Published online before print February 3, 2004
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,2
* Department of Anatomy and Program of Immunology, University of California, San Francisco; and
Inflammatory Diseases Unit, Roche Bioscience, Palo Alto, California
2Correspondence at current address: Thios Pharmaceuticals Inc., 5980 Horton Street #400, Emeryville, CA 94608. E-mail: stefan{at}thiospharm.com
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2-macroglobulin and hevin. The next-abundant transcripts encoded several other protease inhibitors, making this protein class the most prominent in HEC. Several endothelial-specific transcripts were also identified, including those encoding E-selectin, vascular cell adhesion molecule-1, vascular endothelial-junctional adhesion molecule, and platelet-endothelial cell adhesion molecule-1. The library contains a great diversity of transcripts, and studies of the encoded proteins will provide further insight into the complex biology of these specialized endothelial cells.
Key Words: cell adhesion • cell trafficking • lymphocyte homing • expressed sequence tags
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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HEC are unique in their appearance and function as compared with EC of other small vessels [5 6 ]. They have a plump morphology and exhibit a well-developed Golgi complex and extensive, rough endoplasmic reticulum, reflecting a highly active biosynthetic state [6 7 ]. A glycocalyx is found on the luminal surface with discontinuous cell-to-cell junctions with no or poorly organized, tight junctions [8 ]. HEV specifically express molecules that facilitate lymphocyte adhesion and extravasation [1 9 10 ].
Recruitment of lymphocytes across HEV, like other examples of leukocyte trafficking, is thought to involve a cascade of steps that begins with the initial tethering and rolling of lymphocytes along the endothelium. This step is followed by chemokine-mediated integrin activation, leading to firm adhesion of the lymphocyte. The final step is the transmigration of the lymphocyte through the endothelial barrier into the tissue parenchyma [1
]. In peripheral lymph nodes of mice, the initial tethering and rolling are mediated through the interaction of L-selectin on lymphocytes with sulfated carbohydrate determinants presented on a discrete set of sialomucins known as peripheral node vascular addressin (PNAd; reviewed in ref. [4
]). In Peyer’s patch HEV, initial interactions of lymphocytes are mediated by L-selectin or the integrin 4ß7, depending on the activation state of the lymphocyte [11
]. In both settings, the CC chemokine, secondary lymphoid-tissue chemokine (SLC; 6C-kine) is a major activating chemokine for T cells [12
13
].
Although a number of the steps in the lymphocyte extravasation cascade are now well-understood, major gaps exist in our molecular knowledge of this process and of HEC biology in general. In lymph nodes deprived of afferent lymph, HEV lose their conspicuous morphology and functional specializations, suggesting a role for the local environment in maintenance of the HEV phenotype that potentially involves the influence of antigens, cytokines, and the extracellular matrix (ECM) [14 15 ].
A major current approach for gaining insights into the biology of a specific cell type or tissue is through gene expression analysis. Global gene expression profiling via microarray technology is limited by the extent to which the transcriptome is represented in the array. Open gene-profiling techniques such as differential display [16 ], serial analysis of gene expression [17 18 ], and partial sequencing of cDNAs randomly selected from a library [expressed sequence tag (EST) analysis; ref. 19 ] do not require a priori knowledge of the genes of interest. To uncover aspects of HEV biology, Girard and Springer [20 ] have applied a differential display approach to discover genes that are selectively expressed in tonsillar HEC versus human umbilical vein endothelial cell (HUVEC). Miyasaka and co-workers [21 22 ] have performed gene-profiling analyses on HEC by sequencing ESTs from cDNA libraries that were generated from isolated mouse HEC. Here, to further characterize the distinctive molecular features of human HEV, we have undertaken an EST approach by single-pass sequencing of a cDNA library that was enriched in HEC-specific transcripts. This library was generated from human tonsilar HEC mRNA by a polymerase chain reaction (PCR)-based technique known as "PCR-select" [23 ]. The tonsil was from a tonsillitis patient, thus also allowing discovery of genes that are up-regulated in HEV during inflammation. Subtraction was performed with lymphocyte cDNA and HUVEC cDNA to obtain cDNAs that are highly specific to HEC. Our approach has yielded evidence for the selective expression of a considerable number of genes belonging to several different functional classes.
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PCR-select library
HEC isolation from a surgical specimen of human tonsils (pediatric tonsillitis case) and construction of the HUVEC- and peripheral blood lymphocyte-subtracted HEC PCR-select library (pCRII vector) have been described previously [24
25
].
Template preparation and sequencing
An aliquot (20 µl) of the library was plated onto a Luria-Bertani agarose dish with 100 µg/ml ampicillin and cultured overnight at 37°C. Ninety-five colonies were picked with a pipette tip and resuspended in a PCR reaction tube. The same pipette tip was then used to replate the colony on an ordered grid for growth overnight and further screening by colony hybridization. The reaction consisted of 0.2 µM M13 forward primer, 0.2 µM M13 reverse primer, 0.2 mM dNTPs, 2 µl 10x buffer, and 0.2 µl Taq polymerase per 20 µl reaction. The PCR consisted of an initial cycle of 2 min at 95°C and then 40 cycles (20 s at 94°C, 20 s and 55°C, 30 s at 72°C), followed by a final extension of 6 min at 72°C and product analysis by agarose electrophoresis. Only those plasmids yielding amplicons above 200 bp were mini-prepped using the QIAprep 96 Turbo kit (Qiagen, Valencia, CA) and sequenced.
Colony hybridization
After sequencing 704 clones (selected by above PCR screen from the first 1396 plasmids), it became clear that about half of the sequences corresponded to only six abundant transcripts encoding 2-macroglobulin (A2M), hevin (HVN), calgranulin A, autotaxin (ATX), a human ortholog of canine DVS27, and the small proline-rich protein-3 (SPRR-3). To eliminate these redundant sequences, all further clones that passed the PCR screen (running number >1396) were subjected to a colony-hybridization screen. For the probe, we used a mixture of amplicons corresponding to the above cDNAs (except DVS27). These were labeled with the Mega-prime DNA labeling system (Amersham Life Sciences, Little Chalfont, UK). Specifically, colonies that had been replated as an ordered grid and allowed to grow overnight were lifted onto a nitrocellulose filter, denatured, and fixed by UV cross-linking. The filters were then prehybridized in Church buffer, followed by incubation with the probe (2x105 cpm/ml) overnight at 68°C. Positive colonies were identified by autoradiography and discarded.
Sequence analysis
Out of a total of >5000 screened plasmids (running number 1–5789), 1049 clones were analyzed (single pass). Sequences were used as probes in BLAST screening [26
] of public sequence databases as well as mapped to the public human genome using the BLAT algorithm of the Human Genome Browser at the University of California, Santa Cruz (http://genome.ucsc.edu). Each assignment to a known cDNA was confirmed by assembling the matching HEC–ESTs with the assigned cDNA using a contig assembly program (Sequencher, Gene Codes, Ann Arbor, MI). In those cases in which a HEC–EST matched only to a database EST (dbEST), all the overlapping ESTs were identified using the Human Genome Browser at the University of California and were assembled into an EST contig.
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10 µm in diameter, Fig. 1
) and possessed a phase-dense cytoplasm with many inclusions. To define the gene complement specifically expressed in HEC, we used suppression subtractive hybridization [23
] to generate a PCR-select library of sequence tags (HEC–ESTs) that are expressed in human tonsil HEC but not in lymphocytes or HUVEC. This approach uses relatively small cDNA fragments (200–800 bp), which are generated with the restriction enzyme Rsa I. Out of the first 1396 plasmids analyzed, only those clones (704) with inserts of >200 bp were sequenced (single pass). The sequences were assembled and analyzed as described in Materials and Methods.
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Figure 1. Purification of MECA-79+ HEC from human tonsils. Purified MECA-79+ HEC were spun in a cytocentrifuge and stained with hematoxylin. The original bar indicates 10 µM.
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Figure 2. Frequency of ESTs mapping to high-abundance transcripts. Sequences were BLAST-screened against the public sequence databases and assembled into contigs, each representing a different transcript. The figure depicts the number of ESTs (frequency  ) mapping to each of the more abundant transcripts ( 3) as a ratio to the total number of 704 ESTs analyzed in the first phase of the project. Data from the second phase of the project (running number 2000, 350 sequences total) were not included in the figure, as ESTs mapping to high-abundance transcripts had been screened out in this phase. A2M (M11313); HVN (X82157); MRP8, S100 calcium-binding protein A8 (calgranulin A, myeloid-related protein-8, X06234); ATX, (L46720); DVS27, DVS27-related protein "up-regulated in cerebral vasospasm" (AB024518); SPRR3, cornifin-3 (AJ243667); mt 16S rRNA, mitochondrial 16S rRNA (J01415:1671–3229); CysA, cystatin A (stefin 1, X05978); LARC, liver and activation-regulated chemokine; C–C chemokine Exodus 1 [macrophage-inflammatory protein-3 (MIP-3), LARC, U77035]; C7A, complement protein component C7 (J03507); ELAM, E-selectin (M30640, includes six ESTs mapping to noncoding DNA within exon #4 of the E-selectin gene, AL021940); rep DNA, a cluster of overlapping repetitive DNA with no precise match to known human genomic or cDNA sequences; PS-PLA1, phosphatidylserine-specific phospholipase A1 (AF035268); FLJ11311, cDNA FLJ11341 encoding a hypothetical 428 amino acid protein MGC20702 with a C-terminal Src homology 3 (SH3) domain (AK002203); SPINK-5, Kazal-type serine protease inhibitor 5 (lympho-epithelial Kazal-type-related inhibitor, AJ228139); VE-JAM, vascular endothelial junction-associated molecule (JAM-2, AF255910); CH, complement factor H (Y00716); IP10, CXC chemokine interferon (IFN)-inducible protein 10 (IP-10; SCYB10, X02530); DCN, decorin (L01131); THC581572, EST contig transcribed from chr 5p12 encoding a hypothetical 130 amino acid protein with no homology to known proteins (AW271273' AV645808BG179638BF344264BF678902; ' denotes 3'5' orientation); CLIC4, intracellular chloride channel 4 (AL117424); HLA-DR, major histocompatibility (MHC) class II human leukocyte antigen-DR (K01171); mt 12S rRNA, mitochondrial 12S rRNA (J01415:648–1601); mt ATP6, mitochondrial adenosine 5'-triphosphate (ATP) synthase/ATPase subunit 6 (J01415:8527–9207); mt COX1, mitochondrial cytochrome oxidase subunit I (J01415:5904–7445); C4BPA, proline-rich C4b-binding protein (M31452); ProCP, prolylcarboxypeptidase (angiotensinase C, L13977); VCAM, vascular cell adhesion molecule 1 (M30257).
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Table 1. Selection of Genes Expressed in HEC Grouped by Functional Class
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Table 2. Expression of Adhesion Molecules and Chemokines in HEV and HUVEC
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Table 3. Transcripts Specifically Expressed in Human Tonsil HEC
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Figure 3. Chromosomal loci of genes preferentially expressed in HEC. All 406 independent transcripts identified in our cDNA library were mapped to the human genome using the browser at the University of California, Santa Cruz (http://genome.ucsc.edu), in conjunction with literature data when available. Each of the 386 transcripts that could be mapped unambiguously to a single nuclear chromosomal band is represented by a red square. Although HEV genes appear somewhat evenly distributed on all chromosomes, the small chromosomes 18–22 and Y are disproportionately poor in HEV-specific genes. Furthermore, there appear to be hotspots of HEV-specific gene expression such as chromosomal bands 3p24.3, 3q13.3, 5p12, and 5q15.
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50% of the total within the first 704 sequences), the degree of normalization of this library is called into question. In the end, our approach identified 406 transcripts, which we infer are expressed selectively or are overexpressed in human HEC. All but three of these transcripts could be mapped to known cDNAs, ESTs, or genomic DNA sequences, providing evidence of the almost-complete coverage of the human genome in the publicly accessible databases. About 10% of the transcripts are apparently transcribed from introns of known genes or from noncoding genomic DNAs (devoid of long ORFs). Some of these match to ESTs deposited in the public database dbEST. Whether these transcripts reflect incompletely processed RNAs, alternative splice variants, or pseudogenes remains to be determined on a case-by-case basis.
Of the many known proteins encoded by transcripts in our HEC library, the most prominent are protease inhibitors. One possible explanation for the predicted abundance of these proteins would be their participation in defense against proteases secreted by the leukocytes that become intimately associated with HEV during the recruitment process. The preponderance of protease inhibitors appears to be a distinctive feature of HEC, as this class of transcripts did not stand out in gene-profiling of other classes of endothelial cells [44
45
]. In a gene-expression profile on mouse peripheral node HEC performed by Miyasaka and colleagues [22
], 3% of all transcripts encoded proteases and protease inhibitors, whereas in our study, over 20% of the transcripts were in this category. The analysis of mouse HEC did not include subtractive steps, and therefore, 35% of all transcripts encoded proteins with housekeeping functions, and others were not necessarily expressed selectively in HEC as compared with other EC.
As mentioned above, the validity of our approach was confirmed by finding several endothelial-associated molecules (E-selectin, VCAM-1, PECAM-1, and VE-JAM) in the library. E-selectin and VCAM-1, the two most abundantly expressed adhesion molecules in our analysis, were previously reported to be absent from normal HEV [2 37 ], although Hogg and colleagues [39 ] found constitutive VCAM-1 expression on HEV in mouse lymphoid organs. In our study, the HEC were derived from an inflamed tonsil, and thus, expression of these adhesion receptors could have been induced, in accordance with previous reports [33 ]. PECAM-1 is known to be expressed on HUVEC [35 ]. Its occurrence in our subtracted library may reflect a significantly higher gene expression in tonsillar HEC relative to HUVEC. VE-JAM, a member of the JAM family, now designated JAM-B [46 ], was originally identified in this library and shown to be highly restricted to EC including HEC [24 ]. This protein has been implicated in transendothelial migration of lymphocyte subpopulations [24 47 ]. The absence of ICAM-1 and ICAM-2 ESTs is consistent with the expression of these proteins in basal HUVEC [42 ].
As noted above, our library contained abundant transcripts for HVN [20 ], which has previously been shown to be highly enriched in HEC relative to HUVEC through a differential display approach. HVN appears to function in an antiadhesive capacity and could play a role in keeping the endothelial junctions open during lymphocyte diapedesis [9 ]. Another protein implicated in antiadhesion, DCN, was also found in our library [48 ]. MAC25 (also known as angiomodulin, tumor-derived adhesion factor, or insulin-like growth factor-binding protein-7) was identified in the present analysis and also in previous studies of HEC-expressed transcripts in mouse [22 ] and human [41 ]. It is a secreted growth factor-binding protein, which accumulates specifically in tumor blood vessels. One study reported colocalization of MAC25 and MECA-79 antigens on microvillous processes near the cell junctions of HEC [22 41 ], whereas another investigation found MAC25 in a predominantly ablumenal pattern in HEV and reported that it can interact with various ECM components and chemokines [49 ]. This versatile protein may serve several functions in the lymphocyte recruitment cascade.
Chemokines are involved in directing the migration of leukocytes and promoting their activation. Different chemokines are selective for leukocyte subpopulations. It was therefore of great interest that several ESTs corresponding to chemokines were found in our analysis. Exodus-1 (MIP-3, LARC, CCL-20) is the only C–C chemokine found in the library (six matching tags). It is a chemoattractant for CCR6-bearing cells such as dendritic cells and certain subpopulations of memory T cells [50
]. Furthermore, it is induced in epidermal keratinocytes by proinflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor and may play an important role in skin inflammation [51
]. Transcripts encoding two CXC chemokines, IP-10 and IL-8, were also present in the library. IP-10 is induced by proinflammatory stimuli in many cell types including EC [52
]. It is a pleiotropic molecule capable of eliciting potent biological effects, including stimulation of monocytes, natural killer and T cell migration, regulation of T cell and bone marrow progenitor maturation, modulation of adhesion molecule expression, as well as inhibition of angiogenesis [53
]. IL-8 is a neutrophil chemoattractant at nanomolar concentrations. Purified endothelial-derived IL-8 as well as recombinant IL-8 were reported to inhibit neutrophil adhesion to cytokine-activated endothelial monolayers and thus protect these monolayers from neutrophil-mediated damage [54
]. Thus, the endothelial-derived IL-8 may function to attenuate inflammatory events at the interface between vessel wall and blood. Another C–C chemokine SLC (6C-kine, CCL-21), previously shown to be expressed in HEC [12
], was not found in our library. This could easily reflect the sampling limitation inherent to open gene expression profiling in which only a limited number of transcripts are analyzed [18
]. As we characterized only 1049 sequences, differentially expressed genes of low abundance would have a low probability of being detected by our strategy. An additional example of a "missed" transcript in our analysis is provided by the HEC-specific sulfotransferase known as HEC-GlcNAc6ST or L-selectin ligand sulfotransferase [25
55
]. Transcripts corresponding to this gene are known to be differentially expressed in tonsilar HEC relative to HUVEC and lymphocytes [25
]. Yet, we failed to detect any ESTs corresponding to this gene.
HEC have been shown to express surface and soluble factors essential in attracting and repelling immune cells. A transcript with many tags in the library is MRP-8, a potent chemoattractant for myeloid cells [56 ]. It has been speculated to play a role in leukocyte transmigration by increasing the ability of the leukocyte to alter its cellular form. Proinflammatory stimuli such as lipopolysaccharide and IL-1 were shown to up-regulate the mouse MRP-8 ortholog chemotactic protein-10 in murine microvascular endothelial cells [57 ]. However, Hogg and colleagues [58 ] showed endothelium-associated MRP-8 was not synthesized by the EC themselves but rather derived from transmigrating myeloid cells. Our data indicate that HEC can synthesize MRP-8, although we did not find transcripts corresponding to its dimerization partner, MRP-14, found in myeloid cells [59 ]. Over 5% of the tags corresponded to ATX, a member of the family of extracellular phosphodiesterases. Recently, this protein was shown to have lysophospholipase D activity [60 ]. Its various biological activities include promotion of tumor cell motility and growth, augmentation of the metastastic potential of cancer cells, and stimulation of microtubule formation in HUVEC [61 ].
A2M was the most abundant transcript in our library (20% within the first 704 tags). This large plasma glycoprotein is as an irreversible inhibitor of a variety of proteinases [62 ]. The secretion of A2M by HEC, together with other proteinase inhibitors such as CysA [63 ] and SPINK5 [64 ], may again reflect the need of these cells to be protected from proteases released by extravasating lymphocytes. More recently, it has been reported that numerous growth factors, cytokines, and hormones bind to A2M through diverse mechanisms [65 ]. Thus, in HEV, A2M could potentially also serve as a presentation molecule for various protein factors involved in leukocyte diapedesis or in maintenance of the HEC phenotype. EC other than HEC also express this protein, for example, the EC lining endometrial blood vessels [66 ].
An abundant transcript (32 ESTs) of particular interest encodes a human ortholog of a canine protein predicted by the DVS27 mRNA. This latter transcript was previously shown to be highly up-regulated in vasospastic arteries during cerebral hemorrhage [67
]. The canine protein is localized to the nucleus, and DVS27 mRNA levels increase highly in response to IL-1 and IL-1ß [67
]. Baekkevold et al. [68
] have now shown that the human DVS27 transcript in fact encodes a HEC-specific nuclear factor with an N-terminal helix-turn-helix putative DNA-binding domain.
Finally, of note is the strong presence in HEC of transcripts encoding proteins known to be associated with activated keratinocytes and skin disorders, such as cornifins, cytokeratin [31
], sciellin [32
], and SPINK5 [64
]. Additionally, it should be noted in this context that the only C–C chemokine found in our library, MIP-3 or LARC, is also expressed at inflamed epithelial surfaces. The significance of these associations remains to be determined.
Our results complement earlier gene-profiling studies performed on HEC in human and mouse. Applying differential display techniques to a human HEC cDNA library, Girard et al. [41 ] found 22 preferentially expressed cDNAs including those encoding MAC25, HVN, mitochondrial proteins, and the Duffy antigen receptor (DARC). In this study, only HUVEC-derived transcripts but not transcripts derived from lymphocytes were subtracted from the HEC-derived mRNA. Miyasaka and colleagues [21 , 22 ] expression-profiled MECA-79+ and mucosal addressin cell adhesion molecule-1+ (MAdCAM-1+) HEC from mouse mesentheric lymph nodes. These workers again found MAC25 and DARC, as well as a cell-surface protein known as endoglin, among the genes preferentially expressed in MECA-79+ (peripheral-type) HEC [22 ]. Mucosal-type MAdCAM-1+ HEC prominently expressed a secreted glycoprotein designated as leucine-rich HEV glycoprotein [21 ], which is a transforming growth factor-ß-binding protein from the leucine-rich repeat family. It is similar to DCN, which we found in our library.
In summary, this library has revealed a large number of HEC-expressed transcripts and may open new avenues for the investigation of this highly specialized microvasculature. Mechanistic insights will not only help elucidate lymphocyte homing into secondary lymphoid organs but will also parallel processes that occur at sites of inflammation.
Received September 1, 2003; revised December 2, 2003; accepted December 14, 2003.
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4ß7 and LFA-1 in lymphocyte homing to Peyer’s patch-HEV in situ: the multistep model confirmed and refined Immunity 3,99-108[CrossRef][Medline]4ßP/ 4ß7) mediated binding of lymphoma cells to high endothelial venules of mucosa-associated lymph nodes Eur. J. Cell Biol. 61,290-298[Medline]4ß1. Facilitation by JAM3 J. Biol. Chem. 277,27589-27592/CCL20 by epidermal keratinocytes and its role in atopic dermatitis Int. Immunol. 13,95-1033 induces chemokines and adhesion molecules: insight into the role of LT in inflammation and lymphoid organ development J. Immunol. 161,6853-6860
inducible protein 10 kD (IP-10): a novel, pleiotropic member of the C-X-C chemokine superfamily Cytokine Growth Factor Rev. 8,207-219[CrossRef][Medline]2-macroglobulin J. Med. 16,129-224[Medline]2-macroglobulin: evidence for selective modulation of FGF-2-induced angiogenesis Blood 97,3450-34572-Macroglobulin production by the human endometrium J. Clin. Endocrinol. Metab. 80,1021-1026[Abstract]This article has been cited by other articles:
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