High endothelialvenules (HEVs): specialized endotheliumfor lymphocytemigration Jean-Philippe Girard and Timothy A. Springer

High endothelial venules (HEVs? are specialized postcapillary venules found in lymphoid tissues that support high levels of extra- vasation from the . Here, Jean-Philippe Girard and Timothy Springer highlight the unique properties of HEV endotbelium, discuss the mol- ecular mechanisms controlling HE V specialization and review evidence suggesting that HEVs could play an important role in the patbogenesis of chronic inflammatory diseases.

While patrolling the body in search of foreign antigen, The specialized endothelium of HEVs continuously circulate from blood, through Structurd feutures of HEVs lymphoid and other tissues, and back through the lym- The endothelial cells of HEVs have a ‘plump’, al- phatics to the blood. This process, called lymphocyte most cuboidal, appearance very different from the flat recirculation, allows the dissemination of the immune morphology of endothelial cells that line other vessels, response throughout the body, and thereby provides an and are therefore called high endothelial cells by ref- effective immune surveillance for foreign Invaders and erence to their thickness (Fig. 1). Another characteris- alterations in the body’s own cells’J. tic of HIS’s, revealed by light-microscopic examina- The first critical step in lymphocyte migration tion, is the presence of a large number of lymphocytes from circulation into tissue is the adhesion of within their walls. This illustrates the function of HEVs lymphocytes to vascular endothelium. In lymphoid in lymphocyte recruitment, and explains why these ves- organs, lymphocyte adherence and transendothelial sels were implicated in lymphocyte traffic from the time migration occur at specialized postcapillary vascular of their initial description”. sites called high endothelial venules (HEVs). Although At the ultrastructural level, high endothelial cells are HEVs are particularly abundant in the T-cell areas characterized by a prominent Golgi complex, abundant surrounding the B-cell follicles, they serve as the sites polyribosomes and rough endoplasmic reticulum. This of entry both for T and B lymphocytes’. In humans, reveals an intense biosynthetic activity not observed in HEVs are found in all secondary lymphoid organs flat endothelial cells6,‘2. They also contain many mem- (with the exception of , where lymphocyte brane-bound vesicular structures, multivesicular bodies, emigration occurs via the blood sinusoids in the mar- Weibel-Palade bodies and a variety of dense bodies, in- ginal zone), including hundreds of lymph nodes dis- dicating that they are involved in secretion’?. Another persed in the body, and in the phar- important feature of HEVs, revealed by ultrastructural ynx, Peyer’s patches (PI%) in the small intestine, studies, is the existence of discontinuous ‘spot-welded’ appendix, and small aggregates of lymphoid tissue in junctions between high endothelial cells’. These dis- the stomach and large intestine. Moreover, HEV-like continuous junctions differ from the tight junctions vessels are observed in chronically inflamed nonlym- that characterize and arterial endothelium but phoid tissues and are believed to support lymphocyte are similar to the ‘non-occluding’ junctions found in recruitment into these sites4. In contrast to the other postcapillary venules’. This property of HEVs endothelial cells from other vessels, the high endo- probably facilitates the passage of lymphocytes between thelial cells of HEVs have a distinctive appearance, adjacent high endothelial cells and is likely to be one of express specialized ligands for lymphocytes and are the factors allowing massive lymphocyte emigration in able to support high levels of lymphocyte extra- HEVs. vasation5y6. While many reviews have discussed the molecular mechanisms of lymphocyte homing and HE V-specific markers lymphocyte-HEV interactions2,‘-9, most have focused Despite intensive efforts, very few HEV-specific on lymphocytes and the lymphocyte homing receptor markers have been described. The best HEV marker L-selectin, and considerably less attention has been currently available is a carbohydrate epitope recog- given to the HEV side of the interaction. The purpose nized by the monoclonal antibody (mAb) MECA-79 of this review is to highlight the specialized features of (Ref. 13), which stains all HEVs within lymphoid tis- the HEV endothelium that allow lymphocyte emi- sues and does not react with postcapillary venules gration in HEVs to be so efficient, and to discuss the or large vessels in spleen, or nonlymphoid role of the tissue environment in the control of HEV tissues’ ‘+14. MECA-79 mAb inhibits lymphocyte emi- specialization. gration through HEVs into lymph nddes in uivo and endothelial cells17 are not recognized by L-selectin. Simi- larly, CD34 is widely expressed in endothelial cells in most organs, but functions as an L-selectin counter- receptor only when appropriately decorated by specific oligosaccharides zsJ6. Sialic acids are critical for recog- nition since sialidase treatment abolishes both L-selectin binding and lymphocyte adhesion to HEVs in vitro and in vivo16,29. The sLeX molecule has been proposed to function as an HEV ligand for L-selectin30. However, sLeX is expressed in endothelial cells that do not mediate lymphocyte recruitment in uivo31, thereby implying that the biological HEV ligands for L-selectin must be more complex than this simple tetrasaccharide. A 6’-sulfo-sLe” isoform has re- cently been identified as a major capping group of GlyCAM-1 (Ref. 32) and sulfation of both GlyCAMl Fig,. I. Ht$ endothelia~ venule (HEVj in a human tonsr% The (Ref. 331 and CD34 {,Ref. 271 has been shown to be re- ‘plump’ high endothelial cells are stained by in situ hybridrzation quired for L-selectin recognition. Sulfated ligands for with a digoxigenin-labeled cDNA probe encoding hevin, a se- L-selectin have also been detected in rat lymph nodes34. creted protein specifically expressed in HE Vs from human Sulfation may be key to the uniqueness of the HEV tonsils’O. Magnificatzon = x400. ligands. Interestingly, both in humans and rats, the HEV endothelium has been shown to be unique amongst lymphocyte adhesion to lymph node and HEVs vascular endothelium by virtue of its capacity to incor- in vit~o’~J~. Although initially produced against mouse porate large amounts of 35S04 (Refs 4,35). The re- HEVs, the mAb shows a wide crossreactivity among quirement for sulfate in high-affinity L-selectin ligands species I5 . The MECA-79 carbohydrate epitope decorates provides a functional basis for this metabolic special- a family of HEV. counter-receptors for L-selectin, both ization of the HEV endothelium. The critical role of in mouse and human16J7. Another mAb, HECA-452, sulfation in HEV is further reinforced by the fact recognizing a carbohydrate epitope expressed on human that the only HEV-specific mAb currently available, HEVs but not on other vessels, has been described. MECA-79, is also sulfation dependent. Similar to However, unlike MECA-79, this mAb is not HEV spe- L-selectin, MECA-79 recognizes sulfated oligosacchar- cific: HECA-452 recognizes a carbohydrate epitope ides decorating GlyCAM-1 and CD34, and inhibition related to the sialyl-Lewis x and sialyl-Lewis a (sLe” of sulfation of these glycoproteins abrogates recogni- and sLe”) oligosaccharides and, in addition to reacting tion2’. The total absence of crossreactivity of MECA-79 with high endothelium, crossreacts with monocytic cells, with non-HEV cells of the human bodyI suggests that dendritic cells and a subset of skin-homing memory the HEV-specific sulfated oligosaccharides recognized lymphocytes9J8. by MECA-79 and L-selectin are unique structures that Two other HEV markers have been described in the are probably even more complex than 6’-sulfo-sLe”. mouse: (1) the mAb MECA-325 defines an antigen that Clustering of oligosaccharides containing 6’-sulfo-sLe” can be induced in nonlymphoid endothelial cells by on mucin-like domains may be required for recognition interferon y (IFN-y)i9; and (2) the mAb MECA-367 since free oligosaccharides released from GlyCAM-1 recognizes mucosal cell adhesion molecule 1 do not bind with recognizable affinity to L-selectin36, (MAdCAM- )20, a counter-receptor for L-selectin” and and all HEV counter-receptors for L-selectin identified CY$~ integrin 21 that is expressed in mucosal HEVs and to date contain highly O-glycosylated mucin-like do- in venules of intestinal lamina propria but can be in- mains. Recently, MECA-79- ligands for L-selectin were duced in nonmucosal endothelial cells by tumor necrosis identified on KGla hematopoietic cells and neutro- factor cx (TNF-a) and IL-l (Ref. 22). phils25~37~38.CD34 from KGla cells binds less well to L-selectin than CD34 isolated from tonsil stroma, per- HEV-specific O-linked glycosylation of L-selectin haps reflecting an absence of sulfation25. counter-receptors Lymphocyte L-selectin plays a key role in the initial Lymphocyte emigration in HEVs interaction of lymphocytes with HEVs in viva by Molecular mechanisms involved in lymphocyte migration recognizing carbohydrate ligands on the following through HEVs in vivo HEV glycoproteins: glycosylation-dependent cell ad- Lymphocyte migration through HEVs in viuo is a hesion molecule 1 (G~YCAM-~)~~, CD34 (Refs 24,25) very specific and efficient process. Lymphocytes circu- and MAdCAM- (Ref. 17). All of these glycoproteins lating in the blood are able to discriminate between contain highly rigid, extended, serine/threonine-rich, the HEV endothelium and the endothelium lining adja- mucin-like domains that are densely substituted with O- cent nonlymphoid tissuessT39. Approximately 25% of linked oligosaccharides (Fig. 2). Tissue-specific O-linked lymphocytes circulating in HEVs will bind and glycosylation of these three mucin-like proteins is re- emigrate39,40 and it has been estimated that this results quired for L-selectin binding. For example, GlyCAM-1 in as many as 1.4 x lo4 lymphocytes extravasating expressed in lactating mammary gland epithelial cells2* from the blood into a single lymph node (via HEVs) or MAdCAM- expressed in cultured brain-derived every second41. Although it was initially thought that a

Immunology Today 450 Vol. 16 No. 9 1995 HEVs Other vessels L-selectin+ L-selectin- single receptorxounter-receptor pair would explain MECA-79+ MECA-79- the-specifihty of lymphocyte emcgration in HEVs, it is now clear that the molecular mechanisms confering I CD34 1 1 CD34 I this specificity are more complex, with many different GlyCAM- ,l molecules involved in a multistep process (Fig. 3; Ref. 8). Functional inactivation of L-selectin by blocking MAdCAM- antibodies42y43 or by gene knockout44 results in a 99% decrease of lymphocyte migration to peripheral lymph IO nm nodes (PLNs) and a 50% reduction of lymphocyte emi- gration in PP HEVs. This latter result is consistent with the low but significant MECA-79 reactivity with PP f HEVs (Ref. 45) and the expression in PP HEVs of a . MECA-79+ subset of MAdCAM- molecules able to support L-selectin-mediated lymphocyte rolling in vitro (Ref. 17). Moreover, in sheep, pig and rabbit, staining J 3 J of PP HEVs with MECA-79 is as intense as the staining 0 HEV-specific O-linked sulfated oligosaccharides of PLN HEVs, suggesting that, in some species, the re- 0 O-linked oligosaccharides ‘i quirement for L-selectin during lymphocyte emigration / N-linked oligosaccharides in PP’HEVs could be even more criticalIs. Studies with pertussis toxin, which is known to in- Fig. 2. High endothelial venule (HEV)-specific O-linked glycosylation of duce a profound lymphocytosis in humans and mice, counter-receptors for L-selectin. Glycosylation-dependent cell adhesion have shown that a G-protein-mediated activation event molecule I (GlyCAM-1)z3, mucosal addressin cell adhesion molecule 1 is required for lymphocyte emigration in PLN and PP (MAdCAM-1)” and CD34 (Refs 24,2S) are HEV glycoproteins with mucin- HEVs (Ref. 8). Recently, intravital microscopic studies like domains (containing a high percentage of serine and threonine residues) of lymphocyte emigration in mouse PP HEVs have that present dense clusters of O-linked sulfated carbohydrates to lymphocyte revealed that treatment with pertussis toxin has no L-selectin. Although CD34 is expressed in many other vessels in addition to effect on the initial ‘rolling’ interaction of lymphocytes HEVs (Ref. 26), it functions as a high-affinity L-selectin counter-receptor with HEVs but inhibits an activation-dependent ‘stick- only when it is decorated by HEV-specific, sulfated, sialylated, fucosylated, ing’ event required for lymphocyte arrest40. This acti- O-linked oligosaccharides (shown in blue). Sulfation of CD34 is required for high-affinity L-selectin binding and recognition by the HEV-specific mAb vation can be extraordinarily rapid since lymphocyte MECA-79 (Refs 2.5,27). Modified figure reproduced with sticking can occur within l-3 seconds after initiation permission from Ref, 8. of rolling39. Although G-protein-coupled receptors for cytokines of the family have been proposed to play a role in this rapid activation of lymphocyte and this unique topographical distribution is likely to adhesiveness*, the local factors and molecules involved explain why [Ye@,, like L-selectin, is able to mediate in physiological activation of lymphocytes in HEVs lymphocyte attachment and rolling under flow2’. The remain to be defined. c-w,&-vascular cell adhesion molecule 1 (VCAM-1) pair Two lymphocyte integrins involved in the activation- is important for lymphocyte emigration in inflamma- dependent sticking and arrest of lymphocytes in HEVs tion but is not expected to play any role in lymphocyte have now been characterized (Fig. 3). Antibodies binding to HEVs since VCAM-1 is not expressed on against leukocyte function-associated molecule 1 (LFA-1) the latter (Refs 15,46; J-P. Girard and T.A. Springer, (oL&; CDllaKD18) inhibit lymphocyte migration unpublished), except in rat where low levels have been through PLN HEVs by 80% and through PP HEVs by detected4’. 50% (Ref. 43), which is consistent with the constitu- The entire process of lymphocyte sticking to HEVs tive expression of the LFA-1 counter-receptors intercell- (rolling, activation, arrest) takes only a few seconds40, ular adhesion molecule 1 (ICAM-1) and ICAM- on while transendothelial migration and the passage of HEVs. Furthermore, antibodies against OL~P, or its lymphocytes through the HEV basement membrane counter-receptor, MAdCAM-1, can inhibit up to 75% occur in approximately ten minutes48. The molecular of lymphocyte emigration in PP HEVs (Refs 20,43). In mechanisms involved in these two latter steps remain addition to its role in lymphocyte arrest in PP HEVs, poorly characterized. However, modulation of lympho- the a,&-MAdCAM- pair could also mediate the cyte adhesiveness is likely to be critical since migration initial recognition of PP HEVs by some lymphocytes, of lymphocytes through HEV junctions and the base- since a413, has recently been shown to mediate ment membrane requires a succession of adhesive and L-selectin-independent attachment and rolling on de-adhesive states. Hevin, a recently identified major MAdCAM- (Ref. 21). This is more likely to be true secreted component of human HEVs, homologous to for lymphoblasts or memory lymphocytes expressing the extracellular matrix (ECM) adhesion modulator very high levels of a4& than for naive lymphocytes, SPARC (for ‘secreted protein acidic and rich in cys- which are uniformly L-selectinhia4P,l” and probably teine’)‘O, could be one of the factors modulating adhe- absolutely require L-selectin for the initial recognition siveness in HEVs and facilitating lymphocyte trans- of PP HEVs. Interestingly, OL~~,,like L-selectin, is local- endothelial migration. ized on the tips of lymphocyte microvilli, which medi- Other molecules, such as the hyaluronan receptor ate the initial interaction of lymphocytes with HEVs, CD44 (Ref. 49) or the vascular adhesion protein VAP-1

immunology Today 45 1 \/al. 16 No. 9 19% A role for the HE V glycocalyx in lymphocyte emigration? Unlike and other vessels, the luminal sur- face of HEVs is coated by a prominent glycocalyx6. By facilitating the retention (immobilization) of secreted molecules on the luminal surface of HEVs, this thick glycocalyx could play an important role in lymphocyte emigration. For example, the HEV glycocalyx has been proposed to be involved not only in the retention of the L-selectin ligand GlyCAM-1, which is heavily sul- fated and secreted by HEVs, but also in the immobi- lization of the soluble factors predicted to activate lym- tissue phocyte adhesiveness in HEVs (Ref. 7). Other proteins secreted by HEVs, such as the ECM proteins laminin and fibronectin, also seem to be retained in the HEV glycocalyx. Immunohistochemical studies have revealed that, while laminin and fibronectin are only detected in the basement membrane of vessels lined by flat endo- thelial cells, they can be found both at the basal and luminal surface of HEVs (Refs 52,53; J-P. Girard and T.A. Springer, unpublished). Interestingly, antibodies against laminin have been reported to reduce lympho- cyte emigration into PLNs in vivoj4. Moreover, anti- bodies against either fibronectin or the fibronectin re- ceptor, very late antigen 5 (VLA-S), have been found to PLN HEV inhibit significantly the in vitro adhesion of lymphocytes CD34 GIyCAM-1 ? ICAM- ICAM- ? to HEVs in frozen sections of human lymph nodesj3 and the binding of human lymphocytes to cultured rat Step 1 Step 2 Step 3 Step 4 high endothelial cells5’. Therefore, the integrins VLA-5 Attachment Adhesion Sticking Transendothelial (a#,) and VLA-6 (a,&), which are constitutively ex- and rolling triggering and arrest migration pressed on recirculating lymphocytes, and their ECM ligands, fibronectin and laminin, which appear to be Fig. 3. Lymphocyte emigration in high endothelial venules (HEVsl. ia) L!w- retained in the glycocalyx on the luminal surface of phocytes circulating in the blood initiate contact with the hrgh cndothelial HEVs, could play an accessory role in lymphocyte arrest cells via microvilli. This initial adhesion is transzent and is often manrfestcd in HEVs. in rolling of the interacting cells along the HEV endothelmm (step I: attach- ment and rolling). Activation of lymphocyte adhesiveness (step 2: adhesion triggering) results in firm attachment, which becomes stable to physiological Control of HEV specialization by the local shear force (step 3: sticking and arrest). I,ymphocytes then migrate through microenvironment endothelial cell junctions and enter lymphoid tissue after crossing the HEV Components of afferent lymph regulate HEV specialization basal lamina (stage 4: transendothelial migration). The luminal surface of HEVs represent one of the most striking examples HEVs is coated by a prominent glycocalyx and this may play an rmportant of endothelial differentiation. It appears that all of the role in lymphocyte emigration {see text for details). (b) The initial interaction specialized features of HEVs are under the control of of lymphocytes with HEVs in peripheral lymph nodes (PLNsl is mediated b?j the local tissue environment. Indeed, when PLNs are L-selectin, which recognizes the following HE V mu&-like counter-receptors: deprived of afferent lymph, HEVs convert from a high- CD34 and glycosylation-dependent cell adhesion molecule I IGlyCAM-I). to a flat-walled endothelial morphology and lose their Activation of lymphocyte adhesiveness occurs via G-protein-coupled recep- ability to support lymphocyte traffic56,S7. More recently, tors, but the local factors involved in physiological activatton of lymphocytes in HEVs are not yet characterized. The integrin leukocyte function-associated it was shown that ligation of afferent lymphatics also molecule 1 (LFA-1) ((Y&; CDllaICDl8) and rts HEV count&-receptors results in a loss of the luminal staining of HEVs with intercellular adhesion molecule I (ICAM-I) and lCAM-2 play a malor role MECA-79, a tenfold reduction in 35S04 incorporation, in lymphocyte arrest in PLN HEVs. The molecular mechanisms rnz~olt~ed a profound downregulation of GlyCAM-1 mRNA in lymphocyte transendothelial migration remain poorly characterized. levels and a complete inhibition of L-selectin-mediated Modified figure reproduced with permission from Ref. 8. adhesion to HEVs (Refs 58,59). These studies suggest that lymph contains cells or factors that regulate HEV (Ref. 50), have been proposed to play a role in lympho- phenotype and function. One of these factors could be cyte emigration in HEVs, based on results of antigen draining into the lymph from peripheral tis- in vitro assays of lymphocyte adhesion to HEVs. sues, since the lack of antigenic stimulation and subse- However, a recent report showing that CD44 is not quent activation of the PLNs may result in the absence required for lymphocyte recirculation through murine of cytokines required for the induction of HEV charac- HEVs in vivo casts some doubts upon the role of teristics (see below). Consistent with this hypothesis, CD44 as a major lymphocyte receptor for HEVs direct injection of antigen into the node has been (Ref. 51). Therefore, confirmation of a role in lympho- shown to restore the typical HEV morphology in PLNs cyte emigration in HEVs for CD44, VAP-1, or other deprived of afferent lymphs6. molecules identified using in vitro assays, will require The fact that lymph components are required to further in vivo studies. maintain the HEV phenotype illustrates perfectly the critical role of the lymphoid tissue microenvironment when the effects of afferent lymphatic occlusion were in the control of HEV specialization. It also provides reversed, and HEVs had regained their characteristic an explanation for the finding that, although high morphology and phenotype, this population of macro- endothelial cells have been maintained in in vitro cul- phage was not replaced, indicating these macrophages tures60, there has been no report so far that these cells are not required for HEV differentiation”*. By contrast, express the L-selectin counter-receptors GlyCAM-1, arguments in favor of a role of IDCs in HEV differen- MAdCAM- and CD34. By contrast, a recent study tiation are very strong. After reversal of occlusion, the has shown that high endothelial cells rapidly lose the restoration of HEV phenotype and function correlates HEV-specific marker MECA-325 when they are grown with the reappearance of IDCs (Ref. 58). Study of HEV in vitro61. development in lymph node autografts revealed that the first HEVs appeared after the few surviving IDCs Comparison of HEV and blood-brain barrier (BBB) of the graft showed marked proliferation and pro- endotbelium duced amorphous substances, which may induce the Another striking example of modulation of endothe- development of HEVs from kidney capillaries6’. Simi- lial specialization by the local tissue environment is larly, during the development of the human immune provided by the endothelium of the blood-brain bar- system, the appearance of HECA-452+ IDCs in the rier (BBB)62. Special features of this endothelium in- lymph nodes, tonsils and appendix always precedes the clude the presence of tight junctions with very high development of HEVs (Ref. 67). Finally, in chronic electrical resistance between adjacent endothelial cells inflammatory diseases such as rheumatoid arthritis or and the expression of specific markers such as HT7/ autoimmune thyroiditis, the development of HEVs is neurothelin, a member of the immunoglobulin super- also preceded by the infiltration of HECA-452- IDCs family (Fig. 4). The ability of brain endothelial cells to (Refs 68,69). These studies, together with the observa- form a BBB is not intrinsic to these cells but, rather, is tion that ultraviolet irradiation (to which dendritic induced by the brain environment: elegant chick-quail cells are very sensitive) can greatly reduce lymphocyte chimera experiments have revealed that endothelial migration through PLN HEVs (Ref. 57), support the cells from brain capillaries that are permitted to vascu- notion that IDCs play a key role in the induction and larize peripheral tissues become leaky6”; conversely, maintenance of HEV characteristics (Fig. 4). endothelial cells originally derived from peripheral Since HEVs are always associated with dense lym- capillaries form a competent BBB (Ref. 63) and express phocytic infiltration in peripheral lymphoid organs or HT7/neurothelin64 when they vascularize brain tissue. sites of chronic inflammation, and the development of Similarly, transplantation of small lymph node frag- HEVs parallels a large-scale influx of lymphocytes, it ments into kidneys has revealed that capillaries origi- has been suggested that recirculating lymphocytes may nally derived from the renal parenchyma invade the re- be required for HEV differentiation. However, studies sidual autograft and differentiate into HEVs (Ref. 65). of HEV development in athymic or nude mice have These experiments suggest that morphologically and revealed that differentiation of the HEV endothelium is functionally distinct endothelial cell types are not pre- independent of the concentration of lymphocytes in the determined by lineage, and that HEV and BBB endothe- blood’l’. Moreover, the loss of HEV characteristics in lial cells may differentiate under influences from their lymph nodes deprived of afferent lymphatics, despite local microenvironments. normal numbers of recirculating lymphocytes in the In the brain, the cells that are responsible for induc- blood, clearly shows that recirculating lymphocytes are ing endothelial cells to form the tight junctions charac- not sufficient to induce the HEV phenotypes6. Further- teristic of the BBB have been identified. Astrocytes, a more, recent studies suggest that lymphocytes are distinct type of glial cell that surround endothelial cells probably not required for induction of the HEV pheno- and contact the basal lamina via end-feet processes, type: MAdCAM- and MECA-79 are induced on have been shown to induce BBB properties in non-neural HEV-like vessels in the pancreas of IFN-y-transgenic endothelial cells in vivo 66. Astrocyte-conditioned medium severe combined immunodeficiency (SCID) mice: which was found to be sufficient to induce BBB characteris- are deficent in mature T and B lymphocytes”; and tics, including the expression of HT7/neurothelinhL. normal HEVs are present in patients with complete These results emphasize the critical role of the brain DiCeorge syndrome, despite these individuals being microenvironment (astrocytes are the nearest neighbors athymic and devoid of @‘, Therefore, lympho- of brain capillaries) in the control of the specialization cytes are probably not directly involved in the induc- of the BBB endothelium. tion of HEV differentiation and are more likely to play In the case of HEVs, the cell types involved in the an indirect role by allowing a certain level of immune induction and maintenance of the specialized pheno- activity required for the maintenance of the specialized type have not yet been identified. Since subcapsular sinus HEV phenotype. macrophages and interdigitating dendritic cells (IDCs) are depleted from lymph nodes after the occlusion of A role for cytokines and ECM proteins in induction and afferent lymphatics, these cells have been proposed to maintenance of HE V specialization! play a role in the control of the differentiation of the One major difference in the microenvironment of HEV endothelium56-58. However, when the same macro- HEVs and nonlymphoid postcapillary venules is the phage population that is affected by de-afferentiation presence around HEVs of many cytokines generated in was depleted specifically, no effects on the capacity of lymphoid organs during activation of lymphoid cells. HEVs to bind lymphocytes were observed6’. Moreover, Therefore, it is not surprising that cytokines appear to (a) BBB endothelium likely that other microenvironmental factors, particu- larly ECM molecules, are also involved. The compo- sition of the ECM in the vicinity of the high endothe- lial cells has been proposed to influence the permanent HEV phenotype in particular lymphoid organs. This hypothesis is based on the observation that transplan- tation of adult PLNs into a mucosal site, or mesenteric lymph nodes into a peripheral site, did not result in any change of the HEV phenotype, despite probable differences in cytokine and cellular composition of lymph nodes and afferent lymph at mucosal and pe- ripheral sitesT3. It is well known that, in vitro, differen- tiation of endothelial cells can be directed by the ECM on which the cells are grown74. The ECM plays a (b) HEV endotholium crucial role in functional differentiation and cell-type- specific gene expression75 and, in many situations, ECM- induced cell differentiation is reflected in changes in cell shape. For example, ECM induces mammary epithelial cells to adopt a highly columnar morphology required for vectorial secretion of milk and, together with lactogenic hormones, plays a critical role in the induction and maintenance of the differentiated phenotype of these epithelial cells:“. Strikingly, expression of the L-selectin / \ ._.lnminn .. ._ counter-receptor GlyCAM-1, which is generally re- Cvtokines ECM lnterdibitatina stricted to the plump endothelial cells of HEVs in (IFN-y; TNF-a, IL-l, dendrizc cell: II -in\ Lymphoid lymph nodes, is induced in these columnar epithelial ._ ‘V, tissue cells during lactation 28. Therefore, it is tempting to sug- gest that expression of GlyCAM-1 in the highly differ- entiated endothelial cells of HEVs could also require the Fig. 4. Control of endotbelial specialization by the tissue environment. (a) In cooperation of two signals, one originating from ECM the brain, vessels forming the blood-brain barrier (BBB) are charactertzed by proteins and the other from cytokines supplied by the presence of tight junctions with high electrical resistance and the expres- afferent lymph or generated locally. ECM proteins sion of the specific marker HT’llneurothelin. Astrocytes, which contact the important for induction of the plump morphology and basal lamina of these vessels via their end-feet processes, are able to induce these two BBB characteristics in non-neural endothelial cells in vivo. (6) In specialized phenotype of HEV cells could comprise lymphoid tissues, the endothelial cells of high endothelial venules (HEVs) widespread components, such as laminin, that play a have a ‘plump’ morphology, incorporate high levels of sulfate, express spe- key role in differentiation of mammary epithelial cific ligands for lymphocytes defined by the HEV-specific monoclonal anti- cells75, or proteins with more-restricted expression such body (mAb) MECA-79 and are able to support high levels of lymphocyte as the recently identified SPARC-like protein hevin’O. extravasation. All of these characteristics are under the control of the lym- Interestingly, hevin has recently been found to induce a phoid tissue environment. Although the factors and cell types involved in the rounded, plump morphology in endothelial cells cul- induction and maintenance of the specialized HEV phenotype are not well tured in vitro (J-P. Girard and T.A. Springer, unpub- characterized, the important components appear to be cytokines produced lished). However, confirmation of a role for hevin, or locally or supplied by afferent lymph, extracellular matrix (ECM) proteins other ECM proteins, in the differentiation of the HEV surrounding the high endothehal cells and interdigitating dendritic cells. Abbreviations: IFN-y, interferon y; IL-I, interleukin 1; endothelium will require further studies. TNF-ff, tumor necrosis factor LY. HEVs in chronic inflammatory diseases play a critical role in the induction and maintenance HEVs in extralymphoid sites of chronic inflammation in of the HEV phenotype. In mice, MECA-325 and rodents MAdCAM- have been shown to be induced in cul- In the nonobese diabetic (NOD) mouse, which is a tured nonlymphoid endothelial cells by IFN-y (Ref. 19) model for human insulin-dependent diabetes mellitus and TNF-a or IL-1 (Ref. 22), respectively, providing (IDDM), vessels with HEV characteristics (e.g. plump evidence that the specialized phenotype of HEVs could endothelial cells, numerous lymphocytes in the vessel be controlled by local factors associated with immune walls) are observed during inflammation of the pancreas. activity. This possibility is further reinforced by in viva Expression of MECA-79 and MECA-367 (MAdCAM-1) studies in transgenic mice, which have revealed that is induced on these HEV-like vessels76T77during the de- MAdCAM- and, subsequently, MECA-79 are induced velopment of insulitis, whereby lymphocytes infiltrate on HEV-like vessels in the pancreas of mice expressing the pancreatic islets. Staining with MECA-79 is consist- IFN-y or IL-10 in pancreatic p cells71~~2. Together, ent with the induction of functional L-selectin ligands, these studies suggest that cytokines associated with the CD34, MAdCAM- and GlyCAM-1 (Ref. 26). The in- immune response play a key role in the induction and duction of GlyCAM-1 in the inflamed pancreas of NOD maintenance of the specialized HEV phenotype. mice is particularly striking since GlyCAM-1 expression However, cytokines are probably not sufficient by in mice had previously been shown to be restricted to themselves to induce HEV specialization, and it is PLN and mesenteric lymph node (MLN) HEVs (Ref. 23) Together, these results indicate that HEV-like vessels MECA-79 and HECA-452 on these vessels is most pro- induced by chronic inflammation in extralymphoid nounced in association with extensive lymphoid infil- sites appear to be phenotypically similar to HEVs from trates14*69. Therefore, the development of bona fide lymphoid tissues. The induction of MECA-79 and HEVs in the synovial membrane of patients with rheu- MAdCAM- on the endothelium correlates with the matoid arthritis is likely to facilitate large-scale influx expression of their counter-receptors L-selectin and IX& of lymphocytes, leading to amplification and main- on cells infiltrating the islets76. Recent in vivo studies tenance of chronic inflammation. Although a distinct have revealed that these two receptor-counter-receptor endothelial cell recognition system has been proposed pairs, o&-MAdCAM- and L-selectin-MECA-79, play to control lymphocyte emigration into inflamed syn- a major role in the recruitment of lymphocytes from oviumX’, recent studies support the participation of blood into the inflamed pancreas’*. Treatment of NOD several known adhesion molecules (L-selectin, pi, B2 mice with function-blocking mAbs specific for L-selectin and (Yeintegrins) acting in sequence, rather than a single and 0~~integrins resulted in the inhibition of insulitis synovial receptor-counter-receptor pair53,82. and the prevention of autoimmune diabetes. The development of HEVs after prolonged inflam- In addition to pancreatic lesions, NOD mice also matory stimulus is not restricted to diseased synovium, display inflammatory infiltration of the salivary glands, but can also occur in other tissues, particularly the gut and HEV-like vessels expressing MECA-79 but not and thyroid. During chronic inflammation of the gut MAdCAM- were observed in areas of dense lympho- in inflammatory bowel diseases (Crohn’s disease and cytic infiltration “. Conversely, MAdCAM- but not ) or the thyroid in autoimmune thy- MECA-79 has been found to be induced in brain roiditis (Graves’ disease and Hashimoto’s thyroiditis), endothelial cells during chronic relapsing experimental areas of dense lymphocytic infiltration contain vessels allergic encephalomyelitis (EAE) in Biozzi mouse79. The with plump endothelium expressing MECA-79 and differential expression of MECA-79 and MAdCAM- HECA-452 (Refs 14,18,68,83). These observations sug- in HEV-like vessels from different sites suggests that gest that HEVs could play an important role in the the microenvironment in extralymphoid tissues directly pathogenesis of these diseases by mediating abnormal influences the phenotype of the HEVs that are induced lymphocyte recruitment to the gut or the thyroid. in chronic inflammatory conditions. MECA-79’ venules with plump endothelium have also been detected in other sites of chronic inflammation, HE Vs in human chronic inflammatory diseases including many cutaneous inflammatory lesions14. The Vessels with HEV characteristics appear in human presence of MECA-79+ HEV-like vessels in many differ- tissue in association with long-standing chronic inflam- ent human chronic inflammatory diseases indicates that mation (Table 1). Such vessels exhibit plump endothe- L-selectin is likely to play a major role in lymphocyte lial cells, take up and incorporate high levels of 35S04, emigration at chronic inflammatory sites. contain many luminal and intramural lymphocytes (pre- sumably in the process of extravasating) and mediate Concluding remarks in vitro lymphocyte adhesion4. The HEV endothelium is unique amongst vascular In rheumatoid arthritis, it has been observed that the endothelium by virtue of its capacity to recruit large level of sulfate incorporation as well as the ‘plumpness’ numbers of lymphocytes. In the human body, it is esti- (or ‘tallness’) of the endothelium in areas of lympho- mated that as many as 5 X lo6 lymphocytes extrava- cytic infiltration in the synovial membrane are closely sate from the blood through HEVs every second. The related to the concentration of the lymphocytes in specificity and efficiency of this process is explained by the perivascular infiltrates *O. Similarly, expression of specialized features of the HEV endothelium, such as

Immunology Today 455 Vol. 16 No. V IV95 the expression of mu&-like glycoproteins decorated 20 Streeter, P.R., Lake!-Berg, E., Rouse, B.T.N., Bargatze, R.F. with HEV-specific oligosaccharides. In the future, it will and Butcher, E.C. (1988) Nuture 331,41-46 be important to define precisely the molecular mech- 21 Berlin, C., Bargatze, R.F., von Andrian, U.H. et al. anisms involved in the induction and maintenance of ( 1995) Cell 80,4 13-422 the specialized HEV phenotype. HEW-like vessels in- 22 Sikorski, E.E.. Hallmann, R., Berg, E.L. and Butcher, E.C. duced by chronic inflammation in extralymphoid sites i 1993) J. Immunol. 15 1, 5239-5250 23 Lasky, LA.. Singer, M.S., Dowbenko, D. et al. (1992) appear to be phenotypically and functionally similar to Cell 69, 927-938 HEVs from lymphoid tissues. Thus, a better understand- 24 Baumhueter, S., Singer, M.S., Henzel, W. et al. (1993) ing of the mechanisms controlling development and Science 262,436-438 maintenance of HEVs could provide the basis of novel 25 Pm-i, K.D., Finger, E.B., Gaudernack, G. and Springer, T.A. therapeutic approches for the treatment of human .I. Cell Biol. (in press) chronic inflammatory diseases, including rheumatoid 26 Baumhueter, S., Dyhdal, N., Kyle, C. and Lasky, L.A. arthritis and inflammatory bowel diseases, in which ( 1994) Blood 84, 2554-2.565 HEV-like vessels facilitate large-scale influx of lym- 27 Hemmerich, S., Butcher, E.C. and Rosen, S.D. (1994) phocytes, leading to amplification and maintenance of 1. Exp. Med. 180,22 1Y-2226 chronic inflammation. 28 Dowbenko, D., Kikuta, A., Fennie, C., Gillett, N. and Lasky, L.A. ( 1993) ,/. Clin. Invest. 92, 952-960 J-P. Girard was a recipient of fellowships from the 29 Rosen, S.D., Chi, S.I., True, D.D., Singer, M.S. and Association pour la Recherche sur le Cancer (ARC) and the Yednock, T.A. i 198Yi 1. 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Heterogeneity of man organ-specific autoimmune dis- Sandra M. McLachlan eases are caused directly by auto- Basil Rapoport organ-specific antibodies. Among these are Graves’ autoimmune diseases disease (autoantibodies to the thy- Thyroid Molecular Biology Unit, roid-stimulating hormone receptor) Veterans’ Administration Medical and myasthenia gravis (autoand- Center at the University of California, In a recent article in Immunology bodies to the acetylcholine recep- 4 I SO Clement Street, Todayl, Liblau et al. reviewed tor) (reviewed in Refs 2,3, rospec- San Francisco, the role of CD4+ T helper 1 (Thl) tively). Indeed, the incidence of CA 94123, and Th2 cells in the pathogenesis Graves’ disease in the general popu- USA. of organ-specific autoimmune dis- lation is tenfold higher than that of eases. Insulin-dependent diabetes IDDM (-100 versus 10 cases per References mellitus (IDDM) and experimental 100 000 per year)“.‘. 1 Liblau, R.S., Singer, SM. and McDevitt, H.0. (1995) Immunol. autoimmune encephalitis (EAE) Clearly, CD4’ T cells are in- Today 16,34-38 volved in autoantibody generation. were discussed as examples of Thl- 2 Rees Smith, B., McLachlan, SM. mediated organ-specific damage. However, in a disease such as and Furmaniak, J. (19881 Endocr. Rev. They state that ‘similar data exist Graves’ disease, boosting the Th2 9,106-121 for other autoimmune diseases’. In response is likely to be inappro- 3 Lindstrom, J., Shelton. D. and addition, they suggest that immun- priate therapy. Conversely, divert- Fujii, Y. (1988) Adv. Immunol. 42, ization with autoantigens in the ing the Th2 to a Thl response could 233-284 presence of interleukin 4 (IL-4) lead to thyroiditis and hypothy- 4 Larsen, P.R. and Ingbar, S.H. (1992~ may provide an intervention strat- roidism, a form of autoimmune thy- in Williams Textbook of Endocrinology egy in such diseases by diverting roid disease that is even more com- (8th edn) (Wilson, J.D. and Foster, D.W., eds), p. 419, W.B. Saunders the immune response away from mon than Graves’ disease. We wish Company to make a plea that human organ- the Thl cells. 5 Unger, R.H. and Foster, D.W. (1992) This review is representative of specific autoimmune disease be in Williams Textbook of Endocrinology the common implication that organ- perceived as heterogeneous, and not (8th edn) (Wilson, J.D. and Foster, specific autoimmune diseases are as a monolithic group of diseases D.W., eds), p. 1258, W.B. Saunders T-cell mediated. In fact, some hu- directly caused by T cells. Company