![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 15, Issue 5, 2492-2508, May 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||
* The Cancer Research UK Beatson Laboratories, The Beatson Institute for Cancer Research, Glasgow, United Kingdom G61 1BD;
The Division of Immunology, Infection, and Inflammation, Glasgow University, Glasgow, United Kingdom G11 6NT;
Department of Chemistry, Glasgow University, Glasgow, United Kingdom G11 6NT; and
Novartis Forschunginstitut, Vienna, A-1235 Austria
Submitted September 2, 2003;
Revised February 10, 2004;
Accepted February 12, 2004
Monitoring Editor: Howard Riezman
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
; Mackay, 2001; Kunkel and Butcher, 2002). This is achieved by the tightly regulated expression of members of a family of seven-transmembrane (7-TM) chemokine receptors on the surface of leukocytes. Functional differentiation is coupled to chemokine receptor switching to alter intratissue localization and change tissue tropism. Because leukocytes are intimately involved in many diseases, it is of little surprise that chemokines are implicated in pathologies such as chronic inflammatory disease, autoimmunity, atherosclerosis, allergy, allograft rejection, and cancer (Gerard and Rollins, 2001). In addition, chemokine receptors are pirated for cellular entry by HIV, with the ligands blocking viral entry (Simmons et al., 2000). Their importance is emphasized by the association of allelic variants of genes within the chemokine system with variable disease susceptibility, severity, or progression (O'Brien and Moore, 2000; Gonzalez et al., 2002; Cybulsky and Hegele, 2003) and by the analysis of animal models of disease in mice carrying genetic modifications of the chemokine network (Gerard and Rollins, 2001; Power, 2003).
Chemokines show obvious sequence homology, in particular a conserved cysteine motif, which has led to their subdivision (Zlotnik and Yoshie, 2000; Mackay, 2001; Bacon et al., 2002). In the 15 human CXC chemokines, a single nonconserved amino acid separates the first two cysteines, whereas in the 25 human CC chemokines, they are directly juxtaposed. The C chemokines lack one of these first two cysteine residues, whereas three amino acids separate these cysteines in the single CX3C chemokine. In general, this motif limits a chemokine to a receptor subgroup, with six receptors identified for CXC chemokines, 10 for the CC subfamily, and one each for the C and CX3C proteins. These receptors often bind multiple ligands, and many ligands can use multiple receptors, allowing for enhanced flexibility in the regulation of responses. Each of these 18 receptors is found on subsets of leukocytes where they act to define migratory responses to chemokines. However, it is clear that expression is often not restricted to leukocytes (allowing chemokines to exert biological effects on other cell types) and that migration is not the only biological response mediated by chemokines.
Because chemokines play a major role in immune regulation, mechanisms must exist to tightly control their production, distribution, and destruction. Decoy receptors are well characterized in mammalian cytokine biology, providing additional ways to regulate responses (Mantovani et al., 2001). Some viruses encode soluble chemokine-neutralizing proteins among their immunomodulatory armory (Alcami, 2003), but mammalian counterparts have not been described. Instead, specialized 7-TM chemokine decoy receptors may have evolved to fulfill this function, a strategy also apparently used by certain viruses (Bodaghi et al., 1998; Alcami 2003). In mammals, three 7-TM proteins exist that differ in a number of important aspects from the 18 known leukocyte migratory receptors. These are Duffy antigen receptor for chemokines (DARC), CCX-CKR, and D6 (Chaudhuri et al., 1993; Nibbs et al., 1997, 2003; Gosling et al., 2000; Townson and Nibbs, 2002; Lee et al., 2003). Despite structural similarities and high-affinity ligand binding, they cannot couple to the major signaling pathways used by typical chemokine receptors, and no alternative signals have been described. Also, they are predominantly expressed on nonleukocytic cell types and seem unlikely to be directly involved in leukocyte migration. Consequently, transport and/or decoy receptor-like functions have been proposed for these molecules, although little firm evidence exists. For example, DARC expression on red blood cells has been seen as an indication of a sink or reservoir function (Horuk, 1994; Fukuma et al., 2003). Alternatively, DARC expressed by blood vessel endothelial cells (Hadley et al., 1994) may act as a chemokine transporter, presenting tissue-derived chemokines on the luminal surface of blood vessels (Middleton et al., 1997; Lee et al., 2003). We, and others, have proposed decoy and/or DARC-like transport functions for D6 on lymphatic endothelial cells (LECs), a major site of D6 expression (Mantovani et al., 2001; Nibbs et al., 2001, 2003). Indeed, recent work has indicated that despite being "silent," D6 internalizes chemokines and can degrade them over time (Fra et al., 2003). Although this lends support to the decoy receptor hypothesis, it is unclear as to whether ligand degradation is simply a consequence of internalization or a specific property of D6. Also, no mechanistic details behind ligand internalization have been revealed.
Here, we have examined D6-mediated chemokine uptake compared with CCR5, a receptor that also interacts with many of the D6 ligands. We demonstrate that D6-expressing cells internalize CC-chemokine ligand 3 (CCL3) at a rate indistinguishable from CCR5, but unlike CCR5, this is done without reducing the surface levels of receptor, or desensitizing subsequent ligand uptake. This is possible because D6 is constitutively cycling to and from the cell surface in the absence of ligand. In fact, D6 is predominantly an intracellular protein, associated with the early/recycling endosomal compartment with <5% on the surface at any one time. In addition, D6-internalized ligands are more readily retained in the cell than those internalized through CCR5. This is because passage through the acidic endosomal compartment displaces chemokines from D6 but slows their dissociation from CCR5. Thus, by associating with the constitutive recycling endosome pool, and readily disengaging from its ligands at lower pH, D6 can internalize ligand without the need for ligand-induced signaling, and seems specifically designed for targeting CC chemokines for destruction. We also show, using a novel flow cytometric protocol, that interfering individually with the activity of the early endosomal GTPase (rab5), a regulator of clathrin-coated vesicle formation (Eps15), or the endocytosis mediator dynamin I, can all reduce ligand uptake via D6.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Plasmids
Constructs encoding GFP-tagged canine wild-type, S34N, or Q79L rab5, or rat dynamin I (K44A) or rat 
-arrestin-1 (V53D) were generous gifts from S. Ferguson (Robarts Research Institute, London, Canada) (Barlic et al., 1999; Seachrist et al., 2000). Those encoding GFP-tagged mutants of human eps15 (i.e., E
95/295, DIII, and DIII2) were kindly provided by A. Benmerah (Cochin Institute, Paris, France) (Benmerah et al., 1998, 1999).
Antibodies
-Human D6 antibodies are described previously (Nibbs et al., 2001). Other primary antibodies were uncoupled and fluorescein isothiocyanate-coupled -HA (Cambridge Bioscience, Cambridge, United Kingdom), phycoerythrin (PE)-coupled and -uncoupled -CCR5 (R&D Systems, Abingdon, United Kingdom). -CCR5 clone CTC-5 was selected because 1) it bound better to CCR5 than other -CCR5 clones in preliminary studies, and 2) it did not interfere with ligand binding (Lee et al., 1999; our unpublished data). Other antibodies were --arrestin-1, -dynamin I, -rab5, and -rab11 (BD Biosciences, Oxford, United Kingdom), -CD63 (Chemicon, Harrow, United Kingdom), -CD71 (transferrin receptor), and -golgin-97 (Molecular Probes, Leiden, The Netherlands) and -clathrin (Affinity Bioreagents via Cambridge Bioscience). Tetramethylrhodamine isothiocyanate (TRITC)-, Cy3-, and PE-coupled -mouse IgG were all from Sigma (Poole, Dorset, United Kingdom). Antibodies were optimized by preliminary investigations suggested by the suppliers.
Chemokines
The murine (m) CCL3 used was a nonaggregating mutant version, with identical in vitro bioactivity to wild-type murine CCL3 (Graham et al., 1994). Human (h) CCL3-L1 was produced as reported previously (Nibbs et al., 1999) and has identical bioactivity to hCCL3-L1 from R&D Systems. Biotinylated mCCL3 (Bio-CCL3) was synthesized by Albachem (Gladsmuir, East Lothian, United Kingdom) and is identical in sequence to the nonaggregating mutant of mCCL3 described above except it carried an additional biotinylated lysine residue on the C terminus. Other chemokines were from either R&D Systems or Peprotech (London, United Kingdom), were full length according to accepted sites of signal peptidase cleavage, and showed bioactivity on expected receptors. Chemokines were labeled with radioiodine as described previously (Graham et al., 1993).
Radioligand Internalization Assays
mCCL3 Internalization Rate Determination. Cells were harvested by mechanical disruption or brief trypsinization, washed in phosphate-buffered saline (PBS), and 0.5–1 x 106 cells were resuspended in 50 µl of 293 medium containing 12 nM radioligand and incubated at 4°C for 
1 h with regular gentle agitation. Preliminary experiments showed that radioligand binding reaches equilibrium within 30 min. Cells were then spun (2600 rpm, 5 min, 4°C), washed in ice-cold PBS, and resuspended in 200 µl of 293 medium. After shift to 37°C, tubes were spun as described above, the medium removed (which contained minimal radioligand), and the cells washed with either PBS or acid wash (0.2 M acetic acid, 0.5 M NaCl), both ice-cold, for 5 min. Finally, tubes were spun as described above, and the cell pellet counted in a Beckman Gamma 5500B counter (Beckman, High Wycombe, United Kingdom). Ligand internalization was determined by the ratio of radioactivity in acid stripped versus PBS-washed cell pellets.
Ligand Uptake after hCCL3-L1 Pretreatment. Harvested cells were incubated at 5 x 106 cells/ml in binding buffer (293 medium plus 10 mM HEPES, pH 7.4) with or without 200 nM hCCL3-L1 for 1 h on ice and then shifted to 37°C for 45 min. After two washes in 50 ml of ice-cold PBS, cells were resuspended in ice-cold binding buffer to 1 x 107 cells/ml, and 40-µl aliquots were taken. Then, 10 µl of 30 nM 125I-hCCL3-L1, prediluted in binding buffer, was added, and samples were incubated at 37°C for up to 30 min, after which cells were spun (2600 rpm, 5 min, 4°C) and washed with ice-cold PBS. Cell-associated radioactivity was determined using a Beckman Gamma 5500B counter.
Surface Receptor Assessments by Flow Cytometry
For simple flow cytometry, cells were washed with fluorescence-activated cell sorting (FACS) buffer (PBS/2% fetal calf serum) and resuspended in 100 µl of ice-cold FACS buffer. Antibodies were added and the cells left on ice for 30–45 min with occasional gentle agitation. Where -IgG secondary antibodies were required, 1–1.4 ml of ice-cold FACS buffer was added, the samples spun (2600 rpm, 5 min, 4°C), and the cell pellet was resuspended in 50–100 µl of ice cold FACS buffer containing the -IgG antibody and left on ice for 30–45 min with occasional gentle agitation. After the antibody incubation steps, and another cold FACS buffer wash, cells were resuspended in 400 µl of FACS buffer and passed through a FACScan flow cytometer (Becton Dickinson, Cowley, United Kingdom). As a negative control, samples of untransfected cells were used alongside test samples.
SDS-PAGE and Western Blotting
Samples from the radioligand degradation experiments (below) were boiled for 10 min, spun (13,000 rpm, 3 min), and 25 µl electrophoresed on a 4–12% Bis-Tris gradient acrylamide gel (Invitrogen) in 1x MES buffer (Invitrogen). An aliquot of 125I-hCCL3-L1 stock, containing an equivalent number of cpm to that found in 25 µl of the most radioactive test sample, was similarly prepared in 1x LB and loaded on the gel. Samples were run adjacent to MultiMark protein markers (Invitrogen). After electrophoresis, gels were dried onto filter paper and exposed to x-ray film. For -D6 probed Western blots, D6 was purified from L1.2 cells expressing HA, His10-tagged D6 and quantified by bicinchoninic acid assay (Blackburn et al., 2004). Cell lysates from cells treated with or without mCCL3, and with or without cycloheximide (CHX; Sigma Chemical) (20 µg/ml), were prepared in CelLytic-M mammalian cell lysis buffer (Sigma Chemical). An equal volume of HU buffer (8 M urea, 5% SDS, 200 mM Tris-HCl, pH 8, 0.1 mM EDTA, 100 mM dithiothreitol, 0.5% bromphenol blue) was added. Samples were not boiled (this causes D6 aggregation; Blackburn et al., 2004), but they were left at room temperature for 10 min, subjected to SDS-PAGE, electrophoretically transferred onto polyvinylidene difluoride membrane, and blocked overnight in 10% milk/PBS. Blots were incubated with -D6 antibody, washed in PBS/0.1% Tween, visualized with -mouse horseradish peroxidase-coupled secondary antibodies (Amersham Biosciences UK, Little Chalfont, Buckinghamshire, United Kingdom), developed using a WestPico kit (Pierce Chemical, Rockford, IL), and exposed to x-ray film.
Immunostaining Protocols
Cells were grown on fibronectin-treated eight-well chamber slides overnight, washed with PBS, fixed for 10 min in 3.5% paraformaldehyde (PFA), washed twice with PBS, and then incubated for 20 min in 50 mM NH4Cl. After a PBS wash, cells were incubated in PGS (PBS, 0.2% gelatin, 0.05% saponin) for 30 min and then in PGS containing primary antibody for 1 h. After two PGS washes, PGS containing fluorophore-coupled -IgG antibodies was added for 1 h, and cells washed twice with PGS and fixed in 3.5% PFA. Slides were mounted in Vectashield (with or without 4,6-diamidino-2-phenylindole; DAPI) (Vector Laboratories, Peterborough, United Kingdom), a coverslip applied, and sealed with nail varnish.
Immunohistochemistry on Human Tissue Samples
This was performed as published previously (Nibbs et al., 2001) on paraffin-embedded sections obtained from the Glasgow University Pathology Department.
Antibody Feeding and Recycling
Antibody Feeding. Cells on fibronectin-treated eight-well chamber slides were washed twice with PBS and incubated in BM (serum-free 293 medium, 10 mM HEPES pH 7.4, 0.2% bovine serum albumin) at 4°C for at least 30 min. Antibody was then added and cells left at 4°C for a further 30–45 min. Cells were then washed with ice-cold BM (or ice cold BM adjusted to pH 3 where appropriate) and then either directly fixed (10 min in 3.5% PFA) or BM added, and the slides transferred to 37°C in the presence or absence of mCCL3 (200 nM). Cells washed with BM, pH 3, were washed twice with BM to return pH to 7.4 before 37°C incubation. After incubation, cells were washed with PBS, fixed for 10 min in 3.5% PFA, and washed twice in PBS. All slides were then treated as described above for the immunostaining protocols with incubation with 50 mM NH4Cl and then PGS and finally PGS containing fluorophore-coupled antibodies. After two PGS washes, cells were fixed for 10 min in 3.5% PFA and mounted as described above. Control experiments were performed in the absence of saponin.
Antibody Recycling Experiments for Confocal Microscopy Visualization. Cells plated as described above were loaded for up to 1 h at 37°C with antibody, washed with cold BM, pH 3, and then twice with cold BM and incubated in BM containing fluorophore-coupled -IgG antibodies for up to 1 h at 37°C. Cells were washed in PBS, fixed in 3.5% PFA, washed again with PBS, and mounted as described above.
Flow Cytometric Analysis of Recycling. Cells were harvested by mechanical disruption or brief trypsinization, and 5 x 105 cells were loaded with antireceptor antibodies at 37°C for 45–60 min in BM. Cells were then washed twice with cold BM, pH 3, and twice with cold BM, and finally incubated at 37 or 4°C in BM containing PE-coupled -mouse IgG antibodies for up to 90 min. Cells were then washed in 1 ml of PBS and analyzed by flow cytometry using a FACScan flow cytometer.
Confocal Microscopy
All fixed cells were processed on glass slides mounted under a glass coverslip as described above. For live imaging, cells were grown overnight on fibronectin-treated glass-bottomed culture dishes (MatTek, Ashland, MA), the medium was replaced with complete 293 medium containing 10 mM HEPES, pH 7.4, and the cells were examined directly on the confocal microscope fitted with a stage heated to 37°C. All images were captured using a Leica SP-2 confocal (Leica, Milton Keynes, United Kingdom) configured with Leica confocal software, with a 40x or 63x oil immersion objective and digital zoom. Fluorochromes were excited sequentially with lasers at 488 nm (GFP) or 543 nm (Cy3 and TRITC), and with a UV laser to excite DAPI. Images were superimposed using Leica Confocal software and assembled using ThumbsPlus software (Cerious Software, Charlotte, NC). In all experiments, at least eight fields of cells were examined and representative images collected. Serial z-sectioning was used to confirm the intracellular localization, and colocalization, of fluorescence emission where appropriate.
Ligand Degradation Assays
Harvested cells (5 x 105) were incubated at 4°C with radioligand for at least 1 h, washed with ice-cold PBS, resuspended in 100 µl of 293 medium, and incubated at 37°C for up to 2.5 h. In some instances, cells were omitted from the tubes. Where appropriate, 50 mM NH4Cl was included in the 293 medium: cells treated in this way bind radioligand and internalize it upon temperature shift (as revealed by acid stripping) in a manner indistinguishable from nonNH4Cl-treated cells (our unpublished data). In other experiments, 200 nM unlabeled hCCL3-L1 was included during the 37°C incubation. For each time point, triplicate samples were spun (2600 rpm, 5 min, 4°C), the supernatant taken, and the cell pellet washed in 200 µl of 293 medium, which was subsequently combined with the first 100 µl of supernatant. Cell pellets were resuspended in 100 µl of PBS, and 50 µl was counted in a Beckman Gamma 5500B counter. To the remainder, 50 µl of 2x LB (100 mM Tris-HCl, pH 6.5, 4% SDS, 20 mM dithiothreitol, 20% glycerol, 0.2% bromphenol blue) was added in readiness for SDS-PAGE (see above). With the 300 µl of supernatant, 150 µl was taken and an equal volume of 25% trichloroacetic acid (TCA) added. These were incubated at 4°C for 15 min, centrifuged (13,000 rpm, 4°C, 15 min), supernatant taken, and the TCA precipitate washed in 300 µl of ice-cold acetone: the acetone wash was combined with the non-TCA precipitable supernatant. The TCA pellet and non-TCA precipitable material were counted in a Beckman Gamma 5500B counter. The percentage of retrieved counts of associated radioiodine in cells, TCA pellet, and non-TCA precipitable was then calculated. To the remaining 150 µl of supernatant taken from the incubated cells, an equivalent volume of 2x LB was added for SDS-PAGE analysis.
Radioligand Dissociation Assays
Harvested cells (5 x 105) were incubated at 4°C with 12 nM 125I-hCCL3-L1 for at least 1 h, spun (2600 rpm, 5 min, 4°C), and washed with regular agitation for 5–60 min in 1.5 ml of ice-cold 293 medium adjusted to a given pH (2–7) with HCl. Medium was buffered with 10 mM HEPES for pH 6 and above, but below this 10 mM MES was used. Control cells were washed briefly with ice-cold PBS. Cells were retrieved by centrifugation (2600 rpm, 5 min, 4°C) and counted in a Beckman Gamma 5500B counter. Further washing (0.2 M acetic acid, 0.5 M NaCl) was used on some samples to confirm surface localization of radioligand. Each treatment was done in triplicate.
Transient Transfection
HA-D6 cells (2 x 105) were plated into the wells of a six-well dish and incubated for 48 h (37°C, 5% CO2). Effectene (QIAGEN, Valencia, CA) was used for transfection with suppliers' buffers. Except where indicated 1 µg of plasmid DNA was mixed with 100 µl of EC buffer, 3.2 µl of Enhancer added, left for 5 min at room temperature, 10 µl of Effectene added, and the sample mixed and incubated for a further 5–10 min at room temperature. Then, 600 µl of 293 medium was added, and the whole mix was added dropwise to one well of cells bathed in 2 ml of warm 293 medium. Mock transfectants, omitting DNA, were done as a control for each test construct, and pEGFP-N2 was used as an untagged GFP control. Twenty-four hours after transfection, cells were harvested and analyzed for ligand uptake or surface D6 expression.
Flow Cytometric Fluorescent Ligand Uptake Assay
Bio-CCL3 (250 ng) was mixed in PBS with 3 µg of Streptavidin-PE (S-PE) (Molecular Probes) and incubated at room temperature for 45–60 min. Control samples lacked Bio-CCL3 (i.e., S-PE alone). Cells (2–4 x 105) were resuspended in 40 µl of binding buffer, the premixed Bio-CCL3/S-PE or S-PE alone was added, and the cells were incubated at 37°C for 30–150 min with regular gentle agitation. Inclusion of 50 mM NH4Cl only slightly enhances Bio-CCL3/S-PE uptake over this time frame (our unpublished data). Ice-cold FACS buffer (1.4 ml) was added, and the cells were retrieved by centrifugation (2600 rpm, 5 min, 4°C), resuspended in 400 µl of FACS buffer, and passed through a FACScan flow cytometer. For each transient transfection assessed, controls used were untransfected parental HEK293 or mock transfected HA-D6 cells treated with Bio-CCL3/S-PE or S-PE alone. For GFP-tagged constructs, after setting detection parameters to avoid fluorescence bleed-through, GFP- and PE-negative regions were gated using controls (mock-transfected HA-D6 plus Bio-CCL3/S-PE [red only] and GFP-transfected HA-D6 cells plus S-PE [green only]) such that >98% of the cells in these controls fell in the negative gate. More than 98% of all transiently transfected cells, irrespective of the construct used, consistently fell into this negative gate when S-PE alone was used in the incubation. Then data from test samples (transiently transfected plus Bio-CCL3/S-PE) were collected. On analysis, gates of high and low GFP expressers were set, and the number of PE-positive cells was determined and compared with identical gates set on pEGFP-N2 transfectants. For -arrestin-1 and dynamin I constructs, mock-transfected S-PE–treated HA-D6 cells were used to define the PE-negative gate, and transfectants were compared with mock-transfected HA-D6 cells after Bio-CCL3/S-PE uptake for the presence of PE-positive cells. Cell lysates were prepared from transfections as described above. Each sample was done in triplicate, and experiments repeated at least once.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
Surface Levels of D6 Are Not Decreased during Ligand Internalization
For most chemokine receptors, ligand binding increases receptor internalization rate, measured by a reduction in cell surface receptor. hCCL3-L1 or mCCL3 addition, at 37°C, to cultures of cells expressing HA-CCR5 causes a rapid reduction in cell surface receptor detectable with either -HA or -CCR5 antibodies (Figure 1B; our unpublished data). Surprisingly, when these chemokines were individually added to cultures of HEK293 cells expressing HA-D6, this reduction did not occur. Instead, there was a slight, but reproducible, increase in surface D6 levels (Figure 1B) as detected by either -D6 or -HA antibody immunoreactivity. To examine this more closely, we synchronized ligand internalization by loading D6 or CCR5 expressing HEK293 cells at 4°C with a high concentration of hCCL3-L1 (200 nM), a ligand that binds with similar affinity to CCR5 and D6 (Nibbs et al., 1999). Preliminary radioligand binding assays indicated that at this concentration, most available cell surface D6 and CCR5 should be occupied with ligand. On shift to 37°C, the majority of surface ligand is internalized within 10 min (Figure 1A). Despite this, there is no reduction in surface D6 levels compared with unloaded cells over this time frame (or up to an hour at 37°C), as detectable with -D6 or -HA antibodies (Figure 1C). Concentrations of ligand as high as 1 µM were unable to reduce detectable D6 surface protein. On the other hand, surface HA-CCR5 levels are rapidly and extensively down-regulated in a manner coincident with ligand internalization (Figure 1C). Control experiments have demonstrated that there is minimal displacement of any of the antibodies used by hCCL3-L1. In case -D6 or -HA were limiting, we performed studies with a range of higher, supersaturating, concentrations of antibody, or lower numbers of cells. No decreases in surface HA-D6 were detected after hCCL3-L1 or mCCL3 treatment, and a small increase was consistently observed at later time points (our unpublished data). The D6 ligands CCL2, 4, 5, 7, 8, 11, 13, or 14 (all used at 200 nM) did not reduce surface HA-D6 levels, whereas CCL4 or 5 caused a reduction in surface CCR5 detectability (our unpublished data).
To test the functional activity of the receptors after hCCL3-L1 exposure, we examined the ability of hCCL3-L1–treated cells to subsequently take up 125I-hCCL3-L1. There was no reduction in 125I-hCCL-3-L1 uptake by HA-D6 cells after preincubation with 200 nM hCCL3-L1, whereas similarly pretreated HA-CCR5 cells only internalized 50–60% of the amount seen with the untreated cells (Figure 1D). Estimates of receptor number using saturation radioligand binding assays consistently showed no reduction in surface D6 after ligand pretreatment (our unpublished data), although these experiments and others indicated a change in receptor affinity, revealed most clearly when full homologous displacement curves were performed (Supplemental Figure 1). This phenomenon, along with the apparent increase in receptor surface levels described above, does not seem to dramatically alter the rate of ligand internalization at 37°C (Figure 1D), but is intriguing because it represents the first demonstration of a downstream consequence of ligand occupation of D6, suggestive of signaling.
Thus, in contrast to CCR5, D6 can internalize chemokines without undergoing either a reduction in surface receptor levels or desensitization to subsequent chemokine uptake. One possible explanation for this is that D6 constitutively cycles to and from the surface, allowing ligand to enter cells on D6 molecules already destined for internalization. Internalized D6 is replaced by other intracellular D6 molecules predestined for the cell surface. A similar mechanism has been proposed for the cytomegalovirus (CMV) US28 protein (Fraile-Ramos et al., 2001). The following series of experiments lend support to this model for D6.
Most Cellular D6 Is Found in Early and Recycling Endosomes
US28 and other constitutively recycling proteins are found predominantly in endosomes (Fraile-Ramos et al., 2001; Royle and Murrell-Lagnado, 2003), so we investigated the subcellular localization of D6. First, we used biochemical approaches to estimate the percentage of D6 molecules, as a fraction of the full cellular complement, which are on the surface at any one time. Lysates from HA-D6 HEK293 cells were compared, by Western blot analysis, with known quantities of purified D6 (Figure 2A), taking advantage of our recent isolation of pure preparations of D6 (Blackburn et al., 2004). Two bands 46–49 kDa are seen: the upper band is a glycosylated version of the lower with carbohydrate decoration on asparagine 17, a modification not required for function (Blackburn et al., 2004). By comparison of band intensity, we estimate that 105 cells contain 70 ng of HA-D6 protein. Parallel radioligand binding assays revealed, on average, 3 x 105 functional surface receptors per cell. Thus, assuming all surface D6 can bind ligand, 105 cells have 2.5 ng of HA-D6 protein on the surface at any one time, representing only 3.5% of the total.
|
Next, immunofluorescent staining of HA-D6 cells with the -D6 antibody revealed that the majority of the HA-D6 protein is in intracellular vesicles often clustered around the nucleus (Figure 2B). HA-CCR5 was found predominantly on the cell surface as expected (our unpublished observation). These distributions are recapitulated in live stable HEK293 cell transfectants (pools or single cell clones) expressing CCR5 or D6 carrying a C-terminal GFP tag (Figure 2, C and D): GFP was found throughout the cell, as expected, after transfection of expression vectors lacking receptor inserts. It is noteworthy that the fusion proteins had similar mCCL3 or hCCL3-L1 binding affinity to wild-type or HA-tagged proteins and behaved indistinguishably from their HA-tagged counterparts with respect to all the parameters described herein. This differential subcellular distribution is not due to differences in levels of expression, because even when D6-GFP is present at a low level, green fluorescence is predominantly vesicular. In addition, flow cytometric analysis of pools of D6-GFP transfected HEK293 cells stained with -D6 antibodies (detected with PE-coupled -mouse IgG secondary antibodies) showed that as total D6-GFP increased in the cell, the amount detectable on the surface also increased, i.e., there was not a point at which surface expression became saturated (our unpublished data). A similar distribution of endogenous or transfected D6 was observed in other cell lines, and often very little surface D6 was detectable (by flow cytometry and radioligand binding) despite abundant total cellular expression (our unpublished data). This distribution of D6 is reminiscent of granular -D6 immunoreactivity seen in human tissue sections. This can be seen in LECs (Figure 2E; Nibbs et al., 2001) and is particularly manifest in angiosarcoma or Kaposi's sarcoma samples, transformed derivatives of LECs (our unpublished data). These observations provide significant circumstantial evidence that our in vitro systems are recapitulating genuine intracellular distribution in vivo.
To identify the nature of these vesicles, we have stained cells expressing D6-GFP with markers of various vesicular compartments. Unlike US28 (Fraile-Ramos et al., 2001), little D6-GFP is found to colocalize with CD63, a marker of late endosomes and lysosomes (Figure 3A), although occasionally single D6-GFP–containing vesicles seem to stain with -CD63. Instead, markers of early endosomes and the recycling endosomal compartment, namely, rab5 (Figure 3B), the transferrin receptor (Figure 3C), and rab11 (our unpublished data), were consistently found in D6-GFP+ vesicles. A small proportion of D6-GFP+ vesicles also stained with antibodies to clathrin, but the Golgi (visualized with -Golgin 97 antibodies) contained little, if any, D6-GFP (our unpublished data).
|
We next examined chemokine production by the transfectants, screening for all known D6 ligands by reverse transcription-PCR. CCL22 was also included: recent unpublished work has revealed it to be a high-affinity ligand for D6 (previous work used a –2 variant that does not bind D6; Nibbs et al., 1997). CCL2, 7, and 8 mRNA only were detectable at similar very low levels (requiring sensitive reverse transcription-PCR techniques and >30 rounds of amplification) in both transfected and untransfected HEK293 cells. Thus, autocrine ligand production is not responsible for the differences in intracellular distribution observed between D6 and CCR5. Also, D6 distribution is unchanged in the absence of serum, excluding serum proteins as responsible for D6 localization.
D6 Internalizes in the Absence of Chemokine
If D6 undergoes constitutive endocytosis, it should be possible to internalize antibodies against the receptor in the absence of ligand. To test this, we have used antibody feeding techniques, by using either -D6 or -HA antibodies, and cells expressing D6-GFP or HA-D6. Similar results were obtained with all these reagents, and data are shown from D6-GFP cells and -D6 antibodies. Cells were loaded at 4°C with antibody, washed (with buffer at pH 7.4, or pH 3 to strip off antibody), and then either fixed or shifted to 37°Cto encourage internalization and fixed. -D6 location was revealed with Cy3-coupled -mouse IgG antibodies, with or without cell permeabilization with saponin. Without shifting to 37°C, the antibodies remained on the cell surface, with D6-GFP+ vesicles just under the cell surface not colocalizing with Cy3 (Figure 4A). Washing with acidic (pH 3) medium before visualization removed all antibody. However, -D6 antibodies are taken into the cell after 5 min at 37°C, were protected from acid washing, and remained colocalized with D6-GFP+ vesicles around the cell periphery (Figure 4B). After 15 min, the majority of the D6-GFP–containing vesicles are labeled with antibodies (Figure 4C), despite only a small proportion of the total cellular D6-GFP (i.e., those molecules on the surface) being loaded with antibodies at the start of the experiment. This implies that vesicles of internalized D6-GFP, carrying antibodies, rapidly fuse with preexisting intracellular D6-GFP+ vesicles. Antibody uptake was further confirmed by the lack of vesicular staining in nonpermeabilized -D6-fed D6-expressing cells and was dependent on D6 expression because untransfected cells showed no detectable internalization (our unpublished data). The inclusion of 200 nM hCCL3-L1 during these treatments had no discernible effect on the rate of internalization, or the gross distribution of the internalized antibody. Flow cytometric data, detecting surface versus intracellular antibodies associated with antibody-fed cells, support these observations (our unpublished data). Significantly, we have also used flow cytometry to show that -D6 antibody uptake does not alter the size of the surface D6 pool, consistent with constitutive D6 internalization driving -D6 uptake (Supplemental Figure 2).
|
Similar experiments were performed on HA-CCR5 or CCR5-GFP cells by using either -HA or -CCR5 antibodies. As with D6, no internalization of antibody was seen after 4°C loading without shifting to 37°C (Figure 5A). In the absence of exogenous chemokine, very little antibody was internalized after shift to 37°C (Figure 5B, far right). In fact, the distribution of antibody and CCR5-GFP, even after 1 h at 37°C, did not look dissimilar to cells that had not been shifted, although less antibody remained associated with the cells. However, when hCCL3-L1 or mCCL3 was added just before the 37°C incubation, CCR5 and associated antibodies were rapidly internalized and remained colocalized inside the cell (Figure 5B). These data are consistent with the requirement for ligand-induced signaling for significant internalization of CCR5.
|
Internalized D6 Recycles to the Cell Surface
Next, we asked whether internalized D6 recycles to the cell surface. D6-GFP expressing cells were loaded with -D6 antibodies at 37°C for 30–60 min, and surface antibodies were removed by acid stripping. Flow cytometry confirmed that the acid wash removed the majority of the antibodies from the cell surface (see below). Stripped cells were then reincubated at 37°C in the presence of TRITC- or Cy3-coupled -mouse IgG antibodies. If -D6 antibodies return to the surface, these secondary antibodies should become internalized. As shown in Figure 6A, there is significant internalization of the labeled secondary antibody that colocalizes completely with D6-GFP. Large vesicles are formed during these experiments, likely as a result of cross-linking within the D6-GFP/-D6/-mouse IgG complexes disrupting vesicle trafficking. Control experiments showed that secondary antibody uptake is dependent on both D6-GFP and the preincubation step with -D6 antibodies (our unpublished data).
|
To assess recycling rate, similar protocols were used, but the cells were subsequently assessed by flow cytometry. Cells were loaded at 37°C with -D6 antibodies, acid washed, and incubated for varying times at 37 or 4°C, with PE-coupled -mouse IgG. As shown in Figure 6B, acid washing effectively removes surface -D6 antibodies from the cells. However, if the cells are incubated at 37°C they internalize the secondary antibody. Within 90 min, cells exhibit fluorescent levels equivalent to those seen on cells that had simply been incubated with primary and secondary antibodies at 4°C (right-hand column).
These combined data clearly demonstrate that internalized -D6 antibodies return to the surface of the cells and then reinternalize after picking up secondary antibody, results indicative of receptor recycling.
CCL3 Internalized by D6 Is Trapped Inside the Cell and Degraded
Ligands internalized by D6 are reportedly degraded by lysosomes, indicated by the emergence of TCA nonprecipitable radioactivity from D6-expressing cells loaded with radioligand, which can be inhibited by neutralization of vesicle acidification by NH4Cl (Fra et al., 2003). To examine the fate of the internalized ligand in HEK293 cells, HA-D6 cells, loaded with 125I-mCCL3, were shifted to 37°C and the cells and supernatant were analyzed over time. At early time points, most radioactivity (>90%) was associated with the cells, but there was the accumulation of degraded, non-TCA–precipitable products in the supernatant of HA-D6–expressing cells over time (Figure 7A). By 30 min, 30% of the retrieved radioactivity is in the non-TCA precipitable fraction, increasing to 80% after 150 min. In the presence of 50 mM NH4Cl, nearly all the radioactivity remained associated with the cell pellet, consistent with previous observations (Fra et al., 2003). Untransfected cells took up little radiolabel, and there was minimal radioactivity in the non-TCA–precipitable fraction (Figure 7A).
|
SDS-PAGE analysis of the supernatants revealed the appearance over time of diffuse bands with a slower electrophoretic mobility than intact 125I-mCCL3 (Figure 7B). These most likely represent iodinated amino acids or short peptides that do not become shrouded in SDS to allow them to electrophorese effectively on SDS-PAGE. They were coincident with the accumulation of non-TCA precipitable radioactive material and were not seen in supernatants from NH4Cl-treated cells, which kept all their internalized 125I-mCCL3 intact over the 150 min of the experiment. Intracellular 125I-mCCL3 migrated with indistinguishable motility to intact 125I-mCCL3 incubated in the absence of cells (Figure 7B). The smeared high-mobility bands seen in the supernatants were occasionally just detectable in cell lysates, but acid washing before lysate preparation removed these degradation products, suggesting they are attached to the cell surface (our unpublished observations). Next, we examined D6 protein levels during ligand degradation. Cell lysates were made from 5 x 106 HEK293 cells expressing HA-D6 or D6-GFP, cultured continuously in 20 ml of 200 nM mCCL3 for 1, 2, 4, 8, or 24 h in the presence or absence of CHX (Figure 7C; our unpublished data). Over this time frame, there was little, if any, detectable change in D6 protein levels, even in the presence of CHX, and mCCL3 was unable to enhance D6 destruction or bring about a change in the electrophoretic mobility of the receptor. D6 is therefore a stable protein that does not seem to undergo degradation concomitant with D6-mediated ligand destruction. Control blots probed with -actin showed the CHX to be active on these cells (our unpublished data).
This series of experiments confirm and extend previous observations (Fra et al., 2003) and show that ligand internalized by D6 1) is degraded, 2) is released from the cells as soon as it is degraded, 3) is not released from the cells unless it is degraded, 4) involves acidic vesicles in the degradation process, and 5) does not bring about detectable D6 degradation.
Next, we wanted to examine whether these phenomena occurred after ligand internalization by a typical chemokine receptor, CCR5, so experiments were performed side by side on either HA-D6 or HA-CCR5 cells. 125I-hCCL3-L1 was used because its equivalent affinity for D6 and CCR5 (Nibbs et al., 1999) ensures that similar amounts of 125I-hCCL3-L1 are taken up by the two cell lines. Intriguingly, these experiments showed that although internalized ligand is also degraded after CCR5-mediated internalization (and is NH4Cl sensitive), it occurs much more slowly (Figure 8A). This is apparent using either media TCA precipitation (top) or SDS-PAGE of cell lysates (bottom). However, internalized 125I-hCCL3-L1 can be retrieved from CCR5 cells by incubation with unlabeled hCCL3-L1 after the radioligand has been internalized (Figure 8B). Radioligand degradation proceeds at the same rate whether unlabeled hCCL3-L1 is present or not, but the unlabeled hCCL3-L1 successfully chases out internalized 125I-hCCL3-L1 from CCR5 cells only. This 125I-hCCL3-L1 is intact according to its ability to be TCA precipitated (Figure 8B, bottom) and comigrates with intact 125I-hCCL3-L1 by SDS-PAGE (our unpublished data). There is a small amount of intact 125I-hCCL3-L1 displaced from D6 cells by the unlabeled hCCL3-L1 (Figure 8B, top), but this is equivalent to the amount of protein that is still on the cell surface after 125I-hCCL3-L1 loading (as indicated by acid washing). Interestingly, when the competitive elution experiments were repeated in the presence of 50 mM NH4Cl, internalized radioligand was released from D6-expressing cells (Figure 8C). In fact, NH4Cl treatment alone was sufficient to double the amount of intact 125I-hCCL3-L1 release, and this was further enhanced by the presence of excess unlabeled hCCL3-L1. On the other hand, 125I-hCCL3-L1 release from HA-CCR5 cells was reduced by combined unlabeled hCCL3-L1/NH4Cl treatment. In all cases, intracellular and TCA-precipitable radioligand from NH4Cl-treated samples exhibited identical motility in SDS-PAGE to intact 125I-hCCL3-L1 (our unpublished data). Notably, neutralizing vesicle acidity made D6 and CCR5 behave in a similar manner in terms of radioligand release in this assay, a phenomenon investigated further below. As for D6 (Figure 7C), Western blot analysis was done on lysates from mCCL3-treated or untreated CCR5-expressing HEK293 cells, in the presence of CHX. These gave results consistent with previously published data (Signoret et al., 2000), confirming that CCR5 has a half-life of 8 h, which is not noticeably enhanced by ligand, although ligand does reduce the electrophoretic mobility of CCR5 (most likely due to increased receptor phosphorylation) (our unpublished data).
|
These data, taken as a whole, demonstrate that when hCCL3-L1 is internalized into HEK293 cells by D6, rather than by CCR5, its fate is sealed, and it will be targeted for eventual degradation. They also suggest that in an environment high in CCR5/D6 chemokines, D6 would be considerably more effective than CCR5 at neutralizing these molecules. Indeed, head-to-head comparisons of cell lines expressing similar surface levels of D6 or CCR5 show that in continuous culture D6 proves to be a more effective chemokine destroyer (Supplemental Figure 3). Finally, experiments individually using two other ligands for D6 and CCR5, 125I-hCCL4 and 5, showed that as with hCCL3-L1 these ligands are internalized by both receptors, but they are more effectively retained and targeted for degradation when D6 is used (our unpublished data).
Opposing Effects of Lowered pH on hCCL3-L1 Dissociation from D6 and CCR5
The observation that NH4Cl-treated D6- or CCR5-expressing cells behaved in a similar manner in the competitive elution experiments (Figure 8C) led us to hypothesize that the differences between these two receptors may lie in their sensitivity to reduced pH during endosomal passage. We therefore examined the effect of reduced pH on ligand/receptor interactions. D6HA-, D6GFP-, or CCR5GFP-expressing HEK293 cells, loaded with 125I-hCCL3-L1 on ice, were washed at 4°C with PBS or with DMEM adjusted to pH values ranging from 2 to 7. A brief 5-min wash dissociated most ligand from both receptors at pH <4. Under less acidic conditions, there was an indication that D6 held onto 125I-hCCL3-L1 less well than CCR5. These differences were dramatically enhanced when the incubation period was extended (Figure 9). At pH 7, CCR5 and D6 behaved alike, losing 40–60% of surface bound radioligand after 1 h, with D6 consistently releasing slightly more. However, as the pH dropped to levels similar to those encountered in endosomes, progressively less radioligand could be eluted from CCR5, whereas there was a more rapid dissociation from D6. Thus, at pH5, CCR5 holds onto nearly all the loaded ligand for at least an hour, whereas it is essentially all lost from D6 in 30 min. These data suggest that the pH sensitivity of ligand/receptor interaction is responsible for ensuring chemokines internalized by D6 are retained inside cells.
|
Chemokine Internalization by D6 Is Reduced by Genetic Modulation of the rab5 Activity and Eps15 Function
To examine the mechanism of ligand internalization through D6, we used transient transfection methodology to introduce mutant versions of proteins known to interfere with endocytotic pathways and then assessed their impact on ligand uptake through D6. The constructs selected have been widely used and successfully used in the analysis of other chemokine receptors (Barlic et al., 1999; Fan et al., 2003; Venkatesan et al., 2003). To facilitate unbiased, accurate quantitation of ligand uptake relative to expression from the transfected construct, we developed a novel fluorescence-based assay by using GFP-tagged versions of the transfected proteins, and a custom-made biotinylated version of mCCL3, termed Bio-CCL3. This molecule has bioactivity and receptor binding parameters indistinguishable from unmodified mCCL3 and behaves like mCCL3 when radiolabeled and incorporated into the assays described above (our unpublished data). Moreover, we can premix this molecule with fluorescently coupled Streptavidin (such as S-PE) and quantify uptake. An example is shown in Figure 10A, where mock, or untagged GFP-transfected HA-D6 cells show a Bio-CCL3–dependent uptake of S-PE that can be assessed relative to the expression levels of GFP ("low" and "high" gates). HEK293 cells lacking D6 or GFP constructs produce plots like the "Mock (S-PE only)" plot in Figure 10A, irrespective of the presence of Bio-CCL3. Confocal microscopy, in which internalized Bio-CCL3 is detected either by premixing or postinternalization detection methods, shows that premixing does not significantly impact on the rate or extent of ligand uptake (our unpublished data).
|
Using this assay, we first investigated the early endosomal GTPase, rab5. HA-D6–expressing cells were transiently transfected with plasmids expressing GFP-tagged versions of wild-type (wt) rab5, a constitutively active rab5 (Q79L), or a dominant-negative version (S34N), and 24 h later ligand uptake examined by flow cytometry. As shown in Figure 10B, both mutated forms of rab5, particularly when expressed at high levels, caused a reduction in the number of cells able to uptake Bio-CCL3/S-PE during a 1-h incubation relative to control HA-D6 cells expressing an equivalent amount of untagged GFP. Wt rab5 only marginally reduced ligand uptake. Similar experimental analyses were then performed on cells transiently transfected with plasmids encoding forms of Eps15, fused to GFP, known to inhibit clathrinmediated endocytosis and pit assembly (E95/295 and DIII) (Benmerah et al., 1998, 1999). A noninhibitory form of DIII, termed DIII2, was used as a control (Benmerah et al., 1998). Fewer cells expressing E95/295 or DIII were able to internalize ligand, whereas in those expressing DIII2 this was much less pronounced (Figure 10C). In all transient transfectants, shorter incubations with Bio-CCL3/S-PE reduced the amount of ligand taken into the cells, but the impact of the constructs was consistent with the data presented above (our unpublished data). Finally, confocal examination of HA-D6 cells transiently transfected with the above-mentioned constructs revealed the GFP fusion proteins to be subcellularly distributed in a manner consistent with previous reports. The use of confocal microscopy to assess Bio-CCL3 uptake (by premixing with, or postinternalization detection by Streptavidin-Cy3) showed effects of the constructs consistent with the flow cytometric data, but presented considerable difficulties of quantitation (our unpublished data). As a whole, these results demonstrate that modulating rab5 activity or disrupting clathrin-coated vesicle formation can impact on the ability of D6 to internalize ligand.
Ligand Internalization Is Reduced by Dominant-Negative Dynamin I but Not by Dominant-Negative -Arrestin-1
Next, plasmids encoding well-characterized dominant-negative forms of dynamin I (K44A) and -arrestin-1 (V53D) were introduced transiently into HA-D6 cells, and ligand uptake was assessed (Figure 10, D–F). These molecules were not GFP tagged, so increasing amounts of plasmid (1–3 µg) were individually transfected. The amounts used were chosen because of data from pEGFP-N2 transfections, which showed that between 1 and 3 µg of plasmid there is an increase in the number of HA-D6 cells expressing low or high levels of GFP (Figure 10D). In the dynamin I (K44A) and -arrestin-1 (V53D) transfectants, the amount of plasmid-encoded protein was assessed by Western blot analysis (Figure 10E). This reveals robust expression well above levels in untransfected cells (seen only upon prolonged exposure of the blot) and increasing stepwise with the amount of DNA used. Bio-CCL3/S-PE uptake was sensitive to the progressive increase in expression of dynamin I K44A (with up to a 50% reduction in ligand uptake) but was relatively unaffected by the introduction of -arrestin-1 (V53D) plasmid, with only moderate inhibition when very high levels of the protein were expressed (Figure 10F). On the other hand, hCCL3-L1–driven CCR5 internalization was reduced by up to 50% when -arrestin-1 (V53D) was introduced into CCR5-expressing cells, with even the 1 µg transfection showing some inhibition (Supplemental Figure 4). These data suggest that perturbing dynamin I function in HEK293 cells can reduce Bio-CCL3/S-PE uptake through D6 but that this internalization is able to occur in the absence of functional -arrestin-1.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
