A Blocking Antibody to the Hyaluronan Receptor for Endocytosis (HARE) Inhibits Hyaluronan Clearance by Perfused Liver*

Janet A. WeigelDagger , Robert C. RaymondDagger , Carl McGary§, Anil SinghDagger , and Paul H. WeigelDagger ||

From the Dagger  Department of Biochemistry and Molecular Biology and the Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190 and the § Department of Pathology, University of Rochester School of Medicine, Rochester, New York 14642

Received for publication, November 11, 2002, and in revised form, December 19, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hyaluronan (HA) and chondroitin sulfate clearance from lymph and blood is mediated by the hyaluronan receptor for endocytosis (HARE). The purification and molecular cloning (Zhou, B., Weigel, J. A., Saxena, A., and Weigel, P. H. (2002) Mol. Biol. Cell 13, 2853-2868) of this cell surface receptor were finally achieved after we developed monoclonal antibodies (mAbs) against HARE. There are actually two independent isoreceptors for HA, which in rat are designated the 175-kDa HARE and 300-kDa HARE. Only one mAb (number 174) effectively and completely blocked the specific uptake of 125I-HA at 37 °C by rat liver sinusoidal endothelial cells. 125I-HA binding to both the 175-kDa and 300-kDa HARE proteins in a ligand blot assay was almost completely inhibited by <1 µg/ml mAb-174, whereas mouse IgG had little or no effect. MAb-174 also performed very well in Western analysis, indirect fluorescence microscopy, and a variety of immuno-procedures. Immunohistochemistry using mAb-174 localized HARE to the sinusoidal cells of rat liver, spleen, and lymph node. Western analysis using mAb-174 revealed that the sizes of both HARE glycoproteins were the same in these three tissues. 125I-HA was taken up and degraded by excised rat livers that were continuously perfused ex vivo with a recirculating medium. This HA clearance and metabolism by liver, which is a physiological function of HARE, was very effectively blocked by mAb-174 but not by mouse IgG. The results indicate that mAb-174 will be a useful tool to study the functions of HARE and the physiological significance of HA clearance.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

After Meyer and Palmer (1) discovered hyaluronan (HA),1 it was found to be a component of essentially all vertebrate extracellular matrices (ECMs). Fibroblasts, keratinocytes, chondrocytes, and other cells continuously synthesize and secrete HA, which is a linear polymer with a native molecular mass that may exceed 107 Da and is composed of the repeating disaccharide 2-deoxy,2-acetamido-D-glucopyranosyl-beta (1, 4)-D-glucuronopyranosyl-beta (1, 3). Despite its simple structure, HA is involved in many cell functions including migration, differentiation, and phagocytosis (2-6). HA is important in development (4, 7), wound healing (8, 9), angiogenesis (10, 11), and tumor growth and metastasis (12, 13). Although previously believed to be only a structural component in the ECM, HA is now also recognized as an active cell-signaling molecule. Some cell types show distinct physiological responses to HA of different sizes. In particular, some cell types respond physiologically to very small, but not large, HA. Small HA oligosaccharides containing 14-20 sugars stimulate angiogenesis by endothelial cells (10, 11, 14), induce gene expression in activated macrophages (15), and induce NO synthase expression in sinusoidal LECs and Kupffer cells, but not hepatocytes or stellate cells (16).

Cell surface HA receptors identified to date include CD44, RHAMM (CD168), ICAM-1 (CD54), LYVE-1 (5), and an endocytic receptor that is specific for HA and chondroitin sulfate. This latter HA receptor is expressed in LECs, which remove HA and chondroitin sulfate from the blood (2, 17-20). Because this endocytic receptor is also in other tissues and is not a liver-specific HA receptor, it was renamed HARE, the HA receptor for endocytosis (21). Unlike the other cell surface receptors for HA, HARE mediates the rapid endocytosis of HA and chondroitin sulfate via the clathrin-coated pit pathway (19, 22). HARE is, therefore, similar in its mode of action to the transferrin, asialoglycoprotein, mannose, and low density lipoprotein receptors (23).

After the discovery that liver is responsible for HA clearance from the blood (reviewed in Ref. 2), Deaciuc et al. (24, 25) demonstrated the ability of isolated perfused rat liver to take up HA. This uptake process was saturable at an HA concentration of ~0.15 µg/ml with a steady-state uptake rate of ~10 µg HA/h/g wet weight of liver. Deaciuc et al. (24, 25) also showed that galactosamine-induced hepatitis in rats was associated with elevated plasma HA levels and decreased HA clearance ability by perfused liver. These and similar observations by other investigators led to the realization that the HA clearance ability of liver can indicate the functional status of LECs and, thus, the general health of the liver. For example, Itasaka et al. (26) and Rao et al. (27) found that the HA clearance function was a useful parameter for predicting the likely success of human liver transplantation.

We previously identified two large membrane proteins of ~175 kDa and ~300 kDa in rat LECs that were specifically labeled with a photoaffinity derivative of HA (28) and retained specific HA binding activity in a novel ligand blot assay following SDS-PAGE and renaturation (29). The development of specific mAbs raised against the 175-kDa protein enabled these two HARE proteins to be purified from isolated rat LECs (30) and more recently from human spleen (31). After cloning the cDNA for the rat 175HARE, we stably expressed the recombinant protein in SK-Hep-1 cells and found that this smaller HARE species can function as an endocytic recycling receptor with specificity for HA and chondroitin sulfate. The native rat 175HARE is derived by proteolysis from a larger precursor protein (32), probably the largest subunit of the 300HARE. The evidence so far indicates that the 175- and 300-kDa proteins are closely related but functionally independent HARE species.

We noted previously that one of the mAbs generated to the rat HARE, designated mAb-174, inhibits 125I-HA endocytosis by isolated rat LECs and inhibits 125I-HA binding to both HARE species in a ligand blot assay (21). This Ab was also able to immunoprecipitate HARE from isolated LECs. Here we report that mAb-174 also recognizes both HARE species from whole spleen, lymph node, and liver in Western analysis and performs well in immunolocalization and confocal microscopy procedures. More notably, mAb-174 also specifically blocked the uptake and degradation of 125I-HA by isolated perfused liver. This antibody should, therefore, be a valuable reagent in future studies of HARE function.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Media-- 125I-HA was prepared as described (33) using a hexylamine derivative of HA (oligosaccharides of Mr ~70,000). Male Sprague-Dawley rats (200 g) were from Charles River Labs. BSA fraction V was from Intergen. Hanks' balanced salt solution and PBS were formulated according to the Invitrogen catalog formulations. Medium 1 was Eagle's basal medium (Invitrogen no. 41500-018) supplemented with 100 mg/liter succinic acid sodium salt, 75 mg/liter succinic acid, 2.4 g/liter HEPES, and 0.22g/liter NaHCO3. Medium 1/BSA was Medium 1 supplemented with 0.1% BSA (w/v). Collagenase was from Roche Molecular Biochemicals. The preparation of mouse mAbs against the rat HARE was described (21). Tris, SDS, ammonium persulfate, N,N'-methylenebisacrylamide, and SDS-PAGE standards were from Bio-Rad. Rat lymph nodes were a special purchase from Pel-Freez Biologicals. Unless noted otherwise, other chemicals and reagents were from Sigma.

Isolation and Culture of LECs-- Rat livers were perfused with collagenase using a modification of the procedure developed by Seglen (34) as described previously (35). LECs were isolated using differential centrifugation and discontinuous Percoll gradients (36). LECs banding at the 25/50% interface were removed, washed twice with RPMI 1640 (Invitrogen) containing penicillin/streptomycin (100 units each) and 2 mM glutamine and suspended at 1.5-2 × 106 cells/ml. The cells were incubated first on a glass Petri dish for 10 min at room temperature to remove Kupffer cells and then plated on human fibronectin-coated (50 µg/ml) 24-well tissue culture plates for endocytosis experiments or on glass coverslips for microscopy. After incubation at 37 °C for 2 h in a 5% CO2 atmosphere, the cells were washed three times with PBS, once with RPMI 1640, and then incubated in RPMI 1640 without serum at 37 °C for 1 h if they were to be used immediately or with 2% heat-inactivated bovine serum if they were to be cultured overnight before use. The 37 °C pretreatment was performed in all experiments to allow any endogenous cell surface-bound HA, e.g. from the serum, to be removed either by internalization or dissociation and washing.

Clearance of 125I-HA by Perfused Liver-- Rat livers were excised following a standard perfusion protocol (35). During excision and mounting in a recirculation apparatus, they were perfused without recirculation with Buffer 1 (142 mM NaCl, 6.7 mM KCl, and 10 mM HEPES, pH 7.4) for 8-10 min at 35 °C. Washed livers were then pre-perfused for 15 min with recirculation with a recirculation medium (Dulbecco's modified Eagle's medium, Invitrogen catalog no. 41100, without serum but with 60 mM HEPES, pH 7.4, 0.1% (w/v) BSA, and 50 µg/ml goat IgG, Sigma catalog no. I-5256). Then the perfusate was switched to 60 ml of fresh recirculation medium containing 125I-HA (0.25 µg/ml in all experiments) and other additions as noted, and the liver was allowed to take up HA for up to 60 min at 35 °C. Samples (300 µl) of the recirculating perfusate were removed and divided into 50-µl portions for determination of total radioactivity (in duplicate) or HA degradation products (in triplicate). Competitor HA (50 µg/ml) or mouse IgG or mAb-174 IgG (5 µg/ml) was added to the recirculation medium containing the 125I-HA and mixed well before the recirculating perfusion was begun. The first sample taken immediately after starting the perfusion was used to determine the starting values, because there was a substantial (~15%) and reproducible dilution of the recirculating medium by the residual buffer in the liver. In experiments with purified IgGs, these were also included during the 35 °C pre-perfusion treatment at the same concentration used in the experiment.

Degradation of 125I-HA by Perfused Liver-- Degradation of 125I-HA was measured by a cetylpyridinium chloride precipitation assay as described previously (20). Samples (50 µl in triplicate) of recirculation medium containing 125I-HA were added to 250 µl of 1 mg/ml HA in 1.5-ml microfuge tubes. After mixing, 300 µl of 6% (w/v) cetylpyridinium chloride in distilled water was added, and the tubes were mixed by vortexing. After 10 min at room temperature, the samples were centrifuged at 9000 rpm in an Eppendorf model 5417 microcentrifuge at room temperature for 5 min. A sample (300 µl) of the supernatant was taken for determination of radioactivity, and the remainder was removed by aspiration. The tip of the tube containing the precipitate pellet was cut off, put in a gamma  counter tube, and radioactivity was determined. Degradation was measured as the time-dependent increase of non-precipitable radioactivity. >80% of the total radioactivity was precipitable at the beginning of the experiment.

Immunocytochemistry-- Lymph node, liver, and spleen tissues from Sprague-Dawley rats were removed, fixed in 10% neutral buffered formalin, processed, and paraffin embedded overnight on a Tissue Tek V.I.P. processor. Tissue sections (5 µm) were collected on charged slides and dried at 60 °C overnight. The slides were dewaxed three times for 3 min each with xylene followed by four washes for 3 min each with alcohol (100%, 95%, 90%, then 70%) and a single 2-min wash in water at room temperature. The endogenous peroxidase activity was quenched by treatment with 3% hydrogen peroxide for 6 min followed by two 2-min water washes. The tissue sections on the slides were digested for 15 min at 37 °C in pre-warmed 0.1 N HCl containing 0.32 mg/ml pepsin followed by a 2-min water wash and a 2-min PBS wash. The slides were washed with PBS and incubated with the appropriate primary antibody (~1:500) at room temperature for 60 min. After a 1 min PBS wash, the slides were treated with biotinylated horse anti-mouse IgG (1:200) for 30 min at room temperature. After another PBS rinse, the slides were incubated with streptavidin-horseradish peroxidase (1:1000, Jackson Labs) for 30 min, washed once with PBS, and once with distilled water. Color development was for 5 min with 2.0% (v/v) aminoethylcarbazine and hydrogen peroxide (ScyTek Laboratories, Logan Utah) followed by counterstaining with hematoxylin. Slides were viewed with an Olympus BX-40 light microscope equipped with an Olympus DP10 digital camera.

General-- Protein content was determined by the method of Bradford (37) using BSA as standard. Radioiodine was determined using a Packard 5002 gamma  counting system. SDS-PAGE was performed according to the method of Laemmli (38). Western blotting procedures were performed essentially as described by Burnette (39) with minor modifications (21).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To purify and characterize the rat HARE proteins, we developed a panel of eight mAbs raised against the partially purified 175HARE (21, 30). Surprisingly, all of these mAbs also recognized the 300HARE in either Western or immuno-purification assays. We now know that this is because the single 175HARE species and probably the 230-kDa and 250-kDa subunits of the 300HARE are derived by proteolysis from a larger precursor (32). Fig. 1 shows the concentration-dependent blocking of specific uptake of 125I-HA by LECs at 37 °C. Specific internalization was completely inhibited by 5 µg/ml mAb-174, whereas up to 10 µg/ml of three other anti-HARE mAbs inhibited only ~15%. Interestingly, mAb-235 reproducibly inhibited 125I-HA endocytosis by LECs by about 50%. No further inhibition by the other six mAbs (only numbers 28, 30, and 467 are shown) or mouse IgG (not shown) occurred regardless of IgG concentration or duration of treatment.


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Fig. 1.   MAb-174 inhibits 125I-HA uptake by LECs at 37 °C. LECs were plated at 1.9 × 106 cells per well, washed, and pretreated at 37 °C as described under "Experimental Procedures." The cells were washed twice with medium, and then medium containing 1 µg/ml 125I-HA and the indicated amount (up to 10 µg/ml) of mAb-28 (black-triangle), mAb-30 (black-square), mAb-174 (black-down-triangle ), mAb-235 (black-diamond ), or mAb-467 () was added. The dishes were incubated at 37 °C for 60 min, the medium was then aspirated, and the cells were washed three times with PBS. The cells were solubilized in 0.3 N NaOH, and radioactivity and protein content were measured. The values shown are the average of duplicates (which differed by <5%). The control value, with no IgG additions, was set to 100%.

Previously, we reported that mAb-174 blocks 125I-HA binding to both HARE species (21) in a ligand blot assay that detects the activity of these proteins after SDS-PAGE, electrotransfer, and renaturation (29). In dose-response experiments, <1 µg/ml, mAb-174 blocked 125I-HA binding to both the affinity-purified 175HARE and 300HARE proteins, but control IgG up to 10 µg/ml had no effect (not shown). Inhibition of binding to the 175HARE was almost complete; ~88% of the specific binding was blocked at 2-10 µg/ml mAb-174. Although the effect of mAb-174 on 125I-HA binding to the 300HARE was identical to that of the 175HARE below 1 µg/ml, inhibition leveled off at ~50% between 1-10 µg/ml.

Despite the ability of mAb-174 to inhibit HA uptake by LECs at 37 °C or HA binding in the ligand blot assay at room temperature, we unexpectedly found that mAb-174 did not block 125I-HA binding to LECs at 4 °C (Fig. 2). To test the possibility that this inability to inhibit HA binding was due to an inherent difference in HARE between 37 °C and 4 °C (e.g. a conformation change), we fixed LECs so that endocytosis could not occur when the cells were subsequently put at 37 °C. This enabled us to determine whether mAb-174 could block 125I-HA binding per se at 37 °C; this cannot be assessed if endocytosis occurs simultaneously. The results confirmed that mAb-174, but none of the other HARE-specific mAbs tested, could block 125I-HA binding to fixed LECs at 37 °C. We conclude that HARE likely undergoes a conformation change between 37 °C and 4 °C that does not prevent 125I-HA binding to LECs but that alters the epitope recognized by mAb-174 so that it does not block HA binding.


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Fig. 2.   MAb-174 does not inhibit 125I-HA binding to LECs at 4 °C. After plating in 24-well tissue culture plates, LECs were fixed in 1% formaldehyde for 20 min at room temperature. The cells were washed, 5 µg/ml of the indicated IgG in Medium 1/BSA was added to each well, and the plates were put at 37 °C for 45 min. Unfixed cells were incubated in parallel at 4 °C with 5 µg/ml of mAb-174 or mouse IgG. 125I-HA was added at a final concentration of 2 µg/ml to each well, and the plates were incubated at the same temperatures for 30 min. The medium was removed by aspiration, and the wells were washed three times with Medium 1. The cells were then solubilized in 0.3 N NaOH, and radioactivity and protein content were determined. Specific 125I-HA, assessed with a 100-fold excess of unlabeled HA, was 47% in the fixed cells at 37 °C and 55% in the unfixed cells at 4 °C. Results are the mean of triplicates ± standard error. The control value, with no IgG additions, was set to 100%.

We found previously that mAb-174 recognizes the 175HARE and 300HARE proteins in Western analysis of crude LEC membranes (21). Because we had not tested any of our mAbs individually using other tissues, we compared the Western blot reactivity of mAb-174 with the two HARE species from rat lymph node, spleen, and liver. In all three tissues, the HARE proteins were visualized by mAb-174 (Fig. 3). Expression of the 175HARE in lymph node was lower than in the other tissues, although it was detectable (not shown). In addition to its utility as a ligand-blocking antibody and for Western analysis, mAb-174 also worked well in a variety of localization procedures, including indirect fluorescence microscopy (e.g. the same localization pattern was observed previously with a mixture of mAbs; Ref. 21) and immunohistochemistry (Fig. 4). Immunohistochemical localization of HARE using mAb-174 revealed heavy staining of the sinusoidal endothelial cells in liver, spleen, and lymph node (Fig. 4). The medullary sinuses of lymph node and the venous sinusoids of spleen contain large amounts of HARE protein.


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Fig. 3.   MAb-174 recognizes both HARE proteins in Western analysis of rat liver, spleen, and lymph nodes. Tissues were minced in the presence of protease inhibitors and homogenized, and crude membrane pellets were extracted with 2% Nonidet P-40. The extracts were clarified by centrifugation at 14,000 rpm for 10 min, diluted to 0.5% Nonidet P-40, and subjected to affinity chromatography using mAb-30 coupled to CNBr-activated Sepharose (30). The spleen (lanes 2), liver (lanes 3), and lymph node (lanes 4) HARE proteins were eluted with Laemmli sample buffer (38), subjected to SDS-PAGE, and electrotransferred to nitrocellulose. Lanes 1 contain isolated LECs lysed in Laemmli sample buffer. After blocking nonspecific binding sites by treatment with 1% BSA and 10% goat serum in Tris-buffered saline for 4 h at 4 °C, the nitrocellulose membrane was incubated with 1 µg/ml mAb-174 (Panel A) or mouse IgG (Panel B) for 1 h at room temperature. The membranes were then washed in TBST (Tris-buffered saline with 0.05% Tween 20 and 0.05% NaN3.), incubated with 0.8 µg/ml goat anti-mouse IgG conjugated to alkaline phosphatase for 1 h at room temperature, washed, and developed with the BioRad Colorimetric AP detection system. The closed arrows mark the 175 and 300 HARE proteins. The open arrows mark the IgG bands of mAb-30 eluted from the affinity column by the SDS sample buffer.


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Fig. 4.   Immuno-localization of HARE in rat liver, spleen and lymph node. Sections of rat spleen, liver, or lymph node were processed and incubated with mAb-174 ascites fluid (left column) or normal mouse serum (right column) as described under "Experimental Procedures." The bars represent 50 µm.

The fact that mAb-174 effectively inhibits HA binding and uptake by isolated LECs at 37 °C does not necessarily mean that the antibody would be equally able to inhibit HA clearance by these cells in intact liver. To answer this question, we tested the ability of mAb-174 to block 125I-HA uptake and degradation by excised rat livers that were continuously perfused ex vivo. In these experiments we used a concentration of mAb-174 (5 µg/ml) that gave essentially complete inhibition of uptake by LECs. 125I-HA introduced into the perfusion recirculating medium was readily removed; ~50% was cleared within ~20 min (Fig. 5). In the presence of excess unlabeled HA, the removal of 125I-HA was completely blocked. Although control mouse IgG appeared to retard slightly the clearance of 125I-HA, there was no statistically significant difference between most of the data pairs. In contrast, when mAb-174 was present there were very significant differences in 125I-HA removal at all times during the perfusion, and the level of residual uptake was just slightly greater than that in the presence of excess unlabeled HA. The results confirm that mAb-174 effectively blocks 125I-HA uptake by LECs in intact perfused liver.


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Fig. 5.   MAb-174 inhibits 125I-HA uptake by perfused rat liver. Rat livers were perfused ex vivo with recirculation medium containing 0.25 µg/ml 125I-HA with no additions (black-triangle), 5 µg/ml mouse IgG (black-square), 5 µg/ml mAb-174 (), or 50 µg/ml unlabeled HA (black-down-triangle ) as described under "Experimental Procedures." Each point is the mean ± S.D. of duplicates from 3-4 perfused livers (n = 6-8). The values are calculated as the percent of intact 125I-HA remaining in the medium relative to the starting value.

This conclusion was further confirmed by demonstrating the effect of mAb-174 on the appearance of 125I-HA degradation products in the perfusion medium (Fig. 6). 125I-HA removal and processing by perfused liver was so efficient that degradation products were detected within minutes. By 30 min, about 25% of the total 125I-HA in the perfusate had been internalized, degraded, and released back into the medium. Again, no significant differences were seen in the appearance of degradation products when mouse IgG was present, although all of the values were slightly lower. In contrast, mAb-174 substantially reduced the steady-state rate at which 125I-HA degradation products were released. The inhibition by unlabeled HA was essentially complete, indicating that >95% of the observed degradation was mediated by a specific mechanism.


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Fig. 6.   MAb-174 inhibits 125I-HA degradation by perfused rat liver. Rat livers were perfused ex vivo with medium containing 0.25 µg/ml 125I-HA with no additions (black-triangle), 5 µg/ml mouse IgG (black-square), 5 µg/ml mAb-174 (), or 50 µg/ml unlabeled HA (black-down-triangle ) as described under "Experimental Procedures." Each point is the mean ± S.D. of triplicates from 3-4 perfused livers (n = 9-12). The values are calculated as the percent of the initial intact 125I-HA at the beginning of the perfusion.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HA is used in many cosmetic and clinical applications, and new HA-containing materials are currently being developed for medical use. For example, HA is used in ophthalmological surgery (40), to treat patients with osteoarthritis in knee and hip joints (41), to prevent adhesions after surgery (42), and as an aerosol to prevent elastase-mediated injury in pulmonary emphysema (43). Because of the wide range of medical uses for HA, it is very important to understand how HA turnover and clearance are regulated. The present study was undertaken as an initial effort to define the role of HARE in this process.

After LECs were found to contain an endocytic HA receptor (23, 44), others demonstrated that isolated perfused rat liver can remove circulating HA (24, 25, 45), presumably mediated by this HA receptor. The present results verify that this assumption was correct. The quantitative inhibition of 125I-HA uptake and degradation by mAb-174 demonstrates that HARE mediates at least 90% of the liver's clearance ability. In this study, we did not perform an extensive dose response analysis because of the larger number of animals and amount of purified IgG required. Accordingly, we did not necessarily use an optimum dose of mAb-174 for blocking HARE in perfused liver, although the mAb-174 concentration used, 5 µg/ml, gave maximum inhibition of 125I-HA internalization by LECs (Fig. 1).

Using a mixture of anti-HARE mAbs, we found that the small and large rat HARE proteins are highly expressed in the sinusoids of liver, the venous sinuses of the red pulp in spleen, and the medullary sinuses in lymph nodes (21). This distribution was also seen using only mAb-174 (Fig. 4). HARE was not detectable by Western or immuno-cytochemical analysis in brain, lung, heart, muscle, kidney, or intestine. Abundant expression of HARE in the sinusoids of liver and lymphatic tissues is ideal for keeping the systemic HA levels low. Banerji et al. (46) discovered a lymph-specific homologue of CD44, designated LYVE-1, which binds HA and is localized to the luminal face of vessel walls in the lymphatic system. It is not present on blood vessels. Preliminary studies indicate that LYVE-1 and HARE have different and non-overlapping distributions in lymph node and spleen and are not co-localized even within isolated rat LECs.

The rat 175HARE is a 1431-amino acid (156,393 Da) type I membrane protein with a large NH2-terminal extracellular domain (~1323 amino acids), one transmembrane domain (~20 amino acids), and a small COOH-terminal cytoplasmic domain (~88 amino acids). The native protein contains ~25 kDa of N-linked oligosaccharides (30). The recombinant rat 175HARE protein, expressed in SK-Hep-1 cells, is a functional HA receptor able to mediate the specific and continuous endocytosis of 125I-HA through the clathrin coated pit pathway (32). Because the 175HARE is functional in the absence of the 300HARE complex, it is not necessary for these two HARE species to be present in the same cell in order to create a specific, endocytically competent HA receptor. Therefore, the 175HARE and 300HARE are independent isoreceptors for HA. Our present results show that mAb-174 blocks HA binding and endocytosis by both HARE species in intact liver.

A 70-kg person contains about 15 g of HA and turns over about 5 g, or one-third of their total HA, per day (47). About 50% of the total HA is in skin and has a metabolic half-life of <1.5 days (48). Lymph nodes and liver are the major clearance sites for the systemic removal and degradation of HA (2). HA injected intravenously in mammals is rapidly removed from the blood by liver (2, 45, 47), whereas HA introduced into lymph is degraded first by lymph nodes, and the remainder is then removed by the liver. The present model of mammalian HA turnover is that large native HA (~107 Da) in ECMs throughout the body is partially degraded to fragments of ~106 Da that are released into lymphatic vessels and then flow to lymph nodes where ~85% of the total HA is removed and degraded. The remaining HA (~15%) that exits the lymph nodes is ~105 Da and, after entering the blood, is removed by the LECs of liver.

Despite the generation of up to 5 g of HA degradation products per day, the HA clearance systems utilizing HARE in lymph nodes and liver keep the normal steady-state HA concentration in blood very low (i.e. 10-100 ng/ml). Clearance of circulating HA from blood is important for normal health, because blood viscosity could increase to levels that might impair the microcirculation if the concentration of large HA (i.e. >106 Da) increased. Blood HA levels could rise due either to greater HA production or turnover in the body, exceeding the capacity of LECs to remove it, or to a compromised function of HARE in lymph nodes or liver. Elevated blood HA levels are found in several diseases, although in most cases the cause of this elevation is not known. Elevated serum HA is now used as a diagnostic indicator of liver failure. For example, elevated serum HA is a marker for liver fibrosis in hepatitis C virus-associated chronic liver disease (49). Increased ascitic levels of HA occur in liver cirrhosis due to increased HA synthesis by peritoneal cells and decreased uptake by LECs (50). Elevated blood HA levels have also been reported in a variety of other diseases such as rheumatoid arthritis (51), psoriasis (52), and in some cancers (53). Blood HA levels can also predict the functionality of LECs and the likely success of liver transplants (26, 27).

In summary, mAb-174 specifically inhibits the HA-binding and HA-clearance activities of both the small and large HARE proteins in isolated rat LECs and in whole perfused liver. This effective activity-blocking antibody is particularly versatile, because it is also functional in Western analysis and a variety of other immuno-procedures and should be useful in future studies to understand the physiological functions of HARE.

    FOOTNOTES

* This research was supported by NIGMS, National Institutes of Health Grant GM35978.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Pathology, St. Joseph's Hospital, 69 Exchange St., St. Paul, MN 55102.

|| To whom correspondence should be addressed. Tel.: 405-271-1288; Fax: 405-271-3092; E-mail: paul-weigel@ouhsc.edu.

Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M211462200

    ABBREVIATIONS

The abbreviations used are: HA, hyaluronic acid, hyaluronate, or hyaluronan; BSA, bovine serum albumin; ECM, extracellular matrix; HARE, HA receptor for endocytosis; 175HARE, 175-kDa HARE; 300HARE, 300-kDa HARE; ICAM-1, intercellular adhesion molecule-1 (also designated CD54); LEC, sinusoidal liver endothelial cell; LYVE, lymphatic vessel endothelial HA receptor; mAb, monoclonal antibody; PBS, phosphate buffered saline; RHAMM, receptor for HA-mediated motility.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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