1. Immunology and Inflammation
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Blood flow guides sequential support of neutrophil arrest and diapedesis by PILR-β1 and PILR-α

  1. Yu-Tung Li
  2. Debashree Goswami
  3. Melissa Follmer
  4. Annette Artz
  5. Mariana Pacheco-Blanco
  6. Dietmar Vestweber  Is a corresponding author
  1. Max Planck Institute of Molecular Biomedicine, Germany
Research Article
Cite this article as: eLife 2019;8:e47642 doi: 10.7554/eLife.47642
9 figures, 3 videos, 4 tables and 1 additional file

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) for PILR-α-/- and (C) for PILR-β1-/- mice, with expression levels quantified in (B) and (D), respectively. Solid tracks, specific staining; dotted tracks, isotype control. Groups were compared by 2-tailed t-test. (E) qRT-PCR analysis of RNA extracted from peripheral blood of WT (n = 9) or PILR-α-/- (n = 5) or PILR-β1-/- (n = 7) mice for the indicated genes. Groups were analyzed by 1-way ANOVA followed by Holm-Sidak method for multiple comparisons. Error bars, SEM. ****p<0.001.

https://doi.org/10.7554/eLife.47642.002
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 blocking PILR-β-Fc. Dotted black line: secondary antibody only, solid black line: without blocking PILR-β-Fc. (B) MFI quantification of PILRs signals in (A). (C) Quantification of PILRs signal on WT and PILR-α-/- Ly6G+ cells from two experiments.

https://doi.org/10.7554/eLife.47642.003
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 flow cytometry. Results are from at least three mice per genotype.

https://doi.org/10.7554/eLife.47642.005
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) rolling flux fraction, (B) rolling velocity, (C) adherent leukocytes, and (D) extravasated leukocytes. WT: n = 40 vessels from eight mice; PILR-α-/-: n = 37 vessels from seven mice; PILR-β1-/-: n = 35 vessels from seven mice. For (B), 117 cells (WT), 111 cells (PILR-α-/-) and 105 cells (PILR-β1-/-) from the indicated number of vessels were quantified. Groups were analyzed by 1-way ANOVA followed by Tukey’s multiple comparisons. Error bars, SEM. *p<0.05, ***p<.005, ****p<0.001. Hemodynamic parameters are given in Table 1.

https://doi.org/10.7554/eLife.47642.012
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 fluorescently labeled WT PMN in 1:1 ratio and passed over the surfaces at 5 dyn/cm2 at 37°C for 5 min. Adherent PMNs were quantified for n = 45 fields from nine experiments each. (C–E) Bone marrow cells from WT (C), PILR-α-/- (D) or PILR-β1-/- (E) mice were mixed with ICAM-1-Fc/PE-conjugated secondary antibody detection complex. Cell mixtures were pre-treated with non-crosslinking rabbit IgG or crosslinking polyclonal anti-PILRs in pair for 15 min, and then stimulated with 100 ng/ml CXCL-1 for 0 min (unstimulated), 3 and 5 min, followed by fixation and analysis for ICAM-1-Fc binding to Ly6G+ cells by FACS. n = 6 paired samples per treatment per genotype. (F) Experiment described in (C) was repeated except for replacing the chemokine stimulation by 20 ng/ml PMA + 1 μg/ml ionomycin. n = 5 paired samples per treatment. (G–H) Experiments described in (A–B) were repeated in the presence of 1 µM Syk inhibitor PRT-060318 or vehicle control with PILR-α-/- (G) or PILR-β1-/- (H) neutrophils. n = 26 fields each from two experiments (G, control) or 39 fields each from three experiments (G, inhibition) fields. n = 20 fields each from two experiments (H, control) or 30 fields each from three experiments (H, inhibition) fields. Groups were compared by 1-way ANOVA followed by Tukey’s multiple comparisons in (A, B, G, H) or 2-tailed paired t-test in (C–F). Error bars, SEM. *p<0.05, **p<.005, ***p<0.0005, ****p<0.0001.

https://doi.org/10.7554/eLife.47642.020
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, followed by fixation and cell lysis. Syk was immunoprecipitated and analyzed for phospho-Syk (4G10). (B) PMNs from WT (black), PILR-α-/- (red) or PILR-β1-/- (blue) were treated with or without 1 µM Syk-inhibitor PRT-060318 for 30 min. Cells were allowed to adhere to ICAM-1-Fc coated surfaces in the presence of 100 ng/ml CXCL-1 with or without PRT-060318. Adherent cells were counted. n ≥ 12 for each genotype from at least two experiments. Error bars, SEM. Groups were compared by 2-tailed t-test. ns, not significant, *p<0.05, **p<0.01.

https://doi.org/10.7554/eLife.47642.021
Figure 3—figure supplement 1—source data 1

Source data for Figure 3—figure supplement 1A.

https://doi.org/10.7554/eLife.47642.022
Figure 3—figure supplement 1—source data 2

Source data for Figure 3—figure supplement 1B (PILRa-/-).

https://doi.org/10.7554/eLife.47642.023
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
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) extravasation were performed on TNF-α inflamed cremasters of the chimeric mice. Donor-Recipient: For (A–C), WT-WT, n = 39 vessels from five mice; WT-CD99-/-, n = 38 vessels from five mice; PILR-α-/--WT, n = 39 vessels from five mice; PILR-α-/--CD99-/-, n = 34 vessels from four mice. For (D–F), WT-WT, n = 23 vessels from four mice; WT-CD99-/-, n = 28 vessels from five mice; PILR-β1-/--WT, n = 29 vessels from five mice; PILR-β1-/--CD99-/-, n = 36 vessels from five mice. Groups were analyzed by 1-way ANOVA followed by Tukey’s multiple comparisons. Error bars, SEM. *p<0.05, **p<0.01, ***p<.005. Hemodynamic parameters are given in Tables 2 and 3.

https://doi.org/10.7554/eLife.47642.031
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 CHO and CHO-CD99 were allowed to adhere onto PILR-α-Fc or PILR-β-Fc coated surface at RT for 10 min (static conditions). CD99-specific cell adhesion was quantified. n = 30 fields from six experiments. (C) 1:1 Cell mixtures of CHO and CHO-CD99 were passed over flow chamber surfaces coated with PILR-α-Fc (blue) or PILR-β-Fc (red) at RT under stepped shear stress of 3 min interval each (black) and video recorded. CD99-specific cell-surface interactions (≥2 s) were counted every minute. (D) Mean number of CD99-specific interactions under the indicated shear stress (from C) were quantified. n = 8 experiments on PILR-α-Fc coating, n = 9 experiments on PILR-β-Fc coating. (E, F) CHO-CD99 were passed over a surface coated with PILR-α-Fc (blue), PILR-β-Fc (red) or an uncoated control surface under increasing shear stress and video recorded. Transiently interacting (≥50 ms) cells were counted and averaged from six experiments for determining fraction of PILR-specific interacting cell flux. (G) Experiment described as in (E, F) was repeated by passing WT (black), PILR-α-/- (red) and PILR-β1-/- (blue) PMNs through CD99-Fc or control hIgG coated flow chambers. Transiently interacting (≥30 ms) neutrophils were counted and averaged from four experiments for determining the fraction of CD99-specific cell flux interactions. n = 75 measurements per 0.05 dyn/cm2 interval for (E–G). Groups were compared by Mann-Whitney U-test in (B) and 2-tailed t-test in (D). Error bars, SEM. *p<0.05, ***p<.005, ****p<0.001.

https://doi.org/10.7554/eLife.47642.038
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 constant shear stress of 0.2 dyn/cm2 at room temperature for 3 min. Videos were recorded at one frame-per-second. Transiently interacting (≥50 ms) cells were counted and averaged from three experiments per genotype per coating for determining the fraction of PILR-specific cell flux interactions. n = 180 frames per flow. Groups were compared against CHO-WT by Mann-Whitney U-test. ***p<.005, ****p<0.001.

https://doi.org/10.7554/eLife.47642.039
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.

https://doi.org/10.7554/eLife.47642.041
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. Groups were compared by 2-tailed t-test. Error bars, SEM. (B–D) PMN from WT or PILR-β1-/- mice were passed under increasing flow over TNF-α inflamed and CXCL-1 pretreated MDMVEC monolayer under an increasing shear gradient (B) or a shear gradient from 2 to 0 dyn/cm2 (C) or a constant shear at 0.4 dyn/cm2 (D) in 20 min with adhesion events (C) being pooled from five experiments (B, C) or four experiments (D) for each genotype under different shear stresses.

https://doi.org/10.7554/eLife.47642.047
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 (PILRβ1-/-) transwells. Knockouts were compared against WT by 2-way ANOVA followed by Holm-Sidak method for multiple comparisons. (B) WT and PILR-α-/- PMN were allowed to transmigrate through MDMVEC for the indicated times. n = 4 transwells per time point per genotype from two experiments. Genotypes were compared by 2-tailed paired t-test. (C, D) WT and PILR-α-/- PMNs with either genotype fluorescently labeled were mixed and passed over TNF-α inflamed MDMVEC monolayers to allow transendothelial migration at 37°C for 20 min under flow, analyzed by video recording. The fraction of crawling cells (C) and the time needed for diapedesis (D) were determined. n = 14 videos for (C) and n = 31 (WT) or 15 (PILR-α-/-) diapedesis events for (D). Groups were compared by Mann-Whitney U-test. Error bars: SEM. **p<0.01, ***p<.005, ****p<0.001.

https://doi.org/10.7554/eLife.47642.052
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 ICAM-1-Fc coated surface in the presence of 100 ng/ml CXCL-1. n = 25 fields from three experiments. Groups were compared by 2-tailed t-test. **p<0.01, ****p<0.0001.

https://doi.org/10.7554/eLife.47642.053
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 were compared by 2-tailed t-test.

https://doi.org/10.7554/eLife.47642.057
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 surface was measured by flow cytometry. Solid, CD99-Fc, dotted, control. Histograms are representative of two similar experiments.

https://doi.org/10.7554/eLife.47642.056
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.

https://doi.org/10.7554/eLife.47642.062
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)).

https://doi.org/10.7554/eLife.47642.065

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.47642.019

Tables

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.

https://doi.org/10.7554/eLife.47642.016
GenotypeMiceVenulesDiameter (μm)Leukocyte counts (106 cells/ml)Mean blood velocity (mm/s)Newtonian wall shear rate (s−1)
WT84028.7 ± 0.74.34 ± 0.621.22 ± 0.02350 ± 9
PILRα-/-73729.4 ± 0.85.19 ± 0.461.23 ± 0.02343 ± 9
PILRβ1-/-73528.2 ± 0.83.95 ± 0.451.23 ± 0.02357 ± 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.

https://doi.org/10.7554/eLife.47642.036
Donor genotype (neutrophil)Recipient genotype (endothelium)MiceVenulesDiameter (μm)Leukocyte counts (106 cells/ml)Mean blood velocity (mm/s)Newtonian wall shear rate (s−1)
WTWT53925.4 ± 0.63.27 ± 0.191.21 ± 0.00388 ± 8
WTCD99-/-53824.2 ± 0.53.52 ± 0.071.20 ± 0.01404 ± 8
PILRα-/-WT53924.5 ± 0.62.87 ± 0.111.20 ± 0.01403 ± 9
PILRα-/-CD99-/-43423.8 ± 0.63.47 ± 0.121.21 ± 0.01413 ± 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.

https://doi.org/10.7554/eLife.47642.037
Donor genotype (neutrophil)Recipient genotype (endothelium)MiceVenulesDiameter (μm)Leukocyte counts (106 cells/ml)Mean blood velocity (mm/s)Newtonian wall shear rate (s−1)
WTWT42323.3 ± 0.73.13 ± 0.301.24 ± 0.01431 ± 10
WTCD99-/-52723.4 ± 0.85.46 ± 0.381.24 ± 0.01434 ± 12
PILRβ1-/-WT52924.0 ± 0.74.56 ± 0.881.23 ± 0.01416 ± 10
PILRβ1-/-CD99-/-53622.6 ± 0.55.58 ± 0.371.25 ± 0.01443 ± 9
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional 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-CD99PMID: 15280198
AntibodyRabbit anti-PILR-α C-terminus (VD67, polyclonal)This paperFC: 5 μg/ml
AntibodyRabbit anti-PILRs (VD65, crossreactive, polyclonal)This paperFC: 2 μg/ml
Functional assay: 20 μg/ml
AntibodyRat anti-F4/80-FITC (BM8)BiolegendCat# 123107FC: 1:100
AntibodyRat anti-LFA-1-PE (H155-78)BiolegendCat# 141005FC: 1:100
AntibodyRat anti-Ly6G-(APC or FITC) (1A8)BiolegendCat# 127613
Cat# 127605
FC: 1:250
AntibodyRat anti-CD11b-PE (M1/70)BiolegendCat# 101207FC: 1:100
AntibodyArmenian Hamster anti- CD11c-PE (N418)BiolegendCat# 117307FC: 1:100
AntibodyRat anti-CD45-APC (30F11)BiolegendCat# 103111FC: 1:100
AntibodyRat anti-CD182-FITC (SA045E1)BiolegendCat# 149607FC: 1:100
AntibodyMouse anti-NK1.1-PE (PK136)BiolegendCat# 108707FC: 1:100
AntibodyRat anti-B220-PE (RA3-6B2)BD BiosciencesCat# 93992FC: 1:100
AntibodyRat anti-CD4-PE (RM4-5)BD BiosciencesCat# 26589FC: 1:100
AntibodyRat anti-CD162-PE (2PH1)BD PharmingenCat# 555306FC: 1:100
AntibodyRat anti-CD8-FITC (53–6.7)eBioscienceCat# 11-0081-85FC: 1:100
AntibodyRabbit anti-Syk (polyclonal)Thermo FisherCat# PA5-27262IP: 3 μg/ 5×106 PMN
WB: 1:1000
AntibodyMouse anti-phosphotyrosine (4G10)Merck MilliporeCat# 05–321WB: 1 μg/ml
Peptide, recombinant proteinCD99-FcPMID: 15280198
Peptide, recombinant proteinP-sel-FcPMID: 28223280
Peptide, recombinant proteinICAM-1-FcPMID: 28223280
Peptide, recombinant proteinTNF-αPeproTechCat# 315-01A
Commercial assay or kitGeneArt Precision gRNA Synthesis KitThermo FisherA29377
Software, algorithmFlowJo v10FlowJo, LLC
Software, algorithmCytExpertBeckman Coulterv2.3.0.84
FACS data acquisition
Software, algorithmZeiss ZEN (blue edition)ZeissImage/video acquisition
Software, algorithmFiji Image JPMID: 27713081Plugin: Trackmate

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files for the figures have been provided.

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