Pediatric Nephrology

, Volume 20, Issue 9, pp 1306–1314 | Cite as

Impaired neutrophils in children with the typical form of hemolytic uremic syndrome

  • Gabriela C. Fernández
  • Sonia A. Gómez
  • Carolina J. Rubel
  • Leticia V. Bentancor
  • Paula Barrionuevo
  • Marta Alduncín
  • Irene Grimoldi
  • Ramón Exeni
  • Martín A. Isturiz
  • Marina S. Palermo
Original Article

Abstract

Experimental and clinical evidence suggest that activated neutrophils (PMN) could contribute to endothelial damage in Hemolytic Uremic Syndrome (D+HUS). Additionally, while PMN-activating cytokines and PMN-derived products have been found in D+HUS sera, we have demonstrated phenotypic alterations in D+HUS PMN compatible with a deactivation state. Here, we investigated whether D+HUS PMN were actually hyporesponsive, and explored some of the mechanisms probably involved in their derangement. Twenty-two D+HUS children were bled in the acute period, and blood samples from healthy, acute uremic and neutrophilic children were obtained as controls. We evaluated degranulation markers in response to cytokines, intracellular granule content, and reactive oxygen species (ROS) generation in circulating D+HUS and control PMN. The influence of D+HUS-derived plasma and the direct effects of Stx in vitro were evaluated on healthy donors’ PMN. We found that D+HUS PMN presented reduced degranulatory capacity in response to cytokines and intracellular granule content, and decreased ROS generation. D+HUS plasma or Stx did not affect the phenotype and function of healthy donors’ PMN. These results suggest that upon hospitalization D+HUS PMN are functionally impaired and show features of previous degranulation, indicating a preceding process of activation with release of ROS and proteases involved in endothelial damage.

Keywords

D+HUS Patients PMN Cytokines ROS generation 

Notes

Acknowledgements

This study was supported by a “R. Carrillo-A. Oñativia” award from Ministerio de Salud de la Nación, by Fundación Alberto J. Roemmers, and Agencia Nacional de Promoción Científica y Tecnológica, Argentina. The authors thank Marta Felippo, Nora Galassi and Norma Riera for their excellent technical assistance. The authors also thank Fundación de la Hemofilia and Academia Nacional de Medicina for the use of the FACScan flow cytometer, and the Departamento de Hemoterapia of CEMIC for healthy adult blood samples.

References

  1. 1.
    Karmali MA, Petric M, Lim C, Fleming PC, Arbus GS, Lior H (1985) The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J Infect Dis 151:775–782PubMedGoogle Scholar
  2. 2.
    Remuzzi G, Ruggenenti P (1995) The hemolytic uremic syndrome. Kidney Int 48:2–19PubMedGoogle Scholar
  3. 3.
    Paton JC, Paton AW (1998) Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clin Microbiol Rev 11:450–479PubMedGoogle Scholar
  4. 4.
    Keusch GT, Acheson DW (1997) Thrombotic thrombocytopenic purpura associated with Shiga toxins. Semin Hematol 34:106–116PubMedGoogle Scholar
  5. 5.
    Milford D, Taylor CM, Rafaat F, Halloran E, Dawes J (1989) Neutrophil elastases and haemolytic uraemic syndrome. Lancet 2:1153CrossRefGoogle Scholar
  6. 6.
    Fitzpatrick MM, Shah V, Filler G, Dillon MJ, Barratt TM (1992) Neutrophil activation in the haemolytic uraemic syndrome: free and complexed elastase in plasma. Pediatr Nephrol 6:50–53CrossRefPubMedGoogle Scholar
  7. 7.
    Weiss SJ (1989) Tissue destruction by neutrophils. N Engl J Med 320:365–376PubMedGoogle Scholar
  8. 8.
    Lentsch AB, Ward PA (2000) Regulation of inflammatory vascular damage. J Pathol 190:343–348CrossRefPubMedGoogle Scholar
  9. 9.
    Sengelov H, Kjeldsen L, Borregaard N (1993) Control of exocytosis in early neutrophil activation. J Immunol 150:1535–1543PubMedGoogle Scholar
  10. 10.
    Borregaard N, Cowland JB (1997) Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 89:3503–3521PubMedGoogle Scholar
  11. 11.
    Fernandez GC, Rubel C, Barrionuevo P, Lopez L, Ramirez F, Diaz M, Isturiz MA, Palermo MS (2002) Phenotype markers and function of neutrophils in children with hemolytic uremic syndrome. Pediatr Nephrol 17:337–344CrossRefPubMedGoogle Scholar
  12. 12.
    Gianantonio CA, Vitacco M, Mendilaharzu F, Gallo GE, Sojo ET (1973) The hemolytic-uremic syndrome. Nephron 11:174–192PubMedGoogle Scholar
  13. 13.
    Knapp W, Strobl H, Majdic O (1994) Flow cytometric analysis of cell-surface and intracellular antigens in leukemia diagnosis. Cytometry 18:187–198PubMedGoogle Scholar
  14. 14.
    Rubel C, Fernandez GC, Rosa FA, Gomez S, Bompadre MB, Coso OA, Isturiz MA, Palermo MS (2002) Soluble fibrinogen modulates neutrophil functionality through the activation of an extracellular signal-regulated kinase-dependent pathway. J Immunol 168:3527–3535PubMedGoogle Scholar
  15. 15.
    Duner KI (1993) A new kinetic single-stage Limulus amoebocyte lysate method for the detection of endotoxin in water and plasma. J Biochem Biophys Methods 26:131–142CrossRefPubMedGoogle Scholar
  16. 16.
    te Loo DM, Monnens LA, van Der Velden TJ, Vermeer MA, Preyers F, Demacker PN, van Den Heuvel LP, van Hinsbergh VW (2000) Binding and transfer of verocytotoxin by polymorphonuclear leukocytes in hemolytic uremic syndrome. Blood 95:3396–3402PubMedGoogle Scholar
  17. 17.
    Palermo MS, Alves Rosa MF, Van Rooijen N, Isturiz MA (1999) Depletion of liver and splenic macrophages reduces the lethality of Shiga toxin-2 in a mouse model. Clin Exp Immunol 116:462–467CrossRefPubMedGoogle Scholar
  18. 18.
    Rosenbloom AJ, Pinsky MR, Napolitano C, Nguyen TS, Levann D, Pencosky N, Dorrance A, Ray BK, Whiteside T (1999) Suppression of cytokine-mediated beta2-integrin activation on circulating neutrophils in critically ill patients. J Leukoc Biol 66:83–89PubMedGoogle Scholar
  19. 19.
    Rodeberg DA, Bass RC, Alexander JW, Warden GD, Babcock GF (1997) Neutrophils from burn patients are unable to increase the expression of CD11b/CD18 in response to inflammatory stimuli. J Leukoc Biol 61:575–582PubMedGoogle Scholar
  20. 20.
    Vaissiere C, Le Cabec V, Maridonneau-Parini I (1999) NADPH oxidase is functionally assembled in specific granules during activation of human neutrophils. J Leukoc Biol 65:629–634PubMedGoogle Scholar
  21. 21.
    Borregaard N, Heiple JM, Simons ER, Clark RA (1983) Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation. J Cell Biol 97:52–61CrossRefPubMedGoogle Scholar
  22. 22.
    Karlsson A, Dahlgren C (2002) Assembly and activation of the neutrophil NADPH oxidase in granule membranes. Antioxid Redox Signal 4:49–60CrossRefPubMedGoogle Scholar
  23. 23.
    Sassetti B, Vizcarguenaga MI, Zanaro NL, Silva MV, Kordich L, Florentini L, Diaz M, Vitacco M, Sanchez Avalos JC (1999) Hemolytic uremic syndrome in children: platelet aggregation and membrane glycoproteins. J Pediatr Hematol Oncol 21:123–128CrossRefPubMedGoogle Scholar
  24. 24.
    Cohen G, Haag-Weber M, Horl WH (1997) Immune dysfunction in uremia. Kidney Int Suppl 62:S79–82PubMedGoogle Scholar
  25. 25.
    Cohen G, Rudnicki M, Walter F, Niwa T, Horl WH (2001) Glucose-modified proteins modulate essential functions and apoptosis of polymorphonuclear leukocytes. J Am Soc Nephrol 12:1264–1271PubMedGoogle Scholar
  26. 26.
    Wagner JG, Roth RA (1999) Neutrophil migration during endotoxemia. J Leukoc Biol 66:10–24PubMedGoogle Scholar
  27. 27.
    Burtnick LD (1984) Modification of actin with fluorescein isothiocyanate. Biochim Biophys Acta 791:57–62PubMedGoogle Scholar
  28. 28.
    Miller L, Phillips M, Reisler E (1988) Polymerization of actin modified with fluorescein isothiocyanate. Eur J Biochem 174:23–29CrossRefPubMedGoogle Scholar
  29. 29.
    Favazza M, Lerho M, Houssier C (1990) Labelling of histone H5 and its interaction with DNA. 1. Histone H5 labelling with fluorescein isothiocyanate. J Biomol Struct Dyn 7:1291–1300PubMedGoogle Scholar
  30. 30.
    Johnson DA, Taylor P (1982) Site-specific fluorescein-labeled cobra alpha-toxin. Biochemical and spectroscopic characterization. J Biol Chem 257:5632–5636Google Scholar
  31. 31.
    Hentz NG, Richardson JM, Sportsman JR, Daijo J, Sittampalam GS (1997) Synthesis and characterization of insulin-fluorescein derivatives for bioanalytical applications. Anal Chem 69:4994–5000CrossRefPubMedGoogle Scholar
  32. 32.
    Fong JS, Kaplan BS (1982) Impairment of platelet aggregation in hemolytic uremic syndrome: evidence for platelet “exhaustion”. Blood 60:564–570PubMedGoogle Scholar

Copyright information

© IPNA 2005

Authors and Affiliations

  • Gabriela C. Fernández
    • 1
  • Sonia A. Gómez
    • 1
  • Carolina J. Rubel
    • 1
  • Leticia V. Bentancor
    • 1
  • Paula Barrionuevo
    • 1
  • Marta Alduncín
    • 2
  • Irene Grimoldi
    • 2
  • Ramón Exeni
    • 2
  • Martín A. Isturiz
    • 1
  • Marina S. Palermo
    • 1
  1. 1.Department of Immunology, Instituto de Investigaciones HematológicasAcademia Nacional de MedicinaBuenos AiresArgentina
  2. 2.Department of NephrologyHospital Municipal del NiñoBuenos AiresArgentina

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