Murine CD146 is widely expressed on endothelial cells and is recognized by the monoclonal antibody ME-9F1

The endothelium plays an important role in the exchange of molecules, but also of immune cells between blood and the underlying tissue. The endothelial molecule S-Endo 1 antigen (CD146) is preferentially located at endothelial junctions and has been claimed to support endothelial integrity. In this study we show that the monoclonal antibody ME-9F1 recognizes the extracellular portion of murine CD146. Making use of ME-9F1 we found CD146 highly expressed and widely spread on endothelial cells in the analyzed murine tissues. In contrast to humans that express CD146 also on T cells or follicular dendritic cells, murine CD146 albeit at low levels was only found on a subset of NK1.1+ cells. The antibody against murine CD146 is useful for immunomagnetic sorting of primary endothelial cells not only from the liver but from various other organs. In vitro, no evidence was seen that the formation and integrity of endothelial monolayers or the transendothelial migration of T cells was affected by antibody binding to CD146 or by crosslinking of the antigen. This makes the antibody ME-9F1 an excellent tool especially for the ex vivo isolation of murine endothelial cells intended to be used in functional studies

Chemokine Transfer by Liver Sinusoidal Endothelial Cells Contributes to the Recruitment of CD4+ T Cells into the Murine Liver

Leukocyte adhesion and transmigration are central features governing immune surveillance and inflammatory reactions in body tissues. Within the liver sinusoids, chemokines initiate the first crucial step of T-cell migration into the hepatic tissue. We studied molecular mechanisms involved in endothelial chemokine supply during hepatic immune surveillance and liver inflammation and their impact on the recruitment of CD4+ T cells into the liver. In the murine model of Concanavalin A-induced T cell-mediated hepatitis, we showed that hepatic expression of the inflammatory CXC chemokine ligands (CXCL)9 and CXCL10 strongly increased whereas homeostatic CXCL12 significantly decreased. Consistently, CD4+ T cells expressing the CXC chemokine receptor (CXCR)3 accumulated within the inflamed liver tissue. In histology, CXCL9 was associated with liver sinusoidal endothelial cells (LSEC) which represent the first contact site for T-cell immigration into the liver. LSEC actively transferred basolaterally internalized CXCL12, CXCL9 and CXCL10 via clathrin- coated vesicles to CD4+ T cells leading to enhanced transmigration of CXCR4+ total CD4+ T cells and CXCR3+ effector/memory CD4+ T cells, respectively in vitro. LSEC-expressed CXCR4 mediated CXCL12 transport and blockage of endothelial CXCR4 inhibited CXCL12-dependent CD4+ T-cell transmigration. In contrast, CXCR3 was not involved in the endothelial transport of its ligands CXCL9 and CXCL10. The clathrin-specific inhibitor chlorpromazine blocked endothelial chemokine internalization and CD4+ T-cell transmigration in vitro as well as migration of CD4+ T cells into the inflamed liver in vivo. Moreover, hepatic accumulation of CXCR3+ CD4+ T cells during T cell-mediated hepatitis was strongly reduced after administration of chlorpromazine. These data demonstrate that LSEC actively provide perivascularly expressed homeostatic and inflammatory chemokines by CXCR4- and clathrin-dependent intracellular transport mechanisms thereby contributing to the hepatic recruitment of CD4+ T-cell populations during immune surveillance and liver inflammation

recruitment and tolerogenic modulation of T cells within the liver

Die Rekrutierung von T-Zellen spielt eine entscheidende Rolle für die Kompartimentalisierung des Immunsystems: Antigenpräsentation und Aktivierung finden in sekundären lymphatischen Organen statt, im entzündlich veränderten Gewebe werden Effektorfunktionen ausgeübt. In der Mukosa, und vermutlich auch in der Leber, werden immunmodulatorische Vorgänge vermittelt. Da Einwanderung in die Leber damit eine Vorraussetzung für konsekutive Modulation darstellt, wurde im ersten Abschnitt der Arbeit das Wanderungsverhalten von CD4+ T-Zellen analysiert. Wir konnten in der Intravitalmikroskopie und im Homing-Versuch mit radioaktiver Markierung zeigen, dass aktivierte CD4+ T-Zellen in die Leber einwandern, während naive CD4+ T-Zellen in die sekundären lymphatischen Organe rezirkulieren. Dabei akkumulierten aktivierte CD4+ T-Zellen, vermutlich bedingt durch eine differentielle endotheliale Rezeptorausstattung im Leberläppchen, nahezu ausschließlich im Periportalfeld. Auch Th1-Zellen und Th2-Effektor-Zellen fanden sich anders als naive CD4+ T-Zellen präferentiell in der Leber. Sie verteilten sich allerdings überall im Leberläppchen. Th1-Zellen wurden dabei stärker als Th2-Zellen zurückgehalten, was die Bedeutung des Zytokinphänotyps für die Rekrutierung unterstreicht. Neben der Aktivierung, der Differenzierung in Effektor/Memory-Zellen und dem Zytokinphänotyp spielt auch die Präsenz des Antigens selbst zumindest für die Rekrutierung von CD8+ T-Zellen in der Leber eine Rolle. Allerdings ist die TCR-MHC-Bindung alleine nicht ausreichend. Die zusätzliche Interaktion von LFA-1 und ICAM-1 stellt eine notwendige Voraussetzung dar. Im Gegensatz zu CD8+ T-Zellen ist es uns dagegen nicht gelungen, für die Rekrutierung von aktivierten oder CD4+ Effektor-T-Zellen in die gesunde Leber essentielle Adhäsionsmoleküle zu identifizieren. Ein möglicher Grund ist, dass viele Rezeptor-Ligand-Paare redundant oder im Leberläppchen differentiell exprimiert sind. Insgesamt zeigten T-Zellen, die in der intrahepatischen Lymphozytenpopulation stark repräsentiert sind, eine präferentielle Rekrutierung. Wir formulierten daher das Modell der Balancierten Rekrutierung. Es postuliert, dass die intrahepatische Lymphozytenpopulation kontinuierlich durch Immigration, Emigration und intrahepatische Modulation bzw. Deletion geformt wird. Es ist noch nicht abschließend geklärt, ob und durch welche Signale Antigen-erfahrene Zellen moduliert werden können. Im zweiten Teil der Arbeit wurde daher untersucht, welches in vivo Schicksal Effektor-Zellen spontan, unter immunoger oder tolerogener Antigenzufuhr haben. Adoptiv transferierte in vitro-polarisierte Th1-Zellen zeigten spontan eine zeitabhängige Abnahme des Prozentsatzes IFNγ+ Zellen. Da Populationen mit höherer (90%) und niedrigerer (50%) IFNγ+ Zytokinproduzentenfrequenz in vivo identisch überlebten und Nicht-Produzenten keine stärkere Zellteilung aufwiesen, nehmen wir an, dass die Zytokinsynthese auf Einzelzellebene herunterreguliert wird und die Zellen zu einem „quasi-naiven“ Phänotyp zurückkehren. Mittels adoptivem Transfer IFNγ+ ex vivo-sortierter Th1-Zellen konnten wir darüber hinaus nachweisen, dass diese Zellen in der Lage sind, Immunität gegen einen Tumor zu vermitteln. Das TCM/TEM-Paradigma postuliert eine Memory-Zellgeneration aus IFNγ- Effektor-Zellen bzw. TCM wogegen IFNγ+ Zellen als TEM terminal differenziert sind und in vivo zugrunde gehen. Unsere Daten sprechen eher für die Alternativhypothese die besagt, dass Effektor- Zellen temporär ihre Zytokinsynthese einstellen und zu Memory-Zellen differenzieren können. Nach adoptivem Transfer von Th1- und Th2-Zellen und tolerogener Antigenapplikation erfolgte nach einer Expansionsphase eine Zunahme apoptotischer Zellen in der Leber, die für eine intrahepatische Deletion spricht. Während Th1-Zellen eine Abnahme der IFNγ+ Subpopulation zeigten, persistierten IL-4+ Th2-Zellen vor allem in der Leber. Eine Immundeviation ließ sich in diesem Modell nicht induzieren. Die in vitro-Ko- Kultur erbrachte Hinweise, dass die Leber möglicherweise aktiv partizipiert: LSEC förderten das Wachstum von Th2-Zellen, wogegen Th1-Zellen weniger stark expandierten. Die Akkumulation von Zellen, die Zeichen der Apoptose zeigen, in der Leber hat zu der Vermutung Anlass gegeben, dass die Leber passiv sterbende Zellen abräumt. Dagegen gibt es Evidenzen, dass in der Leber selbst auch Apoptose bei aktivierten T-Zellen über Fas/Fas-Ligand-Interaktion induziert werden kann. Unserer Daten legen zudem die Annahme nahe, dass die Leber möglicherweise sogar Antigen-spezifisch T-Zellen modulieren kann. Zusammenfassend formulieren wir ein Modell, das eine Kompartimentalisierung des Immunsystems zugrunde legt: während naive CD4+ T-Zellen in lymphatische Organe migrieren und dort immunogene bzw. tolerogene Signale empfangen, werden Antigen-erfahrenen CD4+ T-Zellen in die Leber rekrutiert, wo sie durch Interaktion mit APC moduliert oder deletiert werden. Man könnte spekulieren, dass eine gestörte hepatische Deletion von Effektor-Zellen beispielsweise über eine Aktivierung von tolerogenen Leber-APC zu einem immunogenen Phänotyp zu einem hepatischen Priming von naiven T-Zellen führen könnte. Diese Vorgänge könnten relevant sein für die Entstehung oder fehlende Suppression von Autoimmunität z.B. bei Autoimmunhepatitis oder systemischer Entzündungsvorgänge.The recruitment of T cells is crucial fort the compartmentalization of the immune system: antigen presentation and activation is induced in secondary lymphoid organs. Effector functions are exerted within inflamed tissues. The mucosa and presumably also in the liver have immunomodulatory functions. By intravital microscopy and in vivo-homingassay we demonstrated that activated CD4+ T cells are preferentially recruited into the liver whereas naive CD4+ T cells recirculate through secondary lymphoid organs. Due to distinct receptor expression within the liver vasculature, activated CD4+ T cells accumulated within the periportal area of the liver lobulus. Th1 and Th2 cells that were also retained at higher numbers compared to naive CD4+ T cells, displayed a scattered distribution within the liver lobulus. More Th1 cells as Th2 cells migrated into the liver underlining the influence of the cytokine phenotype. Apart from activation, memory cell differentiation and cytokine phenotype, the antigen plays a role for recruitment into the liver as demonstrated for CD8+ T cells. However, interaction of the MHC-antigen-complex and the T cell receptor also is not sufficient, LFA-1 and ICAM-1 seem to be mandatory. In contrast to CD8+ T cells, no liver-homing molecules could be identified for CD4+ T cells under homeostatic conditions which might be due to redundant receptor/ligand pairs or differential expression within the lobulus. In summary, T cells that are highly represented within the intraheptic lymphoid population became preferentially recruited suggesting that the intrahepatic T cell pool is shaped by immigration, emigration and intrahepatic modulation, described as “balanced recruitment”. It is still elusive to what extent and by which mechanisms antigen-experienced T cells can be modulated in vivo. Adoptively transferred in vitro-polarized Th1 cells displayed a spontaneous decrease in IFNγ+ cells. Since Th1 cells with a high or a low IFNγ-expression displayed equal survival and proliferation, Th1 cells seem to downregulate their cytokine synthesis. We could also demonstrate that ex vivo-sorted IFNγ+ Th1 cells could reject tumors suggesting that cytokine-producing effector cells can fully differentiate into memory cells instead of undergoing apoptosis as terminal differentiation state. Adoptively transferred Th1 cells underwent apoptosis within the liver after tolerogenic antigen administration whereas Th2 cells preferentially survived suggesting that the liver might participate in systemic tolerance induction by deletion of effector cells. Taken together, the immune system is compartimentalized: naive CD4+ T cells recirculate through secondary lymphoid organs receiving tolerogenic or immunogenic signals whereas effector/memory cells become recruited into parenchymal organs such as the liver undergoing modulation or deletion by interaction with local antigen- presenting cells. It is tempting to speculate that lacking intrahepatic deletion of effector cells might contribute to the pathogenesis of autoimmunity

Enhanced T cell transmigration across the murine liver sinusoidal endothelium is mediated by transcytosis and surface presentation of chemokines

Transmigration through the liver endothelium is a prerequisite for the homeostatic balance of intrahepatic T cells and a key regulator of inflammatory processes within the liver. Extravasation into the liver parenchyma is regulated by the distinct expression patterns of adhesion molecules and chemokines and their receptors on the lymphocyte and endothelial cell surface. In the present study, we investigated whether liver sinusoidal endothelial cells (LSEC) inhibit or support the chemokine-driven transmigration and differentially influence the transmigration of pro-inflammatory or anti-inflammatory CD4(+) T cells, indicating a mechanism of hepatic immunoregulation. Finally, the results shed light on the molecular mechanisms by which LSEC modulate chemokine-dependent transmigration. LSEC significantly enhanced the chemotactic effect of CXC-motif chemokine ligand 12 (CXCL12) and CXCL9, but not of CXCL16 or CCL20, on naive and memory CD4(+) T cells of a T helper 1, T helper 2, or interleukin-10-producing phenotype. In contrast, brain and lymphatic endothelioma cells and ex vivo isolated lung endothelia inhibited chemokine-driven transmigration. As for the molecular mechanisms, chemokine-induced activation of LSEC was excluded by blockage of G(i)-protein-coupled signaling and the use of knockout mice. After preincubation of CXCL12 to the basal side, LSEC took up CXCL12 and enhanced transmigration as efficiently as in the presence of the soluble chemokine. Blockage of transcytosis in LSEC significantly inhibited this effect, and this suggested that chemokines taken up from the basolateral side and presented on the luminal side of endothelial cells trigger T cell transmigration. CONCLUSION: Our findings demonstrate a unique capacity of LSEC to present chemokines to circulating lymphocytes and highlight the importance of endothelial cells for the in vivo effects of chemokines. Chemokine presentation by LSEC could provide a future therapeutic target for inhibiting lymphocyte immigration and suppressing hepatic inflammation

Chemokine expression and internalization by LSEC.

<p>(A) Chemokine mRNA expression was quantified in resting and TNF-α/IFN-γ activated LSEC in relation to GAPDH. (B) LSEC layer were incubated with fluorochrome-labeled CXCL10 or CXCL12 at 37°C or 4°C. Histograms show chemokine uptake determined by flow cytometry. Filled graph, no chemokine at 37°C; thin line overlapping with filled graph, chemokine incubation at 4°C; bold line, chemokine incubation at 37°C. (C) Diagram shows fold increase of geometric mean fluorescence intensity (GMFI) of LSEC incubated with chemokine in relation to GMFI without chemokine incubation at 37°C. Mean values ± SD of 4 independent experiments are shown. (D) LSEC were incubated with fluorochrome-labeled CXCL10 or CXCL12 added to the lower chamber of the transwell for 30 min. Nuclei were stained with DAPI. Representative images of three independent experiments are shown. Bars represent 10 μm. Mean values ± SD of 2–4 independent experiments are shown. ** p< 0.01.</p

Hepatic accumulation of CXCR3⁺ CD4⁺ T cells during T cell-mediated hepatitis after administration of CPZ.

<p>Mice were treated with Con A and received CPZ 60 min after hepatitis induction. (A) Liver samples were stained with anti-CD3 antibody 24 h after hepatitis induction. Portal CD3⁺ T-cell numbers were counted per hpf. Arrows indicate T cells. Representative images and medians of two independent experiments with 5 mice per group. Bars represent 50 μm. (B) NPC were isolated 24 h after hepatitis induction, stained with anti-CD4 and anti-CXCR3 antibody and analyzed by flow cytometry. Representative dot plots and medians of two independent experiments with 3–4 mice per group. * p< 0.05; ** p< 0.01; *** p< 0.001.</p