| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Liver Research Labs Centre for ImmuneRegulation* and the Department ofPathology, University of Birmingham, QueenElizabeth Hospital, Birmingham, United Kingdom; the Medical ResearchCouncil Human Immunology Unit, Institute ofMolecular Medicine, John Radcliffe Hospital, Headington, Oxford, UnitedKingdom; and Millenium PharmaceuticalsIncorporated, Cambridge, Massachusetts
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
The chemokine secondary lymphoid chemokine (SLC), now designated CCL21, is expressed predominantly in lymphoid tissue where it recruits cells bearing its receptor CCR7.8 These cells include DCs, naïve T cells and the recently described central memory T cells that use CCR7 to home to lymphoid tissue, in distinction from effector memory T cells that are excluded from lymph nodes by their lack of CCR7.9-11 That CCL21 plays a critical role in the development and organization of lymph nodes12 is shown by the failure of lymph node development in mice that are deficient in CCL21.13 Moreover, recent animal studies report that CCL21 expression is sufficient for lymphoid neogenesis because tissue-specific expression of a CCL21 transgene12 or induction by lymphotoxin14 results in ectopic lymphoid neogenesis.
CCL21 on high endothelial venules of mesenteric lymph nodes
and Peyer’s patches activates the 
4ß7 integrin on
CCR7+ T cells enabling them to bind to the
mucosal addressin MAdCAM-1.8,15,16
MAdCAM-1
iscritical for the homing of lymphocytes to mucosa-associated
lymphoid tissues;17-19
and sites of mucosal inflammation
where its expression increases promoting the lymphocytic infiltrate of
inflammatory bowel disease.17
MAdCAM-1 has recently been
detected de novo on portal vessels and in lymphoid
aggregates in inflammatory liver diseases associated with inflammatory
bowel disease2,20
suggesting that the recruitment of
mucosal lymphocytes to the liver may be critical in the pathogenesis of
these diseases.
Here we report the induction of CCL21 (previously considered to be a constitutive chemokine) on stromal tissues surrounding portal vessels and in lymphoid aggregates in chronic inflammatory liver disease. CCL21 was also expressed on the endothelium of CD34+ neovessels at the periphery of fibrous septa. Consistent with a role for CCL21 at this site, we detected significant numbers of both CD45RA+ and RA- CCR7+ T cells within the liver of patients with PSC and were able to show that these cells migrate to CCL21 in vitro. Thus increased expression of CCL21 in portal tracts in PSC may be important for the development of chronic inflammation by promoting the recruitment and retention in the liver of lymphocytes that are activated at mucosal sites.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Diseased liver tissue and paired peripheral blood was obtained with consent at the time of liver transplantation. Surplus liver tissue removed from donor organs that had been reduced in size for use in pediatric recipients was used as a nondisease control. All tissues were snap-frozen in liquid nitrogen and stored at -70°C until use. Subsequently 6-µm cryostat sections of 1-cm3 liver blocks were cut for immunohistochemistry and immunofluorescence.21 These sections were air-dried on poly-L-lysine-coated slides (Sigma Chemical Co. Ltd., Poole, Dorset, UK), then fixed for 10 minutes in acetone before staining.
Antibodies
The antibodies used are listed in Table 1
.
|
Dual-color immunohistochemistry was performed as described previously.21 Sections were incubated with all antibodies in Tris-buffered saline (TBS) in 20% normal swine serum. CCL21 was detected by incubating sections with polyclonal goat anti-human rhSLC at room temperature in a humidified container. A mouse anti-human monoclonal antibody to CD11c (a DC marker) incubated at room temperature for 1 hour was also used. Control sections were incubated without primary antibody and with an isotype-matched irrelevant control for the monoclonal antibodies, and with goat anti-rabbit antibody as a control for the polyclonal CCL21 antibody. Subsequently, sections were incubated for 30 to 45 minutes with rabbit anti-goat horseradish peroxidase and rabbit anti-mouse immunoglobulin followed by a wash. A further amplification step using a 30-minute incubation with goat anti-rabbit horseradish peroxidase and mouse alkaline phosphatase anti-alkaline phosphatase was performed. To develop the chromagens, the two substrates were added separately, first, alkaline phosphatase substrate (Sigma Chemical Co. Ltd.) followed by a washing step in TBS for 5 minutes and then peroxidase substrate (diaminobenzidine substrate, Sigma) for 5 to 10 minutes. The sections were counterstained with hematoxylin. Positive CCL21 staining was identified by the presence of a dark brown reaction product whereas positive CD11c staining was identified by the presence of a red reaction product. All washes were performed with Tris-buffered saline.
Immunofluorescence/Confocal Microscopy
Six-µm sections were incubated with goat anti-human CCL21
(as described above) and mouse anti-human CD31/ CD34, PAL-E, and LYVE1.
PAL-E stains vascular endothelium whereas LYVE-1 is absent from
vascular endothelium being confined predominantly to lymphatic
endothelium (Table 1)
.22-24
Sections were incubated with
all antibodies diluted in TBS with 10% fetal calf serum and 1% sodium
azide. After 1 hour, the sections were washed in TBS for 30 minutes in
a water bath. Subsequently fluorescein isothiocyanate and Texas
Red-labeled secondary antibodies (Cambridge Biosciences, Cambridge, UK)
were added to the tissue sections and incubated for 60 minutes in
darkness. After a final 30-minute wash in darkness, the sections were
examined by immunofluorescence microscopy or confocal microscopy. All
washes were performed with TBS.
Liver-Derived Lymphocyte Isolation
Liver-derived lymphocytes were isolated as previously described25 using a combination of mechanical homogenization and enzymatic digestion (with 100U/ml collagenase 1A (Sigma, Poole, Dorset, UK) in RPMI 1640 for 90 minutes at 37°C) followed by density gradient centrifugation.
Peripheral Blood Lymphocytes
Lymphocytes were isolated from peripheral venous blood taken from patients with primary sclerosing cholangitis or from normal volunteers. Umbilical cord blood was also obtained postpartum from Birmingham Women’s Hospital (n = 3). The blood was centrifuged over Lymphoprep (Life Technologies Ltd., Paisley, Scotland) for 30 minutes at 2000 rpm, the lymphocytes harvested, and washed in RPMI 1640 before use.
Flow Cytometry
Cells were stained for three-color analyses as previously
described.25
Where unconjugated antibody was used,
0.5 x 106
cells were incubated with
cytomegalovirus immunoglobulin before incubation with primary
unconjugated monoclonal antibody. Cells were then incubated with a goat
anti-mouse fluorescein-isothiocyanate secondary antibody (DAKO) for 30
to 45 minutes and then blocked for 10 minutes with 10% normal
mouse serum (DAKO). Subsequently incubations with fluorescent-labeled
CD3 (to detect T cells) and other fluorescein
isothiocyanate-, phycoerythrin-,energy-coupled-dye
(ECD)-, or CyChrome-conjugated antibodies (Table 1)
were
performed. All antibody incubations were performed at 4°C for 30
minutes and washes with phosphate-buffered saline (PBS) containing 1%
heat-inactivated fetal calf serum. Cells were fixed in 1%
paraformaldehyde before analysis on a Coulter XL flow cytometer
(Coulter Electronics Ltd., Luton, Beds, UK).
Chemotaxis Assays
Microchemotaxis Chamber Assays of PSC Liver-Derived Lymphocytes
The migration of liver-derived lymphocytes to CCL21 and RANTES was assessed using a 48-well microchemotaxis chamber technique. Briefly, lymphocytes isolated from the explanted livers of patients with PSC were rested overnight in RPMI/10% fetal calf serum before being resuspended at 1.5 x 106/ml in RPMI/0.1% bovine serum albumin. Subsequently, 29 µl of chemokine in RPMI/0.1% bovine serum albumin was placed in the bottom chamber and separated from 50 µl of liver-derived lymphocytes in the top chamber by a Nucleopore Track-Etch Membrane (Corning Costar Corp., Cambridge, MA) with 8-µm pores. After 2 hours at 37°C and 5% CO2, the chemotaxis membrane was removed, washed gently in PBS, and then fixed in methanol before staining with Diff-Quik (Dade Diagnostika GmbH, Munich, Germany). The chemotaxis membrane was then mounted on a slide in distyrene plasticiser and xylene mixture (DPX) before analysis. The numbers of cells on the underside of the chemotaxis membrane were counted. Five high-power fields (x200 magnification) per well and four wells per chemokine were analyzed and compared to control wells containing no chemokine. The average numbers of cells migrating were compared to the control wells and the results are expressed as a chemotactic index. Data represents six separate experiments.
Transwell Assays of PBL
Blood was depleted of monocytes by adding carbonyl iron (10 mg/ml) for 60 minutes at 37°C before removing cells using a Dynal Magnetic Particle Concentrator. The resulting cells were centrifuged over Lymphoprep (Life Technologies Ltd., Paisley, Scotland) harvested, and washed in RPMI 1640. The migration of different subtypes of lymphocyte was assessed using 6.5-mm diameter, 8-µm pore size Transwell inserts (Corning Costar Corp.) as previouslydescribed.26 Briefly, optimum titrations of the chemokines CCL21 (R&D Systems, Abingdon, Oxford, UK) and RANTES (Peprotech EC Ltd., London, UK) were prepared and prewarmed to 37°C in the bottom of the Transwell chamber. The lymphocytes were then resuspended in 0.5% Fraction V bovine serum albumin (Sigma)/RPMI 1640 and 5 x 105 cells were added to the upper chamber of each Transwell insert. After an 18-hour incubation at 37°C, 5% CO2, cells were carefully resuspended from the upper and lower chambers into 250 µl of 0.5% Fraction V bovine serum albumin (Sigma)/RPMI 1640. Control wells containing no chemokine were included in each assay.
Accurate counts of the number of resuspended cells in each
chamber were obtained by diluting 50 µl of the suspended cells in 250
µl of 50% fetal calf serum/PBS. Shortly before analysis on the
Coulter XL flow cytometer, 10 µg of propidium iodide (Sigma) was
added to each sample. The number of viable cells in a fixed volume of
each cell suspension was determined based on cell size and propidium
iodide exclusion. The remaining cells were stained for FACS analysis
according to the protocol described above. The antibodies used for this
were, unconjugated 4ß7 (ACT-1), goat anti-mouse fluorescein
isothiocyanate, CD3-ECD, and CD8-CyChrome. The migration of each subset
was determined as: specific cell migration equals the number of cells
of a specific phenotype in the lower chamber divided by the total
number of cells of that phenotype in both the upper and lower chambers
combined. The results are expressed as chemotactic index that is the
migration of cells relative to the negative control. Data represents
four separate experiments.
Western Immunoblotting
Liver Homogenates and Protein Determination
Liver protein extracts were obtained by homogenization of 5-g pieces of liver tissue in a Teflon homogenizer in 5 to 6 ml of buffer (50 mmol/L Hepes, 100 mmol/L KCl, 3 mmol/L MgCl2, 5 mmol/L ethylenediaminetetraacetic acid, 10 mmol/L NaF, 1 mmol/L sodium orthovanadate, 10% v/v glycerol, 0.1% v/v Tween 20, 5 mmol/L dithiothreitol, 0.1 mmol/L phenylmethyl sulfonyl fluoride, 0.1 mg/ml pepstatin A, 30 µg/ml leupeptin). Homogenates were centrifuged at 10,000 rpm for 10 minutes at 4°C and the supernatants collected in 1-ml aliquots and stored at -80°C. The protein content was determined by the BCA protein assay according to the manufacturer’s instructions (Pierce, Rockford, IL).
Blotting
Protein extracts (40 µg) were resolved on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a nitrocellulose membrane (Hybond C-Extra, Amersham Pharmacia Biotech). The blotted membrane was blocked for 1 hour at room temperature in TBS containing 5% w/v of membrane-blocking reagent (nonfat dried milk), followed by an overnight incubation (18 hours) with the primary antibody to CCL21 (1:300 dilution) in TBS containing 1% of membrane-blocking reagent. After being washed three times with TBS containing 0.1% Tween 20, the membrane was incubated with horseradish peroxidase-conjugated rabbit anti-goat at a dilution of 1:1000 for 1 hour. Protein bands were visualized using the enhanced chemiluminescent detection system (Amersham Pharmacia Biotech) followed by exposure of the membranes to Hyperfilm-ECL (Amersham Pharmacia Biotech) for 15 minutes. Quantification of the protein bands was performed using laser densitometry. Equal protein loading and transfer onto membranes were checked by staining gels and membranes with Coomassie blue. The Western immunoblots were performed at least three times using cell lysates from different liver preparations for each liver type.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
In normal liver CCL21
staining was restricted to small
vessels and occasional cells with a dendritic morphology
within portal tracts (Figures 1, 2, and 3)
. To determine the nature of the CCL21-positive vessels we used
immunofluorescence and confocal
microscopy to co-localize the endothelial
cell markers PAL-E, LYVE-1, CD31, and CD34 with CCL21 (Figure 3)
. CD31
is expressed on all endothelium including lymphatic endothelium whereas
CD34 is primarily restricted to lymphatic endothelium and high
endothelial venules and PAL-E is detected on vascular but not lymphatic
endothelium. LYVE-1 has recently been shown to be expressed by
lymphatic endothelium and sinusoidal endothelium in the liver (R Prevo,
PH Weigel, and DG Jackson, Institute of Molecular Medicine, Oxford,
unpublished observation).23,24,27
It is absent from
vascular endothelium including portal and hepatic venous vascular
endothelium in the liver. These experiments demonstrated that CCL21 was
absent from PAL-E+ portal vessels in normal liver
(Figure 3)
. Dual immunostaining and immunofluorescence with the DC
marker CD11c demonstrated co-localization of CCL21 with CD11c in a
number of portal tract DCs (Figure 2)
. However, many
CD11c+ cells were CCL21-negative indicating that
only a subset of DCs secretes CCL21 (Figure 2)
. Other structures within
the normal liver, particularly vascular and sinusoidal endothelia were
negative. These observations indicate that CCL21 in the normal liver is
involved in the recruitment of DCs via
lymphatics.
|
|
|
In chronic inflammatory liver disease, expression of CCL21
increased and this was particularly marked in PBC and PSC, two diseases
associated with portal lymphoid infiltration and the formation of
lymphoid aggregates (Table 2
and Figure 4
).
|
|
.
Within portal lymphoid aggregates there were scattered cells with the
morphology of DCs (Figure 1E)
and CCL21-positive vessels. These vessels
had the morphology of high endothelial venules. Dual immunostaining for
CCL21 with the endothelial cell markers PAL-E, CD31, and CD34
demonstrated that CCL21 stained a subset of CD34+
and CD31+ vessels. These vessels also stained for
PAL-E but not LYVE-1 indicating that they contain vascular rather than
lymphatic endothelium (Figure 3)
. The morphology and
CD34+ staining of some of the
CCL21+ vessels is consistent with high
endothelial venule-like neovessels, which develop in chronic
inflammation to promote the continuing entry of lymphocytes into areas
of lymphoid neogenesis.28
CCL21 was also demonstrable
albeit weakly on sinusoidal endothelium in a number of cases. In
cirrhosis associated with alcoholic liver disease endothelial and DC
staining for CCL21 were seen much less frequently. CCL21 was
undetectable on cholangiocytes or hepatocytes. In normal spleen tissue
used as a positive control CCL21 was detected on cells in the T cell
area of the periarteriolar sheath (Figure 1H)
.
We subsequently went on to corroborate the above findings by
determining levels of CCL21 protein in normal, PBC, PSC, and alcoholic
liver disease (ALD) liver tissue. Protein extracts were made
from liver tissue homogenates and analyzed by Western blotting and
densitometry. We were able to detect low levels of CCL21 protein in
normal liver tissue and 8 to 10 times higher levels in PSC and PBC
livers (Figure 4)
.
Expression of 4ß7 and CCR7 on Peripheral Blood and
Liver-Derived Lymphocytes
CCR7, the receptor for CCL21, is expressed on naïve T
cells including 4ß7 cells. CCL21 has been shown to activate both
LFA-1 and 4ß7 integrins on naïve lymphocytes. We therefore
analyzed the expression of 4ß7, CD45RA, and CCR7 on
CD3+ lymphocytes from blood and liver tissue.
Analysis of truly naïve T cells obtained from umbilical vein
blood revealed that 91% of the CD3+ T cells in
the neonate were 4ß7+; the majority of these
were also CD45RA+ (Figure 5)
. In normal peripheral blood, 59% of T
cells were CD3+4ß7+
and in patients with PSC, 45% of T cells in blood were
CD3+4ß7+ (Figure 5)
. A
similar proportion of intrahepatic T cells from normal donor liver and
PSC were 4ß7+ although the total number of
lymphocytes in normal liver was much less than in PSC (data not shown).
The liver was enriched for CD45RA-
4ß7+ T cells in patients with PSC in whom
82% of the CD3+4ß7+
cells in peripheral blood were CD45RA+ compared
with 51% of the
CD3+4ß7+ cells in the
liver (Figure 5)
.
|
. Normal PBL contained a mean
of 50% CCR7+ cells whereas 76% of PBL in PSC
were CCR7+. Only 9% of
CD3+ lymphocytes isolated from normal liver were
CCR7+ compared with 20% of intrahepatic T cells
from patients with PSC (Figure 6C)
. CCR7+ T cells
that are CD45RA- represent a population of
central memory cells that have the capability of homing to lymph
nodes.11
The percentage of blood
CCR7+ cells that expressed CD45RA was higher in
normal individuals (73%) but the proportions of intrahepatic T cells
that were CCR7/CD45RA+ were similar (48% and
49%) in normal and PSC liver suggesting that both naïve and
central memory type cells are being recruited during chronic
inflammation (Figure 6)
. However our recent report of
CD45RA+ memory T cells in human liver suggests
that at least some of these naïve CD45RA+
intrahepatic T cells could be memory cells that have reverted to a
CD45RA+ phenotype. Previous antigen priming in
these cells can be determined by the expression of other markers the
best of which is LFA-1. High levels of LFA-1 define memory (determined
by tetramer staining) regardless of CD45RA status.29
We
therefore analyzed LFA-1 levels on the CD45RA+
cells (Figure 6)
. In blood CD45RA+ cells could be
divided into an LFA-1high population (revertants)
and a larger LFA-1low population (true
naïve cells) in both patients [either PSC or ulcerative
colitis (UC)] and organ donors. However when intrahepatic T cells were
analyzed the majority of the CD45RA+ cells were
LFA-1high suggesting that they are antigen-primed
or memory cells. We then attempted to localize
CCR7+ lymphocytes within liver tissue by
immunohistochemistry. Staining was weak but two different anti-CCR7
monoclonal antibody 3D9 and 4H12 both detected occasional positive
cells associated with areas of inflammation and within nodules (Figure 6F)
. Interestingly CCR7 staining was also seen on cells with the
morphology of DCs and on endothelium in neovessels associated with
inflamed stromal areas (Figure 6F)
.
|
To determine whether CCR7 on circulating and intrahepatic
CD3+ T cells is functionally active we tested the
ability of CCL21 to stimulate the migration of blood and liver-derived
lymphocytes in vitro. Because we had limited numbers of
intrahepatic T cells, chemotaxis experiments were done with two
concentrations of CCL21 previously shown to stimulate optimal migration
in PBL (data not shown). We used responses to RANTES as a positive
control because we have previously shown that large numbers of
intrahepatic T cells express CCR5 and migrate to RANTES in
vitro.25,30
Lymphocytes derived from the liver of
patients with PSC were able to migrate to CCL21 in vitro and
the magnitude of the response to CCL21 was comparable to that seen with
RANTES (Figure 7a)
.
|
4ß7+ T
cells from both normal and UC blood but significantly higher rates of
migration were observed with lymphocytes from patients with UC (Figure 7b)
. The ability of
CD3+4ß7+ cells from
patients with UC to respond to CCL21 provides a potential mechanism for
the recruitment of these cells via MAdCAM-1-expressing vessels in
PSC.16 |
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
and -ß
and act as a focus for sustained lymphocyte recruitment and retention
via vessels that resemble the high endothelial venules of lymph
nodes.33,34
Primary sclerosing cholangitis is a chronic
inflammatory disease in which lymphocyte-mediated damage of bile ducts
is associated with expanded portal tracts and the presence of portal
lymphoid aggregates. The disease is strongly associated with
inflammatory bowel disease. We have shown previously that
4ß7+ T cells activated in mucosal sites can
bind to inflamed portal vessels in PSC via MAdCAM-1, which is induced
on these vessels in PSC.2
However, MadCAM-1 expression
alone is probably insufficient to maintain the chronic lymphocytic
infiltrate in PSC. Recent evidence suggests that overexpression of
CCL21 in nonlymphoid tissues can drive neolymphoid
development12
and in light of this and the ability of
CCL21 to activate 4ß7+ adhesion to MAdCAM-1,
we investigated the expression and function of CCL21 in PSC. In normal liver CCL21 was restricted to a few small vessels with the morphology of lymphatics and occasional CD11c+ DCs within portal tracts. CCL21 has been detected on lymphatic vessels in lymph node where it is critical for recruiting DCs from tissue.8,35 Thus CCL21 on lymphatic vessels in normal liver could regulate the trafficking of DCs from the liver to the portal tract, an important pathway for DC emigration from the liver to the draining lymph node.36,37
However, the most striking expression of CCL21 was detected in patients
with chronic inflammatory liver disease, particularly PSC in which
there is an associated expansion of portal tracts with secondary
lymphoid aggregates. In these conditions we detected increased
expression of CCL21 in portal tracts where not only DCs but also
PAL-E-expressing vascular endothelium stained strongly. Further
evidence that these vessels were vascular endothelium is provided by
their failure to stain with the lymphatic endothelial marker LYVE-1.
Many of the CCL21+ PAL-E+
vessels were small neovessels, frequently located at the periphery of
portal tracts and fibrous septa. In addition to being an important
component of evolving fibrosis these small neovessels provide a
potential pathway for the recruitment of T lymphocytes, which are often
present within areas of fibrosis and may be associated with foci of
interface hepatitis extending into the adjacent liver
parenchyma.38
Staining was also detected on
CD34+ vessels, some of which had the morphology
of high endothelial venules. CD34 is characteristically expressed by
high endothelial venules in lymph nodes and by neovessels at sites of
chronic inflammation.28
In some chronic inflammatory
diseases lymphoid neogenesis occurs in response to local cytokines and
provides a site for continued lymphocyte recruitment to
tissue.28,39,40
The presence of CCL21 within these
aggregates could promote the recruitment of CCR7+
DCs and lymphocytes including CD45RA+ and
4ß7+ cells.8,16
In support of
this we detected a high proportion of CCR7+ T
cells within the liver in PSC, 50% of which were
CD45RA+. To demonstrate that CCR7 on these cells
is functional we performed chemotaxis assays with intrahepatic T cells
from patients with PSC and demonstrated that they migrated efficiently
to CCL21 in vitro. Furthermore we were able to show that
CCL21 can preferentially attract 4ß7+
lymphocytes from PBL of patients with UC suggesting that overexpression
of CCL21 in the liver could recruit 4ß7+
mucosal lymphocytes to chronically inflamed portal tracts in PSC.
In a previous study we showed that the endothelium of larger native
portal vessels expressed MAdCAM-1 in cases of PSC and occasionally in
other chronic inflammatory liver diseases. This provides a pathway for
the recruitment of 4ß7+ T cells. In the
present study we were unable to detect CCL21 in the endothelium of
larger portal vessels, but found strong expression of CCL21 in the
smaller neovessels and in surrounding stromal tissues. Because CCL21
contains a glycosaminoglycan-binding domain it is possible that it is
sequestered in the stromal matrix where it could provide a mechanism
for retaining T cells that have migrated across vascular endothelium
thereby promoting the development of perivascular portal-associated
lymphoid tissue.5,41
Our findings in a chronic inflammatory human disease are supported by recent studies in an animal model of chronic hepatic inflammation. Yoneyama and colleagues42 demonstrated the induction of endothelial CCL21 in a murine model of granulomatous hepatitis and reported the development of lymphoid aggregates in response to Propionibacterium. The functional importance of CCL21 in this setting was confirmed because the portal-associated lymphoid tissue diminished after treatment with an anti-CCL21 antibody. The same group have demonstrated similar findings in P. acnes-induced lung inflammation indicating that this might be a common mechanism of neolymphoid tissue development at mucosal sites.43
When CCL21 binds to CCR7 it can activate 4ß7 binding to
MAdCAM-1.16
CCR7 is expressed at high levels on
naïve T cells44
and on a subset of so-called
central memory T cells that lack immediate effector functions but which
can traffic to the lymph node and efficiently stimulate
DCs.11,45
The proportion of circulating T cells expressing
CCR7 was increased in PSC and up to 25% of intrahepatic T cells were
CCR7+. These intrahepatic
CCR7+ T cells were equally divided between
CD45RA+ and CD45RA- cells
suggesting that they include a population of central memory cells
(CCR7+/CD45RA-) that have
undergone differentiation in response to antigen. Whether this antigen
is encountered in the portal-associated lymphoid tissue or in draining
portal lymph nodes is unclear. However, the increased numbers of
CCR7+ CD45RA- cells in
blood in PSC suggests that these cells recirculate.11
The
proportion of CCR7+ cells retrieved from inflamed
liver is likely to be less than those entering tissue because
CCR7+ memory cells lose CCR7 on differentiation
into effector cells in response to antigen, which could occur within
portal-associated lymphoid tissue.45
A high proportion of T cells in blood and liver tissue in PSC were
CD45RA+. This was particularly true for the
CD3+4ß7+ cells of
which 82% were CD45RA+ in blood and 51% in
liver. Thus naïve T cells may also be recruited to the inflamed
liver in response to CCL21. However, the majority of
CD45RA+ cells within the liver were
LFA-1high, which defines a primed/memory
population. Thus the ability of CCL21 to activate integrin binding to
MAdCAM-1, ICAM-1, and VCAM-1, all of which are increased on
endothelium within portal tracts, could result in the retention of
CCR7+ T cells within the liver.2,16
Whether this results in the recruitment of true naïve T cells
that are then fully activated within portal-associated lymphoid tissue
is unknown. It is more likely that the CCR7+
cells recruited are either a population of central memory T cells,
memory cells, or primed cells that have reverted to a
CD45RA+ phenotype. In either case they may be
important for sustaining chronic inflammatory responses in the
liver.46
These findings suggest that CCL21 expressed during chronic inflammatory liver disease promotes the recruitment of both CD45RA+ and CD45RA- T cells. Together with the induction of functionally active MAdCAM-1 in chronic inflammatory liver disease,2,20 and the development of organized lymphoid aggregates these data imply a role for CCL21 in driving the development of organized lymphoid aggregates within the chronically inflamed liver.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by grants from the Medical Research Council (G84 5031), The Digestive Diseases Foundation (CHT 221), the Sir Jules Thorn Trust, and the Wellcome Trust.
Accepted for publication January 18, 2002.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  D H Adams, B Eksteen, and S M Curbishley Immunology of the gut and liver: a love/hate relationship Gut, June 1, 2008; 57(6): 838 - 848. [Full Text] [PDF]
|
|
|
|
 |
|
 A. Manzo, S. Bugatti, R. Caporali, R. Prevo, D. G. Jackson, M. Uguccioni, C. D. Buckley, C. Montecucco, and C. Pitzalis CCL21 Expression Pattern of Human Secondary Lymphoid Organ Stroma Is Conserved in Inflammatory Lesions with Lymphoid Neogenesis Am. J. Pathol., November 1, 2007; 171(5): 1549 - 1562. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Wang, K. G. McDonald, J. S. McDonough, and R. D. Newberry Murine isolated lymphoid follicles contain follicular B lymphocytes with a mucosal phenotype Am J Physiol Gastrointest Liver Physiol, October 1, 2006; 291(4): G595 - G604. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Raine, P. Zaccone, P. Mastroeni, and A. Cooke Salmonella typhimurium Infection in Nonobese Diabetic Mice Generates Immunomodulatory Dendritic Cells Able to Prevent Type 1 Diabetes J. Immunol., August 15, 2006; 177(4): 2224 - 2233. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Heydtmann, D. Hardie, P. L. Shields, J. Faint, C. D. Buckley, J. J. Campbell, M. Salmon, and D. H. Adams Detailed Analysis of Intrahepatic CD8 T Cells in the Normal and Hepatitis C-Infected Liver Reveals Differences in Specific Populations of Memory Cells with Distinct Homing Phenotypes J. Immunol., July 1, 2006; 177(1): 729 - 738. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. K. Lai, P. J. Sun, J. Zhang, A. Jennings, P. F. Lalor, S. Hubscher, J. A. McKeating, and D. H. Adams Expression of DC-SIGN and DC-SIGNR on Human Sinusoidal Endothelium: A Role for Capturing Hepatitis C Virus Particles Am. J. Pathol., July 1, 2006; 169(1): 200 - 208. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Balasubramanian, N. Munshi, M. J. Koziel, Z. Hu, T. J. Liang, J. E. Groopman, and R. K. Ganju Structural proteins of Hepatitis C virus induce interleukin 8 production and apoptosis in human endothelial cells J. Gen. Virol., December 1, 2005; 86(12): 3291 - 3301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Curbishley, B. Eksteen, R. P. Gladue, P. Lalor, and D. H. Adams CXCR3 Activation Promotes Lymphocyte Transendothelial Migration across Human Hepatic Endothelium under Fluid Flow Am. J. Pathol., September 1, 2005; 167(3): 887 - 899. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. G. McDonald, J. S. McDonough, and R. D. Newberry Adaptive Immune Responses Are Dispensable for Isolated Lymphoid Follicle Formation: Antigen-Naive, Lymphotoxin-Sufficient B Lymphocytes Drive the Formation of Mature Isolated Lymphoid Follicles J. Immunol., May 1, 2005; 174(9): 5720 - 5728. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Mori, J. Kim, T. Yamano, H. Takeuchi, S. Huang, N. Umetani, K. Koyanagi, and D. S.B. Hoon Epigenetic Up-regulation of C-C Chemokine Receptor 7 and C-X-C Chemokine Receptor 4 Expression in Melanoma Cells Cancer Res., March 1, 2005; 65(5): 1800 - 1807. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Burman, O. Haworth, D. L. Hardie, E. N. Amft, C. Siewert, D. G. Jackson, M. Salmon, and C. D. Buckley A Chemokine-Dependent Stromal Induction Mechanism for Aberrant Lymphocyte Accumulation and Compromised Lymphatic Return in Rheumatoid Arthritis J. Immunol., February 1, 2005; 174(3): 1693 - 1700. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Heydtmann, P. F. Lalor, J. A. Eksteen, S. G. Hubscher, M. Briskin, and D. H. Adams CXC Chemokine Ligand 16 Promotes Integrin-Mediated Adhesion of Liver-Infiltrating Lymphocytes to Cholangiocytes and Hepatocytes within the Inflamed Human Liver J. Immunol., January 15, 2005; 174(2): 1055 - 1062. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Takeuchi, A. Fujimoto, M. Tanaka, T. Yamano, E. Hsueh, and D. S. B. Hoon CCL21 Chemokine Regulates Chemokine Receptor CCR7 Bearing Malignant Melanoma Cells Clin. Cancer Res., April 1, 2004; 10(7): 2351 - 2358. |
