Cytohesin-1 controls the activation of RhoA and modulates integrin-dependent adhesion and migration of dendritic cells
Blood Quast et al.
113: 5801
Supplemental materials for: Quast et al
Supplemental materials and methods
Oligonucleotide sequences, plasmids
RNAi was performed using oligonucleotides against the following target sequences. Cytohesin-1 (human): 5′-AATGACCTCACTCACACTTTCTT-3′; Cytohesin-1 (mouse): 5′-dNdNTGACCTCACACACACTTTCdNdN-3′. RhoA (human): 5′-TAGGCTGTAACTACTTTATAA-3′ and 5′-TACCTTATAGTTACTGTGTAA -3′; RhoA (mouse): 5′-TTGGATTTCCTAATACTGATA-3′; Rac1 (human): 5′-dNdNGCATTTCCTGGAGAATATAdNdN-3′ and 5′-dNdNGACACGATCGAGAAACTGAdNdN-3′. Renilla Luciferase sequence was used as a control: 5′-AAAAACATGCAGAAAATGCTGTT-3′. All siRNA were purchased from Qiagen or Dharmacon, respectively. Overexpression constructs were cloned in to the pRK5 vector and commonly bear an amino-terminal flag-tag. They contain the wild-type human cytohesin-1, cytohesin-1 E157K, wild-type human RhoA or RhoA T19N cDNA sequences, respectively.23
Generation of human monocyte-derived dendritic cells (Mo-DC)
Mo-DC were prepared from standard buffy coat preparations of healthy blood donors by Ficoll density gradient centrifugation (Pan Biotech). Monocytes were isolated by adhesion to plastic surfaces and cultured in VLE-RPMI medium (Biochrom) supplemented with 10% vol/vol heat-inactivated FCS (Sigma-Aldrich), 20 ng/ml IL-4 and 20 ng/ml GM-CSF (R&D Systems) for 5 to 7 days to allow differentiation into immature DC (imDC). For maturation, DC were stimulated by addition of 1 µg/ml LPS (Sigma-Aldrich) and 50 ng/ml TNF-α (R&D Systems) for 24 to 48 hours. For GTPase Activation assays monocytes were cultured in serum-free CellGrow medium (Cell Genix). Isolation of primary cells was approved by the local Ethics Committee.
Generation of bone marrow-derived DCs (BM-DC)
BM-DC were prepared from bone marrow of six week old C57BL/6 mice. Total bone marrow was filtered with 40 µm pore nylon cell strainers (BD Biosciences Falcon) and plated in 10 cm non-treated petri dishes at 5 × 106 cells in 10 ml IMDM (PAA) supplemented with 10% vol/vol heat-inactivated FCS (Sigma-Aldrich), 2 mM L-Glutamine (Invitrogen), 100 u/ml Penicillin (PAA), 0.1 mg/ml Streptomycin (PAA) and 30% GM-CSF (supernatant derived from the murine Ag-cell line stably transfected to express GM-CSF). The culture medium was half-renewed every three days. At day 8–10 of culture, BM-DC were stimulated to mature by adding 200 ng/ml LPS (Sigma-Aldrich) for 24 h.
Confocal laser scanning microscopy
Glass coverslips were coated with human fibronectin (Harbor Bio-Products, 5 µg/cm2 in PBS, 1 h, RT) and mDC were placed to adhere for 1 h at 37°C/5% CO2. For colocalization studies of cytohesin-1 and CD18, acetone-fixed mDC (−20°C for 10 min) were stained with monoclonal primary antibodies (MHM23 1:200/PBS; undiluted 7H2) for 1 h at RT, washed and incubated with FITC-coupled sheep anti mouse IgG (Jackson Immuno Research, 1:200/PBS) and Cy3-coupled goat anti rat F(ab)2 (Jackson Immuno Research, 1:200/PBS) for 1 h at RT. For localization studies of cytohesin-1 after inhibition of PI3-kinase activity, mDC were incubated with 50 µM PI3-kinase inhibitor LY-294002 (Sigma-Aldrich) for 1 h at 37°C/5% CO2 before immunostaining with anti–Cytohesin-1 mAB 7H2. To quantitate the effects of the PI3-kinase inhibitor, for each experimental condition 200 mDC were visualized and the number of cells showing a CCL19-induced translocation of cytohesin-1 to the plasma membrane was scored. Detection of filamentous actin (F-actin) in mDC was done using Cy3-coupled Phalloidin (Sigma-Aldrich; 100 mM in PBS for 45 min at RT) after fixation of the cells with 4% PFA/PBS for 20 min at RT and permeabilization with 0.1% Triton X-100/PBS for 5 min at RT. To quantitate the number of actin-based membrane protrusions after RNAi, for each experimental condition 200 phalloidin-stained mDC were visualized and the number of cells showing membrane protrusions were scored. Coverslips were mounted with Gelmount (Biomeda), supplemented with 50 mg/ml DABCO anti-fade reagent (Sigma-Aldrich). mDC were analyzed using an Olympus Fluoview 1000 confocal microscope equipped with a Plapo 60×, NA 1.4 oil immersion objective (Olympus). All shown fluorescence staining images are confocal images of a single z-section. The cells shown in Fig. 2A and Fig. S2A, respectively, are focused on the medial z-position of the respective cells, i.e. the cytoplasm and the plasma membrane. The cells in Fig. 7 are focused on the membrane protrusions. The different fluorochromes of secondary antibodies in the co-staining of cytohesin-1 and CD18 were acquired sequentially by the use of a confocal laser scanning microscope.
Static adhesion assays
Static cell adhesion to an ICAM-1 fusion protein was carried out as described previously by Boehm et al.26 with the following modifications. mDC were labeled with 12 µg/ml bisbenzimide H3342 fluorochrome trihydrochloride (Calbiochem) in VLE-RPMI for 30 min at 37°C/5% CO2. Subsequently 5 × 104 cells/well in HBSS were placed on ICAM-1-Fc coated 96-well plates (Nunc, Maxisorp) and stimulated with 50 ng/ml PMA (Sigma-Aldrich) for 60 min at 37°C/5% CO2 to adhere. After 1 h unbound cells were washed off carefully with HBSS and assayed in a fluorescence plate reader (Synergy HT, MWG).
Transfection of Mo-DC with plasmid DNA
Immature human Mo-DC were transfected using the newly developed MicroPorator MP-100 system (Peqlab), which uses a pipette gold-tip as electroporation space instead of the cuvette. For microporation of Mo-DC the MP solution Kit 100µl (Peqlab) was used. The cells were transfected according to manufacturing instructions with following modifications. 5 × 105 immature Mo-DC were transfected with 10µg plasmid (prK5:flag:Cyhtohesin1 WT, prK5:flag:Cyhtohesin1 E157K, prK5:flag:RhoA WT or prK5:flag:RhoA T19N). All samples were co-transfected with 10µg N1:eGFP as a marker for transfection. Pulse conditions were 1500V, 30ms, 1 pulse. 2h after microporation the cells were stimulated with 1µg/ml LPS (Sigma-Aldrich) and 50ng/ml TNFalpha (R&D Systems) to induce maturation. The cells were then cultured overnight in VLE-RPMI (see above) at 37°C and were used for functional assays 24h later. The obtainend transfection efficency was 1–5%.
Static adhesion assay with GFP-transfected Mo-DC
Static cell adhesion assays on ICAM-1-Fc were carried out as described previously (Boehm et al.26) with following modifications. 2 × 105 dendritic cells/well in HBSS were placed on ICAM-1-Fc coated 96-well plates (Nunc, Maxisorp) and stimulated with 50ng/ml PMA (Sigma-Aldrich) for 60 min at 37°C/5% CO2 to adhere. After 1h unbound cells were washed off carefully with HBSS and the adherent GFP-positive cells which represent the transfected population were counted in five fields of view at 10× magnification using fluorescent microscopy using the TE Eclipse microsope (Nikon). The number of GFP-positive cells in the unwashed sample (50–100 cells/field of view) was set to 100%.
BM-DC–T-cell adhesion
To quantify adhesion of BM-DC to T cells, a 96-well adhesion assay was used. T cells were freshly prepared from OT-1 spleens before CD4+ and CD8+ cells were enriched with MACS microbeads (Miltenyi Biotec). 1.5 × 106 OT-1 T cells were immobilized on poly-L-lysine (Sigma-Aldrich; 100 µg/ml for 1h at room temperature) coated 96-wells for 2h in RPMI (PAA) + supplemented with 0.5% vol/vol heat-inactivated FCS (Sigma-Aldrich). OT-1 BM-DC were generated as described above. RNAi of BM-DC was performed as described above. 5 days after transfection BM-DC were used for functional assays. BM-DC were stimulated with 10ng/ml LPS (Sigma-Aldich) for 2h, and subsequently incubated with 1 mg/ml OVA-Protein or without OVA-Protein for 3h. BM-DC were then harvested, washed and subsequently 1 × 105 cells were overlayed on the T cell coated wells in RPMI without serum for 45 min at 37°C/5%CO2. Cells were washed twice, 100% control was left unwashed, and remaining BM-DC were counted using the TE Eclipse microsope (Nikon).
Laminar flow assays
Laminar flow assays were essentially performed as described.22,31 HUVECs or Bend5 cells, respectively, grown to confluence in 35-mm petri dishes were stimulated with 100 U/ml TNF-α (R&D Systems) overnight and incubated with 500 ng/ml human CXCL12 (R&D Systems) or murine CCL21 (Peprotech), respectively, 2h before the assay started. The dishes were assembled in a parallel-plate flow chamber, in which a uniform wall shear stress is generated. The flow chamber was mounted on the stage of a phase-contrast microscope with a 10× objective (Nikon TE Eclipse). For continuous flow assays, mature DC were resuspended at a concentration of 1 × 106/ml in assay medium (HBSS, 10 mM Hepes pH 7.4, 1mM Mg2+, 1mM Ca2+) and drawn through the chamber at controlled flow rates with a syringe pump attached to the outlet. Mo-DC were perfused into the flow chamber at a shear rate of 1.5 dyn/cm2 and six optical fields were analysed for 30 sec each. In contrast, BM-DC were perfused into the flow chamber at a shear rate of 1.5 dyn/cm2 for 6 minutes and six optical fields were analysed after a six minute wash step with eqal flow conditions.
Transwell migration assay
Chemotaxis of mDC was quantified using transwell migration assays. Polycarbonate filters (Costar, Corning, 5 µm pore size) were coated with ICAM-1-Fc supernatant from CV-1 cells over-expressing ICAM-1-Fc fusion protein (approx. 10 µg/ml),26 50 µg/ml fibronectin/PBS (Harbour Bio-Products), 30 µg/ml bovine collagen I/0.5 M acetic acid (Pure Col, Inamed) for 1h at room temperature or left uncoated, respectively. DCs (1 × 105 cells in 300 µl VLE-RPMI/0,5% FCS) were placed to the upper compartment and subsequently incubated at 37°C/5% CO2 to adhere. After 1 h 200 ng/ml CCL19 (R&D Systems) was added to VLE-RPMI/0,5% FCS in the lower compartment. Control assays were performed without chemokine. Transmigrated cells were counted after incubation for 4 h at 37°C/5% CO2.
Migration assay with GFP-transfected Mo-DC
Transwell migration assays were carried out as described above with following modifications. After 4h transmigrated GFP-positive Mo-DC were counted of view at 10× magnification using fluorescent microscopy using the TE Eclipse microsope (Nikon). The number of unstimulated GFP-positive cells was set to 100%.
In vivo migration assays
Mature BM-DC after transfection with control-siRNA or cytohesin-1-siRNA (see above) were labelled with 10 µM TAMRA or 1 µM CFSE (Invitrogen), respectively. To exclude fluorochrome-effects on BM-DC migration, the staining was switched between the experiments. 2.5 × 105 cytohesin-1 knockdown BM-DC and 2.5 × 105 control BM-DC were injected into the hind footpads of C57/BL6 mice. 24–30 h after injection the popliteal lymph nodes were prepared and pressed through a nylon gaze to prepare a single cell suspension. FACS analysis was performed to count the migrated cells. The number of migrated cells transfected with control-siRNA was set to 100%. Animals were kept under specified pathogen-free conditions. Animal care and experiments were done in compliance with institutional guidelines and the German law for Welfare of Laboratory Animals.
GTPase activation assays
A commercial ELISA (G-LISA™, Cytoskeleton) was used according to manufacturing instructions to test the activity of the GTPases RhoA and Rac. Briefly, a RhoA-GTP–binding protein and a Rac-GTP–binding protein, respectively, are linked to the wells of a 96-well plate. Cells after RNAi, overexpression with plasmids or incubation with 25 µM LY-294002 (see above), respectively, were lysed and incubated on the 96-well plate for 30 min at 4°C. During this time, active RhoA and Rac1, respectively, will bind its appropriate binding domain and may subsequently be detected with a specific, HRP-coupled antibody. Absorbance was read at 485 nm using a microplate spectrophotometer (Synergy HT, MWG). For effector protein pulldown assays equilibrated Glutathione Sepharose 4B beads (GE Healthcare) were preincubated for 1h with RTKN(aa7–89)-GST or PAK1(aa67–150)-GST, respectively. HeLa cells were stimulated with EGF (Sigma-Aldrich; 50nM) for 1h at 37°C/5% CO2, lysed and added to the beads. After 90 min incubation on a rotator at 4°C the beads were pelleted and washed with buffer (25mM Tris-HCl, pH 7.5; 30mM MgCl2, 40mM NaCl, 1 mM DTT) before western-blot analysis of GTP-loading of RhoA or Rac1, respectively.
