| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Millennium Pharmaceuticals, Cambridge, MA 02139; and
Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
4
7-) memory
CD4 lymphocytes, including almost all skin memory CD4+
cells expressing the cutaneous lymphocyte Ag (CLA), but weakly or not
expressed in other subsets in thymus and blood. Interestingly, major
fractions of circulating CCR4+ memory CD4 lymphocytes
coexpress the Th1-associated receptors CXC chemokine receptor 3 and
CCR5, suggesting a potential problem in using these markers for Th1 vs
Th2 lymphocyte cells. Moreover, although production of Th2 cytokines in
blood T cells is associated with CCR4+ CD4 lymphocytes,
significant numbers of freshly isolated circulating CCR4+
memory CD4 lymphocytes (including both CLA+ and
CLA- fractions) readily express the Th1 cytokine IFN-
after short-term stimulation. Our results are consistent with a role
for CCR4 as a major trafficking receptor for systemic memory T cells,
and indicate that the patterns and regulation of chemokine receptor
expression in vivo are more complex than indicated by current in vitro
models of Th1 vs Th2 cell generation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Several subsets of CD4 lymphocytes can be defined based on their
expression of adhesion molecules CD62L, cutaneous lymphocyte Ag
(CLA),4
47, and
41, and these various
CD4 lymphocyte subsets traffick to different physiologic sites
(14). For example, CLA+
CD4+ embody memory for cutaneous Ags and are
thought to traffic to the skin (15), while the opposite
subset of
47+
memory CD4 lymphocytes traffics to intestinal sites (16).
Leukocyte adhesion to endothelium involves several overlapping steps of
rolling, activation, and arrest. Exposure of rolling leukocytes to
factors expressed at the adhesion site activates leukocytes and
up-regulates integrin-mediated adhesion. As a result of this
interaction, leukocytes arrest on the endothelium (7, 17).
Leukocyte activation and up-regulation of integrin molecules occur via
a pertussis toxin-sensitive mechanism that is thought to involve
chemokine receptors (7, 18). A growing body of evidence
indicates that leukocyte subsets will also be defined by the expression
of chemokine receptors required to traffic to various physiologic sites
(19).
Several chemokine receptors are expressed on subsets of memory CD4
lymphocytes. For example, CXC chemokine receptor 3 (CXCR3), CCR2, and
CCR5 (20, 21, 22) are all expressed on subsets of memory CD4
lymphocytes, while certain chemokines act selectively on naive T cells
(23) or memory CD4 lymphocytes (24).
Furthermore, several chemokines that interact with these receptors have
been shown to be expressed in inflammatory sites (25) and
lymph nodes (26). Within the memory CD4 lymphocyte
population are subsets of effector T lymphocytes, Th1 and Th2,
classified by the cytokines they secrete (27). Th1
lymphocytes secrete cytokines such as lymphotoxin and IFN- to
promote cellular immune responses, while Th2 lymphocytes release
cytokines such as IL-4 and IL-5 to promote the humoral immunity and
allergic responses (28). Recently, it has been shown that
in vitro derived Th1/Th2 lymphocyte lines differentially express
chemokine receptors. CXCR3 and CCR5 are selectively expressed by in
vitro derived Th1 lymphocytes, while CCR4, CCR8, and CCR3 are expressed
on Th2 lymphocytes (24, 29, 30, 31, 32, 33). Interestingly, the
chemokines macrophage-derived chemokine (MDC) (stimulated T cell
chemoattractant protein-1) for CCR4 and IFN--inducible protein-10
for CXCR3 are induced by cytokines associated with Th1 and Th2
environments, respectively (24, 34, 35). Furthermore, Imai
et al. (33) showed that T cells attracted by thymus and
activation-regulated chemokine (TARC) and MDC predominantly produce
Th2-type cytokines, and that fractionated CCR4+
cells also preferentially produce Th2-type cytokines.
In the studies reported in this work, by using specific anti-CCR4
mAbs, we confirm that CCR4 is differentially expressed on in vitro
derived Th1/Th2 effector lymphocytes and demonstrate that CCR4 is the
major chemokine receptor on CD4 lymphocytes through which MDC and TARC
act. Moreover, we define a large subset of memory CD4 lymphocytes that
express CCR4, which comprise 
50% of memory
CD4+ cells, including nearly all
CLA+ cells. A subset of
CCR4+ cells coexpresses CXCR3 and CCR5 that are
associated with in vitro derived Th1 cells. Although Th2 cytokine
production is linked with expression of CCR4,
CCR4+ memory T cells also contain non-Th2 cells
expressing IFN-. Therefore, contrary to the preferential expression
pattern of CXCR3, CCR5, and CCR4 on T cells that are generated in vitro
in polarized conditions, CCR4 cannot specifically mark Th2 populations
in vivo.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
CCR4 cDNA was obtained by RT-PCR on RNA isolated from a basophilic cell line KG1a using a 5'-oligonucleotide primer and a 3'-oligonucleotide primer, which correspond to the CCR4 sequences and contain flanking HindIII and XbaI sites, respectively. The PCR fragment was subcloned into the HindIII and XbaI sites of pMRB101, in which the inserted gene expression was driven by a CMV promoter. The DNA was stably transfected into a murine pre-B lymphoma cell line (L1.2), as described (36, 37, 38). The cells expressing high levels of CCR4 were selected by serial dilution/subcloning for their ability to chemotax to TARC and MDC. To enhance the receptor expression, the cells were treated at 106/ml with 5 mM butyric acid for 16–18 h before the experiment.
Other cell lines used included stable transfectants of the L1.2 murine pre-B lymphoma cells expressing CCR1–3, CCR5–9, CXCR1-CXCR5, as well as transfectants expressing various orphan G protein-coupled receptors, including BONZO, BOB, LyGPR, APJ, RDC, GPR5, GPR9-6, and AF014958.
Purification of cell populations
Venous blood was collected from volunteer donors, and PBMC were
isolated by Ficoll-Hypaque density-gradient centrifugation, as
described (36). To isolate different subsets of
leukocytes, human peripheral blood was collected in 10% (v/v) 0.1 M
EDTA, layered onto 1-Step Polymorphs gradient (Accurate Chemicals,
Westbury, NY), and centrifuged at 400 x g for 30 min
at room temperature. Neutrophil and mononuclear cell layers were
collected, resuspended in Dulbecco’s PBS without calcium and magnesium
(Life Technologies, Grand Island, NY), and centrifuged for 15 min at
750 x g. RBC were lysed in the neutrophil fraction
by resuspending the pellet in E-Lyse (Cardinal Associates, Santa Fe,
NM) for 5 min on ice. Both cell fractions were washed twice with
ice-cold Dulbecco’s PBS. The mononuclear cells were allowed to adhere
to protein-coated plastic for 2–3 h, and then nonadherent cells were
gently washed off the plate. CD4 cells were purified from mononuclear
cells with the relevant Miltenyi beads using 20 µl of beads for
107 mononuclear cells in PBS/1% BSA/5 mM EDTA at
5 x 107 cells/ml for 30 min at 4°C. They
were then spun down, resuspended in PBS/1% BSA/5 mM EDTA at 5 x
107 cells/ml, and passed over a VS column
(Miltenyi Biotech, Auburn, CA) in a magnetic field to remove nontagged
cells. Cells were removed by forcing 20 ml of PBS/1% BSA/5 mM EDTA
over the VS column, outside the magnetic field.
Preparation of chronically activated Th1 and Th2 lymphocytes
As previously described (39), six-well Falcon
plates were coated overnight with 10 µg/ml anti-CD28 and 2
µg/ml OKT3, and then washed twice with PBS. Umbilical cord blood CD4
lymphocytes (Poietic Systems, Germantown, MD) were cultured at
105-106 cells/ml in DMEM
with 10% FCS and IL-2 (4 ng/ml). IL-12 (5 ng/ml) and anti-IL-4 (1
µg/ml) were used to direct to Th1, while IL-4 (5 ng/ml) and
anti-IFN- (1 µg/ml) were used to direct to Th2. After 4–5
days, the activated Th1 and Th2 lymphocytes were washed once in DMEM
and expanded for 4–7 days in DMEM with 10% FCS and IL-2 (1 ng/ml).
Following this, the activated Th1 and Th2 lymphocytes were restimulated
for 5 days with anti-CD28/OKT3 and cytokines, as described above,
but with the addition of anti-CD95L (1 µg/ml) to prevent
apoptosis. After 4–5 days, the Th1 and Th2 lymphocytes were washed and
then expanded again with IL-2 for 4–7 days. Activated Th1 and Th2
lymphocytes were maintained in this way for a maximum of three
cycles.
Generation of anti-CCR4 mAbs
C57/Black mice were immunized with 107 CCR4/L1.2 transfectants per mouse at 2- to 3-wk intervals for 6–12 times. For the first three immunizations, the transfectants were pretreated with mitomycin C for 30 min before washing and injection to prevent tumor growth in the mice. Spleens were harvested from mice 3–4 days after the last immunization, and cells were fused with the SP2/0 cell line, as described (40). Generally, 3000–5000 hybridomas were generated per fusion and the supernatants were tested for differential staining of CCR4/L1.2 transfectants and other chemokine receptor L1.2 transfectants. Of the 15 fusions performed, two fusions gave rise to anti-CCR4 mAbs, 1G1 (isotype IgG1) and 2B10 (isotype IgG2a), respectively.
Other Abs and reagents
Anti-CD4, -CD8, -19, -49d, -56, -62L, -CLA, -CD45RA, and -CD45RO dye-linked mAbs for immunofluorescence studies were all obtained from PharMingen (San Diego, CA). OKT3, an anti-human CD3 mAb, was obtained from American Type Culture Collection (ATCC, Manassas, VA), and anti-human CD28 mAb was purchased from Becton Dickinson (San Diego, CA). The majority of the anti-chemokine receptor mAbs were generated in LeukoSite (Cambridge, MA) and have the clone names 1D9 (CCR2), 7B11 (CCR3), 2D7 (CCR5), 11A9 (CCR6), and 1C6 (CXCR3). Recombinant human chemokines were obtained from PeproTech (Rocky Hill, NJ) and R&D Systems (Minneapolis, MN). The human endothelial cell line ECV304 was purchased from ATCC. All cytokines were obtained from R&D Systems.
125I-labeled human TARC (125I-TARC) and MDC (125I-MDC) binding
125I-TARC and 125I-MDC were purchased from DuPont-NEN (Boston, MA) and Amersham (Arlington Heights, IL), respectively, and unlabeled chemokines were obtained from PeproTech or R&D Systems. Chemokine binding to target cells was conducted using the following procedure: CCR4/L1.2 cells were washed and resuspended in binding buffer (50 mM HEPES, pH 7.5, 1 mM CaCl2, 5 mM MgCl2, and 0.5% BSA) at 107/ml. For each binding reaction (in a final volume of 100 µl), 25 µl of cell suspension (2.5 x 105 cells) was mixed with 0.1 nM radiolabeled chemokine with or without an appropriate amount of anti-CCR4 mAb or an isotype-matched control mAb. Total binding was in the presence of radiolabeled chemokines only, and nonspecific binding (background) was determined in the presence of 100 nM unlabeled chemokines. The reactions were incubated at room temperature for 45–60 min, and stopped by transferring the mixture to Unifilter-96 GF/B (Packard) plates that were then washed two to three times with binding buffer containing 0.5 M NaCl. The plates were dried and MicroScint scintillation fluid was added before counting. Each sample was assayed in duplicate.
Chemotaxis assay
The 3-µm-pore-diameter Transwell inserts were used either uncoated or coated with 2% gelatin for 2 h. Then 0.45 ml of DMEM with 5% FCS was placed in the lower wells of the chambers, and 2 x 105 ECV304 cells were added to each gelatin-coated insert in 0.2 ml of DMEM/5% FCS. After 2 days, the wells and inserts were washed twice with RPMI containing 0.5% BSA and 10 mM HEPES, and then chemokine was added to the lower well. For chemotaxis of transfectant cells, no ECV304 cells were used to coat the plates. The cells under study were washed once in RPMI and resuspended at 4 x 106 cells/ml for Th1/Th2 lymphocytes and cell lines and at 107 cells/ml for transfectants and resting CD4 lymphocytes in RPMI containing 0.5% BSA and 10 mM HEPES. An aliquot of 200 µl of cell suspension (input of 8 x 105 cells and 2 x 106 cells, respectively) was added to each insert. After 2–4 h, the inserts were removed and the number of cells that had migrated through the ECV304 monolayer to the lower well were counted for 30 s on a Becton Dickinson FACScan with the gates set to acquire the cells of interest. Using this technique, 100% migration would be 25,000 cells for Th1/Th2 cells and 75,000 cells for resting CD4 lymphocytes, in which this number represents the cells in the lower well counted on the FACScan for 1 min. To study the phenotype of migrating cells, identical experiments with CD4 lymphocytes were performed with six-well plates using 24-mm-diameter inserts. Chemotaxis assays were identical with ECV304 migration assays, but fibronectin-coated inserts (10 µg/ml) were used. In all cases, the data points were the result of duplicate wells, with the mean value shown and the error bars representing the sample SD.
Intracellular cytokine staining
Mononuclear cells from human peripheral blood were prepared by
centrifugation on Histopaque-1077 (Sigma, St. Louis, MO). Untouched
CD4+ T cells (purity >97%) were isolated by
depleting non-CD4+ T cells using a magnetic bead
depletion method (Miltenyi Biotec). CD4+ T cells
were stained with Abs to CLA, CD45RA, and CCR4.
CD45RA+ (naive) CCR4-
CLA-, CD45RA- (memory)
CCR4- CLA-,
CD45RA- CCR4+
CLA-, and CD45RA-
CCR4+ CLA+ cells were
sorted using FACStar (Becton Dickinson, San Jose, CA). Sorted cells
were activated for 5 h at 37°C with PMA (50 ng/ml) and ionomycin
(1 µg/ml) in RPMI 1640 medium supplemented with
penicillin/streptomycin, 10% FBS, 50 U/ml IL-2, and 1 µg/ml
brefeldin A. Activated cells were fixed and permeabilized using
Cytofix/Cytoperm solution (PharMingen) and stained with PE-conjugated
isotype control Abs or mAbs to IL-2, IL-5, IFN- (from
PharMingen), and IL-4 (Becton Dickinson). Data were analyzed on
FACSCaliber using CellQuest program (Becton Dickinson).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Two mAbs were raised in C57/Black mice to CCR4/L1.2 transfectants.
Both mAbs reacted with the CCR4/L1.2 transfectants, but not with
parental L1.2 cells. The specificity of these two mAbs for CCR4 was
further extensively examined using a large panel of L1.2 transfectants.
Neither 1G1 nor 2B10 cross-reacted with L1.2 transfectants expressing
CCR1, CCR2, CCR3, CCR5, CCR6, CCR7, and CCR8 (shown in Fig. 1
for 1G1). They also did not cross-react
with L1.2 transfectants expressing CXCR1-CXCR5, or orphan G
protein-coupled receptors including BONZO, BOB, LyGPR, APJ, RDC, GPR5,
GPR9-6, and AF014958 (not shown).
|
Both 1G1 and 2B10 were used to examine the CCR4 expression on
leukocyte subpopulations, and similar results were obtained with both
mAbs. Anti-CCR4 mAb 1G1 was used in the FACS experiments described in
this work. In initial two-color studies of peripheral blood, CCR4 was
found to be expressed on a large subset (20–30%) of CD4 lymphocytes
as well as on a smaller subset (3–5%) of CD8 lymphocytes (Fig. 2A). Although
CCR4dull and CCR4high
subsets of CD4 lymphocytes were evident, CCR4 was expressed at low
levels on CD8 lymphocytes. B lymphocytes, basophils, eosinophils,
neutrophils, and NK cells did not express CCR4 (Fig. 2A and
data not shown). As would be predicted from its expression on blood T
lymphocytes, in examining a large panel of cell lines, CCR4 was found
only on T cell lines (Peer and CEM cells, data not shown).
|
B). The CD4
lymphocytes that expressed CCR4 were mostly of memory phenotype
CD45RA-. As reported in our recent studies
(41), memory CLA+ CD4 lymphocytes,
which traffic to skin, were all CCR4high.
However, CCR4high CLA- and
CCR4dull CLA- CD4
lymphocytes were also evident. In contrast, the subset of memory
47high
CD4 lymphocytes, which traffic to mucosal sites, expressed little or no
CCR4. Moreover, the subset of memory CD4 lymphocytes that expressed the
highest level of CCR4 was predominantly
47-.
Furthermore, the subset of memory CD4 lymphocytes that lacks
4 integrins
(41-
47-)
all expressed high levels of CCR4 (Fig. 2B). CD62L was
expressed on both CCR4+ and
CCR4- subsets of memory CD4 lymphocytes. To further correlate the expression of CCR4 on CD4 lymphocytes with its function, we examined the subset of CD4 lymphocytes that chemotax to MDC by FACS. We used the anti-CCR4 mAb 1G1 in this experiment, as binding of 2B10 to CD4 lymphocytes was inhibited by MDC (data not shown). CCR4+ CD4 lymphocytes were enriched in the population of CD4 lymphocytes that chemotax to MDC (2.6-fold). We also saw an enrichment for CLA+ (4.5-fold) and CD45RA- memory CD4 lymphocytes (2.9-fold) in the migrated population. Among CCR4+ memory CD4 lymphocytes, the majority of the migration to MDC was in the CLA+ CCR4+ subset rather than the CLA- CCR4+ subset (4.5 vs 1.6). Finally, in examining the chemotaxis of 16 cell lines to MDC and TARC, we found that only the two cell lines that expressed CCR4 chemotaxed to MDC and TARC (data not shown). Therefore, expression of CCR4 on T lymphocytes and functional response to MDC/TARC correlate.
CCR4 is expressed on a subset of mature single-positive (SP) CD4+ thymocytes
Because CCR4 transcripts are present in thymus, we examined the
expression of CCR4 on thymocytes. CCR4 was expressed on a small subset
(14%) of thymocytes (Fig. 3A). The expression of CD3 on
all CCR4+ thymocytes indicates that CCR4 is
up-regulated during thymocyte maturation. Another chemokine receptor,
CXCR4, was expressed on immature thymocytes, while CCR9 was expressed
at all stages of T cell development. Due to the expression of CCR4 on
mature thymocytes, we analyzed expression of this chemokine receptor in
immature double-negative
CD4-CD8- and
double-positive CD4+CD8+
thymocytes as well as mature SP CD4 and CD8 thymocytes (Fig. 3B). As expected, CCR4 expression was restricted to mature
SP thymocytes and was mainly expressed on SP CD4 thymocytes. The
majority of SP CD4 thymocytes express CCR4, but a small subset of
CCR4- CD4 SP thymocytes was also evident.
|
The ability of the mAbs 1G1 and 2B10 to inhibit the binding of
TARC (Fig. 4A) and MDC (Fig. 4B) to CCR4 was examined. Both mAbs blocked the
125I-TARC and 125I-MDC
binding to CCR4/L1.2 transfectants, while the isotype-matched control
mAbs had no significant effect. Furthermore, both 1G1 and 2B10
effectively blocked the TARC- and MDC-mediated chemotaxis in CCR4/L1.2
transfectants (Fig. 4, C and D). Of greater
interest, 2B10 and to some extent 1G1 significantly inhibited
chemotaxis of resting CD4 lymphocyte (Fig. 5A) and Th2 lymphocyte (Fig. 5B) to TARC and MDC. Both mAbs had no effect on the
chemotaxis of CD4 lymphocytes to RANTES.
|
|
Although umbilical CD4 lymphocytes did not express CCR4 (data not
shown), chronic activation of these cells in the presence of IL-4 to
generate Th2 lymphocytes resulted in CCR4 expression (Fig. 6), while chronic activation with IL-12
to generate Th1 lymphocytes did not up-regulate CCR4 expression. As
expected, CXCR3 was up-regulated on Th1 lymphocytes, while
47, an integrin used
in lymphocyte trafficking to mucosal sites, and chemokine receptor CCR2
were up-regulated on both Th1 and Th2 lymphocytes (Fig. 6).
|
CCR4 is coexpressed with Th1-associated chemokine receptors CCR5 and CXCR3
The expression of CCR4 on CD4 lymphocytes in relation to other
chemokine receptors known to be expressed on subsets of memory CD4
lymphocytes was examined in three-color experiments (Fig. 7). CCR4 was found on both positive and
negative subsets of CCR2 and CCR6 CD4 lymphocytes. Surprisingly, given
the association of CCR5 and CXCR3 expression with Th1 phenotype by in
vitro studies, a significant population of
CCR5+CCR4+ and
CXCR3+ CCR4+ CD4
lymphocytes was observed in these experiments.
|
As CCR4 is coexpressed on memory CD4 lymphocytes with
Th1-associated chemokine receptors, we next examined the cytokines
produced by activated subsets of memory CD4 lymphocytes defined by
expression of CCR4 and CLA. As expected, naive and memory CD4
lymphocytes secreted IL-2 on stimulation, while production of IL-4,
IL-5, and IFN- was restricted to the memory CD4 lymphocytes (Fig. 8A). Production of IFN- was
associated with the CCR4- memory CD4
lymphocytes, with 51% of CCR4-
CLA- CD4 memory cells producing IFN- on
activation, while only 17% of the CCR4+
CLA- and 10% of the CCR4+
CLA+ memory CD4 lymphocytes produced this
cytokine. Similarly, production of Th2 cytokines IL-4 and IL-5 was
positively associated with CCR4 expression on CD4 lymphocytes, with the
CLA- CCR4+ memory CD4
lymphocytes being the major producers of these two cytokines. However,
while Th2 and Th1 cytokine production was positively and negatively
associated with CCR4 expression, respectively, expression of IFN-
was observed in significant (5–30%) subsets of activated
CCR4+ memory CD4 lymphocytes, and IL-4 production
was also observed in a subset (up to 3%) of freshly isolated
CCR4- memory CD4 lymphocytes on activation.
Studies of additional normal donors for IL-2, IL-4, and IFN-
production were also performed and similar results were observed (Fig. 8B).
|
(81%/24%).
Finally, we sorted CCR4+/-,
CLA+/-, CD45RA+/- CD4 T
cell subsets and stimulated them with anti-CD28 and anti-CD3 in
nonpolarizing culture condition (without added exogenous IL-4 or IL-12
or other neutralizing Abs) for 4 days and then cultured them in IL-2
for 3 days (the first activation cycle). Half the cultured cells were
examined for intracellular cytokine analysis, and the other half were
cultured again (the second activation cycle) and examined for their
cytokine production. Although in vitro differentiated
CCR4+ and CCR4- CD4 T
cells maintained their relative differences in production of IL-4 or
IFN-, CCR4+ CD4 T cells after two cycles of
activation contained not only more T cells producing IL-4, but also
included more T cells producing IFN- than fresh CD4 T cells (Fig. 9). This is also the case for the
CCR4- CD4 T cells. These combined data suggest
that CCR4+ CD4 T cells have potentials to become
Th1 as well as to become Th2 cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Recently, TARC was reported to bind to CCR8 (42). However, in our hands and in other reports (43), TARC and MDC mediated only chemotaxis of CCR4 transfectants and not other chemokine receptor transfectants, including CCR8. A screening of a panel of cell lines in this study indicates that only CCR4+ cell lines chemotax to MDC and TARC. In addition, the majority of chemotaxis of both resting CD4+ cells and chronically activated Th2 lymphocytes to MDC and TARC can be blocked by anti-CCR4 mAbs. We also found that the subset of CD4 lymphocytes that migrated to MDC is enriched for CCR4-expressing cells. These combined results suggest that MDC and TARC predominantly use CCR4 on T cells in mediating chemotaxis of CD4 lymphocytes.
Our studies demonstrate that CCR4 is expressed on a large subset of the SP CD4+ thymocytes that express high levels of TCR, suggesting CCR4 expression is induced on thymocytes at a late stage of T cell development. The restricted expression of CCR4 on SP CD4+ thymocytes correlates with the chemotaxis of this subset of thymocytes to MDC and further demonstrates that expression of CCR4 and chemotaxis to MDC correlate (44). TARC and MDC are also reported to be expressed in the thymus (45, 46) and by APCs (24, 47). It is possible that TARC and MDC are expressed on macrophages, dendritic cells, and epithelial cells in the thymus and attract mature CCR4+ CD4 SP thymocytes to modulate positive and negative selection during T cell development. Interestingly, CD45RA naive T lymphocytes in the periphery do not express CCR4. Therefore, on exit of SP CD4 thymocytes from the thymus to the periphery, CCR4 expression must be down-regulated. Alternatively, those SP CD4 thymocytes that do not express CCR4 in the thymus may represent fully mature thymocytes about to exit the thymus.
The subset of memory CD4 lymphocytes that express CCR4 encompasses
nearly all CD4 lymphocytes that express CLA, but not
47. Indeed, nearly
all CLA+
47-
memory CD4 lymphocytes express high levels of CCR4. However, most
importantly, CCR4 is expressed at high levels by a subset of
CLA-
47-
cells, suggesting that CCR4 may mediate other functions in addition to
its proposed role in trafficking of CLA+ memory
CD4 lymphocytes to skin. It was shown previously that CLA was expressed
by T cells responsible for the Th2 cytokine production in individuals
with skin allergy (48, 49). Nevertheless, in individuals
with allergy at nonskin sites, Th2 lymphocytes were also found in the
memory CLA- CD4 subset. The expression of MDC by
APCs, including activated B cells and the induction of MDC as well as
CCR4 on T lymphocytes by Th2 cytokines (21, 32), indicates
that CCR4 is possibly also involved in local movement of memory CD4
lymphocytes in lymphoid tissue toward dendritic cells and B cells.
Finally, the expression of CCR4 on both CD62L+
and CD62L- memory CD4 lymphocytes indicates that
the memory CCR4+ CD4 subset includes both
effector CD62L- memory CD4 lymphocytes that are
thought to traffick principally to inflammatory sites as well as
resting memory CD62L+ CD4 lymphocytes that can
most likely also traffick to secondary lymphoid organs such as
peripheral lymph nodes.
It was previously shown that MDC and TARC specifically act on in vitro
derived effector Th2 lymphocytes, while CCR4 transcripts are
differentially expressed by in vitro derived Th1/Th2 lymphocytes
(24, 30). Consistent with these observations, we and Imai
et al. (33) found that CCR4 protein is preferentially
expressed on the surface of in vitro derived Th2 cells. However, if
CCR4 were a specific marker for Th2 cells, one would have predicted
that CCR4 would be expressed by a very small subset of memory CD4
lymphocytes, as effector Th2 cells represent only a small subset of
peripheral blood CD4+ cells. Therefore, the
demonstration in this study that CCR4 is expressed on a large subset of
circulating memory CD4 lymphocytes was surprising. As MDC blocks all
staining of leukocytes by the anti-CCR4 mAb 2B10 and both our
anti-CCR4 mAbs stain identical subsets (data no shown), we are
confident that our results represent the distribution of CCR4 on
leukocytes. Imai et al. also showed recently that, using an independent
anti-CCR4 mAb, CCR4 was detected on 20%
CD4+ T cells (33), although the
intensity of their staining was weaker than what we obtained using both
of our mAbs.
Our experiments examining coexpression of CCR4 with other chemokine
receptors on CD4 lymphocytes also gave unexpected results. We found
that CCR4 was expressed on a significant subset of CD4 lymphocytes that
also express chemokine receptor CCR5 and CXCR3, both of which are
selectively expressed on in vitro derived effector Th1 lymphocytes.
More importantly, our data suggest that although CCR4 behaves like a
specific Th2 marker on in vitro derived Th2 lymphocytes, it does not
specifically mark the Th2 population in circulating blood, and in this
sense, neither do CCR5 and CXCR3 specifically mark the Th1 population.
CCR4+ CD4 lymphocytes may represent a larger
subset of memory CD4 lymphocytes, within which are contained the
majority of Th2 lymphocytes. Alternatively, the culture conditions used
to generate in vitro derived Th2 lymphocytes may induce expression of
CCR4. In further studies, we directly examined the cytokines produced
by memory CD4 lymphocytes from blood sorted for expression of CCR4 and
CLA. Although IL-4-producing cells were more frequent in the
CCR4+ than CCR4- subsets,
there tended to be a greater frequency of IFN--producing Th1 cells
among the CCR4- subset. Furthermore, only a
small subset of memory CCR4+ CD4 lymphocytes
produces Th2 cytokines on activation. When freshly sorted
CCR4+ and CCR4- CD4
lymphocytes were activated and expanded before performing intracellular
cytokine staining, the Th2 correlation was still observed. It is
possible that in certain chronic inflammation conditions, the linkage
of CCR4 and Th2 phenotype may increase depending on cytokine expression
profiles of tissue microenvironment. Further studies in patients with
Th2 inflammatory diseases, such as atopic dermatitis and asthma, are
underway to investigate this.
Differences in the results obtained from in vitro derived Th1/Th2 lymphocytes and freshly isolated T lymphocytes in blood have also been observed for expression of adhesion molecules, such as CLA. Although CLA is selectively expressed on in vitro derived Th1 lymphocytes (50), in vivo studies in both normal and atopic dermatitis patients have yielded conflicting data. Under normal conditions, the CLA subsets of memory CD4 lymphocytes produce both Th1 and Th2 cytokines (51), while in atopic dermatitis the CLA subset contains predominantly Th2 lymphocytes (48). In conclusion, our data would indicate that in normal patients there is a linkage of CCR4 with Th2 phenotype, but that this is not at all a tight linkage. Most importantly, our results indicate that this linkage is malleable, and suggest that effector cytokine production and patterns of chemokine receptor expression (at least of CCR4, CXCR3, and CCR5) can be independently controlled and regulated in vivo, thus optimizing the potential for phenotypic control of subset-specific homing to particular sites such as skin.
In summary, in this study we show that CCR4 is expressed on a small
subset of mature SP CD4+ thymocytes and a major
subset of CD4 lymphocytes, which contains all
CLA+
47-
CD4 lymphocytes, as well as a small population of
CLA- memory CD4 lymphocytes. Chemotaxis of CD4
lymphocytes to MDC and TARC correlates with expression of CCR4 and is
blocked by anti-CCR4 mAbs. Therefore, CCR4 represents the major
chemokine receptor through which TARC and MDC act on memory CD4
lymphocytes. Our data indicate that in peripheral blood, subsets of
memory CD4 lymphocytes are present that express CCR4 as well as CXCR3
or CCR5. Also, although there is a linkage between Th2 phenotype and
CCR4 expression in normal blood, distinct populations of
CCR4+ memory CD4 lymphocytes that produce IFN-
on activation are evident. Therefore, our data suggest that the
refinement of Th1 and Th2 is more complex in vivo than in vitro.
Chemokine receptors may play distinct roles at different stages of
memory effector T cell differentiation, very likely reflecting the
different influence and refinement of local immune responses as a
function of tissues, microenvironment, and cytokine milieu.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 C.H.K is a Leukemia Society of America fellow.
3 Address correspondence and reprint requests to Dr. Lijun Wu, Millennium Inc., 75 Sidney Street, Cambridge, MA 02139.
4 Abbreviations used in this paper: CLA, cutaneous lymphocyte Ag; CXCR, CXC chemokine receptor; 125I-MDC, 125I-labeled MDC; 125I-TARC, 125I-labeled TARC; MDC, macrophage-derived chemokine; SP, single positive; TARC, thymus and activation-regulated chemokine.
Received for publication March 17, 2000. Accepted for publication September 29, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in the pathogenesis of the T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis. J. Immunol. 155:5003.[Abstract]
rapidly modulates its receptors and inhibits the anti-CD3 mAb-mediated proliferation of T lymphocytes. J. Immunol. 151:4333.[Abstract]
chemokines costimulate human T lymphocyte activation in vitro. J. Immunol. 156:2095.[Abstract]
47 and LFA-1 in
lymphocyte interactions with Peyer’s patch-HEV in situ: the multi-step
hypothesis confirmed and refined. Immunity. In press.
. J. Immunol. 162:186. and MIP-1. J. Immunol. 161:5663.-Interferon transcriptionally regulates an early-response gene containing homology to platelet proteins. Nature 315:672.[Medline]
on macrophage-derived chemokine production: an amplification circuit of polarized T helper 2 responses. Blood 92:2668. as novel functional ligands for the CCR8 receptor. Eur. J. Immunol. 28:582.[Medline]
are not ligands for human CC-chemokine receptor 8. Eur. J. Immunol. 29:3210.[Medline]
-chemokine TARC is expressed by subsets of dendritic cells and attracts primed CD4+ T cells. Eur. J. Immunol. 29:2684.[Medline]
This article has been cited by other articles:
|
|
 |
|
  T. Kondo and M. Takiguchi Human memory CCR4+CD8+ T cell subset has the ability to produce multiple cytokines Int. Immunol., May 1, 2009; 21(5): 523 - 532. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Knight, A. B. Blazquez, S. Zhang, L. Mayer, H. A. Sampson, and M. C. Berin CD4 T cells activated in the mesenteric lymph node mediate gastrointestinal food allergy in mice Am J Physiol Gastrointest Liver Physiol, December 1, 2007; 293(6): G1234 - G1243. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Mori, K. Ogawa, K. Someya, Y. Kunori, D. Nagakubo, O. Yoshie, F. Kitamura, T. Hiroi, and O. Kaminuma Selective suppression of Th2-mediated airway eosinophil infiltration by low-molecular weight CCR3 antagonists Int. Immunol., August 1, 2007; 19(8): 913 - 921. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-A. Gonzalo, Y. Qiu, J. M. Lora, A. Al-Garawi, J.-L. Villeval, J. A. Boyce, C. Martinez-A, G. Marquez, I. Goya, Q. Hamid, et al. Coordinated Involvement of Mast Cells and T Cells in Allergic Mucosal Inflammation: Critical Role of the CC Chemokine Ligand 1:CCR8 Axis J. Immunol., August 1, 2007; 179(3): 1740 - 1750. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Freeman, V. R. Stolberg, B.-C. Chiu, N. W. Lukacs, S. L. Kunkel, and S. W. Chensue CCR4 Participation in Th Type 1 (Mycobacterial) and Th Type 2 (Schistosomal) Anamnestic Pulmonary Granulomatous Responses J. Immunol., September 15, 2006; 177(6): 4149 - 4158. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Yang, B. E. Castle, A. Hanidu, L. Stevens, Y. Yu, X. Li, C. Stearns, V. Papov, D. Rajotte, and J. Li Sphingosine Kinase 1 Is a Negative Regulator of CD4+ Th1 Cells J. Immunol., November 15, 2005; 175(10): 6580 - 6588. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Bendriss-Vermare, S. Burg, H. Kanzler, L. Chaperot, T. Duhen, O. de Bouteiller, M. D'agostini, J.-M. Bridon, I. Durand, J. M. Sederstrom, et al. Virus overrides the propensity of human CD40L-activated plasmacytoid dendritic cells to produce Th2 mediators through synergistic induction of IFN-{gamma} and Th1 chemokine production J. Leukoc. Biol., October 1, 2005; 78(4): 954 - 966. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Morimoto, Y. Bian, P. Gao, Y. Yashiro-Ohtani, X.-Y. Zhou, S. Ono, H. Nakahara, M. Kogo, T. Hamaoka, and H. Fujiwara Induction of surface CCR4 and its functionality in mouse Th2 cells is regulated differently during Th2 development J. Leukoc. Biol., September 1, 2005; 78(3): 753 - 761. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Carlow, M. J. Williams, and H. J. Ziltener Inducing P-Selectin Ligand Formation in CD8 T Cells: IL-2 and IL-12 Are Active In Vitro but Not Required In Vivo J. Immunol., April 1, 2005; 174(7): 3959 - 3966. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Freeman, B.-C. Chiu, V. R. Stolberg, J. Hu, K. Zeibecoglou, N. W. Lukacs, S. A. Lira, S. L. Kunkel, and S. W. Chensue CCR8 Is Expressed by Antigen-Elicited, IL-10-Producing CD4+CD25+ T Cells, Which Regulate Th2-Mediated Granuloma Formation in Mice J. Immunol., February 15, 2005; 174(4): 1962 - 1970. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Enarsson, M. Brisslert, S. Backert, and M. Quiding-Jarbrink Helicobacter pylori Induces Transendothelial Migration of Activated Memory T Cells Infect. Immun., February 1, 2005; 73(2): 761 - 769. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Jakubzick, H. Wen, A. Matsukawa, M. Keller, S. L. Kunkel, and C. M. Hogaboam Role of CCR4 Ligands, CCL17 and CCL22, During Schistosoma mansoni Egg-Induced Pulmonary Granuloma Formation in Mice Am. J. Pathol., October 1, 2004; 165(4): 1211 - 1221. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R Nissinen, M Leirisalo-Repo, A M Nieminen, L Halme, M Farkkila, T Palosuo, and O Vaarala Immune activation in the small intestine in patients with rheumatoid arthritis Ann Rheum Dis, October 1, 2004; 63(10): 1327 - 1330. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ishida, H. Inagaki, A. Utsunomiya, Y. Takatsuka, H. Komatsu, S. Iida, G. Takeuchi, T. Eimoto, S. Nakamura, and R. Ueda CXC Chemokine Receptor 3 and CC Chemokine Receptor 4 Expression in T-Cell and NK-Cell Lymphomas with Special Reference to Clinicopathological Significance for Peripheral T-Cell Lymphoma, Unspecified Clin. Cancer Res., August 15, 2004; 10(16): 5494 - 5500. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. G. Cronshaw, C. Owen, Z. Brown, and S. G. Ward Activation of Phosphoinositide 3-Kinases by the CCR4 Ligand Macrophage-Derived Chemokine Is a Dispensable Signal for T Lymphocyte Chemotaxis J. Immunol., June 15, 2004; 172(12): 7761 - 7770. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Pilette, J.N. Francis, S.J. Till, and S.R. Durham CCR4 ligands are up-regulated in the airways of atopic asthmatics after segmental allergen challenge Eur. Respir. J., June 1, 2004; 23(6): 876 - 884. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Schaerli, L. Ebert, K. Willimann, A. Blaser, R. S. Roos, P. Loetscher, and B. Moser A Skin-selective Homing Mechanism for Human Immune Surveillance T Cells J. Exp. Med., May 3, 2004; 199(9): 1265 - 1275. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Greiner, C. C. Murphy, F. Willermain, L. Duncan, J. Plskova, G. Hale, J. D. Isaacs, J. V. Forrester, and A. D. Dick Anti-TNF{alpha} Therapy Modulates the Phenotype of Peripheral Blood CD4+ T Cells in Patients with Posterior Segment Intraocular Inflammation Invest. Ophthalmol. Vis. Sci., January 1, 2004; 45(1): 170 - 176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Conroy, L. A. Jopling, C. M. Lloyd, M. R. Hodge, D. P. Andrew, T. J. Williams, J. E. Pease, and I. Sabroe CCR4 blockade does not inhibit allergic airways inflammation J. Leukoc. Biol., October 1, 2003; 74(4): 558 - 563. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ishida, A. Utsunomiya, S. Iida, H. Inagaki, Y. Takatsuka, S. Kusumoto, G. Takeuchi, S. Shimizu, M. Ito, H. Komatsu, et al. Clinical Significance of CCR4 Expression in Adult T-Cell Leukemia/Lymphoma: Its Close Association with Skin Involvement and Unfavorable Outcome Clin. Cancer Res., September 1, 2003; 9(10): 3625 - 3634. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Senechal, P. de Nadai, N. Ralainirina, A. Scherpereel, H. Vorng, P. Lassalle, A.-B. Tonnel, A. Tsicopoulos, and B. Wallaert Effect of Diesel on Chemokines and Chemokine Receptors Involved in Helper T Cell Type 1/Type 2 Recruitment in Patients with Asthma Am. J. Respir. Crit. Care Med., July 15, 2003; 168(2): 215 - 221. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Alferink, I. Lieberam, W. Reindl, A. Behrens, S. Weiss, N. Huser, K. Gerauer, R. Ross, A. B. Reske-Kunz, P. Ahmad-Nejad, et al. Compartmentalized Production of CCL17 In Vivo: Strong Inducibility in Peripheral Dendritic Cells Contrasts Selective Absence from the Spleen J. Exp. Med., March 3, 2003; 197(5): 585 - 599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Agrawal, Z. Vanhorn-Ali, and G. Alkhatib Multiple determinants are involved in HIV coreceptor use as demonstrated by CCR4/CCL22 interaction in peripheral blood mononuclear cells (PBMCs) J. Leukoc. Biol., November 1, 2002; 72(5): 1063 - 1074. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Campbell, M. J. Stinson, F. E. R. Simons, and K. T. HayGlass Systemic chemokine and chemokine receptor responses are divergent in allergic versus non-allergic humans Int. Immunol., November 1, 2002; 14(11): 1255 - 1262. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Zhang, M. P. Soares, Y. Guan, S. Matheravidathu, R. Wnek, K. E. Johnson, A. Meisher, S. A. Iliff, J. S. Mudgett, M. S. Springer, et al. Functional Expression and Characterization of Macaque C-C Chemokine Receptor 3 (CCR3) and Generation of Potent Antagonistic Anti-macaque CCR3 Monoclonal Antibodies J. Biol. Chem., September 6, 2002; 277(37): 33799 - 33810. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B.-C. Chiu, X.-Z. Shang, V. R. Stolberg, E. Komuniecki, and S. W. Chensue Population analysis of CD4+ T cell chemokine receptor transcript expression during in vivo type-1 (mycobacterial) and type-2 (schistosomal) immune responses J. Leukoc. Biol., August 1, 2002; 72(2): 363 - 372. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Lohmann, S. Laue, U. Nietzschmann, T.M. Kapellen, I. Lehmann, S. Schroeder, R. Paschke, and W. Kiess Reduced Expression of Th1-Associated Chemokine Receptors on Peripheral Blood Lymphocytes at Diagnosis of Type 1 Diabetes Diabetes, August 1, 2002; 51(8): 2474 - 2480. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hudak, M. Hagen, Y. Liu, D. Catron, E. Oldham, L. M. McEvoy, and E. P. Bowman Immune Surveillance and Effector Functions of CCR10+ Skin Homing T Cells J. Immunol., August 1, 2002; 169(3): 1189 - 1196. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Nishimura, H. Umehara, T. Nakayama, O. Yoneda, K. Hieshima, M. Kakizaki, N. Dohmae, O. Yoshie, and T. Imai Dual Functions of Fractalkine/CX3C Ligand 1 in Trafficking of Perforin+/Granzyme B+ Cytotoxic Effector Lymphocytes That Are Defined by CX3CR1 Expression J. Immunol., June 15, 2002; 168(12): 6173 - 6180. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Yoshie, R. Fujisawa, T. Nakayama, H. Harasawa, H. Tago, D. Izawa, K. Hieshima, Y. Tatsumi, K. Matsushima, H. Hasegawa, et al. Frequent expression of CCR4 in adult T-cell leukemia and human T-cell leukemia virus type 1-transformed T cells Blood, March 1, 2002; 99(5): 1505 - 1511. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Jopling, I. Sabroe, D. P. Andrew, T. J. Mitchell, Y. Li, M. R. Hodge, T. J. Williams, and J. E. Pease The Identification, Characterization, and Distribution of Guinea Pig CCR4 and Epitope Mapping of a Blocking Antibody J. Biol. Chem., February 22, 2002; 277(9): 6864 - 6873. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Campbell and E. C. Butcher Rapid Acquisition of Tissue-specific Homing Phenotypes by CD4+ T Cells Activated in Cutaneous or Mucosal Lymphoid Tissues J. Exp. Med., January 7, 2002; 195(1): 135 - 141. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Iellem, M. Mariani, R. Lang, H. Recalde, P. Panina-Bordignon, F. Sinigaglia, and D. D'Ambrosio Unique Chemotactic Response Profile and Specific Expression of Chemokine Receptors Ccr4 and Ccr8 by Cd4+Cd25+ Regulatory T Cells J. Exp. Med., September 17, 2001; 194(6): 847 - 854. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
