Purpose: The migration of leukocytes, including monocytes (MNs), is a major contributor to inflam... more Purpose: The migration of leukocytes, including monocytes (MNs), is a major contributor to inflammation in autoimmune diseases such as rheumatoid arthritis (RA). In RA, MN migration to the joint synovial tissue is aided by junctional adhesion molecules (JAMs) expressed by endothelial cells (ECs). The EC JAMs bind integrins found on leukocytes, promoting their ingress. JAM‐C is the ligand for the integrin Mac‐1 (Cd11b/CD18). Like some adhesion molecules, JAM‐C has been found in soluble form, and our recent work has shown that soluble JAM‐C (sJAM‐C) is upregulated in RA synovial fluid. Some soluble adhesion molecules have also previously been shown to induce leukocyte migration. Based on this, we hypothesized that sJAM‐C promotes THP‐1 MN migration by binding Mac‐1 and inducing the phosphorylation of Erk.Methods & Results: To test this hypothesis, we cultured THP‐1 cells to model MN function. Using qPCR (n=2) we found that THP‐1 MNs express the Cd11b portion of Mac‐1. Next, in vitro M...
Junctional adhesion molecule (JAM)-C is a member of the JAM family, expressed by a variety of dif... more Junctional adhesion molecule (JAM)-C is a member of the JAM family, expressed by a variety of different cell types, including human B lymphocytes and some B-cell lymphoma subtypes-in particular, mantle cell lymphoma (MCL). Treatment with anti-JAM-C pAbs reduces homing of human B cells to lymphoid organs in a NOD/SCID mouse model. In the present study, the role of JAM-C in the engraftment of human lymphoma B cells in mice was investigated. Administration of novel anti-JAM-C mAbs reduced tumor growth of JAM-C(+) MCL cells in bone marrow, spleen, liver, and lymph nodes of mice. Treatment with anti-JAM-C antibodies significantly reduced the proliferation of JAM-C-expressing lymphoma B cells. Moreover, the binding of anti-JAM-C antibodies inhibited the phosphorylation of ERK1/2, without affecting other signaling pathways. The results identify for the first time the intracellular MAPK cascade as the JAM-C-driven signaling pathway in JAM-C(+) B cells. Targeting JAM-C could constitute a new...
Intraepithelial lymphocytes (IEL) of the small intestine are anatomically positioned to be in the... more Intraepithelial lymphocytes (IEL) of the small intestine are anatomically positioned to be in the first line of cellular defense against enteric pathogens. Therefore, determining the origin of these cells has important implications for the mechanisms of T cell maturation and repertoire selection. Recent evidence suggests that murine CD8αα intestinal IELs (iIELs) can mature and undergo selection in the absence of a thymus. We analyzed IEL origin by cell transfer, using two congenic chicken strains. Embryonic day 14 and adult thymocytes did not contain any detectable CD8αα T cells. However, when TCR+ thymocytes were injected into congenic animals, they migrated to the gut and developed into CD8αα iIELs, while TCR− T cell progenitors did not. The TCR Vβ1 repertoire of CD8αα+ TCR Vβ1+ iIELs contained only part of the TCR Vβ1 repertoire of total iIELs, and it exhibited no new members compared with CD8+ T cells in the thymus. This indicated that these T cells emigrated from the thymus at ...
Lymphocytes depend on endothelial adhesion molecules such as ICAM-1 and VCAM-1, upregulated with ... more Lymphocytes depend on endothelial adhesion molecules such as ICAM-1 and VCAM-1, upregulated with inflammation, to facilitate transmigration across junctional barriers. New data show that ALCAM replaces VCAM-1 in the CNS during the development of neuroinflammatory diseases such as multiple sclerosis.
We have recently shown that junctional adhesion molecule (JAM)-C-deficient mice have leukocytic p... more We have recently shown that junctional adhesion molecule (JAM)-C-deficient mice have leukocytic pulmonary infiltrates, disturbed neutrophil homeostasis, and increased postnatal mortality. This phenotype was partially rescued when mice were housed in ventilated isolators, suggesting an inability to cope with opportunistic infections. In the present study, we further examined the adaptive immune responses in JAM-C−/− mice. We found that murine conventional dendritic cells express in addition to Mac-1 and CD11c also JAM-B as ligand for JAM-C. By in vitro adhesion assay, we show that murine DCs can interact with recombinant JAM-C via Mac-1. However, this interaction does not seem to be necessary for dendritic cell migration and function in vivo, even though JAM-C is highly expressed by lymphatic sinuses of lymph nodes. Nevertheless, upon immunization and boosting with a protein Ag, JAM-C-deficient mice showed decreased persistence of specific circulating Abs although the initial respons...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2009
Objectives— The molecular mechanisms regulating vascular permeability are only now being elucidat... more Objectives— The molecular mechanisms regulating vascular permeability are only now being elucidated. The junctional adhesion molecule (JAM) JAM-C has been linked to the induction of vascular permeability. We sought to understand the mechanism whereby JAM-C may disrupt junctional integrity in endothelial cells (ECs). Methods and Results— We show here that JAM-C alters permeability through modulation of integrin activity. JAM-C overexpression results in an increase in JAM-C at junctions and an increase in permeability. Conversely, knockdown of JAM-C by siRNA results in a reduction in permeability. JAM-C associates with αvβ3 integrin and regulates its localization and activity. JAM-C also inhibits the activation state of the β 1 integrin although it does not associate with this integrin. These changes induced on the integrins are mediated through regulation of the small GTPase, Rap1b but not Rap1a. Thrombin, a powerful inductor of vascular leak, causes localization of JAM-C into the ju...
T-cell development is dependent on microenvironments generated by stromal cells such as thymic ep... more T-cell development is dependent on microenvironments generated by stromal cells such as thymic epithelial cells (TEC). TEC population, which is phenotypically and functionally heterogeneous, controls the migration, proliferation and selection of differentiating thymocytes. CYR61 (cysteine-rich protein 61) is a matricellular protein reported to promote proliferation and migration of cancer cells. Here we investigated the impact of CYR61 treatment on T-cell development. We found that matricellular protein CYR61 was strongly expressed in TEC and favored TEC/thymocyte interaction by binding to LFA1 and ICAM1. Treatment of thymic lobes with recombinant CYR61 expanded the thymic stromal compartment. Thymocyte-independent TEC proliferation occurred through CYR61 binding to integrin alpha6/beta1 and the activation of Akt signaling pathway. Engraftment of thymic lobes mildly overexpressing CYR61 into athymic nude mice drastically boosted thymic output through the expansion of TEC. Actually, ...
Soluble forms of membrane receptors are emerging candidates as physiological regulators of leukoc... more Soluble forms of membrane receptors are emerging candidates as physiological regulators of leukocyte trafficking. In the present study, we found that the soluble form of the CD38 antigen (sCD38) bears a binding domain of low affinity for a cellular receptor on U937 cells. Cross-linking and peptide-mapping studies confirmed the physical association and the identification of the U937 receptor as a 130 kDa protein. The binding of sCD38 to the receptor was differentially inhibited by several monoclonal antibodies against the CD31 cell-adhesion molecule. Thus the interaction was analysed through direct association of soluble and membrane CD38 with soluble recombinant murine CD31 with three N-terminal and with all six extracellular Ig domains. Cross-linking experiments on U937 intact cells, and ligand blot assays of the immunoprecipitated CD38 molecule, indicated that (i) the recognized epitope is determined by the tertiary structure of the molecule, and that (ii) the binding domain invol...
The rat monoclonal antibody (mAb) termed EA-1 was originally selected for its capacity to block t... more The rat monoclonal antibody (mAb) termed EA-1 was originally selected for its capacity to block the adhesion of T lymphocyte progenitors to mouse thymic endothelium. Here we show that the mAb EA-1 recognizes the alpha 6 chain of alpha 6 beta 1 and alpha 6 beta 4 integrins. Both molecules are present at a high level on the luminal and basolateral side of vascular endothelium and alpha 6 beta 1 integrin is expressed on the highly metastatic cell lines B16/129 (melanoma) and KLN-205 (carcinoma). These lung specific tumors bind preferentially to lung frozen sections, and EA-1 blocked this interaction in vitro. Moreover, mAb EA-1 inhibited experimental metastasis to the lung of B16/129 cells injected intravenously. Metastasis in vivo was blocked when the antibody was injected into mice before or simultaneously with the melanoma cells, as well as when melanoma cells were precoated with EA-1 before injection. We suggest that alpha 6 integrins play a dual role in the metastatic process, mediating the adhesion of tumor cells to the luminal surface of the endothelium and the adhesion to laminin in the subendothelial extracellular matrix during extravasation. Despite the fact that alpha 6 integrins are laminin receptors, EA-1 did not interfere with melanoma cell binding to laminin fragments. Our antibody EA-1 may therefore recognize a binding domain on alpha 6 integrins of a novel ligand involved in cell-cell interaction.
Purpose: The migration of leukocytes, including monocytes (MNs), is a major contributor to inflam... more Purpose: The migration of leukocytes, including monocytes (MNs), is a major contributor to inflammation in autoimmune diseases such as rheumatoid arthritis (RA). In RA, MN migration to the joint synovial tissue is aided by junctional adhesion molecules (JAMs) expressed by endothelial cells (ECs). The EC JAMs bind integrins found on leukocytes, promoting their ingress. JAM‐C is the ligand for the integrin Mac‐1 (Cd11b/CD18). Like some adhesion molecules, JAM‐C has been found in soluble form, and our recent work has shown that soluble JAM‐C (sJAM‐C) is upregulated in RA synovial fluid. Some soluble adhesion molecules have also previously been shown to induce leukocyte migration. Based on this, we hypothesized that sJAM‐C promotes THP‐1 MN migration by binding Mac‐1 and inducing the phosphorylation of Erk.Methods & Results: To test this hypothesis, we cultured THP‐1 cells to model MN function. Using qPCR (n=2) we found that THP‐1 MNs express the Cd11b portion of Mac‐1. Next, in vitro M...
Junctional adhesion molecule (JAM)-C is a member of the JAM family, expressed by a variety of dif... more Junctional adhesion molecule (JAM)-C is a member of the JAM family, expressed by a variety of different cell types, including human B lymphocytes and some B-cell lymphoma subtypes-in particular, mantle cell lymphoma (MCL). Treatment with anti-JAM-C pAbs reduces homing of human B cells to lymphoid organs in a NOD/SCID mouse model. In the present study, the role of JAM-C in the engraftment of human lymphoma B cells in mice was investigated. Administration of novel anti-JAM-C mAbs reduced tumor growth of JAM-C(+) MCL cells in bone marrow, spleen, liver, and lymph nodes of mice. Treatment with anti-JAM-C antibodies significantly reduced the proliferation of JAM-C-expressing lymphoma B cells. Moreover, the binding of anti-JAM-C antibodies inhibited the phosphorylation of ERK1/2, without affecting other signaling pathways. The results identify for the first time the intracellular MAPK cascade as the JAM-C-driven signaling pathway in JAM-C(+) B cells. Targeting JAM-C could constitute a new...
Intraepithelial lymphocytes (IEL) of the small intestine are anatomically positioned to be in the... more Intraepithelial lymphocytes (IEL) of the small intestine are anatomically positioned to be in the first line of cellular defense against enteric pathogens. Therefore, determining the origin of these cells has important implications for the mechanisms of T cell maturation and repertoire selection. Recent evidence suggests that murine CD8αα intestinal IELs (iIELs) can mature and undergo selection in the absence of a thymus. We analyzed IEL origin by cell transfer, using two congenic chicken strains. Embryonic day 14 and adult thymocytes did not contain any detectable CD8αα T cells. However, when TCR+ thymocytes were injected into congenic animals, they migrated to the gut and developed into CD8αα iIELs, while TCR− T cell progenitors did not. The TCR Vβ1 repertoire of CD8αα+ TCR Vβ1+ iIELs contained only part of the TCR Vβ1 repertoire of total iIELs, and it exhibited no new members compared with CD8+ T cells in the thymus. This indicated that these T cells emigrated from the thymus at ...
Lymphocytes depend on endothelial adhesion molecules such as ICAM-1 and VCAM-1, upregulated with ... more Lymphocytes depend on endothelial adhesion molecules such as ICAM-1 and VCAM-1, upregulated with inflammation, to facilitate transmigration across junctional barriers. New data show that ALCAM replaces VCAM-1 in the CNS during the development of neuroinflammatory diseases such as multiple sclerosis.
We have recently shown that junctional adhesion molecule (JAM)-C-deficient mice have leukocytic p... more We have recently shown that junctional adhesion molecule (JAM)-C-deficient mice have leukocytic pulmonary infiltrates, disturbed neutrophil homeostasis, and increased postnatal mortality. This phenotype was partially rescued when mice were housed in ventilated isolators, suggesting an inability to cope with opportunistic infections. In the present study, we further examined the adaptive immune responses in JAM-C−/− mice. We found that murine conventional dendritic cells express in addition to Mac-1 and CD11c also JAM-B as ligand for JAM-C. By in vitro adhesion assay, we show that murine DCs can interact with recombinant JAM-C via Mac-1. However, this interaction does not seem to be necessary for dendritic cell migration and function in vivo, even though JAM-C is highly expressed by lymphatic sinuses of lymph nodes. Nevertheless, upon immunization and boosting with a protein Ag, JAM-C-deficient mice showed decreased persistence of specific circulating Abs although the initial respons...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2009
Objectives— The molecular mechanisms regulating vascular permeability are only now being elucidat... more Objectives— The molecular mechanisms regulating vascular permeability are only now being elucidated. The junctional adhesion molecule (JAM) JAM-C has been linked to the induction of vascular permeability. We sought to understand the mechanism whereby JAM-C may disrupt junctional integrity in endothelial cells (ECs). Methods and Results— We show here that JAM-C alters permeability through modulation of integrin activity. JAM-C overexpression results in an increase in JAM-C at junctions and an increase in permeability. Conversely, knockdown of JAM-C by siRNA results in a reduction in permeability. JAM-C associates with αvβ3 integrin and regulates its localization and activity. JAM-C also inhibits the activation state of the β 1 integrin although it does not associate with this integrin. These changes induced on the integrins are mediated through regulation of the small GTPase, Rap1b but not Rap1a. Thrombin, a powerful inductor of vascular leak, causes localization of JAM-C into the ju...
T-cell development is dependent on microenvironments generated by stromal cells such as thymic ep... more T-cell development is dependent on microenvironments generated by stromal cells such as thymic epithelial cells (TEC). TEC population, which is phenotypically and functionally heterogeneous, controls the migration, proliferation and selection of differentiating thymocytes. CYR61 (cysteine-rich protein 61) is a matricellular protein reported to promote proliferation and migration of cancer cells. Here we investigated the impact of CYR61 treatment on T-cell development. We found that matricellular protein CYR61 was strongly expressed in TEC and favored TEC/thymocyte interaction by binding to LFA1 and ICAM1. Treatment of thymic lobes with recombinant CYR61 expanded the thymic stromal compartment. Thymocyte-independent TEC proliferation occurred through CYR61 binding to integrin alpha6/beta1 and the activation of Akt signaling pathway. Engraftment of thymic lobes mildly overexpressing CYR61 into athymic nude mice drastically boosted thymic output through the expansion of TEC. Actually, ...
Soluble forms of membrane receptors are emerging candidates as physiological regulators of leukoc... more Soluble forms of membrane receptors are emerging candidates as physiological regulators of leukocyte trafficking. In the present study, we found that the soluble form of the CD38 antigen (sCD38) bears a binding domain of low affinity for a cellular receptor on U937 cells. Cross-linking and peptide-mapping studies confirmed the physical association and the identification of the U937 receptor as a 130 kDa protein. The binding of sCD38 to the receptor was differentially inhibited by several monoclonal antibodies against the CD31 cell-adhesion molecule. Thus the interaction was analysed through direct association of soluble and membrane CD38 with soluble recombinant murine CD31 with three N-terminal and with all six extracellular Ig domains. Cross-linking experiments on U937 intact cells, and ligand blot assays of the immunoprecipitated CD38 molecule, indicated that (i) the recognized epitope is determined by the tertiary structure of the molecule, and that (ii) the binding domain invol...
The rat monoclonal antibody (mAb) termed EA-1 was originally selected for its capacity to block t... more The rat monoclonal antibody (mAb) termed EA-1 was originally selected for its capacity to block the adhesion of T lymphocyte progenitors to mouse thymic endothelium. Here we show that the mAb EA-1 recognizes the alpha 6 chain of alpha 6 beta 1 and alpha 6 beta 4 integrins. Both molecules are present at a high level on the luminal and basolateral side of vascular endothelium and alpha 6 beta 1 integrin is expressed on the highly metastatic cell lines B16/129 (melanoma) and KLN-205 (carcinoma). These lung specific tumors bind preferentially to lung frozen sections, and EA-1 blocked this interaction in vitro. Moreover, mAb EA-1 inhibited experimental metastasis to the lung of B16/129 cells injected intravenously. Metastasis in vivo was blocked when the antibody was injected into mice before or simultaneously with the melanoma cells, as well as when melanoma cells were precoated with EA-1 before injection. We suggest that alpha 6 integrins play a dual role in the metastatic process, mediating the adhesion of tumor cells to the luminal surface of the endothelium and the adhesion to laminin in the subendothelial extracellular matrix during extravasation. Despite the fact that alpha 6 integrins are laminin receptors, EA-1 did not interfere with melanoma cell binding to laminin fragments. Our antibody EA-1 may therefore recognize a binding domain on alpha 6 integrins of a novel ligand involved in cell-cell interaction.
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