Involvement of the Lysophosphatidic Acid-Generating Enzyme Autotaxin in Lymphocyte-Endothelial Cell Interactions
2008; Elsevier BV; Volume: 173; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2008.071153
ISSN1525-2191
AutoresTae Nakasaki, Toshiyuki Tanaka, Shinichi Okudaira, Michi Hirosawa, Eiji Umemoto, Kazuhiro Otani, Soojung Jin, Zhongbin Bai, Haruko Hayasaka, Yoshinori Fukui, Katsuyuki Aozasa, Naoya Fujita, Takashi Tsuruo, Keiichi Ozono, Junken Aoki, Masayuki Miyasaka,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoAutotaxin (ATX) is a secreted protein with lysophospholipase D activity that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine. Here we report that functional ATX is selectively expressed in high endothelial venules (HEVs) of both lymph nodes and Peyer's patches. ATX expression was developmentally regulated and coincided with lymphocyte recruitment to the lymph nodes. In adults, ATX expression was independent of HEV-expressed chemokines such as CCL21 and CXCL13, innate immunity signals including those via TLR4 or MyD88, and of the extent of lymphocyte trafficking across the HEVs. ATX expression was induced in venules at sites of chronic inflammation. Receptors for the ATX enzyme product LPA were constitutively expressed in HEV endothelial cells (ECs). In vitro, LPA induced strong morphological changes in HEV ECs. Forced ATX expression caused cultured ECs to respond to lysophosphatidylcholine, up-regulating lymphocyte binding to the ECs in a LPA receptor-dependent manner under both static and flow conditions. Although in vivo depletion of circulating ATX did not affect lymphocyte trafficking into the lymph nodes, we surmise, based on the above data, that ATX expressed by HEVs acts on HEVs in situ to facilitate lymphocyte binding to ECs and that ATX in the general circulation does not play a major role in this process. Tissue-specific inactivation of ATX will verify this hypothesis in future studies of its mechanism of action. Autotaxin (ATX) is a secreted protein with lysophospholipase D activity that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine. Here we report that functional ATX is selectively expressed in high endothelial venules (HEVs) of both lymph nodes and Peyer's patches. ATX expression was developmentally regulated and coincided with lymphocyte recruitment to the lymph nodes. In adults, ATX expression was independent of HEV-expressed chemokines such as CCL21 and CXCL13, innate immunity signals including those via TLR4 or MyD88, and of the extent of lymphocyte trafficking across the HEVs. ATX expression was induced in venules at sites of chronic inflammation. Receptors for the ATX enzyme product LPA were constitutively expressed in HEV endothelial cells (ECs). In vitro, LPA induced strong morphological changes in HEV ECs. Forced ATX expression caused cultured ECs to respond to lysophosphatidylcholine, up-regulating lymphocyte binding to the ECs in a LPA receptor-dependent manner under both static and flow conditions. Although in vivo depletion of circulating ATX did not affect lymphocyte trafficking into the lymph nodes, we surmise, based on the above data, that ATX expressed by HEVs acts on HEVs in situ to facilitate lymphocyte binding to ECs and that ATX in the general circulation does not play a major role in this process. Tissue-specific inactivation of ATX will verify this hypothesis in future studies of its mechanism of action. Much of the efficiency of the immune system depends on continual lymphocyte recirculation through the secondary lymphoid tissues. Lymphocyte recirculation begins with blood lymphocytes interacting with the endothelial wall of high endothelial venules (HEVs) in lymph nodes (LNs) and Peyer's patches (PPs), in a process called lymphocyte rolling. The rolling brings lymphocytes into contact with HEV endothelial cells (ECs), which express a variety of chemokines and adhesion molecules. Lymphocytes bind to these molecules and transmigrate through the HEVs.1von Andrian UH Mempel TR Homing and cellular traffic in lymph nodes.Nat Rev Immunol. 2003; 3: 867-878Crossref PubMed Scopus (1001) Google Scholar, 2Miyasaka M Tanaka T Lymphocyte trafficking across high endothelial venules: dogmas and enigmas.Nat Rev Immunol. 2004; 4: 360-370Crossref PubMed Scopus (359) Google Scholar This interaction between lymphocytes and the molecules on HEV ECs is transient and reversible, and both lymphocytes and HEV ECs show marked and coordinated morphological changes during the process,3Anderson ND Anderson AO Wyllie RG Specialized structure and metabolic activities of high endothelial venules in rat lymphatic tissues.Immunology. 1976; 31: 455-473PubMed Google Scholar indicating that each of the two cell types acts on the other. The molecular details behind these interactions, however, remain to be fully elucidated. Accumulating evidence indicates that a variety of lysophospholipids act on immune cells by transmitting signals through a family of G-protein-coupled receptors to control the cells' differentiation, motility, and survival.4Rosen H Goetzl EJ Sphingosine 1-phosphate and its receptors: an autocrine and paracrine network.Nat Rev Immunol. 2005; 5: 560-570Crossref PubMed Scopus (623) Google Scholar In particular, structurally related lysophospholipid mediators such as sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), which can be generated from lipid stores by cellular enzymatic pathways in macrophages, dendritic cells, mast cells, and platelets, act on lymphocytes and dendritic cells through members of the S1P-receptor family (S1P1∼ S1P5) and the LPA-receptor family (LPA1∼ LPA5), respectively.4Rosen H Goetzl EJ Sphingosine 1-phosphate and its receptors: an autocrine and paracrine network.Nat Rev Immunol. 2005; 5: 560-570Crossref PubMed Scopus (623) Google Scholar, 5Goetzl EJ Wang W McGiffert C Huang MC Graler MH Sphingosine 1-phosphate and its G protein-coupled receptors constitute a multifunctional immunoregulatory system.J Cell Biochem. 2004; 92: 1104-1114Crossref PubMed Scopus (65) Google Scholar, 6Kotarsky K Boketoft A Bristulf J Nilsson NE Norberg A Hansson S Owman C Sillard R Leeb-Lundberg LM Olde B Lysophosphatidic acid binds to and activates GPR92, a G protein-coupled receptor highly expressed in gastrointestinal lymphocytes.J Pharmacol Exp Ther. 2006; 318: 619-628Crossref PubMed Scopus (187) Google Scholar, 7Lee CW Rivera R Gardell S Dubin AE Chun J GPR92 as a new G12/13- and Gq-coupled lysophosphatidic acid receptor that increases cAMP LPA5.J Biol Chem. 2006; 281: 23589-23597Crossref PubMed Scopus (382) Google Scholar Among these ligand-receptor interactions, S1P and its receptor S1P1 are required for thymocyte emigration8Matloubian M Lo CG Cinamon G Lesneski MJ Xu Y Brinkmann V Allende ML Proia RL Cyster JG Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1.Nature. 2004; 427: 355-360Crossref PubMed Scopus (2091) Google Scholar as well as lymphocyte egress from the secondary lymphoid tissues,9Lo CG Xu Y Proia RL Cyster JG Cyclical modulation of sphingosine-1-phosphate receptor 1 surface expression during lymphocyte recirculation and relationship to lymphoid organ transit.J Exp Med. 2005; 201: 291-301Crossref PubMed Scopus (258) Google Scholar, 10Cinamon G Matloubian M Lesneski MJ Xu Y Low C Lu T Proia RL Cyster JG Sphingosine 1-phosphate receptor 1 promotes B cell localization in the splenic marginal zone.Nat Immunol. 2004; 5: 713-720Crossref PubMed Scopus (350) Google Scholar although exactly where and how these signaling molecules function is not fully understood. In addition, the functional significance of the interaction between LPA and its receptors in the immune system is unknown. Autotaxin (ATX), originally identified as a tumor motility-stimulating protein,11Murata J Lee HY Clair T Krutzsch HC Arestad AA Sobel ME Liotta LA Stracke ML cDNA cloning of the human tumor motility-stimulating protein, autotaxin, reveals a homology with phosphodiesterases.J Biol Chem. 1994; 269: 30479-30484Abstract Full Text PDF PubMed Google Scholar is a secreted protein identical to lysophospholipase D (lysoPLD).12Umezu-Goto M Kishi Y Taira A Hama K Dohmae N Takio K Yamori T Mills GB Inoue K Aoki J Arai H Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production.J Cell Biol. 2002; 158: 227-233Crossref PubMed Scopus (803) Google Scholar In vitro, ATX converts lysophosphatidylcholine (LPC) and sphingosylphosphorylcholine to LPA12Umezu-Goto M Kishi Y Taira A Hama K Dohmae N Takio K Yamori T Mills GB Inoue K Aoki J Arai H Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production.J Cell Biol. 2002; 158: 227-233Crossref PubMed Scopus (803) Google Scholar and S1P,13Clair T Aoki J Koh E Bandle RW Nam SW Ptaszynska MM Mills GB Schiffmann E Liotta LA Stracke ML Autotaxin hydrolyzes sphingosylphosphorylcholine to produce the regulator of migration, sphingosine-1-phosphate.Cancer Res. 2003; 63: 5446-5453PubMed Google Scholar respectively. However, a recent study indicates that ATX is a major producer of LPA, but not of S1P, in vivo.14Tanaka M Okudaira S Kishi Y Ohkawa R Iseki S Ota M Noji S Yatomi Y Aoki J Arai H Autotaxin stabilizes blood vessels and is required for embryonic vasculature by producing lysophosphatidic acid.J Biol Chem. 2006; 281: 25822-25830Crossref PubMed Scopus (393) Google Scholar Under physiological conditions, ATX is expressed in the central nervous system and is implicated in oligodendrocyte function.15Fuss B Baba H Phan T Tuohy VK Macklin WB Phosphodiesterase I, a novel adhesion molecule and/or cytokine involved in oligodendrocyte function.J Neurosci. 1997; 17: 9095-9103Crossref PubMed Google Scholar In addition, ATX transcripts have been found in human tonsillar HEVs,16Palmeri D Zuo FR Rosen SD Hemmerich S Differential gene expression profile of human tonsil high endothelial cells: implications for lymphocyte trafficking.J Leukoc Biol. 2004; 75: 910-927PubMed Google Scholar but its protein expression has not been reported in HEVs, let alone its function. While this manuscript was under review, Kanda and colleagues17Kanda H Newton R Klein R Morita Y Gunn MD Rosen SD Autotaxin, an ectoenzyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary lymphoid organs.Nat Immunol. 2008; 9: 415-423Crossref PubMed Scopus (217) Google Scholar published that ATX is involved in lymphocyte trafficking from blood into lymphoid tissues. Here we report that ATX is preferentially expressed in the HEVs of mouse LNs and PPs and that its expression correlates positively with the ability of venules to support lymphocyte trafficking across the endothelial walls. ATX was clearly inducible in chronically inflamed venules that mediate pathological lymphocyte trafficking. Interestingly, LPA receptors were expressed in HEV ECs, and the addition of LPA caused substantial cytoskeletal changes. Forced ATX expression made cultured ECs reactive to LPC, which up-regulated lymphocyte binding to the ECs in a LPA receptor-dependent manner. Because ATX is a major producer of LPA, these results support the idea that ATX and LPA are active participants in lymphocyte trafficking, presumably regulating lymphocyte migratory behaviors locally at HEVs. C57BL/6J and AKR/J mice were purchased from Japan SLC (Hamamatsu, Japan) and CLEA Japan (Tokyo, Japan), respectively. NOD and GFP-transgenic mice18Okabe M Ikawa M Kominami K Nakanishi T Nishimune Y 'Green mice' as a source of ubiquitous green cells.FEBS Lett. 1997; 407: 313-319Abstract Full Text Full Text PDF PubMed Scopus (2265) Google Scholar were kind gifts from Dr. J. Miyazaki (Division of Stem Cell Regulation Research, Osaka University, Osaka, Japan) and Dr. M. Okabe (Research Institute of Microbial Diseases, Osaka University), respectively. The following genetically engineered and mutant mice were backcrossed on a C57BL/6J background for at least four generations, and the backcrossed progeny were used in experiments at 6 to 12 weeks of age: CCR7−/−,19Förster R Schubel A Breitfeld D Kremmer E Renner-Muller I Wolf E Lipp M CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs.Cell. 1999; 99: 23-33Abstract Full Text Full Text PDF PubMed Scopus (1932) Google Scholar DOCK2−/−,20Fukui Y Hashimoto O Sanui T Oono T Koga H Abe M Inayoshi A Noda M Oike M Shirai T Sasazuki T Haematopoietic cell-specific CDM family protein DOCK2 is essential for lymphocyte migration.Nature. 2001; 412: 826-831Crossref PubMed Scopus (370) Google Scholar TLR2−/−,21Akira S Takeda K Toll-like receptor signalling.Nat Rev Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6751) Google Scholar TLR4−/−,21Akira S Takeda K Toll-like receptor signalling.Nat Rev Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6751) Google Scholar MyD88−/−,21Akira S Takeda K Toll-like receptor signalling.Nat Rev Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6751) Google Scholar CXCL13−/−,22Ebisuno Y Tanaka T Kanemitsu N Kanda H Yamaguchi K Kaisho T Akira S Miyasaka M Cutting edge: the B cell chemokine CXC chemokine ligand 13/B lymphocyte chemoattractant is expressed in the high endothelial venules of lymph nodes and Peyer's patches and affects B cell trafficking across high endothelial venules.J Immunol. 2003; 171: 1642-1646PubMed Scopus (0) Google Scholar and plt/plt.23Nakano H Tamura T Yoshimoto T Yagita H Miyasaka M Butcher EC Nariuchi H Kakiuchi T Matsuzawa A Genetic defect in T lymphocyte-specific homing into peripheral lymph nodes.Eur J Immunol. 1997; 27: 215-221Crossref PubMed Scopus (126) Google Scholar Mice were housed at the Institute of Experimental Animal Sciences at Osaka University Medical School, and the experimental protocols were approved by the Ethics Review Committee for Animal Experimentation of the Osaka University Graduate School of Medicine. MAdCAM-1+ HEV ECs were purified from pooled mesenteric LNs as described.24Izawa D Tanaka T Saito K Ogihara H Usui T Kawamoto S Matsubara K Okubo K Miyasaka M Expression profile of active genes in mouse lymph node high endothelial cells.Int Immunol. 1999; 11: 1989-1998Crossref PubMed Scopus (48) Google Scholar The mouse capillary EC line, MBEC425Tatsuta T Naito M Oh-hara T Sugawara I Tsuruo T Functional involvement of P-glycoprotein in blood-brain barrier.J Biol Chem. 1992; 267: 20383-20391Abstract Full Text PDF PubMed Google Scholar was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mmol/L l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10% fetal calf serum (FCS), 1 mmol/L sodium pyruvate, 10 mmol/L HEPES, 0.1 mmol/L nonessential amino acids, and 50 μmol/L 2-mercaptoethanol. To establish cells stably expressing ATX, MBEC4 cells were co-transfected with a mixture of two plasmids, mlysoPLDpCAGGS26Niwa H Yamamura K Miyazaki J Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene. 1991; 108: 193-199Crossref PubMed Scopus (4618) Google Scholar and pSV2-neo, carrying the neomycin (G418) resistance gene, and grown in DMEM containing 750 μg/ml of G418 (Sigma, St. Louis, MO). Individual G418-resistant clones were isolated by limiting dilution and screened by Western blotting and immunocytochemistry with an anti-ATX mAb (4F1)14Tanaka M Okudaira S Kishi Y Ohkawa R Iseki S Ota M Noji S Yatomi Y Aoki J Arai H Autotaxin stabilizes blood vessels and is required for embryonic vasculature by producing lysophosphatidic acid.J Biol Chem. 2006; 281: 25822-25830Crossref PubMed Scopus (393) Google Scholar and by measuring the lysoPLD activities of the culture media. Fatty acid-free bovine serum albumin (BSA), 1-oleoyl-LPC, and Ki16425 were purchased from Sigma. LPA (1-oleoyl-2-hydroxy-sn-3-glycerol-3-phosphate) was purchased from Biomol International (Plymouth Meeting, PA). Total RNA was isolated from various tissues using Isogen (Nippongene, Toyama, Japan) and reverse-transcribed using the Super-Script first-strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA). Oligonucleotide primers for PCR were designed using Primer Express Software (Applied Biosystems, Foster City, CA). The sequences of the oligonucleotides used in PCR were as follows: ATX (mouse), forward, 5′-GGAGAATCACACTGGGTAGATGATG-3′; ATX (mouse), reverse, 5′-ACGGAGGGCGGACAAAC-3′; GAPDH, forward, 5′-GCCAA GGTCATCCATGACAACT-3′; GAPDH, reverse, 5′-GAG GGGCCATCCACAGTCTT. PCR reactions were performed using an ABI Prism 7000 sequence detection system (Applied Biosystems). The transcript number of GAPDH was quantified, and each sample was normalized on the basis of GAPDH content. Formalin-fixed, paraffin-embedded sections (4 μm) were dewaxed, rehydrated, and boiled in 10 mmol/L citrate buffer, pH 6.0. Sections were then washed in Tris-buffered saline and incubated with anti-ATX mAb (4F1) and anti-PNAd mAb (MECA-79)27Streeter PR Rouse BT Butcher EC Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes.J Cell Biol. 1988; 107: 1853-1862Crossref PubMed Scopus (540) Google Scholar or anti-MAdCAM-1 mAb (MECA-89)28Streeter PR Berg EL Rouse BT Bargatze RF Butcher EC A tissue-specific endothelial cell molecule involved in lymphocyte homing.Nature. 1988; 331: 41-46Crossref PubMed Scopus (629) Google Scholar at 4°C overnight, followed by biotin-conjugated goat anti-rat IgG + IgM (Southern Biotechnology Associates, Inc., Birmingham, AL). The sections were further incubated with horseradish peroxidase-conjugated ABC reagent (Vectastain ABC-HRP kit; Vector Laboratories, Burlingame, CA), developed with Metal Enhanced DAB (Pierce, Rockford, IL), and counterstained with Mayer's hematoxylin (Muto Pure Chemicals Co., Tokyo, Japan). In some experiments, biotin-labeled tyramide reagent (Perkin-Elmer Life Sciences, Boston, MA) was used for signal amplification. Total RNA was extracted from mesenteric LNs using TRIzol (Invitrogen). Single-strand cDNA synthesized using the Ready-To-Go kit (GE Health Care UK Ltd., Amersham Place, UK) and a cDNA library of MAdCAM-1+ HEVs29Umemoto E Tanaka T Kanda H Jin S Tohya K Otani K Matsutani T Matsumoto M Ebisuno Y Jang MH Fukuda M Hirata T Miyasaka M Nepmucin, a novel HEV sialomucin, mediates L-selectin-dependent lymphocyte rolling and promotes lymphocyte adhesion under flow.J Exp Med. 2006; 203: 1603-1614Crossref PubMed Scopus (54) Google Scholar were used in PCR analysis with Ex-TaqDNA polymerase (Takara Bio, Otsu, Japan). PCR was performed at 94°C for 2 minutes, 38 cycles at 94°C for 30 seconds, at 57°C for 30 seconds, at 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. The following primer pairs were used: ATX: sense, 5′-TCTAGCATCCCAGAGCACCT-3′, antisense, 5′-GGTCGGTGAGGAAGGATGAA-3′; LPA1: sense, 5′-AAGCAAGCATGTGGTGTGTG-3′, antisense, 5′-ATGTCTATAGGCATACGTGG-3′, LPA2: sense, 5′-GCTAGTACTGAAGCTGATTCC-3′, antisense, 5′-AGCCTAGTCTATGCGGCAAG-3′; LPA3: sense, 5′-GATGAGAGTCCACAGCAACTTG-3′, antisense, 5′-AGATGCGTACGTATACCGCC-3′30Radeke HH von Wenckstern H Stoidtner K Sauer B Hammer S Kleuser B Overlapping signaling pathways of sphingosine 1-phosphate and TGF-β in the murine Langerhans cell line XS52.J Immunol. 2005; 174: 2778-2786Crossref PubMed Scopus (61) Google Scholar; and LPA4: sense, 5′-CACATATAAGGATGGAGTCGC-3′, antisense, 5′-GTCA ACTCAACAGAAGAGGC-3′; CD3ε: sense, 5′-CCTGACAGCAGTAGCCATAATC-3′, antisense, 5′-GCTGTTG AGTCAGCAATGTCC-3′; L-selectin: sense, 5′-GCCATGGTGTTTCCATGGAGATGTGAGGGT-3′, antisense, 5′-ATCATCCATCCTTTCTTGAGATTTCTTGCC-3′; β-actin: sense, 5′-ATGGATGACGATATCGCT-3′, antisense, 5′-ATGAGGTAGTCTGTCAGGT-3′. PCR products were analyzed by agarose gel electrophoresis. Plasmids (pCRII; Invitrogen) containing cDNA fragments of LPA1 (500 bp), LPA4 (366 bp), or MAdCAM-1 (482 bp) were used as templates for RNA probe synthesis. Digoxigenin (DIG)-labeled antisense or sense probes were prepared with T7 and SP6 RNA polymerase (Applied Biosystems), respectively, using the DIG RNA labeling mix (Roche Diagnostics, Basel, Switzerland). Mesenteric and peripheral LNs were fixed with 4% paraformaldehyde for 2 hours, incubated in 30% sucrose in phosphate-buffered saline (PBS) overnight, and embedded in OCT compound. Eight-μm-thick serial frozen sections were fixed in 4% paraformaldehyde for 20 minutes, incubated in 0.1% H2O2, and permeabilized with 50 μg/ml proteinase K for 5 minutes. After an additional fixation with paraformaldehyde, the sections were treated with acetic anhydride in triethanolamine for 10 minutes. The sections were then prehybridized with 50% formamide, 5× standard saline citrate, 1 mg/ml yeast tRNA (Roche Diagnostics), 100 μg/ml heparin, 1× Denhardt's solution, and 0.1% Tween 20 at 60°C for 3 hours, and then hybridized with labeled probe in the same solution overnight at 60°C. After being washed, the sections were incubated with horseradish peroxidase-conjugated anti-DIG (Roche Diagnostics), followed by biotin-labeled tyramide (TSA Biotin System, PerkinElmer Life Sciences) for signal amplification. The hybridized probes were then detected by ABC-alkaline phosphatase (Vector Laboratories) and NBT/BCIP (Roche Diagnostics). Purified MAdCAM-1+ HEV ECs were seeded in collagen type 1-coated eight-well culture slides (Becton Dickinson, Mountain View, CA) (4 × 104 cells/well) and incubated for 3 hours in DMEM containing 20% FCS (Hyclone, Logan, UT). After the nonadherent cells were removed, the remaining ECs were treated for 60 minutes with LPA (15 μmol/L) in fresh DMEM containing FCS stripped with 10% charcoal. Similarly, MBEC4 cells and ATX-expressing MBEC4 cells were seeded in Lab-Tek II chamber slides (Nalge Nunc, Rochester, NY) (0.5 × 104 cells/well), cultured overnight, and treated for 60 minutes with LPA or LPC (10 μmol/L) in fresh DMEM containing 0.1% fatty acid-free BSA. In some experiments, cells were pretreated with 10 μmol/L Ki16425 (Sigma) for 60 minutes at 37°C. After two washes in PBS, the cells were fixed in 4% paraformaldehyde, permeabilized in PBS containing 0.1% Triton X-100, and incubated with Alexa Fluor 594-conjugated MECA89 (2 μg/ml) followed by Alexa Fluor 488-phalloidin (1 U/ml) (Invitrogen) (for MAdCAM-1+ HEV ECs) or Alexa Fluor 488-phalloidin alone (for MBEC4 and ATX-expressing MBEC4 cells). The cells were then observed under a confocal microscope (LSM510META; Carl Zeiss, Jena, Germany; or Radiance 2100; Bio-Rad, Hercules, CA) using Fluoromount-G mounting medium (Southern Biotechnology). Freshly isolated MAdCAM-1+ HEV ECs, MBEC4, and ATX-expressing MBEC4 cells were cultured and stimulated as follows. The MAdCAM-1+ HEV ECs were plated (2.5 × 104 cells/well) in collagen-coated 96-well plates (Becton Dickinson) and allowed to adhere for 3 hours in DMEM containing 20% FCS (Hyclone). After the unbound cells were removed, the remaining cells were starved for 60 minutes by incubation in DMEM containing 0.1% fatty acid-free BSA. MBEC4 cells and MBEC4 transfectants expressing ATX were plated in a flat-bottomed 96-well plate (1 × 104 cells/well) and grown in DMEM containing 10% FCS overnight. The cells were then starved for 16 hours by incubation in DMEM with 0.1% fatty acid-free BSA. These EC preparations were incubated with or without various concentrations of LPA or LPC for 30 to 60 minutes in DMEM with 0.1% fatty acid-free BSA, as indicated. For inhibition studies, the ECs were pretreated with 10 μmol/L Ki16425 for 30 minutes. Spleen cells that did not adhere to plastic were labeled with the fluorescent indicator BCECF-AM as described,31Toyama-Sorimachi N Miyake K Miyasaka M Activation of CD44 induces ICAM-1/LFA-1-independent. Ca2+, Mg(2+)-independent adhesion pathway in lymphocyte-endothelial cell interaction.Eur J Immunol. 1993; 23: 439-446Crossref PubMed Scopus (59) Google Scholar in DMEM with 0.1% fatty acid-free BSA. They were then added to the wells containing the ECs (5 × 105 cells/well) and allowed to bind to the ECs for 30 minutes under static conditions or with rotation (120 rpm). Unbound lymphocytes were removed by inverting the plate, which had been filled with prewarmed FCS-free RPMI 1640 and sealed with Parafilm (Pechinery Plastic Packaging, Inc., Menasha, WI) for 30 minutes at room temperature. The adherent lymphocytes were solubilized with 1% Nonidet P-40 in PBS, and fluorescence intensity was measured by a fluorescence enzyme-linked immunosorbent assay reader (SpectraMax Gemini XS; Molecular Devices, Sunnyvale, CA). The lymphocyte migration assay was performed as previously described.22Ebisuno Y Tanaka T Kanemitsu N Kanda H Yamaguchi K Kaisho T Akira S Miyasaka M Cutting edge: the B cell chemokine CXC chemokine ligand 13/B lymphocyte chemoattractant is expressed in the high endothelial venules of lymph nodes and Peyer's patches and affects B cell trafficking across high endothelial venules.J Immunol. 2003; 171: 1642-1646PubMed Scopus (0) Google Scholar Briefly, spleen cells obtained from GFP-transgenic mice were injected intravenously into mice (2 × 107 cell/mouse) that had received an intraperitoneal injection of a cocktail of anti-ATX mAbs (clones 5E5, S13A9, and S9A9; 150 μg each) or rat IgG (450 μg/mouse; Cappel, Aurora, OH) 3 hours before. The mice were sacrificed 2.5 hours after the lymphocyte injection, and the spleen, mesenteric LNs, brachial LNs, and PPs were harvested. The number of lymphocytes that had migrated into these tissues was determined by flow cytometry. Plasma samples (10 μl) were incubated with 4 mmol/L LPC in buffer containing 100 mmol/L Tris-HCl (pH 9.0), 500 mmol/L NaCl, 5 mmol/L MgCl2, and 0.05% Triton X-100 for 12 hours at 37°C. The liberated choline was detected by an enzymatic photometric method using choline oxidase (Asahi Chemical Industry, Tokyo, Japan), horseradish peroxidase (Toyobo, Tokyo, Japan), and TOOS reagent [N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine] (Dojin, Tokyo, Japan), as reported previously.32Imamura S Horiuti Y Enzymatic determination of phospholipase D activity with choline oxidase.J Biochem (Tokyo). 1978; 83: 677-680PubMed Google Scholar Absorbance was read at 560 nm and converted to nanomoles of choline by comparison to a choline standard curve. A Student's t-test was applied to compare the statistical difference within two groups. Preliminary analysis showed that ATX transcripts appeared at moderate frequencies in the previously generated 3′-directed cDNA libraries of PNAd+ HEV ECs24Izawa D Tanaka T Saito K Ogihara H Usui T Kawamoto S Matsubara K Okubo K Miyasaka M Expression profile of active genes in mouse lymph node high endothelial cells.Int Immunol. 1999; 11: 1989-1998Crossref PubMed Scopus (48) Google Scholar and of MAdCAM-1+ HEV ECs33Saito K Tanaka T Kanda H Ebisuno Y Izawa D Kawamoto S Okubo K Miyasaka M Gene expression profiling of mucosal addressin cell adhesion molecule-1+ high endothelial venule cells (HEV) and identification of a leucine-rich HEV glycoprotein as a HEV marker.J Immunol. 2002; 168: 1050-1059PubMed Google Scholar; they appeared five times among 1558 transcripts obtained from the PNAd+ HEV cDNA library and five times among 2101 transcripts in the MAdCAM-1+ HEV cDNA library. Transcripts of endoglin were found at comparable frequencies in these libraries (data not shown). ATX transcripts are also found in a human tonsil cDNA library enriched for HEV-derived cDNAs by subtraction with lymphocyte cDNAs and umbilical vein endothelial cDNAs.16Palmeri D Zuo FR Rosen SD Hemmerich S Differential gene expression profile of human tonsil high endothelial cells: implications for lymphocyte trafficking.J Leukoc Biol. 2004; 75: 910-927PubMed Google Scholar Real-time PCR analysis showed that ATX mRNA was strongly expressed in the peripheral and mesenteric LNs as well as in the brain and kidney among the mouse tissues tested (Figure 1A). These findings prompted us to look for the expression and localization of ATX proteins in HEVs. As shown in Figure 1B, specific mAb against ATX readily detected its expression in HEVs of peripheral and mesenteric LNs and PPs, but not in non-HEV blood vessels in experimentally naïve mice. Although staining intensities varied in different HEVs, a majority of HEVs of LNs appeared positive for ATX expression, whereas ∼30 to 40% of HEVs of PPs expressed ATX. In mesenteric LNs, both PNAd+ HEVs and MAdCAM-1+ HEVs expressed ATX (Figure 1C). We next examined ATX expression in developing LNs. Although ATX expression was not observed prenatally (Figure 2A), it was detectable 1 day after birth (Figure 2B), and its level increased progressively until postnatal day 7 (Figure 2, C and D), concomitant with the increasing numbers of lymphocytes around HEVs in the peripheral and mesenteric LNs. The appearance of ATX also paralleled to that of PNAd in peripheral LNs (data not shown).34Mebius RE Streeter PR Michie S Butcher EC Weissman IL A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4+ CD3− cells to colonize lymph nodes.Proc Natl Acad Sci USA. 1996; 93: 11019-11024Crossref PubMed Scopus (213) Google Scholar HEV-like venules appear in sites other than peripheral and mesenteric LNs and PPs under certain pathological conditions. For instance, a marked increase in lymphocyte trafficking is associated with the appearance of HEV-specific adhesion molecules and a HEV-like morphology in the cortico-medullary venules of the thymus in aged AKR/J mice that develop thymic hyperplasia and lymphoma.35Michie SA Streeter PR Butcher EC Rouse RV L-selectin and α 4 β 7 integrin homing receptor pathways mediate peripheral lymphocyte traffic to AKR mouse hyperplastic thymus.Am J Pathol. 1995; 147: 412-421PubMed Google Scholar, 36Hiraoka N Petryniak B Nakayama J Tsuboi S Suzuki M Yeh JC Izawa D Tanaka T Miyasaka M Lowe JB Fukuda M A novel, high endothelial venule-specific sulfotransferase expresses 6-sulfo sialyl Lewis(x), an L-selectin ligand displayed by CD34.Immunity. 1999; 11: 79-89Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar We found that a majority of the cortico-medullary venules of hyperplastic thymus from old (>30 weeks) AKR/J mice were PNAd+, as revealed by mAb MECA-79 staining, and ∼60 to 70% of the PNAd+ venules were also positive for ATX expression (Figure 3A). Most of the ATX+ venules appeared PNAd+ (Figure 3A). In co
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