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

• Published in: British journal of haematology Vol. 111; no. 2; pp. 452 - 460
• Main Authors: BARCELLINI, Wilma, CLERICI, Giuliana, MONTESANO, Rosanna, TAIOLI, Emanuela, MORELATI, Fernanda, REBULLA, Paolo, ZANELLA, Alberto
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell 01.11.2000; Blackwell Publishing Ltd
• Subjects: Adult; Aged; Anemia, Hemolytic, Autoimmune - immunology; Anemias. Hemoglobinopathies; Antibody Formation; Autoantibodies - immunology; Binding, Competitive; Biological and medical sciences; Case-Control Studies; Cells, Cultured; Cytokines - immunology; Cytokines - pharmacology; Diseases of red blood cells; Erythrocytes - immunology; Female; General aspects; Hematologic and hematopoietic diseases; Hematology; Humans; Immunoenzyme Techniques; Immunoglobulin G - immunology; Immunopathology; 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; Medical sciences; Middle Aged; Mitogens - pharmacology; Multivariate Analysis; Recombinant Proteins - pharmacology; Stimulation, Chemical; Transforming Growth Factor beta - immunology; Transforming Growth Factor beta - pharmacology; Autoimmunity; Human; Immunopathology; Pathophysiology; Antibody; Cytokine; Idiopathic; Autoantibody; Red blood cell; Th1 lymphocyte; Helper cell; Hemopathy; In vitro; Cell subpopulation; Hemolytic anemia; Immunological investigation; T-Lymphocyte; Immune deviation; Th2 lymphocyte
• ISSN: 0007-1048; 1365-2141
• 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.