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*
Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway; and
Tanabe Research Laboratories, San Diego, CA 92121
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), CC (ß), CXC (
), and
CX3C (
). In addition, about 10 CC, five CXC,
one C, and one CX3C chemokine receptors have been
cloned (reviewed in Refs. 2, 3). The CC chemokine
receptor 4 (CCR4)3 was
found to bind thymus and activation-regulated chemokine (TARC)
(4) as well as macrophage-derived chemokine (MDC)
(5). On the other hand, CCR8 was first cloned in T cells
and in the NK 3.3 cell line and was given the name TER-1
(6). Later work showed that this receptor binds the CC
chemokine I-309, and it was renamed CCR8 (7, 8). The anti-viral/anti-tumor effector NK cells respond to various chemokines (9). Among the recently described chemokines, MDC is chemotactic for NK cells (10), suggesting that they express receptors for this chemokine. However, such expression has not been previously shown. In contrast, CCR4 is expressed in T cells and in particular in the Th2 subtype (11). Similarly, CCR8 is preferentially expressed in Th2 cells (12).
Chemokines play major roles in combating HIV-1 infection. The CC
chemokines macrophage inflammtory protein (MIP)-1, MIP-1ß, and
RANTES inhibit the replication of the M-tropic HIV-1 strains
(13), whereas the CXC chemokine stromal-derived factor
(SDF)-1 inhibits the replication of the T-tropic HIV-1 strains
(14, 15). Recently, I-309 was reported to inhibit the
replication of several HIV-1 strains (16). Also, the
truncated form of MDC inhibits the replication of both HIV-1 strains
(17, 18). One possible mechanism for this inhibition is
the ability of chemokines to activate NK cells (reviewed in Ref.
9). The present work attempts to examine the expression of
chemokine receptors (in particular CCR4 and CCR8 in NK cells) and to
investigate the biological significance of these receptors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Culture medium consisted of RPMI 1640 supplemented with 10% human AB serum (Ullevål Hospital, Oslo, Norway), 10 U/ml penicillin, 100 µg/ml streptomycin, 1 mM L-glutamine, 1% nonessential amino acids (all from Life Technologies, Paisley, U.K.), and 5 x 10-5 M 2-ME (Sigma, St. Louis, MO). Ionomycin was from Biomol (SMS Norway, Olso, Norway).
Preparation of NK cells and their membranes
IL-2-activated NK (IANK) cells were prepared by adherence to plastic flasks for 10 days (in the presence of IL-2) of nylon-wool column nonadherent cells generated from buffy coats of human volunteers (Ullevål Hospital), as previously described (19). Depending on the donor, the plastic adherent cells contained between 17 and 35% CD3+ T cells. Hence, it was necessary to deplete contaminating T cells. This was accomplished by binding these cells twice to M-450 CD3-coupled beads (Dynal, Oslo, Norway). This procedure resulted in more than 95% of the cells expressing the CD56 cell surface marker and less than 5% of the cells expressing the CD3 cell surface marker. IANK cell membranes were prepared in a buffer containing 25 mM Tris, 50 mM NaCl (pH 7.5), 40 µg/ml PMSF, 10 µg/ml leupeptin, 2 µg/ml pepstatin A, and 2 µg/ml aprotinin. After homogenization and sonication, they were centrifuged at 1000 x g for 10 min, and the supernatants were transferred into Beckman tubes and ultracentrifuged at 150,000 x g for 45 min at 4°C. The membranes were transferred into a buffer containing 1% Brij, 25 mM Tris, and 150 mM NaCl.
Pretreatment with pertussis toxin (PT)
PT (Calbiochem-Novabiochem, La Jolla, CA) was activated with 20 mM DTT and 20 mM HEPES for 10 min at 37°C. IANK cells (1 x 106) were either left intact or treated with 2, 20, or 200 ng/ml of activated PT in a final concentration of 10 ml per flask and were incubated for 18 h at 37°C. The cells were collected and washed three times with complete medium. Only preparations that contained more than 95% viable cells were used in the assays described here.
Chemotaxis assay
This procedure has been described in detail (19, 20). In brief, blind-well chemotaxis chambers with a lower-well volume of 200 µl were used. A maximum volume of 200 µl of RPMI medium containing 1% BSA was placed in the lower wells in the presence or absence of various chemokines (MDC, TARC, or I-309; R&D Systems, Abingdon, Oxon, U.K.). Cells (4 x 105) were placed in the upper compartments of Boyden chambers above the filters. The chambers were incubated for 2 h at 37°C in a 5% CO2 incubator. The filters were then removed, dehydrated, and stained with 15% Giemsa stain for 7 min and then mounted on glass slides. Cells in 10 high-power fields from two filters were counted and averaged for each sample. Migration index was calculated as the number of cells migrating toward the concentration gradient of chemokines divided by the number of cells migrating toward medium only.
Measurement of intracellular Ca2+ concentration ([Ca2+]i)
IANK cells were incubated in a buffer containing 145 mM NaCl, 5 mM KCl, 10 mM Na/MOPS, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, 0.25% BSA, 10 mM HEPES (pH 7.4), and 2.5 µM fura-2-acetoxymethyl ester (fura-2-AM) (Calbiochem, La Jolla, CA) for 45 min at 37°C. The cells were washed and resuspended at a concentration of 1 x 105 cells/ml and then incubated with the appropriate concentration of chemokines. A dual excitation luminescence spectrometer system (LS 50, Perkin-Elmer, Buckinghamshire, U.K.) containing 340- and 380-nm band pass filters was used. The emission was determined at 510 nm, and the intensity was measured by using a photomultiplier tube system. The concentration of [Ca2+]i was calculated according to the following formula: Kd (F - Fmin/Fmax - F), where Kd for fura 2 is 225 nM. F, Fmax, and Fmin were determined by measuring the experimental fluorescence, fluorescence in the presence of 10% Triton X, and fluorescence in the presence of 4 mM EGTA plus 10% Triton X, respectively. In experiments in which PT was used, the assays were done in the absence of extracellular calcium to monitor the release of calcium from intracellular stores. In these assays, the fluorescence ratio of bound/free fura 2 was measured.
Immunoblot assay
This was done as described (21). IANK cell
membranes (100 µg) were suspended in 0.5 ml of 1% Brij diluted in 40
mM Tris-HCl, 150 mM NaCl (pH 7.5), and proteolytic enzyme inhibitors
and were incubated with 0.5 µg of goat anti-human CCR4 (Research
Diagnostics, Flanders, NJ) overnight at 4°C with gentle rotation.
Goat IgG (Sigma) was used as a control. These samples were incubated
with 25 µl protein A/G agarose beads for 2 h at 4°C, washed
with 0.1% Brij, and resuspended in 10 µl SDS sample buffer. They
were run on SDS/PAGE, transferred into polyvinylidene difluoride
membranes (Millipore, Bedford, MA), and blocked with 5% BSA for 1
h at room temperature. The membranes were then incubated with 0.5
µg/ml anti-CCR4 for 2 h, washed three times in TBS
containing 0.05% Tweed 20, and incubated with 1:5000 dilution of the
secondary Abs. Development was done by enhanced chemiluminescence
reagents (Pierce, Rockford, IL). For the endocytosis experiments, IANK
cells (4 x 107/ml) were incubated with 50
ng/ml of the particular chemokine (MDC, TARC, or SDF-1; all from R&D
Systems) for 25 min at 37°C before the preparation of membranes from
these cells.
Flow cytometric analysis
For the detection of CCR4 or CCR8, IANK cells were permeabilized first, before introduction of Abs, which detect the carboxy terminal of these receptors. For this purpose, 1 x 106/ml cells were fixed with 4% paraformaldehyde for 1 h at 4°C. They were washed twice in PBS and then incubated (5 x 105) with 150 µl of 1% saponin in PBS for 15 min at room temperature. The samples were centrifuged and resuspended in PBS containing 0.1% saponin and 0.1% FCS. These cells were incubated at 4°C for 45 min with 1 µg goat anti-human CCR4 (Research Diagnostics), goat anti-human CCR8 (made by B.D.), or as a control goat IgG. The cells were washed three times in a buffer containing 0.1% saponin and then incubated with 1:100 dilution of F(ab')2 FITC-conjugated rabbit anti-goat (Southern Biotechnology, Birmingham, AL) for 45 min at 4°C. They were washed three times and then examined in FACScan (Becton Dickinson, Mountain View, CA). In the internalization experiments, the cells (4 x 106/100 µl) were incubated with 5 ng/100 µl MDC for 0–60 min at 37°C. These cells were extensively washed and then examined for the presence of CCR4 as described above.
Phosphorylation of CCR4
IANK cells (4 x 107/ml) were incubated with 50 ng/ml MDC for certain amounts of time (ranging between 0 and 5 min). The reactions were stopped by adding 500 µl ice-cold 2% Brij and were incubated on ice for 30 min. Lysates were collected from these cells and were immunoprecipitated overnight with 500 ng/ml anti-CCR4 or as a control with goat IgG. Protein A/G agarose beads were then added, and the mixtures were incubated for an additional 4 h. They were run on 7.5% SDS-PAGE and were blocked with 5% BSA. The samples were immunoblotted overnight with a 1:50 dilution of mouse anti-phosphoserine/phosphothreonine (Biomedia, Foster City, CA) in 5% BSA. Goat anti-mouse IgG diluted 1:5000 in Tween 20 + TBS was used as a secondary Ab. The same membranes were stripped and reimmunoblotted with 500 ng/ml goat anti-CCR4 and rabbit anti-goat IgG as a secondary Ab.
For the association of G protein-coupled receptor kinases (GRKs) with CCR4 in IANK cell membranes, these cells were either left intact or were stimulated with MDC (as above). Membranes were prepared from these cells and were immunoprecipitated with anti-CCR4. These membranes were run on SDS-PAGE and then immunoblotted with a 1:100 dilution of rabbit anti-human GRK2 or rabbit anti-human GRK3 (Santa Cruz Biotechnology, Santa Cruz, CA). Goat anti-rabbit (Bio-Rad, Hercules, CA) diluted at 1:5000 was used as a secondary Ab.
Analysis of CCR4 and CCR8 expression by multiprobe RNase protection assay (RPA)
Total RNA was prepared by guanidium thiocyanate/cesium chloride
gradients following standard protocols. The specific mRNA for CCR4 and
CCR8 was detected by the human cytokine receptor set 5 (hCR5)
multiprobe template set (RiboQuant; PharMingen, San Diego, CA), which
contains templates for CCR1, CCR2, CCR3, CCR4, CCR5, and CCR8. In
brief, antisense RNA probes were generated from DNA templates using T7
DNA-dependent RNA polymerase in the presence of
[-32P]UTP (specific activity, 10 µCi/µl;
Amersham Pharmacia Biotech, Uppsala, Sweden). Labeled probes were
hybridized with total RNA (10 µg) overnight at 56°C. Unhybridized
RNA was digested with RNase according to PharMingen’s supplied
procedures. RNase-protected probes were resolved on denaturing 5%
polyacrylamide gels. The gels were dried and exposed to film
(BIOMAX MS; Eastman Kodak, Rochester, NY) at -70°C for
3 h.
Statistical analysis
Significant values were determined by using the two-tailed Student t test.
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Results |
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The expression of CCR4 and CCR8 at the mRNA level was examined
utilizing the RPA. hCR5, which has templates for CCR4 and CCR8 among
other chemokine receptors including CCR1, CCR2, CCR3, and CCR5, was
utilized. Results in Fig. 1
show that
IANK cells contain mRNA for CCR4, CCR8, and CCR2 (lane
3). The expression of CCR1 and CCR3 was variable in different
donors and is not shown in this figure. Surprisingly, CCR5 was absent
in IANK cells. Receptors other than CCR4 and CCR8 have not been fully
examined in this report. Yeast tRNA was included as a negative control
and is shown in lane 2 of Fig. 1.
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Fig. 2a shows that IANK
cells express CCR4 and CCR8 as detected by intracellular staining by
Abs that bind the carboxy terminal of these receptors. Because the
amount of anti-CCR8 in our possession was very scarce, we examined
the expression of only CCR4 by other assays such as immunoblotting.
Results in Fig. 2b show that CCR4 is detected by this method
(lane 1). Pretreatment of 4 x
107/ml IANK cells with 50 ng/ml MDC or TARC for
25 min at 37°C significantly reduced the expression of this receptor
(Fig. 2b, lanes 2 and 3,
respectively), suggesting that CCR4 is internalized after pretreatment
with these ligands. SDF-1, which does not bind CCR4, did not induce
the endocytosis of CCR4 (Fig. 2b, lane 4).
To examine in detail the kinetics of CCR4 endocytosis, we pretreated
IANK cells with MDC for various periods of time and examined the
presence of this receptor. Fig. 2c shows that 5 min after
stimulation, this receptor expression was low, with maximal
internalization occurring between 15 and 45 min. Interestingly, this
receptor started to reappear in the cell membranes 60 min after
stimulation. This result suggests that the mechanism of disappearance
of this receptor is due to its internalization and not to other
mechanisms such as impediment of its recognition by the Ab due to
ligand binding.
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Phosphorylation of chemokine receptors is the first step in the
process of endocytosis (reviewed in Ref. 3). Hence, we
examined whether CCR4 is phosphorylated as a result of stimulation with
MDC. Fig. 3 shows that pretreatment of
IANK cells with MDC for 0.5 min resulted in the phosphorylation of CCR4
(upper panel, lane 2) compared with unstimulated
cells (upper panel, lane 1), as detected by Ab to
phosphorylated serine/threonine residues. This phosphorylation was
maximal by 1 min (upper panel, lane 3). It was
also observed after 2 min but disappeared after 5 min of stimulation
with MDC (upper panel, lanes 4 and 5,
respectively). Immunoprecipitating NK cell membranes with goat IgG did
not result in detecting the phosphorylated CCR4. Stripping the
membranes and reimmunoblotting them with anti-CCR4 showed that the
same amount of CCR4 is present in all samples (Fig. 3, lower
panel).
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shows that these
enzymes are not present in IANK cell membranes. However, 0.5 min after
stimulation with MDC, an association between CCR4 and GRK2 (Fig. 4a) but not GRK3 (Fig. 4b) occurred in these
membranes. In contrast, after 1 min of stimulation, both GRK2 and GRK3
were associated with this receptor. Whereas GRK2 dissociated from the
receptor 2 min after incubation with MDC, GRK3 was still associated
with CCR4 at this time point. A similar amount of CCR4 was observed in
these membranes upon stripping and reimmunoblotting with anti-CCR4
(Fig. 4c).
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To investigate the biological significance of the presence of CCR4
and CCR8, we examined the ability of IANK cells to respond to the
ligands recognizing CCR4 and CCR8. Fig. 5
shows that MDC, TARC, and I-309 induced a typical bell-shaped
chemotactic response. As low as 1 pg/ml of TARC induced the chemotaxis
of these cells (p < 0.0001 compared with cells
migrating in the absence of chemokines), whereas 100 pg/ml is the
minimum concentration of MDC and I-309 required to induce the
chemotaxis of IANK cells (p < 0.01 and
p < 0.001, respectively, compared with cells migrating
in the presence of medium only). No significant migration was observed
when chemokines were placed in the upper wells’ "negative
gradients" or in the upper and lower wells’ "no gradients" (data
not shown). Hence, the effect of these chemokines is a chemotactic
rather than a chemokinetic response.
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show
that 2 ng/ml PT did not inhibit the chemotactic response induced by
these chemokines. However, as low as 20 ng/ml inhibited TARC- or
I-309-induced IANK cell chemotaxis (p < 0.004
and p < 0.005, respectively). A dose of 200 ng/ml PT
inhibited MDC, TARC, and I-309-induced chemotaxis
(p < 0.02, p < 0.001, and
p < 0.001, respectively), suggesting that the
chemotactic response induced by these three chemokines is mediated by
PT-sensitive G proteins. MDC, TARC, and I-309 induce the mobilization of [Ca2+]i in IANK cells
Because the pattern of calcium signaling in activated cells
closely correlated to the pattern of receptor expression
(23), we examined the ability of these chemokines to
induce the mobilization of intracellular calcium. Fig. 6 shows that MDC induced a potent calcium
flux in IANK cells, an activity that was desensitized by the addition
of MDC. Similarly, TARC induced a robust calcium flux in these cells,
an activity that was desensitized by the addition of TARC.
Surprisingly, MDC only partially inhibited the calcium flux
response induced by TARC. The concentration of MDC utilized (20 ng/ml)
was enough to desensitize CCR4, as shown in Fig. 6. In contrast,
TARC desensitized the calcium flux response induced by MDC. These
results suggest that TARC in addition to CCR4 may utilize other
receptors. Recent work has shown that in addition to CCR4, TARC
utilizes CCR8 (24). Results in Fig. 6 show that I-309, the
ligand for CCR8, induced a robust calcium flux response in IANK cells.
I-309 only partially inhibited the calcium flux induced by TARC,
whereas TARC completely desensitized the calcium response induced by
I-309. There was no cross-desensitization of the calcium flux response
among MDC and I-309. These results suggest that in IANK cells, TARC
shares the receptors utilized by MDC (CCR4) and I-309 (CCR8). However,
MDC and I-309 do not share the same receptor. Because all three
chemokines induced only a transient calcium flux in IANK cells, we
included ionomycin (ION) as a control. This ionophore induced a
sustained calcium flux in these cells in the presence of extracellular
calcium, suggesting that it facilitates the influx of calcium across
the plasma membranes.
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A), a normal response to this
ionophore when incubated with cells in nominally
Ca2+-free medium (25). Our results
indicate that this ionophore can also induce the release of calcium
from intracellular stores in NK cells. More importantly, PT did not
inhibit the calcium response induced by this ionophore (Fig. 7B). The calcium mobilization effect of MDC and TARC was
completely inhibited upon pretreatment of IANK cells with PT (Fig. 7, C–F). Surprisingly, the calcium flux induced by
I-309 was not affected by prior treatment of these cells with PT (Fig. 7, G and H).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, MIP-1ß, and RANTES and to inhibit the in
vitro replication of the M-tropic HIV-1 (26). In addition,
NK cells that are recruited and activated by the CXC chemokines Mig and
Crg-2 contributed significantly to the eradication of murine vaccinia
virus (27). Also, NK cells respond by migrating toward the
concentration gradients of the anti-HIV-1 chemokines (reviewed in
Ref. 9). In this paper, we have added to the list of
chemokines the CC chemokines TARC, MDC, and I-309. These chemotactic
activities were inhibited upon pretreatment of IANK cells with PT. This
toxin ADP-ribosylates the subunit of members of
Gi and Go proteins and
hence uncouples them from the membranes. Our results suggest that TARC,
MDC, and I-309 receptors are coupled to members of the
Gi or Go proteins (3, 9). Also, we showed that these chemokines induce the
accumulation of intracellular calcium in IANK cells.
Extravasation of NK cells into various tissues and their accumulation
at the sites of HIV-1 replication must be accompanied by the expression
of chemokine receptors in these cells. Also, expression of chemokine
receptors corresponds to the chemotaxis and the calcium signaling
induced by chemokines in T cells (23). In this paper we
have clearly shown by several criteria including flow cytometric,
biochemical, and molecular analyses that NK cells express CCR4.
Messenger RNA for CCR4 or CCR8 was also detected in IANK cells. The
expression of CCR4 and CCR8 explains how TARC, MDC, and I-309 induced
the chemotaxis and the mobilization of intracellular calcium in these
cells. When TER-1 (CCR8) was first cloned, its mRNA was
detected in certain NK cell lines, such as NK 3.3, but not in others,
such as NKL and YT-5, or in activated cells (6). In our
study, it is clear that CCR8 is expressed in NK cells cultured with
IL-2 both at the mRNA and on the surface and in terms of the response
of these cells to the CCR8 ligands TARC and I-309. The difference
between our study and that reported by Napolitano et al.
(6) is that we used adherent IANK cells. These cells may
express different chemokine receptors than nonadherent cells and may
resemble the NK 3.3 chemokine receptor expression. In fact, both
adherent IANK cells and the NK 3.3 cells respond similarly to various
CC chemokines (19). Although not examined in any detail,
we noticed that IANK cells lack mRNA for CCR5, the receptor for
MIP-1ß, MIP-1, and RANTES.
Pretreatment of IANK cells with MDC or with TARC induced the internalization of CCR4. This occurred after 5 min of stimulation and was persistent until 45 min. Several steps must take place before G protein-coupled receptors such as CCR4 are internalized. These include the recruitment of GRKs, which phosphorylate the receptors and consequently bind ß-arrestin (reviewed in Ref. 28). Chemokine receptors form appropriate targets for GRKs because these are serine/threonine kinases, and the carboxy terminal of the chemokine receptors are rich with these residues (reviewed in Refs. 1, 3). In this paper, we have shown that CCR4 is phosphorylated 0.5 min after incubation of NK cells with MDC. GRKs are most probably responsible for phosphorylating CCR4 because we observed that there is a sequential association of GRK2 and GRK3 with CCR4 shortly after stimulation with MDC.
Hence, GRK2 is recruited first, because it is associated with CCR4 0.5 min after incubation with MDC and seems to be responsible for the initial phosphorylation of CCR4 in IANK cells. Thereafter, GRK3 is recruited and remains associated with the phosphorylated receptors for the duration of the phosphorylation process, i.e., up to 2 min. It appears that GRK3 may maintain the receptor in its phosphorylated forms. Both GRK2 and GRK3 are dissociated from the receptor 5 min after stimulation. This is coincided with the loss of phosphorylation. Also, we observed that there is a lapse of time between the phosphorylation of the receptor (within seconds) and its endocytosis (between 5 and 45 min). This is most probably due to the fact that phosphorylated receptors are not internalized right after stimulation. We have evidence showing that after activation with MDC or TARC, phosphorylated CCR4 recruits ß-arrestin and nonreceptor tyrosine kinases. Such recruitment starts 5 min after stimulation (data not shown). These molecules are important for directing phosphorylated receptors toward endocytic pits to start the process of internalization. Interestingly, CCR4 reappeared in NK cell membranes 60 min after stimulation, suggesting that this receptor is not degraded by endogenous proteolytic enzymes but instead is recycled into the membranes. This is important when one considers CCR4 as a target for therapy. Our results support those of others who observed that GRK3 phosphorylates CCR2B (29). In addition, GRK2 and GRK3 were reported to phosphorylate CCR5 and to induce its desensitization and endocytosis upon activation of various cell types with RANTES (22, 30).
It was suggested that in Th2 cells (31) or in L1.2 transfected cells (18), receptors other than CCR4 must bind MDC. Such receptors, if found, are most probably not expressed on NK cells, because in these cells TARC inhibited the calcium mobilization induced by MDC, suggesting that CCR4 is the only receptor utilized by MDC. Alternatively, a novel receptor that binds both MDC and TARC may be identified, which could explain the present results. However, our results suggest that in addition to CCR4, TARC utilizes some other receptors because MDC, the ligand for CCR4, partially inhibited the calcium mobilization response induced by TARC. This receptor is probably CCR8 because I-309, the ligand for CCR8, also partially inhibited the calcium mobilization response induced by TARC. This result supports the findings of Bernardini et al. (24), who showed that TARC utilizes CCR8 in the Jurkat cell line.
In the calcium release assays, we observed that the response induced by
MDC, TARC, or I-309 is sharply declined 10–15 s after stimulation with
these chemokines, suggesting a transient release of calcium from
intracellular sources such as the endoplasmic reticulum. The calcium
channels on these sources are thereafter closed until further
stimulation. This stimulation is facilitated by G proteins, which
recruit and activate phospholipase Cß and hence the inositol 1,4,5
trisphosphate/inositol 1,4,5 trisphosphate receptor pathway or the
ADP-ribosyl cyclase and hence the cyclic ADP-ribose/ryanodine receptor
pathway (32). Both of these pathways are activated by
chemokines in IANK cells (33). The calcium flux response
induced by MDC and TARC is dependent on PT-sensitive G proteins because
PT inhibited this response. Because the heterotrimeric G proteins are
dissociated from IANK cell membranes shortly after stimulation with
chemokines (21), it is not surprising that there is an
initial calcium flux response induced by the three chemokines, which is
desensitized until further association of the various subunits of G
proteins with the receptors. Hence, this response will only be restored
upon the reappearance of the receptor and its reassociation with the
, ß, and subunits of G proteins, waiting for another round of
stimulation. On the other hand, the calcium flux response induced by
I-309 is not inhibited by PT, suggesting that this response is mediated
through PT-insensitive G proteins. The nature of these G proteins is
not known at the present time. This result marked a distinction between
the chemotactic response, which is PT-sensitive, and the calcium
response induced by I-309, supporting the findings of others who
reported such a distinction between the two activities (chemotaxis and
calcium flux) induced by chemokines in T cells (23).
In conclusion, our results are the first to show that NK cells express CCR4 and CCR8, among other chemokine receptors. This, combined with the ability of NK cells to secrete various chemokines, may explain how these cells extravasate into various tissues and may shed some light on the possible involvement of NK cells in eradicating HIV-1-infected cells. Also, our results indicate that CCR4 and CCR8 are not exclusive for Th2 cells.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. A. A. Maghazachi, Department of Anatomy, University of Oslo, P.O. Box 1105, N-0317 Blindern, Oslo, Norway.
3 Abbreviations used in this paper: CCR, CC chemokine receptor; TARC, thymus and activation-regulated chemokine; MDC, macrophage-derived chemokine; MIP, macrophage inflammatory protein; SDF, stromal-derived factor; IANK, IL-2-activated NK; PT, pertussis toxin; fura-2-AM, fura-2-acetoxymethyl ester; GRK, G protein-coupled receptor kinases; RPA, RNase protection assay; hCR5, human cytokine receptor set 5.
Received for publication June 24, 1999. Accepted for publication February 4, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and MIP-1ß as the major HIV-suppressive factors produced by CD8+ T cells. Science 270:1811. into the cell membranes, and their association with Gß after activation of NK cells with chemokines. J. Immunol. 162:3249.This article has been cited by other articles:
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  B. B. Damaj, C. B. Becerra, H. J. Esber, Y. Wen, and A. A. Maghazachi Functional Expression of H4 Histamine Receptor in Human Natural Killer Cells, Monocytes, and Dendritic Cells J. Immunol., December 1, 2007; 179(11): 7907 - 7915. [Abstract] [Full Text] [PDF]
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D. Soler, T. R. Chapman, L. R. Poisson, L. Wang, J. Cote-Sierra, M. Ryan, A. McDonald, S. Badola, E. Fedyk, A. J. Coyle, et al. CCR8 Expression Identifies CD4 Memory T Cells Enriched for FOXP3+ Regulatory and Th2 Effector Lymphocytes J. Immunol., November 15, 2006; 177(10): 6940 - 6951. [Abstract] [Full Text] [PDF]
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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]
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 D. G. Cronshaw, A. Kouroumalis, R. Parry, A. Webb, Z. Brown, and S. G. Ward Evidence that phospholipase C-dependent, calcium-independent mechanisms are required for directional migration of T lymphocytes in response to the CCR4 ligands CCL17 and CCL22 J. Leukoc. Biol., June 1, 2006; 79(6): 1369 - 1380. [Abstract] [Full Text] [PDF]
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 K. J. Carpenter, K. F. Buckland, Z. Xing, and C. M. Hogaboam Intrapulmonary, Adenovirus-Mediated Overexpression of KARAP/DAP12 Enhances Fungal Clearance during Invasive Aspergillosis Infect. Immun., December 1, 2005; 73(12): 8402 - 8406. [Abstract] [Full Text] [PDF]
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 J. Hanna, H. Mussaffi, G. Steuer, S. Hanna, M. Deeb, H. Blau, T. I. Arnon, N. Weizman, and O. Mandelboim Functional aberrant expression of CCR2 receptor on chronically activated NK cells in patients with TAP-2 deficiency Blood, November 15, 2005; 106(10): 3465 - 3473. [Abstract] [Full Text] [PDF]
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K. J. Carpenter and C. M. Hogaboam Immunosuppressive Effects of CCL17 on Pulmonary Antifungal Responses during Pulmonary Invasive Aspergillosis Infect. Immun., November 1, 2005; 73(11): 7198 - 7207. [Abstract] [Full Text] [PDF]
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 A. A. Maghazachi Insights into Seven and Single Transmembrane-Spanning Domain Receptors and Their Signaling Pathways in Human Natural Killer Cells Pharmacol. Rev., September 1, 2005; 57(3): 339 - 357. [Abstract] [Full Text] [PDF]
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C. A. Wysocki, A. Panoskaltsis-Mortari, B. R. Blazar, and J. S. Serody Leukocyte migration and graft-versus-host disease Blood, June 1, 2005; 105(11): 4191 - 4199. [Abstract] [Full Text] [PDF]
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 T. Shimauchi, S. Imai, R. Hino, and Y. Tokura Production of Thymus and Activation-Regulated Chemokine and Macrophage-Derived Chemokine by CCR4+ Adult T-Cell Leukemia Cells Clin. Cancer Res., March 15, 2005; 11(6): 2427 - 2435. [Abstract] [Full Text] [PDF]
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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]
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 C. Qu, E. W. Edwards, F. Tacke, V. Angeli, J. Llodra, G. Sanchez-Schmitz, A. Garin, N. S. Haque, W. Peters, N. van Rooijen, et al. Role of CCR8 and Other Chemokine Pathways in the Migration of Monocyte-derived Dendritic Cells to Lymph Nodes J. Exp. Med., November 15, 2004; 200(10): 1231 - 1241. [Abstract] [Full Text] [PDF]
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 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]
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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]
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