Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1991 Jun;87(6):2012–2017. doi: 10.1172/JCI115230

Shape changes, exocytosis, and cytosolic free calcium changes in stimulated human eosinophils.

P Kernen 1, M P Wymann 1, V von Tscharner 1, D A Deranleau 1, P C Tai 1, C J Spry 1, C A Dahinden 1, M Baggiolini 1
PMCID: PMC296956  PMID: 2040692

Abstract

Essentially pure preparations of normal density eosinophils obtained from patients with hypereosinophilic syndrome (HES) were stimulated with complement factor 5a (C5a), platelet-activating factor (PAF), FMLP and neutrophil-activating peptide (NAP-1/IL-8). Three responses were studied, the transient rise in cytosolic free calcium concentration ([Ca2+]i) (derived from indo-1 fluorescence), shape changes (measured by laser turbidimetry), and exocytosis of eosinophil peroxidase (EPO) (assessed by H2O2/luminol-dependent chemiluminescence). Responses were obtained with all four agonists, but C5a and PAF were by far more potent than FMLP and NAP-1/IL-8, which induced only minor effects. Pretreatment of the cells with pertussis toxin attenuated [Ca2+]i changes, EPO release and, to a lesser extent, shape changes, indicating that GTP-binding proteins of Gi-type are involved in receptor-dependent signal transduction processes leading to these responses. A clear dissociation was observed in the control of the shape change response and EPO exocytosis. The shape change was not affected by Ca2+ depletion or treatment with the protein kinase inhibitor staurosporine, but exocytosis was prevented by Ca2+ depletion and markedly enhanced by staurosporine. The activation of the contractile system, leading to shape changes and motility, thus appears to be independent of the classical signal transduction pathway involving phospholipase C, a [Ca2+]i rise and protein kinase C activation. Exocytosis is, as expected, Ca2+ dependent and appears to be under a negative control involving protein phosphorylations.

Full text

PDF
2012

Selected References

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

  1. Anwar A. R., Kay A. B. The ECF-A tetrapeptides and histamine selectively enhance human eosinophil complement receptors. Nature. 1977 Oct 6;269(5628):522–524. doi: 10.1038/269522a0. [DOI] [PubMed] [Google Scholar]
  2. Badwey J. A., Horn W., Heyworth P. G., Robinson J. M., Karnovsky M. L. Paradoxical effects of retinal in neutrophil stimulation. J Biol Chem. 1989 Sep 5;264(25):14947–14953. [PubMed] [Google Scholar]
  3. Baggiolini M., Dewald B. Cellular models for the detection and evaluation of drugs that modulate human phagocyte activity. Experientia. 1988 Oct 15;44(10):841–848. doi: 10.1007/BF01941181. [DOI] [PubMed] [Google Scholar]
  4. Baggiolini M., Walz A., Kunkel S. L. Neutrophil-activating peptide-1/interleukin 8, a novel cytokine that activates neutrophils. J Clin Invest. 1989 Oct;84(4):1045–1049. doi: 10.1172/JCI114265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barker R. L., Gleich G. J., Pease L. R. Acidic precursor revealed in human eosinophil granule major basic protein cDNA. J Exp Med. 1988 Oct 1;168(4):1493–1498. doi: 10.1084/jem.168.4.1493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bengtsson T., Rundquist I., Stendahl O., Wymann M. P., Andersson T. Increased breakdown of phosphatidylinositol 4,5-bisphosphate is not an initiating factor for actin assembly in human neutrophils. J Biol Chem. 1988 Nov 25;263(33):17385–17389. [PubMed] [Google Scholar]
  7. Berkow R. L., Dodson R. W., Kraft A. S. The effect of a protein kinase C inhibitor, H-7, on human neutrophil oxidative burst and degranulation. J Leukoc Biol. 1987 May;41(5):441–446. doi: 10.1002/jlb.41.5.441. [DOI] [PubMed] [Google Scholar]
  8. Capron M., Kusnierz J. P., Prin L., Spiegelberg H. L., Khalife J., Tonnel A. B., Capron A. Cytophilic IgE on human blood and tissue eosinophils. Int Arch Allergy Appl Immunol. 1985;77(1-2):246–248. doi: 10.1159/000233801. [DOI] [PubMed] [Google Scholar]
  9. Carlson M. G., Peterson C. G., Venge P. Human eosinophil peroxidase: purification and characterization. J Immunol. 1985 Mar;134(3):1875–1879. [PubMed] [Google Scholar]
  10. Colditz I., Zwahlen R., Dewald B., Baggiolini M. In vivo inflammatory activity of neutrophil-activating factor, a novel chemotactic peptide derived from human monocytes. Am J Pathol. 1989 Apr;134(4):755–760. [PMC free article] [PubMed] [Google Scholar]
  11. Connolly T. M., Lawing W. J., Jr, Majerus P. W. Protein kinase C phosphorylates human platelet inositol trisphosphate 5'-phosphomonoesterase, increasing the phosphatase activity. Cell. 1986 Sep 12;46(6):951–958. doi: 10.1016/0092-8674(86)90077-2. [DOI] [PubMed] [Google Scholar]
  12. Deranleau D. A. Real-time models of morphogenetic processes. Experientia. 1988 Aug 15;44(8):679–684. doi: 10.1007/BF01941029. [DOI] [PubMed] [Google Scholar]
  13. Dewald B., Thelen M., Wymann M. P., Baggiolini M. Staurosporine inhibits the respiratory burst and induces exocytosis in human neutrophils. Biochem J. 1989 Dec 15;264(3):879–884. doi: 10.1042/bj2640879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dillon S. B., Verghese M. W., Snyderman R. Signal transduction in cells following binding of chemoattractants to membrane receptors. Virchows Arch B Cell Pathol Incl Mol Pathol. 1988;55(2):65–80. doi: 10.1007/BF02896561. [DOI] [PubMed] [Google Scholar]
  15. Fällman M., Stendahl O., Andersson T. Phorbol ester-induced activation of protein kinase C leads to increased formation of diacylglycerol in human neutrophils. Exp Cell Res. 1989 Mar;181(1):217–225. doi: 10.1016/0014-4827(89)90195-x. [DOI] [PubMed] [Google Scholar]
  16. Gleich G. J., Adolphson C. R. The eosinophilic leukocyte: structure and function. Adv Immunol. 1986;39:177–253. doi: 10.1016/s0065-2776(08)60351-x. [DOI] [PubMed] [Google Scholar]
  17. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  18. Grzeskowiak M., Della Bianca V., Cassatella M. A., Rossi F. Complete dissociation between the activation of phosphoinositide turnover and of NADPH oxidase by formyl-methionyl-leucyl-phenylalanine in human neutrophils depleted of Ca2+ and primed by subthreshold doses of phorbol 12,myristate 13,acetate. Biochem Biophys Res Commun. 1986 Mar 28;135(3):785–794. doi: 10.1016/0006-291x(86)90997-6. [DOI] [PubMed] [Google Scholar]
  19. Hockberger P., Toselli M., Swandulla D., Lux H. D. A diacylglycerol analogue reduces neuronal calcium currents independently of protein kinase C activation. Nature. 1989 Mar 23;338(6213):340–342. doi: 10.1038/338340a0. [DOI] [PubMed] [Google Scholar]
  20. Hugli T. E., Gerard C., Kawahara M., Scheetz M. E., 2nd, Barton R., Briggs S., Koppel G., Russell S. Isolation of three separate anaphylatoxins from complement-activated human serum. Mol Cell Biochem. 1981 Dec 4;41:59–66. doi: 10.1007/BF00225297. [DOI] [PubMed] [Google Scholar]
  21. Kase H., Iwahashi K., Matsuda Y. K-252a, a potent inhibitor of protein kinase C from microbial origin. J Antibiot (Tokyo) 1986 Aug;39(8):1059–1065. doi: 10.7164/antibiotics.39.1059. [DOI] [PubMed] [Google Scholar]
  22. Kiyoto I., Yamamoto S., Aizu E., Kato R. Staurosporine, a potent protein kinase C inhibitor, fails to inhibit 12-O-tetradecanoylphorbol-13-acetate-caused ornithine decarboxylase induction in isolated mouse epidermal cells. Biochem Biophys Res Commun. 1987 Oct 29;148(2):740–746. doi: 10.1016/0006-291x(87)90938-7. [DOI] [PubMed] [Google Scholar]
  23. Klebanoff S. J., Durack D. T., Rosen H., Clark R. A. Functional studies on human peritoneal eosinophils. Infect Immun. 1977 Jul;17(1):167–173. doi: 10.1128/iai.17.1.167-173.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kroegel C., Pleass R., Yukawa T., Chung K. F., Westwick J., Barnes P. J. Characterization of platelet-activating factor-induced elevation of cytosolic free calcium concentration in eosinophils. FEBS Lett. 1989 Jan 16;243(1):41–46. doi: 10.1016/0014-5793(89)81214-1. [DOI] [PubMed] [Google Scholar]
  25. Kroegel C., Yukawa T., Dent G., Venge P., Chung K. F., Barnes P. J. Stimulation of degranulation from human eosinophils by platelet-activating factor. J Immunol. 1989 May 15;142(10):3518–3526. [PubMed] [Google Scholar]
  26. Lackie J. M. The behavioural repertoire of neutrophils requires multiple signal transduction pathways. J Cell Sci. 1988 Apr;89(Pt 4):449–452. doi: 10.1242/jcs.89.4.449. [DOI] [PubMed] [Google Scholar]
  27. Lindley I., Aschauer H., Seifert J. M., Lam C., Brunowsky W., Kownatzki E., Thelen M., Peveri P., Dewald B., von Tscharner V. Synthesis and expression in Escherichia coli of the gene encoding monocyte-derived neutrophil-activating factor: biological equivalence between natural and recombinant neutrophil-activating factor. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9199–9203. doi: 10.1073/pnas.85.23.9199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nakadate T., Jeng A. Y., Blumberg P. M. Comparison of protein kinase C functional assays to clarify mechanisms of inhibitor action. Biochem Pharmacol. 1988 Apr 15;37(8):1541–1545. doi: 10.1016/0006-2952(88)90016-0. [DOI] [PubMed] [Google Scholar]
  29. Omann G. M., Allen R. A., Bokoch G. M., Painter R. G., Traynor A. E., Sklar L. A. Signal transduction and cytoskeletal activation in the neutrophil. Physiol Rev. 1987 Jan;67(1):285–322. doi: 10.1152/physrev.1987.67.1.285. [DOI] [PubMed] [Google Scholar]
  30. Owen W. F., Jr, Rothenberg M. E., Silberstein D. S., Gasson J. C., Stevens R. L., Austen K. F., Soberman R. J. Regulation of human eosinophil viability, density, and function by granulocyte/macrophage colony-stimulating factor in the presence of 3T3 fibroblasts. J Exp Med. 1987 Jul 1;166(1):129–141. doi: 10.1084/jem.166.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Prin L., Capron M., Tonnel A. B., Bletry O., Capron A. Heterogeneity of human peripheral blood eosinophils: variability in cell density and cytotoxic ability in relation to the level and the origin of hypereosinophilia. Int Arch Allergy Appl Immunol. 1983;72(4):336–346. doi: 10.1159/000234893. [DOI] [PubMed] [Google Scholar]
  32. Prin L., Charon J., Capron M., Gosset P., Taelman H., Tonnel A. B., Capron A. Heterogeneity of human eosinophils. II. Variability of respiratory burst activity related to cell density. Clin Exp Immunol. 1984 Sep;57(3):735–742. [PMC free article] [PubMed] [Google Scholar]
  33. Robinson J. M., Heyworth P. G., Badwey J. A. Utility of staurosporine in uncovering differences in the signal transduction pathways for superoxide production in neutrophils. Biochim Biophys Acta. 1990 Oct 15;1055(1):55–62. doi: 10.1016/0167-4889(90)90090-z. [DOI] [PubMed] [Google Scholar]
  34. Rosenberg H. F., Ackerman S. J., Tenen D. G. Human eosinophil cationic protein. Molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity. J Exp Med. 1989 Jul 1;170(1):163–176. doi: 10.1084/jem.170.1.163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rosenberg H. F., Tenen D. G., Ackerman S. J. Molecular cloning of the human eosinophil-derived neurotoxin: a member of the ribonuclease gene family. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4460–4464. doi: 10.1073/pnas.86.12.4460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rothenberg M. E., Owen W. F., Jr, Silberstein D. S., Woods J., Soberman R. J., Austen K. F., Stevens R. L. Human eosinophils have prolonged survival, enhanced functional properties, and become hypodense when exposed to human interleukin 3. J Clin Invest. 1988 Jun;81(6):1986–1992. doi: 10.1172/JCI113547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rothenberg M. E., Petersen J., Stevens R. L., Silberstein D. S., McKenzie D. T., Austen K. F., Owen W. F., Jr IL-5-dependent conversion of normodense human eosinophils to the hypodense phenotype uses 3T3 fibroblasts for enhanced viability, accelerated hypodensity, and sustained antibody-dependent cytotoxicity. J Immunol. 1989 Oct 1;143(7):2311–2316. [PubMed] [Google Scholar]
  38. Sagi-Eisenberg R. GTP-binding proteins as possible targets for protein kinase C action. Trends Biochem Sci. 1989 Sep;14(9):355–357. doi: 10.1016/0968-0004(89)90001-7. [DOI] [PubMed] [Google Scholar]
  39. Salzer W., Gerard C., McCall C. Effect of an inhibitor of protein kinase C on human polymorphonuclear leukocyte degranulation. Biochem Biophys Res Commun. 1987 Oct 29;148(2):747–754. doi: 10.1016/0006-291x(87)90939-9. [DOI] [PubMed] [Google Scholar]
  40. Sanderson C. J. Interleukin-5: an eosinophil growth and activation factor. Dev Biol Stand. 1988;69:23–29. [PubMed] [Google Scholar]
  41. Sha'afi R. I., Molski T. F., Gomez-Cambronero J., Huang C. K. Dissociation of the 47-kilodalton protein phosphorylation from degranulation and superoxide production in neutrophils. J Leukoc Biol. 1988 Jan;43(1):18–27. doi: 10.1002/jlb.43.1.18. [DOI] [PubMed] [Google Scholar]
  42. Sigal C. E., Valone F. H., Holtzman M. J., Goetzl E. J. Preferential human eosinophil chemotactic activity of the platelet-activating factor (PAF) 1-0-hexadecyl-2-acetyl-sn-glyceryl-3-phosphocholine (AGEPC). J Clin Immunol. 1987 Mar;7(2):179–184. doi: 10.1007/BF00916012. [DOI] [PubMed] [Google Scholar]
  43. Smith R. J., Justen J. M., Sam L. M. Function and stimulus-specific effects of phorbol 12-myristate 13-acetate on human polymorphonuclear neutrophils: autoregulatory role for protein kinase C in signal transduction. Inflammation. 1988 Dec;12(6):597–611. doi: 10.1007/BF00914321. [DOI] [PubMed] [Google Scholar]
  44. Tai P. C., Bakes D. M., Barkans J. R., Spry C. J. Plasma membrane antigens on light density and activated human blood eosinophils. Clin Exp Immunol. 1985 May;60(2):427–436. [PMC free article] [PubMed] [Google Scholar]
  45. Tai P. C., Capron M., Bakes D. M., Barkans J., Spry C. J. Monoclonal antibodies to human eosinophil plasma membrane antigens enhance the secretion of eosinophil cationic protein. Clin Exp Immunol. 1986 Mar;63(3):728–737. [PMC free article] [PubMed] [Google Scholar]
  46. Tamaoki T., Nomoto H., Takahashi I., Kato Y., Morimoto M., Tomita F. Staurosporine, a potent inhibitor of phospholipid/Ca++dependent protein kinase. Biochem Biophys Res Commun. 1986 Mar 13;135(2):397–402. doi: 10.1016/0006-291x(86)90008-2. [DOI] [PubMed] [Google Scholar]
  47. Tamura N., Agrawal D. K., Suliaman F. A., Townley R. G. Effects of platelet activating factor on the chemotaxis of normodense eosinophils from normal subjects. Biochem Biophys Res Commun. 1987 Feb 13;142(3):638–644. doi: 10.1016/0006-291x(87)91462-8. [DOI] [PubMed] [Google Scholar]
  48. Taylor D. J., Evanson J. M., Woolley D. E. Contrasting effects of the protein kinase C inhibitor, staurosporine, on cytokine and phorbol ester stimulation of fructose 2,6-bisphosphate and prostaglandin E production by fibroblasts in vitro. Comparative studies using interleukin-1 alpha, tumour necrosis factor alpha, transforming growth factor beta, interferon-gamma and 12-O-tetradecanoylphorbol 13-acetate. Biochem J. 1990 Aug 1;269(3):573–577. doi: 10.1042/bj2690573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Ten R. M., Pease L. R., McKean D. J., Bell M. P., Gleich G. J. Molecular cloning of the human eosinophil peroxidase. Evidence for the existence of a peroxidase multigene family. J Exp Med. 1989 May 1;169(5):1757–1769. doi: 10.1084/jem.169.5.1757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Thelen M., Peveri P., Kernen P., von Tscharner V., Walz A., Baggiolini M. Mechanism of neutrophil activation by NAF, a novel monocyte-derived peptide agonist. FASEB J. 1988 Aug;2(11):2702–2706. [PubMed] [Google Scholar]
  51. Thorpe G. H., Kricka L. J. Enhanced chemiluminescent reactions catalyzed by horseradish peroxidase. Methods Enzymol. 1986;133:331–353. doi: 10.1016/0076-6879(86)33078-7. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. Vadas M. A., David J. R., Butterworth A., Pisani N. T., Siongok T. A. A new method for the purification of human eosinophils and neutrophils, and a comparison of the ability of these cells to damage schistosomula of Schistosoma mansoni. J Immunol. 1979 Apr;122(4):1228–1236. [PubMed] [Google Scholar]
  54. Vegesna R. V., Wu H. L., Mong S., Crooke S. T. Staurosporine inhibits protein kinase C and prevents phorbol ester-mediated leukotriene D4 receptor desensitization in RBL-1 cells. Mol Pharmacol. 1988 May;33(5):537–542. [PubMed] [Google Scholar]
  55. Verghese M. W., Charles L., Jakoi L., Dillon S. B., Snyderman R. Role of a guanine nucleotide regulatory protein in the activation of phospholipase C by different chemoattractants. J Immunol. 1987 Jun 15;138(12):4374–4380. [PubMed] [Google Scholar]
  56. Wardlaw A. J., Moqbel R., Cromwell O., Kay A. B. Platelet-activating factor. A potent chemotactic and chemokinetic factor for human eosinophils. J Clin Invest. 1986 Dec;78(6):1701–1706. doi: 10.1172/JCI112765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Watson S. P., Lapetina E. G. 1,2-Diacylglycerol and phorbol ester inhibit agonist-induced formation of inositol phosphates in human platelets: possible implications for negative feedback regulation of inositol phospholipid hydrolysis. Proc Natl Acad Sci U S A. 1985 May;82(9):2623–2626. doi: 10.1073/pnas.82.9.2623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wolf M., Baggiolini M. The protein kinase inhibitor staurosporine, like phorbol esters, induces the association of protein kinase C with membranes. Biochem Biophys Res Commun. 1988 Aug 15;154(3):1273–1279. doi: 10.1016/0006-291x(88)90277-x. [DOI] [PubMed] [Google Scholar]
  59. Wymann M. P., Kernen P., Bengtsson T., Andersson T., Baggiolini M., Deranleau D. A. Corresponding oscillations in neutrophil shape and filamentous actin content. J Biol Chem. 1990 Jan 15;265(2):619–622. [PubMed] [Google Scholar]
  60. Wymann M. P., Kernen P., Deranleau D. A., Baggiolini M. Respiratory burst oscillations in human neutrophils and their correlation with fluctuations in apparent cell shape. J Biol Chem. 1989 Sep 25;264(27):15829–15834. [PubMed] [Google Scholar]
  61. Wymann M. P., Kernen P., Deranleau D. A., Dewald B., von Tscharner V., Baggiolini M. Oscillatory motion in human neutrophils responding to chemotactic stimuli. Biochem Biophys Res Commun. 1987 Aug 31;147(1):361–368. doi: 10.1016/s0006-291x(87)80130-4. [DOI] [PubMed] [Google Scholar]
  62. Wymann M. P., von Tscharner V., Deranleau D. A., Baggiolini M. The onset of the respiratory burst in human neutrophils. Real-time studies of H2O2 formation reveal a rapid agonist-induced transduction process. J Biol Chem. 1987 Sep 5;262(25):12048–12053. [PubMed] [Google Scholar]
  63. Zucker-Franklin D. Eosinophil function and disorders. Adv Intern Med. 1974;19:1–25. [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES