Participation of the urokinase receptor in neutrophil efferocytosis

Abstract

The urokinase receptor (uPAR) plays an important role in regulation of fibronolysis, cell migration, and adhesion. In this study, we examined whether uPAR plays a role in modulating efferocytosis of neutrophils. Macrophages from uPAR(-/-) mice demonstrated enhanced ability to engulf viable wild-type (WT) neutrophils in vitro and in vivo in the lungs. The increased phagocytic activity of uPAR(-/-) macrophages was abrogated by incubation with soluble uPAR (suPAR), arginine-glycine-aspartic acid (RGD)-containing peptides, or anti-integrin antibodies. There was increased uptake of viable uPAR(-/-) neutrophils by WT macrophages. Incubation of uPAR(-/-) neutrophils with suPAR or anti-integrin antibodies diminished uptake by WT macrophages to baseline. Uptake of uPAR(-/-) neutrophils by uPAR(-/-) macrophages was not enhanced. However, incubation of uPAR(-/-) neutrophils or uPAR(-/-) macrophages, but not both, with suPAR enhanced the uptake of viable uPAR(-/-) neutrophils by uPAR(-/-) macrophages. The adhesion of WT neutrophils to uPAR(-/-) macrophages was higher than to WT macrophages. uPAR(-/-) neutrophils demonstrated increased adhesion to suPAR, which was abrogated by blocking of low-density lipoprotein related protein and integrins. Expression of uPAR on the surface of apoptotic neutrophils was reduced compared with levels on viable neutrophils. These results demonstrate a novel role for uPAR in modulating recognition and clearance of neutrophils.

Figure 1

uPAR^−/− macrophages demonstrate enhanced phagocytosis of viable neutrophils. (A) The phagocytosis of viable WT neutrophils by uPAR^−/− macrophages is significantly increased compared with phagocytic indices when WT neutrophils are incubated with WT macrophages, and is similar to that present when WT macrophages are exposed to apoptotic WT neutrophils. A total of 10⁶ viable or apoptotic WT neutrophils were added into each well of a 96-well plate containing adherent WT or uPAR^−/− macrophage monolayers, and efferocytosis assays performed as described in “In vitro efferocytosis assay.” Phagocytic indices are expressed as the percentage of macrophages containing at least 1 ingested neutrophil. *P < .05, **P < .01, compared with “WT macrophages + viable WT neutrophils.” (B) uPAR is expressed on the surface of WT, but not uPAR^−/− macrophages. WT and uPAR^−/− macrophages were incubated with anti-uPAR antibodies for 1 hour. The cells were then incubated with FITC-conjugated secondary antibodies and flow cytometry performed. (C) uPAR^−/− macrophages actively phagocytose viable WT neutrophils. Viable or apoptotic WT neutrophils were added to WT or uPAR^−/− macrophages. After 60 minutes, cytospin slides were prepared for Wright-Giemsa staining. Slides were viewed with a Labor LUX 12 microscope (Leitz) using a Phaco 2 lens at 40×/0.65. Images were acquired using a Sony cybershot camera model DSC-H2 and were processed with Adobe Photoshop version 7.0 software (Adobe Systems). As shown in the representative figures, the WT viable neutrophils ingested by uPAR^−/− macrophages showed normal nuclear and chromatin patterns, consistent with being viable and not apoptotic.

Figure 2

Modulation of uPAR on the surface of macrophages regulates the phagocytosis of viable neutrophils in vitro and in vivo, and is dependent on macrophage-associated integrins. (A) Blockade of uPAR enhances the ability of WT macrophages to phagocytose viable WT neutrophils. WT macrophages were preincubated without (con) or with 1 μg/mL anti-uPAR antibodies or rabbit IgG for 30 minutes. The medium was then changed to RPMI plus 5% FBS, and viable WT neutrophils were added for 60 minutes and the phagocytic index determined. ***P < .001 compared with the control group. (B) suPAR dose-dependently decreases the ability of uPAR^−/− macrophages to phagocytose viable WT neutrophils. uPAR^−/− macrophages were preincubated without (con) or with suPAR at the indicated concentrations for 30 minutes. The medium was then changed to RPMI plus 5% FBS, and viable WT neutrophils were added for 60 minutes and the phagocytic index determined. **P < .01, ***P < .001 compared with the control group. (C) suPAR binds to the surface of uPAR^−/− and WT macrophages. The cells were incubated with Chromeo 488–labeled mouse suPAR (1 μg/mL) or mouse albumin for 30 minutes and then washed with PBS 5 times. Fluorescent intensity was determined by flow cytometry. (D) uPAR^−/− alveolar macrophages demonstrate enhanced ability to phagocytose viable WT neutrophils in vivo. A total of 10 × 10⁶ viable WT neutrophils were injected intratracheally into WT or uPAR^−/− mice (n = 5 in each group). After 90 minutes, the mice were killed and bronchoalveolar lavage performed with 3 mL PBS. Cytospin slides were prepared using 250 μL bronchoalveolar lavage fluids. Phagocytic indices were determined as described in “In vivo efferocytosis assay.” (E) Exposure to suPAR in vivo abrogates the enhanced ability of uPAR^−/− alveolar macrophages to phagocytose viable WT neutrophils in vivo. A total of 10 μg suPAR or mouse albumin was intratracheally instilled 1 hour before intratracheal administration of 10 × 10⁶ viable WT neutrophils into uPAR^−/− mice (n = 5 in each group). Bronchoalveolar lavage was performed 90 minutes after injection of neutrophils and phagocytic indices determined. n = 5 in each group. **P < .01. ***P < .001. (F) RGD-, but not RAD-containing, peptides abrogate the enhanced activity of uPAR^−/− macrophages to phagocytose viable WT neutrophils. uPAR^−/− macrophages were preincubated with 0.1 or 1 μg/mL RGD or RAD peptides for 30 minutes. The medium was then changed to RPMI plus 5% FBS, and viable WT neutrophils were added for 60 minutes, after which the phagocytic index was determined. **P < .01. ***P < .001. (G) Integrins participate in the enhanced ability of uPAR^−/− macrophages to phagocytose viable WT neutrophils. uPAR^−/− macrophages were preincubated with 1 μg/mL mouse IgG, or antibodies to the integrins α_M, α_V, β₁, β₂, β₃, or β₄ for 30 minutes. The medium was then changed to RPMI plus 5% FBS. and viable WT neutrophils were added for 60 minutes, after which the phagocytic index was determined. *P < .05, **P < .01, compared with the control group treated with IgG.

Figure 3

LRP participates in the enhanced phagocytosis of viable WT neutrophils by uPAR^−/− macrophages. uPAR^−/− macrophages were preincubated without or with 1 μg/mL mouse IgG, anti-LRP antibodies, BSA, or RAP for 30 minutes. The medium was then changed to RPMI plus 5% FBS, and viable WT neutrophils were added for 60 minutes, after which the phagocytic index was determined. **P < .01, compared with the group treated with isotype IgG. ***P < .001, compared with the group treated with BSA.

Figure 4

Phagocytosis of viable uPAR^−/− neutrophils by WT macrophages is increased. (A) Increased phagocytosis of uPAR^−/− neutrophils by WT macrophages. A total of 10⁶ viable WT or uPAR^−/− neutrophils were added to WT macrophages. After 60 minutes of incubation, the phagocytic index was determined. ***P < .001, compared with the phagocytosis with viable WT neutrophils. (B) uPAR is expressed on the surface of WT, but not uPAR^−/− neutrophils. Flow cytometry assays were performed with anti-uPAR antibodies as in Figure 1B. (C) Blockade of uPAR enhances the phagocytosis of viable WT neutrophils by WT macrophages. WT neutrophils were preincubated without (con) or with 1 μg/mL anti-uPAR antibodies or rabbit IgG for 30 minutes. The cells were then resuspended in RPMI plus 5% FBS and added to WT macrophages. After 60 minutes of incubation, the phagocytic index was determined. *P < .05, compared with the group treated with IgG. (D) suPAR abrogates the enhanced phagocytosis of viable WT neutrophils by WT macrophages. Viable uPAR^−/− neutrophils were preincubated without (con) or with 1 μg/mL suPAR or BSA for 30 minutes. The cells was then resuspended in RPMI plus 5% FBS and added to WT macrophages. After 60 minutes of incubation, the phagocytic index was determined. ***P < .001, compared with the group treated with BSA. (E) suPAR binds to the surface of viable neutrophils. The cells were incubated with Chromeo 488-labeled mouse suPAR (1 μg/mL) or mouse albumin for 30 minutes and washed 5 times with PBS. Representative confocal images are shown. (F) Integrins participate in the enhanced phagocytosis of viable uPAR^−/− neutrophils by WT macrophages. uPAR^−/− neutrophils were preincubated with 1 μg/mL mouse IgG or antibodies to the integrins α_M, α_V, β₁, β₂, β₃, or β₄ for 30 minutes. The cells were then resuspended in RPMI plus 5% FBS and added to WT macrophages. After 60 minutes of incubation, the phagocytic index was determined. **P < .01, ***P < .001, compared with the control group treated with IgG.

Figure 5

The presence of uPAR on the surface of either macrophages or neutrophils is required for enhanced phagocytosis of viable neutrophils by macrophages. (A) uPAR^−/− macrophages do not demonstrate enhanced phagocytosis when combined with viable uPAR^−/− neutrophils. A total of 10⁶ viable WT or uPAR^−/− neutrophils were added to uPAR^−/− macrophages and phagocytosis assays were performed. ***P < .001, compared with phagocytosis with viable WT neutrophils. (B) Incubation of uPAR^−/− neutrophils or macrophages, but not both, with suPAR restores the ability of uPAR^−/− macrophages to phagocytose viable uPAR^−/− neutrophils. Viable uPAR^−/− neutrophils alone (neutrophil), uPAR^−/− macrophages alone (macrophage), or both (Both) were preincubated with 1 μg/mL suPAR for 30 minutes. The cells were then resuspended in RPMI plus 5% FBS and added to WT macrophages for phagocytosis assays. Phagocytosis of viable uPAR^−/− neutrophils preincubated with 1 μg/mL BSA by uPAR^−/− macrophages preincubated BSA were used as a control. **P < .01, ***P < .001, compared with the control group treated with BSA. (C) WT and uPAR^−/− macrophages demonstrate similar ability in ingesting carboxylate-modified beads. Carboxylate-modified beads (2 μm) were incubated WT or uPAR^−/− macrophages for 1 hour. The phagocytic index was calculated as the percentage of macrophages that ingested beads. (D) suPAR did not enhance the uptake of beads by uPAR^−/− macrophages. uPAR^−/− macrophages were preincubated without or with 1 μg/mL suPAR, and phagocytosis performed as in panel C.

Figure 6

uPAR^−/− neutrophils demonstrate enhanced adhesion to uPAR. (A) Adhesion of uPAR^−/− neutrophils to uPAR is increased. A total of 10⁶ viable WT and uPAR^−/− neutrophils were added into each well of a 96-well plate precoated with 1 μg/mL BSA or suPAR for 1 hour, and adhesion assays performed as described in “Adhesion assay.” *P < .05. (B) Adhesion of WT neutrophils to uPAR^−/− macrophages is increased. A total of 10⁶ viable WT neutrophils were added into each well of a 96-well plate plated with WT and uPAR^−/− macrophages, and adhesion assays performed as described in “Adhesion assay.” (C) RGD, but not RAD, peptides, abrogate the enhanced adhesion of uPAR^−/− neutrophils to suPAR. uPAR^−/− neutrophils were preincubated with 1 μg/mL BSA (con) or 1 μg/mL RGD or RAD peptides for 30 minutes and adhesion assays performed as in panel A. **P < .01. (D) Antibodies to integrins diminish the enhanced adhesion of viable uPAR^−/− neutrophils to suPAR. uPAR^−/− neutrophils were preincubated with 1 μg/mL mouse IgG or antibodies to the integrins α_M, α_V, β₁, β₂, β₃, or β₄ for 30 minutes. The cells were then resuspended in RPMI 1640 medium and added to suPAR-coated plates for adhesion assays. **P < .01, ***P < .001, compared with the control group treated with control IgG. (E) suPAR and integins are colocalized on the surface of uPAR^−/− macrophages. uPAR^−/− macrophages were incubated with Chromeo 488–labeled mouse suPAR (1 μg/mL) for 30 minutes. The cells were fixed and then stained overnight without or with antibodies to the integrins α_V or β₃. Representative confocal images are shown. (F-K) Anti-integrin antibodies prevent the binding of suPAR to the surface of uPAR^−/− macrophages. Cells were preincubated without (F,G) or with antibodies (1 μg/mL) to the integrins α_V (H), β₃ (I), β₄ (J), or isotype IgG control (K) for 30 minutes. The cells were then incubated with Chromeo 488–labeled mouse suPAR (1 μg/mL) for 1 hour (G-K) and washed 5 times with PBS. Fluorescent intensity was determined by flow cytometry. (L) uPAR^−/− macrophages demonstrate greater Erk phosphorylation after exposure to viable WT neutrophils than do WT macrophages. A total of 4 × 10⁶ viable WT neutrophils were added for the indicated time into each well of a 12-well plate plated with WT or uPAR^−/− macrophages. The cells were then collected and the levels of phosphorylated Erk and total Erk determined by Western blot analysis.

Figure 7

uPAR inhibits the phagocytosis of apoptotic cells. (A,B) Expression of uPAR is decreased in apoptotic neutrophils. Viable or apoptotic neutrophils were stained with anti-uPAR antibodies and expression of uPAR determined by confocal microscopy (A) or flow cytometry (B). The images were acquired using double bidirectional scans of viable and apoptotic neutrophils with a Leica DMIRBE inverted epifluorescence/Nomarski microscope outfitted with Leica TCS NT laser confocal optics (Leica Inc). The levels of fluorescence were averaged using SimplePCI software (Compix). Images were processed using IPLab Spectrum and Adobe Photoshop (Adobe Systems) software. (C) suPAR inhibits phagocytosis of apoptotic cells. A total of 1 μg/mL suPAR or BSA was added to cultures of apoptotic WT neutrophils, and macrophages and phagocytosis assays were performed.