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In vitro quantification of anti‐red blood cell antibody production in idiopathic autoimmune haemolytic anaemia: effect of mitogen and cytokine stimulation
Author(s) -
Barcellini Wilma,
Clerici Giuliana,
Montesano Rosanna,
Taioli Emanuela,
Morelati Fernanda,
Rebulla Paolo,
Zanella Alberto
Publication year - 2000
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/j.1365-2141.2000.02380.x
Subject(s) - cytokine , antibody , immunology , stimulation , red blood cell , immune system , haemolysis , endocrinology , biology , medicine , interleukin 10
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)‐γ was reduced. Multivariate analysis showed that IFN‐γ 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‐γ and IL‐2 production and increased secretion of transforming growth factor (TGF)‐β. 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‐β 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‐β 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.