Advertisement

Measurement of Phospholipid Metabolism in Intact Neutrophils

  • Susan Sergeant
  • Linda C. McPhail
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1124)

Abstract

Phospholipid-metabolizing enzymes are important participants in neutrophil signal transduction pathways. The methods discussed herein describe assays for assessing the activities of phospholipase A2 (PLA2), phospholipase C (PLC), phospholipase D (PLD), and phosphoinositide 3-OH-kinase in intact neutrophils. PLA2 activity is measured as the release of radiolabeled arachidonic acid. PLC activity is measured as the accumulation of inositol 1,4,5-trisphosphate (IP3), a water-soluble product, using a commercially available radioreceptor assay kit. PLD activity is measured as the appearance of its radiolabeled products, phosphatidic acid and phosphatidylethanol. PI3-K activity is measured as the appearance of its radiolabeled product, phosphatidylinositol-3,4,5-trisphosphate.

Keywords

Neutrophil Phospholipase Lipid kinase Phospholipid Signal transduction Lipid second messengers 

Notes

Acknowledgements

Development of the phospholipase D assay was partially supported by National Institutes of Health grant R01 AI-22564 to L.C.M.

References

  1. 1.
    Vadakekalam J, Metz S (1998) Isotopic efflux studies as indices of phospholipase activation. In: Bird IM (ed) Phospholipid Signaling Protocols. Humana Press, Totowa, NJ, pp 175–185CrossRefGoogle Scholar
  2. 2.
    Mueller HW, O’Flaherty JT, Greene DG et al (1984) 1-O-alkyl-linked glycerophospholipids of human neutrophils: distribution of arachidonate and other acyl residues in the ether-linked and diacyl species. J Lipid Res 25:383–388PubMedGoogle Scholar
  3. 3.
    Solodkin-Szaingurten I, Levy R, Hadad N (2007) Differential behavior of sPLA2-V and sPLA2-X in human neutrophils. Biochim Biophys Acta Mol Cell Biol Lipids 1771:155–163CrossRefGoogle Scholar
  4. 4.
    Bauldry SA, Wooten RE (1996) Leukotriene B4 and platelet activating factor production in permeabilized human neutrophils: Role of cytosolic PLA2 in LTB4 and PAF generation. Biochim Biophys Acta Lipids Lipid Metab 1303:63–73CrossRefGoogle Scholar
  5. 5.
    Ayilavarapu S, Kantarci A, Fredman G et al (2010) Diabetes-induced oxidative stress is mediated by Ca2+-independent phospholipase A2 in neutrophils. J Immunol 184:1507–1515PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Ambruso DR, Ellison MA, Thurman GW et al (2012) Peroxiredoxin 6 translocates to the plasma membrane during neutrophil activation and is required for optimal NADPH oxidase activity. Biochim Biophys Acta Mol Cell Res 1823:306–315CrossRefGoogle Scholar
  7. 7.
    Cockcroft S, Stutchfield J (1989) The receptors for ATP and fMetLeuPhe are independently coupled to phospholipases C and A2 via G-protein(s). Relationship between phospholipase C and A2 activation and exocytosis in HL60 cells and human neutrophils. Biochem J 263:715–723PubMedGoogle Scholar
  8. 8.
    Bauldry SA, Wykle RL, Bass DA (1988) 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 263:16787–16795PubMedGoogle Scholar
  9. 9.
    Suire S, Lecureuil C, Anderson KE et al (2012) GPCR activation of Ras and PI3Kγ in neutrophils depends on PLCβ2/β3 and the RasGEF RasGRP4. EMBO J 31:3118–3129PubMedCrossRefGoogle Scholar
  10. 10.
    Jakus Z, Simon E, Frommhold D et al (2009) Critical role of phospholipase Cβ2 in integrin and Fc receptor-mediated neutrophil functions and the effector phase of autoimmune arthritis. J Exp Med 206:577–593PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Di VF, Vicentini LM, Treves S et al (1985) Inositol phosphate formation in fMet-Leu-Phe-stimulated human neutrophils does not require an increase in the cytosolic free Ca2+ concentration. Biochem J 229:361–367Google Scholar
  12. 12.
    Ferretti ME, Nalli M, Biondi C et al (2001) Modulation of neutrophil phospholipase C activity and cyclic AMP levels by fMLP-OMe analogues. Cell Signal 13:233–240PubMedCrossRefGoogle Scholar
  13. 13.
    Skippen A, Swigart P, Cockcroft S (2012) Measurement of phospholipase C by monitoring inositol phosphates using [3H]inositol labeling protocols in permeabilized cells. In: Lambert DG, Rainbow RD (eds) Calcium Signaling Protocols. Humana Press, Totowa, NJ, pp 163–174Google Scholar
  14. 14.
    Berridge MJ, Dawson RM, Downes CP et al (1983) Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides. Biochem J 212:473–482PubMedGoogle Scholar
  15. 15.
    Ali WH, Chen Q, Delgiorno KE et al (2013) Deficiencies of the lipid-signaling enzymes phospholipase D1 and D2 alter cytoskeletal organization, macrophage phagocytosis, and cytokine-stimulated neutrophil recruitment. PLoS ONE 8:e55325PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Norton LJ, Zhang Q, Saqib KM et al (2011) PLD1 rather than PLD2 regulates phorbol-ester-, adhesion-dependent and Fcγ-receptor-stimulated ROS production in neutrophils. J Cell Sci 124:1973–1983PubMedCrossRefGoogle Scholar
  17. 17.
    Agwu DE, McPhail LC, Wykle RL et al (1989) Mass determination of receptor-mediated accumulation of phosphatidate and diglycerides in human neutrophils measured by Coomassie blue staining and densitometry. Biochem Biophys Res Commun 159:79–86PubMedCrossRefGoogle Scholar
  18. 18.
    Wakelam MJO, Powner DJ, Pettitt TR (2008) Determination of phospholipase D, lysophospholipase D and DG kinase signaling pathways in disease states by mass spectrometry. Adv Enzyme Regul 48:254–260PubMedCrossRefGoogle Scholar
  19. 19.
    Agwu DE, McPhail LC, Chabot MC et al (1989) Choline-linked phosphoglycerides. A source of phosphatidic acid and diglycerides in stimulated neutrophils. J Biol Chem 264:1405–1413PubMedGoogle Scholar
  20. 20.
    Bauldry SA, Elsey KL, Bass DA (1992) Activation of NADPH oxidase and phospholipase D in permeabilized human neutrophils. Correlation between oxidase activation and phosphatidic acid production. J Biol Chem 267:25141–25152PubMedGoogle Scholar
  21. 21.
    Pai JK, Liebl EC, Tettenborn CS et al (1987) 12-O-Tetradecanoylphorbol-13-acetate activates the synthesis of phosphatidylethanol in animal cells exposed to ethanol. Carcinogenesis 8:173–178PubMedCrossRefGoogle Scholar
  22. 22.
    Hu T, Exton JH (2005) 1-Butanol interferes with phospholipase D1 and protein kinase Cα association and inhibits phospholipase D1 basal activity. Biochem Biophys Res Commun 327:1047–1051PubMedCrossRefGoogle Scholar
  23. 23.
    Pedruzzi E, Hakim J, Giroud JP et al (1998) Analysis of choline and phosphorylcholine content in human neutrophils stimulated by f-Met-Leu-Phe and phorbol myristate acetate—contribution of phospholipase D and C. Cell Signal 10:481–489PubMedCrossRefGoogle Scholar
  24. 24.
    Wymann MP, Sozzani S, Altruda F et al (2000) Lipids on the move: phosphoinositide 3-kinases in leukocyte function. Immunol Today 21:260–264PubMedCrossRefGoogle Scholar
  25. 25.
    Hawkins P, Stephens L, Suire S et al (2011) PI3K signaling in neutrophils. Curr Top Microbiol Immunol 346:183–202Google Scholar
  26. 26.
    Endemann G, Yonezawa K, Roth RA (1990) Phosphatidylinositol kinase or an associated protein is a substrate for the insulin receptor tyrosine kinase. J Biol Chem 265:396–400PubMedGoogle Scholar
  27. 27.
    Ding J, Vlahos CJ, Liu R et al (1995) Antagonists of phosphatidylinositol 3-kinase block activation of several novel protein kinases in neutrophils. J Biol Chem 270:11684–11691PubMedCrossRefGoogle Scholar
  28. 28.
    Naccache PH, Levasseur S, Lachance G et al (2000) Stimulation of human neutrophils by chemotactic factors is associated with the activation of phosphatidylinositol 3-kinase gamma. J Biol Chem 275:23636–23641PubMedCrossRefGoogle Scholar
  29. 29.
    Wakelam MJO, Clark J (2011) Methods for analyzing phosphoinositides using mass spectrometry. Biochim Biophys Acta Mol Cell Biol Lipids 1811:758–762CrossRefGoogle Scholar
  30. 30.
    van der Kaay J, Cullen PJ, Downes CP (1998) Phosphatidylinositol(3,4,5)trisphosphate (Ptdins(3,4,5)P3) mass measurement using a radioligand displacement assay. In: Bird IM (ed) Phospholipid Signaling Protocols. Humana Press, Totowa, NJ, pp 109–125CrossRefGoogle Scholar
  31. 31.
    van der Kaay J, Batty IH, Cross DAE et al (1997) A novel, rapid, and highly sensitive mass assay for phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and its application to measure insulin-stimulated PtdIns(3,4,5)P3 production in rat skeletal muscle in vivo. J Biol Chem 272:5477–5481PubMedCrossRefGoogle Scholar
  32. 32.
    Cadwallader KA, Condliffe AM, McGregor A et al (2002) Regulation of phosphatidylinositol 3-kinase activity and phosphatidylinositol 3,4,5-trisphosphate accumulation by neutrophil priming agents. J Immunol 169:3336–3344PubMedGoogle Scholar
  33. 33.
    Traynor-Kaplan AE, Thompson BL, Harris AL et al (1989) Transient increase in phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate during activation of human neutrophils. J Biol Chem 264:15668–15673PubMedGoogle Scholar
  34. 34.
    Cockcroft S (1991) Relationship between arachidonate release and exocytosis in permeabilized human neutrophils stimulated with formylmethionyl-leucyl-phenylalanine (fMetLeuPhe), guanosine 5′-[gamma-thio]triphosphate (GTP[S]) and Ca2+. Biochem J 275:127–131PubMedGoogle Scholar
  35. 35.
    Briand SI, Bernier SG, Guillemette G (1998) Monitoring of phospholipase A2 activation in cultured cells using tritiated arachidonic acid. In: Bird IM (ed) Phospholipid signaling protocols. Humana Press, Totowa, NJ, pp 161–166CrossRefGoogle Scholar
  36. 36.
    Anderson R, Steel HC, Tintinger GR (2005) Inositol 1,4,5-triphosphate-mediated shuttling between intracellular stores and the cytosol contributes to the sustained elevation in cytosolic calcium in FMLP-activated human neutrophils. Biochem Pharmacol 69:1567–1575PubMedCrossRefGoogle Scholar
  37. 37.
    Zhang L (1998) Inositol 1,4,5-trisphosphate mass assay. In: Bird IM (ed) Phospholipid signaling protocols. Humana Press, Totowa, NJ, pp 77–87CrossRefGoogle Scholar
  38. 38.
    Fruman DA, Gamache DA, Ernest MJ (1991) Changes in inositol 1,4,5-trisphosphate mass in agonist-stimulated human neutrophils. Agents Actions 34:16–19PubMedCrossRefGoogle Scholar
  39. 39.
    Heilmann I, Perera IY (2013) Measurement of inositol (1,4,5) trisphosphate in plant tissues by a competitive receptor binding assay. In: Munnik T, Heilmann I (eds) Plant Lipid Signaling Protocols. Humana Press, Totowa, NJ, pp 33–41CrossRefGoogle Scholar
  40. 40.
    Arun SN, Xie D, Howard AC et al (2013) Cell wounding activates phospholipase D in primary mouse keratinocytes. J Lipid Res 54:581–591PubMedCrossRefGoogle Scholar
  41. 41.
    Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917PubMedCrossRefGoogle Scholar
  42. 42.
    Thompson NT, Bonser RW, Tateson JE et al (1991) A quantitative investigation into the dependence of Ca2+ mobilisation on changes in inositol 1,4,5-trisphosphate levels in the stimulated neutrophil. Br J Pharmacol 103:1592–1596PubMedCrossRefGoogle Scholar
  43. 43.
    Sergeant S, McPhail LC (2007) Measurement of phospholipid metabolism in intact neutrophils. Methods Mol Biol 412:69–83PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Susan Sergeant
    • 1
  • Linda C. McPhail
    • 1
  1. 1.Department of BiochemistryWake Forest University School of MedicineWinston-SalemUSA

Personalised recommendations