High molecular weight kininogen binds phosphatidylserine and opsonizes urokinase plasminogen activator receptor-mediated efferocytosis

Abstract

Phagocytosis of apoptotic cells (efferocytosis) is essential for regulation of immune responses and tissue homeostasis and is mediated by phagocytic receptors. In this study, we found that urokinase plasminogen activator receptor (uPAR) plays an important role in internalization of apoptotic cells and also characterized the underlying mechanisms. In a flow cytometry-based phagocytic assay, uPAR-deficient macrophages displayed significant defect in internalization but not tethering of apoptotic cells. When uPAR-deficient mice were challenged with apoptotic cells, they exhibited pronounced splenomegaly resulting from accumulation of abundant apoptotic cells in spleen. Overexpression of uPAR in HEK-293 cells enhanced efferocytosis, which was inhibited by Annexin V and phosphatidylserine (PS) liposome, suggesting that uPAR-mediated efferocytosis is dependent on PS. In serum lacking high m.w. kininogen (HK), a uPAR ligand, uPAR-mediated efferocytosis was significantly attenuated, which was rescued by replenishment of HK. As detected by flow cytometry, HK selectively bound to apoptotic cells, but not viable cells. In purified systems, HK was specifically associated with PS liposome. HK binding to apoptotic cells induced its rapid cleavage to the two-chain form of HK (HKa) and bradykinin. Both the H chain and L chain of HKa were associated with PS liposome and apoptotic cells. HKa has higher binding affinity than HK to uPAR. Overexpression of Rac1/N17 cDNA inhibited uPAR-mediated efferocytosis. HK plus PS liposome stimulated a complex formation of CrkII with p130Cas and Dock-180 and Rac1 activation in uPAR-293 cells, but not in control HEK-293 cells. Thus, uPAR mediates efferocytosis through HK interaction with PS on apoptotic cells and activation of the Rac1 pathway.

Figure 1. uPAR is required for the internalization of apoptotic cells in vitro

(A) Absence of uPAR expression in uPAR-deficient macrophages. Macrophages were isolated from WT and uPAR^−/− mice (n=3) and pooled together, respectively, their expression of uPAR was measured by Western blot analysis (upper panel) and flow cytometry (lower panel). The data are representative of three experiments.(B) Internalization of apoptotic cells. Peritoneal macrophages from WT and uPAR^−/− mice were incubated with apoptotic cells at 37°C for 15 minutes, followed by treatment with or without 0.04% trypan blue (TB) to quench the fluorescence of unengulfed apoptotic cells. Phagocytosis of apoptotic cells was analyzed by flow cytometry. Association is indicated in percentage as binding and ingestion (TB:-) and ingestion (TB:+). **, p<0.01(i). Quench of fluorescence signal of CFDA-SE-stained apoptotic cells by TB was confirmed by flow cytometry (ii). (C) Viable or apoptotic neutrophils (5×10⁵) from WT or uPAR^−/− mice were incubated with adherent WT or uPAR^−/− macrophages (5×10⁴) on 96-well plate at 37°C for 15 minutes. After macrophages were treated with TB, phagocytosis was analyzed by flow cytometry. *, p<0.05; ***, p<0.001.

Figure 2. uPAR is required for internalization but not the binding of PS beads

(A) Amnis ImageStream Data Analysis. Peritoneal macrophages from WT and uPAR^−/− mice were incubated with NBD-labeled PS beads at 37°C for 20 minutes. After washing with PBS, macrophages were labeled with PE-F4/80 antibody and evaluated using Amnis ImageStream Data Analysis, as described in the Materials and Methods. Shown are the representative output images after Amnis acquisition (i). ImageStream data were acquired using the Amnis ImageStream Analyzer and the percentage of macrophages that bound (binding) and internalized (internalization) PS-beads were quantitated with the Amnis IDEAS software. (**, p<0.01, ii). (B) As described above, apoptotic cells were used as target instead of PS-beads, peritoneal macrophages from WT and uPAR^−/− mice were labeled with CFDA-SE prior to incubation with PKH26-labeled apoptotic cells at 37°C for 20 minutes. The percentage of macrophages binding (Binding) and internalizing (Internalization) apoptotic cells were analyzed with the Amnis IDEAS software (**, p<0.01).

Figure 3. uPAR-deficient mice exhibit impaired clearance of apoptotic cells

(A) Apoptotic cells (2×10⁷ per mouse) were intravenously injected into WT and uPAR^−/− mice (n=7) every day for 7 days. Shown is the representative gross appearance of spleens on day 8 (i). (ii) The mean spleen index of WT and uPAR^−/− mice that received injection of PBS (AC−) and apoptotic cells (AC+) is shown.**, p<0.01. (B) Spleens of the above experiments were further processed for paraffin-embedded sections. Apoptotic cells were detected by TUNEL method. Nuclei were counterstained with DAPI. Representative images (20×) were shown (i, bars =100 μm). (ii) TUNEL-positive cells were enumerated in 10 randomly chosen high-power fields (hpf). **, p<0.01. (C) Internalization of PS-coated beads or apoptotic neutrophils in vivo. As described in the Materials and Methods, 4×10⁷ NBD-PC-labeled PS-beads (i) or apoptotic neutrophils isolated from WT or uPAR^−/− mice (ii) were intravenously injected into WT and uPAR^−/− mice (n=5). Six hours after injection, spleens were harvested and homogenized, and F4/80-positive macrophages were purified. After treatment with or without TB, internalization of PS-coated beads was analyzed by flow cytometry (i). After treatment with TB, internalization of uPAR^+/+ and uPAR^−/− apoptotic neutrophils was analyzed by flow cytometry (ii). **, p<0.01, ***, p<0.001.

Figure 4. Phosphatidylserine is necessary for uPAR-mediated internalization of apoptotic cells

(A) HEK293 cells were transfected with pIRES2-EGFP plasmid (EGFP-293 cells) or IRES2-EGFP-uPAR plasmid (uPAR-293 cells), their expression of uPAR on membrane was measured by flow cytometry. (B) EGFP-293 cells and uPAR-293 cells were incubated with PKH26-labeled viable or apoptotic cells for 2 hours, respectively. Phagocytosis by green fluorescent 293 cells was evaluated after treatment with TB as described in the legend for Figure 1. ***, p<0.001. (C) suPAR inhibits phagocytosis of apoptotic cells in uPAR-293 cells. uPAR-293 cells were incubated with PKH26-labeled apoptotic cells in the presence of 1 μg/mL suPAR or BSA for 2 hours, which were subjected to the phagocytosis assay. *, p <0.05.(D) After preincubation with 50 μg/mL of unlabeled annexin V or BSA plus 2.5 mM CaCl₂ for 20 minutes, apoptotic cells were labeled with FITC-conjugated annexin V (FITC-AxV), followed by flow cytometric analysis (i). After preincubation with 50 μg/mL of unlabeled annexin V in the presence or absence of 2.5 mM CaCl₂ for 20 minutes, apoptotic cells were co-cultured with uPAR-293 cells, followed by treatment with TB and internalization analysis using flow cytometry (ii). ***, p < 0.001. (E) After preincubation with PBS (Ctrl), 50 nM PC liposome or PS liposome for 20 minutes, apoptotic cells were co-cultured with uPAR-293 cells, followed by treatment with TB and internalization analysis using flow cytometry.***, p < 0.001.

Figure 5. HK binds to apoptotic cells and is required for uPAR-mediated internalization

(A) EGFP-293 cells and uPAR-293 cells were co-cultured with apoptotic cells in the presence 10% FBS or 0% FBS for 2 hours. Internalization of apoptotic cells was analyzed by flow cytometry after treatment with TB as described in the legend for Figure 4. ***, p <0.001.(B) The levels of HK in normal serum (Lane 1, insert) and HK-depleted serum (lane 2, insert) were analyzed by immunoblotting. The uPAR-293 cells were incubated with apoptotic cells in presence of normal serum, HK-depleted serum [HK(−)], HK-depleted serum replenished with 50 nM HK [HK(−)+HK] or HKa [HK(−)+HKa], respectively. After treatment with TB, the internalization of apoptotic cells was evaluated by flow cytometry as described in the legend for Figure 4. ***, p< 0.001. (C) Apoptotic cells were incubated with 100 nM biotinylated HK (HK-B) in PBS with or without 50 μM ZnCl₂ at 4°C for 15 minutes. After washing with PBS containing 0.35% BSA and 50 μM ZnCl₂, the cells were stained with PE-avidin and the fluorescence intensity of was quantitated by flow cytometry. Representative data of HK bound to apoptotic cells in the presence (Zn²⁺ plus) or absence (Zn²⁺ free) of zinc ions (i) and the mean fluorescence intensity (MFI) from triplicate samples (ii) are shown. ***, p <0.001. (D) Mixed apoptotic cells and viable cells were incubated with 50 nM HK-B in Tyrode’s buffer containing 0.35% BSA and 50 μM ZnCl₂ at 4°C for 15 minutes. After washing with PBS containing 50 μM ZnCl₂, the cells were labeled with PE-avidin and non-saturated amounts of FITC-annexin V (AxV). The intensity of PE fluorescence for HK-B bound to apoptotic cells (FITC-AxV-positive) and viable cells (FITC-AxV-negative) was analyzed by flow cytometry (i). Shown in (ii) was the quantification of the percentage of PE-positive cells at the indicated concentrations of HK-B, representing HK-B bound cells (%). (E) SPR assay. PS liposome (i) or PC liposome (ii) at the indicated concentrations was allowed to bind to HK-immobilized CM5 sensor chip to reach equilibrium, followed by dissociation. The response curves were analyzed using BIAevaluation software.

Figure 6. Both of heavy chain and light chain are involved in HK binding to apoptotic cells and PS

(A) HC and LC inhibit the binding of HK to apoptotic cells. Apoptotic cells were incubated with HC and LC at the indicated concentrations at 4°C for 20minutes, followed by addition of biotin-HK (100 nM) and PE-labeled avidin. The rest steps were as described in the legend for Figure 5(B). Shown are the measurements from 3 separate experiments. (B) HK binds to PS liposome via its heavy chain and light chain. (i) Five μ g of HK protein were incubated with PBS (−), 50 nM PC or PS liposomes in Tyrode’s buffer containing 0.35% BSA and 50 μM ZnCl₂ at 4°C for 30 minutes. After ultracentrifugation at 55000 rpm, the pellets were washed twice and solubilized by sample buffer and analyzed by immunoblotting with polyclonal anti-HK antibody. (ii) Before (left panel) or after (left panel) five μg of HK, recombinant heavy chain (HC) or light chain (LC) of HK were incubated with 50 nM PS liposomes. After incubation with PS liposomes, the samples were ultracentrifuged and the pellets were collected. Samples were analyzed by immunoblotting with polyclonal anti-HK antibody. HKa serves as control for molecular weight. (C) In the presence of various peptides at the indicated concentrations, apoptotic cells were incubated with 100 nM biotinylated HK (HK-B), followed by staining with PE-avidin and quantitation by flow cytometry. (D) HK at 30 nM was incubated with apoptotic cells in the presence of prekallikrein (30 nM) at 37°C for the indicated time periods. Apoptotic cells were sonicated and centrifuged at 800 g for 5 minutes. Pellets containing membrane fraction was analyzed by Western blotting using anti-HK Ab. (E) Bradykinin production. Fresh human citrated cell-free plasma was incubated with 5×10⁵ viable cells (Viable), apoptotic cells (Apoptotic) or equal volume of PBS (Ctrl) at 37°C for 30minutes, followed by measurement of bradykinin production. **, p <0.01.

Figure 7. Integrity of lipid rafts and Rac1 activation are required for uPAR mediated-internalization of apoptotic cells

(A) After preincubation with or without 2.5 mM mβCD for 30 minutes, apoptotic cells were co-cultured with EGFP-293 or uPAR-293 cells. The internalization of apoptotic cells was analyzed by flow cytometry as described in the legend for Figure 4. ***, p< 0.001. (B) EGFP-293 or uPAR-293 cells were transfected with empty vector (lane 1) or Rac1/N17 (lane 2) cDNA for 48 hours, the overexpression of Rac1/N17 was detected by Western blotting using anti-Rac1 antibody with β-actin serving as a loading control (i). Internalization of apoptotic cells was analyzed (ii). **, p<0.01. (C) EGFP-293 cells and uPAR-293 cells were starved in DMEM plus 2% FBS for 8 hours, and incubated without (lanes 1 and 3) or with (lane 2 and 4) 1.0 μM PS liposome plus 600 nM HK for 1 hour in basal DMEM containing 0.35% BSA and 50 μM ZnCl₂. Cell lysates were subjected to immunoprecipitation with anti-CrkII antibody (A) and Rac1 activity assay (B), respectively. The immunoprecipitates using anti-CrkII antibody were analyzed by immunoblotting with MoAb against p130Cas and polyclonal antibodies against CrkII and Dock180. Active GTP-loaded form of Rac1 and total Rac1 in cell lysates were detected by immunoblotting. (D) After starvation in DMEM plus 2% FBS for 8 hours, uPAR-293 cells were incubated with PBS (control, Ctrl), 600 nM HK (HK), 1.0 μM PS liposome (PS), or 600 nM HK plus 1.0 μM PS liposome (HK+PS) for 1 hour in DMEM containing 0.35% BSA and 50 μM ZnCl₂. Cell lysates were incubated with GST-PAK1 conjugated with Glutathione Sepharose 4B beads and active GTP-loaded form of Rac1 (Pull-down) and total Rac1 in cell lysates (Lysate) were detected by immunoblotting.