Dynamic changes in endothelial cell adhesion molecule nepmucin/CD300LG expression under physiological and pathological conditions.

Vascular endothelial cells often change their phenotype to adapt to their local microenvironment. Here we report that the vascular endothelial adhesion molecule nepmucin/CD300LG, which is implicated in lymphocyte binding and transmigration, shows unique expression patterns in the microvascular endothelial cells of different tissues. Under physiological conditions, nepmucin/CD300LG was constitutively and selectively expressed at the luminal surface of the small arterioles, venules, and capillaries of most tissues, but it was only weakly expressed in the microvessels of the splenic red pulp and thymic medulla. Furthermore, it was barely detectable in immunologically privileged sites such as the brain, testis, and uterus. The nepmucin/CD300LG expression rapidly decreased in lymph nodes receiving acute inflammatory signals, and this loss was mediated at least in part by TNF-α. It was also down-regulated in tumors and tumor-draining lymph nodes, indicating that nepmucin/CD300LG expression is negatively regulated by locally produced signals under these circumstances. In contrast, nepmucin/CD300LG was induced in the high endothelial venule-like blood vessels of chronically inflamed pancreatic islets in an animal model of non-obese diabetes. Interestingly, the activated CD4(+) T cells infiltrating the inflamed pancreas expressed high levels of the nepmucin/CD300LG ligand(s), supporting the idea that nepmucin/CD300LG and its ligand interactions are locally involved in pathological T cell trafficking. Taken together, these observations indicate that the nepmucin/CD300LG expression in microvascular endothelial cells is influenced by factor(s) that are locally produced in tissues, and that its expression is closely correlated with the level of leukocyte infiltration in certain tissues

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

<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 developmentally regulated during postnatal ontogeny.

<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 is selectively expressed in microvessels.

<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⁺ 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

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

<p>(A) Human pancreatic adenocarcinoma MIA PaCa-2 cells (1.2×10⁷ 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 (1×10⁶ 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 1×10⁵ 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.

<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 apparently absent from immunologically privileged sites.

<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.

<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