Hepatic endothelial CCL25 mediates the recruitment of CCR9+ gut-homing lymphocytes to the liver in primary sclerosing cholangitis.
ABSTRACT Primary sclerosing cholangitis (PSC), a chronic inflammatory liver disease characterized by progressive bile duct destruction, develops as an extra-intestinal complication of inflammatory bowel disease (IBD) (Chapman, R.W. 1991. Gut. 32:1433-1435). However, the liver and bowel inflammation are rarely concomitant, and PSC can develop in patients whose colons have been removed previously. We hypothesized that PSC is mediated by long-lived memory T cells originally activated in the gut, but able to mediate extra-intestinal inflammation in the absence of active IBD (Grant, A.J., P.F. Lalor, M. Salmi, S. Jalkanen, and D.H. Adams. 2002. Lancet. 359:150-157). In support of this, we show that liver-infiltrating lymphocytes in PSC include mucosal T cells recruited to the liver by aberrant expression of the gut-specific chemokine CCL25 that activates alpha4beta7 binding to mucosal addressin cell adhesion molecule 1 on the hepatic endothelium. This is the first demonstration in humans that T cells activated in the gut can be recruited to an extra-intestinal site of disease and provides a paradigm to explain the pathogenesis of extra-intestinal complications of IBD.
Article: Mucosal lymphocytes in the pathogenesis of the hepatic complications of inflammatory bowel disease.Clinical medicine (London, England) 03/2008; 8(1):28-9. · 1.15 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Previous reports investigating the clinical course and management of inflammatory bowel disease (IBD) after orthotopic liver transplant (OLT) have revealed conflicting results. To determine the natural history and course of therapy for liver transplant patients with IBD, we reviewed the records of 35 patients, who underwent OLT between 1985 and 1996 and who had a history of either IBD (29 patients) or primary sclerosing cholangitis (PSC) without evidence of IBD before OLT (6 patients). Of 29 patients with IBD before OLT, 25 had a history of ulcerative colitis (UC) and 4 had Crohn's disease. Six patients had undergone total colectomy, one subtotal colectomy, and three partial colectomy before OLT. Mean follow-up after OLT was 37+/-6.4 months. Immunosuppression included cyclosporine, prednisone, and azathioprine in 34 patients and tacrolimus and prednisone in 1 patient. After OLT, 17 patients (49%) had quiescent disease and were receiving no additional medications other than standard immunosuppression to prevent organ rejection. Five patients (14%) had mild flares controlled with initiation of 5'-aminosalicylates (5'-ASA), and two patients (6%) required an increase in oral prednisone. Only one patient with PSC, without evidence of IBD before OLT, developed IBD after OLT. No patients required intravenous steroids or surgical intervention for active IBD. (1) Standard postOLT immunosuppressive agents in patients undergoing OLT with IBD were able to adequately control disease activity after OLT in the majority of patients. (2) IBD flares after OLT were generally well controlled with aminosalicylates or oral steroids. (3) Aminosalicylates were helpful in the clinical management of IBD, even when patients were taking standard doses of steroids, azathioprine, and cyclosporine.Transplantation 03/1998; 65(3):393-6. · 4.00 Impact Factor
Article: Human Peyer's patch T cells are sensitized to dietary antigen and display a Th cell type 1 cytokine profile.[show abstract] [hide abstract]
ABSTRACT: Animal studies have demonstrated that feeding Ags induces regulatory (Th2, Th3) cells in Peyer's patches (PP), which migrate to the periphery and produce immunomodulatory cytokines such as IL-4, IL-10, or TGF-beta. In this work we have attempted to extend this paradigm to man by analyzing the response of human PP T cells to in vitro challenge with the common dietary Ag beta-lactoglobulin (betalg) of cow's milk. PP T cells stimulated with betalg showed enhanced proliferation compared with blood T cells from the same patient. Increased expression of CD25 and the Th1-associated chemokine receptor CCR5 was also seen on CD4(+) and CD8(+) PP T cells, but not blood T cells, stimulated with betalg. By enzyme-linked immunospot assay and RT-PCR, the PP T cell recall response to betalg and casein was dominated by IFN-gamma, with negligible IL-4, IL-5, IL-10, or TGF-beta. To help explain the PP T cell response to betalg, we examined IL-12 expression. Both IL-12p40 and -p35 transcripts were abundantly expressed in PP, but not in adjacent normal ileal mucosa. Immunoreactive IL-12p40-containing cells were present below the PP dome epithelium. Furthermore, in culture, PP, but not paired PBMC, spontaneously released IL-12p70. These results suggest that the human response to oral Ags in the gut may be different from that in rodents.The Journal of Immunology 12/2000; 165(9):5315-21. · 5.79 Impact Factor
The Journal of Experimental Medicine
J. Exp. Med.
Volume 200, Number 11, December 6, 2004 1511–1517
The Rockefeller University Press • 0022-1007/2004/12/1511/7 $8.00
Brief Definitive Report
Hepatic Endothelial CCL25 Mediates the Recruitment of
CCR9 Gut-homing Lymphocytes to the Liver in
Primary Sclerosing Cholangitis
Patricia F. Lalor,
Allister J. Grant,
Stefan G. Hübscher,
and David H. Adams
Stuart M. Curbishley,
Liver Research Laboratories and
Birmingham, Birmingham B15 2TT, England, UK
Millennium Pharmaceuticals Inc., Cambridge, MA 02139
Department of Rheumatology, Institute for Biomedical Research, University of
Primary sclerosing cholangitis (PSC), a chronic inflammatory liver disease characterized by pro-
gressive bile duct destruction, develops as an extra-intestinal complication of inflammatory
bowel disease (IBD) (Chapman, R.W. 1991.
bowel inflammation are rarely concomitant, and PSC can develop in patients whose colons
have been removed previously. We hypothesized that PSC is mediated by long-lived memory
T cells originally activated in the gut, but able to mediate extra-intestinal inflammation in the
absence of active IBD (Grant, A.J., P.F. Lalor, M. Salmi, S. Jalkanen, and D.H. Adams. 2002.
. 359:150–157). In support of this, we show that liver-infiltrating lymphocytes in PSC
include mucosal T cells recruited to the liver by aberrant expression of the gut-specific chemo-
kine CCL25 that activates
7 binding to mucosal addressin cell adhesion molecule 1 on the
hepatic endothelium. This is the first demonstration in humans that T cells activated in the gut
can be recruited to an extra-intestinal site of disease and provides a paradigm to explain the
pathogenesis of extra-intestinal complications of IBD.
. 32:1433–1435). However, the liver and
Key words:chemokines • integrins • inflammation • hepatitis • colitis
Primary sclerosing cholangitis (PSC) develops in 2.4–
7.5% of patients with inflammatory bowel disease (IBD)
and 70–85% of patients with PSC ultimately develop IBD
(1). Any model to explain the development of hepatic
complications in IBD needs to take into account the fact
that colectomy does not alter the severity or course of
PSC and that liver disease frequently runs an independent
course from inflammation in the bowel (2). Thus, PSC
can develop for the first time many years after IBD has
become quiescent and even after previous proctocolectomy
(3). We proposed that effector T cells generated in the
organized lymphoid tissue of the gut during active IBD (4)
persist as long-lived memory cells that may be recruited to
the liver to trigger hepatic inflammation under specific
Lymphocyte recruitment from the circulation to tissue is
a highly regulated process dependent on sequential interac-
tions with endothelial adhesion molecules and chemokines
(5). Initial transient interactions between lymphocytes and
endothelium tether the lymphocyte and induce it to roll on
the vessel wall where it comes into contact with chemo-
kines. In the presence of an appropriate chemokine, spe-
cific G protein–coupled receptors on the lymphocyte are
activated, triggering high-affinity integrin binding to endo-
thelial ligands leading to arrest and transendothelial migra-
tion into tissue in response to chemotactic signals (6).
Chemokines play a crucial role in orchestrating the re-
cruitment and recirculation of leukocytes to lymphoid organs
and peripheral tissues as well as to sites of inflammation (7).
Tissue-specific combinations of chemokines and endothelial
adhesion molecules provide a molecular “address” that can
be recognized by particular subsets of circulating leukocytes
(8, 9). Thus, the chemokines CCL19 and CCL21 and the
peripheral node addressin are crucial for the structural and
functional organization of secondary lymphoid tissues by
Address correspondence to David H. Adams, Liver Research Laboratories,
Institute for Biomedical Research, University of Birmingham, Queen Eliz-
abeth Hospital, Birmingham B15 2TT, England, UK. Phone: 44-121-
4158700; Fax: 44-121-4158701; email: email@example.com
CCL25-mediated Recruitment in PSC
recruiting and positioning specific subsets of lymphocytes
(10). The best-defined tissue-specific recirculation of mem-
ory lymphocytes occurs in the skin and gut, which provide
distinct barriers to the environment exposed to different
types of antigen (11). The chemokine CCL17 is expressed
by endothelium in the inflamed skin and can trigger the ac-
tivation and adhesion of cutaneous leukocyte antigen posi-
tive skin-specific memory cells expressing high levels of
CCR4 (12); CCR8 is selectively expressed on T cells in
noninflamed skin where it interacts with CCL1 to promote
immune surveillance (13). In contrast, CCL25 is constitu-
tively expressed in epithelium and mucosal vessels in the
small bowel (14), where it interacts with gut-homing B and
T cells expressing its receptor, CCR9. CCR9 is coex-
7 on gut-homing lymphocytes and pro-
motes adhesion to mucosal addressin cell adhesion mole-
cule 1 (MAdCAM-1) on mucosal vessels (6, 15). Several
studies support a role for CCR9 and
homing to the gut. Virtually all small intestinal intraepithe-
lial and lamina propria lymphocytes are CCR9
(16), and the few CCR9 lymphocytes in peripheral blood
expressing high levels of
and increase in number during gut inflammation
secreting B cells in the small intestine are also CCR9
dependent on CCL25 for positioning in the gut (18). Per-
haps the most compelling evidence comes from studies
showing that only dendritic cells from mesenteric lymph
nodes and Peyer’s patches are able to imprint a CCR9
7 phenotype on lymphocytes that directs homing to
the gut after adoptive transfer in vivo (19).
In contrast, little is known about the signals that regulate
lymphocyte homing to the noninflamed liver. CXCR3
ligands are important during inflammation and it has been
proposed that the endothelial adhesion molecule vascular ad-
hesion protein 1 (VAP-1), which is constitutively expressed
on human liver endothelium, may be involved in trafficking
(20). The first evidence of aberrant homing of gut-derived
lymphocytes to the liver came from studies showing that
7 lamina propria lymphocytes bind to liver endothe-
lium in PSC via both constitutively expressed VAP-1 and
aberrantly expressed MAdCAM-1 (21). However, efficient
recruitment of T cells also requires the presence of an appro-
priate chemokine-mediated signal to activate integrin-medi-
ated adhesion and transmigration. Because of the strong as-
sociation between CCL25-CCR9 and gut homing, we
investigated whether CCR9 was involved in the recruitment
of mucosal lymphocytes to the liver in PSC.
7 in lymphocyte
are mostly memory cells
Materials and Methods
Matched liver tissue and peripheral blood was ob-
tained at the time of liver transplantation. Small bowel tissue was
obtained from intestinal resections for Crohn’s disease. All sam-
ples were collected with patient consent and local research ethics
Immunohistochemistry and Dual-Color Coimmunofluorescence.
cryostat sections were fixed in acetone for 10 min. 15 cases of
PSC and 5 cases each of normal liver, primary biliary cirrhosis
(PBC), and alcoholic liver disease were studied. Sections were in-
cubated with 20% goat serum before rabbit anti–human CCL25
polyclonal antibody (P134; 1
g/ml; PeproTech) or mouse anti–
human CXCL12 mAb (MAB350; 8
overnight at 4
C. Control sections were incubated without pri-
mary antibody or rabbit immunoglobulin. Sections were incu-
bated for 20 min with biotinylated secondary antibodies followed
by streptavidin–horseradish peroxidase complex developed with
diaminobenzidine and counterstained with hematoxylin.
Sections for dual immunofluorescence were incubated with
20% goat/rabbit serum for 30 min before primary antibodies
raised against CD68, CD31, Cytokeratin 19 (DakoCytomation),
LYVE-1 (Upstate Biotechnology), or CCL25. Control sections
were incubated without primary antibody. Sections were stained
with goat anti–rabbit FITC (Southern Biotechnology Associates,
Inc.) and goat anti–mouse IgG1 or IgG2a Texas red (Southern
Biotechnology Associates, Inc.) secondary antibodies and nuclei
counterstained with DAPI. Immunofluorescence was assessed us-
ing AxioVision software (Carl Zeiss MicroImaging, Inc.).
Fresh tissue was homogenized, normalized
for total protein using Coomassie blue and loaded on 8% SDS-
PAGE gels. After electrophoresis and transfer onto Hybond
membranes (Amersham Biosciences) blocked with 10% skimmed
milk. CCL25 was detected using goat anti–rabbit horseradish
peroxidase–conjugated antibody detected with the ECL system
RNA was extracted from snap-frozen tis-
sue using RNeasy Mini Kit (QIAGEN). mRNA was transcribed
to cDNA, and real-time PCR was performed on a PE7700 ABI
Prism machine. Each reaction was performed in triplicate using
QuantiTect Probe RT-PCR kit (QIAGEN) according to the
manufacturer’s instructions. Reactions contained 400 nM of
and 200 nM of CCL25-specific Taqman probe, 5
tech). Data are given as fold increase in gene expression normal-
ized to the 18S control and compared with normal skin tissue
(normalized to 1).
Liver- and Gut-derived Lymphocyte Isolation.
mokine receptor expression, liver- and gut-infiltrating lympho-
cytes were isolated by mechanical methods. Tissue was homoge-
nized in a Stomacher 400 circulator and filtered through a fine
mesh, and lymphocytes were separated using 33/77% (vol/vol)
Percoll density gradient centrifugation (Amersham Biosciences).
Lymphocytes were isolated from venous blood
collected into EDTA tubes, diluted 1:1 with PBS, and centri-
fuged over Lymphoprep
Four-Color Flow Cytometry.
with goat immunoglobulin before primary unconjugated mAb
7 (ACT-1; Millennium) for 30 min at 4
were washed in PBS, centrifuged, and labeled with goat anti–
mouse RPE-Cy5. Cells were subsequently stained with fluoro-
chrome-labeled primary mAb against CCR9-RPE (R&D Systems),
CD8-ECD (Beckman Coulter), CD3-RPE-Cy5, CD11a-FITC
(DAKO), CD45RO-FITC or CD45RA-Cy5 (Serotec). Control
samples were labeled with isotype-matched immunoglobulin. Cells
were fixed in 1% paraformaldehyde before analysis on a Coulter
Epics XL flow cytometer (Beckman Coulter) using Summit soft-
Lymphocyte Enrichment, Stimulation, and Intracellular Staining.
Normal PBLs and PSC liver-infiltrating lymphocytes (LILs) were
immunomagnetically enriched to 95% purity for CD45RA, and
g/ml; R&D Systems)
To preserve che-
Lymphocytes were incubated
Eksteen et al.Brief Definitive Report
CD45RA and CCR9, respectively, with EasySep (StemCell
Technologies, Inc.) and either CCR9-RPE (R&D Systems) or
CD45RA-FITC (Serotec) mAb. Purity was confirmed by flow
cytometry. Enriched populations were stimulated with 50 ng/ml
PHA and 500 ng/ml ionomycin, and cytokine export was blocked
after 1 h with 5 mg/ml brefeldin A. Cells were labeled with anti-
CD8ECD before incubation in Permeafix (BD Biosciences) and
labeling with anti-IFN
FITC or anti–IL-2 PE (BD Biosciences).
Viability of cells was assessed using Viaprobe (BD Biosciences).
Unstimulated and isotype-matched mAb were used as controls.
Transwell Chemotaxis of Lymphocytes.
from seven PSC livers was assessed using 6.5-mm diameter, 3-
The migration of LILs
pore transwell inserts (Corning; reference 22). Responses to
CCL5 and CXCL12 were positive controls because large num-
bers of LILs express CCR5 and CXCR4 (23). 100 ng/ml re-
combinant human CCL25, 100 ng/ml rhCCL5, or 100 ng/ml
rhCXCL12 was placed in the bottom of the well, and 5
lymphocytes were added to the upper chamber. Cells were col-
lected from the top and bottom chambers after 2 h and counted
by flow cytometry. Control wells contained medium alone.
Static Adhesion Assay.
Lymphocytes were phenotyped by
flow cytometry for CCR9 expression and used in the static adhe-
sion assay. 18-well Teflon-coated slides (Erie Scientific) were in-
cubated with 10
g/ml rhICAM-1, 10
ing of peripheral blood and tissue infiltrating
lymphocytes. (A) 17% of liver-infiltrating T
cells from PSC patients expressed CCR9?
compared with ?1% normal PBLs and
?2% PBLs from PSC patients (*, P ? 0.01).
5% of PBLs and virtually all lamina propria
CD3? cells in Crohn’s disease expressed
CCR9, whereas ?2% of LILs in PBC were
CCR9?. The graph shows percentage of
CCR9?/CD3? cells as mean ? SEM for
each group. (B) CCR9 is coexpressed with
?4?7 on PSC LIL. (C) CCR9 was ex-
pressed on both CD8 and CD4? LILs in
PSC (CD8 63% vs. CD4 35%; not de-
picted). PSC CD8? LILs are predominantly
memory cells as demonstrated by their ex-
pression of high levels of CD11a (left). Gating
of PSC LILs on CCR9 and CD8 reveals
that all the CCR9? cells are CD11ahigh and
the majority also express CD45RA, con-
firming that they are long-lived memory
cells. (D) PSC LILs were enriched for
CD45RA? and CCR9? and their response
to PHA stimulation was compared with that
of CD45RA? naive T cells from blood.
After 4 h of PHA stimulation, intracellular
cytokine staining of CD45RA? PBLs
showed a marked increase in IL-2, but min-
imal IFN? production, consistent with a
naive phenotype, whereas most CCR9?
CD45RA? (CD11ahigh)-enriched PSC LILs
stained for IFN? consistent with memory
cells. Isotype-matched controls are depicted
as white histograms with positive cytokine
staining as overlapping black histograms.
CCR9 expression and phenotyp-
CCL25-mediated Recruitment in PSC
(prepared as described previously; reference 24), or 1
BSA. Some lymphocytes were preincubated with 100 ng/ml per-
tussis toxin or blocking mAb against
gered by the addition of 10 ng/ml CCL25 or 100 nM/ml
. After nonadherent cells were removed, slides were fixed
and mounted before counting adherent lymphocytes in three
representative high power fields per well.
7. Adhesion was trig-
Results and Discussion
Approximately 20% of LILs in PSC expressed CCR9
2% were detected in livers of organ donors or
patients with other chronic inflammatory liver diseases
(Fig. 1). The mean channel fluorescence of CCR9 on LILs
in PSC was lower than on PBL, suggesting that CCR9
may have been down-regulated on LILs by ligand interac-
tions. However, CCR9 expression on LILs remained func-
tional and was still able to trigger static adhesion and induce
with coexpression of the gut integrin
A functional role for CCR9 in PSC was supported by
the strong CCL25 expression detected in the liver in PSC,
but not in other chronic inflammatory diseases. CCL25 was
detected in portal dendritic cells/macrophages and hepatic
sinusoidal endothelium at areas of interface hepatitis in
PSC, but was absent from other chronic inflammatory dis-
eases (Fig. 2). The specificity of CCL25 staining for PSC
was striking and we confirmed this using Western blotting
with which we were unable to detect CCL25 in other liver
diseases including primary biliary cirrhosis, another inflam-
matory biliary disease. Real-time PCR confirmed a 10-fold
increase in CCL25 mRNA above control tissue. This is the
first report of CCL25 expression outside the thymus or
small intestine, and the lack of significant CCL25 up-regu-
lation in other inflammatory liver diseases suggests that this
expression was strongly associated
(A) The livers of patients with PSC demon-
strated strong sinusoidal staining with
CCL25 ab (A1, brown pigment), staining
was particularly intense in periportal areas in
association with areas of interface hepatitis
(A2), and in portal tracts where macrophages/
dendritic cells (confirmed by CD68 coex-
pression; not depicted) stained strongly
(A3). There was no detectable CCL25
staining of hepatocytes, bile ducts, or vascular
endothelium in normal liver (A4) or other
chronic inflammatory diseases including pri-
mary biliary cirrhosis (A5). Staining with
isotype-matched control antibodies was
negative (A6). (B) Dual color immunofluo-
rescence (B1/B4, yellow merged image)
colocalized staining with CCL25 antibody
(B2/B5, green) and CD31 antibody (B3,
red) or LYVE-1 antibody (B6, red) to sinusoi-
dal endothelial cells. Nulcei were counter-
stained with DAPI blue. (C) Western blotting
confirmed the immunohistochemistry find-
ings with detection of CCL25 in all PSC
livers, but minimal detection in normal or
other chronically inflamed livers. P1-7, PSC;
NL, normal liver; PB1,2, PBC; ALD, alcoholic
liver disease. Protein loading was normalized
with ?-actin staining. (D) Real-time PCR
confirmed a 10-fold increase of CCL25
mRNA in PSC compared with control tissue
(normal skin). *, P ? 0.001. Modest amounts
of CCL25 mRNA were detected in NL and
other liver samples (BAT, biliary atresia;
ALD and PBC) but the levels were not sta-
tistically significant compared with skin.
CCL25 expression in the liver.
Eksteen et al.Brief Definitive Report
aberrant expression is specific to PSC and likely to be in-
volved in the pathogenesis of the disease.
To determine that CCR9 is functionally active on LILs,
we used adhesion and migration assays. CCR9
grated preferentially to CCL25 rather than to CXCL12 or
CCL5 and could also be triggered by CCL25 to bind
immobilized MAdCAM-1 via
CCL25-dependent migration and adhesion were both in-
hibited by pertussis toxin, suggesting that they were medi-
ated by G proteins activated via CCR9. The CCR9
cells in the liver are primed/memory cells as demonstrated
by their expression of high levels of CD11a (Fig. 1). The
majority are also CD45RA
, suggesting that they belong
to the recently described long-lived revertant memory
population (25). We confirmed they are functional mem-
7 integrins (Fig. 3).
ory cells by demonstrating their ability to secrete IFN
response to PHA activation. The presence of functional
4?7 on liver-infiltrating memory T lympho-
cytes is strong evidence that they are of mucosal origin acti-
vated in the gut and subsequently recruited to the liver.
The overlapping expression of endothelial adhesion mol-
ecules and now tissue-specific chemokines between the
liver and the gut suggests that, under particular circum-
stances, effector/memory lymphocytes are able to migrate
to both sites as part of an entero-hepatic T cell recirculation.
Such a mechanism may have evolved to provide surveil-
lance against gut-derived antigens entering the liver via the
portal circulation and is likely to be important in the patho-
genesis of the immune-mediated hepatic complications of
IBD. We propose that T cells activated in the gut during
sion to MAdCAM-1 and chemo-
taxis of PSC LIL. (A) PSC LILs
demonstrated a ?20-fold dose-
dependent increase in adhesion
to MAdCAM-1 under the influ-
ence of 10 ng/ml CCL25 com-
pared with normal LILs (?, P ?
0.002), which was blocked by an
antibody to ?4?7 or preincuba-
tion with pertussis toxin. BSA
served as a mock substrate, and
MnCl2 was used as a nonsignal-
ing trigger of integrin adhesion.
Binding of normal lymphocytes
to ICAM-1 in response to Mn
confirms that these cells could be
triggered to bind via integrins.
There was no significant binding
to MAdCAM-1 in the absence
of CCL25 or Mn. (B) CCL25
(16 ? 7%), CXCL12 (29 ?
10%), and CCL5 (19 ? 6%) all
stimulated chemotaxis of PSC
liver-derived lymphocytes in in-
vasion chambers in vitro (*, P ?
0.02, n ? 7). Phenotyping of
transmigrated lymphocytes by
flow cytometry confirmed that
the CCR9? LILs responded to
CCL25, but not to CXCL12 or
CCL5, suggesting that CCL25 is
the dominant chemokine recruit-
ing CCR9? LIL. (C) Expression
of CXCL12 is restricted to biliary
epithelium (C1) and is not de-
tected on portal (C1, arrow) or
sinusoidal endothelium (C2) in
PSC. No staining was seen in
isotype matched control sections
(C3). Thus, CXCL12 is unlikely
to be important in recruiting
CCR9? cells to the liver, but
might be involved in positioning/
retaining recruited cells around
CCL25 triggers adhe-
CCL25-mediated Recruitment in PSC
episodes of active IBD differentiate into effector cells that
have the ability to bind to both hepatic and mucosal endo-
thelium. Under noninflamed conditions, these cells may be
able to enter the liver via interactions with VAP-1, an adhe-
sion molecule that is constitutively expressed on liver endo-
thelium and up-regulated on inflamed mucosal endothelium
in IBD. Some of the effector T cells generated in the orga-
nized lymphoid tissue of the gut during active IBD (4) will
revert to long-lived memory cells with the ability to recir-
culate to the liver and subsequently to trigger hepatic in-
flammation under the right conditions, even in the absence
of active gut inflammation. The activation and expansion of
these memory cells in the liver may result in the induction
of MAdCAM-1 and CCL25 in the liver, promoting the re-
cruitment of CCR9? ?4?7? mucosal T cells and the devel-
opment of established inflammation (1). This model, in
which long-lived gut-derived memory T cells recirculate
through the liver, could explain why IBD and PSC do not
always occur at the same time and how PSC can be present
for the first time in patients with IBD whose colons have
been removed many years before (1). The alternative hy-
pothesis, in which the extra-intestinal complications of IBD
are driven by activated effector cells that are a consequence
of the continuing release of factors by the inflamed colon,
can explain the joint and skin complications that run con-
comitantly with active bowel disease (26) but not how PSC
can develop de novo after colectomy or why the activity of
liver disease does not parallel bowel inflammation.
PSC is associated with both ulcerative colitis and Crohn’s
disease, and usually develops in patients with colitis, suggest-
ing that colonic inflammation is critical. Because CCL25 is
largely confined to Peyer’s patches and the small bowel mu-
cosa, other gut-specific adhesion molecules might be in-
volved (including CCR10) that may have a role in the re-
cruitment of effector cells to the colon (14). However, recent
papers show significant increases in CCR9? lymphocytes in
the blood during colitis, suggesting that colonic inflammation
activates the CCR9 mucosal T cell pool (16, 17).
Our results demonstrate for the first time aberrant ex-
pression of CCL25 outside the gut or thymus associated
with infiltration of CCR9? T cells into the liver. The abil-
ity of CCL25 to activate migration and ?4?7-mediated ad-
hesion of LILs to MAdCAM-1 suggests that CCL25 and
MAdCAM-1 cooperate in the recruitment of mucosal lym-
phocytes to the liver in PSC.
This work was supported by grants from the Medical Research
Council, the Digestive Disorders Foundation, the European Com-
mission, and a research grant from Pfizer Inc. to A. Miles.
M. Briskin was employed by Millennium Pharmaceuticals Inc.
during this work. The authors have no other potential conflicting
Submitted: 26 May 2004
Accepted: 20 October 2004
1. Chapman, R.W. 1991. Aetiology and natural history of pri-
mary sclerosing cholangitis–a decade of progress? Gut. 32:
2. Grant, A.J., P.F. Lalor, M. Salmi, S. Jalkanen, and D.H. Ad-
ams. 2002. Homing of mucosal lymphocytes to the liver in
the pathogenesis of hepatic complications of inflammatory
bowel disease. Lancet. 359:150–157.
3. Befeler, A.S., T.W. Lissoos, T.D. Schiano, H. Conjeevaram,
K.A. Dasgupta, J.M. Millis, K.A. Newell, J.R. Thistle-
thwaite, and A.L. Baker. 1998. Clinical course and manage-
ment of inflammatory bowel disease after liver transplanta-
tion. Transplantation. 65:393–396.
4. Nagata, S., C. McKenzie, S.L. Pender, M. Bajaj-Elliott, P.D.
Fairclough, J.A. Walker-Smith, G. Monteleone, and T.T.
MacDonald. 2000. Human Peyer’s patch T cells are sensi-
tized to dietary antigen and display a Th cell type 1 cytokine
profile. J. Immunol. 165:5315–5321.
5. von Andrian, U.H., and C.R. Mackay. 2000. T-cell function
and migration. Two sides of the same coin. N. Engl. J. Med.
6. Pachynski, R.K., S.W. Wu, M.D. Gunn, and D.J. Erle.
1998. Secondary lymphoid-tissue chemokine (SLC) stimu-
lates integrin ?4?7-mediated adhesion of lymphocytes to
mucosal addressin cell adhesion molecule-1 (MAdCAM-1)
under flow. J. Immunol. 161:952–956.
7. Salmi, M., and S. Jalkanen. 1999. Molecules controlling lym-
phocyte migration to the gut. Gut. 45:148–153.
8. Campbell, J.J., and E.C. Butcher. 2000. Chemokines in tis-
sue-specific and microenvironment-specific lymphocyte hom-
ing. Curr. Opin. Immunol. 12:336–341.
9. Warnock, R.A., S. Askari, E.C. Butcher, and U.H. von An-
drian. 1998. Molecular mechanisms of lymphocyte homing
to peripheral lymph nodes. J. Exp. Med. 187:205–216.
10. Fan, L., C.R. Reilly, Y. Luo, M.E. Dorf, and D. Lo. 2000.
Cutting edge: ectopic expression of the chemokine TCA4/
SLC is sufficient to trigger lymphoid neogenesis. J. Immunol.
11. Berlin, C., E.L. Berg, M.J. Briskin, D.P. Andrew, P.J.
Kilshaw, B. Holzmann, I.L. Weissman, A. Hamman, and
E.C. Butcher. 1993. ?4?7 integrin mediates binding to the
mucosal vascular addressin MAdCAM-1. Cell. 74:185–195.
12. Campbell, J.J., G. Haraldsen, J. Pan, J. Rottman, S. Qin, P.
Ponath, D.P. Andrew, R. Warnke, N. Ruffing, N. Kassam,
et al. 1999. The chemokine receptor CCR4 in vascular rec-
ognition by cutaneous but not intestinal memory T cells. Na-
13. Schaerli, P., L. Ebert, K. Willimann, A. Blaser, R.S. Roos, P.
Loetscher, and B. Moser. 2004. A skin-selective homing
mechanism for human immune surveillance T cells. J. Exp.
14. Kunkel, E.J., J.J. Campbell, G. Haraldsen, J. Pan, J. Boisvert,
A.I. Roberts, E.C. Ebert, M.A. Vierra, S.B. Goodman, M.C.
Genovese, et al. 2000. Lymphocyte CC chemokine receptor
9 and epithelial thymus-expressed chemokine (TECK) ex-
pression distinguish the small intestinal immune compart-
ment: epithelial expression of tissue-specific chemokines as
an organizing principle in regional immunity. J. Exp. Med.
15. Zabel, B.A., W.W. Agace, J.J. Campbell, H.M. Heath, D.
Parent, A.I. Roberts, E.C. Ebert, N. Kassam, S. Qin, M.
Zovko, et al. 1999. Human G protein-coupled receptor GPR-
9-6/CC chemokine receptor 9 is selectively expressed on in-
testinal homing T lymphocytes, mucosal lymphocytes, and
thymocytes and is required for thymus-expressed chemokine-
Eksteen et al. Brief Definitive Report
mediated chemotaxis. J. Exp. Med. 190:1241–1256.
16. Hosoe, N., S. Miura, C. Watanabe, Y. Tsuzuki, R. Hokari,
T. Oyama, Y. Fujiyama, H. Nagata, and H. Ishii. 2003.
Demonstration of functional role of TECK/CCL25 in T
lymphocyte-endothelium interaction in inflamed and unin-
flamed intestinal mucosa. Am. J. Physiol. Gastrointest. Liver
17. Papadakis, K.A., J. Prehn, S.T. Moreno, L. Cheng, E.A.
Kouroumalis, R. Deem, T. Breaverman, P.D. Ponath, D.P.
Andrew, P.H. Green, et al. 2001. CCR9-positive lympho-
cytes and thymus-expressed chemokine distinguish small
bowel from colonic Crohn’s disease. Gastroenterology. 121:
18. Kunkel, E.J., D.J. Campbell, and E.C. Butcher. 2003. Che-
mokines in lymphocyte trafficking and intestinal immunity.
19. Mora, J.R., M.R. Bono, N. Manjunath, W. Weninger, L.L.
Cavanagh, M. Rosemblatt, and U.H. von Andrian. 2003. Se-
lective imprinting of gut-homing T cells by Peyer’s patch
dendritic cells. Nature. 424:88–93.
20. Lalor, P.F., S. Edwards, G. McNab, M. Salmi, S. Jalkanen,
and D.H. Adams. 2002. Vascular adhesion protein-1 medi-
ates adhesion and transmigration of lymphocytes on human
hepatic endothelial cells. J. Immunol. 169:983–992.
21. Grant, A.J., P.F. Lalor, S.G. Hubscher, M. Briskin, and D.H.
Adams. 2001. MAdCAM-1 expressed in chronic inflamma-
tory liver disease supports mucosal lymphocyte adhesion to
hepatic endothelium. Hepatology. 33:1065–1072.
22. Campbell, J.J., E.P. Bowman, K. Murphy, K.R. Youngman,
M.A. Siani, D.A. Thompson, L. Wu, A. Zlotnik, and E.C.
Butcher. 1998. 6-C-kine (SLC), a lymphocyte adhesion-trig-
gering chemokine expressed by high endothelium, is an ago-
nist for the MIP-3beta receptor CCR7. J. Cell Biol. 141:
23. Shields, P.L., C.M. Morland, M. Salmon, S. Qin, S.G. Hub-
scher, and D.H. Adams. 1999. Chemokine and chemokine
receptor interactions provide a mechanism for selective T cell
recruitment to specific liver compartments within hepatitis
C-infected liver. J. Immunol. 163:6236–6243.
24. Tidswell, M., R. Pachynski, S.W. Wu, S.Q. Qiu, E. Dun-
ham, N. Cochran, M.J. Briskin, P.J. Kilshaw, A.I. Lazarovits,
D.P. Andrew, et al. 1997. Structure-function analysis of the
integrin ?7 subunit: identification of domains involved in ad-
hesion to MAdCAM-1. J. Immunol. 159:1497–1505.
25. Faint, J.M., N.E. Annels, S.J. Curnow, P. Shields, D. Pilling,
A.D. Hislop, L. Wu, A.N. Akbar, C.D. Buckley, P.A. Moss,
et al. 2001. Memory T cells constitute a subset of the human
CD8?CD45RA? pool with distinct phenotypic and migra-
tory characteristics. J. Immunol. 167:212–220.
26. Salmi, M., K. Granfors, R. MacDermott, and S. Jalkanen.
1994. Aberrant binding of lamina propria lymphocytes to
vascular endothelium in inflammatory bowel diseases. Gastro-