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Originally published online as doi:10.1189/jlb.0107052 on August 3, 2007

Published online before print August 3, 2007
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(Journal of Leukocyte Biology. 2007;82:1156-1165.)
© 2007 by Society for Leukocyte Biology

Stabilin-2 is involved in lymphocyte adhesion to the hepatic sinusoidal endothelium via the interaction with {alpha}Mβ2 integrin

Mi-Yeon Jung*,1, Seung-Yoon Park{dagger},1 and In-San Kim*,2

* Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu, Korea; and
{dagger} Department of Biochemistry, School of Medicine, Dongguk University, Kyungju, Korea

2 Correspondence: Department of Biochemistry and Cell Biology, Cell and Matrix Biology Research Institute, School of Medicine, Kyungpook National University, Daegu 700-422, Korea. E-mail: iskim{at}knu.ac.kr


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ABSTRACT
 
Although lymphocyte recirculation to the endothelium plays a critical role in the movement of immune cells from the blood into tissues and sites of inflammation, the mechanisms involved in lymphocyte trafficking via the hepatic circulation have yet to be elucidated fully. In this study, we investigated the role of stabilin-2, which is expressed specifically in the sinusoidal endothelium, in the adhesion of lymphocytes to the hepatic endothelium. Stabilin-2-expressing cells mediate the adhesion of PBLs. This interaction was attributed specifically to the interaction of stabilin-2 with {alpha}Mβ2 integrin. Using mutant stabilin-2 molecules with deletions in the extracellular domain, we mapped the binding site for {alpha}Mβ2 integrin to the fasciclin 1 (FAS1) domains of stabilin-2. The specificity of the interaction between {alpha}Mβ2 integrin and the FAS1 domain was confirmed further by binding assays using neutralizing antibodies. More physiologically, we showed that the down-regulation of stabilin-2 results in the defective binding of lymphocytes to hepatic sinusoidal endothelial cells under conditions of static and physiological flow. Together, these data show that stabilin-2 can reconstitute the lymphocyte–endothelial adhesion cascade under physiological shear stress. We propose a critical role for stabilin-2 in lymphocyte adhesion to specialized endothelia, such as that of the hepatic sinusoid.

Key Words: FAS1 domain • molecules • FEEL-2 • HARE


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INTRODUCTION
 
Multiple molecular interactions between leukocytes and endothelial lining cells play pivotal roles in inflammatory and immune responses. Lymphocyte recruitment from the circulation into tissue is controlled by the interaction between the cell adhesion molecules present on lymphocytes and the endothelial cell surface [1 , 2 ]. The initial reversible and transient adhesion between leukocytes and endothelial cells is achieved principally by the selectins. In the presence of activation signals, the rolling step is followed by firm adhesion mediated by the binding of activated integrins to their ligands [3 ]. Finally, leukocytes transmigrate through the vascular wall into the tissue, following a hierarchy of chemotactic signals toward the inflammation site [1 ]. However, the individual steps in this cascade vary substantially in specialized tissues such as liver, in which the majority of leukocyte adhesion takes place in hepatic sinusoids rather than in the postcapillary venules in response to a chemotactic stimulus [4 ]. In contrast to the vascular endothelial cells, sinusoidal endothelial cells (SECs) show a unique morphology and important differences in the expression of adhesion molecules [5 6 7 ], suggesting that other molecular mechanisms play a specific role in the tethering of lymphocytes in the hepatic sinusoids. Although several adhesion molecules have been suggested, which mediate leukocyte recruitment in specialized vessels, much in this process remains to be elucidated.

The fasciclin 1 (FAS1) domain is an extracellular module of ~140 amino acid residues, which was originally found in an insect cell adhesion molecule (CAM), fasciclin 1, which is expressed in subsets of axon pathways during neuronal development in the grasshopper [8 ]. FAS1 domains are present in numerous secreted and membrane-anchored proteins [9 ]. Unusually for extracellular domains, the FAS1 superfamily consists of members from all phyla and includes mycobacterial-secreted proteins [10 ], plant proteins, such as the major CAM of Volvox, Algal-CAM [11 ], and Arabidopsis arabinogalactans [12 ], several Caenorhabditis elegans proteins of unknown function, and a number of vertebrate proteins. Although the roles of proteins containing the FAS1 domain are largely unknown, there is accumulating evidence that the FAS1 domain is one type of functional domain frequently found in adhesion receptors. FAS1-containing molecules such as βig-h3 and periostin function as cell adhesion substrates via interactions with several integrins [13 14 15 16 ]. Recently, two members of the FAS1 superfamily, stabilin-1 [also known as FAS, epidermal growth factor (EGF)-like, laminin-type EGF-like, and link domain-containing scavenger receptor-1 (FEEL-1) and common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1)] and stabilin-2 also known as FEEL-2 and hyaluronan receptor for endocytosis (HARE), which have similar domain structures, have been characterized [17 ]. They are expressed dominantly in SECs of the spleen, lymph nodes, and liver [18 , 19 ]. They are FAS-like hyaluronan receptor homologues [20 ]. They have been reported to be scavenger receptors, which bind bacteria, endocytose advanced end-products [21 ], and promote capillary angiogenesis in vitro [22 ]. Stabilin-2 has also been described as the endocytic hyaluronan receptor of hepatic SECs [18 , 23 , 24 ]. Furthermore, recent studies have reported that CLEVER-1 mediates lymphocyte adhesion and transmigration through the vascular and lymphatic vessels [25 , 26 ]. These findings led us to speculate that stabilin-2 may function as an adhesion molecule for cell–cell interactions between hematopoietic cells and the endothelial cell surface. In this study, we demonstrate that stabilin-2 mediates the binding of PBLs to the hepatic endothelium through its FAS1 domains. This interaction is preferentially dependent on the {alpha}Mβ2 integrin on the lymphocytes. These data led us to propose a model for heterophilic stabilin-2-mediated cell adhesion in hepatic endothelium.


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MATERIALS AND METHODS
 
Cloning human stabilin-2 cDNA
Partial stabilin-2 cDNA was generated by RT-PCR with primers designed using partial cDNA clones from the human spleen (FLJ00112, DZKZp434E0321, and CD44-like precursor FELL) from the GenBank database. Unidentified exons were identified by analyzing the FLJ00112 sequence of Celera Genomics (Rockville, MD, USA) and were cloned by RT-PCR. The extreme 5' end of stabilin-2 cDNA was determined by 5'-RACE using cDNA from the human spleen. The complete sequence of stabilin-2 cDNA was amplified as five overlapping fragments of 2.0, 1.6, 2.0, 1.2, and 1.8 kb. The fragments were cloned into the pcDNA3.1(–)/Myc–His vector (Invitrogen, Carlsbad, CA, USA) and sequenced (ABI 3700 DNA analyzer, Applied Biosystems, Foster City, CA, USA). The resulting plasmid containing the stabilin-2 cDNA of ~8.5 kb (GenBankTM Accession Number AY311388) was designated pcDNA-stabilin-2.

Cell culture and transfection
L cells (ATCC CCL-1) were grown in DMEM supplemented with 10% heat-inactivated FBS and the appropriate antibiotics. Human hepatic SECs (HHSECs) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and were grown in endothelial cell medium (ScienCell Research Laboratories) supplemented with 10% FBS and endothelial cell growth supplement (ScienCell Research Laboratories). Human PBLs were isolated from the peripheral blood of healthy donors using Ficoll–Hypaque (Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation. For stable transfections, the L cells were transfected in OptiMEM I medium using Lipofectamine (Invitrogen) and were selected with G418 (400 µg/mL). Individual G418-resistant colonies were isolated after 10–12 days of culture. The final clones were designated L/Stab-2/#. Negative control clones were randomly selected from the transfectants with the empty vector (L/Mock). The cells were analyzed for the expression of stabilin-2 using Western blotting and flow cytometry.

Antibodies
mAb directed against the human {alpha}L integrin (Clone 25.3) was purchased from Immunotech (France). mAb directed against human β1 (Clone P5D2), {alpha}M integrin (Clone 44), and {alpha}X integrin (Clone 3.9) were purchased from Chemicon (El Segundo, CA, USA). mAb directed against ICAM-1 (Clone BBA5) and VCAM-1 (Clone BBA3) were purchased from R&D Systems (Minneapolis, MN, USA). To produce anti-human-stabilin-2 antibodies, two His-tagged recombinant proteins corresponding to amino acids 554–655 and 2188–2551 were expressed in Escherichia coli and purified using nickel-nitrilotriacetic acid (Ni-NTA) resin, according to the manufacturer’s instructions. For anti-mouse-stabilin-2 antibody, a GST fusion protein of the cytoplasmic tail encompassing amino acids 2490–2559 was expressed in E. coli and conjugated to glutathione agarose (Amersham Pharmacia, Little Chalfont, UK). The cytoplasmic tail fragment was eluted with thrombin protease (Amersham Pharmacia), according to the manufacturer’s instructions. Antibodies were raised in rabbits by s.c. injection of the protein (200 µg) in CFA (Sigma Chemical Co., St. Louis, MO, USA). Immunizations were repeated four times in IFA (Sigma Chemical Co.) every 2 weeks. The antisera were purified further by Protein A affinity chromatography (Amersham Pharmacia), according to the manufacturer’s protocol.

To produce human anti-stabilin-2 mAb, a His-tagged, recombinant protein corresponding to amino acids 1173–1727 was expressed in E. coli and purified using Ni-NTA resin, according to the manufacturer’s instructions. Six mice were each immunized with 20 µg purified recombinant protein. The mice were boosted twice at 2-week intervals, and blood was drawn from the tail veins 6 weeks after the first immunization. The sera were tested for specific antibody by ELISA and Western blotting. Standard procedures [27 ] were followed for cell fusion and limited dilution cloning. The hybridoma supernatants were screened using an ELISA assay with the recombinant stabilin-2 protein. A consistently positive hybridoma clone (Clone 5G3) was used to produce ascites fluid, as described by Harlow and Lane [27 ]. Antibody titers were monitored by using recombinant stabilin-2 proteins. The specificities of the anti-stabilin-2 antibodies were examined by Western blotting. A single band was detected in the lysates from L cells transfected with a stabilin-2 expression vector. Anti-human-stabilin-2 antibodies did not cross-react with other orthologous proteins, such as mouse and rat stabilin-2, or a paralogous protein, such as human stabilin-1 (data not shown).

Purification of recombinant proteins
To produce recombinant proteins corresponding to the four putative, functional units of stabilin-2, fragments of stabilin-2 cDNA encoding amino acids 66–655, 691–1268, 1303–1883, and 1913–2449 were generated by PCR; and to produce recombinant proteins for each EGF-like repeat domain of stabilin-2, fragments of stabilin-2 cDNA encoding amino acids 66–362, 691–994, 1303–1596, and 1913–2165 were generated by PCR. To produce recombinant proteins corresponding to seven FAS1 domains of stabilin-2, fragments of the stabilin-2 cDNA encoding amino acids 406–508, 554–655, 1030–1130, 1173–1268, 1631–1727, 1778–1883, and 2356–2449 were generated by PCR, cloned into the BamHI and XhoI sites of pET43.1a (Novagen), and designated pET-F1, -F2, -F3, -F4, -F5, -F6, and -F7, respectively. The primers used in this study are shown in Supplemental Table 1. To produce recombinant proteins for each EGF-like repeat domain of stabilin-2, fragments of stabilin-2 cDNA encoding amino acids 97–362, 725–994, 1333–1596, and 1947–2165 were generated by PCR, cloned into the BamHI and XhoI sites of pET43.1a (Novagen), and designated pET-E1, -E2, -E3, and -E4, respectively. These His-tagged, recombinant proteins were expressed in BL-21 cells, harvested, and purified using Ni-NTA resin (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions.

Adhesion assays
HHSECs or L cells, in which stabilin-2 was stably expressed, or mock transfectants were grown to confluence on six-well plates. After two washes, HBSS containing 10% FBS and 1 mM MnCl2 (assay medium) was added. PBLs were isolated from freshly drawn blood using Ficoll (Pharmacia Biotech) gradient centrifugation [28 ]. Lymphocytes were fluorescently labeled with fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Molecular Probes, Eugene, OR, USA) and resuspended in the assay medium to a concentration of 106 cells/ml. Lymphocyte suspension (1 ml) was added to transfectants or a HHSEC monolayer in 1 ml assay medium and incubated for 60 min at 37°C without agitation. After the cells were washed, the number of adherent cells was counted under light and fluorescent microscopes (original magnification, x400). Five fields per sample were counted in each of three independent experiments. For adhesion inhibition studies, the anti-stabilin-2 mAb or isotype-matched control IgG was preincubated with the transfectant monolayer for 30 min at 37°C before the lymphocytes were added. Lymphocytes were preincubated with function-blocking antibodies directed against integrin {alpha} for 30 min at 37°C and then transferred to the transfectant monolayer. For cation studies, PBLs were preincubated for 30 min in cation-free Hepes-Tyrode buffer in the absence or presence of 1 mM Ca2+, Mg2+, or Mn2+ and then transferred to the transfectant monolayer in cation-free Hepes-Tyrode buffer. The number of adherent cells was counted in three experiments, as described above.

For adhesion assays under flow conditions, HHSECs were grown on glass plates. The cells were preincubated with viral particles containing control for stabilin-2 short hairpin (sh)RNA (shStab-2) or scrambled control shRNA (shCont) for 2–3 days and inserted in a parallel wall flow chamber. The PBLs were then gently perfused at 2 dyne/cm2 for 10 min, and the surface-adherent cells were visualized with a charged-coupled device camera. In some experiments, the lymphocytes were preincubated with the indicated recombinant proteins for 30 min before they were added to the flow chamber. To determine the percentage of rolling cells, statically adherent cells, and transmigrated cells, phase contrast video recordings made during lymphocyte perfusion were analyzed as described previously [29 ].

Lentivirus-mediated shRNA production
Three 20-nt shRNA sequences, shStab-2-1: 5'-CAA ACT GGA ATG CAA ATG CC-3', shStab-2-2: 5'-TGG CAA GGA AGG CTG ACC TC-3', and shStab-2-3: 5'-CGC AGC AGT GGA ATT GTC AT-3', corresponding to bases 840–959, 1324–1343, and 3174–3193, respectively, in the stabilin-2 mRNA sequence (Accession Number: NM_017564) were designed. Candidate oligonucleotides were synthesized and cloned into the pSuper/basic vector (OligoEngine, Seattle, WA, USA). pSuper/shStab-2-2 down-regulated stabilin-2 protein expression significantly in stably transfected L cells, and the expression cassette of pSuper/shStab-2-2 was subcloned into the lentiviral vector pRNAT–U6.1/Lenti (GenScript, Piscataway, NJ, USA). shCont was used as the control for shStab-2. All constructs were verified by sequence analysis. Lentiviral transduction was performed according to the manufacturer’s instructions. In brief, the pRNAT–shStab-2 vector was cotransfected with the packaging vector (Invitrogen) into 293FT cells (Invitrogen). The supernatant was collected after 72 h and filtered through a 0.22-µm pore acetate filter. HHSECs were seeded at 2 x 105 cell/mL into 24-well plates. Lentiviral particles and 8 µg/mL Polybrene® (Sigma Chemical Co.) were added to the culture. In our system, using lentivirus-mediated shStab-2, the depletion of stabilin-2 by shRNA-mediated gene silencing was determined to be effective 3 days after infection. pRNAT–shStab-2 and pRNAT–shCont vectors also contained the transgene for GFP to monitor infection efficiency.

Immunofluorescent staining
Cells were cultured overnight on chamber slides, fixed in 4% formaldehyde in PBS for 15 min at room temperature, and permeabilized with 0.1% Triton X-100. Nonspecific binding was minimized by incubating the cells for 1 h in PBS containing 10% goat serum albumin. After extensive washing in PBS, the slides were incubated for 1 h at room temperature with anti-stabilin-2 polyclonal antibody or rabbit IgG as the negative control. The slides were washed three times for 5 min each with PBS at room temperature. Anti-rabbit IgG conjugated with Alexa 568 (Molecular Probes) was added, and the incubation continued for 1 h at room temperature. The slides were washed three times with PBS for 5 min each and treated with a solution of SlowFade (Molecular Probes). The slides were viewed with a Zeiss light microscope using Axioplan2 imaging.

Binding assay for the FAS1 domain
A binding assay was performed as described previously [14 ]. Cells were suspended in medium at a density of 1 x 105 cells/mL, and 1 mL of the cell suspension was preincubated with anti-{alpha}Lβ2 (Clone 25.3), anti-{alpha}Mβ2 (Clone 44), or anti-{alpha}Xβ2 (Clone 3.9) antibody for 30 min at 37°C. The cells were incubated with His-tagged stabilin-2 derivatives in serum-free medium containing 0.1% BSA for 5 h at 4°C. The cells were then washed three times with PBS (pH 7.4) before lysis at 4°C in ice-cold buffer A [10 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.5% SDS, 0.02% sodium azide, 1 mM EDTA, 1 mM PMSF]. The lysates were clarified bycentrifugation at 10,000 g for 10 min at 4°C. Equal amounts of protein were then separated by SDS-PAGE on 8% gel. The amounts of His-tagged proteins were determined by immunoblotting.


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RESULTS
 
Stabilin-2 mediates the adhesion of PBLs
To investigate whether stabilin-2 is involved in the adhesion of PBLs, we generated a cell model for human stabilin-2 surface expression. Mouse fibroblast L cells were transfected with human stabilin-2 cDNA (referred to as L/Stab-2 cells) or empty vector (referred to as L/Mock cells), as they do not express any cadherins and are used widely in cell adhesion studies [30 ]. Using mAb (5G3) directed against human stabilin-2, we detected the surface expression of stabilin-2 on L/Stab-2 cells but not on L/Mock cells by flow cytometry (Fig. 1A ). Immunoblot analysis with polyclonal anti-stabilin-2 antibody showed the expression of stabilin-2 in the membrane fractions but not in the cytosolic fractions of L/Stab-2 cells (Fig. 1B) . In an adhesion assay using human PBLs, lymphocytes significantly bound to L/Stab-2 cells but not to L/Mock cells. PBLs bound to L/Stab-2 cells 4.5 times better than to L/Mock cells (Fig. 1C and 1D) . To confirm whether stabilin-2, on the surfaces of L/Stab-2 cells, can support the adhesion of lymphocytes, a similar adhesion assay was carried out in the presence of anti-stabilin-2 mAb (5G3). Lymphocyte adhesion to L/Stab-2 was inhibited significantly by the anti-stabilin-2 antibody but not by isotype-matched, control IgG (Fig. 1D) . In our preliminary experiments, PBLs did not express any detectable, endogenous stabilin-2 protein in FACS analysis (data not shown), and no expression of stabilin-2 mRNA was detected by RT-PCR, as described previously [31 ]. The adhesion results were pooled from five independent experiments performed in triplicate. Each experiment was analyzed using four independent L/Stab-2 clones and PBLs from three different donors. These data demonstrate that stabilin-2 is a functional adhesion molecule located on the cell surface of transfected cells and can mediate the adhesion of PBLs.


Figure 1
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  		Figure 1. Stabilin-2 mediates the adhesion of PBLs. (A) FACS analysis. L cells transfected with stabilin-2 cDNA (L/Stab-2) or empty vector (L/Mock) were incubated with mAb 5G3 or mouse IgG. The cells were washed, incubated with FITC-conjugated secondary antibody for 45 min on ice, and processed for FACS analysis. (B) Membrane (mb) and cytosolic (cyto) fractions of L/Mock or L/Stab-2 cell lysates were separated by SDS-PAGE. Stabilin-2 was detected as a single band at ~300 kDa, as indicated. A single representative blot is shown. (C) Increased stabilin-2-dependent binding of lymphocytes to stabilin-2 transfectants. Considerably more PBLs (small, round spheres on top of the monolayer) bound to the stabilin-2 transfectants (right panel) than to the mock transfectants (left panel). Original scale bars, 100 µm. (D) Adhesion assays were performed in the presence of mAb 5G3 or isotype-matched control IgG. The results are expressed as mean cell numbers from five randomly selected, high-magnification fields (HMF). The results are the means ± SD of at least three experiments. ANOVA, ***, P < 0.001.

Stabilin-2 mediates the binding of lymphocytes via its FAS1 domains
Stabilin-2 is a large glycoprotein containing four EGF-like repeats (23 EGF domains), seven FAS1 domains, and a Link domain and has four putative, functional units composed of one EGF-like repeat and two FAS1 domains (the fourth unit has a single FAS1 domain and an a Link domain; Fig. 2A ). To identify the region that is responsible for the interaction of stabilin-2 with PBLs, we generated recombinant proteins for the four putative, functional units (Stab-U1, -U2, -U3, and -U4; Fig. 2B ) and examined whether these proteins inhibit the interaction between PBLs and stabilin-2. When PBLs were preincubated with these proteins, their binding was inhibited severely (Fig. 2C) , suggesting that each repeat unit contains a domain involved in binding PBLs and competitively inhibits the binding of PBLs.


Figure 2
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  		Figure 2. Four putative, functional units impaired stabilin-2-mediated lymphocyte tethering. (A) Schematic representation of stabilin-2 protein. The four putative, functional units are indicated. A Nus tag was fused to each recombinant protein to enhance the solubility of the protein. Stab-U1, -U2, and -U3 are composed of one EGF-like repeat (E) and two FAS1 (F) domains. Stab-U4 is composed of an EGF-like repeat, a FAS1 domain, and a Link domain (L). TM, . (B) SDS-PAGE of the recombinant proteins stained with Coomassie brilliant blue G-250. (C) Effect of the four putative, functional units on lymphocyte tethering by L/Stab-2 cells. PBLs were preincubated with the indicated stabilin-2 proteins (10 µM) and added to L/Stab-2 cells. The results are expressed as percentage of adhesion when compared with control Nus protein. The results are the means ± SD of at least three experiments. ANOVA, ***, P < 0.001.

To further identify the domain involved in the heterophilic interaction, we generated recombinant proteins for the FAS1 domain (Stab-F5) and the EGF-like repeat (Stab-E3) in the third unit (Fig. 3A ) and examined their inhibitory effects on the binding of PBLs. Pretreatment with Stab-U3 or Stab-F5 dose-dependently inhibited the binding of PRLs. Conversely, the Stab-E3 protein and the control Nus protein did not affect the binding of PBLs (Fig. 3B) . Six other FAS1 domains in the stabilin-2 protein also inhibited the binding of PBLs significantly (Fig. 3C) . The EGF-like repeats in stabilin-2 had no effect on the binding of PBLs (data not shown). These data demonstrate that the FAS1 domains in stabilin-2 play a key role in the interaction of stabilin 2 with PBLs.


Figure 3
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  		Figure 3. FAS1 domains play a key role in stabilin-2-mediated lymphocyte binding. (A) The recombinant EGF-like repeats and FAS1 domain are depicted and compared with the third putative, functional unit (Stab-U3). (B) Effect of EGF-like repeat-containing proteins on the binding of lymphocytes by L/Stab-2 cells. PBLs were preincubated with three different concentrations (0.1, 1.0, or 10 µM) of the indicated stabilin-2 proteins and added to L/Stab-2 cells. The results are expressed as percentage of adhesion when compared with control protein. The results are the means ± SD of at least three experiments. ANOVA, *, P < 0.05. (C) Effect of recombinant FAS1 domains on the binding of lymphocytes by L/Stab-2 cells. PBLs were preincubated with three different concentrations (0.1, 1.0, or 10 µM) of FAS1 domain proteins and added to L/Stab-2 cells. The results are expressed as percentage of adhesion when compared with control protein. The results are the means ± SD of at least three experiments. ANOVA, ***, P < 0.001.

{alpha}Mβ integrin is a counter-receptor of stabilin-2, supporting the adhesion of lymphocytes
To identify the nature of the cell surface receptor for stabilin-2, we examined the effects of Mn2+, Mg2+, and Ca2+ on stabilin-2-mediated cell adhesion. Lymphocyte adhesion to L/Stab-2 cells was strongly promoted by Mn2+ and to a lesser extent, by Mg2+ but only marginally by Ca2+ (Fig. 4A ). This result suggests that the cell surface receptor for stabilin-2 requires divalent cations for its interaction with ligands. This characteristic is typical of the binding of integrin to its ligand. PBLs showed a strong expression of {alpha}L integrin, some {alpha}M integrin, and little {alpha}X integrin (Fig. 4B , left). To identify a counter-receptor for stabilin-2, the effects of function-blocking mAb directed against leukocyte integrins on the adhesion of lymphocytes to L/Stab-2 cells were examined. The adhesion to L/Stab-2 cells was inhibited specifically by an antibody directed against {alpha}Mβ2 integrin and to a lesser extent, by an antibody directed against {alpha}Lβ2 integrin but not by an antibody directed against {alpha}Xβ2 integrin (Fig. 4B , right). To investigate further whether stabilin-2-mediated PBL adhesion is specifically dependent on the interaction between the FAS1 domain of stabilin-2 and integrin receptors, we studied the binding affinity between the FAS1 domain of stabilin-2 and PBLs in the presence of a specific, function-blocking antibody directed against each integrin. As shown in Figure 4C , recombinant FAS1 protein binds to the PBL surface in a dose-dependent manner, and its binding is inhibited specifically by an antibody directed against {alpha}Mβ2 integrin but not by other anti-integrin antibodies or isotype-matched control IgG (Fig. 4D , left). Integrin antibodies did not cross-react with FAS1 protein (Fig. 4D , right). Taken together, these results suggest that the integrin {alpha}Mβ2 is involved in the adhesion process via the interaction with the FAS1 domain of stabilin-2.


Figure 4
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  		Figure 4. {alpha}Mβ2 integrin is involved in the adhesion process via the interaction with the FAS1 domain of stabilin-2. (A) Lymphocytes were preincubated for 30 min in cation-free Hepes-Tyrode buffer in the absence or presence of 1 mM Ca2+, Mg2+, or Mn2+ and then transferred to stabilin-2 transfectants for the cell adhesion assay. The results are expressed as mean cell numbers from five randomly selected HMF. The results are the means ± SD of at least three experiments. ANOVA, *, P < 0.05; ***, P < 0.001. (B) Expression of integrins on PBLs is analyzed by flow cytometry (left panel). Lymphocytes were preincubated with the following function-blocking mAb directed against leukocyte integrins, at a concentration of 10 µg/mL for 30 min at 37°C, and then added to stabilin-2 transfectants. (right) β1, Anti-integrin β1 subunit antibody (P5D2); {alpha}L, anti-integrin {alpha}L subunit antibody (25.3); {alpha}M, anti-integrin {alpha}M subunit antibody (44); {alpha}X, anti-integrin {alpha}X subunit antibody (3.9). The results are expressed as percentage of adhesion when compared with untreated cells. The results are the means ± SD of at least three experiments. ANOVA, **, P < 0.01; ***, P < 0.001. (C) Dose-dependent binding of His-tagged FAS1 protein to the lymphocyte surface. Cells were incubated with His-tagged FAS1 protein for 5 h at 4°C. After lysis, the amount of His-tagged FAS1 protein associated with the cells was determined by Western blotting with the HRP-conjugated anti-His antibody. Representative results of three independent experiments are shown. (M), Mole. (D) The effect of integrin {alpha}M function-blocking antibody on the binding of His-tagged FAS1 protein to the lymphocyte surface. Cells were preincubated with anti-{alpha}L (25.3), -{alpha}M (44), or -{alpha}X (3.9) antibody. The cells were incubated with His-tagged FAS1 protein in serum-free medium containing 0.1% BSA for 5 h at 4°C. After lysis, the amount of His-tagged FAS1 protein associated with the cells was determined by Western blotting (IB) with the HRP-conjugated anti-His antibody (left panel). Cross-reactivity of integrin antibodies with FAS1 protein was analyzed by Western blot (right panel). Representative results of three independent experiments are shown.

Stabilin-2 mediates lymphocyte adhesion in HHSECs via its FAS1 domain
Stabilin-2 is a large, multifunctional glycoprotein expressed in the SECs of the spleen, lymph nodes, and liver [18 ]. We showed that stabilin-2 is localized to the endothelial cells of the sinus in the liver (Fig. 5A ). As HHSECs are resident cells lining the hepatic sinusoidal wall and are therefore in intimate contact with leukocytes passing through the liver, we hypothesized that stabilin-2 involves the tethering of lymphocytes to SECs. To investigate the functional role of stabilin-2 in a more physiological context, we performed experiments with HHSECs using lentivirus-mediated gene silencing. The HHSECs strongly expressed stabilin-2 (Fig. 5B and 5C) and were transduced with a lentiviral vector expressing shStab-2 or shCont. Flow cytometric analysis confirmed that HHSECs transduced with shCont or shStab-2 showed similar transduction efficiencies (Fig. 5B , left). As shown in Figure 5 , B (right) and C, shStab-2 but not the control shRNA resulted in the effective down-regulation of stabilin-2 protein expression. To analyze whether stabilin-2 is involved in PBL binding to HHSECs, adhesion assays were performed using human PBLs under two different conditions. Under static conditions, lymphocyte binding to HHSECs was decreased significantly by the transduction of shStab-2 (Fig. 5D and 5E) . shCont had no effect. We also performed adhesion assays under nonstatic conditions, mimicking lymphocyte binding to HHSECs under flow conditions in the blood. Lymphocyte binding to HHSECs was inhibited significantly by shStab-2 (Fig. 5F) . To confirm that the FAS1 domain was involved in the heterophilic interaction, we examined the inhibitory effect on the binding of PBLs under flow conditions. The binding of PBLs was inhibited by pretreatment with Stab-F5 and Stab-F6 but not with Stab-E3 or the control Nus (Fig. 5G) . Stab-F5 protein inhibited the binding of lymphocytes in a dose-dependent manner (Fig. 5H) . These data suggest that stabilin-2 plays an important role in lymphocyte binding to HHSECs under physiological condition, and the FAS1 domains of stabilin-2 are crucial for the interaction between stabilin-2 and lymphocytes.


Figure 5
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  		Figure 5. Stabilin-2 mediates lymphocyte binding via its FAS1 domains in HHSECs. (A) Localization of stabilin-2 in human liver tissues using polyclonal antibody (left) and mAb (right) anti-stabilin-2. Original scale bars, 1 µm. (B) Transduction efficiency. At 48 h after infection, the expression of GFP (left panel) and the suppression of stabilin-2 expression (right panel) were analyzed by flow cytometry. (C) Confirmation of shRNA-induced suppression of stabilin-2 expression. HHSECs were infected with a lentivirus containing shStab-2 or shCont. At 72 h after infection, HHSECs were stained with 4'-diamidino-2'-phenylindole (blue) and polyclonal anti-stabilin-2 antibody. Original scale bars, 50 µm. (D) Representative images of lymphocyte binding by HHSECs infected with a lentivirus containing shStab-2 or shCont under static conditions. Original scale bars, 50 µm. (E) Microscopic quantitation of lymphocyte binding by HHSECs infected with a lentivirus containing shStab-2 or shCont under static conditions. The results are expressed as the number of adherent cells per millimeter2. The results are the means ± SD of at least three experiments. ANOVA, ***, P< 0.001. (F) Microscopic quantitation of lymphocyte binding by HHSECs infected with a lentivirus containing shStab-2 or shCont under flow conditions. The results are expressed as the number of adherent cells per millimeter2. The results are the means ± SD of at least three experiments. ANOVA, ***, P < 0.001. (G) Effects of FAS1 domain proteins on the binding of lymphocytes by L/Stab-2 cells. PBLs were preincubated with the indicated stabilin-2 proteins (10 µM) and added to HHSECs. The results are expressed as percentage of adhesion when compared with control Nus protein. The results are the means ± SD of at least three experiments. ANOVA, **, P < 0.01. (H) Effect of FAS1 domains on lymphocyte binding in HHSECs. PBLs were preincubated with two different concentrations (1 or 5 µM) of Stab-F5 protein and perfused into a flow chamber. The results are expressed as percentage of adhesion when compared with control protein. The results are the means ± SD of at least three experiments. ANOVA, *, P < 0.05.

Stabilin-2 acts predominantly at the firm adhesion step rather than rolling and transmigration steps
We analyzed the expression of stabilin-2 on the cell surface of HHSEC treated with TNF-{alpha}, IL-1β, and LPS. None of these factors increased the expression of stabilin-2 consistently (Fig. 6A ). In unstimulated HHSEC, lymphocyte adhesion was inhibited by the blockade of stabilin-2, whereas the blockade of ICAM-1 and VCAM-1 had little effect. Conversely, in TNF-{alpha}-stimulated HHSEC, lymphocyte adhesion was inhibited by the blockade of ICAM-1, VCAM-1, as well as stabilin-2 (Fig. 6B) . Treatment of TNF-{alpha}- or IL-1β-stimulated HHSEC with shStab-2 consistently reduced the total number of adhering lymphocytes by 30–40% (Fig. 6C) . However, the inhibition of stabilin-2 had no effect on the percentage of adherent cells, which rolled or transmigrated on TNF-{alpha}- or IL-1β-stimulated HHSEC (Fig. 6D and 6E) . Similar results were observed in unstimulated HHSEC (data not shown). These data suggest that stabilin-2 acts predominantly at the firm adhesion step rather than rolling and transmigration steps. Furthermore, combined inhibition of stabilin-2 and ICAM-1 significantly reduced lymphocyte adhesion compared to the inhibition of stabilin-2 or ICAM-1 alone, suggesting lymphocyte recruitment in hepatic endothelium is mediated by multiple adhesion molecules (Fig. 6F) .


Figure 6
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  		Figure 6. Stabilin-2 acts predominantly at the firm adhesion step rather than rolling and transmigration steps. (A) The surface expression of stabilin-2 is not increased when HHSECs are treated with TNF-{alpha}, IL-1β, or LPS. (B) The effect of the blockade of stabilin-2, ICAM-1, and VCAM-1 on lymphocyte adhesion to unstimulated or TNF-{alpha}-stimulated HHSECs under flow conditions. The results are expressed as percentage of adhesion when compared with isotype control antibody (for ICAM-1 and VCAM-1) or control-scrambled shRNA (for stabilin-2). The results are the means ± SD of at least three experiments. ANOVA, *, P < 0.05; **, P < 0.01. (C) The effect of shStab-2 on lymphocyte adhesion to TNF-{alpha}- or IL-1β-stimulated HHSECs under flow condition. Total adhesion was normalized to the number of adherent cells per millimeter2; t-test, **, P < 0.01. (D and E) The effect of shStab-2 on lymphocyte rolling and transmigration to TNF-{alpha}- or IL-1β-stimulated HHSECs under flow conditions. Phase contrast video recordings made during lymphocyte perfusion were analyzed off-line to determine the percentages of adherent lymphocytes, which roll (D) or transmigrate (E). The results are the means ± SD of at least three experiments. (F) The combination of the blockade of stabilin-2, ICAM-1, and VCAM-1 on lymphocyte adhesion to TNF-{alpha}-stimulated HHSECs. Total adhesion was normalized to the number of adherent cells per millimeter2. The results are the means ± SD of at least three experiments. ANOVA, **, P < 0.01.


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DISCUSSION
 
The interesting feature of stabilin-2 is its tissue distribution, which is restricted to certain subpopulations of endothelial cells, notably SECs in the spleen, lymph nodes, and liver [18 , 19 , 32 ]. The SECs of the mammalian liver (HHSECs) are highly specialized cells, which support lymphocyte adhesion and recruitment under unique, low shear conditions and have a distinct phenotype compared with that of other vascular endothelial cells [5 ]. HHSECs express different patterns of leukocyte adhesion ligand molecules and few selectin molecules, which are considered the major molecules involved in the tethering of lymphocytes in many tissues [33 , 34 ]. Kubes and colleagues [4 ] suggested a minimal role for selectins in leukocyte recruitment via sinusoids using selectin-deficient animals. These observations suggest that other adhesion molecules play a specific role in the tethering of lymphocytes in the hepatic sinusoids. To date, several adhesion molecules are known to mediate the adhesion of lymphocytes in hepatic endothelium under normal and pathological conditions, including vascular adhesion protein 1 (VAP-1) [29 ], ICAM [35 ], VCAM [36 ], and mucosal addressin cell adhesion molecule-1 [37 ]. Among these, VAP-1 is the most prominent representative, as it constitutively expresses in hepatic endothelium but not nonhepatic endothelium and can mediate the tethering and transmigration of lymphocytes in the absence of selectins [28 , 29 , 38 ]. Functional study of selectin/ICAM-deficient animals also suggests an essential role of ICAM in leukocyte recruitment via sinusoids [4 ]. Blockade of VAP-1 with a blocking antibody showed partial inhibition unless used in combination with the inhibition of other adhesion molecules such as ICAM, suggesting lymphocyte recruitment in hepatic endothelium also mediated by combinations of multiple adhesion molecules. In the present study, we suggest that the FAS1 superfamily member stabilin-2 is a strong candidate for the adhesion molecule involved in the adhesion of lymphocytes in the hepatic sinusoidal endothelium. Four lines of evidence support the proposition that stabilin-2 is involved in adhesion lymphocytes. First, stabilin-2 is known to express selectively in SECs, and our results further confirmed a significant, level expression in hepatic sinusoidal endothelium of the human liver. Second, stabilin-2-transfected L cells could support the binding of lymphocytes, and this binding could be inhibited significantly by an anti-stabilin-2 antibody. Third, knockdown of stabilin-2 resulted in a significant reduction in lymphocyte adhesion in primary HHSECs under static and physiological flow conditions. Fourth, results from experiments using function-blocking integrin antibodies suggest that the {alpha}Mβ2 integrin is involved in the adhesion process via interaction with the FAS1 domain of stabilin-2. Thus, our results suggest that the interaction between stabilin-2 and {alpha}Mβ2 integrin plays an important role in lymphocyte recruitment in the hepatic vasculature.

In this study, we demonstrated that stabilin-2 is involved in lymphocyte adhesion in unstimulated and TNF-{alpha}-stimulated HHSEC. Although the total number of adherent cells was increased in TNF-{alpha}-stimulated HHSEC, the effect of stabilin-2 blockade to lymphocyte adhesion was less marked than that in unstimulated HHSEC. This may reflect increased expression of other adhesion molecules such as ICAM and VCAM. We also analyzed the role of stabilin-2 on individual component of adhesion cascade. It was reported that only a small portion of lymphocytes exhibited sustained rolling, and most of the adherent cells underwent rapid arrest without prior rolling [29 ]. Consistent with this finding, stabilin-2 acts predominantly at the firm adhesion step rather than the rolling step in our experimental condition. In this study, cell surface expression of stabilin-2 is not responsive to factors that increase the expression of other adhesion molecules involved in lymphocyte recruitment. It suggests that stabilin-2, which is constitutively expressed in sinus endothelial cells but does not respond to cytokines, may play a distinct role, which is not attributable to other adhesion molecules, of which expression is constitutively minimal but is highly induced by cytokines.

Stabilin-1 and stabilin-2 are highly expressed in the sinusoids of the spleen, liver, and lymph nodes, as reported previously [18 ]. Recent studies have suggested that stabilin-1, the closest homologue of stabilin-2, mediates cell–cell interactions. Blockade of CLEVER-1 using mAb 3-372 inhibited the lymphocyte traffic in the vascular and lymphatic endothelia and the binding of cancer cells in lymphatic vessels [25 , 39 ]. CLEVER-1 also mediates lymphocyte transmigration in vascular and lymphatic endothelia [26 ]. In this point, stabilin-1 could be involved in lymphocyte recruitment in hepatic sinusoids. Whether two proteins are functionally linked is unclear at present. However, double-knockdown of stabilin-1 and stabilin-2 did not show an additive inhibition compared with knockdown of stabilin-2 alone (data not shown), suggesting one possibility that these receptors are acting at different points in the adhesion cascade, such as rolling and transmigration. Furthermore, stabilin-2 is much more abundant on the cell surface than is stabilin-1 and differs from stabilin-1 by its selective expression in the sinusoidal endothelium but not in other vascular endothelia [19 ], suggesting that stabilin-2 plays a specific role in cell–cell interactions in sinusoids.

Stabilin-2 is a member of the FAS1 superfamily with seven FAS1 domains in its extracellular region. FAS1 domains have been suggested to function as an integrin-binding motif in cell adhesion molecules. Recently, we reported that the FAS1 domain of the TGF-β-induced gene product (βig-h3) contributes to cell adhesion through its interactions with {alpha}3β1 integrin [16 , 40 ] and {alpha}vβ5 integrin [13 ]. We also demonstrated that the YH motif of the FAS1 domains of βig-h3 mediates endothelial-cell adhesion and migration by binding to {alpha}vβ3 [14 ]. Gillan and colleagues [15 ] demonstrated that periostin secreted by epithelial ovarian carcinomas is a ligand for {alpha}vβ3 and {alpha}vβ5 integrins and promotes cell motility. Based on this observation, we assumed that stabilin-2 might also mediate cell–cell interactions via an integrin counter-receptor. An examination of the cation dependence of stabilin-2 adhesion to lymphocytes revealed an Mn2+-enhanced binding component, indicative of integrin involvement. Using neutralizing antibodies, we defined an interaction between the FAS1 domains of stabilin-2 and {alpha}Mβ2 integrin and to a lesser extent, {alpha}Lβ2 integrin in lymphocytes. These findings imply that the FAS1 domain is likely to inhibit the migration of lymphocytes to the site of inflammation and may be useful as a therapeutic reagent to down-regulate the generation of unwanted inflammatory responses.

The hepatic SECs have a major role in the filtration of blood, in that they are for specialized receptor-mediated endocytosis of a number of macromolecules such as hyaluronic acids and advanced glycation end-products [41 ]. This is consistent with previous studies that stabilin-2 reported as the hyaluronan receptor for endocytosis [20 , 23 ]. Stabilin-2 has been suggested as the scavenger receptor that binds to bacteria, and endocytoses advanced end-products [21 , 22 ]. Although the fact that adhesive and scavenging functions of stabilin-2 are linked is unknown, it is possible that the scavenging activity may be involved in regulating the level of waste products with a role in inflammation. Further studies are needed for elucidating the multifunctional roles of stabilin-2 under physiological and pathological conditions.

Furthermore, the extracellular domain of stabilin-2 contains multiple EGF-like domains, which contribute to the adhesive functions of several molecules. The extracellular EGF-like domains of scavenger receptor expressed by endothelial cell-I (SREC-I) and its isoform SREC-II are involved in heterophilic interactions [42 ]. EGF-like repeats mediate the lateral and reciprocal interactions of epithelial cell adhesion molecules in homophilic adhesion [43 ]. Particularly, CED-1 [44 ] and Eater [45 ] are composed of multiple, EGF-like domains in its extracellular domain and reported as a potential phagocytosis receptor for cell corpses and microbial pathogen, respectively. Thus, the domain structure of stabilin-2 places it in this interesting group of adhesion molecules, which appears to mediate important and diverse biological processes.

In conclusion, we have reported a newly identified role for stabilin-2 in the adhesion of lymphocytes in hepatic SECs. Stabilin-2 supports the {alpha}Mβ2-integrin-mediated adhesion of lymphocytes, which requires the FAS1 domains of stabilin-2. Blockade of stabilin-2 function significantly abolished the binding of lymphocytes in primary hepatic SECs under static and flow conditions, which suggests that anti-inflammatory therapies aimed at blocking this interaction could be effective.


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ACKNOWLEDGEMENTS
 
This work was supported by National Research Laboratory Program (M10104000036-0110000-01610); the grant No. RT104-01-01 from the Regional Technology Innovation Program of the MOCIE, Advanced Medical Technology Cluster for Diagnosis and Prediction at Kyungpook National University from MOCIE and the Brain Korea 21 Project in 2007.


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FOOTNOTES
 
1 These authors contributed equally in this work. Back

Received January 22, 2007; revised June 25, 2007; accepted June 26, 2007.


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