Rôle des cellules dendritiques dans le maintien du phénotype endothélial cuboïdal spécialisé dans le recrutement de lymphocytes

Les lymphocytes, cellules effectrices de la réponse immunitaire adaptative, patrouillent dans l'organisme à la recherche d'antigènes étrangers contre lesquels ils sont dirigés. En condition physiologique, ils recirculent continuellement entre la lymphe, le sang, et les organes lymphoïdes secondaires, comme les ganglions lymphatiques. Le transit des lymphocytes naïfs du sang vers le tissu ganglionnaire nécessite une traversée de la paroi vasculaire qui prend place spécifiquement au niveau de veinules post-capillaires: les HEVs (High Endothelial Venules). La lumière de ces vaisseaux sanguins est tapissée de cellules endothéliales cuboïdales exprimant un ensemble de molécules nécessaires au recrutement massif de lymphocytes. En condition physiologique, ce phénotype endothélial hautement différencié est présent exclusivement dans les organes lymphoïdes secondaires, mais peut être induit au sein de tissus subissant une inflammation chronique ou un cancer. Il est donc primordial de comprendre les mécanismes qui régissent l'apparition et le maintien de ce type de vaisseaux afin de pouvoir un jour contrôler l'accès des lymphocytes à un territoire donné. Des études récentes ont mis en évidence un contrôle du phénotype endothélial cuboïdal par le microenvironnement lymphoïde. Néanmoins, les cellules directement impliquées ne sont pas encore caractérisées. Les cellules dendritiques (DC), qui sont amassées autour des HEVs constituaient un bon candidat pour notre étude. Nous avons caractérisé leur rôle grâce à l'utilisation de souris transgéniques CD11c-DTR permettant spécifiquement la déplétion des DCs CD11c+ chez l'adulte suite à l'injection de toxine diphtérique. En absence de DCs, les HEV se dédifférencient pour adopter un phénotype immature similaire à celui observé en période post-natale, provoquant l'abolition de la fonction de recrutement lymphocytaire et une diminution dramatique de la cellularité des ganglions lymphatiques. Nous avons pu suivre en direct ce défaut de recrutement grâce à la technique de microscopie intravitale mettant en évidence des interactions altérées entre les lymphocytes et les veinules du ganglion lymphatique. Des expériences de purification de cellules endothéliales de HEVs et de DCs suivies d'une étape de coculture ont permis de montrer que l'interaction DC/HEV pourrait être directe et impliquer le récepteur LTbetaR exprimé par les HEVs.High Endothelial Venules (HEV) are highly differentiated post-capillary venules specialized in naïve lymphocytes recruitment from the blood. They are located in secondary lymphoid organs and chronic inflammation sites and composed of cuboidal endothelial cells expressing a set of specific molecules allowing them to interact with lymphocytes. Recent studies demonstrate that the lymphoid tissue microenvironment is critical for the maintenance of HEV characteristics, but cell types implicated in this maintenance have not yet been well defined. As dendritic cells (DCs) are drained by lymph flow to lymph nodes where they are found clustered around HEV in the T-cell zone, we addressed the question whether they are responsible for HEV differentiation pressure. Using the CD11c-DTR murine model and bone marrow chimeras, which allow the temporally controlled depletion of all DCs in the mouse, we monitored HEV phenotype and recruitment abilities after various durations of DC depletion. Short term homing assays using naïve fluorescent lymphocytes showed a striking defect of HEV mediated lymphocyte recruitment into lymph node after i.v. injection of cells. Immunofluorescence study of lymph node sections allowed us to observe that HEV topography is highly disturbed and their phenotype is shifted from fully mature adult phenotype to the immature post-natal phenotype. These results, supported by observations of lymphocytes/HEV interactions within the blood flow by intravital microscopy, lead us to conclude that DCs are crucial in the maintenance of HEV phenotype and naïve lymphocyte recruitment into lymph node. Moreover, coculture experiments show that DCs/HEVs interaction could be direct and involve the lymphotoxin-beta receptor on HEV

The IL-1-Like Cytokine IL-33 Is Constitutively Expressed in the Nucleus of Endothelial Cells and Epithelial Cells In Vivo: A Novel ‘Alarmin’?

BACKGROUND: Interleukin-33 (IL-33) is an IL-1-like cytokine ligand for the IL-1 receptor-related protein ST2, that activates mast cells and Th2 lymphocytes, and induces production of Th2-associated cytokines in vivo. We initially discovered IL-33 as a nuclear factor (NF-HEV) abundantly expressed in high endothelial venules from lymphoid organs, that associates with chromatin and exhibits transcriptional regulatory properties. This suggested that, similarly to IL-1alpha and chromatin-associated cytokine HMGB1, IL-33 may act as both a cytokine and a nuclear factor. Although the activity of recombinant IL-33 has been well characterized, little is known yet about the expression pattern of endogenous IL-33 in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that IL-33 is constitutively and abundantly expressed in normal human tissues. Using a combination of human tissue microarrays and IL-33 monoclonal and polyclonal antibodies, we found that IL-33 is a novel nuclear marker of the endothelium widely expressed along the vascular tree. We observed abundant nuclear expression of IL-33 in endothelial cells from both large and small blood vessels in most normal human tissues, as well as in human tumors. In addition to endothelium, we also found constitutive nuclear expression of IL-33 in fibroblastic reticular cells of lymphoid tissues, and epithelial cells of tissues exposed to the environment, including skin keratinocytes and epithelial cells of the stomach, tonsillar crypts and salivary glands. CONCLUSIONS/SIGNIFICANCE: Together, our results indicate that, unlike inducible cytokines, IL-33 is constitutively expressed in normal human tissues. In addition, they reveal that endothelial cells and epithelial cells constitute major sources of IL-33 in vivo. Based on these findings, we speculate that IL-33 may function, similarly to the prototype 'alarmin' HMGB1, as an endogenous 'danger' signal to alert the immune system after endothelial or epithelial cell damage during trauma or infection

Shape and Function of Interstitial Chemokine CCL21 Gradients Are Independent of Heparan Sulfates Produced by Lymphatic Endothelium

Gradients of chemokines and growth factors guide migrating cells and morphogenetic processes. Migration of antigen-presenting dendritic cells from the interstitium into the lymphatic system is dependent on chemokine CCL21, which is secreted by endothelial cells of the lymphatic capillary, binds heparan sulfates and forms gradients decaying into the interstitium. Despite the importance of CCL21 gradients, and chemokine gradients in general, the mechanisms of gradient formation are unclear. Studies on fibroblast growth factors have shown that limited diffusion is crucial for gradient formation. Here, we used the mouse dermis as a model tissue to address the necessity of CCL21 anchoring to lymphatic capillary heparan sulfates in the formation of interstitial CCL21 gradients. Surprisingly, the absence of lymphatic endothelial heparan sulfates resulted only in a modest decrease of CCL21 levels at the lymphatic capillaries and did neither affect interstitial CCL21 gradient shape nor dendritic cell migration toward lymphatic capillaries. Thus, heparan sulfates at the level of the lymphatic endothelium are dispensable for the formation of a functional CCL21 gradient.Peer reviewe

A conduit to amplify innate immunity

In this issue of Immunity, Py et al. (2013) report that upon bacterial infection, a fragment of the matrix protein cochlin is released from the conduits of B cell follicles to trigger protective cytokines in the periphery

HEVs, lymphatics and homeostatic immune cell trafficking in lymph nodes

In search of foreign antigens, lymphocytes recirculate from the blood, through lymph nodes, into lymphatics and back to the blood. Dendritic cells also migrate to lymph nodes for optimal interaction with lymphocytes. This continuous trafficking of immune cells into and out of lymph nodes is essential for immune surveillance of foreign invaders. In this article, we review our current understanding of the functions of high endothelial venules (HEVs), stroma and lymphatics in the entry, positioning and exit of immune cells in lymph nodes during homeostasis, and we highlight the unexpected role of dendritic cells in the control of lymphocyte homing through HEVs

Rôle des cellules dendritiques dans le maintien du phénotype endothélial cuboïdal spécialisé dans le recrutement de lymphocytes

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

IL-33 is abundantly expressed in the nucleus of endothelial cells in human tumor tissues.

<p>Expression of IL-33 in the indicated human tumor tissues was analyzed using immunofluorescence staining. Double staining was performed with IL-33 mAb Nessy-1 (red) and anti-CD31 or anti-vWF polyclonal antibodies (green). DNA was counter-stained with DAPI. Magnification bars: A,B,G,H 50 µm; C,D,E,F 20 µm.</p

IL-33 is a chromatin-associated nuclear factor constitutively expressed in human secondary lymphoid tissues by HEVs and isolated cells in the interfollicular T cells areas.

<p>A: Immunohistochemical staining of a human tonsil section with IL-33 mAb Nessy-1. B–D: Double staining of human tonsils sections with HEV-specific mAb MECA79 (green) and IL-33 mAb Nessy-1 (B, red) or two distinct IL-33 polyclonal antisera, Cter1 (C, red) or Cter2 (D, red). The follicles (fol) are indicated. E and F: Nuclear staining of HEVs and isolated cells in the T cell areas was abrogated by pre-incubating the IL-33 monoclonal (E) and polyclonal (F) antibodies with IL-33 peptides but not control peptides. G and H: Nuclear accumulation of IL-33 in HEV blood vessels and isolated cells was also observed in lymph node (G, IL-33 mAb, red; CD31, green) and appendix (H, IL-33 mAb, red; vWF, green). I and J: Higher magnification of a human tonsil section double-stained with IL-33 and MECA-79 antibodies and counterstained with the DNA-binding dye DAPI. In the nucleus of both HEVs (arrow, upper panel) and isolated cells (arrowhead, lower panel), IL-33 accumulates in nuclear domains that colocalize with dense regions of DAPI staining (J), indicating association with chromatin. Magnification bars: A, I 10 µm; B, G, H 20 µm; J 5 µm; C, D, E, F 60 µm.</p