Volume 136, Issue 2, Pages 705-714 (February 2009) Liver Myofibroblasts Regulate Infiltration and Positioning of Lymphocytes in Human Liver  Andrew P. Holt, Emma L. Haughton, Patricia F. Lalor, Andrew Filer, Christopher D. Buckley, David H. Adams  Gastroenterology  Volume 136, Issue 2, Pages 705-714 (February 2009) DOI: 10.1053/j.gastro.2008.10.020 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Phenotypic characterization of aLMFs isolated from human liver tissue. (A) Immunofluorescent staining of aLMFs isolated from human cirrhotic liver (primary sclerosing cholangitis). Staining was visualized using fluorescein-isothiocyanate or Texas Red–conjugated secondary antibodies. Nuclei were counterstained using 4′,6-diamidino-2-phenylindole. (B) Cell-based enzyme-linked immunosorbent assay quantification of phenotypic markers on 3 different aLMF isolates from diseased livers (cryptogenic cirrhosis, alcoholic liver disease, and metabolic liver disease). Mean ± SEM absorbance from triplicate wells are shown. Background signals from isotype control antibodies were subtracted. Purity of aLMFs was confirmed using the endothelial marker CD31. (C) Indirect immunoperoxidase images from a representative sample of cirrhotic liver myofibroblasts in culture. Positive staining for prolyl-4-hydroxylase (5B5) and αSMA was visualized using a fast red substrate. The cells were negative for CD31. Gastroenterology 2009 136, 705-714DOI: (10.1053/j.gastro.2008.10.020) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 aLMFs and skin fibroblasts secrete different patterns of cytokines and chemokines. Cytokine and chemokine secretion by aLMFs and skin fibroblasts were determined using bead-based, multiplex, enzyme-linked immunosorbent assay analysis of conditioned supernatants. Data shown represent mean ± SEM from 4 aLMF isolates (alcoholic liver disease, cryptogenic, primary sclerosing cholangitis, and metabolic) and 2 skin fibroblast isolates. Cytokines were used at 10 ng/mL and cell monolayers were stimulated for 48 hours before collecting cell supernatants. IL-6 concentration in the conditioned media was confirmed by sandwich enzyme-linked immunosorbent assay. Gastroenterology 2009 136, 705-714DOI: (10.1053/j.gastro.2008.10.020) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 aLMF- and HSC-conditioned supernatants promote strong and rapid lymphocyte chemotaxis both in the presence and absence of proinflammatory cytokine stimulation. (A) Change in lymphocyte chemotaxis relative to control media after 2 hours. Each bar represents mean ± SEM of triplicate samples in 4–6 independent experiments. Each experiment used conditioned media generated from 4 different aLMF isolates (autoimmune hepatitis, metabolic cirrhosis, alcoholic liver disease, and primary sclerosing cholangitis). aLMFs were stimulated with cytokines for 48 hours before collection. Eight different blood donors were used. (B) Automated kinetic chemotaxis assay using fluorescent-labeled lymphocytes to determine chemotaxis towards aLMF (autoimmune hepatitis liver) supernatants in real time. Data are shown as the percentage of chemotaxis relative to control media (mean ± SEM of triplicate samples representative of 4 independent experiments using different lymphocyte donors and aLMFs). (C) Migration responses to pooled supernatants from 3 HSC isolations (all normal liver) after 48 hours prestimulation with cytokines (10 ng/mL) are shown expressed as the percentage of chemotaxis towards untreated HSC supernatants (mean ± SEM of 3 replicate experiments using 3 lymphocyte donors). Mean lymphocyte chemotaxis was determined by counting responses in 13 high-power fields. *P < .05, **P < .01. Gastroenterology 2009 136, 705-714DOI: (10.1053/j.gastro.2008.10.020) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Lymphocyte migration towards aLMF- and HSC-conditioned supernatant is dependent on the presence of CCL2, VEGF, IL-6, and HGF. (A) Supernatants were pooled from 3 aLMF isolations (2 alcoholic liver disease, 1 seronegative hepatitis) after 48 hours treatment with TNFα at 10 ng/mL. Supernatants were incubated with neutralizing or control antibodies for 30 minutes and then placed in the lower well of a modified Boyden chamber. Values represent chemotaxis towards TNFα-treated supernatants expressed as the percentage of chemotaxis towards TNFα-treated, aLMF-conditioned supernatant in the presence of isotype control (IgG control). At least 24 fields of view in 4 independent wells were counted for each sample and each bar represents the mean of 3–9 replicate experiments using 6 lymphocyte donors. The mean total lymphocyte chemotaxis to IgG control-treated aLMFs in 24 high-power fields was 136. (B) Values represent lymphocyte chemotaxis to unconditioned or aLMF-conditioned media in the presence of IgG control or specific neutralizing antibodies. aLMF-conditioned media was pooled from 3 separate aLMF isolations and incubated with IgG control or neutralizing antibodies as described. Data represent mean ± SEM of 3 separate experiments using 3 lymphocyte donors. (C) Values represent lymphocyte chemotaxis to TNFα-stimulated, HSC-conditioned media in the presence of IgG control or specific neutralizing antibodies. HSC-conditioned media was pooled from 3 HSC isolations (all normal liver) and incubated with IgG control or neutralizing antibodies as previously described. Data represent mean ± SEM of 3 separate experiments using 3 lymphocyte donors. *P < .05, ***P < .001. Gastroenterology 2009 136, 705-714DOI: (10.1053/j.gastro.2008.10.020) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Lymphocyte chemotaxis to aLMF-conditioned media is dependent on GPCR-dependent and GPCR-independent cytokine and chemokine signals. Supernatants were collected from 3 separate aLMF isolations (1 alcoholic liver disease, 1 primary sclerosing cholangitis) after 24 hours of treatment with TNFα (10 ng/mL) incubated with neutralizing or control antibodies and then placed in the lower chamber of a modified Boyden chamber. Lymphocytes were left untreated, treated with pertussis toxin, or incubated with antibody directed against CXCR3 for 20 minutes before the assay. Values represent lymphocyte chemotaxis towards TNFα-treated aLMF supernatants using 3 different peripheral blood lymphocyte donors expressed as the percentage of chemotaxis in the presence of isotype control antibody (IgG control). ***P < .001. Gastroenterology 2009 136, 705-714DOI: (10.1053/j.gastro.2008.10.020) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Lymphocyte adhesion and transmigration over aLMF monolayers is increased after stimulation of the monolayer with proinflammatory cytokines. (A) aLMF monolayers from 3 donors (1 alcoholic liver disease, 1 cryptogenic, and 1 metabolic cirrhosis) were stimulated with cytokines (24 hours; 10 ng/mL) before lymphocyte adhesion. Data are shown as mean ± SEM of adherent lymphocytes in 10 high-power fields. Total adhesion (white bars) represents the sum of adherent (phase-bright) or transmigrated cells (phase-dark). Four independent experiments using 3 different aLMF isolates and 4 blood donors were performed. Statistical significance was calculated using independent t tests compared with unstimulated aLMF controls. (B) Representative microscopic images from static adhesion assays comparing lymphocyte adhesion to unstimulated skin monolayers and aLMFs in the presence or absence of cytokine stimulation (24 hours at 10 ng/mL). Phase-dark cells represent cells that have undergone transmigration and statically adherent phase-bright cells are indicated with arrows. Original magnification, 40×. (C) Comparison of lymphocyte adhesion to unstimulated skin fibroblasts (white bars) and aLMFs (black bars). Total adhesion (left hand bars) is the sum of adherent cells (phase-bright) and transmigrated cells (phase-dark). Data are shown as mean ± SEM of adherent lymphocytes in 10 high-power fields using 3 aLMFs (1 alcoholic liver disease, 1 metabolic, and 1 cryptogenic cirrhosis) and 2 skin fibroblast isolates; lymphocytes were obtained from 3 donors. *P < .05. Gastroenterology 2009 136, 705-714DOI: (10.1053/j.gastro.2008.10.020) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Lymphocyte transmigration over TNFα-stimulated aLMF monolayers is ICAM-1 and VCAM-1 dependent. (A) Cell-based enzyme-linked immunosorbent assay quantification of ICAM-1 and VCAM-1 on cultured aLMFs and skin fibroblasts after 24 hours of cytokine stimulation. Data are shown as mean ± SEM of absorbance of triplicate wells in 6 experiments using 3 different aLMF isolates (autoimmune hepatitis, hepatic artery fibrosis, and alcoholic liver disease) and 2 skin fibroblast isolates. Background signals for wells treated with isotype control antibody were subtracted from all test values. (B) Lymphocyte adhesion to TNFα-stimulated (10 ng/mL, 24 h) aLMF monolayers expressed as the percentage of adhesion to aLMF monolayers pretreated with an IgG control. Each bar represents a minimum of 3 replicate experiments using 3 different aLMF isolates (primary sclerosing cholangitis, alcoholic liver disease, and HAT) and 5 independent lymphocyte donors. Where indicated, the monolayers were preincubated with ICAM-1– and/or VCAM-1–blocking antibodies for 40 minutes before the addition of lymphocytes. The mean total lymphocyte adhesion to IgG control-treated aLMFs in 8 high-power fields was 291. *P < .05, **P < .01. Gastroenterology 2009 136, 705-714DOI: (10.1053/j.gastro.2008.10.020) Copyright © 2009 AGA Institute Terms and Conditions

Figure 8 The rate of lymphocyte transmigration and the number of adherent cells is increased after TNFα prestimulation of aLMFs and is dependent on GPCR-dependent and -independent signaling. (A) Real-time chemotactic responses of lymphocytes through aLMF monolayers. Data represent absolute numbers of fluorescently labeled lymphocytes detected in the bottom well at indicated time intervals comparing TNFα and unstimulated aLMF monolayers (primary sclerosing cholangitis) with or without pretreatment of lymphocytes with pertussis toxin (200 ng/mL for 20 minutes). Each data point represents mean ± SEM of 3 replicate experiments and the figure is representative of 1 of 4 experiments repeated using different peripheral blood lymphocyte donors and aLMF isolates. (B) Lymphocyte transmigration through aLMF monolayers is dependent on GPCR signaling but also is reduced by inhibition of HGF, IL-6, and VEGF. Static adhesion assay using 3 separate aLMF isolates (2 alcoholic liver disease, 1 seronegative hepatitis) and 3 peripheral blood lymphocyte donors. Data shown are mean ± SEM of adherent lymphocytes in 10 high-power fields in 3 independent experiments. aLMF monolayers were prestimulated with TNFα (10 ng/mL, 24 h) and incubated with neutralizing antibodies to IL-6, HGF, and VEGF or isotype-matched IgG controls for 30 minutes before use. Where indicated, lymphocytes were pretreated for 20 minutes with pertussis toxin at 200 ng/mL. (C) Lymphocyte adhesion and transmigration over HSC monolayers in the absence of proinflammatory cytokines is dependent on GPCR-linked signals, IL-6, HGF, and VEGF. Static adhesion assay using 3 HSC isolates (all from normal liver) and 3 independent lymphocyte donors. Data shown are mean ± SEM of adherent lymphocytes in 10 high-power fields in 3 independent experiments. HSC monolayers were incubated with neutralizing antibodies to IL-6, HGF, and VEGF or isotype-matched IgG controls for 30 minutes before use. Where indicated, lymphocytes were pretreated for 20 minutes with pertussis toxin at 200 ng/mL. ***P < .001. Gastroenterology 2009 136, 705-714DOI: (10.1053/j.gastro.2008.10.020) Copyright © 2009 AGA Institute Terms and Conditions