Volume 116, Issue 6, Pages 1428-1440 (June 1999) 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  Percy A. Knolle, Edgar Schmitt, Shenciu Jin, Tieno Germann, Rainer Duchmann, Silke Hegenbarth, Guido Gerken, Ansgar W. Lohse  Gastroenterology  Volume 116, Issue 6, Pages 1428-1440 (June 1999) DOI: 10.1016/S0016-5085(99)70508-1 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Antigen-presenting LSECs induce IFN-γ production in a Th1 CD4+ cell clone in the absence of IL-12. LSECs were cultured in flat-bottom microtiter plates at a density of 1 × 105 cells/well. LSECs were kept in culture for 3 days, and antigen-specific CD4+ T cells (LNC.2.F1) (3 × 104/well) were added simultaneously with specific antigen (PPD at a concentration of 10 μg/mL) and IL-12 (50 pg/mL) or anti–IL-12 antibodies or rat immunoglobulin (each at a concentration of 10 μg/mL). After 48 hours, the cell culture supernatants were assayed by sandwich ELISA for the concentration of IFN-γ. Results are ±SD. All experiments were carried out in triplicate, and the experiment shown is representative of 5 separate experiments. Gastroenterology 1999 116, 1428-1440DOI: (10.1016/S0016-5085(99)70508-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Neither IL-12 p35 mRNA nor IL-12 p40 mRNA is produced in significant amounts by LSECs. LSECs (5 × 105/well) were cultured on collagen Typ I–coated 24 well plates. After 5 days, the cells were stimulated with endotoxin (lipopolysaccharide; 1 μg/mL) plus Listeria monozytogenes (5 Listeria/cell) or heat-killed Staphylococcus aureus Cowan-A (SAC; 1:1000). Cells were harvested 18 hours later, and RNA was isolated as described in Materials and Methods. The concentration of (A) IL-12 p35 and (B) p40 mRNA in LSECs (2) was determined by competitive reverse-transcription PCR and semiquantitavely expressed as cDNA titer (see Materials and Methods). Bone marrow cells (▩) were used as a control because this population of APCs is known to produce IL-12 upon stimulation. Gastroenterology 1999 116, 1428-1440DOI: (10.1016/S0016-5085(99)70508-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Only microvascular LSECs but not macrovascular endothelial cells from aorta can induce antigen-specific activation of naive CD4+(6.5+) T cells. Primary cell populations of bone marrow (BM) cells, LSECs, and endothelial cells from aorta were isolated as described in detail in the Materials and Methods. CD4+(6.5+) T cells (1 × 105/well) were added together with hemagglutinin (2 μg/mL) to the respective primary cell populations. To determine activation of naive CD4+(6.5+) T cells after 5 days, cell culture supernatants were tested by specific sandwich ELISA for concentration of IFN-γ. The results are shown ± SD and are representative of 2 separate experiments. Gastroenterology 1999 116, 1428-1440DOI: (10.1016/S0016-5085(99)70508-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 LSECs express CD4, CD11b, and CD11c on the cell surface. LSECs were cultured in collagen I–coated 6-well plates in DMEM/10% fetal calf serum. Three days after isolation, LSECs were gently detached using trypsin-EDTA, stained with fluorochrome-labeled antibodies (CD4-PE/CD11b-FITC/CD11c-FITC) at 3 μg/mL for 45 minutes at 4°C, washed twice in cold PBS, and resuspended in PBS/formalin 1%. Isotype-specific fluorochrome-labeled control antibodies were used at identical concentrations. LSECs were treated with endotoxin (10 ng/mL) for 18 hours before staining with CD11c–fluorescein isothiocyanate. (a) Staining with control antibody, (b) CD11c-FITC staining of LSECs, and (c) CD11c–fluorescein isothiocyanate staining of endotoxin-treated LSECs. Cells (1 × 104) were analyzed in a FACScan using Lysis-II software. The experiment is representative of 4 separate experiments. Gastroenterology 1999 116, 1428-1440DOI: (10.1016/S0016-5085(99)70508-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Endogenous IL-4 production during T-cell priming by antigen-presenting LSECs is responsible for Th0 phenotype in CD4+(6.5+) T cells upon restimulation. CD4+(6.5+) T cells were coincubated with LSECs and hemagglutinin (2 μg/mL). IL-12 protein was added as indicated, and neutralizing antibody to IL-4 or rat Ig were used at a concentration of 10 μg/mL. After 5 days, CD4+(6.5+) T cells were extensively washed and restimulated with anti-CD3ϵ antibody (clone 17A2) (5 μg/mL) cross-linked by goat-antiserum to rat Ig (7.5 μg/mL). Two days after restimulation, cell culture supernatants were tested for IL-4 and IFN-γ concentrations by specific sandwich ELISA. Experiments were always carried out in triplicate. Results of 4 independent experiments are shown ±SD. Gastroenterology 1999 116, 1428-1440DOI: (10.1016/S0016-5085(99)70508-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Antigen-presenting spleen cells induce differentiation of naive CD4+(6.5+) T cells toward Th1 in the presence of LSECs. LSECs were cocultured with 4 × 104 spleen cells/well, and CD4+(6.5+) T cells (1 × 105/well) were added together with hemagglutinin (2 μg/mL). After 5 days, T cells were restimulated with anti-CD3ϵ (5 μg/mL) cross-linked by goat antiserum to rat Ig (7.5 μg/mL). After 48 hours, cell culture supernatants were assayed for the concentration of IFN-γ or IL-4 by specific sandwich ELISAs. Experiments were always carried out in triplicate. Results are ±SD and represent 4 separate and independent experiments. Gastroenterology 1999 116, 1428-1440DOI: (10.1016/S0016-5085(99)70508-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions