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The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1991 Apr;87(4):1139–1145. doi: 10.1172/JCI115110

Immunocytochemical localization of arachidonate 15-lipoxygenase in erythrocytes, leukocytes, and airway cells.

J A Nadel 1, D J Conrad 1, I F Ueki 1, A Schuster 1, E Sigal 1
PMCID: PMC295116  PMID: 2010530

Abstract

In reticulocytes, the enzyme 15-lipoxygenase (15-LO) is believed to contribute to cellular differentiation, and in leukocytes and airway cells 15-LO generates inflammatory mediators. The recent availability of antibodies to 15-LO now allows us to determine which specific cells contain the enzyme, to characterize its subcellular localization, and to determine its expression at the translational level. A polyclonal antibody to recombinant human reticulocyte 15-LO was used with a standard immunofluorescent technique. In rabbit red blood cells, fluorescence appeared during the course of anemia. Early reticulocytes did not fluoresce, but more mature reticulocytes showed increased fluorescent intensity. Late reticulocytes contained little fluorescence. Among human leukocytes, only eosinophils fluoresced. In human trachea, 15-LO immunofluorescence was localized to epithelial cells, and both basal and ciliated cells fluoresced. In all cells studied, fluorescence was localized to the cytoplasm and was variable in degree among cells in each preparation. We conclude that the 15-LO of airway cells and eosinophils is immunologically related to the reticulocyte 15-LO. Furthermore, the variable fluorescence among cells (e.g., in epithelium) and during development (e.g., reticulocytes) suggests a role of 15-LO in cell growth and development.

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  1. Dahlén S. E., Franzén L., Raud J., Serhan C. N., Westlund P., Wikström E., Björck T., Matsuda H., Webber S. E., Veale C. A. Actions of lipoxin A4 and related compounds in smooth muscle preparations and on the microcirculation in vivo. Adv Exp Med Biol. 1988;229:107–130. doi: 10.1007/978-1-4757-0937-7_9. [DOI] [PubMed] [Google Scholar]
  2. Filley W. V., Ackerman S. J., Gleich G. J. An immunofluorescent method for specific staining of eosinophil granule major basic protein. J Immunol Methods. 1981;47(2):227–238. doi: 10.1016/0022-1759(81)90123-x. [DOI] [PubMed] [Google Scholar]
  3. Fruteau de Laclos B., Braquet P., Borgeat P. Characteristics of leukotriene (LT) and hydroxy eicosatetraenoic acid (HETE) synthesis in human leukocytes in vitro: effect of arachidonic acid concentration. Prostaglandins Leukot Med. 1984 Jan;13(1):47–52. doi: 10.1016/0262-1746(84)90101-x. [DOI] [PubMed] [Google Scholar]
  4. Goetzl E. J., Phillips M. J., Gold W. M. Stimulus specificity of the generation of leukotrienes by dog mastocytoma cells. J Exp Med. 1983 Sep 1;158(3):731–737. doi: 10.1084/jem.158.3.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hamberg M., Hedqvist P., Rådegran K. Identification of 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) as a major metabolite of arachidonic acid in human lung. Acta Physiol Scand. 1980 Oct;110(2):219–221. doi: 10.1111/j.1748-1716.1980.tb06656.x. [DOI] [PubMed] [Google Scholar]
  6. Holtzman M. J., Pentland A., Baenziger N. L., Hansbrough J. R. Heterogeneity of cellular expression of arachidonate 15-lipoxygenase: implications for biological activity. Biochim Biophys Acta. 1989 Jun 8;1003(2):204–208. doi: 10.1016/0005-2760(89)90257-9. [DOI] [PubMed] [Google Scholar]
  7. Hunter J. A., Finkbeiner W. E., Nadel J. A., Goetzl E. J., Holtzman M. J. Predominant generation of 15-lipoxygenase metabolites of arachidonic acid by epithelial cells from human trachea. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4633–4637. doi: 10.1073/pnas.82.14.4633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kirsch C. M., Sigal E., Djokic T. D., Graf P. D., Nadel J. A. An in vivo chemotaxis assay in the dog trachea: evidence for chemotactic activity of 8,15-diHETE. J Appl Physiol (1985) 1988 May;64(5):1792–1795. doi: 10.1152/jappl.1988.64.5.1792. [DOI] [PubMed] [Google Scholar]
  9. Kroschwald P., Kroschwald A., Kühn H., Ludwig P., Thiele B. J., Höhne M., Schewe T., Rapoport S. M. Occurrence of the erythroid cell specific arachidonate 15-lipoxygenase in human reticulocytes. Biochem Biophys Res Commun. 1989 Apr 28;160(2):954–960. doi: 10.1016/0006-291x(89)92528-x. [DOI] [PubMed] [Google Scholar]
  10. Kühn H., Brash A. R. Occurrence of lipoxygenase products in membranes of rabbit reticulocytes. Evidence for a role of the reticulocyte lipoxygenase in the maturation of red cells. J Biol Chem. 1990 Jan 25;265(3):1454–1458. [PubMed] [Google Scholar]
  11. Marom Z., Shelhamer J. H., Sun F., Kaliner M. Human airway monohydroxyeicosatetraenoic acid generation and mucus release. J Clin Invest. 1983 Jul;72(1):122–127. doi: 10.1172/JCI110949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. McGuire J., McGee J., Crittenden N., Fitzpatrick F. Cell damage unmasks 15-lipoxygenase activity in human neutrophils. J Biol Chem. 1985 Jul 15;260(14):8316–8319. [PubMed] [Google Scholar]
  13. Murray J. J., Brash A. R. Rabbit reticulocyte lipoxygenase catalyzes specific 12(S) and 15(S) oxygenation of arachidonoyl-phosphatidylcholine. Arch Biochem Biophys. 1988 Sep;265(2):514–523. doi: 10.1016/0003-9861(88)90156-7. [DOI] [PubMed] [Google Scholar]
  14. Nichols R. C., Vanderhoek J. Y. 5-Hydroxyeicosanoids selectively stimulate the human neutrophil 15-lipoxygenase to use endogenous substrate. J Exp Med. 1990 Feb 1;171(2):367–375. doi: 10.1084/jem.171.2.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rapoport S. M., Schewe T., Wiesner R., Halangk W., Ludwig P., Janicke-Höhne M., Tannert C., Hiebsch C., Klatt D. The lipoxygenase of reticulocytes. Purification, characterization and biological dynamics of the lipoxygenase; its identity with the respiratory inhibitors of the reticulocyte. Eur J Biochem. 1979 Jun 1;96(3):545–561. doi: 10.1111/j.1432-1033.1979.tb13068.x. [DOI] [PubMed] [Google Scholar]
  16. Rapoport S., Schmidt J., Prehn S. Maturation of rabbit reticulocytes: susceptibility of mitochondria to ATP-dependent proteolysis is determined by the maturational state of reticulocyte. FEBS Lett. 1985 Apr 22;183(2):370–374. doi: 10.1016/0014-5793(85)80812-7. [DOI] [PubMed] [Google Scholar]
  17. Shak S., Perez H. D., Goldstein I. M. A novel dioxygenation product of arachidonic acid possesses potent chemotactic activity for human polymorphonuclear leukocytes. J Biol Chem. 1983 Dec 25;258(24):14948–14953. [PubMed] [Google Scholar]
  18. Sigal E., Craik C. S., Highland E., Grunberger D., Costello L. L., Dixon R. A., Nadel J. A. Molecular cloning and primary structure of human 15-lipoxygenase. Biochem Biophys Res Commun. 1988 Dec 15;157(2):457–464. doi: 10.1016/s0006-291x(88)80271-7. [DOI] [PubMed] [Google Scholar]
  19. Sigal E., Grunberger D., Cashman J. R., Craik C. S., Caughey G. H., Nadel J. A. Arachidonate 15-lipoxygenase from human eosinophil-enriched leukocytes: partial purification and properties. Biochem Biophys Res Commun. 1988 Jan 15;150(1):376–383. doi: 10.1016/0006-291x(88)90531-1. [DOI] [PubMed] [Google Scholar]
  20. Sigal E., Grunberger D., Craik C. S., Caughey G. H., Nadel J. A. Arachidonate 15-lipoxygenase (omega-6 lipoxygenase) from human leukocytes. Purification and structural homology to other mammalian lipoxygenases. J Biol Chem. 1988 Apr 15;263(11):5328–5332. [PubMed] [Google Scholar]
  21. Sigal E., Grunberger D., Highland E., Gross C., Dixon R. A., Craik C. S. Expression of cloned human reticulocyte 15-lipoxygenase and immunological evidence that 15-lipoxygenases of different cell types are related. J Biol Chem. 1990 Mar 25;265(9):5113–5120. [PubMed] [Google Scholar]
  22. Soberman R. J., Harper T. W., Betteridge D., Lewis R. A., Austen K. F. Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes. J Biol Chem. 1985 Apr 10;260(7):4508–4515. [PubMed] [Google Scholar]
  23. Turk J., Maas R. L., Brash A. R., Roberts L. J., 2nd, Oates J. A. Arachidonic acid 15-lipoxygenase products from human eosinophils. J Biol Chem. 1982 Jun 25;257(12):7068–7076. [PubMed] [Google Scholar]
  24. Widdicombe J. H., Basbaum C. B., Highland E. Ion contents and other properties of isolated cells from dog tracheal epithelium. Am J Physiol. 1981 Nov;241(5):C184–C192. doi: 10.1152/ajpcell.1981.241.5.C184. [DOI] [PubMed] [Google Scholar]
  25. Yazdanbakhsh M., Eckmann C. M., De Boer M., Roos D. Purification of eosinophils from normal human blood, preparation of eosinoplasts and characterization of their functional response to various stimuli. Immunology. 1987 Jan;60(1):123–129. [PMC free article] [PubMed] [Google Scholar]
  26. Ylä-Herttuala S., Rosenfeld M. E., Parthasarathy S., Glass C. K., Sigal E., Witztum J. L., Steinberg D. Colocalization of 15-lipoxygenase mRNA and protein with epitopes of oxidized low density lipoprotein in macrophage-rich areas of atherosclerotic lesions. Proc Natl Acad Sci U S A. 1990 Sep;87(18):6959–6963. doi: 10.1073/pnas.87.18.6959. [DOI] [PMC free article] [PubMed] [Google Scholar]

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