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

The immunopathogenic mechanisms underlying idiopathic autoimmune haemolytic anaemia (AIHA) are still unknown, although regulatory cytokines are though t 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 antibo dy 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 t hat in AIHA patients production of interleukin (IL)-4, IL-6 and IL-13 was s ignificantly increased, whereas that of interferon (IFN)-gamma was reduced. Multivariate analysis showed that IFN-gamma was the only independent facto r significantly associated with the reduced T-helper-1-like cytokine profil e. Patients with active haemolysis showed further reduction of IFN-gamma an d IL-2 production and increased secretion of transforming growth factor (TG F)-beta. In AIHA patients, mitogen stimulation, as well as IL-6, significan tly 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 secr etion in AIHA patients compared with controls. The results suggest that a r educed T-helper-1- and a predominant T-helper-2-like profile and elevated T GF-beta levels might play a role in the immunopathogenesis of AIHA. Further more, our competitive anti-RBC antibody was able to detect anti-RBC antibod y production in some direct antiglobulin test (DAT)-negative AIHA patients.