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Blood, 1 August 2004, Vol. 104, No. 3, pp. 619-625. Prepublished online as a Blood First Edition Paper on April 1, 2004; DOI 10.1182/blood-2003-11-3943.
CHEMOKINES Chemokine receptor CCR7 induces intracellular signaling that inhibits apoptosis of mature dendritic cellsFrom the Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid; Laboratorio de Inmunología, Hospital General Universitario Gregorio Marañón, Madrid; Departamento de Bioquímica and Instituto de Investigaciones Biomédicas Alberto Sols, Facultad de Medicina, Universidad Autónoma de Madrid; and Instituto de Farmacología y Toxicología Consejo Superior de Investigaciones Científicas (CSIC)–Universidad Complutense de Madrid (UCM), Facultad de Medicina, Universidad Complutense, Madrid, Spain.
Acquisition of CCR7 expression is an important phenotype change during dendritic cell (DC) maturation that endows these cells with the capability to migrate to lymph nodes. We have analyzed the possible role of CCR7 on the regulation of the survival of DCs. Stimulation with CCR7 ligands CCL19 and CCL21 inhibits apoptotic hallmarks of serum-deprived DCs, including membrane phosphatidylserine exposure, loss of mitochondria membrane potential, increased membrane blebs, and nuclear changes. Both chemokines induced a rapid activation of phosphatidylinositol 3'-kinase/Akt1 (PI3K/Akt1), with a prolonged and persistent activation of Akt1. Interference with PI3K, Gi, or G protein   subunits abrogated the effects of the chemokines on Akt1 activation and on survival. In contrast, inhibition of extracellular signal-related kinase 1/2 (Erk1/2), p38, or c-Jun N-terminal kinase (JNK) was ineffective. Nuclear factor– B (NFB) was involved in the antiapoptotic effects of chemokines because inhibition of NFB blunted the effects of CCL19 and CCL21 on survival. Furthermore, chemokines induced down-regulation of the NFB inhibitor IB, an increase of NFB DNA-binding capability, and translocation of the NFB subunit p65 to the nucleus. In summary, in addition to its well-established role in chemotaxis, we show that CCR7 also induces antiapoptotic signaling in mature DCs.
Apoptosis, or programmed cell death, is a physiologic process involved in the normal development and maintenance of tissue homeostasis.1 The final stage of this process that leads to the demise of the cell is executed by proteases that degrade vital molecular components of the cell.1 Hallmarks of cells undergoing apoptosis include disruption of mitochondria transmembrane potential, apparition of numerous blebs on the membrane, increased nuclear condensation, and increased appearance of phosphatidylserine (PS) in the outer leaflet of the cell membrane.
Apoptosis is a programmed process that is regulated through a complex mechanism that involves multiple molecular intermediates. Surface receptors may inhibit apoptosis by relaying intracellular signals that either repress proapoptotic molecules and/or stimulate antiapoptotic ones.1 Multiple pathways that inhibit apoptosis use as a common signaling intermediate phosphatidylinositol 3'-kinase (PI3K) and its downstream effector Akt1.1-3 Akt1 phosphorylates and inhibits a variety of proapoptotic regulators and also regulates proteins that promote cell survival.1-3 In this regard, it has been shown that Akt1 may activate I Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that play a fundamental role in the initiation of the immune response.8,9 In the developmental stage called "immature DCs," these cells are mainly in tissues and display low APC ability. Following encounter with foreign antigens, they rapidly undergo a process called maturation. Maturation involves an increase in the antigen-presenting ability and in the migration of the cells to secondary lymphoid organs, where they present antigens to naive T cells and induce specific immune responses. During maturation DCs up-regulate surface expression of chemokine receptor CCR7. Ligands for CCR7, chemokines CCL19 and CCL21, which are constitutively expressed at high levels in lymph nodes (LNs), are powerful attractants that direct DCs to these tissues.10-14
Signaling pathways that emanate from CCR7 in DCs and other leukocytes are just starting to be characterized.15-18 CCR7 and other serpentine-type receptors relay intracellular signals using G protein family members. G proteins are heterodimers, formed by
Reagents and materials
Granulocyte-macrophage colony-stimulating factor (GM-CSF) (Leucomax) was purchased from Schering-Plough (Kenilworth, NJ). DePsipher, interleukin-4 (IL-4), CCL21, and CXCL12 were obtained from R&D Systems (Minneapolis, MN). CCL19 was from PeproTech (Rocky Hill, NJ). Tumor necrosis factor- Cells and culture conditions
Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from healthy donors over a Lymphoprep (Nycomed, Norway), and the monocytes were induced to differentiate to DCs by adding GM-CSF.21-24 Briefly, monocytes (purity of CD14 more than 95%) were resuspended at 0.5 x 106/mL to 1 x 106/mL and cultured in complete medium (RPMI 1640) (Gibco Life Technologies, Paisley, Scotland) containing 10% heat-inactivated fetal calf serum (FCS), HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (25 mM), glutamine (2 mM), penicillin (100 U/mL) and streptomycin (100 µg/mL), GM-CSF (1000 U/mL), and IL-4 (1000 U/mL). Cells were cultured for 6 to 7 days, with cytokine addition every second day to obtain a population of immature DCs. To induce maturation of the cells, GM-CSF, IL-4, and 50 ng/mL TNF- Assays of apoptotic damage DCs were incubated in 0.1% BSA in RPMI plus 20 mM HEPES for 6 hours in the presence or absence of CCL19 or CCL21 and then harvested. At the indicated time points, DCs were stained with FITC-conjugated annexin V and 7-AAD, according to the manufacturer's instructions, and staining assessed by flow cytometry. In these experiments the cells that were apoptotic were those that are annexin V–positive/7-AAD–negative. To analyze mitochondria membrane potential disruption, DCs were incubated with DePsipher and immediately analyzed by flow cytometry or immunofluorescence, according to manufacturer protocol. Apoptotic nuclear morphology was assessed using Hoechst 33342 staining. Scanning electron microscopy and immunofluorescence For scanning electron microscopy, cells were washed in phosphate-buffered saline (PBS) and prefixed in 2.5% glutaraldehyde and then rinsed in 0.1 M cacodylate buffer. After postfixation in 1% osmium tetroxide, tissues were stained in 2% uranyl acetate and then dehydrated in graded ethanols. Scanning electron microscopy (SEM) sections were critical point dried using liquid carbon dioxide and coated with gold. The resulting sections were examined with a scanning electron microscope (Stereoscan 250 MK III; Leica, Deerfield, IL). Immunofluorescence and confocal microscopy analysis were performed as described with slight changes.25,26 Briefly, DCs (50 x 103 cells) suspended in complete medium were incubated at 37° C onto coverslips coated with poly-L-lysine (20 µg/mL). Cells were fixed in 4% paraformaldehyde in PBS (10 minutes at room temperature) and permeabilized with 0.2% Triton X-100 (10 minutes at room temperature). Before processing for immunofluorescence, the cells were treated with 1% BSA (15 minutes) to block unspecific binding. Then, samples were extensively washed with PBS and distilled water. Coverslips were mounted in fluorescent mounting medium (Dako, Carpinteria, CA), and representative fields of cells were photographed through an oil immersion lens (63x). Confocal microscopy was performed using an MRC-1000 Confocal Laser Scanning System (Bio-Rad, Hercules, CA) connected to a Nikon Diaphot 200 inverted microscope (Nikon, Tokyo, Japan). Images of 20 serial vertical cellular sections were acquired every 0.4 µm with the Bio-Rad COMOS graphical user interface and software (Biorad, Hercules, CA). Image analysis was performed using Adobe Photoshop 5.0 (Adobe System) and Image software. Green fluorescent protein (GFP)–expressing cells were analyzed using the FITC channel. Immunoprecipitation of PI3K and kinase assays
Lysis of the cells and subsequent immunoprecipitation was performed as described.25,26 After precipitating PI3K with the anti-p85 subunit antibody, PI3K assays were carried out as described.27 Briefly, DCs (500 x 103 cells) were solubilized in lysis buffer (20 mM Tris [tris(hydroxymethyl)aminomethane]–HCl [pH 7.4], 140 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM sodium orthovanadate, and a protease inhibition cocktail) and immunoprecipitated with anti-PI3K antibody (anti-p85). Kinase reactions were performed in kinase buffer (20 mM Tris-HCl [pH 7.5], 75 mM NaCl, 20 mM HEPES, 10 mM MgCl2, 200 µM adenosine) in the presence of 10 µgofthe substrate L- Cell lysis and Western blot analysis DCs (300 x 103 cells) were stimulated or not with the corresponding chemokines for the indicated period. The stimulation was terminated by solubilizing the cells in 50 µL ice-cold lysis Akt buffer (20 mM Tris-HCl [pH 7.5], 120 mM NaCl, 1% Nonidet P-40 [NP-40], 10% glycerol, 1 mM sodium pyrophosphate, 20 mM NaF, 1 mM sodium orthovanadate, plus a protease inhibitor cocktail [Sigma]). Supernatants were mixed with 5 x sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (500 mM Tris-HCl [pH 6.8], 0.25 mM sodium orthovanadate, 2.5 mM EDTA [ethylenediaminetetraacetic acid], 15% SDS, 5 mM EDTA, 10% 2-mercaptoethanol, 25% glycerol), boiled, and then fractionated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% nonfat milk protein in triethanolamine-buffered saline (TBS), pH 7.5, filters were incubated with the indicated antibodies in 1 x TBST (TBS plus 0.1% Tween 20) and visualized with the appropriate horseradish peroxidase (HRP)–conjugated secondary antibodies (Bio-Rad) and an enhanced chemiluminescence (ECL) substrate (Pierce, Rockford, IL) detection system. Expression constructs and transfections
To overexpress Electrophoretic mobility shift assay (EMSA)
This assay was performed essentially as described.30,31 For competition experiments, unlabeled oligonucleotides (100-fold molar excess) were preincubated with cell extracts at 4° C for 30 minutes before the probe was added. Oligonucleotide probe for NF
Stimulation of dendritic cells with CCL19 and CCL21 inhibits apoptosis of mature DCs To determine whether CCL19 or CCL21 exerted any effect on the viability of DCs, cells were maintained for 6 hours in RPMI plus 0.1%BSA (see "Materials and methods") in the presence or the absence of chemokines. After this time, we assessed the percentage of apoptotic cells estimating DCs that were annexin V–positive/7-AAD–negative. As shown in Figure 1A, after 6 hours almost 40% of control cells were apoptotic. This percentage was consistently reduced to 18% to 20% when the cells were cultured in the presence of 200 ng/mL CCL19 or CCL21 (Figure 1Aii [PDB] ), implying that chemokines reduced between 50% and 60% the percentage of apoptotic cells. The protection conferred by both chemokines was concentration dependent, with a maximal effect reached at 200 ng/mL and no further increase at higher concentrations until 1000 ng/mL. The protective effects of CCL19 or CCL21 were abrogated when the DCs were pretreated with a neutralizing anti-CCR7 monoclonal antibody (mAb 3D12) but not with an isotype control (not shown). The effect of both chemokines was less potent than that elicited by fetal calf serum (Figure 1A). In this case, after 6 hours in complete medium (RPMI including 10% fetal calf serum) less than 5% of the DCs were apoptotic (Figure 1A). Because CXCR4, a homeostatic chemokine receptor that is expressed in mature DCs, may transmit antiapoptotic signals,32,33 we also treated the cells with the CXCR4 ligand CXCL12 (100 ng/mL). Treatment of the DCs with CXCL12 reduced the number of apoptotic cells almost 37%, implying that the protective effect of this chemokine was less potent than that of CCL19 and CCL21, which reduced the number of apoptotic cells from 50% to 60% (Figure 1A and not shown).
We substantiate the results obtained using annexin V, analyzing how CCL19 and CCL21 affected the apparition of other well-known phenotypic changes typical of apoptotic cells—namely, loss of mitochondria membrane potential ( Stimulation of DCs with CCL19- or CCL21-induced activation of PI3K and Akt1
Because PI3K regulates survival of many cell types through its downstream effector Akt1,2 we analyzed whether CCL19 and CCL21 induced activation of PI3K and Akt.1 To analyze PI3K activity, DCs were treated with chemokines for various times and lysed. The lysates were incubated with the anti-PI3K antibody, and the resulting immune complexes were subjected to in vitro kinase assays using L-
We used LY294002, a potent inhibitor of PI3K, to study if inhibition of this kinase affected the survival of DCs. Pretreatment of DCs with the inhibitor abrogated completely PI3K activity (Figure 2A), the phoshorylation of Akt1 (Figure 2C), and the effects of CCL19 and CCL21 on the survival of the cells (Figure 2C). Similar results were obtained when wortmannin, another selective PI3K inhibitor, was used (not shown). Taken together, the data indicate that stimulation of CCR7 induces activation of PI3K/Akt1, with a prolonged activation of Akt1, and that these molecules regulate the survival of the DCs.
CCR7-dependent survival and activation of Akt1 is pertussis toxin sensitive and involves
Chemokine receptors may couple to several G protein family members.19 To analyze the involvement of Gi in the prosurvival signals induced by CCL19 and CCL21, DCs were pretreated with the Gi-selective inhibitor pertussis toxin (PTX). This treatment abrogated the effects of the CCL19 and CCL21 on the survival of DCs and the activation of PI3K (not shown) and Akt1 (Figure 3A), indicating the involvement of a Gi protein in the CCR7-mediated effects. To determine which subunits of trimeric Gi proteins relay the signals from CCR7, we transfected DCs with
Inhibition of MAPK family members Erk1/2, p38, and JNK did not abrogate the effect of CCL19 and CCL21 on the survival of DCs MAPK family members have been implicated in the regulation of apoptosis in different systems.7,34 To investigate if these kinases could mediate the CCR7-dependent effects on survival, we treated DCs with UO126, SB203580, or SP600125, which are selective inhibitors of Erk1/2, p38, or JNK, respectively. Despite the strong inhibition of the activity of the latter kinases (not shown), MAPK inhibitors caused only a slight increase in the basal death of the cells. Furthermore, in these cells, stimulation with CCL19 or CCL21 still caused a reduction in the percentage of apoptosis that was similar to that of control cells (Figure 4). Also, inhibition of different MAPK family members did not abrogate the stimulation of phosphorylation of Akt1 induced by CCL19 or CCL21 (Figure 4). In sum, the results indicate that MAPK family members are not regulating CCR7-mediated survival of DCs under our conditions.
Involvement of NF
NF
To analyze directly if chemokines induced activation of NF
CCR7 plays a fundamental role directing mature DCs to the lymph nodes.10-14 Apart from this well-established function, it is emerging that this receptor plays additional roles, which are important for the physiology of DCs, including regulation of DC cytoarchitecture and endocytosis.15,16 Herein we show that stimulation of DCs with CCR7 ligands CCL19 and CCL21 blocks well-known apoptotic hallmarks of serum-starved DCs, including increased phosphatidyl exposure and membrane blebs on the membrane, nuclear changes, and loss of mitochondria potential (Figure 1). We show that PI3K/Akt1, a signaling axis that regulates apoptosis in a variety of cells settings,1-3 plays an important role in the protective effects elicited by CCR7. First, we observed a potent activation of PI3K/Akt1 that, strikingly, in the case of Akt1 was persistent and prolonged for at least 120 minutes (Figure 2A-B and not shown). Interestingly, the unusual and persistent activation of Akt that we observe has been also observed in lymphocytes for CXCR4, another member of the family of homeostatic chemokine receptors, like CCR7.33 Second, we demonstrated the important role of PI3K/Akt in the CCR7-mediated stimulation of the survival of DCs by showing that interference with these kinases using pharmacologic inhibitors (LY294002 or wortmannin) abrogated the effects of the chemokines (Figure 2C). Because it was shown before that in naive T cells CCL19 induced only transient activation of Akt1,33 the prolonged activation observed in DCs indicates the context dependence of the activation of this kinase. Akt1 phosphorylates/inhibits in the cytoplasm a variety of proapoptotic molecules including caspase 9 and bcl-2 members and also regulates the activation of prosurvival transcriptional factor NF B.2,3 Furthermore, Akt1 can also translocate to the nucleus, where it inhibits proapoptotic transcriptional factors.2,3,39 Therefore, given the number of antiapoptotic targets, the persistent activation of Akt1 induced from CCR7 may have a strong antiapoptotic effect in DCs. In this regard, in other leukocytes it has been shown that Akt can maintain cell survival even in the absence of extrinsic signals.40
We also show that downstream from CCR7, Gi proteins are important to regulate the signaling that converges in PI3K/Akt and survival because pretreatment with pertussis toxin blocked completely the effect of the chemokines (Figure 3A). Furthermore, by transfecting cells with a construct encoding a peptide inhibitor of G
Our results indicate that the transcription factor NF Mature DCs display a variety of molecular mechanisms that increase their survival, including reduced sensibility to proapoptotic signals induced from death receptors and cytotoxic cells.42,43 Our results suggest that signaling from CCR7 may also contribute to the apoptotic-resistant phenotype of mature DCs. The antiapoptotic signaling from CCR7 may have an important impact on the immune response. On their way to the lymph node, chemokine CCL21, that is expressed constitutively by endothelial cells lining lymphatic vessels and high endothelial venules and which it is also secreted, along with CCL19, from lymph node areas, by stimulating CCR7 may contribute to increase the probability of DCs to reach the node regions.11-13 Moreover, as CCL19 and CCL21 are present at high levels in T-cell zones, once DCs reach these regions, CCR7 can also increase the survival of DCs. This could have an important impact on the immune response. Because it has been shown that DCs terminate their life cycle in the lymph node,44-47 an extended longevity, favored by signaling from CCR7, may increase the probability of antigen presentation and stimulation of antigen-specific T cells.
We acknowledge Julia Villarejo and Isabel Treviño for their help, Patricio Aller for advice on Hoechst 33342 staining, Eduardo Muñoz for the p65-GFP construct, and Robert Lefkovitch for providing the dominant negative ARK-CT construct.
Submitted November 18, 2003; accepted March 24, 2004.
Prepublished online as Blood First Edition Paper, April 1, 2004; DOI 10.1182/blood-2003-11-3943.
Supported by grants BFI-2001-228, CAM08.3/0040/2001.1, and PI021058 (J.L.R.-F) and SAF2002-04615-C02-02 (P.S.-M.); Fondo de Investigación Sanitaria and Ramón y Cajal programs (J.L.R.-F.); and a scholarship associated to grant PI021058 (L.R.-B.). N.S.-S. is recipient of a fellowship, Formación del Profesorado Universitario (FPU), conferred by the Ministerio de Educación (Spain).
N.S.-S. and L.R.-B. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: José Luis Rodríguez-Fernández, Centro de Investigaciones Biológicas, CSIC, C/Ramiro de Maeztu, 9, 28040 Madrid, Spain; e-mail: rodrifer{at}cib.csic.es.
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Abstract
Introduction
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