Polarization of CCR7 - Dynamic polarization of CCR7

Chapter 4 Dynamic polarization of CCR7

4.3.3 Polarization of CCR7

What happens to CCR7 once it senses a chemokine gradient? Is it polarizing towards the gradient or not? Are lipid rafts required for CCR7 distribution? These are few of several questions related to CCR7 spatial localization.

First, we let CEM cells that are normally round, adhere to poly-L-lysine coated coverslips showing that CCR7 was homogeneously distributed at the plasma membrane (figure 4.7).

Figure 4.7. CCR7 is equally distributed. CEM cells were adhered to poly-L-lysine coated coverslips, fixed and incubated with an anti-CCR7 antibody. Immunofluorescence was analyzed by confocal microscopy. One representative cell is shown.

When cells were stimulated with either ELC or SLC (figure 4.8), before being attached to poly-L-lysine, cells were polarized and CCR7 was clearly differentially distributed, normally situated in only one part of the membrane. This fact was more dramatic with ELC than with SLC. To observe this difference on cell and CCR7 polarization, cells were incubated after fixation with phalloidin-TRITC in order to analyze actin polymerization. As expected, with ELC more cells showed polymerized actin compared to SLC, nevertheless flow cytometric analysis will be required for a quantitative result.

Figure 4.8. CCR7 is polarized in the presence of chemokines. CEM cells were incubated with (A) ELC or (B) SLC for 5 min, adhered to poly-L-lysine coated coverslips, fixed, permeabilized and incubated with an anti-CCR7 antibody (green) and phalloidin-TRITC (red). Immunofluorescence was analyzed by confocal microscopy.

To confirm our data and to know whether polarized CCR7 is colocalized with lipid raft markers, we used CEM cells stably transfected with CCR7-GFP. For a more physiological environment, this time cells were incubated on fibronectin for 30 min at

37°C before ELC was added. Then cells were fixed and incubated with CTx-Cy3.

Interestingly, control cells were already polarized and showed CCR7-GFP with a completely polarized phenotype in the absence of chemokine (figure 4.9 A). As expected, cells incubated with ELC showed exactly the same pattern (figure 4.9 B), indicating that CCR7 polarization could be mediated by fibronectin rather than being chemokine-mediated. In both cases CTx-Cy3 colocalized with polarized CCR7, indicating that lipid rafts containing GM-1 also play a role in this process.

Figure 4.9. CCR7-GFP is polarized on fibronectin. CEM cells stably expressing CCR7-GFP were incubated for 30 min on fibronectin-coated coverslips. Then, cells were (A) fixed or incubated with ELC (2 µg/ml) for 30 min (B) and then fixed. Then, cells were permeabilized and incubated with cholera toxin Cy3 (red) and analyzed by confocal microscopy.

To analyze cell and receptor polarization in vivo, we performed a needle assay on fibronectin-coated plates using time-lapse microscopy. This assay operates with a needle filled with a chemoattractant, which is constantly released creating a gradient.

Cells are supposed to sense it and migrate towards the chemoattractant release from the needle. To establish this technique, we used HL-60 cells that can be easily

differentiated to neutrophile like cells with 1.2% DMSO over 4 days. The migration assay was performed towards the chemoattractant fMLP, for which neutrophiles express the cognate receptor endogenously. Taking pictures every 20 sec, it was possible to observe that these cells could very easily migrate along the gradient towards the needle (figure 4.10).

Figure 4.10. Neutrophiles can easily migrate towards fMLP. DMSO differentiated HL-60 cells (1.2%

for 4 days) were incubated for 30 min on fibronectin at 37°C. Then a migration assay was performed with a micropipette filled up with fMLP (10nM) that is situated in the upper right side of the pictures.

Pictures were taken every 20 seconds. Arrow: migrated cell, star: needle.

Following the same procedure as before, DMSO differentiated HL-60 cells, which normally do not express CCR7, were transiently transfected with CCR7-GFP.

After 24 hrs of transfection, the same assay was performed but this time the needle

was filled with ELC. Unfortunately, transfected cells were not able to migrate towards the needle (figure 4.11). However in many cases, although no migration was reported, pseudopod formation was observed, suggesting that cells could indeed sense the gradient created by the chemokine but for an unknown reason they were not able to move. Normally, when pseudopod formation was observed CCR7-GFP was polarized and concentrated on these structures (arrows in figure 4.11), strongly suggesting that CCR7-GFP changes its plasma membrane distribution in order to concentrate the gradient sensing machinery at the leading edge.

Figure 4.11. CCR7-GFP is polarized in the presence of ELC. HL-60 cells were differentiated and transfected with CCR7-GFP. After 24 hrs a migration assay on fibronectin was performed towards a needle filled with ELC (1µg/ml). Arrows indicate polarization of CCR7-GFP. The needle is situated in the right middle side of the picture, indicated by a yellow star. This is a representative picture of four different experiments.

4.4. Discussion

How can a migrating cell change its morphology to an asymmetric pattern and which mechanisms are involved are questions that remain largely unknown. In this study, we show asymmetric redistribution of the chemokine receptor CCR7, probably mediated by lipid raft microdomains, on cells stimulated with a chemokine gradient.

Our results suggest that probably raft-associatedCCR7 is accumulating actively at the front of the cell,where lipid rafts recruit signalling molecules involved in gradient sensing, such as PI3Kγ.

Unfortunately, in none of our time-laps experiments we were able to detect migration of differentiated HL 60 cells transiently transfected with CCR7-GFP upon exposing to ELC. When the same cells without transfection were challenged with fMLP, they could migrate towards the needle in a very fast and efficient manner as shown in figure 4.11, demonstrating that our system is working properly. On the other hand chemotaxis experiments on a Transwell^TM filter, with HL-60 cells transiently transfected with CCR7-GFP towards ELC showed that these cells were able to migrate (data not shown), demonstrating that these cells contain the machinery necessary for CCR7 signalling. The reason why these cells were not able to migrate can be due to the transfection conditions, where probably cells were in an inappropriate shape to acquire a migratory phenotype. Alternatively, the failure to migrate can be due to a problem with the ELC gradient. Nevertheless, when CCR7-GFP transfected cells where stimulated with ELC, they were clearly able to polarize and extend pseudopodes enriched with CCR7–GFP as shown in figure 4.12. CCR7-GFP polarization was not really towards the direction of the needle, which can be due to some untransfected cells situated near the needle that could interfere with pseudopod formation towards the needle.

Using CEM cells, we tried to reproduce the same experiment with the needle filled with ELC. In this case, however, cells showed no reaction at all. Nevertheless, an unexpected finding was that when CEM cells stably expressing CCR7-GFP were adhered on fibronectin in order to perform the needle assay experiment, cells without any stimuli were already polarized showing asymmetric CCR7-GFP (data not shown).

We characterized this finding of CEM CCR7-GFP cells on fibronectin, when these cells were fixed thus performing a colocalization assay together with a lipid raft marker, indicating equal location and clear polarization of both. Normally CCR7-GFP was concentrated in only one direction (probably leading edge) and lipid rafts in two, which may represent the leading edge and the uropod (figure 4.10). Nevertheless, studies on cellular polarization have shown that lipid rafts containing the glanglioside GM1 which can be visualized by CTx-B, are polarized only at the uropod (Gomez-Mouton et al., 2001). This finding must be interpreted cautiously due to the polarization of lipid rafts and chemokine receptors that would not be an effect generated by a chemoattractant gradient but may be caused by fibronectin per se.

Moreover, GM1 in our experiments seems to be present in both types of lipid rafts, at the leading edge and at the uropod. To assess whether polarization is an effect generated only by fibronectin, we performed confocal analysis with CCR7-GFP CEM cells fixed on poly-L-lysine. As is shown in figure 4.9, cells without any stimulation showed CCR7-GFP with homogenous distribution at the plasma membrane. In contrast, cells incubated with either ELC or SLC before adhesion to poly-L-lysine and fixation showed that CCR7-GFP acquired also a polarized phenotype. This data suggest that this CCR7 polarization can be an effect generated by the sum of the chemokine gradient and the extracellular matrix, which would help cells to acquire a polarized phenotype.

The hypothesis that lipid rafts form organizing platforms has been reported for other cellular processes as well, like formation of an immunological synapse or axon guidance, which supports a direct role of lipid microdomains in organizing spatial signalling, concentrating the gradient-sensing machinery at the leading edge (Ibanez, 2004). This idea has been strongly supported by direct studies in vivo on CCR5 coupled to GFP, where the chemokine receptor showed a complete distribution at the leading edge colocalizing with lipid rafts in Jurkat T cells when cells were subjected to a chemokine gradient (Gomez-Mouton et al., 2004). Moreover, in HEK293 cells expressing the chemokine receptor CXCR2, where the LLKIL motif at the C-terminus has been described to be essential for receptor polarization (Sai et al., 2004) and the case of CXCR4 where polarization has been observed on leukocytes triggered by immobilized SDF-1 chemokine (van Buul et al., 2003). Furthermore, studies on DMSO differentiated cells during chemotaxis have revealed that the PH domain of the

protein kinase Akt (PHAKT), which is normally in the cytoplasm, is recruited selectively to the membrane at the cell’s leading edge (Servant et al., 2000).

Nevertheless, in some other studies with neutrophiles, it has been shown that these cells did not actively concentrate the chemoattractant receptor C5aR-GFP at the leading edge during chemotaxis but was equally distributed all over the plasma membrane (Servant et al., 1999). Moreover, studies on cAR1-GFP, a cAMP receptor of Dictyostelium discoideum has revealed that cAR1-GFP remain uniformly distributed on the cell surface in a chemotactic gradient (Xiao et al., 1997). In the case of chemokine receptor CCR7 and regarding our previous data, it will probably also be polarized due to its participation in lipid rafts, however experiments must be very carefully monitored. Many reports did not indicate whether the increased fluorescence observed is the result of an increased concentration of receptor molecules per unit area of plasma membrane or the result of increased folding of membranes that is seen at the leading edge (Cassimeris and Zigmond, 1990). Additionally, studies on the chemokine receptor CXCR4 have shown that this receptor is found in lipid raft fractions only in presence of fibronectin, and colocalized with Rac-1, a GTPase involved in migration and therefore in shaping the cytoskeletal architecture (Wysoczynski et al., 2005). In the presence of fibronectin it could also happen that this substrate induces CCR7 recruitment to lipid rafts, which would imply that CCR7 is not being homogenously expressed at the plasma membrane any more. To further examine this possibility, sucrose gradients were performed with CCR7-HA transfected 300-19 cells that were previously incubated with fibronectin, however no difference from the control was found (data not shown). However, these premature B cells do not really adhere to fibronectin. Nevertheless studies on CXCR4-GFP expressed in KG1a cells, which is a human progenitor cell model, have revealed that when cells were incubated on fibronectin coated glass coverslips, polarization of the receptor was found only in the presence of the chemokine. However in this work no experiment was performed clearing CXCR4 participation in lipid rafts on these KG1a cells (van Buul et al., 2003).

Another method used to clarify whether lipid rafts are involved in CCR7-mediated cell migration was by cholesterol depletion experiments using MCD, which is a very problematic molecule that can completely abrogate cellular functions.

Therefore, we titrated the amount of MCD where we found an effect but nevertheless cells were alive and healthy. A very interesting point concerning cholesterol depletion

is the fact that this procedure can completely change the conformation of the receptor affecting the ligand binding, as shown for CCR5 with its ligand MIP-1β (Nguyen and Taub, 2002). To be sure that the effect of MCD was due to lipid raft disruption rather than a conformational change of CCR7 we performed a ligand binding assay with and without MCD were we observed that there was no difference between these two conditions. This data supports our idea that the disruption of lipid rafts impaired signalling and migration mediated by CCR7.

Next we tested the hypothesis that under ligand exposure, lipid rafts would probably recruit even more chemokine receptors. This was at least reported for the chemokine receptor CXCR1, where its lipid raft microenvironment facilitates G protein-dependent signalling (Jiao et al., 2005; Nguyen and Taub, 2002).We performed sucrose gradient analysis in order to quantify the distribution of CCR7.

Surprisingly, we were not able to find any difference when cells were stimulated with the chemokine. A more precise technique such as FRET analysis will be required to resolve this and other debatable points, exploiting all our fluorescent CCR7 fusion proteins, in order to clarify the spatial and temporal activation of CCR7 in vivo that will finally lead to a polarized phenotype and migration.

4.5. Material and methods

4.5.1 Antibodies and reagents

Antibodies were obtained from the following sources: rat anti-human CCR7 (clone 3D12, BD Biosciences PharMingen, San Diego, CA), mouse anti-HA (Sigma, Buchs, Switzerland), mouse anti-GFP (Clontech, Heidelberg, Germany) and anti-mouse HRP-conjugated antibody (DAKO Corp., Hamburg, Germany). Anti-rat Cy3 antibody was obtained from Jackson ImmunoResearch, West Grove, PA. Alexa Fluor 546-labeled transferrin and Alexa Fluor 555- labelled cholera toxin were obtained from Molecular Probes (Leiden, The Netherlands). Human chemokines CCL19 and CCL21 were purchased from PromoCell (Heidelberg, Germany). Methyl-β- cyclodextrin, BSA free lipid, sucrose, phalloidin-TRITC, HRP conjugated cholera toxin was obtained from Sigma (Buchs, Switzerland). Fluo-3/AM was purchased from Calbiochem (La Jolla, CA). FuGENE 6 was purchased from Roche Molecular Biochemicals (Rotkreuz, Switzerland). Coverslips coated with fibronectin were obtained from Becton Dickinson, Heidelberg, Germany.

4.5.2 Cells and transfection

The human embryonic kidney cell line, HEK293, was grown in DMEM (GIBCO BRL/Invitrogen, Basel, Switzerland) with 10% (v/v) fetal bovine serum. HEK293 cells were stably transfected in 10 cm dishes by the calcium phosphate procedure and transiently transfected with Lipofectamin. Cell clones were established by limiting dilution in the presence of 0.8 mg/ml G-418 (GIBCO Invitrogen, Basel, Switzerland).

The pre B cell line 300-19 (Legler et al., 1998; Willimann et al., 1998) was grown in RPMI 1640 (GIBCO Invitrogen, Basel, Switzerland) with 10% (v/v) fetal bovine serum, 0.1% β-mercaptoethanol and 2 mM non-essential amino acids. The T cell line CEM was grown in RPMI 1640 (GIBCO Invitrogen, Basel, Switzerland) with 10%

(v/v) fetal bovine serum. Stable transfection of both cell lines was performed by electroporation and clones were achieved by limiting dilution as described (Legler et al., 1998; Willimann et al., 1998). The human promyelocytic leukemia HL-60 cells were grown in RPMI 1640 with 10% (v/v) fetal bovine serum. To differentiate them

to neutrophiles, cells were cultivated in the presence of 1.2% DMSO for 4 days.

Transient transfection was performed by electroporation.

4.5.3 Construction of expression plasmids

The entire open reading frame of human CCR7 amplified by PCR from SRαpuro-CCR7 (Willimann et al., 1998) using the primers SRαpuro-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) was subcloned into the EcoRI/NotI sites of pcDNA3 (Invitrogen/GIBCO, Grand Island, NY). EGFP, EYFP and ECFP were fused to the N-terminus of CCR7 by PCR amplifying GFP, YFP and CFP from the pEGFP-N1, pEYFP-N1 and pECFP-N1 (Clontech, Palo Alto, CA) respectively 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 AGA CTA CTT GTA CAG CTC GTC). 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.

4.5.4 Chemotaxis

Chemotaxis of 300-19 cells was measured by migration through a polycarbonatefilter of 5 µm pore size in 24-well Transwell^TM chambers (Corning Costar, Cambridge, MA). Cell culture medium (600 µl) containing indicated doses of chemokine, or medium alone as a control for spontaneous migration, was added to the lower chamber; 1x10⁶ cells in 100 µl were added to the upper chamber. After 3 hours of incubation at 37°C a 500 µl aliquotof the cells that migrated to the bottom chamber was counted by flow cytometry acquiring events for a fixedtime period of 60 seconds using the CellQuest software. The number of spontaneously migrated cells was subtracted from the total number of migrated cells and expressed as percent of input cells.

4.5.5 Lipid rafts isolation

HEK293 transfectants from four 10 cm culture dishes expressing CCR7-HA were lysed on ice for 20 min in 2 ml of 1% Triton X-100 in MNE buffer (25 mM MES, pH 6.5, 150 mM NaCl, 2 mM EDTA) and homogenized (10 strokes) with a loose-fitting dounce homogenizer. The homogenates were mixed with 2 ml of 90% sucrose prepared in MNE buffer and placed on the bottom of a centrifuge tube. The samples were then overlaid with 4 ml of 35% sucrose and 4 ml of 5% sucrose in MNE buffer and centrifuged at 40000 rpm in an ultracentrifuge for 20 h. Fractions (1 ml) were collected from the top of the gradient. Total proteins were precipitated with 10% TCA and subjected to Western blot analysis.

4.5.6 Methyl-β- cyclodextrin (MCD) treatment

Cells were washed three times under serum free conditions with RPMI and then 5mM of MCD was added to 10⁷ cells/ml for 30 min at 37°C. Cells were then washed three times with 0.01% BSA free lipid in serum free medium.

4.5.7 Measurements of 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 (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid; pH 7.5) and re-suspended at 1x10⁶ cells/ml. Cells were loaded with 1.5 µl/ml of Fluo-3/AM (4 mM in DMSO, Calbiochem, LaJolla, CA) 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.

4.5.8 Time-lapse videomicroscopy

Real-time cell chemotaxis was studied using time-lapse microscopy. Cells were plated for 30 min at 37°C onfibronectin-coated coverslip chambers. Cell chemotaxisstudies were performed at 37°C using a heating plate and a micromanipulation system (Eppendorf) adapted to themicroscope Axiovert 200M (Carl Zeiss, Jena, Germany).

Stimuli were supplied in a Steril Femtotip II micropipette (Eppendorf, Hamburg, Germany) filled with 100 nM fMLP (Sigma-Aldrich) or 1µg/ml ELC in PBS.

Fluorescence and phase contrastimages were recorded at established time intervals.

Fluorescencewas recorded with a 63 x Plan-Apochromat objective (na=1.4) using the Axiocam MRm camera.

4.5.9 Confocal laser scanning microscopy

Transfected HEK293 cells were grown overnight on coverslips. CEM cells were incubated for 10 min on coverslips coated with poly-L-lysin (Sigma, Buchs, Switzerland) or 30 min on fibronectin-coated coverslips. Cells were washed twice with PBS, 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 Chemie AG, Buchs, Switzerland) for 10 min and washed with 0.2%

Gelatin in PBS. Antibody staining of cells was performed at room temperature for 40 min, cells were washed five times and mounted on glass slides using Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL). Immunofluorescence was analyzed by a LSM 510 confocal microscope (Carl Zeiss, Jena, Germany), with a 63 x Plan-Apochromat objective (na=1.4). Images were acquired using the LSM 510 software (Carl Zeiss).

4.5.10 Western Blotting

Cells were lysed with 1% Triton X-100 in 150 mM NaCl, 50 mM Hepes, 0.1 M EGTA, 2 mM MgCl2 and 10% glycerol. Proteins from total cell lysates were resolved by SDS-PAGE and transferred to Protran nitrocellulose membrane (Schleicher &

Schuell, Dassel, Germany). Membranes were blocked with PBS containing 5% of low-fat dry milk and incubated with the respective antibodies overnight at 4°C or for one hour at room temperature on a rocking plate. After washing, HRP-conjugated secondary antibodies were bound and detected using enhanced chemiluminescence

Im Dokument Characterization of the chemokine receptor CCR7 and its role in cell migration (Seite 97-0)