Volume 127, Issue 3, Pages 870-882 (September 2004) Immune stimulation of hepatic fibrogenesis by CD8 cells and attenuation by transgenic interleukin-10 from hepatocytes  Rifaat Safadi, Masayuki Ohta, Carlos E. Alvarez, M. Isabel Fiel, Meena Bansal, Wajahat Z. Mehal, Scott L. Friedman  Gastroenterology  Volume 127, Issue 3, Pages 870-882 (September 2004) DOI: 10.1053/j.gastro.2004.04.062 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 Reduced fibrosis in IL-10 TG vs. WT animals in 2 models of liver injury. Fibrosis was induced in either WT (A and C) or IL-10 TG (B and D) animals with 4 weeks of either carbon tetrachloride- CCl4 (A and B) or thioacetamide- TAA (C and D), and tissue sections were stained with sirius red, all as described in the Materials and Methods section. Representative tissue sections are shown, with fibrotic septa, which are more established in WT than IL-10 TG animals, highlighted by arrows (magnification ×10). This finding was reproducible for at least 5 different experiments with same number of animals in each subgroup. Gastroenterology 2004 127, 870-882DOI: (10.1053/j.gastro.2004.04.062) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 Reduced relative fibrosis area in IL-10 TG vs. WT animals in 2 models of liver injury and reduced α-SMA expression in whole liver extracts of WT and IL-10 TG mice. (A) Relative fibrosis and α-SMA expression: Upper panel: Animals were treated as described in Figure 1 legend. Relative fibrosis area (expressed as a percentage of total liver area) was assessed by analyzing 36 sirius red-stained liver sections per animal. Each field was acquired at ×10 magnification and then analyzed using a computerized Bioquant morphometry system. There was less relative fibrosis area in the livers of TG animals in both models. P values refer to comparison between wild-type and transgenic animals after CCl4 and TAA, respectively. Lower panel: Whole liver protein lysates were extracted, and 30 μg total protein were loaded per lane and analyzed for α-SMA expression. Decreased α-SMA expression is depicted in lysates from 2 IL-10 TG mice receiving TAA and CCl4, respectively (third and fourth lanes), compared with 2 WT animals also administered TAA and CCl4 (first and second lanes). The experiment was performed in 2 sets of animals with the same result obtained. (B) Reduced expression of fibrogenic mRNA in IL-10 TG mice during liver injury: Either WT or IL-10 TG mice were treated with CCl4 for 4 weeks as described previously, then mRNA was extracted from whole liver lysates and real-time PCR performed to quantify expression of a panel of genes associated with hepatic fibrogenesis, as described in the Materials and Methods section. There was reduced expression of fibrogenic mRNAs in IL-10 TG mice after CCl4 compared with WT mice, including α-smooth muscle actin, TGF-β1, platelet-derived growth factor receptor (PDGFR), collagen α1 (I), ICAM-1, and MMP2. Data depicted represent triplicate values, with similar results obtained in 2 separate experiments. Gastroenterology 2004 127, 870-882DOI: (10.1053/j.gastro.2004.04.062) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 Altered intrahepatic lymphocyte subsets in WT and IL-10 TG mice following liver injury. Using FACS, intrahepatic lymphocytes were isolated, and their composition was analyzed to define patterns that correlated with reduced fibrosis. (A) FACS analysis of intrahepatic lymphocytes: The Y-axis expresses the percentage of α/β and γ/δ T cells from all lymphocytes and of CD4+/CD8+ T cells from α/β T cells. Analyses revealed significant reduction of α/β TCR and CD4+ T cells in naive IL-10 TG compared with WT animals. Following 4 weeks of TAA to induce fibrosis, CD8+ T cells increased and CD4+ T cells decreased in WT animals. However, both subsets were further decreased after TAA administration in IL-10 TG mice (B) Characterization of intrahepatic lymphocytes by cell staining: Intracellular staining of both intrahepatic CD4+ and CD8+ for interferon-γ and IL-4 demonstrates that IL-4–secreting CD4+ T cells are significantly decreased in naive IL-10 TG mice compared with naive WT animals. Compared with naive WT animals, interferon (IFN)-γ–secreting CD4+ T cells were decreased in all other groups. IL-4–secreting CD8+ T cells were significantly increased in fibrotic WT mice compared with naive WT animals and were reduced in the fibrotic TG animals. Unlike the fibrosis-specific alterations, intrahepatic lymphocytes from mice immunized with ovalbumin (OA) showed only CD4+ secretion of IFN-γ. Gastroenterology 2004 127, 870-882DOI: (10.1053/j.gastro.2004.04.062) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 Similar relative fibrosis area in IL-10 TG vs. WT animals in liver injury following irradiation: Relative fibrosis area was assessed by morphometry in TG and WT animals after induction of liver injury by CCl4. Prevention of lymphocyte expansion by irradiation diminished fibrosis equally in both WT and TG groups, suggesting that rIL-10’s antifibrotic effect was mediated by bone marrow-derived cells. Data represents 36 fields from each of 8 mice in each group. Gastroenterology 2004 127, 870-882DOI: (10.1053/j.gastro.2004.04.062) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 5 Adoptive transfer of lymphocytes induces early changes of hepatic fibrosis in naive SCID mice. Mixed lymphocytes were isolated from either naive or CCl4-treated mice and were transferred via IP administration weekly from WT donors to each SCID recipient for 4 weeks. For each subsequent treatment, lymphocytes were isolated from animals with increasing cumulative exposure to CCl4. (A-C) Histology of SCID recipients of lymphocytes from CCl4-treated (A and B) or control (C) mice. Liver sections were stained with sirius red, which demonstrates evidence of early fibrosis by the appearance of slender septa (arrows) in animals following the transfer of lymphocytes from CCl4-treated but not from control mice. No parenchymal disruption or inflammation is apparent. (D) α-Actin immunoblot: Whole liver lysates (30 μg of total protein per lane) of recipient animals were analyzed for α-smooth muscle actin expression by immunoblot. Expression was greater in 2 SCID recipients receiving lymphocytes from animals treated with CCl4 compared with 2 mice receiving lymphocytes from untreated animals. Gastroenterology 2004 127, 870-882DOI: (10.1053/j.gastro.2004.04.062) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 6 Increased liver injury and fibrosis following adoptive transfer of CD8+ T lymphocytes from animals with CCl4 fibrosis. (A) Liver injury: serum aminotransferase levels were assayed in SCID mice receiving either whole mixed lymphocytes, CD4+, or CD8+ T cells from CCl4-treated animals for 4 weeks to assess whether adoptive transfer induced hepatocellular damage. Although all animals receiving lymphocytes had elevated AST, values were significantly greater in CD8+ T-cell recipients compared with SCIDs that were administered either whole lymphocytes or CD4+ T cells. There was no effect of lymphocytes from untreated control animals. All control recipients had normal ALT and AST values. (B) Fibrosis: livers of SCID animals described in panel A were analyzed by morphometry as described in Figure 2 legend, and the relative fibrosis area is depicted. SCID animals receiving CD8+ T cells had significantly more fibrosis than recipients of either whole mixed lymphocytes or CD4+ T cells. Gastroenterology 2004 127, 870-882DOI: (10.1053/j.gastro.2004.04.062) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 7 Adoptive transfer of CD8+ T lymphocytes induces TGF-β1 and collagen I mRNA in recipient livers. mRNA was extracted from whole liver lysates of naive SCID mice after receiving 4 weeks of either whole mixed lymphocytes, CD4+, or CD8+ T cells and analyzed for expression of mRNA levels of TGF-β1 and collagen α1 (I) mRNA by real-time PCR. Both transcripts were elevated, with expression of collagen I significantly higher in CD8+ T-cell recipients. There were 7 animals in each group, and 2 separate experiments were performed with similar results (data from 1 of these 2 experiments is shown). Recipients of lymphocytes from control animals had no change in either transcript. Gastroenterology 2004 127, 870-882DOI: (10.1053/j.gastro.2004.04.062) Copyright © 2004 American Gastroenterological Association Terms and Conditions