Limits...
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.

Show MeSH

Related in: MedlinePlus

Dye transfer between eosinophils and epithelial/endothelial cells. Eosinophils were cocultured with A549 airway epithelial cells or HMVEC-L for 3 h. In each case one of the cell types was labeled and the other unlabeled. Unlabeled cells (eosinophils in (a) and (c), A549 cells in (b) and HMVEC-L in (d)) were gated and analyzed for evidence of dye transfer from the labeled cells. A representative experiment (from 4 to 6 experiments) is shown for each condition. Numbers in the graph indicate % of positive cells. Conditions: (a) transfer from labeled A549 cells to unlabeled eosinophils, (b) transfer from labeled eosinophils cells to unlabeled A549 cells, (c) transfer from labeled HMVEC-L cells to unlabeled eosinophils, and (d) transfer from labeled eosinophils to unlabeled HMVEC-L cells.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4109672&req=5

fig3: Dye transfer between eosinophils and epithelial/endothelial cells. Eosinophils were cocultured with A549 airway epithelial cells or HMVEC-L for 3 h. In each case one of the cell types was labeled and the other unlabeled. Unlabeled cells (eosinophils in (a) and (c), A549 cells in (b) and HMVEC-L in (d)) were gated and analyzed for evidence of dye transfer from the labeled cells. A representative experiment (from 4 to 6 experiments) is shown for each condition. Numbers in the graph indicate % of positive cells. Conditions: (a) transfer from labeled A549 cells to unlabeled eosinophils, (b) transfer from labeled eosinophils cells to unlabeled A549 cells, (c) transfer from labeled HMVEC-L cells to unlabeled eosinophils, and (d) transfer from labeled eosinophils to unlabeled HMVEC-L cells.

Mentions: Dye transfer was observed between A549 and eosinophils in both directions (Figure 3(a) shows transfer from labeled A549 to unlabeled eosinophils and 3B transfer from labeled eosinophils to unlabeled A549 cells). Similarly dye transfer was demonstrated between eosinophils and HMVEC-L in both directions (Figure 3(c) shows transfer from labeled HMVEC-L to unlabeled eosinophils and 3D transfer from labeled eosinophils to unlabeled HMVEC-L). There was no evidence of cell death by the end of the coculture experiments. These experiments showed great variability in the numbers of unlabeled cells that became positive by the end of the experiment. For example, for dye transfer between unlabeled HMVEC-L cells and labeled eosinophils (Figure 3(d)), the percent of endothelial cells containing dye at the end of the experiment ranged from 15% up to 45% in different experiments. Similarly the MFI of endothelial cells containing dye transferred from eosinophils at the end of the experiment had a very wide range. This is the reason we only show a representative experiment out of the 5 experiments we performed. Among these conditions, dye transfer from HMVEC-L to eosinophils appeared to be most efficient (more than 85% or eosinophils became positive after coculture with calcein labeled HMVEC-L, Figure 3(c)). Dye transfer increased with increasing time of coculture and with increasing ratio of labeled to unlabeled cells (data not shown).


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)

Dye transfer between eosinophils and epithelial/endothelial cells. Eosinophils were cocultured with A549 airway epithelial cells or HMVEC-L for 3 h. In each case one of the cell types was labeled and the other unlabeled. Unlabeled cells (eosinophils in (a) and (c), A549 cells in (b) and HMVEC-L in (d)) were gated and analyzed for evidence of dye transfer from the labeled cells. A representative experiment (from 4 to 6 experiments) is shown for each condition. Numbers in the graph indicate % of positive cells. Conditions: (a) transfer from labeled A549 cells to unlabeled eosinophils, (b) transfer from labeled eosinophils cells to unlabeled A549 cells, (c) transfer from labeled HMVEC-L cells to unlabeled eosinophils, and (d) transfer from labeled eosinophils to unlabeled HMVEC-L cells.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Dye transfer between eosinophils and epithelial/endothelial cells. Eosinophils were cocultured with A549 airway epithelial cells or HMVEC-L for 3 h. In each case one of the cell types was labeled and the other unlabeled. Unlabeled cells (eosinophils in (a) and (c), A549 cells in (b) and HMVEC-L in (d)) were gated and analyzed for evidence of dye transfer from the labeled cells. A representative experiment (from 4 to 6 experiments) is shown for each condition. Numbers in the graph indicate % of positive cells. Conditions: (a) transfer from labeled A549 cells to unlabeled eosinophils, (b) transfer from labeled eosinophils cells to unlabeled A549 cells, (c) transfer from labeled HMVEC-L cells to unlabeled eosinophils, and (d) transfer from labeled eosinophils to unlabeled HMVEC-L cells.
Mentions: Dye transfer was observed between A549 and eosinophils in both directions (Figure 3(a) shows transfer from labeled A549 to unlabeled eosinophils and 3B transfer from labeled eosinophils to unlabeled A549 cells). Similarly dye transfer was demonstrated between eosinophils and HMVEC-L in both directions (Figure 3(c) shows transfer from labeled HMVEC-L to unlabeled eosinophils and 3D transfer from labeled eosinophils to unlabeled HMVEC-L). There was no evidence of cell death by the end of the coculture experiments. These experiments showed great variability in the numbers of unlabeled cells that became positive by the end of the experiment. For example, for dye transfer between unlabeled HMVEC-L cells and labeled eosinophils (Figure 3(d)), the percent of endothelial cells containing dye at the end of the experiment ranged from 15% up to 45% in different experiments. Similarly the MFI of endothelial cells containing dye transferred from eosinophils at the end of the experiment had a very wide range. This is the reason we only show a representative experiment out of the 5 experiments we performed. Among these conditions, dye transfer from HMVEC-L to eosinophils appeared to be most efficient (more than 85% or eosinophils became positive after coculture with calcein labeled HMVEC-L, Figure 3(c)). Dye transfer increased with increasing time of coculture and with increasing ratio of labeled to unlabeled cells (data not shown).

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