Lymphocyte-endothelial Cell Interactions Research Articles

Hyaluronan Anchored to Activated CD44 on Central Nervous System Vascular Endothelial Cells Promotes Lymphocyte Extravasation in Experimental Autoimmune Encephalomyelitis

The extravasation of lymphocytes across central nervous system (CNS) vascular endothelium is a key step in inflammatory demyelinating diseases including multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The glycosaminoglycan hyaluronan (HA) and its receptor, CD44, have been implicated in this process but their precise roles are unclear. We find that CD44(-/-) mice have a delayed onset of EAE compared with wild type animals. Using an in vitro lymphocyte rolling assay, we find that fewer slow rolling (<1 μm/s) wild type (WT) activated lymphocytes interact with CD44(-/-) brain vascular endothelial cells (ECs) than with WT ECs. We also find that CD44(-/-) ECs fail to anchor HA to their surfaces, and that slow rolling lymphocyte interactions with WT ECs are inhibited when the ECs are treated with a pegylated form of the PH20 hyaluronidase (PEG-PH20). Subcutaneous injection of PEG-PH20 delays the onset of EAE symptoms by ~1 day and transiently ameliorates symptoms for 2 days following disease onset. These improved symptoms correspond histologically to degradation of HA in the lumen of CNS blood vessels, decreased demyelination, and impaired CD4(+) T-cell extravasation. Collectively these data suggest that HA tethered to CD44 on CNS ECs is critical for the extravasation of activated T cells into the CNS providing new insight into the mechanisms promoting inflammatory demyelinating disease.

Lymphocyte Dynamics on Aligned Endothelial Cells

Immune cells circulating blood vessels recognize the signatures of tissue inflammation in blood vessels and undergo multiple cascades of interactions with inflamed endothelium to eventually leave out the vessels. Parallel plate flow chamber assay has been extensively used to investigate detailed mechanisms of lymphocyte-endothelial cell (ECs) interactions. However, orientation of ECs, which may play significant roles in lymphocyte dynamics on inflamed endothelium, has not been considered in these assays yet. Indeed, healthy blood vessels are composed of endothelium aligned in parallel with the direction of blood flow. To address the importance of ECs alignment on lymphocyte dynamics, we firstly aligned ECs on the substrates containing nanoscale grooves and ridges. Then, dynamics of T cells on the aligned endothelial layers (either parallel or perpendicular to the direction of flow) and randomly oriented ECs were monitored by video microscopy. Firstly, we quantitatively analyzed the direction of T cell crawling under the conditions stated above. Interestingly, the direction of T cell crawling was not significantly affected by the flow direction, but rather biased toward the direction of EC alignment. Indeed, when junctions and nuclei of ECs were visualized, T cells preferentially migrated along the EC the junctions, while avoided crawling on top of EC nucleus. Based on this observation, we hypothesized that topographical structures and mechanical properties of ECs guided crawling of T cells and performed a number of experiments to test the hypothesis. Secondly, we investigated the effect of EC alignment on transendothelial migration (TEM) of T cells. T cells on poorly-aligned ECs underwent TEM more quickly and frequently than T cells on well-aligned ECs. Further analysis revealed that local alignment of EC junctions surrounded by more than three ECs form preferential sites for TEM of T cells.

Downregulation of endothelial adhesion molecules by dimethylfumarate, but not monomethylfumarate, and impairment of dynamic lymphocyte-endothelial cell interactions

Although fumaric acid esters (FAE) have a decade-long firm place in the therapeutic armamentarium for psoriasis, their pleiotropic mode of action is not yet fully understood. While most previous studies have focused on the effects of FAE on leucocytes, we have addressed their activity on macro- and microvascular endothelial cells. As detected both on mRNA and protein levels, dimethylfumarate effected a profound reduction of TNFα-induced expression of E-selectin (CD62E), ICAM-1 (CD54) and VCAM-1 (CD106) on two different endothelial cell populations in a concentration-dependent manner. This reduction of several endothelial adhesion molecules was accompanied by a dramatic diminution of both rolling and firm adhesive interactions between endothelial cells and lymphocytes in a dynamic flow chamber system. Dimethylfumarate, at a concentration of 50 μm, reduced lymphocyte rolling on endothelial cells by 85.9% (P<0.001 compared to untreated controls), and it diminished the number of adherent cells by 88% (P<0.001). In contrast, monomethylfumarate (MMF) influenced neither surface expression of adhesion molecules nor interactions between endothelial cells and lymphocytes. These observations demonstrate that endothelial cells, in addition to the known effects on leucocytes, undergo profound functional changes in response to dimethylfumarate. These changes are accompanied by severely impaired dynamic interactions with lymphocytes, which constitute the critical initial step of leucocyte recruitment to inflamed tissues in psoriasis and other TNF-related inflammatory disorders.

α4β1 integrin-dependent cell sorting dictates T-cell recruitment in oral submucous fibrosis

Aim:The biological mechanism(s) that guide the immunological effectors of lymphocytes to sites of inflammatory response, a feature consistently seen in oral submucous fibrosis (OSF) was evaluated. It is envisaged that endothelial/lymphocyte adhesion cascades involving VCAM-1/α4β1 integrins control the migration of lymphocytes across the vascular endothelium resulting in their homing in these locales.Materials and Methods:The study group comprised 28 OSF cases (M:F = 12:16, age range 18-65 years; mean 55.4 ± 8.5 SD) divided into early (n=17) and advanced (n=11) disease groups. Biopsy specimens of normal buccal mucosa (site compatible) were obtained from 10 healthy volunteers (age and sex matched) who served as control. All the samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. Immunolocalization of β1 subunit associated with α4 integrin was performed by a mouse heterodimer (clone 4B7R, Ig G, R & D Systems Inc., dilution 1:100) using a peroxidase labeled streptavidin–biotin technique. The immunocompetent cell density was expressed as the number of positive cells per mm2. The Mann–Whitney U-test and Fischer exact test were used to evaluate differences. P<0.05 was considered to be significant.Results:The median percentage of “T” lymphocytes with positive integrin α4β1 expression was 77.7 (an interquartile range of 73.3–83.4) for the test cases and for the controls, it was 28.2 (IQR 24.0–38.3). This difference was significant at 0.001 level. For the endothelial cells the positive expression was 82.8 (IQR 77–90.6) and 22.3 (IQR 18.3–29.2) respectively (P<0.001). When the intensity of integrin expression was considered 26/28 cases (96%) and 2/10 (20%) of controls showed intense expression of integrins α4β1 on T lymphocytes (P<0.001). Similarly, 27/28 cases (92.9%) and 2/10 (20%) of controls showed intense expression on endothelial cells (P<0.001). T lymphocyte–endothelial cell interactions were assessed by evaluating the overexpression of integrins on both the endothelial cells and lymphocytes together. The interaction was positive in 15/17 and 11/11 early and advanced OSF cases respectively (P=0.51).Conclusion:Following leukocyte activation, the interaction between leukocyte integrin heterodimers and endothelial superfamily adhesion ligands results in a firm adherence of leukocytes to endothelium, leading to leukocyte migration and homing to sites of mucosal inflammation consistently seen in OSF.

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.

FK778: New Cellular and Molecular Mechanisms of Action

Purpose The new malononitrilamide FK778 is currently being evaluated as an immunosuppressant for organ transplantation. Its main mechanism is inhibition of a pivotal enzyme of pyrimidine biosynthesis. This report revealed new mechanisms of action on different cell types involved in acute and chronic allograft rejection. Methods Purified Brown-Norway rat aortic endothelial cell (EC) cultures were pretreated with several concentrations of FK778. Endothelial adhesion molecule expression (ICAM-1/VCAM-1) stimulated with TNF-α was quantified by immunofluorescence. Purified Lewis rat lymphocytes (LC) incubated with FK778 were stimulated via TCR/CD28 signals, and CD25 expression was quantified using FACS analysis. Uridine addition was used in all assays to reverse the pyrimidine synthesis blockade. Lymphocyte–EC interaction was assessed by micromanipulator-assisted single-cell adhesion assays. Finally, smooth muscle cell (SMC) proliferation and migration was analyzed. Uridine addition was used in all assays to reverse the pyrimidine synthesis blockade. Results TNF-α stimulation and TCR/CD28 co-stimulation significantly increased EC ICAM-1/VCAM-1-expression and LC CD25 surface expression, respectively. These effects were dose-dependently inhibited by FK778 and were not reversed by the addition of uridine. FK778 dose-dependently attenuated LC adhesion to allogeneic EC. The dose-dependent inhibition of SMC proliferation by FK778 was abolished by uridine addition, whereas the inhibitory effect on SMC migration was not affected by uridine supplementation. Conclusions FK778 directly reduced endothelial adhesion molecule up-regulation, inhibited lymphocyte activation, and attenuated lymphocyte-endothelium interactions, critical early steps in graft rejection. These effects were separate from the blockade of pyrimidine synthesis. The antiproliferative potency of FK778 on SMC may be an important mechanism to inhibit the fibroproliferative lesions of chronic organ rejection.

Endogenous Galectn‐1 inhibits lymphocyte‐endothelial cell interactions as determined by small interference RNA

It has recently been suggested that, besides its role on T cell life span, the β-galactoside binding protein Galectin-1 (Gal-1) may play a pivotal role in controlling experimental inflammation by modulating leukocyte-endothelial cell interactions. These studies have so far focused on the effects of exogenously administered Gal-1. The primary objective of this study was to investigate the function of endothelial Gal-1 on lymphocyte-endothelial cell interactions under flow conditions. To achieve this, small interference RNA (siRNA) synthesised by Santa Cruz Biotechnology was used to deplete Gal-1 in human umbilical vein endothelial cells (HUVEC). Cells were transfected with or without non-targeting or Gal-1 specific siRNAs. In all cases, HUVECs were stimulated with TNF-α (10 ng/ml) for 4h, and for 15 min with SDF-1α (20ng/ml) prior to perfusion with freshly isolated lymphocytes (5x105 cells/ml; perfused at 1 dyne/cm2 for 8 min then 10 random frames recorded for off-line analysis). Application of siRNA suppressed Gal-1 levels by 75% at 48h post-transfection as monitored by western blotting. Knockdown of Gal-1 led to a significant increase (86%) in the initial capture of lymphocytes to the endothelium and a subsequent increase in cell rolling and adhesion. These data are strongly indicative that the absence of endothelial Gal-1 favours the initial steps of the inflammatory response. To conclude, knockdown of Gal-1 increases lymphocyte recruitment under flow, indicating that endogenous Gal-1 may act to limit such recruitment during inflammation. This work was funded by the Bart’s and the London Special Trustees (RAB03/Mres)

In vivodemonstration of T lymphocyte migration and amelioration of ileitis in intestinal mucosa of SAMP1/Yit mice by the inhibition of MAdCAM-1

The aetiology of Crohn's disease (CD) remains unknown. Since SAMP1/Yit mice have been reported to develop CD-like spontaneous enteric inflammation, such mice have been studied as an animal model of CD. In this study, using this model we examined T lymphocyte migration in microvessels of intestinal mucosa in vivo and the expression of adhesion molecules by immunohistochemistry. Fluorescence-labelled T lymphocytes isolated from AKR/J (control) mice were injected into the tail veins of recipient mice, and T lymphocyte migration in the postcapillary venules of Peyer's patches, submucosal microvessels, and villus capillaries of the terminal ileum was monitored using an intravital microscope. Adhesion of T lymphocytes was significantly increased in 35 week old SAMP1/Yit mice compared with that in AKR/J or 15 week old SAMP1/Yit mice. Immunohistochemical study showed increased infiltration of CD4, CD8 and beta7-integrin-positive cells and increased expression of MAdCAM-1 and VCAM-1 in the terminal ileum of SAMP1/Yit mice. Antibodies against MAdCAM-1 and VCAM-1 significantly inhibited adhesion of T lymphocytes to microvessels of the terminal ileum, and anti-MAdCAM-1 antibody showed stronger suppressive effect than the anti-VCAM-1 antibody. Periodical administration of anti-MAdCAM-1 antibody twice a week for 7 weeks significantly ameliorated ileitis of SAMP1/Yit mice, but submucosal hypertrophy was not significantly suppressed. Anti-VCAM-1 antibody treatment failed to show significant resolution of ileitis. In addition, anti-MAdCAM-1 antibody treatment also attenuated established ileitis. The results demonstrate that, although MAdCAM-1 and VCAM-1 play an important role in T lymphocyte-endothelial cell interactions in SAMP1/Yit mice, MAdCAM-1 may be a more appropriate target for therapeutic modulation of chronic ileitis.

Role of alpha4 integrin and its ligand VCAM-1 in the specific extravasation of a tumor-specific TH2 clone into tumor tissue that initiates its rejection.

The effectiveness of anticancer immunotherapeutic strategies involving the transfer of tumor-specific T cells depends on appropriate lymphocyte-endothelial cell interactions that facilitate the migration of lymphocytes into tumor. Here, we investigated the molecular mechanisms underlying the migration of the antigen-specific Th2 CD4(+) T-cell clone YS1093 into S1509a tumor tissue. YS1093 is specific for the S1509a tumor but does not recognize the S713a tumor. Transfer of YS1093 cells into mice bearing both S1509a and S713a tumors caused only the S1509a tumor to regress. This regression was markedly inhibited by pretreating YS1093 cells with an anti-alpha4 integrin MAb and administering an anti-VCAM-1 MAb at T-cell transfer. Since vascular endothelial cells in S1509a tumor tissues express VCAM-1 and the MHC class II (I-E(k)) molecule restricting YS1093 activity, labeled YS1093 cells migrated specifically into the S1509a tumor, and this migration was also blocked by the anti-TCRbeta F(ab')(2) and anti-I-E(k) MAbs. Furthermore, in vitro assays revealed that anti-CD3 MAb-mediated TCR cross-linkage initiated the binding of alpha4 integrin on YS1093 cells to VCAM-1. This adhesive activity was completely blocked by the anti-alpha4 integrin MAb. These results strongly suggest that i.v.-transferred YS1093 cells act in tumor regression by specifically recognizing their tumor antigen peptide in the context of I-E(k) on vascular endothelial cells in the S1509a tumor, which activates the binding of alpha4 integrin to VCAM-1 on the endothelial cells, facilitating YS1093 extravasation into the tumor. It is likely that this initial migration of specific CD4(+) T cells into tumor tissues promotes the subsequent infiltration into the tumor of other immunocytes that effect tumor destruction.

Contributions from self-renewal and trafficking to the uterine NK cell population of early pregnancy.

Uterine NK (uNK) cells are abundant in human and murine uteri during decidualization. It is unclear whether precursors of uNK (pre-uNK) cells self-renew or are recruited from other sites. To assess self-renewal of pre-uNK cells, uterine segments from NK cell-competent mice were grafted orthotopically into NK/uNK cell-deficient or wild-type mice. Only in wild-type recipients did decidualized grafts contain uNK cells, indicating that pre-uNK cells do not self-renew in uterus. To identify pre-uNK cell sources, thymus, bone marrow, lymph node, or spleen cells were grafted from virgin or pregnant NK cell-competent donors into mated NK/uNK cell-deficient recipients. Cells from secondary lymphoid tissues of pregnant donors gave high level uNK cell reconstitution, which was independent of chemokine receptors CCR2 or CCR5. Pregnancy-induced changes to lymphocyte-endothelial cell interactions were documented using adhesion of human lymphocytes to frozen mouse tissue sections under shear. A dynamic increase was observed in L-selectin- and alpha(4) integrin-dependent adhesion of CD56(bright) NK cells to decidualizing uterus and in human PBL adhesion to lymph node endothelium. These data support a model that attributes the dramatic increases in human and murine uNK cells during decidualization to precursor cell recruitment.

Nitric oxide modulates T-lymphocyte migration in Peyer's patches and villous submucosa of rat small intestine

Background & Aims: Although nitric oxide (NO) is known to influence the recruitment of neutrophils in inflamed tissue, its role in lymphocyte-endothelial cell interactions remains poorly understood. The objectives of this study were to assess the effects of NO synthesis inhibition on T-lymphocyte migration in microvessels of rat small intestine and to define the role of adhesion molecules in this process. Methods: T lymphocytes collected from rat intestinal lymph were labeled with carboxyfluorescein diacetate succinimidyl ester and injected into the jugular vein of recipient rats. The migration of T lymphocytes into normal and N G -nitro- L-arginine methyl ester ( L-NAME)-treated intestinal microvessels was monitored by using an intravital microscope. Results: L-NAME significantly increased rolling and adherence of lymphocytes in postcapillary venules of Peyer's patches and submucosal venules without significantly decreasing red blood cell velocity. The subsequent appearance of lymphocytes in the initial lymphatics was also accelerated by L-NAME. Anti–α4-integrin antibody markedly inhibited the L-NAME–induced lymphocyte-endothelial cell interaction. Anti–P-selectin monoclonal antibody also significantly attenuated these adhesive interactions in both vascular regions. Conclusions: These data suggest that NO is an important modulator of lymphocyte migration in Peyer's patches and in nonlymphoid regions of the intestine. GASTROENTEROLOGY 1998;115:618-627

Cloning and Characterization of Mouse Vascular Adhesion Protein-1 Reveals a Novel Molecule with Enzymatic Activity

Human vascular adhesion protein-1 (VAP-1) is a sialylated endothelial cell adhesion molecule mediating the initial L-selectin-independent interactions between lymphocytes and endothelial cells in man. In this work we cloned and characterized mouse VAP-1 (mVAP-1) and produced an anti-mVAP-1 mAb against a recombinant mVAP-1 fusion protein. The isolated cDNA encodes a novel 84.5-kDa mouse molecule. The anti-mVAP-1 mAb stained high endothelial venules in peripheral lymph nodes, and smooth muscle cells and lamina propria vessels in gut. During immunoblotting, this anti-mVAP-1 mAb recognized a 110/220-kDa Ag, suggesting that mVAP-1 is a dimer. Since mVAP-1 has significant sequence identity to members of a family of enzymes called the copper-containing amine oxidases, we showed that mVAP-1 possesses monoamine oxidase activity. Thus, mVAP-1 is the first mouse membrane-bound amine oxidase identified at the molecular level. Based on the 83% identity between the isolated cDNA and human VAP-1 cDNA, the expression pattern, the molecular mass, and the enzyme activity against monoamines, the cloned molecule represents a mouse homologue of human VAP-1. Cloning of mVAP-1 provides a valuable tool for in vivo studies of the significance of VAP-1 for lymphocyte-endothelial cell interactions and of the possible relationship between leukocyte adhesion and amine oxidase activity.

IV. Lymphocyte trafficking in the intestine and liver

Naive lymphocytes patrol continuously between the blood and different lymphatic tissues to sample the whole body for foreign antigens. During inflammation, leukocyte recruitment into tissue is enhanced to promote the recruitment of a range of effector cells into the affected area. The complex recirculatory pathways that underlie this process are governed by adhesion receptors on blood-borne leukocytes and by their specific ligands expressed on the luminal aspect of endothelial cells lining the vessels. Gut-associated lymphatic tissues are positioned strategically at the major port of entry for foreign antigens. They form a functionally unified entity that utilizes both the afferent and efferent arms of the immune response to respond to the large array of antigens entering via the gut under normal conditions as well as during inflammation. Once antigens have been absorbed from the gut, they may enter the portal vein and the liver where the immune response can be further regulated by the resident immune cells of the liver. Thus the gut and liver form an important barrier to enteral antigens, and leukocyte recruitment to these sites will need to be carefully regulated to ensure effective immune surveillance. In this article, we describe the current concepts of lymphocyte adhesion in these two organs as revealed by animal models. Subsequently, we discuss how well these principles apply to the lymphocyte-endothelial cell interactions in humans and what additional insights can be obtained from human studies.

Expression of ICAM-1 enhances in vivo lymphocyte adhesion in a murine fibrosarcoma.

ICAM-1 is essential for lymphocyte-endothelial cell interactions. We have demonstrated that increased expression of ICAM-1 in tumors results in an enhanced response to adoptive immunotherapy. We undertook this study to determine whether increased expression of ICAM-1 results in increased lymphocyte adhesion in vivo. Parental MCA-105 tumor cells were cotransfected with ICAM-1 and the NeoR plasmid. A neomycin resistant clone (Cl149) was selected and increased expression of ICAM-1 confirmed by FACS analysis. Tumor fragments (MCA-105 or Cl149) were placed in a dorsal skin-fold chamber on day 0 in C57BL/6 mice. Lymphocytes were fluorescently labeled using 0.5% acridine orange and activity recorded on videotape at 700x magnification. Lymphocyte activity was quantitated over 30 second intervals in postcapillary venules as either passing or rolling/sticking (R/S). The % R/S was calculated for each category and evaluated using chi 2 analysis. Whereas 38% of lymphocytes were classified as R/S in normal tissue, 32% were classified as R/S (P > .05) in the MCA-105 tumor. However, in the ICAM-1 transfected CL149, there was significantly greater R/S at 53% (P < .05). These data demonstrate increased lymphocyte adhesion in tumors with enhanced expression of ICAM-1 by direct in vivo observations and may partially explain the salutary effect of increased ICAM-1 expression on adoptive immunotherapy. This suggests the possible application of adhesion molecule expression in the cellular therapy of cancer.

Expression of ICAM‐1 enhances in vivo lymphocyte adhesion in a murine fibrosarcoma

Background and Objectives ICAM-1 is essential for lymphocyte-endothelial cell interactions. We have demonstrated that increased expression of ICAM-1 in tumors results in an enhanced response to adoptive immunotherapy. We undertook this study to determine whether increased expression of ICAM-1 results in increased lymphocyte adhesion in vivo. Methods Parental MCA-105 tumor cells were cotransfected with ICAM-1 and the NeoR plasmid. A neomycin resistant clone (Cl149) was selected and increased expression of ICAM-1 confirmed by FACS analysis. Tumor fragments (MCA-105 or Cl149) were placed in a dorsal skinfold chamber on day 0 in C57BL/6 mice. Lymphocytes were fluorescently labeled using 0.5% acridine orange and activity recorded on videotape at 700× magnification. Lymphocyte activity was quantitated over 30 second intervals in postcapillary venules as either passing or rolling/sticking (R/S). The % R/S was calculated for each category and evaluated using χ2 analysis. Results Whereas 38% of lymphocytes were classified as R/S in normal tissue, 32% were classified as R/S (P > .05) in the MCA-105 tumor. However, in the ICAM-1 transfected CL149, there was significantly greater R/S at 53% (P < .05). Conclusions These data demonstrate increased lymphocyte adhesion in tumors with enhanced expression of ICAM-1 by direct in vivo observations and may partially explain the salutary effect of increased ICAM-1 expression on adoptive immunotherapy. This suggests the possible application of adhesion molecule expression in the cellular therapy of cancer. J. Surg. Oncol. 1977;66:39–44. © 1997 Wiley-Liss, Inc.

Lymphocyte-endothelial cell interactions in multiple sclerosis: disease specificity and relationship to circulating tumour necrosis factor-alpha and soluble adhesion molecules.

This study addressed two questions; first, whether the supranormal adherence of blood lymphocytes from patients with multiple sclerosis (MS) to endothelial cell monolayers treated with tumour necrosis factor-alpha (TNF alpha) was a feature common to other inflammatory disorders; and second, whether the adherence properties of blood lymphocytes from MS patients were related to changes in disease activity and to levels of circulating TNF alpha and soluble adhesion molecules. In the first part of the investigation, lymphocytes from 14 patients with MS were more adherent to TNF alpha-treated endothelial cells (P < 0.01) than those from healthy controls, whereas the adherence properties of lymphocytes from 12 patients with rheumatoid arthritis, eight patients with psoriasis and ten patients with neurological diseases other than MS were normal. In the second phase of the work, measurement of the adhesive properties of lymphocytes isolated at monthly intervals from a further six MS patients over a 5-8 month period, found that changes in binding to TNF alpha-treated endothelial cells, directly paralleled changes in circulating levels of TNF alpha (r = 0.77; P < 0.001) and soluble vascular cell adhesion molecule-I (sVCAM-1) r = 0.67; P = 0.001). An increase in disease activity, measured by T2-weighted and gadolinium-enhanced magnetic resonance imaging of the central nervous system (CNS), occurred in two patients and was associated with heightened lymphocyte adhesiveness and a rise in serum TNF alpha levels. Further analysis of the 34 serum samples from the six MS patients revealed a direct relationship between the concentration of sL-selectin and soluble intercellular adhesion molecule-I (sICAM-I) (r = 0.65; P < 0.001) and between sL-selectin and sTNF alpha (r = 0.42; P < 0.02). These findings support the view that disease activity in MS is associated with an increased adhesive interaction of blood lymphocytes with vascular endothelium at inflammatory sites within the CNS.

Substance P promotes lymphocyte-endothelial cell adhesion preferentially via LFA-1/ICAM-1 interactions

Substance P (SP), an 11 amino acid peptide, is released by C and A delta sensory nerve fibers during tissue insult and inflammation. We investigated the effect of SP on the expression and avidity of adhesion molecules, on lymphocytes and endothelial cells, which are central to the inflammatory cascade. Using in vitro adhesion assays we found that pretreatment of murine endothelial cells with SP enhanced their adhesiveness to splenocytes, the murine T cell hybridoma EL4 and nylon-enriched primary murine T cell in a dose and time dependent manner, the optimum dose being 10 −10 M and the optimum time 6 h. SP at 10 −10 M was also able to stimulate the splenocytes, EL4 T cells and primary T cells to enhance their adhesiveness for endothelial cells. The increased adhesiveness was associated with enhanced expression of ICAM-1 on endothelial cells and increased avidity of LFA-1 on lymphocytes. Further SP was chemotactic for T cells. These data suggest that SP modulates lymphocyte-endothelial cell interactions by preferentially upregulating LFA-1 and ICAM-1 interactions.

Endotoxin stimulates lymphocyte-endothelial interactions in rat intestinal Peyer's patches and villus mucosa

Although lymphocyte-endothelial cell interactions represent a key step in controlling the recruitment of lymphocytes into gut-associated tissues, its dynamic process in microvessels of lymphoid (Peyer's patches) and nonlymphoid (villus) regions of the small bowel remains poorly understood. We monitored the migration of fluorescence-labeled T lymphocytes into normal and lipopolysaccharide (LPS)-inflamed rat intestinal microvessels using intravital microscopy. In Peyer's patches, T lymphocytes selectively adhered to postcapillary venules, although such selectivity was not observed in submucosal venules of villi. T lymphocytes exhibited rolling behavior followed by firm adhesion in microvessels of both the Peyer's patches and the villi, with both types of adhesive interaction being mediated by alpha 4-integrins. The enhanced rolling and adherence of lymphocytes observed in Peyer's patches and submucosal venules of villi of LPS-treated rats were preceded by a reduction in shear rate and were mediated largely by alpha 4-integrins and partly by beta 2-integrins. In capillaries of intestinal mucosa, lymphocyte adherence occurred without rolling and was independent of alpha 4-integrins. LPS also significantly increased adherence of lymphocytes to villus capillaries, which was not mediated by either alpha 4- or beta 2-integrin. These observations demonstrate significant heterogeneity of lymphocyte-endothelial cell interactions within different regions of the intestinal mucosa.

Human vascular adhesion protein 1 (VAP-1) is a unique sialoglycoprotein that mediates carbohydrate-dependent binding of lymphocytes to endothelial cells.

The regulated interactions of leukocytes with vascular endothelial cells are crucial in controlling leukocyte traffic between blood and tissues. Vascular adhesion protein-1 (VAP-1) is a novel, human endothelial cell molecule that mediates tissue-selective lymphocyte binding. Two species (90 and 170 kD) of VAP-1 exist in lymphoid tissues. Glycosidase digestions revealed that the mature 170-kD form of VAP-1 expressed on the lumenal surfaces of vessels is a heavily sialylated glycoprotein. The sialic acids are indispensable for the function of VAP-1, since the desialylated form of VAP-1 no longer mediates lymphocyte binding. We also show that L-selectin is not required for binding of activated lymphocytes to VAP-1 under conditions of shear stress. The 90-kD form of VAP-1 was only seen in an organ culture model, and may represent a monomeric or proteolytic form of the larger species. These data indicate that L-selectin negative lymphocytes can bind to tonsillar venules via the VAP- 1-mediated pathway. Moreover, our findings extend the role of carbohydrate-mediated binding in lymphocyte-endothelial cell interactions beyond the known selectins. In conclusion, VAP-1 naturally exists as a 170-kD sialoglycoprotein that uses sialic acid residues to interact with its counter-receptors on lymphocytes under nonstatic conditions.

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 approximately 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.