B and T lymphocyte subsets enter peripheral lymph nodes and Peyer's patches without preference in vivo: no correlation occurs between their localization in different types of high endothelial venules and the expression of CD44, VLA-4, LFA-1, ICAM-1, CD2 or L-selectin

Many lymphocytes enter tissues such as peripheral lymph nodes and Peyer's patches through high endothelial venules (HEV). It is known that HEV differ in the expression of adhesion molecules as lymphocyte subsets do. Through the interaction of these molecules B and T lymphocyte subsets are thought to be preferentially directed into lymphoid organs. However, it is unclear which role these mechanisms play in vivo, since there are no studies demonstrating that blood lymphocyte subsets preferentially interact with different types of HEV in vivo. Therefore, in the present study the frequency of B, T, CD4⁺ and CD8⁺ lymphocytes in the wall of the HEV of rat peripheral lymph nodes and Peyer's patches was analyzed by immunohistology. In addition, the expression of CD44, VLA-4, LFA-1, ICAM-1, CD2 and L-selectin on B and T lymphocyte subsets of the blood was determined by flow cytometry. Although B and T lymphocytes showed significantly different levels of expression for each adhesion molecule investigated, the relation of B and T lymphocytes within the HEV of peripheral lymph nodes and Peyer's patches was strikingly comparable (38.0 ± 5.2% vs. 40.6 ± 5.7% and 62.0 ± 5.2% vs. 59.4 ± 5.7%, respectively). The same was true for CD4⁺ and CD8⁺ cells. Thus, although HEV and the blood lymphocyte subsets differ markedly in their expression pattern of adhesion molecules, the existing levels are sufficient to mediate comparable entrance of B and T lymphocyte subsets into both types of HEV.     

Involvement of the lysophosphatidic acid-generating enzyme autotaxin in lymphocyte-endothelial cell interactions

Autotaxin (ATX) is a secreted protein with lysophospholipase D activity that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine. Here we report that functional ATX is selectively expressed in high endothelial venules (HEVs) of both lymph nodes and Peyer’s patches. ATX expression was developmentally regulated and coincided with lymphocyte recruitment to the lymph nodes. In adults, ATX expression was independent of HEV-expressed chemokines such as CCL21 and CXCL13, innate immunity signals including those via TLR4 or MyD88, and of the extent of lymphocyte trafficking across the HEVs. ATX expression was induced in venules at sites of chronic inflammation. Receptors for the ATX enzyme product LPA were constitutively expressed in HEV endothelial cells (ECs). In vitro, LPA induced strong morphological changes in HEV ECs. Forced ATX expression caused cultured ECs to respond to lysophosphatidylcholine, up-regulating lymphocyte binding to the ECs in a LPA receptor-dependent manner under both static and flow conditions. Although in vivo depletion of circulating ATX did not affect lymphocyte trafficking into the lymph nodes, we surmise, based on the above data, that ATX expressed by HEVs acts on HEVs in situ to facilitate lymphocyte binding to ECs and that ATX in the general circulation does not play a major role in this process. Tissue-specific inactivation of ATX will verify this hypothesis in future studies of its mechanism of action.     

B and also T lymphocytes migrate via gut lymph to all lymphoid organs and the gut wall,but only IgA+ cells accumulate in the lamina propria of the intestinal mucosa

In pigs the lymphocytes emigrating from the intestinal wall were collected by cannulating the lymphatics, labeled in vitro using a fluorescent dye and retransfused. The injection of 6.6 ± 4.2 × 10⁸ cells resulted in a labeling index between 1.5 % in intestinal lymph, 0.2 % in the spleen and lymph nodes, ∼ 0.1 % in the intestinal lamina propria and 0.003 % in intraepithelial lymphocytes. About 25 % of the injected cells were present in the blood and 1 % was recovered in the lymph. T cells were found in similar proportions in the injected and the recovered cells in the organs (70 – 80 %). The proportion of IgA⁺ cells among the immigrated cells in the intestinal lamina propria ranged from 5 to 8 %, which in absolute numbers was up to 60 % of the injected IgA⁺ cells. T and IgM⁺ cells did not show a higher accumulation in any organ. These experiments in conventional, unrestrained animals revealed that (1) T cells immigrate into the intestinal lamina propria, (2) preferential migration of IgA⁺ cells from gut lymph to the intestinal lamina propria is obvious under in vivo conditions and (3) the immigrated IgA⁺ cells represent a very small population which is difficult to detect when analyzed in relative numbers.     

Impaired recirculation of B lymphocytes in chronic lymphocytic leukemia

Lymphocytes of the peripheral blood and thoracic duct lymph were studied in 4 patients with chronic lymphocytic leukemia (CLL),1 patient with lymphosarcoma (LSA) and 3 patients with nonhematolo-gical diseases (controls). When stimulated in vitro with phytohemagglu-tinin (PHA) lymph lymphocytes of CLL patients responded markedly as determined by ¹⁴[C]thymidine incorporation, whereas blood lymphocytes showed a delayed and diminished response. The response of blood and lymph lymphocytes of the LSA and control patients was equal. Purified rabbit antisera against κ,λ, μ and γ-chains were labeled with ²⁵I and the labeled cells assessed by autoradiography. In CLL patients, the percentage of lymphocytes bearing κ and μ determinants was higher in the blood than in the lymph. Controls showed a much lower percentage of lymphocytes with immunoglobulins, which was equal in blood and lymph. Furthermore, the membrane dynamics of HL-A-anti-HL-A complexes on the surface of blood and lymph lymphocytes were studied by means of membrane fluorescence. In CLL, the percentage of lymph lymphocytes showing “cap formation” within 2 h was higher in the lymph than in the blood. Using autotransfusion of [³H-]cytidine-labeled blood lymphocytes, it is shown that the recovery of labeled cells in the lymph of CLL patients within 48 h is diminished compared to controls. It is concluded that in CLL, the leukemic cells are B cells whose capacity to recirculate from blood to lymph through the postcapillary venules is impaired. Only a minor population of PHA-responsive T cells appears to recirculate normally. Consequently, the concentration of T cells is higher in the lymph than in the blood and the leukemic B lymphocytes accumulate in the vascular pool. The impaired ability for recirculation and “cap formation” suggests a membrane abnormality of the CLL cell.     

The ontogeny of human lymphocyte recirculation: high endothelial cell antigen (HECA-452) and CD44 homing receptor expression in the development of the immune system

In the present report we have studied the expression of a lymphocyte homing receptor, the CD44 antigen, and of HECA-452, a high endothelial-specific antigen, during the development of the human immune system. We found that prothymocyte immigrants of the thymus already expressed the CD44 antigen. Similarly, the first peripheral T lymphocytes in fetal lymph nodes, tonsils and gut-associated lymphoid tissue were also CD44+. Cortical thymocytes and germinal center cells were CD44-. CD44 antigen expression was, thus, not limited to mature recirculating lymphocytes. This suggests that CD44 may not only be involved in recirculation of mature lymphocytes but also in the migration of prothymocytes to their site of maturation, i.e. the thymus. High endothelial venules (HEV) were not demonstrable at the early onset of lymphocyte immigration into the developing lymphoid organs. However, when large-scale influx of lymphocytes occurred, it paralleled HEV development. HECA-452 antigen expression preceded the morphological transformation of endothelium into a HEV phenotype. Expression of this antigen therefore, independently reflected the specialized nature of high endothelium. In a patient with complete DiGeorge's syndrome normal HEV developed, indicating that the presence of T lymphocytes is not a requirement for HEV development. Interestingly, a subpopulation of venules located in the thymic medulla near the cortico-medullary junction expressed the HECA-452 antigen. These vessels, which had flat or intermediately high endothelium, are probably involved in lymphocyte migration to the thymus.     

Disappearance and reappearance of high endothelial venules and immigrating lymphocytes in lymph nodes deprived of afferent lymphatic vessels: a possible regulatory role of macrophages in lymphocyte migration

Interruption of the afferent lymphatic vessels of the popliteal lymph node resulted in the disappearance of high endothelial venules (HEV) and immigrating lymphocytes within 3 weeks. HEV showed several characteristic morphological changes: the endothelial cells became flattened and less pyroninophilic, the chromatine became condensed and protein synthetizing and secretory cell organelles became scarce. At the same time the number of macrophages in the lymph node was severely reduced. Injection of sheep red blood cells into such lymph nodes, 6 weeks after operation, resulted in reappearance of HEV and immigrating lymphocytes, and development of many plasma cells and some germinal centres. Injection of lipopolysaccharide into the operated lymph nodes resulted in the appearance of many plasma cells and a few poorly developed germinal centres; HEV and immigrating lymphocytes, however, remained almost absent. The results show a relationship between the immigration of lymphocytes and the activity of the endothelial cells in the HEV. The activation of the latter may occur by mediators released by antigen-stimulated macrophages and T cells. Moreover, the morphological features of the HEV are independent of the presence of recirculating lymphocytes.     

Education of human lymphocytes against mouse cells: specific recognition of H-2 antigens.

Xeno-sensitization of human peripheral blood lymphocytes (PBL) against mouse lymphoid cells has been studied in vivo in a local graft-vs. -host (GVH) assay and in vitro in a mixed lymphocyte culture-cell-mediated lympholysis system. Human PBL were injected into the footpads of mice rendered unresponsive by total-body irradiation, and these were subsequently tested for the presence of cytotoxic cells in the draining popliteal lymph node (LN). In spite of a definite PBL-induced LN proliferation, no cytotoxic activity was detected against mouse target cells. In contrast with the GVH assay, human PBL collected after 7 days of in vitro culture in the presence of irradiated mouse cells, were strongly cytotoxic against mouse target cells. The antigenic specificities recognized by in vitro educated cells were primarily those coded for by the H-2 complex. Only target cells with an H-2 haplotype identical to that of the sensitizing mouse strain or with at least an H-2 D end in common with that strain were killed by xeno-sensitized lymphocytes. Mouse target cells derived from congenic resistant strains remained unaffected. It was verified that in vitro educated PBL depleted of B cells retained their cytotoxic effect, indicating that non-B cells and probably T cells were involved in the response.     

Rapid decrease in lymphocyte adherence to high endothelial venules in lymph nodes deprived of afferent lymphatic vessels

Occlusion of the afferent lymph flow to the lymph node (LN) results in both flattening of the endothelium of high endothelial venules (HEV) and a severe decrease in numbers of lymphocytes in transit across the walls of the flattened HEV. In the present study we have used the in vitro lymphocyte-binding assay to investigate the ability of HEV in rat LN to bind lymphocytes at various time points after occlusion of the afferent lymph flow. In addition the specificity of T and B lymphocyte adherence to HEV of such operated LN was studied. In normal LN, lymphocytes adhered to virtually all HEV using the in vitro binding assay. However, 1 and 2 weeks after operation lymphocytes bound to only 50-60% of the HEV and by 3-6 weeks 20-30%. The total numbers of lymphocytes bound to these HEV had also diminished to 10% of the control value 3-6 weeks after operation. Morphometric analysis showed that this was not only due to a reduction in the area of HEV endothelium available for lymphocyte adherence by flattening of the high endothelial cells, but also to a strong decrease in the numbers of bound lymphocytes per unit area high endothelium. In spite of the reduction in numbers of adhering lymphocytes the T/B cell ratio did not change. The results show that the reduction in lymphocyte binding of HEV in operated LN is a rapid event, probably due to loss of high endothelial cell determinants involved in binding of lymphocytes. The decrease in lymphocyte binding clearly precedes flattening of HEV endothelium suggesting that the height of high endothelial cells is of secondary importance to lymphocyte adherence.     

The interaction of high endothelial venules with T and B cells in peripheral lymph nodes after antigenic stimulation

The influence of an ongoing immune response on the specific interaction of high endothelial venules (HEV) in peripheral lymph nodes with T and B cells was determined using short-term homing experiments and an in vitro HEV adherence assay. It was demonstrated that stimulation with various antigens did not change the receptor specificity of the HEV. However, the retention and/or the efflux of immigrant cells in the stimulated lymph nodes had changed.     

Control mechanism of lymphocyte traffic Altered migration of 51Cr-labeled mouse lymph node cells pretreated in vitro with phospholipases

Glycoproteins have so far been the only surface components thought to be of importance in the process of recognition between circulating lymphocytes and endothelial cells of the lymph node post-capillary venules and thus in the control of lymphocyte traffic. In this paper the effect of in vitro treatment of ⁵¹Cr-labeled mouse lymph node cells with phospholipases (PL) (A and C) on their migration into syngeneic recipients was investigated. PL-A and PL-C-treated cells migrated differently from control (untreated) cells. Diminished numbers of PL-A-treated lymphocytes were found in the lymph nodes at 1, 6 and 24 h after cell transfer with a simultaneous increase in the lungs at 1 h, and spleen at 6 h after transfer. PL-C-treated cells remained in the blood longer than untreated cells. We conclude that factors other than the integrity of surface glycoproteins are involved in the control of lymphocyte traffic. The roles played by cell adhesion, in particular cell-to-cell interactions in regulating the rate of cell migration, are discussed.     

The migratory behavior of murine CD4+ cells of memory phenotype

Lymphocyte differentiation is connected with profound alterations in the migratory pattern of lymphocytes. Whereas naive cells predominantly recirculate through lymphoid tissues, activated lymphocytes acquire an increased preference for immigration into non-lymphoid tissues and a reduced capacity for recirculation via high endothelial venules (HEV). A variety of data had indicated that memory-related subpopulations of cells in man and sheep, classified by the low expression of the CD45RA isotype, also lack the capacity to recirculate via HEV. However, recent data in the rat called these results into question. We therefore analyzed the migration properties of murine CD4⁺ T cell subpopulations defined by several markers used to distinguish memory from naive CD4⁺ cells in mice, namely CD45RB, L-selectin and CD44. Our data clearly show that the majority of putative memory cells expressing either low levels of CD45RB, low levels of L-selectin or high levels of CD44 display a strongly reduced capacity for direct entry into lymphoid tissues, including the spleen, from the blood stream. The accumulation in peripheral lymph nodes is further reduced by treatment with anti-L-selectin antibody, which blocks their entry via HEV. This indicates that memory CD4⁺ T cells are not excluded from crossing lymph node HEV, and that the numbers of cells entering the node via this route exceed the numbers entering via the afferent lymph, at least in the absence of local inflammation. Concomitantly, a strongly enhanced localization of cells of the memory phenotype is observed in lung and liver as compared with naive cells. Trafficking to specific sites such as skin or gut mucosa is not a prominent feature of the total population of memory cells. The trafficking to lung and liver and an increased ability to bind to dendritic cells, demonstrable in in vitro adhesion assays, suggest a more sessile phenotype of most memory cells. With respect to these properties, memory cells have a surprizing similarity to fully activated lymphocytes.     

Blockade of both L-selectin and alpha4 integrins abrogates naive CD4 cell trafficking and responses in gut-associated lymphoid organs

The recirculation of naive lymphocytes from blood to lymph that is initiated in high endothelial venules (HEV) of secondary lymphoid organs such as lymph nodes and Peyer's patches (PP) is regulated by multiple interactions of adhesion receptor/counter-receptor pairs involving both selectins and integrins. We showed previously that blocking of only L-selectin is sufficient to ablate trafficking of naive CD4 cells and the development of their responses in peripheral lymph nodes but not in PP where alpha4beta7 integrins are thought to primarily regulate entry. However, although antibody to alpha4 integrins partially inhibited homing of naive CD4 cells to PP and not to lymph nodes, there was no effect on the development primary responses in these tissues or spleens. Since previous studies indicate that both alpha4beta7 integrins and L-selectin regulate adhesion of naive cells to PP HEV, we examined the effect a blockade of both adhesion pathways on the recirculation of naive CD4 cells. There was no detectable homing of naive CD4 cells to PP or lymph nodes when interactions with both receptors were inhibited, resulting in a profound depletion of naive CD4 cells and loss of antigen responses in these sites. In contrast, increased numbers of naive CD4 cells and responses of higher magnitude were found in the spleen. The results demonstrate recirculation of naive CD4 cells through tissues where entry is controlled through HEV is essential for the local generation of primary responses.      

A Method for Tracking the Migration of Blood Lymphocytes

A method has been devised for labeling whole blood with the fluorescent dye fluorescein isothiocyanate (FITC) so the migration of blood lymphocytes can be studied in the sheep. Although lymphocytes can be purified from blood using density gradient media or elutriation it is difficult to obtain a large number of cells, because many cells are usually lost during the purification steps. It is desirable to label at least 10^8–10⁹ lymphocytes for lymphocyte tracking studies, because a smaller number is difficult to subsequently detect and quantitate in blood and lymph even using flow cytometry. Also, it is desirable to minimize the in vitro manipulation of lymphocytes, because dead or damaged lymphocytes will not recirculate. By labeling all the cellular components of a sample of whole blood rather than first purifying the lymphocytes we have been able to satisfy both of these criteria. Although labeled blood cells of all types are reinjected into the animal, the lymphocytes are easily distinguishable from other cells using a flow cytometer. In these studies between 2.4–12.4×10⁸ lymphocytes were injected intravenously, and they were detectable in the blood and lymph for at least 10 days. The recovery of FITC-labeled (FITC⁺) lymphocytes in efferent lymph is comparable to that of lymphocytes labeled with other fluorescent or radioactive markers. The presence of labeled non-lymphoid cells in the animal makes this technique impractical for studies of lymphocyte localization within histologic sections. However, it is useful for studies in animals in which lymphatic vessels can be cannulated and the blood-to-lymph recirculation of labeled lymphocytes monitored, and it also may be applicable for studies in which lymphoid organ suspensions are analyzed using flow cytometry.     

The migration of lymphocytes across specialized vascular endothelium: VIII. Physical and chemical conditions influencing the surface morphology of lymphocytes and their ability to enter lymph nodes

The introductory review amplifies the finding that simply holding lymphocytes in vitro reversibly compromises their ability to enter lymph nodes from the blood, although entry into the spleen is unaffected. The differential migration of T and B lymphocytes from the blood, lymphocyte traffic in athymic rats, and the secretion of a sulphated glycoconjugate by high endothelial cells in lymph nodes are also discussed. Original data are presented concerning the effects of varying the conditions under which lymphocytes are held in vitro (time, temperature, medium, centrifugation) on their ability to enter lymph nodes and also on their surface morphology. In general, conditions that reduced the number of microvilli and induced surface blebbing also tended to affect the delicate function of crossing specialized vascular endothelium; but there was no simple relationship between morphology and migratory behavior. The localization of lymphocytes to the bone marrow was augmented by holding them in vitro, and this effect was greater after holding at room temperature (RT) than at 0°C, in contrast to impaired entry into lymph nodes. Small amounts of heparin (10 units) injected along with lymphocytes significantly reduced early localization in lymph nodes. These findings have practical implications for the design of lymphocyte traffic experiments and are relevant to the mechanism of lymphocyte attachment to vascular endothelium, since the well-known effect of trypsinizing lymphocytes can be reproduced by maintenance in vitro.     

Differential regulation of tissue-specific lymph node high endothelial venule cell adhesion molecules by tumour necrosis factor and transforming growth factor-beta 1.

Lymphocytes migrate from blood into lymph nodes (LN) of rats specifically at segments of venules lined by high endothelium (HEV). We have previously shown that pretreatment of LN HEV cells with pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), augments their adhesiveness for thoracic duct lymphocytes (TDL). Here we report that a mouse monoclonal antibody, 3C10, recognized tissue-specific endothelial determinants on rat LN HEV cells and blocked their adhesiveness for TDL and EL-4J cells transfected with rat L-selectin. In contrast, 3C10 antibody did not inhibit lymphocyte attachment to Peyer's patch (PP) frozen sections or cultured PP HEV cells. The antibody immunoprecipitated from LN HEV cells two proteins with apparent molecular weights of 90,000 and 50,000. The expression of 3C10 antigen on LN HEV cells was increased by incubation with TNF-alpha or IFN-gamma. Furthermore, pretreatment of cytokine-stimulated LN HEV cells with 3C10 antibody blocked TDL binding in a dose-dependent manner. In contrast, 3C10 antigen expression on LN HEV cells was significantly decreased following incubation of cells with transforming growth factor-beta 1 (TGF-beta 1). In addition, TGF-beta 1 also abrogated the adhesiveness of LN HEV cells stimulated with TNF-alpha, IFN-gamma or both cytokines. Together, these data suggest that endothelial determinants recognized by the 3C10 antibody are tissue-specific ligands for lymphocyte adhesion and cytokines such as TNF-alpha and TGF-beta differentially regulate their expression and function.     

CD4+ T cells of both the naive and the memory phenotype enter rat lymph nodes and Peyer's patches via high endothelial venules: Within the tissue their migratory behavior differs

It is thought that naive T cells predominantly enter lymphoid organs such as lymph nodes (LN) and Peyer's patches (PP) via high endothelial venules (HEV), whereas memory T cells migrate mainly into non-lymphoid organs. However, direct evidence for the existence of these distinct migration pathways in vivo is incomplete, and nothing is known about their migration through the different compartments of lymphoid organs. Such knowledge would be of considerable interest for understanding T cell memory in vivo. In the present study we separated naive and memory CD4⁺ T cells from the rat thoracic duct according to the expression of the high and low molecular weight isoforms of CD45R, respectively. At various time points after injection into congenic animals, these cells were identified by quantitative immunohistology in HEV, and T and B cell areas of different LN and PP. Three major findings emerged. First, both naive and memory CD4⁺ T cells enter lymphoid organs via the HEV in comparable numbers. Second, naive and memory CD4⁺ T cells migrate into the B cell area, although in small numbers and continuously enter established germinal centers (GC) with a bias for memory CD4⁺ T cells. Third, memory CD4⁺ T cells migrate faster through the T cell area of lymphoid organs than naive CD4⁺ T cells. Thus, our study shows that memory CD4⁺ T cells are not excluded from the HEV route. In addition, “memory” might depend in part on the ability of T cells to specifically enter the B cell area and GC and to screen large quantities of lymphoid tissues in a short time.     

Early deficit of lymphocytes in Wiskott-Aldrich syndrome: possible role of WASP in human lymphocyte maturation

Wiskott–Aldrich syndrome (WAS) is an X‐linked platelet/immunodeficiency disease. The affected gene encodes WASP, a multidomain protein that regulates cytoskeletal assembly in blood cells. Patients have recurring infections, and their lymphocytes exhibit deficient proliferative responses in vitro. We report an evaluation of peripheral blood lymphocytes of 27 WAS patients, aged one month to 55 years. Whereas NK cells were normal, a significant deficit of T and B lymphocytes was observed. The number of lymphocytes was already decreased in infant patients, suggesting deficient output. Both CD4 and CD8 T lymphocytes were affected; the decrease was most pronounced for naïve T cells. Naïve CD4 lymphocytes of patients showed normal expression of Bcl‐2, and Ki‐67, and normal survival in vitro, suggesting that their in vivo survival and proliferation are normal. The collective data suggest that the patients’ lymphocyte deficit results from deficient output, likely due to abnormal lymphocyte maturation in the thymus and bone marrow. We propose that WASP plays an important role not only in the function of mature T lymphocytes, but also in the maturation of human T and B lymphocytes and that impaired lymphocyte maturation is central to the aetiology of WAS immunodeficiency.     

The human peripheral lymph node vascular addressin. An inducible endothelial antigen involved in lymphocyte homing.

The extravasation of blood-borne lymphocytes into organized lymphoid tissues and sites of chronic inflammation is directed in part by interactions of lymphocyte surface adhesion molecules, known as homing receptors, with tissue-selective endothelial ligands called vascular addressins. In mice and humans, lymphocyte L-selectin and the peripheral lymph node addressin (PNAd) form a homing receptor-endothelial ligand pair involved in lymphocyte traffic to peripheral lymph node (PLN). We have examined the tissue distribution and function of human PNAd, using monoclonal antibody MECA-79 and in vitro assays of L-selectin-dependent lymphocyte binding. We demonstrate that PNAd is expressed by human high endothelial venules (HEV) in lymphoid tissues which support lymphocyte adhesion via a PLN-associated recognition system. MECA-79 inhibits adhesion to these HEV of a cell line that binds predominantly via the PLN-homing receptor, L-selectin, but has no effect on adhesion by a mucosal HEV-binding cell line. Furthermore, MECA-79 blocks binding of human peripheral blood mononuclear cells to both PLN and tonsil HEV, but not significantly to HEV in the appendix. In addition, we demonstrate PNAd induction on venules at chronic inflammatory sites in humans, particularly sites with severe or long-standing chronic inflammatory involvement. These results confirm that PNAd functions as a PLN vascular addressin in humans, and that in addition to directing normal lymphocyte recirculation to lymph nodes and tonsils, this addressin likely participates in lymphocyte recruitment to sites of chronic inflammation.     

Different forms of human vascular adhesion protein-1 (VAP-1) in blood vessels in vivo and in cultured endothelial cells: implications for lymphocyte-endothelial cell adhesion models

Vascular endothelium plays a pivotal role in controlling leukocyte extravasation from the blood into the tissues. Vascular adhesion protein-1 (VAP-1) is a novel endothelial cell molecule which mediates lymphocyte binding to the vascular lining (Salmi, M., and Jalkanen, S., Science 1992. 257: 1407). In this study, we analyzed endothelial cell type-specific differences of VAP-1. In vivo, VAP-1 is a 90/170-kDa molecule which is mainly expressed on the lumenal surface and in cytoplasmic granules of peripheral lymph node-type postcapillary venules (high endothelial venules, HEV). In tonsil HEV, VAP-1 is modified with abundant sialic acids. VAP-1 is also detectable in the cytoplasm of human umbilical vein endothelial cells (HUVEC) and in an endothelial cell hybrid EaHy-926, although both cell types lack detectable surface VAP-1. Cultured endothelial cells do not express MECA-79-defined peripheral lymph node addressins either. VAP-1 was not translocated onto the endothelial cell surface after stimulation with multiple cytokines, mitogens or secretagogues which induced expression of other known endothelial adhesion molecules. Biochemical analyses revealed that VAP-1 is a ～ 180-kDa protein in these endothelial cell types. Digestions with neuraminidase, O-glycanase and N-glycanase, as well as treatment of cells with tunicamycin and benzyl-N-acetylgalactosaminide, did not alter the molecular mass of VAP-1 in EaHy-926. Pulse-chase experiments showed that VAP-1 is directly synthesized as a 180-kDa molecule without any detectable precursors. Thus, in cultured endothelial cells, VAP-1 is a 180-kDa protein which is devoid of post-translational modifications, and in particular, lacks the sialic acids crucial for the function of VAP-1 in tonsil vessels. Notably, the endothelial cell types commonly used as a model in studying lymphocyte-endothelial cell interactions lack surface expression of VAP-1 and peripheral node addressins, and hence are inherently of limited use in analyses of the initial adhesion of lymphocytes.