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The Journal of Immunology, 2005, 174: 6927-6933.
Copyright © 2005 by The American Association of Immunologists

Cross-Desensitization among CXCR1, CXCR2, and CCR5: Role of Protein Kinase C-{epsilon}1

Mohd W. Nasser*, Robin J. Marjoram*, Stephan L. Brown{dagger} and Ricardo M. Richardson2,*,{dagger}

* Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, NC 27707; and {dagger} Department of Biochemistry, Meharry Medical College, Nashville, TN 37208


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The IL-8 (or CXCL8) chemokine receptors, CXCR1 and CXCR2, activate protein kinase C (PKC) to mediate leukocyte functions. To investigate the roles of different PKC isoforms in CXCL8 receptor activation and regulation, human mononuclear phagocytes were treated with CXCL8 or CXCL1 (melanoma growth-stimulating activity), which is specific for CXCR2. Plasma membrane association was used as a measure of PKC activation. Both receptors induced time-dependent association of PKC{alpha}, -{beta}1, and -{beta}2 to the membrane, but only CXCR1 activated PKC{epsilon}. CXCL8 also failed to activate PKC{epsilon} in RBL-2H3 cells stably expressing CXCR2. {Delta}CXCR2, a cytoplasmic tail deletion mutant of CXCR2 that is resistant to internalization, activated PKC{epsilon} as well as CXCR1. Expression of the PKC{epsilon} inhibitor peptide {epsilon}V1 in RBL-2H3 cells blocked PKC{epsilon} translocation and inhibited receptor-mediated exocytosis, but not phosphoinositide hydrolysis or peak intracellular Ca2+ mobilization. {epsilon}V1 also inhibited CXCR1-, CCR5-, and {Delta}CXCR2-mediated cross-regulatory signals for GTPase activity, Ca2+ mobilization, and internalization. Peritoneal macrophages from PKC{epsilon}-deficient mice (PKC{epsilon}–/–) also showed decreased CCR5-mediated cross-desensitization of G protein activation and Ca2+ mobilization. Taken together, the results indicate that CXCR1 and CCR5 activate PKC{epsilon} to mediate cross-inhibitory signals. Inhibition or deletion of PKC{epsilon} decreases receptor-induced exocytosis and cross-regulatory signals, but not phosphoinositide hydrolysis or peak intracellular Ca2+ mobilization, suggesting that cross-regulation is a Ca2+-independent process. Because {Delta}CXCR2, but not CXCR2, activates PKC{epsilon} and cross-desensitizes CCR5, the data further suggest that signal duration leading to activation of novel PKC may modulate receptor-mediated cross-inhibitory signals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chemokines are a large family of chemotactic cytokines that induce leukocyte chemoattraction and activation at sites of inflammation (1, 2). These functions are mediated via specific cell surface G protein-coupled receptors (GPCRs)3 (3). Chemokine receptors couple predominantly to Gi to activate phospholipase C and increase intracellular Ca2+, activation of phospholipase D (PLD), generation of diacylglycerides, and activation of protein kinase C (PKC) (4, 5). Chemokines and chemokine receptors are redundant in their interactions (4). Because individual receptors mediate multiple and distinct signaling pathways upon activation, cross-desensitization among multiple chemokines seems to play an important role in limiting their signal redundancy (6). CXCL8 activates two receptors, CXCR1 and CXCR2, to mediate cellular responses. CXCR1 is specific for CXCL8, whereas CXCR2 also interacts with CXCL1, epithelial cell-derived neutrophil attractant 78 (CXCL5), and neutrophil-activating peptide-2 (CXCL7). CXCR1, but not CXCR2, activates PLD, superoxide production, and cross-desensitization pathways (7, 8). Studies with leukocytes and transfected RBL-2H3 indicated that cross-desensitization requires sustained PKC activation, which, in the case of CXCR2, was prevented by rapid receptor phosphorylation and internalization (9, 10).

PKC is a family of at least 10 isoforms that can be divided into three groups: the classical PKCs (or calcium and diacylglycerol (DAG) dependent) {alpha}, {beta}I, {beta}II, and {gamma}; the novel (or calcium independent) {delta}, {epsilon}, {eta}, and {theta}; and the atypical (or calcium and DAG independent) {zeta} and {iota} (11, 12). The specificity of many signal transduction pathways relies on the sequential activation of PKC by Ca2+ and DAG (13). Several leukocyte responses, including secretion and respiratory burst, are mediated through PKC-activating pathways (14, 15, 16, 17). In this study, human mononuclear phagocytes, murine peritoneal macrophages, and stably transfected RBL-2H3 cells were used to investigate the roles of different PKC isoforms in the ability of CXCR1, CXCR2, and CCR5 to mediate cross-regulatory signals. The data from this study demonstrate that CXCR1 and CCR5, but not CXCR2, activate PKC{epsilon} to cross-desensitize cellular responses and limit signal redundancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

[35S]Guanosine-5',0-(3-thiotriphosphate) ([35S]GTP{gamma}S), [32P]GTP, and myo-[2-3H]inositol were purchased from DuPont-NEN. IL-8 (CXCL8, monocyte derived), melanoma growth-stimulating activity (human CXCL1), keratinocyte-derived chemokine (KC; murine CXCL1), MIP-1{beta} (human CCL4), and RANTES (CCL5, human and murine) were purchased from PeproTech. Geneticin (G418) and all tissue culture reagents were purchased from Invitrogen Life Technologies. PKC and {beta}-actin Abs were purchased from Santa Cruz Biotechnology. 12CA5 mAb and protease inhibitors were purchased from Roche. Anti-human IL-8RA (CXCR1) and IL-8RB (CXCR2) Abs were purchased from BD Pharmingen. HRP-conjugated sheep anti-mouse Ab and ECL were purchased from Amersham Biosciences. Mono-Poly resolving medium Ficoll-Hypaque density and cytochrome c were purchased from ICN. Indo-1/AM and pluronic acid were purchased from Molecular Probes. Superoxide dismutase, zymosan A, PMA, GDP, GTP, GTP{gamma}S, ATP, and M2-Flag Ab were purchased from Sigma-Aldrich. All other reagents were obtained from commercial sources. cDNA encoding the tail-deleted mutant of CXCR2, {Delta}CXCR2 (331T), was provided by Dr. A. Richmond (Vanderbilt University, Nashville, TN). Plasmids containing a flag epitope tag followed by the sequence encoding aa 2–144 of the PKC{epsilon} ({epsilon} V1) or PKC {delta} ({delta}V1) were gifts from Drs. I. Mook Jung (Ajou University School of Medicine, Suwan, Korea) and D. Mochly-Rosen (Stanford University School of Medicine, Stanford, CA). cDNA encoding the GFP-tagged PKC{epsilon} was provided by Dr. M. G. Caron (Duke University, Durham, NC). PKC{epsilon}-deficient mice were provided by Dr. R. O. Messing (University of California, San Francisco, CA).

Construction of epitope-tagged CXCR1, CXCR2, and CCR5

Nucleotides encoding the nine-amino acid (YPYDVPDYA) hemagglutinin (CXCR1) or the octapeptide (DYKDDDDK) Flag (CCR5) epitope sequences were inserted between the N-terminal initiator methionine and the second amino acid of each cDNA by PCR as described previously (10, 18). The resulting PCR products were cloned into the eukaryotic expression vector pcDNA3, and the receptors were sequenced to confirm the intended mutations and lack of secondary mutations.

Cell culture and transfection

RBL-2H3 cells were maintained as monolayer cultures in DMEM supplemented with 15% heat-inactivated FBS, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 mg/ml) (19). RBL-2H3 cells (107 cells) were transfected by electroporation with 20 µg of pcDNA3 containing the receptor cDNAs, and geneticin-resistant cells were cloned into single cells by FACS analysis (10, 18, 19). Levels of protein expression were monitored by FACS analysis and Western blotting, using 12CA5 (hemagglutinin), M2 (Flag), and CXCR1- and CXCR2- specific Abs.

Isolation of human mononuclear phagocytes and murine peritoneal macrophages

Mononuclear phagocytes were isolated from heparinized human blood on a multiple density gradient as described previously (20, 21). The murine macrophages were isolated from the mouse peritoneal cavity as described previously (22). Briefly, mice were injected i.p. with zymosan A (1 mg/ml sterile saline). After 36 h, animals were killed by carbon dioxide exposure, peritoneal cavities were lavaged with 5 ml of RPMI 1640 containing 2 mM EDTA, and differential cell counts were performed using a Neubauer hemocytometer and a light microscope. All experiments were approved by and conformed to the guidelines of the appropriate committees at North Carolina Central University.

Western blot analysis

Human mononuclear phagocytes (5 x 106) were treated with or without ligands for 1–5 min and washed three times with ice-cold PBS, and membranes were prepared and assayed for protein concentration as described previously (20). Membrane proteins (~50 µg) were resolved in 10% SDS-PAGE, transferred to nitrocellulose membrane, and probed with mouse mAbs against PKC {alpha}, {beta}I, {beta}II, {epsilon} or {beta}-actin. Abs were detected with HRP-conjugated sheep anti-mouse Ab and ECL. For RBL cells expressing the epitope-tagged {epsilon}V1 or {delta}V1 peptides, cell lysates were resolved in 12% SDS-PAGE, and nitrocellulose membranes were probed with M2 anti-Flag Ab.

Confocal microscopy studies

Transfected cells were plated overnight onto 35-mm plastic dishes containing a centered, 1-cm, glass-bottom well. Cells were placed in 1 ml of MEM buffered with 20 mM HEPES and treated with CXCL8 (100 nM) and PMA (100 nM) in the same medium. Images were collected with a laser scanning confocal microscope (LSM-410; Zeiss) (23).

Intracellular calcium measurement

For calcium mobilization, RBL-2H3 cells (5 x 106) or murine peritoneal macrophages (107) were washed with HEPES-buffered saline and loaded with 1 µM Indo-I/AM in the presence of 1 µM pluronic acid for 30 min at room temperature. Then the cells were washed and resuspended in 1.5 ml of buffer. The increase in intracellular calcium in the presence or the absence of ligands was measured as described previously (18, 19, 20). The percentage of homologous desensitization is determined as the percentage of Ca2+ mobilization elicited by a second dose of the same ligand. The percentage of heterologous desensitization is the percentage of Ca2+ mobilization obtained after pretreatment with PMA. The percentage of cross-desensitization is the percentage of Ca2+ mobilization elicited by a second dose of a different ligand compared with that when this ligand was used as the first dose.

Phosphoinositide (PI) hydrolysis.

RBL-2H3 cells were subcultured overnight in 96-well culture plates (50,000 cells/well) in inositol-free medium supplemented with 10% dialyzed FBS and 1 µCi/ml [3H]inositol. The generation of inositol phosphates (IPs) was determined as previously reported (18, 19).

Secretion of {beta}-hexosaminidase

RBL-2H3 cells were subcultured overnight in 96-well culture plates (50,000 cells/well). Cells were washed with HEPES-buffered saline containing 0.1% BSA, then treated with and without agonist, and {beta}-hexosaminidase release was assessed as previously reported (18, 19).

GTPase activity and [35S]GTP{gamma}S binding

Cells (107) were treated with the appropriate concentrations of stimulants, and membranes were prepared as previously described (18, 20). [35S]GTP{gamma}S binding and GTPase activity using 10–20 µg of membrane preparations were conducted as described previously (10, 18, 20).

Radioligand binding assays and receptor internalization

RBL-2H3 cells were subcultured overnight in 24-well plates (0.5 x 106 cells/well) in growth medium. Cells were then rinsed with DMEM supplemented with 20 mM HEPES, pH 7.4, and 10 mg/ml BSA and incubated in the same medium containing CCL5 or CXCL8 (100 nM) for 60 min. For radioligand binding, cells were placed on ice, washed three times with ice-cold PBS containing 10 mg/ml BSA, and incubated for 2–4 h in DMEM (250 µl) containing the radiolabeled ligand (0.1 nM). Reactions were stopped with 1 ml of ice-cold PBS, and cells washed five times. Cells were then solubilized with RIPA buffer (200 µl) and dried under vacuum, and bound radioactivity was counted. Nonspecific radioactivity bound was determined in the presence of 500 nM unlabeled ligand (10).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CXCR1- and CXCR2-mediated PKC activation

To determine the ability of CXCR1 and CXCR2 to activate PKCs, mononuclear phagocytes isolated from blood were treated with 100 nM CXCL8, which activates CXCR1 and CXCR2, or with CXCL1, which is specific for CXCR2, for different periods of time. Plasma membrane association of PKC was determined by Western blotting using anti-PKC{alpha}, -{beta}I-, -{beta}II-, and -{epsilon}-specific Abs. As shown in Fig. 1, both CXCL8 and CXCL1 induced time-dependent association of PKC{alpha}, -{beta}I, and {beta}II to the membrane. The time course of CXCL8-mediated PKC association, however, was more sustained than that of CXCL1. Interestingly, CXCL8, but not CXCL1, showed a robust increase in PKC{epsilon} association to the plasma membrane (~1–5 min; Fig. 1A). Mouse peritoneal macrophages pretreated with murine CXCL1 also showed no increase in PKC{epsilon} association to the plasma membrane compared with untreated cells (data not shown).



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  		FIGURE 1. CXCR1 and CXCR2 mediated PKC activation in human mononuclear phagocytes and RBL-2H3 cells. A, Human mononuclear phagocytes purified from blood were stimulated with CXCL8 (100 nM) and CXCL1 (100 nM) for different periods of time. Reactions were stopped with ice-cold PBS. Cells (5 x 106) were washed twice with PBS, and membranes were prepared as described in Materials and Methods. Membrane proteins (~50 µg/lane) were separated by SDS-PAGE, and PKC{alpha}, -{beta}I, -{beta}II, and -{epsilon} were analyzed by Western blotting using specific anti-PKC Abs. The experiment was repeated with four donors, with similar results. B, RBL-2H3 cells stably expressing CXCR1 (RBL-CXCR1) or CXCR2 (RBL-CXCR2) were treated with CXCL8 (100 nM) for different periods of time and assayed for PKC{epsilon} association to the plasma membrane as described above. Data shown are representative of one of three experiments.

 
Because CXCL8 activates both CXCR1 and CXCR2 in leukocytes (8), we further investigated the specificity of the CXCL8 receptors in mediating PKC{epsilon} activation using RBL-2H3 cells stably expressing CXCR1 (RBL-CXCR1) or CXCR2 (RBL-CXCR2). As shown in Fig. 1B, CXCL8 activation of CXCR1 showed a sustained increase in PKC{epsilon} association to the plasma membrane. A slight and transient increase in CXCR2-mediated PKC{epsilon} association was detected at 30–60 s in both mononuclear phagocytes and RBL-2H3 cells (Fig. 1).

Role of signal strength in CXCL8-mediated PKC{epsilon} activation

Upon activation by CXCL8, CXCR2 was shown to mediate shorter cellular responses than CXCR1 due to rapid receptor down-regulation (9, 10). To determine the role of signal length in the different abilities of the receptors to activate PKC{epsilon}, CXCR1, CXCR2, or {Delta}CXCR2 (a C terminus-truncated and internalization-resistant mutant of CXCR2) were transfected in RBL-2H3 cells along with a GFP-tagged PKC{epsilon}. CXCL8-mediated PKC{epsilon} translocation to the cell membrane was studied by fluorescence microscopy. As shown in Fig. 2, CXCR1, but not CXCR2, induced PKC{epsilon} translocation to the cell membrane. {Delta}CXCR2, which was previously shown to mediate greater cellular activation than CXCR2 and trigger signal for cross-regulation (9, 10), induced PKC{epsilon} translocation in response to CXCL8 (Fig. 2). PMA-mediated PKC{epsilon} activation in all three cell lines.



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  		FIGURE 2. CXCL8 and PMA mediated PKC{epsilon} translocation. RBL-2H3 cells coexpressing GFP-tagged PKC{epsilon} along with CXCR1, CXCR2, or {Delta}CXCR2 were plated overnight onto an eight-well coverglass plate (0.2 x 106 cells/well). Cells were placed in 0.2 ml of MEM buffered with 20 mM HEPES and treated with CXCL8 (100 nM) or PMA (100 nM) in the same medium for 5 min. Cells were fixed with 4% paraformaldehyde, and images were collected with a Zeiss laser scanning confocal microscope (LSM-410). Shown are representative confocal microscopic images of three (CXCR1 and {Delta}CXCR2) to five (CXCR2) different experiments.

 
The specificity of PKC{epsilon} was also assessed using a Flag-tagged PKC{epsilon} fragment, {epsilon}V1 (24, 25). {epsilon}V1 is a specific binding region to the receptor-activated C-kinase, which specifically inhibits PKC{epsilon} translocation to the plasma membrane (24, 25). RBL cells stably coexpressing CXCR1 and CCR5 (ACCR5) (10) were transfected with vector expressing {epsilon}V1 or {delta}V1 or with vector alone. The expression of {epsilon}V1 (Fig. 3A, lanes 2 and 3) and {delta}V1 (Fig. 3A, lanes 4 and 5) fragments was confirmed by Western blot using the Flag M2-specific Ab. {epsilon}V1 expression, but not {delta}V1 or vector alone, blocked CXCL8- and CCL5-induced PKC{epsilon} translocation to the plasma membrane (Fig. 3B).



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  		FIGURE 3. Effects of the PKC inhibitor fragments {epsilon}V1 and {delta}V1 on PKC{epsilon} activation. A, RBL cells stably expressing CXCR1 and CCR5 (ACCR5) and GFP-tagged PKC{epsilon} were transfected with pBabe vector alone (lane 1) or vector containing the {epsilon}V1 (lanes 2 and 3) or {delta}V1 (lanes 4 and 5) fragments. The expression of {epsilon}V1 and {delta}V1 fragments was confirmed by Western blot using the Flag M2-specific Ab. B, Cells were plated overnight onto an eight-well coverglass plate (0.2 x 106 cells/well) treated with CXCL8 (100 nM) or CCL5 (100 nM) for 5 min. Cells were fixed with 4% paraformaldehyde, and images were collected with a Zeiss laser scanning confocal microscope (LSM-410). Shown are representative confocal microscopic images of three different experiments.

 
Role of PKC{epsilon} in CXCR1- and CCR5-mediated cellular responses

Expression of the {epsilon}V1 fragment in ACCR5 had no effect on CXCR1-mediated (CXCL8) and CCR5-mediated (CCL5 and MIP-1{beta} or CCL4) PI hydrolysis (Fig. 4A) or peak intracellular Ca2+ mobilization (Fig. 4B), but inhibited secretion of {beta}-hexosaminidase by ~50% (Fig. 4, C and D). Expression of the {delta}V1 fragment or vector alone had no significant effect on receptor-mediated PI hydrolysis, secretion of {beta}-hexosaminidase, or Ca2+ mobilization (Fig. 4 and data not shown).



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  		FIGURE 4. Effects of {epsilon}V1 and {delta}V1 on CXCR1- and CCR5-mediated cellular responses. A, For the generation of IPs ([3H]IPs), ACCR5 RBL-2H3 cells (50,000 cells/well) expressing pBabe vector alone or vector containing {epsilon}V1 or {delta}V1 fragments were cultured overnight in the presence of [3H]inositol (1 µCi/ml). Cells were preincubated (10 min, 37°C) with HEPES-buffered saline containing 10 mM LiCl in a total volume of 200 µl and stimulated with 100 nM CXCL8, CCL5, or MIP-1{beta}, for 10 min. Supernatant was used to determine the release of [3H]IPs. Data are represented as the fold stimulation over the basal level. The experiment was repeated three times with similar results. B, For intracellular Ca2+ mobilization, ACCR5 RBL-2H3 cells (~5 x 106 cells/assay) expressing the {epsilon}V1 fragment or vector alone were loaded with Indo-1, and CXCL8 or CCL5 (10 nM)-stimulated Ca2+ mobilization was measured. Representative tracings of three experiments are shown. C and D, For secretion, ACCR5 RBL-2H3 cells (50,000 cells/well) expressing pBabe vector alone or vector containing {epsilon}V1 or {delta}V1 fragments were seeded as indicated in A and stimulated with different concentrations of CXCL8 or CCL5 for 10 min. Supernatant (15 µl) was removed, and {beta}-hexosaminidase release was measured as described in Materials and Methods. Data are presented as a percentage of the total {beta}-hexosaminidase in the cells. The data shown are the mean ± SD from three independent experiments performed in triplicate. Statistically significant results relative to control or untreated cells were calculated using Student’s t test (INSTAT; GraphPad): *, p < 0.01; **, p < 0.001.

 
Role of PKC{epsilon} in CXCR1- and CCR5-mediated cross-desensitization

To determine the role of PKC{epsilon} in CXCL8- and CCL5-induced cross-desensitization, {epsilon}V1 fragment or vector alone was expressed in ACCR5 and BCCR5 (RBL cells stably coexpressing CXCR2 and CCR5) (10) RBL cells. Ligand-induced GTPase activity in membrane was measured. As shown in Fig. 5, CXCL8 and CCL5 pretreatment of ACCR5 (Fig. 5, A and B) and BCCR5 (Fig. 5, C and D) cells homologously desensitized GTPase activity to the same agonist, relative to control or untreated cells. In ACCR5 cells expressing the vector alone (Fig. 5A), but not BCCR5 cells (Fig. 5C), and pretreated with CXCL8, CCL5-mediated GTPase activity was reduced by ~45%. ACCR5 (Fig. 5A) and BCCR5 (Fig. 5C) pretreated with CCL5 also exhibited a 50–60% decrease in CXCL8-mediated GTPase activity. Expression of the {epsilon}V1 fragment in ACCR5 cells (Fig. 5B) significantly reduced CXCL8-mediated cross-desensitization of CCL5 (~25%). CCL5-mediated cross-desensitization of CXCL8 was also diminished in ACCR5 (~20%; Fig. 5B) and BCCR5 (7%; Fig. 5D) cells expressing the {epsilon}V1 fragment. As expected, CCL5-mediated GTPase activity was resistant to cross-desensitization by CXCL8 in BCCR5 cells that express the CXCR2 receptor (Fig. 5, C and D).



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  		FIGURE 5. Effect of the {epsilon}V1 fragment in cross-desensitization of GTPase activity. RBL-2H3 cells expressing CXCR1 and CCR5 (ACCR5; A and B) or CXCR2 and CCR5 (BCCR5; C and D) were transfected with pBabe vector (A and C) or vector containing {epsilon}V1 fragment (B and D). Cells were pretreated with CXCL8 (100 nM) or CCL5 (100 M) for ~5 min. Membranes were prepared and assayed for agonist-stimulated GTPase activity. Data are presented as a percentage of maximum stimulation, which is the net maximal stimulation obtained with untreated cells. The data shown are the mean ± SD from three independent experiments performed in triplicate. Statistically significant results relative to control or untreated cells were calculated using Student’s t test (INSTAT; GraphPad): *, p < 0.01; **, p < 0.001.

 
The effect of {epsilon}V1 on CXCL8- and CCL5-induced cross-desensitization of intracellular Ca2+ mobilization was also determined in ACCR5 cells expressing {epsilon}V1 vs control or vector alone. As shown in Table I, {epsilon}V1 had no effect on CXCL8- and CCL5-mediated homologous (CXCL8->CXCL8 and CCL5->CCL5) and heterologous (PMA->CXCL8 and PMA->CCL5) desensitization of Ca2+ mobilization. {epsilon}V1 expression, however, diminished CXCL8- and CCL5-mediated cross-desensitization of Ca2+ mobilization to each other by ~70% (50 vs 16% for CXCL8->CCL5) and ~83% (41 vs 7% for CCL5->CXCL8), respectively (Table I).


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  		Table I. Effect of PKC{epsilon} in cross-desensitization among CCR5 and CXCR1a

 
Role of PKC{epsilon} in receptor-mediated cross-internalization

CXCL8 stimulation of CXCR1 and {Delta}CXCR2, but not CXCR2, was previously shown to cross-internalize CCR5 (10). We next determined the role of PKC{epsilon} in CXCR1-mediated cross-internalization of CCR5. ACCR5 cells were treated with CXCL8 or CCL5 for 60 min, and 125I-labeled CCL5 binding was performed to measure receptor internalization. As shown in Fig. 6, pretreatment of ACCR5-RBL cells expressing the vector alone with either CXCL8 or CCL5 caused ~64 and ~45% internalizations of CCR5, respectively. Expression of the {epsilon}V1 fragment diminished CCL5-mediated homologous internalization of CCR5 (~40%) and CXCL8-mediated cross-internalization (~3%). CXCR1 was resistant to cross-internalization by CCL5 (data not shown) (10).



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  		FIGURE 6. Effect of the {epsilon}V1 fragment on CXCL8-mediated cross-internalization of CCR5. ACCR5 RBL cells (0.5 x 106 cells/well) expressing the vector or vector containing the {epsilon}V1 fragment were pretreated with 100 nM of either CXCL8 or CCL5 for 60 min at 37°C. Cells were then washed and assayed for 125I-labeledCCL5 binding. The values are presented as a percentage of the total, which is defined as the total amount of 125I-labeledCCL5 bound to control (untreated) cells. The data shown are the mean ± SD from two independent experiments performed in triplicate. Statistically significant results relative to control or untreated cells were calculated using Student’s t test (INSTAT; GraphPad): *, p < 0.01; **, p < 0.001.

 
In vivo role of PKC{epsilon} in cross-desensitization between CCR5 and CXCR2

The role of PKC{epsilon} in cross-desensitization was also determined in murine peritoneal macrophages from PKC{epsilon}-deficient mice (PKC{epsilon}–/–) vs control animals (PKC{epsilon}+/+) (26). Because mice only expresses a human homologue of CXCR2, KC, the murine homologue of CXCL1 was used (27). CCL5 and CXCL1 homologously desensitized Ca2+ mobilization in response to a second dose of the same ligand in both PKC{epsilon}–/– and PKC{epsilon}+/+ (Fig. 7). CCL5-mediated cross-desensitization of Ca2+ mobilization to CXCL1, however, was markedly decreased in macrophages from PKC{epsilon}–/– (Fig. 7, right panel) relative to wild-type mice PKC{epsilon}+/+ (Fig. 7, left panel). CXCL1-mediated GTP{gamma}S binding in membrane was also resistant to cross-desensitization by CCL5 in PKC{epsilon}–/– mice (Fig. 8B) relative to PKC{epsilon}+/+ control mice (Fig. 8A). CXCL1 pretreatment had no effect on CCL5-mediated Ca2+ mobilization or GTP{gamma}S binding in both PKC{epsilon}–/– and PKC{epsilon}+/+ mice (Fig. 7 and data not shown).



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  		FIGURE 7. CCL5-mediated cross-desensitization of CXCR2 in murine macrophages deficient in PKC{epsilon} expression. For intracellular Ca2+ mobilization, zymosan-elicited peritoneal macrophages (107 cells) were loaded with Indo-1 and stimulated with 10 nM CXCL1 or CCL5. Cells were rechallenged 3 min later with a second dose of the indicated ligand. Each tracing represents an analysis from a single mouse of the indicated PKC{epsilon} genotype. The data shown are representative of three experiments.

 


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  		FIGURE 8. Cross-desensitization of CXCR2-induced GTP{gamma}S binding in membranes from PKC{epsilon}–/– macrophages. For [35S]GTP{gamma}S binding, zymosan-elicited peritoneal macrophages (107 cells) from wild-type, PKC{epsilon}+/+ (A) and PKC{epsilon}-deficient, PKC{epsilon}–/– (B), mice were treated with or without CCL5 (100 nM) as described in Materials and Methods. Membranes were prepared and assayed for CXCL1 (100 nM)-stimulated [35S]GTP{gamma}S binding. Results shown are representative of one of four experiments performed in triplicate. Statistically significant results relative to control or untreated cells were calculated using Student’s t test (INSTAT; GraphPad): *, p < 0.01; **, p < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Most chemokines and their receptors have redundant specificity, in that many chemokines activate more than one chemokine receptor, and many chemokine receptors are activated by multiple chemokines. Studies with neutrophils and RBL cells coexpressing different combinations of chemokine receptors indicated that cross-desensitization among multiple chemokines may be important in limiting their signal redundancy at the site of inflammation and infection (6). To date, the molecular events underlying receptor cross-desensitization are poorly understood. One of the sites of cross-desensitization appears to be at the level of receptor/G protein coupling and involves cross-phosphorylation of the receptor by a PKC-dependent mechanism, followed by receptor uncoupling from its G protein (6, 9). The second one is an as yet unidentified modulation of a downstream component leading to decreased activation of phospholipase C (6, 9). CXCL8 activates CXCR1 and CXCR2, but only CXCR1 mediates receptor cross-phosphorylation and cross-desensitization (9, 10). This study was designed to investigate the roles of different PKC isoforms in the distinct ability of CXCL8 receptors to mediate cross-desensitization. The data from this study demonstrated that CXCR1, but not CXCR2, activates PKC{epsilon} to mediate receptor cross-desensitization and cross-internalization. This contention is supported by the following observations. First, in human mononuclear phagocytes, CXCL8, but not CXCL1, increased PKC{epsilon} association with the plasma membrane (Fig. 1). Second, in RBL cells stably coexpressing GFP-tagged PKC{epsilon} and either CXCR1 or CXCR2, CXCL8 activation of CXCR1, but not CXCR2, induced PKC{epsilon} translocation to the membrane (Fig. 2). Third, expression of the PKC{epsilon} inhibitor peptide {epsilon}V1 in ACCR5-RBL cells blocked PKC{epsilon} translocation to the plasma membrane and reduced CXCL8-mediated cross-desensitization of both intracellular Ca2+ mobilization and GTPase activity to CCL5 as well as cross-internalization of CCR5 (Table I and Figs. 3, 5, and 6).

Previous studies from our laboratory and others suggested that signal duration resulting in activation of PLD and sustained production of DAG is important for chemokine receptors to mediate leukocyte cytotoxicity and activate cross-inhibitory pathways (7, 9, 28, 29). The data presented in this study further support this contention. First, inhibition of PKC{epsilon} in RBL cells had no effect in CXCL8- or CCL5-mediated PI hydrolysis or peak intracellular Ca2+ mobilization, but diminished receptor-mediated exocytosis by ~50% (Fig. 4 and Table I). Second, peritoneal macrophages from PKC{epsilon}–/– mice also showed no change in CCL5-induced peak intracellular Ca2+ mobilization (Fig. 7), but exhibited a significant decrease in superoxide production relative to PKC{epsilon}+/+ macrophages (data not shown). Third, the phosphorylation- and desensitization-resistant mutant of CXCR2, {Delta}CXCR2, which generated longer signals (i.e., GTPase activity, PI hydrolysis, and Ca+2 mobilization) (9, 10) than CXCR2 and activated PLD, induced PKC{epsilon} translocation and cross-desensitization of the Ca2+ response and GTPase activity to CCR5 (Fig. 2 and data not shown).

Of interest is that PKC{epsilon} inhibition had no effect on the receptor homologous and heterologous desensitization of Ca2+ mobilization and G protein activation, but inhibited cross-desensitization (Table I and Figs. 5, 7, and 8). Homologous desensitization is mediated predominantly via GPCR kinase-dependent mechanisms (30, 31). The heterologous and cross-desensitizations of chemoattractant receptors, however, are both PKC dependent (6, 30, 31). Thus, the lack of effect of PKC{epsilon} inhibition on heterologous desensitization probably indicates that receptor heterologous and cross-desensitizations are two independent signaling events mediated via different PKC-dependent pathways. Heterologous phosphorylation and desensitization are early signaling events, which occur via transient activation of PKCs, whereas cross-desensitization requires prolonged receptor stimulation and sustained PKC activation. Supporting this contention is the finding that upon activation by CXCL8, both CXCR1 and CXCR2 induced transient activation of PKC{alpha}, -{beta}1, and -{beta}2 and underwent heterologous desensitization (Fig. 1 and Table I). Furthermore, {Delta}CXCR2, but not CXCR2, which is resistant to receptor internalization, mediated signal for cross-desensitization of CCR5 (Fig. 5) (9, 10).

Zhang et al. (32) demonstrated that CCR1 predominantly activates PKC{delta} to cross-desensitize the response to the opioid receptor. In RBL-2H3 cells, however, expression of the {delta}V1 fragment had no significant effect on CXCL8-mediated cross-desensitization of Ca2+ mobilization and GTPase activity to CCR5. One possible explanation could be that CCR1 and CXCR1 activate different novel PKC isoforms to mediate cross-desensitization. Second, as indicated by the authors, because PKC{epsilon} translocated predominantly to the Golgi in NIH-3T3 cells pretreated with PMA, its involvement in CCR1 cross-desensitization was not assessed (33). As shown in Fig. 2, however, both CXCL8 and PMA induced PKC{epsilon} translocation from the cytosol to the membrane in RBL-2H3 cells. PMA also induced PKC{epsilon} translocation to the membrane in human mononuclear phagocytes and alveolar macrophages (34, 35).

The data from this study, however, do not exclude the participation of other PKC isoforms in CXCL8-mediated receptor cross-desensitization. First, staurosporine reversed CXCL8-mediated cross-desensitization of CCR5 by ~100%, whereas inhibition or deletion of PKC{epsilon} only caused an ~80% decrease in receptor cross-desensitization (9, 36). Second, previous studies have shown that PKC{alpha} and PKC{beta} are important for chemoattractant receptor-mediated activation and regulation of exocytosis and respiratory burst (14, 15, 16, 17). Thus, it is possible that sustained activation of both classical and novel PKCs by DAG is required for receptor to cause cross-phosphorylation, cross-desensitization, and cross-internalization of susceptible receptors as well as modification of downstream effector activities.

In summary, the results indicate that CXCR1 and CCR5, but not CXCR2, activate PKC{epsilon} to mediate cross-desensitization of intracellular Ca2+ mobilization and GTPase activity and cross-internalization. {Delta}CXCR2, which generates longer signals relative to CXCR2, activates PKC{epsilon} and mediates cross-desensitization. Thus, chemokines interacting with different receptors, based on signal strength, may activate selective PKC isoforms to mediate receptor cross-regulation and limit signal redundancy at sites of inflammation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI38910 and CA92077 and U.S. Department of Veterans Affairs Grant 626/151. Back

2 Address correspondence and reprint requests to Dr. Ricardo M. Richardson, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707. E-mail address: mrrichardson{at}nccu.edu Back

3 Abbreviations used in this paper: GPCR, G protein-coupled receptor; DAG, diacylglycerol; GTP{gamma}S, guanosine-5',0-(3-thiotriphosphate); IP, inositol phosphate; KC, keratinocyte-derived chemokine; PI, phosphoinositide; PKC, protein kinase C; PLD, phospholipase D. Back

Received for publication November 22, 2004. Accepted for publication March 15, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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