Expression profile of active genes in mouse lymph node high endothelial cells
Dai Izawa1,
Toshiyuki Tanaka1,
Koichi Saito1,
Hideki Ogihara1,
Takeo Usui1,
Shoko Kawamoto2,
Kenichi Matsubara3,
Kosaku Okubo2 and
Masayuki Miyasaka1
1 Department of Bioregulation, Biomedical Research Center, Osaka University Graduate School of Medicine, and
2 Institute for Molecular and Cellular Biology, Osaka University, 2-2 Yamada-oka, Suita 565-0871, Japan
3 Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-01, Japan
Correspondence to:
M. Miyasaka
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Abstract
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High endothelial venules (HEV) allow rapid and selective lymphocyte trafficking from the blood into secondary lymphoid tissues. Here we report the expression profile of active genes in mouse high endothelial cells (HEC). HEC were first purified from mouse lymph nodes (LN) by magnetic cell sorting with MECA-79 mAb and a 3'-directed cDNA library that faithfully represents the composition of mRNA was constructed. A total of 1495 cDNA sequences were obtained from randomly selected clones. Based on their sequence identity, they were grouped into 754 different species [gene signatures (GS)] of which 335 GS were identified in GenBank. Among the previously identified genes, expression of several endothelial cell surface molecules including endoglin and ICAM-1 was detected in HEC. Comparison of the gene expression profile with that of purified CD31+ flat endothelial cells identified several molecules, such as KC chemokine and Duffy antigen/receptor for chemokines, that are known to be selectively expressed in activated endothelial cells or post-capillary venules. Interestingly, mac25/TAF, which is known to be expressed specifically in tumor vessels and implicated in the regulation of cell adhesion, was highly and selectively expressed in HEC in mouse LN, suggesting that it may participate in regulating HEC-specific functions. Comparison with the expression profiles obtained from 35 different cell types showed at least 22 GS that were apparently specific to HEC. Our results illustrate the expression differences between HEC and CD31+ flat endothelial cells, and will be useful for the identification and characterization of genes specific for HEC.
Keywords: 3'-directed cDNA library, gene expression profile, gene signature, high endothelial venule, lymphocyte homing, mac25
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Introduction
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Most naive lymphocytes preferentially migrate into secondary lymphoid tissues such as lymph nodes (LN) and Peyer's patches by trafficking across specialized postcapillary venules called high endothelial venules (HEV) (for a review, see 1). The endothelial cells of HEV (HEC) are characterized by their distinct cuboidal morphology and discontinuous junctions between adjacent HEC, and also by their luminal presentation of ligands for the leukocyte adhesion molecule L-selectin (1).
While the interaction between L-selectin and its ligand is important in the early steps of lymphocyteHEV adhesive interactions, recent studies have indicated that the molecular mechanism that directs the specificity of lymphocyteHEC interaction is much more complex than initially thought. For instance, in addition to lymphocytes, peripheral blood mononuclear cells also express L-selectin and roll along HEC, but only lymphocytes are arrested by and transmigrate through HEC (2). Therefore, it is likely that HEC express a variety of regulatory molecules such as adhesion molecules, chemoattractants/repellents and extracellular matrix proteins, which may play important roles in the downstream events following primary rolling. However, these molecules remain to be fully characterized.
In an attempt to identify the molecules expressed specifically in HEV, Girard and Springer (3) purified HEC from human tonsils by immunomagnetic selection with HEC-specific mAb MECA-79 and isolated a gene encoding an anti-adhesive secreted protein hevin. This prompted us to examine genes active in mouse HEC by using a method called gene expression profiling. This method entails construction of a 3'-directed cDNA library that faithfully represents the original composition of mRNA species and random sequencing of cDNA clones each of which consists of short nucleotides sequences just upstream of poly(A) tail (4). These short sequences are called gene signatures (GS), since they are unique to individual genes. Active genes are then identified by their sequences and the relative abundance of each transcripts can be estimated by the frequency of appearance of the corresponding GS in the library (4). The resulting gene expression profile hence describes quantitative aspects of the genes active in HEC. This approach has been useful in the identification of cell type-specific genes by comparing the expression profiles obtained from various cell types (5).
Here, we report, for the first time, a list of genes actively expressed in mouse HEC. By comparing the gene expression profile of HEC with those from other cell types, including CD31+ flat endothelial cells, T cells and B cells, we found a set of genes expressed in HEC but not in CD31+ flat endothelial cells. By making comparison with other 3'-directed cDNA libraries already obtained from 35 different cell types, we also found that mouse HEC express a few novel genes previously not registered in the public Expressed Sequence Tag (EST) database. These data will be useful in the future to further dissect the molecular mechanisms by which HEC allow only lymphocytes to bind to and transmigrate through them.
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Methods
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Animals, antibodies and reagents
The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Osaka University Medical School. Specific pathogen-free male C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). The following antibodies were obtained from commercial sources: rat anti-mouse CD34 (RAM34; PharMingen, San Diego, CA), FITC-conjugated rat anti-mouse macrophage (F4/80; Caltag, San Francisco, CA), FITC-conjugated rat anti-mouse CD31 (MEC 13.3; PharMingen) and FITC- or biotin-conjugated goat anti-rat Ig (IgM + IgG, H + L; Southern Biotechnology, Birmingham, AL). Hybridoma cell lines secreting anti-mouse endothelial cell mAb MJ7/18 (6) and MECA-32 (7) were obtained from the Developmental Studies Hybridoma Bank. MECA-79 (8) was kindly provided by Dr E. C. Butcher (Stanford University, CA). Biotin-conjugated MECA-79 was prepared in our laboratory. Anti-VCAM-1 [M/K-1 (9)] was a generous gift from Dr K. Miyake (Saga Medical School, Saga). Hybridoma cell lines were maintained with RPMI 1640 (ICN, Costa Mesa, CA) containing 10 mM HEPES, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol, 10 mM NaHCO3, 2 mM L-glutamine, 1% (v/v) 100xnon-essential amino acids (ICN), 100 U/ml penicillin, 100 µg/ml streptomycin, (Gibco/BRL, Grand Island, NY) and 10% heat-inactivated FCS (Dainippon, Osaka, Japan). Purified MECA-79+ cells, purified CD31+ cells, a mouse fibroblastic L cell line and mouse endothelioma F-2 (10) were maintained in an endothelial cell culture medium consisting of DMEM (Sigma, St Louis, MO) containing the same supplements described above, except for 20% heat-inactivated FCS (lot. 11152365; HyClone, Logan, UT).
Purification of HEC
Purification of HEC from mouse LN was performed using the method described by Girard and Springer (3) with some modifications. Peripheral and mesenteric LN were pooled and minced in washing medium (RPMI 1640 containing 10% FCS). The minced tissues were gently squeezed between two glass slides, suspended in washing medium and allowed to settle for a few minutes. The lymphocyte-rich supernatant was discarded and the remaining stromal elements were digested in a washing medium containing 0.1% type I collagenase (Worthington, Freehold, NJ) and 2 µg/ml DNase I (Sigma) for 45 min at 37°C under continuous stirring (45 r.p.m.). After treatment with collagenase/DNase, the partially dissociated cells were slowly passed through a 22-gauge needle a few times. After a brief centrifugation, the cell pellet was resuspended in ice-cold PBS containing 0.2% BSA (BSA/PBS) and overlaid onto a 40% FCS cushion, and allowed to form a sediment over 1 h on ice at 1 g. After removal of the lymphocyte-rich upper layer, the remaining cells were centrifuged and then digested in PBS containing 0.2% of trypsin (Gibco/BRL) at 37°C for up to 15 min until the cells were dispersed. Digestion was stopped by adding FCS to a final concentration of 10% and cells were treated with 10 µg/ml of DNase I. The material was then passed through a 22-gauge needle twice, followed by filtration through 0.1 mm nylon mesh to obtain a single-cell suspension. HEC were further purified by two rounds of MACS (Miltenyi Biotec, Bergisch Gladbach, Germany). For this purpose, stromal cells were first labeled with biotin-conjugated MECA-79 (10 µg/ml) for 15 min, washed with BSA/PBS, and then stained with a mixture of 10-fold diluted streptavidin-conjugated microbeads (Miltenyi Biotech) and 20-fold diluted streptavidin-conjugated phycoerythrin (PE; Becton Dickinson, Franklin Lakes, NJ) in BSA/PBS. After washing with BSA/PBS, the cells were resuspended in a separation buffer (PBS containing 0.5% BSA and 5 mM EDTA) and purified using a VS separation column (Miltenyi Biotech). Magnetic cell separation was performed once again using a RS separation column. All procedures of magnetic separation were carried out at 4°C.
Isolation of CD31+ endothelial cells
Minced hearts of newborn mice (n = 40) were digested with 0.5% collagenase at 37°C for 15 min and allowed to stand at 1 g for 3 min. The supernatant was collected and washed by endothelial cell culture medium. The remaining undissociated tissue was digested as above a further 3 times. Dissociated cells were purified for CD31+ cells by a MACS RS separation column using FITC-conjugated anti-mouse CD31 and anti-FITC antibody-conjugated microbeads (Miltenyi Biotec). After 5 days of culture, magnetically separated cells (81.2% CD31+) were used for the 3'-directed cDNA library construction.
Immunoperoxidase staining of cytocentrifuged preparation
Purified MECA-79+ cells suspended in PBS containing 10% BSA (105 cells/ml) were cytocentrifuged at 300 g for 10 min, air-dried and fixed in methanol. The cells were then incubated with MECA-79 (2 µg/ml) and washed with PBS containing 0.02% Tween 20. Cells were then stained with biotin-conjugated goat anti-rat Ig and ABC reagent (Vector, Burlingame, CA) using Metal Enhanced DAB (Pierce, Rockford, IL).
RT-PCR analysis
Total RNA from LN or purified HEC was reverse transcribed by using SuperScript II reverse transcriptase (Gibco/BRL) with oligo(dT)18 as a primer. Equal amounts of the reverse transcribed material (6 ng of total RNA equivalent) were used as templates for amplification. To detect GlyCAM-1 mRNA, we used the sense primer 5'-CTGCCTGGGTCCAAAGATGAA-3' and antisense primer 5'-TGACTTCGTGATACGACTGGC-3'. As a control, we used the sense primer 5'-ATGGATGACGATATCGCT-3' and antisense primer 5'-ATGAGGTAGTCTGTCAGGT-3' for mouse ß-actin. PCR amplification was performed with 20 pmol of each primer and 1 U of EX-Taq polymerase (Takara, Otsu, Japan) in a final volume of 50 µl for 3 min at 94°C followed by 17 cycles of denaturation (94°C, 30 s), annealing (60°C, 30 s) and extension (72°C, 60 s). Total RNA samples form cultured endothelial cells and fibroblastic L cells were prepared and reverse transcribed as above. Mouse mac25 mRNA was detected using the sense primer 5'-ATCACTCTGGAGTTCAGC-3' and antisense primer 5'-AAGAGAAGTGTGTCAGGC-3'. PCR amplification was carried out as above for 3 min at 93°C followed by 27 thermal cycles of denaturation (94°C, 30 s), annealing (58°C, 30 s) and extension (72°C, 60 s).
Culture of HEC and phenotypic analysis
Purified MECA-79+ cells were seeded onto a plastic dish and adherent cells were selected after 20 h of incubation. Adherent cells were used for phenotypic analysis after 2030 days of culture. For flow cytometry, cells were detached using cold PBS containing 5 mM EDTA and incubated with optimal concentrations of FITC-conjugated or unlabeled primary antibodies on ice for 15 min. After washing, unlabeled primary antibodies were labeled with FITC-conjugated goat anti-rat Ig. Fluorescently labeled cells were analyzed on an Epics-XL flow cytometer (Coulter, Hialeah, FL). Acetylated low-density lipoprotein (LDL) (Dil-acLDL; Biomedical Technologies, Stoughton, MA) uptake and lymphocyte binding were examined as previously described (11).
Library construction and sequencing analysis
Purified MECA-79+ cells (6x105 cells) were lysed with Tri-Zol reagent (Gibco/BRL) to prepare total RNA. After DNase I digestion, total RNA was further purified with RNeasy RNA purification kit (Qiagen, Hilden, Germany). Using 130 ng total RNA from purified MECA-79+ cells, a 3'-directed cDNA library was constructed and analyzed as described previously (4). Inserted cDNA sequences less than 20 bp and those that had >5% ambiguous nucleotides were eliminated. The cDNA sequences (1495 sequences qualified) were then grouped according to their sequence identity. In each group, the sequence with the fewest ambiguous bases was searched against GenBank (Re. 110) for gene identification using the FastA program. In our analysis, we excluded those GS species that were apparently derived from non-lymphoid tissues. In particular, we found extremely small pieces of salivary glands that were often trapped among cervical LN and non-specifically bound to paramagnetic beads during MACS separation. Thus, there was a small contamination of salivary glandular cells in the MECA-79+ cell preparation. Based on this observation, we eliminated from our analysis those GS species known to be expressed in salivary glands. The 3'-directed cDNA libraries were similarly prepared from CD31+ flat endothelial cells (1680 sequences qualified), CD4+ T cells (1154 sequences qualified), CD8+ T cells (1471 sequences qualified) and B220+ B cells (1075 sequences qualified), and compared with the above mentioned library from HEC. Information about above-mentioned cDNA libraries and those from other cell types is available from our bodymap server (http://bodymap.ims.u-tokyo.ac.jp/).
In situ hybridization
Polyester wax (BDH, Poole, UK) embedded sections were prepared as described previously (12). Briefly, paraformaldehyde-fixed LN were washed with PBS, embedded and stored at 4°C until use. Dried sections (8 µm) were treated in 100% ethanol for 1 min, 100% methanol for 3 min and 100% chloroform for 1 min at room temperature, and used immediately for hybridization. Digoxigenin-labeled single-strand cDNA probe for mac25 was prepared as follows. cDNA was first amplified with sense and antisense primers (5'-ATCACTCTGGAGTTCAGC-3' and 5'-AAGAGAAGTGTGTCAGGC-3'), and the PCR product was used as a template to synthesize digoxigenin-labeled single-strand cDNA probes. The PCR product (200 ng) was mixed with 20 pmol of the antisense or sense primer, 0.5 U of EX-Taq polymerase (Takara) and 5 µl of DigDNA labeling mixture (Boehringer Mannheim, Mannheim, Germany) in a volume of 25 µl, and thermal cycling reaction was performed (93°C for 3 min followed by 25 cycles of 94°C for 45 s, 50°C for 30 s and 72°C for 1 min). Each specimen was incubated at 58°C for 16 h with 80 µl of 5xSSC containing 50% formamide and 2.5 µl of digoxigenin-labeled probe. After triplicate washing in 2xSSC with 50% formamide at 58°C for 20 min each, the detection of the digoxigenin-labeled probe was performed according to the instructions provided by the manufacturer (DIG nucleic acid detection kit; Boehringer Mannheim).
Northern blot analysis
A Multiple Tissue Northern Blot filter (Clontech, Palo Alto, CA) was hybridized with 32P-labeled XcmIBsgI fragment of mac25 cDNA (a kind gift of Drs S. Komatsu and Y. Hayashizaki, Riken, Tsukuba, Japan). Hybridization was carried out at 68°C for 1 h with ExperssHyb solution (Clontech). The blot was then washed at room temperature for 40 min in 2xSSC containing 0.05% SDS and then at 50°C for 40 min in 0.1x SSC with 0.1% SDS. The filter was exposed to X-ray film at 80°C with intensifying screens. The ß-actin probe was purchased from Clontech.
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Results
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Isolation of HEC from mouse LN
In the first step, we prepared highly purified HEC from mouse LN as a source of cDNA library. For this purpose, a stromal cell-rich fraction was obtained from mouse LN as described in Methods, and subjected to collagenase/DNase digestion and then to immunomagnetic cell sorting after staining with HEC-specific mAb MECA-79 (8). At the end of the separation step, at least 90% of cells in the sorted fraction were clearly MECA-79+, while only 3.9% of cells in the initial lymphocyte-depleted stromal cell-rich fraction was MECA-79+ (Fig. 1A
). In agreement with this result, immunoperoxidase staining of cytocentrifuged preparations of magnetically sorted cells showed that the majority of cells were large, non-lymphoid type bearing MECA-79+ molecules on the cell surface (Fig. 1B
). Semi-quantitative RT-PCR analysis showed that mRNA for GlyCAM-1 expressed specifically in HEC (13) was significantly enriched in the magnetically sorted cell fraction (Fig. 1C
). Further phenotypic analysis with flow cytometry showed that the sorted cells were positive for endoglin (6), CD31, CD34, VCAM-1 and MECA-32 (7) expression as summarized in Table 1
. They were positive in acetylated LDL uptake and active in lymphocyte binding and lymphocyte transmigration in vitro. Therefore, we considered these cells to be suitable as a source of cDNA library.

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Fig. 1. Purification of MECA-79+ mouse HEC. (A) Flow cytometry of MECA-79+ cells of LN stromal cell fraction (broken line) and immunomagnetically purified HEC fraction (solid line). Biotin-conjugated MECA-79 binding was detected with PE-conjugated streptavidin. (B) Immunoperoxidase staining of cytocentrifuged preparation of purified HEC with MECA-79. Bar, 10 µm (x1000). (C) Semi-quantitative RT-PCR analysis of purified HEC. Equal amounts of total RNA (6 ng) from LN and immunomagnetically purified HEC were subjected to RT-PCR for GlyCAM-1 and ß-actin mRNA. The PCR products were electrophoresed on agarose gel and stained with CYBR Green. Lanes 1 and 4, whole LN; lanes 2, 3, 5, and 6, purified HEC prepared in two different experiments (lanes 2 and 5 or lanes 3 and 6) were examined.
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Construction of a 3'-directed cDNA library from mouse HEC
To examine the expression profile of active genes in HEC, we constructed a 3'-directed cDNA library (4) from the purified MECA-79+ cells. No in vitro culture was used to amplify MECA-79+ cells before RNA preparation so as not to alter their metabolic state or phenotype. The 3'-directed library consisted of short cDNA fragments bearing MboI site on the 5' end and poly(A) tail on the 3' end, and faithfully represented the composition of mRNA. We subjected these cDNAs to cycle sequencing reactions and collected 1495 cDNA sequences. These sequences were regarded as representing chromosomal gene transcripts and hence were termed as GS. We then compared their sequence identity (sequences with >90% identity were regarded as identical and accordingly grouped together) and found that they were composed of 754 independent GS species. Subsequently, we categorized these species according to their functions, as shown in Table 2
, together with the frequency of their appearance. Among them, 335 GS species represented by 926 sequences were derived from known genes registered in the GenBank and 419 GS species represented by 569 sequences were apparently derived from novel genes. Among the known genes, about one-third were those involved in housekeeping functions (Table 2
, group A). In particular, GS species representing ribosomal proteins were frequently found, consistent with the observation that HEC have numerous mature ribosomes in their cytoplasm and are very active in protein synthesis (1). The rest of the known genes were those with more specialized function as listed in Table 2
(group B).
Comparison of expression profile of HEC with those of other cell types
In the next step, we compared the expression profile of known genes in HEC with those obtained from other cell types such as CD31+ flat endothelial cells, CD4+ T cells, CD8+ T cells and B220+ B cells. We categorized those GS representing known genes and recurrently appearing (>2 times) in the HEV library according to their functions and cellular localization, and also their frequency of appearance, and compared them with those in other libraries (Table 3
). The data showed that the expression profile of HEC was somewhat similar to that of CD31+ flat endothelial cells but quite distinct from those of CD4+ T cells, CD8+ T cells and B220+ B cells. For example, HEC and CD31+ flat endothelial cells commonly expressed a few molecules such as endoglin (6), CD32 (14), CD63 (15), L6 antigen (16) and ICAM-1, but shared little with other lymphocyte subsets. Of note was that HEC expressed a few molecules that were not found in CD31+ flat endothelial cells, such as follistatin-like protein mac25 (17,18), TAG7 (19), GlyCAM-1 (13), Duffy antigen/receptor for chemokines (DARC) (20) and
-1 protease inhibitor (21). In an attempt to identify novel genes specific to HEC, we then compared the gene expression profile of HEC with the previously obtained profiles from 35 different cell types. Among the GS that were apparently specific to HEC, those appearing more than twice in the HEC-derived cDNA library are listed in Table 4
. Homology search against dbEST showed that, among the 22 apparently HE-specific GS, four GS were not registered in dbEST and hence probably were derived from novel genes.
In situ hybridization analysis of mRNA for follistatin-like protein mac25 in mouse LN
mac25 found in our HEV library has a structural similarity to hevin also found in HEV by Girard and Springer (3) in that both have a follistatin-like structure (17). To verify the HEV-specific expression of mac25 mRNA within mouse LN, a digoxigenin-labeled antisense or sense probe was hybridized with tissue sections from mouse LN. As shown in Fig. 2
(A), the antisense probe of mac25 selectively hybridized with HEV (Fig. 2A
), whereas the sense probe did not (Fig. 2B
), indicating that mac25 mRNA was selectively expressed in HEV within mouse LN. No detectable hybridization signal was observed in flat endothelial cells of blood vessels in the mouse LN (Fig. 2A
).

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Fig. 2. In situ hybridization analysis of mac25 expression in mouse LN. LN sections were hybridized with digoxigenin-labeled single-stranded antisense (A) or sense (B) cDNA probe for mac25 mRNA. Note the positive staining of HEV in the parafollicular area. Bars, 50 µm (x200).
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Expression of mac25 mRNA in various mouse tissues and endothelial cell lines
Since mac25 mRNA expression was previously reported in some tissues other than LN such as spleen and kidney (17) in adult mice, we further investigated the tissue expression of mac25 mRNA by Northern blot analysis. In agreement with the previous report, mouse mac25 mRNA was detected in a variety of adult mouse tissues (Fig. 3A
). The 1.3 kb mac25 mRNA was abundantly expressed in the heart and kidney. The mac25 mRNA was also present in the spleen, lung, liver and testis at lower levels, but absent in the brain and skeletal muscle. These results indicated that mac25 mRNA is also expressed in non-HEV cells in various tissues but, within LN, it is restricted to HEV. We then examined the expression of mac25 mRNA in cultured cell lines including those of endothelial origin, some of which support high levels of lymphocyte binding and transmigration (Table 1
) (11) by RT-PCR. Notably, mac25 mRNA was expressed in those endothelial cell lines that support lymphocyte binding and transmigration such as KOP2.16 and HEC but not in those with little lymphocyte binding/transmigration ability such as F-2 (Fig. 3B
). However, the observation that a fibroblastic L cell line expressed mac25 mRNA (Fig. 3B
) in the absence of the lymphocyte binding/transmigration ability suggests that there is probably no causal relationship between the mac25 expression and the ability to support lymphocyte binding and transmigration.

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Fig. 3. Northern blot and RT-PCR analysis of mac25 mRNA expression. (A) Northern blot analysis of mac25 (upper panel) and ß-actin (lower panel) transcripts using mouse multiple tissue blots. (B) PCR was carried out using first-strand cDNAs prepared from KOP2.16, cultured HEC, F-2 or L cells. The products corresponding to mac25 (upper panel) and ß-actin (lower panel) were separated by 2% agarose gel electrophoresis.
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Discussion
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In an attempt to understand the molecular basis of highly specific trafficking of lymphocytes across HEC, we investigated in the present study the active genes expressed in HEC and documented for the first time the gene expression profile of mouse MECA-79+ HEC. By randomly selecting and sequencing cDNA from a non-biased library, we first performed a semi-quantitative analysis of transcripts in HEC. As shown in Table 2
, approximately one-third of transcripts in HEC were those related to protein synthesis and energy production, corresponding to intense biosynthetic activities in HEC (1). We also detected the expression of other endothelial cell surface molecules such as endoglin and ICAM-1 in HEC. Among them, transcripts of endoglin were found 7 times in the HEC library but only once in CD31+ flat endothelial cell library. Since endoglin is a membrane-bound glycoprotein that binds transforming growth factor (TGF)-ß1 and -ß3 and regulates signaling via type I and II TGF-ß receptors (22), it is possible that TGF signals are involved in the regulation of the phenotype and/or function of HEC.
Expression of DARC in HEC but not CD31+ flat endothelial cells may merit further investigation. DARC is a promiscuous chemokine receptor capable of binding various chemokines (20). Although this molecule was initially thought to be a scavenger receptor for chemokines, recent studies have suggested that DARC may play a role in chemokine presentation on endothelial cells (23). Therefore, the possibility can be raised that DARC participates in selective trafficking of lymphocytes across HEC through presenting adhesion-inducing chemokines such as SLC produced by HEC itself (24) or cells in LN. Although transcripts for KC chemokine were found in our HEC cDNA library, the chemokine is chemotactic for neutrophils (25) and the significance of the present observation is currently unknown.
We also identified in HEC a selective expression of
-1 protease inhibitor which can inhibit neutrophil-derived elastase (21). Since
-1 protease inhibitor has been reported to abolish neutrophil binding to extracellular matrix (21), it would be interesting to examine whether
-1 protease inhibitor is involved in negatively regulating neutrophil binding to and transmigration through HEC.
Our analysis also showed that another adhesion-related protein mac25 is strongly expressed in HEC in mouse LN and also in endothelial cell lines which support high levels of lymphocyte transmigration. Mouse mac25, originally identified as a follistatin-like protein (17), is a mouse homolog of tumor-derived adhesion factor (TAF) in humans. TAF can bind to type IV collagen and is implicated in the organization of endothelial cells into tubular structures in vitro (18). Interestingly, TAF is known to be localized specifically in the basement membrane of tumor vessels and has been suggested to play a role in abnormalities often seen in the tumor endothelium such as increased vascular permeability (18). Considering that HEV has a leaky property allowing not only small mol. wt substances but also lymphocytes to pass through, it would be interesting to examine whether mac25/TAF plays a role in this regard in HEV.
By comparing the gene expression profile of HEC with those of other cell types and also with a public database, our results showed that a few novel genes are specifically expressed in HEC. Although the collection of EST of mouse cDNA sequences is expanding rapidly, four GS that were found only in the HEC cDNA library in this analysis have not yet been registered in the EST database, suggesting that these GS are unique to HEC. Further investigation should establish the exact identity of genes from which these GS were derived.
The present study has the following limitations so that no general conclusions about physiological characteristics of HEC can be drawn at this stage. First, the number of active genes identified in HEC is still limited. Second, expression monitoring by random sequencing is not sufficiently sensitive to accurately determine the abundance of mRNAs with relatively low expression levels. For instance, the transcript of the recently cloned high endothelial-specific sulfotransferase gene (26) was not identifiable with our expression profiling method although its expression could be detected by RT-PCR analysis in our purified HEC. While an emerging technology with arrays of unique fragments of cDNA immobilized at high density on solid support (27) may overcome the above-mentioned limitations to a certain extent, it is not applicable to previously unidentified genes. Thus, a high throughput sequencing strategy for gene expression profiling is urgently needed. In summary, our study identified for the first time a number of genes active in mouse MECA-79+ HEC. The new findings should help elucidate the function of HEC. Analysis of a cDNA library similarly established from purified HEC expressing MAdCAM-1 (28) is currently underway in our laboratory and, together with the present results, it will provide further insight into the physiology of HEC in vivo.
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Acknowledgments
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We thank Dr E. C. Butcher and Dr K. Miyake for providing mAb MECA-79 and M/K-1, respectively. We also thank Drs S. Komatsu and Y. Hayashizaki for mouse mac25 cDNA. This work was supported in part by a Grant-in-Aid for COE Research of the Ministry of Education, Science, Sports and Culture, Japan, a Grant-in-Aid for Scientific Research on Priority Areas of the Ministry of Education, Science, Sports and Culture, Japan, and a grant from the Science of Technology Agency, Japan.
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Abbreviations
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DARC Duffy antigen/receptor for chemokines |
EST Expressed Sequence Tag |
GS gene signature |
HEC high endothelial cell |
HEV high endothelial venule |
LDL low-density lipoprotein |
LN lymph node |
PE phycoerythrin |
TAF tumor-derived adhesion factor |
TGF transforming growth factor |
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Notes
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Transmitting editor: K. Takatsu
Received 19 March 1999,
accepted 1 September 1999.
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