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Cholinergic epithelial cell with chemosensory traits in murine thymic medulla.

Panneck AR, Rafiq A, Schütz B, Soultanova A, Deckmann K, Chubanov V, Gudermann T, Weihe E, Krasteva-Christ G, Grau V, del Rey A, Kummer W - Cell Tissue Res. (2014)

Bottom Line: ChAT expression was confirmed by in situ hybridization.They did not express neuroendocrine (chromogranin A, PGP9.5) or thymocyte (CD3) markers but rather thymic epithelial (CK8, CK18) markers and were immunoreactive for components of the taste transduction cascade such as Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel (TRPM5), and phospholipase Cβ2.This cell might participate in intrathymic infection-sensing mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy and Cell Biology, Justus-Liebig-University Giessen, Aulweg 123, 35385, Giessen, Germany.

ABSTRACT
Specialized epithelial cells with a tuft of apical microvilli ("brush cells") sense luminal content and initiate protective reflexes in response to potentially harmful substances. They utilize the canonical taste transduction cascade to detect "bitter" substances such as bacterial quorum-sensing molecules. In the respiratory tract, most of these cells are cholinergic and are approached by cholinoceptive sensory nerve fibers. Utilizing two different reporter mouse strains for the expression of choline acetyltransferase (ChAT), we observed intense labeling of a subset of thymic medullary cells. ChAT expression was confirmed by in situ hybridization. These cells showed expression of villin, a brush cell marker protein, and ultrastructurally exhibited lateral microvilli. They did not express neuroendocrine (chromogranin A, PGP9.5) or thymocyte (CD3) markers but rather thymic epithelial (CK8, CK18) markers and were immunoreactive for components of the taste transduction cascade such as Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel (TRPM5), and phospholipase Cβ2. Reverse transcription and polymerase chain reaction confirmed the expression of Gα-gustducin, TRPM5, and phospholipase Cβ2. Thymic "cholinergic chemosensory cells" were often in direct contact with medullary epithelial cells expressing the nicotinic acetylcholine receptor subunit α3. These cells have recently been identified as terminally differentiated epithelial cells (Hassall's corpuscle-like structures in mice). Contacts with nerve fibers (identified by PGP9.5 and CGRP antibodies), however, were not observed. Our data identify, in the thymus, a previously unrecognized presumptive chemosensitive cell that probably utilizes acetylcholine for paracrine signaling. This cell might participate in intrathymic infection-sensing mechanisms.

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Cholinergic medullary epithelial cells express components of the canonical taste transduction cascade. a–c Most ChAT-eGFP-positive epithelial medullary cells are immunoreactive (arrows) for the downstream signaling components of the canonical bitter, sweet, and umami taste transduction cascade, namely Gα-gustducin (α-gustducin), phospholipase Cβ2 (PLCβ2), and transient receptor potential melastatin-like subtype 5 channel (TRPM5). Insets in a and b Preabsorption controls (Preab) in which the primary antibody had been saturated with cognate peptide prior to application to the tissue section. Arrowheads in a and in inset in c indicate single cells displaying only one marker. Bars 20 μm. d, e Ultrastructural immunohistochemistry, pre-embedding technique. PLCβ2-immunoreactive (d) and TRPM5-immunoreactive (e) epithelial cells extending processes (arrows). a, b Male aged 25 weeks. Inset in a, b Male aged 16 weeks. c Male aged 31 weeks. Inset Female aged 25 weeks. d, e 31 weeks. Bars 5 μm. f, g RT-PCR, ethidium-bromide-stained agarose gels. Gα-gustducin-specific (α-gust), PLCβ2-specific (weakly), and TRPM5-specific products were amplified from whole thymus (f) and thymic cells isolated by aid of TRPM5-antibody and magnetic beads (g). Arrowhead indicates 200 bp marker. PCR was conducted with (+) and without (-; serving as negative control) reverse transcription of RNA. β-Actin served as a housekeeping gene
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Fig4: Cholinergic medullary epithelial cells express components of the canonical taste transduction cascade. a–c Most ChAT-eGFP-positive epithelial medullary cells are immunoreactive (arrows) for the downstream signaling components of the canonical bitter, sweet, and umami taste transduction cascade, namely Gα-gustducin (α-gustducin), phospholipase Cβ2 (PLCβ2), and transient receptor potential melastatin-like subtype 5 channel (TRPM5). Insets in a and b Preabsorption controls (Preab) in which the primary antibody had been saturated with cognate peptide prior to application to the tissue section. Arrowheads in a and in inset in c indicate single cells displaying only one marker. Bars 20 μm. d, e Ultrastructural immunohistochemistry, pre-embedding technique. PLCβ2-immunoreactive (d) and TRPM5-immunoreactive (e) epithelial cells extending processes (arrows). a, b Male aged 25 weeks. Inset in a, b Male aged 16 weeks. c Male aged 31 weeks. Inset Female aged 25 weeks. d, e 31 weeks. Bars 5 μm. f, g RT-PCR, ethidium-bromide-stained agarose gels. Gα-gustducin-specific (α-gust), PLCβ2-specific (weakly), and TRPM5-specific products were amplified from whole thymus (f) and thymic cells isolated by aid of TRPM5-antibody and magnetic beads (g). Arrowhead indicates 200 bp marker. PCR was conducted with (+) and without (-; serving as negative control) reverse transcription of RNA. β-Actin served as a housekeeping gene

Mentions: The majority of ChAT-eGFP-positive epithelial medullary cells were immunoreactive for the downstream signaling components of the canonical bitter, sweet, and umami taste transduction cascade, i.e., Gα-gustducin, PLCβ2, and TRPM5, although eGFP-positive cells without additional immunoreactivities were also observed (Fig. 4a–c). Few cells with immunoreactivity for PLCβ2 or TRPM5 that did not express ChAT-eGFP were present, whereas Gα-gustducin-immunoreactive cells lacking ChAT-eGFP were practically absent. Ultrastructural immunohistochemistry revealed that medullary PLCβ2- and TRPM5-immunoreactive cells were epithelial cells extending long processes (Fig. 4d, e).Fig. 4


Cholinergic epithelial cell with chemosensory traits in murine thymic medulla.

Panneck AR, Rafiq A, Schütz B, Soultanova A, Deckmann K, Chubanov V, Gudermann T, Weihe E, Krasteva-Christ G, Grau V, del Rey A, Kummer W - Cell Tissue Res. (2014)

Cholinergic medullary epithelial cells express components of the canonical taste transduction cascade. a–c Most ChAT-eGFP-positive epithelial medullary cells are immunoreactive (arrows) for the downstream signaling components of the canonical bitter, sweet, and umami taste transduction cascade, namely Gα-gustducin (α-gustducin), phospholipase Cβ2 (PLCβ2), and transient receptor potential melastatin-like subtype 5 channel (TRPM5). Insets in a and b Preabsorption controls (Preab) in which the primary antibody had been saturated with cognate peptide prior to application to the tissue section. Arrowheads in a and in inset in c indicate single cells displaying only one marker. Bars 20 μm. d, e Ultrastructural immunohistochemistry, pre-embedding technique. PLCβ2-immunoreactive (d) and TRPM5-immunoreactive (e) epithelial cells extending processes (arrows). a, b Male aged 25 weeks. Inset in a, b Male aged 16 weeks. c Male aged 31 weeks. Inset Female aged 25 weeks. d, e 31 weeks. Bars 5 μm. f, g RT-PCR, ethidium-bromide-stained agarose gels. Gα-gustducin-specific (α-gust), PLCβ2-specific (weakly), and TRPM5-specific products were amplified from whole thymus (f) and thymic cells isolated by aid of TRPM5-antibody and magnetic beads (g). Arrowhead indicates 200 bp marker. PCR was conducted with (+) and without (-; serving as negative control) reverse transcription of RNA. β-Actin served as a housekeeping gene
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Fig4: Cholinergic medullary epithelial cells express components of the canonical taste transduction cascade. a–c Most ChAT-eGFP-positive epithelial medullary cells are immunoreactive (arrows) for the downstream signaling components of the canonical bitter, sweet, and umami taste transduction cascade, namely Gα-gustducin (α-gustducin), phospholipase Cβ2 (PLCβ2), and transient receptor potential melastatin-like subtype 5 channel (TRPM5). Insets in a and b Preabsorption controls (Preab) in which the primary antibody had been saturated with cognate peptide prior to application to the tissue section. Arrowheads in a and in inset in c indicate single cells displaying only one marker. Bars 20 μm. d, e Ultrastructural immunohistochemistry, pre-embedding technique. PLCβ2-immunoreactive (d) and TRPM5-immunoreactive (e) epithelial cells extending processes (arrows). a, b Male aged 25 weeks. Inset in a, b Male aged 16 weeks. c Male aged 31 weeks. Inset Female aged 25 weeks. d, e 31 weeks. Bars 5 μm. f, g RT-PCR, ethidium-bromide-stained agarose gels. Gα-gustducin-specific (α-gust), PLCβ2-specific (weakly), and TRPM5-specific products were amplified from whole thymus (f) and thymic cells isolated by aid of TRPM5-antibody and magnetic beads (g). Arrowhead indicates 200 bp marker. PCR was conducted with (+) and without (-; serving as negative control) reverse transcription of RNA. β-Actin served as a housekeeping gene
Mentions: The majority of ChAT-eGFP-positive epithelial medullary cells were immunoreactive for the downstream signaling components of the canonical bitter, sweet, and umami taste transduction cascade, i.e., Gα-gustducin, PLCβ2, and TRPM5, although eGFP-positive cells without additional immunoreactivities were also observed (Fig. 4a–c). Few cells with immunoreactivity for PLCβ2 or TRPM5 that did not express ChAT-eGFP were present, whereas Gα-gustducin-immunoreactive cells lacking ChAT-eGFP were practically absent. Ultrastructural immunohistochemistry revealed that medullary PLCβ2- and TRPM5-immunoreactive cells were epithelial cells extending long processes (Fig. 4d, e).Fig. 4

Bottom Line: ChAT expression was confirmed by in situ hybridization.They did not express neuroendocrine (chromogranin A, PGP9.5) or thymocyte (CD3) markers but rather thymic epithelial (CK8, CK18) markers and were immunoreactive for components of the taste transduction cascade such as Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel (TRPM5), and phospholipase Cβ2.This cell might participate in intrathymic infection-sensing mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy and Cell Biology, Justus-Liebig-University Giessen, Aulweg 123, 35385, Giessen, Germany.

ABSTRACT
Specialized epithelial cells with a tuft of apical microvilli ("brush cells") sense luminal content and initiate protective reflexes in response to potentially harmful substances. They utilize the canonical taste transduction cascade to detect "bitter" substances such as bacterial quorum-sensing molecules. In the respiratory tract, most of these cells are cholinergic and are approached by cholinoceptive sensory nerve fibers. Utilizing two different reporter mouse strains for the expression of choline acetyltransferase (ChAT), we observed intense labeling of a subset of thymic medullary cells. ChAT expression was confirmed by in situ hybridization. These cells showed expression of villin, a brush cell marker protein, and ultrastructurally exhibited lateral microvilli. They did not express neuroendocrine (chromogranin A, PGP9.5) or thymocyte (CD3) markers but rather thymic epithelial (CK8, CK18) markers and were immunoreactive for components of the taste transduction cascade such as Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel (TRPM5), and phospholipase Cβ2. Reverse transcription and polymerase chain reaction confirmed the expression of Gα-gustducin, TRPM5, and phospholipase Cβ2. Thymic "cholinergic chemosensory cells" were often in direct contact with medullary epithelial cells expressing the nicotinic acetylcholine receptor subunit α3. These cells have recently been identified as terminally differentiated epithelial cells (Hassall's corpuscle-like structures in mice). Contacts with nerve fibers (identified by PGP9.5 and CGRP antibodies), however, were not observed. Our data identify, in the thymus, a previously unrecognized presumptive chemosensitive cell that probably utilizes acetylcholine for paracrine signaling. This cell might participate in intrathymic infection-sensing mechanisms.

Show MeSH
Related in: MedlinePlus