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Gene Expression Profiling of Mucosal Addressin Cell Adhesion Molecule-1⁺ High Endothelial Venule Cells (HEV) and Identification of a Leucine-Rich HEV Glycoprotein as a HEV Marker¹

Koichi Saito^*, Toshiyuki Tanaka^*, Hidenobu Kanda^*, Yukihiko Ebisuno^*, Dai Izawa^*, Shoko Kawamoto, Kosaku Okubo and Masayuki Miyasaka^2,*

^* Laboratory of Molecular and Cellular Recognition, Osaka University Graduate School of Medicine; and Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
High endothelial venule (HEV) cells support lymphocyte migration from the peripheral blood into secondary lymphoid tissues. Using gene expression profiling of mucosal addressin cell adhesion molecule-1⁺ mesenteric lymph node HEV cells by quantitative 3'-cDNA collection, we have identified a leucine-rich protein, named leucine-rich HEV glycoprotein (LRHG) that is selectively expressed in these cells. Northern blot analysis revealed that LRHG mRNA is 1.3 kb and is expressed in lymph nodes, liver, and heart. In situ hybridization analysis demonstrated that the mRNA expression in lymph nodes is strictly restricted to the HEV cells, and immunofluorescence analysis with polyclonal Abs against LRHG indicated that the LRHG protein is localized mainly to HEV cells and possibly to some lymphoid cells surrounding the HEVs. LRHG cDNA encodes a 342-aa protein containing 8 tandem leucine-rich repeats of 24 aa each and has high homology to human leucine-rich ₂-glycoprotein. Similar to some other leucine-rich repeat protein family members, LRHG can bind extracellular matrix proteins that are expressed on the basal lamina of HEVs, such as fibronectin, collagen IV, and laminin. In addition, LRHG binds TGF-. These results suggest that LRHG is likely to be multifunctional in that it may capture TGF- and/or other related humoral factors to modulate cell adhesion locally and may also be involved in the adhesion of HEV cells to the surrounding basal lamina.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Postnatally, circulating lymphocytes show a certain tissue tropism distinguishing venular endothelial cells in different sites of the body. This behavior is partly determined by the expression by the lymphocytes of adhesion molecules, such as L-selectin and ₄₇ integrin, that can specifically recognize endothelial adhesion receptors expressed by venular endothelial cells in a tissue-specific manner (reviewed in Refs. 1 –3). These vascular counterreceptors for lymphocyte adhesion molecules are termed vascular addressins, because they provide geographical cues or address codes to circulating lymphocytes (1, 4), and those expressed on high endothelial venule (HEV)³ cells in lymph nodes (LNs) and Peyer’s patches have been the most extensively studied. These vascular addressins include the peripheral lymph node addressins (PNAd), which consist of core proteins that display sulfated mucin-type carbohydrates, such as GlyCAM-1 (5), CD34 (6), and podocalyxin (7), and the mucosal addressin, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) (8). It is generally believed that PNAd interacts with L-selectin, directing lymphocytes to the peripheral LNs (5), whereas MAdCAM-1 interacts with ₄₇ integrin, directing lymphocytes to mesenteric LNs and Peyer’s patches (9).

HEV cells express not only vascular addressins but also certain chemokines that enable their specific interaction with lymphocytes. In particular, a CC chemokine, secondary lymphoid tissue chemokine (SLC) (10), is produced by HEV cells and can rapidly activate the _L2 (LFA-1) (11) and ₄₇ (12) integrins on lymphocytes, allowing the lymphocytes to adhere firmly to and transmigrate across the HEV. plt/plt mice, which are genetically deficient in SLC expression by HEV, show extremely impaired migration of T cells and dendritic cells into the LNs and spleen (13), indicating the prime importance of this molecule in the trafficking of certain leukocyte subsets into lymphoid tissues. HEV cells also express a variety of other chemokines, including EBI-1 ligand chemokine (14) and B lymphocyte chemoattractant (15), but their functional significance remains to be clarified.

Given the redundancy observed in chemokines and adhesion molecules (16), it is not difficult to imagine that HEV cells express many more chemokines and adhesion molecules than we know of presently. To address this possibility, intensive investigation has been performed to identify novel molecules expressed specifically in HEVs, and a number of such molecules have been found, including hevin (17), HEV-specific N-acetylglucosamine 6-sulfotransferase (18, 19), junctional adhesion molecule (JAM)-2 (20), vascular endothelial JAM (21), and endoglycan (22). In addition, although not novel, other molecules that were originally reported to be present in nonlymphoid tissues have been found in LNs, particularly in HEV cells. These include mac25/angiomodulin/IGFBP-rP1 (23, 24) and a promiscuous chemokine receptor, Duffy Ag/receptor for chemokine (DARC) (23, 24).

To identify novel molecules in HEV, we previously performed an unbiased gene expression analysis in mouse HEV cells obtained from peripheral LNs (23). In our previous analysis, we prepared a 3'-directed cDNA library that faithfully represented the original mRNA composition of highly purified MECA-79 (PNAd)-positive mouse HEV cells and analyzed 1500 3'-cDNA sequences randomly selected from the library. Subsequently, by comparing these sequences with those obtained from 35 cell types, we found that MECA-79⁺ peripheral LN HEV cells exhibit a unique gene expression profile that includes a few novel genes (23).

In the present study, we extended this analysis to MECA-367 (MAdCAM-1)-positive HEV cells and herein report a list of genes selectively expressed in mouse mesenteric HEV cells. The expression profiling analysis also allowed us to identify as a novel HEV marker a 342-aa protein that contains tandem arrays of the leucine-rich repeat (LRR) motif. This protein apparently belongs to the LRR superfamily (25) and is likely to be a mouse homolog of leucine-rich ₂-glycoprotein, a protein previously identified in human plasma (26). Because the mRNA and protein product are abundantly and selectively expressed in HEV, we designated this molecule leucine-rich HEV glycoprotein (LRHG). LRHG interacted with various extracellular matrix (ECM) proteins, similar to other LRR proteins, and also bound TGF-. These findings suggest that LRHG may be involved in the regulation of HEV-ECM interactions as well as in modulating the adhesive properties of lymphoid cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals and reagents

All animal experiments were performed under the experimental protocol approved by the Ethics Review Committee for Animal Experimentation of Osaka University Graduate School of Medicine. Male C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). MECA-79 (27) and MECA-367 mAbs (4) were kindly provided by Dr. E. C. Butcher (Stanford University, Stanford, CA). The following ECM proteins were obtained from commercial sources: mouse type IV collagen, natural mouse laminin, mouse fibronectin, human type I and type III collagen, and human vitronectin were purchased from Life Technologies (Gaithersburg, MD); human type V and type VI collagen were from Southern Biotechnology (Birmingham, AL); mouse type II collagen was from Elastin Products (Owensville, MO); human fibronectin was from ICN Pharmaceuticals (Aurora, OH); recombinant human TGF-RII/Fc chimera (rhTGFR) and monoclonal anti-human TGF1 Ab (anti-TGF- mAb) were purchased from R&D Systems (Minneapolis, MN).

Construction of cDNA library and sequencing

HEV cells were isolated from mouse mesenteric LNs using the MECA-367 mAb by immunomagnetic selection, and total RNA was prepared from purified MECA-367⁺ cells as described by Izawa et al. (23). Using 160 ng total RNA from purified MECA-367⁺ cells, a 3'-directed cDNA library was constructed and analyzed as described previously (23).

Cell culture

The mouse LN-derived endothelial cell lines KOP2.16 (28), HEC367-1, and HEC367-2 were maintained in DMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 20% heat-inactivated FCS (HyClone Laboratories, Logan, UT), 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 1% (v/v) 100x nonessential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin. The mouse endothelial cell lines, F-2 (29) and SVEC4-10 (30), and a mouse fibroblast L cell line were maintained in DMEM containing 10% heat-inactivated FCS (Dainippon Pharmaceutical, Osaka, Japan) and the same supplements described above.

Lymphocyte-endothelial cell adhesion assay

Purified MECA-367⁺ cells were plated at semiconfluent density in six-well plates. LN cells were then added to the wells and incubated for 2 h at 37°C. Nonadherent lymphocytes were removed by gentle washing, and then lymphocyte adhesion to and transmigration underneath the endothelial cells were observed by microscopy.

Northern blot analysis

Mouse poly(A)⁺ multitissue Northern blots (Clontech, Palo Alto, CA) were hybridized with a ³²P-labeled LRHG or -actin probe (1.0 x 10⁶ cpm/ml) using ExpressHyb hybridization buffer (Clontech). RNA from various mouse tissues was isolated using TRIzol (Life Technologies) according to the manufacturer’s instructions. The samples were fractionated on a 0.8% agarose gel containing 17% formaldehyde and transferred to Hybond-N⁺ nylon membranes (Amersham Pharmacia Biotech, Uppsala, Sweden). The filters were hybridized with the LRHG or -actin probe as described above. In certain tissues, such as skeletal muscle, kidney, and testis, the -actin probe detected not only the standard 2-kb band but also smaller bands (1.6–1.8 kb), which represent -actin isoforms.

cDNA library screening and cloning of LRHG cDNA

A cDNA fragment of LRHG was labeled with HRP using the ECL direct nucleic acid labeling and detection system (Amersham) and was used to screen a mouse liver 5'-STRECH PLUS TriplEx cDNA library (Clontech). Approximately 1.2 x 10⁵ PFU were immobilized on Hybond-N⁺ nylon membrane and then hybridized with the HRP-labeled probe (700 pg/ml) according to the manufacturer’s instructions.

RT-PCR analysis

First-strand cDNA synthesis from total RNA (1 µg) was performed using Ready-To-Go (Amersham) with an oligo(dT) primer. PCR was conducted using a sense primer (5'-GATGATGGCTGGGGTGTGCTG-3') and an antisense primer (5'-AACTGCTTTGGTGACCCCTGAAAC-3') specific to mouse LRHG and ExTaq polymerase (TaKaRa, Otsu, Japan) under the following conditions: 94°C for 1 min; 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, 27 cycles; 72°C for 5 min. As a control, a primer pair for mouse -actin (5'-ATGGATGACGATATCGC-3' and 5'-ATGAGGTAGTCTGTCAGGT-3') was used. PCR products were analyzed by agarose gel electrophoresis.

In situ hybridization

The LRHG cDNA in pTriplEx plasmid was transcribed into digoxigenin-labeled antisense RNA with T3 polymerase (Stratagene, La Jolla, CA) or sense RNA with T7 polymerase (Toyobo, Osaka, Japan), using the DIG RNA Labeling Mix (Boehringer Mannheim, Mannheim, Germany). Frozen mesenteric LN sections (10 µm) from C57BL/6 mice were hybridized with digoxigenin-labeled RNA probe (10 ng/µl) and reacted with 1.5 U/ml alkaline phosphatase-conjugated anti-digoxigenin (Boehringer Mannheim).

Production of recombinant LRHG and its purification

An open reading frame of LRHG cDNA was inserted into a pBAD-myc-His vector (Invitrogen, Carlsbad, CA). The resulting expression plasmid containing LRHG-myc-His was transfected into Top10 Escherichia coli (Invitrogen). The recombinant protein was purified using the Xpress System (Invitrogen). Briefly, the E. coli cell lysates were sonicated and subjected to rapid freeze-thaw cycles. The rLRHG protein was purified from the cleared lysate using Ni²⁺-charged columns following the manufacturer’s recommendations. This rLRHG was used in the ECM-binding assay.

To isolate inclusion bodies from LRHG-transformants, the E. coli were suspended in 10 mM KH₂PO₄ (pH 7.0) and 1 mM EDTA (lysis buffer) and sonicated. After centrifugation, the pellet was resuspended in lysis buffer containing 0.5% Triton X-100. Insoluble materials were then washed with H₂O and solubilized in 8 M urea. The rLRHG was purified using a Ni²⁺-charged agarose column and also an anti-myc mAb-conjugated agarose column (Santa Cruz Biotechnology, Santa Cruz, CA). This rLRHG preparation was used in the TGF--binding assay.

Generation of rabbit polyclonal Abs against mouse LRHG

Polyclonal Abs were raised against rLRHG protein by s.c. immunization of rabbits with the protein (100 µg), which had been emulsified in TiterMax Gold (CytRx, Norcross, GA). The polyclonal IgG was affinity purified from immunized rabbit serum using a protein G (Amersham) column and a column conjugated with a LRHG peptide (PADTVHLSVEFS, corresponding to aa 60–71).

Immunohistochemistry

Immunostaining of frozen sections was performed as previously described (31). Briefly, mesenteric LN cryosections that were fixed in acetone and then in 4% paraformaldehyde in PBS were incubated with the anti-LRHG polyclonal Ab. The sections were then incubated with biotin-conjugated anti-rabbit IgG, followed by alkaline phosphatase-conjugated ABC reagent (Vector Laboratories, Burlingame, CA). After gentle fixation in 1% glutaraldehyde, the sections were stained using Vector Red (Vector Laboratories) as a substrate. For two-color staining, the sections were further incubated with FITC-conjugated MECA-367 or MECA-79 mAbs. Purified MECA-367⁺ cells were spun in a cytocentrifuge, fixed with methanol, and incubated with biotin-conjugated MECA-367 mAb. After a washing, the cells were stained with ABC reagent (Vector) and Metal Enhanced DAB (Pierce, Rockford, IL).

LRHG binding to ECM proteins

Various ECM proteins (10 µg/ml) dissolved in 0.1 M Tris-HCl (pH 7.4), 50 mM NaCl were immobilized onto 96-well microtiter plates (Sumilon H; Sumitomo Bakelite, Tokyo, Japan) at 4°C (50 µl/well) (32). The wells were blocked with 3% BSA and incubated with myc-His-tagged LRHG or myc-His-tagged mac25 (provided by D. Nagakubo of our laboratory) or myc-His-tagged L-selectin (provided by H. Kawashima of our laboratory; 10 µg/ml). Binding of the rLRHG was detected with HRP-conjugated anti-myc mAb (Invitrogen) and o-phenylenediamine as a substrate.

Binding of ¹²⁵I-TGF- to recombinant LRHG

Recombinant myc-His-tagged LRHG, myc-His-tagged L-selectin, rhTGFR, and anti-TGF- mAb dissolved in PBS (10 µg/ml) were immobilized onto 96-well microtiter plates (50 µl/well) at 4°C. After blocking with PBS containing 1% BSA and 0.05% Tween 20 (TPBS), ¹²⁵I-TGF- (Amersham) was added to each well, and the plates were incubated for 4 h at 37°C. The wells were then washed with TPBS, and the bound radioactivity was counted. To verify binding specificity, rLRHG was first incubated with anti-myc mAb-conjugated agarose (Santa Cruz Biotechnology) or Ni²⁺-charged agarose (Invitrogen). Unbound materials were immobilized onto 96-well microtiter plates, and the binding assay was performed as described above.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Purification of MECA-367⁺ HEV cells from mouse mesenteric LN

We first obtained an HEV-enriched stromal cell fraction from mouse mesenteric LNs, as we described previously (23). This stromal cell fraction was substantially enriched with HEV cells; 7.5% of the cells were HEV cells as assessed by immunofluorescence staining with the MAdCAM-1-specific mAb, MECA-367 (normally, <0.01% of the LN cells are MAdCAM-1⁺). After gentle trypsin digestion, the stromal cell fraction was further subjected to two rounds of immunomagnetic cell sorting with MACS using the MECA-367 mAb, and, as assessed by flow cytometry, over 90% of the sorted cells were MECA-367⁺ (Fig. 1A). Immunoperoxidase staining of the sorted cells showed that the majority of cells were large nonlymphoid cells expressing MAdCAM-1 (Fig. 1B). In addition, when these cells were plated on culture dishes, mesenteric LN lymphocytes bound to and migrated underneath them avidly, indicating that they have the phenotype and function consistent with their identification as HEV cells (Fig. 1C).

View larger version (68K): [in this window] [in a new window]   FIGURE 1. Purification of MECA-367+ HEV cells from mouse mesenteric LN. A, Flow cytometric analysis of purified MECA-367+ HEV cells () and the stromal cell fraction (·····). B, Purified MECA-367+ HEV cells were spun in a cytocentrifuge and stained with the MECA-367 mAb. Black bar, 10 µm. (C) Lymphocyte binding to purified MECA-367+ HEV cells. Purified MECA-367+ HEV cells were cultured, and added lymphocytes avidly transmigrated underneath them. White bar, 100 µm.

To investigate the gene expression profile of HEV cells, we constructed a 3'-directed cDNA library from the purified MECA-367⁺ HEV cells as we had previously done with MECA-79⁺ HEV cells (23) and subjected these cDNAs to cycle sequencing reactions to collect a total of 2101 cDNA sequences. These short 3'-cDNA sequences are called gene signatures (GSs), because they are unique to individual genes (33). The GS sequences with >90% identity were regarded as identical. Accordingly, they were grouped together and subsequently classified into 1304 independent GSs. By examining these sequences against the GenBank database, we found that 427 of the GSs were derived from genes that had been previously reported, and 877 of them were from unidentified genes. Among the known genes, about one-third encoded ribosomal proteins (Table I), consistent with the notion that HEV cells have intense biosynthetic activity (34).

Further comparison of the gene expression profile of the MECA-367⁺ HEV-derived cDNA library with expression profiles obtained from 35 tissues and cell types and the GenBank database revealed several hitherto unidentified genes in the mouse to be highly expressed in MECA-367⁺ HEV cells. Because one of them (GS12070) was also highly expressed in the liver (see below), we isolated a full length cDNA from a mouse liver cDNA library and determined its complete nucleotide sequence. The full length cDNA was 1.3 kb long and contained a single open reading frame that began at nt 21 and terminated at nt 1049, encoding a putative protein of 342 aa (Fig. 2). Because a cDNA obtained from the MECA-367⁺ HEV cells had an identical nucleotide sequence (data not shown), we reasoned that the same protein was expressed in the liver and MECA-367⁺ HEV cells. The deduced amino acid sequence contained tandem arrays of 8 LRRs; this motif is found in >60 proteins and is thought to be involved in protein-protein interactions (25). As seen in other LRR protein family members, each LRR of this protein contained a well-conserved 11-residue segment (LxxLxLxxN/CxL). This LRR protein had four potential N-linked glycosylation sites, three potential O-linked glycosylation sites, and no apparent transmembrane domain. Although some LRR proteins are proteoglycans, e.g., decorin and biglycan, this LRR protein had no potential glycosaminoglycan attachment sites, such as serine-glycine and serine-alanine pairs. It shared an extremely similar domain structure and high amino acid homology (67%) with human leucine-rich ₂-glycoprotein, a protein of unknown function initially identified in human plasma (26), and hence was likely to be its mouse homolog. Because the high expression of this unique protein in HEV has not been reported before, we designated this protein LRHG.

View larger version (44K): [in this window] [in a new window]   FIGURE 2. Deduced LRHG amino acid sequence. A, Alignment of deduced amino acid sequence of LRHG and human leucine-rich 2-glycoprotein. The identical amino acid residues are shown by a bar. The predicted signal peptide sequence is underlined. Putative N-glycosylation sites are in black boxes, and putative O-glycosylation sites are in circles. The LRR domains are in boxes and conserved sheets in the LRR domains are indicated by arrows. B, Schematic illustration of the LRHG protein. The boxes represent the LRR domains. LRHG has eight LRRs. The numbers indicate the positions of amino acid residues. Nc, putative N-glycosylation site; Oc, putative O-glycosylation site. The nucleotide sequence has been submitted to the EMBL Data Library/GenBank/DDBJ databases with the accession number AB055885.

View larger version (46K): [in this window] [in a new window]   FIGURE 3. Northern blot analysis of LRHG distribution in mouse tissues. A, A mouse tissue poly(A) RNA blot was probed with 32P-labeled LRHG or -actin cDNA. The sizes of the RNA standards are indicated in kilobases (k). See Materials and Methods for an explanation of the multiple -actin bands seen in certain tissues. B, The total RNA (30 µg) from various mouse tissues was separated in a 0.8% agarose gel and hybridized with a 32P-labeled LRHG or -actin probe. The molecular size of ribosomal RNA (28S and 18S) is indicated. MLN, mesenteric LN.

View larger version (72K): [in this window] [in a new window]   FIGURE 4. In situ hybridization analysis of the LRHG in mouse mesenteric LN. Sections were hybridized with a digoxigenin-labeled LRHG antisense riboprobe (A), MAdCAM-1 antisense riboprobe (B), LRHG antisense riboprobe (C), LRHG sense probe (D), MAdCAM-1 antisense probe (E), and GlyCAM-1 antisense riboprobe (F). The sections were then incubated with alkaline phosphatase-conjugated antidigoxigenin, and the digoxigenin-labeled compounds were detected as described in Materials and Methods. Bar, 100 µm.

View larger version (42K): [in this window] [in a new window]   FIGURE 5. RT-PCR analysis of LRHG expression in various cells. Agarose gel electrophoresis analysis of cDNA fragments amplified by PCR using LRHG-specific or -actin primers. LRHG mRNA was detected in purified MECA-367+ HEV cells and purified MECA-79+ HEV cells but not in other cell lines (HEC367-1, HEC367-2, F2, KOP2.16, SVEC4-10, and L cells)

View larger version (51K): [in this window] [in a new window]   FIGURE 6. Immunohistochemical analysis of LRHG expression in mouse mesenteric LN. Double staining of mouse mesenteric LN cryosections with Abs against LRHG (A, C, and F) and MECA-367 (B and D) or MECA-79 (G). E and H show the combined images of C and D and F and G, respectively. The sections were fixed and incubated first with LRHG-specific Ab and then with biotin-conjugated anti-rabbit Ab, enhanced by ABC reagent, and stained by Vector Red (red). Next, the sections were incubated with FITC-conjugated MECA-367 or MECA-79 (green). White bar, 50 µm.

Because some of the LRR proteins that are structurally related to LRHG, such as decorin and biglycan, bind various ECM proteins (42), we next sought to determine whether LRHG could also bind ECM proteins. For this purpose, myc-His-tagged LRHG was added to wells containing immobilized ECM proteins, and after a washing, LRHG binding was examined using an anti-myc mAb. As shown in Fig. 7, LRHG bound to fibronectin, laminin, and various types of collagens moderately, whereas another HEV protein, mac25, bound to type IV collagen strongly (39). A control protein L-selectin that was also myc-His-tagged, similar to LRHG and mac25, did not bind to any of the ECM proteins examined. These results demonstrate that LRHG can bind various ECM proteins, particularly those that accumulate in the basal lamina of vascular beds, and suggest that LRHG may participate in regulating the adhesive interactions of HEV cells with the surrounding ECM proteins in the basal lamina.

View larger version (24K): [in this window] [in a new window]   FIGURE 7. Binding of LRHG to various ECM proteins. Microtiter plates coated with various ECM proteins were incubated with myc-His-tagged LRHG, mac25, or L-selectin protein for 1 h at room temperature. The amount of bound myc-His-tagged protein was determined by ELISA as described in Materials and Methods. Data are shown with standard deviations of triplicate determinations. FN, Fibronectin; LN, laminin; VN, vitronectin; COL, collagen.

LRHG binds TGF-

Certain LRR proteins have been reported to bind TGF- (43). Therefore, we sought to determine whether LRHG can also bind TGF-. Recombinant myc-His-tagged LRHG was immobilized onto a plastic support and subjected to a binding assay with ¹²⁵I-labeled TGF-. As shown in Fig. 8A, myc-His-tagged LRHG protein bound TGF-, whereas this binding was not as strong as that of rhTGFR or anti-TGF- mAb that was used as a positive control in this experiment. As shown in Fig. 8B, LRHG binding to TGF- was specific and not mediated by a minor contaminant(s) in the LRHG preparation we used, because absorption of the recombinant protein with anti-myc-conjugated beads or a Ni²⁺-charged column abrogated the TGF- binding. Another LRR protein, decorin, also bound TGF- (data not shown) as previously demonstrated (43).

View larger version (23K): [in this window] [in a new window]   FIGURE 8. Binding of TGF- to rLRHG. A, 125I-labeled TGF- binding to immobilized rLRHG. rL-selectin was used as an irrelevant myc-His protein. Anti-TGF- mAb and rhTGFR were used as positive controls. B, rLRHG was absorbed with anti-myc-agarose (myc-) or Ni2+-charged agarose (His-) and immobilized on 96-well ELISA plates, and binding assays were performed. The binding is expressed as a percent of 125I-labeled TGF- bound to rLRHG.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Of 2101 sequences that we examined in the MECA-367⁺ HEV cell library, 1304 were apparently derived from independent genes, and >60% were previously unreported, indicating that HEV cells express a large number of unique genes, a majority of which are as yet uncharacterized. Of the known genes, MECA-367⁺ HEV cells expressed typical endothelial markers such as endoglin, ICAM-1, and L6 Ag, confirming their endothelial origin. MECA-367⁺ HEV cells also expressed some genes in common with MECA-79⁺ HEV cells but not CD31⁺ endothelial cells; these common genes may represent HEV-specific genes or genes that are expressed in activated endothelial cells. These include the genes for SLC, KC, mac25, DARC, TAG7, ApoE, and transglutaminase. SLC is constitutively expressed in LN HEV cells (11) and mice deficient in SLC in lymphoid tissues (13) and those deficient in its receptor CCR7 (44) show a selective defect in the migration of T cells and dendritic cells. DARC and mac25 expression in LN HEV cells was previously reported (23, 24). However, the expression of an inflammatory chemokine, KC, and an inflammation-related cytokine, TAG7, in HEV has not been reported previously. Both of these molecules attract and activate nonlymphoid-type inflammatory cells (36, 37), although these cell types do not migrate across HEVs under normal conditions. Currently, we do not know whether these molecules are expressed in HEVs at the protein level, but if so, there must be a mechanism whereby the function of these inflammatory mediators is abrogated. It is interesting that a putative scavenger for chemokines that binds KC, DARC (38), is expressed in HEV cells. ApoE also seems to be highly expressed in both MECA-367⁺ HEV cells and MECA-79⁺ HEV cells, but not in CD31⁺ flat endothelial cells. Although ApoE is a ligand for several lipoprotein receptors and is known to play a major role in the hepatic clearance of remnant lipoproteins (45), it also stimulates the incorporation of ³⁵SO₄ and the production of heparan sulfate in endothelial cells (41). Because intensive incorporation of ³⁵SO₄ and production of heparan sulfate proteoglycans are observed in HEV cells in vivo (3), an increased expression of ApoE may be functionally related to the unique biosynthetic activity of HEV cells. Transglutaminase is another protein expressed in both types of HEV cells but not in CD31⁺ flat endothelial cells. This protein is expressed in human endothelial cells, and its down-regulation leads to alterations in spreading and adhesion (46), although its biological significance remains unknown.

Several genes were found only in MECA-367⁺ HEV cells, and not in MECA-79⁺ HEV cells, normal endothelial cells, or T and B lymphocytes. They include those encoding MAdCAM-1, VE-cadherin, lactadherin, Ly-6C.2, IL-2R -chain, SPI3, cyclin D1, -amylase-2, and MyD118. However, except for MAdCAM-1, the transcripts of these genes appeared at such low to moderate frequencies that the significance of this observation is currently unclear. Also, we do not know whether the proteins encoded by these genes are selectively expressed in MECA-367⁺ HEV cells. Further examination is required to verify the differential expression of these various molecules in HEV cells.

The present study demonstrated that a leucine-rich protein, LRHG, is an HEV marker in LNs and adds it to a growing list of novel molecules expressed preferentially in HEV. LRHG belongs to the LRR family; it bears eight LRR in tandem arrays and has anextremely similar domain structure to and high amino acid homology (67%) with human leucine-rich ₂-glycoprotein, an LRR family member initially identified in human plasma (26). Judging from the extent of the homology, LRHG is likely to be a mouse homolog of this protein. The human leucine-rich ₂-glycoprotein is a secretory protein with characteristics of an acute phase protein, in that its plasma level increases in the early stage of inflammation (47), but otherwise its function is unknown.

Although in situ hybridization analysis showed clearly that the mRNA expression of LRHG is restricted to HEV cells (Fig. 4, A and C), immunohistochemical analysis with a polyclonal Ab showed LRHG staining in several layers of lymphocytes surrounding the HEVs as well as in HEV cells (Fig. 6). The staining in the lymphocytes became fainter the farther away they were from the HEVs (Fig. 6, C and F), indicating that the LRHG protein may be secreted from HEV cells where it binds lymphocytes immigrating into the LN from HEVs; it may then be lost from the surface of the lymphocytes as the lymphocytes migrate further into the LN cortex. Preliminary studies indicate that LRHG binds to a certain type of lymphoid cell, although its exact phenotype remains unclear (K. Saito and T. Tanaka, unpublished observation).

Girard et al. (17) have identified an adhesion-regulating secretory protein, hevin, in HEVs, that has an antiadhesive effect on endothelial cells. Our preliminary studies indicate that LRHG does not have obvious antiadhesive properties (K. Saito and T. Tanaka, unpublished observation). Rather, LRHG binds to various ECM proteins such as fibronectin, laminin, and collagen that are abundant in the basal lamina of HEVs; hence, it may serve to mediate adhesion between HEV cells and the adjacent basal lamina.

LRHG appears to be a TGF--binding LRR protein, similar to decorin and biglycan. The myc-His-tagged rLRHG bound TGF-. At present, we do not know whether TGF- binds to the LRR domain of LRHG. Nevertheless, the ability of LRHG to bind TGF- is interesting because TGF- is a cytokine that negatively regulates cell adhesion. TGF- inhibits lymphocyte adhesion to TNF--stimulated or IFN--stimulated endothelial cells (48). Although little is known about the expression of TGF in the LN paracortex, including in HEVs, it is interesting to speculate that LRHG localized to the HEV area serves as an anchoring molecule for TGF- or the like, thus helping the cytokine to form a concentration gradient around HEVs to regulate lymphocyte adhesiveness and migration in this area. Newly immigrating lymphocytes are likely to be adhesive to the parenchymal ECM, because their integrins have been recently activated by chemokines on the surface of HEVs (11). For these cells to successfully leave the HEV area to migrate further to the appropriate anatomical compartments, including T-dependent areas and follicular regions in the cortex, their adhesion to the ECM may have to be down-regulated by some mechanism(s).

Collectively, a variety of molecules that have been implicated in the regulation of cell adhesion are uniquely expressed in HEV cells. Further studies with LRHG and other molecules differentially expressed in HEV cells, and those molecules apparently expressed selectively in mesenteric but not peripheral LN HEV cells, may help elucidate the complex mechanism of tissue-specific lymphocyte trafficking across HEVs and the subsequent positioning of different lymphocyte subsets into various microcompartments in the LN.

	Acknowledgments

	Footnotes

 
¹ This work was supported in part by a Grant-in-Aid for COE Research from the Ministry of Education, Science, Sports and Culture, Japan; a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture, Japan; and a grant from the Science of Technology Agency, Japan.

² Address correspondence and reprint requests to Dr. Masayuki Miyasaka, Laboratory of Molecular and Cellular Recognition, Osaka University Graduate School of Medicine C8, 2-2, Yamada-oka, Suita, 565-0871, Japan. E-mail address: mmiyasak{at}orgctl.med.osaka-u.ac.jp

³ Abbreviations used in this paper: HEV, high endothelial venule; ApoE, apolipoprotein E; ECM, extracellular matrix; GlyCAM-1, glycosylation-dependent cell adhesion molecule-1; MAdCAM-1, mucosal addressin cell adhesion molecule-1; LN, lymph node; LRHG, leucine-rich HEV glycoprotein; LRR, leucine-rich repeat; PNAd, peripheral node addressin; SLC, secondary lymphoid tissue chemokine; JAM, junctional adhesion molecule; DARC, Duffy Ag/receptor for chemokine; rhTGFR, recombinant human TGF-RII/Fc chimera; GS, gene signature; MyD118, myeloid differentiation 118; SPI3, serine proteinase inhibitor 3; VE, vascular endothelial.

Received for publication April 10, 2001. Accepted for publication November 30, 2001.

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