Potentials of the Circulating Pruritogenic Mediator Lysophosphatidic Acid in Development of Allergic Skin Inflammation in Mice
2014; Elsevier BV; Volume: 184; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2014.01.029
ISSN1525-2191
AutoresYoshibumi Shimizu, Yoshiyuki Morikawa, Shinichi Okudaira, Shigenobu Kimoto, Tamotsu Tanaka, Junken Aoki, Akira Tokumura,
Tópico(s)Pharmacological Effects of Natural Compounds
ResumoItching and infiltration of immune cells are important hallmarks of atopic dermatitis (AD). Although various studies have focused on peripheral mediator–mediated mechanisms, systemic mediator–mediated mechanisms are also important in the pathogenesis and development of AD. Herein, we found that intradermal injection of lysophosphatidic acid (LPA), a bioactive phospholipid, induces scratching responses by Institute of Cancer Research mice through LPA1 receptor– and opioid μ receptor–mediating mechanisms, indicating its potential as a pruritogen. The circulating level of LPA in Naruto Research Institute Otsuka Atrichia mice, a systemic AD model, with severe scratching was found to be higher than that of control BALB/c mice, probably because of the increased lysophospholipase D activity of autotaxin (ATX) in the blood (mainly membrane associated) rather than in plasma (soluble). Heparan sulfate proteoglycan was shown to be involved in the association of ATX with blood cells. The sequestration of ATX protein on the blood cells by heparan sulfate proteoglycan may accelerate the transport of LPA to the local apical surface of vascular endothelium with LPA receptors, promoting the hyperpermeability of venules and the pathological uptake of immune cells, aggravating lesion progression and itching in Naruto Research Institute Otsuka Atrichia mice. Itching and infiltration of immune cells are important hallmarks of atopic dermatitis (AD). Although various studies have focused on peripheral mediator–mediated mechanisms, systemic mediator–mediated mechanisms are also important in the pathogenesis and development of AD. Herein, we found that intradermal injection of lysophosphatidic acid (LPA), a bioactive phospholipid, induces scratching responses by Institute of Cancer Research mice through LPA1 receptor– and opioid μ receptor–mediating mechanisms, indicating its potential as a pruritogen. The circulating level of LPA in Naruto Research Institute Otsuka Atrichia mice, a systemic AD model, with severe scratching was found to be higher than that of control BALB/c mice, probably because of the increased lysophospholipase D activity of autotaxin (ATX) in the blood (mainly membrane associated) rather than in plasma (soluble). Heparan sulfate proteoglycan was shown to be involved in the association of ATX with blood cells. The sequestration of ATX protein on the blood cells by heparan sulfate proteoglycan may accelerate the transport of LPA to the local apical surface of vascular endothelium with LPA receptors, promoting the hyperpermeability of venules and the pathological uptake of immune cells, aggravating lesion progression and itching in Naruto Research Institute Otsuka Atrichia mice. Atopic dermatitis (AD) is a chronically relapsing skin disorder with pruritic and eczematous skin lesions. In animal models of AD and a large population of patients with AD, specific leukocytes, including eosinophils and lymphocytes, become increasingly recruited from the blood into lesional skin areas, leading to release of peripheral mediators involved in pruritus.1Boguniewicz M. Leung D.Y. Atopic dermatitis: a disease of altered skin barrier and immune dysregulation.Immunol Rev. 2011; 242: 233-246Crossref PubMed Scopus (746) Google Scholar, 2Jin H. He R. Oyoshi M. Geha R.S. Animal models of atopic dermatitis.J Invest Dermatol. 2009; 129: 31-40Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar The pathogenesis and development of AD are thought to be regulated by complex arrays of genes and environmental factors. Among the animal models of AD, NC/Nga mice commonly develop an eczematous AD-like skin lesion around the face under conventional conditions because of mite allergens.3Matsuda H. Watanabe N. Geba G.P. Sperl J. Tsudzuki M. Hiroi J. Matsumoto M. Ushio H. Saito S. Askenase P.W. Ra C. Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC/Nga mice.Int Immunol. 1997; 9: 461-466Crossref PubMed Scopus (680) Google Scholar Unlike the NC/Nga mouse, the Naruto Research Institute Otsuka Atrichia (NOA) mouse, a hair-deficient mutant, develops ulcerative skin lesions in a specific pathogen-free environment.4Kondo T. The NOA mouse: a new hair-deficient mutant (a possible animal model of allergic dermatitis).Mouse Genome. 1997; 95: 698-700Google Scholar In NOA mice, atopic lesions expand to cover a wide area of the animal's skin and are exacerbated by aging. These mouse models display AD-like symptoms, including itching, elevated serum IgE level, increased numbers of mast cells, eosinophils, and lymphocytes in the dermis, and a type 2 helper T-cell–biased immune response. These lines of evidence suggest that the animals are a useful model for both examining mechanisms of the pathogenesis and aggression and acquiring a fundamental basis for novel treatments of AD. Lysophosphatidic acid (LPA) is a physiologically important phospholipid mediator acting on a wide range of animal cells, including several inflammatory cells through G protein–coupled LPA receptors.5Tokumura A. A family of phospholipid autacoids: occurrence, metabolism and bioactions.Prog Lipid Res. 1995; 34: 151-184Crossref PubMed Scopus (166) Google Scholar, 6Choi J.W. Herr D.R. Noguchi K. Yung Y.C. Lee C.W. Mutoh T. Lin M.E. Teo S.T. Park K.E. Mosley A.N. Chun J. LPA receptors: subtypes and biological actions.Annu Rev Pharmacol Toxicol. 2010; 50: 157-186Crossref PubMed Scopus (649) Google Scholar LPA was shown to induce chemokinesis of Jurkat T cells7Zheng Y. Kong Y. Goetzl E.J. Lysophosphatidic acid receptor-selective effects on Jurkat T cell migration through a Matrigel model basement membrane.J Immunol. 2001; 166: 2317-2322Crossref PubMed Scopus (87) Google Scholar and to promote invasion and polarization of mouse lymphoma lines via binding to and activation of LPA receptors.8Stam J.C. Michiels F. Van der Kammen R.A. Moolenaar W.H. Collard J.G. Invasion of T-lymphoma cells: cooperation between Rho family GTPases and lysophospholipid receptor signaling.EMBO J. 1998; 17: 4066-4074Crossref PubMed Scopus (203) Google Scholar In addition, LPA was shown to enhance infiltration of guinea pig and mouse eosinophils into bronchoalveolar lavage fluid in vivo,9Hashimoto T. Yamashita M. Ohata H. Momose K. Lysophosphatidic acid enhances in vivo infiltration and activation of guinea pig eosinophils and neutrophils via a Rho/Rho-associated protein kinase-mediated pathway.J Pharmacol Sci. 2003; 91: 8-14Crossref PubMed Scopus (53) Google Scholar, 10Zhao Y. Tong J. He D. Pendyala S. Evgeny B. Chun J. Sperling A.I. Natarajan V. Role of lysophosphatidic acid receptor LPA2 in the development of allergic airway inflammation in a murine model of asthma.Respir Res. 2009; 10: 114Crossref PubMed Scopus (51) Google Scholar and to induce migration and oxygen radical production of human eosinophils in vitro.11Idzko M. Laut M. Panther E. Sorichter S. Dürk T. Fluhr J.W. Herouy Y. Mockenhaupt M. Myrtek D. Elsner P. Norgauer J. Lysophosphatidic acid induces chemotaxis, oxygen radical production, CD11b up-regulation, Ca2+ mobilization, and actin reorganization in human eosinophils via pertussis toxin-sensitive G proteins.J Immunol. 2004; 172: 4480-4485Crossref PubMed Scopus (52) Google Scholar Its circulating level is tightly controlled by a balance of LPA-producing with LPA-degrading enzyme activities in mammals.12Tokumura A. Metabolic pathways and physiological and pathological significances of lysolipid phosphate mediators.J Cell Biochem. 2004; 92: 869-881Crossref PubMed Scopus (47) Google Scholar, 13Ren H.M. Panchatcharam M. Mueller P. Escalante-Alcalde D. Morris A.J. Smyth S.S. Lipid phosphate phosphatase (LPP3) and vascular development.Biochim Biophys Acta. 2013; 1831: 126-132Crossref PubMed Scopus (37) Google Scholar In mice, the production of LPA in the blood is assumed to be largely due to the lysophospholipase D (lysoPLD) activity of autotaxin (ATX) because the plasma LPA level of heterozygous mice (ATX+/−) is approximately half that of wild-type (WT) mice.14Tanaka M. Okudaira S. Kishi Y. Ohkawa R. Iseki S. Ota M. Nojim 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 (385) Google Scholar Recently, Lundequist and Boyce15Lundequist A. Boyce J.A. LPA5 is abundantly expressed by human mast cells and important for lysophosphatidic acid induced MIP-1β release.PLoS One. 2011; 6: e18192Crossref PubMed Scopus (36) Google Scholar demonstrated that LPA5 is abundantly expressed by human mast cells and partially involved in LPA-induced calcium mobilization and macrophage inflammatory protein-1β release. In addition, LPA was shown to induce plasma exudation and proliferation of human mast cells.16Hashimoto T. Ohata H. Honda K. Lysophosphatidic acid (LPA) induces plasma exudation and histamine release in mice via LPA receptors.J Pharmacol Sci. 2006; 100: 82-87Crossref PubMed Scopus (33) Google Scholar, 17Bagga S. Price K.S. Lin D.A. Friend D.S. Austen K.F. Boyce J.A. Lysophosphatidic acid accelerates the development of human mast cells.Blood. 2004; 104: 4080-4087Crossref PubMed Scopus (63) Google Scholar The evidence allowed us to hypothesize that an increased concentration of circulating LPA is involved in the pathological accumulation of inflammatory cells to the skin lesion sites and contributes to the development of systemic AD. Heparinase III from Flavobacterium heparinum, EDTA-2K, and fatty acid–free bovine serum albumin (BSA) were from Sigma-Aldrich (St. Louis, MO). Heparin was from Wako Pure Chemicals Industries (Osaka, Japan). Dioctanoylglycerol pyrophosphate (8:0 DGPP), 1-pentadecanoyl-lysophosphatidylcholine (15:0-LPC), 1-palmitoyl-LPC (16:0), 1-heptadecanoyl-LPC (17:0), and sphingosine 1-phosphate (S1P; d17:1) were from Avanti Polar Lipids (Alabaster, AL). 1-Linoleoyl-LPC (18:2) was prepared from 1,2-dilinoleoyl-PC (Avanti Polar Lipids) with porcine pancreas phospholipase A2 (Sigma-Aldrich), as described previously.18Tanaka T. Ikita K. Ashida T. Motoyama Y. Yamaguchi Y. Satouchi K. Effects of growth temperature on the fatty acid composition of the free-living nematode Caenorhabditis elegans.Lipids. 1996; 31: 1173-1178Crossref PubMed Scopus (96) Google Scholar 1-Pentadecanoyl-LPA, 1-heptadecanoyl-LPA, and 1-linoleoyl-LPA were prepared from 1-pentadecanoyl-LPC, 1-heptadecanoyl-LPC, and 1-linoleoyl-LPC, respectively, with Streptomyces chromofuscus phospholipase D (PLD; Sigma-Aldrich), as described previously.19Tokumura A. Iimori M. Nishioka Y. Kitahara M. Sakashita M. Tanaka S. Lysophosphatidic acids induce proliferation of cultured vascular smooth muscle cells from rat aorta.Am J Physiol. 1994; 267: C204-C210PubMed Google Scholar 1-Oleoyl-18:1-lysophosphatidylmethanol (LPM) was prepared from 1-oleoyl-LPC (Avanti Polar Lipids) by transphosphatidylation with Actinomadura sp. PLD (Seikagaku Biobusiness, Tokyo, Japan).20Kokusho Y. Kato S. Machida H. Iwasaki S. Purification and properties of phospholipase D from Actinomadura sp. strain No. 362.Agric Biol Chem. 1987; 51: 2515-2524Crossref Scopus (24) Google Scholar In brief, 18:1-LPC (15 mg) was dissolved in 2 mL of diethyl ether, 0.5 mL of 10% methanol, and 0.2 mL of 0.22 mol/L CaCl2. The reaction was started by adding 20 U of PLD dissolved in 0.4 mL of 0.2 mol/L sodium acetate. The reaction mixture was stirred vigorously for 3 hours at 65°C. The product, 18:1-LPM, was separated on a silica gel thin-layer plate with a mixture of chloroform/methanol/H2O (65:35:5, v/v/v), and extracted from the corresponding zone. 1-Linolenoyl-LPA (18:3) was prepared from 1,2-dilinolenoyl-PC (Avanti Polar Lipids) with phospholipase A2 and PLD reactions, as previously described. S32826 was from Calbiochem (San Diego, CA) and Cayman Chemical (Ann Arbor, MI). HA130 was from Calbiochem. Ki16425 was from Cayman Chemical. Histamine dihydrochloride and naloxone hydrochloride were from Alexis-Enzo Life Sciences (Farmingdale, NY). The NOA mouse, a hair-deficient mutant, was established as an inbred strain in 1997 by Kondo.4Kondo T. The NOA mouse: a new hair-deficient mutant (a possible animal model of allergic dermatitis).Mouse Genome. 1997; 95: 698-700Google Scholar The NC/Nga mice were established as an inbred strain by Kondo et al21Kondo K. Differences in hematopoietic death among inbred strains of mice.in: Bond V.P. Sugahara T. Comparative Cellular and Species Radiosensitivity. Igaku Shoin, Tokyo1969: 20-22Google Scholar on the basis of Japanese fancy mice. Male BALB/c and NOA mice obtained from Clea Japan (Tokyo) were housed under specific pathogen-free conditions. Male NC/Nga mice were obtained from Japan SLC (Shizuoka) and housed under conventional conditions. Female Institute of Cancer Research (ICR) mice were obtained from Japan SLC and housed under specific pathogen-free conditions. Mice were given free access to a standard chow (DC-8; Clea Japan) and water. Mice were handled in accordance with the Guidelines for Animal Experimentation of the Tokushima University School of Medicine (Tokushima, Japan), and all animal experiments were approved by the Tokushima University Animal Experiment Committee. Mouse blood was collected from the vena cava or eye socket after 12 hours of fasting. Mouse blood anticoagulated with 1 mg/mL EDTA-2K or 3 U/mL heparin was centrifuged at 1200 × g for 25 minutes at 4°C to obtain the plasma and hemocyte fraction. Mouse platelet-rich plasma (PRP) was prepared by centrifugation of blood at 50 × g for 20 minutes at 20°C. Hematological variables, such as the white blood cell (WBC) count, red blood cell count, hemoglobin concentration (g/dL), hematocrit percentage, mean cell volume (fL), mean cell hemoglobin (pg), mean cell hemoglobin concentration (g/dL), and platelet count, were recorded by an automated hematological analyzer (Celltac-α MEK-6358; Nihon Kohden, Tokyo). Subpopulation analysis of WBCs was performed using the mouse-specified profile on a veterinary hematological analyzer (Sysmex XT-2000iV; Sysmex Corp, Kobe, Japan). LPC, LPA, and S1P were extracted from EDTA-treated mouse plasma using a modified Bligh and Dyer method. In brief, 0.1 mL of the plasma was vortex mixed with a mixture of chloroform-methanol-water (1:2:0.6, v/v/v; 1.8 mL) for 1 minute. Specific amounts of internal standards (17:0-LPA, 17:0-LPC, and C17-S1P) were added to the mixture. The mixtures were separated into two phases by addition of 0.5 mL each of chloroform and water containing 20 mg KCl. After adjusting the pH of the aqueous phase to 2 to 3 with 5 N HCl, the chloroform-rich lower layer was separated and dried under a stream of N2 gas. Extracted lipids were reconstituted with a small volume of solvent used as a mobile phase for reverse-phase liquid chromatography (LC) and analyzed by an LC-tandem mass spectrometry (MS/MS) system with 4000Q-TRAP (AB SCIEX, Foster City, CA) equipped with an Agilent 1100 high-performance LC pump (Agilent Technologies, Waldbronn, Germany) and an HTS-PAL autosampler (CTC Analytics, Zwingen, Switzerland). LPCs in the lipid extract were separated on an Ascentis Express C18 column (2.7 μm, 2.1 × 150 mm; Supelco, Bellefonte, PA) developed with an isocratic solvent system of methanol-water mixture (19:1, v/v) containing 5 mmol/L ammonium formate at 0.2 mL/minute. Acidic polar phospholipids, such as LPA and S1P, in the lipid extract were separated on an ODS-100Z column (5 μm, 2.0 × 150 mm; Tosoh, Tokyo) developed with an isocratic solvent system of methanol-water mixture (19:1, v/v) containing 5 mmol/L ammonium formate at 0.2 mL/minute. For quantification, LPC was analyzed by positive-ion electrospray ionization (ESI) with multiple reaction monitoring of parent [(M + H)+]/daughter [(phosphocholine)+] at m/z 184, where M is the molecular weight of the parent species. LPAs were quantified in ESI− with multiple reaction monitoring of parent [(M−H)−]/daughter ions [(cyclic glycerophosphate)−] at m/z 153. S1P was analyzed in ESI− mode using a combination of deprotonated molecular ions and a fragmented ion at m/z 79 (PO3−). Quantification was accomplished by referencing peak areas to those of the internal standards. Analytical conditions for MS/MS were described previously.22Tokumura A. Carbone L.D. Yoshioka Y. Morishige J. Kikuchi M. Postlethwaite A. Watsky M.A. Elevated serum levels of arachidonoyl-lysophosphatidic acid and sphingosine 1-phosphate in systemic sclerosis.Int J Med Sci. 2009; 6: 168-176Crossref PubMed Scopus (110) Google Scholar Plasma from 0.1 mL heparin-anticoagulated blood samples was added to a tube in which 15:0-LPA from an aliquot of stock solution had been dried. The final concentration of 15:0-LPA was 0.002 mmol/L. The plasma sample was gently vortex mixed and incubated for 12 or 24 hours at 37°C. After incubation, 200 pmol 17:0-LPA was added to the incubation mixture as an internal standard, and lipids, including unmetabolized 15:0-LPA, were extracted by the method of Bligh and Dyer after acidification of the aqueous phase to pH 2 to 3. 15:0-LPA in the lipid extract was quantified in reference to the internal standard (17:0-LPA) by LC-MS/MS, as previously described. Heparin-anticoagulated blood samples (0.1 mL) were added to the tube with 15:0- or 18:3-LPA at a final concentration of 0.06 mmol/L. The samples were gently vortex mixed and incubated for 1 or 10 minutes at 37°C. After incubation, 6000 pmol 17:0-LPA was added to each sample as an internal standard. Unmetabolized 15:0- or 18:3-LPA was extracted from the incubated mixture by the method of Bligh and Dyer after acidification of the aqueous phase (pH 2 to 3), and followed by LC-MS/MS of unmetabolized LPA, as previously described. LysoPLD activity was assayed essentially as described previously.23Tokumura A. Majima E. Kariya Y. Tominaga K. Kogure K. Yasuda K. Fukuzawa K. Identification of human plasma lysophospholipase D, a lysophosphatidic acid-producing enzyme, as autotaxin, a multifunctional phosphodiesterase.J Biol Chem. 2002; 277: 39436-39442Crossref PubMed Scopus (610) Google Scholar First, 0.04 mL heparin-anticoagulated venous blood or plasma sample was diluted with 0.098 mL saline. Second, 0.06 mL of 16:0- or 18:2-LPC solution (0.167 or 0.5 mmol/L), dispersed in saline buffer containing 0.25% fatty acid–free BSA, was added to the diluted blood or plasma sample. The final concentrations of LPC were 0.05 or 0.15 mmol/L. Third, 0.002 mL of dimethyl sulfoxide (DMSO) solution of an ATX inhibitor (S32826 or HA130) or vehicle alone was added to the mixture. The final concentration of DMSO was 1%. The mixture was incubated for various times at 37°C. After incubation, it was centrifuged at 8250 × g for 1 minute at 4°C, and 0.2 mL of 7.5 mmol/L 3-(4-hydroxyphenyl)propionic acid, 2.6 mL 0.1 mol/L Tris-HCl (pH 8.5), and 0.1 mL 2 U/mL horseradish peroxidase were added to 0.1-mL aliquots of the reaction mixture. After the addition of 0.01 mL of 300 U/mL choline oxidase solution, λex fluorescence was determined at 320 nm and λem at 404 nm. For determination of ATX antigen levels, 0.03 mL of 10-fold diluted plasma, blood, or hemocyte fraction samples was mixed with 0.01 mL of sample-loading buffer (250 mmol/L Tris-HCl, pH 6.8, 8% SDS, 5% 2-mercaptoethanol, 40% glycerol, and a trace amount of bromophenol blue), heated at 90°C for 5 minutes, and separated by 10% SDS-PAGE. Separated proteins in the gels were electrophoretically transferred onto polyvinylidene difluoride membranes (ATTO, Tokyo) at 108 mA for 90 minutes. After blocking the membranes with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20, membranes were incubated with an antibody raised against human ATX (3D1, 150-fold diluted) overnight at room temperature, as described previously.24Tanaka M. Kishi Y. Takanezawa Y. Kakehi Y. Aoki J. Arai H. Prostatic acid phosphatase degrades lysophosphatidic acid in seminal plasma.FEBS Lett. 2004; 571: 197-204Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar After washing with Tris-buffered saline containing 0.05% Tween 20, the membrane was incubated with horseradish peroxidase–conjugated goat anti-rat IgG (American Qualex, San Clemente, CA; diluted 2000-fold) for 2 hours. After washing, the immunoreactive bands were detected by enhanced chemiluminescence Prime Western blot detection reagent (GE Healthcare Life Sciences, Piscataway, NJ) with an LAS-4000 mini image analyzer (Fujifilm, Tokyo). The intensity of the band was quantified with use of the software Multi Gauge, version 3.2 (Fujifilm). The scratching behavior with 5- to 6-week-old female ICR mice was observed according to the method described.25Andoh T. Nagasawa T. Satoh M. Kuraishi Y. Substance P induction of itch-associated response mediated by cutaneous NK1 tachykinin receptors in mice.J Pharmacol Exp Ther. 1998; 286: 1140-1145PubMed Google Scholar, 26Inagaki N. Nagao M. Igeta K. Kawasaki H. Kim J.F. Nagai H. Scratching behavior in various strains of mice.Skin Pharmacol Appl Skin Physiol. 2001; 14: 87-96Crossref PubMed Scopus (95) Google Scholar, 27Hashimoto T. Ohata H. Momose K. Itch-scratch responses induced by lysophosphatidic acid in mice.Pharmacology. 2004; 72: 51-56Crossref PubMed Scopus (66) Google Scholar The hair was clipped over the rostral part of the back 1 day before intradermal injection of the pruritogen. On the test day, mice were individually placed in a 12 × 20 × 16-cm observation cage to acclimate them for approximately 60 minutes. After acclimation, 50, 100, or 150 nmol per site histamine, 150 nmol per site 18:2-LPA, or 150 nmol per site 18:1-LPM was injected intradermally, without anesthesia, in a volume of 0.05 mL of saline containing 0.25% fatty acid–free BSA. Mice were held in a mouse holder (KN-330; Natsume Seisakusho Co, Ltd, Tokyo) during injection to prevent struggling. Immediately after injection, the mice were put back into the observation cage and videotaped with no one present. Usually, mice scratch with their paws several times a second; therefore, a series of these scratches was counted as one scratch event. Naloxone hydrochloride (1 mg/kg) was s.c. administered near the injection site 10 to 12 minutes before pruritogen addition, in a volume of 0.05 mL.28Yamamoto A. Sugimoto Y. Involvement of peripheral mu opioid receptors in scratching behavior in mice.Eur J Pharmacol. 2010; 649: 336-341Crossref PubMed Scopus (24) Google Scholar DGPP (1 mg/kg) and Ki16425 (1 mg/kg) were co-administered with the pruritogen. DGPP and naloxone hydrochloride were dissolved in saline containing 0.25% fatty acid–free BSA. Ki16425 was dissolved in ethanol and diluted with saline containing 0.25% fatty acid–free BSA. Statistical evaluation of the data was performed by one-way analyses of variance for independent or correlated samples, followed by Tukey's test or Student's t-test for paired and correlated samples. P < 0.05 was considered statistically significant. To examine the involvement of circulating LPA in the pathogenesis of AD-like skin lesions, we measured the plasma level of LPA in young mice (aged 5 weeks, no lesions) and old mice (aged>14 weeks, 3 to 5 months, severe lesions). Although no significant difference in the plasma levels of LPA was observed in 5-week-old WT controls (BALB/c) and NOA mice (Figure 1A), NOA mice developed an age-dependent increase in the plasma level of LPA versus WT mice (1394 ± 195 versus 656 ± 19.6 pmol/mL; P < 0.05), and >14-week-old NC/Nga mice have a lower plasma level of LPA than age-matched WT mice (Figure 1B). Molecular species analysis by LC-MS/MS showed that the plasma levels of 16:1-, 18:2-, 20:5-, and 22:6-LPAs were significantly higher in NOA mice (aged>14 weeks) than in WT mice (aged>14 weeks). However, there were no significant differences in the levels of other LPA species (Figure 1C). We also analyzed the plasma levels of S1P, another important member of the lysophospholipid mediator family, and its dihydro-form, sphinganine 1-phosphate. The plasma level of dihydro-S1P in NOA mice (aged>14 weeks) was lower than that of WT mice (aged>14 weeks). However, there were no differences in the plasma level of S1P (Figure 1D). Interestingly, plasma levels of S1P and dihydro-S1P in NC/Nga mice were lower than those in NOA and WT mice (Figure 1D). Because the plasma level of LPA was significantly higher in NOA mice than in WT mice, we speculated that activities of LPA-degrading enzymes [soluble lysophospholipase A (LPL) and membrane-bound lipid phosphate phosphatase (LPP)] in the blood circulation of NOA mice were lower than those of WT mice. We estimated blood cell–associated and soluble LPA-degrading activities by measuring the degradation rate of exogenous 15:0-LPA (or 18:3-LPA) after its incubation with diluted whole blood or plasma. As shown in Figure 2A , there was no difference between the soluble LPL activities toward 15:0-LPA in NOA and WT mice. Unexpectedly, LPP activity in the whole blood of NOA mice was significantly higher than that of WT mice (Figure 2B). Interestingly, LPP activity toward 18:3-LPA was significantly lower than that toward 15:0-LPA at some time points, as shown in Figure 2B, indicating the substrate preference of the LPP in mouse blood for saturated LPAs over unsaturated LPAs. Because our results indicated that the higher plasma LPA level in aged NOA mice compared with that of aged WT mice was not due to increased LPA-degrading activity, we next measured the LPA-producing activity. LPA in the circulating blood of mice is produced from lysophospholipids, such as LPC, by lysoPLD activity of ATX.29Aoki J. Taira A. Takanezawa Y. Kishi Y. Hama K. Kishimoto T. Mizuno K. Saku K. Taguchi R. Arai H. Serum lysophosphatidic acid is produced through diverse phospholipase pathways.J Biol Chem. 2002; 277: 48737-48744Crossref PubMed Scopus (361) Google Scholar First, we measured the plasma level of LPC, a physiological precursor of LPA, by LC-MS/MS. Although no significant difference in the plasma levels of LPC was observed in WT and NOA mice at 5 weeks of age (Figure 3A), NOA mice developed an age-dependent decrease in the plasma level of LPC versus WT mice (273 ± 22.8 versus 449 ± 23.4 nmol/mL; P 14 weeks) were significantly lower than those of WT mice (aged>14 weeks) (Figure 3C). Similar results were obtained for NC/Nga mice (Figure 3, B and C). Consistent with these results, activities of lysoPLD toward endogenous LPCs in the diluted plasma in NOA and NC/Nga mice were significantly lower than those of WT mice (Figure 3D). However, no significant differences were found in the lysoPLD activities toward exogenous 0.15 mmol/L 16:0-LPC between NOA and WT mice and 18:2-LPC among NOA, NC/Nga, and WT mice (Figure 3D). Although the plasma LPA level was greatly increased in aged NOA mice with severe lesions compared with aged WT mice, reduced and unaltered soluble lysoPLD activity of ATX was observed toward endogenous and exogenous LPCs, respectively, in NOA mice compared with WT mice. Recently, ATX was shown to bind to cells via cell-surface molecules, including integrins30Fulkerson Z. Wu T. Sunkara M. Kooi C.V. Morris A.J. Smyth S.S. Binding of autotaxin to integrins localizes lysophosphatidic acid production to platelets and mammalian cells.J Biol Chem. 2011; 286: 34654-34663Crossref PubMed Scopus (106) Google Scholar, 31Kanda H. Newton R. Klein R. Morita Y. Gunn M.D. Rosen S.D. Autotaxin, an ectoenzyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary lymphoid organs.Nat Immunol. 2008; 9: 415-423Crossref PubMed Scopus (211) Google Scholar and heparan sulfate proteoglycans (HSPGs).32Houben A.J. van Wijk X.M. van Meeteren L.A. van Zeijl L. van de Westerlo E.M. Hausmann J. Fish A. Perrakis A. van Kuppevelt T.H. Moolenaar W.H. The polybasic insertion in autotaxin α confers specific binding to heparin and cell surface heparan sulfate proteoglycans.J Biol Chem. 2013; 288: 510-519Crossref PubMed Scopus (42) Google Scholar To determine whether ATX also acts as a membrane-bound form in the mouse blood circulation, we first measured the time dependency of lysoPLD activity with the whole blood of a WT mouse. The lysoPLD activity toward exogenous 0.15 mmol/L 18:2-LPC in the blood was higher than those in plasma and blood incubated with 0.15 and 0.05 mmol/L 18:2-LPC, respectively (Figure 4A). Consistent with human plasma and fetal calf serum lysoPLD activity,29Aoki J. Taira A. Takanezawa Y. Kishi Y. Hama K. Kishimoto T. Mizuno K. Saku K. Taguchi R. Arai H. Serum lysophosphatidic acid is produced through diverse phospholipase pathways.J Biol Chem. 2002; 277: 48737-48744Crossref PubMed Scopus (361) Google Scholar, 33Tokumura A. Miyake M. Yoshimoto O. Shimizu M. Fukuzawa K. Metal-ion stimulation and inhibition of lysophospholipase D which generates bioactive lysophosphatidic acid in rat plasma.Lipids. 1998; 33: 1009-1015Crossref PubMed Scopus (52) Google Scholar addition of a divalent cation chelator, such as EDTA, to the diluted blood almost completely inhibited the blood lysoPLD activity (Figure 4B). Co-addition of Ca2+, Mn2+, Co2+, Zn2+, Ni2+, or Cu2+ with EDTA to diluted plasma or blood at equimolar concentrations masked the inhibitory effect of EDTA, whereas Ba2+ and Mg2+ failed to do so (Figure 4B). The blood lysoPLD activity in NOA mice toward 16:0- or 18:2-LPC for 6 hours was significantly higher than that in WT mice (Figure 4C). In addition, the blood lysoPLD activity toward 18:2-LPC was significantly higher than that toward 16:0-LPC in both NOA and WT mice (Figure 4C). LysoPLD activity in the plasma from WT was inhibited in a dose-dependent manner by the addition of the ATX-selective inhibitor HA130 or S32826 (Figure 4D). Although the lysoPLD activities in th
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