Blood flow guides sequential support of neutrophil arrest and diapedesis by PILR-β1 and PILR-α
- Max Planck Institute of Molecular Biomedicine, Germany
Figures
Figure 1 with 2 supplements
Characterization of PILR-α-/- and PILR-β1-/-neutrophils for surface expression of molecules involved in extravasation.
Ly6G+ bone marrow neutrophils from WT, PILR-α-/- or PILRβ1-/- mice were analyzed for indicated antigens by FACS. Representative histograms of at least three independent experiments are shown in (A) …
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Figure 1—source data 1
Source data for Figure 1B and D.
- https://doi.org/10.7554/eLife.47642.010
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Figure 1—source data 2
Source data for Figure 1E.
- https://doi.org/10.7554/eLife.47642.011
Figure 1—figure supplement 1
Quantification of PILR subtypes neutrophils.
(A) Ly6G+ WT and PILR-α-/- bone marrow cells were stained with a primary antibody recognizing both PILR-α and PILR-β in the absence (black) or presence of 2.5-fold (red) or 5-fold (orange) excess of …
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Figure 1—figure supplement 1—source data 1
Source data for Figure 1—figure supplement 1B-C.
- https://doi.org/10.7554/eLife.47642.004
Figure 1—figure supplement 2
Characterization of immune subsets in PILR-α-/- and PILR-β1-/-mice.
WT (black), PILR-α-/- (red) and PILR-b1-/- (blue) CD45+ leukocytes from spleen (A), bone marrow (B) or peripheral blood (C) were stained by antibodies against the indicated antigens and analyzed by …
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Figure 1—figure supplement 2—source data 1
Source data for Figure 1—figure supplement 2A.
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Figure 1—figure supplement 2—source data 2
Source data for Figure 1—figure supplement 2B (F4/80+).
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Figure 1—figure supplement 2—source data 3
Source data for Figure 1—figure supplement 2B (Ly6G+).
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Figure 1—figure supplement 2—source data 4
Source data for Figure 1—figure supplement 2C.
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Figure 2
Impact of PILRs on neutrophil extravasation in the inflamed cremaster.
WT, PILR-α-/- and PILR-β1-/- mice were stimulated with intrascrotal TNF-α for 2 hr. Cremaster muscles were exposed and examined for neutrophil extravasation by intravital microscopy (IVM) for (A) …
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Figure 2—source data 1
Source data for Figure 2A, C and D.
- https://doi.org/10.7554/eLife.47642.013
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Figure 2—source data 2
Source data for Figure 2B.
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Figure 2—source data 3
Source data for Figure 2A-D (Table 1).
- https://doi.org/10.7554/eLife.47642.015
Figure 3 with 1 supplement
PILR-β1 is required for CD99-stimulated neutrophil adhesion on ICAM-1-Fc in vitro.
Flow chamber surfaces were coated with P-selectin-Fc, ICAM-1-Fc and CXCL-1, and co-coated with either CD99-Fc or human IgG1 control (hIgG). PILR-α-/- (A) or PILR-β1-/- (B) PMN were mixed with …
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Figure 3—source data 1
Source data for Figure 3A.
- https://doi.org/10.7554/eLife.47642.025
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Figure 3—source data 2
Source data for Figure 3B.
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Figure 3—source data 3
Source data for Figure 3C-E.
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Figure 3—source data 4
Source data for Figure 3F.
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Figure 3—source data 5
Source data for Figure 3G.
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Figure 3—source data 6
Source data for Figure 3H.
- https://doi.org/10.7554/eLife.47642.030
Figure 3—figure supplement 1
PILRs modulate β2 integrin activity via Syk.
(A) PMN were pre-treated with non-crosslinking rabbit IgG or crosslinking polyclonal anti-PILRs for 15 min, and then stimulated with 100 ng/ml CXCL-1 for 0 s (unstimulated), 30 s, 60 s and 75 s, …
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Figure 3—figure supplement 1—source data 1
Source data for Figure 3—figure supplement 1A.
- https://doi.org/10.7554/eLife.47642.022
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Figure 3—figure supplement 1—source data 2
Source data for Figure 3—figure supplement 1B (PILRa-/-).
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Figure 3—figure supplement 1—source data 3
Source data for Source data for Figure 3—figure supplement 1B (PILRb1-/-).
- https://doi.org/10.7554/eLife.47642.024
Figure 4
PILR-β1, but not PILR-α, is essential for CD99-stimulated support of neutrophil adhesion in vivo.
WT or CD99-/- mice received bone marrow transplantation from WT (A–F) or PILR-α-/- (A–C) or PILR-β1-/- (D–F). IVM measurement of (A, D) rolling flux fraction, (B, E) adhesion and (C, F) …
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Figure 4—source data 1
Source data for Figure 4A–C.
- https://doi.org/10.7554/eLife.47642.032
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Figure 4—source data 2
Source data for Figure 4A–C (Table 2).
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Figure 4—source data 3
Source data for Figure 4D–F.
- https://doi.org/10.7554/eLife.47642.034
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Figure 4—source data 4
Source data for Figure 4D–F (Table 3).
- https://doi.org/10.7554/eLife.47642.035
Figure 5 with 2 supplements
Shear stress enhances the interaction of CD99 with PILR-β.
(A) CHO cells overexpressing CD99 (CHO-CD99) were constructed and analyzed for surface expression of CD99. Solid tracks, anti-CD99; dotted tracks, secondary antibody only. (B) 1:1 Cell mixtures of …
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Figure 5—source data 1
Source data for Figure 5B.
- https://doi.org/10.7554/eLife.47642.043
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Figure 5—source data 2
Source data for Figure 5C-D.
- https://cdn.elifesciences.org/articles/47642/elife-47642-fig5-data2-v2.csv
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Figure 5—source data 3
Source data for Figure 5E-F.
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Figure 5—source data 4
Source data for Figure 5G.
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Figure 5—figure supplement 1
CD99-specific interaction with coated PILR-Fc under constant flow.
Parental CHO cells (CHO-WT) or CHO cells expressing full-length CD99 (CHO-CD99FL) were passed over a surface coated with PILR-α-Fc (blue) or PILR-β-Fc (red), or over an uncoated control surface at a …
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Figure 5—figure supplement 1—source data 1
Source data for Figure 5—figure supplement 1.
- https://doi.org/10.7554/eLife.47642.040
Figure 5—figure supplement 2
CD99-Fc and full-length CD99 produced in CHO cells are glycosylated.
(A) CD99-Fc was treated with 0 or 0.3 U/ml sialidase at 37°C for 1 hr and analyzed by Western blot. (B) CHO-CD99 was similarly treated with sialidase. Lysate was prepared and analyzed by Western blot.
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Figure 5—figure supplement 2—source data 1
Source data for Figure 5—figure supplement 2A-B.
- https://doi.org/10.7554/eLife.47642.042
Figure 6
Shear switches on PILR-β1-stimulated adhesion of neutrophils to endothelial monolayers under flow.
(A) PMN from WT or PILR-β1-/- mice were allowed to adhere onto TNF-α inflamed and CXCL-1 pretreated MDMVEC under stasis. Adherent cells were counted. n = 17 fields per genotype from two experiments. …
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Figure 6—source data 1
Source data for Figure 6A.
- https://doi.org/10.7554/eLife.47642.048
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Figure 6—source data 2
Source data for Figure 6B.
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Figure 6—source data 3
Source data for Figure 6C.
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Figure 6—source data 4
Source data for Figure 6D.
- https://doi.org/10.7554/eLife.47642.051
Figure 7 with 3 supplements
PILR-α supports crawling and diapedesis.
(A) Correlation between adhesion and transmigration of WT, PILR-α-/- and PILR-β1-/- PMNs in static transendothelial cell migration (TEM) assays through MDMVEC. n = 96 (WT), 54 (PILR-α-/-) and 41 …
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Figure 7—source data 1
Source data for Figure 7A.
- https://doi.org/10.7554/eLife.47642.059
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Figure 7—source data 2
Source data for Figure 7B.
- https://doi.org/10.7554/eLife.47642.060
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Figure 7—source data 3
Source data for Figure 7C-D.
- https://doi.org/10.7554/eLife.47642.061
Figure 7—figure supplement 1
PILR-α suppresses adhesion on endothelial monolayers.
(A) WT or PILR-α-/- PMNs were allowed to adhere onto TNF-α stimulated MDMVEC and adherent cells were counted. n = 16 fields from two experiments. (B) WT or PILR-α-/- PMNs were allowed to adhere on …
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Figure 7—figure supplement 1—source data 1
Source data for Figure 7—figure supplement 1A.
- https://doi.org/10.7554/eLife.47642.054
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Figure 7—figure supplement 1—source data 2
Source data for Figure 7—figure supplement 1B.
- https://doi.org/10.7554/eLife.47642.055
Figure 7—figure supplement 2
Effect of PILR-α on leukocyte crawling requires endothelial surface.
Transmigration of WT and PILR-α- /- PMNs towards 40 ng/ml CXCL-1 through polycarbonate transwell filters (3 µm pore size) coated with 2 µg/ml ICAM-1-Fc for 1 hr at 37°C. n = 6 transwells. Groups …
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Figure 7—figure supplement 2—source data 1
Source data for Figure 7—figure supplement 2.
- https://doi.org/10.7554/eLife.47642.058
Figure 7—figure supplement 3
PILR-α on cellular surface is unavailable for CD99-Fc binding due to blockade by cis-interaction with sialylated entities.
CHO- WT (black) or overexpressing PILR-α (CHO-PILR-α, blue) were treated with 0 or 0.3 U/ml sialidase at 37°C for 30 min. Cells were washed and probed for CD99-Fc binding. CD99-Fc binding on cell …
Figure 8
Graphical depiction.
An antagonistic pair of receptors, PILR-β1 and PILR-α, supports neutrophil arrest and seamless transition to crawling via modulating β2 integrin activity at different steps during extravasation.
Author response image 1
Transmigration of WT and PILR-α-/- PMNs towards 40ng/ml CXCL1 through polycarbonate transwell filters (3μm pore size) coated with 2μg/ml ICAM-1-Fc for 1 h at 37°C.
(P value is 0.0658 (2-tailed t-test)).
Videos
Video 1
Representative IVM video of a vessel in WT in Figure 2.
https://doi.org/10.7554/eLife.47642.017
Video 2
Representative IVM video of a vessel in PILR-α-/- in Figure 2.
https://doi.org/10.7554/eLife.47642.018
Video 3
Representative IVM video of a vessel in PILR-β1-/- in Figure 2.
https://doi.org/10.7554/eLife.47642.019Tables
Table 1
Hemodynamic parameters of mice in full knockout characterization in Figure 2.
Genotypes, number of mice, number of venules, venule diameters, leukocyte counts, blood velocities and Newtonian wall shear stresses are shown as mean ± SEM.
| Genotype | Mice | Venules | Diameter (μm) | Leukocyte counts (106 cells/ml) | Mean blood velocity (mm/s) | Newtonian wall shear rate (s−1) |
|---|---|---|---|---|---|---|
| WT | 8 | 40 | 28.7 ± 0.7 | 4.34 ± 0.62 | 1.22 ± 0.02 | 350 ± 9 |
| PILRα-/- | 7 | 37 | 29.4 ± 0.8 | 5.19 ± 0.46 | 1.23 ± 0.02 | 343 ± 9 |
| PILRβ1-/- | 7 | 35 | 28.2 ± 0.8 | 3.95 ± 0.45 | 1.23 ± 0.02 | 357 ± 10 |
Table 2
Hemodynamic parameters of bone marrow transplanted chimeric mice for intravital microscopy in Figure 4A–C.
Genotypes of donors and recipients, number of mice, number of venules, venule diameters, leukocyte counts, blood velocities and Newtonian wall shear stresses are shown as mean ± SEM.
| Donor genotype (neutrophil) | Recipient genotype (endothelium) | Mice | Venules | Diameter (μm) | Leukocyte counts (106 cells/ml) | Mean blood velocity (mm/s) | Newtonian wall shear rate (s−1) |
|---|---|---|---|---|---|---|---|
| WT | WT | 5 | 39 | 25.4 ± 0.6 | 3.27 ± 0.19 | 1.21 ± 0.00 | 388 ± 8 |
| WT | CD99-/- | 5 | 38 | 24.2 ± 0.5 | 3.52 ± 0.07 | 1.20 ± 0.01 | 404 ± 8 |
| PILRα-/- | WT | 5 | 39 | 24.5 ± 0.6 | 2.87 ± 0.11 | 1.20 ± 0.01 | 403 ± 9 |
| PILRα-/- | CD99-/- | 4 | 34 | 23.8 ± 0.6 | 3.47 ± 0.12 | 1.21 ± 0.01 | 413 ± 10 |
Table 3
Hemodynamic parameters of bone marrow transplanted chimera mice for intravital microscopy in Figure 4D–F.
Genotypes of donors and recipients, number of mice, number of venules, venule diameters, leukocyte counts, blood velocities and Newtonian wall shear stresses are shown as mean ± SEM.
| Donor genotype (neutrophil) | Recipient genotype (endothelium) | Mice | Venules | Diameter (μm) | Leukocyte counts (106 cells/ml) | Mean blood velocity (mm/s) | Newtonian wall shear rate (s−1) |
|---|---|---|---|---|---|---|---|
| WT | WT | 4 | 23 | 23.3 ± 0.7 | 3.13 ± 0.30 | 1.24 ± 0.01 | 431 ± 10 |
| WT | CD99-/- | 5 | 27 | 23.4 ± 0.8 | 5.46 ± 0.38 | 1.24 ± 0.01 | 434 ± 12 |
| PILRβ1-/- | WT | 5 | 29 | 24.0 ± 0.7 | 4.56 ± 0.88 | 1.23 ± 0.01 | 416 ± 10 |
| PILRβ1-/- | CD99-/- | 5 | 36 | 22.6 ± 0.5 | 5.58 ± 0.37 | 1.25 ± 0.01 | 443 ± 9 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | CD99-/- | PMID: 28223280 | ||
| Genetic reagent (M. musculus) | PILR-α-/- | This paper | ||
| Genetic reagent (M. musculus) | PILR-β1-/- | This paper | ||
| Cell line (C. griseus) | CHO-CD99 | PMID: 15280198 | ||
| Antibody | Rabbit anti-PILR-α C-terminus (VD67, polyclonal) | This paper | FC: 5 μg/ml | |
| Antibody | Rabbit anti-PILRs (VD65, crossreactive, polyclonal) | This paper | FC: 2 μg/ml Functional assay: 20 μg/ml | |
| Antibody | Rat anti-F4/80-FITC (BM8) | Biolegend | Cat# 123107 | FC: 1:100 |
| Antibody | Rat anti-LFA-1-PE (H155-78) | Biolegend | Cat# 141005 | FC: 1:100 |
| Antibody | Rat anti-Ly6G-(APC or FITC) (1A8) | Biolegend | Cat# 127613 Cat# 127605 | FC: 1:250 |
| Antibody | Rat anti-CD11b-PE (M1/70) | Biolegend | Cat# 101207 | FC: 1:100 |
| Antibody | Armenian Hamster anti- CD11c-PE (N418) | Biolegend | Cat# 117307 | FC: 1:100 |
| Antibody | Rat anti-CD45-APC (30F11) | Biolegend | Cat# 103111 | FC: 1:100 |
| Antibody | Rat anti-CD182-FITC (SA045E1) | Biolegend | Cat# 149607 | FC: 1:100 |
| Antibody | Mouse anti-NK1.1-PE (PK136) | Biolegend | Cat# 108707 | FC: 1:100 |
| Antibody | Rat anti-B220-PE (RA3-6B2) | BD Biosciences | Cat# 93992 | FC: 1:100 |
| Antibody | Rat anti-CD4-PE (RM4-5) | BD Biosciences | Cat# 26589 | FC: 1:100 |
| Antibody | Rat anti-CD162-PE (2PH1) | BD Pharmingen | Cat# 555306 | FC: 1:100 |
| Antibody | Rat anti-CD8-FITC (53–6.7) | eBioscience | Cat# 11-0081-85 | FC: 1:100 |
| Antibody | Rabbit anti-Syk (polyclonal) | Thermo Fisher | Cat# PA5-27262 | IP: 3 μg/ 5×106 PMN WB: 1:1000 |
| Antibody | Mouse anti-phosphotyrosine (4G10) | Merck Millipore | Cat# 05–321 | WB: 1 μg/ml |
| Peptide, recombinant protein | CD99-Fc | PMID: 15280198 | ||
| Peptide, recombinant protein | P-sel-Fc | PMID: 28223280 | ||
| Peptide, recombinant protein | ICAM-1-Fc | PMID: 28223280 | ||
| Peptide, recombinant protein | TNF-α | PeproTech | Cat# 315-01A | |
| Commercial assay or kit | GeneArt Precision gRNA Synthesis Kit | Thermo Fisher | A29377 | |
| Software, algorithm | FlowJo v10 | FlowJo, LLC | ||
| Software, algorithm | CytExpert | Beckman Coulter | v2.3.0.84 FACS data acquisition | |
| Software, algorithm | Zeiss ZEN (blue edition) | Zeiss | Image/video acquisition | |
| Software, algorithm | Fiji Image J | PMID: 27713081 | Plugin: Trackmate |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.47642.063
