our LEC HABP or any other protein.

Amino acid sequencing of our approximately 270-kd LECHABP showed that it is a novel protein, unrelated to anysequenced HABPs. Polyclonal antibodies raised against thisprotein recognized both the upper and lower bands onimmunoblots, suggesting that the 2 proteins are related. Thispattern differed to that of immunoblots probed with the 2F8anti-mouse macrophage SR antibody, indicating that the LECHABP is not related to this SR, which is also expressed onLECs.39 The anti–LEC HABP antibody specifically stainscultured LECs, but not cultured hepatocytes, and stains onlythe liver sinusoids, indicating that the purification protocol iscapable of purifying sinusoidal LEC-specific proteins fromwhole liver. Furthermore, monensin arrested the antibody-stained protein at the level of early endosomes. This suggeststhat the HABP is involved in the early events of endocytosis,as is expected for an HA receptor on LECs.

The HABP antibody was also capable of inhibiting LECsfrom binding (at 4°C and 37°C) and degrading (at 37°C)metabolically 3H-labeled HA (3.85 3 106 d). It was noted thatthe nonimmune IgG could inhibit the binding of 3H-HA byup to 18% at 4°C, but had no effect on endocytosis. Fabfragments of rabbit IgG have been shown to interact nonspe-cifically with HA at 4°C40; such an interaction may be ofimportance at these lower temperatures when the receptorwould have a reduced affinity for its ligand. Other investiga-tors have in fact reported that nonimmune Fab fragments(0.4 mg/mL) could inhibit the binding of 3H-HA to LECs by60% at 6°C.38

The antibody did not inhibit denatured collagen binding/endocytosis by LECs, indicating that the antibody is not toxicand that the antibody inhibition of HA binding is probablynot caused by steric hindrance effected by the binding of theantibody to an irrelevant cell surface protein. However, theantibody effectively inhibits the endocytosis and degradationof 2 SR ligands, namely 125I-FSA and 125I-PINP. This finding isconsistent with those of Eskild et al.,19 who showed that FSA,a ligand for LEC-SR, blocks the uptake of CS by the LEC HAreceptor. Furthermore, we have shown a specific interactionof the HABP with an affinity matrix coupled with PINP, an SRligand. It was noted that, although the antibody inhibitedPINP binding to LECs at 4°C and markedly reduced degrada-tion of this ligand at 37°C, it did not decrease the amount ofcell-associated PINP at 37°C. A possible explanation is that at37°C an additional PINP-binding activity is present on the

surface of LECs, which is not involved in endocytosis. Thatthe antibody does not inhibit the binding of a third SR ligand,namely 125I-nidogen, may be a reflection of the differentaffinities the LEC SR(s) have for these 3 ligands, or that thereare other receptors, not recognized by the antibody, that canbind SR ligands. Further inhibition studies with other SRligands are currently underway.

The term ‘‘scavenger receptor’’ is used to describe agrowing family of structurally unrelated proteins that have acommon affinity for negatively charged ligands and modifiedproteins such as oxidized and acetylated low-density lipopro-teins (LDLs).41 In the liver, a number of SRs are known to beexpressed, such as SR-A,39 SR-B1,42 CD36,43 LOX-1,44 andCD68/macrosialin.45 In addition, the LEC HABP should nowbe included in this group; partial amino acid sequences fromthis protein do not match any previously described SRs.SR-As are expressed at low, but detectable, levels on LECs.39

However, cultured LECs from SR-A knockout mice arecapable of binding and degrading modified LDLs, albeit at30% to 50% of wild type levels. Furthermore, the liver uptakeand clearance of modified LDLs from the blood do not differbetween wild type and SR-A knock-out mice,46,47 suggestingthe presence of alternative pathways/receptors that can com-pensate fully for the loss of SR-A. It is possible that the LECHABPs reported here might be partly responsible for theclearance of SR ligands in SR-A knock-out mice.46,47

Uptake of a fourth ligand, mannan, was also inhibited by20%, suggesting that the LEC HABPs also have some affinityfor this polysaccharide, which is cleared predominantly bythe mannose receptor on LECs.48 Mannan is produced on thesurface of certain gram-positive bacteria and yeast, suggestingthat the LEC HABPs may play a role in the clearance ofmicrobes or their lysis products from the blood.

In conclusion, the results presented in this report showthat a novel cell-surface endocytic protein on LECs withaffinity for HA is a likely candidate for an LEC HA receptor.The same protein has affinity for a number of SR ligands,suggesting that it has a function in clearing these moleculesfrom the blood as well. Collectively, the described (andyet-to-be-described) LEC-SRs have an affinity for modifiedLDLs,49,50 which play a role in the pathology of atherosclero-sis,51 and advanced glycation end products–modified pro-teins,52 which have been implicated in the pathology ofAlzheimer’s disease,53 diabetic complications,54 atherosclero-sis,55,56 and hemodialysis-related amyloidosis.57 The clear-ance of these ligands from the circulation by LECs is probablyan essential process for the maintenance of health. It istherefore of interest to examine further the relationship ofLEC-SRs to the LEC HA receptor, the latter receptor beingresponsible for the clearance of large amounts of HA (about10-100 mg/d in humans58) from the bloodstream. Thesestudies will help to elucidate the full SR profile present on thesurface of LECs, an important part of the hepatic reticuloen-dothelial system.

Acknowledgment: The authors acknowledge the expertamino-acid sequencing services of Dr. Bo Ek (Department ofPlant Biology, Swedish Agricultural University, Uppsala, Swe-den) and the technical help of Kjetil Elvevold, Kajsa Lilja, andAnne-Marie Gustafson. The authors are grateful for theinvaluable advice of Professors Torvard Laurent, J. RobertFraser, Ulf Lindahl, and Ulf Eriksson and Dr. Baldur Svein-bjørnsson.

1284 MCCOURT ET AL. HEPATOLOGY November 1999

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