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. 1994 Sep 1;180(3):945–958. doi: 10.1084/jem.180.3.945

Isolation of a nitric oxide inhibitor from mammary tumor cells and its characterization as phosphatidyl serine

PMCID: PMC2191656  PMID: 8064242

Abstract

Macrophages from mice bearing large D1-DMBA-3 mammary tumors have a decreased capacity to kill tumor targets. This effect is due to an impaired ability to produce nitric oxide (NO) in response to lipopolysaccharide (LPS) stimulation. Here we report that the DA-3 tumor cell line, derived from the in vivo adenocarcinoma D1-DMBA-3, produces a factor that inhibits both NO production/release and cytotoxicity of LPS-activated peritoneal exudate macrophages (PEM). However, other complex macrophage functions such as phagocytosis, superoxide production, mitochondrial dehydrogenase activity, and synthesis of proteins were not reduced by this factor. The NO inhibitor has been found to be lipid in nature. Lipid extracts from DA-3 cell culture supernatants were purified by repeated silica gel column chromatography. The active molecule was unambiguously characterized as phosphatidyl serine (PS) by fast atom bombardment tandem mass spectrometry. Preliminary results indicate a lack of induced NO synthase (iNOS) activity in the lysates of LPS-activated PEM pretreated with PS. The ubiquity of PS in the inner leaflet of biological membranes and its NO inhibitory property, suggest that this phospholipid may be one of the long elusive molecules responsible for regulating physiological levels of NO in the host and hence preventing cellular dysfunction and/or tissue damage. Furthermore, the possible overexpression and shedding of PS by DA-3 tumor cells may represent a novel mechanism to impair macrophage cytotoxicity, a host function that contributes to the protection against developing neoplasms.

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Selected References

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  1. Alam S. Q., Alam B. S., Chen T. W. Activities of fatty acid desaturases and fatty acid composition of liver microsomes in rats fed beta-carotene and 13-cis-retinoic acid. Biochim Biophys Acta. 1984 Feb 9;792(2):110–117. doi: 10.1016/0005-2760(84)90210-8. [DOI] [PubMed] [Google Scholar]
  2. Anthony L. S., Morrissey P. J., Nano F. E. Growth inhibition of Francisella tularensis live vaccine strain by IFN-gamma-activated macrophages is mediated by reactive nitrogen intermediates derived from L-arginine metabolism. J Immunol. 1992 Mar 15;148(6):1829–1834. [PubMed] [Google Scholar]
  3. Barker E., Mueller B. M., Handgretinger R., Herter M., Yu A. L., Reisfeld R. A. Effect of a chimeric anti-ganglioside GD2 antibody on cell-mediated lysis of human neuroblastoma cells. Cancer Res. 1991 Jan 1;51(1):144–149. [PubMed] [Google Scholar]
  4. Bogdan C., Vodovotz Y., Paik J., Xie Q. W., Nathan C. Traces of bacterial lipopolysaccharide suppress IFN-gamma-induced nitric oxide synthase gene expression in primary mouse macrophages. J Immunol. 1993 Jul 1;151(1):301–309. [PubMed] [Google Scholar]
  5. COHN Z. A. The fate of bacteria within phagocytic cells. I. The degradation of isotopically labeled bacteria by polymorphonuclear leucocytes and macrophages. J Exp Med. 1963 Jan 1;117:27–42. doi: 10.1084/jem.117.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Caselli E., Baricordi O. R., Melchiorri L., Bellini F., Ponzin D., Bruni A. Inhibition of DNA synthesis in peripheral blood mononuclear cells treated with phosphatidylserines containing unsaturated acyl chains. Immunopharmacology. 1992 May-Jun;23(3):205–213. doi: 10.1016/0162-3109(92)90027-a. [DOI] [PubMed] [Google Scholar]
  7. Cenci E., Romani L., Mencacci A., Spaccapelo R., Schiaffella E., Puccetti P., Bistoni F. Interleukin-4 and interleukin-10 inhibit nitric oxide-dependent macrophage killing of Candida albicans. Eur J Immunol. 1993 May;23(5):1034–1038. doi: 10.1002/eji.1830230508. [DOI] [PubMed] [Google Scholar]
  8. Chiu D., Lubin B., Shohet S. B. Erythrocyte membrane lipid reorganization during the sickling process. Br J Haematol. 1979 Feb;41(2):223–234. doi: 10.1111/j.1365-2141.1979.tb05851.x. [DOI] [PubMed] [Google Scholar]
  9. DITTMER J. C., LESTER R. L. A SIMPLE, SPECIFIC SPRAY FOR THE DETECTION OF PHOSPHOLIPIDS ON THIN-LAYER CHROMATOGRAMS. J Lipid Res. 1964 Jan;5:126–127. [PubMed] [Google Scholar]
  10. Ding A., Nathan C. F., Graycar J., Derynck R., Stuehr D. J., Srimal S. Macrophage deactivating factor and transforming growth factors-beta 1 -beta 2 and -beta 3 inhibit induction of macrophage nitrogen oxide synthesis by IFN-gamma. J Immunol. 1990 Aug 1;145(3):940–944. [PubMed] [Google Scholar]
  11. Doe W. F., Henson P. M. Macrophage stimulation by bacterial lipopolysaccharides. I. Cytolytic effect on tumor target cells. J Exp Med. 1978 Aug 1;148(2):544–556. doi: 10.1084/jem.148.2.544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Drapier J. C., Pellat C., Henry Y. Generation of EPR-detectable nitrosyl-iron complexes in tumor target cells cocultured with activated macrophages. J Biol Chem. 1991 Jun 5;266(16):10162–10167. [PubMed] [Google Scholar]
  13. Ferrari M., Fornasiero M. C., Isetta A. M. MTT colorimetric assay for testing macrophage cytotoxic activity in vitro. J Immunol Methods. 1990 Aug 7;131(2):165–172. doi: 10.1016/0022-1759(90)90187-z. [DOI] [PubMed] [Google Scholar]
  14. Fidler I. J., Poste G. The cellular heterogeneity of malignant neoplasms: implications for adjuvant chemotherapy. Semin Oncol. 1985 Sep;12(3):207–221. [PubMed] [Google Scholar]
  15. Fu Y. X., Watson G., Jimenez J. J., Wang Y., Lopez D. M. Expansion of immunoregulatory macrophages by granulocyte-macrophage colony-stimulating factor derived from a murine mammary tumor. Cancer Res. 1990 Jan 15;50(2):227–234. [PubMed] [Google Scholar]
  16. Gilbreath M. J., Hoover D. L., Alving C. R., Swartz G. M., Jr, Meltzer M. S. Inhibition of lymphokine-induced macrophage microbicidal activity against Leishmania major by liposomes: characterization of the physicochemical requirements for liposome inhibition. J Immunol. 1986 Sep 1;137(5):1681–1687. [PubMed] [Google Scholar]
  17. Girolomoni G., Pastore S., Zacchi V., Cavani A., Marconi A., Giannetti A. Phosphatidylserine enhances the ability of epidermal Langerhans cells to induce contact hypersensitivity. J Immunol. 1993 May 15;150(10):4236–4243. [PubMed] [Google Scholar]
  18. Gordesky S. E., Marinetti G. V. The asymetric arrangement of phospholipids in the human erythrocyte membrane. Biochem Biophys Res Commun. 1973 Feb 20;50(4):1027–1031. doi: 10.1016/0006-291x(73)91509-x. [DOI] [PubMed] [Google Scholar]
  19. Green S. J., Meltzer M. S., Hibbs J. B., Jr, Nacy C. A. Activated macrophages destroy intracellular Leishmania major amastigotes by an L-arginine-dependent killing mechanism. J Immunol. 1990 Jan 1;144(1):278–283. [PubMed] [Google Scholar]
  20. Heck D. E., Laskin D. L., Gardner C. R., Laskin J. D. Epidermal growth factor suppresses nitric oxide and hydrogen peroxide production by keratinocytes. Potential role for nitric oxide in the regulation of wound healing. J Biol Chem. 1992 Oct 25;267(30):21277–21280. [PubMed] [Google Scholar]
  21. Hibbs J. B., Jr, Taintor R. R., Vavrin Z. Macrophage cytotoxicity: role for L-arginine deiminase and imino nitrogen oxidation to nitrite. Science. 1987 Jan 23;235(4787):473–476. doi: 10.1126/science.2432665. [DOI] [PubMed] [Google Scholar]
  22. Hibbs J. B., Jr, Taintor R. R., Vavrin Z., Rachlin E. M. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem Biophys Res Commun. 1988 Nov 30;157(1):87–94. doi: 10.1016/s0006-291x(88)80015-9. [DOI] [PubMed] [Google Scholar]
  23. Hibbs J. B., Jr, Vavrin Z., Taintor R. R. L-arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells. J Immunol. 1987 Jan 15;138(2):550–565. [PubMed] [Google Scholar]
  24. Jensen N. J., Tomer K. B., Gross M. L. Fast atom bombardment and tandem mass spectrometry of phosphatidylserine and phosphatidylcholine. Lipids. 1986 Sep;21(9):580–588. doi: 10.1007/BF02534056. [DOI] [PubMed] [Google Scholar]
  25. Kwon N. S., Stuehr D. J., Nathan C. F. Inhibition of tumor cell ribonucleotide reductase by macrophage-derived nitric oxide. J Exp Med. 1991 Oct 1;174(4):761–767. doi: 10.1084/jem.174.4.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  27. Ladisch S., Gillard B., Wong C., Ulsh L. Shedding and immunoregulatory activity of YAC-1 lymphoma cell gangliosides. Cancer Res. 1983 Aug;43(8):3808–3813. [PubMed] [Google Scholar]
  28. Ladisch S., Wu Z. L., Feig S., Ulsh L., Schwartz E., Floutsis G., Wiley F., Lenarsky C., Seeger R. Shedding of GD2 ganglioside by human neuroblastoma. Int J Cancer. 1987 Jan 15;39(1):73–76. doi: 10.1002/ijc.2910390113. [DOI] [PubMed] [Google Scholar]
  29. Liew F. Y., Millott S., Parkinson C., Palmer R. M., Moncada S. Macrophage killing of Leishmania parasite in vivo is mediated by nitric oxide from L-arginine. J Immunol. 1990 Jun 15;144(12):4794–4797. [PubMed] [Google Scholar]
  30. Lubin B., Chiu D., Bastacky J., Roelofsen B., Van Deenen L. L. Abnormalities in membrane phospholipid organization in sickled erythrocytes. J Clin Invest. 1981 Jun;67(6):1643–1649. doi: 10.1172/JCI110200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Miller F. R., Miller B. E., Heppner G. H. Characterization of metastatic heterogeneity among subpopulations of a single mouse mammary tumor: heterogeneity in phenotypic stability. Invasion Metastasis. 1983;3(1):22–31. [PubMed] [Google Scholar]
  32. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992 Sep;6(12):3051–3064. [PubMed] [Google Scholar]
  33. Pick E., Mizel D. Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader. J Immunol Methods. 1981;46(2):211–226. doi: 10.1016/0022-1759(81)90138-1. [DOI] [PubMed] [Google Scholar]
  34. Roof R. W., Luescher I. F., Unanue E. R. Phospholipids enhance the binding of peptides to class II major histocompatibility molecules. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1735–1739. doi: 10.1073/pnas.87.5.1735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rothman J. E., Lenard J. Membrane asymmetry. Science. 1977 Feb 25;195(4280):743–753. doi: 10.1126/science.402030. [DOI] [PubMed] [Google Scholar]
  36. Ruco L. P., Meltzer M. S. Macrophage activation for tumor cytotoxicity: development of macrophage cytotoxic activity requires completion of a sequence of short-lived intermediary reactions. J Immunol. 1978 Nov;121(5):2035–2042. [PubMed] [Google Scholar]
  37. SVENNERHOLM L. Quantitative estimation of sialic acids. II. A colorimetric resorcinol-hydrochloric acid method. Biochim Biophys Acta. 1957 Jun;24(3):604–611. doi: 10.1016/0006-3002(57)90254-8. [DOI] [PubMed] [Google Scholar]
  38. Savill J., Fadok V., Henson P., Haslett C. Phagocyte recognition of cells undergoing apoptosis. Immunol Today. 1993 Mar;14(3):131–136. doi: 10.1016/0167-5699(93)90215-7. [DOI] [PubMed] [Google Scholar]
  39. Schini V. B., Durante W., Elizondo E., Scott-Burden T., Junquero D. C., Schafer A. I., Vanhoutte P. M. The induction of nitric oxide synthase activity is inhibited by TGF-beta 1, PDGFAB and PDGFBB in vascular smooth muscle cells. Eur J Pharmacol. 1992 Jun 17;216(3):379–383. doi: 10.1016/0014-2999(92)90434-6. [DOI] [PubMed] [Google Scholar]
  40. Schroit A. J., Tanaka Y., Madsen J., Fidler I. J. The recognition of red blood cells by macrophages: role of phosphatidylserine and possible implications of membrane phospholipid asymmetry. Biol Cell. 1984;51(2):227–238. doi: 10.1111/j.1768-322x.1984.tb00303.x. [DOI] [PubMed] [Google Scholar]
  41. Schulz G., Cheresh D. A., Varki N. M., Yu A., Staffileno L. K., Reisfeld R. A. Detection of ganglioside GD2 in tumor tissues and sera of neuroblastoma patients. Cancer Res. 1984 Dec;44(12 Pt 1):5914–5920. [PubMed] [Google Scholar]
  42. Sotomayor E. M., Fu Y. X., Lopez-Cepero M., Herbert L., Jimenez J. J., Albarracin C., Lopez D. M. Role of tumor-derived cytokines on the immune system of mice bearing a mammary adenocarcinoma. II. Down-regulation of macrophage-mediated cytotoxicity by tumor-derived granulocyte-macrophage colony-stimulating factor. J Immunol. 1991 Oct 15;147(8):2816–2823. [PubMed] [Google Scholar]
  43. Stuehr D. J., Kwon N. S., Gross S. S., Thiel B. A., Levi R., Nathan C. F. Synthesis of nitrogen oxides from L-arginine by macrophage cytosol: requirement for inducible and constitutive components. Biochem Biophys Res Commun. 1989 Jun 15;161(2):420–426. doi: 10.1016/0006-291x(89)92615-6. [DOI] [PubMed] [Google Scholar]
  44. Stuehr D. J., Nathan C. F. Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med. 1989 May 1;169(5):1543–1555. doi: 10.1084/jem.169.5.1543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tanaka Y., Schroit A. J. Insertion of fluorescent phosphatidylserine into the plasma membrane of red blood cells. Recognition by autologous macrophages. J Biol Chem. 1983 Sep 25;258(18):11335–11343. [PubMed] [Google Scholar]
  46. Tsunawaki S., Sporn M., Ding A., Nathan C. Deactivation of macrophages by transforming growth factor-beta. Nature. 1988 Jul 21;334(6179):260–262. doi: 10.1038/334260a0. [DOI] [PubMed] [Google Scholar]
  47. Unanue E. R., Allen P. M. The basis for the immunoregulatory role of macrophages and other accessory cells. Science. 1987 May 1;236(4801):551–557. doi: 10.1126/science.2437650. [DOI] [PubMed] [Google Scholar]
  48. Utsugi T., Schroit A. J., Connor J., Bucana C. D., Fidler I. J. Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes. Cancer Res. 1991 Jun 1;51(11):3062–3066. [PubMed] [Google Scholar]
  49. Verkleij A. J., Zwaal R. F., Roelofsen B., Comfurius P., Kastelijn D., van Deenen L. L. The asymmetric distribution of phospholipids in the human red cell membrane. A combined study using phospholipases and freeze-etch electron microscopy. Biochim Biophys Acta. 1973 Oct 11;323(2):178–193. doi: 10.1016/0005-2736(73)90143-0. [DOI] [PubMed] [Google Scholar]
  50. Wikstrand C. J., Longee D. C., McLendon R. E., Fuller G. N., Friedman H. S., Fredman P., Svennerholm L., Bigner D. D. Lactotetraose series ganglioside 3',6'-isoLD1 in tumors of central nervous and other systems in vitro and in vivo. Cancer Res. 1993 Jan 1;53(1):120–126. [PubMed] [Google Scholar]

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