1. Bile duct ligation in rats induces biliary expression of cytokine-induced neutrophil chemoattractant 
Jacqueline M. Saito*, Jacquelyn J. Maher‡ 
Gastroenterology 
Volume 118, Issue 6, Pages (June 2000)
DOI: /S (00)
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2. Fig. 1
Neutrophil alterations after BDL. Circulating neutrophil counts at various intervals after BDL (■) or sham operation (○). The number of neutrophils increases within 12 hours of both BDL and sham operation, but subsequently increases specifically in BDL rats. Values represent mean ± SEM; n = 3-5. *P < 0.01, BDL vs. sham.
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3. Fig. 2
Neutrophils in liver tissue after BDL. The number of neutrophils present in liver tissue at various intervals after BDL (■) or sham operation (○). Neutrophils were identified by immunostaining with anti-Gr antibody (see Materials and Methods). Cells were counted by direct visualization in high-power (20×) fields centered on portal tracts. Values represent mean ± SEM for 3 fields. *P < 0.05, BDL vs. sham.
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4. Fig. 3
Distribution of neutrophils in BDL liver. Photomicrographs illustrate neutrophils, stained with anti-Gr antibody, in (A and C) BDL or (B and D) sham-operated rats. (A and B) At 24 hours postoperatively, neutrophils are concentrated in the portal tracts of BDL liver but not sham liver. (C and D) Eight days after surgery, neutrophils remain abundant in the portal tracts of BDL liver but are scattered sparsely in sham liver. High-power views of BDL liver at 48 hours show neutrophil transmigration into periportal tissue. Neutrophils concentrate around small proliferating bile ductules (E) as well as larger intrahepatic bile ducts (F). Original magnification 4× (A-D); 10× (E and F).
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5. Fig. 3
Distribution of neutrophils in BDL liver. Photomicrographs illustrate neutrophils, stained with anti-Gr antibody, in (A and C) BDL or (B and D) sham-operated rats. (A and B) At 24 hours postoperatively, neutrophils are concentrated in the portal tracts of BDL liver but not sham liver. (C and D) Eight days after surgery, neutrophils remain abundant in the portal tracts of BDL liver but are scattered sparsely in sham liver. High-power views of BDL liver at 48 hours show neutrophil transmigration into periportal tissue. Neutrophils concentrate around small proliferating bile ductules (E) as well as larger intrahepatic bile ducts (F). Original magnification 4× (A-D); 10× (E and F).
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6. Fig. 4
Induction of CINC mRNA in liver after BDL. Autoradiogram illustrates CINC mRNA in whole liver at various intervals after BDL or sham operation. mRNA signals were obtained by ribonuclease protection, with GAPDH serving as an internal control. In normal liver, CINC mRNA is undetectable (not shown); within 3 hours of laparotomy, CINC mRNA is markedly induced in both BDL and sham livers. CINC mRNA disappears from sham liver between 24 and 48 hours but remains up-regulated in BDL livers for at least 48 hours. Results are representative of data from 3 independent groups of rats. tRNA, negative control; LPS Ctrl, positive control liver from LPS-treated rat.
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7. Fig. 5
Immunolocalization of CINC in BDL liver. Photomicrographs illustrate the distribution of CINC in rat liver 24 hours after (A and C) BDL or (B and D) sham operation. CINC was identified by immunoperoxidase staining as described in Materials and Methods. In BDL liver, CINC localizes to portal tracts and specifically to the periductular zones. In sham liver, faint CINC staining is present diffusely in hepatocytes. Original magnification 10× (A and B); 20× (C and D).
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8. Fig. 6
Localization of CINC mRNA in liver tissue by in situ hybridization. Matched bright-field and dark-field images of livers centered on portal tracts. (A and B) BDL liver hybridized with antisense CINC probe at 12 hours; (C and D) sham liver hybridized with antisense CINC probe at 12 hours; (E and F) BDL liver hybridized with sense CINC probe at 12 hours; (G and H) BDL liver hybridized with antisense CINC probe at 48 hours. CINC mRNA localizes specifically to bile ducts within 12 hours of BDL. In sham liver at 12 hours, CINC mRNA is expressed faintly and diffusely in hepatocytes. No CINC mRNA is detectable in BDL liver hybridized with a sense cRNA. By 48 hours after BDL, CINC mRNA is less apparent in portal tracts but is expressed strongly in individual cells scattered in the periportal region. (Original magnification 10×.)
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9. Fig. 7
CINC secretion by NRCs. NRCs were cultured in defined medium and treated with LPS or cytokines for 24 hours. CINC was measured in culture medium by ELISA (see Materials and Methods). Basal CINC secretion by NRCs measured 1.9 ng · mL−1 · day−1; CINC secretion increased significantly in response to LPS, TNF-α, and IL-1β. Values represent mean ± SEM; n = 3. *P < vs. control. †P < 0.05 vs. control.
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10. Fig. 8
Effect of bile on NRC CINC secretion. NRCs were incubated for 24 hours in defined medium containing rat bile in various concentrations (0%-50%, vol/vol). At the end of the incubation period, CINC was quantitated in culture medium by ELISA. Data represent NRC CINC secretion under control conditions without bile (□), in response to bile from normal rats (●), and in response to bile from 8-day BDL rats (■). Before incubation with NRCs, bile contained less than 2 ng/mL CINC (not shown). Bile from normal rats failed to stimulate CINC secretion except at 50%, where it caused a 2.9-fold increase. Bile from 8-day BDL rats stimulated CINC secretion at concentrations of 20% and 50%. At 50%, induction of CINC reached 12 times control values. Data represent mean ± SEM; n = 3-4. *P < vs. 0% bile; #P < 0.05 vs. 20% normal bile; §P < 0.01 vs. 50% normal bile.
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11. Fig. 9
Effect of LPS/cytokine inhibition on the NRC response to bile. Bile from 8-day rats was diluted to 30% (vol/vol) in culture medium and treated either with polymyxin (100 U/mL), polymyxin + anti–TNF-α (20 μg/mL), or polymyxin + anti–IL-1β (30 μg/mL). Treated and untreated bile were then added to cultured NRCs, and CINC secretion was measured after 24 hours. Control cells were incubated for 24 hours in defined medium without bile. NRCs incubated in culture medium containing 30% bile (■) secreted 3 times more CINC than cells incubated in culture medium alone (●). The stimulatory effect of bile was not abrogated by treatment with either polymyxin or a combination of polymyxin and anticytokine antibodies (▨). Values represent mean ± SEM. *P < 0.05 vs. control.
Gastroenterology  , DOI: ( /S (00) )
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