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. 2017 Nov;31(11):4759-4769.
doi: 10.1096/fj.201700280R. Epub 2017 Jul 12.

Endothelial cell SHP-2 negatively regulates neutrophil adhesion and promotes transmigration by enhancing ICAM-1-VE-cadherin interaction

Meiping Yan  1 , Xinhua Zhang  1 , Ao Chen  1 , Wei Gu  1 , Jie Liu  1 , Xiaojiao Ren  1 , Jianping Zhang  1 , Xiaoxiong Wu  1 , Aaron T Place  2 , Richard D Minshall  2   3 , Guoquan Liu  4
Affiliations

Endothelial cell SHP-2 negatively regulates neutrophil adhesion and promotes transmigration by enhancing ICAM-1-VE-cadherin interaction

Meiping Yan et al. FASEB J. 2017 Nov.

Abstract

Intercellular adhesion molecule-1 (ICAM-1) mediates the firm adhesion of leukocytes to endothelial cells and initiates subsequent signaling that promotes their transendothelial migration (TEM). Vascular endothelial (VE)-cadherin plays a critical role in endothelial cell-cell adhesion, thereby controlling endothelial permeability and leukocyte transmigration. This study aimed to determine the molecular signaling events that originate from the ICAM-1-mediated firm adhesion of neutrophils that regulate VE-cadherin's role as a negative regulator of leukocyte transmigration. We observed that ICAM-1 interacts with Src homology domain 2-containing phosphatase-2 (SHP-2), and SHP-2 down-regulation via silencing of small interfering RNA in endothelial cells enhanced neutrophil adhesion to endothelial cells but inhibited neutrophil transmigration. We also found that VE-cadherin associated with the ICAM-1-SHP-2 complex. Moreover, whereas the activation of ICAM-1 leads to VE-cadherin dissociation from ICAM-1 and VE-cadherin association with actin, SHP-2 down-regulation prevented ICAM-1-VE-cadherin association and promoted VE-cadherin-actin association. Furthermore, SHP-2 down-regulation in vivo promoted LPS-induced neutrophil recruitment in mouse lung but delayed neutrophil extravasation. These results suggest that SHP-2-via association with ICAM-1-mediates ICAM-1-induced Src activation and modulates VE-cadherin switching association with ICAM-1 or actin, thereby negatively regulating neutrophil adhesion to endothelial cells and enhancing their TEM.-Yan, M., Zhang, X., Chen, A., Gu, W., Liu, J., Ren, X., Zhang, J., Wu, X., Place, A. T., Minshall, R. D., Liu, G. Endothelial cell SHP-2 negatively regulates neutrophil adhesion and promotes transmigration by enhancing ICAM-1-VE-cadherin interaction.

Keywords: LPS; inflammation; leukocyte; lung injury.

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Figures

Figure 1.
Figure 1.
SHP-2 interacts with ICAM-1 in vitro and in vivo. A) EA.hy926 cells were transfected with mouse wild-type ICAM-1 cDNA and immunoprecipitation (IP) with Ab to SHP-2, followed by Western blot analysis with anti–SHP-2 or anti–ICAM-1. Total protein levels of ICAM-1 and SHP-2 were also determined by Western blot. B) Mouse lung was lysed, then IP was performed with Ab to SHP-2, followed by Western blot analysis with anti–SHP-2 and anti–ICAM-1. Whole-lung protein levels of ICAM-1 and SHP-2 were determined by Western blot. Three independent experiments were performed.
Figure 2.
Figure 2.
SHP-2 mediates ICAM-1 activation-induced Src phosphorylation. HUVECs were cotransfected with mouse wild-type (WT) ICAM-1 cDNA and SHP-2 siRNA or control (Ctrl) siRNA for 48 h, then ICAM-1 XL was performed. A) Expression of SHP-2 in transfected cells was determined by Western blot analysis (Student’s unpaired t test, 2-tailed). B–E) Western blot analysis of phosphorylation of Src at Y419 (human; B), total protein levels of Src (C), phosphorylation of ICAM-1 at Y512 (D), and total protein levels of ICAM-1 (E) were determined. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as loading control, and the blots of GAPDH were derived from the same samples. Data are presented as means ± sem of 3 independent experiments and data analysis was performed by Bonferroni post-tests after 2-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 3.
Figure 3.
SHP-2 differentially regulates neutrophil adhesion and transmigration. A) Neutrophils that were isolated from fresh mouse blood were labeled, activated, and added to HUVECs that were transfected with mouse ICAM-1 cDNA and control or SHP-2 siRNA. After 2–3 h, HUVECs were washed and lysed, then neutrophil fluorescence was measured. B) Cells were treated as in panel A, then plated on Transwell inserts, fMLP was added to the bottom chamber, and freshly isolated neutrophils that were labeled with fluorescent dye were added to the upper chamber. Neutrophils that transmigrated into the bottom chamber were collected and measured. C) Cotransfected HUVECs were grown on coverslips for 48 h, stimulated with rat anti-mouse ICAM-1 mAb, TNF-α, or left untreated, and then fixed and stained for VE-cadherin (green in merged image). Arrows indicate gap formation. Data are presented as means ± sem of 3 independent experiments and data analysis performed by 2-tailed Student’s unpaired t test. **P < 0.01. Scale bar, 20 μm.
Figure 4.
Figure 4.
SHP-2 interacts with ICAM-1 and the VE-cadherin–β-catenin complex. A) EA.hy926 cells that were transfected with mouse ICAM-1 cDNA were crosslinked and lysed. Immunoprecipitation (IP) of proteins from cell lysates with Abs to SHP-2 was performed, followed by Western blot analysis with anti–SHP-2, anti–ICAM-1, anti–β-catenin, and anti–VE-cadherin Abs. Total protein levels of ICAM-1, SHP-2, β-catenin, and VE-cadherin were also determined by Western blot. B) EA.hy926 cell were treated as in panel A, followed by IP with Ab to VE-cadherin and Western blot analysis with anti–ICAM-1 and anti–VE-cadherin Abs. Total protein levels of ICAM-1 and VE-cadherin were determined by Western blot. C) EA.hy926 cell were cotransfected with mICAM-1 cDNA and SHP-2 siRNA or control (Ctrl) siRNA, and freshly isolated and activated neutrophils were added to EA.hy926 cell for 2 h, then cells were washed twice and lysed. Cell lysates were immunoprecipitated with Ab to VE-cadherin and Western blot analysis with anti–ICAM-1 and anti–VE-cadherin Abs. Then, ICAM-1–VE-cadherin expression was quantitated. Total protein levels of ICAM-1 and VE-cadherin were also determined by Western blot. In panels A–C, 3 independent experiments were performed. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Data are presented as means ± sem, and data analysis was performed by Bonferroni post-tests after 2-way ANOVA in panel C. ***P < 0.001.
Figure 5.
Figure 5.
SHP-2 mediates the interaction of ICAM-1 and the VE-cadherin–β-catenin complex. A) Immunoprecipitation (IP) with Ab to anti–SHP-2 Ab was performed by using EA.hy926 cell lysates after transfection with mouse ICAM-1 wild-type (WT), ICAM-1 Y518F, and ICAM-1 Y518D cDNA for 48 h, followed by Western blot analysis with anti–SHP-2, anti–ICAM-1, anti–VE-cadherin, and anti–β-catenin Abs. Whole-cell lysates (right). Whole protein levels of ICAM-1, SHP-2, VE-cadherin, and β-catenin were determined by Western blot. B) EA.hy926 cells were transfected with SHP-2 siRNA, control (Ctrl) siRNA, and SHP-2 cDNA, then IP of proteins from EA.hy926 cell lysates anti–ICAM-1 Ab was performed, followed by Western blot analysis with anti–SHP-2, anti–ICAM-1, anti–VE-cadherin, and anti–β-catenin Abs. Protein levels of ICAM-1, SHP-2, VE-cadherin, and β-catenin were determined by Western blot. C) EA.hy926 cells that were transfected with mouse ICAM-1 WT cDNA and control or SHP-2 siRNA for 48 h were crosslinked and lysed. IP of proteins from cell lysates with VE-cadherin Ab was performed, followed by Western blot analysis with anti-actin and anti–VE-cadherin Abs. Whole protein levels of actin and VE-cadherin were determined by Western blot. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. In panels A–C, 3 independent experiments were performed.
Figure 6.
Figure 6.
SHP-2 regulates neutrophil extravasation in vivo. Adult (age 8–10 wk) C57BL/6 mice were injected with control (Ctrl) or SHP-2 siRNA in liposomes. At 44 h after injection, mice were injected with LPS (8 mg/kg i.p.), and later lung tissue and BAL fluids were collected at 0, 2, 4, and 8 h. A) Down-regulation of SHP-2 by siRNA silencing was estimated by Western blot. B–D) Lung tissue MPO activity (B), BAL MPO activity (C), and protein contents in BAL were determined (D; n = 3 mice for each group). The blots of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were derived from the same samples. Data are presented as means ± sem of 3 independent experiments, and data analysis was performed by Bonferroni post-tests after 2-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 7.
Figure 7.
LPS-induced degradation of VE-cadherin in mouse lungs after SHP-2 down-regulation. Adult (age 8–10 wk) C57BL/6 mice were treated as in Fig. 6. Lungs were collected and lysed for total protein level by Western blot. Expression of ICAM-1 (A), phosphorylation levels of Src at Y419 (human; B), and protein levels of VE-cadherin (C) were determined by Western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as loading control and the blots of GAPDH were derived from the same samples (n = 3 mice for each group). Ctrl, control. Data are presented as means ± sem of 3 independent experiments and data analysis was performed by Bonferroni post-tests after 2-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure 8.
Figure 8.
Schematic view of neutrophil–endothelial adhesion and transmigration. A) Under resting conditions, ICAM-1, SHP-2, and VE-cadherin form a complex in endothelial cells. ICAM-1 distributes uniformly on cells and VE-cadherin also localizes in sequence. B) In contrast to resting conditions, neutrophil–endothelial adhesion leads to ICAM-1–SHP-2–VE-cadherin complex disassembly, an increase in VE-cadherin–actin interaction, ICAM-1 cluster formation, and VE-cadherin behaving like a curtain.
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