Skip to main content

Advertisement

SpringerLink
Log in

Immune monitoring of patients with septic shock by measurement of intraleukocyte cytokines

  • Original
  • Published:
Intensive Care Medicine Aims and scope Submit manuscript

Abstract

Objective

To assess the immune competence of patients presenting with septic shock by measuring on-line the production of intracellular cytokines by circulating leukocytes.

Design and setting

Prospective study in a 18-bed medical intensive care unit of a university hospital.

Patients and participants

21 patients with septic shock, and 11 volunteers.

Interventions

Single-step isolation of leukocytes from whole blood obtained within the first 24 h after admission. Leukocytes were fixed immediately or after treatment with lipopolysaccharide (LPS) and/or heterologous plasma.

Measurements and results

Leukocytes were permeabilized, and the intracellular cytokine expression of TNF-α and IL-10 was quantified by immunostaining and flow cytometry. LPS treatment significantly increased monocyte intracellular cytokine TNF-α and IL-10 as well as lymphocyte intracellular cytokine IL-10 in normal leukocytes. Septic monocytes and granulocytes had nonstimulated intracellular cytokine TNF-α concentrations lower than those measured in volunteers and were severely hyporesponsive to LPS. These phenotypic changes were correlated with disease severity and could be reproduced by treatment of normal leukocytes with plasma from patients with septic shock.

Conclusions

Intracellular cytokine staining is a simple and rapid method to assess in situ and on-line the inflammatory balance and responsiveness of leukocyte subpopulations and could therefore represent a useful monitoring tool to assess the immune competence of critically ill patients. This study identifies the cellular source of cytokines in whole blood and confirms prior reports showing that septic phagocytes are characterized by a predominant anti-inflammatory phenotype, with hyporesponsiveness to LPS, depending on a plasma deactivation factor.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference (1992) Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med 20:864–874

    PubMed  Google Scholar 

  2. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR (2001) Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 29:1303–1310

    Article  CAS  PubMed  Google Scholar 

  3. Martin GS, Mannino DM, Eaton S, Moss M (2003) The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 348:1546–1554

    Article  PubMed  Google Scholar 

  4. Hotchkiss RS, Karl IE (2003) The pathophysiology and treatment of sepsis. N Engl J Med 348:138–150

    Article  CAS  PubMed  Google Scholar 

  5. Cohen J (2002) The immunopathogenesis of sepsis. Nature 420:885–891

    Article  CAS  PubMed  Google Scholar 

  6. Abraham E (1999) Why immunomodulatory therapies have not worked in sepsis. Intensive Care Med 25:556–566

    Article  CAS  PubMed  Google Scholar 

  7. Munford RS, Pugin J (2001) Normal responses to injury prevent systemic inflammation and can be immunosuppressive. Am J Respir Crit Care Med 163:316–321

    CAS  PubMed  Google Scholar 

  8. Volk HD, Reinke P, Docke WD (2000) Clinical aspects: from systemic inflammation to ‘immunoparalysis.’ Chem Immunol 74:162–177

    Google Scholar 

  9. Haveman JW, Muller Kobold AC, Tervaert JW, van den Berg AP, Tulleken JE, Kallenberg CG, The TH (1999) The central role of monocytes in the pathogenesis of sepsis: consequences for immunomonitoring and treatment. Neth J Med 55:132–141

    Article  CAS  PubMed  Google Scholar 

  10. Pugin J, Ricou B, Steinberg KP, Suter PM, Martin TR (1996) Proinflammatory activity in bronchoalveolar lavage fluids from patients with ARDS, a prominent role for interleukin-1. Am J Respir Crit Care Med 153:1850–1856

    CAS  PubMed  Google Scholar 

  11. Pugin J, Verghese G, Widmer MC, Matthay MA (1999) The alveolar space is the site of intense inflammatory and profibrotic reactions in the early phase of acute respiratory distress syndrome. Crit Care Med 27:304–312

    Article  CAS  PubMed  Google Scholar 

  12. Tracey KJ, Cerami A (1993) Tumor necrosis factor: an updated review of its biology. Crit Care Med 21:S415–S422

    CAS  PubMed  Google Scholar 

  13. Randow F, Syrbe U, Meisel C, Krausch D, Zuckermann H, Platzer C, Volk HD (1995) Mechanism of endotoxin desensitization: involvement of interleukin 10 and transforming growth factor beta. J Exp Med 181:1887–1892

    Article  CAS  PubMed  Google Scholar 

  14. Brandtzaeg P, Osnes L, Ovstebo R, Joo GB, Westvik AB, Kierulf P (1996) Net inflammatory capacity of human septic shock plasma evaluated by a monocyte-based target cell assay: identification of interleukin-10 as a major functional deactivator of human monocytes. J Exp Med 184:51–60

    Article  CAS  PubMed  Google Scholar 

  15. Sfeir T, Saha DC, Astiz M, Rackow EC (2001) Role of interleukin-10 in monocyte hyporesponsiveness associated with septic shock. Crit Care Med 29:129–133

    Article  CAS  PubMed  Google Scholar 

  16. Fumeaux T, Pugin J (2002) Role of interleukin-10 in the intracellular sequestration of human leukocyte antigen-DR in monocytes during septic shock. Am J Respir Crit Care Med 166:1475–1482

    Article  PubMed  Google Scholar 

  17. Gogos CA, Drosou E, Bassaris HP, Skoutelis A (2000) Pro- versus anti-inflammatory cytokine profile in patients with severe sepsis: a marker for prognosis and future therapeutic options. J Infect Dis 181:176–180

    Article  CAS  PubMed  Google Scholar 

  18. Dissel JT van, van Langevelde P, Westendorp RG, Kwappenberg K, Frolich M (1998) Anti-inflammatory cytokine profile and mortality in febrile patients. Lancet 351:950–953

    Article  PubMed  Google Scholar 

  19. Park WY, Goodman RB, Steinberg KP, Ruzinski JT, Radella F 2nd, Park DR, Pugin J, Skerrett SJ, Hudson LD, Martin TR (2001) Cytokine balance in the lungs of patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 164:1896–1903

    CAS  PubMed  Google Scholar 

  20. Munoz C, Misset B, Fitting C, Bleriot JP, Carlet J, Cavaillon JM (1991) Dissociation between plasma and monocyte-associated cytokines during sepsis. Eur J Immunol 21:2177–2184

    CAS  PubMed  Google Scholar 

  21. Le Gall JR, Lemeshow S, Saulnier F (1993) A new Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study. JAMA 270:2957–2963

    PubMed  Google Scholar 

  22. Cabioglu N, Bilgic S, Deniz G, Aktas E, Seyhun Y, Turna A, Gunay K, Esen F (2002) Decreased cytokine expression in peripheral blood leukocytes of patients with severe sepsis. Arch Surg 137:1037–1043

    Article  CAS  PubMed  Google Scholar 

  23. Mueller A, Kreuzfelder E, Nyadu B, Lindemann M, Rebmannn V, Majetschak M, Obertacke U, Schade UF, Nast-Kolb D, Grosse-Wilde H (2003) Human leukocyte antigen-DR expression in peripheral blood mononuclear cells from healthy donors influenced by the sera of injured patients prone to severe sepsis. Intensive Care Med 29:2285–2290

    Article  PubMed  Google Scholar 

  24. Spolarics Z, Siddiqi M, Siegel JH, Garcia ZC, Stein DS, Denny T, Deitch EA (2003) Depressed interleukin-12-producing activity by monocytes correlates with adverse clinical course and a shift toward Th2-type lymphocyte pattern in severely injured male trauma patients. Crit Care Med 31:1722–1729

    Article  CAS  PubMed  Google Scholar 

  25. Maino VC, Picker LJ (1998) Identification of functional subsets by flow cytometry: intracellular detection of cytokine expression. Cytometry 34:207–215

    Article  CAS  PubMed  Google Scholar 

  26. Estcourt C, Rousseau Y, Sadeghi HM, Thieblemont N, Carreno MP, Weiss L, Haeffner-Cavaillon N (1997) Flow-cytometric assessment of in vivo cytokine-producing monocytes in HIV-infected patients. Clin Immunol Immunopathol 83:60–67

    Article  CAS  PubMed  Google Scholar 

  27. Berg AP van den, Twilhaar WN, van Son WJ, van der Bij W, Klompmaker IJ, Slooff MJ, The TH, de Leij LH (1998) Quantification of immunosuppression by flow cytometric measurement of intracellular cytokine synthesis. Transpl Int 11 [Suppl 1]:S318–S321

  28. McNerlan SE, Rea IM, Alexander HD (2002) A whole blood method for measurement of intracellular TNF-alpha, IFN-gamma and IL-2 expression in stimulated CD3+ lymphocytes: differences between young and elderly subjects. Exp Gerontol 37:227–234

    Article  CAS  PubMed  Google Scholar 

  29. Pietschmann P, Gollob E, Brosch S, Hahn P, Kudlacek S, Willheim M, Woloszczuk W, Peterlik M, Tragl KH (2003) The effect of age and gender on cytokine production by human peripheral blood mononuclear cells and markers of bone metabolism. Exp Gerontol 38:1119–1127

    Article  CAS  PubMed  Google Scholar 

  30. Prussin C, Metcalfe DD (1995) Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J Immunol Methods 188:117–128

    Article  CAS  PubMed  Google Scholar 

  31. Desch CE, Kovach NL, Present W, Broyles C, Harlan JM (1989) Production of human tumor necrosis factor from whole blood ex vivo. Lymphokine Res 8:141–146

    CAS  PubMed  Google Scholar 

  32. Finch-Arietta MB, Cochran FR (1991) Cytokine production in whole blood ex vivo. Agents Actions 34:49–52

    CAS  PubMed  Google Scholar 

  33. De Groote D, Zangerle PF, Gevaert Y, Fassotte MF, Beguin Y, Noizat-Pirenne F, Pirenne J, Gathy R, Lopez M, Dehart I et al (1992) Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation. Cytokine 4:239–248

    Article  PubMed  Google Scholar 

  34. Desfaits AC, Serri O, Renier G (1998) Normalization of plasma lipid peroxides, monocyte adhesion, and tumor necrosis factor-alpha production in NIDDM patients after gliclazide treatment. Diabetes Care 21:487–493

    CAS  PubMed  Google Scholar 

  35. Elborn JS, Norman D, Delamere FM, Shale DJ (1992) In vitro tumor necrosis factor-alpha secretion by monocytes from patients with cystic fibrosis. Am J Respir Cell Mol Biol 6:207–211

    CAS  PubMed  Google Scholar 

  36. Anand M, Chodda SK, Parikh PM, Nadkarni JS (1998) Dysregulated cytokine production by monocytes from chronic lymphocytic leukemia patients. Cancer Biother Radiopharm 13:43–48

    CAS  PubMed  Google Scholar 

  37. Granchi D, Ciapetti G, Stea S, Savarino L, Filippini F, Sudanese A, Zinghi G, Montanaro L (1999) Cytokine release in mononuclear cells of patients with Co-Cr hip prosthesis. Biomaterials 20:1079–1086

    Article  CAS  PubMed  Google Scholar 

  38. Peters AM, Jager FS, Warneke A, Muller K, Brunkhorst U, Schedel I, Gahr M (1991) Cytokine secretion by peripheral blood monocytes from human immunodeficiency virus-infected patients is normal. Clin Immunol Immunopathol 61:343–352

    CAS  PubMed  Google Scholar 

  39. Shinohara K, Ayame H, Tanaka M, Matsuda M, Ando S, Tajiri M (1991) Increased production of tumor necrosis factor-alpha by peripheral blood mononuclear cells in the patients with aplastic anemia. Am J Hematol 37:75–79

    CAS  PubMed  Google Scholar 

  40. Dubravec DB, Spriggs DR, Mannick JA, Rodrick ML (1990) Circulating human peripheral blood granulocytes synthesize and secrete tumor necrosis factor alpha. Proc Natl Acad Sci U S A 87:6758–6761

    CAS  PubMed  Google Scholar 

  41. Dibbs Z, Thornby J, White BG, Mann DL (1999) Natural variability of circulating levels of cytokines and cytokine receptors in patients with heart failure: implications for clinical trials. J Am Coll Cardiol 33:1935–1942

    Article  CAS  PubMed  Google Scholar 

  42. Vassilakopoulos T, Karatza M-H, Katsaounou P, Kollintza A, Zakynthinos S, Roussos C (2003) Antioxidants attenuate the plasma cytokine response to exercise in humans. J Appl Physiol 94:1025–1032

    CAS  PubMed  Google Scholar 

  43. Godot V, Harraga S, Deschaseaux M, Bresson-Hadni S, Gottstein B, Emilie D, Vuitton DA (1997) Increased basal production of interleukin-10 by peripheral blood mononuclear cells in human alveolar echinococcosis. Eur Cytokine Netw 8:401–408

    CAS  PubMed  Google Scholar 

  44. Suarez A, Castro P, Alonso R, Mozo L, Gutierrez C (2003) Interindividual variations in constitutive interleukin-10 messenger RNA and protein levels and their association with genetic polymorphisms. Transplantation 75:711–717

    Article  CAS  PubMed  Google Scholar 

  45. Kilpinen S, Huhtala H, Hurme M (2002) The combination of the interleukin-1alpha (IL-1alpha-889) genotype and the interleukin-10 (IL-10 ATA) haplotype is associated with increased interleukin-10 (IL-10) plasma levels in healthy individuals. Eur Cytokine Netw 13:66–71

    CAS  PubMed  Google Scholar 

  46. Volk HD, Thieme M, Heym S, Docke WD, Ruppe U, Tausch W, Manger D, Zuckermann S, Golosubow A, Nieter B et al (1991) Alterations in function and phenotype of monocytes from patients with septic disease: predictive value and new therapeutic strategies. Behring Inst Mitt 88:208–215

    PubMed  Google Scholar 

  47. Schultz MJ, Olszyna DP, de Jonge E, Verbon A, van Deventer SJ, van der Poll T (2000) Reduced ex vivo chemokine production by polymorphonuclear cells after in vivo exposure of normal humans to endotoxin. J Infect Dis 182:1264–1267

    Article  CAS  PubMed  Google Scholar 

  48. Schleiffenbaum B, Fehr J (1990) The tumor necrosis factor receptor and human neutrophil function. Deactivation and cross-deactivation of tumor necrosis factor-induced neutrophil responses by receptor down-regulation. J Clin Invest 86:184–195

    CAS  PubMed  Google Scholar 

  49. McCall CE, Grosso-Wilmoth LM, LaRue K, Guzman RN, Cousart SL (1993) Tolerance to endotoxin-induced expression of the interleukin-1 beta gene in blood neutrophils of humans with the sepsis syndrome. J Clin Invest 91:853–861

    CAS  PubMed  Google Scholar 

  50. Stephan F, Yang K, Tankovic J, Soussy CJ, Dhonneur G, Duvaldestin P, Brochard L, Brun-Buisson C, Harf A, Delclaux C (2002) Impairment of polymorphonuclear neutrophil functions precedes nosocomial infections in critically ill patients. Crit Care Med 30:315–322

    Article  PubMed  Google Scholar 

  51. Marie C, Muret J, Fitting C, Losser MR, Payen D, Cavaillon JM (1998) Reduced ex vivo interleukin-8 production by neutrophils in septic and nonseptic systemic inflammatory response syndrome. Blood 91:3439–3446

    CAS  PubMed  Google Scholar 

  52. Cavaillon JM (2002) “Septic plasma”: an immunosuppressive milieu. Am J Respir Crit Care Med 166:1417–1418

    Article  PubMed  Google Scholar 

  53. Taniguchi T, Koido Y, Aiboshi J, Yamashita T, Suzaki S, Kurokawa A (1999) Change in the ratio of interleukin-6 to interleukin-10 predicts a poor outcome in patients with systemic inflammatory response syndrome. Crit Care Med 27:1262–1264

    Article  CAS  PubMed  Google Scholar 

  54. Oberholzer C, Oberholzer A, Clare-Salzler M, Moldawer LL (2001) Apoptosis in sepsis: a new target for therapeutic exploration. FASEB J 15:879–892

    Article  CAS  PubMed  Google Scholar 

  55. Westendorp RG, Langermans JA, Huizinga TW, Elouali AH, Verweij CL, Boomsma DI, Vandenbroucke JP, Vandenbrouke JP (1997) Genetic influence on cytokine production and fatal meningococcal disease. Lancet 349:170–173

    Article  CAS  PubMed  Google Scholar 

  56. Stüber F (2000) Impact of genomic variation on inflammatory processes and sepsis. In: Eichacker PQ, Pugin J (eds) Evolving concepts in sepsis and septic shock. Kluwer, Boston

  57. Kiecolt-Glaser JK, Preacher KJ, MacCallum RC, Atkinson C, Malarkey WB, Glaser R (2003) Chronic stress and age-related increases in the proinflammatory cytokine IL-6. Proc Natl Acad Sci USA 100:9090–9095

    Article  CAS  PubMed  Google Scholar 

  58. Zanni F, Vescovini R, Biasini C, Fagnoni F, Zanlari L, Telera A, Di Pede P, Passeri G, Pedrazzoni M, Passeri M (2003) Marked increase with age of type 1 cytokines within memory and effector/cytotoxic CD8+ T cells in humans: a contribution to understand the relationship between inflammation and immunosenescence. Exp Gerontol 38:981–987

    Article  CAS  PubMed  Google Scholar 

  59. Gabriel P, Cakman I, Rink L (2002) Overproduction of monokines by leukocytes after stimulation with lipopolysaccharide in the elderly. Exp Gerontol 37:235–247

    Article  CAS  PubMed  Google Scholar 

  60. Marik PE, Zaloga GP (2001) The effect of aging on circulating levels of proinflammatory cytokines during septic shock. Norasept II Study Investigators. J Am Geriatr Soc 49:5–9

    Article  CAS  PubMed  Google Scholar 

  61. Volk HD, Reinke P, Docke WD (1999) Immunological monitoring of the inflammatory process: which variables? When to assess? Eur J Surg Suppl 584:70–72

    PubMed  Google Scholar 

  62. Heagy W, Hansen C, Nieman K, Cohen M, Richardson C, Rodriguez JL, West MA (2000) Impaired ex vivo lipopolysaccharide-stimulated whole blood tumor necrosis factor production may identify ‘septic’ intensive care unit patients. Shock 14:271–276

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors acknowledge the financial support of the Stanley Thomas Johnson Foundation and the Institutional Fund Projet de Recherche et Développement of the University Hospital of Geneva.

Author information

Authors and Affiliations

  1. Laboratory of the Division of Medical Intensive Care, Departments of Internal Medicine and of Microbiology and Molecular Medicine, University Hospital and Faculty of Medicine, 24 rue Micheli-du-Crest, 1211, Geneva 14, Switzerland

    Thierry Fumeaux, Julien Dufour, Sabine Stern & Jérôme Pugin

Authors
  1. Thierry Fumeaux
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Julien Dufour
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sabine Stern
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jérôme Pugin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jérôme Pugin.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fumeaux, T., Dufour, J., Stern, S. et al. Immune monitoring of patients with septic shock by measurement of intraleukocyte cytokines. Intensive Care Med 30, 2028–2037 (2004). https://doi.org/10.1007/s00134-004-2429-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00134-004-2429-8

Keywords

Search

Navigation