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.

Connecting liver and gut: murine liver sinusoidal endothelium induces gut tropism of CD4+ T cells via retinoic acid.

UNLABELLED: Gut-activated T cells migrating into the liver can cause extraintestinal manifestations of inflammatory bowel disease. T cells acquire a gut-homing phenotype dependent on retinoic acid (RA) provided by intestinal dendritic cells (DC). We investigated whether liver antigen-presenting cells can induce gut tropism supporting an enterohepatic lymphocyte circulation. Priming of CD4(+) T cells by liver sinusoidal endothelial cells (LSEC) supported migration into gut and gut-associated lymphoid tissue. As observed for T cells primed by intestinal DCs, this gut tropism depended on α(4) ß(7) integrin and CC chemokine receptor 9 (CCR9) expression by LSEC-primed CD4(+) T cells. The induction of gut-homing molecules was mediated by RA, a derivate of vitamin A that is stored in large amounts within the liver. LSECs expressed functional retinal dehydrogenases and could convert vitamin A to RA. Conversely, the lack of signaling via the RA receptor prevented the expression of α(4) ß(7) integrin and CCR9 on LSEC-primed CD4(+) T cells, consequently reducing their in vivo migration to the intestine. Other liver antigen-presenting cells failed to support high expression of α(4) ß(7) integrin on CD4(+) T cells, thus, the potential to induce gut homing is restricted to LSECs. CONCLUSION: The capacity to promote gut tropism via vitamin A use is not unique for intestinal DCs but is also a feature of LSECs. Our data support the assumption that CD4(+) T cells can migrate from the liver to the gut as one branch of a postulated enterohepatic lymphocyte circulation.

Failure of CD4 T-cells to respond to liver-derived antigen and to provide help to CD8 T-cells.

CD4 T-cell help is required for the induction of efficient CD8 T-cells responses and the generation of memory cells. Lack of CD4 T-cell help may contribute to an exhausted CD8 phenotype and viral persistence. Little is known about priming of CD4 T-cells by liver-derived antigen. We used TF-OVA mice expressing ovalbumin in hepatocytes to investigate CD4 T-cell priming by liver-derived antigen and the impact of CD4 T-cell help on CD8 T-cell function. Naïve and effector CD4 T-cells specific for ovalbumin were transferred into TF-OVA mice alone or together with naïve ovalbumin-specific CD8 T-cells. T-cell activation and function were analyzed. CD4 T-cells ignored antigen presented by liver antigen-presenting cells (APCs) in vitro and in vivo but were primed in the liver-draining lymph node and the spleen. No priming occurred in the absence of bone-marrow derived APCs capable of presenting ovalbumin in vivo. CD4 T-cells primed in TF-OVA mice displayed defective Th1-effector function and caused no liver damage. CD4 T-cells were not required for the induction of hepatitis by CD8 T-cells. Th1-effector but not naïve CD4 T-cells augmented the severity of liver injury caused by CD8 T-cells. Our data demonstrate that CD4 T-cells fail to respond to liver-derived antigen presented by liver APCs and develop defective effector function after priming in lymph nodes and spleen. The lack of CD4 T-cell help may be responsible for insufficient CD8 T-cell function against hepatic antigens.

Priming of CD4+ T cells by liver sinusoidal endothelial cells induces CD25low forkhead box protein 3- regulatory T cells suppressing autoimmune hepatitis.

UNLABELLED: Elucidating cellular mechanisms that maintain the intrahepatic immune balance is crucial to our understanding of viral or autoimmune liver diseases and allograft acceptance. Liver sinusoidal endothelial cells (LSECs) play an important role in modifying local immune responses to tolerance in major histocompatibility complex (MHC) I-restricted models, whereas their contribution in the MHCII context is still controversial. In an MHCII chimeric mouse model that excludes MHCII-mediated antigen presentation by professional antigen-presenting cells, we demonstrated that LSECs prime CD4(+) T cells to a CD45RB(low) memory phenotype lacking marker cytokine production for effector cells that was stable in vivo following immunogenic antigen re-encounter. Although these cells, which we term T(LSEC), had the capacity to enter lymph nodes and the liver, they did not function as effector cells either in a delayed-type hypersensitivity reaction or in a hepatitis model. T(LSEC) inhibited the proliferation of naïve CD4(+) T cells in vitro although being CD25(low) and lacking expression of forkhead box protein (FoxP)3. Furthermore, these cells suppressed hepatic inflammation as monitored by alanine aminotransferase levels and cellular infiltrates in a T cell-mediated autoimmune hepatitis model in vivo. CONCLUSION: T(LSEC) first described here might belong to the expanding group of FoxP3(-) regulatory T cells. Our findings strengthen the previously discussed assumption that CD4(+) T cell priming by nonprofessional antigen-presenting cells induces anti-inflammatory rather than proinflammatory phenotypes. Because recruitment of CD4(+) T cells is increased upon hepatic inflammation, T(LSEC) might contribute to shifting antigen-dependent immune responses to tolerance toward exogenous antigens or toward endogenous self-antigens, especially under inflammatory conditions.

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

UNLABELLED: 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.

Sustained delayed-type hypersensitivity reaction after in vivo priming but successful induction of unresponsiveness after adoptive transfer of CD4+ effector T cells.

Potential reasons for weak effects of oral tolerance in the primed immune system are still under discussion. In the present study, impacts of oral antigen uptake were studied in adoptive transfer models using T cell receptor transgenic CD4(+) T cells allowing analysis of antigen-specific donor cells on single cell level. After in vivo priming and subsequent feeding, an antigen-specific delayed-type hypersensitivity reaction was sustained. Concomitantly, donor cells preferentially found in the draining lymph nodes remained at equal numbers. In contrast, adoptively transferred Th1 cells that migrated preferentially into spleen and liver became fewer upon feeding accompanied by a suppressed delayed-type hypersensitivity reaction. Thus, antigen-experienced cells did not seem to become generally resistant to tolerogenic stimuli. Our data suggest that besides a permanent inflammatory stimulus provided by the persisting antigen, diverse tissue distribution of in vivo-induced compared to adoptively transferred effector cells might interfere with oral tolerance in the experienced immune system.

In vivo modulation of antigen-experienced cells in response to high-dose oral antigen: deletion but no evidence for alterations in the cytokine phenotype.

Whether also antigen-experienced CD4(+) T cell populations undergo modulations upon oral antigen uptake supporting systemic unresponsiveness is still not fully understood. Using an adoptive transfer model with chicken ovalbumin (OVA)-specific T cells, we demonstrated that absolute numbers of transferred ex vivo-isolated CD4(+) memory T cells and in vitro-polarized T(h)1 cells considerably decrease within spleen and liver upon repetitive OVA feeding. As a consequence, these mice did not mount a delayed-type hypersensitivity reaction after OVA challenge. OVA-specific T(h)1 cells re-isolated from the liver showed augmented signs of apoptosis. However, there was no evidence that transferred effector or memory T cells acquired a regulatory phenotype, became anergic or underwent immune deviation. Our data suggest that oral antigen application does not induce alterations in the phenotype of CD4(+) effector and memory T cells. Instead, deletion of antigen-experienced CD4(+) T cells preferentially within the liver might be a major mechanism contributing to antigen-specific systemic unresponsiveness upon oral antigen uptake.

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.

Differential priming of CD8 and CD4 T-cells in animal models of autoimmune hepatitis and cholangitis.

UNLABELLED: The pathogenesis of autoimmune liver diseases is poorly understood. Animal models are necessary to investigate antigen presentation and priming of T-cells in the context of autoimmunity in the liver. Transgenic mouse models were generated in which the model antigen ovalbumin is expressed in hepatocytes (TF-OVA) or cholangiocytes (ASBT-OVA). Transgenic OT-I (CD8) or OT-II (CD4) T-cells specific for ovalbumin were adoptively transferred into TF-OVA and ASBT-OVA mice to induce in vivo priming of antigen-specific T-cells. T-cell migration and activation, as well as induction of liver inflammation, were studied. OT-I T-cells preferentially located to the liver of both mouse strains whereas no migration of OT-II T-cells to the liver was observed. OT-I T-cells proliferated in the liver of TF-OVA mice and the liver and liver draining lymph nodes of ASBT-OVA mice. OT-II CD4 T-cells were activated in spleen and liver draining lymph node of TF-OVA mice but not in ASBT-OVA mice. Transfer of OT-I T-cells led to histologically distinct inflammatory conditions in the liver of ASBT-OVA and TF-OVA mice and caused liver injury as determined by the elevation of serum alanine aminotransferase. CONCLUSION: An antigen expressed in hepatocytes is presented to CD8 and CD4 T-cells, whereas the same antigen expressed in cholangiocytes is presented to CD8 but not CD4 T-cells. In both models, activation of CD8 T-cells occurs within the liver and causes liver inflammation. The models presented here are valuable to investigate the priming of T-cells in the liver and their role in the development of autoimmune disease of the liver.

Adoptively transferred Th1 cell populations lose IFNgamma+ cells by cytokine down-regulation on single-cell level.

Against the background of effector T cell heterogenity in terms of their in situ cytokine expression, IFNgamma production has been argued to define distinct Th1 lineages: whereas IFNgamma- Th1 cells survive and differentiate in vivo, IFNgamma+ Th1 cells eventually undergo apoptosis. Alternatively, lineage commitment might not be directly associated with the actual IFNgamma production. To address this issue, we adoptively transferred in vitro-polarized Th1 cell populations. Although absolute numbers of total Th1 cells after 3 days in vivo remained unchanged, numbers of IFNgamma+ within the Th1 cells declined by approximately 50%. This was not affected by the initial frequencies of IFNgamma+ cells within the transferred Th1 cell populations and by the presence of the antigen. Arguing against positive selection of IFNgamma non-producers in vivo, cell division rates of IFNgamma+ and IFNgamma- Th1 cells were comparable. Our data suggest that the 'loss' of IFNgamma+ cells within the transferred Th1 cell population might be rather caused by down-regulation of the cytokine expression on single-cell level than by deletion of individual IFNgamma+ cells. Thus, our findings are more in line with the hypothesis that actual cytokine expression does not define distinct differentiation states and polarization-specific genes remain accessible also in IFNgamma- Th1 effector cells.

Early intrahepatic antigen-specific retention of naïve CD8+ T cells is predominantly ICAM-1/LFA-1 dependent in mice.

We have previously shown that naïve CD8+ T cells recognizing their cognate antigen within the liver are retained and undergo activation in situ, independent from lymphoid tissues. Intrahepatic primary T cell activation results in apoptosis and may play a crucial role in the ability of the liver to induce tolerance. Although adhesion molecules required for intrahepatic retention of T cells that have undergone previous extra-hepatic activation have been characterized, adhesive interactions involved in selective antigen-dependent intrahepatic retention of naïve CD8+ T cells have not been investigated. By adoptively transferring radiolabeled T cell receptor (TCR)-transgenic CD8+ T cells into recipient animals ubiquitously expressing the relevant antigen, we show that 40% to 60 % of donor antigen-specific naïve CD8+ T cells were retained in the liver within 1 hour after transfer, despite ubiquitous expression of the antigen. Intravital microscopy showed that most donor naïve T cells slowed down and were irreversibly retained intrahepatically within the first few minutes after adoptive transfer, strongly suggesting that they were directly activated by liver cells in situ. This process was largely dependent on LFA-1 and ICAM-1, but was independent of blocking with antibodies against VCAM-1, alpha4 integrin, P-selectin, VAP-1, and beta1 integrin. ICAM-2 seemed to play only a minor role in this process. Interestingly, LFA-1 expressed by both donor T cells and liver cells was involved in retention of the antigen-reactive T cells. In conclusion, LFA-1-dependent intrahepatic T cell retention and activation are linked events that may play a crucial role in the establishment of liver-induced antigen-specific tolerance.

The composition of intrahepatic lymphocytes: shaped by selective recruitment?

Intrahepatic lymphocytes have a distinct subset composition and phenotype. Compared with lymphoid tissues, the frequency of natural killer (NK) cells, NKT cells and gammadelta T cells among total lymphocytes is increased within the liver, and alphabeta T cells are predominantly effector/memory cells. Divergent hypotheses on the origin of intrahepatic T cells have emerged to explain this; in these hypotheses, either local development or selective recruitment of cells into the liver dominates. This Opinion highlights findings showing that the migratory preferences of lymphocyte subsets reflect their representation within the liver surprisingly well, suggesting that the composition of intrahepatic lymphocytes, in the absence of inflammation, is largely shaped by the dynamics of cell entry and exit into and from the liver.

The spectrum of lymphoid subsets preferentially recruited into the liver reflects that of resident populations.

Intrahepatic lymphocytes (IHL) differ phenotypically from cells found in the peripheral blood or in lymphoid organs. The liver contains T-cells that are also found in lymphoid organs but a higher proportion of these T-cells compared to those in lymphoid organs express activation or memory markers and very few naïve T-cells are present within the liver. Furthermore, subsets such as NK and NK T-cells, which are detected at comparably lower levels within the lymphoid organs are increased within the liver. To investigate whether a preferential recruitment of certain lymphoid subsets from the circulation contributes to the composition of intrahepatic lymphocytes, we compared their frequency in the liver with their organ tropisms. CFSE-labeled murine lymphoid cells were injected intravenously and their distribution within liver and spleen was analyzed after 24 h. Especially CD45RB(low) memory T-cells, NK and NK T-cells, which are also present at high proportions within IHL, became predominantly recruited into the liver. In contrast, subsets such as naïve CD62L(high) T-cells and B-cells, which are predominantly represented within the lymphoid organs, preferentially migrated into the spleen. These findings indicate that the pattern of migratory preferences reflects the representation of various subsets within the intrahepatic lymphocytes surprisingly well, suggesting that the composition of intrahepatic lymphocytes is largely shaped by the dynamics of entry and exit of cells into the organ.

Immunomodulatory effects of the liver: deletion of activated CD4+ effector cells and suppression of IFN-gamma-producing cells after intravenous protein immunization.

The liver is tolerogenic in many situations, including as an allograft and during the response to allogeneic MHC expressed on hepatocytes. The majority of data that address this issue focus on endogenous Ags. Little is known about CD4(+) T cells and their fate under tolerizing conditions, especially with respect to fully differentiated CD4(+) effector T cells. In this study, we used the adoptive transfer of populations of TCR-transgenic CD4(+) T cells, skewed toward the Th1 or Th2 phenotype, to test whether either apoptotic or immune deviation mechanisms apply to cytokine-producing CD4(+) T cells that enter the liver. After transfer, Th1 and Th2 cells could be detected up to 25 days in lymphoid organs and the liver. Intravenous high dose Ag application resulted in accumulation, proliferation, and subsequent deletion of effector cells within the liver. Th1 cells lost their capacity to produce cytokines, whereas IL-4 expression was sustained within Th2 cells from the liver. However, there was no evidence for a deviation of Th1-programmed cells toward a Th2 (IL-4) or regulatory T cell (IL-10) pattern of cytokine expression. We used isolated populations of liver-derived APCs to test whether the liver had the capacity to impose a bias toward IL-4 expression in T cells. These experiments showed that liver sinusoidal endothelial cells selectively suppress the expansion of IFN-gamma-producing cells, yet they promote the outgrowth of IL-4-expressing Th2 cells, creating an immune suppressive milieu within this organ. These data suggest that presentation of Ags in the liver leads to modulation of immune response in terms of quantity and quality.

Differentiation-dependent and subset-specific recruitment of T-helper cells into murine liver.

It has been suggested that the liver traps and deletes activated and potentially harmful T cells, especially of the CD8(+) subset, providing mechanisms to limit systemic immune responses. It is unknown whether this also applies to CD4(+) T cells. In this study, we show that activated stages of CD4(+) T cells were trapped in the liver on intraportal injection. Intravital microscopy showed an immediate adhesion of activated CD4(+) T cells within periportal sinusoids after intraportal injection. Furthermore, we detected high frequencies of interferon gamma (IFN-gamma)-- (Th1) and interleukin 4 (IL-4)-- (Th2) synthesizing effector cells in the liver. Transfer experiments were performed to identify those phenotypes showing specific retention in the liver. Our data show that effector stages and activated cells in general are more efficiently recruited into the liver than resting CD4(+) T cells, similar to what has previously been shown for CD45RB(low) memory cells. In addition, we observed a certain preference for Th1-polarized cells to be trapped by the liver. However, the actual cytokine-producing cells did not specifically enrich among the total population. In conclusion, these data indicate that the liver acts as a filter for activated and memory/effector cells. Cells trapped in the liver might subsequently undergo modulatory influences exerted by the postulated specific microenvironment of the liver.