Blockade but Not Overexpression of the Junctional Adhesion Molecule C Influences Virus-Induced Type 1 Diabetes in Mice

Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing beta-cells in the pancreas. Recruitment of inflammatory cells is prerequisite to beta-cell-injury. The junctional adhesion molecule (JAM) family proteins JAM-B and JAM–C are involved in polarized leukocyte transendothelial migration and are expressed by vascular endothelial cells of peripheral tissue and high endothelial venules in lympoid organs. Blocking of JAM-C efficiently attenuated cerulean-induced pancreatitis, rheumatoid arthritis or inflammation induced by ischemia and reperfusion in mice. In order to investigate the influence of JAM-C on trafficking and transmigration of antigen-specific, autoaggressive T-cells, we used transgenic mice that express a protein of the lymphocytic choriomeningitis virus (LCMV) as a target autoantigen in the β-cells of the islets of Langerhans under the rat insulin promoter (RIP). Such RIP-LCMV mice turn diabetic after infection with LCMV. We found that upon LCMV-infection JAM-C protein was upregulated around the islets in RIP-LCMV mice. JAM-C expression correlated with islet infiltration and functional beta-cell impairment. Blockade with a neutralizing anti-JAM-C antibody reduced the T1D incidence. However, JAM-C overexpression on endothelial cells did not accelerate diabetes in the RIP-LCMV model. In summary, our data suggest that JAM-C might be involved in the final steps of trafficking and transmigration of antigen-specific autoaggressive T-cells to the islets of Langerhans.     

The junctional adhesion molecule-C promotes neutrophil transendothelial migration in vitro and in vivo

The third member of the family of junctional adhesion molecules (JAMs), JAM-3, also called JAM-C, was recently shown to be a novel counter-receptor on platelets for the leukocyte beta(2)-integrin Mac-1 (alphaMbeta(2), CD11b/CD18). Here, new functional aspects of the role of endothelial cell JAM-C were investigated. Endothelial cells express JAM-C, which is predominantly localized within junctions at interendothelial contacts, since it codistributes with a tight junction component, zonula occludens-1. Whereas JAM-C does not participate in neutrophil adhesion to endothelial cells, it mediates neutrophil transmigration in a Mac-1-dependent manner. In particular, inhibition of JAM-C significantly reduced neutrophil transendothelial migration, and the combination of JAM-C and platelet/endothelial cell adhesion molecule-1 blockade almost completely abolished neutrophil transendothelial migration in vitro. In vivo, inhibition of JAM-C with soluble mouse JAM-C resulted in a 50% reduction of neutrophil emigration in the mouse model of acute thioglycollate-induced peritonitis. Thus, JAM-C participates in neutrophil transmigration and thereby provides a novel molecular target for antagonizing interactions between vascular cells that promote inflammatory vascular pathologies.     

Murine junctional adhesion molecules JAM-B and JAM-C mediate endothelial and stellate cell interactions during hepatic fibrosis

Classical junctional adhesion molecules JAM-A, JAM-B and JAM-C influence vascular permeability, cell polarity as well as leukocyte recruitment and immigration into inflamed tissue. As the vasculature becomes remodelled in chronically injured, fibrotic livers we aimed to determine distribution and role of junctional adhesion molecules during this pathological process. Therefore, livers of naïve or carbon tetrachloride-treated mice were analyzed by immunohistochemistry to localize all 3 classical junctional adhesion molecules. Hepatic stellate cells and endothelial cells were isolated and subjected to immunocytochemistry and flow cytometry to determine localization and functionality of JAM-B and JAM-C. Cells were further used to perform contractility and migration assays and to study endothelial tubulogenesis and pericytic coverage by hepatic stellate cells. We found that in healthy tissue, JAM-A was ubiquitously expressed whereas JAM-B and JAM-C were restricted to the vasculature. During fibrosis, JAM-B and JAM-C levels increased in endothelial cells and JAM-C was de novo generated in myofibroblastic hepatic stellate cells. Soluble JAM-C blocked contractility but increased motility in hepatic stellate cells. Furthermore, soluble JAM-C reduced endothelial tubulogenesis and endothelial cell/stellate cell interaction. Thus, during liver fibrogenesis, JAM-B and JAM-C expression increase on the vascular endothelium. More importantly, JAM-C appears on myofibroblastic hepatic stellate cells linking them as pericytes to JAM-B positive endothelial cells. This JAM-B/JAM-C mediated interaction between endothelial cells and stellate cells stabilizes vessel walls and may control the sinusoidal diameter. Increased hepatic stellate cell contraction mediated by JAM-C/JAM-C interaction may cause intrahepatic vasoconstriction, which is a major complication in liver cirrhosis.     

Genetic evidence for functional redundancy of Platelet/Endothelial cell adhesion molecule-1 (PECAM-1): CD31-deficient mice reveal PECAM-1-dependent and PECAM-1-independent functions

Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31), a member of the Ig superfamily, is expressed strongly at endothelial cell-cell junctions, on platelets, and on most leukocytes. CD31 has been postulated to play a role in vasculogenesis and angiogenesis, and has been implicated as a key mediator of the transendothelial migration of leukocytes. To further define the physiologic role of CD31, we used targeted gene disruption of the CD31 gene in embryonic stem cells to generate CD31-deficient mice. CD31-deficient mice (CD31KO) are viable and born at the expected Mendelian frequency, remain healthy, and exhibit no obvious vascular developmental defects. In response to inflammatory challenge, polymorphonuclear leukocytes of CD31KO mice are arrested between the vascular endothelium and the basement membrane of inflammatory site mesenteric microvessels, confirming a role for CD31 in the migration of neutrophils through the subendothelial extracellular matrix. Normal numbers of leukocytes are recovered from inflammatory sites in CD31KO mice, however, suggesting that the defect in leukocyte migration across basal lamina observed in the absence of CD31 may be compensated for by the use of other adhesion molecules, or possibly an increased rate of migration. Homing of T lymphocytes in vivo is normal, and CD31KO mice are able to mount a cutaneous hypersensitivity response normally. In addition, CD31-mediated homophilic adhesion does not appear to play a role in platelet aggregation in vitro. This study provides genetic evidence that CD31 is involved in transbasement membrane migration, but does not play an obligatory role in either vascular development or leukocyte migration.     

Junctional adhesion molecule C (JAM-C) dimerization aids cancer cell migration and metastasis

Most cancer deaths result from metastasis, which is the dissemination of cells from a primary tumor to distant organs. Metastasis involves changes to molecules that are essential for tumor cell adhesion to the extracellular matrix and to endothelial cells. Junctional Adhesion Molecule C (JAM-C) localizes at intercellular junctions as homodimers or more affine heterodimers with JAM-B. We previously showed that the homodimerization site (E66) in JAM-C is also involved in JAM-B binding. Here we show that neoexpression of JAM-C in a JAM-C-negative carcinoma cell line induced loss of adhesive property and pro-metastatic capacities. We also identify two critical structural sites (E66 and K68) for JAM-C/JAM-B interaction by directed mutagenesis of JAM-C and studied their implication on tumor cell behavior. JAM-C mutants did not bind to JAM-B or localize correctly to junctions. Moreover, mutated JAM-C proteins increased adhesion and reduced proliferation and migration of lung carcinoma cell lines. Carcinoma cells expressing mutant JAM-C grew slower than with JAM-C WT and were not able to establish metastatic lung nodules in mice. Overall these data demonstrate that the dimerization sites E66-K68 of JAM-C affected cell adhesion, polarization and migration and are essential for tumor cell metastasis.     

Endothelial cell P-selectin mediates a proinflammatory and prothrombogenic phenotype in cerebral venules of sickle cell transgenic mice

Whereas the adhesion of leukocytes and erythrocytes to vascular endothelium has been implicated in the vasooclusive events associated with sickle cell disease, the role of platelet-vessel wall interactions in this process remains undefined. The objectives of this study were to: 1) determine whether the adhesion of platelets and leukocytes in cerebral venules differs between sickle cell transgenic (betaS) mice and their wild-type (WT) counterparts (C57Bl/6) under both resting and posthypoxic conditions, and 2) define the contributions of P-selectin to these adhesion processes. Animals were anesthetized, and platelet and leukocyte interactions with endothelial cells of cerebral postcapillary venules were monitored and quantified using intravital fluorescence microscopy in WT, betaS, and chimeric mice produced by transplanting bone marrow from WT or betaS mice into WT or P-selectin-deficient (P-sel(-/-)) mice. Platelet and leukocyte adhesion to endothelial cells in both unstimulated and posthypoxic betaS mice were significantly elevated over WT levels. Chimeric mice involving bone marrow transfer from betaS mice to P-sel(-/-) mice exhibited a profound attenuation of both platelet and leukocyte adhesion compared with betaS bone marrow transfer to WT mice. These findings indicate that betaS mice assume both an inflammatory and prothrombogenic phenotype, with endothelial cell P-selectin playing a major role in mediating these microvascular responses.     

Junctional adhesion molecules are required for melanoma cell lines transendothelial migration in vitro

One of the main steps of metastasis is extravasation, a phenomenon well described in lymphocytes but remaining to be fully uncovered for melanoma. Junctional adhesion molecules (JAMs) control the transendothelial migration of leukocytes. To date, the role of the JAM proteins, notably JAM-A and JAM-C, has not been examined in melanoma. Here, we compared two melanoma tumor cell lines, A375 and SLM8 cells, the A375 cell line being four times more efficient than the SLM8 cells in the crossing of the endothelial monolayer. We show evidence of the differential expression of JAM-A and JAM-C in these cell lines with JAM-C mainly expressed in the A375 cell line, and JAM-A detected preferentially in the SLM8 cells. To further dissect the respective roles of these proteins, we used both siRNA and blocking antibodies to decrease JAM-A and JAM-C expression.     

Transgenic mice expressing different levels of soluble platelet/endothelial cell adhesion molecule-IgG display distinct inflammatory phenotypes.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31), expressed on the surfaces of leukocytes and concentrated in the junctions between endothelial cells plays an important role in transendothelial migration of neutrophils and monocytes. Soluble recombinant PECAM-IgG injected i.v. into mice blocks acute leukocyte emigration by 80%. To study the role of PECAM in models of chronic inflammation, we generated transgenic mice constitutively expressing soluble full-length murine PECAM as an IgG chimera. Three founder lines expressed this transgene and constitutively secreted murine PECAM-IgG into the plasma where it was maintained at characteristic concentrations for each line. All mice had similar hematologic profiles to wild-type littermates and were healthy when maintained in the standard laboratory animal facility. Both the leukocytes and the endothelium of mice of all transgenic lines expressed the same levels of endogenous PECAM-1 as wild-type littermates. Similarly, there were no detectable differences in the expression of several other common leukocyte and endothelial cell adhesion molecules. Mice that produced moderate (10-20 microg/ml) concentrations of PECAM-IgG demonstrated a severely blunted acute inflammatory response, despite mobilizing appropriate numbers of circulating leukocytes. Surprisingly, mice that constitutively produced high (400-1,000 microg/ml) concentrations of PECAM-IgG were unresponsive to its anti-inflammatory effects. This is the first demonstration that a soluble form of a cell adhesion molecule can be stably expressed and retain efficacy in vivo over prolonged periods. This approach is applicable to many other extracellular molecules. However, the plasma concentrations of such constitutively produced inhibitors may greatly influence the resulting phenotype.     

Neutrophil transmigration under shear flow conditions in vitro is junctional adhesion molecule-C independent

Endothelial cell junctional adhesion molecule (JAM)-C has been proposed to regulate neutrophil migration. In the current study, we used function-blocking mAbs against human JAM-C to determine its role in human leukocyte adhesion and transendothelial cell migration under flow conditions. JAM-C surface expression in HUVEC was uniformly low, and treatment with inflammatory cytokines TNF-alpha, IL-1beta, or LPS did not increase its surface expression as assessed by FACS analysis. By immunofluorescence microscopy, JAM-C staining showed sparse localization to cell-cell junctions on resting or cytokine-activated HUVEC. Surprisingly, staining of detergent-permeabilized HUVEC revealed a large intracellular pool of JAM-C that showed little colocalization with von Willebrand factor. Adhesion studies in an in vitro flow model showed that functional blocking JAM-C mAb alone had no inhibitory effect on polymorphonuclear leukocyte (PMN) adhesion or transmigration, whereas mAb to ICAM-1 significantly reduced transmigration. Interestingly, JAM-C-blocking mAbs synergized with a combination of PECAM-1, ICAM-1, and CD99-blocking mAbs to inhibit PMN transmigration. Overexpression of JAM-C by infection with a lentivirus JAM-C GFP fusion protein did not increase adhesion or extent of transmigration of PMN or evoke a role for JAM-C in transendothelial migration. These data suggest that JAM-C has a minimal role, if any, in PMN transmigration in this model and that ICAM-1 is the preferred endothelial-expressed ligand for PMN beta(2) integrins during transendothelial migration.     

Schwann cell-specific JAM-C-deficient mice reveal novel expression and functions for JAM-C in peripheral nerves

Junctional adhesion molecule-C (JAM-C) is an adhesion molecule expressed at junctions between adjacent endothelial and epithelial cells and implicated in multiple inflammatory and vascular responses. In addition, we recently reported on the expression of JAM-C in Schwann cells (SCs) and its importance for the integrity and function of peripheral nerves. To investigate the role of JAM-C in neuronal functions further, mice with a specific deletion of JAM-C in SCs (JAM-C SC KO) were generated. Compared to wild-type (WT) controls, JAM-C SC KO mice showed electrophysiological defects, muscular weakness, and hypersensitivity to mechanical stimuli. In addressing the underlying cause of these defects, nerves from JAM-C SC KO mice were found to have morphological defects in the paranodal region, exhibiting increased nodal length as compared to WTs. The study also reports on previously undetected expressions of JAM-C, namely on perineural cells, and in line with nociception defects of the JAM-C SC KO animals, on finely myelinated sensory nerve fibers. Collectively, the generation and characterization of JAM-C SC KO mice has provided unequivocal evidence for the involvement of SC JAM-C in the fine organization of peripheral nerves and in modulating multiple neuronal responses.     

Blocking Junctional Adhesion Molecule C Enhances Dendritic Cell Migration and Boosts the Immune Responses against Leishmania major

The recruitment of dendritic cells to sites of infections and their migration to lymph nodes is fundamental for antigen processing and presentation to T cells. In the present study, we showed that antibody blockade of junctional adhesion molecule C (JAM-C) on endothelial cells removed JAM-C away from junctions and increased vascular permeability after L. major infection. This has multiple consequences on the output of the immune response. In resistant C57BL/6 and susceptible BALB/c mice, we found higher numbers of innate immune cells migrating from blood to the site of infection. The subsequent migration of dendritic cells (DCs) from the skin to the draining lymph node was also improved, thereby boosting the induction of the adaptive immune response. In C57BL/6 mice, JAM-C blockade after L. major injection led to an enhanced IFN-γ dominated T helper 1 (Th1) response with reduced skin lesions and parasite burden. Conversely, anti JAM-C treatment increased the IL-4-driven T helper 2 (Th2) response in BALB/c mice with disease exacerbation. Overall, our results show that JAM-C blockade can finely-tune the innate cell migration and accelerate the consequent immune response to L. major without changing the type of the T helper cell response.     

Human Thy-1 (CD90) on activated endothelial cells is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18)

Leukocyte recruitment in response to inflammatory signals is in part governed by interactions between endothelial cell receptors belonging to the Ig superfamily and leukocyte integrins. In our previous work, the human Ig superfamily glycoprotein Thy-1 (CD90) was identified as an activation-associated cell adhesion molecule on human dermal microvascular endothelial cells. Furthermore, the interaction of Thy-1 with a corresponding ligand on monocytes and polymorphonuclear cells was shown to be involved in the adhesion of these leukocytes to activated Thy-1-expressing endothelial cells. In this study, we have identified the specific interaction between human Thy-1 and the leukocyte integrin Mac-1 (CD11b/CD18; alphaMbeta2) both in cellular systems and in purified form. Monocytes and polymorphonuclear cells were shown to adhere to transfectants expressing human Thy-1 as well as to primary Thy-1-expressing human dermal microvascular endothelial cells. Furthermore, leukocyte adhesion to activated endothelium as well as the subsequent transendothelial migration was mediated by the interaction between Thy-1 and Mac-1. This additional pathway in leukocyte-endothelium interaction may play an important role in the regulation of leukocyte recruitment to sites of inflammation.     

Neisseria meningitidis infection of human endothelial cells interferes with leukocyte transmigration by preventing the formation of endothelial docking structures

Neisseria meningitidis elicits the formation of membrane protrusions on vascular endothelial cells, enabling its internalization and transcytosis. We provide evidence that this process interferes with the transendothelial migration of leukocytes. Bacteria adhering to endothelial cells actively recruit ezrin, moesin, and ezrin binding adhesion molecules. These molecules no longer accumulate at sites of leukocyte-endothelial contact, preventing the formation of the endothelial docking structures required for proper leukocyte diapedesis. Overexpression of exogenous ezrin or moesin is sufficient to rescue the formation of docking structures on and leukocyte migration through infected endothelial monolayers. Inversely, expression of the dominant-negative NH(2)-terminal domain of ezrin markedly inhibits the formation of docking structures and leukocyte diapedesis through noninfected monolayers. Ezrin and moesin thus appear as pivotal endothelial proteins required for leukocyte diapedesis that are titrated away by N. meningitidis. These results highlight a novel strategy developed by a bacterial pathogen to hamper the host inflammatory response by interfering with leukocyte-endothelial cell interaction.     

Cutting edge: combined treatment of TNF-alpha and IFN-gamma causes redistribution of junctional adhesion molecule in human endothelial cells.

Proinflammatory cytokines such as TNF-alpha and IFN-gamma induce cell adhesion molecules in endothelial cells and promote transmigration of leukocytes across endothelial cells. However, when those two were administered together, leukocyte transmigration paradoxically decreased. We cloned a human and bovine homologue of the junctional adhesion molecule (JAM), a novel molecule at the tight junction, and examined the effects of proinflammatory cytokines on JAM in HUVECs. The combined treatment of TNF-alpha plus IFN-gamma caused a disappearance of JAM from intercellular junctions. However, flow cytometry, cell ELISA, and subcellular fractionation analysis demonstrated that the amount of JAM was not reduced. This suggested that JAM changed its distribution in response to proinflammatory cytokines. This redistribution of JAM might be involved in a decrease in transendothelial migration of leukocytes at inflammatory sites.     

PECAM-1 (CD 31) mediates transendothelial leukocyte migration in experimental colitis

Transendothelial migration of circulating leukocytes into the colonic wall is a key step in the development of the inflammatory infiltrate in inflammatory bowel disease (IBD). The platelet-endothelial cell adhesion molecule-1 PECAM-1 (CD31) is expressed in the tight junction area of endothelial cells, where it is supposed to support the transmigration process. The aim of this study was to determine the role of PECAM-1 in experimental IBD and to show whether blockade of PECAM-1 has therapeutic effects. Chronic colitis was induced in female BALB/c mice by cyclic oral administration of dextran sodium sulfate (DSS) 3% (wt/vol). Expression of PECAM-1 was visualized by immunohistochemistry. In the treatment group animals received 1 mg/kg anti-PECAM-1 (2H8) ip daily starting on day 26. On day 30 leukocyte adhesion and migration was measured during N(2)O-isoflurane anesthesia in the distal colon by intravital microscopy. Disease activity index (DAI), histology, and MPO levels were compared with healthy and diseased controls. PECAM-1 was expressed in colitic mice. Chronic DSS colitis was characterized by a marked increase in rolling, adherent, and transmigrated leukocytes compared with healthy controls. Immunoblockade of PECAM-1 reduced leukocyte transmigration significantly and also diminished leukocyte rolling and sticking in an indirect manner. It also resulted in a significantly diminished DAI and MPO levels, as well as an amelioration of the histological inflammation score. PECAM-1 plays an important role in transendothelial leukocyte migration in DSS colitis. PECAM-1 could be a novel target for antibody-based treatment in IBD.     

Dual interaction of JAM-C with JAM-B and alpha(M)beta2 integrin: function in junctional complexes and leukocyte adhesion

The junctional adhesion molecules (JAMs) have been recently described as interendothelial junctional molecules and as integrin ligands. Here we show that JAM-B and JAM-C undergo heterophilic interaction in cell-cell contacts and that JAM-C is recruited and stabilized in junctional complexes by JAM-B. In addition, soluble JAM-B dissociates soluble JAM-C homodimers to form JAM-B/JAM-C heterodimers. This suggests that the affinity of JAM-C monomers to form dimers is higher for JAM-B than for JAM-C. Using antibodies against JAM-C, the formation of JAM-B/JAM-C heterodimers can be abolished. This liberates JAM-C from its vascular binding partner JAM-B and makes it available on the apical side of vessels for interaction with its leukocyte counter-receptor alpha(M)beta2 integrin. We demonstrate that the modulation of JAM-C localization in junctional complexes is a new regulatory mechanism for alpha(M)beta2-dependent adhesion of leukocytes.     

L-selectin facilitates emigration and extravascular locomotion of leukocytes during acute inflammatory responses in vivo

L-selectin has been shown to be important in mediating leukocyte recruitment during inflammatory responses. Although there are numerous in vitro studies demonstrating that engagement of L-selectin leads to the activation of several signaling pathways potentially contributing to subsequent adhesion, emigration, or even migration through the interstitium, whether this actually induces cellular events in vivo is completely unknown. Therefore, we used intravital microscopy to visualize the role of L-selectin in downstream leukocyte adhesion, emigration, and interstitial migration events in wild-type and L-selectin-deficient (L-selectin(-/-)) mice. The cremaster muscle was superfused with the chemotactic inflammatory mediators platelet-activating factor or KC. Leukocyte rolling, adhesion, and emigration in postcapillary venules were examined, and the migration of emigrated leukocytes was recorded continuously using time-lapse videomicroscopy. Platelet-activating factor increased leukocyte adhesion to a similar level in both wild-type and L-selectin(-/-) mice. In contrast, both the number of emigrated leukocytes and the distance of extravascular migration were significantly reduced in L-selectin(-/-) mice. A similar pattern was observed in response to the superfusion of KC. Because superfusion of these mediators induced chemokinesis, we developed a new in vivo chemotaxis assay using slow release of KC from an agarose gel positioned 350 microm from a postcapillary venule. These experiments showed that L-selectin(-/-) leukocytes were also severely impaired in their ability to respond to a directional cue. These findings indicate that L-selectin is important in enabling leukocytes to respond effectively to chemotactic stimuli in inflamed tissues.     

Integrin activation by chemokines: relevance to inflammatory adhesion cascade during T cell migration

The adhesive function of integrins is regulated through cytoplasmic signaling induced by several stimuli, whose process is designated "inside-out signaling". A large number of leukocytes are rapidly recruited to the sites of inflammation where they form an essential component of the response to infection, injury, autoimmune disorders, allergy, tumor invasion, atherosclerosis and so on. The recruitment of leukocytes into tissue is regulated by a sequence of interactions between the circulating leukocytes and the endothelial cells. Leukocyte integrins play a pivotal role in leukocyte adhesion to endothelial cells. During the process, the activation of integrins by various chemoattractants, especially chemokines, is essential for integrin-mediated adhesion in which a signal transduced to the leukocyte converts the functionally inactive integrin to an active adhesive configuration. We have proposed that H-Ras-sensitive activation of phosphoinositide 3 (PI 3)-kinase and subsequent profilin-mediated actin polymerization, can be involved in chemokine-induced integrin-dependent adhesion of T cells. The present review documents the relevance of cytoplasmic signaling and cytoskeletal assembly to integrin-mediated adhesion induced by chemoattractants including chemokines during inflammatory processes. In contrast, various adhesion molecules are known to transduce extracellular information into cytoplasm, which leads to T cell activation and cytokine production from the cells, designated "outside-in signaling". Such a bi-directional "cross-talking" among adhesion molecules and cytokines is most relevant to inflammatory processes by augmenting immune cell migration from circulation into inflamed tissue such as rheumatoid arthritis, tumor invasion, Beh?et's disease and atherosclerosis.