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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

Recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8) attenuates HL-60 cell migration. (A) Differentiated HL-60 cells (3×105) were pre-stimulated with different doses (1,000, 500, 250 and 125 ng/ml) of rhMFG-E8 or PBS for 2 h, and then plated in 500 µl volume at the Boyden chamber inserts. The outer compartment of the inserts contained 500 µl of RPMI medium with 50 ng/ml of recombinant human interleukin-8 (IL-8) as a chemoattractant. After 1.5 h of incubation, the upper surface of the filter was swabbed with cotton-tipped applicators to remove non-migratory cells. Migrated cells were fixed with 4% paraformaldehyde (PFA) and stained with propidium iodide (PI) (1 µg/ml). A total of 6 random microscopic fields per well were counted. Scale bar, 100 µm. (B) The average number of migrated dHL-60 cells is plotted in a bar diagram where the results are expressed as the means ± SE obtained from 6 fields/group of 3 independent experiments. *P<0.05 vs. PBS treatment.
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f1-ijmm-36-01-0018: Recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8) attenuates HL-60 cell migration. (A) Differentiated HL-60 cells (3×105) were pre-stimulated with different doses (1,000, 500, 250 and 125 ng/ml) of rhMFG-E8 or PBS for 2 h, and then plated in 500 µl volume at the Boyden chamber inserts. The outer compartment of the inserts contained 500 µl of RPMI medium with 50 ng/ml of recombinant human interleukin-8 (IL-8) as a chemoattractant. After 1.5 h of incubation, the upper surface of the filter was swabbed with cotton-tipped applicators to remove non-migratory cells. Migrated cells were fixed with 4% paraformaldehyde (PFA) and stained with propidium iodide (PI) (1 µg/ml). A total of 6 random microscopic fields per well were counted. Scale bar, 100 µm. (B) The average number of migrated dHL-60 cells is plotted in a bar diagram where the results are expressed as the means ± SE obtained from 6 fields/group of 3 independent experiments. *P<0.05 vs. PBS treatment.

Mentions: To examine the effects of rhMFG-E8 on neutrophil migration, the dHL-60 cells were pre-treated with various concentrations of rhMFG-E8 for 2 h. The cells were then allowed to proceed for migration towards rhIL-8 as a chemoattractant using a Boyden chamber. As shown in Fig. 1A and B, pre-treatment of the dHL-60 cells with rhMFG-E8 led to a significantly decrease in their migration towards rhIL-8 in a dose-dependent manner. Since the most notable decrease in their migration occurred following stimulation with rhMFG-E8 at the doses of 500 and 1,000 ng/ml, among these 2 doses we decided to utilize the lesser dose of 500 ng/ml of rhMFG-E8 protein for the subsequent in vitro experiments.


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)

Recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8) attenuates HL-60 cell migration. (A) Differentiated HL-60 cells (3×105) were pre-stimulated with different doses (1,000, 500, 250 and 125 ng/ml) of rhMFG-E8 or PBS for 2 h, and then plated in 500 µl volume at the Boyden chamber inserts. The outer compartment of the inserts contained 500 µl of RPMI medium with 50 ng/ml of recombinant human interleukin-8 (IL-8) as a chemoattractant. After 1.5 h of incubation, the upper surface of the filter was swabbed with cotton-tipped applicators to remove non-migratory cells. Migrated cells were fixed with 4% paraformaldehyde (PFA) and stained with propidium iodide (PI) (1 µg/ml). A total of 6 random microscopic fields per well were counted. Scale bar, 100 µm. (B) The average number of migrated dHL-60 cells is plotted in a bar diagram where the results are expressed as the means ± SE obtained from 6 fields/group of 3 independent experiments. *P<0.05 vs. PBS treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-ijmm-36-01-0018: Recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8) attenuates HL-60 cell migration. (A) Differentiated HL-60 cells (3×105) were pre-stimulated with different doses (1,000, 500, 250 and 125 ng/ml) of rhMFG-E8 or PBS for 2 h, and then plated in 500 µl volume at the Boyden chamber inserts. The outer compartment of the inserts contained 500 µl of RPMI medium with 50 ng/ml of recombinant human interleukin-8 (IL-8) as a chemoattractant. After 1.5 h of incubation, the upper surface of the filter was swabbed with cotton-tipped applicators to remove non-migratory cells. Migrated cells were fixed with 4% paraformaldehyde (PFA) and stained with propidium iodide (PI) (1 µg/ml). A total of 6 random microscopic fields per well were counted. Scale bar, 100 µm. (B) The average number of migrated dHL-60 cells is plotted in a bar diagram where the results are expressed as the means ± SE obtained from 6 fields/group of 3 independent experiments. *P<0.05 vs. PBS treatment.
Mentions: To examine the effects of rhMFG-E8 on neutrophil migration, the dHL-60 cells were pre-treated with various concentrations of rhMFG-E8 for 2 h. The cells were then allowed to proceed for migration towards rhIL-8 as a chemoattractant using a Boyden chamber. As shown in Fig. 1A and B, pre-treatment of the dHL-60 cells with rhMFG-E8 led to a significantly decrease in their migration towards rhIL-8 in a dose-dependent manner. Since the most notable decrease in their migration occurred following stimulation with rhMFG-E8 at the doses of 500 and 1,000 ng/ml, among these 2 doses we decided to utilize the lesser dose of 500 ng/ml of rhMFG-E8 protein for the subsequent in vitro experiments.

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