Junctional adhesion molecule C (JAM-C) is a component of endothelial cell (EC) tight junctions that has been implicated in a number of endothelial functions, such as angiogenesis and trafficking of leukocytes through the endothelium during inflammation. Work within our lab has identified that loss of JAM-C at EC junctions results in increased reverse transendothelial migration (rTEM) of neutrophils back into the circulation, a response that has been associated with the dissemination of inflammation to distant organs. Whilst the mechanism by which JAM-C is lost or redistributed away from EC junctions has begun to be elucidated, little is known about how loss of endothelial JAM-C impacts the functions of ECs. As such, this thesis aimed to investigate the effect of JAM-C deficiency on EC functions to unravel possible molecular and cellular mechanisms of mediating neutrophil rTEM. To address the effect of JAM-C deficiency on EC functions, an in vitro RNA interference (RNAi) approach was used to efficiently knock-down (KD) JAM-C in human umbilical vein ECs (HUVECs). Importantly, KD of JAM-C did not affect expression of other key EC junctional markers such as JAM-A and VE-Cadherin and cell proliferation and apoptosis were similarly unaffected. Gene expression profiling using microarrays revealed that JAM-C depleted HUVECs exhibited a pro-inflammatory phenotype under basal conditions that was characterised by increased expression of pro-inflammatory genes such as ICAM1 and IL8. Following IL-1β-induced inflammation, no difference in expression of pro-inflammatory genes was detected between control and JAM-C KD HUVECs. However, protein levels of secreted chemokines such as IL-8 were reduced in JAM-C KD HUVECs following stimulation with IL-1β. This was corroborated by in vivo studies demonstrating reduced levels of secreted chemokines in the plasma of mice where JAM-C was conditionally deleted from ECs. A novel finding of this work is the demonstration that JAM-C KD HUVECs exhibit increased autophagy under basal conditions. This might provide a potential mechanism for the reduced chemokine secretion that is observed in this system, whereby chemokines are preferentially trafficked for autophagosome-mediated degradation. Taken together, these findings indicate a multi-functional role for JAM-C in regulating EC homeostasis under basal conditions. JAM-C KD ECs respond aberrantly to inflammatory stimuli by secreting reduced chemokine levels, a consequence that could provide novel insights into the mechanisms of neutrophil rTEM under conditions of endothelial JAM-C loss.

The immunoglobulin superfamily (IgSF) is a large protein superfamily of membrane and soluble proteins that influence recognition, binding, and adhesion. Among members of this family are cell adhesion molecules (CAMs), which form cell-cell contact points that play key roles in development, cell polarization, and cellular fate. Cadherins (CADs) are calcium-dependent proteins of the adherens junction (AJ), and polarize epithelium and endothelium. The tight junction (TJ) is a multiprotein junctional complex whose function is to control the permeability of the paracellular pathway. At the membrane level, TJs are composed of three types of proteins: claudins (CLDNs), occludin (OCLN) and junctional adhesion molecules (JAMs). JAMs are members of the IgSF while CLDN and OCLN are 4-α-helix membrane proteins. Although JAMs are part of the TJ and reside in the same ultrastructure, they are similar to CADs in their secondary, tertiary, and quaternary protein structure. Crystallographic studies of CADs in the presence of calcium yielded trans interactions that resulted in cell-cell contacts. In the absence of calcium, CADs form cis interactions that do not form cell-cell interactions. The crystal structure of JAM-A, has a quaternary organization of a cis dimer. In spite of the many similarities, a link between CADs and JAMs remains unclear. Beyond this point, the association between JAMs, CLDNs, and OCLN in the TJ is vaguely understood. The JAM family (JAM-A, -B, -C and 4) and their tissue-specific distribution indicate that they are key to understanding the TJ’s function and the interplay with the AJ. JAM-A has been used as a prototype for the other three members of the family, but based on current evidence we hypothesized that these proteins may display unique properties to support TJ’s function in a given tissue. Are JAMs affected by calcium just as CADs? Do CLDNs and OCLN make direct contact with JAMs? Do JAMs coordinate the interplay between TJ and AJ? We designed a strategy based on recombinant proteins and biophysical methods to answer these questions. First, we fused the extracellular domain of each JAM to maltose-binding protein (MBP). Our results indicate that JAM proteins have similar secondary structures, but unique tertiary structures. Surface Plasmon Resonance experiments showed that JAM proteins favored heterotypic compared to homotypic interactions. Second, we addressed the effects of cations (Ca2+, Mg2+, Cu2+, Fe2+, Fe3+, and Zn2+) on JAM-A. The exposure of JAM-A to the resulted in changes in its secondary, tertiary structure, and homotypic binding affinity. Finally, we addressed whether cations had an effect on the other TJ components and if there is an interplay with E-CAD. We determined that in the assembly of a simple TJ and AJ, JAM-A and E-CAD are calcium-dependent, while CLDN1 and OCLN are calcium independent. We conclude that TJ components such as CLDN1 and OCLN may work as anchors to maintain cell-cell interactions while JAM-A and E-CAD would be regulated by cations in order to accommodate other homeostatic functions.

No description available.

L’adhésion, la migration cellulaire et l’environnement stromal sont intimement liés pour garantir l’homéostasie du système immuno-hématopoïétique. Néanmoins, nos connaissances des mécanismes responsables du maintien de ce processus fonctionnel restent fragmentaires. Notre étude a permis de mieux caractériser le stroma ganglionnaire et médullaire dans lesquels nous avons démontré de nouveaux rôles immuno-régulateurs des molécules d’adhésion jonctionnelle JAM-B et JAM-C. Dans la zone T des ganglions lymphatiques, les cellules réticulaires fibroblastiques (FRC) sécrètent des composés de la matrice extracellulaire et des chimiokines homéostatiques, nécessaires à la migration intranodale des lymphocytes T naïfs. La génération de nouveaux anticorps monoclonaux a permis d’identifier une diversité phénotypique et fonctionnelle au sein de la population FRC. L’un d’entre eux reconnaît la Thrombomoduline permettant d’identifier une population de FRC exprimant les protéines JAM-C et PDGFRα. Cette population cellulaire, dénommée FRCDP (Double Positive) sécrète des chimiokines homéostatiques, ce qui la distingue de la population FRCDN (Double Negative). Les souris sauvages traitées avec l’anticorps anti-JAM-C présentent une diminution significative du taux intranodal des chimiokines CXCL12, CCL19, CCL21 affectant la recirculation des cellules T naïves. De façon similaire, les cellules stromales des niches hématopoïétiques fournissent un environnement fonctionnel, nécessaire à l’homéostasie du système hématopoïétique. Les molécules d’adhésion sont connues pour contrôler ces mécanismes. JAM-C est exprimée à la surface des cellules souches hématopoïétiques (CSH) mais son rôle dans l’hématopoïèse reste inconnu. Notre étude montre que la molécule JAM-B est exprimée par l’environnement médullaire et interagit spécifiquement avec JAM-C sur les CSH. Les souris déficientes pour le gène jam-b présentent une diminution du nombre de CSH quiescentes et une réponse accrue aux agents mobilisants, démontrant ainsi que le couple JAM-B/JAM-C est nécessaire au maintien et à la rétention des CSH dans la moelle osseuse. / Homeostasis of the immune and hematopoietic system is dependent of cell adhesion, cell migration and stromal environment. Nevertheless, the molecular mechanisms involved in the crosstalk between hematopoietic and stromal cells have remained elusive. Our studies allowed to better characterize lymph node (LN) and bone marrow (BM) stromal compartments through the demonstration that expression of junctional adhesion molecules (JAM) in these compartments is necessary for the maintenance of immune and hematopoietic homeostasis. In the T cell zone (LN), extracellular matrix and homeostatic chemokines are secreted by fibroblastic reticular cells (FRC) which control naive T cell migration. We have identified new FRC subsets using a monoclonal antibody based approach to identify new cell surface markers of stromal cells. We have found that the FRC population expressing JAM-C, Thrombomodulin and PDGFRα (FRCDP, for Double Positive) secretes homeostatic chemokines such as CCL21, CCL19 and CXCL12. In contrast, FRCDN (Double Negative) that lack JAM-C and Thrombomodulin expression do not. Functionally, we have shown that JAM-C controls the secretion of CCL21, CCL19 and CXCL12 by FRCDP and that anti-JAM-C treated mice exhibit a decrease of intranodal chemokine contents. These results suggest that JAM-C may regulate homeostasis through the control of homeostatic chemokine secretion. We therefore asked the question whether similar function for JAM-C or its ligand JAM-B may be identified in the bone marrow. In the BM, Hematopoietic Stem Cells (HSC) are maintained quiescent and undifferentiated in specific stromal structures called HSC niches. HSC/niche interactions via adhesion molecules and chemokines are known to be active player of HSC homeostasis. Recently, JAM-C expression by HSC has been reported, but its role in hematopoiesis has remained elusive. We have demonstrated that HSC interact with JAM-B expressed by BM stromal cells in a JAM-C dependent manner. Moreover, we have observed a decreased pool of quiescent HSC in jam-b deficient mice. Finally, we have found that jam-b deficient mice exhibit an increase in intramedullary CXCL12 content and an exacerbated response to mobilizing agents. Collectively, these data demonstrate that JAM-B and JAM-C play a dual function in lymph node and bone marrow microenvironments through the regulation of leuko-stromal adhesion and chemokine secretion.

Umbilical cord blood (UCB) is used as a source of haematopoietic stem cells (HSCs) for transplantation but shows defective homing to the bone marrow niche and delayed haematological reconstitution. Following transplantation, HSCs will home to the bone marrow in response to the CXCL12 chemokine, adhere to the bone marrow sinusoidal endothelial cells and then migrate into and lodge in bone marrow niches. In addition to CXCR4, a variety of molecules have been described as being important in these processes. In this laboratory, junctional adhesion molecule-A (JAM-A) was shown to be expressed on human UCB CD133⁺/CD34⁺ cells and regulated by hypoxia. In this thesis, further phenotypic studies show that this molecule is most highly expressed on human CD41a⁺ megakaryocytes and CD14⁺ monocytes/macrophages in UCB. JAM-A was also found to be expressed on all human UCB CD133⁺ cells, which have been shown by others to encompass the HSCs and early myeloid-lymphoid precursors and on the majority of CD34⁺ haematopoietic progenitor cells (HPCs). While it is also present on bone marrow sinusoidal endothelium (BMEC), JAM-A is not detected on cultured bone marrow mesenchymal stromal cells (MSCs). JAM-A blockade, silencing and overexpression experiments showed that JAM-A contributes to, but is not solely responsible for, the adhesion of CD34⁺ haematopoietic progenitor cells to IL-1β activated BMEC-60 cells and fibronectin. Lack of significance in cell migration suggested that JAM-A is more likely to act as an adhesion molecule or a regulator of adhesion rather than as a migratory molecule in such cells. Further functional studies using the proximity ligation assay highlight a potential association of JAM-A with CXCR4 and the adhesion molecules, tetraspanin CD82 and integrin β1. Mechanistic studies were commenced to establish if JAMA could modulate CXCR4 signalling following CXCL12 stimulation, but time constraints prevented these from being completed. These preliminary experiments which were carried out first in the Jurkat cell line lacking JAM-A or transduced to express JAM-A, however, suggest that JAM-A may modulate CXCL12-induced Rap1 phosphorylation and ERK1/2 phosphorylation. The former pathway is important for integrin function and the latter pathway is important in cell adhesion. The results described here, although requiring finalisation, support the hypothesis that JAM-A acts as an adhesion molecule and also may fine tune CXCR4 and integrin mediated functions on human CD34⁺ cells, thereby potentially regulating engraftment of these cells to the bone marrow niche.