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Departments of Immunology (C.A.H., R.H.), Molecular Pharmacology (M.L., S.R.), Cell Sciences (M.R.), Drug Metabolism and Pharmacology (L.D.), and Chemistry (I.I.), Berlex Biosciences, Richmond, California; Departamento de Inmunologiía y Oncología, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain (L.K., J.G., G.M., C.M.-A.); and Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania (M.J.B., R.W.D.)
Received May 11, 2005; accepted October 12, 2005
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i, because it could be blocked by treatment with pertussis toxin. The CCR8 agonist activated cells expressing murine CCR8, eliciting their chemotaxis and inducing phosphorylation of extracellular signal-regulated kinase ERK1/2. Like CCL1, ZK 756326 inhibited human immunodeficiency virus (HIV) fusion of cells expressing CD4 and CCR8. Finally, unlike mCCL1, ZK 756326 bound to and activated a form of mCCR8 that was mutated to eliminate O-linked sulfation at tyrosines 14 and 15. Therefore, ZK 756326 is most probably not binding in the same manner as CCL1 but can activate the switch mechanism involved in transducing signaling events. In summary, we have identified a nonpeptide agonist of CCR8. This compound may be useful in evaluating the physiological role of CCR8 in HIV infection, as well as in the general study of CCR8 biology without the constraints inherent to the use of protein agonists such as its natural ligand.
). Drugs that target these receptors make up greater than 45% of all known marketed medicines (Drews, 1996). Considerable evidence exists that points to a major role of chemokines in the pathophysiology of a number of autoinflammatory diseases, including multiple sclerosis, rheumatoid arthritis, atherosclerosis, dermatitis, organ transplant rejection, etc. (Gerard and Rollins, 2001). Thus, chemokine receptor antagonists could prove to be useful therapeutics in treating these and other inflammatory diseases. Based on these considerations, there has been a veritable explosion of activity to discover potent, selective, clinically useful chemokine receptor antagonists (Dhanak et al., 2001a,b; Dorn et al., 2001; Finke et al., 2001a,b,c; Forbes et al., 2000; Horuk et al., 2001; Ichiyama et al., 2003; Widdowson et al., 2004), and this has generated more than 250 patent applications and nine human clinical trials (Horuk, 2003).
We have studied the CC chemokine receptor CCR8 for several reasons. In an earlier study, we showed that CCR8, when coexpressed with CD4, can function as an HIV coreceptor for diverse viral strains. The ligand for CCR8, CCL1 (I-309), was a potent inhibitor of HIV-mediated cell-cell fusion and virus infection (Horuk et al., 1998). In addition to its potential interest as an HIV coreceptor, CCR8-deficient mice have been shown to have an impaired Th2 immune response, suggesting a potential role for this receptor in allergic disease and asthma (Chensue et al., 2001). However, reports from several other laboratories have been unable to confirm these findings, and there is a suspicion that this might reflect strain-dependent differences in the CCR8-deficient animals (Chung et al., 2003; Goya et al., 2003). Nevertheless, based on the potential interest in CCR8 as a therapeutically relevant target, we embarked on a high-capacity receptor binding screen to discover nonpeptide antagonists of CCR8.
Analysis of compounds that were identified in a screen testing inhibition of CCL1 binding showed that one of the nonpeptides was a CCR8 receptor agonist able to stimulate a number of biological responses on both human and mouse CCR8. Here, we present a detailed characterization of 2-{2-[4-(3-phenoxybenzyl)piperazin-1-yl]ethoxy}ethanol (ZK 756326) activity on human and mouse CCR8. This is, to our knowledge, the first description of a nonpeptide agonist of a chemokine receptor, and it might be especially valuable in helping to decipher the ligand/receptor contact points required for chemokine receptor signaling.
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Materials and Methods |
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CCR8-Expressing Cells. The human malignant glioma U87 MG cells expressing CCR8 were obtained as described previously (Horuk et al., 1998). Stable cells were selected in minimal essential medium with nonessential amino acids (Invitrogen, Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum and 300 µg/ml G418. For binding assays, the cells were harvested and washed once with phosphate-buffered saline (PBS). Human embryonic kidney (HEK) 293 cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium (Cambrex Bio Science Verviers S.p.r.l., Verviers, Belgium) containing 10% (v/v) fetal bovine serum and supplemented with glutamine, penicillin, and streptomycin. The mouse thymic lymphoma BW5147.3 (TIB-47) cell line was obtained from the American Type Culture Collection and cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (Sigma, St. Louis, MO), 1 mM sodium pyruvate, 2 mM L-glutamine, 10 mM HEPES, pH 7.4, and antibiotics (Sigma).
Chemokine Binding Studies. U87 MG cells expressing human CCR8 (60,000 cells/well) were incubated with 0.2 mg/well wheat germ agglutinin-coated SPA beads (GE Healthcare, Little Chalfont, Buckinghamshire, UK) for 30 min at room temperature in binding buffer [PBS/Ca/Mg, 0.1% (w/v) bovine serum albumin (BSA), and 30 µg/ml bacitracin]. Compounds (concentrations varied as indicated in text) or 100 nM unlabeled CCL1 (nonspecific binding) were added to the cell/bead mixture. Finally, 100 pM 125I-CCL1 (specific activity, 2200 Ci/mmol; approximately 20,000-25,000 cpm/well) was added to the mixture and incubated at room temperature for 2 h. Bound 125I-CCL1 was measured by scintillation counting (Wallac TriLux; PerkinElmer Life and Analytical Sciences).
Cytosolic Ca2+ Measurements on U87 Cell Lines. U87 MG cells expressing CCR8 were plated on poly-D-lysine-coated black 96-well plates (BD Biosciences, Franklin Lakes, NJ) at 10,000 cells/well and were cultured overnight. Cells were then loaded with Calcium 3 (Molecular Devices, Sunnyvale, CA), a Ca2+-sensitive non-wash fluorescence dye, for 60 min at 37°C in Hanks' balanced salts solution (Invitrogen) containing 20 mM HEPES, 3.2 mM CaCl2, 1% (v/v) fetal bovine serum, and 2.5 mM probenecid. Changes in intracellular free-Ca2+ concentration were measured with Fluorometric Imaging Plate Reader (FLIPR 3) (Molecular Devices) immediately after the addition of agonist at room temperature. Cross-desensitization experiments were performed by a first addition of the agonist (CCL1 at 30 nM or ZK 756326 at 3 µM), immediately followed by a second addition of 100 nM CCL1.
Measurement of Extracellular Acidification with the Microphysiometer. Extracellular acidification was measured in a microphysiometer as described previously (Liang et al., 2000a). Human or mouse U87-CCR8 cells were seeded in polycarbonate cell capsules (Molecular Devices) at 1 x 105 cells/capsule and incubated for 48 h at 5% CO2, 37°C. Capsules were washed with a modified RPMI 1640 medium (containing 1 mM sodium phosphate, supplemented with 1 mg/ml BSA) and loaded into the chambers of the microphysiometer (Molecular Devices). The chambers were perfused with the low buffering media at a rate of 100 µl/min. After stabilization, the cells were perfused with the indicated concentrations of chemokine and/or compound for 6 min, during which the rate of acidification was measured. Data shown are representative of at least two separate experiments.
Transient Transfection of mCCR8 Variants and Calcium Mobilization Studies. Mammalian expression plasmids containing the Myc-tagged coding sequences of wild-type or YFFY mutant mouse CCR8 cDNAs have been described previously (Gutierrez et al., 2004). Plasmids were prepared using the Jetstar Maxi Kit 50 (Genomed, Bad Oeynhausen, Germany) and transiently transfected into HEK 293 cells with Lipofectamine Plus (Invitrogen) according to manufacturers' instructions. Forty-eight hours after transfection, HEK 293 cells were harvested, washed, and incubated (30 min) with 3 µM Indo-1 AM (Invitrogen). Cells were washed twice, resuspended at 3 x 106 cell/ml, and stored on ice until use. All incubations and washes were in Hanks' balanced salt solution with 0.5% (w/v) BSA, 10 mM HEPES, and 0.8 mM CaCl2. Aliquots of the cell suspension were heated for 5 min at 37°C, excited with light at 335 nm, and stimulated with purified mCCL1 (TCA-3) (BD Biosciences PharMingen; San Diego, CA) or ZK 756326. Intracellular Ca2+ levels were assessed as Indo-1 fluorescence at 390 nm and recorded as a time trace in an F2500 fluorometer (Hitachi, Tokyo, Japan).
Chemotaxis. Migration assays with BW5147.3 cells were performed in Transwell inserts (Corning Life Sciences, Acton, MA) with a 5 µm-pore diameter. Cells were resuspended in RPMI with 1% (w/v) BSA and 25 mM HEPES, pH 7.4 (3 x 105 cells/well), and 100-µl aliquots were loaded into upper inserts. Samples of mCCL1 and ZK 756326 prepared in 600 µl of the same medium were placed in the lower wells. After incubation (2 h at 37°C), inserts were removed, and the migrated cells were counted in an EPICS XL flow cytometer (Beckman Coulter, Fullerton, CA). Duplicate wells were used for each point. A migration index was established as the ratio of the number of cells that had migrated in response to the chemokine or to ZK 756326 divided by the number of cells that had migrated in response to buffer alone.
Western Blot Analysis of Extracellular Signal-Related Kinase (ERK) Activation. Exponentially growing BW5147.3 cells were serum-starved for 16 h (overnight) in depletion medium [Dulbecco's modified Eagle's medium, 1% (w/v) BSA, 1 mM sodium pyruvate, and 2 mM L-glutamine, 10 mM HEPES, pH 7.4, and antibiotics]. Cells were centrifuged, washed with the same medium, and resuspended. Cells (4 x 106/tube) were preincubated (10 min at 37°C) and stimulated with mCCL1 or ZK 756326 in a final volume of 0.2 ml. At different time points after agonist exposure, cells were immediately washed in ice-cold PBS and lysed in radioimmunoprecipitation assay buffer [20 mM Tris-HCl, pH 8, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% (v/v) Nonidet P-40, 0.5% deoxycholate, 0.1% (w/v) sodium dodecyl sulfate, and protease inhibitors]. Cell lysates were electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose. Western blotting was performed using an antiphosphospecific ERK-mitogen-activated protein kinase monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) following the manufacturer's instructions. Blots were then stripped and probed with an anti-total ERK polyclonal antibody, used at 1:1000 (Cell Signaling Technology, Beverly, MA) for normalization. Antibody staining was evaluated by enhanced chemiluminescence detection (GE Healthcare).
HIV Cell-Cell Fusion Assay. The assay was performed as described previously (Doranz et al., 1996). Target cells (quail QT6) were cotransfected with plasmids expressing CD4, the indicated chemokine receptor, and a luciferase expression plasmid under the control of the T7 promoter (T7-luc; Promega). Effector cells (QT6) were infected with a recombinant vaccinia expressing the T7 polymerase (vTF1.1) and transfected with an Env expression plasmid. The effector cells were added to the targets approximately 18 h after transfection and were allowed to interact for a period of 7.5 h at 37°C. Productive cell-cell fusion was assessed by assaying for T7 polymerase driven production of luciferase.
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2A, in which IC50 values of 5.4, 4,4, 34.8, 16, 5.9, and <20 µM (at 20 µM 65% inhibition), respectively, were observed. The compound is unlikely to be an agonist on these biogenic amine receptors, because when tested at concentrations up to 10 µM on a representative receptor, 5-HT1A, it showed no agonist activity in a GTP
S binding assay.
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Because ZK 756326 could block binding of CCL1 with CCR8, we tested whether it could also inhibit the functional activation of CCR8 by CCL1. To determine this, we measured agonist-induced Ca2+ mobilization in U87 cells expressing CCR8. The cells responded in a concentration-dependent manner to CCL1 (data not shown). However, to our surprise, we discovered that ZK 756326 was itself an agonist of CCR8, inducing a concentration-dependent calcium response (Fig. 3). The peak response of the CCR8-expressing cells to ZK 756326 was reached in the submicromolar to micromolar range, suggesting a reduced potency compared with CCL1. However, sample traces indicate that the receptor is stimulated in a similar manner (Fig. 4). As predicted from the binding competition studies, the compound had no inherent agonist activity on CCR4 (Fig. 3) or on other chemokine receptors including CXCR3, CXCR4, and CCR5 (data not shown). Thus, the agonist like properties of the compound seemed to be CCR8-specific.
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After ligand stimulation, chemokine receptors undergo desensitization whereby subsequent ligand challenges generate a reduced response. Using the calcium release assay, ligands can be tested for cross-desensitization, an indication that the ligands act on the same receptor population. The CCR8 ligand CCL1 (30 nM) was able to desensitize the receptor to subsequent challenge with CCL1 (100 nM) (Fig. 4A). ZK 756326 at a concentration of 3 µM was able to inhibit the subsequent signaling elicited by 100 nM CCL1 in a similar manner, suggesting that it was acting specifically on CCR8.
Signaling of CCL1 through CCR8 has previously been shown to be pertussis toxin sensitive, an indication of signaling through the G-protein Gi (Goya et al., 1998). The U87-CCR8 cell line was incubated with pertussis toxin and challenged with CCL1 (as indicated in the Fig. 4B legend). Pertussis toxin treatment reduced signaling of both CCL1 and ZK 756326, completely eliminating the CCL1-induced calcium response and reducing the ZK 756326-induced calcium response by >75% (Fig. 4B). In additional experiments, pretreatment with pertussis toxin eliminated all signaling by ZK 756326. These data further indicate that the agonist-induced responses of ZK 756326 are specifically mediated through CCR8 and require G-protein coupling.
The ability of receptors to induce increases in intracellular calcium relies on a subset of G-proteins. However, it is well documented that chemokine receptors can activate G-protein-induced pathways independent of calcium release. The microphysiometer, by measuring extracellular acidification rates, can detect many of these signaling pathways. Thus, we compared the agonist activity of ZK 756326 and CCL1 in the microphysiometer. CCL1 induced a concentration-dependent response in this system, with an EC50 value of 31 nM (Fig. 5A). ZK 756326 elicited similar maximal responses with an EC50 value of 254 nM (Fig. 5A). The microphysiometer responses to CCL1 and ZK 756326 both were inhibited by pertussis toxin (data not shown).
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; Link et al., 1992; Cotte et al., 1998; Liang et al., 2000b). To test whether ZK 756326 had agonist activity on murine CCR8, a U87 cell line expressing murine CCR8 was generated (U87-mCCR8). When tested in the microphysiometer, the murine CCR8 ligand mCCL1 was able to stimulate mCCR8 in a potent, concentration-responsive manner. As with the human CCR8 receptor, mCCR8 was activated by mCCL1 with an EC50 value of 12 nM (Fig. 5B). The ZK 756326 compound was also able to induce signaling in the U87-mCCR8 cells (Fig. 5D). This response was concentration-dependent, with an IC50 value of 2.6 µM, although the peak response was lower in the murine CCR8 cell lines. Therefore, as measured in the microphysiometer, ZK 756326 demonstrated activity on both human and mouse CCR8. This is in contrast to our previous work with CCR1 receptor antagonists, where we observed strong species selectivity, with the potency for rodent receptors 100- to 200-fold less than that seen on the human receptor (Liang et al., 2000b; Horuk et al., 2001). Others have reported similar species selectivity for small-molecule antagonists acting on other chemokine receptors (Gladue et al., 2003).
There is a wealth of evidence that chemokine signaling activates a variety of intracellular kinases (Ganju et al., 1998; Kampen et al., 2000; Tilton et al., 2000; Bonacchi et al., 2001). For example, at least two reports have indicated that the intracellular kinase ERK may be involved in CCR8 signaling (Louahed et al., 2003; Spinetti et al., 2003). Thus, we used ERK phosphorylation as an additional measurement of the functional agonism of ZK 756326 for CCR8 and tested whether it could induce phosphorylation of intracellular kinases. The BW5147.3 murine thymoma cell line, which naturally expresses mCCR8 at low levels (approximately 1700 copies of CCR8/cell; unpublished results), was used for these assays. Cells were stimulated with mCCL1 (30 nM) or ZK 756326 (15 µM) at concentrations determined to give near-maximal response in the microphysiometer assays. Cell lysates were prepared and analyzed by Western blot with antiphospho-ERK 1/2 (Fig. 6). ERK phosphorylation was observed with either chemokine or ZK 756326, with an early peak in phosphorylation occurring between 2 and 5 min, and a second peak at 120 min.
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; Floridi et al., 2003). When tested in a transwell migration assay, the murine CCR8 agonist mCCL1 could induce chemotaxis, attracting cells with a peak response at 400 pM (Fig. 7). This concentration response was biphasic, which is typical of chemokine-induced cell migration. It is noteworthy that ZK 756326 was also able to mimic this specific chemokine function and attracted cells in a chemotactic manner, with an observed peak migration at a concentration of 2.4 µM (Fig. 7).
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We have previously shown that a double tyrosine-to-phenylalanine mutation at amino acid residues 14 and 15 of mouse CCR8 (described previously as the YFFY mutant) severely impaired its ability to mediate calcium transients when stimulated with mCCL1 (Gutierrez et al., 2004). Thus, it was interesting to determine whether the nonpeptide CCR8 agonist ZK 756326 would mimic mCCL1 in this aspect. HEK 293 cells were transiently transfected with plasmids encoding wild-type or YFFY mutant mCCR8; 48 h later they were tested for receptor expression at the cell surface. The expression levels of wild-type and mutant CCR8 were identical (data not shown), and calcium transients were measured. As expected, cells transfected with wild-type mCCR8 responded readily to 1 nM mCCL1 (Fig. 8A, top trace) and to 18 µM ZK 756326 (Fig. 8A, bottom trace). As shown previously (Gutierrez et al., 2004), 10 nM mCCL1 did not induce an increase in intracellular calcium in cells transfected with the YFFY mutant (Fig. 8B, top trace). However, they showed a strong response to 18 µM ZK 756326 (Fig. 8B, bottom trace), similar to that of wild-type mCCR8. Both the wild type and the YFFY mutant of mCCR8 were sensitive and responded similarly to lower concentrations of ZK 756326; as expected, HEK 293 cells transfected with void vector did not respond to mCCL1 or ZK 756326 (data not shown).
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In addition to binding and signaling by CCL1, CCR8 can serve as a coreceptor for HIV-1, and CCL1 can inhibit this process (Horuk et al., 1998). To determine whether ZK 756326 could mimic the HIV-1 inhibition of CCL1, we tested it in an Env-mediated cell-cell fusion assay (Fig. 9). Various Env-expressing effector cells were allowed to elicit membrane fusion with target cells expressing CD4 and CCR8. In this assay, effector cells also express T7 RNA polymerase, whereas target cells bearing CD4 and a coreceptor contain a luciferase plasmid under control of the T7 promoter. Fusion between target and effector cells results in cytoplasmic mixing and the subsequent production of luciferase, whose enzymatic activity can be easily quantified. We found that a number of HIV-1 Envs, both CCR5-tropic (ADA, YU2, JRFL, SF162) and dual-tropic (580, 148, 89.6, UGO24), were able to produce membrane fusion via CCR8. Cell-cell fusion mediated by all of these Envs was inhibited specifically by ZK 756326 (10 µM) to well below the IC50 level, with most Envs now showing fusion at between 4 and 25% (Fig. 9). When cells expressing CD4 and CCR5 were used as targets, ZK 756326 did not inhibit fusion, confirming its specificity for CCR8 (data not shown).
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Discussion |
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In previous work, we showed that mCCL1 does not stimulate calcium flux on YFFY-expressing cells because it does not bind to the mutant receptor (Gutierrez et al., 2004). The fact that ZK 756326 stimulates this mutant as well as wild-type mCCR8 suggests that the manner in which ZK 756326 and mCCL1 bind the receptor is different and that ZK 756326 binding to mCCR8 is independent of tyrosines 14 and 15. Because both tyrosines 14 and 15 in mCCR8 are O-sulfated (Gutierrez et al., 2004), it has been proposed that the negative charges contributed by these sulfate groups play an important role in the binding of mCCL1, which is a cationic polypeptide with a pI of 9.5 (Gutierrez et al., 2004). At neutral pH, ZK 756326 is predicted to have a slightly positive net charge (+1), which would not drive the type of charge interactions predicted to occur between mCCL1 and the receptor sulfate groups. In addition, because the molecular mass of ZK 756326 (356 Da) is considerably smaller than that of mCCL1 (7.8 kDa), ZK 756326 would not be expected to interact with mCCR8 using the same residues. It has been reported that other small-molecule ligands for GPCRs interact at the heptahelical bundle near the extracellular side of transmembrane domains (Onuffer and Horuk, 2002; Paterlini, 2002). The fact that ZK 756326 is insensitive to mutations at the N-terminal domain of mCCR8 further suggests that it does not interact with this region and supports the idea that it could interact exclusively with the putative transmembrane site. Further work will be needed to precisely define this site.
The coreceptors used by HIV to infect cells are attractive targets for antiviral therapy. Small molecule inhibitors of both CCR5 and CXCR4 have been developed and some are in human clinical trials. Whether inhibitors of other viral coreceptors will prove to be useful for antiviral therapy remains to be determined. So far, there is no compelling evidence that coreceptors other than CCR5 or CXCR4 are important in mediating HIV-1 infection of relevant primary cell types, although there is some evidence that CCR8 may be important for thymocyte depletion and infection by HIV-1. However, if small-molecule inhibitors potently block HIV-1 infection mediated by CCR5 or CXCR4, HIV-1 may evolve to use other coreceptors to infect cells. An example of unexpected coreceptor use in the face of strong selective pressure is provided by SIVrcm infection of red-capped mangabeys. Many red-capped mangabeys are CCR5-negative because of a naturally occurring polymorphism in the CCR5 open-reading frame. Viruses isolated from these infected animals use CCR2 as their major coreceptor to infect cells. Thus, development of small-molecule inhibitors that target alternative HIV coreceptors could play a role in antiviral therapy in some circumstances.
It is interesting to note that many of the compounds identified in the binding screen displayed specific CCR8 agonist activity in the calcium assay. These results emphasize the need for consideration of primary functional assays when screening for agonistic activity. There may be situations in which a chemokine receptor agonist could be favorable; i.e., CCR5 activation by an agonist will lead to potential inhibition by HIV infection and therefore justify the use of a functional primary assay. At this time, we cannot say whether this is a characteristic of the receptor or simply a bias that exists in the nonpeptide library used to screen this receptor. It has been reported that AMD-3100, a drug developed as a CXCR4 antagonist, did show at least partial agonist activity on CXCR4 (Zhang et al., 2002), although there is controversy surrounding this activity. In the calcium response assays, ZK 756326 is acting as a full agonist, given that the peak response generated by the compound is similar to that seen with CCL1. In other ways, the ZK 756326-mediated signaling of CCR8 is similar to that of the natural ligand: inhibition by pertussis toxin and the induction of ERK1/2 phosphorylation. To our knowledge, this is the first example of a small molecule acting as an agonist at a chemokine receptor.
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Footnotes |
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ABBREVIATIONS: GPCR, G-protein-coupled receptor; HIV, human immunodeficiency virus; ZK 756326, 2-{2-[4-(3-phenoxybenzyl)piperazin-1-yl]ethoxy}ethanol; PBS, phosphate-buffered saline; HEK, human embryonic kidney; BSA, bovine serum albumin; ERK, extracellular signal-related kinase; Env, HIV-1 envelope protein; 5-HT, 5-hydroxytryptamine; AMD-3100, 1,1'-(1,4-phenylenebis(methylene))bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride.
1 Current affiliation: Abgenix Inc., Fremont, CA. 
2 Current affiliation: Genetrix SL, Madrid, Spain.
Address correspondence to: Christopher Haskell, Berlex Biosciences, De-partment of Immunology, PO Box 4099, Richmond, CA 94804. E-mail: christopher_haskell{at}berlex.com
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References |
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1 in Rex 16 cells, adrenergic 
-). Three minutes after initial stimulation, cells were rechallenged with CCL1 (100 nM) (arrow). Response traces were set to zero (relative fluorescent units) at 8 s, immediately after the first addition. These traces are representative of three assays. B, pertussis toxin treatment. Cells were pretreated with pertussis toxin (200 ng/ml, 1 h, 37°C) (—) or buffer (- -) and then challenged with CCL1 (30 nM) (+) or ZK 756326 (3 mM) (
