Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Apr 1;113(1):137–146. doi: 10.1083/jcb.113.1.137

Cytoplasmic lipid bodies of neutrophils: formation induced by cis- unsaturated fatty acids and mediated by protein kinase C

PMCID: PMC2288908  PMID: 1901065

Abstract

Lipid bodies, nonmembrane-bound cytoplasmic inclusions, serve as repositories of esterified arachidonate and are increased in cells associated with inflammatory reactions. We have evaluated stimuli and mechanisms responsible for lipid body formation within human polymorphonuclear leukocytes (PMNs). Arachidonic acid and oleic acid stimulated dose-dependent formation of lipid bodies over 0.5-1 h. Other C20 and C18 fatty acids were less active and demonstrated rank orders as follows: cis-unsaturated fatty acids were much more active than trans-fatty acids, and activity diminished with decreasing numbers of double bonds. Lipid bodies elicited in vitro with cis-fatty acids were ultrastructurally identical to lipid bodies present in PMNs in vivo. Lipid body induction was not because of fatty acid-elicited oxidants or fatty acid-induced ATP depletion. Cis-fatty acid-induced activation of protein kinase C (PKC) was involved in lipid body formation as evidenced by the capacity of other PKC activators, 1-oleoyl-2-acetyl- glycerol and two active phorbol esters, phorbol myristate acetate, and phorbol 12,13 dibutyrate, but not an inactive phorbol, to induce lipid body formation. The PKC inhibitor, 1-O-hexadecyl-2-O-methyl-glycerol, inhibited PMN lipid body formation induced by oleic and arachidonic acids and by 1-oleoyl-2-acetyl-glycerol and phorbol myristate acetate. Other PKC inhibitors (staurosporine, H-7) also inhibited lipid body formation. Formation of lipid bodies in PMNs is a specific cellular response, stimulated by cis-fatty acids and diglycerides and apparently mediated by PKC, which results in the mobilization and deposition of lipids within discrete, ultrastructurally defined cytoplasmic domains.

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agwu D. E., McPhail L. C., Chabot M. C., Daniel L. W., Wykle R. L., McCall C. E. Choline-linked phosphoglycerides. A source of phosphatidic acid and diglycerides in stimulated neutrophils. J Biol Chem. 1989 Jan 25;264(3):1405–1413. [PubMed] [Google Scholar]
  2. Ahnfelt-Rønne I., Olsen U. B. Leukotriene production in rat peritoneal leukocytes requires intact energy metabolism. Biochem Pharmacol. 1985 Sep 1;34(17):3095–3100. doi: 10.1016/0006-2952(85)90153-4. [DOI] [PubMed] [Google Scholar]
  3. Arslan P., Corps A. N., Hesketh T. R., Metcalfe J. C., Pozzan T. cis-Unsaturated fatty acids uncouple mitochondria and stimulate glycolysis in intact lymphocytes. Biochem J. 1984 Jan 15;217(2):419–425. doi: 10.1042/bj2170419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Badwey J. A., Curnutte J. T., Karnovsky M. L. cis-Polyunsaturated fatty acids induce high levels of superoxide production by human neutrophils. J Biol Chem. 1981 Dec 25;256(24):12640–12643. [PubMed] [Google Scholar]
  5. Badwey J. A., Curnutte J. T., Robinson J. M., Berde C. B., Karnovsky M. J., Karnovsky M. L. Effects of free fatty acids on release of superoxide and on change of shape by human neutrophils. Reversibility by albumin. J Biol Chem. 1984 Jun 25;259(12):7870–7877. [PubMed] [Google Scholar]
  6. Bass D. A., Gerard C., Olbrantz P., Wilson J., McCall C. E., McPhail L. C. Priming of the respiratory burst of neutrophils by diacylglycerol. Independence from activation or translocation of protein kinase C. J Biol Chem. 1987 May 15;262(14):6643–6649. [PubMed] [Google Scholar]
  7. Bass D. A., McPhail L. C., Schmitt J. D., Morris-Natschke S., McCall C. E., Wykle R. L. Selective priming of rate and duration of the respiratory burst of neutrophils by 1,2-diacyl and 1-O-alkyl-2-acyl diglycerides. Possible relation to effects on protein kinase C. J Biol Chem. 1988 Dec 25;263(36):19610–19617. [PubMed] [Google Scholar]
  8. Bauldry S. A., Wykle R. L., Bass D. A. Phospholipase A2 activation in human neutrophils. Differential actions of diacylglycerols and alkylacylglycerols in priming cells for stimulation by N-formyl-Met-Leu-Phe. J Biol Chem. 1988 Nov 15;263(32):16787–16795. [PubMed] [Google Scholar]
  9. Cabot M. C., Jaken S. Structural and chemical specificity of diacylglycerols for protein kinase C activation. Biochem Biophys Res Commun. 1984 Nov 30;125(1):163–169. doi: 10.1016/s0006-291x(84)80349-6. [DOI] [PubMed] [Google Scholar]
  10. Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U., Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem. 1982 Jul 10;257(13):7847–7851. [PubMed] [Google Scholar]
  11. Chien K. R., Sen A., Reynolds R., Chang A., Kim Y., Gunn M. D., Buja L. M., Willerson J. T. Release of arachidonate from membrane phospholipids in cultured neonatal rat myocardial cells during adenosine triphosphate depletion. Correlation with the progression of cell injury. J Clin Invest. 1985 Jun;75(6):1770–1780. doi: 10.1172/JCI111889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cohen H. J., Chovaniec M. E. Superoxide production by digitonin-stimulated guinea pig granulocytes. The effects of N-ethyl maleimide, divalent cations; and glycolytic and mitochondrial inhibitors on the activation of the superoxide generating system. J Clin Invest. 1978 Apr;61(4):1088–1096. doi: 10.1172/JCI109008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Coimbra A., Lopes-Vaz A. The presence of lipid droplets and the absence of stable sudanophilia in osmium-fixed human leukocytes. J Histochem Cytochem. 1971 Sep;19(9):551–557. doi: 10.1177/19.9.551. [DOI] [PubMed] [Google Scholar]
  14. Daniel L. W., Small G. W., Schmitt J. D., Marasco C. J., Ishaq K., Piantadosi C. Alkyl-linked diglycerides inhibit protein kinase C activation by diacylglycerols. Biochem Biophys Res Commun. 1988 Feb 29;151(1):291–297. doi: 10.1016/0006-291x(88)90592-x. [DOI] [PubMed] [Google Scholar]
  15. Dell K. R., Severson D. L. Effect of cis-unsaturated fatty acids on aortic protein kinase C activity. Biochem J. 1989 Feb 15;258(1):171–175. doi: 10.1042/bj2580171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dougherty R. W., Dubay G. R., Niedel J. E. Dynamics of the diradylglycerol responses of stimulated phagocytes. J Biol Chem. 1989 Jul 5;264(19):11263–11269. [PubMed] [Google Scholar]
  17. Dvorak A. M., Dvorak H. F., Peters S. P., Shulman E. S., MacGlashan D. W., Jr, Pyne K., Harvey V. S., Galli S. J., Lichtenstein L. M. Lipid bodies: cytoplasmic organelles important to arachidonate metabolism in macrophages and mast cells. J Immunol. 1983 Dec;131(6):2965–2976. [PubMed] [Google Scholar]
  18. Dvorak A. M., Hammel I., Schulman E. S., Peters S. P., MacGlashan D. W., Jr, Schleimer R. P., Newball H. H., Pyne K., Dvorak H. F., Lichtenstein L. M. Differences in the behavior of cytoplasmic granules and lipid bodies during human lung mast cell degranulation. J Cell Biol. 1984 Nov;99(5):1678–1687. doi: 10.1083/jcb.99.5.1678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fantone J., Jester S., Loomis T. Metmyoglobin promotes arachidonic acid peroxidation at acid pH. J Biol Chem. 1989 Jun 5;264(16):9408–9411. [PubMed] [Google Scholar]
  20. Fridovich S. E., Porter N. A. Oxidation of arachidonic acid in micelles by superoxide and hydrogen peroxide. J Biol Chem. 1981 Jan 10;256(1):260–265. [PubMed] [Google Scholar]
  21. Fujita I., Irita K., Takeshige K., Minakami S. Diacylglycerol, 1-oleoyl-2-acetyl-glycerol, stimulates superoxide-generation from human neutrophils. Biochem Biophys Res Commun. 1984 Apr 30;120(2):318–324. doi: 10.1016/0006-291x(84)91256-7. [DOI] [PubMed] [Google Scholar]
  22. Jemelin M., Frei J. Leukocyte energy metabolism. 3. Anaerobic and aerobic ATP production and related enzymes. Enzymol Biol Clin (Basel) 1970;11(4):298–323. [PubMed] [Google Scholar]
  23. Kramer I. M., van der Bend R. L., Tool A. T., van Blitterswijk W. J., Roos D., Verhoeven A. J. 1-O-hexadecyl-2-Q-methylglycerol, a novel inhibitor of protein kinase C, inhibits the respiratory burst in human neutrophils. J Biol Chem. 1989 Apr 5;264(10):5876–5884. [PubMed] [Google Scholar]
  24. Lambeth J. D., Burnham D. N., Tyagi S. R. Sphinganine effects on chemoattractant-induced diacylglycerol generation, calcium fluxes, superoxide production, and on cell viability in the human neutrophil. Delivery of sphinganine with bovine serum albumin minimizes cytotoxicity without affecting inhibition of the respiratory burst. J Biol Chem. 1988 Mar 15;263(8):3818–3822. [PubMed] [Google Scholar]
  25. Liles W. C., Meier K. E., Henderson W. R. Phorbol myristate acetate and the calcium ionophore A23187 synergistically induce release of LTB4 by human neutrophils: involvement of protein kinase C activation in regulation of the 5-lipoxygenase pathway. J Immunol. 1987 May 15;138(10):3396–3402. [PubMed] [Google Scholar]
  26. McIntyre T. M., Reinhold S. L., Prescott S. M., Zimmerman G. A. Protein kinase C activity appears to be required for the synthesis of platelet-activating factor and leukotriene B4 by human neutrophils. J Biol Chem. 1987 Nov 15;262(32):15370–15376. [PubMed] [Google Scholar]
  27. Murakami K., Chan S. Y., Routtenberg A. Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids. J Biol Chem. 1986 Nov 25;261(33):15424–15429. [PubMed] [Google Scholar]
  28. Niedel J. E., Kuhn L. J., Vandenbark G. R. Phorbol diester receptor copurifies with protein kinase C. Proc Natl Acad Sci U S A. 1983 Jan;80(1):36–40. doi: 10.1073/pnas.80.1.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. O'Flaherty J. T., Schmitt J. D., McCall C. E., Wykle R. L. Diacylglycerols enhance human neutrophil degranulation responses: relevancy to a multiple mediator hypothesis of cell function. Biochem Biophys Res Commun. 1984 Aug 30;123(1):64–70. doi: 10.1016/0006-291x(84)90380-2. [DOI] [PubMed] [Google Scholar]
  30. Peters-Golden M., Shelly C. Inhibitory effect of exogenous arachidonic acid on alveolar macrophage 5-lipoxygenase metabolism. Role of ATP depletion. J Immunol. 1988 Mar 15;140(6):1958–1966. [PubMed] [Google Scholar]
  31. Phillips W. A., Mossmann H., Ferber E. Changes in the incorporation of free fatty acids upon the stimulation of human polymorphonuclear leukocytes. J Leukoc Biol. 1986 Mar;39(3):267–284. doi: 10.1002/jlb.39.3.267. [DOI] [PubMed] [Google Scholar]
  32. Rider L. G., Dougherty R. W., Niedel J. E. Phorbol diesters and dioctanoylglycerol stimulate accumulation of both diacylglycerols and alkylacylglycerols in human neutrophils. J Immunol. 1988 Jan 1;140(1):200–207. [PubMed] [Google Scholar]
  33. Robinson J. M., Karnovsky M. L., Karnovsky M. J. Glycogen accumulation in polymorphonuclear leukocytes, and other intracellular alterations that occur during inflammation. J Cell Biol. 1982 Dec;95(3):933–942. doi: 10.1083/jcb.95.3.933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rottenberg H., Hashimoto K. Fatty acid uncoupling of oxidative phosphorylation in rat liver mitochondria. Biochemistry. 1986 Apr 8;25(7):1747–1755. doi: 10.1021/bi00355a045. [DOI] [PubMed] [Google Scholar]
  35. Sekiguchi K., Tsukuda M., Ase K., Kikkawa U., Nishizuka Y. Mode of activation and kinetic properties of three distinct forms of protein kinase C from rat brain. J Biochem. 1988 May;103(5):759–765. doi: 10.1093/oxfordjournals.jbchem.a122343. [DOI] [PubMed] [Google Scholar]
  36. Sekiguchi K., Tsukuda M., Ogita K., Kikkawa U., Nishizuka Y. Three distinct forms of rat brain protein kinase C: differential response to unsaturated fatty acids. Biochem Biophys Res Commun. 1987 Jun 15;145(2):797–802. doi: 10.1016/0006-291x(87)91035-7. [DOI] [PubMed] [Google Scholar]
  37. Serhan C. N., Broekman M. J., Korchak H. M., Marcus A. J., Weissmann G. Endogenous phospholipid metabolism in stimulated neutrophils differential activation by FMLP and PMA. Biochem Biophys Res Commun. 1982 Aug;107(3):951–958. doi: 10.1016/0006-291x(82)90615-5. [DOI] [PubMed] [Google Scholar]
  38. Shearman M. S., Naor Z., Sekiguchi K., Kishimoto A., Nishizuka Y. Selective activation of the gamma-subspecies of protein kinase C from bovine cerebellum by arachidonic acid and its lipoxygenase metabolites. FEBS Lett. 1989 Jan 30;243(2):177–182. doi: 10.1016/0014-5793(89)80125-5. [DOI] [PubMed] [Google Scholar]
  39. Smith R. J., Sam L. M., Justen J. M., Leach K. L., Epps D. E. Human polymorphonuclear neutrophil activation with arachidonic acid. Br J Pharmacol. 1987 Jul;91(3):641–649. doi: 10.1111/j.1476-5381.1987.tb11258.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Spragg R. G., Hinshaw D. B., Hyslop P. A., Schraufstätter I. U., Cochrane C. G. Alterations in adenosine triphosphate and energy charge in cultured endothelial and P388D1 cells after oxidant injury. J Clin Invest. 1985 Oct;76(4):1471–1476. doi: 10.1172/JCI112126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tanaka T., Makino R., Iizuka T., Ishimura Y., Kanegasaki S. Activation by saturated and monounsaturated fatty acids of the O2- -generating system in a cell-free preparation from neutrophils. J Biol Chem. 1988 Sep 25;263(27):13670–13676. [PubMed] [Google Scholar]
  42. Tou J. S. Activation of the metabolism of the fatty acyl group in granulocyte phospholipids by phorbol myristate acetate. Biochim Biophys Acta. 1981 Sep 24;665(3):491–497. doi: 10.1016/0005-2760(81)90262-9. [DOI] [PubMed] [Google Scholar]
  43. Tyagi S. R., Tamura M., Burnham D. N., Lambeth J. D. Phorbol myristate acetate (PMA) augments chemoattractant-induced diglyceride generation in human neutrophils but inhibits phosphoinositide hydrolysis. Implications for the mechanism of PMA priming of the respiratory burst. J Biol Chem. 1988 Sep 15;263(26):13191–13198. [PubMed] [Google Scholar]
  44. Weller P. F., Ackerman S. J., Nicholson-Weller A., Dvorak A. M. Cytoplasmic lipid bodies of human neutrophilic leukocytes. Am J Pathol. 1989 Nov;135(5):947–959. [PMC free article] [PubMed] [Google Scholar]
  45. Weller P. F., Dvorak A. M. Arachidonic acid incorporation by cytoplasmic lipid bodies of human eosinophils. Blood. 1985 May;65(5):1269–1274. [PubMed] [Google Scholar]
  46. Weller P. F., Monahan-Earley R. A., Dvorak H. F., Dvorak A. M. Cytoplasmic lipid bodies of human eosinophils. Subcellular isolation and analysis of arachidonate incorporation. Am J Pathol. 1991 Jan;138(1):141–148. [PMC free article] [PubMed] [Google Scholar]
  47. Willingham M. C., Rutherford A. V. The use of osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells. J Histochem Cytochem. 1984 Apr;32(4):455–460. doi: 10.1177/32.4.6323574. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES