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Type 4A cAMP-specific phosphodiesterase is stored in granules of human neutrophils and eosinophils

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Abstract

Persistent elevations of cAMP levels are generally accompanied by an inhibition of granulocyte functions. Phosphodiesterases play a critical role in regulating intracellular levels of cAMP. The expression of three isoforms of type 4 cAMP-specific phosphodiesterase (PDE4) in neutrophils suggests diversity of isoform localization and targeting in regulating cell function. The sites of cAMP regulation in granulocytes by the PDE4A isoform were investigated by immunoelectron microscopy. PDE4A was localized uniformly in all granule classes of eosinophils, but was restricted in neutrophils to a subset of myeloperoxidase (MPO)-containing granules that were round or elongated with a central crystalloid core. Granulocytes were stimulated with fMLP to investigate the sites of PDE4A targeting during cell activation. In neutrophils, fMLP induced a rapid (1 min) translocation of granules containing PDE4A to the plasmalemma, where some PDE4A and MPO were exocytosed. In these cells, PDE4A labeling within granules was focal and no longer homogeneous. While immunogold labeling of PDE4A was reduced after fMLP stimulation, staining of MPO-containing granules remained high. Extracellular release of PDE4A was also observed in eosinophils stimulated with fMLP. Morphometry revealed that Au labeling was significantly reduced within 1 min, and that there was a shift in PDE4A localization within eosinophil granules from the crystalline core to the matrix. Fluctuations of cAMP levels and ectoprotein kinase activity with PKA properties occur in blood under normal and pathological conditions. The exclusive localization of PDE4A within granules of neutrophils and eosinophils suggests that PDE4A may function to downregulate cAMP signaling at the cell membrane and/or in the extracellular space at the time of granule release.

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References

  • Agwu DE, McCall CE, McPhail LC (1991) Regulation of phospholipase D-induced hydrolysis of choline-containing phosphoglycerides by cyclic AMP in human neutrophils. J Immunol 146:3895–3903

    CAS  PubMed  Google Scholar 

  • Baillie GS, Huston E, Scotland G, Hodgkin M, Gall I, Peden AH, MacKenzie C, Houslay ES, Currie R, Pettitt TR, Walmsley AR, Wakelam MJO, Warwicker J, Houslay MD (2002) TAPAS-1, a novel microdomain within the unique N-terminal region of the PDE4A1 cAMP-specific phosphodiesterase that allows rapid, Ca2+-triggered membrane association with selectivity for interaction with phosphatidic acid. J Biol Chem 277:28298–22002

    Article  CAS  PubMed  Google Scholar 

  • Bainton DF, Miller LJ, Kishimoto TK, Springer TA (1987) Leukocyte adhesion receptors are stored in peroxidase-negative granules of human neutrophils. J Exp Med 166:1641–1653

    CAS  PubMed  Google Scholar 

  • Beard MB, Huston E, Campbell L, Gall I, McPhee I, Yarwood S, Scotland G, Houslay MD (2002) In addition to the SH3 binding region, multiple regions within the N-terminal noncatalytic portion of the cAMP-specific phosphodiesterase, PDE4A5, contribute to its intracellular targeting. Cell Signal 14:453–465

    Article  CAS  PubMed  Google Scholar 

  • Beil WJ, Weller PF, Tzizik DM, Galli SJ, Dvorak AM (1993) Ultrastructural immunogold localization of tumor necrosis factor-α to the matrix compartment of eosinophil secondary granules in patients with idiopathic hypereosinophilic syndrome. J Histochem Cytochem 41:1611

    CAS  PubMed  Google Scholar 

  • Bengis-Garber C, Gruener N (1996) Protein kinase A down-regulates the phosphorylation of p46 Phox in human neutrophils: a possible pathway for inhibition of the respiratory burst. Cell Signal 8:291–296

    Article  CAS  PubMed  Google Scholar 

  • Borregaard N, Cowland JB (1997) Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 89:3503–3521

    CAS  PubMed  Google Scholar 

  • Coffey RG (1992) Effects of cyclic nucleotides on granulocytes. In: Coffey RG (ed) Granulocyte responses to cytokines: basic and clinical research, vol. Marcel Dekker, New York, pp 301–338

  • Damiano VV, Kucich U, Murer E, Laudenslager N, Weinbaum G (1988) Ultrastructural quantitation of peroxidase- and elastase-containing granules in human neutrophils. Am J Pathol 131:235–245

    CAS  PubMed  Google Scholar 

  • Dent G, Giembycz MA, Rabe KF, Barnes PJ (1991) Inhibition of eosinophil cyclic nucleotide PDE activity and opsonized zymosan-stimulated respiratory burst by "type IV"-selective PDE inhibitors. Br J Pharmacol 103:1339–1346

    CAS  PubMed  Google Scholar 

  • Derian CK, Santulli RJ, Rao PE, Solomon HF, Barret JA (1995) Inhibition of chemotactic peptide-induced neutrophil adhesion to vascular endothelium by cAMP modulators. J Immunol 154:308–317

    CAS  PubMed  Google Scholar 

  • Dubucquoi S, Desreumaux P, Janin A, Klein O, Goldman M, Tavernier J, Capron A, Capron M (1994) Interleukin 5 synthesis by eosinophils: association with granules and immunoglobulin-dependent secretion. J Exp Med 179:703–708

    CAS  PubMed  Google Scholar 

  • Dubyak GR, Cowen DS, Lazarus HN (1990) Activation of the inositol phospholipid signaling system by receptors for extracellular ATP in human neutrophils, monocytes, and neutrophil/monocyte progenitor cells. Ann N Y Acad Sci 603:218–238

    Google Scholar 

  • Dusenbery KE, Mendiola JR, Skubitz KM (1988) Evidence for ecto-protein kinase activity on the surface of human neutrophils. Biochem Biophys Res Comm 153:7–13

    CAS  PubMed  Google Scholar 

  • Egesten A, Breton-Gorius J, Guichard J, Gullberg U, Olsson I (1994) The heterogeneity of azurophil granules in neutrophil promyelocytes: immunogold localization of myeloperoxidase, cathepsin G, elastase, proteinase 3, and bactericidal/permeability increasing protein. Blood 83:2985–2994

    CAS  PubMed  Google Scholar 

  • Ehrlich YH, Hogan MV, Pawlowska Z, Naik U, Kornecki E (1990) Ectoprotein kinase in the regulation of cellular responsiveness to extracellular ATP. Ann N Y Acad Sci 603:401–416

    CAS  PubMed  Google Scholar 

  • Emes CH, Crawford N (1982) Ecto-protein kinase activity in rabbit peritoneal polymorphonuclear leukocytes. Biochim Biophys Acta 717:98–104

    Article  CAS  PubMed  Google Scholar 

  • Goding JW (2000) Ecto-enzymes: physiology meets pathology. J Leukoc Biol 67:285–311

    CAS  PubMed  Google Scholar 

  • Gordon JL (1986) Extracellular ATP: effects, sources and fate. Biochem J 233:309–319

    CAS  PubMed  Google Scholar 

  • Grady PG, Thomas LL (1986) Characterization of cyclic-nucleotide phosphodiesterase activities in resting and N-formylmethionylleucylphenylalanine-stimulated human neutrophils. Biochim Biophys Acta 885:282–293

    Article  CAS  PubMed  Google Scholar 

  • Harvath L, Robbins JD, Russell AA, Seamon KB (1991) cAMP and human neutrophil chemotaxis. J Immunol 146:224–232

    CAS  PubMed  Google Scholar 

  • Hatmi M, Gavaret JM, Elalamy I, Vargaftig BB, Jacquemin C (1996) Evidence for cAMP-dependent platelet ectoprotein kinase activity that phosphorylates platelet glycoprotein IV (CD36). J Biol Chem 271:24776–24780

    Article  CAS  PubMed  Google Scholar 

  • Houslay MD, Scotland G, Erdogan S, Huston E, Mackenzie S, McCallum JF, McPhee I, Pooley L, Rena G, Ross A, Beard M, Peder A, Begg F, Wilkinson I, Yarwood S, Ackerman C, Houslay ES, Hoffman R, Engels P, Sullivan M, Bolger G (1997) Intracellular targeting, interaction with Src homology 3 (SH3) domains and rolipram-detected conformational switches in cAMP-specific PDE4A phosphodiesterase. Biochem Soc Trans 25:374–381

    CAS  PubMed  Google Scholar 

  • Iannone MA, Wolberg G, Zimmerman TP (1989) Chemotactic peptide induces cAMP elevation in human neutrophils by amplification of the adenylate cyclase response to endogenously produced adenosine. J Biol Chem 264:20177–20180

    CAS  PubMed  Google Scholar 

  • Kubler D, Pyerin W, Bill O, Hotz A, Sonka J, Kinzel V (1989) Evidence for ecto-protein kinase activity that phosphorylates kemptide in a cyclic AMP-dependent mode. J Biol Chem 264:14549–14555

    CAS  PubMed  Google Scholar 

  • Levi-Schaffer F, Lacy P, Severs NJ, Newman TM, North J, Gomperts B, Kay AB, Moqbel R (1995) Association of granulocyte-macrophage colony-stimulating factor with the crystalloid granules of human eosinophils. Blood 85:2579–2586

    CAS  PubMed  Google Scholar 

  • Madden VJ (1998) Microwave processing of cell monolayers in situ for post-embedding immunocytochemistry with retention of ultrastructure and antigenicity. Microsc Microanal 4:854–855

    Google Scholar 

  • Manning CD, McLaughlin MM, Livi GP, Cieslinski LB, Torphy TJ, Barnette MS (1996) Prolonged beta adrenoreceptor stimulation up-regulates cAMP phosphodiesterase activity in human monocytes by increasing mRNA and protein for phosphodiesterases 4A and 4B. J Pharmacol Exp Ther 276:810–818

    CAS  PubMed  Google Scholar 

  • Michel JJ, Scott JD (2002) AKAP mediated signal transduction. Annu Rev Pharmacol Toxicol 42:235–257

    Article  CAS  PubMed  Google Scholar 

  • Newsholme SJ, Schwartz L (1993) cAMP-specific phosphodiesterase inhibitor, rolipram, reduces eosinophil infiltration evoked by leukotrienes or by histamine in guinea pig conjunctiva. Inflammation 17:25–31

    CAS  PubMed  Google Scholar 

  • Nielson CP, Vestal RE, Sturm RJ, Heaslip R (1990) Effects of selective phosphodiesterase inhibitors on the polymorphonuclear leukocyte respiratory burst. J Allergy Clin Immunol 86:801–808

    CAS  PubMed  Google Scholar 

  • Nourshargh S, Hoult JRS (1986) Inhibition of human neutrophil degranulation by forskolin in the presence of phosphodiesterase inhibitors. Eur J Pharmacol 122:205–212

    Article  CAS  PubMed  Google Scholar 

  • O'Connell JC, McCallum JF, McPhee I, Wakefield J, Houslay ES, Wishart W, Bolger G, Frame M, Houslay MD (1996) The SH3 domain of Src tyrosyl protein kinase interacts with the N-terminal splice region of the PDE4A cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5). Biochem J 318:255–262

    CAS  PubMed  Google Scholar 

  • Ottonello L, Morone MP, Dapino P, Dallegri F (1995a) Cyclic AMP-elevating agents down-regulate the oxidative burst induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) in adherent neutrophils. Clin Exp Immunol 101:502–506

    CAS  PubMed  Google Scholar 

  • Ottonello L, Morone MP, Dapino P, Dallegri F (1995b) Tumour necrosis factor alpha-induced oxidative burst in neutrophils adherent to fibronectin: effects of cyclic AMP-elevating agents. Br J Haematol 91:566–570

    CAS  PubMed  Google Scholar 

  • Peachell PT, Undem BJ, Schleimer RP, R. W. MacGlashan J, Lichtenstein LM, Cieslinski LB, Torphy TJ (1992) Preliminary identification and role of phosphodiesterase isozymes in human basophils. J Immunol 148:2503–2510

    CAS  PubMed  Google Scholar 

  • Plaut M, Marone G, Thomas LL, Lichtenstein LM (1980) Cyclic nucleotides in immune responses and allergy. Adv Cyclic Nucleotide Res 12:161–171

    CAS  PubMed  Google Scholar 

  • Pryzwansky KB (1987) Human leukocytes as viewed by stereo high voltage electron microscopy. Blood Cells 12:505–530

    CAS  PubMed  Google Scholar 

  • Pryzwansky KB, Breton-Gorius J (1985) Identification of a subpopulation of primary granules in human neutrophils based upon maturation and distribution. Lab Invest 53:664–671

    CAS  PubMed  Google Scholar 

  • Pryzwansky KB, Merricks EP (1998) Chemotactic peptide-induced changes of intermediate filament organization in neutrophils during granule secretion: role of cyclic guanosine monophosphate. Mol Biol Cell 9:2933–2947

    CAS  PubMed  Google Scholar 

  • Pryzwansky KB, Steiner AL, Spitznagel JK, Kapoor CL (1981) Compartmentalization of cyclic AMP during phagocytosis by human neutrophilic granulocytes. Science 211:407–410

    CAS  PubMed  Google Scholar 

  • Pryzwansky KB, Wyatt TA, Reed W, Ross GD (1991) Phorbol ester induces transient focal concentrations of functional, newly expressed CR3 in neutrophils at sites of specific granule exocytosis. Eur J Cell Biol 54:61–75

    CAS  PubMed  Google Scholar 

  • Pryzwansky KB, Kidao S, Merricks EP (1998) Compartmentalization of PDE-4 and cAMP-dependent protein kinase in neutrophils and macrophages during phagocytosis. Cell Biochem Biophys 28:251–275

    CAS  PubMed  Google Scholar 

  • Pryzwansky K, Madden VJ, Jones SK (2001) Type 4A cAMP-specific phosphodiesterase (PDE-4A) is localized within granules of human granulocytes and is exocytosed during fMLP stimulation. FASEB J 15:A1172

    Google Scholar 

  • Rice WG, J. M. Kinkade J, Parmley RT (1986) High resolution of heterogeneity among human neutrophil granules: physical, biochemical, and ultrastructural properties of isolated fractions. Blood 68:541–555

    CAS  PubMed  Google Scholar 

  • Rice WG, Ganz T, Kinkade JM, Selsted ME, Lehrer RI, Parmley RT (1987) Defensin-rich granules of human neutrophils. Blood 70:757–765

    CAS  PubMed  Google Scholar 

  • Santamaria LF, Palacios JM, Belta J (1997) Inhibition of eotaxin-mediated human eosinophil activation and migration by the selective cyclic nucleotide phosphodiesterase type 4 inhibitor rolipram. Br J Pharmacol 121:1150–1154

    CAS  PubMed  Google Scholar 

  • Scotland G, Houslay MD (1995) Chimeric constructs show that the unique N-terminal domain of the cyclic AMP phosphodiesterase RD1 (RNPDE4A1A; rPDE-IVA1) can confer membrane association upon the normally cytosolic protein chloramphenicol acetyltransferase. Biochem J 308:673–681

    CAS  PubMed  Google Scholar 

  • Shakur Y, Wilson M, Pooley L, Lobban M, Griffiths SL, Campbell AM, Beattie J, Daly C, Houslay MD (1995) Identification and characterization of the type IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum. Biochem J 306:801–809

    CAS  PubMed  Google Scholar 

  • Simchowitz L, Fischbein LC, Spilberg I, Atkinson JP (1980) Induction of a transient elevation in intracellular levels of adenosine-3'-5'-cyclic monophosphate by chemotactic factors: an early event in human neutrophil activation. J Immunol 125:1482–1491

    Google Scholar 

  • Skubitz KM, Goueli SA (1991) Basic fibroblast growth factor is a substrate for phosphorylation by human neutrophil ecto-protein kinase activity. Biochem Biophys Res Comm 174:49–55

    CAS  PubMed  Google Scholar 

  • Skubitz KM, Ehresmann DD, Ducker TP (1991) Characterization of human neutrophil ecto-protein kinase activity released by kinase substrates. J Immunol 147:638–650

    CAS  PubMed  Google Scholar 

  • Smith KJ, Scotland G, Beattie J, Trayer IP, Houslay MD (1996) Determination of the structure of the N-terminal splice region of the cyclic AMP-specific phosphodiesterase RD1 (RNPDE4A1) by H NMR and identification of the membrane association domain using chimeric constructs. J Biol Chem 271:16703–16711

    Article  CAS  PubMed  Google Scholar 

  • Smolen JE, Geosits SJ (1984) Human neutrophil phosphodiesterase. Inflammation 2:193–199

    Google Scholar 

  • Smolen JE, Weissmann G (1981) Stimuli which provoke secretion of azurophil enzymes from human neutrophils induce increments in adenosine cyclic 3'-5'-monophosphate. Biochim Biophys Acta 672:197–206

    Article  CAS  PubMed  Google Scholar 

  • Souness JE, Carter CM, Diocee BK, Hassall GA, Wood LJ, Turner NC (1991) Characterization of guinea-pig eosinophil phosphodiesterase activity. Biochem Pharmacol 42:937–945

    Article  CAS  PubMed  Google Scholar 

  • Takenawa T, Ishitoya J, Nagai Y (1986) Inhibitory effect of prostaglandin E2, forskolin, and dibutyryl cAMP on arachidonic acid release and inositol phospholipid metabolism in guinea pig neutrophils. J Biol Chem 261:1092–1098

    CAS  PubMed  Google Scholar 

  • Teshima R, Saito Y, Ikebuchi H, DeSilva NR, Morita Y, Nakanishi M, Sawda J, Kitani S (1997) Effect of an ectokinase inhibitor, K252b, on degranulation and Ca2+ signals of RBL-2H3 cells and human basophils. J Immunol 159:964–969

    CAS  PubMed  Google Scholar 

  • Underwood DC, Osborn RR, Novak LB, Matthews JK, Newsholme SJ, Undem BJ, Hand JM, Torphy TJ (1993) Inhibition of antigen-induced bronchoconstriction and eosinophil infiltration in the guinea pig by the cyclic AMP-specific phosphodiesterase inhibitor, rolipram. J Pharmacol Exp Ther 266:306–313

    CAS  PubMed  Google Scholar 

  • Verghese MW, Fox K, McPhail LC, Snyderman R (1985) Chemoattractant-elicited alterations of cAMP levels in human polymorphonuclear leukocytes require a Ca2+-dependent mechanism which is independent of transmembrane activation of adenylate cyclase. J Biol Chem 260:6769–6775

    CAS  PubMed  Google Scholar 

  • Verghese MW, McConnell RT, Lenhard JM, Hamacher L, Jin S-LC (1995a) Regulation of distinct cyclic AMP-specific phosphodiesterase (phosphodiesterase type 4) isozymes in human monocytic cells. Mol Pharmacol 47:1164–1171

    CAS  PubMed  Google Scholar 

  • Verghese MW, McConnell RT, Strickland AB, Gooding RC, Stimpson SA, Yarnall DP, Taylor JD, Furdon PJ (1995b) Differential regulation of human monocyte-derived TNFα and IL-1β by Type IV cAMP-phosphodiesterase (cAMP-PDE) inhibitors. J Pharmacol Exp Ther 272:1313–1320

    CAS  PubMed  Google Scholar 

  • Wang P, Wu P, Ohleth KM, Egan RW, Billah MM (1999) Phosphodiesterase 4B2 is the predominant phosphodiesterase species and undergoes differential regulation of gene expression in human monocytes and neutrophils. Mol Pharmacol 56:170–174

    CAS  PubMed  Google Scholar 

  • Ward PA, Walker BAM, Hagenlocker BE (1990) Functional consequences of interactions between human neutrophils and ATP, ATPγS, and adenosine. Ann N Y Acad Sci 603:108–119

    CAS  PubMed  Google Scholar 

  • Wright CD, Kuipers PJ, Kobylarz-Singer D, Devall LJ, Klinkefus BA, Weishaar RE (1990) Differential inhibition of human neutrophil functions. Biochem Pharmacol 40:699–707

    CAS  PubMed  Google Scholar 

  • Wyatt TA, Lincoln TM, Pryzwansky KB (1993) Regulation of human neutrophil degranulation by LY83583 and l-arginine: role of cGMP-dependent protein kinase. Am J Physiol Cell Physiol 34:C201–C211

    Google Scholar 

  • Ydrenius L, Majeed M, Rasmusson BJ, Stendahl O, Sarndahl E (2000) Activation of cAMP-dependent protein kinase is necessary for actin rearrangements in human neutrophils during phagocytosis. J Leukoc Biol 67:520–528

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Susan Jones for technical assistance, and Dr. Sharon Wolda of ICOS Corporation for antibodies to PDE4A.

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  1. Department of Pathology and Laboratory Medicine, Brinkhous-Bullitt Building, University of North Carolina, Chapel Hill, NC 27599-7525, USA

    Katherine B. Pryzwansky

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  1. Katherine B. Pryzwansky
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  2. Victoria J. Madden
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Correspondence to Katherine B. Pryzwansky.

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This study was supported by NSF grant #MCB-9421731 and University of North Carolina Faculty Research Grant 3-13645

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Pryzwansky, K.B., Madden, V.J. Type 4A cAMP-specific phosphodiesterase is stored in granules of human neutrophils and eosinophils. Cell Tissue Res 312, 301–311 (2003). https://doi.org/10.1007/s00441-003-0728-y

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