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• W2040300892 abstract "Sphingosine-1-phosphate (S1P) lyase catalyzes the degradation of S1P, a potent signaling lysosphingolipid. Mice with an inactive S1P lyase gene are impaired in the capacity to degrade S1P, resulting in highly elevated S1P levels. These S1P lyase-deficient mice have low numbers of lymphocytes and high numbers of neutrophils in their blood. We found that the S1P lyase-deficient mice exhibited features of an inflammatory response including elevated levels of pro-inflammatory cytokines and an increased expression of genes in liver associated with an acute-phase response. However, the recruitment of their neutrophils into inflamed tissues was impaired and their neutrophils were defective in migration to chemotactic stimulus. The IL-23/IL-17/granulocyte-colony stimulating factor (G-CSF) cytokine-controlled loop regulating neutrophil homeostasis, which is dependent on neutrophil trafficking to tissues, was disturbed in S1P lyase-deficient mice. Deletion of the S1P4 receptor partially decreased the neutrophilia and inflammation in S1P lyase-deficient mice, implicating S1P receptor signaling in the phenotype. Thus, a genetic block in S1P degradation elicits a pro-inflammatory response but impairs neutrophil migration from blood into tissues. Sphingosine-1-phosphate (S1P) lyase catalyzes the degradation of S1P, a potent signaling lysosphingolipid. Mice with an inactive S1P lyase gene are impaired in the capacity to degrade S1P, resulting in highly elevated S1P levels. These S1P lyase-deficient mice have low numbers of lymphocytes and high numbers of neutrophils in their blood. We found that the S1P lyase-deficient mice exhibited features of an inflammatory response including elevated levels of pro-inflammatory cytokines and an increased expression of genes in liver associated with an acute-phase response. However, the recruitment of their neutrophils into inflamed tissues was impaired and their neutrophils were defective in migration to chemotactic stimulus. The IL-23/IL-17/granulocyte-colony stimulating factor (G-CSF) cytokine-controlled loop regulating neutrophil homeostasis, which is dependent on neutrophil trafficking to tissues, was disturbed in S1P lyase-deficient mice. Deletion of the S1P4 receptor partially decreased the neutrophilia and inflammation in S1P lyase-deficient mice, implicating S1P receptor signaling in the phenotype. Thus, a genetic block in S1P degradation elicits a pro-inflammatory response but impairs neutrophil migration from blood into tissues. Sphingosine 1-phosphate (S1P) 3The abbreviations used are: S1P, sphingosine-1-phosphate; DKO, double knockout; P6, postnatal day 6; P18, postnatal day 18; RT-qPCR, real-time-quantitative PCR; APC, allophycocyanin; PerCP, peridinin chlorophyll protein; PE, phycoerythrin; MCP, monocyte chemoattractant protein; G-CSF, granulocyte-colony stimulating factor; GO, gene ontology; CBA, Cytometric Bead Array; MFI, mean fluorescence intensity. is a sphingolipid signaling molecule that exerts important physiologic functions through its interaction with a family of G protein-coupled receptors (S1P1–5) (1Rosen H. Goetzl E.J. Nat. Rev. Immunol. 2005; 5: 560-570Crossref PubMed Scopus (623) Google Scholar, 2El Alwani M. Wu B.X. Obeid L.M. Hannun Y.A. Pharmacol. Ther. 2006; 112: 171-183Crossref PubMed Scopus (136) Google Scholar, 3Kono M. Allende M.L. Proia R.L. Biochim. Biophys. Acta. 2008; 1781: 435-441Crossref PubMed Scopus (50) Google Scholar, 4Rivera J. Proia R.L. Olivera A. Nat. Rev. Immunol. 2008; 8: 753-763Crossref PubMed Scopus (520) Google Scholar). S1P is synthesized by the phosphorylation of sphingosine by either of two sphingosine kinases, Sphk1 and Sphk2 (5Kim R.H. Takabe K. Milstien S. Spiegel S. Biochim. Biophys. Acta. 2009; 1791: 692-696Crossref PubMed Scopus (153) Google Scholar, 6Hannun Y.A. Obeid L.M. Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2484) Google Scholar). After its formation, S1P may be either dephosphorylated back to sphingosine by the action of two specific S1P phosphatases, Sgpp1 and Sgpp2, or permanently degraded by the S1P lyase Sgpl1 to the nonsphingolipid substrates hexadecenal and phosphoethanolamine (7Fyrst H. Saba J.D. Nat. Chem. Biol. 2010; 6: 489-497Crossref PubMed Scopus (267) Google Scholar). Alternatively, S1P can be exported out of the cell where it is able to interact with the S1P receptors (5Kim R.H. Takabe K. Milstien S. Spiegel S. Biochim. Biophys. Acta. 2009; 1791: 692-696Crossref PubMed Scopus (153) Google Scholar, 8Skoura A. Hla T. J. Lipid Res. 2009; 50: S293-S298Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). S1P is found highly enriched in the circulation-in both blood and lymph-while its concentration in tissues remains very low by comparison, as the result of the combined synthetic and degradative activities involved in the S1P biosynthetic pathway (9Hla T. Venkataraman K. Michaud J. Biochim. Biophys. Acta. 2008; 1781: 477-482Crossref PubMed Scopus (153) Google Scholar). Blood S1P is produced by erythrocytes (10Hänel P. Andréani P. Gräler M.H. FASEB J. 2007; 21: 1202-1209Crossref PubMed Scopus (311) Google Scholar, 11Ito K. Anada Y. Tani M. Ikeda M. Sano T. Kihara A. Igarashi Y. Biochem. Biophys. Res. Commun. 2007; 357: 212-217Crossref PubMed Scopus (159) Google Scholar, 12Pappu R. Schwab S.R. Cornelissen I. Pereira J.P. Regard J.B. Xu Y. Camerer E. Zheng Y.W. Huang Y. Cyster J.G. Coughlin S.R. Science. 2007; 316: 295-298Crossref PubMed Scopus (735) Google Scholar), but other cells such as mast cells and platelets can also secrete S1P (13Yatomi Y. Ohmori T. Rile G. Kazama F. Okamoto H. Sano T. Satoh K. Kume S. Tigyi G. Igarashi Y. Ozaki Y. Blood. 2000; 96: 3431-3438Crossref PubMed Google Scholar, 14Jolly P.S. Bektas M. Watterson K.R. Sankala H. Payne S.G. Milstien S. Spiegel S. Blood. 2005; 105: 4736-4742Crossref PubMed Scopus (55) Google Scholar). Endothelial cells can contribute to the circulating S1P pool (15Venkataraman K. Thangada S. Michaud J. Oo M.L. Ai Y. Lee Y.M. Wu M. Parikh N.S. Khan F. Proia R.L. Hla T. Biochem. J. 2006; 397: 461-471Crossref PubMed Scopus (173) Google Scholar, 16Venkataraman K. Lee Y.M. Michaud J. Thangada S. Ai Y. Bonkovsky H.L. Parikh N.S. Habrukowich C. Hla T. Circ. Res. 2008; 102: 669-676Crossref PubMed Scopus (389) Google Scholar) and are likely responsible for the S1P that is present in the lymph (12Pappu R. Schwab S.R. Cornelissen I. Pereira J.P. Regard J.B. Xu Y. Camerer E. Zheng Y.W. Huang Y. Cyster J.G. Coughlin S.R. Science. 2007; 316: 295-298Crossref PubMed Scopus (735) Google Scholar). The proper compartmentalization of S1P in circulation and in tissues is important for the trafficking and positioning of lymphocytes. The egress of lymphocytes out of primary and secondary lymphoid organs is dependent on S1P receptors on lymphocytes (17Allende M.L. Dreier J.L. Mandala S. Proia R.L. J. Biol. Chem. 2004; 279: 15396-15401Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 18Matloubian M. Lo C.G. Cinamon G. Lesneski M.J. Xu Y. Brinkmann V. Allende M.L. Proia R.L. Cyster J.G. Nature. 2004; 427: 355-360Crossref PubMed Scopus (2091) Google Scholar, 19Kabashima K. Haynes N.M. Xu Y. Nutt S.L. Allende M.L. Proia R.L. Cyster J.G. J. Exp. Med. 2006; 203: 2683-2690Crossref PubMed Scopus (162) Google Scholar, 20Walzer T. Chiossone L. Chaix J. Calver A. Carozzo C. Garrigue-Antar L. Jacques Y. Baratin M. Tomasello E. Vivier E. Nat. Immunol. 2007; 8: 1337-1344Crossref PubMed Scopus (322) Google Scholar, 21Allende M.L. Zhou D. Kalkofen D.N. Benhamed S. Tuymetova G. Borowski C. Bendelac A. Proia R.L. FASEB J. 2008; 22: 307-315Crossref PubMed Scopus (57) Google Scholar, 22Pereira J.P. Xu Y. Cyster J.G. PLoS One. 2010; 5: e9277Crossref PubMed Scopus (83) Google Scholar, 23Allende M.L. Tuymetova G. Lee B.G. Bonifacino E. Wu Y.P. Proia R.L. J. Exp. Med. 2010; 207: 1113-1124Crossref PubMed Scopus (134) Google Scholar), which recognize the higher concentrations of S1P around exit points leading to the blood and lymph (12Pappu R. Schwab S.R. Cornelissen I. Pereira J.P. Regard J.B. Xu Y. Camerer E. Zheng Y.W. Huang Y. Cyster J.G. Coughlin S.R. Science. 2007; 316: 295-298Crossref PubMed Scopus (735) Google Scholar, 24Schwab S.R. Pereira J.P. Matloubian M. Xu Y. Huang Y. Cyster J.G. Science. 2005; 309: 1735-1739Crossref PubMed Scopus (654) Google Scholar, 25Zachariah M.A. Cyster J.G. Science. 2010; 328: 1129-1135Crossref PubMed Scopus (157) Google Scholar). When the activity of S1P lyase is inhibited or deleted as in the S1P lyase-knock-out (Sgpl1−/−) mice, compartmental S1P concentrations are altered, blocking lymphocyte egress and resulting in lymphophenia (24Schwab S.R. Pereira J.P. Matloubian M. Xu Y. Huang Y. Cyster J.G. Science. 2005; 309: 1735-1739Crossref PubMed Scopus (654) Google Scholar, 26Vogel P. Donoviel M.S. Read R. Hansen G.M. Hazlewood J. Anderson S.J. Sun W. Swaffield J. Oravecz T. PLoS One. 2009; 4: e4112Crossref PubMed Scopus (138) Google Scholar, 27Weber C. Krueger A. Münk A. Bode C. Van Veldhoven P.P. Gräler M.H. J. Immunol. 2009; 183: 4292-4301Crossref PubMed Scopus (51) Google Scholar). In S1P lyase-deficient mice, neutrophils are highly elevated in blood, in contrast to their low lymphocyte numbers (26Vogel P. Donoviel M.S. Read R. Hansen G.M. Hazlewood J. Anderson S.J. Sun W. Swaffield J. Oravecz T. PLoS One. 2009; 4: e4112Crossref PubMed Scopus (138) Google Scholar). Here we report that the S1P lyase-null mice have an elevated pro-inflammatory response with impaired migration of neutrophils into tissues resulting in an abnormal neutrophil homeostatic regulatory loop. These results implicate S1P lyase activity as a regulator of inflammatory responses and neutrophil trafficking. Sgpl1−/− mice were obtained from Philip Soriano, Mount Sinai School of Medicine, New York, and have been described previously (28Schmahl J. Raymond C.S. Soriano P. Nat. Genet. 2007; 39: 52-60Crossref PubMed Scopus (158) Google Scholar, 29Bektas M. Allende M.L. Lee B.G. Chen W. Amar M.J. Remaley A.T. Saba J.D. Proia R.L. J. Biol. Chem. 2010; 285: 10880-10889Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). LysMcre mice, S1pr4+/− mice and Tlr4−/− mice were obtained from The Jackson Laboratory, Bar Harbor, ME. The Sgpl1−/− and control Sgpl1+/+ mice were generated from Sgpl1+/− matings. To specifically delete the S1P1 receptor from granulocytes and macrophages, we established S1Pr1fl/fl mice (30Allende M.L. Yamashita T. Proia R.L. Blood. 2003; 102: 3665-3667Crossref PubMed Scopus (319) Google Scholar) carrying a lysozyme promoter-driven Cre recombinase transgene (Gr-S1pr1KO mice) derived from LysMcre mice (31Clausen B.E. Burkhardt C. Reith W. Renkawitz R. Förster I. Transgenic Res. 1999; 8: 265-277Crossref PubMed Scopus (1592) Google Scholar). The following double knock-out (DKO) mice were produced through cross-breeding: Sgpl1−/− Gr-S1pr1KO, Sgpl1−/− S1pr4−/− and Sgpl1−/− Tlr4−/−. Because Sgpl1−/− mice die around 3 to 5 weeks of age (26Vogel P. Donoviel M.S. Read R. Hansen G.M. Hazlewood J. Anderson S.J. Sun W. Swaffield J. Oravecz T. PLoS One. 2009; 4: e4112Crossref PubMed Scopus (138) Google Scholar, 28Schmahl J. Raymond C.S. Soriano P. Nat. Genet. 2007; 39: 52-60Crossref PubMed Scopus (158) Google Scholar, 29Bektas M. Allende M.L. Lee B.G. Chen W. Amar M.J. Remaley A.T. Saba J.D. Proia R.L. J. Biol. Chem. 2010; 285: 10880-10889Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), all mice were analyzed at postnatal day 18 (P18) unless specified. Mice were housed in a clean conventional facility that excluded specific mouse pathogens. Mice were genotyped by multiplex PCR from tail snips using the set of primers and conditions for each mouse line listed below. Sgpl1: 5′-CGCTCAGAAGGCTCTGAGTCATGG-3′, 5′-CATCAAGGAAACCCTGGACTACTG-3′, 5′-CCAAGTGTACCTGCTAAGTTCCAG-3′; conditions were previously described (29Bektas M. Allende M.L. Lee B.G. Chen W. Amar M.J. Remaley A.T. Saba J.D. Proia R.L. J. Biol. Chem. 2010; 285: 10880-10889Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). S1pr1loxP: 5′-GAGCGGAGGAAGTTAAAAGTG-3′, 5′-CCTCCTAAGAGATTGCAGCAA-3′; conditions were previously described (30Allende M.L. Yamashita T. Proia R.L. Blood. 2003; 102: 3665-3667Crossref PubMed Scopus (319) Google Scholar). Cre: 5′-GCCTGCATTACCGGTCGATGC-3′, 5′-CAGGGTGTTATAAGCAATCCC-3′; the following conditions were used: denaturation, 94 °C × 5 min; amplification, 94 °C × 1 min, 60 °C × 1 min, 72 °C × 1 min (35 cycles); extension, 72 °C × 3 min. The Cre allele produces a band of about 500 bp. S1pr4: 5′-CCCCGTAGAGGCTCAGGATAGCCAC-3′, 5′-GGCCTACGTGGTCAACGTGCTGC-3′. 5′-GACGAGTTCTTCTGAGGGGATCGATC-3′; the following conditions were used: denaturation, 94 °C × 5 min; amplification, 94 °C × 1 min, 60 °C × 30 s, 72 °C × 1.5 min (35 cycles); extension, 72 °C × 2 min. The S1pr4+/+ allele produces a band of about 380 bp and the S1pr4−/− allele produces a band of 600 bp. Tlr4: 5′-CAGGGTTGTACTTTAGGAGAGAGAGAAAGC-3′, 5′-GCTGCCCGGATCATCCAGG-3′, 5′-CCACCCATATTGCCTATACTCATTAGTTG-3′, 5′-GCCATGCCATGCCTTGTCTTCA-3′; the following conditions were used: denaturation, 95 °C × 10 min; amplification, 95 °C × 30 s, 57 °C × 1 min, 72 °C × 1 min (40 cycles); extension, 72 °C × 7 min. The Tlr4+/+ allele produces a band of about 410 bp and the Tlr4−/− allele produces a band of 290 bp. Total bone marrow cells were isolated from Sgpl1+/+ and Sgpl1−/− mice by flushing the femur and tibia from both legs two times with 1 ml of PBS. Cells were injected i.v. into lethally irradiated Rag2−/− mice (Taconic, Germantown, NY). Transplanted mice were analyzed 8 weeks after the procedure. Sgpl1+/+, and Sgpl1−/− mice were injected intraperitoneal with 10 mg/kg of body weight of LPS from Escherichia coli 055:B5 (Sigma-Aldrich) and observed for 5 days. In other experiments, mice were euthanized 60, 90, and 120 min after LPS injection. Sgpl1+/+ and Sgpl1−/− mice were injected intraperitoneal with 300 μl of 4% thioglycollate (Sigma-Aldrich). After 4 h, mice were euthanized and blood collected by heart puncture. Cells that were recruited into the peritoneal cavity were collected by lavage using 1 ml of ice-cold PBS three times. Total bone marrow cells were isolated from mice by flushing the femur and tibia from both legs two times with 1 ml of PBS. To obtain total leukocytes, spleen and mesenteric lymph nodes were dissected and mechanically disaggregated. Single-cell suspensions were obtained using a 40-μm cell strainer. Peripheral blood was obtained by cardiac puncture. Red blood cells were removed from blood samples and splenic single-cell suspensions by ammonium chloride lysis. For some experiments, neutrophils were sorted from total bone marrow cells using anti-Ly-6G magnetic microbeads (Miltenyi Biotec, Auburn, CA). The absolute number of each cell subpopulation was determined by flow cytometry using CALTAG counting beads (Invitrogen, Carlsbad, CA). For bone marrow and spleen, the absolute cell counts were normalized by the individual body weight expressed in grams. Alternatively, blood cell counts were determined in the Department of Laboratory Medicine at the National Institutes of Health. Tissues from Sgpl1+/+ and Sgpl1−/− mice were fixed and embedded in paraffin. Sections were stained with H&E. A comprehensive histologic evaluation of the sections was performed by the National Institutes of Health Division of Veterinary Resources Pathology Service. Cells were diluted in 1% BSA-PBS and incubated with anti-FcγR antibody (BD Biosciences, San Jose, CA) to block binding of conjugated antibodies to FcγR (BD Biosciences). Antibodies anti-mouse Gr-1 (FITC- and allophycocyanin [APC]-conjugated), anti-CD11b (phycoerythrin (PE)- and FITC-conjugated), anti-CD4 (peridinin chlorophyll protein [PerCP]-conjugated), anti-CD8 (PE-conjugated), anti-B220 (APC-conjugated), anti-CD62L (APC-conjugated), anti-CD11a (PE-conjugated), anti-CD49d (PE-conjugated), anti-CD18 (FITC-conjugated), anti-IL-17 (PE-conjugated), and anti-Foxp3 (FITC-conjugated) were purchased from BD Biosciences. After cells were labeled with the appropriate antibodies for 30 min on ice and fixed in 1% paraformaldehyde in PBS, they were subjected to flow cytometry on a FACScalibur (BD Biosciences). Data were analyzed using the FlowJo software (Tree Star, Ashland, OR). Myeloid cells in bone marrow were identified as Gr-1high CD11b+ and Gr-1low CD11b+. Neutrophils in blood and spleen were identified as Gr-1high CD11b+ cells and monocytes as Gr-1low/− CD11b+ cells. T lymphocytes were identified as CD4+ or CD8+ cells and B lymphocytes as B220+ cells. Th-17 cells were detected by determining the intracellular production of IL-17 in CD4+ cells. Lymphocytes were isolated from mesenteric lymph nodes, activated with 50 ng/ml phorbol 12-myristate 13-acetate and 500 ng/ml ionomycin for 2 h and incubated with BD GolgiPlug (BD Biosciences) for 2 h. Cells were then stained with anti-CD4 PerCP-conjugated antibody on ice for 30 min, fixed, and permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences), and stained with IL-17 PE-conjugated and Foxp3 FITC-conjugated antibodies. Th-17 cells were identified as CD4+ IL-17+ Foxp3− cells. For bromodeoxyuridine (BrdU) labeling, mice were injected intraperitoneally once with 100 μl of a 10 mg/ml BrdU solution (BD Biosciences) and analyzed 48 h later. BrdU-positive cells were detected using a FITC-BrdU Flow kit (BD Biosciences) by flow cytometry. Serum levels of corticosterone were determined by ELISA (AssayPro, St. Charles, MO). Serum levels of cytokines IL-12, TNF, IFN-g, monocyte chemoattractant protein (MCP)-1, IL-10, and IL-6 were determined using the BD Cytometric Bead Array (CBA, BD Biosciences). Serum IL-17 was detected by ELISA (R&D Systems, Minneapolis, MN). Serum granulocyte-colony stimulating factor (G-CSF) was detected using the mouse cytokine/chemokine LINCOplex kit from Millipore (St. Charles, MO). IL-23 expression was studied by determining the levels of the Il23a subunit mRNA by real-time-quantitative PCR (RT-qPCR) as described below. The concentration of shed, soluble CD62L (s-CD62L) in serum from Sgpl1+/+ and Sgpl1−/− mice was determined by ELISA (R&D Systems). The response of neutrophils toward formyl-methionyl-leucyl-phenylalanine (fMLP) was studied using 6.5 mm Transwell inserts with a 5-μm pore size (Corning, Cambridge, MA). A splenocyte suspension in RPMI 1640 plus 0.4 mg/ml fatty acid-free (FAF)-BSA (Sigma-Aldrich) (medium + FAF-BSA) was added to each insert in a well containing 600 μl of a solution of 1 μm fMLP (SigmaAldrich) prepared in medium + FAF-BSA. Wells containing medium + FAF-BSA without fMLP were used as controls. After 3 h at 37 °C, cells in the bottom of the wells were harvested, counted, and analyzed by flow cytometry. For RT-qPCR, total RNA was purified from bone marrow neutrophils and from liver using TRIZOL (Invitrogen). The mRNA expression levels of mouse Tnf (Mm00443258_m1), Vcam-1 (Mm00449197_m1), Saa1 (Mm00656927_g1), Saa3 (Mm00441203_m1), Sell (Mm00441291_m1), Il23a (Mm00518984_m1), S1pr1 (Mm00514644_m1), S1pr2 (Mm01177794), S1pr3 (Mm00515669_m1), S1pr4 (Mm00468695_s1), and S1pr5 (Mm00474763_m1) genes were determined by RT-qPCR using Assay-on-Demand probes and primers (Applied Biosystems, Foster City, CA) on an ABI Prism 7700 Sequence Detection System (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (Mm99999915_g1) mRNA level was used as an internal control. For microarray analysis, RNA purified from Sgpl1+/+ and Sgpl1−/− livers was prepared and analyzed on Affymetrix GeneChip Mouse Genome 430 2.0 arrays as described (29Bektas M. Allende M.L. Lee B.G. Chen W. Amar M.J. Remaley A.T. Saba J.D. Proia R.L. J. Biol. Chem. 2010; 285: 10880-10889Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The National Center for Biotechnology Information Gene Expression Omnibus accession number for the microarray data is GSE18745. Genes that belong to the Gene Ontology (GO) category “acute inflammation” were compared between Sgpl1+/+ and Sgpl1−/− mRNA samples using a heat map generated using the Partek Genomic Suite 6.5 software (Partek Inc., St. Louis, MO). Sphingolipids in serum were measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) by the Lipidomics Core at the Medical University of South Carolina on a Thermo Finnigan (Waltham, MA) TSQ 7000 triple quadrupole mass spectrometer, operating in a multiple reaction monitoring-positive ionization mode as described (32Bielawski J. Szulc Z.M. Hannun Y.A. Bielawska A. Methods. 2006; 39: 82-91Crossref PubMed Scopus (424) Google Scholar). Statistical significance was determined using the Mann-Whitney or Student's t test. In all cases, values of p < 0.05 were considered statistically significant. Sgpl1−/− mice generally die soon after weaning (26Vogel P. Donoviel M.S. Read R. Hansen G.M. Hazlewood J. Anderson S.J. Sun W. Swaffield J. Oravecz T. PLoS One. 2009; 4: e4112Crossref PubMed Scopus (138) Google Scholar, 28Schmahl J. Raymond C.S. Soriano P. Nat. Genet. 2007; 39: 52-60Crossref PubMed Scopus (158) Google Scholar, 29Bektas M. Allende M.L. Lee B.G. Chen W. Amar M.J. Remaley A.T. Saba J.D. Proia R.L. J. Biol. Chem. 2010; 285: 10880-10889Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), necessitating studies on young mice, at P18 unless otherwise indicated. The Sgpl1−/− mice were severely lymphopenic, with deficiencies of both T (CD4+, CD8+) cells and B (B220+) cells in blood (supplemental Fig. S1, A and C). Low numbers of T and B cells were also apparent in the spleen of the Sgpl1−/− mice (supplemental Fig. S1, B and D). The lymphocyte deficiency in the Sgpl1−/− mice has been attributed to a number of possible sources, including a block in the egress of recirculating lymphocytes from secondary lymphoid organs and to defects in the development of T cells and B cells (27Weber C. Krueger A. Münk A. Bode C. Van Veldhoven P.P. Gräler M.H. J. Immunol. 2009; 183: 4292-4301Crossref PubMed Scopus (51) Google Scholar). In contrast to the severe lymphopenia, blood levels of neutrophils, and monocytes were highly elevated in the Sgpl1−/− mice (Fig. 1A) (26Vogel P. Donoviel M.S. Read R. Hansen G.M. Hazlewood J. Anderson S.J. Sun W. Swaffield J. Oravecz T. PLoS One. 2009; 4: e4112Crossref PubMed Scopus (138) Google Scholar). Sgpl1+/− mice had normal blood cell counts indicating that both Sgpl1 alleles must to be deleted to produce this immune phenotype (supplemental Fig. S2A). We next determined the numbers and percentages of neutrophils and monocytes in the spleen and their precursors in the bone marrow. Compared with the WT mice, the percentages of splenic neutrophils and monocytes were dramatically elevated in Sgpl1−/− compared with WT mice (Fig. 1C). The total numbers of neutrophils and monocytes in spleen, when normalized by body weight, were also significantly increased in the Sgpl1−/− mice compared with WT mice (Fig. 1D). We enumerated myeloid cells, the precursors of neutrophils and monocytes, in bone marrow and found a substantial increase of both immature and mature myeloid cells in the Sgpl1−/− mice when expressed as either percentage of total cells or absolute cell numbers normalized by body weight (Fig. 1, E and F). The ratio of immature to mature myeloid cells was similar between Sgpl1−/− and WT mice suggesting differentiation of neutrophil precursors in the bone marrow of Sgpl1−/− mice was normal. To establish whether increased granulopoiesis was responsible for the elevated neutrophil numbers in Sgpl1−/− mice, we labeled proliferating precursor cells in bone marrow by BrdU incorporation and determined the appearance of BrdU+ neutrophils in blood after 48 h. The numbers of BrdU+ neutrophils generated in Sgpl1−/− mice were significantly higher than in WT mice (Fig. 1B). These data indicate that increased granulopoiesis was occurring in the Sgpl1−/− mice. We also determined the circulating ceramide, sphingosine, and S1P levels at P6 and P18 by HPLC-MS/MS analysis (supplemental Fig. S3). Both ceramide and S1P levels were substantially elevated at P6 and P18 in the Sgpl1−/− mice compared with WT mice. With increasing age, ceramide and S1P levels increased in the Sgpl1−/− mice. Sphingosine remained at similar low levels at both ages tested. Sgpl1+/− mice had serum sphingolipid levels similar to WT mice (supplemental Fig. S2B). To rule out stress-induced changes on peripheral lymphocytes and neutrophils, we measured circulating corticosterone levels and found that there was no significant difference between the values of Sgpl1−/− and WT mice (supplemental Fig. S4). Neutrophilia can be associated with inflammation. Microarray analysis of P18 liver mRNA using Affymetrix mouse genome GeneChips revealed that the GO category of “acute inflammation” was significantly different between Sgpl1−/− and WT mice (p < 0.001). Acute-phase reactants, serum amyloids (Saa1–4), orosomucoids (Orm1–3), and LPS binding protein (Lbp) were highly elevated in the livers of the Sgpl1−/− mice compared with those of WT mice, indicative of an acute inflammatory process (Fig. 2A). We next determined the progression of inflammatory changes in Sgpl1−/− mice by examining the mRNA expression of some genes related to inflammation in liver at P6 and P18. An increase in Tnf, Saa1, and Saa3 was detected by RT-qPCR at both ages in the Sgpl1−/− livers compared with those of WT mice (Fig. 2B). We also detected an increase in the expression level of the endothelial adhesion molecule Vcam-1 in the P18 Sgpl1−/− livers when compared with controls (Fig. 2B). Vcam-1 is elevated during the endothelium response to inflammatory stimuli (33Barreiro O. Martín P. González-Amaro R. Sánchez-Madrid F. Cardiovasc. Res. 2010; 86: 174-182Crossref PubMed Scopus (66) Google Scholar). Together, these results indicate the presence of a pro-inflammatory process in the liver of Sgpl1−/− mice that intensifies with age. In the serum of Sgpl1−/− mice, we detected a significant increase in the levels of the pro-inflammatory cytokines TNF, IFN-γ, MCP-1, and IL-6 compared with levels observed in WT mice (Fig. 2C), demonstrating that the inflammation was systemic. Sgpl1+/− mice did not display any elevated serum cytokine levels (supplemental Fig. S2C). Injection of Sgpl1−/− mice with a dose of LPS, which was not lethal to WT mice, caused their death by 8 h (supplemental Fig. S5A). Within 120 min after the LPS injection, the Sgpl1−/− mice expressed substantially higher levels of TNF, IL-6, and MCP-1 in serum compared with the WT mice injected with LPS (supplemental Fig. S5B). Serum ceramide, sphingosine, and S1P concentrations were not significantly elevated over the respective baseline values in either Sgpl1−/− or control mice after LPS treatment during the same time period (supplemental Fig. S5C). The pro-inflammatory state of the Sgpl1−/− mice raised the possibility that stimulation of the innate immune system by opportunistic bacterial infection, perhaps as a result of their severe lymphocyte deficiency, might be inducing systemic inflammation. We found no histologic evidence for the presence of infection in the tissue of Sgpl1−/− mice compared with WT mice (supplemental Fig. S6). We therefore studied the effect of deleting the Toll-like receptor-4 (Tlr4) in Sgpl1−/− mice (Fig. 3). Tlr4 is a receptor that detects the LPS of Gram-negative bacterial cell walls, and triggers a cascade of events that activates the transcription of many pro-inflammatory genes involved in the innate immune response (34Beutler B.A. Blood. 2009; 113: 1399-1407Crossref PubMed Scopus (642) Google Scholar). If the inflammatory response in Sgpl1−/− mice was the result of a Gram-negative bacterial infection, the inflammation in the Sgpl1−/− Tlr4−/− double knock-out (DKO) mice should be suppressed relative to the Sgpl1−/− mice. We found that the Sgpl1−/− Tlr4−/− DKO and Sgpl1−/− mice had similarly depressed lymphocyte and elevated neutrophil blood counts (Fig. 3A) and similar elevations of pro-inflammatory cytokines in serum (Fig. 3B) and of mRNA levels for Tnf and Vcam-1 in liver (Fig. 3C). Whereas the Sgpl1−/− mice were hypersensitive to LPS, dying by 8 h after being challenged with a nonlethal dose of LPS, the Sgpl1−/− Tlr4−/− DKO mice were resistant (supplemental Fig. S5A). These results indicate that the pro-inflammatory condition of the Sgpl1−/− mice was unlikely to be the result of an activation of the innate immune system by an opportunistic Gram-negative bacterial infection. We next sought to determine if the neutrophilia was correlated with large numbers of neutrophils infiltrating into the liver of Sgpl1−/− mice, as would be expected based on the acute inflammation exhibited by this tissue (Fig. 2, A and B). Surprisingly, we found that the liver parenchyma in Sgpl1−/− mice appeared similar to Sgpl1+/+ mice and did not exhibit extensive neutrophil infiltration (Fig. 4, A and B); however, we did observe abnormally high accumulations of neutrophils that appeared to be confined at the hepatic sinusoids in Sgpl1−/− mice compared with Sgpl1+/+ mice (Fig. 4, C and D). These results suggested a possible impairment of neutrophil recruitment from the blood into the inflamed tissue in the Sgpl1−/− mice. A survey of other Sgpl1−/− tissues showed that they were spared from substantial neutrophil infiltration (supplemental Fig. S6, C–L). As reported previously (26Vogel P. Donoviel M.S. Read R. Hansen G.M. Hazlewood J. Anderson S.J. Sun W. Swaffield J. Oravecz T. PLoS One. 2009; 4: e4112Crossref PubMed Scopus (138) Google Scholar), the Spgl1−/− lungs contained a slight increase in macrophages within alveoli (supplemental Fig. S6, A and B). To establish whether Sgpl1 deficiency altered the ability of neutrophils to migrate to sites of inflammation, we injected mice intraperitoneal with thioglycollate and examined the influx of neutrophils into" @default.
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• W2040300892 date "2011-03-01" @default.
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• W2040300892 title "Sphingosine-1-phosphate Lyase Deficiency Produces a Pro-inflammatory Response While Impairing Neutrophil Trafficking" @default.
• W2040300892 cites W1519007189 @default.
• W2040300892 cites W1595293335 @default.
• W2040300892 cites W1645222688 @default.
• W2040300892 cites W188314664 @default.
• W2040300892 cites W1954792542 @default.
• W2040300892 cites W1965704337 @default.
• W2040300892 cites W1966452335 @default.
• W2040300892 cites W1967180851 @default.
• W2040300892 cites W1967802627 @default.
• W2040300892 cites W1975275391 @default.
• W2040300892 cites W1979620916 @default.
• W2040300892 cites W1982334431 @default.
• W2040300892 cites W1983241545 @default.
• W2040300892 cites W1984290711 @default.
• W2040300892 cites W1988204787 @default.
• W2040300892 cites W1989138941 @default.
• W2040300892 cites W1989607865 @default.
• W2040300892 cites W1990827391 @default.
• W2040300892 cites W1991558659 @default.
• W2040300892 cites W1997014789 @default.
• W2040300892 cites W2005271761 @default.
• W2040300892 cites W2006683926 @default.
• W2040300892 cites W2006985918 @default.
• W2040300892 cites W2010203793 @default.
• W2040300892 cites W2015949769 @default.
• W2040300892 cites W2019209482 @default.
• W2040300892 cites W2030726997 @default.
• W2040300892 cites W2035809865 @default.
• W2040300892 cites W2037155515 @default.
• W2040300892 cites W2042753405 @default.
• W2040300892 cites W2047607345 @default.
• W2040300892 cites W2050640165 @default.
• W2040300892 cites W2058353078 @default.
• W2040300892 cites W2062242135 @default.
• W2040300892 cites W2063704442 @default.
• W2040300892 cites W2069968649 @default.
• W2040300892 cites W2072478343 @default.
• W2040300892 cites W2077215651 @default.
• W2040300892 cites W2078702640 @default.
• W2040300892 cites W2081280923 @default.
• W2040300892 cites W2084756176 @default.
• W2040300892 cites W2086687315 @default.
• W2040300892 cites W2088325902 @default.
• W2040300892 cites W2093738866 @default.
• W2040300892 cites W2106187425 @default.
• W2040300892 cites W2110910354 @default.
• W2040300892 cites W2114784330 @default.
• W2040300892 cites W2115088294 @default.
• W2040300892 cites W2119802141 @default.
• W2040300892 cites W2123274906 @default.
• W2040300892 cites W2127704524 @default.
• W2040300892 cites W2129274801 @default.
• W2040300892 cites W2130902918 @default.
• W2040300892 cites W2132579946 @default.
• W2040300892 cites W2135160600 @default.
• W2040300892 cites W2137583122 @default.
• W2040300892 cites W2144885443 @default.
• W2040300892 cites W2145985395 @default.
• W2040300892 cites W2146556036 @default.
• W2040300892 cites W2146981926 @default.
• W2040300892 cites W2151403120 @default.
• W2040300892 cites W2152181595 @default.
• W2040300892 cites W2155952015 @default.
• W2040300892 cites W2157818339 @default.
• W2040300892 cites W2159421712 @default.
• W2040300892 cites W2159573784 @default.
• W2040300892 cites W2162744849 @default.
• W2040300892 cites W2162783323 @default.
• W2040300892 cites W2162838509 @default.
• W2040300892 cites W2163240674 @default.
• W2040300892 cites W2171053963 @default.
• W2040300892 cites W2266998221 @default.
• W2040300892 cites W4246164535 @default.
• W2040300892 doi "https://doi.org/10.1074/jbc.m110.171819" @default.
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