Human NK Cells Express CC Chemokine Receptors 4 and 8 and Respond to Thymus and Activation-Regulated Chemokine, Macrophage-Derived Chemokine, and I-309

Culture medium and other reagents

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 × 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 × g for 10 min, and the supernatants were transferred into Beckman tubes and ultracentrifuged at 150,000 × 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 × 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 × 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 × 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: K_d (F − F_min/F_max − F), where K_d for fura 2 is 225 nM. F, F_max, and F_min 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 × 10⁷/ml) were incubated with 50 ng/ml of the particular chemokine (MDC, TARC, or SDF-1α; 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 × 10⁶/ml cells were fixed with 4% paraformaldehyde for 1 h at 4°C. They were washed twice in PBS and then incubated (5 × 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 × 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 × 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 [α-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.

Detection of mRNA for CCR8 and CCR4 in IANK cells

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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FIGURE 1.

Detection of CCR4 and CCR8 mRNA in IANK cells utilizing RPA. hCR5, which detects mRNA for CCR4 and CCR8 among other chemokine receptors, has been utilized. Detection of mRNA was as described in the procedures supplied by PharMingen. Lane 1, mRNA of RNase- unprotected probes. Lane 2, Yeast tRNA as a control. Lane 3, mRNA of protected probes in IANK cells. Arrows indicate the presence of mRNA for CCR4 and CCR8. Other chemokine receptors marked in the figure have not been examined in detail.

Detection of surface expression of CCR4 and CCR8

Fig. 2⇓a 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. 2⇓b show that CCR4 is detected by this method (lane 1). Pretreatment of 4 × 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. 2⇓b, lanes 2 and 3, respectively), suggesting that CCR4 is internalized after pretreatment with these ligands. SDF-1α, which does not bind CCR4, did not induce the endocytosis of CCR4 (Fig. 2⇓b, 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. 2⇓c 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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FIGURE 2.

Expression of CCR4 and CCR8 in IANK cells. a, Expression of CCR4 and CCR8 was determined by flow cytometric analysis. Goat anti-human CCR4 or goat anti-human CCR8 was introduced inside permeabilized IANK cells, respectively. The cells were washed and then incubated with F(ab′)₂ FITC-conjugated rabbit anti-goat as a secondary Ab. Filled columns represent the background controls in the presence of goat IgG plus the secondary Ab. b, Expression of CCR4 by immunoblot assay. IANK cell membranes were immunoprecipitated with anti-CCR4 and then immunoblotted with anti-CCR4 (lane 1). In lanes 2-4, the cells (4 × 10⁷/ml) were incubated first with 50 ng/ml of TARC, MDC, or SDF-1α, respectively, for 25 min at 37°C before the membranes were prepared from these cells. These membranes were immunoprecipitated and then immunoblotted with anti-CCR4 as above. Lane 5 shows membranes immunoprecipitated with goat IgG as a control. c, Time kinetic study of CCR4 internalization. IANK cells (4 × 10⁷/ml) were stimulated with 50 ng/ml MDC for 0, 5, 15, 30, 45, and 60 min. These cells were examined for the presence of CCR4 as determined in a.

Phosphorylation of CCR4

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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FIGURE 3.

MDC induces the phosphorylation of CCR4 in IANK cells. IANK cells (4 × 10⁷/ml) were stimulated with 50 ng/ml MDC for 0–5 min. Lysates were prepared from these cells and were immunoprecipitated overnight with goat anti-CCR4 or with goat IgG as a control. They were run on SDS-PAGE and then immunoblotted with anti-phosphoserine/phosphothreonine (α-p). In the lower panel, the membranes were stripped and reimmunoblotted with anti-CCR4.

The serine/threonine kinases GRK2 and GRK3 have been implicated in phosphorylating chemokine receptors such as CCR5 (22). To address the possibility that CCR4 phosphorylation may be related to the activity of these enzymes, we examined whether CCR4 is associated with GRK2 or GRK3. Fig. 4⇓ 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. 4⇓a) but not GRK3 (Fig. 4⇓b) 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. 4⇓c).

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FIGURE 4.

Association of CCR4 with GRK2 and GRK3 shortly after stimulation of IANK cells with MDC. IANK cells were stimulated with MDC as described in Fig. 2⇑. Membranes were prepared from these cells and were immunoprecipitated with goat anti-CCR4 or with goat IgG as a control. These membranes were immunoblotted with rabbit anti-human GRK2 (a) or rabbit anti-human GRK3 (b). Goat anti-rabbit Ab was used as a secondary Ab. The membranes were stripped and reimmunoblotted with goat anti-CCR4 (c). Rabbit anti-goat Ab was used as a secondary Ab.

MDC, TARC, and I-309 induce the chemotaxis of IANK cells

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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FIGURE 5.

Effect of CC chemokines on the in vitro motility of IANK cells. Various concentrations (1–10,000 pg/ml) of MDC, TARC, or I-309 were placed in the lower wells of Boyden chambers, whereas 4 × 10⁵ IANK cells were placed in the upper wells. Migration index was calculated as the number of cells migrating in the presence of chemokines divided by the number of cells migrating in the presence of medium only (open columns). Mean ± SD of six separate experiments. The effect of PT on MDC, TARC, and I-309 is shown in the lower right panel. In these experiments, IANK cells (1 × 10⁶/ml) were either left intact (untreated) or were incubated with 2, 20, or 200 ng/ml activated PT overnight at 37°C. These cells were washed three times and placed in the upper wells of Boyden chambers. In the lower wells, 100 pg/ml of MDC, TARC, or I-309 was placed. Migration index was calculated as the number of cells migrating in the presence of chemokines divided by the number of cells migrating in the absence of chemokines (control; open columns). Mean ± SD of four separate experiments.

Almost all receptors for chemokines are coupled to heterotrimeric G proteins (1, 2, 3, 9). To investigate whether this is true for TARC, MDC, or I-309, we pretreated IANK cells with various concentrations of PT for 18 h at 37°C. Results in Fig. 5⇑ 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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FIGURE 6.

MDC, TARC, and I-309 induce the mobilization of [Ca²⁺]_i in fura 2-AM-labeled IANK cells. The concentrations of chemokines used in this assay were 20 ng/ml MDC, 16 ng/ml TARC, and 20 ng/ml I-309. Ionomycin (ION) was used at 20 μM. Arrows indicate the times at which chemokines were added. Changes in the cytoplasmic level of Ca²⁺ were determined in a 90-s span.

To investigate whether the calcium flux response induced by these chemokines is PT-sensitive, we pretreated IANK cells with 200 ng/ml PT for 18 h before examining them in the calcium assay. Ionomycin was used as a control. In these assays, extracellular calcium was not added to the buffer. Ionomycin induced a transient calcium flux, which returned to a baseline level (Fig. 7⇓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. 7⇓B). 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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FIGURE 7.

Effect of PT on the mobilization of intracellular calcium induced by MDC, TARC, and I-309. IANK cells (1 × 10⁶/ml) were either left untreated (UNT) or were pretreated with 200 ng/ml of activated PT for 18 h at 37°C. The cells were extensively washed, labeled with fura 2-AM, and then examined in the calcium release assay. Ionomycin (ION) was used at 20 μM. The concentrations of chemokines are the same as in Fig. 6⇑. Arrows indicate the times at which these reagents were added. One of three representative experiments.