SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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 CD45RBlow 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 TLSEC, 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. TLSEC inhibited the proliferation of naïve CD4+ T cells in vitro although being CD25low 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: TLSEC 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, TLSEC might contribute to shifting antigen-dependent immune responses to tolerance toward exogenous antigens or toward endogenous self-antigens, especially under inflammatory conditions. (HEPATOLOGY 2009.)

Under physiological conditions, the liver seems rather to support the induction of peripheral tolerance than to establish immunity.1 In particular, liver sinusoidal endothelial cells (LSECs) are believed to shift the hepatic immune balance toward tolerance by presenting major histocompatibility complex (MHC) I–restricted antigens, thereby inducing anergy and tolerance.2, 3 LSECs constitutively express MHCII and costimulatory molecules4 and take up exogenous antigens.2 Naïve CD4+ T cells migrate into the liver5, 6 and interact with LSECs.7 This raises the question whether LSECs also prime CD4+ T cells. Coculturing naïve CD4+ T cells with LSECs resulted in stimulation of the T cells.8 Another recent study using LSEC preparations depleted of cells of hematopoietic origin has created doubts about the priming capacity of LSECs for naïve CD4+ T cells in the absence of professional antigen-presenting cells (APCs).9 Priming of naïve CD4+ T cells by other nonprofessional APCs such as aortic endothelial cells or naïve B cells induces distinct T cell populations lacking cytokine production and displaying suppressive capacities.10, 11 In murine models of hepatic antigen expression, specific CD4+ T cells could acquire either a CD25high forkhead box protein (FoxP)3+ regulatory phenotype12 or an anergic phenotype with effector functions upon antigen re-encounter.13

In the present study, we investigated the phenotype of CD4+ T cells primed by LSECs (TLSEC) using bone marrow chimeric mice expressing MHCII exclusively on nonhematopoietic cells (MHCII chimeras) to exclude the effects of professional APCs. Furthermore, we studied the in vivo stability of the TLSEC phenotype and the contribution of TLSEC to immunity or suppression of inflammation in a delayed-type hypersensitivity (DTH) reaction and a transfer model for autoimmune hepatitis.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Animals.

Female wild-type (WT) C57BL/6, CD90.1+, chicken ovalbumin (OVA) T cell receptor transgenic OT-II mice14 and OT-I mice15 were obtained from the Bundesamt für Risikobewertung (Berlin, Germany). MHCII−/− mice16, 17 were bought from Taconic (Germantown, NY). TF-OVA mice expressing membrane-bound OVA in hepatocytes18 were bred at the Charité Campus Benjamin Franklin animal facility. All animals received humane care according to the criteria published by the National Institutes of Health (Bethesda, MD).

MHCII Chimeras.

WT recipient mice were lethally irradiated and received a single intravenous injection of 3 to 5 × 106 MHCII−/− bone marrow cells. Starting 1 week prior and ending 4 weeks after transplantation, bone marrow recipient mice continuously received 0.4% enrofloxacin (Bayer, Leverkusen, Germany). After 6 weeks, MHCII and CD4 expression on blood cells was checked by flow cytometry to confirm the reconstitution with MHCII−/− bone marrow. Within the livers of MHCII chimeras CD146+ LSECs remained MHCII+, whereas recipient APCs of hematopoietic origin were replaced by MHCII−/− donor cells (Supporting Fig. 1). LSECs were isolated from MHCII chimeras 8 and 10 weeks after bone marrow transplantation.

Preparation of LSECs, Spleen APCs, and T Cell Populations.

LSECs were isolated ex vivo using anti-CD146 as described,19 resulting in 96.35 ± 0.80% purity as determined by uptake of 1 μg/mL acetylated low-density lipoprotein-Bodipy (Molecular Probes, Leiden, The Netherlands).20 LSECs preparations contained 3.65 ± 0.80% of nonendothelial cells, including approximately 2% T cells, around 1% APCs and 0.13 ± 0.12% CD11c+ dendritic cells. Ex vivo sorting of LSECs from WT mice and MHCII chimeras resulted in comparable purity, phenotype, and vitality. LSECs from both WT and MHCII chimeras displayed no alterations in their MHCII expression and only a slight decrease in intercellular adhesion molecule-1 expression levels upon in vitro coculture with naïve OVA-specific CD4+ T cells (Supporting Fig. 2).

Spleens were disrupted and passed through a fine mesh, and CD90+ cells were depleted by magnetic cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany). Naïve CD4+ T cells were prepared from lymph nodes and spleens of OT-II mice by magnetic cell sorting, giving more than 99% CD4+ CD62Lhigh cells. Residual MHCII+ APCs within the T cell preparations were subsequently depleted. Prior to coculture experiments, T cells were labeled with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) (MoBiTec, Göttingen, Germany). CD8+ T cells were purified from lymph nodes and spleens of OT-I mice by way of magnetic cell sorting (Miltenyi Biotec).

In Vitro Coculture.

For in vitro priming, LSECs were cultured overnight and rinsed to remove dead cells. LSECs or CD90-depleted spleen APCs (1 × 106) were cocultured with 5 × 105 MHCII-depleted naïve CFSE-labeled CD4+ T cells in the presence of 5 μg/mL OVA peptide (Humboldt-Universität, Berlin, Germany). Experiments including LSECs from MHCII chimeras were set up in 96-well cell culture plates using 2 × 105 LSECs or spleen APCs and 1 × 105 CD4+ T cells. Cell culture reagents were from Sigma (Deisenhofen, Germany).

Intracellular Staining.

Cells were restimulated with phorbol myristate acetate and ionomycin,6 fixed in 2% paraformaldehyde (Sigma) and subsequently stained with directly labeled antibodies specific for interferon (IFN)-γ, interleukin (IL)-4, IL-10, IL-2, and for surface markers (all from BD Biosciences, Heidelberg, Germany). A FoxP3 staining kit was obtained from eBiosciences (San Diego, CA). Cells were analyzed using a FACSCalibur flow cytometer and the Cell-Quest Pro software (BD Biosciences).

In Vitro Suppression Assay.

Suppression assays were performed as described.21 Briefly, 1 × 105 OVA-specific CD4+ T cells, consisting of suppressor or control and CFSE-labeled naïve CD25-depleted responder cells at various ratios, were cultured with 2 × 105 irradiated CD90-depleted spleen APCs in the presence of 1 μg/mL anti-CD3. Cells were harvested at day 3, counterstained for surface markers, and the proliferation of responder cells was quantified by flow cytometric determination of CFSE mean fluorescence intensity.22

In Vivo Homing Assay.

Radioactively labeled cells were applied to quantify organ-specific distribution of total injected cells as described.5 Cells (2 × 107) were labeled with 20 μCi 51Cr (GE Healthcare-Buchler, Braunschweig, Germany) as outlined and defined numbers were injected into syngeneic mice. After 24 hours of migration, animals were killed and a panel of organs removed. The specific radioactivity of these organs and of the remaining body was counted using a Wizard gamma counter (Wallac, Turku, Finland). Total recovered radioactivity mirrors the number of all viable donor cells, because 51Cr is released by dead cells and excreted through the kidneys. Thus, the percentage of organ-specific radioactivity in relation to the total recovered radioactivity reflects the percentage of cells that have migrated into the respective organs.

Adoptive Transfer and Induction of a DTH Reaction.

TLSEC or CD4+ T cells primed by spleen APCs (TSAPC) were harvested on day 6 of coculture and labeled with CFSE, and 3 to 6 × 106 cells were adoptively transferred into WT mice. One day after transfer, 250 ng OVA peptide in 5 μL incomplete Freund's adjuvant or the adjuvant alone were injected into the left or right hind footpad, respectively. Footpad swelling was determined 24, 48, and 72 hours after antigen application. After the last measurement, mice were killed and cells re-isolated from various organs were analyzed with flow cytometry. Donor cells bearing the congenic marker CD90.1 were distinguished from CD90.2-positive recipient cells by staining for CD4 and CD90.1.

OVA-Transgenic Hepatitis Model.

Antigen-dependent and T cell–mediated autoimmune hepatitis was induced in TF-OVA mice as described.18 OVA-specific CD8+ (4 × 106) and 4 × 106 naïve CD4+ T cells or TLSEC were adoptively transferred into TF-OVA mice. Serum samples drawn on the indicated days were stored at −20°C, and alanine aminotransferase (ALT) levels were subsequently quantified on a Roche modular analyzer (Grenzach-Wyhlen, Germany).

For histological analysis, liver samples were fixed for 24 hours with 4% paraformaldehyde and embedded in paraffin. Four-micrometer sections were stained with hematoxylin-eosin. Immunofluorescence was performed using frozen liver sections incubated with anti-CD8 (eBiosciences) and secondary Alexa Fluor 555–labeled anti-rat antibody (Invitrogen, Karlsruhe, Germany) followed by nuclear staining with 4',6-diamidino-2-phenylindole (Sigma). Sections were mounted with Fluoromount (DAKO, Glostrup, Denmark) and images were taken with a Zeiss Image Z1 microscope (Carl Zeiss MicroImaging, Heidelberg, Germany).

Data Analysis.

Data were analyzed and statistical significance was determined using SPSS (SPSS, Chicago, IL) and the GraphPad Prism 3 software for Macintosh (GraphPad Software, San Diego, CA). Mann-Whitney tests were performed unless indicated otherwise.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

CD4+ T Cells Primed by LSECs Acquire a Phenotype Lacking Production of Marker Cytokines for T Helper 1, T Helper 2, and IL-10–Producing Cells.

To determine whether LSECs are able to prime CD4+ T cells, CFSE-labeled naïve CD4+ T cells were cocultured on LSECs from WT or MHCII chimeras and proliferation was quantified from the incremental decrease of CFSE mean fluorescence intensity. LSECs from MHCII chimeras induced cell division in naïve CD4+ T cells, albeit at a significantly lower rate compared with LSECs from WT mice. No proliferation was observed in the absence of LSECs, ruling out priming by residual APCs within the T cell preparation (Fig. 1A). Priming by LSECs resulted in lower T cell numbers compared with cultures with professional spleen APCs for antigen presentation (3.1 ± 0.8 × 105). In addition and as a result of weaker proliferation, significantly fewer T cells were obtained from cultures with LSECs from MHCII chimeras compared with WT mice. Without additional APCs, only very few T cells survived, resulting in numbers significantly lower compared with cultures containing LSECs (Fig. 1B).

thumbnail image

Figure 1. Effect of LSECs from MHCII chimeras on the in vitro proliferation of naïve OVA-specific CD4+ T cells. LSECs from WT mice or MHCII chimeras were used to stimulate CFSE-labeled naïve OVA-specific CD4+ T cells. All cultures received 5 μg/mL OVA peptide. Cells were harvested at day 6. (A) T cell proliferation was determined by flow cytometry as the decrease in cellular CFSE content. Plots are representative of 7 to 10 independent experiments in duplicate or triplicate. The mean CFSE fluorescence intensity is indicated (upper panel). The relative decrease of the mean CFSE fluorescence intensity compared with undivided cells (dotted line) was determined (lower panel). The median values of 7 to 10 independent experiments are shown. *P < 0.05. ***P < 0.001. (B) Absolute numbers of CD4+ T cells harvested at day 6 of coculture were quantified. The median values of 7 to 10 independent experiments are shown. **P < 0.01.

Download figure to PowerPoint

Next, we analyzed the phenotype acquired by naïve OVA-specific CD4+ T cells that were primed by LSECs. After priming with OVA peptide presented by spleen APCs or LSECs from WT mice, about 40% of the CD4+ T cells produced IL-2. Because LSECs from the MHCII chimeras were significantly less efficient in the induction of IL-2–producing cells, residual professional APC might have been responsible for the effect in the wild-type setting. Spleen APCs generated substantial proportions of IFN-γ+ T helper 1 cells, whereas LSECs, irrespective of their origin, induced significantly fewer IFN-γ+ cells (Fig. 2A). Independently of the type of APCs in the coculture systems, the frequencies of IL-4+ and IL-10+ T cells were negligible, at about 0.2% and 0.3%, respectively (Fig. 2B). Taken together, in the absence of professional APCs, LSECs were indeed capable of inducing proliferation of naïve CD4+ T cells, resulting in cells, designated TLSEC, that did not produce marker cytokines for effector cells.

thumbnail image

Figure 2. Cytokine production of OVA-specific CD4+ T cells primed by LSECs from MHCII chimeras. LSECs from WT mice or MHCII chimeras were used to stimulate naïve OVA-specific CD4+ T cells. All cultures received 5 μg/mL OVA peptide and were harvested at day 6. Frequencies of (A) IL-2+ and IFN-γ+ or (B) IL-4+ and IL-10+ CD4+ T cells were determined by way of intracytoplasmic staining. Representative plots and mean values ± standard deviation from 5 to 7 independent experiments in duplicate or triplicate are shown. **P < 0.01.

Download figure to PowerPoint

TLSEC Suppress the Proliferation of Naïve CD4+ T Cells In Vitro Although Being CD25low and Lacking FoxP3 Expression.

The lack of cytokine production after priming by LSECs suggested that TLSEC might have acquired regulatory properties. Indeed, both TLSEC and ex vivo–isolated CD4+CD25high regulatory T cells (Treg) significantly suppressed the proliferation of naïve CD4+ responder T cells. TLSEC displayed a slightly weaker effect than Treg. Naïve CD4+ T cells had no suppressive capacities (Fig. 3).

thumbnail image

Figure 3. Proliferation of naïve CD4+ responder T cells in the presence of TLSEC. Naïve CFSE-labeled CD4+ T cells were stimulated by anti-CD3 and spleen APCs in the presence of increasing frequencies of TLSEC, ex vivo–isolated Treg, or naïve CD4+ T cells. After 3 days, cells were harvested and the proliferation of the responder T cells was determined according to the mean CFSE fluorescence intensity. Representative plots for four experiments with 10% suppressor cells (upper panel) are shown. Data from four independent experiments with triplicates were analyzed by stepwise backward linear regression (lower panel). *P < 0.05. ***P < 0.001.

Download figure to PowerPoint

To determine whether TLSEC belong to the CD25high FoxP3+ Treg population, Treg marker expression was analyzed. Naïve CD62L+ CD4+ T cells contained very few CD25high FoxP3+ cells. Ex vivo–isolated Treg coexpressed CD25 and FoxP3. TSAPC were CD25+ but FoxP3; thus, CD25 was predominantly up-regulated upon activation. TLSECdisplayed a comparably lower activation-dependent CD25 expression. The FoxP3+ subfraction was typically below 5% (Fig. 4) and 1.7% to 2.1% of the cells were αE-integrin+. In conclusion, TLSEC, although being CD25low FoxP3, displayed suppressive capacities in vitro.

thumbnail image

Figure 4. Expression of CD25 and FoxP3 by TSAPC and TLSEC. Naïve CD4+ T cells, ex vivo–isolated Treg, TSAPC, or TLSEC were stained for CD25 and FoxP3 expression and analyzed by multiparameter flow cytometry. Plots are representative of three independent experiments.

Download figure to PowerPoint

TLSEC Migrate into Lymphoid Organs, Liver, and Intestine.

As an important prerequisite for potential suppressive in vivo effects, tissue immigration of radioactively labeled TLSEC was analyzed in homing assays. TSAPC were predominantly found within the spleen and the liver, whereas only very few cells migrated into the lymph nodes or the intestine, displaying the typical migration pattern of effector T cells. In contrast, TLSEC were recruited into the liver, albeit at lower numbers compared with TSAPC, and into lymph nodes and the intestine (Fig. 5A)—an intermediate homing pattern between that of naïve and effector T cells. Tissue immigration is determined by adhesion molecules on T cells and their counterparts on endothelial cells. Thus, expression of lymph node and gut homing molecules was analyzed. Upon priming, both TSAPC and TLSEC acquired a CD45RBlow and CC motif chemokine receptor 7low memory phenotype. TSAPC partially down-regulated CD62L and displayed low expression of α4β7. In contrast, TLSEC were CD62Lhigh and up-regulated α4β7, presumably accounting for their preferential immigration into lymph nodes and intestine compared with TSAPC (Fig. 5B).

thumbnail image

Figure 5. Homing properties and expression of adhesion molecules by TSAPC and TLSEC. (A) Radioactively labeled TLSEC or TSAPC were injected into syngeneic mice, and after 24 hours organ-specific radioactivity reflecting the percentage of total donor cells that have migrated into an organ was determined. The mean values ± standard deviation of three independent experiments with 3 to 5 mice per group are shown. *P < 0.05. ***P < 0.001. (B) TSAPC or TLSEC were harvested at day 6, stained for CD45RB, CC motif chemokine receptor 7, CD62L, and α4β7, and were analyzed with flow cytometry. Plots are representative of three independent experiments. Thin line, negative control; thick line, specific antibody. The percentages of positive cells are given.

Download figure to PowerPoint

TLSEC Do Not Function as Effector Cells In Vivo and Display a Stable Phenotype After Immunogenic Subcutaneous Antigen Application.

As demonstrated by the in vivo homing assays, TLSEC entered peripheral lymph nodes and thus should be able to exert local effects after subcutaneous antigen application. To investigate the functional in vivo capacities of TLSEC and, secondly, to monitor the stability of their distinct phenotype, especially following immunogenic in vivo antigen re-encounter, a DTH reaction was used. OVA-specific TLSEC or TSAPC were adoptively transferred into WT mice and a DTH reaction was induced with OVA peptide. TSAPC supported a significant OVA-specific footpad swelling compared with the control footpad, whereas in the presence of equal numbers of TLSEC the animals did not mount an antigen-dependent DTH reaction (Fig. 6A). Both TLSEC and TSAPC equally proliferated upon antigen application within the OVA-draining popliteal lymph node but not in the control lymph node (Fig. 6B). To determine the phenotypic stability after immunogenic antigen re-exposure in vivo, effector cytokine expression of TLSEC re-isolated from various organs was analyzed with flow cytometry. As demonstrated by intracytoplasmic cytokine staining, TSAPC displayed a weak increase of their IFN-γ expression, whereas TLSEC maintained their overall low IFN-γ production within the spleen (data not shown) and draining lymph node (Fig. 6C). Taken together, in contrast to TSAPC, TLSEC, although they encountered their antigen in vivo, did not function as effector cells and displayed a stable IFN-γlow phenotype upon antigen rechallenge.

thumbnail image

Figure 6. OVA-specific DTH reaction, in vivo proliferation, and IFN-γ production of TSAPC or TLSEC. TLSEC or matching numbers of TSAPC were adoptively transferred. OVA peptide in incomplete Freund's adjuvant was injected into the left hind footpad the following day, and the adjuvant alone injected into the right hind footpad served as a control. (A) Footpad thickness was measured before and at several time points after injection. Footpad swelling is expressed as the ratio of the footpad thickness of the OVA-treated versus the respective control footpad (fold increase). Median of 8 to 9 mice from three independent experiments. *P < 0.05 compared with baseline ratio. (B) Three days after induction of a DTH reaction, the mice were killed, and donor cells were re-isolated from the control and the OVA-draining popliteal lymph node. Proliferation of CD90.1+ donor cells was quantified by incremental decrease in cellular CFSE content. (C) Percentages of IFN-γ+ cells within CD90.1+ donor cells were determined by way of intracytoplasmic cytokine staining. Data from one out of three independent experiments with 2 to 3 mice per group are shown.

Download figure to PowerPoint

TLSEC Suppress Hepatic Inflammation in an OVA-Specific Autoimmune Hepatitis Model.

TLSEC displayed suppressive capacities in vitro and phenotypic stability in vivo, and were capable of homing into the liver. These findings raised the question whether TLSEC support or suppress intrahepatic inflammation. These aspects were studied in an OVA-specific autoimmune hepatitis model using TF-OVA mice. In these animals expressing membrane-bound OVA within hepatocytes, adoptive transfer of OVA-specific CD8+ and naïve CD4+ T cells resulted in increased ALT levels, reflecting hepatic inflammation. In contrast, following simultaneous transfer of CD8+ T cells and TLSEC, ALT levels were significantly lower compared with the latter group, suggesting that TLSEC did not induce hepatitis (Fig. 7A), whereas TSAPC transferred together with CD8+ T cells amplified the disease (data not shown). In line with data from the DTH reaction, these findings show that TLSEC, in contrast to TSAPC, do not function as effector cells either in inflammation in the footpad or in the liver.

thumbnail image

Figure 7. Course of OVA-specific hepatitis reflected by ALT levels and histology after adoptive transfer of antigen-specific CD8+, naïve CD4+ T cells, and/or TLSEC. OVA-specific CD8+ and naïve CD4+ T cells, CD8+ T cells and TLSEC or CD8+, and naïve CD4+ T cells together with TLSEC were adoptively transferred into TF-OVA mice. (A) Serum ALT levels were determined at days 5 and 15 and normalized to the value at day 0. The mean values ± standard error of the mean from three independent experiments with 2 to 5 mice per group are shown. *P < 0.05. (B) Liver samples taken at day 5 were stained with hematoxylin-eosin (upper panel) or by way of immunofluorescence for CD8 (red, lower panel) and for the nuclei (blue, lower panel) (original magnification ×100). The images are representative of three independent experiments.

Download figure to PowerPoint

To further investigate whether TLSEC have the capacity to suppress intrahepatic inflammation, naïve OVA-specific CD8+ and CD4+ T cells were transferred to induce an inflammation, and TLSEC were injected in parallel. Under these conditions, the ALT increase was significantly lower compared with the group that did not receive TLSEC. At later time points, ALT levels dropped to base levels in both groups (Fig. 7A). Local infiltrations of mononuclear cells are another important parameter for T cell–mediated hepatic damage. Few scattered mononuclear cells were found within the liver parenchyma, and their numbers did not differ between groups (data not shown). In contrast, mononuclear cells were predominantly found in the periportal area, where their numbers were reduced upon additional transfer of TLSEC compared with mice that received CD8+ and naïve CD4+ T cells (Fig. 7B, upper panel). Few CD4+ T cells were detectable with immunofluorescence (data not shown), whereas periportal infiltrates consisted predominantly of CD8+ T cells (Fig. 7B, lower panel). Thus, TLSEC suppressed hepatic inflammation as monitored by periportal CD8+ T cell infiltration and the course of ALT levels in vivo.

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

LSECs, which represent a large, unique intrahepatic APC population, promote CD8+ T cell tolerance.2, 3 In contrast, the contribution of LSECs to intrahepatic CD4+ T cell priming and local immune regulation has been controversial.8, 9 Standard LSEC preparations contained small fractions of dendritic cells that could support proliferation even at very low numbers (data not shown). LSECs from MHCII chimera, used in the present study to exclude CD4+ T cell priming by professional APCs, induced proliferation and differentiation into CD45RBlow cytokine-negative TLSEC. LSEC preparations from WT mice promoted higher IL-2 production, resulting in increased proliferation and cell counts. However, residual professional APCs did not influence the phenotype of the TLSEC. Thus, our data support the assumption that not only do LSECs modulate CD8+ T cells as previously shown, but they can also participate in CD4+ T cell priming.

Recent publications have described the induction of regulatory T cells with unusual phenotypes. IL-10–secreting regulatory T cells do not express FoxP3 but have regulatory functions comparable to ex vivo–isolated CD25high Treg.23 Priming of CD4+ T cells by naïve B cells promotes development of TofB cells, which retain CD62L expression and suppress the proliferation of naïve CD4+ T cells in vitro and allograft rejection but remain FoxP3.11 Chronic antigen stimulation in vivo induces FoxP3 CD4+ T cells, known as dtg CD25 TR, which do not produce IL-2 or IFN-γ but exert regulatory functions on naïve CD4+ T cells in vitro and suppress a CD8+ T cell–mediated colitis.24 Indeed, similarly to IL-10+ regulatory T cells, TofB cells, and dtg CD25 TR cells, TLSEC displayed in vitro suppressive capacities although being CD25low, FoxP3, and αE-integrin. Unlike in vivo–differentiated anergic CD4+ T cells that do not have suppressive capacities and can be induced to produce effector cytokines,13 but like unusual regulatory T cells such as dtg CD25 TR,24 TLSEC displayed a stable phenotype after immunogenic antigen re-encounter in vivo. TLSEC remained IFN-γlow, and in contrast to IFN-γ+ TSAPCeffector cells they did not mediate a DTH reaction. Lacking effector function, in contrast to IFN-γ+ TSAPC, was not the consequence of lower cell numbers in the draining lymph nodes or reduced cell division. Against the background of these studies and of our own findings for TLSEC, FoxP3 seems not to be essential for the function of (induced) regulatory T cells as previously assumed.23–25 TLSEC expressed low levels of CD25 and secreted IL-2 but did not produce transforming growth factor-β, IL-5, IL-17 (data not shown), or IL-10. This argues against, for example, consumption of IL-2 as a mechanism of suppression, as discussed for FoxP3 IL-10 regulatory T cells.23 Pointing to a crucial role of direct cell–cell contact, in preliminary experiments TLSEC did not suppress responding T cell proliferation if separated by a transwell membrane (data not shown). As to the mechanisms of in vivo suppression of OVA-specific hepatitis, our data show that TLSEC reduced periportal infiltration by CD8+ T cells—the cell population responsible for tissue damage and concomitant ALT release by in vivo cytolysis of OVA-bearing cells in this model.18 Thus, the absence of an increase in ALT is most likely the consequence of reduced CD8+ T cell infiltration, and the suppressive in vivo effect of TLSEC might at least partially be due to an inhibition of CD8+ T cell immigration.

Our data showing that TLSEC do not mediate a DTH reaction in vivo nicely fit the recent finding that CD8+ T cells primed by LSECs likewise acquire a CD25low L-selectinhigh phenotype without effector cytokine production and without cytotoxic activity.26 This study describes B7-H1 as being crucial for tolerogenic CD8+ T cell maturation. In contrast, blocking experiments (Supporting Fig. 3) suggested that B7–H1 interactions do not play a major role in the differentiation of CD4+ TLSEC, whereas they are mandatory for CD8+ T cells. Activation of LSECs by bacterial lipoproteins and tumor necrosis factor-α–induced up-regulation of MHCII and of costimulatory molecules but did not alter the TLSEC phenotype (data not shown). It is tempting to speculate that priming of CD4+ T cells by nonprofessional APCs such as LSECs might generally lead to regulatory T cells with unusual phenotypes. However, the exact mechanisms of regulatory T cells induction still remain elusive.

Recently, evidence has been accumulating that migration patterns and tissue localization are important determinants of the in vivo function of regulatory T cells. TofB cells migrate like naïve CD4+ T cells, which are preferentially recruited into lymph nodes and, consequently, suppress the priming of T cells during immune responses in vivo.11 Naïve-like αE-integrin CD25+ Treg mainly recirculate through lymph nodes, whereas their memory-like αE-integrin+ counterpart efficiently enters inflamed sites27 and modulates established immune reactions.28 Corresponding to a higher CD62L expression compared with TSAPC and dendritic cells,11 TLSEC entered the lymph nodes, enabling them to exert effects on T cell priming. Importantly and in contrast to TofB and αE-integrin regulatory T cells, TLSEC also displayed a preference for intestine and liver. This distinct migration pattern is most likely the consequence of an up-regulation of α4β7. The α4β7 integrin is crucial for gut homing29 and α4-integrin has been demonstrated to be important for the recruitment of T helper 1 cells into the inflamed liver.30 In line with these homing capacities, TLSEC did indeed suppress intrahepatic inflammation as reflected by ALT levels and histology in an autoimmune hepatitis model.

In homeostasis, approximately 10% to 15% of naïve CD4+ T cells applied to the tail vein and up to 20% injected into the portal vein5, 6 become trapped within the liver. Under these conditions, tolerogenic priming of CD4+ T cells by LSECs presenting, for example, exogenous food antigens might occur but might nonetheless be a rare event. In contrast, in the inflamed liver leukocyte rolling and (antigen-dependent) adhesion is increased, particularly within the sinusoids.7, 31 Therefore, CD4+ T cell priming by LSECs resulting in FoxP3-suppressive T cells could contribute to the shift from an immune reaction to the maintenance of tolerance under inflammatory conditions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We are grateful to Dörte Huscher for support in statistical analysis; Uta Lauer and Heidi Hecker-Kia for excellent technical assistance with anti-CD146 hybridoma culture and antibody conjugation; and Balint Szilagyi for fruitful discussions on the role of α4β7 integrin.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • 1
    Racanelli V, Rehermann B. The liver as an immunological organ. HEPATOLOGY 2006; 43(Suppl): S54S62.
  • 2
    Limmer A, Ohl J, Kurts C, Ljunggren HG, Reiss Y, Groettrup M, et al. Efficient presentation of exogenous antigen by liver endothelial cells to CD8+ T cells results in antigen-specific T-cell tolerance. Nat 2000; 6: 13481354.
  • 3
    Limmer A, Ohl J, Wingender G, Berg M, Jungerkes F, Schumak B, et al. Cross-presentation of oral antigens by liver sinusoidal endothelial cells leads to CD8 T cell tolerance. Eur J Immunol 2005; 35: 29702981.
  • 4
    Knolle PA, Gerken G. Local control of the immune response in the liver. Immunol Rev 2000; 174: 2134.
  • 5
    Hamann A, Klugewitz K, Austrup F, Jablonski-Westrich D. Activation induces rapid and profound alterations in the trafficking of T cells. Eur J Immunol 2000; 30: 32073218.
  • 6
    Klugewitz K, Topp SA, Dahmen U, Kaiser T, Sommer S, Kury E, et al. Differentiation-dependent and subset-specific recruitment of T-helper cells into murine liver. HEPATOLOGY 2002; 35: 568578.
  • 7
    Wong J, Johnston B, Lee SS, Bullard DC, Smith CW, Beaudet AL, et al. A minimal role for selectins in the recruitment of leukocytes into the inflamed liver microvasculature. J Clin Invest 1997; 99: 27822790.
  • 8
    Knolle PA, Schmitt E, Jin S, Germann T, Duchmann R, Hegenbarth S, et al. Induction of cytokine production in naive CD4(+) T cells by antigen- presenting murine liver sinusoidal endothelial cells but failure to induce differentiation toward Th1 cells. Gastroenterology 1999; 116: 14281440.
  • 9
    Katz SC, Pillarisetty VG, Bleier JI, Shah AB, DeMatteo RP. Liver sinusoidal endothelial cells are insufficient to activate T cells. J Immunol 2004; 173: 230235.
  • 10
    Ma W, Pober JS. Human endothelial cells effectively costimulate cytokine production by, but not differentiation of, naive CD4+ T cells. J Immunol 1998; 161: 21582167.
  • 11
    Reichardt P, Dornbach B, Rong S, Beissert S, Gueler F, Loser K, et al. Naive B-cells generate regulatory T-cells in the presence of a mature immunological synapse. Blood 2007; 110: 15191529.
  • 12
    Luth S, Huber S, Schramm C, Buch T, Zander S, Stadelmann C, et al. Ectopic expression of neural autoantigen in mouse liver suppresses experimental autoimmune neuroinflammation by inducing antigen-specific Tregs. J Clin Invest 2008; 118: 34033410.
  • 13
    Knoechel B, Lohr J, Zhu S, Wong L, Hu D, Ausubel L, et al. Functional and molecular comparison of anergic and regulatory T lymphocytes. J Immunol 2006; 176: 64736483.
  • 14
    Barnden MJ, Allison J, Heath WR, Carbone FR. Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements. Immunol Cell Biol 1998; 76: 3440.
  • 15
    Hogquist KA, Jameson SC, Heath WR, Howard JL, Bevan MJ, et al. T cell receptor antagonist peptides induce positive selection. Cell 1994; 76: 1727.
  • 16
    Cosgrove D, Gray D, Dierich A, Kaufman J, Lemeur M, Benoist C, et al. Mice lacking MHC class II molecules. Cell 1991; 66: 10511066.
  • 17
    Grusby MJ, Johnson RS, Papaioannou VE, Glimcher LH. Depletion of CD4+ T cells in major histocompatibility complex class II-deficient mice. Science 1991; 253: 14171420.
  • 18
    Derkow K, Loddenkemper C, Mintern J, Kruse N, Klugewitz K, Berg T, et al. Differential priming of CD8 and CD4 T-cells in animal models of autoimmune hepatitis and cholangitis. HEPATOLOGY 2007; 46: 11551165.
  • 19
    Schrage A, Loddenkemper C, Erben U, Lauer U, Hausdorf G, Jungblut PR, et al. Murine CD146 is widely expressed on endothelial cells and is recognized by the monoclonal antibody ME-9F1. Histochem Cell Biol 2008; 129: 441451.
  • 20
    Voyta JC, Via DP, Butterfield CE, Zetter BR. Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol 1984; 99: 20342040.
  • 21
    Lehmann J, Huehn J, de la Rosa M, Maszyna F, Kretschmer U, Krenn V, et al. Expression of the integrin alpha Ebeta 7 identifies unique subsets of CD25+ as well as CD25-regulatory T cells. Proc Natl Acad Sci U S A 2002; 99: 1303113036.
  • 22
    de St Groth BF, Smith AL, Koh WP, Girgis L, Cook MC, Bertolino P. Carboxyfluorescein diacetate succinimidyl ester and the virgin lymphocyte: a marriage made in heaven. Immunol Cell Biol 1999; 77: 530538.
  • 23
    Vieira PL, Christensen JR, Minaee S, O'Neill EJ, Barrat FJ, Boonstra A, et al. IL-10-secreting regulatory T cells do not express Foxp3 but have comparable regulatory function to naturally occurring CD4+CD25+ regulatory T cells. J Immunol 2004; 172: 59865993.
  • 24
    Hansen W, Westendorf AM, Reinwald S, Bruder D, Deppenmeier S, Groebe L, et al. Chronic antigen stimulation in vivo induces a distinct population of antigen-specific Foxp3 CD25 regulatory T cells. J Immunol 2007; 179: 80598068.
  • 25
    Lin W, Haribhai D, Relland LM, Truong N, Carlson MR, Williams CB, et al. Regulatory T cell development in the absence of functional Foxp3. Nat Immunol 2007; 8: 359368.
  • 26
    Diehl L, Schurich A, Grochtmann R, Hegenbarth S, Chen L, Knolle PA. Tolerogenic maturation of liver sinusoidal endothelial cells promotes B7-homolog 1-dependent CD8(+) T cell tolerance. HEPATOLOGY 2007; 47: 296305.
  • 27
    Huehn J, Siegmund K, Lehmann JC, Siewert C, Haubold U, Feuerer M, et al. Developmental stage, phenotype, and migration distinguish naive- and effector/memory-like CD4+ regulatory T cells. J Exp Med 2004; 199: 303313.
  • 28
    Siegmund K, Feuerer M, Siewert C, Ghani S, Haubold U, Dankof A, et al. Migration matters: regulatory T-cell compartmentalization determines suppressive activity in vivo. Blood 2005; 106: 30973104.
  • 29
    Mora JR, Bono MR, Manjunath N, Weninger W, Cavanagh LL, Rosemblatt M, et al. Selective imprinting of gut-homing T cells by Peyer's patch dendritic cells. Nature 2003; 424: 8893.
  • 30
    Bonder CS, Norman MU, Swain MG, Zbytnuik LD, Yamanouchi J, Santamaria P, et al. Rules of recruitment for Th1 and Th2 lymphocytes in inflamed liver: a role for alpha-4 integrin and vascular adhesion protein-1. Immunity 2005; 23: 153163.
  • 31
    Nishimura T, Ohta A. A critical role for antigen-specific Th1 cells in acute liver injury in mice. J Immunol 1999; 162: 65036509.

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_23191_sm_supplfig1.tif565KSupplemental Figure 1
HEP_23191_sm_supplfig2.tif786KSupplemental Figure 2
HEP_23191_sm_supplfig3.tif1081KSupplemental Figure 3

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.