S U M M A R Y We have previously reported the TLR4 expression in human intestinal lymphatic vessels. In the study here, microarray analysis showed the expression of the TLR4, MD-2, CD14, MyD88, TIRAP, TRAM, IRAK1, and TRAF6 genes in cultured human neonatal dermal lymphatic microvascular endothelial cells (LEC). The microarray analysis also showed that LEC expressed genes of IL-6, IL-8, VCAM-1, and ICAM-1, and the real-time quantitative PCR analysis showed that mRNA production was increased by lipopolysaccharide (LPS). The LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 production in LEC was suppressed by the introduction of TLR4-specific small interfering RNA, and also by anti-TLR4, nobiletin, and CAPE pretreatment. These findings suggest that LEC has TLR4-mediated LPS recognition mechanisms that involve at least activation of NF-kB, resulting in increased expression of IL-6, IL-8, VCAM-1, and ICAM-1. Both the LPS effect on the gene expression and also the suppression by nobiletin and CAPE pretreatment on the protein production were larger in IL-6 and in VCAM-1 than in IL-8 and in ICAM-1 in LEC. The signal transduction of NF-kB and AP-1-dependent pathway may be more critical for the expression of IL-6 and VCAM-1 than that of IL-8 and ICAM-1 in LEC. LYMPHATIC VESSELS express not only platelet-endothelial adhesion molecule-1 (PECAM-1) but also the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in inflamed and uninflamed human small intestine and tongue, and lymphatic endothelium enhances VCAM-1 and ICAM-1 production with tumor necrosis factor-a (TNF-a) (Sawa et al. 1999(Sawa et al. ,2007Ebata et al. 2001). It is well established that VCAM-1 and ICAM-1 induction needs a signaling pathway that involves protein kinase C and p38 mitogenactivated protein kinases (MAPKs)-mediated activation of transcription factors nuclear factor-kappaB (NF-kB) and activator protein 1 (AP-1) (Voraberger et al. 1991;Ahmad et al. 1998;Ishizuka et al. 1998;Oertli et al. 1998;Kobuchi et al. 1999;Lawson et al. 1999;Roebuck 1999). It is thought that lymphatic endothelium has signal transduction pathways to induce leukocyte adhesion molecule expression through receptors for inflammatory cytokines. However, it has not been established whether lymphatic endothelium has the expression mechanisms of immunological functional molecules independent of the inflammatory cytokines.The toll-like receptor (TLR) initiates a series of innate immune mechanisms against various microorganism infections by sensing the presence of pathogen-associated molecular patterns (PAMPs) like lipopolysaccharide (LPS), which is the major component of the outer surface of Gram-negative bacteria. The LPS stimulates leukocyte and blood endothelium through the LPS recognition systems, binding with CD14 and transferring to TLR4 and MD-2 complex, followed by a myleoid differentiation primary response protein (MyD88)-dependent or -independent pathway that culminates in the early-or late-phase activation of the IkB kinase (IKK) complex and MAPK...

The clinical study for lingual disease requires the detailed investigation of the lingual lymphatic network and lymphatic marker-positive cells. Recently, it has been reported that several tissue cells and leukocytes express lymphatic markers, LYVE-1 and podoplanin. This study was aimed to clarify the lingual distribution of cells expressing LYVE-1 and podoplanin. In the mouse tongue, podoplanin is expressed in nerve sheaths, lingual gland myoepithelial cells, and lymphatic vessels. LYVE-1 is expressed in the macrophage marker Mac-1-positive cells as well as lymphatic vessels, while factor-VIII was detected in only blood endothelial cells. α-SMA was detected in vascular smooth muscle and myoepithelial cells. Therefore, identification of lymphatic vessels in lingual glands, the combination of LYVE-1 and factor-VIII, or LYVE-1 and Mac-1 is useful because myoepithelial cells express podoplanin and α-SMA. The immunostaining of factor-VIII on lymphatic vessels was masked by the immunostaining to LYVE-1 or podoplanin because lymphatic vessels express factor-VIII to a far lesser extent than blood vessels. Therefore, except for the salivary glands, the combination of podoplanin and α-SMA, or factor-VIII is useful to identify lymphatic vessels and blood vessels with smooth muscle, or blood capillaries.

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We have recently reported that salivary gland cells express the lymphatic endothelial cell marker podoplanin. The present study was aimed to immunohistochemically investigate the expression of the myoepithelial cell marker α-smooth muscle actin (SMA) on podoplanin-positive cells in mouse parotid and sublingual glands, and to elucidate podoplanin localization in salivary gland myoepithelial cells by immunoelectron microscopic study. The distribution of myoepithelial cells expressing podoplanin and α-SMA was examined by immunofluorescent staining, and the localization of reaction products of anti-podoplanin antibody was investigated by pre-embedded immunoelectron microscopic method. In immunohistochemistry, the surfaces of both the mucous acini terminal portion and ducts were covered by a number of extensive myoepithelial cellular processes expressing podoplanin, and the immunostaining level with anti-podoplanin antibody to myoepithelial cells completely coincided with the immunostaining level with anti-α-SMA antibody. These findings suggest that podoplanin is a salivary gland myoepithelial cell antigen, and that the detection level directly reflects the myoepithelial cell distribution. In immunoelectron microscopic study, a number of reaction products with anti-podoplanin antibody were found at the Golgi apparatus binding to the endoplasmic reticulum in the cytoplasm of myoepithelial cells between sublingual gland acinar cells, and were also found at the myoepithelial cell membrane. These findings suggest that salivary gland myoepithelial cells constantly produce podoplanin and glycosylate at the Golgi apparatus, and transport them to the cell membrane. Podoplanin may be involved in maintaining the homeostasis of myoepithelial cells through its characteristic as a mucin-type transmembrane glycoprotein.