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A critical role for pannexin-1 in activation of innate immune cells of the choroid plexus.

Maslieieva V, Thompson RJ - Channels (Austin) (2014)

Bottom Line: Here we have developed a novel technique for studying epiplexus cells in acutely isolated, live and intact choroid plexus.We show that epiplexus cells are potently activated by exogenous ATP, increasing their motility within the tissue.Furthermore, ATP acts at least in part through the P2X4 ionotropic purinergic receptor.

View Article: PubMed Central - PubMed

Affiliation: Hotchkiss Brain Institute; Department of Cell Biology and Anatomy; University of Calgary; Calgary, AB Canada.

ABSTRACT
Epiplexus cells are a population of innate immune cells in the choroid plexus of the brain ventricles. They are thought to contribute to the immune component of the blood-cerebrospinal-fluid-barrier (BCSFB). Here we have developed a novel technique for studying epiplexus cells in acutely isolated, live and intact choroid plexus. We show that epiplexus cells are potently activated by exogenous ATP, increasing their motility within the tissue. This ATP-induced chemokinesis required activation of pannexin-1 channels, which are expressed by the epithelial cells of the choroid plexus and not the epiplexus cells themselves. Furthermore, ATP acts at least in part through the P2X4 ionotropic purinergic receptor. Thus, the resident immune cells of the choroid plexus appear to be in communication with the epithelial cells through pannexin-1 channels.

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Figure 3. Panx1 channels are involved in epiplexus cells activation by exogenous 100 µM ATP. Panx1 channels are robustly expressed on choroidal epithelium, but were rarely detected in epiplexus cells. Western blotting analysis confirmed presence of Panx1 in the CP. The Panx1 blockers, 500 µM probenecid and 100 µM 10panx significantly decreased ATP-triggered chemokinesis. (A and B) Immunofluorescent staining for Panx1 in the CP and on an individual IB4-positive epiplexus cell. (C) Detection of Panx1 protein by western blotting. (D) Normalized average distance and statistical analysis, each symbol represents a single cell and all cells from the experiments are shown; (E) normalized summative distance; (F) slope of normalized summative distance where each symbol represents a single isolated CP. Scale bars are 50 μm (A) and 5 μm (B).
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Figure 3: Figure 3. Panx1 channels are involved in epiplexus cells activation by exogenous 100 µM ATP. Panx1 channels are robustly expressed on choroidal epithelium, but were rarely detected in epiplexus cells. Western blotting analysis confirmed presence of Panx1 in the CP. The Panx1 blockers, 500 µM probenecid and 100 µM 10panx significantly decreased ATP-triggered chemokinesis. (A and B) Immunofluorescent staining for Panx1 in the CP and on an individual IB4-positive epiplexus cell. (C) Detection of Panx1 protein by western blotting. (D) Normalized average distance and statistical analysis, each symbol represents a single cell and all cells from the experiments are shown; (E) normalized summative distance; (F) slope of normalized summative distance where each symbol represents a single isolated CP. Scale bars are 50 μm (A) and 5 μm (B).

Mentions: To test the responsiveness of epiplexus cells to purinergic signaling, we bath applied ATP. We reasoned that unlike focal applications, bath exposure to ATP would more closely mimic an infection or injury. The movement of epiplexus cells was first determined under baseline conditions (without exogenously applied ATP) by manually tracking the movement of somas at 5 min intervals (Fig. 2A and B; Video 1). Over a 95 min imaging period epiplexus cells were largely quiescent (Figs. 2and3; Video 1) with a mean normalized (i.e., baseline subtracted) movement of 0.05 ± 0.15 μm/5 min (n = 124 cells from 5 CPs). Note that in Figure 2C and F the summative distance (i.e., running sum of distance traveled at 5 min intervals) could appear negative if the cells were active during the early baseline but became subsequently quiescent (see Materials and Methods).


A critical role for pannexin-1 in activation of innate immune cells of the choroid plexus.

Maslieieva V, Thompson RJ - Channels (Austin) (2014)

Figure 3. Panx1 channels are involved in epiplexus cells activation by exogenous 100 µM ATP. Panx1 channels are robustly expressed on choroidal epithelium, but were rarely detected in epiplexus cells. Western blotting analysis confirmed presence of Panx1 in the CP. The Panx1 blockers, 500 µM probenecid and 100 µM 10panx significantly decreased ATP-triggered chemokinesis. (A and B) Immunofluorescent staining for Panx1 in the CP and on an individual IB4-positive epiplexus cell. (C) Detection of Panx1 protein by western blotting. (D) Normalized average distance and statistical analysis, each symbol represents a single cell and all cells from the experiments are shown; (E) normalized summative distance; (F) slope of normalized summative distance where each symbol represents a single isolated CP. Scale bars are 50 μm (A) and 5 μm (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048302&req=5

Figure 3: Figure 3. Panx1 channels are involved in epiplexus cells activation by exogenous 100 µM ATP. Panx1 channels are robustly expressed on choroidal epithelium, but were rarely detected in epiplexus cells. Western blotting analysis confirmed presence of Panx1 in the CP. The Panx1 blockers, 500 µM probenecid and 100 µM 10panx significantly decreased ATP-triggered chemokinesis. (A and B) Immunofluorescent staining for Panx1 in the CP and on an individual IB4-positive epiplexus cell. (C) Detection of Panx1 protein by western blotting. (D) Normalized average distance and statistical analysis, each symbol represents a single cell and all cells from the experiments are shown; (E) normalized summative distance; (F) slope of normalized summative distance where each symbol represents a single isolated CP. Scale bars are 50 μm (A) and 5 μm (B).
Mentions: To test the responsiveness of epiplexus cells to purinergic signaling, we bath applied ATP. We reasoned that unlike focal applications, bath exposure to ATP would more closely mimic an infection or injury. The movement of epiplexus cells was first determined under baseline conditions (without exogenously applied ATP) by manually tracking the movement of somas at 5 min intervals (Fig. 2A and B; Video 1). Over a 95 min imaging period epiplexus cells were largely quiescent (Figs. 2and3; Video 1) with a mean normalized (i.e., baseline subtracted) movement of 0.05 ± 0.15 μm/5 min (n = 124 cells from 5 CPs). Note that in Figure 2C and F the summative distance (i.e., running sum of distance traveled at 5 min intervals) could appear negative if the cells were active during the early baseline but became subsequently quiescent (see Materials and Methods).

Bottom Line: Here we have developed a novel technique for studying epiplexus cells in acutely isolated, live and intact choroid plexus.We show that epiplexus cells are potently activated by exogenous ATP, increasing their motility within the tissue.Furthermore, ATP acts at least in part through the P2X4 ionotropic purinergic receptor.

View Article: PubMed Central - PubMed

Affiliation: Hotchkiss Brain Institute; Department of Cell Biology and Anatomy; University of Calgary; Calgary, AB Canada.

ABSTRACT
Epiplexus cells are a population of innate immune cells in the choroid plexus of the brain ventricles. They are thought to contribute to the immune component of the blood-cerebrospinal-fluid-barrier (BCSFB). Here we have developed a novel technique for studying epiplexus cells in acutely isolated, live and intact choroid plexus. We show that epiplexus cells are potently activated by exogenous ATP, increasing their motility within the tissue. This ATP-induced chemokinesis required activation of pannexin-1 channels, which are expressed by the epithelial cells of the choroid plexus and not the epiplexus cells themselves. Furthermore, ATP acts at least in part through the P2X4 ionotropic purinergic receptor. Thus, the resident immune cells of the choroid plexus appear to be in communication with the epithelial cells through pannexin-1 channels.

Show MeSH