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Published online before print February 3, 2004
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* Laboratory for Psychoneuroimmunology, University Medical Center Utrecht, The Netherlands;
Departamento de Biología Molecular, Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autonóma de Madrid, Spain; and
Howard Hughes Medical Institute Laboratories, Departments of Cell Biology and Medicine, Duke University Medical Center, Durham, North Carolina
1Correspondence: University Medical Center, Room KC03.068.0, Lundlaan 6, 3584 EA, Utrecht, The Netherlands. E-mail: c.heijnen{at}wkz.azu.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50%. However, the physiological relevance of reduced GRK2 levels in lymphocytes is not known. Here, we investigated whether reduced GRK2 expression changes the chemotactic response of T cells to the chemokines CCL3, CCL4, and CCL5. Activated T cells from GRK2+/– mice, which have a 50% reduction in GRK2 protein levels, showed a significant 40% increase in chemotaxis toward the CCR5 ligand CCL4. In addition, chemotaxis toward the CCR1 and CCR5 ligands CCL3 and CCL5 was also increased. Binding of CCL4 to activated T cells from GRK2+/– and wild-type (WT) mice was similar, but agonist-induced CCR5 phosphorylation was attenuated in GRK2+/– cells. Moreover, the calcium response and phosphorylation of protein kinase B and extracellular-regulated kinase in response to CCL4 were significantly increased in GRK2+/– T cells, showing that signaling is increased when the level of GRK2 is reduced. GRK2+/– and WT cells do become refractory to restimulation with CCL4. In conclusion, a 50% decrease in T cell GRK2 expression results in increased responsiveness to CCL3, CCL4, and CCL5, suggesting that the 50% reduction in lymphocyte GRK2 level as observed during inflammation can have functional consequences for the response of these cells to chemokines.
Key Words: receptor regulation • knockout mice • migration • G protein-coupled receptor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies in overexpression systems have shown that the extent of agonist-induced desensitization of GPCR depends on the intracellular availability of GRKs and ß-arrestins [10 11 ]. In addition, several studies have shown that inactivation of the GPCR desensitization machinery can enhance and/or prolong physiological responses to GPCR ligands in whole animals and specific organ systems [12 13 14 15 16 ].
GRK2 is expressed in many tissues, and particularly high expression levels have been described in cells of the immune system [17 18 ]. Moreover, T lymphocyte activation is accompanied by alterations in GRK2 activity and GRK2 expression [17 19 ]. We have previously shown that induction of an autoimmune disease in rats results in changes in GRK2 protein levels in the immune system. GRK activity as well as GRK2 protein expression were reduced in immune cells during the acute phase of adjuvant arthritis in the rat [20 ] as well as during relapsing progressive experimental autoimmune encephalomyelitis (EAE) [21 ]. In addition, we observed a 50% decrease in the level of GRK2 in peripheral blood mononuclear cells (PBMC) of humans with rheumatoid arthritis [22 ] and multiple sclerosis (unpublished results).
In model systems overexpressing GRK and GPCR, it has been shown that GRK2 can phosphorylate a large number of GPCR that are relevant for immune functioning, including several chemokine receptors, prostaglandin receptors, adrenergic receptors, opioid receptors, muscarinic receptors, and the substance P receptor [1 ]. However, it is not known whether the regulated expression of GRK2 in the immune system is of physiological importance. In addition, physiologically relevant GRK2 substrate receptors in the immune system have not yet been widely examined.
Chemokine/chemoattractant receptors are expressed in immune cells, including macrophages, dendritic cells, and (activated) lymphocytes. These GPCR play an important role in directing the migration of leukocytes to inflamed tissue [23 24 25 ]. In cell lines transfected with chemokine receptors and GRK, it has been shown that agonist-induced phosphorylation and/or desensitization of chemokine/chemoattractant receptors, such as BLT-1, CCR2b, and CCR5, are promoted by GRKs [26 27 28 29 30 ]. Moreover, it has been shown in transfected cell lines that overexpression of GRK results in reduced receptor signaling [26 27 28 ].
In view of our previous observations that GRK2 levels are reduced during arthritis and EAE, the aim of our study was to get more insight into the physiological importance of reduced GRK2 levels in normal T cells. To address this issue, we used activated T cells from GRK2+/– mice and showed that the level of GRK2 in these cells is reduced by 50%, which makes it an excellent model to investigate functional consequences of a 50% reduction in GRK2 level. We analyzed chemotaxis and signaling in response to the CCR5 ligand CCL4 [macrophage-inflammatory protein-1ß (MIP-1ß)]. In addition, we analyzed the chemotactic response to the CCR5/CCR1 ligands CCL3 (MIP-1
) and regulated on activation, normal T expressed and secreted (RANTES; CCL5).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T cell activation
Splenocytes were obtained by dispersion through filter chambers (NPBI, Emmer-Compascuum, The Netherlands). Subsequently, red blood cells were lysed, and splenocytes were resuspended in culture medium (RPMI 1640; Gibco, Grand Island, NY) supplemented with 5% fetal calf serum (FCS; Gibco), 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-mercaptoethanol. Splenic T cells were isolated by incubation of splenocyte cell suspensions on nylon wool columns for 45 min at 37°C. Cells were cultured at 2.5 x 106 cells/well in 24-well plates, coated with 1 µg/ml anti-CD3 for 48 h at 37°C. Subsequently, interleukin (IL)-2 was added to the cultures for another 72–96 h. In all assays, these activated T cells were used.
GRK expression
Splenocytes and activated T cells were lysed in ice-cold radio immunoprecipitation assay (RIPA) buffer [20 mM Hepes, pH 7.5, 1% Triton X-100, 150 mM NaCl, 10 mM EDTA, 2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 20 µg/ml leupeptin, and 200 µg/ml benzamidine] for 30 min at 4°C. Brains were lysed using tissue lysis buffer [50 mM Tris, pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycolate, 0.1% sodium dodecyl sulfate (SDS)]. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and analyzed for GRK and ß-arrestin expression by immunoblot as described previously [22
]. Immunoreactivity was detected by enhanced chemiluminescence (ECL; Amersham Int., Buckinghamshire, UK). Autoradiographs were scanned using a GS-700 imaging densitometer (Bio-Rad Laboratories, Hercules, CA).
Chemotaxis assay
T cell migration was analyzed using a transwell system with polycarbonate membranes and pore size of 5 µm (Costar, Corning, NY). Activated T cells were suspended at 5 x 106 cells/ml in RPMI 1640 supplemented with 0.5% FCS, and 100 µl cell suspension was placed in the top chamber. Medium containing varying concentrations of CCL3, CCL4, or CCL5 (600 µl; MIP-1, MIP-1ß, and RANTES, R&D Systems, The Netherlands) was added to the lower well. After incubation at 37°C for 2 h, cells were harvested from the lower well and counted by fluorescein-activated cell sorter (FACS) for 2 min. Migration was expressed as percent of cell input. To determine chemokinesis, the same concentration of ligand was added to upper and lower well.
Competitive CCL4 binding assay
Human CCL4 (MIP-1ß, NEN, Boston, MA) labeled with 125I was used as the radioligand, and murine CCL4 (MIP-1ß, R&D Systems) was used as competitive ligand. Activated T cells (5x105) were incubated for 90 min at 30°C with 0.1 nM radiolabeled ligand and increasing concentrations of the competitor in RPMI containing 20 mM Hepes and 0.5% bovine serum albumin (BSA) in a total volume of 150 µl. The reaction was stopped by the addition of ice-cold phosphate-buffered saline, supplemented with 0.5% BSA and 0.3 M NaCl, followed by rapid filtration through Whatman GF/C glass fiber filters (Whatman, Inc., Clifton, NJ) under vaccuum and additional washing. Cell-associated radioactivity was assessed in a 
-scintillation counter. Nonspecific binding was determined in the presence of 100 nM unlabeled CCL4. Curve fit, receptor number (Bmax), and dissociation constant [inhibitory concentration (IC)50] were calculated using Graphpad Prism software.
CCR5 phosphorylation
Activated T cells were metabolically labeled with 32Pi (Amersham Int.; 40 µCi/ml) for 3 hours. Cells were stimulated with 100 nM CCL4 for 5 min. After treatment, cells were lysed in lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1% SDS, 10 mM NaF) with protease inhibitors. CCR5 was immunoprecipitated using anti-CCR5 antibody (R&D Systems) and proteinA/G sepharose. Immunoprecipitates were dissociated and resolved by SDS-PAGE. Radioactively labeled receptors were visualized by autoradiography, and autoradiographs were scanned using a GS-700 imaging densitometer (Bio-Rad Laboratories).
Calcium signaling
Activated T cells were loaded with Fluo-3-AM(Molecular Probes, Eugene, OR) and stimulated with chemokine in assay buffer (145 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 1 mM CaCl2, 0.5 mM MgSO4.7H2O, 5 mM glucose, and 10 mM Hepes, pH 7.4). Changes in mean fluorescence intensity (MFI) were monitored using a FACSCalibur (Becton Dickinson, San Jose, CA). Traces represent MFI for 200–300 cells per second sampled in 5-s periods.
Measurement of phosphorylated protein kinase B (PKB) and ERK
Activated T cells were starved for 4 h in serum-free medium to minimize basal kinase activity, and cells (5x106 per point) were stimulated with increasing concentrations of CCL4 in a total volume of 200 µl at 37°C. The reaction was stopped on ice, and cells were lysed in 100 µl RIPA buffer (supplemented with 10 mM NaF and 1 mM Na3VO4). Proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and probed with antiphospho-ERK (Santa Cruz Biotechnology, Santa Cruz, CA) or antiphospho-PKB (Ser 473; Cell Signaling Technology, Beverly, MA). ECL (Amersham Int.) detected immunoreactivity. Blots were then stripped and reprobed for total cellular ERK-2 or PKB with monoclonal anti-ERK-2 (Santa Cruz Biotechnology) or polyclonal anti-PKB (a gift from Prof. Johannes L. Bos, Dept. Physiological Chemistry, University Medical Centre Utrecht) antibodies. Autoradiographs were scanned using a GS-700 imaging densitometer (Bio-Rad Laboratories). Quantitation of phospho-ERK/phospho-PKB was normalized to the total amount of ERK/PKB present, respectively.
Response to restimulation with CCL4
Cells were stimulated with CCL4 for 5–30 min, washed extensively on ice, and restimulated with CCL4 for 1 min. The reaction was stopped by addition of and equal volume of 6% paraformaldehyde/0.6% saponin in saline, and polymerized actin was stained by addition of fluorescein isothiocyanate (FITC)–phalloidin (final concentration, 0.2 µm). Intensity of fluorescence was determined by FACS analysis.
Data analysis
Data are expressed as mean and SEM and were confirmed in at least three independent experiments. Two-way ANOVA analyzed data for dose-response curves. P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50% reduction in GRK2 protein levels in the spleen (Fig. 1
). The reduction in GRK2 does not affect the expression of its cofactor ß-arrestin (Fig. 1)
. In addition, GRK2 expression is also decreased by 50% in nonimmune organs such as the brain (Fig. 1)
and heart [15
] of GRK2+/– mice.
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50% in cells from GRK2+/– mice (Fig. 1)
. ß-Arrestin levels were similar in activated T cells from WT and GRK2+/– mice (Fig. 1)
. In addition, no difference was observed in levels of GRK3, GRK5, and GRK6 between activated T cells from WT and GRK2+/– animals (data not shown).
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40% increase in migration to the optimal concentration of 10 nM CCL4 (WT: 16.9±0.9%; GRK2+/–: 23.5±1.5%; P<0.01; Fig. 2A
). No differences in chemokinesis were observed between WT and GRK2+/– cells when 10 nM CCL4 was added to the upper and lower chamber (WT: 11±0.3%; GRK2+/–: 12.0±0.4%). Moreover, spontaneous migration in the absence of chemokine did not differ between WT and GRK2+/– cells (Fig. 2)
.
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) and CCL5 (RANTES) was assessed. As is shown in Figure 2B
and 2C
, migration to the optimal concentration of CCL3 (1 nM) or to CCL5 was also increased significantly by 30% in the GRK2+/–mice (P<0.01 for CCL3 and CCL5, Fig. 2
).
CCL4 binding to activated T cells from GRK2+/– mice
It is possible that the increased chemotactic response to CCL4 is associated with alterations in the expression of CCL4 binding sites on cells from GRK2+/– mice. Therefore, we performed receptor-binding experiments with radiolabeled CCL4. As is shown in Figure 3
, competition-binding curves for binding of CCL4 to activated T cells from GRK2+/– and WT mice were comparable. There was no difference in IC50 and Bmax for binding CCL4 to WT and GRK2+/– T cells (Table 3
). Therefore, we conclude that the reduction in GRK2 level does not result in altered expression of receptors for CCL4.
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CCL4-induced CCR5 phosphorylation
To investigate whether reduced GRK2 has consequences for CCR5 phosphorylation, we labeled T cells metabolically with 32Pi, stimulated the cells with CCL4, and immunoprecipitated CCR5. Incubation of T cells with CCL4 for 5 min stimulates phosphorylation of the receptor. However, CCL4-induced phosphorylation of CCR5 was attenuated when we used cells from GRK2 +/– animals (Fig. 6
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50% (ref. [22
] and unpublished observations). T cells from GRK2+/– mice express 50% of the GRK2 protein level that is present in T cells from WT mice and therefore provide an excellent model to study the functional consequences of the reduction in GRK2 level we observed in inflammatory autoimmune diseases. Our data demonstrate that the 50% reduction in GRK2 protein expression in GRK2+/– T cells results in a significant 30–40% increase in the chemotactic response to the chemokines CCL3, CCL4, and CCL5. Moreover, we show that the CCL4-induced increase in calcium level, PKB phosphorylation, and ERK-2 phosphorylation is significantly enhanced, whereas CCR5 phosphorylation is attenuated in GRK2+/– cells. Surprisingly, however, GRK2+/– T cells do become refractory to restimulation similar to WT cells. It is remarkable that the 50% decrease in GRK2 expression is sufficient to result in significant increases in receptor signaling and in chemotaxis in T lympohcytes. It has been shown before that the GRK2 protein level in cardiac tissue from these heterozygous GKR2+/– mice is also reduced by 50%. More importantly, the 50% reduction in cardiac GRK2 also has functional consequences, as it leads to increased contractile function of the heart [15 ]. These observations are especially important in view of the fact that many GPCR can be phosphorylated in overexpression systems by more than one GRK. It is known that T cells not only express GRK2 but also substantial levels of GRK3, GRK5, and GRK6 [17 34 ]. In cells transfected with CCR5 and GRK, it has been shown that not only GRK2 but also GRK3, GRK5, and GRK6 can phosphorylate this receptor [29 ], suggesting a redundancy in the system. Nevertheless, our data show that in cells naturally expressing GRK2, even a 50% reduction in the level of GRK2 can be sufficient to have functional consequences, suggesting that the other members of the GRK family or other kinases that can phosphorylate CCR5 do not fully compensate for the effect of reduced GRK2 on CCL4-induced chemotaxis, CCR5 phosphorylation, and signaling. A lack of redundancy in the GPCR/GRK interaction in physiological systems has also been described for other GPCR/GRK combinations. For example, in GRK5–/– mice, muscarinic receptor desensitization is impaired, and these mice display behavioral supersensitivity to muscarinic agonists [35 ]. In addition, in GRK3–/– mice, airway responsiveness to muscarinic cholinergic activation is increased [14 ]. In tissue culture, M2 and M3 subtypes of the muscarinic receptor can be desensitized by GRK2 and GRK3 [36 ].
The observed increase in the response to CCL4 is not a result of effects of reduced GRK2 levels on expression of receptors for CCL4, as the number and affinity of CCL4 binding sites did not differ between WT and GRK2+/– cells. Moreover, we have evidence showing that all activated T cells from WT and GRK2+/– animals respond to CCL4 with increased actin polymerization (data not shown). Therefore, the increased response of T cells from GRK2+/– animals to CCL4 cannot be explained by an increased number of CCL4-responsive cells or by increased CCL4 binding.
In overexpression systems, it has been shown that GRK2 can phosphorylate CCR5, the natural receptor for CCL4 [29 ]. Moreover, it has been shown that GRK2 overexpression promotes agonist-induced desensitization of CCR5 [30 ]. We show that CCL4-induced calcium signaling and PKB and ERK phosphorylation are all increased in GRK2+/– cells and that agonist-induced phosphorylation of CCR5 is attenuated. These data are in agreement with the concept of impaired CCR5 desensitization in GRK2+/– cells. However, when we performed classical desensitization experiments in which we examined the response of cells to restimulation, we observed that WT and GRK2+/– cells become refractory to restimulation with CCL4. These data suggest that there are two at least partially independent processes involved in CCR5 desensitization. It is interesting that we have recently described a similar discrepancy between enhanced signaling and normal refractoriness to restimulation with agonist in polymorphonuclear leukocytes (PMN) deficient for GRK6. GRK6–/– PMN respond to leukotriene B4 (LTB4) with prolonged calcium signaling and increased chemotaxis but also become normally refractory to restimulation with LTB4 [37 ].
Huttenrauch et al. [38 ] described signaling and internalization of CCR5 mutants that lack one or more C-terminal phosphorylation sites. These authors showed that mutation of any three C-terminal serine residues in CCR5 completely abolishes ß-arrestin binding and receptor internalization. However, mutation of specific serine residues is required to result in increased signaling. Based on these results, it was concluded that CCR5 internalization is dependent on phosphorylation of any two C-terminal serine residues, whereas desensitization is independently regulated by the phosphorylation of distinct serine residues. Although we cannot directly relate our data to the study by Huttenrauch et al. [38 ], it is conceivable that the phosphorylation of the residues involved in desensitization is impaired at low GRK2 levels, whereas phosphorylation of residues involved in arrestin binding and receptor internalization takes place normally at low GRK2. In addition, other GRKs or other kinases may be responsible for normal unresponsiveness to restimulation with CCR5 agonists in cells with low GRK2. In this respect, it is important to note that although we did detect attenuated agonist-induced CCR5 phosphorylation by GRK2+/– T cells, we cannot exclude that the phosphorylation of some specific residues occurs normally.
It may well be possible that processes independent of receptor desensitization also contribute to increased chemotaxis to CCL3, CCL4, and CCL5 in GRK2+/– cells. For example, it has been shown that GRK2 can bind and phosphorylate tubulin [7 8 ]. Chemoattractant-induced tubulin polymerization plays a major role in regulation of cellular motility [39 40 ]. Knowing that GRK2 binds and phosphorylates tubulin, decreased GRK2 expression might affect cytoskeletal plasticity resulting in altered chemotaxis.
In our experiments, not only the response of GRK2+/– cells to CCL4 is increased but also the response to CCL3 and CCL5. Although CCL3, CCL4, and CCL5 can all bind to and activate CCR5, it may well be possible that the increased response of GRK2+/– T cells to CCL3 and CCL5 is also mediated by CCR1. We do not know at present whether CCR1 is also a GRK2 substrate. Additional evidence for a crucial role of GRK in regulation of T cell chemotaxis comes from a study by Fong et al. [34 ], who investigated signaling and chemotaxis in response to CXCL12 (SDF-1) in GRK6–/– T cells. Comparison of the results obtained with GRK6–/– T cells and our results in GRK2+/– T cells reveals an important difference. We observed an increase in chemotaxis in cells with a 50% reduction in GRK2 level, whereas Fong et al. [34 ] reported that the complete absence of GRK6 results in a decrease in chemotaxis. Thus, although both studies suggest an important role for GRK in T cell chemotaxis, depending on the type of GRK and/or chemokine investigated, chemotaxis is increased or decreased. It should be noted, however, that as a result of the lethality of homozygous GRK2–/– animals, we do not know whether the complete absence of GRK2 would also lead to impaired chemotaxis.
In summary, we have shown here that a 50% reduction in the level of GRK2 results in increased chemotaxis of T cells to the CCR5 ligand CCL4 and the CCR5/CCR1 ligands CCL3 and CCL5. Moreover, we demonstrate that the reduced levels of GRK2 result in increased, CCL4-induced calcium signaling and PKB and ERK phosphorylation. In view of these data, we propose that the changes in cellular level of GRK2 that occur during inflammatory autoimmune disease may have important consequences for regulation of immune cell trafficking.
Received April 4, 2003; revised November 14, 2003; accepted January 9, 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) is significantly increased compared with WT mice (
). Data represent means (±SE) from three independent experiments, using six to eight animals per group from at least three different litters. **, P < 0.01.
