13 research outputs found

    Nepmucin/CD300LG is rapidly down-regulated by <i>in vitro</i> culture of LN ECs.

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    <p>LN stromal cells that contain ECs were seeded on a collagen I-coated plate, and adherent cells were collected at time 0 or after 5-hours <i>in vitro</i> culture. The expression of <i>nepmucin, CD31, ICAM-1,</i> and <i>DARC,</i> was examined by quantitative PCR. Data represent the mean ± SD (n = 3 per group). **<i>p</i><0.01, ***<i>p</i><0.005.</p

    Nepmucin/CD300LG expression is decreased in tumors and tumor-draining lymph nodes.

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    <p>(A) Human pancreatic adenocarcinoma MIA PaCa-2 cells (1.2×10<sup>7</sup> cells) were subcutaneously injected into the flank of SCID mice that had been treated with an anti-IL-2Rβ mAb. Eighteen days later, nepmucin expression was examined in the tumor tissues. (B) Nepmucin expression in liver metastatic tumors was examined after LM8G5 osteosarcoma cells (1×10<sup>6</sup> cells) were intravenously injected into the ileocolic vein of C3H/HeN mice. (C, D) Axillary LNs were harvested from C57BL/6 mice that had been subcutaneously inoculated with 1×10<sup>5</sup> B16-F10 melanoma cells. The nepmucin and CD31 expressions were examined by immunohistochemistry (C) and quantitative PCR (D) at the indicated time points after inoculation. Data show representative images, n = 3–4 mice per group. **<i>p</i><0.01, n.s., not significant. Scale bars, 100 µm.</p

    Nepmucin/CD300LG shows heterogeneous expression patterns in distinct compartments of the thymus and spleen.

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    <p>(A-E) Frozen sections of thymus (A), spleen (B), liver (C), kidney (D), and small intestine (E) were stained with an anti-nepmucin mAb (Alexa Fluor 594; red) and anti-PV-1 mAb (Alexa Fluor 647; blue). Cryosections of the thymus (A) and small intestine (E) were further incubated with Hoechst 33342 (white). In the thymus (A), the cortico-medullary junction is indicated by a dotted line. Med: medulla, Cor: Cortex, CA: central artery, WP: white pulp, RP: red pulp, CV: central vein, GL: glomerulus. Scale Bars, 100 µm.</p

    Nepmucin/CD300LG expression is developmentally regulated during postnatal ontogeny.

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    <p>The thymus was collected on P0.5 (A) and spleens were collected on P0.5, 7.5, 10.5, and 14.5, as well as from adult mice (B). Tissue sections were stained with an anti-MAdCAM-1 mAb (Alexa Fluor 488; green), anti-nepmucin mAb (Alexa Fluor 594; red), and anti-PV-1 mAb (Alexa Fluor 647; blue). In the spleen at P7.5-10.5, nepmucin was detected predominantly in microvessels leading to the marginal sinus (arrows). From P14.5 to adulthood, nepmucin was also found in the marginal sinus ECs (arrowheads). Scale bars, 100 µm.</p

    Nepmucin/CD300LG expression is apparently absent from immunologically privileged sites.

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    <p>Frozen sections of the indicated tissues were incubated with an anti-nepmucin mAb or anti-CD31 mAb, followed by HRP-conjugated anti-rat IgG. The reaction was then developed with DAB substrate. Arrows indicate blood vessels in the brain. No nepmucin expression was observed in these tissues. Scale bars, 100 µm.</p

    Nepmucin/CD300LG expression is down-regulated by inflammatory signals.

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    <p>(A, B) Draining LNs and contralateral LNs were harvested at the indicated time points after CFA was subcutaneously injected into the right footpad. In (A), the expressions of nepmucin and CD31 were analyzed by immunofluorescence staining. In (B), the nepmucin mRNA expression was assessed by quantitative PCR using primers that specifically recognize <i>nepmucin</i> gene. Data represent the mean ± SD (n = 3 per group) from three independent experiments. In (C), the nepmucin and CD31 expressions were examined by immunofluorescence staining (upper panel) and quantitative PCR (lower panel) of the draining LNs two days after the footpad injection of TNF-α (200 ng) or PBS. Data show representative images, n = 3-4 mice per group. *<i>p</i><0.5, **<i>p</i><0.01, ***<i>p</i><0.005, n.s., not significant. Scale bars, 100 µm.</p

    E-NPP3 controls plasmacytoid dendritic cell numbers in the small intestine

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    <div><p>Extracellular adenosine 5’-triphosphate (ATP) performs multiple functions including activation and induction of apoptosis of many cell types. The ATP-hydrolyzing ectoenzyme ecto-nucleotide pyrophosphatase/phosphodiesterase 3 (E-NPP3) regulates ATP-dependent chronic allergic responses by mast cells and basophils. However, E-NPP3 is also highly expressed on epithelial cells of the small intestine. In this study, we showed that E-NPP3 controls plasmacytoid dendritic cell (pDC) numbers in the intestine through regulation of intestinal extracellular ATP. In <i>Enpp3</i><sup>-/-</sup> mice, ATP concentrations were increased in the intestinal lumen. pDC numbers were remarkably decreased in the small intestinal lamina propria and Peyer’s patches. Intestinal pDCs of <i>Enpp3</i><sup>-/-</sup> mice showed enhanced cell death as characterized by increases in annexin V binding and expression of cleaved caspase-3. pDCs were highly sensitive to ATP-induced cell death compared with conventional DCs. ATP-induced cell death was abrogated in <i>P2rx7</i><sup>-/-</sup> pDCs. Accordingly, the number of intestinal pDCs was restored in <i>Enpp3</i><sup>-/-</sup> <i>P2rx7</i><sup>-/-</sup> mice. These findings demonstrate that E-NPP3 regulates ATP concentration and thereby prevents the decrease of pDCs in the small intestine.</p></div

    Nepmucin/CD300LG is selectively expressed in microvessels.

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    <p>(A-C) Whole-mount images of vasculature in the uvea (A), diaphragm (B), and trachea (C). Mice were intravenously injected with Alexa Fluor 594-conjugated anti-nepmucin mAb (red) and Alexa Fluor 647-conjugated anti-PV-1 mAb (blue), and transcardially perfused with 4% PFA. Collected tissues were observed using a laser-scanning confocal microscope, and Z-series images of each tissue were projected into one plane to show the vascular structures. (D, E) Mice were intravenously injected with Alexa Fluor 647-conjugated anti-nepmucin mAb (blue). After fixation, collected tissues were stained with Cy3-conjugated anti-α-smooth muscle actin mAb (red). The arterioles (D) and venules (E) were identified by the wrapping morphology of α-smooth muscle actin-expressing perivascular cells. Nepmucin-expressing vascular fragments enwrapped by α-smooth muscle actin<sup>+</sup> cells are indicated by arrows. (F) A frozen section of tissue containing the ventral aorta and inferior vena cava was stained with Alexa Fluor 594-conjugated anti-nepmucin mAb (red) and FITC-conjugated anti-CD31 mAb (green). Note that nepmucin staining was absent in the aorta (AO) and vena cava (VC), whereas it was detectable in the capillaries of the surrounding adipose tissues. Ar: arteriole, Ve: venule. Scale bars, 100 µm.</p

    P2X7-dependent induction of pDC apoptosis in vivo.

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    <p><b>(A, B)</b> Frequencies of annexin V-positive (A) and active caspase-3-positive (B) cells gated on CD45<sup>+</sup> PDCA-1<sup>+</sup> CD11c<sup>med</sup> pDCs from the PPs and SILP of wild-type, <i>Enpp3</i><sup><i>-/-</i></sup>, <i>P2rx7</i><sup><i>-/-</i></sup>, and <i>Enpp3</i><sup><i>-/-</i></sup> <i>P2rx7</i><sup><i>-/-</i></sup> mice (n = 7 per groups in a, and n = 5 per groups in b). Representative histograms are shown (left) and the means ± SD of the percentages of annexin V-positive (A) and active caspase-3-positive cells (B) are shown (right). *<i>p</i> < 0.05, **<i>p</i> < 0.01 <b>(c)</b> Frequency and number of PDCA-1<sup>+</sup> CD11c<sup>med</sup> pDCs in the PPs and SILP from wild-type, <i>Enpp3</i><sup><i>-/-</i></sup>, <i>P2rx7</i><sup><i>-/-</i></sup>, and <i>Enpp3</i><sup><i>-/-</i></sup> <i>P2rx7</i><sup><i>-/-</i></sup> (n = 13 per groups) mice. Representative dot plots are shown (left) and the means ± SD of the percentages of pDCs are shown (right). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p

    Enhancement of ATP-induced apoptosis of pDCs.

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    <p><b>(A, B)</b> Cells isolated from MLNs were treated with the indicated concentrations of ATP for 3 h. Annexin V-positive cells (A) and active caspase-3-positive cells (B) among PDCA-1<sup>+</sup> CD11c<sup>med</sup> pDCs and PDCA-1<sup>-</sup> CD11c<sup>high</sup> cDCs were analyzed by flow cytometry. Representative histograms are shown (left) and the means ± SD (n = 3) of the percentages of annexin V-positive (A) and active caspase-3-positive cells (B) are shown (right). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. <b>(C)</b> Frequency of active caspase-3-positive cells among CD45<sup>+</sup> PDCA-1<sup>+</sup> CD11c<sup>med</sup> pDCs and and PDCA-1<sup>-</sup> CD11c<sup>high</sup> cDCs from the PPs and SILP of mice injected with 300 μl PBS (n = 6) or 1 mM ATP-γS (n = 5) into their small intestinal lumen. Representative histograms are shown (left) and the means ± SD of the percentages of positive cells are shown (right). *<i>p</i> < 0.05, **<i>p</i> < 0.01, NS: not significant.</p
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