Opposite Fate of Endocytosed CCR7 and Its
Ligands: Recycling versus Degradation
Carolina Otero, Marcus Groettrup and Daniel F. Legler
This information is current as
of July 31, 2017.
Subscription
Permissions
Email Alerts
This article cites 62 articles, 41 of which you can access for free at:
http://www.jimmunol.org/content/177/4/2314.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2006 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
References
J Immunol 2006; 177:2314-2323; ;
doi: 10.4049/jimmunol.177.4.2314
http://www.jimmunol.org/content/177/4/2314
The Journal of Immunology
Opposite Fate of Endocytosed CCR7 and Its Ligands:
Recycling versus Degradation1
Carolina Otero, Marcus Groettrup, and Daniel F. Legler2
The chemokine receptor CCR7 and its ligands CCL19 and CCL21 play a crucial role for the homing of lymphocytes and dendritic
cells to secondary lymphoid tissues. Nevertheless, how CCR7 senses the gradient of chemokines and how migration is terminated
are poorly understood. In this study, we demonstrate that CCR7(-GFP) is endocytosed into early endosomes containing transferrin
receptor upon CCL19 binding, but less upon CCL21 triggering. Internalization of CCR7 was independent of lipid rafts but relied
on dynamin and Eps15 and was inhibited by hypertonic sucrose, suggesting clathrin-dependent endocytosis. After chemokine
removal, internalized CCR7 recycled back to the plasma membrane and was able to mediate migration again. In contrast,
internalized CCL19 was sorted to lysosomes for degradation, showing opposite fate for endocytosed CCR7 and its ligand. The
Journal of Immunology, 2006, 177: 2314 –2323.
Biotechnology Institute Thurgau, University of Konstanz, Tägerwilen, Switzerland;
and Department of Biology, Division of Immunology, University of Konstanz, Konstanz, Germany
Received for publication November 10, 2005. Accepted for publication May
24, 2006.
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 the German Research Foundation (DFG, TR-SFB 11),
the Thurgauische Stiftung für Wissenschaft und Forschung, and the State Secretariat
for Education and Research (Section 16 law of research). D.F.L. is a recipient of a
career development award from the Dr. Max Cloëtta Foundation, Zürich, Switzerland.
2
Address correspondence and reprint requests to Dr. Daniel F. Legler, Biotechnology
Institute Thurgau, University of Konstanz, Konstanzerstrasse 19, CH-8274 Tägerwilen, Switzerland. E-mail address: [email protected]
Copyright © 2006 by The American Association of Immunologists, Inc.
is still sparsely investigated (15–20). In particular, information on
how CCR7-mediated migration is stopped after a cell has arrived
at its final destination within the lymph node has remained unclear.
CCL19 and CCL21 are both produced by stroma cells within the
T cell zone (12). Remarkably, CCL21 is transcytosed to high endothelial venules (HEV)3 (21) and mediates LFA-1-mediated arrest of the recruited T lymphocytes (22–24). Thus, T lymphocytes
and dendritic cells that home to the T zone of lymph nodes seem
first to be recruited to HEV by CCL21, but then the CCL21 signal
must be overcome by an attraction signal provided by CCL19/
CCL21 derived from the T zone. B cells within the lymph node
that have seen an Ag migrate directionally toward the B zone–T
zone boundary along a gradient of CCL21 (and eventually CCL19)
to encounter T cells (25).
One way of rendering a cell unresponsive to chemokines is receptor internalization. Chemokine receptor endocytosis is best described for the HIV coreceptors CCR5 and CXCR4 but follows
distinct mechanisms (26). Remarkably, binding of CXCL12 to
CXCR4 leads to the ubiquitylation of the receptor followed by its
degradation in lysosomes (27, 28). In contrast, endocytosed CCR5
is recycled back to the plasma membrane (29 –31). Strikingly,
CCR7 internalization was observed by CCL19 triggering, but not
by stimulation with CCL21 (32), although binding affinities and
G-protein activation are comparable (4, 18). Of note, CCR7 desensitization through receptor phosphorylation and ␤-arrestin
binding was enhanced by CCL19 stimulation, compared with
CCL21 (18), whereas T cell polarization mediated by the chemokines was indistinguishable (17). However, up to now, the mechanism of CCR7 signaling and trafficking remains largely unclear,
and there is currently no information on the fate of CCL19 after
CCR7 endocytosis.
Cell surface receptors can be internalized by two segregated
pathways: clathrin-dependent and clathrin-independent, lipid raft/
caveolae-dependent endocytosis (33, 34). The classical clathrindependent pathway is well characterized. Clathrin-coated pits at
the plasma membrane bud and pinch off in a dynamin- and adaptor
protein (such as Eps15)-dependent manner to form clathrin-coated
vesicles. After endocytosis, clathrin-coated vesicles are uncoated
3
Abbreviations used in this paper: HEV, high endothelial venule; EGFP, enhanced
GFP; HA, hemagluttinin; MCD, methyl-␤-cyclodextrin; VSV, vesicular stomatitis
virus; wt, wild type.
0022-1767/06/$02.00
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
L
eukocyte traffic is fundamental for immune regulation
and, hence, is highly coordinated. Tissue- and microenvironment-selective leukocyte homing is the basis for this
organization. It is now well established that cell migration is orchestrated mainly by chemokines which, together with adhesion
molecules, deliver the key signal that allows leukocytes to transmigrate from the bloodstream into the tissue (1–3). All chemokines
act through signaling via seven-transmembrane domain G proteincoupled cell surface receptors, but migration properties vary
greatly among different cell types. The central role of the chemokine receptor CCR7 and its ligands CCL19 (ELC, Exodus-3, MIP3␤, and CK␤11) and CCL21 (SLC, Exodus-2, 6Ckine, and
TCA-4) in the homing to secondary lymphoid organs is undisputed. CCR7 is highly expressed on naive T cells and to a lower
level on B cells. A transient increase in CCR7 expression is found
upon T cell activation (4), whereas T cell differentiation toward
effector cells is accompanied by the down-regulation of the receptor on the cell surface (5). However, in dendritic cells, CCR7 expression is induced upon maturation (6 – 8). Mice lacking CCR7
show delayed kinetics in Ab responses, delayed-type hypersensitivity reactions, and morphological abnormalities in secondary
lymphoid organs as a consequence of an impaired homing of mature dendritic cells and lymphocytes (9, 10). The fact that CCR7
ligands are mandatory for the homing to secondary lymphoid organs has been demonstrated in plt/plt mice lacking CCL19 and
CCL21 (11–14).
Although CCR7 and its ligands are essential for eliciting a potent cellular immune response, CCR7 signaling and its regulation
The Journal of Immunology
and fuse with the early endosomes, the central control organelles
for sorting receptors. Either receptors recycle back to the plasma
membrane via recycling endosomes or are directed to late endosomes and lysosomes for degradation. Alternatively, receptors can
be endocytosed in a lipid raft/caveolae-dependent manner. This
pathway is ill defined but largely depends on cellular cholesterol
(33–35).
In this study, we investigated the route of internalization and the
trafficking of CCR7 by monitoring a newly generated GFP-tagged
CCR7. In addition, we tracked CCL19 after receptor binding by a
chemokine-Fc chimera. Analysis of CCR7 endocytosis and investigations on the routes of CCL19 and CCL21 after receptor triggering is critical for a better understanding of how immune cells,
such as dendritic cells and lymphocytes, sense a chemokine gradient originating in secondary lymphoid organs.
Materials and Methods
Abs and reagents
Cells and transfection
The human embryonic kidney cell line, HEK293, was grown in DMEM
(Invitrogen Life Technologies) with 10% (v/v) FBS. HEK293 cells were
stably transfected in 10-cm dishes by the calcium phosphate procedure.
Cell clones were established by limiting dilution in the presence of 0.8
mg/ml G-418 (Invitrogen Life Technologies). The pre B cell line 300-19
(4, 36) was grown in RPMI 1640 (Invitrogen Life Technologies) with 10%
(v/v) FBS, 10% 2-ME, and 2 mM nonessential amino acids. Stable transfection was performed by electroporation, and clones were achieved by
limiting dilution as described (4, 36). The human T cell line CEM was
cultured in RPMI 1640 supplemented with 10% (v/v) FBS.
Human PBL were isolated from healthy donors by separation on FicollPaque (Pharmacia Biotech), followed by depletion of monocytes using
anti-CD14-conjugated magnetic microbeads (Miltenyi Biotec).
Cells (2 ⫻ 106/ml) were cultured in RPMI 1640 supplemented with 10%
(v/v) FBS, 10% 2-ME, and 2 mM nonessential amino acids. PBL were
activated for 4 – 6 days with 200 U/ml IL-2 and 1 ␮g/ml PHA-L
(Sigma-Aldrich).
Construction of expression plasmids
The entire open reading frame of human CCR7 was amplified by PCR from
SR␣puro-CCR7 (4) using the primers CCR7se2 (5⬘-ATA GAA TTC CGT
CAT GGA CCT GGG GAA AC, restriction site underlined) and CCR7as
(5⬘-TAT GCG GCC GCT GGG GAG AAG GTG GTG) and subcloned
into the EcoRI/NotI sites of pcDNA3 (Invitrogen Life Technologies). Enhanced GFP (EGFP) was fused to the N terminus of CCR7 by PCR amplifying GFP from pEGFP-N1 (Clontech) and subcloning into the XhoI/
XbaI sites of pcDNA3-CCR7 using the primers EGFPse (5⬘-AAA CTC
GAG CAG TGA GCA AGG GCG AGG) and EGFPas (5⬘-AAA TCT
AGACTA CTT GTA CAG CTC GTC). A VSV-tagged CCR7 was cloned
by PCR amplification of CCR7 using the primers CCR7se3 (5⬘-TAT GAA
TTC GAC CTG GGG AAA CCA ATG AAA AGC) and CCR7as3 (5⬘TAA TCT AGA CTA TGG GGA GAA GGT GGT G) and subcloning into
the EcoRI/XbaI sites of pCR3-VSV (MT044; provided by M. Thome, University of Lausanne, Epalinges, Switzerland). The CCR7-HA construct was
made by replacing the GFP (XhoI/XbaI) with the annealed oligonucleotides
CCR7-HAse (5⬘-TCG AGC ATA CCC ATA CGA CGT CCC AGA CTA
CGC TTA GT) and CCR7-HAas (5⬘-CTA GAC TAA GCG TAG TCT
GGG ACG TCG TAT GGG TAT GC) coding for the HA tag. 300-19 cells
expressing wild-type (wt), VSV- or HA-tagged CCR7 migrated similarly in
response to CCL21, indicating that the tags do not affect CCR7 functions
(data not shown).
Human CCL19 and CCL21 were amplified by PCR using the following
primers: SLCse (5⬘-ATA ATA GGA TCC ACA GAC ATG GCT CAG
TCA C), SLCas (5⬘-TAT TAA GAA TTC TGG CCC TTT AGG GGT
CTG), ELCse (5⬘-ATAT AAG CTTCCC TCC ATG GCC CTG) and
ELCas (5⬘-TTAT GAA TTC ACT GCT GCG GCG CTT C) and pCR2SLC and pCR2-ELC (provided by O. Yoshie, Kinki University, Osaka,
Japan) as template. Amplified DNAs were subcloned into the HindIII/
EcoRI sites of pCR3-Fc (PS521; provided by P. Schneider (University of
Lausanne, Eplinges, Switzerland).
GFP-tagged dynamin II (wt, K44A) constructs (37) and GFP-tagged Eps
15 constructs (38) were obtained from U. Greber (University of Zurich,
Zurich, Switzerland).
Flow cytometry
Cells were washed twice with FACS buffer (PBS containing 2% FBS and
5 mM EDTA) and, where required, incubated with the respective Abs for
30 min at 4°C. Cells were washed twice and fluorescence was acquired by
a FACScan II using CellQuest software (BD Biosciences). Data were analyzed with the FlowJo software (Tree Star).
Chemotaxis
Chemotaxis of 300-19 cells was measured by migration through a polycarbonate filter of 5-␮m pore size in 24-well Transwell chambers (Corning
Costar). Cell culture medium (600 ␮l) containing indicated doses of chemokine, or medium alone as a control for spontaneous migration, was
added to the lower chamber; a total of 1 ⫻ 105 cells in 100 ␮l was added
to the upper chamber. After 3 h of incubation at 37°C, a 500-␮l aliquot of
the cells that migrated to the bottom chamber was counted by flow cytometry acquiring events for a fixed time period of 60 s using CellQuest software. The number of migrated cells was expressed as percentage of input
cells.
Chemokine-mediated changes in intracellular free calcium
concentrations
Cells were washed twice with Ca2⫹ buffer (145 mM NaCl, 5 mM KCl, 1
mM Na2HPO4, 1 mM MgCl2, 5 mM glucose, 1 mM CaCl2, and 10 mM
HEPES (pH 7.5)) and resuspended at 1 ⫻ 106 cells/ml. Cells were loaded
with 1.5 ␮l/ml Fluo-3/AM (4 mM in DMSO) for 30 min at 37°C. Cells
were washed, and chemokine-induced calcium mobilization-related fluorescence changes of Fluo-3 were measured by flow cytometry.
Confocal laser scanning microscopy
Transfected HEK293 cells were grown overnight on coverslips. 300-19
cells were incubated for 1 h on coverslips coated with poly-L-lysin (SigmaAldrich). If not stated otherwise, cells were treated with 2 ␮g/ml CCL19 or
CCL21, 3 ␮g/ml biotinylated CCL19, 10 ␮g/ml CCL19-Fc or CCL21-Fc,
50 ␮g/ml transferrin, or 50 nM lysotracker. Cells were washed twice with
PBS and fixed for 10 min with 4% paraformaldehyde followed by three
washing steps with PBS. For intracellular stainings, cells were permeabilized with 0.2% Triton X-100 (Fluka) for 10 min and washed with 0.2%
gelatin in PBS. Ab staining of cells was preformed at room temperature for
40 min, and cells were washed five times and mounted on glass slides using
Fluoromount-G (Southern Biotechnology Associates). Immunofluorescence was analyzed by a confocal microscope (LSM 510; Zeiss) with a
⫻63 Plan-Apochromat objective (aperture ⫽ 1.4). Images were acquired
using LSM 510 software (Zeiss).
Western blotting
Cells were lysed with 1% Triton X-100 in 150 mM NaCl, 50 mM HEPES,
0.1 M EGTA, 2 mM MgCl2, 10% glycerol containing leupeptin, aprotinin,
and pepstatin (1 ␮g/ml each; Roche). Proteins from total cell lysates were
resolved by SDS-PAGE and transferred to Protran nitrocellulose membrane (Schleicher & Schuell Microscience). Membranes were blocked with
PBS containing 5% of low-fat dry milk and incubated with the respective
Abs overnight at 4°C or for 1 h at room temperature on a rocking plate.
After washing, HRP-conjugated secondary Abs were bound and detected
using ECL (Pierce/Socochim).
Results
Differential endocytosis of CCR7 by CCL19 and CCL21
The homing of lymphocytes and dendritic cells largely depends on
the attraction of the cells by the chemokines CCL19 and CCL21.
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
Abs were obtained from the following sources: PE-Cy5-labeled rat antihuman CCR7 (clone 3D12; BD Pharmingen), goat anti-human IgG1HRP
(Santa Cruz Biotechnology); streptavidin-Cy3 and streptoavidin-FITC
(Jackson ImmunoResearch Laboratories); mouse anti-phospho ERK-1/2
and mouse anti-total ERK-1/2 (Cell Signaling Technology); mouse antihemagluttinin (HA), (Sigma-Aldrich); HRP-conjugated goat anti-mouse
IgG (DakoCytomation); and mouse anti-proteasome subunit C7 (␣1; provided by Dr. K. Scherrer, Paris, France). Alexa Fluor 546-labeled transferrin and lysotracker Red DND-99 were from Molecular Probes. Human
chemokines CCL19 and CCL21 were purchased from PromoCell. Monobiotinylated human CCL19 was from RMF Dictagene. Streptavidin-peroxidase, filipin III, methyl-␤-cyclodextrin (MCD), sucrose, cycloheximide,
chloroquine, and protein A-Sepharose were obtained from Sigma-Aldrich.
Fluo-3/AM was purchased from Calbiochem.
2315
2316
OPPOSITE FATE OF CCR7 AND CCL19 TRAFFICKING
At its final destination, the migratory signal needs to be shut off,
which normally occurs by chemokine receptor down-modulation
or receptor desensitization. To unravel the mechanism how CCR7
is silenced, we investigated the internalization of CCR7 in IL-2and PHA-activated human PBL. CCR7 endocytosis by CCL19 was
readily observed in a concentration- and time-dependant manner
(Fig. 1). Cell surface expression of CCR7 on T cells was already
reduced by 15% at a CCL19 concentration of 30 ng/ml. More than
60% of CCR7 was internalized in the presence of 3 ␮g/ml CCL19
(Fig. 1A). Endocytosis was rapid, because after 2 min of chemokine addition, 30% of CCR7 disappeared from the plasma membrane. Maximal internalization was reached after 30 min of incubation (Fig. 1B). Interestingly, CCR7 endocytosis by CCL21 was
observed only at high chemokine concentrations (⬎300 ng/ml) and
reached a maximum of ⬃25% (Fig. 1A). Our data on CCL19mediated endocytosis of CCR7 are largely in agreement with a
previous study by Bardi et al. (32), although they did not find any
internalization by CCL21 in T cells at all.
To further investigate CCR7 localization and trafficking, we fused
the enhanced GFP to the C terminus of human CCR7. We stably
expressed CCR7-GFP in the murine pre-B cell line 300-19, a cell
line that does not respond to CCL19 and CCL21 (4, 36). CCR7GFP transfected cells readily migrated in response to CCL21, similar to 300-19 cells expressing wt CCR7, whereas CCR7-GFPpositive cells did not migrate in a Transwell chemotaxis assay in
the absence of chemokines (Fig. 2A). The phosphorylation of the
ERK-1 and ERK-2 is an early and transient event after chemokine
triggering (16). To test whether CCR7-GFP is fully functional, we
stimulated 300-19-transfected cells for various time points with
CCL21 and analyzed the phosphorylation of ERK-1/2 by Western
blot analysis. Identical kinetics and potency of ERK-1/2 activation
upon CCL21 triggering was observed for cells expressing CCR7GFP and wt CCR7 (Fig. 2B). 300-19 cells are ideal for testing
chemotaxis but not for morphological and trafficking studies.
Therefore, we stably transfected the human embryonic kidney cell
line HEK293 with CCR7-GFP. CCL21 stimulation of HEK293
cells expressing CCR7-GFP resulted in the phosphorylation of
ERK-1/2 (Fig. 2C), comparable to 300-19 transfectants. As expected, CCR7-GFP mainly localized to the plasma membrane of
both transfected cell lines as assessed by confocal microscopy
(Fig. 2D). These data provide clear evidence that CCR7-GFP is
fully functional.
FIGURE 2. Characterization of CCR7-GFP. A, Migration of the murine
pre-B cell line 300-19 stably transfected with either human CCR7 wt or
CCR7-GFP in response to CCL21 (1 ␮g/ml) was assessed in a Transwell
chemotaxis assay. After 3 h of incubation at 37°C, cells in the lower chamber were collected and counted by flow cytometry. Mean values and SD of
three independent experiments are depicted as percentage of migrated cells.
B, 300-19 cells expressing either CCR7 wt or CCR7-GPF were incubated
with 2 ␮g/ml CCL21 for indicated time points and lyzed, and the activation
of ERK-1/2 was determined by Western blot analysis using an Ab recognizing the phosphorylated forms of ERK-1 and ERK-2 (pERK-1/2). An Ab
against total ERK-2 (tERK-2) was used to ensure equal protein loading. C,
The activation of ERK-1/2 was confirmed in HEK293 cells expressing
CCR7-GFP. D, Cell surface expression of transfected CCR7-GFP in
300-19 and HEK293 cells was determined by confocal microscopy. Scale
bars,10 ␮m.
CCR7-GFP colocalizes with early and recycling endosomes but
not with lysosomes
FIGURE 1. Internalization of CCR7 in human PBL. A, Human PBL
were cultured for 4 – 6 days in the presence of IL-2 and PHA and incubated
with graded concentrations of CCL19 (circles) or CCL21 (triangles) for 30
min at 37°C. Cell surface expression of CCR7 was determined by flow
cytometry using the mAb 3D12 against CCR7 which can still recognize the
epitope in the presence of bound ligand. B, The same T cells were incubated for various time points with 3 ␮g/ml chemokines, and cell surface
expression of CCR7 was determined by flow cytometry. Mean values of
three independent experiments are shown. Error bars are ⬍1%.
To investigate the intracellular trafficking of CCR7, we stimulated
HEK293 cells expressing CCR7-GFP with CCL19 and CCL21.
After 5 min of CCL19 stimulation, CCR7-GFP was endocytosed
and appeared as punctuated structures within the cell (Fig. 3A).
Some CCR7-GFP was still present at the plasma membrane, confirming our data from primary T cells. Upon CCL21 stimulation,
CCR7-GFP remained mainly at the plasma membrane, although
intracellular CCR7-GFP spots were reproducibly observed (data
not shown), confirming that CCL19 triggers CCR7 internalization
more efficiently than does CCL21. Internalized G protein-coupled
receptors are generally degraded in lysosomes or recycled back to
the plasma membrane via early and recycling endosomes. To investigate these two possibilities for CCR7, we incubated HEK293CCR7-GFP cells with CCL19 together with Alexa Fluor 546-labeled transferrin. The trafficking of the iron transport protein
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
Generation of a fluorescent fully functional CCR7
The Journal of Immunology
2317
transferrin is one of the best studied processes. Upon ligand binding, transferrin receptor is internalized by clathrin-coated pits giving rise to clathrin-coated vesicles. Endocytosed transferrin receptor, together with transferrin, then fuse with recycling endosomes
and are directed back to the plasma membrane (39). Extensive
colocalization of transferrin and CCR7-GFP was observed after 5
min and 3 h of CCL19 and transferrin stimulation (Fig. 3, A and B),
suggesting that CCR7-GFP localizes in endosomes. To discriminate recycling from early endosomes, HEK293-CCR7-GFP cells
were incubated with CCL19 and Alexa Fluor 546-labeled transferrin for 5 min, washed to remove unbound ligands, and further
incubated for 15 min at 37°C in the absence of ligands. Confocal
microscopy studies revealed that endocytosed CCR7-GFP colocalized with transferrin (data not shown), providing clear evidence
that the spotted distribution of CCR7-GFP represents recycling
endosomes. Furthermore, we investigated whether CCR7-GFP
also resides in lysosomes. To this end, we stimulated CCR7-GFP
expressing cells with CCL19 for 3 h. Analysis by confocal microscopy demonstrated that CCR7-GFP did not colocalize with
lysotracker, a marker for late endosomes and lysosomes (Fig. 3C).
Also, shorter or prolonged incubations with CCL19 (up to 9 h)
revealed the same results (data not shown), indicating that CCR7GFP is not sorted to the degradative pathway.
Endocytosed CCR7 is recycled and not degraded
To formally prove that CCR7 is indeed recycled, we incubated
HEK293 cells expressing CCR7-GFP with CCL19 or CCL21 for
30 min at 37°C. Subsequently, the excess of chemokine was removed, and cells were incubated for 1 h in the absence of chemokine at 37°C to allow receptor recycling. Cell surface expression of
CCR7 was determined by flow cytometry using a CCR7 specific
mAb (Fig. 4A). As expected, CCR7-GFP surface expression was
reduced after incubation of the cells with CCL19, and to a lesser
extent also with CCL21. After washing off chemokines, endocytosed CCR7 reappeared at the plasma membrane (Fig. 4A), demonstrating that CCR7 is either recycled after internalization or de
novo synthesized. Similar results also were obtained with 300-19
cells expressing HA-tagged CCR7 (Fig. 4B). To discriminate be-
FIGURE 4. Internalized CCR7 recycles back to the plasma membrane
and can mediate migration. (A) HEK293 cells expressing CCR7-GFP were
stimulated with 2 ␮g/ml CCL19, CCL21 or medium for 30 min at 37°C
and surface expression of CCR7 was determined by FACS analysis using
a CCR7 specific Ab (3D12). Where indicated, cells were washed extensively to remove non-bound chemokines and subsequently incubated for
1 h in the absence of chemokines permitting the recycling of CCR7-GFP
back to the plasma membrane. Mean values and SEM of three independent
experiments are shown as percent of CCR7 endocytosis or percent of recycled CCR7. B, CCR7 endocytosis and recycling in 300-19 cells expressing HA-tagged CCR7 was performed as described in A. In addition, cells
were pretreated with 50 ␮g/ml cycloheximide for 1 h and kept in the
presence of cycloheximide for the entire experiment to block de novo synthesis of CCR7 (gray bars). C, CEM T cells were incubated or not for 30
min with 2 ␮g/ml CCL19 to induce CCR7 endocytosis, washed, and subjected to a Transwell chemotaxis assay. In addition, internalized CCR7 was
allowed to recycle back to the plasma membrane by incubation for 1 h in
the absence of chemokine before the chemotaxis assay. To prevent de novo
synthesis of CCR7, CEM cells were pre-incubated for 1 h and kept in the
presence of 50 ␮g/ml cycloheximide (gray bars). Cells were allowed to
migrate in response to 1 ␮g/ml CCL19 for 3 h and quantified by flow
cytometry.
tween recycling and de novo synthesis, we pretreated 300-19CCR7-HA cells with cycloheximide for 1 h to prevent protein
synthesis, followed by CCL19 triggering. As depicted in Fig. 4B,
surface expression of CCR7 after endocytosis and recycling was
comparable between untreated and cycloheximide-treated cells,
providing clear evidence that CCR7 is recycled rather then newly
synthesized. To investigate whether recycled CCR7 can mediate
chemotaxis, we incubated CEM cells that endogenously express
CCR7 with CCL19 for 30 min to internalize CCR7 and allowed
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
FIGURE 3. Internalized CCR7-GFP colocalizes with recycling endosomes but not with lysosomes. HEK293 cells expressing CCR7-GFP were
incubated with 2 ␮g/ml CCL19 for 5 min (A) or 3 h (B and C) at 37°C in
the presence of either 50 ␮g/ml Alexa Fluor 546-labeled transferrin (A and
B) or 50 nM lysotracker (C). Cells were fixed, and the fluorescence was
analyzed by confocal microscopy. Scale bars, 10 ␮m.
2318
monomeric state after reduction and of ⬃80 kDa in a nonreduced
dimeric form as judged by SDS-PAGE followed by Coomassie
brilliant blue staining (data not shown). The biological activity of
the chemokine Fc fusion proteins was tested by the ability to mobilize intracellular free calcium and to induce chemotaxis. 300-19
cells expressing CCR7 were loaded with Fluo-3/AM and subsequently exposed to the chemokines, and the calcium-dependent
change in fluorescence was measured over time. Challenging 30019-CCR7 cells with CCL19, CCL21, or the corresponding chemokine-Fc-fusion proteins elucidated comparable transient rises in
[Ca2⫹]i (Fig. 6A), indicating that both CCL19-Fc and CCL21-Fc
are functional. No mobilization of [Ca2⫹]i was observed in parental 300-19 cells lacking CCR7, indicating that the rise in [Ca2⫹]i
was specific. Additionally, the chemotactic activity of CCL19-Fc
and CCL21-Fc were tested in a Transwell chemotaxis assay. As
shown in Fig. 6B, 300-19 cells expressing CCR7 migrated normally in response to CCL19-Fc, CCL19, and CCL21. For an unknown reason, only marginal migration toward CCL21-Fc was
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
the receptor to recycle back to the plasma membrane for 1 h. As
expected, cells with internalized CCR7 did not migrate in response
to CCL19, whereas cells with reexpressed CCR7 migrated toward
CCL19 (Fig. 4C). Similar results were obtained with 300-19 cells
expressing either CCR7-GFP or CCR7-HA (data not shown). Recycled rather than newly synthesized CCR7 was responsible for
chemotaxis, because pretreatment of CEM cells with cycloheximide did not hamper migration (Fig. 4C). Furthermore, recycled
CCR7 elicited the mobilization of cytosolic free calcium upon
CCL19 stimulation (data not shown), providing clear evidence that
recycled CCR7 is biologically functional.
To examine the overall rate of CCR7 degradation, potentially by
a nonlysosomal pathway, we performed a degradation assay in the
presence of both chemokines. HEK293-CCR7-GFP cells were incubated for up to 9 h with CCL19 or CCL21 in the presence or
absence of cycloheximide, to monitor on the one hand the steadystate level of the protein and, in contrast, the impact of de novo
synthesis. Using flow cytometry, we found no evidence for CCR7
degradation, because the fluorescence derived from CCR7-GFP
was not reduced upon chemokine triggering (Fig. 5A). To corroborate these data, we also investigated the degradation of CCR7 by
Western blotting. HEK293 cells expressing CCR7-HA were incubated with CCL19 or CCL21 for up to 6 h at 37°C, and the amount
of CCR7-HA was determined from total cell lysates using an antiHA Ab (Fig. 5B). Again, we found no evidence for CCR7 degradation, suggesting that the half-life of the receptor is very long.
OPPOSITE FATE OF CCR7 AND CCL19 TRAFFICKING
Generation of functional recombinant CCL19-Fc and CCL21-Fc
chemokine fusion proteins
To monitor the fate of CCL19 and CCL21 once they bound to
CCR7, we generated expression constructs encoding for chemokines fused to the Fc part of human IgG1 as there are no good Abs
against the chemokines available. We expressed human CCL19-Fc
and human CCL21-Fc in HEK293 cells and purified the recombinant fusion proteins from the supernatants over protein-A columns.
Both proteins were purified with a relative mass of ⬃40 kDa in a
FIGURE 5. Internalized CCR7 is not degraded. A, HEK293-CCR7GFP cells were incubated for different time periods with 2 ␮g/ml CCL19
(circles) or CCL21 (triangles) in the presence (open symbols) or absence
(closed symbols) of cycloheximide. Total GFP-derived fluorescence was
measured by flow cytometry. B, HEK293 cells stably expressing
CCR7-HA were incubated with 2 ␮g/ml CCL19 or CCL21 for up to 6 h.
Cells were lyzed, and the total amount of CCR7-HA was determined by
Western blotting using an anti-HA Ab. The ␣1 proteasome subunit was
used as a loading control.
FIGURE 6. Generation of functional CCL19-Fc and CCL21-Fc. A, Parental 300-19 cells (bottom panel) or cells stably expressing CCR7 were
loaded with Fluo-3/AM and stimulated with 4 ␮g/ml purified CCL19-Fc or
CCL21-Fc and chemokine-mediated changes in intracellular free calcium
concentrations were recorded over time by flow cytometry. For comparison, [Ca2⫹]i changes in response to 2 ␮g/ml untagged CCL19 and CCL21
were measured. The arrowheads indicate the time point of chemokine addition (B). The migration of 300-19 cells expressing CCR7 in response to
CCL19, CCL21 (1 ␮g/ml), and chemokine-Fc fusion proteins (10 ␮g/ml)
was measured in Transwell chemotaxis assays. After 3 h of incubation,
cells migrated to the lower wells were collected and counted by flow cytometry. As a control, cell migration in the absence of chemokine was
determined.
The Journal of Immunology
observed at different concentrations of chemokine (Fig. 6B and
data not shown).
CCL19-Fc is internalized together with CCR7 but then sorted to
lysosomes for degradation
The functional recombinant proteins enabled us to study the intracellular trafficking and interaction of both chemokines with CCR7.
CCL19-Fc induced internalization of CCR7-GFP similar to
CCL19; and CCL19-Fc colocalized with CCR7-GFP as shown by
confocal microscopy (Fig. 7A). Consistent with CCL21,
CCL21-Fc also induced some internalization of CCR7 (data not
shown). Remarkably, after incubation of HEK293 cells expressing
CCR7-GFP with CCL19-Fc for 30 min at 37°C, followed by washing off the chemokine and an additional incubation for 6 h in the
absence of the chemokine, most of the CCL19-Fc staining disappeared and vaguely colocalized with CCR7-GFP, which recycled
back to the plasma membrane (Fig. 7B). Similar results were obtained with monobiotinylated CCL19 (data not shown). To address
the trafficking of endocytosed chemokine, we stimulated CCR7transfected HEK293 cells with CCL19-Fc together with Alexa
Fluor 546-labeled transferrin. After 30 min of incubation,
CCL19-Fc partially colocalized with transferrin (Fig. 7C), indicating that CCL19-Fc localizes in early endosomes like CCR7. However, not all intracellular CCL19-Fc spots colocalized with endosomes. To address the origin of these additional compartments, we
stimulated CCR7-expressing cells with CCL19-Fc for 8 h in the
presence of lysotracker. As depicted in Fig. 7D, CCL19-Fc also
partially colocalized with lysosomes. To prove that CCL19-Fc is
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
FIGURE 7. CCL19-Fc is internalized
together with CCR7 and colocalizes with
both transferrin and lysosomes. A,
HEK293 cells expressing CCR7-GFP
were incubated with 10 ␮g/ml CCL19-Fc
for 30 min at 37°C. Cells were fixed and
permeabilized with Triton X-100.
CCL19-Fc was visualized using a biotinylated anti-human IgG Ab and streptavidin-Cy3. The localization of CCR7-GFP
and CCL19-Fc was determined by confocal microscopy. B, HEK293-CCR7GFP cells were stimulated with
CCL19-Fc for 30 min as in (A), washed
extensively and cultured in the absence
of chemokines for additional 6 h. C,
HEK293 cells stably transfected with
VSV-CCR7 were incubated with
CCL19-Fc for 30 min at 37°C in the
presence of Alexa Fluor 546-labeled
transferrin. CCL19-Fc was monitored
with a biotinylated anti-human IgG Ab
and streptavidin-FITC. D, HEK293 cells
expressing VSV-CCR7 were incubated
with CCL19-Fc and lysotracker for 8 h.
Representative images are shown. Scale
bars, 10 ␮m. E, HEK293 cells expressing
CCR7-HA were incubated with 10 ␮g/ml
CCL19-Fc (upper panel) or 3 ␮g/ml
monobiotinylated CCL19 (lower panel)
for 30 min at 37°C in the presence or
absence of 200 ␮M chloroquine. Cells
were washed, incubated for indicated
time periods in the presence or absence
of chloroquine, washed, and lyzed, and
proteins were separated by SDS-PAGE.
CCL19-Fc was detected by Western blotting using an anti-human IgG Ab coupled
to HRP. Biotinylated CCL19 was detected using streptavidinHRP.
2319
2320
OPPOSITE FATE OF CCR7 AND CCL19 TRAFFICKING
indeed degraded in lysosomes, we stimulated HEK293 cells expressing CCR7-HA with CCL19-Fc for 30 min in the presence or
absence of chloroquine. Chemokines were removed and cells were
further incubated for 3 and 6 h in the presence or absence of chloroquine. CCL19-Fc was degraded after 3 and 6 h (Fig. 7E). Treatment of the cells with the lysosomotrophic agent chloroquine and
subsequent incubation with CCL19-Fc significantly inhibited chemokine degradation, providing clear evidence for CCL19-Fc degradation in lysosomes (Fig. 7E). To exclude that the degradation
was due to the Fc part, we repeated the experiments with a chemically synthesized monobiotinylated CCL19, where a single amino
acid was biotinylated. In fact, biotinylated CCL19 also was degraded (Fig. 7E) comparable to CCL19-Fc. Chloroquine treatment
abolished the degradation of the biotinylated chemokine leading to
an accumulation of CCL19 in intracellular compartments (data not
shown).
CCR7 endocytosis is mediated by clathrin-coated pits
FIGURE 8. Endocytosis of CCR7 in human T cells is prevented by
sucrose treatment but not by cholesterol depletion. IL-2- and PHA-activated PBL were treated or not with 5 ␮g/ml filipin III, 2 mg/ml MCD, or
0.4 M sucrose. After 1 h of incubation at 37°C, cells were stimulated for
30 min with 3 ␮g/ml CCL19 or CCL21 or were left untreated. Cells were
transferred to 4°C and washed, and the surface expression of CCR7 was
determined by flow cytometry. Mean values and SD of at least three independent experiments are shown.
FIGURE 9. CCR7 internalization is dynamin II and Eps15 dependent.
HEK293 cells stably expressing VSV-CCR7 were transiently transfected
with GFP-tagged Dyn II wt (A), Dyn II K44A (B), Eps15 wt (C) and Eps15
mt (D). After 16 h of transfection, the cells were incubated with 10 ␮g/ml
CCL19-Fc for 30 min at 37°C, fixed, and permeabilized. CCL19-Fc was
visualized by a biotinylated anti-human IgG Ab and streptavidin-Cy3.
CCR7 internalization by CCL19-Fc in GFP positive and negative cells was
analyzed by confocal microscopy. Scale bars, 10 ␮m.
GFP-tagged dynamin II (Fig. 9A). However, expression of a GFPtagged dominant-negative mutant of dynamin II (dyn II K44A)
abolished the internalization of CCR7 (Fig. 9B). In contrast, Eps15
has until very recently (46, 47) been implicated only in clathrincoated pits assembly (38). Internalization of CCR7 was readily
observed after CCL19-Fc-mediated stimulation of CCR7-positive
cells transfected with Eps15-GFP (Fig. 9C). Remarkably, overexpressing a GFP-tagged dominant-negative form of Eps15 (Eps15
E⌬95/295) completely inhibited CCR7 internalization (Fig. 9D).
These data, in conjunction with the finding that sucrose treatment
abolished endocytosis, strongly suggest that CCR7 is internalized
through the clathrin-dependent pathway.
Taken together, we demonstrate that CCL19 is more efficient
than CCL21 in CCR7 internalization. CCR7 and its ligands are
most likely endocytosed together through clathrin-coated pits. In
early endosomes, the CCR7-ligand complex may dissociates and
CCR7 and its ligand follow different routes. The chemokine, which
is no longer used, is eliminated by lysosomal degradation. However, the receptor recycles back to the plasma membrane, ready to
bind a new ligand, permitting cell migration toward the source of
chemokine within the draining lymph node.
Discussion
There is no doubt that CCR7 and its ligands are essential for the
homing of dendritic cells and T lymphocytes to lymph nodes, Peyer’s Patches and the spleen (2, 9, 10, 48, 49). Ag-loaded DCs and
circulating T lymphocytes enter secondary lymphoid organs by
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
Two principal pathways of membrane receptor internalization are
known (33). The best-studied pathway is clathrin-dependent endocytosis with the respective machinery of adaptor proteins and
GTPases. The other pathway depends mainly on cholesterol-rich
membrane microdomains, also termed lipid rafts, and referred to as
clathrin-independent endocytosis, or in cells expressing caveolin,
also caveolae-dependent endocytosis (34). Both pathways can be
specifically inhibited. The formation of clathrin-coated pits can be
blocked under hypertonic conditions using 0.4 M sucrose (40).
Clathrin-independent endocytosis can be inhibited by sequestering
cellular cholesterol by MCD or filipin (41– 43). The pathway of
CCR7 internalization has not yet been investigated. To do so, we
pretreated IL-2 and PHA-activated PBL with filipin, MCD, or sucrose; incubated the cells with chemokines for 30 min at 37°C; and
measured the surface expression of CCR7 by flow cytometry.
Blocking the clathrin-independent pathway by filipin had no effect
on CCR7 endocytosis by CCL19 or CCL21 (Fig. 8). These data
were corroborated by treatment with MCD (Fig. 8) at a concentration that hampered TCR signaling (data not shown). Inhibition
of the clathrin-dependent pathway by sucrose abolished CCR7 endocytosis by CCL19 and CCL21 (Fig. 8), suggesting that CCR7
endocytosis is mediated by clathrin-coated pits.
To further characterize the endocytic pathway, we investigated
on the role of dynamin II and Eps15 in CCR7 internalization by
confocal microscopy. Dynamin II is involved in the formation of
both clathrin-coated and caveolar vesicles (44, 45). As expected, a
normal rate of CCR7 endocytosis was detected after CCL19-Fc
triggering in HEK293-CCR7 cells transiently transfected with
The Journal of Immunology
taxis and flux calcium in response to CCL19. Nevertheless, we
also investigated whether CCR7 is degraded. However, we neither
found colocalization with lysosomes nor evidence for degradation
as measured by the fluorescence intensity of CCR7-GFP over time
and by assessing the protein levels over time by Western blotting
(Figs. 3 and 5). Thus, in contrast with CXCR4, which is rapidly
degraded and has a half-life of ⬃3 h (27), CCR7 has a very long
half-life and recycles (Fig. 5). This finding is important because,
for example, dendritic cells that are infected in the periphery with
a virus need to migrate over a significant distance after sensing
CCR7 ligands for the first time. Viral infection of dendritic cells is
often accompanied by an inhibition of translation (57). Consequently, if CCR7 would be degraded after endocytosis-like
CXCR4, infected dendritic cells lacking surface expression of
CCR7 would be insensitive to CCL19/CCL21 and would never
make it into the lymph node. Under these conditions, an efficient
priming of lymphocytes would not occur because the Ags would
remain in the periphery.
The fate of chemokines after receptor internalization has remained enigmatic. More than a decade ago, even before the first
CC chemokine receptor was cloned, Wang et al. (58) identified the
first hint of chemokine degradation after endocytosis. Internalized
125
I-labeled CCL2 was progressively released into the culture supernatant of monocytes in a degraded form. CCL2 degradation was
inhibited by ammonium chloride, implicating lysosomal degradation (58). Later, it has been postulated, based on indirect evidence,
that CCR5, one of the receptors for CCL2, is recycled back to the
plasma membrane together with APO-CCL5, but not with CCL5
(29). In contrast, iodinated CCL3L1 was shown to be slowly degraded in an ammonium chloride-dependent manner after internalization via CCR5 (59). Interestingly, the membrane-anchored chemokine CX3CL1 is expressed at two different locations within the
cell, diffuse at the plasma membrane and punctuated in juxtanuclear compartments, and continuously cycles between the cell
surface and the endomembrane storage compartment in a SNAREdependent manner (60). The situation for the putative chemokine
receptor D6 that scavenges a large variety of inflammatory CC
chemokines is clearer. Internalized 125I-labeled CCL2 and
CCL3L1 by D6 were rapidly degraded in an ammonium chloridedependent manner (59, 61). Noteworthy, CCL19 is not scavenged
by D6 (62), and CCL21 scavenging was not addressed; thus, information about internalized CCR7 ligands is missing. Using a
CCL19-Fc fusion protein, we could, for the first time, investigate
the trafficking of a CCR7 ligand. CCL19 is internalized together
with CCR7 and localized in early endosomes (Fig. 7). There,
CCL19 dissociated from the receptor, which was recycled, and
was sorted to lysosomes for degradation as shown by confocal
microscopy (Fig. 7). Degradation of CCL19 by lysosomes was
further supported by the fact that chloroquine treatment abolished
CCL19 degradation as assessed by Western blotting (Fig. 7).
Taken together, we provide strong evidence that CCR7 after
CCL19 triggering is internalized via clathrin-coated pits and is
transported to early endosomes followed by its recycling back to
the plasma membrane where it can participate again in chemokine
gradient sensing. In contrast, CCL19 dissociates from the endocytosed receptor, presumably in early endosomes, and is sorted to
lysosomes for degradation. This may be of fundamental importance for inducing an efficient and potent immune response, because CCR7 is critical for the homing of lymphocytes and dendritic cells to secondary lymphoid organs. The fact that CCR7 is
recycled, rather than degraded, may be essential for virally infected dendritic cells to maintain the capacity to sense the chemokine gradient until the cells reach their final destination, even if
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
sensing CCL21 presented on HEV. Thereafter, they migrate to the
T zone where CCL19, as well as CCL21, are expressed, facilitating
the contact between naive T lymphocytes and Ag-loaded DCs and,
hence, the priming of T cells. For this event, fine-tuning of cell
migration may be critical.
One important way of modulating chemokine receptor responsiveness is receptor endocytosis after ligand binding. Studies on
chemokine receptor endocytosis moved into focus because chemokine-induced internalization of cell surface receptors was a major defense mechanism of chemokine-mediated inhibition of HIV
infection. Thus, endocytosis has been studied most intensively on
the HIV coreceptors CCR5 and CXCR4. CXCR4 internalization is
induced by its ligand CXCL12, but also by phorbol esters (50, 51).
CXCL12-mediated endocytosis occurs via clathrin-coated pits and
depends on Rab5 and Eps15 (26). After ligand binding, CXCR4 is
monoubiquitylated, endocytosed, and subsequently sorted to lysosomes for degradation (27). Interestingly, receptor mutants that are
not ubiquitylated internalize normally (27), but CXCR4 ubiquitylation by AIP4 is required for sorting to lysosomes and its degradation (28). However, CXCR4 also was shown to recycle back to
the plasma membrane (26, 50). In contrast, CCR5 internalization
does not occur by phorbol esters (52), but only by its ligands (26).
However, data on the routes of CCR5 internalization are controversial. CCR5 endocytosis was shown to be clathrin dependent
(29, 53). Furthermore, intracellular CCR5 colocalized with fluorescent-labeled transferrin (54) and ␤-arrestin (55, 56). In contrast,
cholesterol depletion by nystatin and filipin affected CCR5 endocytosis, and CCR5 was found to colocalize with caveolin, suggesting a role of caveolae/lipid rafts in this process (26, 55). Although
different routes of CCR5 endocytosis have been described, there is
consensus that CCR5 is recycled back to the cell surface (29, 54, 55).
The mechanism of CCR7 endocytos is poorly investigated. In
this study, we demonstrate that CCR7 in PBL is rapidly internalized after binding of CCL19 and to a lesser extent also by its
second ligand CCL21 (Fig. 1). This is intriguing because CCR7 is
the only chemokine receptor that is able to turn on different signaling pathways depending on different ligands. Our data are essentially in agreement with previous findings by Bardi et al. (32),
who observed CCL19- but not CCL21-mediated endocytosis of
CCR7 in activated lymphocytes . However, they also found borderline internalization of CCR7 by CCL21 in naive T cells, dendritic cells, and transfectants (32). The differential behavior of
CCL19 and CCL21 is striking because both chemokines have similar binding affinities and induce G protein activation, calcium mobilization, and chemotaxis with equal potency (4, 18). To further
characterize the trafficking of CCR7, we generated a CCR7-GFP
fusion protein that fulfills all functional properties of wt CCR7
(Fig. 2). We provide evidence that CCR7 is internalized by clathrin-coated pits, because overexpression of dominant-negative mutants of dynamin II and Eps15 blocked CCL19-induced endocytosis of CCR7 (Fig. 9). For a long time, Eps15 has been described
to be specific for clathrin-dependent endocytosis (33, 38). However, it has been shown recently that Eps15 also may be involved
in clathrin-independent endocytosis (46, 47). As hypertonic sucrose treatment also abolished CCR7 internalization (Fig. 8) is it
reasonable to assume that CCR7 uses clathrin-coated pits for entering the cells. After endocytosis, clathrin-coted vesicles containing CCR7 fuse with early endosomes, as shown by colocalization
with transferrin (Fig. 3). Endosomes are the key control organelles
for sorting where the decision is shaped whether receptors are
directed to late endosomes and lysosomes for degradation or are
recycled back to the plasma membrane (33). Internalized CCR7
followed the route of recycling back to the plasma membrane (Fig.
4 and Ref. 32), and recycled CCR7 was able to mediate chemo-
2321
2322
viral infection inhibits translation and, hence, neosynthesis
of CCR7.
Acknowledgments
We are grateful to Drs. Urs Greber, Osamu Yoshie, Pascal Schneider, and
Margot Thome for plasmids, and Matthias Langhorst for assistance in image processing.
Disclosures
The authors have no financial conflict of interest.
References
22. Gunn, M. D., K. Tangemann, C. Tam, J. G. Cyster, S. D. Rosen, and
L. T. Williams. 1998. A chemokine expressed in lymphoid high endothelial
venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc.
Natl. Acad. Sci. USA 95: 258 –263.
23. Warnock, R. A., J. J. Campbell, M. E. Dorf, A. Matsuzawa, L. M. McEvoy, and
E. C. Butcher. 2000. The role of chemokines in the microenvironmental control
of T versus B cell arrest in Peyer’s patch high endothelial venules. J. Exp. Med.
191: 77– 88.
24. Stein, J. V., A. Rot, Y. Luo, M. Narasimhaswamy, H. Nakano, M. D. Gunn,
A. Matsuzawa, E. J. Quackenbush, M. E. Dorf, and U. H. von Andrian. 2000. The
CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lymphoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-associated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node
high endothelial venules. J. Exp. Med. 191: 61–76.
25. Okada, T., M. J. Miller, I. Parker, M. F. Krummel, M. Neighbors, S. B. Hartley,
A. O’Garra, M. D. Cahalan, and J. G. Cyster. 2005. Antigen-engaged B cells
undergo chemotaxis toward the T zone and form motile conjugates with helper T
cells. PLoS Biol. 3:e150.
26. Venkatesan, S., J. J. Rose, R. Lodge, P. M. Murphy, and J. F. Foley. 2003.
Distinct mechanisms of agonist-induced endocytosis for human chemokine receptors CCR5 and CXCR4. Mol. Biol. Cell 14: 3305–3324.
27. Marchese, A., and J. L. Benovic. 2001. Agonist-promoted ubiquitination of the G
protein-coupled receptor CXCR4 mediates lysosomal sorting. J. Biol. Chem. 276:
45509 – 45512.
28. Marchese, A., C. Raiborg, F. Santini, J. H. Keen, H. Stenmark, and J. L. Benovic.
2003. The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G
protein-coupled receptor CXCR4. Dev. Cell 5: 709 –722.
29. Signoret, N., A. Pelchen-Matthews, M. Mack, A. E. Proudfoot, and M. Marsh.
2000. Endocytosis and recycling of the HIV coreceptor CCR5. J. Cell Biol. 151:
1281–1294.
30. Signoret, N., T. Christophe, M. Oppermann, and M. Marsh. 2004. pH-independent endocytic cycling of the chemokine receptor CCR5. Traffic 5: 529 –543.
31. Mueller, A., and P. G. Strange. 2004. Mechanisms of internalization and recycling of the chemokine receptor, CCR5. Eur. J. Biochem. 271: 243–252.
32. Bardi, G., M. Lipp, M. Baggiolini, and P. Loetscher. 2001. The T cell chemokine
receptor CCR7 is internalized on stimulation with ELC, but not with SLC. Eur.
J. Immunol. 31: 3291–3297.
33. Le Roy, C., and J. L. Wrana. 2005. Clathrin- and non-clathrin-mediated endocytic
regulation of cell signalling. Nat. Rev. Mol. Cell Biol. 6: 112–126.
34. Pfeffer, S. 2003. Membrane domains in the secretory and endocytic pathways.
Cell 112: 507–517.
35. Aguilar, R. C., and B. Wendland. 2005. Endocytosis of membrane receptors: two
pathways are better than one. Proc. Natl. Acad. Sci. USA 102: 2679 –2680.
36. Legler, D. F., M. Loetscher, R. S. Roos, I. Clark-Lewis, M. Baggiolini, and
B. Moser. 1998. B cell-attracting chemokine 1, a human CXC chemokine expressed in lymphoid tissues, selectively attracts B lymphocytes via BLR1/
CXCR5. J. Exp. Med. 187: 655– 660.
37. Cao, H., H. M. Thompson, E. W. Krueger, and M. A. McNiven. 2000. Disruption
of Golgi structure and function in mammalian cells expressing a mutant dynamin.
J. Cell Sci. 113 (Pt 11): 1993–2002.
38. Benmerah, A., M. Bayrou, N. Cerf-Bensussan, and A. Dautry-Varsat. 1999. Inhibition of clathrin-coated pit assembly by an Eps15 mutant. J. Cell Sci. 112 (Pt
9): 1303–1311.
39. Johnson, A. O., R. N. Ghosh, K. W. Dunn, R. Garippa, J. Park, S. Mayor,
F. R. Maxfield, and T. E. McGraw. 1996. Transferrin receptor containing the
SDYQRL motif of TGN38 causes a reorganization of the recycling compartment
but is not targeted to the TGN. J. Cell Biol. 135: 1749 –1762.
40. Heuser, J. E., and R. G. Anderson. 1989. Hypertonic media inhibit receptormediated endocytosis by blocking clathrin-coated pit formation. J. Cell Biol. 108:
389 – 400.
41. Harder, T., R. Kellner, R. G. Parton, and J. Gruenberg. 1997. Specific release of
membrane-bound annexin II and cortical cytoskeletal elements by sequestration
of membrane cholesterol. Mol. Biol. Cell 8: 533–545.
42. Keller, P., and K. Simons. 1998. Cholesterol is required for surface transport of
influenza virus hemagglutinin. J. Cell Biol. 140: 1357–1367.
43. Orlandi, P. A., and P. H. Fishman. 1998. Filipin-dependent inhibition of cholera
toxin: evidence for toxin internalization and activation through caveolae-like domains. J. Cell Biol. 141: 905–915.
44. Oh, P., D. P. McIntosh, and J. E. Schnitzer. 1998. Dynamin at the neck of caveolae mediates their budding to form transport vesicles by GTP-driven fission from
the plasma membrane of endothelium. J. Cell Biol. 141: 101–114.
45. De Camilli, P., K. Takei, and P. S. McPherson. 1995. The function of dynamin
in endocytosis. Curr. Opin. Neurobiol. 5: 559 –565.
46. Chen, H., and P. De Camilli. 2005. The association of epsin with ubiquitinated
cargo along the endocytic pathway is negatively regulated by its interaction with
clathrin. Proc. Natl. Acad. Sci. USA 102: 2766 –2771.
47. Sigismund, S., T. Woelk, C. Puri, E. Maspero, C. Tacchetti, P. Transidico,
P. P. Di Fiore, and S. Polo. 2005. Clathrin-independent endocytosis of ubiquitinated cargos. Proc. Natl. Acad. Sci. USA 102: 2760 –2765.
48. Moser, B., and P. Loetscher. 2001. Lymphocyte traffic control by chemokines.
Nat. Immunol. 2: 123–128.
49. Cyster, J. G. 1999. Chemokines and cell migration in secondary lymphoid organs.
Science 286: 2098 –2102.
50. Amara, A., S. L. Gall, O. Schwartz, J. Salamero, M. Montes, P. Loetscher,
M. Baggiolini, J. L. Virelizier, and F. Arenzana-Seisdedos. 1997. HIV coreceptor
downregulation as antiviral principle: SDF-1␣-dependent internalization of the
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
1. Moser, B., M. Wolf, A. Walz, and P. Loetscher. 2004. Chemokines: multiple
levels of leukocyte migration control. Trends Immunol. 25: 75– 84.
2. Campbell, D. J., C. H. Kim, and E. C. Butcher. 2003. Chemokines in the systemic
organization of immunity. Immunol. Rev. 195: 58 –71.
3. Muller, G., U. E. Hopken, and M. Lipp. 2003. The impact of CCR7 and CXCR5
on lymphoid organ development and systemic immunity. Immunol. Rev. 195:
117–135.
4. Willimann, K., D. F. Legler, M. Loetscher, R. S. Roos, M. B. Delgado,
I. Clark-Lewis, M. Baggiolini, and B. Moser. 1998. The chemokine SLC is expressed in T cell areas of lymph nodes and mucosal lymphoid tissues and attracts
activated T cells via CCR7. Eur. J. Immunol. 28: 2025–2034.
5. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two
subsets of memory T lymphocytes with distinct homing potentials and effector
functions. Nature 401: 708 –712.
6. Sallusto, F., P. Schaerli, P. Loetscher, C. Schaniel, D. Lenig, C. R. Mackay,
S. Qin, and A. Lanzavecchia. 1998. Rapid and coordinated switch in chemokine
receptor expression during dendritic cell maturation. Eur. J. Immunol. 28:
2760 –2769.
7. Sozzani, S., P. Allavena, G. D’Amico, W. Luini, G. Bianchi, M. Kataura, T. Imai,
O. Yoshie, R. Bonecchi, and A. Mantovani. 1998. Differential regulation of chemokine receptors during dendritic cell maturation: a model for their trafficking
properties. J. Immunol. 161: 1083–1086.
8. Gunn, M. D. 2003. Chemokine mediated control of dendritic cell migration and
function. Semin. Immunol. 15: 271–276.
9. Forster, R., A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf, and
M. Lipp. 1999. CCR7 coordinates the primary immune response by establishing
functional microenvironments in secondary lymphoid organs. Cell 99: 23–33.
10. Ohl, L., M. Mohaupt, N. Czeloth, G. Hintzen, Z. Kiafard, J. Zwirner,
T. Blankenstein, G. Henning, and R. Forster. 2004. CCR7 governs skin dendritic
cell migration under inflammatory and steady-state conditions. Immunity 21:
279 –288.
11. Gunn, M. D., S. Kyuwa, C. Tam, T. Kakiuchi, A. Matsuzawa, L. T. Williams, and
H. Nakano. 1999. Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization.
J. Exp. Med. 189: 451– 460.
12. Luther, S. A., H. L. Tang, P. L. Hyman, A. G. Farr, and J. G. Cyster. 2000.
Coexpression of the chemokines ELC and SLC by T zone stromal cells and
deletion of the ELC gene in the plt/plt mouse. Proc. Natl. Acad. Sci. USA 97:
12694 –12699.
13. Vassileva, G., H. Soto, A. Zlotnik, H. Nakano, T. Kakiuchi, J. A. Hedrick, and
S. A. Lira. 1999. The reduced expression of 6Ckine in the plt mouse results from
the deletion of one of two 6Ckine genes. J. Exp. Med. 190: 1183–1188.
14. Nakano, H., and M. D. Gunn. 2001. Gene duplications at the chemokine locus on
mouse chromosome 4: multiple strain-specific haplotypes and the deletion of
secondary lymphoid-organ chemokine and EBI-1 ligand chemokine genes in the
plt mutation. J. Immunol. 166: 361–369.
15. Scandella, E., Y. Men, D. F. Legler, S. Gillessen, L. Prikler, B. Ludewig, and
M. Groettrup. 2004. CCL19/CCL21-triggered signal transduction and migration
of dendritic cells requires prostaglandin E2. Blood 103: 1595–1601.
16. Tilton, B., L. Ho, E. Oberlin, P. Loetscher, F. Baleux, I. Clark-Lewis, and
M. Thelen. 2000. Signal transduction by CXC chemokine receptor 4: stromal
cell-derived factor 1 stimulates prolonged protein kinase B and extracellular signal-regulated kinase 2 activation in T lymphocytes. J. Exp. Med. 192: 313–324.
17. Bardi, G., V. Niggli, and P. Loetscher. 2003. Rho kinase is required for CCR7mediated polarization and chemotaxis of T lymphocytes. FEBS Lett. 542: 79 – 83.
18. Kohout, T. A., S. L. Nicholas, S. J. Perry, G. Reinhart, S. Junger, and
R. S. Struthers. 2004. Differential desensitization, receptor phosphorylation, ␤-arrestin recruitment, and ERK1/2 activation by the two endogenous ligands for the
CC chemokine receptor 7. J. Biol. Chem. 279: 23214 –23222.
19. Riol-Blanco, L., N. Sanchez-Sanchez, A. Torres, A. Tejedor, S. Narumiya,
A. L. Corbi, P. Sanchez-Mateos, and J. L. Rodriguez-Fernandez. 2005. The chemokine receptor CCR7 activates in dendritic cells two signaling modules that
independently regulate chemotaxis and migratory speed. J. Immunol. 174:
4070 – 4080.
20. Sanchez-Sanchez, N., L. Riol-Blanco, G. De La Rosa, A. Puig-Kroger,
J. Garcia-Bordas, D. Martin, N. Longo, A. Cuadrado, C. Cabanas, A. L. Corbi, et
al. 2004. Chemokine receptor CCR7 induces intracellular signaling that inhibits
apoptosis of mature dendritic cells. Blood 104: 619 – 625.
21. Carlsen, H. S., G. Haraldsen, P. Brandtzaeg, and E. S. Baekkevold. 2005. Disparate lymphoid chemokine expression in mice and men: no evidence of CCL21
synthesis by human high endothelial venules. Blood 106: 444 – 446.
OPPOSITE FATE OF CCR7 AND CCL19 TRAFFICKING
The Journal of Immunology
51.
52.
53.
54.
55.
56.
chemokine receptor CXCR4 contributes to inhibition of HIV replication. J. Exp.
Med. 186: 139 –146.
Signoret, N., J. Oldridge, A. Pelchen-Matthews, P. J. Klasse, T. Tran, L. F. Brass,
M. M. Rosenkilde, T. W. Schwartz, W. Holmes, W. Dallas, et al. 1997. Phorbol
esters and SDF-1 induce rapid endocytosis and down modulation of the chemokine receptor CXCR4. J. Cell Biol. 139: 651– 664.
Signoret, N., M. M. Rosenkilde, P. J. Klasse, T. W. Schwartz, M. H. Malim,
J. A. Hoxie, and M. Marsh. 1998. Differential regulation of CXCR4 and CCR5
endocytosis. J. Cell Sci. 111 (Pt 18): 2819 –2830.
Signoret, N., L. Hewlett, S. Wavre, A. Pelchen-Matthews, M. Oppermann, and
M. Marsh. 2005. Agonist-induced endocytosis of CC chemokine receptor 5 is
clathrin dependent. Mol. Biol. Cell 16: 902–917.
Mack, M., B. Luckow, P. J. Nelson, J. Cihak, G. Simmons, P. R. Clapham,
N. Signoret, M. Marsh, M. Stangassinger, F. Borlat, et al. 1998. Aminooxypentane-RANTES induces CCR5 internalization but inhibits recycling: a novel inhibitory mechanism of HIV infectivity. J. Exp. Med. 187: 1215–1224.
Mueller, A., E. Kelly, and P. G. Strange. 2002. Pathways for internalization and
recycling of the chemokine receptor CCR5. Blood 99: 785–791.
Kraft, K., H. Olbrich, I. Majoul, M. Mack, A. Proudfoot, and M. Oppermann.
2001. Characterization of sequence determinants within the carboxyl-terminal
domain of chemokine receptor CCR5 that regulate signaling and receptor internalization. J. Biol. Chem. 276: 34408 –34418.
2323
57. Freigang, S., H. C. Probst, and M. van den Broek. 2005. DC infection promotes
antiviral CTL priming: the “Winkelried” strategy. Trends Immunol. 26: 13–18.
58. Wang, J. M., A. Hishinuma, J. J. Oppenheim, and K. Matsushima. 1993. Studies
of binding and internalization of human recombinant monocyte chemotactic and
activating factor (MCAF) by monocytic cells. Cytokine 5: 264 –275.
59. Weber, M., E. Blair, C. V. Simpson, M. O’Hara, P. E. Blackburn, A. Rot,
G. J. Graham, and R. J. Nibbs. 2004. The chemokine receptor D6 constitutively
traffics to and from the cell surface to internalize and degrade chemokines. Mol.
Biol. Cell 15: 2492–2508.
60. Liu, G. Y., V. Kulasingam, R. T. Alexander, N. Touret, A. M. Fong, D. D. Patel,
and L. A. Robinson. 2005. Recycling of the membrane-anchored chemokine,
CX3CL1. J. Biol. Chem. 280: 19858 –19866.
61. Fra, A. M., M. Locati, K. Otero, M. Sironi, P. Signorelli, M. L. Massardi,
M. Gobbi, A. Vecchi, S. Sozzani, and A. Mantovani. 2003. Cutting edge: scavenging of inflammatory CC chemokines by the promiscuous putatively silent
chemokine receptor D6. J. Immunol. 170: 2279 –2282.
62. Bonecchi, R., M. Locati, E. Galliera, M. Vulcano, M. Sironi, A. M. Fra,
M. Gobbi, A. Vecchi, S. Sozzani, B. Haribabu, et al. 2004. Differential recognition and scavenging of native and truncated macrophage-derived chemokine
(macrophage-derived chemokine/CC chemokine ligand 22) by the D6 decoy receptor. J. Immunol. 172: 4972– 4976.
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017