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* Kan Research Institute, Kyoto, Japan;
Department of Internal Medicine, Osaka Dental University, and
Department of Microbiology, Kinki University School of Medicine, Osaka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells, and terminally differentiated CD8+ T cells.
In addition, soluble fractalkine preferentially induced migration of
cytotoxic effector lymphocytes. Furthermore, interaction of cytotoxic
effector lymphocytes with membrane-bound fractalkine promoted
subsequent migration to the secondary chemokines, such as macrophage
inflammatory protein-1
/CC ligand 4 or IL-8/CXC ligand 8. Thus,
fractalkine expressed on inflamed endothelium may function as a
vascular regulator for cytotoxic effector lymphocytes, regardless of
their lineage and mode of target cell recognition, through its ability
to capture them from blood flow and to promote their emigration in
response to other chemokines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fractalkine/CX3C ligand 1 is the sole member of the CX3C subfamily and is an exceptional molecule as a chemokine for being a transmembrane protein consisting of a chemokine domain with a unique CXXXC motif atop an extended mucin-like stalk (7). In the classic pathway of leukocyte emigration, leukocytes tether and roll on the surface of endothelium by weak interactions between selectins and selectin ligands. This allows leukocytes to be exposed to locally produced chemokines being presented on the surface of endothelium through binding to glycosaminoglycans. Signals transduced via chemokine receptors then activate integrins and induce firm adhesion of rolling leukocytes on the endothelium. Finally, leukocytes start diapedesis through the endothelium and into tissues along the gradient of chemokines (8, 9, 10). Previously, we and others have shown that fractalkine and its specific receptor CX3CR1 represent a novel type of leukocyte trafficking regulator, performing both adhesive and chemotactic functions (11, 12, 13). The membrane-bound fractalkine rapidly induces firm adhesion of CX3CR1-expressing cells under both static and flow conditions without requiring selectin-mediated rolling or activation of integrins (11, 12, 13). In addition, soluble fractalkine released from the cell surface by proteolytic cleavage (14) induces calcium mobilization, integrin activation, and migration of CX3CR1-expressing cells similar to other soluble chemokines (11, 12, 13). Thus, through both membrane-bound and soluble forms, fractalkine is likely to play important roles in trafficking of cells expressing CX3CR1 (11).
The expression of fractalkine was originally demonstrated on human
umbilical endothelial cells upon treatment with TNF-
or IL-1 in
vitro (7). Enhanced expression of fractalkine and
infiltration of CD16+ NK cells were also
demonstrated in crescent glomerulonephritis of human patients
(15). Recently, a polymorphism in CX3CR1, which reduces
its binding activity to fractalkine, was reported to increase the risk
of HIV diseases, but to reduce the risk of atherosclerosis
(16, 17, 18). Collectively, fractalkine and its receptor,
CX3CR1, are likely to involved in tissue accumulation of leukocytes
through inflamed endothelium.
Cytotoxic lymphocytes, which include a diverse variety of cells such as
NK cells, CD8+ T cells, and T cells, are
the major effector cells in both innate and acquired immunity against
intracellular pathogens and tumor cells (19). Even though
the roles of chemokines and chemokine receptors in the trafficking
regulations of CD4+ Th cells have been fairly
well understood recently, similar trafficking regulations remain mostly
unknown for cytotoxic lymphocytes (2). In the present
study we have demonstrated that surface expression of CX3CR1 defines
PBL commonly possessing high levels of intracellular perforin and
granzyme B, and that soluble fractalkine preferentially attracts these
perforin- and granzyme B-positive lymphocytes. Thus, fractalkine and
CX3CR1 are likely to play important roles in trafficking of
cytotoxic effector lymphocytes regardless of their lineage and mode of
target cell recognition. Furthermore, we have demonstrated that the
membrane-bound fractalkine enhances their migration to other
chemokines. These results suggest that fractalkine regulates tissue
emigration of cytotoxic effector lymphocytes through inflamed
endothelium.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mAbs to human CX3CR1 were generated from WKY/Ncrj rats immunized
with CX3CR1 transfectants by a standard method. Two mAbs, 2A9-1 (IgG2b)
and 1F2-2 (IgG2a), were obtained. There specificity was examined by
flow cytometry using a panel of murine L1.2 cells expressing all known
human chemokine receptors (CCR1
10, CXCR16, XCR1, and CX3CR1).
Both mAbs reacted only with murine L1.2 cells expressing CX3CR1. FITC-,
PE-, PC5-, or allophycocyanin-labeled anti-CD3 (UCHT1,
IgG1), anti-CD4 (13B8.2, IgG1), anti-CD8 (B9.11, IgG1),
anti-CD11a (S25.3.1, IgG1), anti-CD14 (RMO52, IgG2a),
anti-CD16 (3G8, IgG1), anti-CD19 (J4.119, IgG1), anti-CD27
(1A4-CD27, IgG1), anti-CD28 (CD28.2, IgG1), anti-CD45RA (HI100,
IgG2b), anti-CD57 (NC1, IgM), anti-CD62 ligand (Dreg56, IgG1),
anti-TCR (BMA031, IgG2b), and anti-TCR (IMMU510,
IgG1) were purchased from Coulter (Hialeah, FL). FITC- or R-PE-labeled
anti-CD3 (UCHT1, IgG1), anti-CD4 (MT310, IgG1), anti-CD8
(DK25, IgG1), anti-CD11b (2LPM19c, IgG1), anti-CD14 (TUK14,
IgG2a), anti-CD25 (ACT-1, IgG1), anti-CD45RA (4KB5, IgG1),
anti-CD45RO (UCHL1, IgG2a), anti-CD95 (DX2, IgG1), and
anti-CCR2 (no. 48607.211, IgG2b) were purchased from DAKO (Kyoto,
Japan). PerCP-labeled anti-CD4 (SK3, IgG1) and anti-CD8
(SK1, IgG1) were purchased from BD Biosciences (Mountain View, CA).
R-PE-labeled anti-CCR5 (2D7, IgG2a), anti-CXCR1 (5A12, IgG2b),
anti-CXCR2 (6C6, IgG1), and anti-CXCR3 (1C6, IgG1) as well as
CyChrome-labeled anti-CD45RO (UCHL1, IgG2a) were purchased from BD
PharMingen (San Diego, CA). mAbs to human CCR7 (6B3, IgG1) described
previously (20) were provided by Dr. Hasegawa. PBMC
prepared from healthy adult donors by the standard method using
Ficoll-Paque (Pharmacia, Uppsala, Sweden) were stained first with
anti-CX3CR1 (2A9-1) and then with FITC-conjugated goat anti-rat
IgG(H + L) F(ab')2 (mouse serum absorbed, no.
CLCC40101; Cedarlane Laboratories, Hornsby, Canada). In some
experiments another anti-CX3CR1 mAb IF2-2 was used to confirm the
results. After incubation with 1% normal rat serum for 20 min, the
cells were further stained with labeled mAbs to various surface Ags.
After washing, cells were analyzed on FACSCalibur (BD Biosciences).
Intracellular staining
After staining for surface CX3CR1 (see above), cells were
resuspended and fixed with IntraPrep Reagent 1 (Coulter) at room
temperature for 15 min. After washing once with PBS, cells were
permeabilized with IntraPrep Reagent 2 at room temperature for 5 min.
Cells were then incubated with PE-labeled anti-perforin (G9,
IgG2b; BD PharMingen) or PE-labeled anti-granzyme B (CLB-GB11,
IgG1; Research Diagnostics, Flanders, NJ) at room temperature
for 15 min in the dark. For negative controls, PE-labeled isotype
controls purchased from BD PharMingen were used. After washing once
with PBS, cells were resuspended in PBS/0.5% formaldehyde and analyzed
on FACSCalibur (BD Biosciences).
Chemotaxis assay
Migration of PBL through ECV304 cells was conducted as described previously (11). In brief, ECV304 cells were seeded into Transwell culture inserts (Costar, Cambridge, MA) with pore size of 5 µm (2 x 105 cells/insert) and cultured for 48–72 h in medium 199 supplemented with 10% FCS. Chemokines diluted to 10 nM in the migration medium (RPMI 1640/medium 199 (1:1), 0.5% BSA, and 20 mM HEPES, pH 7.4) were added to 24-well tissue culture plates (600 µl/well), and inserts coated with ECV304 cells were placed. PBMC were then added to the upper chambers (106 cells in 100 µl). After incubation at 37°C for 4 h, cells migrated into the lower chambers were stained for various markers as stated above and analyzed by flow cytometry.
Isolation of CX3CR1+ and CX3CR1- CD8+ T cell subsets
CD8+ T cells were prepared from PBMC by two rounds of positive selection using the MACS system (Miltenyi Biotec). In brief, PBMC at 4 x 107 cells/70 µl MACS buffer (PBS containing 1% FCS and 5 mM EDTA) were incubated with the FcR blocking reagent (20 µl for 4 x 107 cells) and anti-CD8 microbeads (10 µl for 4 x 107 cells) at 4°C for 20 min. After washing, cells were resuspended in MACS buffer (107 cells/400 µl), and selection was conducted using LS column and Midi MACS magnet according to the manufacturer’s instruction. The resulting CD8+ T cells were >97% CD8+CD3+CD16- as determined by flow cytometry. Purified CD8+ T cells were next stained with anti-CX3CR1 mAb 2A9-1 and then with FITC-conjugated goat anti-rat IgG(H + L) F(ab')2. CX3CR1+ and CX3CR1- populations with >95% purity were obtained through sorting on FACSVantage (BD Biosciences).
Cytotoxicity assay
Cytotoxicity was determined using the anti-CD3 mAb-mediated redirected Eu3+ release assay as described previously (21). In brief, FcR-bearing P815 target cells (2 x 106) were suspended in 1 ml labeling buffer: the mixture of 880 µl HEPES buffer (50 mM HEPES (pH 7.4), 93 mM NaCl, 5 mM KCl, and 2 mM MgCl2), 140 µl dextran sulfate stock solution (HEPES buffer containing 0.5% dextran; m.w., 500,000), and 80 µl europium (Eu) stock solution (mixture of 1.52 ml Eu atomic absorption standard solution from Aldrich, 0.5 ml 100 mM diethylene-triamine-penta-acetic acid (DTPA) solution and 7.98 ml HEPES buffer). After incubation at 37°C for 20 min, cells were washed twice with repairing buffer (HEPES buffer containing 2 mM CaCl2 and 10 mM glucose) and three times with RPMI 1640 containing 10% FCS. Subsets of purified CD8+ T cells (see above) were mixed with 5 x 103 labeled P815 cells at various E:T cell ratios in the presence or the absence of anti-CD3 mAb (UCHT1, Genzyme, Cambridge, MA). After incubation at 37°C for 3 h, supernatants (60 µl) of triplicate cultures were collected and mixed with 140 µl enhancement solution (Wallac/Berthord, Gaithersburg, MD). After shaking for 5 min, fluorescence was measured with the time-resolved fluorometer (ARVO-1240sx; Wallac/Berthold). Specific cytotoxicity was determined according to the formula: % specific lysis = 100 x ((experimental Eu release - spontaneous Eu release)/(maximal Eu release - spontaneous Eu release)).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have generated two novel rat mAbs, 2A9-1 and 1F2-2, against
human CX3CR1. We confirmed their specificity to human CX3CR1 by using a
panel of transfectants stably expressing all known chemokine receptors,
including CCR1 10, CXCR1 6, XCR1, and CX3CR1 (data not shown).
Using these mAbs, we examined expression of CX3CR1 on PBL from healthy
adult donors by flow cytometry (Table I
).
Essentially identical results were obtained by two different
anti-CX3CR1 Abs (2A9-1 and 1F2-2). As shown in Fig. 1a, most
CD16+ NK cells (>90%) and a substantial
fraction of CD3+ T cells (10–30%) expressed
CX3CR1 with similar intensities. In contrast,
CD19+ B cells were essentially negative. Among T
cells, CX3CR1 were expressed on 40% of
CD8+ T cells, 5% of
CD4+ T cells, and 70% of T cells (Fig. 1b). CX3CR1 was also expressed on 40–85% of
CD56+CD16-CD3-
NK cells and 50–80% of
CD3+CD56+CD16-
NKT cells (data not shown).
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c). However, 40% of
CD45RA-CD27- terminally
differentiated memory/effector CD4+ T cells
expressed CX3CR1. Similarly, CD8+ T cells can be
divided into four subpopulations according to differential expression
of CD45RA and CD27 (23, 24):
CD45RA+CD27+ naive
subset, CD45RA-CD27+ and
CD45RA-CD27- memory
subsets, and CD45RA+CD27-
fully differentiated effector subset (Fig. 1c). CX3CR1 was
hardly expressed on
CD45RA+CD27+ naive
cells. In contrast, 30%
CD45RA-CD27+ and 50%
CD45RA-CD27- memory
cells expressed CX3CR1. Furthermore, most (90%)
CD45RA+CD27-
CD8+ T cells, which correspond to terminally
differentiated effector CD8+ T cells
(24), expressed CX3CR1. These results strongly suggest
that CX3CR1 expression is closely associated with the development of
effector functions in CD4+ and
CD8+ T cells. Expression of various surface markers on CX3CR1-expressing lymphocytes
To further characterize the phenotypes of cells expressing CX3CR1,
CD8+ T cells, CD4+ T cells,
T cells, and CD16+ NK cells were analyzed
for coexpression of CX3CR1 and various functional and/or activation
makers. As shown in Fig. 2, CD57 (HNK-1)
and CD11b, which are good markers for cytotoxic lymphocytes
(23), were mostly coexpressed with CX3CR1 on all three T
cell subsets and CD16+ NK cells. In contrast,
costimulatory molecules such as CD27 and CD28 were mostly negative or
only intermediate on CX3CR1-expressing subsets. CD95 (Fas), which is
intermediate in terminally differentiated effector
CD8+ T cells (22), was mostly
expressed on CX3CR1-expressing cells at intermediate levels. CD25 (an
activation maker) was mostly negative on CX3CR1-expressing subsets.
CD62 ligand, which is essential for lymphocyte homing to lymph nodes
(25), was mostly negative on CX3CR1-expressing
CD8+ T cells, CD4+ T cells,
and CD16+ NK cells. Notably, however, the
majority of CX3CR1-expressing T cells were positive for CD62
ligand. CD11a, which is involved in trafficking to inflamed tissues and
thus correlates well with terminally differentiated effector T cells
(24), was consistently expressed at high levels in
CX3CR1-expressing CD8+ T cells,
CD4+ T cells, T cells, and
CD16+ NK cells. Collectively, the surface
phenotypes of CX3CR1-expressing cells, regardless of their major
lineage and mode of target cell recognition, are very similar to those
previously reported for terminally differentiated effector
CD8+ T cells (24). Thus,
CX3CR1-expressing lymphocytes are likely to represent a group of fully
differentiated effector lymphocytes ready to migrate into inflamed
tissues.
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Cytotoxic effector lymphocytes such as NK cells, cytotoxic
CD8+ T cells (CTLs) and the majority of T
cells contain specialized cytoplasmic granules storing the pore-forming
protein (perforin) and serine proteases (granzymes), which are released
from these cells upon activation to mediate target cell lysis
(26). We therefore examined CD16+ NK
cells, CD4+ T cells, CD8+ T
cells, and T cells for surface expression of CX3CR1 and
intracellular staining of perforin and granzyme B. As shown in Fig. 3, surface expression of CX3CR1 highly
correlated with intracellular staining of perforin and granzyme B in
all four classes of lymphocytes. Thus, CX3CR1 is commonly expressed on
circulating cytotoxic effector lymphocytes containing intracellular
cytotoxic granules regardless of their lineage and mode of target cell
recognition.
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To test the immediate cytotoxic activity of CX3CR1-expressing T
cells, we purified CD8+ T cells by positive
selection and sorted them into CX3CR1+ and
CX3CR1- fractions. We measured the cytotoxic
activities of the original and sorted populations by anti-CD3
mAb-mediated redirected cytotoxicity assay (27). As shown
in Fig. 4, CX3CR1+CD8+ T cells indeed
showed much greater cytotoxic activity than presorted
CD8+ T cells. In contrast,
CX3CR1-CD8+ T cells only
showed marginal cytotoxic activity. We confirmed that the cytotoxic
activity was dependent on anti-CD3 mAb and was not affected by the
anti-CX3CR1 mAb used for cell sorting (data not shown). Thus,
circulating CD8+ T lymphocytes with immediate
cytotoxic activity selectively express CX3CR1.
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The selective expression of CX3CR1 on circulating lymphocytes
containing cytotoxic granules suggests that fractalkine plays an
important role in the recruitment of cytotoxic lymphocytes from the
blood. To support this idea, we conducted Transwell chemotaxis assays
using soluble fractalkine and stained input and migrated T cells for
intracellular perforin and granzyme B. Soluble fractalkine attracted
CD16+ NK cells, CD8+ T
cells, and CD4+ T cells with efficiencies
consistent to their percent positivity of CX3CR1 (data not shown)
(11). Although original CD8+ T cells
were heterogeneous in terms of intracellular contents of perforin and
granzyme B, the cells attracted to soluble fractalkine displayed high
strong staining for intracellular perforin and granzyme B (Fig. 5a). Similar results were
obtained for CD4+ T cells (data not shown). If
spontaneous migration was subtracted, soluble fractalkine almost
exclusively attracted cells possessing intracellular perforin and
granzyme B (Fig. 5b). We also confirmed that neutralizing
mAbs to fractalkine (3A5 and 3H7), but not control mouse IgG,
effectively blocked fractalkine-induced migration of
CD16+ NK cells and granzyme B-positive
CD8+ and CD4+ T cells (Fig. 5c).
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We next examined coexpression of CX3CR1 and other chemokine
receptors, namely, CCR5, CXCR3, CXCR1, CXCR2, CCR2, and CCR7. As shown
in Fig. 6, even though most
CD16+ NK cells expressed CX3CR1 and CXCR1, only
small fractions were found to coexpress CCR5 and CXCR3. In contrast,
most CX3CR1-expressing T cells coexpressed CCR5, while
coexpression of CXCR3 or CXCR1 was variable from donor to donor. In the
case of CD8+ and CD4+ T
cells, substantial fractions of CX3CR1-expressing cells coexpressed
CCR5 and CXCR1 at high to intermediate levels, while only minor
fractions were found to coexpress CXCR3 or CCR2. In contrast, CCR7, the
chemokine receptor essential for lymphocyte homing to secondary
lymphoid tissues (2) was clearly negative on
CX3CR1-expressing cells regardless of their lineages. CXCR2 was
negative on any lymphocyte subsets examined, although it was clearly
positive on both monocytes and granulocytes. These results indicate
that expression of CX3CR1 is regulated quite differently from other
chemokine receptors.
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The membrane-bound fractalkine has been shown to mediate firm
adhesion and activation of CX3CR1-expressing cells (11, 12, 28, 29). However, it is not clear how membrane-bound fractalkine
affects their migration. We therefore performed transmigration assay
using ECV304 cells and fractalkine-transfected ECV304 (ECV-FKN) cells
to examine its effects on migration of CX3CR1-expressing lymphocytes to
soluble fractalkine and the second chemokine. We have previously shown
that TNF-activated HUVECs and ECV-FKN cells express similar levels of
fractalkine, indicating that ECV-FKN cells express physiologically
relevant levels of fractalkine (12). Since a substantial
fraction of CX3CR1-expressing CD8+ T cells
coexpress CCR5, but not CCR2 (Fig. 6), we chose macrophage inflammatory
protein-1
(MIP-1)3/CC ligand
4 (the ligand for CCR5) as a test chemokine and macrophage chemotactic
protein-1 (MCP-1)/CC ligand 2 (the ligand for CCR2) as a negative
control.
As shown in Fig. 7a,
transmigration of CD8+ T cells to soluble
fractalkine was reduced by the expression of membrane-bound fractalkine
(ECV-FKN). This suggests that adhesion by the membrane-bound form of
fractalkine is dominant over cell migration to the soluble form of
fractalkine. In contrast, transmigration of CD8+
T cells to MIP-1, but not to MCP-1, was significantly enhanced in
the presence of membrane-bound fractalkine (Fig. 7a).
Furthermore, only CD8+ T cells carrying granzyme
B (the CX3CR1+ fraction) showed enhanced
migration to MIP-1 in the presence of membrane-bound fractalkine
(Fig. 7b). The enhancing effects were completely blocked by
a neutralizing mAb to fractalkine (3A5), but not by control IgG. The
enhancing effects were not observed when PBMCs were added to the upper
chamber together with 10 nM soluble fractalkine or preactivated with
300 nM soluble fractalkine (data not shown). In contrast, migration of
CD8+ T cells without granzyme B (the
CX3CR1- fraction) to MIP-1 or migration
induced by MCP-1, the latter being selective for the granzyme
B- fraction, was not affected by the presence of
membrane-bound fractalkine (Fig. 7b). The enhanced migration
of granzyme B-positive CD8+ T cells was mainly
due to augmentation of maximal response to MIP-1 by membrane-bound
fractalkine (Fig. 7c).
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(30), we further examined the effect of membrane-bound
fractalkine on migration of CD16+ NK cells to
IL-8/CXC ligand 8 (the ligand of CXCR1). As shown in Fig. 8a, migration of
CD16+ NK cells to IL-8 was also strongly enhanced
by membrane-bound fractalkine. The enhancing effects were again
effectively blocked by a neutralizing mAb to fractalkine (3A5). The
enhanced migration of CD16+ NK cells was mainly
due to augmentation of the maximal response to IL-8 by membrane-bound
fractalkine (Fig. 8c). Thus, membrane-bound fractalkine not
only mediates the adhesion of CX3CR1-expressing cells, but also
enhances their migration to the secondary chemokines.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells,
CD8+ T cells, and a minor fraction of
CD4+ T cells, are essential for host defense
against intracellular pathogens and altered self cells
(19). Their excessive activities, however, lead to
organ-specific autoimmune diseases (31, 32, 33). They are
commonly endowed with cytotoxic mechanisms such as cytoplasmic granules
containing perforin and granzymes and surface expression of
death-signaling Fas ligand (19, 26, 34). In the present
study we have clearly demonstrated that the fractalkine receptor CX3CR1
is selectively expressed on various lineages of lymphocytes with high
contents of intracellular perforin and granzyme B (Fig. 3). Most
CD16+ NK cells and the majority of T
cells, which play important roles in innate immunity as immediate
effector killer cells (19), express CX3CR1 along with
cytoplasmic perforin and granzyme B (Figs. 1 and 3). Similarly,
CX3CR1-expressing CD8+ T cells, but not those
without surface CX3CR1, were of terminally differentiated phenotypes,
armed with perforin and granzyme B, and displayed strong cytotoxic
activity (Figs. 3 and 4). Furthermore, CD4+ T
cells expressing CX3CR1 were also terminally differentiated and
contained intracellular perforin and granzyme B (Figs. 1 and 3),
demonstrating that CX3CR1 is a good surface marker for
CD4+ T cells with cytotoxic activity. Predominant
expression of Fas ligand on terminally differentiated
CD8+ T cells with remarkable lytic activity has
also been demonstrated (24). Thus, we propose to call
CX3CR1-expressing T cells, which are terminally differentiated subsets
with cytotoxic activity, cytotoxic effector T cells
(TCE).
Previously, we and others have shown that soluble fractalkine induces
migration of NK cells and CD8+ T cells, and that
the membrane-bound fractalkine mediates efficient capture, firm
adhesion, and activation of CX3CR1-expressing cells in both static and
flow conditions (11, 12, 13). In the present study we have
further demonstrated that soluble fractalkine is a selective
chemoattractant for lymphocytes displaying cytotoxic effector
phenotypes (Fig. 5). In addition, the membrane-bound fractalkine
enhances migration of CX3CR1-expressing cells to other chemokines, such
as MIP-1 and IL-8, acting on the same cells (Fig. 7). Even though it
remains to be seen whether the membrane-bound fractalkine indeed
enhances chemotaxis of cells expressing CX3CR1 to other chemokines in
vivo, we propose that fractalkine provides an initial clue for
circulating NK cells and TCE to emigrate through
inflamed endothelium as P- and E-selectin mediate selective recruitment
of Th1 cells by inducing their rolling under flow (35). At
present we do not know the exact molecular mechanisms of this
enhancement, but integrin activation and cell polarization induced by
membrane-bound fractalkine may have promoting effects on migratory
responses to secondary chemokines (12, 28). In this
context it is interesting to examine whether P- and E-selectin also
promote the migration of Th1 cells toward Th1-directed chemokines even
in the static condition. Thus, fractalkine expressed on inflamed
endothelium plays dual functions: 1) as a selective capturing molecule
for rapidly circulating killer effector lymphocytes in the blood, and
2) as a subsequent cosignaling molecule promoting their migration to
secondary chemokines.
Sallusto et al. (36) have shown that surface expression of
CCR7, the chemokine receptor that promotes lymphocyte homing to
secondary lymphoid tissues, divides human memory T cells into two
functionally distinct subsets. CCR7- memory T
cells, termed effector memory T cells, preferentially migrate to
inflamed tissues and display immediate effector functions. In contrast,
CCR7+ T memory cells, termed central memory T
cells, lack immediate effector functions and preferentially home to
secondary lymphoid tissues, where they are efficiently stimulated by
dendritic cells and differentiate into CCR7-
effector cells upon secondary stimulation (36). It has
been also reported that CD8+ cytotoxic T cells
start to express perforin and granzymes during differentiation to
memory/effector stages after antigenic stimulation (19).
Consistently, we have observed that most CX3CR1+
T cells express CD11a, and cytoplasmic perforin and granzyme B, but
lack L-selectin and/or CCR7 (Figs. 2, 3, and 6), indicating that
expression of CX3CR1 defines the terminally differentiated
TCE ready to infiltrate into inflamed
tissues.
Recent studies have shown that the expression of some chemokine
receptors correlates with Th1 or Th2 polarization and/or with
tissue-selective migration. We found that the majorities of
CX3CR1-expressing CD8+ T cells and
CD4+ T cells were also found to coexpress CCR5
and, less frequently, CXCR3 (Fig. 6), the chemokine receptors known to
be highly selective for Th1 cells (2), suggesting that
CX3CR1-expressing CD8+ and
CD4+ T cells partly overlap with Tc1 and Th1,
respectively. In this regard, Fraticelli et al. (35)
reported that fully polarized Th1 cells selectively expressed CX3CR1
and migrated to fractalkine. Most T cells were also found to
coexpress CCR5 and CXCR3 (Fig. 6). In contrast, except for CX3CR1 and
CXCR1, CD16+ NK cells were found to be negative
for most other chemokine receptors (Fig. 6). Therefore, CX3CR1 and
CXCR1 (Fig. 7) seem to be the major chemokine receptors involved in
trafficking of CD16+ NK cells. Consistently,
Campbell et al. (30) reported that
CD16+ NK cells were the predominant population in
peripheral blood migrating to IL-8 and fractalkine.
Recently, the second membrane-type chemokine, CXC ligand 16, has been
described (37, 38). In humans its receptor CXCR6/Bonzo is
expressed on fractions of CD45RO+
CD8+ and CD4+ T cells as
well as small subsets of CD16+ NK cells and
T cells and is coordinately regulated with CCR5
(39). A recent study has further shown that CXCR6 is
expressed by subsets of Th1 and Tc1, but not by Th2 or Tc2 cells
(40). In contrast to the almost complete match of surface
expression of CX3CR1 and possession of preformed perforin and granzyme
B in various lymphocyte lineages, including CD8+
T cells (Fig. 3), however, only an average 34% of granzyme
A+ CD8+ T cells were shown
to be positive for CXCR6 (40). Thus, we assume that, like
CCR5, CXCR6 is expressed only on subsets of CX3CR1-expressing cytotoxic
effector lymphocytes and on noncytotoxic effector cells. It remains to
be seen whether there are any differences between the
CXCR6+CX3CR1+ and
CXCR6-CX3CR1+ subsets of
cytotoxic lymphocytes in terms of their functional property and tissue
migration (40, 41).
In conclusion, we have demonstrated that fractalkine and CX3CR1 are likely to play dual roles in the recruitment of effector lymphocytes with full cytotoxic activity, suggesting important pathophysiological roles of fractalkine in vascular and tissue injuries (42). CX3CR1 is also valuable as a highly selective surface marker for terminally differentiated TCE, whose selective dysfunction is likely to be associated with various chronic diseases, such as HIV-1 infection (30).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Toshio Imai, Kan Research Institute, Science Center Building 3, Kyoto Research Park, 1 Chydoji Awata-cho, Simogyo-ku, Kyoto 600-8815, Japan. E-mail address: t-imai{at}kan.gr.jp
3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; DTPA, diethylene-triamine-penta-acetic acid; MCP, macrophage chemotactic protein; TCE, cytotoxic effector T cell.
Received for publication December 19, 2001. Accepted for publication April 16, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 F. Montecucco and F. Mach Common inflammatory mediators orchestrate pathophysiological processes in rheumatoid arthritis and atherosclerosis Rheumatology, January 1, 2009; 48(1): 11 - 22. [Abstract] [Full Text] [PDF]
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G. Murphy, N. Caplice, and M. Molloy Fractalkine in rheumatoid arthritis: a review to date Rheumatology, October 1, 2008; 47(10): 1446 - 1451. [Abstract] [Full Text] [PDF]
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B. Olsson, B. Ridell, L. Carlsson, S. Jacobsson, and H. Wadenvik Recruitment of T cells into bone marrow of ITP patients possibly due to elevated expression of VLA-4 and CX3CR1 Blood, August 15, 2008; 112(4): 1078 - 1084. [Abstract] [Full Text] [PDF]
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 A. Pachot, M.-A. Cazalis, F. Venet, F. Turrel, C. Faudot, N. Voirin, J. Diasparra, N. Bourgoin, F. Poitevin, B. Mougin, et al. Decreased Expression of the Fractalkine Receptor CX3CR1 on Circulating Monocytes as New Feature of Sepsis-Induced Immunosuppression J. Immunol., May 1, 2008; 180(9): 6421 - 6429. [Abstract] [Full Text] [PDF]
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N. Mizutani, T. Sakurai, T. Shibata, K. Uchida, J. Fujita, R. Kawashima, Y. I. Kawamura, N. Toyama-Sorimachi, T. Imai, and T. Dohi Dose-Dependent Differential Regulation of Cytokine Secretion from Macrophages by Fractalkine J. Immunol., December 1, 2007; 179(11): 7478 - 7487. [Abstract] [Full Text] [PDF]
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V. Bjerkeli, J. K. Damas, B. Fevang, J. C. Holter, P. Aukrust, and S. S. Froland Increased expression of fractalkine (CX3CL1) and its receptor, CX3CR1, in Wegener's granulomatosis possible role in vascular inflammation Rheumatology, September 1, 2007; 46(9): 1422 - 1427. [Abstract] [Full Text] [PDF]
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 Y. Zeng, N. Huebener, S. Fest, S. Weixler, U. Schroeder, G. Gaedicke, R. Xiang, A. Schramm, A. Eggert, R. A. Reisfeld, et al. Fractalkine (CX3CL1)- and Interleukin-2-Enriched Neuroblastoma Microenvironment Induces Eradication of Metastases Mediated by T Cells and Natural Killer Cells Cancer Res., March 1, 2007; 67(5): 2331 - 2338. [Abstract] [Full Text] [PDF]
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 L. Ziegler-Heitbrock The CD14+ CD16+ blood monocytes: their role in infection and inflammation J. Leukoc. Biol., March 1, 2007; 81(3): 584 - 592. [Abstract] [Full Text] [PDF]
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S. Ueha, M. Murai, H. Yoneyama, M. Kitabatake, T. Imai, T. Shimaoka, S. Yonehara, S. Ishikawa, and K. Matsushima Intervention of MAdCAM-1 or fractalkine alleviates graft-versus-host reaction associated intestinal injury while preserving graft-versus-tumor effects J. Leukoc. Biol., January 1, 2007; 81(1): 176 - 185. [Abstract] [Full Text] [PDF]
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Y. Le Priol, D. Puthier, C. Lecureuil, C. Combadiere, P. Debre, C. Nguyen, and B. Combadiere High Cytotoxic and Specific Migratory Potencies of Senescent CD8+CD57+ Cells in HIV-Infected and Uninfected Individuals J. Immunol., October 15, 2006; 177(8): 5145 - 5154. [Abstract] [Full Text] [PDF]
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N. Kobayashi, T. Kondo, H. Takata, S. Yokota, and M. Takiguchi Functional and phenotypic analysis of human memory CD8+ T cells expressing CXCR3. J. Leukoc. Biol., August 1, 2006; 80(2): 320 - 329. [Abstract] [Full Text] [PDF]
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 K. Furuichi, J.-L. Gao, and P. M. Murphy Chemokine Receptor CX3CR1 Regulates Renal Interstitial Fibrosis after Ischemia-Reperfusion Injury Am. J. Pathol., August 1, 2006; 169(2): 372 - 387. [Abstract] [Full Text] [PDF]
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H. Lin, M. Nieda, J. F. Hutton, V. Rozenkov, and A. J. Nicol Comparative gene expression analysis of NKT cell subpopulations J. Leukoc. Biol., July 1, 2006; 80(1): 164 - 173. [Abstract] [Full Text] [PDF]
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 G. D. Norata, K. Garlaschelli, M. Ongari, S. Raselli, L. Grigore, and A. L. Catapano Effects of Fractalkine Receptor Variants on Common Carotid Artery Intima-Media Thickness Stroke, June 1, 2006; 37(6): 1558 - 1561. [Abstract] [Full Text] [PDF]
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 Y. Chen, S. R. Green, F. Almazan, and O. Quehenberger The Amino Terminus and the Third Extracellular Loop of CX3CR1 Contain Determinants Critical for Distinct Receptor Functions Mol. Pharmacol., March 1, 2006; 69(3): 857 - 865. [Abstract] [Full Text] [PDF]
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J. Harcourt, R. Alvarez, L. P. Jones, C. Henderson, L. J. Anderson, and R. A. Tripp Respiratory Syncytial Virus G Protein and G Protein CX3C Motif Adversely Affect CX3CR1+ T Cell Responses J. Immunol., February 1, 2006; 176(3): 1600 - 1608. [Abstract] [Full Text] [PDF]
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Y. Watanabe, T. Nakayama, D. Nagakubo, K. Hieshima, Z. Jin, F. Katou, K. Hashimoto, and O. Yoshie Dopamine Selectively Induces Migration and Homing of Naive CD8+ T Cells via Dopamine Receptor D3 J. Immunol., January 15, 2006; 176(2): 848 - 856. [Abstract] [Full Text] [PDF]
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 C. N. Davis and J. K. Harrison Proline 326 in the C Terminus of Murine CX3CR1 Prevents G-Protein and Phosphatidylinositol 3-Kinase-Dependent Stimulation of Akt and Extracellular Signal-Regulated Kinase in Chinese Hamster Ovary Cells J. Pharmacol. Exp. Ther., January 1, 2006; 316(1): 356 - 363. [Abstract] [Full Text] [PDF]
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 J. K. Damas, A. Boullier, T. Waehre, C. Smith, W. J. Sandberg, S. Green, P. Aukrust, and O. Quehenberger Expression of Fractalkine (CX3CL1) and its Receptor, CX3CR1, Is Elevated in Coronary Artery Disease and Is Reduced During Statin Therapy Arterioscler Thromb Vasc Biol, December 1, 2005; 25(12): 2567 - 2572. [Abstract] [Full Text] [PDF]
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O. Gasser, A. Missiou, C. Eken, and C. Hess Human CD8+ T cells store CXCR1 in a distinct intracellular compartment and up-regulate it rapidly to the cell surface upon activation Blood, December 1, 2005; 106(12): 3718 - 3724. [Abstract] [Full Text] [PDF]
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F. Suzuki, T. Nanki, T. Imai, H. Kikuchi, S. Hirohata, H. Kohsaka, and N. Miyasaka Inhibition of CX3CL1 (Fractalkine) Improves Experimental Autoimmune Myositis in SJL/J Mice J. Immunol., November 15, 2005; 175(10): 6987 - 6996. [Abstract] [Full Text] [PDF]
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 R. A. Tripp, C. Oshansky, and R. Alvarez Cytokines and Respiratory Syncytial Virus Infection Proceedings of the ATS, August 1, 2005; 2(2): 147 - 149. [Abstract] [Full Text] [PDF]
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 M Hasegawa, S Sato, T Echigo, Y Hamaguchi, M Yasui, and K Takehara Up regulated expression of fractalkine/CX3CL1 and CX3CR1 in patients with systemic sclerosis Ann Rheum Dis, January 1, 2005; 64(1): 21 - 28. [Abstract] [Full Text] [PDF]
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T. Nanki, Y. Urasaki, T. Imai, M. Nishimura, K. Muramoto, T. Kubota, and N. Miyasaka Inhibition of Fractalkine Ameliorates Murine Collagen-Induced Arthritis J. Immunol., December 1, 2004; 173(11): 7010 - 7016. [Abstract] [Full Text] [PDF]
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 M. B. Sukkar, R. Issa, S. Xie, U. Oltmanns, R. Newton, and K. F. Chung Fractalkine/CX3CL1 production by human airway smooth muscle cells: induction by IFN-{gamma} and TNF-{alpha} and regulation by TGF-{beta} and corticosteroids Am J Physiol Lung Cell Mol Physiol, December 1, 2004; 287(6): L1230 - L1240. [Abstract] [Full Text] [PDF]
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C. Hess, T. K. Means, P. Autissier, T. Woodberry, M. Altfeld, M. M. Addo, N. Frahm, C. Brander, B. D. Walker, and A. D. Luster IL-8 responsiveness defines a subset of CD8 T cells poised to kill Blood, December 1, 2004; 104(12): 3463 - 3471. [Abstract] [Full Text] [PDF]
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 J. Barlic, D. H. McDermott, M. N. Merrell, J. Gonzales, L. E. Via, and P. M. Murphy Interleukin (IL)-15 and IL-2 Reciprocally Regulate Expression of the Chemokine Receptor CX3CR1 through Selective NFAT1- and NFAT2-dependent Mechanisms J. Biol. Chem., November 19, 2004; 279(47): 48520 - 48534. [Abstract] [Full Text] [PDF]
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A. M. Vine, A. G. Heaps, L. Kaftantzi, A. Mosley, B. Asquith, A. Witkover, G. Thompson, M. Saito, P. K. C. Goon, L. Carr, et al. The Role of CTLs in Persistent Viral Infection: Cytolytic Gene Expression in CD8+ Lymphocytes Distinguishes between Individuals with a High or Low Proviral Load of Human T Cell Lymphotropic Virus Type 1 J. Immunol., October 15, 2004; 173(8): 5121 - 5129. [Abstract] [Full Text] [PDF]
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M. Daoudi, E. Lavergne, A. Garin, N. Tarantino, P. Debre, F. Pincet, C. Combadiere, and P. Deterre Enhanced Adhesive Capacities of the Naturally Occurring Ile249-Met280 Variant of the Chemokine Receptor CX3CR1 J. Biol. Chem., May 7, 2004; 279(19): 19649 - 19657. [Abstract] [Full Text] [PDF]
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S. Y. Ahn, C.-H. Cho, K.-G. Park, H. J. Lee, S. Lee, S. K. Park, I.-K. Lee, and G. Y. Koh Tumor Necrosis Factor-{alpha} Induces Fractalkine Expression Preferentially in Arterial Endothelial Cells and Mithramycin A Suppresses TNF-{alpha}-Induced Fractalkine Expression Am. J. Pathol., May 1, 2004; 164(5): 1663 - 1672. [Abstract] [Full Text] [PDF]
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 T. Nakayama, K. Hieshima, D. Nagakubo, E. Sato, M. Nakayama, K. Kawa, and O. Yoshie Selective Induction of Th2-Attracting Chemokines CCL17 and CCL22 in Human B Cells by Latent Membrane Protein 1 of Epstein-Barr Virus J. Virol., February 15, 2004; 78(4): 1665 - 1674. [Abstract] [Full Text] [PDF]
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 Q. Zhang, K. Shimoya, K. Temma, T. Kimura, T. Tsujie, M. Shioji, K. Wasada, O. Fukui, S. Hayashi, T. Kanagawa, et al. Expression of fractalkine in the Fallopian tube and of CX3CR1 in sperm Hum. Reprod., February 1, 2004; 19(2): 409 - 414. [Abstract] [Full Text] [PDF]
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G. Ferlazzo and C. Munz NK Cell Compartments and Their Activation by Dendritic Cells J. Immunol., February 1, 2004; 172(3): 1333 - 1339. [Full Text] [PDF]
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H. Umehara, E. T. Bloom, T. Okazaki, Y. Nagano, O. Yoshie, and T. Imai Fractalkine in Vascular Biology: From Basic Research to Clinical Disease Arterioscler Thromb Vasc Biol, January 1, 2004; 24(1): 34 - 40. [Abstract] [Full Text] [PDF]
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A. Garin, N. Tarantino, S. Faure, M. Daoudi, C. Lecureuil, A. Bourdais, P. Debre, P. Deterre, and C. Combadiere Two Novel Fully Functional Isoforms of CX3CR1 Are Potent HIV Coreceptors J. Immunol., November 15, 2003; 171(10): 5305 - 5312. [Abstract] [Full Text] [PDF]
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J. Barlic, J. M. Sechler, and P. M. Murphy IL-15 and IL-2 oppositely regulate expression of the chemokine receptor CX3CR1 Blood, November 15, 2003; 102(10): 3494 - 3503. [Abstract] [Full Text] [PDF]
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E. Lavergne, B. Combadiere, O. Bonduelle, M. Iga, J.-L. Gao, M. Maho, A. Boissonnas, P. M. Murphy, P. Debre, and C. Combadiere Fractalkine Mediates Natural Killer-Dependent Antitumor Responses in Vivo Cancer Res., November 1, 2003; 63(21): 7468 - 7474. [Abstract] [Full Text] [PDF]
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G. Valbuena, W. Bradford, and D. H. Walker Expression Analysis of the T-Cell-Targeting Chemokines CXCL9 and CXCL10 in Mice and Humans with Endothelial Infections Caused by Rickettsiae of the Spotted Fever Group Am. J. Pathol., October 1, 2003; 163(4): 1357 - 1369. [Abstract] [Full Text] [PDF]
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S. Y. Thomas, R. Hou, J. E. Boyson, T. K. Means, C. Hess, D. P. Olson, J. L. Strominger, M. B. Brenner, J. E. Gumperz, S. B. Wilson, et al. CD1d-Restricted NKT Cells Express a Chemokine Receptor Profile Indicative of Th1-Type Inflammatory Homing Cells J. Immunol., September 1, 2003; 171(5): 2571 - 2580. [Abstract] [Full Text] [PDF]
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J.-Q. Gao, Y. Tsuda, K. Katayama, T. Nakayama, Y. Hatanaka, Y. Tani, H. Mizuguchi, T. Hayakawa, O. Yoshie, Y. Tsutsumi, et al. Antitumor Effect by Interleukin-11 Receptor {alpha}-Locus Chemokine/CCL27, Introduced into Tumor Cells through a Recombinant Adenovirus Vector Cancer Res., August 1, 2003; 63(15): 4420 - 4425. [Abstract] [Full Text] [PDF]
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A. Gismondi, J. Jacobelli, R. Strippoli, F. Mainiero, A. Soriani, L. Cifaldi, M. Piccoli, L. Frati, and A. Santoni Proline-Rich Tyrosine Kinase 2 and Rac Activation by Chemokine and Integrin Receptors Controls NK Cell Transendothelial Migration J. Immunol., March 15, 2003; 170(6): 3065 - 3073. [Abstract] [Full Text] [PDF]
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 K. Shimoya, Q. Zhang, K. Tenma, Y. Ota, K. Hashimoto, Y. Shizusawa, T. Kimura, M. Koyama, and Y. Murata Fractalkine (FRK) levels in amniotic fluid and its production during pregnancy Mol. Hum. Reprod., February 1, 2003; 9(2): 97 - 101. [Abstract] [Full Text] [PDF]
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