High endothelial venules (HEVs): specialized endothelium for lymphocyte migration

High endothelial venules (HEVs) are specialized postcapillary venules found in lymphoid tissues that support high levels of lymphocyte extravasation from the blood. Here, Jean-Philippe Girard and Timothy Springer highlight the unique properties of HEV endothelium, discuss the molecular mechanisms controlling HEV specialization and review evidence suggesting that HEVs could play an important role in the pathogenesis of chronic inflammatory diseases.     

High endothelial differentiation in human lymphoid and inflammatory tissues defined by monoclonal antibody HECA-452.

Lymphocyte traffic into lymph nodes and into mucosa-associated lymphoid tissues is regulated by specialized postcapillary high endothelial venules (HEVs). The authors have produced a rat monoclonal antibody, HECA-452, that detects a human endothelial cell differentiation antigen selectively expressed on high endothelium. In immunoperoxidase studies, HECA-452 intensely stains all HEVs within lymphoid organs. In normal nonlymphoid tissues the antibody stains no vascular endothelium. The antibody, in addition to reacting with high endothelium, cross-reacts with a set of monocytic cells. In pathologic states such as autoimmune thyroiditis and Crohn's disease, known for the development of dense, frequently organized, lymphocytic infiltrates, HECA-452 detects HEV-like vessels containing luminal and intramural lymphocytes, presumably in the process of extravasating. The antigen was not expressed at detectable levels by venules in less heavily infiltrated chronic inflammatory sites nor in acutely inflamed tissues. In lymphoid malignancies, the only vessels stained were morphologically characteristic HEVs in association with areas of residual normal lymphoid tissue or reactive lymphocytic infiltrates. The specificity of HECA-452 for high endothelial cells confirms the highly specialized nature of these vessels and will permit studies of the regulation of high endothelial cell differentiation in vivo and in vitro. The HECA-452 antigen is preserved in paraffin sections of sublimate formaldehyde- and also routinely formalin-fixed tissues. Thus, HECA-452 will be widely applicable for the immunohistologic detection of endothelium specialized for the support of highly increased lymphocyte extravasation in inflammatory sites.     

Heterogeneity of endothelial cells: the specialized phenotype of human high endothelial venules characterized by suppression subtractive hybridization

High endothelial venules (HEVs) are specialized postcapillary venules, found in lymphoid organs and chronically inflamed tissues, that support high levels of lymphocyte extravasation from the blood. Molecular characterization of HEV endothelial cells (HEVECs) has been hampered by difficulties in their purification and in vitro maintenance. To overcome these limitations, we developed a strategy combining the use of freshly purified HEVECs ( approximately 98% positive for the HEV-specific marker MECA-79) and the recently described polymerase chain reaction (PCR)-based cDNA subtraction cloning procedure called suppression subtractive hybridization (SSH). Subtracted probes prepared by SSH from small amounts of total RNA were used to screen a HEVEC cDNA library. This resulted in cloning of 22 cDNAs preferentially expressed in HEVECs, which encode the promiscuous chemokine receptor DARC, mitochondrial components, and matricellular proteins. The latter included hevin, thrombospondin-1, and mac25/IGFBP-rP1, which is a secreted growth factor-binding protein previously found to accumulate specifically in tumor blood vessels. Biochemical and histochemical analysis confirmed the identification of mac25 and DARC as novel markers of the HEVECs. Ultrastructural immunolocalization revealed a noticeable association of mac25 and MECA-79 antigens with microvillous processes near the endothelial cell junctions, suggesting a role for mac25 in the control of lymphocyte emigration. This study shows that PCR-based SSH is useful for cloning of differentially expressed genes in very small samples.     

Plasmacytoid dendritic cells employ multiple cell adhesion molecules sequentially to interact with high endothelial venule cells--molecular basis of their trafficking to lymph nodes

Plasmacytoid dendritic cells (pDCs) are natural type I IFN-producing cells found in lymphoid tissues, where they support both innate and adaptive immune responses. They emigrate from the blood to lymph nodes, apparently through high endothelial venules (HEVs), but little is known about the mechanism. We have investigated the molecular mechanisms of pDC migration using freshly isolated DCs and HEV cells. We found that pDCs bound avidly to HEV cells and then transmigrated underneath them. Two observations suggested that these binding and migration steps are differentially regulated. First, treatment of pDCs with pertussis toxin blocked transmigration but not binding. Second, pDCs were able to bind but not to transmigrate under non-HEV endothelial cells, although the binding was observed to both HEV and non-HEV endothelial cells. Antibody inhibition studies indicated that the binding process was mediated by alphaL and alpha4 integrins on pDCs and by intercellular adhesion molecule (ICAM)-1, ICAM-2 and vascular cell adhesion molecule-1 on HEVs. The transmigration process was also mediated by alphaL and alpha4 integrins on pDCs, with junctional adhesion molecule-A on HEV cells apparently serving as an additional ligand for alphaL integrin. These data show for the first time that pDCs employ multiple adhesion molecules sequentially in the processes of adhesion to and transmigration through HEVs.     

Human endomucin: distribution pattern,expression on high endothelial venules,and decoration with the MECA-79 epitope

Endomucin is a typical sialomucin that we recently identified on the surface of mouse endothelial cells and on putative hematopoetic clusters of the dorsal aorta in the embryo. We have generated a panel of monoclonal antibodies (mAbs) against the extracellular part of human endomucin and polyclonal antibodies against the cytoplasmic part. Using immunohistochemistry endomucin was specifically detected on endothelial cells of blood and lymphatic vessels of all analyzed human tissues. In addition, the polyclonal antibodies stained the epithelium of the epidermis as well as epithelial and myoepithelial cells of the eccrine and apocrine glands in the skin. This nonendothelial staining could only be seen with a subset of mAbs if the staining procedure was amplified. Although high endothelial venules (HEVs) were not significantly stained with mAbs against endomucin, the polyclonal antibodies clearly detected endomucin on HEVs in lymphatic organs of the mouse and human, suggesting HEV-specific glycosylation affecting recognition by the mAbs. Indeed, endomucin isolated from human and mouse lymphoid organs carried the MECA-79 epitope that defines a set of L-selectin ligands on HEVs called peripheral node addressins. We conclude that human and mouse endomucin are endothelial sialomucins with the potential to function as L-selectin ligands.     

Helicobacter-induced chronic active lymphoid aggregates have characteristics of tertiary lymphoid tissue

Susceptible strains of mice that are naturally or experimentally infected with murine intestinal helicobacter species develop hepatic inflammatory lesions that have previously been described as chronic active hepatitis. The inflammatory infiltrates in some models of chronic autoimmunity or inflammation resemble tertiary lymphoid organs hypothesized to arise by a process termed lymphoid organ neogenesis. To determine whether hepatic inflammation caused by infection with helicobacter could give rise to tertiary lymphoid organs, we used fluorescence-activated cell sorting, immunohistochemistry, and in situ hybridization techniques to identify specific components characteristic of lymphoid organs in liver tissue sections and liver cell suspensions from helicobacter-infected mice. Small venules (high endothelial venules [HEVs]) in inflammatory lesions in Helicobacter species-infected livers were positive for peripheral node addressin. Mucosal addressin cell adhesion molecule also stained HEVs and cells with a staining pattern consistent with scattered stromal cells. The chemokines SLC (CCL 21) and BLC (CXCL13) were present, as were B220-positive B cells and T cells. The latter included a na?ve (CD45lo-CD62Lhi) population. These findings suggest that helicobacter-induced chronic active hepatitis arises through the process of lymphoid organ neogenesis.     

Endomucin, a sialomucin expressed in high endothelial venules, supports L-selectin-mediated rolling

Lymphocyte homing to lymph nodes is regulated by transient but specific interactions between lymphocytes and high endothelial venules (HEVs), the initial phase of which is mainly governed by the leukocyte adhesion molecule L-selectin, which recognizes sulfated and sialylated O-linked oligosaccharides displayed on sialomucin core proteins. One of the sialomucin proteins, endomucin, is predominantly expressed in vascular endothelial cells of a variety of tissues including the HEVs of lymph nodes; however, whether it functions as a ligand for L-selectin remains to be formally proven. Here we show that the endomucin splice isoform a is predominantly expressed in PNAd+ HEVs and MAdCAM-1+ HEVs, as seen in non-HEV-type vascular endothelial cells. Using affinity purification with soluble L-selectin, we found that HEV endomucin is specifically modified with L-selectin-reactive oligosaccharides and can bind L-selectin as well as an HEV-specific mAb, MECA-79. Our results also indicated that a 90-100 kDa endomucin species is preferentially decorated with L-selectin-reactive sugar chains, whereas an 80 kDa species represents conventional forms expressed in non-HEV-type vascular endothelial cells in lymph nodes. Furthermore, a CHO cell line expressing endomucin together with a specific combination of carbohydrate-modifying enzymes [core-2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT), alpha-1,3-fucosyltransferase VII (FucTVII) and L-selectin ligand sulfotransferase (LSST)] showed L-selectin-dependent rolling under flow conditions in vitro. These results suggest that when endomucin is appropriately modified by a specific set of glycosyltransferases and a sulfotransferase, it can function as a ligand for L-selectin, and that the endomucin expressed in HEVs may represent another sialomucin ligand for L-selectin.     

Immunohistochemical study of stromal and vascular components of tonsillar polyps: high endothelial venules as participants of the polyp's lymphoid tissue

Tonsillar polyps are nonneoplastic lesions usually composed of variable amounts of lymphoid and vascular and connective tissues. All of them are generally assumed to be hamartomatous proliferations, but the profile of vascular and connective components has yet to be explored. The vascular system of the tonsils is complex and includes highly specialized structures (i.e., high endothelial venules (HEVs)) involved in lymphocyte homing into lymphoid tissues. In 14 tonsillar polyps and 26 control tonsils, an immunohistochemical study was performed using CD34 (blood vessels and HEVs), MECA-79 (HEVs), D2-40 (lymphatic vessels), Ki-67, collagens I and III, fibronectin, and tenascin-C. The polyps showed increased total lymphatic area, whereas the number of blood vessels and lymphatics and the blood vascular area did not differ significantly from those of control tonsils. Rare Ki-67+ endothelial cells were found. In the polyps, we detected, possibly for the first time, HEVs amid lymphoid tissue, and that the amount of the latter correlated positively with HEV density. The polyps also presented lesser amounts of fibronectin and collagens I and III than in normal tonsils, which were distributed in a disorganized fashion. Tenascin-C expression was uncommon in the polyps and control tonsils. Tonsillar polyps are composed of disorganized connective tissue and lymphatic channels which can be considered hamartomatous proliferations. However, the lymphoid component is possibly reactive due to its relationship with the HEVs. The highly differentiated phenotype of the HEVs and their complex biology are not in agreement with what would be expected for a component of hamartomatous nature.     

Novel regulators of lymphocyte trafficking across high endothelial venules

The physiological recruitment of circulating lymphocytes from the blood into secondary lymphoid tissues is an essential homeostatic mechanism for the immune system because it allows lymphocytes to encounter efficiently both their specific cognate antigen and the regulatory cells with which they need to interact, to initiate, maintain, and terminate immune responses appropriately. This constitutive lymphocyte trafficking is mediated by high endothelial venules (HEVs), which are present in secondary lymphoid tissues other than the spleen. There is growing evidence that lymphocyte trafficking across HEVs involves at least three steps, namely, (i) tethering/rolling, (ii) arrest/firm adhesion/intraluminal crawling, and (iii) transendothelial migration (TEM). Although the mechanisms underlying the first two steps have been determined relatively well, the mechanism regulating TEM is only partially understood. In particular, the molecular mechanism driving lymphocyte movement from the apical to the basolateral surface of the endothelial cells (ECs) of HEVs remains ill defined. This step is crucial for successful lymphocyte extravasation, and is thus an important target for therapeutic intervention in various immunological diseases. Here, we review the molecular mechanisms governing lymphocyte-HEV interactions, and highlight possible roles for two HEV proteins, i.e., nepmucin/CD300g and autotaxin, in lymphocyte TEM.     

大鼠肠系膜淋巴结高内皮微静脉内蛋白多糖的分布

目的　研究蛋白多糖在大鼠肠系膜淋巴结高内皮微静脉(HEVs)中的分布,探讨蛋白多糖在淋 巴细胞归巢过程中的调节作用。方法　阳性胶体铁染色—酶连续阻断法,光镜和电镜观察蛋白多糖于HEVs 内的淋巴细胞、内皮细胞、基膜上的分布。结果　HEVs的基膜和邻接基膜的淋巴细胞胞膜呈强阳性染色,能 被透明质酸酶、肝素酶、软骨素酶ABC阻断;电镜显示,胶体颗粒主要排列于基膜的内、外侧及穿越基膜的淋 巴细胞胞膜上,内皮细胞、穿内皮细胞的淋巴细胞和腔内的淋巴细胞不着色。结论　蛋白多糖于大鼠肠系膜 淋巴结HEVs内主要分布于基膜和穿基膜的淋巴细胞胞膜上,可能对归巢淋巴细胞穿越HEVs管壁有调节作 用。     

Microvascular organization of human palatine tonsils

We describe the three-dimensional organization of the microvasculature of human palatine tonsils as revealed by the vascular corrosion casting/scanning electron microscope method and light microscopy of sections. The tonsillar arteries travel in the connective tissue septa and give off many branches. They further branch into arterioles which in turn enter the follicle and the interfollicular region. These arterioles, giving off capillaries en route, reach the subepithelial region where they break up into sinusoidal capillaries. The subepithelial capillary network overlying the follicle protrudes hemispherically towards the crypt, while that overlying the interfollicular region has many switch-back loops of capillaries projected towards the crypt. The subepithelial sinusoids gather into the high endothelial venules (HEVs) which, collecting capillaries in the follicle and the interfollicular region en route, course down into the interfollicular region alongside the follicle. The HEVs surround the lateral and basal surfaces of the follicle and ultimately lead into the ordinary veins in the septa. The subepithelial sinusoids seem to be involved in taking up immunoglobulins secreted by plasma cells and any other substances released by lymphocytes and/or macrophages as well as supplying the tissues with necessary oxygen and nutrients. That the HEVs are downstream to the subepithelial sinusoids suggests that some substances which are taken up into the sinusoids and transported to the postcapillary venules induce differentiation of HEVs and maintain them.     

Oxygen radicals are not a prerequisite for neutrophil-mediated increased vascular permeability

The neutrophil granulocyte is considered to play a key role in the inflammatory process, contributing to the increased microvascular permeability and tissue damage seen at inflammatory sites. The mechanism underlying this process is unknown, but studies in vitro using cultured endothelium and blood polymorphonuclear granulocytes (PMNs) point to an involvement of oxygen-free radicals. In this study, we have used the hamster cheek pouch microcirculatory model to evaluate the impact of radical scavenging enzymes, superoxide dismutase (SOD), and catalase on the microvascular inflammatory reactions induced by leukotriene B4 (LTB4), which is known to induce PMN-dependent plasma leakage in this model. The variables studied were leukocyte adhesion in postcapillary venules, macromolecular permeability as leakage of fluorescent dextran, and emigration of PMNs. SOD and catalase were given in high doses as intravenous infusion and also as superfusion over the cheek pouch. All studied variables increased dramatically upon superfusion of LTB4 (4 nM) over the cheek pouch preparation and remained elevated throughout the 30-minute exposure period. Treatment with SOD and/or catalase did not influence this inflammatory response to LTB4. Nor did SOD and catalase influence the plasma leakage and emigration of PMNs caused by superfusion of the synthetic chemotactic peptide, formylmethionylleucylphenylalanine (400 nM) which from other animal models is known to induce a PMN-dependent plasma leakage. Although free radicals might contribute to the tissue damage seen at some inflammatory sites, we conclude that they are not crucially involved in the interaction between the PMN and the microvascular endothelium induced by the inflammatory mediators LTB4 and formylmethionylleucylphenylalanine, and are thus not a prerequisite for PMN-dependent microvascular permeability.     

Complexity and differential expression of carbohydrate epitopes associated with L-selectin recognition of high endothelial venules.

Carbohydrate ligands for lymphocyte L-selectin are expressed on high endothelial venules (HEVs) in peripheral lymph nodes and sites of chronic inflammation and mediate the recruitment of lymphocytes from the blood into these tissues. In the mouse, these ligands, collectively termed the peripheral lymph node addressin (PNAd), have been shown to contain fucose, sialic acid, and sulfate and to include several HEV glycoproteins including GlyCAM-1, CD34, and MAdCAM-1. Monoclonal antibody (MAb) MECA-79, which binds a sulfate-dependent epitope, recognizes PNAd in both mouse and man. In humans, only CD34 has been identified among the glycoprotein species that react with MECA-79. Although P-selectin is highly expressed in tonsil HEVs, it was not found to react with MECA-79 or to support L-selectin-mediated lymphocyte rolling. To further characterize human PNAd, MAbs were developed against purified PNAd immunoisolated from human tonsil. MAbs JG-1, JG-5, JG-9, and JG-10, like MECA-79, bind HEVs in human tonsil and react similarly in Western blots, and JG-9 and JG-10 also block lymphocyte rolling on purified PNAd. In addition, by competitive ELISA on purified tonsil PNAd, all MAbs were found to react with overlapping epitopes. However, JG-1, JG-5, JG-9, and JG-10 do not recognize mouse PNAd, and unlike MECA-79, they recognize determinants that are sensitive to neuraminidase. Strikingly, the epitope recognized by JG-1, although abundant in tonsil and peripheral lymph node, is absent from appendix HEVs or HEVs in some samples of chronically inflamed skin, even though these HEVs are MECA-79 reactive. Moreover, although JG-5 and JG-9 react well with tonsil, peripheral lymph node, and inflamed skin HEVs, they react only with occasional endothelial cells in appendix tissues. These findings point to significant diversity in the carbohydrate determinants expressed by HEVs and recognized by L-selectin and demonstrate their differential representation in different sites in vivo. These antibodies should be useful in probing the precise structure of human L-selectin ligands.     

大鼠肠系膜淋巴结内淋巴细胞归巢的形态学特征及淋巴细胞功能相关抗原-1、血小板内皮细胞黏附分子-1的表达

目的探讨淋巴细胞穿越高内皮微静脉（HEVs）的形态学表现及淋巴细胞功能相关抗原-1（LFA-1）、血小板内皮细胞粘附分子-1（PECAM-1）的调节作用。方法用光镜、透射电镜和免疫组织化学方法观察大鼠肠系膜淋巴结高内皮微静脉和淋巴细胞穿越其管壁的形态学表现,分析LFA-1、PECAM-1的表达。结果高内皮微静脉中位于内皮细胞内、细胞间和细胞连接间隙内有大量淋巴细胞存在,淋巴细胞以突起或焊点样细胞膜突起与内皮细胞接触,在基底膜内外板之间的透明层也可见淋巴细胞存在;LFA-1主要表达于高内皮微静脉管腔内与内皮细胞接触的淋巴细胞上;PECAM-1主要表达于内皮细胞和穿越其中的淋巴细胞上。结论1．淋巴细胞首先以突起与内皮细胞接触,再通过内皮细胞内和细胞间穿越内皮细胞进入细胞连接间隙,继而穿越基底膜到血管周围鞘,进人淋巴组织;2．LFA-1参与了淋巴细胞与内皮细胞的牢固黏附;3．PECAM-1可能与淋巴细胞跨内皮迁移有关。     

The narrowing of high endothelial venules of the rat lymph node

The lymph node contains blood vessels of a special type, termed "high endothelial venules" (HEVs), which are involved in the process of lymphocyte recirculation. In standard tissue sections, many HEVs exhibit a nearly closed or closed lumen containing small lymphocytes but few, if any, erythrocytes. The question arose as to whether the appearance of HEVs in tissue sections is influenced by the routine method of animal sacrifice and/or of tissue processing. Therefore, the present work investigated the effects on HEVs of sacrificing rats as well as of excising and fixing their nodes with various procedures. It was observed that procedures involving animal bleeding or blood loss from nodes increase the percentage of HEVs exhibiting a nearly closed or closed lumen. The results further revealed that the endothelial thickness and other morphological features of HEVs are modified by this artifactual narrowing of HEVs. The possible significance of the phenomenon is discussed.     

Periarteriolar localization of mast cells promotes oriented interstitial migration of leukocytes in the hamster cheek pouch.

As studied by intravital microscopy, mast cell-dependent inflammatory reactions evoked by antigen or compound 48/80 in the hamster cheek pouch involved leakage of plasma and emigration of leukocytes exclusively from the venules. The leukocyte diapedesis and subsequent tissue migration induced by antigen or compound 48/80 were oriented from the venules towards adjacent arterioles. In contrast, leukocyte emigration induced by a mast cell-independent stimulus, leukotriene B4, did not show preferential orientation towards arterioles. Moreover, mast cells were abundant in the hamster cheek pouch, and they were localized predominantly along arterioles, rather than along venules. Because mast cells are considered to be the source of the chemotactic mediators causing the leukocyte emigration, the periarteriolar mast cell localization may be of functional significance by creating chemotactic gradients between arterioles and venules, thereby promoting oriented and effective interstitial migration of leukocytes. Whether or not a similar mechanism is operative in other species and tissues remains to be established, however, arteriolar predominance of mast cells was observed also in rat calvarial periosteum and in mouse skin.     

蛋白多糖在大鼠小肠集合淋巴小结高内皮微静脉中的分布

目的 研究蛋白多糖(PG)在大鼠小肠集合淋巴小结高内皮微静脉(HEVs)中的分布，探讨PG对淋巴细胞归巢的调节作用。方法 阳性胶体铁染色-酶连续阻断法，光镜和电镜观察PG在HEVs内的淋巴细胞、内皮细胞、基膜上的分布。结果 HEVs的基膜和邻接基膜的淋巴细胞的细胞膜呈强阳性着色，能被透明质酸酶、肝素酶、硫酸软骨素酶ABC阻断；电镜显示，胶体颗粒主要排列于基膜的内、外侧及穿越基膜的淋巴细胞的细胞膜上，内皮细胞、穿内皮细胞的淋巴细胞和腔内的淋巴细胞不着色。结论 分布在HEVs的基膜和穿基膜的淋巴细胞的细胞膜上的PG，主要是硫酸乙酰肝素蛋白多糖、硫酸软骨素蛋白多糖和透明质酸，可能与归巢淋巴细胞穿越基膜密切相关。     

Microvascular architecture of rat nasal associated lymphoid tissue

The blood vascular architecture of the rat nasal associated lymphoid tissue (NALT) was studied by scanning electron microscopy of corrosion casts. To examine the correlation of the vasculature with the distribution of lymphocyte subsets, the NALT was also studied by light microscopy of immuno-stained samples. The NALT was supplied by a branch of the inferior nasal artery which arose from the sphenopalatine artery. This branch reached the bottom of the NALT and ramified arterioles to the follicles and the parafollicular regions. These arterioles ascended toward the subepithelial region, giving off capillaries en route to form a coarse plexus within the follicles and the parafollicular regions. The arterioles reached the subepithelial region and formed a subepithelial capillary network consisting of a single layer of flat meshwork. The follicular, parafollicular and subepithelial capillaries anastomosed one another. The capillaries in each region were gathered into collecting venules, which in turn drained into high endothelial venules (HEVs) in the parafollicular region. The HEVs ran through the parafollicular regions around the follicular perimeters, and flowed into ordinary veins to leave the NALT. Lymphocytes labeled with an anti-T cell antibody were mainly distributed in the parafollicular regions, where HEVs were situated. B cells were mostly observed in the follicular and dome areas. The microvascular structure and its correlation with lymphocyte subset domains in the NALT were essentially similar to those in other mucosa associated lymphoid tissues (MALTs) such as tonsils and Peyer's patches.