Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0106035 on April 13, 2006

Published online before print April 13, 2006
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(Journal of Leukocyte Biology. 2006;79:1369-1380.)
© 2006 by Society for Leukocyte Biology

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

Darran G. Cronshaw*, Andreas Kouroumalis*, Richard Parry*, Adam Webb*, Zarin Brown{dagger} and Stephen G. Ward*,1

* Department of Pharmacy & Pharmacology, University of Bath, Claverton Down, Avon, United Kingdom; and
{dagger} Novartis Horsham Research Centre, West Sussex, United Kingdom

1Correspondence: Department of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath, Avon, UK BA2 7AY. E-mail: S.G.Ward{at}bath.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophage-derived chemokine [CC chemokine ligand 22 (CCL22)] and thymus- and activation-regulated chemokine (CCL17) mediate cellular effects, principally by binding to their receptor CC chemokine receptor 4 (CCR4) and together, constitute a multifunctional chemokine/receptor system with homeostatic and inflammatory roles within the body. This study demonstrates that CCL22 and CCL17 stimulate pertussis toxin-sensitive elevation of intracellular calcium in the CEM leukemic T cell line and human peripheral blood-derived T helper type 2 (Th2) cells. Inhibition of phospholipase C (PLC) resulted in the abrogation of chemokine-mediated calcium mobilization. Chemokine-stimulated calcium responses were also abrogated completely by the inhibition of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor-mediated calcium release. Chemotactic responses of CEM and human Th2 cells to CCL17 and CCL22 were similarly abrogated by inhibition of PLC and inhibition of novel, Ca2+-independent/diacylglycerol-dependent protein kinase C (PKC) isoforms. Inhibition of Ins(1,4,5)P3 receptor-mediated calcium release from intracellular stores had no effect on chemotactic responses to CCR4 ligands. Taken together, this study provides compelling evidence of an important role for PLC and diacylglycerol-dependent effector mechanisms (most likely involving novel PKC isoforms) in CCL17- and CCL22-stimulated, directional cell migration. In this regard, CCL22 stimulates phosphatidylinositol-3 kinase-independent phosphorylation of the novel {delta} isoform of PKC at threonine 505, situated within its activation loop—an event closely associated with increased catalytic activity.

Key Words: chemokines • signaling • chemotaxis • PKC


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines are a superfamily of low molecular weight proteins that possess similar secondary and tertiary structures and based on the arrangement of their two highly conserved amino-terminal cysteine residues, can be further divided into four subfamilies: CC, CXC, XC, and CX3C [1 ]. The CC chemokines thymus- and activation-regulated chemokine/CC chemokine ligand 17 (CCL17) and macrophage-derived chemokine/CCL22 can be classified as homeostatic and inflammatory chemokines and exert their effects via the CC chemokine receptor 4 (CCR4). Owing to the fact CCR4 expression was initially thought to be exclusive to T helper cell type 2 (Th2) cells, early pathological roles for CCR4 concentrated on known Th2-mediated conditions, most notably allergic inflammation of the airways [2 3 4 ]. Other diseases in which CCR4 and/or CCL22/CCL17 have been implicated include atopic dermatitis [5 , 6 ], endotoxic shock [7 ], rheumatoid and juvenile rheumatoid arthritis [8 ], fulminant hepatic failure [9 ], Hodgkin’s lymphoma [10 ], T cell non-Hodgkin lymphoma [11 ], and pulmonary fibrosis [12 ]. Despite the considerable evidence that CCL17 and CCL22 exert their biochemical and functional responses via interaction with CCR4, both chemokines are also known to bind the scavenging, nonsignaling receptors, Duffy antigen receptor for chemokines and D6 [13 , 14 ]. Moreover, CCL17 has been reported to bind to eosinophils in the absence of detectable CCR4 expression, suggesting the putative existence of a novel, as-yet unidentified receptor for CCL17 [15 ].

Chemokine receptors are pertussis toxin (PTX)-sensitive G{alpha}i protein-coupled seven-transmembrane receptors [1 , 16 , 17 ]. A number of studies have demonstrated the activation of various signaling pathways for most chemokines and in multiple cell types, including elevation of cytosolic intracellular calcium concentration ([Ca2+]i) as well as activation of the phosphoinositide-3 kinase (PI-3K) and extracellular signal-regulated kinase/mitogen-activated protein kinase pathways [17 ]. The majority of research focusing on signaling pathways required for directional migration in response to chemoattractant stimulation has focused on neutrophils (plus other myeloid cells) and Dictyostelium. It is widely appreciated that neutrophils and Dictyostelium require the activity of various PI-3K isoforms for chemoattractant gradient-sensing and directional cell migration [18 19 20 21 22 ]. However, the situation in T lymphocytes appears to be more complex with growing evidence that PI-3K is a dispensable signal for directed T cell migration in several settings including CCR4-mediated T lymphocyte migration [23 24 25 ]. Moreover, migration of neutrophils PI-3K{gamma}–/– mice, in response to chemokines is not completely abrogated [21 ], suggesting that neutrophil migration may also require PI-3K-independent component pathways [26 ].

Given that PI-3K appears to be a nonessential signal for CCR4-mediated T lymphocyte migration [23 ], we explored signaling pathways that may underpin the chemotactic responses of T lymphocytes to CCL17 and CCL22. In this study, we demonstrate that CCL17 and CCL22 elicit robust elevation of intracellular calcium, which is dependent on phospholipase C (PLC) activation. It is interesting that although chemokine-stimulated T cell migration is similarly dependent on PLC activation, the chemotactic responses are resistant to inhibition of intracellular calcium elevation. However, extensive pharmacological analysis revealed that migratory responses to CCL17 and CCL22 most likely involve other downstream effectors of PLC, namely activation of diacylglycerol (DAG)-dependent effectors, such as the novel DAG-dependent protein kinase C (PKC) isoforms (e.g., {delta}, {epsilon}, {theta}, and µ isoforms). In this regard, we show that CCL22 stimulates phosphorylation of the novel {delta} PKC isoform at threonine 505 (Thr-505) within its activation loop, an event that is known to be associated with increased catalytic activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Recombinant human (rh)CCL22 was purchased from R&D Systems (Abingdon, UK). The phycoerythrin-conjugated mouse anti-CCR4 monoclonal antibody was purchased from BD PharMingen (Oxford, UK). PTX, PD98059, rottlerin, RO-32-0432, Gö6976, U73122, U73343, and 2-aminoethoxydiphenyl borane (2-APB) were acquired from Calbiochem (Nottingham, UK). A phospho-specific rabbit polyclonal antibody, which recognizes phosphor-Thr-505-PKC{delta} (Catalog No. 9374), was purchased from Cell Signaling Technologies (New England Biolabs, Hitchin, UK). The anti-PKC{delta} rabbit polyclonal antibody (sc-213) was purchased from Santa Cruz Biotechnology (CA). Pluronic F-127 and Fluo-4 AM were purchased from Molecular Probes (Eugene, OR). Brilliant Black and Probenecid were purchased from Sigma-Aldrich (Gillingham, UK). Cell culture reagents were purchased from Life Technologies (Paisley, UK). Solvents were purchased from Fisher Scientific (Loughborough, UK). All other reagents were purchased from Sigma-Aldrich.

Cell culture
The human Caucasian acute T lymphoblastoid leukemia cell line CEM (Novartis, Horsham, UK) was cultured in humidified incubators in 5% CO2 at 37°C in RPMI-1640 medium, supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin.

In vitro generation of Th2 cells
Heparinized blood samples from healthy adult volunteers were separated on a Ficoll-paque (1.077) density gradient. Peripheral blood mononuclear cells were removed from the gradient, and naïve CD4+ T cells (CD4+ CD45RA+) were isolated using magnetic cell separation. Briefly, this involved magnetically labeling cells with CD4+ MicroBeads and separating (positive selection) on a column, which is placed in the magnetic field of an AutoMACS separator (beads and machine from Miltenyi Biotec, Germany). Cells were washed, and the process was repeated for positive selection of CD45RA+ cells within the CD4+ cell fraction. Isolated CD4+ CD45RA+ cells were cultured in humidified incubators in 5% CO2 at 37°C in RPMI-1640 medium, supplemented with 10% (v/v) FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1% minimal essential medium vitamins, 10 µg/ml insulin, 5.5 µg/ml transferrin, 6.7 ng/ml selenium, 50 µM 2-mercaptoethanol (2-ME), and 20 U/ml rh interleukin (IL)-2. Cells were polarized to a Th2-like phenotype by the addition of rhIL-4 (200 ng/ml) and neutralizing anti-IL-12 (1 µg/ml) and anti-interferon-{gamma} (1 µg/ml) antibodies (R&D Systems). Activation of cells was initiated by culturing in anti-CD3 (1 µg/ml)-coated 12-well plates with the above-mentioned media for the first 5 days. Cells were then washed and again cultured in the above media, except with 50 U/ml rhIL-2 (Chemicon, Chandlers Ford, UK), in 175 cm2 tissue-culture flasks. Cells were used between Weeks 3 and 4 post-isolation.

Chemotaxis assay
Chemotaxis assays were conducted in 96-well chemotaxis chambers (NeuroProbe, Gaithersburg, MD) carrying Transwell-permeable supports with polyvinylpyrrolidine-free polycarbonate membranes used (5 µm pore size). The lower chambers of each well were filled with 365 µl agonist at the appropriate concentration required [diluted in RPMI 1640+0.1% bovine serum albumin (BSA)] and carefully overlaid with the polycarbonate membrane. Cells were washed twice and resuspended in RPMI-1640 media + 0.1% BSA at 1 x 106 cells/ml, and 200 µl cell suspension was added to the upper chambers. The chambers were incubated for 90 min in a 5% CO2-humidified incubator at 37°C, and cells migrating across the membrane into the lower chamber were determined as described previously using 20 µl Cell Titer 96 AQueous reagent (Promega, Southampton, UK) following the manufacturer’s instructions [27 ]. Treatment of cells with PTX, U73122, U73343, 2-APB, rottlerin, Gö6976, and Ro-32-0432 had no detectable effect on the ability of cells to bioreduce the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium compound in the CellTiter 96 AQueous One solution compared with the untreated cells (data not shown). Unless otherwise stated, results are expressed as the mean chemotactic index (±SEM; n=5), which is the ratio of cells migrating toward chemokine versus untreated cells randomly migrating across the membrane (with or without the presence of inhibitor as appropriate). Statistical analysis is performed using ANOVA and Student’s t-test with a Bonferroni correction where necessary.

Measurement of cytosolic-free [Ca2+]I
Agonists were prepared in assay buffer (Hanks’ balanced saline solution/20 mM HEPES without phenol Red indicator+0.1% BSA w/v) at 3x required final concentration and aliquoted (60 µl) into a round-bottom 96-well plate. Fluo-4 (50 µg) was dissolved in 22 µl dimethyl sulfoxide (DMSO) followed by the addition of 22 µl pluronic F-127 acid (20% solution in DMSO). The Fluo-4 mix was then added to 22 ml assay buffer with 100 µM brilliant Black (100 µM) and 2.5 mM probenecid (loading buffer). CEM/Th2 cells were washed in assay buffer and resuspended at 1 x 106 cells/ml in loading buffer for 30 min. If antagonists were used, then cells were resuspended in assay buffer with inhibitor for required preincubation time (taking into account the subsequent 30 min Fluo-4 incubation time) before the addition of the Fluo-4 mix with probenecid and brilliant Black. Cell suspension (100 µl; 100,000 cells) was added to each well of a black-walled, clear-bottomed, poly-D-lysine-coated plate (Corning Costar UK Ltd., High Wycombe) and incubated for 30 min at 37°C, 5% CO2. The presence of brilliant Black (a quenching agent) in the loading buffer negates the need for washing. After loading, the plate was centrifuged at 1200 revolutions per minute for 5 min at room temperature. The cell plate was loaded into the fluorometric imaging plate reader (FLIPR; Molecular Devices Ltd., Wokingham, UK), a signal test was taken (allowing the determination of any effect of the inhibitors upon Fluo-4 loading of the cells), and laser power was adjusted to obtain a basal level of ~10,000 fluorescence intensity units. The cells were then excited at 488 nm using the FLIPR laser, and fluorescence emission was determined using a charged-coupled device (CCD) camera with a band-pass interference filter (510–560 nm). Fluorescence readings were taken at 1-s intervals for 60 s, and a further 150 readings were taken at 2-s intervals. Agonist (50 µl) was added (dispense speed, 30 µl/s) after 1 min using the FLIPR. Raw fluorescence data were exported for each well and tabulated versus time within Microsoft Excel (Microsoft Corp., Redmond, WA). Data were then imported into Origin 6.0 (Microcal Software Inc., Northampton, MA), the peak response over basal was determined as a fold increase, and curves were fitted with the logistic nonlinear curve-fit equation: y = (A1–A2)/1 + (X/X0)P.

Immunoblotting
Cells were stimulated (2x106 cells/ml; 1 ml/sample) and incubated at 37°C in RPMI 1640 + 0.1% BSA. Reactions were terminated by the addition of 200 µl 1x sample buffer [62.5 mM Tris, 2% sodium dodecyl sulfate (SDS), 5% mercaptoethanol (v/v), 10% glycerol (w/v), and 0.02% bromophenol blue]. The samples were then boiled for 5 min, and the solubilized proteins were electrophoresed through 10% polyacrylamide/SDS gels and transferred by electroblotting onto nitrocellulose membranes, which were incubated for 1 h with 25 ml blocking solution [1% nonfat milk/0.05% sodium azide in Tris-buffered saline (TBS); 10 mM Tris (pH 7.5), 100 mM NaCl], and then incubated overnight with 10 ml 1 in 1000 dilution (in TBS+0.1% Tween) of appropriate antibody, as described previously [23 ]. When necessary, membranes were stripped for reprobing by incubation in stripping buffer [62.5 mM Tris (pH 6.8), 2% SDS, and 100 mM 2-ME] at 60°C for 30 min.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CCL22- and CCL17-induced elevation of [Ca2+]i in CEM cells is rapid, transient, and concentration-dependent
Most chemokine receptors stimulate elevation of intracellular calcium following activation [1 , 17 ]. CCR4 has been shown to mobilize calcium in a range of cell types including platelets, natural killer cells, and Th2 cells [28 29 30 ]. We have previously demonstrated that the CEM leukemic T cell line expresses CCR4 [23 ]. Using this cell model, we now demonstrate that CCL17 and CCL22 can elicit rapid intracellular elevation of [Ca2+]i in the CEM leukemic T cell line (Fig. 1A and 1B ). This concentration-dependent mobilization is transient, and a return to basal levels was achieved at approximately 90 s poststimulation. It is also apparent that CCL22 is a more potent activator of calcium flux than CCL17 (Fig. 1C) , which correlates with previous observations concerning their relative potency at eliciting PKB phosphorylation and other signaling/functional responses [23 ]. Chemokines predominantly signal through G protein-coupled receptors, which are coupled to the G{alpha}i heterotrimeric G proteins, although other G{alpha} proteins have been demonstrated to couple to some chemokine receptors [16 , 17 ]. Pretreatment of CEM cells overnight (for 16 h) with 100 ng/ml PTX abolished CCL22- or CCL17-stimulated increases in [Ca2+]i (data not shown). This is consistent with signaling events mediated by G{alpha}I-coupled CCR4, although the involvement of Go, which is also PTX-sensitive, and/or other novel receptors cannot be entirely ruled out.


Figure 1
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  		Figure 1. CCL17 and CCL22 elevate intracellular calcium in the CEM leukemic T cell line. CEM cells were washed and resuspended in loading buffer, labeled with Fluo-4, and aliquoted at 80,000 cells/well onto poly-L-lysine-coated 96-well plates and stimulated with CCL17 or CCL22 as indicated. The cells were then excited at 488 nm using the FLIPR laser as described in Materials and Methods, and change in fluorescence emission of Fluo-4 was determined in response to (A) CCL22 or (B) CCL17. Data are derived from a single experiment representative of at least three others. (C) A comparison of peak calcium responses to CCL22 and CCL17 stimulation of CEM cells with results expressed as fold increase over unstimulated (vehicle) cells. Data are mean ± SEM of four separate experiments.

 
Inhibition of PLC attenuates CCL22- and CCL17-induced calcium mobilization
One of the major mechanisms by which calcium release from the endoplasmic reticulum stores is controlled, involves the hydrolysis of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] by PLC resulting in the formation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which interacts with the Ins(1,4,5)P3 receptor (IP3R) [31 ]. PLCß can be activated by the Gß{gamma} subunits after chemokine receptor ligation [20 , 32 , 33 ]. To determine whether the increase in [Ca2+]i following ligation of the CCR4 receptor requires the enzymatic function of PLC isoforms, we used U73122, a broad spectrum pharmacological inhibitor of PLC isoforms, reported to have an inhibitory concentration of 50% (IC50) between 500 nM and 2.1 µM [34 ]. The CCL22-induced elevation of [Ca2+]i was inhibited by pretreatment of CEM cells with U73122 in a concentration-dependent manner (Fig. 2A ). Similarly, CCL17-stimulated calcium mobilization was also sensitive to U73122 pretreatment (Fig. 2B) . A related compound U73343, which is an inactive analog of U73122, had no effect on chemokine-induced intracellular calcium mobilization (Fig. 2C and 2D) . These observations are consistent with the involvement of PLC in the mechanisms responsible for chemokine-stimulated elevation of [Ca2+]i. We therefore sought to explore whether Ins(1,4,5)P3 was the mediator of this response. 2-APB has been described as an inhibitor of calcium release from IP3R stores without affecting binding of Ins(1,4,5)P3 to IP3R and has an IC50 of 42 µM [35 ]. Treatment of CEM cells with 2-APB led to the complete abrogation of CCL22 (Fig. 3A )- and CCL17 (Fig. 3B) -induced elevation of [Ca2+]i.


Figure 2
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  		Figure 2. Inhibition of PLC attenuates the CCL22- and CCL17-induced calcium mobilization in CEM cells, which were washed and resuspended in loading buffer and pretreated with U73122 for 1 h at indicated concentrations. Cells were then labeled with Fluo-4 and aliquoted out at 80,000 cells/well onto poly-L-lysine-coated 96-well plates as described in Materials and Methods. The cells were then excited at 488 nm using the FLIPR laser, and change in fluorescence emission of Fluo-4 was determined in response to CCL22 (A) or CCL17 (B) stimulation, using a CCD camera with a band-pass interference filter (510–560 nm). Results are expressed as fold increase over unstimulated (vehicle/inhibitor) cells (±SEM), and curve-fitting was performed using the logistic nonlinear curve-fit equation in Origin. Alternatively, cells were pretreated with the inactive analog U73343 for 1 h prior to stimulation with 30 µM CCL22 (C) or 30 µM CCL17 (D). These results are expressed as fold increase over unstimulated (vehicle/inhibitor) cells (±SEM). Results are representative of at least three separate experiments.

 

Figure 3
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  		Figure 3. CCL17- and CCL22-induced elevation of [Ca2+]i requires calcium release from IP3R-sensitive stores. CEM cells were washed and resuspended in loading buffer and pretreated with 2-APB (75 µM) for 1 h. Cells were then labeled with Fluo-4 and aliquoted out at 80,000 cells/well onto poly-L-lysine-coated 96-well plates, as described in Materials and Methods. The cells were then excited at 488 nm using the FLIPR laser, and change in fluorescence emission of Fluo-4 was determined in response to CCL22 (A) or CCL17 (B) stimulation using a CCD camera with a band-pass interference filter (510–560 nm). Results are expressed as fold increase over unstimulated (vehicle/inhibitor) cells (±SEM), and curve-fitting was performed using the logistic nonlinear curve-fit equation in Origin. Results are representative of at least three separate experiments.

 
Effect of PLC inhibition on CCL22-mediated, directed T cell migration
Having established that the PLC inhibitor abrogated increases in [Ca2+]i elicited by CCL17 and CCL22, we next evaluated the effect of U73122 on chemokine-stimulated, directional cell migration. U73122 pretreatment of CEM cells inhibited CCL22-induced chemotaxis in a concentration-dependent manner with complete abrogation occurring at 3 µM U73122 (Fig. 4A ). Sensitivity of CCL22-stimulated CEM cell chemotaxis to U73122 therefore indicates that the hydrolytic products of PLC activity, namely Ins(1,4,5)P3 and/or DAG, are likely to be required for CCR4-mediated migration. To further investigate the importance of Ins(1,4,5)P3-mediated calcium release in cell migration events, we pretreated CEM cells with 2-APB at concentrations that we previously demonstrated were able to abrogate CCL22-mediated intracellular calcium mobilization (Fig. 3A) . It is interesting that 2-APB had no impact on CCL22-induced chemotaxis (Fig. 4B) , indicating that Ins(1,4,5)P3-mediated calcium release is not required for directed cell migration in response to CCL22.


Figure 4
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  		Figure 4. CCL22-mediated CEM cell chemotaxis requires PLC activation but not calcium mobilization. CEM cells (2x105 cells/200 µl) were added to the upper wells of a 96-well chemotaxis chamber, the lower wells of which contained 1 nM CCL22. Cells were preincubated with indicated concentrations of U73122 (A) or 2-APB (B) for 1 h as described in Materials and Methods. Chemotaxis, across a 5-µm membrane, was determined after 1.5 h incubation at 37°C in 5% CO2. The data are derived from a single experiment with quintuplicate replicates, which is representative of three other experiments. Data are expressed as the mean chemotactic index (±SEM), which is the ratio of cells migrating toward CCL22 versus cells randomly migrating (±inhibitor). Data were analyzed by ANOVA and Student’s t-test with a Bonferroni correction to compare responses in the presence and absence of U73122 (*, P<0.05; **, P<0.000005). The horizontal line at a chemotactic index of 1 represents unstimulated basal migration and is shown for ease of comparison with stimulated levels.

 
Effect of PKC inhibition on CCL22-mediated, directed T cell migration
Thus far, this study has demonstrated that Ins(1,4,5)P3-regulated calcium release is a dispensable signal for cell migration, although inhibition of PLC, which is responsible for Ins(1,4,5)P production, has a significant effect on migration. One explanation for this data could be that DAG (the other major product of PLC activity) plays a key role in regulating migration. The most widely studied DAG effectors are members of the PKC family. It has been demonstrated that various PKC isoforms are required for chemotaxis of various cell types in response to a variety of stimuli [36 , 37 ]. Gö6976 is a selective inhibitor for the conventional PKCs (i.e., {alpha}, ß, and {gamma}) and effective at nanomolar concentrations, while novel PKC isoform activity (i.e., {delta}, {epsilon}, {eta}, and {theta}) is unaffected, even at micromolar concentrations [38 ]. Pretreatment of CEM cells with Gö6976, at concentrations up to 300 nM, had no influence on CCL22-induced, directed cell migration (Fig. 5A ). As the conventional PKC isoforms require calcium and DAG for activation, the lack of effect of Gö6976 on cell migration compares favorably with the lack of requirement for Ins(1,4,5)P3-dependent calcium mobilization for CCL22-induced CEM cell chemotaxis.


Figure 5
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  		Figure 5. Conventional PKC isoform activity is not required for CCL22-mediated CEM cell chemotaxis. CEM cells (2x105 cells/200 µl) were added to the upper wells of a 96-well chemotaxis chamber, the lower wells of which contained 1 nM CCL22. Cells were preincubated for 30 min with indicated concentrations of Gö6976 for 30 min (A), RO-32-0432 (B), or rottlerin (C), as described in Materials and Methods. Chemotaxis, across a 5-µm membrane, was determined after 1.5 h incubation at 37°C in 5% CO2. The data are derived from a single experiment with quintuplicate replicates, which are representative of three other experiments. Data are expressed as the mean chemotactic index (±SEM), which is the ratio of cells migrating toward CCL22 versus cells randomly migrating (±inhibitor). Data were analyzed by ANOVA and Student’s t-test (P<0.05) to compare responses in the presence and absence of Gö6976. The horizontal line at a chemotactic index of 1 represents unstimulated basal migration and is shown for ease of comparison with stimulated levels.

 
The novel PKC isoforms ({delta}, {epsilon}, {eta}, and {theta}) require DAG for their activation. A number of inhibitors display inhibitory effects toward the conventional and novel PKC isoforms. One of these, RO-32-0432, has been described as a broad-spectrum PKC isoform inhibitor, although it does display a tenfold- and fourfold-more selective action for PKC{alpha} (IC50=9 nM) and PKCß (IC50=28 nM) over PKC{epsilon} (IC50=108 nM), respectively [39 ]. Pretreatment of CEM cells with RO-32-0432 completely ablated CCL22-driven CEM cell chemotaxis (Fig. 5B) . Rottlerin is another PKC inhibitor that displays up to tenfold-greater selectivity for PKC{delta} (IC50=3–6 µM) over other isoforms (IC50=30–42 µM) [40 ]. Rottlerin inhibited CCL22-mediated CEM cell chemotaxis in a concentration-dependent manner, and complete inhibition was observed at 10 µM rottlerin (Fig. 5C) .

Inhibition of PLC and PKC abrogates CCL17- and CCL22-stimulated chemotaxis of Th2 cells
Having elucidated the sensitivity of CCL22-stimulated chemotaxis of a transformed T cell line to inhibitors of PLC and PKC isoforms, we sought to explore whether these pathways were also required for migration responses in normal human T cells. CCL22 and CCL17 were able to elicit intracellular calcium mobilization in Th2 cells, and these responses were once again inhibited by the PLC inhibitor U73122 (Fig. 6A and 6B ).


Figure 6
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  		Figure 6. Th2 cell chemotaxis in response to CCR4 ligands requires PLC and PKC activation. Cells were washed and resuspended in loading buffer and pretreated with U73122 (1 µM) for 1 h. Cells were then labeled with Fluo-4 and aliquoted out at 90,000 cells/well onto poly-L-lysine-coated 96-well plates, as described in Materials and Methods. The cells were then excited at 488 nm using the FLIPR laser, and change in fluorescence emission of Fluo-4 was determined in response to CCL22 (A) or CCL17 (B) stimulation, using a CCD camera with a band-pass interference filter (510–560 nm). Results are expressed as fold increase over unstimulated (vehicle/inhibitor) cells (±SEM), and curve-fitting was performed using the logistic nonlinear curve-fit equation in Origin. Results are representative of three separate experiments. For chemotactic studies, Th2 cells were generated, as described in Materials and Methods, washed three times in RPMI, and resuspended in RPMI/0.1% BSA for 1 h at 37°. Cells were preincubated with U73122, U73343 (3 µM), or 75 µM 2-APB (C) for 1 h or 10 µM RO-32-0432 (D) for 30 min at indicated concentrations. Cells (1x105 cells/25 µl) were added to the upper wells of a 96-well chemotaxis chamber, the lower wells of which contained 100 nM CCL22 or 100 nM CCL17, as described in Materials and Methods. Chemotaxis, across a 5-µm membrane, was determined after 1.25 h incubation at 37°C in 5% CO2. The data are derived from a single experiment with triplicate replicates, which is representative of three other experiments. Data are expressed as the mean chemotactic index (±SEM), which is the ratio of cells migrating toward CCL22 versus cells randomly migrating. Data were analyzed by ANOVA and Student’s t-test with a Bonferroni correction to compare responses in the presence and absence of inhibitor (*, P<0.005; **, P<0.0005). The horizontal line at a chemotactic index of 1 represents unstimulated basal migration and is shown for ease of comparison with stimulated levels.

 
Likewise, CCL22- and CCL17-mediated Th2 cell chemotaxis was abrogated completely by pretreatment with the PLC inhibitor U73122 (Fig. 6C) . The inactive analog U73343 had no effect on chemokine-mediated chemotaxis of Th2 cells (Fig. 6C) . Similarly, chemotactic responses were insensitive to abrogation of Ins(1,4,5)P3-mediated elevation of [Ca2+]i by 2-APB (Fig. 6C) . However, the broad-spectrum PKC inhibitor RO-32-0432 abolished CCL17- and CCL22-induced Th2 cell chemotaxis (Fig. 6D) . Hence, the sensitivity of CCL17- and CCL22-stimulated Th2 chemotactic responses to pharmacological manipulation of PLC and PKC correlates closely with that observed in CEM cells.

CCL22 stimulates PKC{delta} phosphorylation in CEM and Th2 cells
The sensitivity of chemotactic responses of CEM and Th2 cells to rottlerin, which displays greater selectivity for PKC{delta}, prompted us to determine the effects of CCL22 on PKC{delta} activation. Several priming phosphorylations at highly conserved serine/threonine phosphorylation motifs in all PKC isoforms have been identified and are believed to play a key role in catalytic activation [41 ]. Accordingly, we monitored phosphorylation of PKC{delta} on Thr-505 (an event associated with increased catalytic activity) as an indirect measure of its activation in response to CCL22. Indeed, CCL22 stimulated phosphorylation of PKC{delta} at Thr-505 within the activation loop in a concentration (Fig. 7A )- and time (Fig. 7B) -dependent manner. Similar responses were observed with CCL17 (data not shown). The phosphorylation of Thr-505 has been reported to be mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK-1), an enzyme thought to lie downstream of PI-3K [42 ]. To ascertain the requirement of PI-3K activation in CCL22-stimulated Thr-505 phosphorylation, we examined the effect of the PI-3K inhibitor LY294002. It is interesting that pretreatment with LY294002 had only a modest inhibitory effect on phosphorylation of PKC{delta} in response to CCL22 (Fig. 7A) .


Figure 7
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  		Figure 7. CCL22 stimulates PKC{delta} phosphorylation in CEM (A) and Th2 (B) cells, which were aliquoted at 2 x 106 cells/ml and were then left unstimulated (control) or stimulated at 37°C with CCL22 at the concentrations indicated (A) or with 10 nM CCL22 for the indicated times (B). CEM cells were also pretreated with vehicle or 10 µM LY294002 for 10 min prior to addition of CCL22 (5 min) at the concentrations indicated (A). Cells were then lysed by the addition of 1x sample buffer as described in Materials and Methods. Proteins were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and immunoblotted with a phospho-specific PKC{delta} antibody with affinity for the Thr-505-phosphorylated form of PKC{delta}, and protein was visualized with enhanced chemiluminescence. The blot was stripped and reprobed with anti-pan PKC{delta} antibody to verify loading levels and efficiency of protein transfer. Results are representative of at least three separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using leukemic CEM cells and human Th2 cells, we demonstrate that the CCR4 ligands CCL22 and CCL17 are able to stimulate the rapid and transient elevation of [Ca2+]i, which is sensitive to PTX and appears to be dependent on PLC activation and release of calcium from intracellular IP3R-sensitive stores. It is interesting that the elevation of [Ca2+]i is redundant with respect to cell migration in response to CCL22 or CCL17. However, other downstream effectors of PLC, namely DAG-dependent PKC isoforms, do appear to be important for chemotactic events elicited by these two chemokines. In this regard, chemokine stimulation results in phosphorylation of the novel {delta} isoform of PKC at Thr-505 within its activation loop—an event associated with increased catalytic activity [43 ].

The PLC inhibitor U73122 completely attenuated the CCL17- and CCL22-stimulated calcium responses. The ß{gamma} G protein subunits are known to be capable of stimulating the PLCß2 and -ß3 isoforms, which are activated by chemokine receptors [32 , 44 ]. It is curious that studies with mice deficient in PLCß1 and -ß3 appeared to suggest that the PLC pathway is not required for chemotaxis in neutrophils, although the role in T lymphocytes was not investigated thoroughly [20 ]. The activation of PLC-sensitive signaling pathways has been demonstrated for a number of chemokines, including CXC chemokine ligand 8 (CXCL8)/CXC chemokine receptor 2 (CXCR2), CCL15/CCR1, and CCL2/CCR2b [16 , 17 ]. Two recent studies using the PLC inhibitor U73122 have demonstrated the importance of PLC activation for CXCL12/CXCR4- and CXCL11/CXCR3-mediated chemotaxis of T cells [25 , 45 ]. There have been many roles for calcium in cell motility and polarity proposed, from the regulation of actin-binding proteins to the stimulation of myosin II-based contraction [46 ]. Although some studies report that transient increases in calcium mobilization are required for chemotaxis of CXCR4-stimulated haemopoietic stem cells and CCR7-stimulated dendritic cells [47 , 48 ], the situation in T cells seems rather different. For example, chemotaxis of T lymphocytes in response to CCL5 does not rely on elevation of [Ca2+]i [49 ], an observation that correlates with the data presented in this study, demonstrating that calcium mobilization appears to be a dispensable signal for CCL17- and CCL22-mediated T cell chemotaxis.

Our data indicate that Ins(1,4,5)P3-mediated calcium release is probably not required for CCL17- and CCL22-stimulated cell migration. Conversely, evidence presented here indicates that DAG [the other product of PLC-mediated PI(4,5)P2 hydrolysis] likely plays a key role in chemotactic responses. Use of antisense oligodeoxyribonucleotides to specifically reduce PKCß expression has previously provided evidence that PKCß is required for monocyte chemotaxis in response to CCL2 [36 ]. In addition, the atypical calcium and DAG-independent {zeta} PKC isosform has been shown to be essential for neutrophil CXCL8-mediated chemotaxis [37 ]. However, PKCß is a conventional PKC isoform that requires DAG and calcium for its activity. Based on the pharmacological evidence presented in this study concerning calcium-independent mechanisms contributing to chemotactic mechanisms, PKCß seems an unlikely candidate for involvement in T cell migratory responses to CCL17 and CCL22. This is confirmed by the lack of effect of the conventional PKC inhibitor Gö6976 on this migratory event. Although a role for the atypical PKC{zeta} in CCR4-mediated CEM cell migration cannot be ruled out, it seems more likely that a novel DAG-dependent PKC isoform contributes toward CEM and Th2 cell chemotactic machinary. This conclusion would seem to be confirmed by the sensitivity of CCL22-induced T cell migration to the broad-spectrum PKC inhibitor RO-32-0432 and particularly, the inhibitory effects of rottlerin, which is believed to have greater selectivity for PKC{delta}. This correlates with our observation that stimulation of CCR4 results in phosphorylation of Thr-505 within the PKC{delta} activation loop and observations that PKC{delta} has a role in the chemotaxis of human osteogenic sarcoma cells following CCL15/CCR1 interactions [50 , 51 ].

A series of priming phosphorylations at highly conserved serine/threonine phosphorylation motifs in all PKC isoforms locks the enzyme in a closed, stabilized, catalytically competent, and protease/phosphatase-resistant conformation. Conventional PKC activation loop phosphorylation has been attributed to the master kinase PDK-1, which complexes with the C-terminal of the membrane-localized, unphosphorylated enzyme [43 ]. Conventional PKCs are then believed to autophosphorylate on a conserved proline-flanked "turn motif" and a hydrophobic FXXFS/TF/Y motif. Novel PKC isoforms undergo similar priming phosphorylations, and Thr-505 (in the activation loop), Ser-643 (in the hydrophobic region), and Ser-662 (in the C-terminal region) are the key sites for phosphorylation in PKC{delta} [41 ]. The notion that CCL17 and CCL22 stimulate PKC{delta} phosphorylation via activation of PDK-1, which in turn leads to increased PKC{delta} catalytic activity required for chemotactic responses, may initially appear to contradict our previous findings that CCR4-mediated migration is independent of PI-3K-dependent signals [23 ]. This is compounded by reports that PKC{delta} is often a functional enzyme in the absence of Thr-505 phosphorylation, and this event has been reported to be required for the stability of the enzyme rather than its subsequent enzyme activation [42 , 43 ]. However, there are at least two lines of evidence to support a role for PI-3K-independent phosphorylation and activation of PKC{delta} in response to CCL17 and CCL22. First, in some settings, PDK-1 phosphorylates conventional PKC isoforms by a mechanism that is independent of PI-3K [52 ]. This is supported by our observation that pretreatment with the PI-3K inhibitor LY294002 had only a modest, inhibitory effect on CCL22-stimulated phosphorylation of PKC{delta} at Thr-505. Second, the catalytic activity of membrane-associated, allosterically activated PKC{delta} can be increased by Thr-505 phosphorylation, mediated by other PKC isoforms in several systems [41 , 53 ]. Given that chemoattractants can stimulate several Src tyrosine kinases, it is also interesting to note that PKC{delta} contains multiple sites for tyrosine posphorylation, some of which have been demonstrated to influence its activation [41 ]. Moreover, the conserved domain 2 of PKC{delta} is a phosphotyrosine-binding domain, which allows for greater adaptability and diversity for integrating with other signaling events [54 ].

It is not well understood how PKC isoforms regulate cell motility/migration, although it is likely that PKC isoforms regulate changes in integrin affinity and lateral mobility [55 ] and/or exert effects on actin reorganization/polymerization [56 , 57 ] and phosphorylation of myosin light chain (MLC) [58 , 59 ]. Indeed, epidermal growth factor-induced MLC phosphorylation requires PKC{delta} [59 ]. During lymphocyte function-associated antigen-1-mediated locomotion of activated T cells, PKCßI and PKC{delta} associate with the microtubules in the uropod, the trailing extension of the migrating T cells. It is curious that although PKC{delta} is associated with the microtubule-organizing center (MTOC) but not the microtubules, PKCß1 is located with the MTOC and along the microtubules in the trailing cell extensions [60 ]. This may be indicative of discrete functions for individual PKC isoforms in T cell migration. Several PKC isoform knockout mice exist, including those for the ß, {delta}, and {zeta} isoforms. Although these exhibit an immune phenotype, mainly at the B cell level, no major defect in T cell migration in these mice has been reported so far, possibly as a result of redundancy in function between individual isoforms [61 ].

The PKD family of enzymes is a distant relative to the PKC family and requires DAG but not calcium for activation. One study has suggested a role for PKD in fibroblast-directed cell migration [62 ], and various studies have shown that PKD lies downstream of PLC, PKC{delta}, and Rho/Rho-associated kinase [63 64 65 ]. However, it has also been reported that Gö6976 can act as an inhibitor of PKD [66 ]. Given that Gö6976 exhibits no effect on CCL22- or CCL17-stimulated cell migration, it seem unlikely that PKD has a role to play in CCR4-mediated T cell chemotaxis. In addition to PKC isoforms, DAG can influence several additional signaling pathways including activation of Rap-guanine nucleotide exchange factors (GEFs), calDAG-GEFs, which are regulated by calcium and/or DAG, via distinct binding domains [67 ]. The Rap-guanosine-triphosphatase seems to be of fundamental importance for integrin activation by antigen receptors and chemokines and is essential for chemotaxis [68 69 70 ]. Indeed, a recent study demonstrated that CXCL12-mediated B cell migration was dependent on Rap and PLC activation but not calcium [71 ].

One important consideration when interpreting the pharmacological strategies described here is the potential for off-target effects of the inhibitors and whether they might contribute to observed effects of the inhibitors. Certainly, the PLC inhibitor U73122 is known to inhibit Ca2+-adenosine-triphosphatases [72 ], and the PKC inhibitor rottlerin can uncouple mitochondrial respiration from oxidative phosphorylation [73 ]. Although these off-target effects cannot be discounted entirely, the pharmacological data presented in this study indicate that although chemotactic responses of CEM and human Th2 cells to CCL17 and CCL22 ligation are reliant on PLC, they are independent of Ins(1,4,5)P3-mediated calcium release. Rather, these migratory responses most likely involve downstream DAG-dependent effectors of PLC. Although the most obvious candidate effectors are members of the novel PKC isoform family (particularly PKC{delta}), a role for additional DAG effectors such as PKD and CalDAG-GEFs cannot be excluded. RNA interference knockdown of PKC{delta} and use of T cells derived from PKC{delta} null mice are the subject of on-going work and will help further delineate the role of PKC{delta} in T cell chemotactic responses to CCL17, CCL22, as well as other chemokine/receptor interactions. Migrating leukocytes must navigate through complex chemoattractant fields and must migrate from one chemoattractant source to another. Migrating neutrophils have been reported to display "memory" of their recent environment, such that cells’ perception of the relative strength of orienting signals is influenced by their history [74 ]. This allows combinations of chemoattractants to guide leukocytes in a step-by-step manner to their destinations within tissues. The biochemical basis for this chemotactic memory is unclear, but tailored migratory responses involving heterogeneity in signaling events activated by chemoattractant receptors would favor this process [75 ]. The existence of multiple signaling pathways would also facilitate tailored migratory responses according to a particular physiological environment, allowing for fine-tuning of cell migration.


   ACKNOWLEDGEMENTS
 
D. G. C. was the recipient of a Biotechnology and Biological Sciences Research Council–Co-operative Awards in Sciences of the Environment (BBSRC-CASE) studentship in association with Novartis. This work was also supported by a Wellcome Trust University award to S. G. W. (Ref. 006195) and a BBSRC project grant (Ref. C12884).

Received January 18, 2006; revised February 13, 2006; accepted February 28, 2006.


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 MATERIALS AND METHODS
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 DISCUSSION
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