title: In vitro quantification of anti-red blood cell antibody production in idiopathic autoimmune haemolytic anaemia: effect of mitogen and cytokine stimulation.
abstract: 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.
authors: Divisione di Ematologia, Epidemiology Unit and Centro Trasfusionale e di Immunologia dei Trapianti, IRCCS Ospedale Maggiore di Milano, Milan, Italy. wbarcel@polic.cilea.it
authors: W
authors: W
authors: Barcellini
authors: G
authors: G
authors: Clerici
authors: R
authors: R
authors: Montesano
authors: E
authors: E
authors: Taioli
authors: F
authors: F
authors: Morelati
authors: P
authors: P
authors: Rebulla
authors: A
authors: A
authors: Zanella
mesh_terms: D000328:Adult; D000368:Aged; D000744:Anemia, Hemolytic, Autoimmune / Q000276:immunology*; D000917:Antibody Formation; D001323:Autoantibodies / Q000276:immunology*; D001667:Binding, Competitive; D016022:Case-Control Studies; D002478:Cells, Cultured; D016207:Cytokines / Q000276:immunology* / Q000494:pharmacology; D004912:Erythrocytes / Q000276:immunology*; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000276:immunology*; D007371:Interferon-gamma / Q000276:immunology; D016753:Interleukin-10 / Q000494:pharmacology; D018793:Interleukin-13 / Q000276:immunology / Q000494:pharmacology; D007376:Interleukin-2 / Q000276:immunology; D015847:Interleukin-4 / Q000276:immunology / Q000494:pharmacology; D015850:Interleukin-6 / Q000276:immunology / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D008934:Mitogens / Q000494:pharmacology; D015999:Multivariate Analysis; D011994:Recombinant Proteins / Q000494:pharmacology; D013268:Stimulation, Chemical; D016212:Transforming Growth Factor beta / Q000276:immunology / Q000494:pharmacology
chemical_list: D001323:Autoantibodies; D016207:Cytokines; D007074:Immunoglobulin G; D018793:Interleukin-13; D007376:Interleukin-2; D015850:Interleukin-6; D008934:Mitogens; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D016753:Interleukin-10; D015847:Interleukin-4; D007371:Interferon-gamma