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Connexin 43 expression on peripheral blood eosinophils: role of gap junctions in transendothelial migration.

Vliagoftis H, Ebeling C, Ilarraza R, Mahmudi-Azer S, Abel M, Adamko D, Befus AD, Moqbel R - Biomed Res Int (2014)

Bottom Line: Gap junctions mediate direct communication between adjacent cells and may represent a new way of communication between immune cells distinct from communication through cytokines and chemokines.Cx43 is localized not only in the cytoplasm but also on the plasma membrane.The gap junction inhibitors 18-a-glycyrrhetinic acid and octanol did not have a significant effect on dye transfer but reduced dye exit from eosinophils.

View Article: PubMed Central - PubMed

Affiliation: Pulmonary Research Group, Department of Medicine, University of Alberta, 550 HMRC, Edmonton, AB, Canada T6G 2S2.

ABSTRACT
Eosinophils circulate in the blood and are recruited in tissues during allergic inflammation. Gap junctions mediate direct communication between adjacent cells and may represent a new way of communication between immune cells distinct from communication through cytokines and chemokines. We characterized the expression of connexin (Cx)43 by eosinophils isolated from atopic individuals using RT-PCR, Western blotting, and confocal microscopy and studied the biological functions of gap junctions on eosinophils. The formation of functional gap junctions was evaluated measuring dye transfer using flow cytometry. The role of gap junctions on eosinophil transendothelial migration was studied using the inhibitor 18-a-glycyrrhetinic acid. Peripheral blood eosinophils express Cx43 mRNA and protein. Cx43 is localized not only in the cytoplasm but also on the plasma membrane. The membrane impermeable dye BCECF transferred from eosinophils to epithelial or endothelial cells following coculture in a dose and time dependent fashion. The gap junction inhibitors 18-a-glycyrrhetinic acid and octanol did not have a significant effect on dye transfer but reduced dye exit from eosinophils. The gap junction inhibitor 18-a-glycyrrhetinic acid inhibited eosinophil transendothelial migration in a dose dependent manner. Thus, eosinophils from atopic individuals express Cx43 constitutively and Cx43 may play an important role in eosinophil transendothelial migration and function in sites of inflammation.

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Effect of Cx inhibitors on dye transfer and transendothelial migration. (a) Unlabeled HMVEC-L cells were cocultured with labeled eosinophils (Eosin) or HMVEC-L (HMVEC-L). The graph shows % decrease in mean fluorescent intensity of unlabeled HMVEC-L in the presence of various concentrations of 18-a-glycyrrhetinic acid compared to mean fluorescent intensity in the absence of 18-a-glycyrrhetinic acid (n = 5). (b) Residual dye in labeled eosinophils or HMVEC-L following coculture with unlabeled HMVEC-L in the presence or absence of 18-a-glycyrrhetinic acid. The graph shows “% change mean fluorescent intensity (amount of dye)” in labeled eosinophils or HMVEC-L in the presence of various concentrations of 18-a-glycyrrhetinic acid compared to dye content in the absence of 18-a-glycyrrhetinic acid (n = 4). (c) Effect of various concentrations of 18-a-glycyrrhetinic acid on eosinophil transmigration through endothelial monolayers grown on transwells without fibronectin. (d) Effect of 18-a-glycyrrhetinic acid in transmigration of eosinophils or neutrophils through endothelial monolayers grown on transwells coated with FN.
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fig4: Effect of Cx inhibitors on dye transfer and transendothelial migration. (a) Unlabeled HMVEC-L cells were cocultured with labeled eosinophils (Eosin) or HMVEC-L (HMVEC-L). The graph shows % decrease in mean fluorescent intensity of unlabeled HMVEC-L in the presence of various concentrations of 18-a-glycyrrhetinic acid compared to mean fluorescent intensity in the absence of 18-a-glycyrrhetinic acid (n = 5). (b) Residual dye in labeled eosinophils or HMVEC-L following coculture with unlabeled HMVEC-L in the presence or absence of 18-a-glycyrrhetinic acid. The graph shows “% change mean fluorescent intensity (amount of dye)” in labeled eosinophils or HMVEC-L in the presence of various concentrations of 18-a-glycyrrhetinic acid compared to dye content in the absence of 18-a-glycyrrhetinic acid (n = 4). (c) Effect of various concentrations of 18-a-glycyrrhetinic acid on eosinophil transmigration through endothelial monolayers grown on transwells without fibronectin. (d) Effect of 18-a-glycyrrhetinic acid in transmigration of eosinophils or neutrophils through endothelial monolayers grown on transwells coated with FN.

Mentions: In particular for Figure 4(a), unlabeled HMVEC-L cells were cocultured with labeled eosinophils for 1 h in the presence of 18-a-glycyrrhetinic acid or just diluent. The MFI of the HMVEC-L cells at the end of the coculture, as a consequence of dye transferred from labeled eosinophils, was then calculated with flow cytometry. We then expressed the MFI of cells cocultured in the presence of 18-a-glycyrrhetinic acid as a percent of the MFI of cells cocultured in the presence of diluent and subtracted this number from 100, since dye transfer would be expected to be lower in the presence of 18-a-glycyrrhetinic acid. This number was labeled as “decrease in mean fluorescence intensity” and is shown in Figure 4(a).


Connexin 43 expression on peripheral blood eosinophils: role of gap junctions in transendothelial migration.

Vliagoftis H, Ebeling C, Ilarraza R, Mahmudi-Azer S, Abel M, Adamko D, Befus AD, Moqbel R - Biomed Res Int (2014)

Effect of Cx inhibitors on dye transfer and transendothelial migration. (a) Unlabeled HMVEC-L cells were cocultured with labeled eosinophils (Eosin) or HMVEC-L (HMVEC-L). The graph shows % decrease in mean fluorescent intensity of unlabeled HMVEC-L in the presence of various concentrations of 18-a-glycyrrhetinic acid compared to mean fluorescent intensity in the absence of 18-a-glycyrrhetinic acid (n = 5). (b) Residual dye in labeled eosinophils or HMVEC-L following coculture with unlabeled HMVEC-L in the presence or absence of 18-a-glycyrrhetinic acid. The graph shows “% change mean fluorescent intensity (amount of dye)” in labeled eosinophils or HMVEC-L in the presence of various concentrations of 18-a-glycyrrhetinic acid compared to dye content in the absence of 18-a-glycyrrhetinic acid (n = 4). (c) Effect of various concentrations of 18-a-glycyrrhetinic acid on eosinophil transmigration through endothelial monolayers grown on transwells without fibronectin. (d) Effect of 18-a-glycyrrhetinic acid in transmigration of eosinophils or neutrophils through endothelial monolayers grown on transwells coated with FN.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109672&req=5

fig4: Effect of Cx inhibitors on dye transfer and transendothelial migration. (a) Unlabeled HMVEC-L cells were cocultured with labeled eosinophils (Eosin) or HMVEC-L (HMVEC-L). The graph shows % decrease in mean fluorescent intensity of unlabeled HMVEC-L in the presence of various concentrations of 18-a-glycyrrhetinic acid compared to mean fluorescent intensity in the absence of 18-a-glycyrrhetinic acid (n = 5). (b) Residual dye in labeled eosinophils or HMVEC-L following coculture with unlabeled HMVEC-L in the presence or absence of 18-a-glycyrrhetinic acid. The graph shows “% change mean fluorescent intensity (amount of dye)” in labeled eosinophils or HMVEC-L in the presence of various concentrations of 18-a-glycyrrhetinic acid compared to dye content in the absence of 18-a-glycyrrhetinic acid (n = 4). (c) Effect of various concentrations of 18-a-glycyrrhetinic acid on eosinophil transmigration through endothelial monolayers grown on transwells without fibronectin. (d) Effect of 18-a-glycyrrhetinic acid in transmigration of eosinophils or neutrophils through endothelial monolayers grown on transwells coated with FN.
Mentions: In particular for Figure 4(a), unlabeled HMVEC-L cells were cocultured with labeled eosinophils for 1 h in the presence of 18-a-glycyrrhetinic acid or just diluent. The MFI of the HMVEC-L cells at the end of the coculture, as a consequence of dye transferred from labeled eosinophils, was then calculated with flow cytometry. We then expressed the MFI of cells cocultured in the presence of 18-a-glycyrrhetinic acid as a percent of the MFI of cells cocultured in the presence of diluent and subtracted this number from 100, since dye transfer would be expected to be lower in the presence of 18-a-glycyrrhetinic acid. This number was labeled as “decrease in mean fluorescence intensity” and is shown in Figure 4(a).

Bottom Line: Gap junctions mediate direct communication between adjacent cells and may represent a new way of communication between immune cells distinct from communication through cytokines and chemokines.Cx43 is localized not only in the cytoplasm but also on the plasma membrane.The gap junction inhibitors 18-a-glycyrrhetinic acid and octanol did not have a significant effect on dye transfer but reduced dye exit from eosinophils.

View Article: PubMed Central - PubMed

Affiliation: Pulmonary Research Group, Department of Medicine, University of Alberta, 550 HMRC, Edmonton, AB, Canada T6G 2S2.

ABSTRACT
Eosinophils circulate in the blood and are recruited in tissues during allergic inflammation. Gap junctions mediate direct communication between adjacent cells and may represent a new way of communication between immune cells distinct from communication through cytokines and chemokines. We characterized the expression of connexin (Cx)43 by eosinophils isolated from atopic individuals using RT-PCR, Western blotting, and confocal microscopy and studied the biological functions of gap junctions on eosinophils. The formation of functional gap junctions was evaluated measuring dye transfer using flow cytometry. The role of gap junctions on eosinophil transendothelial migration was studied using the inhibitor 18-a-glycyrrhetinic acid. Peripheral blood eosinophils express Cx43 mRNA and protein. Cx43 is localized not only in the cytoplasm but also on the plasma membrane. The membrane impermeable dye BCECF transferred from eosinophils to epithelial or endothelial cells following coculture in a dose and time dependent fashion. The gap junction inhibitors 18-a-glycyrrhetinic acid and octanol did not have a significant effect on dye transfer but reduced dye exit from eosinophils. The gap junction inhibitor 18-a-glycyrrhetinic acid inhibited eosinophil transendothelial migration in a dose dependent manner. Thus, eosinophils from atopic individuals express Cx43 constitutively and Cx43 may play an important role in eosinophil transendothelial migration and function in sites of inflammation.

Show MeSH
Related in: MedlinePlus