Volume 127, Issue 3, Pages 892-902 (September 2004) Inducible histamine protects mice from P. acnes-primed and LPS-induced hepatitis through H2-receptor stimulation  Minori Yokoyama, Akira Yokoyama, Shuji Mori, Hideo K. Takahashi, Tadashi Yoshino, Takeshi Watanabe, Takehiko Watanabe, Hiroshi Ohtsu, Masahiro Nishibori  Gastroenterology  Volume 127, Issue 3, Pages 892-902 (September 2004) DOI: 10.1053/j.gastro.2004.06.020 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 Survival rates of wild-type, HDC−/− and H2R−/− mice with P. acnes-primed LPS-induced hepatitis. The mice received an IV injection of 1 mg P. acnes suspended in 100 μL saline, followed by an IV injection of 0.5 μg LPS in 100 μL saline 7 days later. The wild-type mice were treated with d-chlorpheniramine (4 mg/kg, SC) (CHL), famotidine (5 mg/kg, SC) (FAM) or ranitidine (10 mg/kg, SC, twice) (RAN) (A), and HDC−/− mice were treated with histamine (4 mg/kg, SC, twice) (HA) (B). The histamine receptor antagonists or histamine were injected into mice 10 minutes before and 2 hours after the challenge with LPS. (C) The survival rate of H2R−/− mice was compared with the respective wild-type mice (n = 10). Gastroenterology 2004 127, 892-902DOI: (10.1053/j.gastro.2004.06.020) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 Time course changes in HDC activity and the levels of histamine and tele-methylhistamine in the liver of wild-type mice treated with P. acnes alone (○), P. acnes plus LPS (■), and HDC−/− mice treated with P. acnes plus LPS (▵) (A-C). The results are the means ± SEM of 5 mice. *P < 0.05, **P < 0.01 compared with the value by treatment with P. acnes alone. The HDC protein in the liver of wild-type mice treated with saline, P. acnes alone, or P. acnes plus LPS was detected at 6 hours by Western blotting, and the results were quantified by image scanner were normalized by β-actin (D). Western blotting shows the typical results of 3 mice from each group. The results of quantification are the means ± SEM of 3–5 mice (E). *P < 0.05 compared with the value with P. acnes alone. Gastroenterology 2004 127, 892-902DOI: (10.1053/j.gastro.2004.06.020) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 Localization of HDC-immunoreactive cells in the liver of wild-type mice with P. acnes–primed LPS-induced hepatitis. For double immunofluorescence staining, the frozen tissue sections were fixed with paraformaldehyde. Then, the sections were incubated with anti-HDC and anti-CD68 (A and B) or anti-HDC and anti-CD3 (C) antibodies followed by DTAF (HDC)- and Cy3 (CD68 or CD3)-labeled secondary antibodies, respectively. Arrowhead indicates each HDC-positive cell. The pictures represent the typical staining from different liver sections of the same mouse. We observed liver sections from 3 mice. Bars = 20 μm. Gastroenterology 2004 127, 892-902DOI: (10.1053/j.gastro.2004.06.020) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 Histologic changes in the liver of wild-type and HDC−/− mice. Six hours after LPS challenge, the mice were fixed with 10% formalin, and the liver tissues were processed for histologic examinations. (A) Hematoxylin-eosin staining of liver sections from wild-type mice (a, P. acnes alone; b, P. acnes plus LPS; c, P. acnes plus LPS plus d-chlorpheniramine; d, P. acnes plus LPS plus famotidine). (B) Hematoxylin-eosin staining of liver sections from HDC−/− mice (a, P. acnes plus LPS; b, P. acnes plus LPS plus histamine). (C) Detection of apoptotic cells by TUNEL staining from the same specimens as in A (a, P. acnes alone; b, P. acnes plus LPS; c, P. acnes plus LPS plus d-chlorpheniramine; d, P. acnes plus LPS plus famotidine). (D) Detection of apoptotic cells by TUNEL staining from the same specimens as in B (a, P. acnes plus LPS; b, P. acnes plus LPS plus histamine). The histamine receptor antagonists or histamine were injected into mice 10 minutes before and 2 hours after the challenge with LPS. Arrowheads indicate the apoptotic cells. Bars = 100 μm. Gastroenterology 2004 127, 892-902DOI: (10.1053/j.gastro.2004.06.020) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 5 Changes in plasma AST and ALT levels in wild-type and HDC−/− mice with P. acnes-primed LPS-induced hepatitis. (A and B) Time course changes in plasma AST and ALT levels in wild-type mice treated with P. acnes alone (○) or P. acnes plus LPS (▴) and in HDC−/− mice treated with P. acnes plus LPS (□) or treated with P. acnes plus LPS plus histamine (4 mg/kg, SC) (♦). Histamine was injected into mice 10 minutes before and 2 hours after the challenge with LPS. The results are the means ± SEM of 5 mice. *P < 0.05, **P < 0.01 compared with the value by treatment with P. acnes alone. (C and D) The plasma AST and ALT levels in wild-type mice treated with d-chlorpheniramine (4 mg/kg, SC) (CHL) or famotidine (5 mg/kg, SC) (FAM) 6 hours after the challenge with LPS. Histamine receptor antagonists were injected into mice 10 minutes before and 2 hours after the challenge with LPS. The results are the means ± SEM of 5 mice. *P < 0.05 compared with the value by treatment with P. acnes alone. #P < 0.05 compared with the value by treatment with P. acnes plus LPS. Gastroenterology 2004 127, 892-902DOI: (10.1053/j.gastro.2004.06.020) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 6 Expression of pro-IL-18 and TNF-α in the liver of wild-type, HDC−/−, and H2R−/− mice treated with P. acnes and LPS. In the left panel, mice were administered d-chlorpheniramine (4 mg/kg, SC) (CHL), famotidine (5 mg/kg, SC) (FAM), or histamine (4 mg/kg, SC) (HA) 10 minutes before and 2 hours after the challenge with LPS. Three hours after LPS challenge, mice were killed, and liver samples were prepared for electrophoresis. Pro-IL-18 (A) and TNF-α (B) were detected by Western blotting, and the results quantified by image scanner were normalized by β-actin. Upper panel shows the typical results of 2 mice from each group. The lower panel results are the means ± SEM of 4 or 5 mice. *P < 0.05, **P < 0.01 compared with the value in the respective wild-mice treated with P. acnes and LPS. ##P < 0.01 compared with the value in the HDC−/− mice treated with P. acnes and LPS. Gastroenterology 2004 127, 892-902DOI: (10.1053/j.gastro.2004.06.020) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 7 Effects of d-chlorpheniramine and famotidine on plasma cytokine response in P. acnes-primed LPS-induced hepatitis in wild-type mice. d-Chlorpheniramine (4 mg/kg, SC; ▴) or famotidine (5 mg/kg, SC; ■) was administered to mice 10 minutes before and 2 hours after the challenge with LPS, and the plasma cytokines were determined at the time points indicated. Results are means ± SEM of 5 mice. *P < 0.05, **P < 0.01 compared with the value of saline control (○). Gastroenterology 2004 127, 892-902DOI: (10.1053/j.gastro.2004.06.020) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 8 Changes in plasma cytokine levels in HDC−/− and H2R−/−mice in P. acnes-primed LPS-induced hepatitis. In HDC−/− mice, histamine (4 mg/kg, SC) (HA) was administered to mice 10 minutes before and 2 hours after the challenge with LPS, and plasma cytokines were determined 3 hours after the LPS challenge. Results are the means ± SEM of 5 mice. *P < 0.05, **P < 0.01 compared with the value in the respective wild-type mice. #P < 0.05, ##P <0.01 compared with the value by treatment with P. acnes and LPS. Gastroenterology 2004 127, 892-902DOI: (10.1053/j.gastro.2004.06.020) Copyright © 2004 American Gastroenterological Association Terms and Conditions