ABSTRACT
Modulation of the immunosuppressive tumor microenvironment (TME) is essential for enhancing the anti-tumor effects of immune checkpoint inhibitors (ICIs). Adhesion molecules and enzymes such as vascular adhesion protein-1 (VAP-1), which are expressed in some cancers and tumor vascular endothelial cells, may be involved in the generation of an immunosuppressive TME. In this study, the role of VAP-1 in TME was investigated in 2 murine colon cancer models and human cancer cells. Intraperitoneal administration of the VAP-1-specific inhibitor U-V296 inhibited murine tumor growth by enhancing IFN-γ-producing tumor antigen-specific CD8+ T cells. U-V296 exhibited significant synergistic anti-tumor effects with ICIs. In the TME of mice treated with U-V296, the expression of genes associated with M2-like macrophages, Th2 cells (Il4, Retnla, and Irf4), angiogenesis (Pecam1), and fibrosis (Acta2, Loxl2) were significantly decreased, and the Th1/Th2 balance was increased. H2 O2 , an enzymatic product of VAP-1, which promoted the production of IL-4 by mouse Th2 and inhibited IFN-γ by mouse Th1 and human tumor-infiltrating lymphocytes, was decreased in tumors and CD31+ tumor vascular endothelial cells in the TMEs of mice treated with VAP-1 inhibitor. TCGA database analysis showed that VAP-1 expression was a negative prognostic factor in human cancers, exhibiting a significant positive correlation with IL-4, IL4R, and IL-13 expression and a negative correlation with IFN-γ expression. These results indicated that VAP-1 is involved in the immunosuppressive TMEs through H2 O2 -associated Th2/M2 conditions and may be an attractive target for the development of combination cancer immunotherapy with ICIs.
Subject(s)
Amine Oxidase (Copper-Containing)/antagonists & inhibitors , Cell Adhesion Molecules/antagonists & inhibitors , Immune Checkpoint Inhibitors/pharmacology , Neoplasms/immunology , Neoplasms/therapy , Amine Oxidase (Copper-Containing)/immunology , Animals , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Cell Adhesion Molecules/immunology , Cell Line, Tumor , Endothelial Cells/drug effects , Endothelial Cells/immunology , Female , Immunotherapy/mortality , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Macrophages/drug effects , Macrophages/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Th1 Cells/drug effects , Th1 Cells/immunology , Th2 Cells/drug effects , Th2 Cells/immunology , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunologyABSTRACT
OBJECTIVE: Recently we characterized five mouse monoclonal antibodies that allow the specific and sensitive detection of human diamine oxidase (DAO). To understand differences in binding characteristics and recognition of enzyme variants, we mapped the antibody binding sites. METHODS: Fragments of human DAO were expressed as glutathione-S-transferase fusion proteins that were used for testing antibody binding on immunoblots. Combined information from species cross-reactivity, sequence comparison and binding site-prediction software were used to localize the epitope recognized by each antibody. RESULTS: All five monoclonal DAO antibodies bound to linear epitopes between the N3 and enzymatic domains of the 732 amino acid protein. The binding sites could be mapped onto amino acid regions V262-E278 and P279-R288, respectively, which exhibit considerable sequence variation in mammals explaining the fact that the human DAO antibodies do not cross-react with DAO from other species. The antibodies efficiently bind only denatured human DAO but not the native protein. CONCLUSIONS: Characterization of the binding sites of the DAO antibodies revealed that the antibodies bind two adjacent epitopes and exhibit similar binding characteristics and species cross-reactivity. As the epitopes do not overlap any of the amino acid substitutions described for clinically significant DAO gene polymorphisms, our antibodies will also be useful for analyses of the mutant DAO proteins.
Subject(s)
Amine Oxidase (Copper-Containing)/metabolism , Antibodies, Monoclonal/pharmacology , Binding Sites , Amine Oxidase (Copper-Containing)/genetics , Amine Oxidase (Copper-Containing)/immunology , DNA, Complementary/genetics , Epitopes , Escherichia coli/genetics , Humans , Plasmids , Protein BindingABSTRACT
INTRODUCTION: Primary sclerosing cholangitis (PSC) is a progressive inflammatory liver disease characterised by relentless liver fibrosis and a high unmet need for new therapies. Preventing fibrosis represents an important area of interest in the development of vital new drugs. Vascular adhesion protein-1 (VAP-1) drives inflammation in liver disease, and provision of an antibody against VAP-1 blunts fibrosis in murine models of liver injury. METHODS AND ANALYSIS: BUTEO is a single-arm, two-stage, open-label, multi-centre, phase II clinical trial. Up to 59 patients will receive treatment with anti-VAP monoclonal antibody, BTT1023, over a 78-day treatment period. Adults with PSC and a serum alkaline phosphatase (ALP) of at least 1.5 times the upper limit of normal will be included. Our primary outcome measure is a reduction in ALP by >25% from baseline to Day 99. Secondary outcome measures include safety and tolerability, changes pre therapy/post therapy in circulating serum VAP-1 as well as imaging findings. The first patient participant was recruited on 08 September 2015. ETHICS AND DISSEMINATION: This protocol has been approved by the Research Ethics Committee (REC, reference 14/EM/1272). The first REC approval date was 06 January 2015 with three subsequent approved amendments. This article refers to protocol V3.0, dated 16 March 2016. Results will be disseminated via peer-reviewed publication and presentation at international conferences. TRIAL REGISTRATION: The trial is registered with the European Medicines agency (EudraCT: 2014-002393-37), the National Institute for Health Research (Portfolio ID: 18051) and ISRCTN: 11233255. The clinicaltrials.gov identifier is NCT02239211. Pre-results.
Subject(s)
Alkaline Phosphatase/blood , Antibodies, Monoclonal/therapeutic use , Cholangitis, Sclerosing/drug therapy , Liver/physiopathology , Adolescent , Adult , Aged , Amine Oxidase (Copper-Containing)/immunology , Cell Adhesion Molecules/immunology , Female , Humans , Male , Middle Aged , Research Design , Treatment Outcome , United Kingdom , Young AdultABSTRACT
BACKGROUND: It has been suggested that polymorphisms of histamine metabolising enzymes can be a risk factor for developing histamine-involving diseases. The aim of the present study is to research the possible association between two functional single nucleotide polymorphisms (SNPs): C314T in the Histamine-N-Methyl Transferase gene and C2029G in the Diamine Oxidase gene, with the severity of allergic rhinitis and the number of allergic diseases, in a group of allergic Mexican children. METHODS: We studied 154 unrelated allergic children. SNPs were analysed by RT-PCR. The total serum IgE was measured by chemiluminescence and the serum histamine by ELISA. We used logistic regression analysis to determine OR. RESULTS: Patients carrying the mutant allele for any SNP had more risk to develop higher rhinitis severity or a bigger number of allergic diseases. Haplotype analysis revealed that this effect is synergistic. In patients carrying one or two mutant alleles, serum histamine levels were higher than those of patients carrying only wild alleles. Serum IgE levels were not associated with the presence of mutant alleles. CONCLUSION: The presence of these SNPs in patients with allergic rhinitis can lead to higher serum histamine, therefore to a higher risk of developing more severe symptoms or more associated allergic diseases, even if the serum IgE remains low
No disponible
Subject(s)
Humans , Male , Female , Child, Preschool , Child , Adolescent , Rhinitis, Allergic/drug therapy , Rhinitis, Allergic/epidemiology , Histamine N-Methyltransferase , Histamine N-Methyltransferase/immunology , Amine Oxidase (Copper-Containing)/analysis , Amine Oxidase (Copper-Containing)/immunology , Hypersensitivity, Immediate/immunology , Rhinitis, Allergic/immunology , Logistic Models , 28599 , Genotype , Eczema/complications , Eczema/immunology , Rhinitis/immunology , Asthma/immunologyABSTRACT
Amine oxidase, copper containing 3 (AOC3, also known as vascular adhesion protein-1 (VAP-1)) is an endothelial adhesion molecule that contributes to the extravasation of neutrophils, macrophages, and lymphocytes to sites of inflammation. However, the role of AOC3/VAP-1 in allergic responses remains unknown. Here, we studied eosinophil and CD4+ T-cell recruitment to the airways using AOC3/VAP-1-deficient mice. In an OVA-triggered asthma model, AOC3/VAP-1 slightly contributed to the accumulation of leukocytes in lungs in an age-dependent manner. We then established a new model to kinetically measure recruitment of OVA-specific CD4+ T cells to different airway immune compartments during the priming and effector phases of an adaptive immune response. The results showed that in the absence of AOC3/VAP-1, recruitment of antigen-specific CD4+ T cells to draining bronchial lymph nodes is reduced by 89% on day 3 after tracheal allergen exposure, but this difference was not observed on day 6. The dispersal of effector cells to lung and tracheal mucosa is AOC3/VAP-1 independent. Thus, in allergic airway reactions, AOC3/VAP-1 transiently contributes to the antigen-specific, CD4+ T-cell traffic to secondary lymphatic tissues, but not to airway mucosa or lung parenchyma. Our results suggest a largely redundant function for AOC3/VAP-1 in allergic inflammatory responses of the airways.
Subject(s)
Amine Oxidase (Copper-Containing)/immunology , Asthma/immunology , CD4-Positive T-Lymphocytes/immunology , Cell Adhesion Molecules/immunology , Lung/immunology , Trachea/immunology , Adaptive Immunity , Adoptive Transfer , Amine Oxidase (Copper-Containing)/biosynthesis , Amine Oxidase (Copper-Containing)/genetics , Animals , CD4-Positive T-Lymphocytes/transplantation , Cell Adhesion/immunology , Cell Adhesion Molecules/biosynthesis , Cell Adhesion Molecules/genetics , Eosinophils/immunology , Humans , Inflammation/immunology , Leukocytes/immunology , Lymph Nodes/cytology , Lymph Nodes/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Ovalbumin , Respiratory Mucosa/cytology , Respiratory Mucosa/immunologyABSTRACT
UNLABELLED: Vascular adhesion protein-1 (VAP-1) is an endothelial glycoprotein mediating leukocyte trafficking from blood to sites of inflammation. BTT-1023 is a fully human monoclonal anti-VAP-1 antibody developed to treat inflammatory diseases. In this study, we preclinically evaluated radioiodinated BTT-1023 for inflammation imaging. METHODS: Rabbits were intravenously injected with radioiodinated BTT-1023. Distribution and pharmacokinetics were assessed by PET/CT up to 72 h after injection. Human radiation dose estimates for (124)I-BTT-1023 were extrapolated. Additionally, rabbits with chemically induced synovitis were imaged with (123)I-BTT-1023 SPECT/CT. RESULTS: Radioiodinated BTT-1023 cleared rapidly from blood circulation and distributed to liver and thyroid. Inflamed joints were delineated by SPECT/CT. The estimated human effective dose due to (124)I-BTT-1023 was 0.55 mSv/MBq, if blockage of thyroid uptake is assumed. CONCLUSION: The radioiodinated BTT-1023 was able to detect mild inflammation in vivo. Clinical (124)I-BTT-1023 PET studies with injected radioactivity of 0.5-0.7 MBq/kg may be justified.
Subject(s)
Amine Oxidase (Copper-Containing)/metabolism , Antibodies, Monoclonal , Cell Adhesion Molecules/metabolism , Molecular Imaging/methods , Neovascularization, Pathologic/diagnostic imaging , Amine Oxidase (Copper-Containing)/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacokinetics , Cell Adhesion Molecules/immunology , Humans , Inflammation/diagnostic imaging , Inflammation/metabolism , Inflammation/physiopathology , Iodine Radioisotopes , Multimodal Imaging , Positron-Emission Tomography , Rabbits , Radiation Dosage , Tomography, X-Ray ComputedABSTRACT
UNLABELLED: Hepatitis induced by concanavalin A (Con A) in mice is well known to be a T-lymphocyte-mediated injury. It has been reported that T helper (Th)1 and Th2 lymphocytes use α4 integrin and vascular adhesion protein (VAP)-1, respectively, to adhere within the hepatic sinusoids. Therefore, we investigated whether inhibition of these molecules ameliorates or worsens the Con A-induced hepatic injury in vivo. Vehicle or antibody to α4 integrin or VAP-1 was intravenously administered 30 minutes before Con A administration. In control mice Con A markedly increased the serum alanine aminotransferase (ALT) level in a dose-dependent manner, and induced a massive infiltration of CD3, particularly interleukin (IL)-4 producing CD4 T cells and liver injury. Both parameters were reduced by anti-VAP-1 antibody despite antibody only blocking the adhesion, not the amine oxidase activity of VAP-1. Both activities of VAP-1 were eliminated in VAP-1-deficient mice and both Con A-induced liver injury and CD4 T-cell infiltration were eradicated. In contrast to anti-VAP-1, anti-α4 integrin antibody reduced interferon-gamma (IFN-γ)-producing CD3 T cells but this worsened Con A hepatitis, suggesting inhibition of a suppressor cell. Con A induced the recruitment of CD49d(+) monocytic myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) into the liver. Anti-α4 integrin dramatically blocked the influx of MDSCs but not Tregs. CONCLUSION: Our findings show that VAP-1 and α4 integrin have opposing effects in Con A-induced hepatic injury, which is associated with blocking the recruitment of CD4 lymphocytes and monocytic MDSCs, respectively. Moreover, these data provide the rationale for a potential therapeutic approach to target adhesion molecules in autoimmune hepatitis.
Subject(s)
Amine Oxidase (Copper-Containing)/immunology , Antibodies, Anti-Idiotypic/immunology , Antibodies, Anti-Idiotypic/therapeutic use , Cell Adhesion Molecules/immunology , Chemical and Drug Induced Liver Injury/drug therapy , Concanavalin A/adverse effects , Integrin alpha4/immunology , Alanine Transaminase/blood , Amine Oxidase (Copper-Containing)/deficiency , Amine Oxidase (Copper-Containing)/genetics , Animals , CD4-Positive T-Lymphocytes/pathology , Cell Adhesion Molecules/deficiency , Cell Adhesion Molecules/genetics , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Disease Models, Animal , Dose-Response Relationship, Drug , Liver/metabolism , Liver/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , T-Lymphocytes, Regulatory/pathology , Treatment OutcomeABSTRACT
Pulmonary metastasis is a frequent cause of poor outcome in cancer patients. The formation of pulmonary metastasis is greatly facilitated by recruitment of myeloid cells, which are crucial for tumor cell survival and extravasation. During inflammation, homing of myeloid cells is mediated by endothelial activation, raising the question of a potential role for endothelial activation in myeloid cell recruitment during pulmonary metastasis. Here, we show that metastatic tumor cell attachment causes the induction of the endothelial activation markers vascular cell adhesion molecule-1 (VCAM-1) and vascular adhesion protein-1 (VAP-1). Induction of VCAM-1 is dependent on tumor cell-clot formation, decreasing upon induction of tissue factor pathway inhibitor or treatment with hirudin. Furthermore, inhibition of endothelial activation with a VCAM-1 blocking antibody or a VAP-1 small molecule inhibitor leads to reduced myeloid cell recruitment and diminished tumor cell survival and metastasis without affecting tumor cell adhesion. Simultaneous inhibition of VCAM-1 and VAP-1 does not result in further reduction in myeloid cell recruitment and tumor cell survival, suggesting that both act through closely related mechanisms. These results establish VCAM-1 and VAP-1 as mediators of myeloid cell recruitment in metastasis and identify VAP-1 as a potential target for therapeutic intervention to combat early metastasis.
Subject(s)
Amine Oxidase (Copper-Containing)/immunology , Cell Adhesion Molecules/immunology , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Lung/pathology , Myeloid Cells/pathology , Vascular Cell Adhesion Molecule-1/immunology , Animals , Blood Coagulation , Cell Adhesion , Cell Line, Tumor , Endothelial Cells/immunology , Endothelial Cells/pathology , Humans , Lung/immunology , Lung Neoplasms/blood , Lung Neoplasms/immunology , Macrophages/immunology , Macrophages/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, SCID , Myeloid Cells/immunologyABSTRACT
Diamine oxidase (DAO) oxidatively deaminates histamine and other diamines. Due to the lack of antibodies for human DAO, many findings on this enzyme had not been confirmed in man. Therefore, we produced a series of monoclonal antibodies by immunizing mice with human DAO protein fragments expressed in vitro. Five different monoclonal antibodies specific for human DAO were obtained that do not recognize any other human protein and can detect DAO with 100-fold greater sensitivity than the most sensitive enzymatic assays currently available. Using these antibodies allowed confirming the expression and cellular localization of DAO in various human tissues such as kidney, intestine and placenta where the presence of the enzyme had previously been deduced from activity measurement and DAO mRNA analysis. Due to the high sensitivity of the novel monoclonal antibodies, DAO was also detected at sites that previously evaded unequivocal proof of DAO enzymatic activity such as the urine. On the other hand, with these antibodies it was possible to show that DAO is normally not present in human liver and blood serum. The new monoclonal antibodies not only allow a comprehensive quantitative evaluation of the expression of DAO at the cellular level in man but will also facilitate sensitive analyses of disease-associated alterations of this enzyme.
Subject(s)
Amine Oxidase (Copper-Containing)/immunology , Amine Oxidase (Copper-Containing)/metabolism , Antibodies, Monoclonal/metabolism , Amine Oxidase (Copper-Containing)/chemistry , Amine Oxidase (Copper-Containing)/genetics , Animals , Antibody Affinity , Antibody Specificity , Enzyme-Linked Immunosorbent Assay , Female , Humans , Intestines/enzymology , Kidney/enzymology , Mice , Mice, Inbred BALB C , Peptide Fragments/immunology , Placenta/enzymology , Pregnancy , Serum/enzymology , SwineABSTRACT
UNLABELLED: Non-steroidal anti-inflammatory drugs (NSAIDs) are the drugs most frequently involved in hypersensitivity drug reactions. Histamine is released in the allergic response to NSAIDs and is responsible for some of the clinical symptoms. The aim of this study is to analyze clinical association of functional polymorphisms in the genes coding for enzymes involved in histamine homeostasis with hypersensitivity response to NSAIDs. We studied a cohort of 442 unrelated Caucasian patients with hypersensitivity to NSAIDs. Patients who experienced three or more episodes with two or more different NSAIDs were included. If this requirement was not met diagnosis was established by challenge. A total of 414 healthy unrelated controls ethnically matched with patients and from the same geographic area were recruited. Analyses of the SNPs rs17740607, rs2073440, rs1801105, rs2052129, rs10156191, rs1049742 and rs1049793 in the HDC, HNMT and DAO genes were carried out by means of TaqMan assays. The detrimental DAO 16 Met allele (rs10156191), which causes decreased metabolic capacity, is overrepresented among patients with crossed-hypersensitivity to NSAIDs with an OR â=â1.7 (95% CI â=â1.3-2.1; Pc â=â0.0003) with a gene-dose effect (Pâ=â0.0001). The association was replicated in two populations from different geographic areas (Pc â=â0.008 and Pc â=â0.004, respectively). CONCLUSIONS AND IMPLICATIONS: The DAO polymorphism rs10156191 which causes impaired metabolism of circulating histamine is associated with the clinical response in crossed-hypersensitivity to NSAIDs and could be used as a biomarker of response.
Subject(s)
Amine Oxidase (Copper-Containing)/genetics , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Drug Hypersensitivity/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Alleles , Amine Oxidase (Copper-Containing)/immunology , Case-Control Studies , Child , Child, Preschool , Drug Hypersensitivity/immunology , Female , Gene Frequency , Genotype , Humans , Immunoglobulin E/immunology , Male , Middle Aged , Polymorphism, Single Nucleotide , Young AdultABSTRACT
Vascular adhesion protein-1 (VAP-1) is an endothelial, cell surface-expressed oxidase involved in leukocyte traffic. The adhesive function of VAP-1 can be blocked by anti-VAP-1 Abs and small-molecule inhibitors. However, the effects of VAP-1 blockade on antitumor immunity and tumor progression are unknown. In this paper, we used anti-VAP-1 mAbs and small-molecule inhibitors of VAP-1 in B16 melanoma and EL-4 lymphoma tumor models in C57BL/6 mice. Leukocyte accumulation into tumors and neoangiogenesis were evaluated by immunohistochemistry, flow cytometry, and intravital videomicroscopy. We found that both anti-VAP-1 Abs and VAP-1 inhibitors reduced the number of leukocytes in the tumors, but they targeted partially different leukocyte subpopulations. Anti-VAP-1 Abs selectively inhibited infiltration of CD8-positive lymphocytes into tumors and had no effect on accumulation of myeloid cells into tumors. In contrast, the VAP-1 inhibitors significantly reduced only the number of proangiogenic Gr-1(+)CD11b(+) myeloid cells in melanomas and lymphomas. Blocking of VAP-1 by either means left tumor homing of regulatory T cells and type 2 immune-suppressing monocytes/macrophages intact. Notably, VAP-1 inhibitors, but not anti-VAP-1 Abs, retarded the growth of melanomas and lymphomas and reduced tumor neoangiogenesis. The VAP-1 inhibitors also reduced the binding of Gr-1(+) myeloid cells to the tumor vasculature. We conclude that tumors use the catalytic activity of VAP-1 to recruit myeloid cells into tumors and to support tumor progression. Small-molecule VAP-1 inhibitors therefore might be a potential new tool for immunotherapy of tumors.
Subject(s)
Amine Oxidase (Copper-Containing)/antagonists & inhibitors , Amine Oxidase (Copper-Containing)/immunology , Cell Adhesion Molecules/antagonists & inhibitors , Cell Adhesion Molecules/immunology , Cell Migration Inhibition/immunology , Growth Inhibitors/therapeutic use , Lymphoma, T-Cell/immunology , Melanoma, Experimental/immunology , Myeloid Cells/immunology , Myeloid Cells/pathology , Adjuvants, Immunologic/therapeutic use , Allylamine/analogs & derivatives , Allylamine/therapeutic use , Amine Oxidase (Copper-Containing)/biosynthesis , Animals , Antibodies, Blocking/therapeutic use , Antibodies, Monoclonal/therapeutic use , Cell Adhesion Molecules/biosynthesis , Cell Line, Tumor , Cell Migration Inhibition/drug effects , Enzyme Inhibitors/therapeutic use , Female , Lymphoma, T-Cell/pathology , Lymphoma, T-Cell/therapy , Melanoma, Experimental/blood supply , Melanoma, Experimental/pathology , Melanoma, Experimental/therapy , Mice , Mice, Inbred C57BL , Myeloid Cells/drug effects , Semicarbazides/therapeutic useABSTRACT
Leukocytes migrate from the blood into areas of inflammation by interacting with various adhesion molecules on endothelial cells. Vascular adhesion protein-1 (VAP-1) is a glycoprotein expressed on inflamed endothelium where it plays a dual role: it is both an enzyme that oxidizes primary amines and an adhesin that is involved in leukocyte trafficking to sites of inflammation. Although VAP-1 was identified more than 15 years ago, the counterreceptor(s) for VAP-1 on leukocytes has remained unknown. Here we have identified Siglec-10 as a leukocyte ligand for VAP-1 using phage display screenings. The binding between Siglec-10 and VAP-1 was verified by different adhesion assays, and this interaction was also consistent with molecular modeling. Moreover, the interaction between Siglec-10 and VAP-1 led to increased hydrogen peroxide production, indicating that Siglec-10 serves as a substrate for VAP-1. Thus, the Siglec-10-VAP-1 interaction seems to mediate lymphocyte adhesion to endothelium and has the potential to modify the inflammatory microenvironment via the enzymatic end products.
Subject(s)
Amine Oxidase (Copper-Containing)/metabolism , Cell Adhesion Molecules/metabolism , Cell Adhesion/physiology , Chemotaxis, Leukocyte/physiology , Lectins/metabolism , Receptors, Cell Surface/metabolism , Amine Oxidase (Copper-Containing)/chemistry , Amine Oxidase (Copper-Containing)/immunology , Animals , CHO Cells , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/immunology , Cricetinae , Cricetulus , Endothelium/immunology , Endothelium/metabolism , Humans , Lectins/chemistry , Lectins/immunology , Ligands , Lymphocytes/metabolism , Mice , Mice, Knockout , Peptide Library , Protein Binding , Protein Structure, Quaternary , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/immunology , Recombinant ProteinsABSTRACT
Cancer growth is regulated by several nonmalignant cell types, such as leukocytes and endothelial cells, which reside in the stroma of the tumor. Vascular adhesion protein-1 (VAP-1) is an amine oxidase enzyme that is expressed on the surface of endothelial cells. It supports leukocyte traffic into inflamed tissues, but nothing is known about its possible role in cancer biology in vivo. Here, we report that B16 melanoma and EL-4 lymphoma remain smaller in VAP-1-deficient mice than in wild-type controls. We found an unexpected defect in tumor angiogenesis in the absence of VAP-1. VAP-1 also selectively enhanced the recruitment of Gr-1+CD11b+ myeloid cells into the tumors. Generation of mice expressing enzymatically inactive VAP-1 showed that the oxidase activity of VAP-1 was necessary to support neoangiogenesis, myeloid cell recruitment, and tumor growth in vivo. These data describe VAP-1 as the first adhesion molecule known to be involved in the recruitment of Gr-1+CD11b+ myeloid cells into tumors. They also suggest that VAP-1 is a potential new tool for immunotherapy of tumors that could be exploited to reduce tumor burden by controlling the traffic of Gr-1+CD11b+ myeloid cells.
Subject(s)
Amine Oxidase (Copper-Containing)/physiology , Cell Adhesion Molecules/physiology , Lymphoma/pathology , Melanoma, Experimental/pathology , Myeloid Cells/pathology , Amine Oxidase (Copper-Containing)/antagonists & inhibitors , Amine Oxidase (Copper-Containing)/immunology , Amine Oxidase (Copper-Containing)/metabolism , Animals , CD11b Antigen/biosynthesis , CD11b Antigen/immunology , Cell Adhesion Molecules/antagonists & inhibitors , Cell Adhesion Molecules/immunology , Cell Adhesion Molecules/metabolism , Cell Growth Processes/physiology , Female , Lymphoma/immunology , Male , Melanoma, Experimental/blood supply , Melanoma, Experimental/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myeloid Cells/immunology , Neovascularization, Pathologic/pathology , Oxidoreductases/metabolism , Receptors, Chemokine/biosynthesis , Receptors, Chemokine/immunologyABSTRACT
AIM: To study the levels of serum soluble intercellular adhesion molecule-1 (sICAM-1), plasma D-lactate and diamine oxidase (DAO) in patients with inflammatory bowel disease (IBD), and the potential clinical significance. METHODS: Sixty-nine patients with IBD and 30 healthy controls were included in this study. The concentration of sICAM-1 was detected with enzyme-linked immunosorbent assay, the level of D-lactate and DAO was measured by spectroscopic analysis, and the number of white blood cells (WBC) was determined by routine procedure. RESULTS: The levels of sICAM-l, DAO, and WBC in IBD patients were significantly higher than those in the control group (P < 0.01). sICAM-l in IBD patients was found to be closely related to the levels of DAO and D-lactate (212.94 +/- 69.89 vs 6.35 +/- 2.35, P = 0.000), DAO 212.94 +/- 69.89 vs 8.65 +/- 3.54, P = 0.000) and WBC (212.94 +/- 69.89 vs 7.40 +/- 2.61, P = 0.000), but no significant difference was observed between patients with ulcerative colitis and patients with Crohn's disease. The post-treatment levels of sICAM-l, D-lactate and WBC were significantly lower than before treatment (sICAM-l 206.57 +/- 79.21 vs 146.21 +/- 64.43, P = 0.000), (D-lactate 1.46 +/- 0.94 vs 0.52 +/- 0.32, P = 0.000) and (WBC 7.24 +/- 0.2.33 vs 5.21 +/- 3.21, P = 0.000). CONCLUSION: sICAM-1, D-lactate and DAO are closely related to the specific conditions of IBD, and thus could be used as a major diagnostic index.
Subject(s)
Amine Oxidase (Copper-Containing) , Inflammatory Bowel Diseases , Intercellular Adhesion Molecule-1 , Lactic Acid , Adolescent , Adult , Amine Oxidase (Copper-Containing)/blood , Amine Oxidase (Copper-Containing)/immunology , Female , Humans , Inflammatory Bowel Diseases/blood , Inflammatory Bowel Diseases/immunology , Intercellular Adhesion Molecule-1/blood , Intercellular Adhesion Molecule-1/immunology , Lactic Acid/blood , Lactic Acid/immunology , Leukocyte Count , Male , Middle Aged , Young AdultABSTRACT
Inflammatory cytokines are released in response to stress, tissue damage, and infection. Acutely, this response is adaptive; however, chronic elevation of inflammatory proteins can contribute to health problems including cardiovascular, endocrine, mood, and sleep disorders. Few studies have examined how sleep deprivation acutely affects inflammatory markers, which was the aim of the current study. Nineteen healthy men and women aged 28.05+/-8.56 (mean+/-SD) were totally sleep deprived for 40 h under constant routine conditions. Pro-inflammatory markers: intracellular adhesion molecule-1 (ICAM-1), E-selectin, vascular adhesion molecule-1 (VCAM-1), c-reactive protein (CRP), interleukin-6 (IL-6), and interleukin-1beta (IL-1beta), and the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1ra) were assayed in plasma. Daytime levels during baseline (hours 1-15 of scheduled wakefulness) were compared to daytime levels during sleep deprivation (hours 25-39 of scheduled wakefulness), thus controlling for circadian phase within an individual. Repeated measures ANOVA with planned comparisons showed that 40 h of total sleep deprivation induced a significant increase in E-selectin, ICAM-1, IL-1beta, and IL-1ra, a significant decrease in CRP and IL-6, and no significant change in VCAM-1. Alterations in circulating levels of pro- and anti-inflammatory cytokines and cell adhesion molecules during sleep deprivation were consistent with both increased and decreased inflammation. These findings suggest that one night of sleep loss triggers a stress response that includes stimulation of both pro- and anti-inflammatory proteins in the healthy young subjects tested under our experimental conditions.
Subject(s)
Acute-Phase Reaction/immunology , Cell Adhesion Molecules/blood , Cytokines/blood , Sleep Deprivation/immunology , Acute-Phase Reaction/blood , Adaptation, Physiological/immunology , Adult , Amine Oxidase (Copper-Containing)/blood , Amine Oxidase (Copper-Containing)/immunology , Analysis of Variance , Biomarkers/blood , C-Reactive Protein/analysis , Cell Adhesion Molecules/immunology , E-Selectin/blood , E-Selectin/immunology , Female , Humans , Intercellular Adhesion Molecule-1/blood , Intercellular Adhesion Molecule-1/immunology , Interleukin 1 Receptor Antagonist Protein/blood , Interleukin-1beta/blood , Interleukin-6/blood , Male , Reference Values , Sleep Deprivation/blood , Stress, Physiological/blood , Stress, Physiological/immunology , Time FactorsABSTRACT
Vascular adhesion protein-1 (VAP-1) is an endothelial cell molecule which controls leukocyte infiltration into tissues. Elevated serum soluble VAP-1-levels have been described in certain diseases with an inflammatory component. VAP-1 expression or function has not previously been studied in multiple sclerosis (MS). We report here that the concentration of soluble VAP-1 in serum is significantly higher in multiple sclerosis patients with ongoing inflammatory activity, as demonstrated by gadolinium-enhancing MRI lesions, when compared to patients with no gadolinium-enhancing lesions (555+/-195 vs. 388+/-102 ng/ml, p=0.0068). We propose that VAP-1 might participate in controlling leukocyte entry into inflamed brain.
Subject(s)
Amine Oxidase (Copper-Containing)/blood , Amine Oxidase (Copper-Containing)/immunology , Cell Adhesion Molecules/blood , Cell Adhesion Molecules/immunology , Chemotaxis, Leukocyte/immunology , Multiple Sclerosis, Relapsing-Remitting/blood , Multiple Sclerosis, Relapsing-Remitting/immunology , Adult , Aged , Blood-Brain Barrier/physiopathology , Cell Adhesion/immunology , Central Nervous System/immunology , Central Nervous System/pathology , Central Nervous System/physiopathology , Endothelial Cells/immunology , Female , Gadolinium , Humans , Leukocytes/immunology , Magnetic Resonance Imaging , Male , Middle Aged , Multiple Sclerosis, Relapsing-Remitting/diagnosis , Predictive Value of Tests , Up-Regulation/immunologyABSTRACT
Human vascular adhesion protein-1 (VAP-1) is a homodimeric 170-kDa sialoglycoprotein that is expressed on the surface of endothelial cells and functions as a semicarbazide-sensitive amine oxidase and as an adhesion molecule. Blockade of VAP-1 has been shown to reduce leukocyte adhesion and transmigration in in vivo and in vitro models, suggesting that VAP-1 is a potential target for anti-inflammatory therapy. In this study we have constructed mouse-human chimeric antibodies by genetic engineering in order to circumvent the potential problems involved in using murine antibodies in man. Our chimeric anti-VAP-1 antibodies, which were designed to lack Fc-dependent effector functions, bound specifically to cell surface-expressed recombinant human VAP-1 and recognized VAP-1 in different cell types in tonsil. Furthermore, the chimeric antibodies prevented leukocyte adhesion and transmigration in vitro and in vivo. Hence, these chimeric antibodies have the potential to be used as a new anti-inflammatory therapy.
Subject(s)
Amine Oxidase (Copper-Containing)/antagonists & inhibitors , Amine Oxidase (Copper-Containing)/physiology , Antibodies, Blocking/therapeutic use , Cell Adhesion Molecules/antagonists & inhibitors , Cell Adhesion Molecules/physiology , Cell Migration Inhibition , Inflammation Mediators/antagonists & inhibitors , Inflammation Mediators/physiology , Amine Oxidase (Copper-Containing)/genetics , Amine Oxidase (Copper-Containing)/immunology , Amino Acid Sequence , Animals , Antibodies, Blocking/genetics , Binding Sites, Antibody , Cell Adhesion/immunology , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/immunology , Cell Movement/immunology , Endothelium, Vascular/physiology , Flow Cytometry , Humans , Inflammation Mediators/immunology , Mice , Mice, Transgenic , Molecular Sequence Data , RatsABSTRACT
Vascular adhesion protein 1 (VAP-1) is an endothelial adhesion molecule with an enzymatic activity. It deaminates biogenic amines, resulting in the formation of aldehydes and hydrogen peroxide. During the enzymatic reaction a transient Schiff base is formed between endothelial VAP-1 and its leukocytic ligand, and this interaction is important for lymphocyte adhesion. VAP-1 monomer has six potential N-linked, and three putative O-linked glycosylation sites and an SSSS sequence potentially forming an attachment site for an adjacent O-linked site. In this work we modeled the carbohydrate decorations on a structural model of VAP-1, and studied which of those potential glycosylation sites are utilized, and whether those decorations accessible to a lymphocyte ligand are important in lymphocyte adhesion and enzymatic activity of VAP-1. We show that, unlike the O-linked attachment sites, all six N-linked glycosylation sites are in use. Furthermore, mutation of the N-linked attachment sites strategically located on the top of the molecule reduces lymphocyte adhesion in non-static conditions, and enhances the catalytic activity of membrane-bound human VAP-1 in static conditions, suggesting that glycosylation regulates the functional properties of VAP-1.
Subject(s)
Amine Oxidase (Copper-Containing)/physiology , Cell Adhesion Molecules/physiology , Amine Oxidase (Copper-Containing)/chemistry , Amine Oxidase (Copper-Containing)/genetics , Amine Oxidase (Copper-Containing)/immunology , Animals , CHO Cells , Cell Adhesion/immunology , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/immunology , Cricetinae , Endothelial Cells/cytology , Endothelial Cells/enzymology , Endothelial Cells/immunology , Flow Cytometry , Glycosylation , Immunoblotting , Isoelectric Focusing , Lymphocytes/cytology , Lymphocytes/enzymology , Lymphocytes/immunology , Models, Molecular , Mutagenesis, Site-Directed , Rats , Thoracic Surgery, Video-Assisted , TransfectionABSTRACT
The oxidative deamination of methylated putrescine by a diamine oxidase activity (DAO) is an important step in the biosynthesis of nicotine in tobacco and tropane alkaloids in several Solanaceous plants. A polyclonal rabbit antiserum was previously developed to a purported purified DAO enzyme from Nicotiana tabacum. The antiserum bound to a single 53 kDa protein and immunoprecipitated 80% of DAO activity from tobacco root extracts. In an effort to obtain DAO cDNAs, this antiserum was used to screen a tobacco cDNA expression library and three distinct immunoreactive cDNA clones were isolated. These cDNAs encoded predicted proteins that were either identical or nearly identical to predicted S-adenosylhomocysteine hydrolase (SAHH) from two Nicotiana species. Thus, the rabbit antiserum was not specific to DAO, even though it immunodepleted the majority of DAO activity from root extracts. Alternative hypotheses to explain the DAO immunodepletion results (such as poisoning of DAO activity or that SAHH is a bifunctional enzyme) were tested and ruled out. Therefore, we hypothesize that SAHH associates with DAO as part of a larger multienzyme complex that may function in planta as a nicotine metabolic channel.
Subject(s)
Adenosylhomocysteinase/genetics , Amine Oxidase (Copper-Containing)/genetics , Nicotiana/enzymology , Adenosylhomocysteinase/immunology , Adenosylhomocysteinase/metabolism , Amine Oxidase (Copper-Containing)/immunology , Amine Oxidase (Copper-Containing)/metabolism , Animals , Binding, Competitive/immunology , Immune Sera/immunology , Phylogeny , Plant Extracts/immunology , Plant Extracts/metabolism , Plant Proteins/genetics , Plant Proteins/immunology , Plant Proteins/metabolism , Plant Roots/enzymology , Plant Roots/genetics , Protein Binding/immunology , Rabbits , Nicotiana/geneticsABSTRACT
Lymphocytes leave the blood using a sequential adhesion cascade. Vascular adhesion molecule-1 (VAP-1) is a surface-expressed endothelial glycoprotein, which belongs to a distinct subgroup of monoamine oxidases. We show here that catalytic activity of VAP-1 on primary endothelial cells directly regulates lymphocyte rolling under defined laminar shear. VAP-1 seems to bind to a primary amino group presented on the lymphocyte surface and oxidatively deaminate it in a reaction, which results in the formation of a transient covalent bond between the two cell types. Instead, soluble reaction products (aldehydes and hydrogen peroxide) are not needed for the VAP-1-dependent rolling. Enzymatic regulation of lymphocyte adhesion to endothelium provides a previously unrecognized rapid way of controlling the extravasation process.