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Chemokine Biology Laboratory, Department of Molecular Biosciences, Adelaide University, Adelaide, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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/CCL4, but
up-regulation of CCR6 did not result in altered chemotactic
responsiveness to macrophage-inflammatory protein-3
/CCL20,
suggesting an alternative function for this
receptor. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokines can be divided into two major (CC and CXC) and two minor (C and CX3C) families, based on a cysteine signature motif (3). Alternatively, they can be classified as homeostatic (constitutive) or inflammatory (inducible) according to their function (4, 5). Homeostatic chemokines are expressed constitutively and appear to be responsible for the trafficking of lymphocytes under conditions of homeostasis. In contrast, inflammatory chemokines are specifically up-regulated at sites of inflammation and are thought to play a role in the recruitment of lymphocytes to peripheral tissues in response to immunological challenge.
CCR5 is a receptor for the inflammatory chemokines RANTES/CCL5,
macrophage-inflammatory protein (MIP)-1/CCL3, and MIP-1/CCL4
(6), while CCR6 is the unique receptor for the
inflammatory chemokine MIP-3/CCL20 (7, 8). Within both
the CD4+ and CD8+ T cell
subsets, CCR5 and CCR6 are expressed primarily on memory cells, with
little or no expression in the naive population (9, 10, 11, 12).
Selective expression in previously activated, rather than naive, T
cells is emerging as a general phenomenon of inflammatory chemokine
receptors and suggests that T cells up-regulate the expression of
inflammatory chemokine receptors upon activation. We and others
(13, 14, 15) have recently shown this to be the case with
another inflammatory chemokine receptor, CXCR3. In contrast,
stimulation of T cells with mitogens, phorbol esters, anti-CD3, or
anti-CD3 plus anti-CD28 does not up-regulate the expression of
CCR5 (10, 12, 16) or CCR6 (9, 10); in some
studies the expression of these receptors is decreased (8, 17). However, a recent report by Riley et al. (18)
revealed that the nature of the in vitro stimulation can profoundly
affect the regulation of CCR5, in that costimulation through CD28 and
CTLA-4 exerted paradoxical effects on CCR5 expression.
Up-regulation of CCR5 and CCR6 expression on T cell activation appears to occur in vivo, as these receptors are expressed on previously activated T cells in the blood, as discussed above, and on T cells accumulating in inflamed tissues such as synovium (13), psoriatic skin (19), and multiple sclerosis lesions (20). However, the methods of polyclonal T cell stimulation commonly used in in vitro studies do not appear to be sufficient or appropriate to induce the expression of these receptors. Accordingly, we have undertaken a detailed assessment of CCR5 and CCR6 expression on CD4+ T cells using a physiologically relevant in vitro culture system, the MLR. This culture system is dependent on DC for the majority of APC function and results in the proliferation of naive as well as memory CD4+ T cells. We have previously used this culture system to demonstrate increased CXCR3 expression and a concomitant decrease in CXCR4 expression on activated CD4+ T cells (15). We now extend these findings to show that CCR5 and CCR6 are up-regulated on CD4+ T cells following allogeneic activation, although the kinetics and functional outcome of receptor up-regulation differ notably from the patterns observed for CXCR3 and CXCR4.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-CD4 (clone OKT4) was a gift from Dr. R. Roy (Center de Recherche en Rheumatologie et Immunologie, Université Laval, Quebec, Canada) and isotype controls were gifts from Prof. H. Zola (Child Health Research Institute, Adelaide, Australia). Anti-CCR5 (clone 2D7) was purchased from BD PharMingen (San Diego, CA) in both an unlabeled and a PE-conjugated format, while anti-CCR6 (clone 53103.111), anti-CXCR3 (clone 49801.111), and biotinylated anti-CXCR4 (clone 44716.111) were purchased from R&D Systems (Minneapolis, MN). PE-labeled Abs to CD45RA (clone F8-11-13) and CD45RO (clone UCHL1) were obtained from Serotec (Oxford, U.K.), and PE-conjugated anti-CD25 (clone M-A251) was obtained from BD PharMingen. Secondary detection reagents (fluorescein-, PE-, and PECy5-conjugated streptavidin and PE-conjugated anti-mouse Ig) were obtained from Rockland Immunochemicals (Gilbertsville, PA). Where required, primary Abs were labeled with FITC (Sigma, Castle Hill, Australia) or aminohexanoyl-biotin-N-hydroxysuccinimide ester (Zymed Laboratories, South San Francisco, CA), both as previously described (21).
PBMC isolation and MLR
Mononuclear cells were prepared from healthy volunteers, and MLR cultures were set up as previously described (15).
CFSE labeling
PBMC were suspended to 2 x 107 cells/ml in PBS containing 0.1% human AB serum, and CFSE (Molecular Probes, Eugene, OR) was added to a final concentration of 10 µM. After incubation at 37°C for 10 min, the staining reaction was quenched by the addition of a large volume of complete medium for 5 min, followed by two washes in the same medium.
Immunofluorescence staining and flow cytometric analysis
Cells were suspended to 4 x 106 viable cells/ml in PBS containing 2% pooled human AB serum and 0.04% sodium azide (staining buffer). FcRs were blocked by incubation for 30 min at room temperature with 50 µg/million cells human IgG (Sigma), then 50 µl of blocked cells were aliquoted into round-bottom tubes. For most experiments, cells were mixed with saturating concentrations of the unlabeled primary Ab, incubated for 30 min at room temperature, and washed once with 3 ml of staining buffer before addition of biotin- or PE-conjugated anti-mouse detection Ab. Following a 30-min incubation on ice, cells were washed, and free binding sites on the detection Ab were blocked by incubation with 20 µg/tube mouse gammaglobulin (Rockland Immunochemicals) for 20 min at room temperature. Fluorescein- and PE-conjugated primary Abs as well as PECy5-conjugated streptavidin if required were then added and incubated for 30 min on ice. Cells were washed with 3 ml of staining buffer followed by 3 ml of serum-free staining buffer and fixed in 200 µl of paraformaldehyde (1% in PBS; BDH Laboratory Supplies, Poole, U.K.). A slightly different approach was adopted when using CXCR3 and CXCR4 Abs, which were directly biotinylated. Cells were mixed with all of the required primary Abs, incubated at room temperature for 30 min, washed, incubated with PECy5-conjugated streptavidin, then washed and fixed as described above. Labeled cells were analyzed on a FACScan and data were analyzed using CellQuest 3.1 software (BD Biosciences, Mountain View, CA). For all analyses, lymphocytes were gated using forward and side angle light scatter characteristics, and background staining observed using isotype-matched control Abs was subtracted from all percentages calculated. In some experiments ethidium monoazide staining (10 µl of a 50 µg/ml solution/million cells, incubated under fluorescent light for 15 min, followed by washing in staining buffer) was used to determine that viability within the lymphocyte gate was at least 98% (data not shown).
Detection of intracellular chemokine receptor protein by flow cytometry
Preparation of permeabilized and nonpermeabilized cells was conducted as previously described (15), and the treated cells were processed for flow cytometry as usual, except that cells were maintained in staining buffer containing 0.02% saponin to ensure that complete membrane permeabilization was maintained throughout.
Cytospin preparation and immunofluorescence microscopy
Cells were spun onto glass slides, and immunofluorescence staining was performed as previously described (15).
RNA extraction and RT-PCR
RNA purification from day 9 cell pellets and RT-PCR were conducted as previously described (15). The following primers were used in this study: GAPDH, 5'-TCCTTGGAGGCCATGTAGGCCAT-3' and 5'-TGATGACATCAAGAAGGTGGTGAAG-3'; CCR5, 5'-TGACATCTACCTGCTCAACC-3' and 5'-CCTGTGCCTCTTCTTCTCAT-3'; and CCR6, 5'-TCATCTGCCTTGTTGTGTGG-3' and 5'-TGTCGTTATCTGCGGTCTCA-3'. PCR products were resolved on 2% agarose gels, stained with SYBR-gold (Molecular Probes) and visualized/analyzed using a Molecular Imager FX (Bio-Rad, Hercules, CA). The band intensity values for each receptor were expressed as a ratio relative to band intensity for the GAPDH PCR product amplified from the same template.
Chemotaxis assay
Cells were suspended to 5 x 106
viable cells/ml in RPMI 1640 containing 0.5% BSA (RPMI-BSA). Synthetic
MIP-1/CCL4 and MIP-3/CCL20 (kind gifts of Prof. I. Clark-Lewis,
Biomedical Research Center, University of British Columbia, Vancouver,
Canada) were diluted to 200 ng/ml or 1 µg/ml, respectively, in
RPMI-BSA, and 600 µl was added to the lower chambers of a Transwell
plate (6.5-mm diameter filter, 5-µm pore size; Corning Glass,
Corning, NY). After adding 100 µl of cells to the upper chambers, the
assay was incubated for 3 h at 37°C, and cells were collected
from the lower chamber after extensive washing of the filter underside.
In most experiments the total number of cells in the lower chamber was
quantified by duplicate hemocytometer counts, and this value was
divided by the number of input cells to calculate the percent
migration. To correct for any variations in spontaneous migration, the
data are expressed as the migration index, calculated by dividing the
percentage of migration obtained in the presence of chemokine by the
percentage of migration for negative controls. For the dose-response
studies using MIP-3/CCL20, cells were first fluorescently labeled by
incubation with calcein (Molecular Probes; 40 nM final concentration in
RPMI-BSA) for 30 min at 37°C, followed by three washes in RPMI-BSA.
Labeled cells were used in Transwell assays as described above, and
then the cells in the lower chamber were quantified by transferring
them to a 96-well microtiter tray and measuring fluorescent emission on
the Molecular Imager FX. Following quantification, cells were washed in
staining buffer and incubated with biotin-conjugated anti-CD4 for
30 min on ice. After washing with 3 ml of staining buffer,
PE-conjugated streptavidin was added and incubated for 30 min on ice,
and the cells were washed, fixed, and analyzed by flow cytometry as
described above. The percentage of migration of
CD4+ T cells was calculated by multiplying each
fluorescence value by the percentage of cells positive for CD4 in that
sample, then inserting the values into the following formula:
(Y -
Ymin)/Ymax),
where Ymin is the value obtained in
the absence of chemokine, Ymax is the
value obtained for 100 µl of cells added directly to the lower
chamber of the Transwell, and Y is the relevant experimental
value.
Statistical analyses
Statistical analyses were performed using a two-tailed Student’s t test; p < 0.05 was considered significant. Data in the text are presented as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokine receptor expression on CD4+ T
cells was quantified by flow cytometry for allogeneic and syngeneic
(control) MLR cultures over a 12-day period (Fig. 1
). CD4+ T cells in
syngeneic cultures did not undergo any notable changes in CCR5
expression over the time course, although a modest, steady decline in
CCR6 expression was evident. In contrast, culture under allogeneic
conditions resulted in a marked increase in the percentage of
CD4+ T cells expressing CCR5 or CCR6 over the 9-
to 12-day period. The percentage of CD4+ T cells
expressing CCR5 was significantly greater in allogeneic compared with
syngeneic cultures at 9 days (p < 0.05), while
a significant difference in the percentage of
CD4+ T cells expressing CCR6 was detected at both
9 and 12 days (p < 0.01 and p
< 0.05, respectively). Of interest, CD8+ T cells
from the same cultures did not undergo any significant change in the
expression of either CCR5 or CCR6, even though CFSE studies indicated
that proliferation of CD8+ T cells did occur in
the MLR (data not shown).
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, C–F. It can
be seen that the CD4+ T cell population from
allogeneic cultures not only contains more CCR5+
and CCR6+ cells, but that the
level of expression of these receptors,
particularly CCR5, is also higher, as evidenced by a shift in
fluorescence intensity. Furthermore, we have previously shown that
CD4+ T cells activated in the MLR are
characterized by increased expression of CD4, and that the
CD4high phenotype correlates well with cellular
activation and division (15). Accordingly, these plots
demonstrate that the up-regulation of CCR5 and CCR6 is restricted to
the CD4high population. Expression of CCR5 and CCR6 is coordinated with the acquisition of an activated/memory phenotype
More detailed flow cytometric analysis at 9 days revealed that
CD4+ T cells expressing CCR5 or CCR6 were
generally characterized by an activated/memory phenotype (Fig. 2 and Table I).
CD4+ T cells that expressed CCR5 or CCR6 were
mostly positive for CD45RO and negative for CD45RA, a phenotype typical
of memory T cells. Furthermore, most of these
CCR5+ and CCR6+ cells also
coexpressed the activation marker CD25. Expression of an additional
activation marker, CD69, showed a less complete correlation with CCR5
and CCR6 expression (data not shown), possibly due to decreased
expression of this early activation marker at a relatively late time
point, such as day 9.
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To determine whether CCR5 and CCR6 were expressed on the same or
distinct subpopulations of activated CD4+ T
cells, cells from day 9 allogeneic MLR cultures were colabeled with Abs
to CD4, CCR5, and CCR6. As illustrated in Fig. 3, four distinct subpopulations could be
identified within the total CD4+ population on
the basis of CCR5 and CCR6 expression. The double-negative
(CCR5-CCR6-) population
was the largest (see total CD4+, R1 in
Fig. 3); however, by separating the total CD4+
population into resting (CD4normal, R2) and
activated (CD4high, R3) subpopulations, it was
observed that most of this double-negative population resided in the
CD4normal subpopulation, indicating that the
double-negative cells were mostly resting T cells that had not been
activated in the MLR. The activated (CD4high) T
cell population contained a small number of double-negative cells and
CCR6 single-positive cells and larger populations of CCR5
single-positive and double-positive
(CCR5+CCR6+) cells.
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To determine whether the increased expression of CCR5 and CCR6 was
due to increased levels of mRNA for the receptors, RT-PCR analysis was
performed on cell pellets collected from allogeneic and syngeneic
cultures after a 9-day incubation period. As illustrated in Fig. 4, the levels of mRNA for both CCR5 and
CCR6 were significantly higher in allogeneic compared with syngeneic
cultures (p < 0.05 for both), suggesting that
enhanced transcription of the CCR5 and CCR6 genes resulted in the
increased levels of surface protein detected by flow cytometry.
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, A and B). The
lack of intracellular CCR5 was confirmed by immunofluorescence
microscopy (Fig. 5C), which demonstrated that CCR5 staining
was primarily restricted to the cell surface despite the inclusion of a
permeabilization step before labeling. Because lymphocytes have only a
small amount of cytoplasm, it was possible that low level intracellular
staining may be mistaken for surface staining. However, the patterns of
CCR5 staining were virtually identical with those observed when
nonpermeabilized cells were labeled with anti-CD4 (data not shown),
suggesting that CCR5 is, in fact, localized exclusively on the cell
surface.
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50% of the
CD4+ T cells stained positively for CCR6
following permeabilization, suggesting that a subpopulation of cells
constitutively expresses CCR6 intracellularly in the absence of surface
expression, and that following alloactivation some, but not all, of
these cells begin expressing CCR6 on the surface. The intracellular
localization of CCR6 protein was confirmed by immunofluorescence
microscopy, as illustrated in Fig. 5C. In both syngeneic and
allogeneic cultures a punctate distribution of protein was evident
throughout the cell. Surprisingly, surface staining around the
circumference of the cells was not obvious. However, many of the
pockets of staining appear to be localized close to the cell surface,
suggesting that extracellular CCR6 protein may be arranged in distinct
foci on the cell membrane. In contrast to CXCR3 and CXCR4, the regulation of CCR5 and CCR6 expression is more dependent on time in culture than cell division status
Some programs of T cell gene expression, such as the up-regulation of cytokine genes, are intimately dependent on the initiation and progression of cell division that occur in response to TCR triggering and costimulation (22). To determine the relationship between the up-regulation of CCR5 and CCR6 and cell division, studies using the intracellular tracker dye CFSE were undertaken. Cells were labeled with the dye before culture, then analyzed by flow cytometry 6 or 9 days later. Cell division is associated with a progressive 2-fold loss in CFSE fluorescence intensity (23), thus enabling the expression patterns of chemokine receptors on CD4+ T cells that have undergone a defined number of cell divisions to be compared.
As shown in Fig. 6A, the
percentage of cells positive for CCR5 on day 6 increased progressively
over the first two rounds of cell division (the difference between
populations that had divided zero and two times was statistically
significant; p < 0.005), but then remained relatively
stable over subsequent division cycles. Even after seven or more rounds
of cell division, there were no further increases in CCR5 expression on
day 6. However, after another 3 days in culture, a striking
up-regulation of CCR5 was observed in the divided population
(p < 0.001 compared with the undivided
population; note that it was not possible to accurately discriminate
the number of cell division cycles at 9 days; accordingly, all divided
cells were considered as a single population). These observations
suggest that there are two distinct phases to CCR5 up-regulation: one
that occurs immediately following the initiation of cell division and
another that occurs many days after the T cell activation process is
initiated.
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B). However, by day 9 the
percentage of divided cells staining positively for CCR6 had increased
significantly (p < 0.005 compared with the
undivided population), indicating that CCR6 up-regulation is highly
dependent on the time that has elapsed since cellular activation and
occurs as a late activation event, temporally distinct from the cell
division process.
These results suggest that, unlike the regulation of the
cytokine-producing phenotype, the regulation of chemokine receptor
expression is partially (CCR5) or completely (CCR6) dependent on the
time that has elapsed since the initial TCR-triggering event. To
determine whether this phenomenon applied to the regulation of other
chemokine receptors, similar studies were performed using Abs to CXCR3
and CXCR4. Remarkably, the up-regulation of CXCR3 (Fig. 6C)
and the down-regulation of CXCR4 (Fig. 6D) occurred in close
parallel with the cell division process on day 6, and no further
changes in receptor expression were noted on day 9.
Allogeneic activation results in increased migration toward CCR5, but not CCR6, ligands
The most apparent reason for increased CCR5 and CCR6 expression on
activated T cells would be to enable migration along gradients of
inflammatory chemokines in peripheral tissues. To test whether the
up-regulation of CCR5 and CCR6 expression enabled activated T cells to
migrate more efficiently toward the relevant chemokine ligands, cells
from day 9 allogeneic and syngeneic cultures were subject to Transwell
chemotaxis assays. As shown in Fig. 7A, migration toward the CCR5
ligand MIP-1/CCL4 was virtually undetectable in syngeneic cultures,
but increased significantly following allogeneic activation
(p < 0.005). In marked contrast, cells from
allogeneic and syngeneic cultures displayed an almost identical
migratory response to the CCR6 ligand MIP-3/CCL20
(p > 0.05). To confirm this negative result
with respect to MIP-3/CCL20, more detailed chemotaxis studies were
undertaken. A highly sensitive, fluorescent-based assay was used to
test migration at a range of MIP-3/CCL20 concentrations, and the
cells were labeled with anti-CD4 before and after migration, such
that the specific response of CD4+ T cells could
be assessed in the absence of other potentially contaminating
lymphocyte populations (Fig. 7B). In agreement with the data
presented in Fig. 7A, these studies confirmed the lack of
enhanced migration toward MIP-3/CCL20 in allogeneic compared with
syngeneic cultures. At the lowest concentration of MIP-3/CCL20
tested, there appeared to be slightly enhanced chemotaxis in the
allogeneic population compared with that in the syngeneic controls, but
this difference was not statistically significant
(p > 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we have documented significant and consistent up-regulation of CCR5 and CCR6 during the allogeneic activation of primary T cells. It is likely that the activation of T cells by APC, as occurs in the MLR, provides T cells with various signals in addition to stimulation through the TCR that are required for the induction of novel patterns of chemokine receptor expression. If these signals are absent or inappropriate, as may be the case with lectin, anti-CD3, or anti-CD3 plus anti-CD28 stimulation, up-regulation will not be observed. In support of this concept, HIV infection studies have demonstrated that the infection of CD4+ T cells with R5 virus isolates is increased following activation by APC in vitro or in vivo, but is decreased following in vitro stimulation with anti-CD3 plus anti-CD28 (18). These findings suggest that the up-regulation of CCR5, which is required for the entry of R5 viruses, only occurs following T cell interaction with APC and not in response to APC-independent in vitro stimulation.
The signals required for up-regulation of CCR5 and CCR6 could include physiologically relevant costimulatory pathways and immunomodulatory cytokines. In this regard it has been demonstrated that costimulation through CD28 down-regulates CCR5 expression, whereas simultaneous costimulation through a combination of CD28 and CTLA-4 results in CCR5 up-regulation (18). Cytokines also clearly play a critical role in the regulation of CCR5 and CCR6 expression. Although T cell activation using anti-CD3 alone does not have a significant effect on CCR5 or CCR6 expression (9, 14), the combination of anti-CD3 plus IL-2 induces a dramatic up-regulation of CCR5 expression levels (11, 13, 24). Moreover, IL-2 alone is sufficient to up-regulate the expression of CCR5 (11, 12, 17, 24), although the ability of IL-2 to up-regulate CCR6 is unclear (8, 9, 17). Thus, it is possible that the lack of up-regulation of CCR5, and possibly CCR6, in response to anti-CD3 treatment is due to insufficient production of IL-2 in the cultures. Notably, IL-15, a cytokine with properties similar to those of IL-2, has also been shown to up-regulate CCR5 and CCR6 expression (17, 25).
In the present study, the expression of CCR5 and CCR6 on CD4+ T cells activated in the MLR showed a good correlation with the acquisition of an activated/memory phenotype. However, the inverse was not necessarily true, in that a proportion of activated/memory cells remained negative for CCR5 and, in particular, CCR6. Colabeling activated CD4+ T cells for both CCR5 and CCR6 confirmed that CCR5 was expressed on a greater percentage of cells and revealed that three distinct subpopulations could be identified on the basis of CCR5 and CCR6 expression: a small population of CCR6+CCR5- cells and much larger populations of CCR5+CCR6- and CCR5+CCR6+ cells. Interestingly, another inflammatory chemokine receptor, CXCR6, shows almost perfect coexpression with CCR5, but only around half of the CXCR6+ T cells coexpressed CCR6 (17), providing further evidence that the expression of CCR6 in the memory population is more restricted than that of other inflammatory chemokine receptors. It is not clear at this stage whether the groups of activated T cells defined by CCR5 and CCR6 expression represent functionally distinct subpopulations or simply reflect the heterogeneity of the T cell response. Future studies aimed at isolating each subpopulation by cell sorting may allow the populations to be compared in terms of, for example, their trafficking properties in vivo or their cytokine-producing phenotype.
In keeping with the increased surface expression of CCR5 and CCR6, the
level of mRNA for each receptor was higher in allogeneic compared with
syngeneic cultures. However, intracellular stores of CCR6, but not
CCR5, were detected in CD4+ T cells, suggesting
the existence of more complex regulatory mechanisms. Thus, whether the
increased surface expression of CCR6 is due to translocation of
preformed receptor protein from the cytoplasm or to enhanced de novo
protein production resulting from increased transcription is not clear
at this stage. However, it is interesting to note that only 50% of
CD4+ T cells expressed CCR6 intracellularly, and
that this was not altered upon allogeneic activation. Therefore, a
possible scenario is that only a proportion of
CD4+ T cells are able to produce CCR6, and that
these cells do so constitutively and store the protein intracellularly.
Upon activation, transcription of the CCR6 gene is increased,
saturating the mechanisms that maintain CCR6 inside the cell and
enabling expression on the cell surface. This increased level of
transcription would presumably be maintained as the cells return to a
resting state, thereby enabling memory T cells to continue expressing
the receptor on the surface. This scenario is in keeping with the
observation that only 50% of the activated
CD4+ T cells that up-regulated CCR5 also
up-regulated CCR6.
The time-course studies presented in Fig. 1 suggested that the
up-regulation of CCR5 and CCR6 expression was a late event, peaking
around days 9–12. This was confirmed by the CFSE studies. On day 6,
up-regulation of these receptors was either minimal (CCR5) or
undetectable (CCR6) regardless of the number of rounds of division the
cells had undergone. In contrast, a striking up-regulation of both CCR5
and CCR6 was detectable on the divided population on day 9, a time at
which cellular proliferation is markedly reduced (15).
This observation suggests that the up-regulation of CCR6 and, to a
lesser extent, CCR5 is only initiated after a defined period of time
has elapsed since activation and initiation of cell division. Despite
the temporal distinction, however, cell division is clearly a
prerequisite for up-regulated expression of CCR5 and CCR6, as the
undivided population did not alter the expression of these
receptors.
Delaying chemokine receptor up-regulation until after T cells have completed the cell division process may be a mechanism to prevent activated T cells leaving the secondary lymphoid tissue until they have undergone proliferation and differentiation in an appropriate microenvironment. However, such a mechanism is clearly not a universal theme in chemokine biology, as both CXCR3 and CXCR4 were subject to changes in expression that began immediately upon initiation of division and progressed in coordination with subsequent divisions. Additionally, there was a small increase in the expression of CCR5, but not CCR6, after the first two rounds of cell division on day 6. It is possible that chemokine receptors that are modulated immediately upon cellular activation play a role distinct from those that are modulated several days after initiation of cell division. One possibility is that these early receptors are required for altered microenvironmental positioning of dividing T cells within the secondary lymphoid tissue, for example, to provide B cell help. Of relevance, the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 are expressed in the lymph nodes of rats during experimental autoimmune encephalomyelitis,4 and ligands for CCR5 have also been identified in the draining lymph node during elicitation of contact hypersensitivity (26). The expression of these chemokines and others (27) in lymph nodes under inflammatory conditions suggests a functional role within secondary lymphoid tissue.
The most apparent reason for increased expression of CCR5 and CCR6 on
activated T cells is to enable these cells to begin sensing
inflammatory chemokine gradients produced in peripheral tissues in
response to infection and thereby move specifically to the appropriate
site. In keeping with this hypothesis, allogeneic activation resulted
in activated T cells being able to respond chemotactically to the
CCR5-specific ligand MIP-1/CCL4. In contrast,
CD4+ T cells from syngeneic cultures were already
able to respond to MIP-3/CCL20, presumably due to the expression of
CCR6 on a reasonable proportion of T cells in the starting population,
and allogeneic activation did not increase the chemotactic response
further.
The lack of increased migration toward MIP-3/CCL20 following
allogeneic activation suggests that the up-regulation of CCR6 plays an
alternative function. While a number of possibilities exist, integrin
activation is a likely candidate. MIP-3/CCL20 induces the adhesion
of memory T cells to ICAM (28), and moreover, a recent
study by Fitzhugh et al. (29) suggests that the
interaction between MIP-3/CCL20 and CCR6 is a critical factor in the
ability of memory T cells to firmly adhere to endothelial cells, a
prerequisite for extravasation. Alternatively, it is conceivable that
signaling through MIP-3 is required for the induction of alternative
patterns of protein synthesis that are required for activated T cell
function, such as the initiation of distinct cytokine secretion
pathways or the expression of altered cell surface molecules. Finally,
the possibility remains that the purpose of CCR6 up-regulation is to
mediate chemotaxis toward MIP-3, but that a further signal, such as
that provided by a cytokine, is required to couple the newly acquired
receptor to the appropriate intracellular signaling cascades. In this
context it is interesting to note that while CCR6 is expressed at
readily detectable levels by freshly isolated B cells, no chemotactic
or calcium flux response to MIP-3 is observed (9).
Together, the present study and our previous study of chemokine
receptor expression in the MLR (15) demonstrate that four
chemokine receptors are regulated on CD4+ T cells
in response to allogeneic activation. However, there are notable
differences in the kinetics and functional outcome of regulation, the
proportion of activated cells that modulate the receptors, and the
molecular mechanisms by which changes in surface expression are
achieved. Based on the proportion of divided cells expressing each
receptor, there appears to be a hierarchy in the extent of receptor
regulation, with CXCR3 and CXCR4 being modulated on the majority of
divided cells (81 and 91% respectively), CCR5 on 65% of the
divided population, and CCR6 on 30% of divided cells. Two of the
receptors, CXCR3 and CXCR4, are modulated in close parallel with cell
division, while in the case of CCR5 and CCR6, division and receptor
regulation are temporally distinct. The up-regulation of CCR5, CCR6,
and CXCR3 is associated with an increase in mRNA levels, while, in
contrast, the down-regulation of CXCR4 protein is not mirrored by a
corresponding decrease in CXCR4 transcripts. CXCR3 and CCR6, but not
the other two receptors, are stored intracellularly, although even here
there are differences; the percentage of CD4+ T
cells expressing intracellular CXCR3 is nearly 100%, while only around
half that express CCR6 intracellularly. Finally, while the regulation
of CCR5, CXCR3, and CXCR4 resulted in altered chemotactic
responsiveness toward the respective ligands, the up-regulation of CCR6
did not result in enhanced migration toward MIP-3/CCL20. Thus,
although activated T cells express a number of different chemokine
receptors, the variability observed with regard to their expression and
function suggests that each may play a unique biological role and its
expression be fine-tuned by distinct mechanisms.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Shaun R. McColl, Chemokine Biology Laboratory, Department of Molecular Biosciences, Adelaide University, Adelaide 5005, Australia. E-mail: shaun.mccoll{at}adelaide.edu.au
3 Abbreviations used in this paper: DC, dendritic cell; MIP, macrophage-inflammatory protein.
4 S. R. McColl, S. Mahalingham, M. Staykova, L. A. Tylaska, K. E. Fisher, C. A. Strick, R. P. Gladue, K. S. Neote, and D. O. Willenborg. Cloning and characterization of rat I-TAC/CXCL11 and other CXCR3 ligands in experimental autoimmune encephalomyelitis in the rat. Submitted for publication.
Received for publication August 6, 2001. Accepted for publication November 1, 2001.
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References |
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, and MIP-1. J. Biol. Chem. 271:17161. and is highly expressed in human dendritic cells. J. Exp. Med. 186:837.. J. Immunol. 162:186./CCL20 and CC chemokine receptor 6 in psoriasis. J. Immunol. 164:6621. and IP-10 are expressed in demyelinating brain lesions. Proc. Natl. Acad. Sci. USA 96:6873. and MIP-1. J. Immunol. 161:5663.This article has been cited by other articles:
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  A. Liston, R. E. Kohler, S. Townley, S. Haylock-Jacobs, I. Comerford, A. C. Caon, J. Webster, J. M. Harrison, J. Swann, I. Clark-Lewis, et al. Inhibition of CCR6 Function Reduces the Severity of Experimental Autoimmune Encephalomyelitis via Effects on the Priming Phase of the Immune Response J. Immunol., March 1, 2009; 182(5): 3121 - 3130. [Abstract] [Full Text] [PDF]
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J. F. Camargo, M. P. Quinones, S. Mummidi, S. Srinivas, A. A. Gaitan, K. Begum, F. Jimenez, S. VanCompernolle, D. Unutmaz, S. S. Ahuja, et al. CCR5 Expression Levels Influence NFAT Translocation, IL-2 Production, and Subsequent Signaling Events during T Lymphocyte Activation J. Immunol., January 1, 2009; 182(1): 171 - 182. [Abstract] [Full Text] [PDF]
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 H. J. P. M. Koenen, R. L. Smeets, P. M. Vink, E. van Rijssen, A. M. H. Boots, and I. Joosten Human CD25highFoxp3pos regulatory T cells differentiate into IL-17-producing cells Blood, September 15, 2008; 112(6): 2340 - 2352. [Abstract] [Full Text] [PDF]
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 K. G. McDonald, J. S. McDonough, C. Wang, T. Kucharzik, I. R. Williams, and R. D. Newberry CC Chemokine Receptor 6 Expression by B Lymphocytes Is Essential for the Development of Isolated Lymphoid Follicles Am. J. Pathol., April 1, 2007; 170(4): 1229 - 1240. [Abstract] [Full Text] [PDF]
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 J. H. Lee, H. J. Kang, J.-S. Woo, S. W. Chae, S. H. Lee, S. J. Hwang, and H.-M. Lee Up-regulation of Chemokine Ligand 20 in Chronic Rhinosinusitis. Arch Otolaryngol Head Neck Surg, May 1, 2006; 132(5): 537 - 541. [Abstract] [Full Text] [PDF]
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 P. Ghadjar, S. E. Coupland, I.-K. Na, M. Noutsias, A. Letsch, A. Stroux, S. Bauer, H. J. Buhr, E. Thiel, C. Scheibenbogen, et al. Chemokine Receptor CCR6 Expression Level and Liver Metastases in Colorectal Cancer J. Clin. Oncol., April 20, 2006; 24(12): 1910 - 1916. [Abstract] [Full Text] [PDF]
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 J. D. Holland, M. Kochetkova, C. Akekawatchai, M. Dottore, A. Lopez, and S. R. McColl Differential functional activation of chemokine receptor CXCR4 is mediated by g proteins in breast cancer cells. Cancer Res., April 15, 2006; 66(8): 4117 - 4124. [Abstract] [Full Text] [PDF]
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Y. Hayakawa and M. J. Smyth CD27 Dissects Mature NK Cells into Two Subsets with Distinct Responsiveness and Migratory Capacity J. Immunol., February 1, 2006; 176(3): 1517 - 1524. [Abstract] [Full Text] [PDF]
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G. F. Debes, M. E. Dahl, A. J. Mahiny, K. Bonhagen, D. J. Campbell, K. Siegmund, K. J. Erb, D. B. Lewis, T. Kamradt, and A. Hamann Chemotactic Responses of IL-4-, IL-10-, and IFN-{gamma}-Producing CD4+ T Cells Depend on Tissue Origin and Microbial Stimulus J. Immunol., January 1, 2006; 176(1): 557 - 566. [Abstract] [Full Text] [PDF]
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R. Varona, V. Cadenas, L. Gomez, C. Martinez-A, and G. Marquez CCR6 regulates CD4+ T-cell-mediated acute graft-versus-host disease responses Blood, July 1, 2005; 106(1): 18 - 26. [Abstract] [Full Text] [PDF]
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H. W. van Deventer, W. O'Connor Jr., W. J. Brickey, R. M. Aris, J. P.Y. Ting, and J. S. Serody C-C Chemokine Receptor 5 on Stromal Cells Promotes Pulmonary Metastasis Cancer Res., April 15, 2005; 65(8): 3374 - 3379. [Abstract] [Full Text] [PDF]
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K. A. Latham, K. B. Whittington, R. Zhou, Z. Qian, and E. F. Rosloniec Ex Vivo Characterization of the Autoimmune T Cell Response in the HLA-DR1 Mouse Model of Collagen-Induced Arthritis Reveals Long-Term Activation of Type II Collagen-Specific Cells and Their Presence in Arthritic Joints J. Immunol., April 1, 2005; 174(7): 3978 - 3985. [Abstract] [Full Text] [PDF]
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M. Kleinewietfeld, F. Puentes, G. Borsellino, L. Battistini, O. Rotzschke, and K. Falk CCR6 expression defines regulatory effector/memory-like cells within the CD25+CD4+ T-cell subset Blood, April 1, 2005; 105(7): 2877 - 2886. [Abstract] [Full Text] [PDF]
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S. K. Lundy, S. A. Lira, J. J. Smit, D. N. Cook, A. A. Berlin, and N. W. Lukacs Attenuation of Allergen-Induced Responses in CCR6-/- Mice Is Dependent upon Altered Pulmonary T Lymphocyte Activation J. Immunol., February 15, 2005; 174(4): 2054 - 2060. [Abstract] [Full Text] [PDF]
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B. Bahbouhi, A. Landay, and L. Al-Harthi Dynamics of cytokine expression in HIV productively infected primary CD4+ T cells Blood, June 15, 2004; 103(12): 4581 - 4587. [Abstract] [Full Text] [PDF]
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I. Louis, G. Dulude, S. Corneau, S. Brochu, C. Boileau, C. Meunier, C. Cote, N. Labrecque, and C. Perreault Changes in the lymph node microenvironment induced by oncostatin M Blood, August 15, 2003; 102(4): 1397 - 1404. [Abstract] [Full Text] [PDF]
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N. W. Lukacs, A. L. Miller, and C. M. Hogaboam Chemokine Receptors in Asthma: Searching for the Correct Immune Targets J. Immunol., July 1, 2003; 171(1): 11 - 15. [Full Text] [PDF]
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R. E. Kohler, A. C. Caon, D. O. Willenborg, I. Clark-Lewis, and S. R. McColl A Role for Macrophage Inflammatory Protein-3{alpha}/CC Chemokine Ligand 20 in Immune Priming During T Cell-Mediated Inflammation of the Central Nervous System J. Immunol., June 15, 2003; 170(12): 6298 - 6306. [Abstract] [Full Text] [PDF]
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 J. Reibman, Y. Hsu, L. C. Chen, B. Bleck, and T. Gordon Airway Epithelial Cells Release MIP-3{alpha}/CCL20 in Response to Cytokines and Ambient Particulate Matter Am. J. Respir. Cell Mol. Biol., June 1, 2003; 28(6): 648 - 654. [Abstract] [Full Text] [PDF]
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A. Ito, A. Matejuk, C. Hopke, H. Drought, J. Dwyer, A. Zamora, S. Subramanian, A. A. Vandenbark, and H. Offner Transfer of Severe Experimental Autoimmune Encephalomyelitis by IL-12- and IL-18-Potentiated T Cells Is Estrogen Sensitive J. Immunol., May 1, 2003; 170(9): 4802 - 4809. [Abstract] [Full Text] [PDF]
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R. Mo, J. Chen, Y. Han, C. Bueno-Cannizares, D. E. Misek, P. A. Lescure, S. Hanash, and R. L. Yung T Cell Chemokine Receptor Expression in Aging J. Immunol., January 15, 2003; 170(2): 895 - 904. [Abstract] [Full Text] [PDF]
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A. Glatzel, D. Wesch, F. Schiemann, E. Brandt, O. Janssen, and D. Kabelitz Patterns of Chemokine Receptor Expression on Peripheral Blood {gamma}{delta} T Lymphocytes: Strong Expression of CCR5 Is a Selective Feature of V{delta}2/V{gamma}9 {gamma}{delta} T Cells J. Immunol., May 15, 2002; 168(10): 4920 - 4929. [Abstract] [Full Text] [PDF]
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