Liver sinusoidal endothelial cells depend on mannose receptor-mediated recruitment of lysosomal enzymes for normal degradation capacity

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Liver sinusoidal endothelial cells (LSECs) are largely responsible for the removal of circulating lysosomal enzymes (LE) via mannose receptor (MR)-mediated endocytosis. We hypothesized that LSECs rely on this uptake to maintain their extraordinarily high degradation capacity for other endocytosed material. Circulatory half-life studies of 125I-cathepsin-D in MR knockout (MR−/−) and wild-type mice, and endocytosis studies in LSEC cultures, showed a total dependence on the MR for effective clearance of cathepsin-D. Radioiodinated formaldehyde-treated serum albumin, a ligand for the LSEC scavenger receptors, was used to study catabolism of endocytosed material in MR−/− and wild-type mice. The plasma clearance, liver uptake, and the starting point for release of degradation products to blood, were similar in both experimental groups, indicating normal endocytosis and intracellular transport of scavenger receptor ligands in MR−/− mice. However, the rate of formaldehyde-treated serum albumin catabolism in the liver of the MR deficient animals was reduced to approximately 50% of wild-type values. A similar reduction in intracellular degradation was recorded in LSEC cultures from MR−/− mice compared to wild-type controls. In accordance with this, MR−/− LSECs had markedly and significantly reduced enzyme activities for four out of five LE tested, i.e., cathepsin-D, α-mannosidase, β-hexosaminidase and arylsulfatase, but not acid phosphatase, compared to wild-type controls. Immunoblot analysis showed that the content of pro-cathepsin-D relative to total cathepsin-D in wild-type LSECs was less than one-fifth of that in hepatocytes, indicating lower endogenous LE production in the LSECs. Conclusion: We show for the first time that LSEC depend on MR-mediated recruitment of LE from their surroundings for effective catabolism of endocytosed macromolecules. (HEPATOLOGY 2008;48:2007–2015.)

Mammalian liver sinusoidal endothelial cells (LSEC) are specialized scavenger endothelial cells1, 2 that remove soluble macromolecular waste products from tissue turnover via several high affinity endocytic receptors, including the mannose receptor (MR),3, 4 and different scavenger receptors (SRs).5–7

The MR is a C-type lectin with roles in immunity8 and glycoprotein homeostasis.9 In mammals, the receptor is expressed in several cell types, including most tissue macrophages,10 subsets of immature dendritic cells,8 LSECs,3, 4, 11, 12 and lymph node sinusoidal endothelial cells.11 However, MR expression on Kupffer cells (KCs) is still debated: it is absent in human KCs,11 but present in mouse KCs,13 although expression was far higher in mouse LSECs. Low MR expression in KCs compared to LSECs was also reported in rat.4

The MR has several functional domains responsible for its broad ligand-binding specificity. These include eight C-type lectin-like domains (which bind carbohydrates with terminal D-mannose, L-fucose, or N-acetyl-D-glucosamine14), an outer cysteine-rich domain (which binds some sulfated sugars15), and a fibronectin type II repeat (which binds collagens16, 17). Other endogenous ligands for the MR include tissue plasminogen activator,18 the C-terminal propeptide of type I procollagen,19 neutrophil-derived myeloperoxidase,20 and many lysosomal hydrolases.21–23 When these are injected into the circulation of mice and rats, they are efficiently cleared from blood predominantly by LSECs.3, 4, 19, 21, 22, 24, 25

To cope with the daily burden of grams of endocytosed material, LSECs are geared for rapid intracellular transport to degradation organelles,26 high capacity degradation of ligands, and release of degraded low molecular weight material to the surroundings.27 Effective catabolism is dependent on a large pool of active lysosomal enzymes (LE), and it has been shown that LSECs from rat28 and pig12 contain considerably higher LE activities per gram cell protein than KCs and hepatocytes (HCs). Because LSECs also have a major role as scavenger cells for LE via the MR, we hypothesized that this function is an important way for the cell to recruit LE. This is an alternative route of accumulating LE compared to the classical pathway in which newly synthesized enzymes are trapped by the mannose 6-phosphate receptors (MPRs) in the trans-Golgi network, before transport and release into late endosomes.29 However, the issue of LSEC reliance on a steady supply of LE via the MR has been difficult to resolve.

By the use of a MR-knockout mouse model (MR−/−) it was recently shown that these animals have elevated levels of LE in blood as compared to wild-type mice,9 indicating that the MR has an important role in blood glycoprotein clearance and homeostasis. In the present study, using the same knockout model, we set out to investigate the importance of MR-mediated uptake of LE in LSECs for normal catabolism of endocytosed material. We show that LSECs depend on a functional MR to maintain a physiological level of LE necessary for effective degradation of macromolecules.

Abbreviations

cpm, counts per minute; FSA, formaldehyde-treated serum albumin; HC, hepatocyte; KC, Kupffer cell; LSEC, liver sinusoidal endothelial cell; LE, lysosomal enzymes; MPR, mannose 6-phosphate receptor; MR, mannose receptor; MR−/−, mannose receptor knockout; NPC, nonparenchymal cell; PBS, phosphate buffered saline; SD, standard deviation; SR, scavenger receptor; TRITC, tri-rhodamine-B-isothiocyanate.

Materials and Methods

Chemicals and Reagents.

Triton X-100, tri-rhodamine-B-isothiocyanate (TRITC), and bovine spleen cathepsin-D (C3138) were from Sigma Chemicals Co. (St. Louis, MO). Culture medium Roswell Park Memorial Institute 1640, supplemented with 20 mM sodium bicarbonate, 0.006% penicillin, and 0.01% streptomycin was from Gibco BRL (Roskilde, Denmark). Bovine collagen type I (Vitrogen 100) was from Cohesion Technologies (Palo Alto, CA). Formaldehyde-treated serum albumin (FSA) was prepared as described.30 Rabbit anti-mouse cathepsin-D antiserum31 was a kind gift from Professor Regina Pohlmann, Münster University, Germany, and Professor Kurt von Figura, Georg-August University, Göttingen, Germany.

Animals.

Wild-type C57BL/6 mice were from Harlan (France). MR−/− C57BL/6 mice were kindly provided by Professor Michel Nussenzweig, The Rockefeller University, New York, NY. The MR−/− mice were further backcrossed for three generations with wild-type C57BL/6 at the Animal House, University of Tromsø, Norway, before breeding homozygous MR−/− mice for experiments. The MR−/− status was tested by polymerase chain reaction.9 The animals were fed a standard chow (Scanbur BK, Nittedal, Norway) ad libitum. Animals in different experimental groups were age-matched, gender-matched, and weight-matched. Experimental protocols were approved by the Norwegian Animal Research Authority in accordance with The Norwegian Animal Experimental and Scientific Purposes Act, 1986.

Statistics and Pharmacokinetic Analyses

The statistical calculations (Mann-Whitney test and independent samples t test) were performed with the SPSS statistical package for Windows version 14.0 (SPSS Inc. Chicago, IL). Clearance kinetics were analyzed as described.32

Cells.

HCs and LSECs from MR−/− and wild-type mice were isolated as described.33 Primary cultures of HCs and LSECs were established on tissue culture plates (Falcon, Becton Dickinson, Plymouth, UK) precoated with 0.1% collagen type I and maintained in serum-free Roswell Park Memorial Institute 1640 medium at 37°C at 5% CO2. The LSEC and HC cultures were washed 40 minutes and 1 hour after seeding, respectively, after which both cell types were allowed to continue spreading for approximately 1 hour before use. The purified LSEC cultures contained 93%-97% LSECs, and 1%-2% each of HCs, KCs, and stellate cells, from both animal groups. The purity of HC cultures was >90%, with most contaminating cells being LSECs. The plating efficiency of cells isolated from MR−/− and wild-type mice was also identical. Seeding of 3-5 × 105 LSECs/cm2 gave confluent monolayer cultures of approximately 1-1.5 ×105 cells/cm2. To check the viability and responsiveness of cultured LSECs from MR−/− and wild-type mice, 2 hours of endocytosis of 125I-FSA was measured and found to be equal in both groups (see Endocytosis Experiments and Results).

Ligand Labeling Procedures.

FSA and cathepsin-D in phosphate buffered saline (PBS) were labeled with carrier-free Na125I.25 The resulting radioactivities were approximately 2.5 × 106 counts per minute (cpm)/μg 125I-FSA, and 5 × 106 cpm/μg 125I-cathepsin-D. Injected 125I-cathepsin-D was mixed with cold cathepsin-D to give a final concentration of 10 μg protein/100 μL/106 cpm.

Cathepsin-D was conjugated with Alexa-Fluor 488-5-TFP (Invitrogen, Oslo, Norway) according to the manufacturer's protocol, and TRITC-FSA was prepared by incubating FSA and TRITC in Na2CO3 buffer (0.5 mol/L, pH 9.5) at a protein:dye ratio of 4:1 at 4°C overnight, and dialyzed against PBS.

Endocytosis Experiments.

Mouse LSECs (3-5 × 105 cells seeded per cm2) or HCs (5 × 105 cells seeded per cm2) cultures were established in 48-well tissue culture plates. Endocytosis experiments with 125I-labeled ligands were performed under serum-free conditions within 4 hours of plating, as described.25

Primary cultures of mouse nonparenchymal cells (NPCs) (from the LSEC-enriched, KC-enriched, and stellate cell–enriched liver cell suspension after removal of HCs by low-speed differential centrifugation33) were established on 0.1% collagen-coated glass coverslips and incubated for 10 minutes with 10 μg/mL TRITC-FSA to tag LSEC lysosomes only.12 The NPC cultures contained mostly LSECs, and approximately 10%-15% each of KCs and stellate cells. The cells were washed and allowed to process the ligand for 30 minutes, before 10 minutes incubation with 2 μg/mL Alexa-Fluor488-cathepsin-D, and 100 μg/mL unlabeled FSA to block binding to SRs, which is induced by the Alexa-Fluor488-modification of cathepsin-D. The cells were then washed, chased for 30 minutes, fixed in 10% formalin, and subjected to fluorescence microscopy.

Blood Clearance and Organ Distribution of Radiolabeled Ligands.

125I-FSA (2 μg), or 125I-cathepsin-D (10 μg) in 100 μL 0.9% NaCl, were injected into the tail vein of anesthetized MR−/− and wild-type mice. Immediately thereafter blood samples (10 μL) were collected from the tail tip and analyzed for degraded/nondegraded ligand.25

At the end of the experiment, the animals were sacrificed with 100% CO2, and perfused intracardially with PBS to remove free tracer from the vasculature, after which the organs were analyzed for radioactivity.25

Assay of Lysosomal Enzymes.

Cell cultures were solubilized in 0.1% Triton X-100 and stored at −70°C until assayed. Protein, acid phosphatase, α-mannosidase, β-hexosaminidase, and arylsulfatase assays were as described.12 Cathepsin-D was assayed according to the manufacturer's assay kit instructions (Sigma; CS0800).

Western Blot Analysis.

LSEC and HC cultures were solubilized in radioimmunoprecipitation assay buffer with proteinase inhibitors25 and immunoblotted6 with rabbit anti-mouse cathepsin-D antiserum (1:100, 4°C, overnight) and donkey anti-rabbit antibody horseradish peroxidase conjugate (GE Healthcare, Uppsala, Sweden; 1:20000, room temperature, 45 minutes). Immunoreactive protein bands were visualized with Amersham's Electrochemical Luminescence kit (ECL kit; GE Healthcare).

Results

MR−/− Mice Have Markedly Reduced Ability to Clear Cathepsin-D.

Lee et al.9 found elevated blood levels of several LE in MR−/− mice, indicating that the MR is involved in the plasma clearance of LE. Consistent with this observation we found that blood clearance of 125I-cathepsin-D (10 μg) was significantly impaired in MR−/− mice compared to wild types (Fig. 1). Semilogarithmic decay plots (not shown) revealed a biphasic pattern of elimination of 125I-cathepsin-D in wild-type mice only; 63% (standard deviation [SD] = 7.2) of the radioactivity was eliminated during an initial rapid α-phase with a t1/2 of 0.90 minute (SD = 0.11), while the remainder was eliminated with a t1/2 of 8.9 minute (SD = 4.4) during the terminal β-phase. In the MR−/− mice, a distinct initial α-phase of ligand elimination could not be calculated, and the t1/2 of the terminal β-phase was significantly slower (22.0 minutes, SD = 2.3, P < 0.01) than in the wild types.

Figure 1.

Circulatory half-life of cathepsin-D. Wild-type (WT, n = 3) and MR−/− (n = 3) mice were injected intravenously with 10 μg 125I-cathepsin-D, and blood samples taken at the indicated time points were analyzed for 125I-labeled degradation products and intact ligand.25 Radioactivity in blood 0.5 minute after injection was taken as 100%. 125I-labeled degradation products were not detected in blood samples during the monitoring period.

Whole organ analysis of wild-type and MR−/− mice 30 minutes after injection of 125I-cathepsin-D (Fig. 2) showed the following distribution of radioactivity: wild-type: liver, 46%; kidneys, 5%; body, 27%; blood, 15%; MR−/−: liver, 9%; kidneys, 12%; body, 29%; blood, 44%. There was a significant (P < 0.01) reduction of liver uptake, with a corresponding increase in blood levels (P < 0.01), and an (nonsignificant) increased kidney clearance of cathepsin-D in the MR−/− mice compared to the wild types.

Figure 2.

Anatomical distribution of cathepsin-D. The wild-type (WT, shaded bars, n = 3) and MR−/− (open bars, n = 3) mice used in the circulatory half-life study (Fig. 1) were killed 30 minutes after intravenous injection of 10 μg 125I-cathepsin-D and analyzed for organ and tissue radioactivity. Recovered radioactivity in all tissues at this time point was taken as 100%, and represented >95% of injected radioactivity. Error bars represent the standard error of the mean (SEM). *Statistically significant (P < 0.01) difference between WT and MR−/− tissue radioactivity.

LSECs from MR−/− Mice Lack the Ability to Endocytose Cathepsin-D.

When trace amounts of 125I-cathepsin-D (0.02 μg/mL) were incubated with LSEC cultures for 2 hours, the ligand was endocytosed by LSECs from wild-types only (Fig. 3A,B), and coincubation with 50 mM D-mannose completely inhibited the uptake, indicating no alternative route for MR-mediated uptake.

Figure 3.

Endocytosis of cathepsin-D in liver sinusoidal endothelial cell (LSEC) cultures. Primary LSEC cultures from (A) wild-type and (B) MR−/− mice were incubated with 125I-cathepsin-D (0.02 μg/mL) with or without 50 mM D-mannose for 2 hours at 37°C. Endocytosis is presented as a percentage of total ligand radioactivity added to the cultures, and results are means of triplicate experiments. Error bars represent SD. Abbreviations: CathD, cathepsin-D; FSA, formaldehyde-treated serum albumin (a ligand for LSEC SRs).

NPC cultures, containing LSECs, KCs, and stellate cells, were challenged with Alexa-Fluor488-cathepsin-D (Fig. 4). In wild-type NPC cultures, cathepsin-D was only seen in vesicles in LSECs, as identified by their accumulation of TRITC-FSA (Fig. 4B,D).12 Similar experiments with MR−/−-NPC cultures showed no uptake of cathepsin-D in any cell type (Fig. 4A,C).

Figure 4.

Endocytosis of cathepsin-D in nonparenchymal cell (NPC) cultures. NPC cultures from (A,C) MR−/− and (B,D) wild-type (WT) mice were established on glass coverslides. In (A) and (B), the SR ligand, TRITC-FSA (bright red vesicles), was administered to the cultures for 10 minutes at 37°C to tag LSEC endosomes/lysosomes, before the cells were allowed to transport the ligand intracellularly for 30 minutes. (C,D) Then the NPC cultures were pulsed for 10 minutes with 2 μg/mL AlexaFluor-488-cathepsin-D, washed, and chased for 30 minutes before fixation. The micrograph imaging settings were equal in all parallels.

To study endocytosis of cathepsin-D in HCs, MR−/− mouse HCs were used. This was necessary because HC cultures always contain a small fraction (up to 10%) of contaminating LSECs, and wild-type LSECs endocytose 125I-cathepsin-D, which interfere with the HC assay. Incubation of HC cultures with 125I-cathepsin-D (0.02 μg/mL) for 3 hours at 37°C resulted in only 2% culture-associated radioactivity (n = 3, results not shown). Coincubation of 125I-cathepsin-D with either 50 mM D-mannose or 50 mM D-galactose had no inhibitory effect.

Lysosomal Enzyme Content Is Significantly Lower in LSECs from MR−/− Mice Than in Wild-Type LSEC.

Freshly isolated HCs and LSECs from wild-type and MR−/− mice were assayed for β-hexosaminidase, acid phosphatase, α-mannosidase, arylsulfatase, and cathepsin-D.

LSECs from MR−/− mice showed significantly reduced β-hexosaminidase, α-mannosidase, arylsulfatase, cathepsin-D, but not acid phosphatase enzyme activity, compared to wild-type LSECs (Table 1).

Table 1. Enzyme Activities in Liver Sinusoidal Endothelial Cells and Hepatocytes
Lysosomal enzymeWild-Type MouseMR−/− MouseRelative Enzyme Activity in Wild-Type (WT) Versus MR−/− Cells
LSECHCLSECHCLSECWT/LSECMR−/−HCWT/HCMR−/−
  • One unit (U) = 1017 substrate molecules transformed per gram of cell protein per minute. One UMCA = 1 nmol MCA (7-methoxy-coumarin-4-acetic acid) released per gram of cell protein per minute. Values are mean ± SD. Statistical analysis was done using a nonparametric test for comparing two means (Mann-Whitney test).

  • *

    P value < 0.05.

  • P value < 0.01.

  • nonsignificant result.

β-Hexosaminidase677 U SD = 255, n = 850.6 U SD = 19.3, n = 8148 U SD = 90.3, n = 777.5 U SD = 65.5, n = 74.570.65
Acid phosphatase212 U SD = 134, n = 846.9 U SD = 36.3, n = 7126 U SD = 69.1, n = 7122 U SD = 103, n = 71.680.38
α-Mannosidase0.57 U SD = 0.27, n = 70.38 U SD = 0.2, n = 70.18 U SD = 0.11, n = 40.34 U SD = 0.24, n = 53.14*1.08
Arylsulfatase2.28 U SD = 2.53, n = 70.17 U SD = 0.08, n = 70.12 U SD = 0.05, n = 50.81 U SD = 0.69, n = 719.70.21
Cathepsin-D14563 UMCA SD = 6085, n = 51153 UMCA SD = 263, n = 4404 UMCA SD = 397, n = 32254 UMCA SD = 427, n = 336.0*0.51*

In contrast, when comparing the LE content in HCs, the activity of all tested enzymes, except α-mannosidase, was increased in the HCs from MR−/− mice compared to wild-type; however, the difference was statistically significant only for cathepsin-D (Table 1).

In wild-type mice, LSECs showed significantly higher LE activity per gram cell protein than HCs (P < 0.05). The relative enzyme activity in LSEC:HC, per gram cell protein, for β-hexosaminidase, acid phosphatase, α-mannosidase, arylsulfatase, and cathepsin-D, was 13.4:1, 4.5:1, 1.5:1, 13.4:1, and 12.6:1, respectively.

LSECs from MR−/− Mice Show Normal Endocytosis but Significantly Reduced Degradation Capacity for Denatured Albumin.

We examined how reduced LE activities in MR−/− LSECs affected endocytosis and intracellular degradation of macromolecules endocytosed via other receptors, by examining blood clearance and in vitro LSEC endocytosis of a commonly used SR ligand, FSA (Figs. 5–7). FSA is cleared from blood almost exclusively by LSECs.34

Figure 5.

Blood clearance and subsequent in vivo degradation of formaldehyde-treated serum albumin (FSA) in (A,B) wild-type and (C,D) MR−/− mice. After intravenous injection of 125I-FSA (2 μg) into wild-type (n = 4) and MR−/− mice (n = 4), blood samples were collected for 60 minutes, and analyzed for 125I-labeled degradation products and intact ligand. (A,C) Nondegraded ligand in blood; (B,D) degradation products released into the blood. Results are given in cpm/μL blood. Slopes in (B) and (D) represent the average release rate of degradation products. The four individual wild-type and MR−/− mice are represented by the symbols □ ▵ ◊ ○,.

Figure 6.

Anatomical distribution of formaldehyde-treated serum albumin (FSA) in wild-type and MR−/− mice. (A) Anatomical distribution of 125I-FSA (2 μg), 10 minutes after intravenous injection into wild-type (filled bars, n = 5) and MR−/− mice (open bars, n = 5). In (B) the animals used in the circulatory half-life studies (Fig. 5) were analyzed for anatomical distribution of 125I-FSA (2 μg), 60 minutes after intravenous injection into wild-type (filled bars, n = 4) and MR−/− mice (open bars, n = 4). The sum of recovered radioactivity in the listed tissues was taken as 100%, and represented >95% of the injected dose in both experiments. The results are given in % of total recovered radioactivity + standard error of the mean (SEM). In (B) statistical significant (P < 0.01) difference between wild-type and MR−/− tissue radioactivity is shown by *. During the 60-minute monitoring period, radioactivity escaped from the animal by blood sampling only; organ-associated radioactivity in liver therefore represents endocytosed but not released degraded material, whereas the blood radioactivity represents mostly degradation products, which may leak into tissues. This would explain why carcass and gastrointestinal (GI)-tract radioactivity was highest in wild-type animals.

Figure 7.

Endocytosis of formaldehyde-treated serum albumin (FSA) in LSEC. LSEC cultures from wild-type (WT, n = 11) and MR−/− mice (n = 7) were incubated with 125I-FSA (0.1 μg/mL) for 2 hours at 37°C. Cell-associated radioactivity (filled bars), and degraded ligand radioactivity (open bars) are shown. Total endocytosed radioactivity represents the sum of cell-associated and degraded ligand radioactivity. The results, given as a percentage of total endocytosed radioactivity after 2 hours, are means + standard error of the mean (SEM). The difference in intracellular degradation of FSA was statistically significant (P < 0.01). The average endocytosis of 125I-FSA in the WT LSEC cultures was 31.2% (SD = 8.2, n = 11) of added ligand and in the MR−/− LSEC cultures it was 29.1% (SD = 11.5, n = 7). Each experiment was done in duplicate.

The circulatory half-life and subsequent liver uptake of 125I-FSA (2μg), was similar in wild-type and MR−/− mice (Figs. 5A,C, and 6A). Moreover, the release of degradation products to blood occurred almost simultaneously; 12.9 minutes (SD = 1.9) postinjection in wild-type (Fig. 5B) and 14.1 minutes (SD = 1.2) postinjection in MR−/− mice (Fig. 5D). However, the MR−/− mouse degradation rate of FSA was only half that of wild-types (Fig. 5B,D). The slope (cpm/minute) of the curve for release of radioactive degradation products was calculated at 15-40 minutes postinjection, and found to be 8.3 (SD = 0.63) in wild-type mice and 3.95 (SD = 0.44) in MR−/− mice (P < 0.01).

Consistent with the observation that wild-type mice degrade FSA twice as quickly as MR−/− mice, the livers of MR−/− mice retained almost double the radioactivity as wild-type livers 60 minutes after intravenous injection of 125I-FSA (Fig. 6B).

Furthermore, in LSEC cultures, the total uptake of 125I-FSA (0.1 μg/mL) after 2 hours incubation at 37°C was almost identical in cells isolated from MR−/− and wild-type animals (Fig. 7), whereas ligand degradation was significantly lower in MR−/− LSECs; the percentage of released 125I-degradation products being 39% of the wild-type.

The Amount of Pro-Cathepsin-D Is Markedly Lower in Mouse LSECs Than in HCs.

To compare the relative amount of pro-cathepsin-D to total cathepsin-D in LSECs and HCs, cell extracts from wild-type mice were analyzed with immunoblotting using a rabbit anti-mouse cathepsin-D antiserum (Fig. 8). The immunoblots showed very little pro-cathepsin-D in LSECs compared to in HCs when equal amounts of total cell protein were loaded, approximately 2% and 11% in LSECs and HCs, respectively (as measured by pixel density of bands from three different experiments; Fuji Multigauge software). The strongest band in LSECs corresponds to the light fragment (∼14 kDa) while in the HCs the strongest is the intermediate fragment (∼46 kDa).

Figure 8.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis of cathepsin-D and pro-cathepsin-D expression in LSEC and hepatocytes (HC). LSEC 1 and LSEC 2 represent cell extracts from two different wild-type mice, and the HC represents mixed cell extracts from these two mice. Cell protein loading: 10 μg/lane. A rabbit anti-mouse cathepsin-D antiserum was used to visualize the bands. Pro-cathepsin-D was approximately 51 kDa, and the cathepsin-D polypeptides were approximately 46 kDa, 28 kDa, and 14 kDa, respectively.

Discussion

Cells target LE to lysosomes via different routes. In the classical pathway,29 newly synthesized mannose-6-phosphate-tagged enzymes are transported from the trans-Golgi network to endosomal compartments by the two MPR receptors, MPR-46 and MPR-300, the latter of which is also involved in endocytosis of mannose-6-phosphate-tagged LE from the plasma membrane. The routing of LE to extracellular surroundings may be a result of normal processes such as missorting of newly synthesized enzymes, or release during inflammation, damage, repair, or remodeling of tissues. As LE, in addition to their high-mannose-type-oligosaccharides (which may be phosphorylated), contain hybrid-type-oligosaccharides and complex-type-oligosaccharides, they are potential ligands for several receptors, including MPR-300, the asialoglycoprotein receptor and the MR.35, 36 MR-independent and MPR-independent LE uptake pathways also exist.37 The present study focuses on MR recruitment of LE in LSECs, and presents evidence that the cells rely on this receptor for supply of LE.

The MR is expressed on a subset of cells, namely those with high endocytic activity, such as macrophages, immature monocyte derived- and dermal dendritic cells, and scavenger endothelial cells including LSECs.2, 8, 10 The term scavenger endothelial cells has been introduced1 to describe specialized types of endothelial cells in vertebrates, functionally characterized by their extraordinarily high endocytic capacity for soluble macromolecules, as compared to other endothelia. The anatomical location may vary across vertebrate classes1; in mammals, these cells are located in the liver where they make up the whole population of LSECs. LSECs from rat and pig have a very high content of LE,12, 28 and the present study in mice also showed significantly higher activity per g cell protein in LSECs than in HCs for the five LE tested. The relative specific activity in mouse LSEC:HC for three of these enzymes, namely acid phosphatase, arylsulfatase and cathepsin-D (5:1; 13.4:1, and 12.6:1, respectively) was almost identical to that in rat,28 i.e., 4.5:1, 15.4:1 and 12.8:1, respectively.

Because LE are resistant to intracellular degradation (as evidenced by the lack of degradation products in Fig. 1), we suggest that MR-mediated uptake of enzymes benefits LSECs with improved lysosomal degradation capacity, making them less dependent on other pathways for LE recruitment or production. In order to test this hypothesis we used an MR knockout mouse model9 to study the circulatory fate of injected cathepsin-D, in vitro endocytosis of cathepsin-D in LSEC cultures, LSEC enzyme content and degradative capacity of a non-MR ligand, and the endogenous cathepsin-D profile in LSECs compared to HCs.

We found that the liver is the most important scavenger organ for circulating cathepsin-D, as the high uptake observed in livers of control mice was drastically reduced in MR−/− mice (P = 0.01), with no significant compensatory uptake in other organs. An initial rapid α-phase of cathepsin-D clearance, indicating high-affinity ligand-receptor binding, was only observed in wild-type controls, and the half-life of the terminal β-phase was markedly slower in the MR−/− mice, indicating that there is no effective alternative to the MR for elimination of blood-borne cathepsin-D. This is in accordance with Lee et al.,9 who reported that mice deficient in the MR had elevated blood levels of eight out of nine LE (cathepsin-D was not measured), suggesting that the MR is of major importance for the plasma homeostasis of these molecules.

In rat liver, LSEC is reported to be the major cell type involved in endocytosis of soluble MR ligands3, 4, 19, 21, 22, 24, 25 but their role in the uptake of cathepsin-D has not been previously reported. Fluorescence microscopy of wild-type and MR−/− NPC cultures incubated with Alexa-Fluor488-cathepsin-D showed that wild-type LSEC was the only cell type involved in ligand uptake. Correspondingly, when LSEC cultures from wild-type and MR−/− mice were challenged with trace amounts of 125I-cathepsin-D for 2 hours, the ligand was effectively taken up by wild-type cells only, suggesting that no alternative receptors are involved in LSEC endocytosis of cathepsin-D. Uptake or binding of cathepsin-D in HC cultures was very low and high doses of mannose or galactose had no inhibitory effect, suggesting that HCs possess a low affinity binding mechanism for cathepsin-D unrelated to the MR or asialoglycoprotein receptor. The cathepsin-D used in our study was extracted from bovine spleen, and a small (unknown) fraction of the ligand may be phosphorylated, as reported in cathepsin-D from pig spleen,38 thus some uptake via MPR-300 cannot be excluded. This receptor has been reported to be present in HCs but absent in LSECs.39–41

Although plasma levels of LE were elevated in MR−/− mice,9 these authors found no significant difference between tissue LE levels in wild-type and MR−/− mice, and they therefore suggested that intracellular stores of LE are normal in the livers of MR-deficient animals. However, in the liver, the MR is expressed primarily on LSECs,3, 11, 13 which only make up a few percent of liver cell mass. Although these cells are the primary clearance sites of many plasma LE their relative contribution to the total liver pool of LE is probably swamped by the much larger HC-pool. In the present study, we found significantly reduced activities for β-hexosaminidase, α-mannosidase, arylsulfatase and cathepsin-D in the MR−/− LSECs. Acid phosphatase levels were also reduced, but the difference was not statistically significant. In contrast, the activities of the enzymes, except α-mannosidase, were increased in HCs from the MR−/− mice; the differences were statistically significant only for cathepsin-D due to large individual variations. However, this tendency may explain the finding by Lee et al.9 that liver LE levels were within the normal range in MR−/− mice on a whole-organ basis.

The increased LE activity in MR−/− mouse HCs warrants further study. It has been reported that up to 20% of newly synthesized cathepsin-D in HCs is excreted from the cells.37 With reduced clearance mechanisms in adjacent LSECs, as with MR−/− mice, more enzyme may be reabsorbed by the HCs. Alternatively, LSECs in MR−/− mice, sensing a deficiency of LE, may secrete mediators to stimulate LE production in the adjacent HCs.

When monitoring the blood clearance and anatomical distribution of the SR ligand FSA, we found that liver uptake and starting point of degradation product release were similar in wild-type and MR−/− mice. However, FSA degradation rate was significantly lower in the latter, suggesting impaired catabolism of endocytosed ligand. This finding was confirmed in endocytosis studies of FSA in wild-type and MR−/− LSEC cultures. Taken together these studies indicate that uptake and intracellular transport of SR ligands to degradation organelles is normal in MR-deficient LSECs, but that the lysosomal degradation capacity for internalized proteins is reduced due to the lower LE content in these cells.

To investigate the relative proportion of endogenously produced cathepsin-D in LSEC, we immunoblotted extracts of freshly isolated HCs and LSECs with anti-cathepsin-D. In mouse, cathepsin-D is synthesized as a 51 kDa pro-polypeptide that is cleaved into intermediate 38-45 kDa forms, which can be processed further into the final mature heavy (∼30 kDa) and light (14-16 kDa) forms linked by noncovalent interactions.31 We found that pro-cathepsin-D represented approximately 11% and 2% of the total cathepsin-D in HCs and LSECs, respectively. Interestingly, the strongest stained band in the LSEC lanes corresponds to the light fragment (∼14 kDa), whereas in HC, the band of the intermediate fragment (∼46 kDa) was most intensely stained. This suggests that endogenous production of cathepsin-D is of minor importance in LSECs compared to in HCs.

In conclusion, we present novel evidence that the LSEC MR is essential for cathepsin-D clearance, and that these cells rely on MR-mediated uptake of LE to maintain their high lysosomal degradation capacity. In addition to a general waste clearance function, recruitment of LE to degradative organelles may be an important function of the MR, as many cells that express this receptor (e.g., macrophages, and scavenger endothelia) share the common feature in that they are highly effective in lysosomal degradation.

Acknowledgements

We thank Dr. Michel Nussenzweig, The Rockefeller University, for his generous gift of MR−/− mice, and Drs. Regina Pohlmann, Münster University, and Kurt von Figura, Georg-August University, Germany, for their kind gift of cathepsin-D antiserum, and Ivana Malovic for skilled technical assistance.

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