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

Co-localization of CXCL12 and CXCL10 with components of the endocytic pathway.

<p>LSEC were incubated with AlexaFluor 647-labeled CXCL12 or CXCL10 present in the lower chamber of the transwell. LSEC were stained with (A) anti-EEA1, (B) anti-TfR or (C) anti-LAMP-1 antibody. (D) LSEC were incubated with AlexaFluor 488-labeled AcLDL present in the lower chamber of the transwell and stained with anti-LAMP-1 antibody. (E) LSEC were incubated with AlexaFluor 647-labeled chemokine and AcLDL-AlexaFluor 488 present in the lower chamber of the transwell. Representative images of two independent experiments are shown. Bars represent 5 μm.</p

Receptor-mediated chemokine internalization by LSEC.

<p>(A) Expression of CXCR3 and CXCR4 mRNA in resting and <i>in vitro</i> activated LSEC was quantified in relation to GAPDH. (B) LSEC were treated with AMD3100 prior to incubation with fluorochrome-labeled CXCL12 for 60 min and analyzed by flow cytometry. (C) LSEC were treated with AMD3100 present in the lower chamber of the transwell prior to incubation with AlexaFluor 647-labeled CXCL12 added to the lower chamber for 60 min. Nuclei were stained with DAPI. Representative images of three independent experiments are shown. Bars represent 20 μm. (D) LSEC were treated with AMD3100 present in the lower chamber of the transwell prior pre-incubation with CXCL12 added to the lower chamber for 120 min. After removal of unbound chemokine, transmigration assays with total CD4⁺ T cells were performed. (E) LSEC from CXCR3^-/- mice were incubated with fluorochrome-labeled CXCL10 for 120 min. (F) CXCR3-deficient LSEC were pre-incubated with CXCL10 present in the lower chamber of the transwell for 120 min. Transmigration assays with effector/memory CD4⁺ T cells were performed. Mean values ± SD of 2–4 independent experiments are shown. * p< 0.05; ** p< 0.01; ns, not significant.</p

Hepatic chemokine expression during T cell-mediated hepatitis.

<p>Con A was intravenously injected into C57BL/6 mice. (A) Quantitative RT-PCR analysis of CXCL9, CXCL10 and CXCL12 mRNA expression in liver tissue of healthy or Con A-treated mice was performed. The chemokine expression was quantified in relation to GAPDH as a housekeeping gene. Mean values ± SD of 4–5 mice per group are shown. (B) Plasma ALT levels were determined at indicated time points. Medians of three independent experiments with 4–5 mice per group are shown. (C) Liver samples were stained with anti-CD146 and anti-CXCL9 antibody 12 h after hepatitis induction. Nuclei were stained with DAPI. Arrows indicate co-localization of CXCL9 and CD146. Bars represent 50 μm. Images are representative of three independent experiments. * p< 0.05; *** p< 0.001; ns, not significant.</p

Migration of CD4⁺ T cells into the inflamed and healthy liver after administration of CPZ.

<p>C57BL/6 mice were treated with Con A and received CPZ 7 h after hepatitis induction. Healthy mice also received CPZ. Radioactively labeled total CD4⁺ T cells were intravenously transferred into mice 120 min after administration of CPZ. Liver-specific radioactivity in relation to total radioactivity of the body was determined after 60 min migration time. Mean values ± SD of 4 independent experiments with three mice per group are shown. ** p< 0.01; *** p< 0.001; ns, not significant.</p

Priming of naive CD4 T-cells by liver-derived antigen <i>in vivo.</i>

<p>OT-II T-cells were purified from the lymph nodes and spleen of OT-II mice and labeled with CFSE. Four million cells were transferred intravenously into splenectomized TF-OVA mice (A), MHC-II^−/− → TF-OVA chimeras (B), or TF-OVA mice (control). Cells from the indicated organs were isolated 44 (A) or 68 hours (B) after cell transfer and analyzed for the presence of proliferating OT-II T-cells by detection of CFSE-dilution. All plots depict data gated on CD4⁺ cells. Events to the right of the vertical line represent the undivided population. Events at the far left of the plot represent unlabeled endogenous cells. Representative results from n = 4 bone-marrow chimeras and n = 4 control mice (A), and n = 6 splenectomized and n = 4 control mice (B) are shown.</p

CD4 T-cells primed by liver-derived antigen display deficient Th1-effector function.

<p>(A) Four million CFSE-labeled OT-II T-cells were transferred intravenously into TF-OVA mice or into B6 control mice. After 68 hours, non-parenchymal cells were purified from liver and spleen and incubated <i>in vitro</i> with PMA/ionomycin. Production of Interferon-γ and IL-2 was analyzed after 4 hours. Representative results are depicted (n = 6). (B) Mice were treated as in (A), but cells were analyzed for CD25 and FoxP3 expression immediately after purification of cells from the indicated organs. Representative results are depicted from n = 6 mice in each group. All plots depict data gated on CD4⁺CFSE⁺ cells. CD25/FoxP3 plots on the right depict the frequency of CD25⁺FoxP3⁺ double positive cells.</p

Priming of CD4 T-cells by endogenous antigen in the inflamed liver.

<p>(A) Eight million OT-I T-cells were transferred intravenously into TF-OVA mice (+ Hepatitis), or mice were left untreated (- Hepatitis). ALT levels were determined at day 6. Values from individual mice and mean ± SEM are depicted (*** p<0.0001 by Mann-Whitney test). (B) Four million CFSE-labeled OT-II T-cells were transferred intravenously into TF-OVA mice at day 6 after transfer of OT-I T-cells (+ Hepatitis) or into untreated TF-OVA mice (- Hepatitis). Non-parenchymal cells were isolated from liver and spleen analyzed for the presence of CFSE⁺ cells. The absolute number of CFSE⁺ cells in liver or spleen was determined 20 hours after transfer of OT-II T-cells. Cumulative results are depicted from n = 6 mice per group (mean ± SEM, ** p<0.005 by Student's t-test). (C) Mice were treated as in (B). Proliferation was analyzed 20 and 44 hours after transfer of OT-II T-cells. Events to the right of the vertical line represent the undivided population. Events at the far left of the plot represent unlabeled endogenous cells. Representative results are depicted from n = 6 mice in each group. All plots display data gated on CD4⁺Vα2⁺ cells.</p

Effector CD4 T-cells accumulate in the liver of TF-OVA mice.

<p>(A) Effector OT-II T-cells with a Th1-phenotype were generated <i>in vitro</i> with the cognate peptide antigen in the presence of IL-12, Interferon-γ, and anti-IL-4 antibody. After 6 days in culture, cells were restimulated with PMA/ionomycin and stained for Interferon-γ and IL-4. (B) Four million CFSE-labeled naïve or effector OT-II T-cells were transferred into TF-OVA or B6 control mice. Non-parenchymal cells from the indicated organs were isolated after 20 or 68 hours, and analyzed for the presence of proliferating OT-II T-cells by CFSE dilution. All plots depict data gated on CD4⁺Vα2⁺ cells. Events to the right of the vertical line represent the undivided population. Events at the far left of the plot represent unlabeled endogenous cells. Representative results from n = 4–6 mice in each group are shown. (C) Cells from the indicated organs were isolated at the indicated days after transfer of 4 million naïve or effector OT-II T-cells, and numbers of CD4⁺Vα2⁺ cells were enumerated. Data shown are derived from n = 3–6 mice per group at each time point. Note the different scales for spleen and liver/liver lymph node (mean ± SEM; * p<0.05, *** p<0.001 by Student's t-test). (D) Liver sections from TF-OVA and B6 mice were stained with H&E 6 days after transfer of 4 million effector OT-II T-cells. Representative images from n = 6 mice per group (magnification 100x) are depicted.</p