Molecules controlling lymphocyte migration to the gut.
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Cell Adhesion Molecules↗
Biomedical subjects
M Salmi
Publications and source records attributed to M Salmi.
Isolation, structural characterization, and chromosomal mapping of the mouse vascular adhesion protein-1 gene and promoter.
Vascular adhesion protein-1 (VAP-1) is an endothelial cell adhesion molecule which mediates lymphocyte binding to endothelial cells. The cloning of a mouse VAP-1 (mVAP-1) cDNA revealed that mVAP-1 is a novel 110/220 kDa transmembrane molecule with significant identity to copper-containing amine oxidases. In this work the nucleotide sequence and primary structure of the mVAP-1 gene was determined and the promoter region was structurally characterized. The isolated approximately 14.4-kb mVAP-1 gene consists of 4 exons and 3 introns. Primer extension analysis and 5' rapid amplification of cDNA ends revealed multiple transcription initiation sites in different tissues suggesting that the mVAP-1 transcription is differently regulated in different tissues. Analysis of the sequence immediately upstream of the detected transcription initiation sites showed no canonical TATA or CCAAT elements, but putative regulatory elements were found close to the detected transcription start sites. The cloning of the mVAP-1 gene reveals the first insight into the genomic organization of murine amine oxidases and will, by targeted disruption of the gene, allow us to understand better the importance of VAP-1 in leukocyte trafficking and monoamine oxidase activity for the function of the immune system.
Amine Oxidase (Copper-Containing)↗
Circulating form of human vascular adhesion protein-1 (VAP-1): increased serum levels in inflammatory liver diseases.
Vascular adhesion protein-1 (VAP-1) is a dimeric 170-kDa endothelial transmembrane molecule that under normal conditions is most strongly expressed on the high endothelial venules of peripheral lymph nodes and on hepatic endothelia. It is a glycoprotein that mediates tissue-selective lymphocyte adhesion in a sialic acid-dependent manner. In this study, we report the detection of a soluble form of VAP-1 in circulation. We developed a quantitative sandwich ELISA using novel anti-VAP-1 mAbs and used it to determine the levels of soluble VAP-1 (sVAP-1) in the serum of healthy individuals and in patients with inflammatory diseases. In healthy persons, circulating sVAP-1 concentrations were 49 to 138 ng/ml. Immunoblotting studies revealed that the apparent molecular mass of dimeric sVAP-1 is slightly (approximately 10 kDa) higher than that of transmembrane VAP-1 under nonreducing conditions. In contrast, the electrophoretic mobilities of monomeric sVAP-1 and transmembrane VAP-1 were similar after reduction and boiling. Adhesion assays showed that the circulating sVAP-1 modulates lymphocyte binding to endothelial cells. Inflammation can cause an elevation of serum sVAP-1 levels, because sVAP-1 concentrations in patients with certain liver diseases were two- to fourfold higher than those in normal individuals. In contrast, rheumatoid arthritis and inflammatory bowel diseases were not associated with elevated levels of sVAP-1. These findings indicate that there is a functionally active, soluble form of VAP-1 in circulation and suggest that the serum level of sVAP-1 might be a useful marker of disease activity in inflammatory liver diseases.
Adult↗
Cloning of vascular adhesion protein 1 reveals a novel multifunctional adhesion molecule.
Vascular adhesion protein 1 (VAP-1) is a human endothelial sialoglycoprotein whose cell surface expression is induced under inflammatory conditions. It has been shown previously to participate in lymphocyte recirculation by mediating the binding of lymphocytes to peripheral lymph node vascular endothelial cells in an L-selectin-independent fashion. We report here that the VAP-1 cDNA encodes a type II transmembrane protein of 84.6 kD with a single transmembrane domain located at the NH2-terminal end of the molecule and six potential N-glycosylation sites in the extracellular domain. In vivo, the protein exists predominantly as a homodimer of 170-180 kD. Ax endothelial cells transfected with a VAP-1 cDNA express VAP-1 on their cell surface and bind lymphocytes, and the binding can be partially inhibited with anti-VAP-1 mAbs. VAP-1 has no similarity to any currently known adhesion molecules, but has significant identity to the copper-containing amine oxidase family and has a monoamine oxidase activity. We propose that VAP-1 is a novel type of adhesion molecule with dual function. With the appropriate glycosylation and in the correct inflammatory setting, its expression on the lumenal endothelial cell surface allows it to mediate lymphocyte adhesion and to function as an adhesion receptor involved in lymphocyte recirculation. Its primary function in other locations where it is expressed, such as smooth muscle, may depend on its inherent monoamine oxidase activity.
Amine Oxidase (Copper-Containing)↗
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.
Amine Oxidase (Copper-Containing)↗
The role of two distinct endothelial molecules, vascular adhesion protein-1 and peripheral lymph node addressin, in the binding of lymphocyte subsets to human lymph nodes.
Lymphocyte binding to high endothelial venules (HEV) in noninflamed peripheral lymph nodes (PLN) relies heavily on two endothelial adhesion molecules called vascular adhesion protein-1 (VAP-1) defined by mAb 1B2 and the peripheral lymph node addressins (PNAd) defined by mAb MECA-79. Data from several different groups indicate that these two molecules share several characteristics in expression, biochemical structure, and function, raising the possibility that VAP-1 may be identical to the 170- and 90-kDa species of PNAd glycoproteins. In this study, we show that many PLN HEV coexpress these two molecules. In parallel SDS-PAGE analyses, the m.w. of the 90- and 170-kDa forms of these molecules are indistinguishable. Nevertheless, we show by different metabolic labelings, by reciprocal cross-precipitations, and by immunofluorescence stainings of newly established VAP-1 transfectants that the 90- and 170-kDa species of PNAd and VAP-1 are distinct molecules. In functional terms, VAP-1 is strikingly selective in mediating PLN HEV adhesion of CD8-positive, but not of CD4-positive T cells. In contrast, PNAd contributes to the adhesion of both CD4-positive and CD8-positive cells to these vessels. Together, these data show that initial adhesion of CD8-positive lymphocytes to PLN HEV requires a PNAd- and a VAP-1-dependent step that are both essential and may occur simultaneously or sequentially.
Amine Oxidase (Copper-Containing)↗
Endothelial ligands and homing of mucosal leukocytes in extraintestinal manifestations of IBD.
Crohn's disease and ulcerative colitis are quite often complicated with manifestations in extraintestinal organs like joints, eye, and skin. Although the etiopathogenesis of these nonmucosal complications remains unsettled, they all share the characteristic feature of inappropriate leukocyte recruitment in nonlymphatic organs. Under normal conditions, lymphocytes recirculate between the blood and lymphoid organs in search of their cognate antigens, whereas polymorphonuclear leukocytes are excluded from tissues. On inflammation, the leukocyte trafficking changes dramatically. Granulocytes infiltrate into the inflammatory focus very rapidly, and they are followed by lymphocytes, especially activated immunoblasts and memory cells, which now also leave the vasculature at nonlymphoid tissues. Leukocyte extravasation from the blood into the tissue is a multistep process governed by sequential interactions between adhesion molecules expressed on the surface of leukocytes and their ligands on the luminal side of the endothelial cells lining the vessels. In this review, we describe the recirculation routes of mucosal lymphocytes in physiologic conditions, as well as the changes seen in mucosal and extramucosal homing in inflammatory bowel disease (IBD). We present a working model of how adhesive molecular interactions between mucosal immune cells and endothelial cells may explain the pathogenesis of the development of inflammatory cell infiltrate in distant organs in IBD, and how this information may help to plan new antiadhesive therapeutic strategies.
Animals↗
Cell adhesion and migration. 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.
Cell Adhesion↗
Vascular adhesion protein 1 (VAP-1) mediates lymphocyte subtype-specific, selectin-independent recognition of vascular endothelium in human lymph nodes.
Interactions between lymphocyte surface receptors and their ligands on vascular endothelial cells regulate the exit of lymphocytes from the circulation. Distinct subsets of mononuclear cells bind to high endothelial venules (HEVs) in different lymphoid organs to a different extent, but the molecular mechanisms behind this selectivity have remained poorly characterized. Here we show that vascular adhesion protein-1 (VAP-1) mediates subtype-specific binding of CD8-positive T cells and natural killer cells to human endothelium. VAP-1-dependent, oligosaccharide-dependent peripheral lymph node (PLN) HEV adhesion under shear was independent of L-selectin, P-selectin glycoprotein ligand 1, and alpha4 integrins, the known lymphocyte receptors involved in the initial recognition of endothelial cells. PLN HEV adhesion was also critically dependent on peripheral lymph node vascular addressins (PNAds), but lymphocyte L-selectin was absolutely required for PNAd binding. Most lymphocytes relied on both PNAd and VAP-1 in HEV binding. The overlapping function of L-selectin ligands and VAP-1 in PLN introduces a new control point into the lymphocyte extravasation process. Finally, intravital microscopy revealed that VAP-1 is involved in initial interactions between human lymphocytes and endothelial cells in inflamed rabbit mesenterial venules in vivo. In conclusion, VAP-1 is a novel contact-initiating ligand that discriminates between different subpopulations of mononuclear cells and is an appealing target for selective modulation of adhesion of CD8- and CD16-positive effector cells.
Adult↗
Ly-6C regulates endothelial adhesion and homing of CD8(+) T cells by activating integrin-dependent adhesion pathways.
Ly-6C belongs to the Ly-6 family of glycosyl phosphatidylinositol-anchored surface glycoproteins and is expressed on a subset of mature CD8(+) T cells. Ly-6C ligation can mediate T cell activation and causes interleukin 2 secretion in cytolytic T cell clones. We characterize herein a new mAb 1G7.G10 against Ly-6C that recognizes an epitope involved in lymphocyte adhesion and in lymphocyte homing. Pretreatment of lymph node lymphocytes and of purified CD8(+) T cells (but not of lymphocytes depleted of CD8(+) T cells) with 1G7.G10 reduced their in vitro binding to lymph node high endothelial venules by 28% and 34%, respectively. This effect was bypassed by cross-linking Ly-6C molecules with 1G7.G10 and a second-step antibody. The in vivo homing of (donor) CD8(+) T lymphocytes to lymph nodes was reduced by Ly-6C blocking with 1G7. G10 (whole antibody) or with its fragments [F(ab) or F(ab)2] by 20% or by 32% and 48%, respectively. Cross-linking of Ly-6C in vitro induced very late antigen-4 and lymphocyte function-associated antigen 1-mediated aggregation of CD8(+) T cells, suggesting that ligand binding to Ly-6C leads to activation of integrins. This activation may facilitate homing of Ly-6C+ CD8(+) T cells in vivo.
Animals↗
Regulation of CD44v6-containing isoforms during proliferation of normal and malignant epithelial cells.
CD44 is a family of molecules involved in cell-cell and cell-matrix interactions. Various isoforms of CD44 arise by insertion of one or more of the variant exons into the common backbone shared by all forms of CD44. In this work, we studied the expression of CD44 and exon v6-containing CD44 isoforms (CD44v6) in several nonmalignant and malignant conditions and the possibilities for regulating the expression of CD44v6. In primary squamocellular carcinomas of the head and neck, CD44 and CD44v6 were down-regulated in poorly differentiated tumors, whereas these molecules were uniformly expressed in the normal squamocellular epithelium, in proliferating skin diseases, and in nonmalignant tumors. When CD44v6 expression of original tumors and that of squamocellular carcinoma cell lines derived from them were compared, no CD44v6 up-regulation could be observed on in vitro growing cells. Moreover, several regulators were unable to up-regulate CD44v6 expression on cultured cell lines in vitro. When the same cell lines formed tumors after s.c. injection into severe combined immunodeficient mice, some of them up-regulated their CD44v6 expression. These data suggest that cell lines at certain differentiation stages can be induced to express CD44v6. Our results further indicate that CD44v6 positivity cannot be used as a universal indicator of tumor metastasis. Instead, the down-regulation of CD44v6 in squamocellular tumors is a sign of malignant transformation of the epithelium.
Adult↗
Homing of mucosal leukocytes to joints. Distinct endothelial ligands in synovium mediate leukocyte-subtype specific adhesion.
Inflammation and infection of the gut can be followed by reactive arthritis at a distant joint. Leukocyte recruitment into synovium is essential for this process, but nothing is known about the endothelial adhesion molecules in synovial membrane which direct the homing of activated, gut-derived leukocytes to joints. Here we analyzed the expression of the known endothelial adhesion molecules in inflamed synovium and their function in binding of mucosal leukocytes. Intercellular adhesion molecule-1 (ICAM-1/CD54) and vascular adhesion protein-1 (VAP-1) were most prominently expressed in synovial vessels. All other adhesion molecules were found at lower levels in inflamed synovia, except mucosal addressin which was absent. Binding of macrophages isolated from lamina propria of the gut to synovial endothelium was almost entirely P-selectin-dependent. In contrast, small intestinal lymphocytes and immunoblasts both relied mainly on VAP-1 in recognition of synovial vessels. Thus, endothelial P-selectin and VAP-1 mediate binding of mucosal effector cells to synovium in a leukocyte subtype-selective manner. Antiadhesive therapy against these inducible molecules should ablate the pathogenetic cascade leading to inappropriate homing of leukocytes to joints in arthritis.
Amine Oxidase (Copper-Containing)↗
Origin and function of circulating CD44 in non-Hodgkin's lymphoma.
Soluble isoforms of various adhesion molecules have recently been found in the circulation, but the physiologic effects of such molecules are still unconfirmed. Our earlier study suggests that the serum level of the 70- to 80-kDa form of CD44 (sCD44) parallels the clinical treatment response in patients with lymphoma. In the present study we investigated the origin and the function of sCD44 in non-Hodgkin's lymphoma. Both peripheral blood and tumor lymphocytes were able to shed soluble CD44 in cell culture. In a SCID mouse model, transplanted Burkitt lymphoma (Namalwa) cells transfected with human CD44 shed soluble CD44. In binding studies sCD44 was able to adhere to hyaluronate and fibronectin, and moreover, sCD44 was able to block the binding of hyaluronate to CD44 on the cell surface and to block the binding of lymphocytes to high endothelial venules, suggesting that sCD44 retains its biological activity although it does not contain the cytoplasmic tail. In conclusion, sCD44 is biologically active and is at least partially shed by lymphoma cells in non-Hodgkin's lymphoma patients.
Animals↗
Function of lymphocyte homing-associated adhesion molecules on human natural killer and lymphokine-activated killer cells.
We have analyzed the ability of fresh and rIL-2-activated human NK cells to interact with high endothelial venules (HEV) that are known to support physiologic lymphocyte extravasation, and examined the role of different adhesion molecules in this process. In in vitro HEV-binding assays, NK cells bound to both peripheral lymph node (pLN) and mucosal HEV. Activation by rIL-2 slightly decreased adherence to pLN HEV, but increased adherence to mucosal high endothelium. Markedly fewer NK cells than PBL expressed L-selectin, and the expression was diminished further upon treatment with rIL-2. Inhibition studies showed, however, that L-selectin was the most important single molecule to mediate adhesion to pLN HEV. Binding to mucosal HEV was mediated mainly by CD44 and alpha 4 integrin, and the expression level of these molecules was increased by rIL-2, paralleling the results in HEV-binding assays. Higher m.w. forms of CD44, representing differentially glycosylated/variant forms of CD44, were more abundant on large granular lymphocytes than on unseparated PBL. We conclude that, despite weak recirculatory capacity, NK cells or a subpopulation of NK cells with the correct adhesion molecules can interact with and bind to high endothelial cells. Lymphokines can modulate the expression of adhesion molecules that NK cells utilize for HEV binding. Our results suggest that activation of NK cells with IL-2 may facilitate the extravasation of lymphokine-activated killer cells, especially to mucosal sites, whereas homing to peripheral lymphoid tissues may be diminished. This should be taken into consideration when procedures for lymphokine-activated killer cell immunotherapy are planned.
Endothelium, Lymphatic↗
Differential regulation and function of CD73, a glycosyl-phosphatidylinositol-linked 70-kD adhesion molecule, on lymphocytes and endothelial cells.
CD73, otherwise known as ecto-5'-nucleotidase, is a glycosyl-phosphatidylinositol-linked 70-kD molecule expressed on different cell types, including vascular endothelial cells (EC) and certain subtypes of lymphocytes. There is strong evidence for lymphocyte CD73 having a role in several immunological phenomena such as lymphocyte activation, proliferation, and adhesion to endothelium, but the physiological role of CD73 in other cell types is less clear. To compare the biological characteristics of CD73 in different cell types, we have studied the structure, function, and surface modulation of CD73 on lymphocytes and EC. CD73 molecules on lymphocytes are shed from the cell surface as a consequence of triggering with an anti-CD73 mAb, mimicking ligand binding. In contrast, triggering of endothelial CD73 does not have any effect on its expression. Lymphocyte CD73 is susceptible to phosphatidylinositol phospholipase, whereas only a small portion of CD73 on EC could be removed by this enzyme. Furthermore, CD73 on EC was unable to deliver a tyrosine phosphorylation inducing signal upon mAb triggering, whereas triggering of lymphocyte CD73 can induce tyrosine phosphorylation. Despite the functional differences, CD73 molecules on lymphocytes and EC were practically identical structurally, when studied at the protein, mRNA, and cDNA level. Thus, CD73 is an interesting example of a molecule which lacks structural variants but yet has a wide diversity of biological functions. We suggest that the ligand-induced shedding of lymphocyte CD73 represents an important and novel means of controlling lymphocyte-EC interactions.
5'-Nucleotidase↗
CD73 mediates lymphocyte binding to vascular endothelium in inflamed human skin.
Inflammatory diseases of the skin are characterized by abundant lymphocytic infiltrates at the site of inflammation, which are critical for the perpetuation of chronic disease. Lymphocytes gain entry to the site of inflammation by the use of adhesion molecules, which recognize their counterparts on vascular endothelial cells. CD73 is a lymphocyte differentiation antigen, which has recently been shown to mediate lymphocyte binding to cultured endothelial cells. Here, we have examined its expression and function in inflammatory situations using inflammatory skin diseases as a model. In several idiopathic and allergic disorders of the skin, a vast majority of the skin-infiltrating lymphocytes were found to express CD73. However, on the circulating lymphocytes of these patients the expression of CD73 does not differ from that of healthy individuals. Of the peripheral blood lymphocytes (PBL) of patients, 13 % are CD73+; of these, 9 % express the cutaneous lymphocyte antigen (CLA), 32 % express CD45RO, and 86 % are L-selectin+. Only 1% of PBL express both CLA and CD73. In contrast, most skin-infiltrating lymphocytes express both molecules, which led us to investigate the role of CD73 in the skin-homing behavior of these cells. In the frozen-section adhesion assay, when PBL were treated with the anti-CD73 monoclonal antibody 4G4, their binding to the vascular endothelium in inflamed skin was inhibited by 70 %. Taken together, our results demonstrate that CD73+ lymphocytes preferentially accumulate into inflamed skin and, most importantly, that CD73 is involved in lymphocyte binding to vessels in inflamed skin. In the future, these findings may offer new means to treat inflammatory disorders of the skin.
5'-Nucleotidase↗
HLA-B27 modulates intracellular survival of Salmonella enteritidis in human monocytic cells.
Human major histocompatibility complex class I allele HLA-B27 is associated with a group of diseases called spondyloarthropathies. In reactive arthritis (ReA), the disease is triggered by certain infections, e.g. gastroenteritis caused by Salmonella. The host/microbe interaction is abnormal in susceptible individuals leading to inefficient elimination of arthritis-triggering bacteria, fragments of them, or both, after the initial infection. Using transfected human monocytic U937 cell lines, we demonstrate that the expression of the HLA-B27 antigen does not influence the uptake of S. enteritidis into U937 cells in vitro. Interestingly, HLA-B27 remarkably impairs the elimination of S. enteritidis within the HLA-B27 transfected U937 cells. The impaired elimination of ReA-triggering microbes by HLA-B27+ monocytes may offer an explanation for the persistence of ReA-triggering microbes in susceptible HLA-B27+ individuals. This modulation of the host/microbe interaction by HLA-B27 may have an important role in the pathogenesis of ReA.
Cell Count↗
Organ-selective regulation of vascular adhesion protein-1 expression in man.
Vascular adhesion protein-1 (VAP-1) is an endothelial molecule which mediates lymphocyte binding to endothelium in peripheral lymph nodes and at certain sites of inflammation. The expression of VAP-1 in vivo is strongly up-regulated in inflamed tissues, such as gut and skin. The purpose of this work was to examine the factors responsible for this induction of VAP-1. Since the expression of VAP-1 could not be induced in cultured endothelial cells with a large panel of mediators, we used an organ culture technique for the investigation of the regulation of VAP-1 expression in a more physiological micromilieu. Indeed, we found that the expression of endothelial VAP-1 could be up-regulated in human tonsillar tissue with interleukin (IL)-1, IL-4, tumor necrosis factor (TNF-alpha), interferon (IFN)-gamma and lipopolysaccharide, whereas histamine, thrombin, dibutyryl cAMP, N-formyl-Met-Leu-Phe (fMLP) and phorbol 12-myristate 13-acetate (PMA) had no effect. The induced VAP-1 protein was similar in molecular weight to the non-induced VAP-1, suggesting that VAP-1 synthesized de novo carries appropriate carbohydrate moieties. In contrast to tonsil organ culture, similar inductions performed with human appendix showed no up-regulation of VAP-1 expression, indicating that the regulation of VAP-1 expression exhibits organ-selective characteristics. Furthermore, in these tissues the smooth muscle cells, which constitutively express VAP-1, could not be stimulated to alter their level of expression of this molecule. In conclusion, the expression of VAP-1 can be markedly up-regulated with several mediators in tonsil but not in appendix organ culture, whereas cultured endothelial cells cannot be induced to express VAP-1. These results indicate that the expression of VAP-1 is regulated in a tissue- and cell type-selective manner, and a correct micromilieu is required for the up-regulation to occur.
Amine Oxidase (Copper-Containing)↗