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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 1. Identification of epiplexus cells as immune cells. (A) Scheme of the experimental approach. (B) CPs are located in all four brain ventricles. (C) An acutely isolated and intact CP from the lateral ventricle. Scale bar is approximately 1 mm. (D) A living epiplexus cell resting on the choroidal epithelium, visualized with transmitted light (left) or by labeling with Alexa Fluor 488 isolectin B4 conjugate from Griffonia simplicifolia (center); EC – epiplexus cell, EP – choroidal epithelium, BV – blood vessel, LV – lumen of the lateral ventricle. Scale bar is 20 μm. (E) Immunofluorescent staining for immune cell markers, Iba1 and IB4 in the CP. Note the significant co-localization (yellow cells in the merge image). Scale bar is 20 μm.
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Figure 1: Figure 1. Identification of epiplexus cells as immune cells. (A) Scheme of the experimental approach. (B) CPs are located in all four brain ventricles. (C) An acutely isolated and intact CP from the lateral ventricle. Scale bar is approximately 1 mm. (D) A living epiplexus cell resting on the choroidal epithelium, visualized with transmitted light (left) or by labeling with Alexa Fluor 488 isolectin B4 conjugate from Griffonia simplicifolia (center); EC – epiplexus cell, EP – choroidal epithelium, BV – blood vessel, LV – lumen of the lateral ventricle. Scale bar is 20 μm. (E) Immunofluorescent staining for immune cell markers, Iba1 and IB4 in the CP. Note the significant co-localization (yellow cells in the merge image). Scale bar is 20 μm.

Mentions: To understand the physiology of epiplexus cells, we developed an acutely isolated, intact CP preparation (Fig. 1A-C). The CP was rapidly removed from rat brain and submerged in artificial CSF (aCSF). The CP was then labeled with Alexa Fluor 488 isolectin B4 conjugate (IB4), a fluorescent marker for microglia and macrophages.21,34-36 IB4 labeled a population of cells that were evenly distributed across the apical surface (Fig. 1D and E). The IB4-positive cells had large visible processes. To identify these IB4-positive cells as putative epiplexus cells, we fixed and co-labeled the CP with antibodies against Iba1 (ionized calcium binding adaptor molecule 1), a specific marker for cells of the monocytic lineage.37,38 All IB4 positive cells in the CP co-labeled with Iba1 (Fig. 1E), confirming their identity as epiplexus cells. We then took advantage of the IB4-fluorescence to investigate epiplexus cell responsiveness to exogenous purines by imaging their motility in response to purinergic receptor agonists and antagonists.


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

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

Figure 1. Identification of epiplexus cells as immune cells. (A) Scheme of the experimental approach. (B) CPs are located in all four brain ventricles. (C) An acutely isolated and intact CP from the lateral ventricle. Scale bar is approximately 1 mm. (D) A living epiplexus cell resting on the choroidal epithelium, visualized with transmitted light (left) or by labeling with Alexa Fluor 488 isolectin B4 conjugate from Griffonia simplicifolia (center); EC – epiplexus cell, EP – choroidal epithelium, BV – blood vessel, LV – lumen of the lateral ventricle. Scale bar is 20 μm. (E) Immunofluorescent staining for immune cell markers, Iba1 and IB4 in the CP. Note the significant co-localization (yellow cells in the merge image). Scale bar is 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Figure 1. Identification of epiplexus cells as immune cells. (A) Scheme of the experimental approach. (B) CPs are located in all four brain ventricles. (C) An acutely isolated and intact CP from the lateral ventricle. Scale bar is approximately 1 mm. (D) A living epiplexus cell resting on the choroidal epithelium, visualized with transmitted light (left) or by labeling with Alexa Fluor 488 isolectin B4 conjugate from Griffonia simplicifolia (center); EC – epiplexus cell, EP – choroidal epithelium, BV – blood vessel, LV – lumen of the lateral ventricle. Scale bar is 20 μm. (E) Immunofluorescent staining for immune cell markers, Iba1 and IB4 in the CP. Note the significant co-localization (yellow cells in the merge image). Scale bar is 20 μm.
Mentions: To understand the physiology of epiplexus cells, we developed an acutely isolated, intact CP preparation (Fig. 1A-C). The CP was rapidly removed from rat brain and submerged in artificial CSF (aCSF). The CP was then labeled with Alexa Fluor 488 isolectin B4 conjugate (IB4), a fluorescent marker for microglia and macrophages.21,34-36 IB4 labeled a population of cells that were evenly distributed across the apical surface (Fig. 1D and E). The IB4-positive cells had large visible processes. To identify these IB4-positive cells as putative epiplexus cells, we fixed and co-labeled the CP with antibodies against Iba1 (ionized calcium binding adaptor molecule 1), a specific marker for cells of the monocytic lineage.37,38 All IB4 positive cells in the CP co-labeled with Iba1 (Fig. 1E), confirming their identity as epiplexus cells. We then took advantage of the IB4-fluorescence to investigate epiplexus cell responsiveness to exogenous purines by imaging their motility in response to purinergic receptor agonists and antagonists.

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
Related in: MedlinePlus