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MFG-E8 inhibits neutrophil migration through αvβ₃-integrin-dependent MAP kinase activation.

Aziz M, Yang WL, Corbo LM, Chaung WW, Matsuo S, Wang P - Int. J. Mol. Med. (2015)

Bottom Line: Treatment with rhMFG‑E8 resulted in a significant inhibition of dHL‑60 cell migration in a dose‑dependent manner.There was a 46% decrease in CXCR2 expression in the rhMFG‑E8‑treated dHL‑60 cells, which was associated with a 32% increase in GRK2 expression.The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL‑60 cell migration which was significantly inhibited treatment with rhMFG‑E8.

View Article: PubMed Central - PubMed

Affiliation: Center for Translational Research, The Feinstein Institute for Medical Research and Department of Surgery, Hofstra North Shore‑LIJ School of Medicine, Manhasset, NY, USA.

ABSTRACT
We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFG‑E8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFG‑E8 attenuates neutrophil migration. Recombinant human MFG‑E8 (rhMFG‑E8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL‑60, was treated with rhMFG‑E8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin‑8 (IL‑8) as the chemoattractant. Surface CXCR2 and intracellular G protein‑coupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogen‑activated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFG‑E8 resulted in a significant inhibition of dHL‑60 cell migration in a dose‑dependent manner. There was a 46% decrease in CXCR2 expression in the rhMFG‑E8‑treated dHL‑60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL‑60 cells, treatment with rhMFG‑E8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 10‑30 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL‑60 cell migration which was significantly inhibited treatment with rhMFG‑E8. Furthermore, blocking the MFG‑E8 receptors, αvβ3/αvβ5‑integrins, by anti‑αv‑integrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFG‑E8‑induced inhibition of dHL‑60 cell migration. Finally, treatment of the dHL‑60 cells with SB203580 and PD98059 neutralized the rhMFG‑E8‑induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFG‑E8 through which it inhibits neutrophil migration through αvβ3-integrin-dependent MAP kinase activation.

No MeSH data available.


Related in: MedlinePlus

Expression of CXCR2 and G protein-coupled receptor kinase 2 (GRK2) in recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8)-treated neutrophils. (A) Differentiated HL-60 (dHL-60) cells (1.5×106 cells) treated with rhMFG-E8 (500 ng/ml) for 2 h were surface-stained with PE-CXCR2 and then subjected to flow cytometric analysis. Data were analyzed by Flowjo software with 15,000 events per sample. Isotype controls and Fc receptor blocker were used for all the samples. The representative histograms for PBS and rhMFG-E8-treated dHL-60 cells obtained from 3 independent experiments are shown. (B) Bar diagram representing the mean fluorescence intensities (MFI) of the PBS- and rhMFG-E8-treated samples are shown. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments. *P<0.05 vs. PBS treatment. (C and D) To examine the expression of intracellular GRK2 levels, dHL-60 cells (5×106 cells) treated with rhMFG-E8 (500 ng/ml) for 2 h were first surface-stained with PE-CXCR2, and then to examine intracellular GRK2 expression, cells were fixed and permeabilized with Intraprep, followed by staining with FITC-GRK2 antibodies. After washing, the stained cells were subjected to flow cytometry using a FACSVerse flow cytometer. Appropriate isotype controls and Fc receptor blocker were used for all the samples. Representative histogram and the bar diagrams indicating the MFI of PBS and rhMFG-E8-treated samples are shown. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments. *P<0.05 vs. PBS treatment. (E) Differentiated HL-60 cells (1.5×106/ml) were placed into 1.5 ml microfuge tubes with Opti-MEM and then stimulated with either rhMFG-E8 (500 ng/ml) or PBS for different periods of time. Following incubation, the cell lysates were harvested and then subjected to western blot analysis using rabbit anti-GRK2 monoclonal antibody. Results were normalized to β-actin as an internal control and are expressed as the fold induction in comparison to the 0 min time point. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments. *P<0.05 vs. 0 min.
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f2-ijmm-36-01-0018: Expression of CXCR2 and G protein-coupled receptor kinase 2 (GRK2) in recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8)-treated neutrophils. (A) Differentiated HL-60 (dHL-60) cells (1.5×106 cells) treated with rhMFG-E8 (500 ng/ml) for 2 h were surface-stained with PE-CXCR2 and then subjected to flow cytometric analysis. Data were analyzed by Flowjo software with 15,000 events per sample. Isotype controls and Fc receptor blocker were used for all the samples. The representative histograms for PBS and rhMFG-E8-treated dHL-60 cells obtained from 3 independent experiments are shown. (B) Bar diagram representing the mean fluorescence intensities (MFI) of the PBS- and rhMFG-E8-treated samples are shown. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments. *P<0.05 vs. PBS treatment. (C and D) To examine the expression of intracellular GRK2 levels, dHL-60 cells (5×106 cells) treated with rhMFG-E8 (500 ng/ml) for 2 h were first surface-stained with PE-CXCR2, and then to examine intracellular GRK2 expression, cells were fixed and permeabilized with Intraprep, followed by staining with FITC-GRK2 antibodies. After washing, the stained cells were subjected to flow cytometry using a FACSVerse flow cytometer. Appropriate isotype controls and Fc receptor blocker were used for all the samples. Representative histogram and the bar diagrams indicating the MFI of PBS and rhMFG-E8-treated samples are shown. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments. *P<0.05 vs. PBS treatment. (E) Differentiated HL-60 cells (1.5×106/ml) were placed into 1.5 ml microfuge tubes with Opti-MEM and then stimulated with either rhMFG-E8 (500 ng/ml) or PBS for different periods of time. Following incubation, the cell lysates were harvested and then subjected to western blot analysis using rabbit anti-GRK2 monoclonal antibody. Results were normalized to β-actin as an internal control and are expressed as the fold induction in comparison to the 0 min time point. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments. *P<0.05 vs. 0 min.

Mentions: CXCR2, a surface receptor for the chemokine, IL-8, plays a crucial role in IL-8-dependent neutrophil migration (15,16,37). The dHL-60 cells stimulated with rhMFG-E8 showed a significant downregulation in CXCR2 expression at their cell surface (Fig. 2A and B), which was further linked to the decrease in neutrophil migration following stimulation with rhMFG-E8. Since intracellular GRK2 serves as a negative regulator of surface CXCR2 expression in neutrophils, we wished to assess intracellular GRK2 expression in the dHL-60 cells following stimulation with rhMFG-E8. Of note, we observed a significant upregulation in intracellular GRK2 expression in the dHL-60 cells stimulated with rhMFG-E8, as revealed by flow cytometric analysis (Fig. 2C and D). Consistently, western blot analysis of GRK2 protein expression also revealed the reproducible findings of its upregulation upon rhMFG-E8 stimulation in a time-dependent manner (Fig. 2E), suggesting that the inhibition of CXCR2 expression in dHL-60 cells may be mediated through the upregulation of GRK2 expression.


MFG-E8 inhibits neutrophil migration through αvβ₃-integrin-dependent MAP kinase activation.

Aziz M, Yang WL, Corbo LM, Chaung WW, Matsuo S, Wang P - Int. J. Mol. Med. (2015)

Expression of CXCR2 and G protein-coupled receptor kinase 2 (GRK2) in recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8)-treated neutrophils. (A) Differentiated HL-60 (dHL-60) cells (1.5×106 cells) treated with rhMFG-E8 (500 ng/ml) for 2 h were surface-stained with PE-CXCR2 and then subjected to flow cytometric analysis. Data were analyzed by Flowjo software with 15,000 events per sample. Isotype controls and Fc receptor blocker were used for all the samples. The representative histograms for PBS and rhMFG-E8-treated dHL-60 cells obtained from 3 independent experiments are shown. (B) Bar diagram representing the mean fluorescence intensities (MFI) of the PBS- and rhMFG-E8-treated samples are shown. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments. *P<0.05 vs. PBS treatment. (C and D) To examine the expression of intracellular GRK2 levels, dHL-60 cells (5×106 cells) treated with rhMFG-E8 (500 ng/ml) for 2 h were first surface-stained with PE-CXCR2, and then to examine intracellular GRK2 expression, cells were fixed and permeabilized with Intraprep, followed by staining with FITC-GRK2 antibodies. After washing, the stained cells were subjected to flow cytometry using a FACSVerse flow cytometer. Appropriate isotype controls and Fc receptor blocker were used for all the samples. Representative histogram and the bar diagrams indicating the MFI of PBS and rhMFG-E8-treated samples are shown. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments. *P<0.05 vs. PBS treatment. (E) Differentiated HL-60 cells (1.5×106/ml) were placed into 1.5 ml microfuge tubes with Opti-MEM and then stimulated with either rhMFG-E8 (500 ng/ml) or PBS for different periods of time. Following incubation, the cell lysates were harvested and then subjected to western blot analysis using rabbit anti-GRK2 monoclonal antibody. Results were normalized to β-actin as an internal control and are expressed as the fold induction in comparison to the 0 min time point. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments. *P<0.05 vs. 0 min.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4494603&req=5

f2-ijmm-36-01-0018: Expression of CXCR2 and G protein-coupled receptor kinase 2 (GRK2) in recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8)-treated neutrophils. (A) Differentiated HL-60 (dHL-60) cells (1.5×106 cells) treated with rhMFG-E8 (500 ng/ml) for 2 h were surface-stained with PE-CXCR2 and then subjected to flow cytometric analysis. Data were analyzed by Flowjo software with 15,000 events per sample. Isotype controls and Fc receptor blocker were used for all the samples. The representative histograms for PBS and rhMFG-E8-treated dHL-60 cells obtained from 3 independent experiments are shown. (B) Bar diagram representing the mean fluorescence intensities (MFI) of the PBS- and rhMFG-E8-treated samples are shown. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments. *P<0.05 vs. PBS treatment. (C and D) To examine the expression of intracellular GRK2 levels, dHL-60 cells (5×106 cells) treated with rhMFG-E8 (500 ng/ml) for 2 h were first surface-stained with PE-CXCR2, and then to examine intracellular GRK2 expression, cells were fixed and permeabilized with Intraprep, followed by staining with FITC-GRK2 antibodies. After washing, the stained cells were subjected to flow cytometry using a FACSVerse flow cytometer. Appropriate isotype controls and Fc receptor blocker were used for all the samples. Representative histogram and the bar diagrams indicating the MFI of PBS and rhMFG-E8-treated samples are shown. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments. *P<0.05 vs. PBS treatment. (E) Differentiated HL-60 cells (1.5×106/ml) were placed into 1.5 ml microfuge tubes with Opti-MEM and then stimulated with either rhMFG-E8 (500 ng/ml) or PBS for different periods of time. Following incubation, the cell lysates were harvested and then subjected to western blot analysis using rabbit anti-GRK2 monoclonal antibody. Results were normalized to β-actin as an internal control and are expressed as the fold induction in comparison to the 0 min time point. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments. *P<0.05 vs. 0 min.
Mentions: CXCR2, a surface receptor for the chemokine, IL-8, plays a crucial role in IL-8-dependent neutrophil migration (15,16,37). The dHL-60 cells stimulated with rhMFG-E8 showed a significant downregulation in CXCR2 expression at their cell surface (Fig. 2A and B), which was further linked to the decrease in neutrophil migration following stimulation with rhMFG-E8. Since intracellular GRK2 serves as a negative regulator of surface CXCR2 expression in neutrophils, we wished to assess intracellular GRK2 expression in the dHL-60 cells following stimulation with rhMFG-E8. Of note, we observed a significant upregulation in intracellular GRK2 expression in the dHL-60 cells stimulated with rhMFG-E8, as revealed by flow cytometric analysis (Fig. 2C and D). Consistently, western blot analysis of GRK2 protein expression also revealed the reproducible findings of its upregulation upon rhMFG-E8 stimulation in a time-dependent manner (Fig. 2E), suggesting that the inhibition of CXCR2 expression in dHL-60 cells may be mediated through the upregulation of GRK2 expression.

Bottom Line: Treatment with rhMFG‑E8 resulted in a significant inhibition of dHL‑60 cell migration in a dose‑dependent manner.There was a 46% decrease in CXCR2 expression in the rhMFG‑E8‑treated dHL‑60 cells, which was associated with a 32% increase in GRK2 expression.The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL‑60 cell migration which was significantly inhibited treatment with rhMFG‑E8.

View Article: PubMed Central - PubMed

Affiliation: Center for Translational Research, The Feinstein Institute for Medical Research and Department of Surgery, Hofstra North Shore‑LIJ School of Medicine, Manhasset, NY, USA.

ABSTRACT
We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFG‑E8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFG‑E8 attenuates neutrophil migration. Recombinant human MFG‑E8 (rhMFG‑E8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL‑60, was treated with rhMFG‑E8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin‑8 (IL‑8) as the chemoattractant. Surface CXCR2 and intracellular G protein‑coupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogen‑activated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFG‑E8 resulted in a significant inhibition of dHL‑60 cell migration in a dose‑dependent manner. There was a 46% decrease in CXCR2 expression in the rhMFG‑E8‑treated dHL‑60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL‑60 cells, treatment with rhMFG‑E8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 10‑30 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL‑60 cell migration which was significantly inhibited treatment with rhMFG‑E8. Furthermore, blocking the MFG‑E8 receptors, αvβ3/αvβ5‑integrins, by anti‑αv‑integrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFG‑E8‑induced inhibition of dHL‑60 cell migration. Finally, treatment of the dHL‑60 cells with SB203580 and PD98059 neutralized the rhMFG‑E8‑induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFG‑E8 through which it inhibits neutrophil migration through αvβ3-integrin-dependent MAP kinase activation.

No MeSH data available.


Related in: MedlinePlus