1. Volume 125, Issue 4, Pages 1060-1076 (October 2003)
The chemokine CCL21 modulates lymphocyte recruitment and fibrosis in chronic hepatitis C1  
Andrea Bonacchi, Ilaria Petrai, Raffaella M.S Defranco, Elena Lazzeri, Francesco Annunziato, Eva Efsen, Lorenzo Cosmi, Paola Romagnani, Stefano Milani, Paola Failli, Giacomo Batignani, Francesco Liotta, Giacomo Laffi, Massimo Pinzani, Paolo Gentilini, Fabio Marra 
Gastroenterology 
Volume 125, Issue 4, Pages (October 2003)
DOI: /S (03)

2. Figure 1
Expression of the chemokine receptor CCR7 is increased in patients with chronic hepatitis C. (A ) Total RNA was isolated from specimens of normal human liver (NHL, lanes 3–5) or from patients with chronic HCV-related hepatitis (CHC, lanes 6–9). Ten micrograms of RNA was analyzed by RNase protection assay as described in Materials and Methods. In lane 1, total RNA isolated from human peripheral blood mononuclear cells was run as a positive control; in lane 2, transfer RNA was run as a negative control. (B) Relative expression of CCR7 to the housekeeping gene GAPDH as assessed by densitometry. ∗P < 0.05.
Gastroenterology  , DOI: ( /S (03) )

3. Figure 2
Hepatic gene expression of CCL21 during chronic hepatitis C. (A ) Expression of CCL21 was assessed by in situ hybridization, and CCL21 gene expression was barely detectable in normal human liver. (B) During chronic hepatitis C, CCL21 expression was markedly up-regulated in the enlarged portal tracts and in the active fibrous septae (arrows). (C ) Up-regulated expression was also detected at the periphery of inflammatory lymphoid follicles within the portal tracts (arrows). (D) No signal was observed when specimens of chronic hepatitis C were hybridized with the sense probe (negative control). (Original magnification 60×.)
Gastroenterology  , DOI: ( /S (03) )

4. Figure 3
Up-regulated expression of CCL21 during chronic hepatitis C. (A ) Protein expression of CCL21 was found in a few star-shaped cells in the portal tracts of normal human liver. (B) Increased expression was present in the portal tracts in patients with chronic hepatitis C, especially at the septum/nodule interface (short arrows) or in correspondence to inflammatory lymphoid follicles (long arrows). (C ) Expression of CCL21 was present at the periphery of the follicles, similar to CCL21 gene expression. D shows the negative control. pt, portal tract; f, inflammatory lymphoid follicle. (Original magnification A, B, D, 120×; C, 240×.)
Gastroenterology  , DOI: ( /S (03) )

5. Figure 4
Colocalization of CCL21 expression with different cellular markers. (A-F) Serial sections from livers with chronic hepatitis C were immunostained with antibodies against (A ) CCL21, (B) CD3, (C ) CD8, (D) CD11c, (E ) α-smooth muscle actin, or (F ) factor VIII. An enlarged portal tract with an inflammatory lymphoid follicle (f) is shown. Expression of CCL21 is evident in areas where (C ) CD8+ T lymphocytes and (D) mature dendritic cells accumulate. (B) CCL21-expressing cells are also surrounded by α-smooth muscle actin—positive cells. (G) Combined immunofluorescence for CCL21 (left panel, green) and CD11c (right panel, red). The middle panel shows the overlaid pictures, and yellow color indicates signal colocalization. (Original magnification 240×.)
Gastroenterology  , DOI: ( /S (03) )

6. Figure 5
Expression of CCR7 in patients with different grades or stages of chronic hepatitis C. (A ) RNA isolated from normal liver tissue (n = 3) or percutaneous liver biopsy specimens obtained from patients with chronic hepatitis C (CHC) and mild inflammation and fibrosis (Ishak’s HAI grade ≤3, stage ≤2; n = 8), moderate-severe inflammation and mild fibrosis (HAI grade ≥4, stage ≤2; n = 5), or moderate-severe inflammation and fibrosis (HAI grade ≥4, stage ≥3, n = 6) were analyzed by RNase protection assay for expression of CCR7 or the housekeeping gene GAPDH. The upper panel shows representative autoradiograms, and the lower panel shows densitometry data from all patients. ∗P < 0.05 vs. normal human liver (NHL) (one-way analysis of variance and Tukey’s test). (B) A section obtained from a percutaneous liver biopsy performed in a patient with chronic hepatitis C, and no cirrhosis was immunostained with antibodies against CCL21. Specific signal appears as dark gray (arrows).
Gastroenterology  , DOI: ( /S (03) )

7. Figure 6
Expression of CCR7 in patients with primary biliary cirrhosis. RNA isolated from normal liver tissue (n = 3) or from tissue obtained from patients with primary biliary cirrhosis at the time of orthotopic liver transplantation (n = 5) was analyzed by RNase protection assay for expression of CCR7 or the housekeeping gene GAPDH. The upper panel shows representative autoradiograms, and the lower panel shows data obtained from densitometry. ∗P < 0.05 vs. normal human liver (NHL) (Student t test).
Gastroenterology  , DOI: ( /S (03) )

8. Figure 7
Expression of the chemokine receptors CCR7 and CXCR3 by peripheral and intrahepatic CD8+ T lymphocytes. (A ) PBLs or IHLs from control subjects (black bars) or from patients with chronic hepatitis C (white bars) were analyzed by flow cytometry for expression of CD3, CD8, CCR7, and CXCR3. °P < 0.05 vs. IHLs; P < 0.05 vs. chronic hepatitis C. (B) CD3+, CD8+ IHLs from control subjects (black bars) or from patients with chronic hepatitis C (white bars) were analyzed for expression of CCR7 or CXCR3 alone (CCR7s and CXCR3s) or in combination (CCR7/CXCR3). ∗P < 0.05 vs. chronic hepatitis C.
Gastroenterology  , DOI: ( /S (03) )

9. Figure 8
Expression of functional CCR7 on activated HSCs. (A ) Cultured HSCs were detached from the dish and analyzed by flow cytometry with antibodies directed against the chemokine receptors CXCR3, CCR2, or CCR7 as indicated (white area). Signal from isotype-specific controls is indicated by the gray area. (B) HSCs were seeded on glass coverslips and loaded with the fluorescent indicator Fura-2. After addition of 100 ng/mL CCL21 (time 0), fluorescence was recorded every 12 seconds as described in Materials and Methods. (C ) Time course of the average calcium concentration in a group of HSCs.
Gastroenterology  , DOI: ( /S (03) )

10. Figure 9
CCL21 induces migration but not proliferation of HSCs. (A ) Confluent HSCs were serum deprived for 48 hours, and cell migration was measured using modified Boyden chambers with increasing concentrations of CCL21 or 10 ng/mL PDGF (positive control) as agonists. (B) Serum-starved HSCs were incubated with the indicated concentrations of CCL21, 10 ng/mL PDGF, or 10% serum. After 20 hours, cells were pulsed with [3H]thymidine and DNA synthesis was measured as described in Materials and Methods. (C ) Conditions were identical to those described for B. After 96 hours, cell number was measured by crystal violet staining.
Gastroenterology  , DOI: ( /S (03) )

11. Figure 10
CCL21 accelerates in vitro wound healing. Confluent, culture-activated HSCs were serum deprived for 48 hours. A wound was produced in the monolayer with a pipette tip, and the cells were exposed to (A ) serum-free medium alone, (B) 10 ng/mL CCL21, (C ) 100 ng/mL CCL21, or (D) 10% fetal bovine serum. Twenty-four hours later, the cells were fixed and colored and the wounds were photographed.
Gastroenterology  , DOI: ( /S (03) )

12. Figure 11
CCL21 activates the Ras/ERK cascade in HSCs. (A ) Serum-starved HSCs were incubated with 100 ng/mL CCL21 for the indicated time points. Total proteins were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and analyzed by immunoblotting using the indicated antibodies. The molecular weight markers are shown on the left. (B) The experiment was performed exactly as described in A. Fifty micrograms of protein was immunoprecipitated with anti-ERK antibodies, and the immunobeads were used for ERK assay as described in Materials and Methods. (C ) Serum-starved HSCs were exposed to increasing concentrations of CCL21 for 10 minutes. Total proteins were analyzed by immunoblotting using the indicated antibodies.
Gastroenterology  , DOI: ( /S (03) )

13. Figure 12
CCL21 activates Src and Akt. (A ) Serum-starved HSCs were incubated with 100 ng/mL CCL21 for the indicated time points. Total proteins were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and analyzed by immunoblotting using the indicated antibodies. (B) Serum-starved HSCs were exposed to increasing concentrations of CCL21 for 10 minutes. Total proteins were analyzed by immunoblotting using the indicated antibodies. (C ) HSCs were incubated with the MEK inhibitor PD98059 (30 μmol/L), the inhibitor of Src, PP1 (5 μmol/L), or the PI3-K inhibitor LY (10 μmol/L) before stimulating migration with 100 ng/mL CCL21.
Gastroenterology  , DOI: ( /S (03) )

14. Figure 13
Activation of transcription factors by CCL21 in HSCs. (A and B) Serum-starved HSCs were incubated with 100 ng/mL CCL21 for the indicated time points. Total proteins were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and analyzed by immunoblotting using the indicated antibodies. (C ) Serum-starved HSCs were incubated with 100 ng/mL CCL21 for the indicated time points. Ten micrograms of nuclear extracts was used for electrophoretic mobility shift assay with a radiolabeled consensus oligonucleotide for the transcription factor NF-κB. The arrow indicates migration of the specific complex.
Gastroenterology  , DOI: ( /S (03) )

15. Figure 14
Up-regulation of CCL2 and CXCL8 expression by CCL21. (A ) Serum-starved HSCs were incubated with 100 ng/mL CCL21 for the indicated time points. Total RNA was analyzed by RNase protection assay as described in Materials and Methods. The increase in chemokine expression relative to unstimulated samples, as assessed by densitometry, is shown in the bottom barograms. (B) Serum-starved HSCs were incubated with 10 or 100 ng/mL CCL21, as indicated, for 24 hours, and the concentration of CCL2 in the supernatant was measured by enzyme-linked immunosorbent assay. Mean ± SEM of 3 experiments. ∗P < 0.05 vs. unstimulated cells.
Gastroenterology  , DOI: ( /S (03) )