Peripheral Node Addressin Research Articles

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Induction of peripheral lymph node addressin in human gastric mucosa infected byHelicobacter pylori

Helicobacter pylori infects over half the world's population and is a leading cause of peptic ulcer and gastric cancer. H. pylori infection results in chronic inflammation of the gastric mucosa, and progression of chronic inflammation leads to glandular atrophy and intestinal metaplasia. However, how this chronic inflammation is induced or maintained is not well known. Here, we show that chronic inflammation caused by H. pylori infection is highly correlated with de novo synthesis of peripheral lymph node addressin (PNAd) presented on high-endothelial venule (HEV)-like vessels. The number of HEV-like vessels dramatically increases as chronic inflammation progresses. We found that the PNAd is bound by L-selectin.IgM chimeric protein, and decorated by NCC-ST-439 antibody, which is suggested to recognize both nonsulfated and 6-sulfated sialyl Lewis X on core 2 branched O-glycans, and MECA-79 antibody, which reacts with 6-sulfo N-acetyllactosamine on extended core 1 O-glycans. These results indicate that PNAd on HEV-like vessels present in the gastric mucosa subsequent to H. pylori infection is similar to those on HEVs present in the secondary lymphoid organs, which are essential for lymphocyte circulation. Moreover, eradication of H. pylori is associated with the disappearance of HEV-like vessels in the gastric mucosa. By contrast, very few PNAd were found in the gastric mucosa of patients with chemical gastritis caused by nonsteroidal antiinflammatory drugs. These results strongly suggest that PNAd in HEV-like vessels plays a critical role in lymphocyte recruitment during chronic inflammation induced by H. pylori infection.

Aberrant B1 cell migration into the thymus results in activation of CD4 T cells through its potent antigen‐presenting activity in the development of murine lupus

B1 cells have different origin and function from conventional B (B2) cells and are considered to be involved in autoantibody production in the development of autoimmune disease. We found that B1 cells preferentially accumulated in the target organs including thymus in aged BWF1 mice, a murine model for systemic lupus erythematosus, and that B lymphocyte chemoattractant (BLC/CXCL13) expression was increased in the thymus before the onset of lupus nephritis, while stromal cell-derived factor-1 (SDF-1/CXCL12) and secondary lymphoid tissue chemokine (SLC/CCL21) expression remained unchanged. Adhesion molecules such as peripheral node addressin (PNAd), ICAM-1, and VCAM-1 were also expressed on endothelial cells in the enlarged thymic perivascular space (PVS) in aged BWF1 mice. BLC protein and PNAd were co-localized on these high-endothelial-venules-like vessels in enlarged PVS. B1 cells expressed higher level of costimulatory molecules and showed a potent antigen-presenting activity in allogeneic mixed lymphocyte reaction comparable to splenic dendritic cells. Interestingly, B1 cells stimulated proliferation of autologous thymic CD4 T cells in the presence of IL-2. These results indicate that aberrant B1 cell trafficking into the thymus due to ectopic high expression of BLC may result in an activation of self-reactive T cells in the development of murine lupus.

IκB Kinase Complex α Kinase Activity Controls Chemokine and High Endothelial Venule Gene Expression in Lymph Nodes and Nasal-Associated Lymphoid Tissue

The lymphotoxin (LT) beta receptor plays a critical role in secondary lymphoid organogenesis and the classical and alternative NF-kappaB pathways have been implicated in this process. IKKalpha is a key molecule for the activation of the alternative NF-kappaB pathway. However, its precise role and target genes in secondary lymphoid organogenesis remain unknown, particularly with regard to high endothelial venules (HEV). In this study, we show that IKKalpha(AA) mutant mice, who lack inducible kinase activity, have hypocellular lymph nodes (LN) and nasal-associated lymphoid (NALT) tissue characterized by marked defects in microarchitecture and HEV. In addition, IKKalpha(AA) LNs showed reduced lymphoid chemokine CCL19, CCL21, and CXCL13 expression. IKKalpha(AA) LN- and NALT-HEV were abnormal in appearance with reduced expression of peripheral node addressin (PNAd) explained by a severe reduction in the HEV-associated proteins, glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), and high endothelial cell sulfotransferase, a PNAd-generating enzyme that is a target of LTalphabeta. In this study, analysis of LTbeta(-/-) mice identifies GlyCAM-1 as another LTbeta-dependent gene. In contrast, TNFRI(-/-) mice, which lose classical NF-kappaB pathway activity but retain alternative NF-kappaB pathway activity, showed relatively normal GlyCAM-1 and HEC-6ST expression in LN-HEV. In addition, in this communication, it is demonstrated that LTbetaR is prominently expressed on LN- and NALT-HEV. Thus, these data reveal a critical role for IKKalpha in LN and NALT development, identify GlyCAM-1 and high endothelial cell sulfotransferase as new IKKalpha-dependent target genes, and suggest that LTbetaR signaling on HEV can regulate HEV-specific gene expression.

A novel model for lymphocytic infiltration of the thyroid gland generated by transgenic expression of the CC chemokine CCL21.

Lymphocytic infiltrates and lymphoid follicles with germinal centers are often detected in autoimmune thyroid disease (AITD), but the mechanisms underlying lymphocyte entry and organization in the thyroid remain unknown. We tested the hypothesis that CCL21, a chemokine that regulates homeostatic lymphocyte trafficking, and whose expression has been detected in AITD, is involved in the migration of lymphocytes to the thyroid. We show that transgenic mice expressing CCL21 from the thyroglobulin promoter (TGCCL21 mice) have significant lymphocytic infiltrates, which are topologically segregated into B and T cell areas. Although high endothelial venules expressing peripheral lymph node addressin were frequently observed in the thyroid tissue, lymphocyte recruitment was independent of L-selectin or lymphotoxin-alpha but required CCR7 expression. Taken together, these results indicate that CCL21 is sufficient to drive lymphocyte recruitment to the thyroid, suggest that CCL21 is involved in AITD pathogenesis, and establish TGCCL21 transgenic mice as a novel model to study the formation and function of lymphoid follicles in the thyroid.

A stroma-derived defect in NF-kappaB2-/- mice causes impaired lymph node development and lymphocyte recruitment.

The NF-kappaB family of transcription factors is vital to all aspects of immune function and regulation in both the hemopoietic and stromal compartments of immune environments. Recent studies of mouse models deficient for specific members of the NF-kappaB family have revealed critical roles for these proteins in the process of secondary lymphoid tissue organogenesis. In this study, we investigate the role of NF-kappaB family member NF-kappaB2 in lymph node development and lymphocyte recruitment. Inguinal lymph nodes in nfkappab2(-/-) mice are reduced in size and cellularity, most notably in the B cell compartment. Using in vitro and in vivo lymph node grafting assays, we show that the defect resides in the stromal compartment. Further examination of the nfkappab2(-/-) inguinal lymph nodes revealed that expression of peripheral node addressin components CD34 and glycosylation-dependent cell adhesion molecule-1 along with the high endothelial venule-restricted sulfotransferase HEC-GlcNAc6ST was markedly reduced. Furthermore, expression of the lymphocyte homing chemokines CCL19, CCL21, and CXCL13 was down-regulated. These data highlight the role of NF-kappaB2 in inguinal lymph node organogenesis and recruitment of lymphocytes to these organs due to its role in up-regulation of essential cell adhesion molecules and chemokines, while suggesting a potential role for NF-kappaB2 in organization of lymph node endothelium.

HCV RECURRENCE AFTER LIVER TRANSPLANTATION IS ASSOCIATED WITH THE DEVELOPMENT OF INTRAHEPATIC LYMPHOID FOLLICLES DESPITE IMMUNESUPPRESSION

P796 Aims: Chronic inflammation in the liver can lead to the development of lymphoid tissue in the portal tracts. Some of these lymphoid infiltrates are highly organized, containing all characteristic components of secondary lymphoid organs, with B-lymphocytes localized in follicles and with T-cells more diffusely distributed in surrounding areas. One could speculate that these ectopic lymphoid structures fulfill similar functions as secondary lymphoid organs in the initiation and perpetuation of local immune responses. In the current study we investigated the formation of portal tract-associated lymphoid tissue (PALT) after liver transplantation, particularly in patients with hepatitis C virus (HCV) recurrence. In this setting, we can investigate a possible effect of immunosuppressive regimens on the neogenesis of lymphoid tissue induced by HCV infection of the liver graft. Results: First, the explant livers of chronic HCV patients after orthotopic liver transplantation were analysed by immunohistochemistry for the presence of PALT structures. In 9 out of 10 patients, B-cell follicles and T-cell areas were observed. Approximately one-third of all portal tracts which were examined contained B-cell follicles. Of these follicles, 4% contained a network of follicular dendritic cells (FDC) indicating of presence of an active germinal center. Most of the paracortical T cell areas contained blood vessels resembling high endothelial venules that are found in lymph nodes. These endothelial cells stained positive for the Peripheral Node Addressin (PNAD), a ligand for the adhesion receptor L-selectin. All of the microvascular endothelium in the PALT, but not the sinusoidal endothelium, stained positive for CD34 indicating neovascularization in the PALT. Next, post-transplantation biopsies of the HCV patients were analyzed. All these patients suffered from re-infection of the graft by HCV. More then a third of these patients developed PALT which contained B-cell follicles. Follicles were not found earlier as 18 months after transplantation. In this small number of post-transplantation follicles, however, no FDC-network was observed. This suggest that B-cell organization in response to HCV recurrence is relatively slow and renders inactive follicles. None of the control (HCV-negative) transplantation patients developed PALT, even at later time points after transplantation. Conclusions: HCV infection can result in the formation of ectopic lymphoid compartments in the liver graft. Immunosuppressive drugs do not prevent the formation of these PALT, although the number of B-cell follicles found in post-transplantation biopsies was significantly lower compared to liver biopsies of chronic HCV patients with end-stage liver disease. However, the role of PALT in chronic inflammatory liver diseases, like viral hepatitis, and the significance for HCV-recurrence remains unknown.

Detection of ectopic B-cell follicles with germinal centers in the meninges of patients with secondary progressive multiple sclerosis.

Multiple sclerosis (MS) is characterized by synthesis of oligoclonal immunoglobulins and the presence of B-cell clonal expansions in the central nervous system (CNS). Because ectopic lymphoid tissue generated at sites of chronic inflammation is thought to be important in sustaining immunopathological processes, we have investigated whether structures resembling lymphoid follicles could be identified in the CNS of MS patients. Sections from post-mortem MS brains and spinal cords were screened using immunohistochemistry for the presence of CD20+ B-cells, CD3+ T-cells, CD138+ plasma cells and CD21+, CD35+ follicular dendritic cells, and for the expression of lymphoid chemokines (CXCL 13, CCL21) and peripheral node addressin (PNAd). Lymphoid follicle-like structures containing B-cells, T-cells and plasma cells, and a network of follicular dendritic cells producing CXCL13 were observed in the cerebral meninges of 2 out of 3 patients with secondary progressive MS, but not in relapsing remitting and primary progressive MS. We also show that proliferating B-cells are present in intrameningeal follicles, a finding which is suggestive of germinal center formation. No follicle-like structures were detected in parenchymal lesions. The formation of ectopic lymphoid follicies in the meninges of patients with MS could represent a critical step in maintaining humoral autoimmunity and in disease exacerbation.

Developing neonatal rabbit appendix, a primary lymphoid organ, is seeded by immature blood-borne B cells: evidence for roles for CD62L/PNAd, CCR7/CCL21, α4β1 and LFA-1

Young rabbit appendix is a homologue of chicken bursa of Fabricius; both are crucial sites for preimmune B-cell repertoire development. We describe here some of the molecules involved in the multi-step recruitment of blood-borne B cells into neonatal rabbit developing appendix. Sialyl-Lewis-x, CD62L and integrins such as LFA-1 and α4β1 were detected on B cells in peripheral blood. Peripheral lymph node addressin (PNAd), a CD62L counter-receptor was observed on appendix HEV. We also detected chemokine receptor CCR7 on peripheral blood B cells and one of the CCR7 ligands, CCL21, on appendix HEV but not in appendix follicles. Higher levels of CXCR5 expression compared to CCR7 on appendix B cells suggest that CXCR5 may be involved in recruitment of B cells into follicles. The proportions of appendix B cells expressing CD62L, sialyl-Lewis-x and α4β1 declined between day 3 and 4 weeks after birth while percentages of Lewis-x + appendix B cells increased. These changes correlate with the stage of repertoire diversification by gene conversion in both rabbits and chickens. The cross-reactivity of antibodies to mouse or human adhesion molecules described in this study indicates that some of the structures of these important molecules are conserved across species.

A novel endothelial L-selectin ligand activity in lymph node medulla that is regulated by alpha(1,3)-fucosyltransferase-IV.

Lymphocytes home to peripheral lymph nodes (PLNs) via high endothelial venules (HEVs) in the subcortex and incrementally larger collecting venules in the medulla. HEVs express ligands for L-selectin, which mediates lymphocyte rolling. L-selectin counterreceptors in HEVs are recognized by mAb MECA-79, a surrogate marker for molecularly heterogeneous glycans termed peripheral node addressin. By contrast, we find that medullary venules express L-selectin ligands not recognized by MECA-79. Both L-selectin ligands must be fucosylated by α(1,3)-fucosyltransferase (FucT)-IV or FucT-VII as rolling is absent in FucT-IV+VII−/− mice. Intravital microscopy experiments revealed that MECA-79–reactive ligands depend primarily on FucT-VII, whereas MECA-79–independent medullary L-selectin ligands are regulated by FucT-IV. Expression levels of both enzymes paralleled these anatomical distinctions. The relative mRNA level of FucT-IV was higher in medullary venules than in HEVs, whereas FucT-VII was most prominent in HEVs and weak in medullary venules. Thus, two distinct L-selectin ligands are segmentally confined to contiguous microvascular domains in PLNs. Although MECA-79–reactive species predominate in HEVs, medullary venules express another ligand that is spatially, antigenically, and biosynthetically unique. Physiologic relevance for this novel activity in medullary microvessels is suggested by the finding that L-selectin–dependent T cell homing to PLNs was partly insensitive to MECA-79 inhibition.

Coordinate regulation of lymphocyte-endothelial interactions by pregnancy-associated hormones.

Precursors of uterine NK cells home to the uterus during early pregnancy from multiple lymphohemopoietic sources. In mouse uterine tissue, pregnancy markedly up-regulates both L-selectin- and alpha(4) integrin-dependent adhesion pathways for circulating human CD56(bright) cells, the phenotype of human uterine NK cells. Based on roles for these adhesion molecules in lymphocyte homing, we examined effects of pregnancy or the steroid hormones 17beta-estradiol or progesterone on lymphocyte-endothelial interactions in secondary lymphoid tissues and in uterus. From preimplantation gestation day 3, specialized high endothelial venules in peripheral lymph nodes and Peyer's patches supported elevated L-selectin and alpha(4)beta(7) integrin-dependent lymphocyte adhesion under shear throughout pregnancy, as compared with high endothelial venules of virgin or postpartum donors. Squamous endothelium from nonlymphoid tissue was not affected. Pregnancy-equivalent endothelial responses were observed in lymph nodes and Peyer's patches from ovariectomized mice receiving 17beta-estradiol and/or progesterone replacement therapy. Adhesion of human CD56(bright) cells to uteri from pregnant or hormone-treated ovariectomized mice was enhanced through L-selectin- and alpha(4) integrin-dependent mechanisms and involved multiple vascular adhesion molecules including mucosal addressin cell adhesion molecule-1, VCAM-1, and peripheral lymph node addressin. Analysis of Tie2-green fluorescence protein transgenic mice demonstrated that CD56(bright) cells adhered primarily to vascular endothelium within the decidua basalis. Microdomain localization of adhesion involving large clusters of lymphocytes was induced on uteri from natural matings, but not pseudopregnancy. Steroid hormones also had independent effects on L-selectin function in splenic lymphocytes that mimicked physiological stimulation induced by pregnancy or fever-range temperatures. These results provide the first evidence for coordinated, organ-specific, steroid hormone-induced changes in lymphocyte homing mechanisms that could contribute to local and systemic immune responses during pregnancy.

Morphological analysis of leucocyte transmigration in the pleural cavity.

The role that pleural mesothelial cells play in leucocyte transmigration into the pleural cavity was investigated in lipopolysaccharide-stimulated mice. Changes in mesothelial cell morphology and changes in expression of adhesion molecules on mesothelial cells and leucocytes were analysed by light microscopy, immunohistochemistry, transmission electron microscopy (TEM) and immuno-scanning electron microscopy (immuno-SEM). After stimulation, the mesothelial cells separated completely from one another before leucocyte penetration across the mesothelial layer occurred. These changes occurred primarily in the immediate vicinity of ribs, where a large number of leucocytes accumulated. Immuno-SEM showed that the expression of intercellular adhesion molecule-1 (ICAM-1) on the parietal pleural mesothelial cells was significantly up-regulated by lipopolysaccharide stimulation, and that of vascular cell adhesion molecule-1 (VCAM-1) was induced. Both were restricted to the microvilli of the mesothelial cells. By contrast, expression of intercellular adhesion molecule-2 (ICAM-2), platelet/endothelial cell adhesion molecule-1 (PECAM-1), mucosal addressin cell adhesion molecule-1 (MAdCAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1), peripheral node addressin (PNAd) and fibronectin were not detected. Lymphocyte function associated antigen-1 (LFA-1), macrophage-1 molecule (Mac-1) and very late appearing antigen-4 (VLA-4), all ligands of ICAM-1 and VCAM-1, were present on the transmigrated neutrophils and macrophages. These findings demonstrate that the immediate vicinity of ribs is a source of leucocyte migration into the pleural space.

Gene therapy to target dendritic cells from blood to lymph nodes.

Peripheral lymph nodes (PLN) are strategic microenvironments where antigen-presenting dendritic cells (DC), loaded with environmental antigens, and naive lymphocytes meet to initiate immune responses. The unique capacity of DC to induce primary immune responses has led to their use in clinical medicine; however, delivering DC to lymph nodes is problematic. Intravenously injected DC cannot access to PLN, while DC injected into tissue migrate inefficiently through lymphatics to PLN. We achieved DC targeting to T-cell areas of PLN by endowing DC with a novel receptor for peripheral node addressin (PNAd), an adhesion molecule present on the lymph node venular endothelium. This novel receptor is a chimeric E/L-selectin (ELS) that, we have previously shown, binds to PNAd. DC were genetically modified by retroviral transduction to express ELS. ELS expression was targeted to tips of microvilli, and mediated rolling of DC on PNAd both in vivo and in vitro. Such genetically engineered DC could extravasate directly from blood through the lymph node endothelium as opposed to nontransduced DC. This study provides evidence that the trafficking of DC can be modified using gene transfer technologies. More efficient delivery of DC to PLN should assist the development of improved vaccination strategies.

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 naïve (CD45lo-CD62Lhi) population. These findings suggest that helicobacter-induced chronic active hepatitis arises through the process of lymphoid organ neogenesis.

Ectopic LT alpha beta directs lymphoid organ neogenesis with concomitant expression of peripheral node addressin and a HEV-restricted sulfotransferase.

Lymph node (LN) function depends on T and B cell compartmentalization, antigen presenting cells, and high endothelial venules (HEVs) expressing mucosal addressin cell adhesion molecule (MAdCAM-1) and peripheral node addressin (PNAd), ligands for naive cell entrance into LNs. Luminal PNAd expression requires a HEV-restricted sulfotransferase (HEC-6ST). To investigate LTαβ's activities in lymphoid organogenesis, mice simultaneously expressing LTα and LTβ under rat insulin promoter II (RIP) control were compared with RIPLTα mice in a model of lymphoid neogenesis and with LTβ−/− mice. RIPLTαβ pancreata exhibited massive intra-islet mononuclear infiltrates that differed from the more sparse peri-islet cell accumulations in RIPLTα pancreata: separation into T and B cell areas was more distinct with prominent FDC networks, expression of lymphoid chemokines (CCL21, CCL19, and CXCL13) was more intense, and L-selectin+ cells were more frequent. In contrast to the predominant abluminal PNAd pattern of HEV in LTβ−/− MLN and RIPLTα pancreatic infiltrates, PNAd was expressed at the luminal and abluminal aspects of HEV in wild-type LN and in RIPLTαβ pancreata, coincident with HEC-6ST. These data highlight distinct roles of LTα and LTαβ in lymphoid organogenesis supporting the notion that HEC-6ST–dependent luminal PNAd is under regulation by LTαβ.

Sulphated endothelial ligands for L-selectin in lymphocyte homing and inflammation.

Lymphocytes from the blood home to secondary lymphoid tissues through a process of tethering, rolling, firm adhesion and transmigration. Tethering and rolling of lymphocytes is mediated by the interaction of L-selectin on lymphocytes with sulphated ligands expressed by the specialized endothelial cells of high endothelial venules (HEVs). The sulphate-dependent monoclonal antibody MECA79 stains HEVs in peripheral lymph nodes and recognizes the complex of HEV ligands for L-selectin termed peripheral node addressin. High endothelial cell GlcNAc-6-sulphotransferase/L-selectin ligand sulphotransferase is a HEV-expressed sulphotransferase that contributes to the formation of the MECA79 epitope and L-selectin ligands on lymph node HEVs. MECA79-reactive vessels are also common at sites of chronic inflammation, suggesting mechanistic parallels between lymphocyte homing and inflammatory trafficking.

Aggregation of Antigen-Specific T Cells at the Inoculation Site of Mature Dendritic Cells

Cellular immune responses are initiated by direct interaction of naive T cells with professional antigen-presenting cells, i.e., dendritic cells. In general, this interaction takes place in secondary lymphoid organs to which both naive T cells and mature dendritic cells preferentially home. This physiologic scenario differs substantially, however, from therapeutic dendritic-cell-based vaccinations used to treat human cancer. In fact, only a small fraction of intradermally injected dendritic cells migrate to the draining lymph node and the majority of cells remain at the site of inoculation. These sites are characterized by a distinct oligoclonal T cell infiltrate comprising both L-Selectin+/CD45RA+ and L-Selectin+/CD45RA- cells. Blood vessels expressing peripheral lymph node addressin represent possible entry channels for such naive and central memory T cells, the former probably attracted by dendritic cell-CK1 produced by the injected dendritic cells. In situ staining with multimeric peptide/major histocompatibility complex class I complexes revealed that infiltrating T cells specifically recognize peptide epitopes presented by the injected dendritic cells. Thus, the fraction of dendritic cells not migrating to secondary lymphatic tissue after therapeutic inoculation nevertheless seem to be involved in a specific immune modulation.

Mucosal addressin expression and binding-interactions with naive lymphocytes vary among the cranial, oral, and nasal-associated lymphoid tissues.

The head and neck lymph nodes (LN)--or cranial, oral, and nasal-associated lymphoid tissue (CONALT)--help disseminate activated lymphocytes to produce salivary immune responses, especially after intranasal immunization. To elucidate the mechanisms that induce immunity at these sites, we investigated the interactions between addressins and homing-receptors that allow for lymphocyte binding to high endothelial venules (HEV) of CONALT. In vivo lymphocyte trafficking to CONALT was mediated primarily through interactions between peripheral node addressin (PNAd) and L-selectin, whereas interactions between mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and alpha 4 beta 7 played a role in retention in cervical LN (CLN). Upon immunofluorescent staining for PNAd and MAdCAM-1, nearly all HEV in CONALT expressed PNAd, with varying MAdCAM-1 expression among these LN. The parotid gland LN (PRLN) and submaxillary gland LN (SMLN) rely exclusively upon PNAd-L-selectin interactions for naive-lymphocyte binding, whereas the CLN utilize PNAd-L-selectin interactions and MAdCAM-1-alpha 4 beta 7 interactions for binding. Intense staining of non-HEV-expressed vascular cell adhesion molecule-1 (VCAM-1) was observed in PRLN, whereas SMLN and CLN displayed less VCAM-1 but showed intense staining for diffuse MAdCAM-1. This study suggests that though PNAd-L-selectin interactions play an important role in the trafficking of lymphocytes throughout CONALT, varying MAdCAM-1 and VCAM-1 addressin expression and usage impart important differences among the PRLN, SMLN, and CLN.

Immunolocalization of peripheral lymph node addressins in normal and neoplastic human thymuses.

Peripheral lymph node addressin is a specific L-selectin ligand of the high endothelial venules that plays an important role in lymphocyte homing to lymph nodes. Tissue selective migration of lymphocytes through this pathway to the thymus has also been proposed. In this work, peripheral lymph node addressin expression was investigated immunohistochemically with a monoclonal antibody, clone MECA-79, in formaldehyde-fixed, paraffin-embedded tissue sections of 5 normal neonatal thymuses, 25 thymomas, 3 thymic carcinomas, and 2 thymic lymphoid hyperplasias. In normal thymuses, peripheral lymph node addressin expression was found in the endothelium of corticomedullary and medullary vessels surrounded by perivascular space. In type B thymomas and thymic lymphoid hyperplasias, peripheral lymph node addressin was detected in the vessels with perivascular spaces, at the medullary differentiation areas, and in paralymphoid follicles, respectively. However, in type A thymomas and thymic carcinomas, MECA-79-positive vessels were restricted to the remnants of pre-existing thymic tissue, and they were absent from the neoplastic areas. These findings suggest that in normal and most neoplastic thymuses, peripheral lymph node addressin is expressed by regions of vascular endothelium corresponding to postcapillary venules that may serve as a pathway for homing of recirculating lymphocytes to the thymus.

L-selectin Dimerization Enhances Tether Formation to Properly Spaced Ligand

Selectin counterreceptors are glycoprotein scaffolds bearing multiple carbohydrate ligands with exceptional ability to tether flowing cells under disruptive shear forces. Bond clusters may facilitate formation and stabilization of selectin tethers. L-selectin ligation has been shown to enhance L-selectin rolling on endothelial surfaces. We now report that monoclonal antibodies-induced L-selectin dimerization enhances L-selectin leukocyte tethering to purified physiological L-selectin ligands and glycopeptides. Microkinetic analysis of individual tethers suggests that leukocyte rolling is enhanced through the dimerization-induced increase in tether formation, rather than by tether stabilization. Notably, L-selectin dimerization failed to augment L-selectin-mediated adhesion below a threshold ligand density, suggesting that L-selectin dimerization enhanced adhesiveness only to properly clustered ligand. In contrast, an epidermal growth factor domain substitution of L-selectin enhanced tether formation to L-selectin ligands irrespective of ligand density, suggesting that this domain controls intrinsic ligand binding properties of L-selectin without inducing L-selectin dimerization. Strikingly, at low ligand densities, where L-selectin tethering was not responsive to dimerization, elevated shear stress restored sensitivity of tethering to selectin dimerization. This is the first indication that shear stress augments effective selectin ligand density at local contact sites by promoting L-selectin encounter of immobilized ligand.