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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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CLSM for Cx43 expression on freshly isolated human peripheral blood eosinophils. Staining with isotype control antibody (top panels) and mouse anti-Cx43 antibody (lower panels) is shown. Left panels show antibody staining alone and middle panels Cx43 staining along with DAPI to visualize the nuclei. The right panels show transmitted light images from the same slides.
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fig2: CLSM for Cx43 expression on freshly isolated human peripheral blood eosinophils. Staining with isotype control antibody (top panels) and mouse anti-Cx43 antibody (lower panels) is shown. Left panels show antibody staining alone and middle panels Cx43 staining along with DAPI to visualize the nuclei. The right panels show transmitted light images from the same slides.

Mentions: To localize Cx43 protein in eosinophils we performed CLSM. All freshly isolated peripheral blood eosinophils expressed Cx43 (Figure 2) by CLSM. Staining in eosinophils was present on the plasma membrane as well as in the cytoplasm, but the cytoplasm staining was predominant. Therefore, eosinophils express Cx43 on the cytoplasmic membrane but they have a large pool of cytoplasmic Cx43.


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)

CLSM for Cx43 expression on freshly isolated human peripheral blood eosinophils. Staining with isotype control antibody (top panels) and mouse anti-Cx43 antibody (lower panels) is shown. Left panels show antibody staining alone and middle panels Cx43 staining along with DAPI to visualize the nuclei. The right panels show transmitted light images from the same slides.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: CLSM for Cx43 expression on freshly isolated human peripheral blood eosinophils. Staining with isotype control antibody (top panels) and mouse anti-Cx43 antibody (lower panels) is shown. Left panels show antibody staining alone and middle panels Cx43 staining along with DAPI to visualize the nuclei. The right panels show transmitted light images from the same slides.
Mentions: To localize Cx43 protein in eosinophils we performed CLSM. All freshly isolated peripheral blood eosinophils expressed Cx43 (Figure 2) by CLSM. Staining in eosinophils was present on the plasma membrane as well as in the cytoplasm, but the cytoplasm staining was predominant. Therefore, eosinophils express Cx43 on the cytoplasmic membrane but they have a large pool of cytoplasmic Cx43.

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