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(Journal of Leukocyte Biology. 2003;73:201-207.)
© 2003 by Society for Leukocyte Biology

The chemokine receptor CCR8 mediates rescue from dexamethasone-induced apoptosis via an ERK-dependent pathway

Gaia Spinetti*,{dagger}, Giovanni Bernardini*,{dagger}, Grazia Camarda*, Antonella Mangoni*, Angela Santoni{dagger}, Maurizio C. Capogrossi* and Monica Napolitano*

* Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy; and
{dagger} Department of Experimental Medicine and Pathology, University of Rome, "La Sapienza," Italy

Correspondence: Monica Napolitano, M.D., Ph.D., Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata-IRCCS, 00167, Via Monti di Creta 104, Rome, Italy. E-mail: m.napolitano{at}idi.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several chemokines have been shown to regulate cellular apoptosis following discrete stimuli. It was previously demonstrated that the CC chemokine CCL1 (I-309) rescues thymic lymphoma cells from apoptosis by unknown mechanisms. The aim of our study was to characterize the role of the CC chemokine receptor 8 (CCR8), the only described receptor for CCL1, in the rescue of murine thymic lymphoma cells and murine thymocytes from dexamethasone (dex)-induced apoptosis. We show here that the CCR8-restricted agonist Kaposi sarcoma-associated herpesvirus-encoded chemokine viral macrophage-inflammatory protein-1 (vMIP-1) rescues thymic lymphoma cells from dex-induced apoptosis, similar to CCL1, and that such rescue is extracellular-regulated kinase-dependent. Although it has been hypothesized that the rescuing effect of CCL1 from apoptosis could be CCR8-mediated, here, we formally demonstrate the role of such receptor as its selective antagonist encoded by the MC148 gene of molluscum contagiosum virus MC148/vMCC-I inhibits v-MIP-1- and CCL1-induced rescue activity. In addition, CCR8 ligands inhibit dex-induced apoptosis of murine thymocytes with potential implications for thymic selection.

Key Words: viral • kinases • thymocytes • cell death


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucocorticoid hormones such as dexamethasone (dex) are physiological inducers of apoptosis of lymphoid cells and have been shown to play an important role in the regulation of the T helper cell type 1 (Th1)/Th2 balance [1 2 3 ]. Dex induces apoptosis of CD4+/CD8+ thymocytes, T cell hybridomas, T lymphocytes, and other cell types [1 ]. The interplay between T cell receptor (TCR) signaling following self-antigen/major histocompatibility complex recognition and dex in the thymus leads to inhibition of activation-induced cell death resulting in proper positive selection [4 5 6 ]. Therefore, interfering with dex-induced cell death may be important in the modulation of immune responses. Several molecules, including interferon-{alpha}, substance P, interleukin (IL)-9, IL-4, and the CC chemokine CCL1, were shown to inhibit glucocorticoid-induced apoptosis [7 8 9 10 ].

Chemokines are key modulators of leukocyte chemotaxis, proliferation, and differentiation and play a role in transendothelial migration of leukocytes [11 ]. In addition to CCL1, several members of the chemokine superfamily, including CXCL8 (IL-8), CXCL2 (growth regulated oncogene ß), CCL5 (regulated on activation, normal T expressed and secreted) [12 13 14 ], as well as the ligand-receptor pair CXCL12 (stromal cell-derived factor-1)/CXCR4, act as regulators of survival/apoptosis of multiple cell types including T lymphocytes [15 ], neurons [16 ], and endothelial cells [17 ].

The CC chemokine receptor 8 (CCR8) is mainly expressed in lymphoid tissues [18 19 20 ], and its restricted ligands are represented by the eukaryotic CC chemokine CCL1 [19 , 21 ] and the Kaposi sarcoma herpesvirus (KSHV)-encoded viral macrophage-inflammatory protein-1 (vMIP-1) [22 , 23 ]. The receptor is highly expressed in the thymus [18 19 20 ] by CD4 + single positive and CD4 + CD8 + double positive thymocytes and by Th2 cells [24 , 25 ]. Although there is evidence of the role played by CCR8 in the regulation of Th2 responses [24 , 25 ], the characterization of the biological activities exerted by the CCR8 ligand/receptor pair in the thymus has been poorly elucidated. CCR8 was shown to function as a HIV-1 coreceptor in primary human thymocytes for several HIV-1 strains [26 ], suggesting a role for CCR8 in virally induced thymic pathogenesis.

CCL1 and its murine homologue, mCCL1 (TCA3), are expressed by activated T lymphocytes and monocytes [27 ], apolipoprotein A-stimulated endothelium, atherosclerotic plaques [28 ], and adult T cell leukemia (ATL) cells [29 ]. It is interesting that CCL1 overexpression in ATL induces cell survival [29 ]. CCL1 attracts Th2 lymphocytes and endothelial cells [20 , 30 31 32 ] and protects thymic lymphoma cells from dex-induced apoptosis [10 ].

The goal of our project was to investigate whether CCR8-mediated signals could regulate cell survival of thymic lymphoma cells and murine thymocytes. In this report, we show that vMIP-1 and CCL1 are able to trigger a CCR8-mediated rescue from dex-induced apoptosis by an extracellular-regulated kinase (ERK)-dependent mechanism and that a selective CCR8 antagonist blocks such rescue, thus pointing at the involvement of CCR8 in cell survival.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and reagents
BW5147 cells were obtained from American Type Culture Collection (LGC, Middlesex, UK) and were cultured in complete medium (CM): Iscove’s modified Dulbecco’s medium (IMDM; Gibco-BRL, Life Technologies, Grand Island, NY) supplemented with 1.5 mM L-glutamine, 0.2 mM L-asparagine, 0.5 mM L-arginine, 50 µM ß-mercaptoethanol, and 10% fetal bovine serum (FBS).

Human CCL1, vMIP-1, and molluscum contagiosum virus encoded (vMCV-type II)/MC148/vMCC-I were purchased from R&D Systems (Minneapolis, MN) and human CCL2, from Pepro Tech (London, UK). PD98059 was purchased from Calbiochem (La Jolla, CA). Dex and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO).

Mice
Female CD1 mice were purchased from Charles River (Calco, Milan, Italy). Thymocytes were obtained from 4- to 5-week-old mice. Briefly, the animals were killed by cervical dislocation, and the cell suspension derived from the minced thymus was washed in phosphate-buffered saline (PBS), filtered using a sterile gauze, and resuspended in RPMI-1640 medium supplemented with 2% FBS. Cells were then stained with anti-CD4 (Caltag, Burlingame, CA) and anti-CD8 (PharMingen, San Diego, CA) antibodies following standard procedures and were analyzed by flow cytometry to assess thymocyte purity.

Binding assay
BW5147 cells (106 cells/sample) were washed twice in cold binding buffer (BB): RPMI medium, 10 mM HEPES, and 0.1% bovine serum albumin and were resuspended in 200 µl BB. Cells were then incubated with 0.2 nM [125I]CCL1 (2000 Ci/mmol; Amersham Intl., Buckinghamshire, UK) and increasing amounts of unlabeled CCL1 or vMIP-1 (0.16–40 nM) for 3 h at 4°C in 1.5 ml Eppendorf tubes. Cells were then carefully layered on a cushion of 200 µl FBS in 0.5 ml thin Eppendorf tubes and were centrifuged at 1500 rpm at 4°C in a microfuge. The supernatant was vacuum-aspirated, the pellet was washed twice with cold BB, and radioactivity was counted. Data were analyzed using the LIGAND program, and the graphs were derived using the GraphPad Prism program (GraphPad Software, San Diego, CA) and expressed as the average of triplicates. Three independent experiments were allowed to calculate the range of the dissociation constant.

Chemotaxis assay
Chemotaxis was performed using Transwell inserts with 5-µm pore-size filters (Corning Costar Corp., Cambridge, MA) as follows. Chemokines (0.1–30 nM) were added to the lower chamber in 0.6 ml migration medium (MM): IMDM, 0.5% FBS, and 25 mM HEPES. BW5147 cells were washed twice and resuspended at 107 cells/ml in MM, and 100 µl of the cell suspension was placed in the upper chamber and was incubated for 2 h at 37°C in a 5% CO2. Then, cells in the lower chamber were counted. Each sample was assessed in duplicate.

Checkerboard analysis experiments were performed as above, but chemokines were added to the upper and lower wells at the dose of 3 nM.

Intracellular calcium concentration measurement
BW5147 (5x106/ml) cells were washed once in CM and loaded with 2.5 µM of the fluorescent calcium probe Fluo-4/acetoxymethyl ester in the presence of 1 µM Pluronic F-127 (Molecular Probes, Eugene, OR). After incubating the cells for 40 min at 37°C in the dark with frequent, gentle agitation, cells were washed twice in CM and placed on ice in the dark. For each sample, 0.3 x 106 cells were treated with chemokines (5 nM) and analyzed on a FACScan equipped with Cell Quest software (Becton Dickinson, Mountain View, CA). For desensitization studies, chemokines were added 300–500 s following the first stimulation.

Cell survival
Propidium iodide (PI) staining
BW5147 cells (0.15x106/ml) were cultured for 24 h in complete IMDM. Cells were then treated with 250 nM dex or DMSO (vehicle) in the presence or absence of 5 nM chemokines. The optimal dose of dex was chosen after performing a dose-response (25–1000 nM) curve. After 12, 24, or 48 h of stimulation, cells were harvested, washed once in PBS, and stained with a hypotonic PI solution (50 µg/ml PI, 0.1% Triton, 0.1% Na citrate) for 4 h in the dark at +4°C and were then analyzed by flow cytometry using a FACscan (Becton Dickinson). The percentage of dex-induced-specific apoptosis by chemokines was calculated as follows [6 ]: % apoptosis = M1 value of dex-treated sample – M1 value of DMSO control sample/M2 value of the DMSO control sample x 100. M1 and M2 values represent percentage of hypodiploid and normal dyploid DNA contents, respectively.

Cell death detection by enzyme-linked immunosorbent assay (ELISA) kit
A cell death detection ELISA kit (Boehringer Mannheim, Indianapolis, IN) was used as an independent assay to measure apoptosis. Briefly, BW5147 treated for 24 h in the presence or absence of dex (250 nM) or DMSO (250 nM) and chemokines (5 nM) was harvested in lysis buffer, and the cytoplasmic fraction was added to streptavidin-coated microtiter plates to then be incubated with antihistone and anti-DNA-labeled antibodies. Photometric analysis of the colometric reaction was allowed to quantify nucleosome-bound DNA fragments according to the manufacturer’s instruction: 100% apoptosis = optical density (O.D.) value of dex-treated sample – O.D. value of DMSO-treated sample in the absence of chemokines. The inhibitory effect of chemokines was calculated by dividing the O.D. value in the presence of chemokines by the value in the absence of factors x 100. The addition of chemokines did not alter the O.D. values of DMSO-treated samples. Each sample was measured in duplicate, and each experiment was performed at least four times. The viral antagonist vMCV-type II/MC148/vMCC-I (R&D Systems) at the concentration of 50 nM was preincubated 15 min before the addition of the chemokines to the culture. The mitogen-activated protein kinase (MAPK) kinase (MEK)-1 inhibitor PD98059 (Calbiochem) was added simultaneously to dex or DMSO and chemokines and was used at the concentration of 50 µM.

Annexin-V staining
Mouse thymocytes (7.5x105/ml) were treated for 16 h in the presence or absence of 10 nM dex or DMSO and 10 nM chemokines. The dose of 10 nM dex was chosen as the optimal concentration, between 1 and 250 nM, which would induce 25–40% basal apoptosis onto which the effect of chemokines was tested. Apoptosis was measured by treating thymocytes with Annexin-V fluorescein isothiocyanate (FITC; PharMingen) as follows: Each sample was washed twice with binding buffer (0.01 M HEPES/NaOH, pH 7.4, 0.14 mM NaCl, 2.5 mM CaCl2) incubated for 15 min at room temperature with Annexin-V FITC, and then was analyzed by flow cytometry. Percent-specific apoptosis was calculated using the following formula: % of Annexin-V-positive cells (dex–DMSO values)/% of Annexin-V-negative cells (DMSO value) x 100 in the presence or absence of chemokines.

Western blot analysis of ERK-MAPK activation
BW5147 (107 cells/ml) cells were stimulated in CM at 37°C with CCL1 or vMIP-1 (80 nM) in a final volume of 0.5 ml. A concentration-dependent effect of vMIP-1 on ERK activation was also assessed at doses ranging from 1 to 80 nM. MC148/vMCC-I, at the concentration of 25 and 100 nM, was preincubated 1 h before the addition of vMIP-1 (5 nM). Following agonist exposure, cells were immediately washed in PBS containing 1 mM sodium orthovanadate and 25 mM sodium fluoride and were lysed in Triton X-100 solubilization buffer for 30 min (1% Triton X-100, 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mg/ml aprotinin, 5 mg/ml leupeptin). Whole cell lysates (50 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto nitrocellulose. Western blotting was performed using an antiphospho-specific ERK-MAPK monoclonal antibody (E10, New England Biolabs, Beverly, MA) following the manufacturer’s instructions. The blot was then stripped and probed with an antitotal ERK polyclonal antibody, used at 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA) for normalization. Antibody staining was evaluated by ehanced chemiluminescence detection (Amersham Pharmacia Biotech).

Statistical analysis
Results were analyzed by one-way ANOVA and by Student’s t-test. A value of P < 0.05 was considered statistically significant. Results are reported as the mean value ± SE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
vMIP-1 and CCL1 act on a shared receptor on BW5147 cells
As vMIP-1 is a restricted CCR8 ligand, we tested whether CCL1 and vMIP-1 may act via a common receptor on BW5147 cells, a T lymphoma cell line that is rescued by CCL1 from dex-induced apoptosis [10 ] by performing binding, calcium mobilization, and chemotaxis assays.

We first characterized CCL1 and vMIP-1-binding sites by radioligand-binding competition to BW5147 cells. Increasing amounts of unlabeled CCL1 or vMIP-1 efficiently displaced radiolabeled CCL1 binding (Fig. 1A and 1B ). The calculated IC50 were 5–7 nM and 5 nM, respectively, indicating that vMIP-1 could compete for the same sites on BW5147 cells likely to be represented by CCR8, highly expressed by this cell line [19 , 20 ].



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  		Figure 1. CCL1 and vMIP-1 act on a common receptor in BW5147 cells as demonstrated by binding, chemotaxis, and calcium assays. (A) Increasing amount of unlabeled CCL1 displaced binding of iodinated CCL1 to BW5147 cells with an IC50 of 5–7 nM. (B) Unlabeled vMIP-1 displaces CCL1 binding in heterologous displacement studies with an IC50 of 5 nM. Shown is a representative experiment out of three. (C) Checkerboard analysis of CCL1 and vMIP-1-induced BW5147 chemotaxis. CCL1 and vMIP-1 reciprocally inhibited chemotaxis at the optimal dose of 3 nM. Shown is the mean of three to five experiments ± SE. (D) Reciprocal cross-desensitization of CCL1 and vMIP-1-induced calcium mobilization. CCL1 and vMIP-1, used at the concentration of 40 nM, induce calcium mobilization in BW5147 cells as measured by fluorescein-activated cell sorter analysis. The addition of chemokines after ~400 s from the first challenge induced a reciprocal cross-desensitization between CCL1 and vMIP-1 as shown in the middle and bottom panels. The experiments were done three times.

 
We next tested whether CCL1 and vMIP-1 could induce chemotaxis of BW5147 cells in a Transwell migration assay. CCR8 ligands were chemotactic for these cells in a dose-dependent manner (0.1–30 nM) with maximal activity at 1–3 nM, and a control chemokine, CCL2, had no effect in the same range of doses (data not shown). CCL1 and vMIP-1 showed a reciprocal inhibition using a checkerboard chemotaxis assay, further suggesting that they may act via CCR8 (Fig. 1C) .

Cross-desensitization of calcium signals in response to chemokines has been widely used to assess activation of a shared receptor [33 ]. CCL1 and vMIP-1, at 40 nM, induced a rapid mobilization of intracellular calcium in BW5147 as shown in Figure 1D . Cells stimulated first with CCL1 did not respond to the subsequent challenge with vMIP-1 and vice versa.

vMIP-1 rescues cells from dex-induced apoptosis; the CCR8 antagonist MC148/MCC-I blocks CCL1 and vMIP-1-induced rescue
CCL1 was previously reported to inhibit dex-induced apoptosis in thymic lymphoma cells [10 ]. Such biological activity is possibly mediated by CCR8, as it is the only described receptor for CCL1 [19 , 21 ]. As we demonstrated that CCL1 and vMIP-1 act on a shared receptor on BW5147 cells, we tried to assess whether vMIP-1 could rescue cells from dex-induced apoptosis, thus strengthening the hypothesis that CCR8 may be involved in survival. vMIP-1, at the optimal concentration of 5 nM, inhibited cellular apoptosis at 24 h by ~60%, as evaluated by PI staining, and at 12 and 48 h, such rescue was undetectable (Fig. 2A ).



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  		Figure 2. Rescue from dex-induced apoptosis by vMIP-1. Treatment with MC148/MCC-I blocks the protective effect exerted by CCR8 ligands. (A) Effect of vMIP-1 (gray bars), CCL1 (light gray bars), or CCL2 (solid bars) used at the concentration of 5 nM or none (open bars) on the apoptosis induced by dex (250 nM) at 12, 24, and 48 h, as measured by the PI staining technique. The inhibitory activity exerted by the CCR8-restricted ligands (50–60%) was present at 24 h following stimulation and at 12 and 48 h, was undetectable. CCL2, a chemokine that does not bind to CCR8, had no effect on dex-induced apoptosis. The results are the mean ± SE derived from five experiments. (B) Independent evaluation of DNA fragmentation performed by the use of an ELISA kit specific for nucleosome formation after 24 h of treatment as above. Shown is the percentage of apoptosis. The results are the mean ± SE derived from four experiments. (C) Effect of the selective CCR8 antagonist MC148/MCC-I on the apoptosis induced by dex in the presence or absence of CCL1 and vMIP-1 using an ELISA kit specific for nucleosome formation. The addition of 50 nM MC148/MCC-I (open bars) to the samples blocked the rescue activity shown by CCL1 and vMIP-1 on 24 h dex-induced apoptosis (solid bars). In fact, % of apoptosis of dex-treated samples (solid bars) is strongly diminished in the presence of CCL1 or vMIP-1 (P<0.01), and in dex/MCC-I-treated samples (open bars), the difference does not reach statistical significance (P>0.05). The results are the mean ± SE of three independent experiments.

 
An independent evaluation of DNA fragmentation, performed using an ELISA kit specific for nucleosome formation, gave similar results following 24 h of treatment; i.e., CCL1 and vMIP-1 inhibited apoptosis by ~60%, and CCL2 (MCP-1) was inactive (Fig. 2B) . Specific apoptosis (100%) corresponds to dex-induced apoptosis in the absence of chemokines.

MC148/MCC-I is a CC chemokine encoded by the poxvirus Molluscum contagiosum, which displaces CCL1 binding to CCR8, thus acting as a selective CCR8 antagonist, without affecting the function of any other chemokine receptor [34 ]. As shown in Figure 2C , the addition of MC148/MCC-I to the culture blocks the rescue from dex-induced apoptosis exerted by CCL1 and vMIP-1, thus demonstrating a role for CCR8 in cell survival.

Rescue of BW5147 cells from dex-induced apoptosis is dependent on ERK activation
Activation of the MEK/ERK cascade is able to inhibit glucocorticoid receptor (GR)-mediated cell death in T cell hybridomas, thymocyte cell lines, and primary T cells [4 ]. CCL1 (Fig. 3A ) and vMIP-1 (Fig. 3B) induce ERK2-MAPK phosphorylation in BW5147 cells with peak activity at about 0.5 min after stimulation, and dex treatment by itself has no effect on ERK2 activation as shown in Figure 3C . We then showed that vMIP-1 activates ERK2 at doses of chemokines able to rescue cells from apoptosis (5 nM); see Figure 3D . Further, we showed that MC148/MCC-I treatment partially inhibits vMIP-1-induced ERK2 phosphorylation (Fig. 3E) . PD98059, an inhibitor of MAPK/ERK-activating kinase MEK-1, completely blocks such phosphorylation (not shown). Treatment with MC148/MCC-I alone does not induce ERK2 phosphorylation (not shown).



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  		Figure 3. ERK2-MAPK phosphorylation by CCR8 ligands. Rescue from dex-induced apoptosis is sensitive to treatment with the MEK-1 inhibitor PD98059. (A) ERK2-MAPK phosphorylation induced by 80 nM CCL1 and (B) 80 nM vMIP-1 in BW5147 cells peaked at 0.5 min after stimulation. Shown is the phosphorylated form of ERK2 and total ERK of a representative experiment out of three. (C) Treatment with 250 nM dex did not significantly affect ERK2-MAPK activation, in the absence or presence of chemokines. (D) Dose-response effect of vMIP-1 on ERK2 activation (1–80 nM) assessed at 1 and 3 min after stimulation. (E) Effect of MC148/MCC-I (25, 100 nM) on vMIP-1-induced ERK phosphorylation. Lane a, Not stimulated; lane b, vMIP-1; lane c, vMIP-1 + MC148/MCC-I, 25 nM; lane d, vMIP-1 + MC148/MCC-I, 100 nM. (F) CCL1 and vMIP-1 (5 nM) inhibition of 24 h dex-induced apoptosis is blocked by treatment with PD98059 (50 µM), as measured by an ELISA kit method. Shown is the mean ± SE of three independent experiments.

 
Therefore, we investigated whether triggering of this signaling cascade may be required for the CCR8-mediated protection from apoptosis. Here, we show that treatment with PD98059, at a dose able to block ERK phosphorylation, strongly inhibited the rescue from cellular apoptosis (Fig. 3F) . These results indicate that CCL1 and vMIP-1 rescue BW5147 cells from dex-induced apoptosis by activating an ERK-dependent pathway.

CCR8 ligands rescue murine thymocytes from dex-induced apoptosis
As dex is an important modulator of thymocyte cell death, we tested whether CCR8 ligands may counteract glucocorticoid-induced apoptosis in freshly isolated murine thymocytes. It is interesting that CCL1 and vMIP-1, at 10 nM, exerted a 40–50% rescue from apoptosis on 4- to 5-week-old thymocytes from CD1 mice treated for 16 h with 10 nM dex as shown in Figure 4A and 4B . This finding suggests that CCR8-mediated rescue from apoptosis may be relevant in the regulation of immune responses.



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  		Figure 4. CCR8 ligands rescue murine thymocytes from dex-induced apoptosis. (A) Shown is the % of apoptosis of 4- to 5-week-old murine thymocytes after 16 h treatment with 10 nM dex in the absence (none) or presence of 10 nM CCL1 or vMIP-1 as measured by Annexin-V staining. CCR8 ligands exerted 40–50% rescue from dex-induced apoptosis. The results represent the mean ± SE of four independent experiments. (B) Representative experiment of Annexin-V-stained thymocytes as described in A. Shown is the variation in fluorescence intensity in DMSO (upper panel)- and in dex (lower panel)-treated samples in the absence of chemokines (gray area) or in the presence of CCL1 (dotted line) or vMIP-1 (solid line).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we show that the inhibition of dex-induced apoptosis by the CC chemokines CCL1 and vMIP-1 is CCR8-mediated, as these chemokines act on a shared receptor on thymic lymphoma cells and as the selective CCR8 antagonist MC148/MCC-I inhibits their protective effect. In addition, we show that the CCR8-triggered survival signal in BW5147 cells depends on ERK activation. Finally, we demonstrate that freshly isolated murine thymocytes are protected by CCR8 ligands from dex-induced apoptosis.

Glucocorticoids regulate the homeostasis of the immune system, inhibiting interleukin gene expression and triggering apoptosis of thymocytes, T cell hybridomas, and T lymphocytes [1 2 3 ]. In thymocytes, the cross-talk between TCR and glucocorticoid signaling is involved in the development of the antigen-specific T cell repertoire [4 5 6 ].

Several chemokines and chemokine receptors are known to be expressed within the thymus and mediate chemotactic and HIV coreceptor functions in thymocytes [35 36 37 38 39 ]. Further, they affect dex-induced apoptosis in thymic lymphomas [10 ] and block cycloheximide or Fas-mediated apoptosis [40 ].

We and others have previously demonstrated that CCR8 is highly expressed by the thymus and by CD4+ and CD4+/CD8+ thymocytes [18 19 20 , 36 ]. In this study, we show a protective effect of vMIP-1 and CCL1 on dex-induced apoptosis of murine thymocytes. This finding suggests that CCR8-mediated signals may be important in thymic selection by modulating apoptotic cell death in response to glucocorticoids.

We and others reported that CCR8 is a marker of polarized Th2 cells [20 , 24 ], that CCL1 induces their functional activation, and that CCR8 knockout mice have an impaired Th2 cytokine production and a decreased eosinophil recruitment [25 ]. It is intriguing to hypothesize that CCR8 triggering may promote an antiapoptotic effect in the presence of dex, also in Th2 cells, and that this may represent a regulatory mechanism of immune responses. The resistance to glucocorticoid-induced apoptosis of natural killer 1.1+ cells [41 ], which play an important role in Th2 responses and are CCR8-expressing cells [20 ], further supports such hypothesis.

In the present study, we have shown that ERK activation by CCR8 ligands is required for their protective effect, as the MEK1 inhibitor PD98059 blocks dex-induced rescue from apoptosis by CCL1 and vMIP-1.

The induction of MAPK activation by chemokines affects multiple biological activities, including cell migration, adhesion, and proliferation [42 , 43 ]. In several reports, activation of MEK-ERK plays a critical role in mediating antiapoptotic signals [44 , 45 ]. Triggering ERK-dependent pathways could promote cell survival by increasing transcription of cell survival-related genes and inactivating components of the cell death machinery [46 ].

MAPK-ERK and c-Jun N-terminal kinase finely regulate GR activity [47 ]. For example, TCR activation of the ERK cascade via ras inhibits T cell death induced by glucocorticoids, and a constitutively active mutant of MEK1 protects T cell hybridomas from glucocorticoid-induced apoptosis [4 ].

As ERK1/2 inhibit GR-induced transcriptional enhancement of proapoptotic genes [47 ], the activation of this pathway by chemokines may be an important step in the interplay between chemokine and glucocorticoid signaling.

The inhibitory effect of vMIP-1 on dex-induced apoptosis of murine-thymic lymphoma cells and thymocytes that we report in this study is in line with examples of RNA and DNA viruses, including pox- and herpesviruses, able to subvert host-immune functions by molecular mimicry of genes that are crucially involved in the control of immune responses, cell growth/apoptosis, or differentiation [48 , 49 ]. Their expression may help to establish a long-lasting latency in infected cells, as is the case for herpesviruses [50 ]. Viral inhibitors of apoptosis may be necessary to avoid premature death of host cells, thus allowing a proper spreading of infection. KSHV is known to express open reading frame (ORF)16 [50 ], whose cellular homologue is represented by bcl-2, and ORF71, which encodes a death-effector domain-containing protein homologous to cellular FLICE-inhibitory proteins (FLIPs), also known as v-FLIP, hypothesized to inhibit Fas-mediated apoptosis [51 ]. Therefore, vMIP-1 may be considered another KSHV-encoded regulator of apoptosis that may potentially exert a control on cell survival of T cell infiltrates in KS lesions, which have a predominant type II cytokine profile and abundantly express CCR8 [52 ].

In conclusion, this study shows that CCR8-mediated signals rescue thymic lymphoma cells and murine thymocytes from dex-induced apoptosis, thus pointing at a role for this chemokine receptor in cell survival with potential implications for the regulation of immune functions and neoplastic growth.


   ACKNOWLEDGEMENTS
 
This work was partially supported by grant #1/RF99.18 from Ministero della Sanità and from Associazione per la Ricerca sul Cancro (AIRC) to M. N. We are grateful to M. Rosado, F. Mainiero, and S. Sebastiani for helpful suggestions and to D. Porcelli and R. Romagnoli for technical help.

Received March 4, 2002; revised October 8, 2002; accepted October 17, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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