1. Innate immune system plays a critical role in determining the progression and severity of acetaminophen hepatotoxicity 
Zhang-Xu Liu, Sugantha Govindarajan, Neil Kaplowitz 
Gastroenterology 
Volume 127, Issue 6, Pages (December 2004)
DOI: /j.gastro
Copyright © 2004 American Gastroenterological Association Terms and Conditions

2. Figure 1
Effect of depletion of both NK and NKT cells by anti-NK1.1 on APAP-induced liver injury. Normal B6 mice were administered intraperitoneally a toxic dose of APAP (500 mg/kg) on day 0. In anti-NK1.1–treated mice, 300 μg of anti-NK1.1 (PK136) MAb or control IgG2a was injected intraperitoneally daily starting 2 days before APAP administration until termination of experiment. (A) Serum ALT values: mice were bled at the indicated times after APAP administration and serum ALT values were measured. The number of mice at the beginning of the experiments were 22 for APAP alone, 20 for the anti-NK1.1–treated group, and 8 for the control mouse IgG2a-treated group. *P < .05 vs. APAP-treated control mice. The mean ± SE from different groups are plotted. □, APAP; ▨, APAP + IgG2a; ▩, APAP + αNK1.1. (B) Histologic analysis of liver sections after staining with H&E. Mice were challenged with APAP (500 mg/kg) with or without anti-NK1.1 treatment, and the livers were harvested at 24 hours after APAP and prepared for histology. Necrotic area is indicated by arrows. (C) Mouse survival: survival was followed-up for 4 days after APAP challenge (500 mg/kg). Data shown are pooled from 3 repeated experiments with similar results. The numbers (n) represent the number of mice used at the beginning of experiments for each group. *P < .05 vs. APAP-treated control mice (APAP). ■, APAP; □, APAP + αNK1.1.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

3. Figure 2
Serum ALT values after APAP treatment (500 mg/kg, intraperitoneally) in NK or NKT cell-depleted mice. (A) Serum ALT values were determined at the indicated times in B6 control mice and antibody-treated mice (8 mice per group) after APAP challenge. A total of 15 μL of anti-AsGM1 antiserum or control rabbit Ig was injected intraperitoneally daily starting 2 days before APAP until termination of experiment. □, APAP; ▨, APAP + rabbit Ig; ▩, APAP + αAsGM1. (B) B6 wild-type mice (wt), B6-CD1d-deficient mice (CD1d−/−), and CD1d−/− mice with control rabbit Ig treatment (CD1d−/−+ rabbit Ig) or anti-AsGM1 treatment (CD1d−/−+ αAsGM1) were challenged with APAP (500 mg/kg) and serum ALT values were measured at the indicated times. Mean ± SE from groups of 6–8 mice are plotted. *P < .05 vs. CD1d−/− mice. □, Wild type; ▨, CD1D−/−; ▩, CD1D-/- + rabbit Ig; 阸, CD1D−/− + αAsGM1.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

4. Figure 3
Effect of depletion of NK1.1+ cells on hepatic leukocytes after APAP challenge. (A) Liver leukocytes were isolated at the indicated times from APAP (500 mg/kg)-challenged mice with or without anti-NK1.1 treatment. Total number of hepatic leukocytes per liver from 3–5 mice per group are plotted as mean ± SE. *P < .05 vs. nontreated control mice at time 0; **P < .05 vs. APAP-treated control mice at 24 hours. □, APAP; ▨, APAP + αNK1.1. (B) Representative flow cytometry data of liver leukocytes. Livers from groups of 3 normal control B6 mice and APAP (500 mg/kg)-challenged mice with or without anti-NK1.1 treatment were harvested at 24 hours and pooled for isolation of hepatic leukocytes. The cells were double-stained with PE-conjugated anti-NK1.1, anti-Gr-1, and FITC-conjugated anti-CD3, anti-Mac-1, and anti-F4/80 antibodies and analyzed for T cells (CD3+ NK1.1−), NK cells (CD3−NK1.1+), NKT cells (CD3+ NK1.1+), neutrophils (Mac-1+ Gr-1+), and macrophages (F4/80+) by flow cytometry. Percentages for individual cell populations are shown. (C) Absolute number of each cell type per liver at 4 hours (left panel) and 24 hours (right panel) after APAP challenge. Liver leukocytes were isolated at indicated times after APAP (500 mg/kg) challenge with or without pretreatment of anti-NK1.1 MAb, and analyzed by 2-parameter flow cytometry. The number per liver for individual cell type were calculated by multiplying the percentage of individual cell type by the total number of isolated liver leukocytes per liver. Pooled results from 3 experiments are plotted as mean ± SE. *P < .05 vs. respective cell type in nontreated B6 mice; **P < .05 vs. neutrophils (PMN) in APAP-treated B6 mice. □, T; ▨, NK; ▩, NKT; 阸, PMN; , MØ.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

5. Figure 4
Wright-Giemsa staining of liver leukocytes. Aliquots of liver leukocytes from normal control mice and APAP (500 mg/kg)-challenged mice at 24 hours were cytospun onto glass slides and stained with Wright-Giemsa (1000×). Arrows identify neutrophils.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

6. Figure 5
Effect of depletion of NK1.1+ cells on expression of IFN-γ and chemokines in the liver of APAP-challenged mice. (A) Induction of mRNA for cytokines and chemokines. Liver tissue was harvested from normal control mice and APAP (500 mg/kg)-challenged mice with or without anti-NK1.1 treatment at the indicated times after APAP challenge. RNA was extracted and analyzed by reverse-transcription PCR. Data shown are from 3 experiments with similar results. (B) Contribution of NK1.1+ cells (NK and NKT) to IFN-γ production. Hepatic leukocytes were isolated from nontreated control mice and APAP-challenged mice with or without pretreatment of anti-NK1.1 MAb at 24 hours after APAP challenge (500 mg/kg). Cells were pooled from 3 mice per group and intracellular IFN-γ was detected in hepatic leukocytes by flow cytometric analysis as described in the Materials and Methods section. (C) Serum ALT levels in GKO mice. B6 wild-type (wt) control mice and GKO mice were challenged with APAP (500 mg/kg). Serum was harvested as indicated and assayed for ALT. Mean ± SE from groups of 5–8 mice at indicated times are plotted. *P < .05 vs. APAP-treated wild-type mice. □, wild type; ▨, GKO.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

7. Figure 6
Effect of depletion of NK1.1+ cells on FasL expression in the liver. (A) Induction of mRNA for Fas and FasL. Liver tissue was harvested from normal control mice and APAP (500 mg/kg)-challenged mice with or without anti-NK1.1 treatment at the indicated times after APAP challenge. RNA was extracted and analyzed by reverse-transcription PCR. (B) FasL-expressing cells in hepatic leukocytes. Hepatic leukocytes were isolated from control mice and APAP-challenged (500 mg/kg) mice with or without anti-NK1.1 treatment at 24 hours after APAP challenge. Cells were stained with biotinylated anti-mouse FasL MAb followed by PE-conjugated streptavidin and then analyzed by flow cytometry. Data shown are representative of 3 experiments. (C) FasL expression on individual cell types. Hepatic leukocytes were isolated from control mice and APAP-challenged (500 mg/kg) mice at 24 hours after APAP challenge and stained with biotinylated anti-FasL MAb followed by PE-conjugated anti-mouse NK1.1, Gr-1, FITC-conjugated anti-mouse CD3, Mac-1, F4/80 MAbs, and Cy-chrome-conjugated streptavidin. Expression of FasL was analyzed on the gated NK cells (CD3−NK1.1+), NKT cells (CD3+NK1.1+), macrophages (F4/80+), and neutrophils (PMN) (Gr-1+Mac-1+) by multicolor flow cytometry. Numbers indicate percentages of FasL-staining cells. Broken lines indicate the background staining of isotype-matched control IgG; solid lines indicate the staining of FasL.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

8. Figure 7
APAP-induced liver injury in Fas-deficient (lpr) and FasL-deficient (gld) mice. (A) Serum ALT values were determined in wild-type mice, lpr mice, and gld mice at the indicated times after APAP (500 mg/kg) challenge. The data shown are representative of 3 experiments with similar results. Mean ± SE from groups of 5–8 mice at the indicated times are plotted. * and **P < .05 vs. wild-type control mice (wt) □, wild type; ▨, lpr; ▩, gld. (B) Mouse survival after APAP (500 mg/kg) challenge. In all studies, survival was followed-up for 4 days after APAP challenge. Data shown represent pooled results from 3 different experiments. Individual experiments consisted of 6–8 mice per group and showed similar results. *P < .05 vs. wild-type control mice (wt). □, wild type; ▾, lpr; ■, gld.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

9. Figure 8
APAP metabolism in the liver. (A) Hepatic GSH concentrations in APAP-challenged mice. Normal B6 mice and anti-NK1.1 MAb-treated mice were challenged with APAP (500 mg/kg). Liver tissues from groups of 3 mice at the indicated times after APAP challenge were harvested and total hepatic GSH level was measured. □, APAP; ▨, APAP + αNK1.1. (B) APAP covalent binding to hepatic proteins at 2 and 4 hours after toxic dose of APAP (500 mg/kg) in B6 mice, anti-NK1.1–treated mice, CD1d-deficient mice (CD1d−/−), and Fas-deficient (lpr) mice. Liver homogenates of APAP-challenged mice were analyzed by Western blot using an anti-APAP antiserum. Untreated B6 mice were used as controls. Arrows indicate the multiple bands of covalent binding to hepatic proteins.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions