Role for CXCR6 in Recruitment of Activated CD8+ Lymphocytes to Inflamed Liver
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免疫学杂志 2005年第1期
Abstract
Hepatic infiltration of activated CD8 lymphocytes is a major feature of graft-vs-host disease (GvHD). Chemoattractant cytokines and their receptors are key regulators of lymphocyte trafficking, but the involvement of chemoattractant receptors in the physiologic recruitment of cells into the inflamed liver has not been defined. The present study examines the role of the chemokine receptor CXCR6, which is highly expressed by liver-infiltrating CD8 T cells. Hepatic accumulation of donor CD8, but not donor CD4, lymphocytes was significantly reduced in GvHD induced by transfer of CXCR6–/–, H-2Db lymphocytes into BDF1, H-2Dbxd recipients. To determine whether altered recruitment contributes to the reduced accumulation, CXCR6–/– or wild-type splenic lymphocytes participating in an active GvHD response were isolated and transferred i.v. into secondary recipients with active GvHD, and the short term (6-h) recruitment of transferred cells to the inflamed liver was assessed. CXCR6–/– CD8 (but not CD4) cells displayed a significant (33%) reduction in liver localization, whereas frequencies in blood of CXCR6–/– and wild-type CD8 cells were similar. Proliferation and apoptosis of liver-infiltrating donor CD8 cells were unaffected. We conclude that CXCR6 helps mediate the recruitment of activated CD8 lymphocytes in GvHD-induced hepatitis and may be a useful target to treat pathological inflammation in the liver.
Introduction
Donor T cell recruitment into target tissues is a prominent component of graft-vs-host disease (GvHD),6 and a major complication in allogenic bone marrow transplantation ( 1). Recruitment of activated lymphocytes is a multistep process in which the expression of specific adhesion molecules and chemokine receptors endows lymphocytes with the ability to access extravascular tissues ( 2). Chemokine receptors play a central role in lymphocyte recruitment by rapidly transmitting intracellular signals and triggering increased integrin affinity to adhesion molecule ligands displayed by endothelial cells ( 3). At present, >20 chemokine receptors have been described ( 4). The role of specific chemokine receptors in attracting activated lymphocytes appears to be both stimulus and organ dependent, contributing to inflammatory and homeostatic tissue localization of lymphocyte subsets ( 5). In GvHD the liver is a key target organ, characterized by massive lymphocyte accumulation ( 6), which may reflect enhanced recruitment and/or proliferation.
Acute GvHD is initiated by donor T cells recognizing and reacting to host allo-Ags and is characterized by a Th1-polarized response ( 7). Different combinations of chemokine receptors are associated with Th1- vs Th2-dependent immune reactions ( 8). Accordingly, it has been shown that the chemokine receptor CXCR6 (Bonzo/STRL33/TYMSTR) is expressed on T cells of Th1 phenotype ( 9, 10, 11, 12, 13). CXCR6, originally described as a novel simian immunodeficiency virus receptor ( 14) and fusion cofactor for HIV-1 strains ( 15), is expressed at very low levels in naive CD8 cells ( 16, 17), but can be up-regulated in T cells by activated dendritic cells (DC) ( 9, 10, 11). Moreover, it has been shown that many Th1-polarized cells ( 13) and in vitro-activated CD8 cells ( 16) are attracted by the CXCR6 ligand, CXC ligand 16 (CXCL16; SR-PSOX/SexCKine). In target tissues, CXCR6-expressing cells are readily found in the liver of patients with alcohol- or hepatitis C-induced liver cirrhosis ( 9, 18). CXCL16 is abundantly expressed in lymphoid and nonlymphoid tissues, such as the liver ( 13, 16, 19). Furthermore, the kinetics of DC-mediated up-regulation also suggest a role of CXCR6 in facilitating effector T cell migration into sites of pathological inflammation; CXCR6 is up-regulated, whereas CCR7, a chemokine receptor involved in homing to lymph nodes, is down-regulated after prolonged incubation with DC under Th1-polarizing conditions ( 11). However, the role of CXCR6 in lymphocyte recruitment in vivo is not known.
The aim of this study was to assess the contribution of CXCR6 to the recruitment of activated lymphocytes into inflamed liver. For this purpose we developed a short term, in vivo recruitment assay of activated lymphocytes into the inflamed livers of mice undergoing active GvHD and compared the recruitment of CXCR6–/– vs CXCR6+/+ lymphocytes. Our results show that CXCR6 has a significant role in recruiting activated CD8, but not CD4, lymphocytes to the inflamed liver in GvHD-induced hepatitis.
Materials and Methods
Animal model of GvHD
C57BL/6J (B6, H-2b; The Jackson Laboratory), C57BL/6J-IghaThy1aGpi1a (Thy1.1, H-2b; The Jackson Laboratory), and CXCR6-GFP knockin mice (CXCR6–/–, backcrossed onto C57BL/6 more than eight generations, H-2b) were used as donors. CXCR6-GFP mice have the CXCR6-coding sequence replaced with a sequence for enhanced GFP, which is regulated through the CXCR6 promoter ( 17). B6D2F1/J mice (BDF1, H-2bxd; The Jackson Laboratory) were used as recipients or as donors for syngeneic control experiments. All mice were 8–12 wk old and housed under conventional conditions in the animal facility at the Veterans Affairs Palo Alto Health Care System. All experiments were approved by the local ethics committee at Stanford University. Nonlethal GvHD was induced as previously described ( 20). Briefly, 50 x 106 donor lymphocytes isolated from the spleen were transferred i.v. into recipient mice on day 0. The splenocytes from C57BL/6 and CXCR6–/– were similar with respect to the expression of activation markers (CD69, CD25, CD44, and CD45RB) and adhesion molecules (L-selectin and 47) and cell subset distribution (CD4, CD8, NK, NKT, and B cells; data not shown). On day 7, recipient mice were killed by CO2 inhalation, and the liver, spleen, and blood were harvested. In indicated experiments, syngeneic lymphocytes were labeled with 0.5 μM CFSE (Molecular Probes) at 37°C for 10 min before injection.
Short term recruitment assay
GvHD was induced in BDF1 mice by transferring lymphocytes from B6-Thy1.2, congenic B6-Thy1.1, or CXCR6–/–-Thy1.2 mice as described above. This congenic model for identification of donor lymphocytes was chosen because using CXCR6–/– mice expressing GFP as donors excluded CFSE labeling of donor cells. On day 8 after transfer, lymphocytes were isolated from the spleen of GvHD mice induced by Thy1.2 cells (wild-type (WT) or CXCR6–/–), and 20 x 106 cells were subsequently transferred to GvHD mice induced by Thy1.1 cells (short term recruitment mice). Six hours after the second transfer, the short term recruitment mice were killed, and cells from the liver, spleen, and blood were harvested. This 6-h window allowed us to compare the recruitment of Thy1.2 cells with or without CXCR6 into inflamed livers. The phenotypes of the 20 x 106 lymphocytes injected from WT and CXCR6–/– mice were similar with respect to the expression of activation markers, adhesion molecules, and cell subset distribution described above (data not shown). A liver recruitment ratio was calculated as the absolute number of cells recruited to the liver divided by the number of injected cells. Short term recruitment was determined for CD8+H-2Dd–Thy1.2+, CD4+H-2Dd–Thy1.2+, and CD19+H-2Dd–IgDb+ cell subsets. Additionally, the level of defined donor cell subsets circulating in the blood was analyzed.
Isolation of liver-infiltrating lymphocytes (LIL)
After mice were killed, the liver was perfused through the portal vein with 5 ml of PBS. The liver was harvested, dispersed through a stainless steel mesh (70-μm pore size), and washed with HBSS containing 10% bovine calf serum. The dispersed cells were mixed with a 33% Percoll (Amersham Biosciences) solution containing 100 U/ml heparin (Sigma-Aldrich) and centrifuged for 20 min (800 x g) at room temperature. Next, the cell pellet was resuspended in double-distilled water for 20 s to lyse RBC, and the lysis was stopped with 2x PBS. The isolated lymphocytes were then resuspended in HBSS (2% bovine calf serum), counted in a hemocytometer, and kept on ice until staining for flow cytometric analysis.
RT-PCR and real-time PCR
Total RNA was extracted from whole liver tissue using an RNeasy kit according to the manufacturer’s instructions (Qiagen). RNA concentration and purity were determined by measuring absorbance spectrophotometrically at 260 and 280 nm. RT-PCR was performed with ThermoScript RT-PCR System (Invitrogen Life Technologies). Mouse -actin served as an internal control gene. The PCR profile was 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, followed by a final extension of 7 min at 72°C. The primer sequences of CXCR6, CXCL16, and -actin were: CXCR6 (forward), 5'-TAC GAT GGG CAC TAC GAG GGA G-3'; CXCR6 (reverse), 5'-GCA AAG AAA CCA ACA GGG AGA CCA C-3'; CXCL16 (forward), 5'-GCT TTG GAC CCT TGT CTC TTG C-3'; CXCL16 (reverse), 5'-GTG CTG AGT GCT CTG ACT ATG TGC-3'; -actin (forward), 5'-ATG TTT GAG ACC TTC AAC ACC-3'; and -actin (reverse), 5'-TCT CCA GGG AGG AAG AGG AT-3'. For real-time PCR of CXCR6 and CXCL16 from normal and GvHD liver, liver tissue was harvested, and total RNA was isolated as described above and converted to cDNA using the Reverse Transcription System (Promega) following the manufacturer’s instructions. 15Sa was used as an endogenous control for relative quantification using the comparative threshold cycle method. All reactions were performed in triplicate on an Mx3000P Real-Time PCR System (Stratagene) using 2x SYBR Green and the following primers: 15S (forward), 5'-GAAGCACGGATACATTGGTGA3'; 15S (reverse), 5'-TGTTCTGCCATTTCTCTAGGT-3'; CXCR6 (forward), 5'-CCCTGTACTTTATGCCTTTG-3'; CXCR6 (reverse), 5'-CTTGGAACTGTCCTCAGAAG-3'; CXCL16 (forward), 5'-CCTTGTCTCTTGCGTTCTTCC-3'; CXCL16 (reverse), 5'-TCCAAAGTACCCTGCGGTATC-3'; CXCL10 (forward), 5'-CCAAGTGCTGCCGTCATTTTC-3'; and CXCL10 (reverse), 5'-GGCTCGCAGGGATGATTTCAA-3'. The chemokine or chemokine receptor expression in normal liver was given an arbitrary value of 1, and their expression in GvHD liver was expressed as fold up-regulation from normal.
Flow cytometry
Isolated cells (1–2 x 106) were stained with the following rat anti-mouse Abs conjugated to FITC, PE, PerCP, PE-indotricarbocyanine, allophycocyanin, allophycocyanin-indotricarbocyanine, or biotin: H-2Dd (clone 34-2-12), CD4 (clone RM4-5), CD8 (clone 53-6.7), CD44 (clone IM7), CD45RB (clone 16A), CD25 (clone PC61), CD69 (clone H1.2F3), L-selectin (clone Mel-14), 47 (clone DATK 32), Thy1.2 (clone 53-2.1), CD19 (clone 1D3), IgDb (clone 217-170), and NK1.1 (clone PK136). The above-mentioned Abs were obtained from BD Pharmingen. NeutrAvidin Cascade Blue was purchased from Molecular Probes. To study proliferation, we used a BrdU Flow Kit (BD Biosciences, San Jose, CA) following the manufacturer’s instructions. Briefly, 1 mg of BrdU was injected i.p. 2 h before death, and the isolated lymphocytes were subsequently permeabilized for detection of intracellular BrdU. Four-color flow cytometry was performed using a FACSCalibur (BD Biosciences), and seven-color flow cytometry was performed with LSR II (BD Biosciences). The data were acquired and analyzed on CellQuest software (version 3.3) for FACSCalibur and on FACSDiVa software (version 2.1) and CellQuest-Pro software (version 4.0) for LSR II. Data are presented as the mean ± SEM. Student’s t test was used for between-group comparisons, and significance was set at p < 0.05.
Migration assay
LIL were isolated from GvHD livers induced by WT, CXCR6+/–, and CXCR6–/– splenocytes at day 7 of disease. LIL (0.5–1 x 106) were placed in the upper chamber of Transwell inserts (5-μm pore size; Corning Costar) and migrated to chemokines at their optimal concentrations for 2 h at 37°C as described previously ( 21). After migration, polystyrene beads (Polysciences) were added to each bottom well as an internal standard, and an aliquot was collected for analysis by flow cytometry for calculation of the percent migrated. Triplicate wells were pooled and stained with Abs to H-2Dd and CD8 for calculating donor CD8 migration. The data presented are the percent migration of the indicated subsets, determined by measuring the number of cells of each subset in the starting and migrated populations. The chemokine CXCL12 was purchased from PeproTech, and CXCL16 was obtained from R&D Systems.
Results
CXCR6 is up-regulated in GvHD hepatitis
We initially asked whether CXCR6 and CXCL16 are relevant candidates to study in liver homing by examining their expression under normal and inflamed conditions. We examined livers from control and/or GvHD mice 7 days after lymphocyte transfer. CXCR6 and CXCL16 mRNA were amplified by PCR from total RNA, and we found clear evidence of CXCR6 and CXCL16 mRNA expression in both healthy controls and GvHD animals (data not shown). We also quantitatively analyzed the expression of CXCR6 and CXCL16 and found an 2-fold increase in mRNA expression in GvHD-inflamed liver compared with normal liver, as assessed by real-time PCR (mean ± SEM fold increase: CXCR6, 1.6 ± 0.1; CXCL16, 1.9 ± 0.1; Fig. 1a; n = 4). Next, we asked whether CXCR6 was expressed on transferred lymphocytes infiltrating the liver. For this purpose, we used heterozygous GFP knockin mice, in which one allele of the CXCR6 gene has been replaced by enhanced GFP, to examine the expression of CXCR6 in normal (syngeneic transfer) and inflamed (allogeneic transfer) liver ( 17). We analyzed GFP expression in LIL from day 7 of GvHD induced by CXCR6-GFP (CXCR6+/–) lymphocytes in allogeneic and syngeneic hosts. We observed an increase in the percentage of GFPhigh lymphocytes among donor CD4 and CD8 lymphocytes in the liver of allogeneic vs syngeneic recipients. The percentages of CD8+GFPhigh donor lymphocytes among CD8 LIL were 24 ± 3 and 60 ± 2% in syngeneic and allogeneic recipients, respectively (Fig. 1b; n = 3–5; p < 0.05). Similarly, the percentage of CD4+GFPhigh donor lymphocytes among CD4 LIL increased from 9 ± 1% in syngeneic recipients to 42 ± 2% in allogeneic recipients (Fig. 1b; n = 3–5; p < 0.05). Because GFP is under the control of the CXCR6 promoter, these findings suggest that donor lymphocytes up-regulate CXCR6 before or during accumulation in the liver. Furthermore, we observed that a larger percentage of the donor cells were GFPhigh in the GvHD liver as opposed to secondary lymphoid tissues such as the spleen (Fig. 1c), consistent with the possibility of up-regulation of CXCR6 in secondary lymphoid organs and migration of these cells to effector sites.
FIGURE 1. CXCR6/CXCL16 expression and in vitro migration to CXCL16. a, GVHD was induced in BDF1 (H-2bxd) mice by the transfer of splenocytes from C57BL/6 (H-2b) mice. On day 7 of disease, livers were harvested from GvHD and normal BDF1 mice, mRNA was isolated, and real-time PCR was performed. Samples were normalized to 15Sa rRNA, and chemokine and chemokine receptor expression from normal liver was assigned a relative value of 1.0. Data are expressed as the mean ± SEM (n = 4). b, GvHD was induced in BDF1 (allogeneic) mice by transfer of splenocytes from heterozygous CXCR6-GFP (CXCR6+/–, H-2b) mice. The same splenocytes were transferred into Thy1.1 (syngeneic, H-2b) mice as a negative control. GFP expression as a marker for CXCR6 expression was assessed by flow cytometry on donor LIL subsets. Representative histograms are shown. Histograms are scaled to more easily compare the percentage of positive lymphocytes. c, GFP (CXCR6) expression on donor lymphocyte subsets is compared in liver () and spleen (). d, CXCR6 message was amplified from C57BL/6 (B6) and CXCR6-GFP (CXCR6–/–) livers. -Actin message was amplified as a control. The primers used are described in Materials and Methods. e, Liver-infiltrating lymphocytes from day 7 of GVHD hepatitis induced by WT, CXCR6+/–, or CXCR6–/– donor cells were migrated to CXCL16 and CXCL12. A representative experiment of three independent experiments is shown.
Absence of CXCR6 results in reduced effector CD8 cell accumulation in liver
We next asked whether the absence of CXCR6 would influence the efficiency of transferred effector T cells to accumulate in the liver in a model of GvHD. Liver infiltration of donor T cells in GvHD was induced by transfer of WT (B6 into BDF1) or CXCR6-deficient (CXCR6–/– into BDF1) donor lymphocytes. We confirmed that these CXCR6 gene-targeted mice do not express CXCR6, as shown by RT-PCR (Fig. 1d), and thus constitute an ideal tool for investigating the role of CXCR6. We also confirmed in vitro that activated CXCR6–/– CD8 lymphocytes fail to migrate to the CXCR6 ligand CXCL16, but are still able to migrate to another chemokine, CXCL12, which was used as a positive control (Fig. 1e). CXCR6+/– mice have an intermediate phenotype (Fig. 1e). Subsequent comparisons were made between GvHD induced by WT and CXCR6–/– splenocytes. In our in vivo analysis on day 7, both groups of mice displayed marked hepatitis; the numbers of LIL were 6.1 ± 1.1 x 106 and 6.8 ± 1.9 x 106 cells/liver in GvHD generated by WT and CXCR6–/– lymphocytes, respectively. In comparison, syngeneic controls had 1.9 ± 0.3 x 106 cells/liver. Importantly, there were far fewer donor CD8 T cells in GvHD induced by CXCR6–/– cells (54% decrease in donor-derived CD8+ lymphocytes compared with WT GvHD, 172 ± 18 x 103 vs 79 ± 3 x 103 (mean ± SEM); n = 7; p < 0.05; Fig. 2a). No difference was observed in the number of donor cells infiltrating the spleen (B6, 1.7 ± 0.3 x 106; CXCR6–/–, 1.7 ± 0.4 x 106; Fig. 2b), suggesting that this reduction in liver CD8 cells is not a generalized phenomenon characterized by decreased donor cells in all compartments. The difference in liver extended to a reduction in the percentage of donor CD8 cells (39% reduction) among total CD8 cells (i.e., (donor CD8)/(total CD8) x 100; B6, 38 ± 5%; CXCR6–/–, 23 ± 4%; Fig. 2c; p < 0.05). However, no statistically significant difference was found in donor CD4 cells (Fig. 2d). Considered together, the decrease in donor CXCR6–/– CD8 lymphocytes in percentage and absolute numbers indicate that there was a selective decrease in the CXCR6–/– cell subset, not only an overall decrease in total LIL. These findings stress the importance of CXCR6 in the accumulation of pathological antihost CD8 T cells to a target organ of GvHD.
FIGURE 2. Lymphocyte accumulation in tissues. The absolute numbers of donor CD8 lymphocytes (CD8+H-2Dd – in liver (a) and spleen (b) 7 days after adoptive transfer are shown. GvHD was induced in BDF1 mice by transfer of splenocytes from WT and CXCR6-deficient (CXCR6–/–) C57BL/6 mice. Transfer of syngeneic CFSE-labeled BDF1 splenocytes into BDF1 mice served as the negative control (Control). The absolute number of donor lymphocytes was calculated by multiplying the percentage of CD8+H-2Dd –(allogeneic) and CD8+CFSE+ (syngeneic) cells in the small lymphocyte gate by the absolute number of lymphocytes recovered. The percentages of CD8 (c) and CD4 (d) lymphocytes of donor origin in liver and spleen were calculated by dividing the number of CD8+H-2Dd – and CD4+H-2Dd –lymphocytes by the total number of CD8+ and CD4+ lymphocytes in the respective tissues. The percentage of syngeneic donor CD8 lymphocytes was calculated by dividing the number of CD8+CFSE+ by the total number of CD8+ lymphocytes in the respective tissues. Data are shown as the mean ± SEM (n = 7). p < 0.05, WT vs CXCR6–/– groups.
Decreased accumulation of CXCR6–/– cells in liver is due to decreased recruitment
The decreased accumulation of antihost CXCR6–/– CD8 T cells could reflect decreased recruitment to the liver; however, altered proliferation or survival could also play a role. To differentiate between these possibilities, we developed a short term recruitment assay that minimizes the potential for proliferative or survival signals to influence the accumulation of donor lymphocytes in the liver. With this assay we were able to track donor cells infiltrating liver and lymphoid organs during a 6-h period after injection by determining the number of donor H-2Dd–Thy1.2+ cells in various compartments. We chose 6 h for the short term recruitment assay based on initial experiments that demonstrated the ability to detect migrating, short term recruitment donor cells in the liver in the midst of on-going GvHD hepatitis (data not shown). Importantly, essentially all donor CD8 T cells (H-2Db), whether CXCR6–/– or WT, were of the previously activated phenotype, as shown by the expression of (CD44high, CD45RBlow, and L-selectinlow); thus, the donor CD8 T cells appeared to share a similar phenotype and state of activation. Spleen cells from day 8 GvHD mice were injected i.v. into recipients (H-2Dbxd) also undergoing day 8 GvHD. After collecting cells from various tissues, we calculated a liver recruitment ratio, defined as the absolute number of CD8 or CD4 short term recruitment donor cells (CD8+H-2Dd–Thy1.2+ or CD4+H-2Dd–Thy1.2+) harvested from a specific compartment divided by the absolute number of such donor cells injected multiplied by 100. By performing this short term recruitment assay using donor lymphocytes from WT GvHD and CXCR6-deficient GvHD, we found that the liver recruitment ratio of donor CXCR6–/– CD8 cells was decreased by 33% (B6, 12 ± 2%; CXCR6–/–, 8 ± 1%; n = 8; p < 0.05; Fig. 3a), which corresponds well with the 39% decrease in the percentage of donor CD8 lymphocytes infiltrating the liver as described above (Fig. 2c). These findings suggest that CXCR6 supports recruitment, rather than proliferation, of activated CD8 cells in GvHD. It is noteworthy that the liver recruitment ratios of other CXCR6-deficient donor cells, such as Thy1.2+CD4+ T lymphocytes and IgDb+CD19+ naive B lymphocytes, were not different from those of WT animals (Fig. 3a). IgDb+CD19+ naive B lymphocytes were used as an internal negative control, because previous reports, which we confirmed in this study, have shown that naive B lymphocytes do not express CXCR6 ( 16, 17). Moreover, because naive B lymphocytes are a relatively rare population in GvHD liver, their localization may be considered a basal or background level. Of note, the recruitment of lymphocytes to recipient spleen, including CD8 cells, was not affected by CXCR6 deficiency (data not shown). These data suggest that the reduction of CXCR6–/– CD8 T cells in the liver is due to decreased recruitment rather than altered proliferation.
FIGURE 3. Short term recruitment of donor lymphocytes in the liver. GvHD was induced by adoptively transferring WT B6-Thy1.2, CXCR6–/–-Thy1.2 and B6-Thy1.1 (congenic) lymphocytes into separate BDF1 mice. On day 8 after adoptive transfer, lymphocytes from WT and CXCR6-deficient (CXCR6–/–) GvHD mice were transferred into congenic GvHD mice, and liver lymphocytes were isolated 6 h later. A liver recruitment ratio was calculated for different cell subsets (CD8, CD4, and CD19) in the liver: [cell subset]recruited/[cell subset]injected. b, Circulating levels of donor lymphocytes on day 8 after adoptive transfer. Blood was harvested by cardiac puncture in anesthetized, short term recruitment mice, stained, and analyzed by flow cytometry for the percentage of specific cell subsets (CD8+H-2Dd–Thy1.2+ or CD4+H-2Dd–Thy1.2+) among leukocytes. Data are shown as the mean ± SEM (n = 8). p < 0.05.
Reduced hepatic recruitment of CXCR6–/– CD8 lymphocytes could also reflect decreased availability in the blood. To address this, we examined the percentage of relevant cells circulating in the blood and found that the levels of CXCR6–/– vs WT CD8 cells (CD8 donor cells from the second transfer, i.e., CD8+H-2Dd–Thy1.2+; Fig. 3b; n = 8) in the blood of recipient mice 6 h after injection (i.e., at the end of the short term recruitment assays) were similar, suggesting that CXCR6 deficiency did not significantly alter availability in the blood.
Although little proliferation is likely to occur during the 6-h assay, to rule out any possible effect of proliferation on cell numbers, we compared the proliferation of CXCR6–/– vs WT donor T cell populations by measuring the incorporation of BrdU into LIL. We injected BrdU i.p. 2 h before harvesting LIL from day 7 WT and CXCR6-deficient GvHD mice. As shown above (Fig. 2a), the absolute number of CXCR6–/– donor lymphocytes infiltrating the liver was again decreased (Fig. 4a), but the percentage of WT donor vs CXCR6–/– CD8 lymphocytes incorporating BrdU was almost identical (WT B6, 14 ± 1%; CXCR6–/–, 16 ± 3%; Fig. 4b; n = 4–5), suggesting that the absence of CXCR6 does not affect the proliferative capacity of the donor CD8 cells in the liver.
FIGURE 4. Proliferation and apoptosis of donor lymphocytes in the liver. GvHD was induced by adoptively transferring wild-type B6-Thy1.2 and CXCR6–/–-Thy1.2 lymphocytes into separate BDF1 mice. On day 7, liver lymphocytes were isolated, and BrdU incorporation was determined in LIL by flow cytometry. a, A representative histogram showing BrdU staining of donor CD8 lymphocytes in the liver from wild-type (gray line) and CXCR6–/– (solid line) GvHD. b, Percentage of BrdU-positive donor CD8 lymphocytes is shown for WT () and CXCR6–/– () lymphocytes. c, Percentage of annexin V- and 7-AAD-positive donor CD8 lymphocytes is shown for WT () and CXCR6–/– () lymphocytes. Data are shown as the mean ± SEM (n = 4–8). Annexin, p = 0.17; 7-AAD, p = 0.87.
A third potential contributing factor to the accumulation of cells in the liver is apoptosis. It has been well established that a large number of activated cells programmed for apoptosis accumulate in the liver ( 22). To assess the possibility that CXCR6/CXCL16 may be solely providing a survival signal in the liver and thus accumulating more cells in WT GvHD, we examined donor LIL for annexin V and 7-aminoactinomycin D (7-AAD) staining to assess the number of cells undergoing apoptosis. With our method of isolating LIL, we found few of the harvested cells to be undergoing apoptosis or necrosis and little difference between the WT and CXCR6-deficient donor cells in terms of their apoptotic markers (annexin, 3 ± 0.6% of WT B6; 2 ± 0.5% of CXCR6–/– (p = 0.17); 7-AAD, 2 ± 0.2% of WT B6 vs 2 ± 0.4% of CXCR6–/– (p = 0.87); Fig. 4c; n = 4–8). These data thus support an important role for CXCR6 in CD8 cells homing into the liver.
Discussion
In this study we examined the in vivo role of the chemokine receptor CXCR6 in a model of lymphocyte recruitment to the liver during GvHD hepatitis. First, we examined the expression of CXCR6 on LIL using mice heterozygous for a GFP knockin mutation; the endogenous CXCR6 promoter drives the expression of GFP, allowing the detection of CXCR6+ cells by green fluorescence ( 17). Using these splenocytes as donors, we found few T cells infiltrating the liver under normal conditions over a 1-wk period, in contrast to the significant increase observed under inflamed conditions, concomitant with an increase in the percentage of lymphocytes expressing CXCR6. The increase in frequency of CXCR6+ T cells was most prominent in target tissues of inflammation (i.e., liver) compared with secondary lymphoid organs (i.e., spleen). This is consistent with past findings showing that CXCR6 is up-regulated upon activation and is found in abundance on T cells in Th1-mediated inflammation as in hepatitis C-induced ( 9, 18) and alcohol-induced cirrhosis and rheumatoid arthritis ( 9). Next, we examined expression of the ligand, CXCL16, and found it under both normal and inflamed conditions in the liver by RT-PCR. Our finding of CXCL16 up-regulation of mRNA expression during GvHD is consistent with the findings of others who have reported up-regulation during inflammation in the liver ( 16, 19) and in heart valves ( 23). CXCL16 is also observed in normal bone marrow, where it may participate in the homing of plasma cells to the bone marrow ( 24).
After confirming the expression of the receptor-ligand pair in GVHD hepatitis, we examined the functional significance of the pair by focusing on the natural course of GvHD in mice induced by transferred CXCR6-deficient cells. Transferring splenocytes from homozygous CXCR6-GFP mice, we examined the severity of disease by analyzing the frequency of donor cells infiltrating the liver. We found that the lack of CXCR6 on donor cells resulted in fewer CD8 LIL, in both percentage and absolute numbers, suggesting a specific decrease in the pathologically relevant cell population. Because this observation was made 1 wk after donor cell transfer, we could not exclude the possibility that CXCR6 was required for lymphocyte proliferation or amplification of the inflammatory reaction. We therefore examined the role of CXCR6 in recruitment more closely using a short term recruitment assay. By focusing our attention on a 6-h window, we were able to minimize the effect of proliferation and survival while concentrating on actual homing. With this short term recruitment assay we again found a subset-specific decrease in the donor CD8 T cells, demonstrating a specific role for CXCR6 in activated CD8 T cell localization from blood into the inflamed liver.
A role for CXCR6 and CXCL16 in cell recruitment from blood has been suggested previously based on their ability to mediate chemotaxis ( 13, 16) and firm adhesion (24, 25) of different cell populations in vitro. In its soluble form, CXCL16 could be transported and presented luminally by endothelial cells to mediate chemokine-dependent activation of surface integrins on lymphocytes. Indeed, CXCL16-dependent firm adhesion of 41+ lymphocytes on VCAM-1+ endothelial cells has been suggested to be important in firm adhesion of lymphocytes to neocapillaries of infected cardiac heart valves ( 23). In its transmembrane form, CXCL16 may be able to function as a structural adhesion molecule, anchoring activated CD8 lymphocytes, which may promote their retention in the site of inflammation ( 24, 25). CXCL16 joins fractalkine (CX3C ligand 1) as the only other transmembrane chemokine that can function in both its soluble and its membrane-bound form to regulate cell migration and adhesion ( 26, 27).
A role for CXCR6/CXCL16 in providing a survival signal could also be put forth, because CXCL16 is up-regulated on DC under activating conditions ( 16) with a concomitant increase in CXCR6 on cocultured T cells ( 9). Other chemokines, such as stromal cell-derived factor-1, have been shown to be survival factors for CD4 T cells ( 28). However, in our analysis of markers of proliferation (BrdU incorporation) and apoptosis (annexin V and 7-AAD) on donor CD8 LIL, we found no significant difference in the percentage of positive cells, suggesting no proliferative or survival advantage of WT cells over CXCR6-deficient cells. Our current studies have concentrated on short term recruitment, but additional studies will be necessary to critically examine possible additional roles of CXCR6/CXCL16 in the liver. One factor we have not been able to address directly in the current studies is the rate of exit of homed cells from the liver. Detailed studies conducted in sheep estimate the total output of lymphocytes from the liver through the afferent lymphatics to be 1 x 106 cells/h under normal conditions ( 29). However, under activated conditions (i.e., after the administration of i.v. endotoxin), afferent lymphatic flow from the liver to the liver-draining lymph node peaked at 6 h, but cellular output decreased until 6 h and subsequently peaked by 18 h. Furthermore, cell transfer experiments of alloantigen-stimulated lymphocytes into normal rats showed progressive accumulation of cells in the liver from 6–9 h, but peak accumulation in the liver-draining lymph node occurred later, at 15 h ( 30). The results of both studies suggest that at 6 h after injection, the liver is rapidly accumulating cells, and cellular exit is likely to have little effect on cell numbers at this early time point.
The residual accumulation of CXCR6–/– CD8 cells in the liver indicates that other mechanisms compensate for the CXCR6 deficit. Other investigators have suggested a role of CCR5 in mediating recruitment of CD8 donor T lymphocytes to the liver in GvHD ( 31, 32, 33). Their studies examined total lymphocyte accumulation at different time points, but did not exclude a potential contribution of concomitant effects on proliferation; nevertheless, CCR5 and CXCR6 may both participate in the accumulation of CD8 lymphocytes in GvHD-induced hepatitis. Indeed, CCR5-, CXCR3-, and CXCR6-expressing lymphocytes have been found in liver infiltrates of patients with hepatitis C ( 18, 34), and combinations of these three chemokine receptors can be coexpressed on activated T cells ( 8, 17). Experiments have also supported an important role of CXCR3 in many target organs of GvHD, including the liver, as shown by improved liver histology in GvHD induced by CXCR3-deficient lymphocytes compared with WT ( 35). Beyond being partially redundant for recruitment into the liver, multiple chemokine receptors may also play a role in navigation within the liver. Others have stressed the concept of a "symphony" of chemokines mediating an inflammatory process ( 36) and have emphasized that combinations of chemokine receptors, rather than individual receptors, act together to define the trafficking of differentiated effector/memory lymphocyte populations ( 8).
In conclusion, our novel data demonstrate that CXCR6 is an important mediator of the accumulation of activated CD8 T cells in the liver in GVHD-induced hepatitis. The reduced accumulation of CXCR6–/– CD8 cells appears to reflect, at least in large part, a significant defect in their ability to migrate into inflamed liver from blood. Thus, we conclude that CXCR6 plays an important role in the hepatic homing and accumulation of activated CD8 T lymphocytes and may act in concert with other chemokine receptors to orchestrate pathological inflammation in the liver.
Acknowledgments
We thank C. Crumpton and L. Rott for their expertise at the flow cytometry core facility, L. Ohler and G. Sperinde for their expertise in real-time PCR, and C. Arnold and G. Debes for their constructive criticism and suggestions on the paper.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by grants from the National Institutes of Health, the Department of Veterans Affairs (to E.C.B.), and the FACS Core Facility of the Stanford Digestive Diseases Center. T.S. was supported by a K08 grant from the National Institutes of Health. H.T. was supported by the Swedish Research Council (2001-6576, 2002-955, 2002-8012, and 2003-4661), Crafoordska stiftelsen, Blanceflors stiftelse, Einar och Inga Nilssons stiftelse, Harald och Greta Jaenssons stiftelse, Greta och Johan Kocks stiftelser, Fr?ken Agnes Nilssons stiftelse, Magnus Bergvalls stiftelse, Mossfelts stiftelse, Nanna Svartz stiftelse, Ruth och Richard Julins stiftelse, Svenska L?kares?llskapet, and Teggers stiftelse.
2 Address correspondence and reprint requests to Dr. Tohru Sato, Veterans Affairs Palo Alto Health Care System, 3801 Miranda Avenue, MC154B, Palo Alto, CA 94304. E-mail address: tsato@stanford.edu
3 T.S. and H.T. contributed equally to this work.
4 Current address: Department of Surgery, Lund University, Malmo University Hospital, S-205 02, Malmo, Sweden.
5 Current address: Dalhousie University, Sir Charles Tupper Medical Building, Room 7C, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5.
6 Abbreviations used in this paper: GvHD, graft-vs-host disease; 7-AAD, 7-aminoactinomycin D; CXCL, CXC ligand; DC, dendritic cell; LIL, liver-infiltrating lymphocyte; WT, wild type.
Received for publication March 23, 2004. Accepted for publication October 22, 2004.
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Hepatic infiltration of activated CD8 lymphocytes is a major feature of graft-vs-host disease (GvHD). Chemoattractant cytokines and their receptors are key regulators of lymphocyte trafficking, but the involvement of chemoattractant receptors in the physiologic recruitment of cells into the inflamed liver has not been defined. The present study examines the role of the chemokine receptor CXCR6, which is highly expressed by liver-infiltrating CD8 T cells. Hepatic accumulation of donor CD8, but not donor CD4, lymphocytes was significantly reduced in GvHD induced by transfer of CXCR6–/–, H-2Db lymphocytes into BDF1, H-2Dbxd recipients. To determine whether altered recruitment contributes to the reduced accumulation, CXCR6–/– or wild-type splenic lymphocytes participating in an active GvHD response were isolated and transferred i.v. into secondary recipients with active GvHD, and the short term (6-h) recruitment of transferred cells to the inflamed liver was assessed. CXCR6–/– CD8 (but not CD4) cells displayed a significant (33%) reduction in liver localization, whereas frequencies in blood of CXCR6–/– and wild-type CD8 cells were similar. Proliferation and apoptosis of liver-infiltrating donor CD8 cells were unaffected. We conclude that CXCR6 helps mediate the recruitment of activated CD8 lymphocytes in GvHD-induced hepatitis and may be a useful target to treat pathological inflammation in the liver.
Introduction
Donor T cell recruitment into target tissues is a prominent component of graft-vs-host disease (GvHD),6 and a major complication in allogenic bone marrow transplantation ( 1). Recruitment of activated lymphocytes is a multistep process in which the expression of specific adhesion molecules and chemokine receptors endows lymphocytes with the ability to access extravascular tissues ( 2). Chemokine receptors play a central role in lymphocyte recruitment by rapidly transmitting intracellular signals and triggering increased integrin affinity to adhesion molecule ligands displayed by endothelial cells ( 3). At present, >20 chemokine receptors have been described ( 4). The role of specific chemokine receptors in attracting activated lymphocytes appears to be both stimulus and organ dependent, contributing to inflammatory and homeostatic tissue localization of lymphocyte subsets ( 5). In GvHD the liver is a key target organ, characterized by massive lymphocyte accumulation ( 6), which may reflect enhanced recruitment and/or proliferation.
Acute GvHD is initiated by donor T cells recognizing and reacting to host allo-Ags and is characterized by a Th1-polarized response ( 7). Different combinations of chemokine receptors are associated with Th1- vs Th2-dependent immune reactions ( 8). Accordingly, it has been shown that the chemokine receptor CXCR6 (Bonzo/STRL33/TYMSTR) is expressed on T cells of Th1 phenotype ( 9, 10, 11, 12, 13). CXCR6, originally described as a novel simian immunodeficiency virus receptor ( 14) and fusion cofactor for HIV-1 strains ( 15), is expressed at very low levels in naive CD8 cells ( 16, 17), but can be up-regulated in T cells by activated dendritic cells (DC) ( 9, 10, 11). Moreover, it has been shown that many Th1-polarized cells ( 13) and in vitro-activated CD8 cells ( 16) are attracted by the CXCR6 ligand, CXC ligand 16 (CXCL16; SR-PSOX/SexCKine). In target tissues, CXCR6-expressing cells are readily found in the liver of patients with alcohol- or hepatitis C-induced liver cirrhosis ( 9, 18). CXCL16 is abundantly expressed in lymphoid and nonlymphoid tissues, such as the liver ( 13, 16, 19). Furthermore, the kinetics of DC-mediated up-regulation also suggest a role of CXCR6 in facilitating effector T cell migration into sites of pathological inflammation; CXCR6 is up-regulated, whereas CCR7, a chemokine receptor involved in homing to lymph nodes, is down-regulated after prolonged incubation with DC under Th1-polarizing conditions ( 11). However, the role of CXCR6 in lymphocyte recruitment in vivo is not known.
The aim of this study was to assess the contribution of CXCR6 to the recruitment of activated lymphocytes into inflamed liver. For this purpose we developed a short term, in vivo recruitment assay of activated lymphocytes into the inflamed livers of mice undergoing active GvHD and compared the recruitment of CXCR6–/– vs CXCR6+/+ lymphocytes. Our results show that CXCR6 has a significant role in recruiting activated CD8, but not CD4, lymphocytes to the inflamed liver in GvHD-induced hepatitis.
Materials and Methods
Animal model of GvHD
C57BL/6J (B6, H-2b; The Jackson Laboratory), C57BL/6J-IghaThy1aGpi1a (Thy1.1, H-2b; The Jackson Laboratory), and CXCR6-GFP knockin mice (CXCR6–/–, backcrossed onto C57BL/6 more than eight generations, H-2b) were used as donors. CXCR6-GFP mice have the CXCR6-coding sequence replaced with a sequence for enhanced GFP, which is regulated through the CXCR6 promoter ( 17). B6D2F1/J mice (BDF1, H-2bxd; The Jackson Laboratory) were used as recipients or as donors for syngeneic control experiments. All mice were 8–12 wk old and housed under conventional conditions in the animal facility at the Veterans Affairs Palo Alto Health Care System. All experiments were approved by the local ethics committee at Stanford University. Nonlethal GvHD was induced as previously described ( 20). Briefly, 50 x 106 donor lymphocytes isolated from the spleen were transferred i.v. into recipient mice on day 0. The splenocytes from C57BL/6 and CXCR6–/– were similar with respect to the expression of activation markers (CD69, CD25, CD44, and CD45RB) and adhesion molecules (L-selectin and 47) and cell subset distribution (CD4, CD8, NK, NKT, and B cells; data not shown). On day 7, recipient mice were killed by CO2 inhalation, and the liver, spleen, and blood were harvested. In indicated experiments, syngeneic lymphocytes were labeled with 0.5 μM CFSE (Molecular Probes) at 37°C for 10 min before injection.
Short term recruitment assay
GvHD was induced in BDF1 mice by transferring lymphocytes from B6-Thy1.2, congenic B6-Thy1.1, or CXCR6–/–-Thy1.2 mice as described above. This congenic model for identification of donor lymphocytes was chosen because using CXCR6–/– mice expressing GFP as donors excluded CFSE labeling of donor cells. On day 8 after transfer, lymphocytes were isolated from the spleen of GvHD mice induced by Thy1.2 cells (wild-type (WT) or CXCR6–/–), and 20 x 106 cells were subsequently transferred to GvHD mice induced by Thy1.1 cells (short term recruitment mice). Six hours after the second transfer, the short term recruitment mice were killed, and cells from the liver, spleen, and blood were harvested. This 6-h window allowed us to compare the recruitment of Thy1.2 cells with or without CXCR6 into inflamed livers. The phenotypes of the 20 x 106 lymphocytes injected from WT and CXCR6–/– mice were similar with respect to the expression of activation markers, adhesion molecules, and cell subset distribution described above (data not shown). A liver recruitment ratio was calculated as the absolute number of cells recruited to the liver divided by the number of injected cells. Short term recruitment was determined for CD8+H-2Dd–Thy1.2+, CD4+H-2Dd–Thy1.2+, and CD19+H-2Dd–IgDb+ cell subsets. Additionally, the level of defined donor cell subsets circulating in the blood was analyzed.
Isolation of liver-infiltrating lymphocytes (LIL)
After mice were killed, the liver was perfused through the portal vein with 5 ml of PBS. The liver was harvested, dispersed through a stainless steel mesh (70-μm pore size), and washed with HBSS containing 10% bovine calf serum. The dispersed cells were mixed with a 33% Percoll (Amersham Biosciences) solution containing 100 U/ml heparin (Sigma-Aldrich) and centrifuged for 20 min (800 x g) at room temperature. Next, the cell pellet was resuspended in double-distilled water for 20 s to lyse RBC, and the lysis was stopped with 2x PBS. The isolated lymphocytes were then resuspended in HBSS (2% bovine calf serum), counted in a hemocytometer, and kept on ice until staining for flow cytometric analysis.
RT-PCR and real-time PCR
Total RNA was extracted from whole liver tissue using an RNeasy kit according to the manufacturer’s instructions (Qiagen). RNA concentration and purity were determined by measuring absorbance spectrophotometrically at 260 and 280 nm. RT-PCR was performed with ThermoScript RT-PCR System (Invitrogen Life Technologies). Mouse -actin served as an internal control gene. The PCR profile was 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, followed by a final extension of 7 min at 72°C. The primer sequences of CXCR6, CXCL16, and -actin were: CXCR6 (forward), 5'-TAC GAT GGG CAC TAC GAG GGA G-3'; CXCR6 (reverse), 5'-GCA AAG AAA CCA ACA GGG AGA CCA C-3'; CXCL16 (forward), 5'-GCT TTG GAC CCT TGT CTC TTG C-3'; CXCL16 (reverse), 5'-GTG CTG AGT GCT CTG ACT ATG TGC-3'; -actin (forward), 5'-ATG TTT GAG ACC TTC AAC ACC-3'; and -actin (reverse), 5'-TCT CCA GGG AGG AAG AGG AT-3'. For real-time PCR of CXCR6 and CXCL16 from normal and GvHD liver, liver tissue was harvested, and total RNA was isolated as described above and converted to cDNA using the Reverse Transcription System (Promega) following the manufacturer’s instructions. 15Sa was used as an endogenous control for relative quantification using the comparative threshold cycle method. All reactions were performed in triplicate on an Mx3000P Real-Time PCR System (Stratagene) using 2x SYBR Green and the following primers: 15S (forward), 5'-GAAGCACGGATACATTGGTGA3'; 15S (reverse), 5'-TGTTCTGCCATTTCTCTAGGT-3'; CXCR6 (forward), 5'-CCCTGTACTTTATGCCTTTG-3'; CXCR6 (reverse), 5'-CTTGGAACTGTCCTCAGAAG-3'; CXCL16 (forward), 5'-CCTTGTCTCTTGCGTTCTTCC-3'; CXCL16 (reverse), 5'-TCCAAAGTACCCTGCGGTATC-3'; CXCL10 (forward), 5'-CCAAGTGCTGCCGTCATTTTC-3'; and CXCL10 (reverse), 5'-GGCTCGCAGGGATGATTTCAA-3'. The chemokine or chemokine receptor expression in normal liver was given an arbitrary value of 1, and their expression in GvHD liver was expressed as fold up-regulation from normal.
Flow cytometry
Isolated cells (1–2 x 106) were stained with the following rat anti-mouse Abs conjugated to FITC, PE, PerCP, PE-indotricarbocyanine, allophycocyanin, allophycocyanin-indotricarbocyanine, or biotin: H-2Dd (clone 34-2-12), CD4 (clone RM4-5), CD8 (clone 53-6.7), CD44 (clone IM7), CD45RB (clone 16A), CD25 (clone PC61), CD69 (clone H1.2F3), L-selectin (clone Mel-14), 47 (clone DATK 32), Thy1.2 (clone 53-2.1), CD19 (clone 1D3), IgDb (clone 217-170), and NK1.1 (clone PK136). The above-mentioned Abs were obtained from BD Pharmingen. NeutrAvidin Cascade Blue was purchased from Molecular Probes. To study proliferation, we used a BrdU Flow Kit (BD Biosciences, San Jose, CA) following the manufacturer’s instructions. Briefly, 1 mg of BrdU was injected i.p. 2 h before death, and the isolated lymphocytes were subsequently permeabilized for detection of intracellular BrdU. Four-color flow cytometry was performed using a FACSCalibur (BD Biosciences), and seven-color flow cytometry was performed with LSR II (BD Biosciences). The data were acquired and analyzed on CellQuest software (version 3.3) for FACSCalibur and on FACSDiVa software (version 2.1) and CellQuest-Pro software (version 4.0) for LSR II. Data are presented as the mean ± SEM. Student’s t test was used for between-group comparisons, and significance was set at p < 0.05.
Migration assay
LIL were isolated from GvHD livers induced by WT, CXCR6+/–, and CXCR6–/– splenocytes at day 7 of disease. LIL (0.5–1 x 106) were placed in the upper chamber of Transwell inserts (5-μm pore size; Corning Costar) and migrated to chemokines at their optimal concentrations for 2 h at 37°C as described previously ( 21). After migration, polystyrene beads (Polysciences) were added to each bottom well as an internal standard, and an aliquot was collected for analysis by flow cytometry for calculation of the percent migrated. Triplicate wells were pooled and stained with Abs to H-2Dd and CD8 for calculating donor CD8 migration. The data presented are the percent migration of the indicated subsets, determined by measuring the number of cells of each subset in the starting and migrated populations. The chemokine CXCL12 was purchased from PeproTech, and CXCL16 was obtained from R&D Systems.
Results
CXCR6 is up-regulated in GvHD hepatitis
We initially asked whether CXCR6 and CXCL16 are relevant candidates to study in liver homing by examining their expression under normal and inflamed conditions. We examined livers from control and/or GvHD mice 7 days after lymphocyte transfer. CXCR6 and CXCL16 mRNA were amplified by PCR from total RNA, and we found clear evidence of CXCR6 and CXCL16 mRNA expression in both healthy controls and GvHD animals (data not shown). We also quantitatively analyzed the expression of CXCR6 and CXCL16 and found an 2-fold increase in mRNA expression in GvHD-inflamed liver compared with normal liver, as assessed by real-time PCR (mean ± SEM fold increase: CXCR6, 1.6 ± 0.1; CXCL16, 1.9 ± 0.1; Fig. 1a; n = 4). Next, we asked whether CXCR6 was expressed on transferred lymphocytes infiltrating the liver. For this purpose, we used heterozygous GFP knockin mice, in which one allele of the CXCR6 gene has been replaced by enhanced GFP, to examine the expression of CXCR6 in normal (syngeneic transfer) and inflamed (allogeneic transfer) liver ( 17). We analyzed GFP expression in LIL from day 7 of GvHD induced by CXCR6-GFP (CXCR6+/–) lymphocytes in allogeneic and syngeneic hosts. We observed an increase in the percentage of GFPhigh lymphocytes among donor CD4 and CD8 lymphocytes in the liver of allogeneic vs syngeneic recipients. The percentages of CD8+GFPhigh donor lymphocytes among CD8 LIL were 24 ± 3 and 60 ± 2% in syngeneic and allogeneic recipients, respectively (Fig. 1b; n = 3–5; p < 0.05). Similarly, the percentage of CD4+GFPhigh donor lymphocytes among CD4 LIL increased from 9 ± 1% in syngeneic recipients to 42 ± 2% in allogeneic recipients (Fig. 1b; n = 3–5; p < 0.05). Because GFP is under the control of the CXCR6 promoter, these findings suggest that donor lymphocytes up-regulate CXCR6 before or during accumulation in the liver. Furthermore, we observed that a larger percentage of the donor cells were GFPhigh in the GvHD liver as opposed to secondary lymphoid tissues such as the spleen (Fig. 1c), consistent with the possibility of up-regulation of CXCR6 in secondary lymphoid organs and migration of these cells to effector sites.
FIGURE 1. CXCR6/CXCL16 expression and in vitro migration to CXCL16. a, GVHD was induced in BDF1 (H-2bxd) mice by the transfer of splenocytes from C57BL/6 (H-2b) mice. On day 7 of disease, livers were harvested from GvHD and normal BDF1 mice, mRNA was isolated, and real-time PCR was performed. Samples were normalized to 15Sa rRNA, and chemokine and chemokine receptor expression from normal liver was assigned a relative value of 1.0. Data are expressed as the mean ± SEM (n = 4). b, GvHD was induced in BDF1 (allogeneic) mice by transfer of splenocytes from heterozygous CXCR6-GFP (CXCR6+/–, H-2b) mice. The same splenocytes were transferred into Thy1.1 (syngeneic, H-2b) mice as a negative control. GFP expression as a marker for CXCR6 expression was assessed by flow cytometry on donor LIL subsets. Representative histograms are shown. Histograms are scaled to more easily compare the percentage of positive lymphocytes. c, GFP (CXCR6) expression on donor lymphocyte subsets is compared in liver () and spleen (). d, CXCR6 message was amplified from C57BL/6 (B6) and CXCR6-GFP (CXCR6–/–) livers. -Actin message was amplified as a control. The primers used are described in Materials and Methods. e, Liver-infiltrating lymphocytes from day 7 of GVHD hepatitis induced by WT, CXCR6+/–, or CXCR6–/– donor cells were migrated to CXCL16 and CXCL12. A representative experiment of three independent experiments is shown.
Absence of CXCR6 results in reduced effector CD8 cell accumulation in liver
We next asked whether the absence of CXCR6 would influence the efficiency of transferred effector T cells to accumulate in the liver in a model of GvHD. Liver infiltration of donor T cells in GvHD was induced by transfer of WT (B6 into BDF1) or CXCR6-deficient (CXCR6–/– into BDF1) donor lymphocytes. We confirmed that these CXCR6 gene-targeted mice do not express CXCR6, as shown by RT-PCR (Fig. 1d), and thus constitute an ideal tool for investigating the role of CXCR6. We also confirmed in vitro that activated CXCR6–/– CD8 lymphocytes fail to migrate to the CXCR6 ligand CXCL16, but are still able to migrate to another chemokine, CXCL12, which was used as a positive control (Fig. 1e). CXCR6+/– mice have an intermediate phenotype (Fig. 1e). Subsequent comparisons were made between GvHD induced by WT and CXCR6–/– splenocytes. In our in vivo analysis on day 7, both groups of mice displayed marked hepatitis; the numbers of LIL were 6.1 ± 1.1 x 106 and 6.8 ± 1.9 x 106 cells/liver in GvHD generated by WT and CXCR6–/– lymphocytes, respectively. In comparison, syngeneic controls had 1.9 ± 0.3 x 106 cells/liver. Importantly, there were far fewer donor CD8 T cells in GvHD induced by CXCR6–/– cells (54% decrease in donor-derived CD8+ lymphocytes compared with WT GvHD, 172 ± 18 x 103 vs 79 ± 3 x 103 (mean ± SEM); n = 7; p < 0.05; Fig. 2a). No difference was observed in the number of donor cells infiltrating the spleen (B6, 1.7 ± 0.3 x 106; CXCR6–/–, 1.7 ± 0.4 x 106; Fig. 2b), suggesting that this reduction in liver CD8 cells is not a generalized phenomenon characterized by decreased donor cells in all compartments. The difference in liver extended to a reduction in the percentage of donor CD8 cells (39% reduction) among total CD8 cells (i.e., (donor CD8)/(total CD8) x 100; B6, 38 ± 5%; CXCR6–/–, 23 ± 4%; Fig. 2c; p < 0.05). However, no statistically significant difference was found in donor CD4 cells (Fig. 2d). Considered together, the decrease in donor CXCR6–/– CD8 lymphocytes in percentage and absolute numbers indicate that there was a selective decrease in the CXCR6–/– cell subset, not only an overall decrease in total LIL. These findings stress the importance of CXCR6 in the accumulation of pathological antihost CD8 T cells to a target organ of GvHD.
FIGURE 2. Lymphocyte accumulation in tissues. The absolute numbers of donor CD8 lymphocytes (CD8+H-2Dd – in liver (a) and spleen (b) 7 days after adoptive transfer are shown. GvHD was induced in BDF1 mice by transfer of splenocytes from WT and CXCR6-deficient (CXCR6–/–) C57BL/6 mice. Transfer of syngeneic CFSE-labeled BDF1 splenocytes into BDF1 mice served as the negative control (Control). The absolute number of donor lymphocytes was calculated by multiplying the percentage of CD8+H-2Dd –(allogeneic) and CD8+CFSE+ (syngeneic) cells in the small lymphocyte gate by the absolute number of lymphocytes recovered. The percentages of CD8 (c) and CD4 (d) lymphocytes of donor origin in liver and spleen were calculated by dividing the number of CD8+H-2Dd – and CD4+H-2Dd –lymphocytes by the total number of CD8+ and CD4+ lymphocytes in the respective tissues. The percentage of syngeneic donor CD8 lymphocytes was calculated by dividing the number of CD8+CFSE+ by the total number of CD8+ lymphocytes in the respective tissues. Data are shown as the mean ± SEM (n = 7). p < 0.05, WT vs CXCR6–/– groups.
Decreased accumulation of CXCR6–/– cells in liver is due to decreased recruitment
The decreased accumulation of antihost CXCR6–/– CD8 T cells could reflect decreased recruitment to the liver; however, altered proliferation or survival could also play a role. To differentiate between these possibilities, we developed a short term recruitment assay that minimizes the potential for proliferative or survival signals to influence the accumulation of donor lymphocytes in the liver. With this assay we were able to track donor cells infiltrating liver and lymphoid organs during a 6-h period after injection by determining the number of donor H-2Dd–Thy1.2+ cells in various compartments. We chose 6 h for the short term recruitment assay based on initial experiments that demonstrated the ability to detect migrating, short term recruitment donor cells in the liver in the midst of on-going GvHD hepatitis (data not shown). Importantly, essentially all donor CD8 T cells (H-2Db), whether CXCR6–/– or WT, were of the previously activated phenotype, as shown by the expression of (CD44high, CD45RBlow, and L-selectinlow); thus, the donor CD8 T cells appeared to share a similar phenotype and state of activation. Spleen cells from day 8 GvHD mice were injected i.v. into recipients (H-2Dbxd) also undergoing day 8 GvHD. After collecting cells from various tissues, we calculated a liver recruitment ratio, defined as the absolute number of CD8 or CD4 short term recruitment donor cells (CD8+H-2Dd–Thy1.2+ or CD4+H-2Dd–Thy1.2+) harvested from a specific compartment divided by the absolute number of such donor cells injected multiplied by 100. By performing this short term recruitment assay using donor lymphocytes from WT GvHD and CXCR6-deficient GvHD, we found that the liver recruitment ratio of donor CXCR6–/– CD8 cells was decreased by 33% (B6, 12 ± 2%; CXCR6–/–, 8 ± 1%; n = 8; p < 0.05; Fig. 3a), which corresponds well with the 39% decrease in the percentage of donor CD8 lymphocytes infiltrating the liver as described above (Fig. 2c). These findings suggest that CXCR6 supports recruitment, rather than proliferation, of activated CD8 cells in GvHD. It is noteworthy that the liver recruitment ratios of other CXCR6-deficient donor cells, such as Thy1.2+CD4+ T lymphocytes and IgDb+CD19+ naive B lymphocytes, were not different from those of WT animals (Fig. 3a). IgDb+CD19+ naive B lymphocytes were used as an internal negative control, because previous reports, which we confirmed in this study, have shown that naive B lymphocytes do not express CXCR6 ( 16, 17). Moreover, because naive B lymphocytes are a relatively rare population in GvHD liver, their localization may be considered a basal or background level. Of note, the recruitment of lymphocytes to recipient spleen, including CD8 cells, was not affected by CXCR6 deficiency (data not shown). These data suggest that the reduction of CXCR6–/– CD8 T cells in the liver is due to decreased recruitment rather than altered proliferation.
FIGURE 3. Short term recruitment of donor lymphocytes in the liver. GvHD was induced by adoptively transferring WT B6-Thy1.2, CXCR6–/–-Thy1.2 and B6-Thy1.1 (congenic) lymphocytes into separate BDF1 mice. On day 8 after adoptive transfer, lymphocytes from WT and CXCR6-deficient (CXCR6–/–) GvHD mice were transferred into congenic GvHD mice, and liver lymphocytes were isolated 6 h later. A liver recruitment ratio was calculated for different cell subsets (CD8, CD4, and CD19) in the liver: [cell subset]recruited/[cell subset]injected. b, Circulating levels of donor lymphocytes on day 8 after adoptive transfer. Blood was harvested by cardiac puncture in anesthetized, short term recruitment mice, stained, and analyzed by flow cytometry for the percentage of specific cell subsets (CD8+H-2Dd–Thy1.2+ or CD4+H-2Dd–Thy1.2+) among leukocytes. Data are shown as the mean ± SEM (n = 8). p < 0.05.
Reduced hepatic recruitment of CXCR6–/– CD8 lymphocytes could also reflect decreased availability in the blood. To address this, we examined the percentage of relevant cells circulating in the blood and found that the levels of CXCR6–/– vs WT CD8 cells (CD8 donor cells from the second transfer, i.e., CD8+H-2Dd–Thy1.2+; Fig. 3b; n = 8) in the blood of recipient mice 6 h after injection (i.e., at the end of the short term recruitment assays) were similar, suggesting that CXCR6 deficiency did not significantly alter availability in the blood.
Although little proliferation is likely to occur during the 6-h assay, to rule out any possible effect of proliferation on cell numbers, we compared the proliferation of CXCR6–/– vs WT donor T cell populations by measuring the incorporation of BrdU into LIL. We injected BrdU i.p. 2 h before harvesting LIL from day 7 WT and CXCR6-deficient GvHD mice. As shown above (Fig. 2a), the absolute number of CXCR6–/– donor lymphocytes infiltrating the liver was again decreased (Fig. 4a), but the percentage of WT donor vs CXCR6–/– CD8 lymphocytes incorporating BrdU was almost identical (WT B6, 14 ± 1%; CXCR6–/–, 16 ± 3%; Fig. 4b; n = 4–5), suggesting that the absence of CXCR6 does not affect the proliferative capacity of the donor CD8 cells in the liver.
FIGURE 4. Proliferation and apoptosis of donor lymphocytes in the liver. GvHD was induced by adoptively transferring wild-type B6-Thy1.2 and CXCR6–/–-Thy1.2 lymphocytes into separate BDF1 mice. On day 7, liver lymphocytes were isolated, and BrdU incorporation was determined in LIL by flow cytometry. a, A representative histogram showing BrdU staining of donor CD8 lymphocytes in the liver from wild-type (gray line) and CXCR6–/– (solid line) GvHD. b, Percentage of BrdU-positive donor CD8 lymphocytes is shown for WT () and CXCR6–/– () lymphocytes. c, Percentage of annexin V- and 7-AAD-positive donor CD8 lymphocytes is shown for WT () and CXCR6–/– () lymphocytes. Data are shown as the mean ± SEM (n = 4–8). Annexin, p = 0.17; 7-AAD, p = 0.87.
A third potential contributing factor to the accumulation of cells in the liver is apoptosis. It has been well established that a large number of activated cells programmed for apoptosis accumulate in the liver ( 22). To assess the possibility that CXCR6/CXCL16 may be solely providing a survival signal in the liver and thus accumulating more cells in WT GvHD, we examined donor LIL for annexin V and 7-aminoactinomycin D (7-AAD) staining to assess the number of cells undergoing apoptosis. With our method of isolating LIL, we found few of the harvested cells to be undergoing apoptosis or necrosis and little difference between the WT and CXCR6-deficient donor cells in terms of their apoptotic markers (annexin, 3 ± 0.6% of WT B6; 2 ± 0.5% of CXCR6–/– (p = 0.17); 7-AAD, 2 ± 0.2% of WT B6 vs 2 ± 0.4% of CXCR6–/– (p = 0.87); Fig. 4c; n = 4–8). These data thus support an important role for CXCR6 in CD8 cells homing into the liver.
Discussion
In this study we examined the in vivo role of the chemokine receptor CXCR6 in a model of lymphocyte recruitment to the liver during GvHD hepatitis. First, we examined the expression of CXCR6 on LIL using mice heterozygous for a GFP knockin mutation; the endogenous CXCR6 promoter drives the expression of GFP, allowing the detection of CXCR6+ cells by green fluorescence ( 17). Using these splenocytes as donors, we found few T cells infiltrating the liver under normal conditions over a 1-wk period, in contrast to the significant increase observed under inflamed conditions, concomitant with an increase in the percentage of lymphocytes expressing CXCR6. The increase in frequency of CXCR6+ T cells was most prominent in target tissues of inflammation (i.e., liver) compared with secondary lymphoid organs (i.e., spleen). This is consistent with past findings showing that CXCR6 is up-regulated upon activation and is found in abundance on T cells in Th1-mediated inflammation as in hepatitis C-induced ( 9, 18) and alcohol-induced cirrhosis and rheumatoid arthritis ( 9). Next, we examined expression of the ligand, CXCL16, and found it under both normal and inflamed conditions in the liver by RT-PCR. Our finding of CXCL16 up-regulation of mRNA expression during GvHD is consistent with the findings of others who have reported up-regulation during inflammation in the liver ( 16, 19) and in heart valves ( 23). CXCL16 is also observed in normal bone marrow, where it may participate in the homing of plasma cells to the bone marrow ( 24).
After confirming the expression of the receptor-ligand pair in GVHD hepatitis, we examined the functional significance of the pair by focusing on the natural course of GvHD in mice induced by transferred CXCR6-deficient cells. Transferring splenocytes from homozygous CXCR6-GFP mice, we examined the severity of disease by analyzing the frequency of donor cells infiltrating the liver. We found that the lack of CXCR6 on donor cells resulted in fewer CD8 LIL, in both percentage and absolute numbers, suggesting a specific decrease in the pathologically relevant cell population. Because this observation was made 1 wk after donor cell transfer, we could not exclude the possibility that CXCR6 was required for lymphocyte proliferation or amplification of the inflammatory reaction. We therefore examined the role of CXCR6 in recruitment more closely using a short term recruitment assay. By focusing our attention on a 6-h window, we were able to minimize the effect of proliferation and survival while concentrating on actual homing. With this short term recruitment assay we again found a subset-specific decrease in the donor CD8 T cells, demonstrating a specific role for CXCR6 in activated CD8 T cell localization from blood into the inflamed liver.
A role for CXCR6 and CXCL16 in cell recruitment from blood has been suggested previously based on their ability to mediate chemotaxis ( 13, 16) and firm adhesion (24, 25) of different cell populations in vitro. In its soluble form, CXCL16 could be transported and presented luminally by endothelial cells to mediate chemokine-dependent activation of surface integrins on lymphocytes. Indeed, CXCL16-dependent firm adhesion of 41+ lymphocytes on VCAM-1+ endothelial cells has been suggested to be important in firm adhesion of lymphocytes to neocapillaries of infected cardiac heart valves ( 23). In its transmembrane form, CXCL16 may be able to function as a structural adhesion molecule, anchoring activated CD8 lymphocytes, which may promote their retention in the site of inflammation ( 24, 25). CXCL16 joins fractalkine (CX3C ligand 1) as the only other transmembrane chemokine that can function in both its soluble and its membrane-bound form to regulate cell migration and adhesion ( 26, 27).
A role for CXCR6/CXCL16 in providing a survival signal could also be put forth, because CXCL16 is up-regulated on DC under activating conditions ( 16) with a concomitant increase in CXCR6 on cocultured T cells ( 9). Other chemokines, such as stromal cell-derived factor-1, have been shown to be survival factors for CD4 T cells ( 28). However, in our analysis of markers of proliferation (BrdU incorporation) and apoptosis (annexin V and 7-AAD) on donor CD8 LIL, we found no significant difference in the percentage of positive cells, suggesting no proliferative or survival advantage of WT cells over CXCR6-deficient cells. Our current studies have concentrated on short term recruitment, but additional studies will be necessary to critically examine possible additional roles of CXCR6/CXCL16 in the liver. One factor we have not been able to address directly in the current studies is the rate of exit of homed cells from the liver. Detailed studies conducted in sheep estimate the total output of lymphocytes from the liver through the afferent lymphatics to be 1 x 106 cells/h under normal conditions ( 29). However, under activated conditions (i.e., after the administration of i.v. endotoxin), afferent lymphatic flow from the liver to the liver-draining lymph node peaked at 6 h, but cellular output decreased until 6 h and subsequently peaked by 18 h. Furthermore, cell transfer experiments of alloantigen-stimulated lymphocytes into normal rats showed progressive accumulation of cells in the liver from 6–9 h, but peak accumulation in the liver-draining lymph node occurred later, at 15 h ( 30). The results of both studies suggest that at 6 h after injection, the liver is rapidly accumulating cells, and cellular exit is likely to have little effect on cell numbers at this early time point.
The residual accumulation of CXCR6–/– CD8 cells in the liver indicates that other mechanisms compensate for the CXCR6 deficit. Other investigators have suggested a role of CCR5 in mediating recruitment of CD8 donor T lymphocytes to the liver in GvHD ( 31, 32, 33). Their studies examined total lymphocyte accumulation at different time points, but did not exclude a potential contribution of concomitant effects on proliferation; nevertheless, CCR5 and CXCR6 may both participate in the accumulation of CD8 lymphocytes in GvHD-induced hepatitis. Indeed, CCR5-, CXCR3-, and CXCR6-expressing lymphocytes have been found in liver infiltrates of patients with hepatitis C ( 18, 34), and combinations of these three chemokine receptors can be coexpressed on activated T cells ( 8, 17). Experiments have also supported an important role of CXCR3 in many target organs of GvHD, including the liver, as shown by improved liver histology in GvHD induced by CXCR3-deficient lymphocytes compared with WT ( 35). Beyond being partially redundant for recruitment into the liver, multiple chemokine receptors may also play a role in navigation within the liver. Others have stressed the concept of a "symphony" of chemokines mediating an inflammatory process ( 36) and have emphasized that combinations of chemokine receptors, rather than individual receptors, act together to define the trafficking of differentiated effector/memory lymphocyte populations ( 8).
In conclusion, our novel data demonstrate that CXCR6 is an important mediator of the accumulation of activated CD8 T cells in the liver in GVHD-induced hepatitis. The reduced accumulation of CXCR6–/– CD8 cells appears to reflect, at least in large part, a significant defect in their ability to migrate into inflamed liver from blood. Thus, we conclude that CXCR6 plays an important role in the hepatic homing and accumulation of activated CD8 T lymphocytes and may act in concert with other chemokine receptors to orchestrate pathological inflammation in the liver.
Acknowledgments
We thank C. Crumpton and L. Rott for their expertise at the flow cytometry core facility, L. Ohler and G. Sperinde for their expertise in real-time PCR, and C. Arnold and G. Debes for their constructive criticism and suggestions on the paper.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by grants from the National Institutes of Health, the Department of Veterans Affairs (to E.C.B.), and the FACS Core Facility of the Stanford Digestive Diseases Center. T.S. was supported by a K08 grant from the National Institutes of Health. H.T. was supported by the Swedish Research Council (2001-6576, 2002-955, 2002-8012, and 2003-4661), Crafoordska stiftelsen, Blanceflors stiftelse, Einar och Inga Nilssons stiftelse, Harald och Greta Jaenssons stiftelse, Greta och Johan Kocks stiftelser, Fr?ken Agnes Nilssons stiftelse, Magnus Bergvalls stiftelse, Mossfelts stiftelse, Nanna Svartz stiftelse, Ruth och Richard Julins stiftelse, Svenska L?kares?llskapet, and Teggers stiftelse.
2 Address correspondence and reprint requests to Dr. Tohru Sato, Veterans Affairs Palo Alto Health Care System, 3801 Miranda Avenue, MC154B, Palo Alto, CA 94304. E-mail address: tsato@stanford.edu
3 T.S. and H.T. contributed equally to this work.
4 Current address: Department of Surgery, Lund University, Malmo University Hospital, S-205 02, Malmo, Sweden.
5 Current address: Dalhousie University, Sir Charles Tupper Medical Building, Room 7C, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5.
6 Abbreviations used in this paper: GvHD, graft-vs-host disease; 7-AAD, 7-aminoactinomycin D; CXCL, CXC ligand; DC, dendritic cell; LIL, liver-infiltrating lymphocyte; WT, wild type.
Received for publication March 23, 2004. Accepted for publication October 22, 2004.
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