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While the sphingosine-1-phosphate (S1P)/sphingosine-1-phosphate receptor-1 (S1PR1) axis is critically important for lymphocyte egress from lymphoid organs, S1PR1-activation also occurs in vascular endothelial cells (ECs), including those of the high-endothelial venules (HEVs) that mediate lymphocyte immigration into lymph nodes (LNs). To understand the functional significance of the S1P/S1PR1-Gi axis in HEVs, we generated Lyve1;Spns2Δ/Δ conditional knockout mice for the S1P-transporter Spinster-homologue-2 (SPNS2), as HEVs express LYVE1 during development. In these mice HEVs appeared apoptotic and were severely impaired in function, morphology and size; leading to markedly hypotrophic peripheral LNs. Dendritic cells (DCs) were unable to interact with HEVs, which was also observed in Cdh5CRE-ERT2;S1pr1Δ/Δ mice and wildtype mice treated with S1PR1-antagonists. Wildtype HEVs treated with S1PR1-antagonists in vitro and Lyve1-deficient HEVs show severely reduced release of the DC-chemoattractant CCL21 in vivo. Together, our results reveal that EC-derived S1P warrants HEV-integrity through autocrine control of S1PR1-Gi signaling, and facilitates concomitant HEV-DC interactions.

Lymph nodes (LNs) are highly confined environments with a cell-dense three-dimensional meshwork, in which lymphocyte migration is regulated by intracellular contractile proteins. However, the molecular cues directing intranodal cell migration remain poorly characterized. Here we demonstrate that lysophosphatidic acid (LPA) produced by LN fibroblastic reticular cells (FRCs) acts locally to LPA2 to induce T-cell motility. In vivo, either specific ablation of LPA-producing ectoenzyme autotaxin in FRCs or LPA2 deficiency in T cells markedly decreased intranodal T cell motility, and FRC-derived LPA critically affected the LPA2-dependent T-cell motility. In vitro, LPA activated the small GTPase RhoA in T cells and limited T-cell adhesion to the underlying substrate via LPA2. The LPA-LPA2 axis also enhanced T-cell migration through narrow pores in a three-dimensional environment, in a ROCK-myosin II-dependent manner. These results strongly suggest that FRC-derived LPA serves as a cell-extrinsic factor that optimizes T-cell movement through the densely packed LN reticular network.

We previously demonstrated that electroacupuncture (EA) stimulation both produced antinociception and attenuated intrathecal (i.t.) morphine analgesia, suggesting that EA is capable of inducing two opposing systems, that is, opioid and anti-opioid mechanisms. This study examined the involvement of cholecystokinin (CCK) in the anti-opioid effects following EA in the spinal cord. EA was applied to commonly used acupoints for antinociception, ST-36 located 5-mm lateral to the anterior tubercle of the tibia, and analgesia was assessed by the hind-paw pressure test in male Sprague-Dawley rats. I.t. administration of CCK (0.01 – 10 µg) attenuated i.t. morphine analgesia (10 µg) dose-dependently. The attenuation of morphine analgesia following EA was reversed by i.t. proglumide, a CCK-receptor antagonist (0.01 µg). CCK-like immunoreactivity was increased in lamina I and II in the dorsal horn, and expression of spinal CCK mRNA increased after EA. Moreover, i.t. pretreatment with the neurokinin-1 (NK1)-receptor antagonist L-703,606 (18 µg) reversed both EA- and CCK-induced attenuation of morphine analgesia. These results suggest that CCK-mediated neural systems in the spinal cord may be involved in the attenuation of morphine analgesia following EA and that substance P-induced activation of NK1 receptors may be responsible for the downstream neuronal transmission of the CCK-mediated neuronal system. Keywords:: electroacupuncture, morphine, anti-opioid, cholecystokinin, neurokinin

Leukocyte microvilli are flexible projections enriched with adhesion molecules. The role of these cellular projections in the ability of T cells to probe antigen-presenting cells has been elusive. In this study, we probe the spatial relation of microvilli and T-cell receptors (TCRs), the major molecules responsible for antigen recognition on the T-cell membrane. To this end, an effective and robust methodology for mapping membrane protein distribution in relation to the 3D surface structure of cells is introduced, based on two complementary superresolution microscopies. Strikingly, TCRs are found to be highly localized on microvilli, in both peripheral blood human T cells and differentiated effector T cells, and are barely found on the cell body. This is a decisive demonstration that different types of T cells universally localize their TCRs to microvilli, immediately pointing to these surface projections as effective sensors for antigenic moieties. This finding also suggests how previously reported membrane clusters might form, with microvilli serving as anchors for specific T-cell surface molecules. [ABSTRACT FROM AUTHOR]

Naive lymphocytes continuously migrate from the blood into lymph nodes (LNs) via high endothelial venules (HEVs). To extravasate from the HEVs, lymphocytes undergo multiple adhesion steps, including tethering, rolling, firm adhesion and transmigration. We previously showed that autotaxin (ATX), an enzyme that generates lysophosphatidic acid (LPA), is highly expressed in HEVs, and that the ATX/ LPA axis plays an important role in the lymphocyte transmigration across HEVs. However, the detailed mechanism underlying this axis's involvement in lymphocyte transmigration has remained ill-defined. Here, we show that two LPA receptors, LPA4 and LPA6, are selectively expressed on HEV endothelial cells (ECs) and that LPA4 plays a major role in the lymphocyte transmigration across HEVs in mice. In the absence of LPA4 expression, lymphocytes accumulated heavily within the HEV EC layer, compared to wild-type (WT) mice. This accumulation was also observed in the absence of LPA6 expression, but it was less pronounced. Adoptive transfer experiments using WT lymphocytes revealed that the LPA4 deficiency in ECs specifically compromised the lymphocyte transmigration process, whereas the effect of LPA6 deficiency was not significant. These results indicate that the signals evoked in HEV ECs via the LPA4 and LPA6 differentially regulate lymphocyte extravasation from HEVs in the peripheral LNs. [ABSTRACT FROM AUTHOR]

Plasmacytoid dendritic cells (pDCs) are natural type I IFN-producing cells found in lymphoid tissues, where they support both innate and adaptive immune responses. They emigrate from the blood to lymph nodes, apparently through high endothelial venules (HEVs), but little is known about the mechanism. We have investigated the molecular mechanisms of pDC migration using freshly isolated DCs and HEV cells. We found that pDCs bound avidly to HEV cells and then transmigrated underneath them. Two observations suggested that these binding and migration steps are differentially regulated. First, treatment of pDCs with pertussis toxin blocked transmigration but not binding. Second, pDCs were able to bind but not to transmigrate under non-HEV endothelial cells, although the binding was observed to both HEV and non-HEV endothelial cells. Antibody inhibition studies indicated that the binding process was mediated by αL and α4 integrins on pDCs and by intercellular adhesion molecule (ICAM)-1, ICAM-2 and vascular cell adhesion molecule-1 on HEVs. The transmigration process was also mediated by αL and α4 integrins on pDCs, with junctional adhesion molecule-A on HEV cells apparently serving as an additional ligand for αL integrin. These data show for the first time that pDCs employ multiple adhesion molecules sequentially in the processes of adhesion to and transmigration through HEVs. [ABSTRACT FROM AUTHOR]