|
Brief Definitive Report |
Address correspondence to David H. Adams, Liver Research Laboratories, Institute for Biomedical Research, University of Birmingham, Queen Elizabeth Hospital, Birmingham B15 2TT, England, UK. Phone: 44-121-4158700; Fax: 44-121-4158701; email: d.h.adams{at}bham.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: chemokines integrins inflammation hepatitis colitis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Lymphocyte recruitment from the circulation to tissue is a highly regulated process dependent on sequential interactions 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 chemokines. In the presence of an appropriate chemokine, specific G proteincoupled receptors on the lymphocyte are activated, triggering high-affinity integrin binding to endothelial ligands leading to arrest and transendothelial migration into tissue in response to chemotactic signals (6).
Chemokines play a crucial role in orchestrating the recruitment 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 recruiting and positioning specific subsets of lymphocytes (10). The best-defined tissue-specific recirculation of memory 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 activation and adhesion of cutaneous leukocyte antigen positive 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 constitutively 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 coexpressed with 4ß7 on gut-homing lymphocytes and promotes adhesion to mucosal addressin cell adhesion molecule 1 (MAdCAM-1) on mucosal vessels (6, 15). Several studies support a role for CCR9 and
4ß7 in lymphocyte homing to the gut. Virtually all small intestinal intraepithelial and lamina propria lymphocytes are CCR9+
4ß7+ (16), and the few CCR9+ lymphocytes in peripheral blood expressing high levels of
4ß7+ are mostly memory cells and increase in number during gut inflammation (17). IgA-secreting B cells in the small intestine are also CCR9+ and dependent on CCL25 for positioning in the gut (18). Perhaps 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+
4ß7+ 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 adhesion 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 4ß7+ lamina propria lymphocytes bind to liver endothelium 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 appropriate chemokine-mediated signal to activate integrin-mediated adhesion and transmigration. Because of the strong association between CCL25-CCR9 and gut homing, we investigated whether CCR9 was involved in the recruitment of mucosal lymphocytes to the liver in PSC.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunohistochemistry and Dual-Color Coimmunofluorescence.
6-µm 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 incubated with 20% goat serum before rabbit antihuman CCL25 polyclonal antibody (P134; 1 µg/ml; PeproTech) or mouse antihuman CXCL12 mAb (MAB350; 8 µg/ml; R&D Systems) overnight at 4°C. Control sections were incubated without primary antibody or rabbit immunoglobulin. Sections were incubated for 20 min with biotinylated secondary antibodies followed by streptavidinhorseradish 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 antirabbit FITC (Southern Biotechnology Associates, Inc.) and goat antimouse IgG1 or IgG2a Texas red (Southern Biotechnology Associates, Inc.) secondary antibodies and nuclei counterstained with DAPI. Immunofluorescence was assessed using AxioVision software (Carl Zeiss MicroImaging, Inc.).
Western Blotting.
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 antirabbit horseradish peroxidaseconjugated antibody detected with the ECL system (Amersham Biosciences).
Real-Time PCR.
RNA was extracted from snap-frozen tissue 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 CCL25-specific 5'-CCACACCCAAGGTGTCTTTGA-3' and 5'-GAGCACAGCCCACCCAAT-3' primers (AltaBioscience) and 200 nM of CCL25-specific Taqman probe, 5'-FAM-ACTGCTGCCTGGCCTACCACTACCC-TAMRA-3'; Eurogentech). Data are given as fold increase in gene expression normalized to the 18S control and compared with normal skin tissue (normalized to 1).
Liver- and Gut-derived Lymphocyte Isolation.
To preserve chemokine receptor expression, liver- and gut-infiltrating lymphocytes were isolated by mechanical methods. Tissue was homogenized 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).
PBL Isolation.
Lymphocytes were isolated from venous blood collected into EDTA tubes, diluted 1:1 with PBS, and centrifuged over Lymphoprep (Life Technologies).
Four-Color Flow Cytometry.
Lymphocytes were incubated with goat immunoglobulin before primary unconjugated mAb against 4ß7 (ACT-1; Millennium) for 30 min at 4°C. Cells were washed in PBS, centrifuged, and labeled with goat antimouse RPE-Cy5. Cells were subsequently stained with fluorochrome-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 software (DakoCytomation).
Lymphocyte Enrichment, Stimulation, and Intracellular Staining.
Normal PBLs and PSC liver-infiltrating lymphocytes (LILs) were immunomagnetically enriched to 95% purity for CD45RA, and 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 antiIL-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.
The migration of LILs from seven PSC livers was assessed using 6.5-mm diameter, 3-µm pore transwell inserts (Corning; reference 22). Responses to CCL5 and CXCL12 were positive controls because large numbers of LILs express CCR5 and CXCR4 (23). 100 ng/ml recombinant human CCL25, 100 ng/ml rhCCL5, or 100 ng/ml rhCXCL12 was placed in the bottom of the well, and 5 x 106 lymphocytes were added to the upper chamber. Cells were collected 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 adhesion assay. 18-well Teflon-coated slides (Erie Scientific) were incubated with 10 µg/ml rhICAM-1, 10 µg/ml rhMAdCAM-1 (prepared as described previously; reference 24), or 1 µg/ml BSA. Some lymphocytes were preincubated with 100 ng/ml pertussis toxin or blocking mAb against 4ß7. Adhesion was triggered by the addition of 10 ng/ml CCL25 or 100 nM/ml MnCl2. After nonadherent cells were removed, slides were fixed and mounted before counting adherent lymphocytes in three representative high power fields per well.
![]() |
Results and Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
PSC is associated with both ulcerative colitis and Crohn's disease, and usually develops in patients with colitis, suggesting that colonic inflammation is critical. Because CCL25 is largely confined to Peyer's patches and the small bowel mucosa, other gut-specific adhesion molecules might be involved (including CCR10) that may have a role in the recruitment 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 expression of CCL25 outside the gut or thymus associated with infiltration of CCR9+ T cells into the liver. The ability of CCL25 to activate migration and 4ß7-mediated adhesion of LILs to MAdCAM-1 suggests that CCL25 and MAdCAM-1 cooperate in the recruitment of mucosal lymphocytes to the liver in PSC.
![]() |
Acknowledgments |
---|
M. Briskin was employed by Millennium Pharmaceuticals Inc. during this work. The authors have no other potential conflicting financial interests.
Submitted: 26 May 2004
Accepted: 20 October 2004
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|