The chemokine CCL21 modulates lymphocyte recruitment and fibrosis in chronic hepatitis C

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GASTROENTEROLOGY 2003;125:1060–1076

he Chemokine CCL21 Modulates Lymphocyte Recruitmentnd Fibrosis in Chronic Hepatitis C

NDREA BONACCHI,* ILARIA PETRAI,* RAFFAELLA M. S. DEFRANCO,* ELENA LAZZERI,‡

RANCESCO ANNUNZIATO,* EVA EFSEN,* LORENZO COSMI,* PAOLA ROMAGNANI,‡

TEFANO MILANI,‡ PAOLA FAILLI,§ GIACOMO BATIGNANI,‡ FRANCESCO LIOTTA,* GIACOMO LAFFI,*ASSIMO PINZANI,* PAOLO GENTILINI,* and FABIO MARRA*

ipartimento di *Medicina Interna, ‡Fisiopatologia Clinica, and §Farmacologia Preclinica e Clinica, University of Florence, Florence, Italy

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ackground & Aims: The chemokines CCL19 and CCL21ind CCR7, which is involved in the organization ofecondary lymphoid tissue and is expressed duringhronic tissue inflammation. We investigated the ex-ression of CCL21 and CCR7 in chronic hepatitis C. Theffects of CCL21 on hepatic stellate cells (HSCs) werelso studied. Methods: Expression of CCL21 was as-essed by in situ hybridization and immunohistochem-stry. CCR7 on T cells was analyzed by flow cytometry.ultured human HSCs were studied in their activatedhenotype. Results: In patients with chronic hepatitis C,xpression of CCL21 and CCR7 was up-regulated. CCL21as detected in the portal tracts and around inflamma-ory lymphoid follicles, in proximity to T lymphocytes andendritic cells, which contributed to expression of thishemokine. Expression of CCR7 was also increased inatients with primary biliary cirrhosis. Intrahepatic CD8�

lymphocytes isolated from patients with chronic hep-titis C had a significantly higher percentage of positivityor CCR7 than those from healthy controls, and thexpression of CCR7 was associated with that of CXCR3.ultured HSCs expressed functional CCR7, the activa-ion of which stimulated cell migration and acceleratedound healing in an in vitro model. Exposure of HSCs toCL21 triggered several signaling pathways, includingxtracellular signal–regulated kinase, Akt, and nuclearactor �B, resulting in induction of proinflammatoryenes. Conclusions: Expression of CCL21 during chronicepatitis C is implicated in the recruitment of T lympho-ytes and the organization of inflammatory lymphoidissue and may promote fibrogenesis in the inflamedreas via activation of CCR7 on HSCs.

ore than 70% of patients infected with hepatitis Cvirus (HCV) develop a chronic disease, often lead-

ng to progressive liver dysfunction and the eventualeed for transplantation. Several studies in the last de-ade have characterized the immune response that occursuring chronic hepatitis C. It is believed that the lack of

strong and/or multispecific response against viral

pitopes leads to the inability to clear the virus, resultingn chronic inflammation and liver injury.1 In this con-ext, hepatocellular damage is mediated by cytotoxic Tymphocytes and possibly other cells of the innate im-une response. Interestingly, the bulk of intrahepaticD8� T cells during active liver damage is non–antigen

pecific, suggesting the importance of virus-independentechanisms of inflammatory cell recruitment.1 Thus,

nderstanding the molecular mechanisms leading toymphocyte infiltration within the liver is crucial toevise better therapeutic strategies for this disease.The crosstalk among resident and infiltrating cells

ithin a given tissue is critically dependent on cytokinesnd chemokines.2 Chemokines are a family of cytokinesharacterized by the ability to induce migration of dif-erent cell types, including leukocytes, through an actionn specific receptors.3 Chemokine receptors belong to thetrans-membrane domain group of G protein–coupled

eceptors and activate different intracellular signalingathways based on the type of receptor and the targetell. Indeed, numerous studies have shown that chemo-ine receptors are expressed by endothelial cells, fibro-lasts, pericytes, and epithelial and cancer cells.3 Ingreement with this observation, it has been shown thathe chemokine system plays an important role in theegulation of many physiologic and pathologic condi-ions, including leukocyte homeostasis and recruitmento injured tissues, development, fibrosis, angiogenesis,nd cancer. The chemokines CCL21 (also known as SLCr exodus 2) and CCL19 (ELC or MIP-3�) bind theeceptor CCR7 and are involved in the organization of

Abbreviations used in this paper: ERK, extracellular signal–regulatedinase; HAI, histologic activity index; HSC, hepatic stellate cell; IHL,ntrahepatic lymphocyte; NF-�B, nuclear factor �B; PBL, peripherallood lymphocyte; PDGF, platelet-derived growth factor; PI3-K, phos-hatidylinositol 3-kinase; RNase, ribonuclease.

© 2003 by the American Gastroenterological Association0016-5085/03/$30.00

doi:10.1053/S0016-5085(03)01194-6

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normal lymphoid tissue during development.4 CCL21 isexpressed by high endothelial venules in lymph nodesand Peyer’s patches and by stromal cells in the T-cellareas of secondary lymphoid organs.4 Conversely, CCR7is expressed by naive and central memory T cells and bydendritic cells on maturation.5–7 Mice lacking the ex-pression of CCL21 or with targeted disruption of theCCR7 gene show profound alterations in the architectureof lymph nodes, suggesting that this system plays amajor role in the de novo formation of lymphoid tis-sue.8,9

The presence of chronic inflammation and persistentliver damage during chronic hepatitis C is often accom-panied by the development of fibrosis and cirrhosis.These events result from chronic activation of a “wound-healing” process in a setting in which removal of theinjuring agent cannot be achieved. Hepatic stellate cells(HSCs), together with other liver-derived myofibroblasts,represent the key cellular elements in the liver wound-healing process and in the development of hepatic fibro-sis.10 Following injury, HSCs undergo a process knownas “activation,” acquiring a myofibroblast-like phenotypewith the ability to proliferate, contract, migrate, andproduce extracellular matrix components.10 In addition,activated HSCs regulate the recruitment of inflammatorycells through the secretion of chemotactic factors, includ-ing chemokines such as CCL2 (monocyte chemoattrac-tant protein 1) and CXCL8 (interleukin 8).11,12 Recentdata indicate that chemokines can also exert direct pro-fibrogenic actions through the activation of chemokinereceptors expressed by HSCs.13,14 Thus, the chemokinesystem has the capability to coordinate several phases ofwound healing, including inflammation and tissue re-pair.

The aims of this study were to assess the expression ofCCL21 in the liver of patients with chronic hepatitis Cand to evaluate the possible role of this chemokine andits cognate receptor, CCR7, in the pathogenesis of lym-phocyte recruitment and fibrosis. Our data indicate thatCCL21 expression is involved in the recruitment of Tlymphocytes and the organization of newly formed lym-phoid tissue in the liver of patients with chronic HCV-related hepatitis, and that activation of CCR7 on HSCsmay contribute to the accumulation of matrix-producingcells in the inflamed areas.

Materials and MethodsReagents

Monoclonal antibodies against CCR7 (clone 2H4),CD8 (clone SK1), CD4 (clone SK3), CD3 (clone SK7 for flowcytometry or UCHT1 for immunohistology), and CD11c

(clone S-HCL-3) were from Becton Dickinson (San Diego,CA). Purified, fluorochrome-conjugated isotype control andfluorochrome-conjugated anti-isotype monoclonal antibodieswere purchased from Southern Biotechnology Associates (Bir-mingham, AL). Fluorescent secondary antibodies used in im-munofluorescence were from Molecular Probes (Eugene, OR).Goat polyclonal antibodies against CCL21 were purchasedfrom R&D Systems (Minneapolis, MN). Monoclonal antibod-ies against �–smooth muscle actin (clone 1A4) and alkalinephosphatase– conjugated anti-goat antibodies were purchasedfrom Sigma Chemical Co. (St. Louis, MO). Polyclonal anti-bodies against factor VIII were from Dako (Glostrup, Den-mark). Polyclonal antibodies against the phosphorylated formof extracellular signal–regulated kinase (ERK), Raf-1, Bad(Ser112), I�B, and Akt (Ser473) were from New EnglandBioLabs (Beverly, MA). Polyclonal anti-ERK antibodies usedfor Western blotting and polyclonal anti-Akt antibodies werefrom Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonalanti-ERK antibodies used for immune complex kinase assay,phospho-specific antibodies against activated Src (Y416), andmonoclonal anti-Src antibodies were from Upstate Biotechnol-ogy Inc. (Lake Placid, NY). Human recombinant CCL21,CCL19, and platelet-derived growth factor (PDGF)-BB werepurchased from Peprotech (Rocky Hill, NJ). Radionuclideswere purchased from ICN (Costa Mesa, CA). All other reagentswere of analytical grade.

Tissues

Normal liver tissue (8 samples) used for all experi-ments was obtained during surgical liver resection for second-ary liver cancer. The tissue was obtained at a minimum of 5 cmfrom the tumor, and normal histology was assessed by routineexamination. In 3 of these subjects, a blood sample was alsoobtained the day before surgery for analysis of peripheral bloodlymphocytes (PBLs). The expression pattern of chemokinereceptors in these subjects did not differ from that observed inPBLs obtained from an age-matched group of healthy controls(data not shown).

Samples from patients with chronic hepatitis C and cirrhosisused for in situ hybridization and immunohistochemistry wereobtained from livers explanted for orthotopic transplantation(n � 3) or resected for hepatocellular carcinoma (n � 3) at aminimum of 5 cm from the tumor. For ribonuclease (RNase)protection assay, we used the previously indicated samples orspecimens obtained by percutaneous liver biopsy (n � 19)performed in patients with chronic hepatitis C at differentgrades and stages. In addition, we analyzed total RNA ex-tracted from liver tissue derived from patients with end-stageliver disease due to primary biliary cirrhosis (n � 5) or chronichepatitis B (n � 3). The source of these latter tissues was liverexplanted for orthotopic transplantation.

For analysis of intrahepatic lymphocytes (IHLs) and PBLs, 4patients with chronic hepatitis C undergoing percutaneousneedle biopsy were studied. A fragment of the liver specimenand a blood sample were obtained on the same day. Allpatients had no history of alcohol intake or other known causes

October 2003 CCL21 IN CHRONIC INFLAMMATION AND FIBROSIS 1061

of liver damage. The specimens were snap frozen in liquidnitrogen until analyzed. The procedures followed in the studywere in accordance with the ethical standards of the RegionalCommittee on Human Experimentation.

Cloning and Sequencing of CCL19 andCCL21 Probes

Messenger RNA (mRNA) was extracted from humanthymus, reversed to first-strand complementary DNA, andamplified by reverse-transcription polymerase chain reaction aspreviously described.15 The primers used were as follows:CCL19: forward, 5�-ATGGCCCTGCTACTGGCC; reverse,5�-CAATGCTTGACTCGGACT; CCL21: forward, 5�-ATG-GCTCAGTCACTGGCT; reverse, 5�-GGCCCTTTAGGGG-TCTGT. The DNA fragments of 340 and 400 base pairs,respectively, were subcloned in PGEM-T (Promega, Madison,WI) according to the manufacturer’s instructions. Sequencingof the amplified products was performed by automated se-quencing.

In Situ Hybridization

This technique was performed as described else-where.15 Briefly, 10-�m frozen sections were cut and fixed in4% paraformaldehyde for 20 minutes. After a prehybridizationtreatment (0.2N HCl, 0.125 mg/mL pronase, 4% paraformal-dehyde, acetic anhydride 1:400 in 0.1 mol/L triethanolaminebuffer, pH 8), sections were dehydrated in increasing ethanolconcentrations. Thirty microliters of hybridization solution(4� standard saline citrate, 1� Denhardt’s solution, 10%dextran sulfate, 0.1 mg/mL sheared herring sperm DNA, and1 mg/mL yeast transfer RNA) containing 8 � 105 cpm of35S-labeled RNA probe was applied on each section and cov-ered with Parafilm (American National Can, Menasha, WI).Sense and antisense RNA-radiolabeled probes were synthesizedusing SP6 or T7 RNA polymerases as appropriate (RiboprobeGemini System; Promega) in the presence of �-35S-thiouridi-netriphosphate (1300 mCi/mmol; NEN-Du Pont, Paris,France). RNA probes were extracted with phenol/chloroform,ethanol precipitated, and subsequently subjected to alkalinedigestion. Hybridization was performed at 52°C for 16 hours.After that, sections were washed and autoradiography wasperformed. Exposure time varied from 15 to 20 days. Sectionswere developed in D19, fixed in Kodak fixative (EastmanKodak, Rochester, NY), counterstained with H&E/phloxine,and mounted with Permount (ProSciTech, Thuringowa Cen-tral Qld, Australia). Negative controls consisted of sectionshybridized with a sense RNA probe.

Immunohistochemistry

These experiments were performed on frozen sectionsas described in detail elsewhere.16,17 Dried sections were se-quentially incubated with the primary antibody and, afterwashing, with mouse monoclonal anti-rabbit antibodies andfinally with affinity-purified rabbit anti-mouse antibodies. Atthe end of the incubation, sections were washed twice inTris-buffered saline and then incubated with APAAP (alkaline

phosphatase anti alkaline phosphatase) and developed. Nega-tive controls were treated with omission of the primary anti-body or its substitution with nonimmune rabbit immuno-globulins. When a goat antibody was used as a primaryantibody, alkaline phosphatase– conjugated anti-goat immu-noglobulins were used directly. When monoclonal antibodieswere used as a primary antibody, incubation with monoclonalanti-rabbit antibodies was omitted.

Immunofluorescence

Six-micrometer-thick sections were cut from frozentissue, allowed to dry on glass slides, and sequentially fixed for30 minutes in acetone and chloroform. Sections were thenwashed 3 times for 5 minutes in PBST (50 mmol/L Tris-HCl,pH 7.4, 150 mmol/L NaCl, 0.1% Triton X-100), incubatedfor 60 minutes with blocking buffer (5.5% horse serum inPBST), and then incubated with primary antibodies (1:100dilution in phosphate-buffered saline containing 3% bovineserum albumin) for 20 hours at 4°C. After washing with PBSTcontaining 0.1% bovine serum albumin for 15 minutes, sec-tions were incubated with secondary antibodies diluted inPBST/3% bovine serum albumin for 1 hour at room temper-ature and washed 3 times for 5 minutes in PBST and once fora few seconds in deionized water. Finally, slides were mountedusing mounting medium and a coverslip and viewed with afluorescence microscope (Leica, Wetzlar, Germany).

Cell Culture

Human HSCs were isolated from wedge sections ofliver tissue unsuitable for transplantation by collagenase/pro-nase digestion and centrifugation on Stractan gradients. Pro-cedures used for cell isolation and characterization have beenextensively described elsewhere.18 All of the experiments wereperformed on cells cultured on uncoated plastic dishes, show-ing an “activated” or “myofibroblast-like” phenotype.

Isolation of IHLs and Flow Cytometry

Liver fragments were gently disaggregated through astainless-steel mesh (Medimachine; Becton Dickinson) to ob-tain single-cell suspensions from which mononuclear cells wereseparated by centrifugation on Ficoll-Hypaque gradient. Aftersaturation of nonspecific binding sites with total rabbit im-munoglobulin G, mononuclear cells were incubated for 20minutes on ice with specific or isotype-control antibodies. Inthe indirect staining, this step was followed by a secondincubation on ice with an appropriate anti-isotype– conjugatedantibody (Southern Biotechnology Associates). Finally, cellswere washed and analyzed on a BD-LSR cytofluorimeter usingCellQuest software (Becton Dickinson). In all cytofluorometricanalyses, 104 events were acquired for each sample. A similarprocedure was followed for analysis of cultured HSCs.

Intracellular Calcium Concentration

Digital video imaging of intracellular-free calciumconcentration ([Ca2�]i) in individual human HSCs was per-formed as described previously.19 Human HSCs were grown to

1062 BONACCHI ET AL. GASTROENTEROLOGY Vol. 125, No. 4

subconfluence in complete culture medium on round glasscoverslips (25-mm diameter, 0.2-mm thick) for 72 hours andthen incubated for 48 hours in serum-free, insulin-free me-dium. Cells were then loaded with 4 �mol/L Fura-2AM and15% Pluronic F-127 for 30 minutes at 22°C. [Ca 2�]i wasmeasured in Fura-2–loaded cells in HEPES-NaHCO3 buffercontaining 140 mmol/L NaCl, 3 mmol/L KCl, 0.5 mmol/LNaH2PO4, 12 mmol/L NaHCO3, 1.2 mmol/L MgCl2, 1.0mmol/L CaCl2, 10 mmol/L HEPES, and 10 mmol/L glucose,pH 7.4. Ratio images (340/380 nm) were collected every 3seconds, and calibration curves were obtained for each cellpreparation.19 CCL21 or CCL19 (10 or 100 ng/mL) were addeddirectly to the perfusion chamber immediately after recordingthe [Ca2�]i basal value and maintained throughout the dura-tion of the experiment.

Cell Proliferation

Measurement of thymidine incorporation was per-formed on serum-starved, confluent HSCs incubated with ago-nists for 24 hours and then pulsed with [3H]thymidine. DNAsynthesis was measured as described elsewhere.20 Measurementof cell number was conducted in serum-starved, subconfluentcells plated on 12-well dishes and exposed to CCL21 or otherconditions for 5 days. Cells were then washed with phosphate-buffered saline and incubated with 0.5% (wt/vol) crystal violetin 20% methanol for 5 minutes at room temperature. After 3washes with phosphate-buffered saline, the monolayer wasincubated with 0.1 mol/L sodium citrate, pH 4.2, for 1 hourat room temperature. At the end of incubation, an aliquot ofthis solution was transferred to a 96-well dish and read at 565nm. All assays were performed in triplicate.

Cell Migration

Confluent HSCs were serum starved for 48 hours andthen washed, trypsinized, and resuspended in serum-free me-dium containing 1% albumin at a concentration of 3 � 105

cells/mL. Chemotaxis was measured in modified Boyden cham-bers equipped with 8-�m pore filters (Poretics, Livermore,CA) coated with rat tail collagen (Collaborative BiomedicalProducts, Bedford, MA) as previously described.21 When in-hibitors were used, cultured cells were incubated with thedrugs to be tested or with their vehicle for 15 minutes beforetrypsinization and equal concentrations were added in theBoyden chamber.

Wound-Healing Assay

HSCs were plated on 6-well culture dishes, grown tosubconfluency, and serum starved for 24 hours. The monolayerwas discontinued by producing 3 parallel scratches with asterile pipette tip. After 24 hours, the cells were washed withphosphate-buffered saline and stained with 0.5% (wt/vol) crys-tal violet in 20% methanol.

RNase Protection Assay

Total RNA was isolated from frozen liver tissue usingNucleospin columns (Mackerey-Nagel, Durer, Germany).

RNA from cultured HSCs was isolated with RNAFast (Mo-lecular Systems, San Diego, CA). Integrity of RNA waschecked by agarose electrophoresis. A total of 2–10 �g of totalRNA were used for RNase protection assay using a commer-cially available kit from BD Pharmingen, San Diego, CA.32P-labeled complementary RNA was transcribed from Mul-tiProbe templates (BD Pharmingen) according to the manu-facturer’s instructions. After hybridization, protected frag-ments were separated on a sequencing gel as previouslydescribed.22 The specific signals were quantitated by densi-tometry.

Preparation of Nuclear Extracts and GelMobility Shift Assay

Nuclear extracts were prepared according to Andrewand Faller.23 Gel mobility shift analysis was performed usingnuclear factor �B (NF-�B) consensus oligonucleotides (Pro-mega) as described elsewhere.20 Briefly, 5 �g of nuclear ex-tracts was incubated for 30 minutes at room temperature in abuffer containing 35 mmol/L HEPES, pH 7.8, 0.5 mmol/Lethylenediaminetetraacetic acid, 0.5 mmol/L dithiothreitol,10% glycerol, 10 �g/mL poly-dI-dC, 0.28 mmol/L spermi-dine, and 100,000 cpm of 32P-labeled oligonucleotide probe.The DNA-protein complexes were separated by polyacryl-amide gel electrophoresis in 0.5� Tris/borate/ethylenedia-minetetraacetic acid. At the end of the run, the gel was driedand autoradiographed.

CCL2 Enzyme-Linked Immunosorbent Assay

Serum-deprived subconfluent HSCs were exposed toincreasing concentrations of CCL21 for 24 hours. At the end ofincubation, aliquots of the conditioned medium were dilutedwith nonconditioned medium and assayed for CCL2 using acommercially available enzyme-linked immunosorbent assaykit (Biosource, Camarillo, CA).

Preparation of Cell Lysates and WesternBlotting

Confluent, serum-starved HSCs were treated with theappropriate conditions, quickly placed on ice, and washed withice-cold phosphate-buffered saline. The monolayer was lysed inRIPA buffer (20 mmol/L Tris-HCl, pH 7.4, 150 mmol/LNaCl, 5 mmol/L ethylenediaminetetraacetic acid, 1% NonidetP-40, 1 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonylfluoride, 0.05% wt/vol aprotinin). Insoluble proteins werediscarded by high-speed centrifugation at 4°C. Protein con-centration in the supernatant was measured in triplicate usinga commercially available assay (Pierce, Rockford, IL). Equalamounts of total cellular proteins were separated by sodiumdodecyl sulfate/polyacrylamide gel electrophoresis and ana-lyzed by Western blot as previously described.14

ERK Assay

ERK was immunoprecipitated from 250 �g of totalcell lysate using polyclonal anti-ERK antibodies and protein


A/Sepharose. After washing, the immunobeads were incubatedin a buffer containing 10 mmol/L HEPES, pH 7.4, 10 mmol/LMgCl2, 0.5 mmol/L dithiothreitol, 0.5 mmol/L Na3VO4, 25�mol/L adenosine triphosphate, 1 �Ci [32P]adenosinetriphosphate, and 0.4 mg/mL myelin basic protein for 30minutes at 30°C. At the end of the incubation, the reactionwas stopped by addition of Laemmli buffer and run on 15%sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Af-ter electrophoresis, the gel was dried and autoradiographed.

Data Presentation and Statistical Analysis

Autoradiograms and autoluminograms are representa-tive of at least 3 experiments with comparable results. Unlessotherwise indicated, bar graphs show the mean SE of datafrom a representative experiment. Statistical analysis was per-formed by using Student t test or 1-way analysis of variancewith Tukey’s test for multiple comparisons as appropriate.

ResultsIncreased Expression of CCR7 and CCL21During Chronic Hepatitis C

We first tested whether mRNA levels for thisreceptor were increased in patients with chronic hepatitisC and fibrosis. Gene expression of CCR7 was barelydetectable in normal human liver tissue and clearly up-regulated in patients with chronic hepatitis C, in whicha statistically significant 3-fold increase was observed(Figure 1). We next evaluated the expression of CCL21

and CCL19, the 2 known ligands of CCR7, in controlliver tissue and during chronic hepatitis C. In situ hy-bridization using a probe for CCL21 showed little, if any,signal in control liver tissue (Figure 2A). In contrast, inthe liver of patients with chronic hepatitis C, mRNAtranscripts for CCL21 were markedly up-regulated (Fig-ure 2B and C). Hybridization signal was evident in theenlarged portal tract and along fibrotic septa that sur-round regenerating nodules (Figure 2B). In addition,CCL21 mRNA was expressed at the periphery of theinflammatory lymphoid follicles that are frequently ob-served in the portal tract of patients with chronic hepa-titis C (Figure 2C). Of note, expression of this chemokinewas almost absent in the internal portion of the follicle.

We next performed immunostaining of liver tissuewith antibodies directed toward human CCL21 (Figure3). In control tissue, only a few scattered cells with aspindle-shaped appearance were immunostained in theportal tracts. Analysis of tissues from patients withchronic hepatitis C clearly confirmed the increased ex-pression of CCL21 and the pattern of distribution pre-viously observed by in situ hybridization. In fact, evidentimmunostaining was found in the portal tract, along thefibrotic septa (Figure 3B), and at the periphery of in-flammatory lymphoid follicles (Figure 3C). Similar towhat was observed by in situ hybridization, CCL21expression was more marked at the periphery of thefollicles. We also analyzed liver expression of CCL19, theother CCR7-binding chemokine, by in situ hybridiza-

Figure 1. Expression of the chemokine receptor CCR7 is increased inpatients with chronic hepatitis C. (A) Total RNA was isolated fromspecimens of normal human liver (NHL, lanes 3–5) or from patientswith chronic HCV–related hepatitis (CHC, lanes 6–9). Ten microgramsof RNA was analyzed by RNase protection assay as described inMaterials and Methods. In lane 1, total RNA isolated from humanperipheral blood mononuclear cells was run as a positive control; inlane 2, transfer RNA was run as a negative control. (B) Relativeexpression of CCR7 to the housekeeping gene GAPDH as assessedby densitometry. *P � 0.05.

Figure 2. Hepatic gene expression of CCL21 during chronic hepatitisC. (A) Expression of CCL21 was assessed by in situ hybridization, andCCL21 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 pe-riphery of inflammatory lymphoid follicles within the portal tracts(arrows). (D) No signal was observed when specimens of chronichepatitis C were hybridized with the sense probe (negative control).(Original magnification 60�.)


tion. Surprisingly, no signal was present either in controlliver tissue or in tissue obtained from patients withchronic hepatitis C (data not shown). Thus, up-regulatedexpression of CCR7 in the liver is associated with in-creased levels of only 1 of its cognate ligands, namelyCCL21.

To obtain information on the cell types involved inCCL21 expression or targeted by its action, serialsections of liver specimens from patients with chronichepatitis C were immunostained with different anti-bodies directed against specific cellular markers andcompared with the expression pattern observed afterstaining with CCL21 (Figure 4). We focused on portaltracts with inflammatory lymphoid follicles, where themost marked and characteristic expression of CCL21could be observed (see Figures 2 and 3). T lympho-cytes identified by CD3 staining were presentthroughout the portal tracts and in the inflammatorylymphoid follicles (Figure 4B). In contrast, CD20� Blymphocytes were present at the center of the follicles,as observed in secondary lymphoid tissue (data notshown). Interestingly, CD8�T lymphocytes were moreabundant at the edges of the follicles, adjacent to theareas where CCL21 expression is more marked (Figure4C), suggesting that these cells could be the target ofthe action of this chemokine. Staining for CD11c, amarker of activated dendritic cells, was detected in thesame areas (Figure 4D). These data indicate that ex-pression of CCL21 in the areas of dense inflammatory

infiltration during chronic hepatitis C is associatedwith the presence of activated dendritic cells andCD8� T lymphocytes. In contrast, no colocalizationcould be observed with factor VIII, indicating thatendothelial cells do not significantly contribute to theexpression of this chemokine during chronic hepatitisC (Figure 4F). An intriguing finding is the clearcolocalization of CCL21 expression with �–smoothmuscle actin—positive cells (Figure 4E). Because�–smooth muscle actin is a marker of activated stel-late cells and liver myofibroblasts, this finding sug-gests that expression of CCL21 could be related to thebiology of cells responsible for liver tissue repair andfibrogenesis.

We tried more direct experimental approaches toestablish the role played by the different cell types inthe expression of CCL21. Unfortunately, a combina-tion of immunohistochemistry and in situ hybridiza-tion was not successful, because the CCL21 signaltended to be less evident when these techniques wereused together. To establish the possible role of acti-

Figure 3. Up-regulated expression of CCL21 during chronic hepatitisC. (A) Protein expression of CCL21 was found in a few star-shapedcells in the portal tracts of normal human liver. (B) Increased expres-sion was present in the portal tracts in patients with chronic hepatitisC, especially at the septum/nodule interface (short arrows) or incorrespondence 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�.)

Figure 4. Colocalization of CCL21 expression with different cellularmarkers. (A–F) Serial sections from livers with chronic hepatitis Cwere immunostained with antibodies against (A) CCL21, (B) CD3, (C)CD8, (D) CD11c, (E) �–smooth muscle actin, or (F ) factor VIII. Anenlarged portal tract with an inflammatory lymphoid follicle (f) isshown. Expression of CCL21 is evident in areas where (C) CD8� Tlymphocytes and (D) mature dendritic cells accumulate. (B) CCL21-expressing cells are also surrounded by �–smooth muscle actin—positive cells. (G) Combined immunofluorescence for CCL21 (leftpanel, green) and CD11c (right panel, red). The middle panel showsthe overlaid pictures, and yellow color indicates signal colocalization.(Original magnification 240�.)


vated dendritic cells in the expression of CCL21, weperformed double immunofluorescence using antibod-ies directed against CCL21 and CD11c (Figure 4G). Aclear, albeit partial, colocalization was present in sev-eral cells, as indicated by the yellow color in theoverlaid pictures. The possible contribution of acti-vated HSCs was also evaluated, assessing the ability ofthese cells in culture to express CCL21. However,little, if any, signal was present when HSC cultureswere immunostained with anti-CCL21 antibodies orwhen mRNA was analyzed by reverse-transcriptionpolymerase chain reaction (data not shown). Thus,activated dendritic cells, together with other cellularelements, participate in CCL21 expression in thissetting.

Expression of CCR7 in Different Grades andStages of Chronic Hepatitis C and in OtherForms of Chronic Liver Disease

Data from immunohistochemistry and in situ hy-bridization as previously described were obtained in tis-sue from patients with end-stage liver disease associatedwith chronic HCV-related hepatitis. To establishwhether the components of the CL21/CCR7 axis werealso up-regulated in earlier stages of chronic hepatitis C,we analyzed total RNA isolated from specimens of 19patients with chronic hepatitis C undergoing percutane-ous liver biopsy. These patients were subdivided accord-ing to the presence of mild inflammation and fibrosis(Ishak’s24 histologic activity index [HAI ] grade �3,stage �2; n � 8), moderate-severe inflammation andmild fibrosis (HAI grade �4, stage �2; n � 5), ormoderate-severe inflammation and fibrosis (HAI grade�4, stage �3, n � 6). As shown in Figure 5A, the levelsof CCR7 expression were significantly higher only intissues from patients with higher degrees of inflamma-tion than in normal liver tissue, irrespective of the pres-ence of fibrosis. Moreover, when expression of CCL21 wasanalyzed in specimens obtained by percutaneous biopsyfrom patients with low-stage hepatitis, up-regulated ex-pression of this chemokine was still observed (Figure5B). These data indicate that, in patients with chronichepatitis C, up-regulation of CCL21 and CCR7 expres-sion occurs independently of the presence of cirrhosis andthat chronic inflammation critically contributes to thesefindings.

We also tested whether up-regulation of CCR7 wasalso present in other forms of chronic liver disease. Whencompared with normal human liver, expression of CCR7was up-regulated in samples from patients with end-stage primary biliary cirrhosis (Figure 6) although lessmarkedly than in chronic hepatitis C. Although CCR7

expression was also higher in patients with chronic hep-atitis B virus–related chronic liver disease than in normalliver (0.20 0.09 vs. 0.09 0.06 CCR7/GAPDHdensitometry units), the difference did not reach statis-tical significance (P � 0.26).

Increased Expression of CCR7 in Liver-Infiltrating Lymphocytes During ChronicHepatitis C

Because T cells play a major role in the patho-genesis of liver damage in HCV-infected patients, wetested surface expression of CCR7 in T lymphocytesisolated from control liver tissue and from the liver of

Figure 5. Expression of CCR7 in patients with different grades orstages of chronic hepatitis C. (A) RNA isolated from normal livertissue (n � 3) or percutaneous liver biopsy specimens obtained frompatients with chronic hepatitis C (CHC) and mild inflammation andfibrosis (Ishak’s HAI grade �3, stage �2; n � 8), moderate-severeinflammation and mild fibrosis (HAI grade �4, stage �2; n � 5), ormoderate-severe inflammation and fibrosis (HAI grade �4, stage �3,n � 6) were analyzed by RNase protection assay for expression ofCCR7 or the housekeeping gene GAPDH. The upper panel showsrepresentative autoradiograms, and the lower panel shows densitom-etry data from all patients. *P � 0.05 vs. normal human liver (NHL)(one-way analysis of variance and Tukey’s test). (B) A section obtainedfrom a percutaneous liver biopsy performed in a patient with chronichepatitis C, and no cirrhosis was immunostained with antibodiesagainst CCL21. Specific signal appears as dark gray (arrows).


patients with chronic hepatitis C. These data were com-pared with those of PBLs of the same subjects. In thesame samples, we also analyzed the expression ofCXCR3, a chemokine receptor previously shown to beup-regulated in liver-infiltrating cells during chronichepatitis C and considered critical in driving the recruit-ment of activated lymphocytes to sites of chronic tissueinflammation, especially in the presence of a type 1–po-larized response.25,26 When CD8� PBLs were comparedwith CD8� IHLs, an opposite behavior was present forCCR7 and CXCR3. In fact, expression of CCR7 wassignificantly lower in CD8� IHLs than in CD8� PBLs inboth patients with hepatitis C and control subjects (Fig-ure 7A). Conversely, CXCR3 was more abundant inCD8� IHLs than in CD8� PBLs, although this differ-ence was statistically significant only for patients withhepatitis C. Comparison of the 2 groups of subjects didnot show statistically significant differences in the per-centage of CD8� PBLs expressing CCR7 or CXCR3(Figure 7A), although a trend toward a more frequentexpression of CCR7 was observed in patients with hep-atitis. In contrast, CD8� IHLs from patients withchronic hepatitis C showed a significantly higher per-centage of positivity for both CCR7 and CXCR3 thanCD8� IHLs of control subjects (Figure 7A). Analysis ofCD4� T lymphocytes again showed that CCR7-express-ing cells were found at higher levels in PBLs than in

IHLs, whereas CXCR3 was more expressed in IHLs thanin PBLs (Table 1). However, no significant differences inthe percentage of expression of CCR7 or CXCR3 werefound comparing control subjects and patients withchronic hepatitis C.

Figure 6. Expression of CCR7 in patients with primary biliary cirrho-sis. RNA isolated from normal liver tissue (n � 3) or from tissueobtained from patients with primary biliary cirrhosis at the time oforthotopic liver transplantation (n � 5) was analyzed by RNase pro-tection assay for expression of CCR7 or the housekeeping geneGAPDH. The upper panel shows representative autoradiograms, andthe lower panel shows data obtained from densitometry. *P � 0.05vs. normal human liver (NHL) (Student t test).

Figure 7. Expression of the chemokine receptors CCR7 and CXCR3by peripheral and intrahepatic CD8� T lymphocytes. (A) PBLs or IHLsfrom control subjects (black bars) or from patients with chronic hep-atitis C (white bars) were analyzed by flow cytometry for expression ofCD3, CD8, CCR7, and CXCR3. °P � 0.05 vs. IHLs; P � 0.05 vs.chronic hepatitis C. (B) CD3�, CD8� IHLs from control subjects (blackbars) or from patients with chronic hepatitis C (white bars) wereanalyzed for expression of CCR7 or CXCR3 alone (CCR7s andCXCR3s) or in combination (CCR7/CXCR3). *P � 0.05 vs. chronichepatitis C.

Table 1. Expression of the Chemokine Receptors CCR7 andCXCR3 by Peripheral and Intrahepatic CD4�

T Lymphocytes

PBLs IHLs

Control HCV Control HCV

CCR7 68.2 3.5a 71.3 3.3a 26.8 8.2 24.5 4.2CXCR3 42.7 12.4 35.2 3.9a 71.5 5.0 80.6 5.5

NOTE. PBLs or IHLs from control subjects or from patients withchronic hepatitis C (HCV) were analyzed by flow cytometry for expres-sion of CD3, CD4, CCR7, and CXCR3.aP � 0.05 vs. IHLs of the same group.


It has recently been shown that activated T lympho-cytes can maintain the expression of CCR7 during theactivation process, together with that of other chemokinereceptors, such as CXCR3 or CCR5, which appear atlater stages of activation.27–29 For this reason, we deter-mined whether CD8� T cells expressed CCR7 alone or incombination with CXCR3 (Figure 7B). Only a very lowpercentage of CD8� IHLs expressed CCR7 alone,whereas most CCR7-expressing cells also coexpressedCXCR3. As a result, the presence of double-positiveCD8� T cells was significantly higher in patients withchronic hepatitis. On the other hand, CXCR3 was ex-pressed alone in most IHLs, both from control subjectsand from patients with hepatitis, and a significantlyhigher percentage of CXCR3-expressing CD8� T cellswas found in patients with HCV-related hepatitis (Fig-ure 7B). Together, these data indicate that, in patientswith chronic hepatitis, a higher prevalence of CCR7-expressing IHLs is selectively found in the CD8� com-partment and that nearly all of these T cells coexpressCXCR3.

Expression of Functional CCR7 by CulturedHuman HSCs

HSCs express several chemokine receptors, in-cluding CXCR3 and CCR5.14,30 The expression ofCCL21 was closely colocalized with that of �–smoothmuscle actin, a marker of activated HSCs and myofibro-blasts (Figure 4), suggesting that cells responsible forliver tissue repair and fibrogenesis could be exposed tothe action of this chemokine. To establish whether thebiology of activated HSCs could be modulated byCCL21, we analyzed the expression and functionality ofCCR7 in cultured human HSCs. Using RNase protec-tion assay of total RNA isolated from cultured humanHSCs, we observed the presence of specific transcripts forCCR7 (data not shown). In addition, the expression ofCCR7 on the surface of activated HSCs was shown byflow cytometry (Figure 8A). As previously reported,these cells also expressed CXCR3, but not CCR2, thatwere used as positive and negative controls, respectively.To test whether CCR7 expressed by HSCs is functional,we evaluated the ability of CCL21 to induce an increasein [Ca2�

i] in these cells (Figure 8B and C). Intracellularcalcium concentration increased �1 minute after expo-sure to CCL21 and became approximately 6-fold greaterthan basal. Similar results on calcium influx were ob-tained using CCL19, the other CCR7-binding chemo-kine, as an agonist (data not shown). Thus, culturedhuman HSCs express functional CCR7.

Analysis of the Biologic Effects of CCL21on Cultured HSCs

Because an effect of chemokines is to induce mi-gration of target cells, we tested whether recombinantCCL21 was chemotactic for HSCs in vitro (Figure 9A). Adose-dependent stimulation of HSC migration was ob-served in response to CCL21, with the maximal effect ata concentration of 100 ng/mL, which induced a 5-foldincrease compared with unstimulated cells. Exposure tohigher concentrations of CCL21 resulted in a lower che-motactic effect, a finding observed with other chemoat-tractants, including PDGF. Interestingly, the chemotac-tic effect of CCL21 on HSCs was achieved at lowerconcentrations than required to induce migration of lym-phocytes.31 Chemokines are also able to induce prolifer-ation of specific cell types, and we evaluated the mito-genic effect of CCL21 on HSCs (Figure 9). WhereasPDGF and fetal bovine serum, used as positive controls,induced a marked increase in [3H]thymidine uptake andcell number, no effects were observed in cells exposed toconcentrations of CCL21 that effectively induce cell mi-gration.

HSCs and liver myofibroblasts are effectors of thereparative response in the liver, and therefore we testedthe effects of CCL21 in an in vitro model of woundhealing. A stimulation of wound closure was producedby addition of CCL21 at concentrations able to activateCCR7 (Figure 10B and C). As expected, serum (Figure10D) or PDGF (data not shown) were markedly effectivein promoting wound healing. These data indicate thatactivation of CCR7 present on the surface of HSCs isassociated with the induction of biologic activities thatmay favor wound healing and fibrogenesis.

Binding of CCL21 to CCR7 Leads toActivation of Multiple Protein Kinasesin HSCs

The activation of chemokine receptors triggersseveral different signaling pathways, which representsthe molecular basis for the biologic effect of chemokines.Exposure of cultured HSCs to CCL21 for different peri-ods of time resulted in increased phosphorylation ofRaf-1, a protein involved in activation of the Ras/ERKpathway (Figure 11A). Peak activation was observed10–20 minutes after the addition of CCL21 with areturn to basal levels within 2 hours after stimulation. Inthe ERK cascade, Raf-1 is the activator of MEK 1/2,which in turn phosphorylates and activates ERK 1/2.Using phospho-specific antibodies, we observed an in-creased phosphorylation of both ERK isoforms, namelyp44ERK-1 and p42ERK-2 with a similar time course as


Figure 8. Expression of func-tional CCR7 on activatedHSCs. (A) Cultured HSCs weredetached from the dish and an-alyzed by flow cytometry withantibodies directed against thechemokine receptors CXCR3,CCR2, or CCR7 as indicated(white area). Signal from iso-type-specific controls is indi-cated by the gray area. (B)HSCs were seeded on glasscoverslips and loaded with thefluorescent indicator Fura-2. Af-ter addition of 100 ng/mLCCL21 (time 0), fluorescencewas recorded every 12 sec-onds as described in Materialsand Methods. (C) Time courseof the average calcium concen-tration in a group of HSCs.


Raf-1 (Figure 11A). Moreover, ERK phosphorylationinduced by CCL21 was associated with increased cata-lytic activity, as assessed using an immune complexkinase assay (Figure 11B). One of the downstream targetsof ERK is represented by p90RSK, which in turn phos-phorylates and inactivates the proapoptotic protein Badon serine 112. Accordingly, exposure of activated HSCsto CCL21 resulted in long-lasting phosphorylation ofBad on serine 112 (Figure 11A). Dose-response experi-ments (Figure 9C) indicated that peak activation of ERKwas obtained with the same concentration (i.e., 100ng/mL) that maximally affected HSC migration (seeFigure 9).

The nonreceptor tyrosine kinase Src plays an im-portant role in the downstream signaling of several Gprotein– coupled receptors. Therefore, we assessed theability of CCL21 to activate Src in HSCs. Activation ofCCR7 was associated with an increase in Src phos-phorylation on the activation-specific tyrosine 416(Figure 12A). Phosphatidylinositol 3-kinase (PI3-K)/Akt is another signaling pathway frequently activatedby G protein– coupled receptors and is implicated inthe regulation of several cellular functions modulatedby chemokines, such as migration and cell survival.Akt phosphorylation on the activation-specific residueserine 473 was markedly increased in HSCs exposed toCCL21 (Figure 12B). Taken together, these data in-dicate that activation of CCR7 in HSCs leads to

activation of multiple intracellular signaling path-ways, including Ras/ERK, Src, and PI3K/Akt.

To explore the relevance of the signaling pathwaysactivated by CCL21 in mediating the biologic effects ofCCR7 activation in HSCs, we used specific pharmaco-logic inhibitors of the different pathways. CCL21-in-duced migration of HSCs was markedly inhibited bydrugs interfering with the activation of ERK, Src, orPI3K (Figure 12C). Thus, activation of these pathways isnecessary for CCR7-related chemotaxis of HSCs.

CCL21 Activates Nuclear Signaling

Similar to other cytokines, the ability of che-mokines to modulate the biology of target cells isdependent on changes in gene transcription. The ef-fects of CCL21 in HSCs were assessed on 2 nuclearsignaling pathways, such as Stat3 and NF-�B. Acti-vation of Stat3 by tyrosine phosphorylation results indimerization of cytosolic Stat monomers and theirtranslocation to the nucleus, where they act as tran-scription factors. In HSCs exposed to CCL21, a rapidand reversible increase in tyrosine phosphorylation ofStat3, as assessed by phospho-specific antibodies, wasobserved (Figure 13A). This effect was comparable tothat induced by PDGF, a well-established activator ofStat3.

NF-�B is another transcription factor that has mul-tiple effects on the biology of different cells, including

Figure 9. CCL21 induces migration but not proliferation of HSCs. (A)Confluent HSCs were serum deprived for 48 hours, and cell migrationwas measured using modified Boyden chambers with increasing con-centrations of CCL21 or 10 ng/mL PDGF (positive control) as ago-nists. (B) Serum-starved HSCs were incubated with the indicatedconcentrations of CCL21, 10 ng/mL PDGF, or 10% serum. After 20hours, cells were pulsed with [3H]thymidine and DNA synthesis wasmeasured as described in Materials and Methods. (C) Conditionswere identical to those described for B. After 96 hours, cell numberwas measured by crystal violet staining.

Figure 10. CCL21 accelerates in vitro wound healing. Confluent,culture-activated HSCs were serum deprived for 48 hours. A woundwas produced in the monolayer with a pipette tip, and the cells wereexposed to (A) serum-free medium alone, (B) 10 ng/mL CCL21, (C)100 ng/mL CCL21, or (D) 10% fetal bovine serum. Twenty-four hourslater, the cells were fixed and colored and the wounds were photo-graphed.


HSCs. Activation of NF-�B requires phosphorylationof the inhibitory protein I�B, which is subsequentlydegraded through the ubiquitin-proteasome pathway.CCL21 induced a rapid phosphorylation of I�B (Fig-ure 13B), which lasted for at least 60 minutes afteraddition of the cytokine. When nuclear extracts ofHSCs treated with CCL21 were analyzed by electro-phoretic mobility shift assay using a consensus oligo-nucleotide for NF-�B, a retarded complex was ob-served that was more evident after 30 minutes ofstimulation (Figure 13C).

Induction of NF-�B–Regulated ChemokineGenes by CCL21

Activation of NF-�B is implicated in the tran-scription of several genes involved in inflammatory re-sponses, including chemokines. To establish whetheractivation of NF-�B by agonists of CCR7 has any effecton NF-�B–regulated genes, we analyzed the expressionof CXCL8 (known as interleukin 8) and CCL2 (monocytechemoattractant protein 1) in HSCs exposed to CCL21(Figure 14A). mRNA levels for both chemokines wereincreased by CCL21 treatment, with the effect moremarked for CXCL8. Increased mRNA levels for thesechemokines were associated with a significant increase inthe accumulation of CCL2 in HSC culture supernatant

Figure 11. CCL21 activates the Ras/ERK cascade in HSCs. (A) Se-rum-starved HSCs were incubated with 100 ng/mL CCL21 for theindicated time points. Total proteins were separated by sodium do-decyl sulfate/polyacrylamide gel electrophoresis and analyzed by im-munoblotting using the indicated antibodies. The molecular weightmarkers are shown on the left. (B) The experiment was performedexactly as described in A. Fifty micrograms of protein was immuno-precipitated with anti-ERK antibodies, and the immunobeads wereused for ERK assay as described in Materials and Methods. (C)Serum-starved HSCs were exposed to increasing concentrations ofCCL21 for 10 minutes. Total proteins were analyzed by immunoblot-ting using the indicated antibodies.

Figure 12. CCL21 activates Src and Akt. (A) Serum-starved HSCswere incubated with 100 ng/mL CCL21 for the indicated time points.Total proteins were separated by sodium dodecyl sulfate/polyacryl-amide gel electrophoresis and analyzed by immunoblotting using theindicated antibodies. (B) Serum-starved HSCs were exposed to in-creasing concentrations of CCL21 for 10 minutes. Total proteins wereanalyzed by immunoblotting using the indicated antibodies. (C) HSCswere incubated with the MEK inhibitor PD98059 (30 �mol/L), theinhibitor of Src, PP1 (5 �mol/L), or the PI3-K inhibitor LY294002 (10�mol/L) before stimulating migration with 100 ng/mL CCL21.


(Figure 14B). Inhibition of NF-�B activation by theproteasome inhibitor MG-132 blocked the effects ofCCL21 on CCL2 secretion (data not shown). Taken to-gether, these results show that activation of NF-�B byCCL21 is accompanied by proinflammatory actions onHSCs such as the induction of other chemokines.

DiscussionDuring chronic HCV infection, tissue damage is

associated with persistent recruitment of inflammatorycells and activation of mesenchymal cells, resulting inthe deposition of extracellular matrix and progressivefibrosis. Accumulating data indicate that cell-to-cell

communication via secretion of cytokines is one of thecrucial mechanisms modulating the hepatic wound-heal-ing response. In this study, we have identified the axiscomprised by the chemokine receptor CCR7 and itsligand, CCL21, as a novel system involved in the recruit-ment of T lymphocytes and the development of fibrosisduring chronic hepatitis C. CCL21 was markedly up-regulated in the liver of patients with chronic hepatitisand was associated with increased liver infiltration ofCCR7-expressing cells and increased CCR7 gene expres-sion. CCR7 is expressed by naive cells and some subsetsof activated T lymphocytes and by dendritic cells as partof the maturation process that renders them capable tomigrate to lymph nodes to present the antigen.5–7 Inpatients with chronic hepatitis C, expression of CCL21was localized in areas of inflammation in the enlargedportal tracts and characteristically surrounded the in-flammatory lymphoid follicles that have been described

Figure 13. Activation of transcription factors by CCL21 in HSCs. (Aand B) Serum-starved HSCs were incubated with 100 ng/mL CCL21for the indicated time points. Total proteins were separated by sodiumdodecyl sulfate/polyacrylamide gel electrophoresis and analyzed byimmunoblotting using the indicated antibodies. (C) Serum-starvedHSCs were incubated with 100 ng/mL CCL21 for the indicated timepoints. Ten micrograms of nuclear extracts was used for electro-phoretic mobility shift assay with a radiolabeled consensus oligonu-cleotide for the transcription factor NF-�B. The arrow indicates migra-tion of the specific complex.

Figure 14. Up-regulation of CCL2 and CXCL8 expression by CCL21.(A) Serum-starved HSCs were incubated with 100 ng/mL CCL21 forthe indicated time points. Total RNA was analyzed by RNase protec-tion assay as described in Materials and Methods. The increase inchemokine expression relative to unstimulated samples, as as-sessed 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 thesupernatant was measured by enzyme-linked immunosorbent assay.Mean SEM of 3 experiments. *P � 0.05 vs. unstimulated cells.


in the liver of patients with this disease.32 The expressionpattern of CCL21 and the cellular distribution in theinflammatory lymphoid follicles, with T lymphocytesdistributed throughout the follicle and B cells in thecentral area, strikingly recall secondary lymphoid tissue.CD11c-positive, activated dendritic cells were found atthe periphery of the follicle, in close proximity to thearea showing CCL21 immunostaining. In addition, thesecells costained with CCL21 in double immunofluores-cence experiments, indicating that mature dendritic cellstake part in the expression of CCL21 during chronichepatitis C, although other cell types are likely to con-tribute.33 Taken together, these data suggest that CCL21expression in the liver of patients with chronic hepatitisC may contribute to the formation and organization of a“tertiary” lymphoid tissue, which has been described indifferent conditions of chronic inflammation.34,35 Severallines of evidence support this view. In a murine model ofgranulomatous liver disease caused by injection of Pro-pionibacterium acnes, formation of granulomas takes placein the sinusoids, whereas the portal tracts are occupied bya dense follicular infiltrate that has been named “portal-associated lymphoid tissue.”35 In this model, CD11c-positive, mature dendritic cells express CCR7 and arerecruited to the portal tracts, and neutralization ofCCL21 by passive immunization prevents the formationof this lymphoid tissue. This series of events is likely tobe common to several conditions of chronic hepaticinflammation, because expression of CCL21 and forma-tion of portal-associated lymphoid tissue has been re-cently described in the liver of patients with primarysclerosing cholangitis.33 The role of CCL21 is also sug-gested by the observation that transgenic expression oflymphotoxin � in the kidney or pancreas is associatedwith newly formed lymphoid tissue, whereas expressionof CCL21 has a pattern similar to that observed in theportal tracts during hepatitis C.36 Finally, ectopic expres-sion of CCL21 was shown to be sufficient to organizelymphoid neogenesis even at sites different from second-ary lymphoid tissue.37

Analysis of the expression pattern of chemokine recep-tors during chronic hepatitis C has shown a markedincrease in the expression of CXCR3, CCR5, and CCR6in liver-infiltrating T lymphocytes, where these receptorswere generally coexpressed.25,38 Expression of CXCR3and CCR5 is acquired by activated T cells, particularly inconditions of type 1 polarization, such as that associatedwith secretion of high amounts of tumor necrosis factor� and interferon gamma.26 In this study, we confirmedthat most liver T cells express CXCR3 and identified apopulation of CCR7-expressing IHLs that are the target

of the chemotactic action of CCL21. The percentage of Tcells that express CCR7 was not different comparing theCD4� population in control tissue and chronic hepatitis,whereas a significantly higher percentage of positivitywas found in CD8� IHLs from patients with hepatitis C.The selective enrichment in CCR7 and CXCR3 expres-sion in the CD8� population is a relevant finding, be-cause this subset of T cells has been shown to play acritical role in the immunopathogenesis of hepatitis Cthrough both direct cytotoxicity and cytokine secretion.1

Along these lines, it is interesting to observe that CD8�

cells are located at the periphery of the inflammatorylymphoid follicle, where CCL21 expression is moremarked, another finding that recalls the structure ofsecondary lymphoid tissue.

The association between overexpression of CCR7 andliver inflammation is underscored by the observation thatpatients with chronic hepatitis C and more evident portaland periportal inflammation had higher expression levelsof this chemokine than those with modest leukocyteinfiltration, irrespective of the presence of advanced fi-brosis. The fact that the presence of fibrosis is lessrelevant than inflammation is also supported by theobservation that the increase in CCR7 expression wasmodest in patients with end-stage liver disease due tochronic hepatitis B virus infection. It should be notedthat increased expression of CCR7 and CCL21 is notunique to chronic hepatitis C, because similar findingshave been reported in patients with primary biliarycirrhosis or primary sclerosing cholangitis.33

Acquisition of CXCR3 or CCR5 occurs during theprocess of T-cell activation and is associated with aneffector phenotype. Although the presence of CCR7 wasbelieved to define memory cells without any effectorphenotype, recent studies performed on circulating andtissue-infiltrating lymphocytes show that, althoughCCR7 is progressively lost during differentiation, thepresence of CCR7 does not rule out an effector functionand T cells may simultaneously express both CCR7 andtissue-homing chemokine receptors (e.g., CCR5 orCXCR3) during this process.27–29 Based on these obser-vations, it may be proposed that during chronic liverinjury, such as that caused by HCV, the liver becomes anadditional site of lymphocyte activation, where T cellsare recruited via CCR7 to inflammatory lymphoid folli-cles and thereby activated, leading to acquisition ofCXCR3 expression.

If accumulation of inflammatory cells is a relevantevent for the pathogenesis of liver damage during chronichepatitis C, progression to fibrosis and cirrhosis requirespersistent activation of the cells responsible for tissue


healing within the liver. Recent studies have shown thatthe inflammatory and reparative phases of liver woundhealing are strictly connected by common cellular andmolecular mechanisms. Besides being major effectors ofliver fibrogenesis, HSCs are also capable of regulatinginflammatory cell trafficking via expression of severalcytokines and chemokines.10–12 Chemokines are not onlycritical for leukocyte recruitment but modulate the func-tion of several tissue-repairing cells, including HSCs.This concept is clearly supported by the present study,where we have identified another system that connectsinflammation and fibrosis during chronic hepatitis C.The areas of CCL21 expression were adjacent to thosewhere activated HSCs and/or myofibroblasts are located,and HSCs were found to express functional CCR7, theactivation of which induces cell migration and accelerateswound healing. Together, the available data suggestthat, in patients with chronic hepatitis C, similar mo-lecular mechanisms drive the accumulation of inflamma-tory cells and HSCs to discrete areas, such as the portaltracts, thus determining the localization of the fibrogenicresponse.

An additional novel aspect of the present study isrepresented by the elucidation of some of the signalingpathways downstream of CCR7 activation in HSCs. Thesignaling mechanisms of this chemokine receptor hadbeen only partially analyzed using T lymphocytes ortransfected cells.39 We show that activation of CCR7leads to induction of several signaling pathways, includ-ing activation of Ras/ERK and PI3-K/Akt, which hasbeen previously shown to modulate the chemotactic re-sponse of HSCs to chemokines.14 We also provide evi-dence for activation of Src, a nonreceptor tyrosine kinasethat participates in the propagation of signals generatedby different G protein– coupled receptors particularly forthe induction of tyrosine phosphorylation.40 Along theselines, we have observed CCL21-mediated tyrosine phos-phorylation of Stat3, which results in activation of thislatent transcription factor. It is likely that Stat3 activa-tion is dependent on the kinase activity of Src, becausecells overexpressing an oncogenic form of Src show acti-vation of several Stat proteins, including Stat3.41 How-ever, the possible role of other kinases, such as those ofthe Jak family, cannot presently be ruled out. The bio-logic significance of Stat3 activation in HSCs is presentlyundefined. Stat3 participates in signaling by cytokinereceptors of the interleukin-6 superfamily, and recentlySaxena et al. have shown that leptin, which inducesexpression of procollagen, also activates this pathway inrat HSCs.42 In other systems, activation of the Jak/Statpathway has been associated with induction of collagen

expression, but whether this effect may be expanded toHSCs remains to be ascertained.

The activation of NF-�B by CCL21 is an interestingobservation that is likely to have an important impact onthe biology of HSCs. Activation of NF-�B had not beenpreviously shown for CCR7, whereas other groups haverecently shown that this pathway is triggered by virus-encoded chemokine receptors.43 Activation of NF-�B iscritical to induce transcription of several proinflamma-tory genes, including those encoding for chemokines,and to generate cell-survival signals. Activation ofNF-�B by CCL21 has indeed a biologic counterpart inHSCs, because it resulted in increased expression of 2NF-�B–dependent chemokine genes such as CCL2 andCXCL8. These findings underscore yet another relevantaspect of CCL21 biology in its ability to generate localamplification of the inflammatory response via inductionof NF-�B. In addition, NF-�B, together with otherpathways such as Akt and ERK, may promote survival ofHSCs, thus contributing to the maintenance of fibrosis.Accordingly, in glomerular mesangial cells, CCL21 hasrecently been shown to reduce Fas-mediated apoptosis.44

Whether this protection from programmed cell death isshared by HSCs deserves further investigation.

In summary, this study provides evidence for the roleof a system comprised by the chemokine CCL21 and itsreceptor, CCR7, in the recruitment of lymphocytes tothe inflamed liver during chronic hepatitis C and theformation of inflammatory lymphoid tissue. By its ac-tions on HSCs, these molecules provide a link betweenthe inflammatory and reparative phases of liver woundhealing.

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Received September 14, 2002. Accepted July 10, 2003.Address requests for reprints to: Fabio Marra, M.D., Ph.D., Diparti-

mento di Medicina Interna, Viale Morgagni 85, I-50134 Florence, Italy.e-mail: [email protected]; fax: (39) 055-417123.

Supported by grants from the Italian Ministry for University andResearch, the Research Fund of the University of Florence, the ItalianMinistry of Health, and the Italian Liver Foundation. E.E. was supportedin part by the Tode Travel Grant, the Direktør Madsen’s Grant, and Fhv.Direktør Nielsen’s Grant (Denmark).

The authors thank Wanda Delogu and Nadia Navari for skillfultechnical help, Dr. Roberto G. Romanelli for help in collecting liverbiopsy specimens, and Dr. Mario Strazzabosco (Ospedali Riuniti diBergamo, Italy) for providing part of the tissue samples with primarybiliary cirrhosis.


The chemokine CCL21 modulates lymphocyte recruitment and fibrosis in chronic hepatitis C

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