In vitro quantification of anti-red blood cell antibody production in idiopathic autoimmune haemolytic anaemia: effect of mitogen and cytokine stimulation

Br J Haematol. 2000 Nov;111(2):452-60. doi: 10.1046/j.1365-2141.2000.02380.x.


The immunopathogenic mechanisms underlying idiopathic autoimmune haemolytic anaemia (AIHA) are still unknown, although regulatory cytokines are thought to play an important role. We investigated cytokine production by mitogen-stimulated whole blood cultures from 21 patients with AIHA and from 22 age- and sex-matched controls. In parallel experiments, we studied the effect of mitogen and cytokine stimulation on anti-red blood cell (RBC) IgG antibody production, assessed as both binding on autologous RBCs and secretion in culture supernatants. To quantify anti-RBC antibody, we set up a sensitive and quantitative solid phase competitive immunoassay. The results showed that in AIHA patients production of interleukin (IL)-4, IL-6 and IL-13 was significantly increased, whereas that of interferon (IFN)-gamma was reduced. Multivariate analysis showed that IFN-gamma was the only independent factor significantly associated with the reduced T-helper-1-like cytokine profile. Patients with active haemolysis showed further reduction of IFN-gamma and IL-2 production and increased secretion of transforming growth factor (TGF)-beta. In AIHA patients, mitogen stimulation, as well as IL-6, significantly increased autologous anti-RBC-binding relative to unstimulated cultures. Mitogen stimulation and addition of IL-4, IL-6, IL-10, IL-13 and TGF-beta significantly increased both autologous anti-RBC binding and antibody secretion in AIHA patients compared with controls. The results suggest that a reduced T-helper-1- and a predominant T-helper-2-like profile and elevated TGF-beta levels might play a role in the immunopathogenesis of AIHA. Furthermore, our competitive anti-RBC antibody was able to detect anti-RBC antibody production in some direct antiglobulin test (DAT)-negative AIHA patients.

MeSH terms

  • Adult
  • Aged
  • Anemia, Hemolytic, Autoimmune / immunology*
  • Antibody Formation
  • Autoantibodies / immunology*
  • Binding, Competitive
  • Case-Control Studies
  • Cells, Cultured
  • Cytokines / immunology*
  • Cytokines / pharmacology
  • Erythrocytes / immunology*
  • Female
  • Humans
  • Immunoenzyme Techniques
  • Immunoglobulin G / immunology*
  • Interferon-gamma / immunology
  • Interleukin-10 / pharmacology
  • Interleukin-13 / immunology
  • Interleukin-13 / pharmacology
  • Interleukin-2 / immunology
  • Interleukin-4 / immunology
  • Interleukin-4 / pharmacology
  • Interleukin-6 / immunology
  • Interleukin-6 / pharmacology
  • Male
  • Middle Aged
  • Mitogens / pharmacology
  • Multivariate Analysis
  • Recombinant Proteins / pharmacology
  • Stimulation, Chemical
  • Transforming Growth Factor beta / immunology
  • Transforming Growth Factor beta / pharmacology


  • Autoantibodies
  • Cytokines
  • Immunoglobulin G
  • Interleukin-13
  • Interleukin-2
  • Interleukin-6
  • Mitogens
  • Recombinant Proteins
  • Transforming Growth Factor beta
  • Interleukin-10
  • Interleukin-4
  • Interferon-gamma