Recent studies about autoimmune diseases in animal models and in humans focused their attention on lymphocyte activation and in vitro cytokine production. The respective contribution of the Th1 and Th2 cytokines to the pathogenesis of autoimmune diseases is still a matter of debate. In this study the role of IL-2, IL-4, IFN-gamma, IL-10 and IL-12 cytokines were investigated by examining their spontaneous and mitogen-induced (OKT3 and PHA or LPS) synthesis and T-cells proliferative response by peripheral blood mononuclear cells to determine their role in the pathogenesis of AIHA. Thirteen patients affected by AIHA, idiopathic or associated with other diseases, and 13 healthy subjects, randomly selected from a group of blood donors, were investigated. This study indicated that AIHA is characterised by increased basal synthesis of IL-4 and decreased levels of IFN-gamma compared with healthy controls (p < 0,01). These results suggest that there is a basal decrease of Th1 cytokine and an increase of the Th2 ones. Enhanced IL-2 levels in AIHA patients are likely due to the necessity of a T-cell proliferation stimulus rather than produced as Th1 prevalent stimulation. Furthermore, it has been observed a significant increase in IL-12 production in LPS stimulated cultures from healthy controls, but not in AIHA patients, that shows IL-10 increased levels, which could cause a secondary decrease in IFN-gamma production and a stimulation of Th2 differentiation. These observations indicate that decreased production of Th1-type cytokines and prevalent Th2 ones leading to autoantibodies production in AIHA may be secondary to the imbalance between IL-10 and IL-12. These results strongly suggest that manipulation of the cytokine network, i.e. IL-10/IL-12 balance, maintained by cells of the innate immune system, can have a strong effect on the incidence of AIHA and their modulation might be useful for a therapeutic control of the disorder.

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In animal and human autoimmune hemolytic anemia (AIHA) immunologic tolerance loss against RBC self-antigens could be originated by several mechanisms: ignored self-antigens' epitopes, polyclonal lymphocyte activation, molecular mimicry between self- and foreign antigens, central or peripheral tolerance errors, or immunoregulatory disturbances including the alteration of a cytokine network. To identify the immunologic factors contributing to autoimmune onset and maintenance, several murine strains (such as NZB and NZB/NZW) that spontaneously develop a complex autoimmune syndrome, including AIHA, have been extensively studied. In human AIHA, the respective roles of IL-2, IL-4, IFN-gamma, IL-10, and IL-12 were investigated by examining the spontaneous and mitogen-induced (OKT3 or LPS) production of these cytokines. ELISA methods were used in PBMCs to evaluate whether the manipulation of IL-10/IL-12 balance can have an effect on the incidence of autoimmune diseases and whether this might be useful for the control of AIHA. Results affirmed that AIHA is a disease that exhibits an increased basal synthesis of IL-4 and decreased levels of IFN-gamma by AIHA PBMCs compared with controls and that there is a basal increase of Th2 cytokines. Th1-type cytokine decrease in the basal state occurred in parallel with an increase of constitutive IL-10 production and an IL-12 decrease. In conclusion, decreased production of Th1-type cytokines and the production of autoantibodies in AIHA may be secondary to the imbalance between IL-10 and IL-12, and the neutralization of IL-10 may be efficacious in diminishing the clinical pathology associated with Th2 subset prevalence. In the same way, the treatment with IL-12 could offer a second and independent level of blockade against the consequences of the overstimulation of B cells associated with AIHA.

We studied 16 patients affected by autoimmune hemolytic anaemia (AIHA), both idiopathic and associated with other diseases (B and T lymphoma, B hepatitis, gastric carcinoma, systemic lupus erythematosus) or alpha-methyldopa therapy, in order to value T- and B-cell activation. We determined the count of T- and B-cell subsets in peripheral blood, the proliferative response of peripheral blood lymphocytes (PBL) to phytohemagglutinin (PHA) and to pokeweed mitogen (PWM), the percentage of CD25+ cells in culture and interleukin (IL)-1alpha, IL-2, IL-4, tumor necrosis factor (TNF)alpha and soluble IL-2 receptor (sIL-2R) levels in sera and in culture. Except for an increase in CD4+ and CD8+ T cell number in a case of AIHA associated with a T lymphoma and an increase in the percentage of CD5+ and PCA1+ B cells in two cases of AIHA associated with B lymphoma and with SLE, no further data showed a relationship with the disease possibly associated with AIHA, so both idiopathic and secondary AIHA cases were analyzed together. CD4+ T cells were reduced in number in 9 cases, while CD8+ T cells were reduced in 6 cases. The percentage of CD5+ B cells was increased in 5 cases. The percentage of PCA1+ cells was increased in all cases (mean +/- sd: 18 +/- 22 vs 0,2 +/- 1 in controls). The average PBL proliferative response to PHA was reduced (S.I. 71 +/- 55 vs 138 +/- 45 in controls) as well as that to PWM (S.I. 27 +/- 21 vs 75 +/- 24 in controls), despite IL-2 high levels, in all cases, in both sera (mean +/- sd: 648 +/- 351 pg/ml vs 16 +/- 4 pg/ml in controls) and culture supernatants (mean +/- sd: 1045 +/- 677 pg/ml vs 195 +/- 51 pg/ml in controls). In PHA stimulated cultures the percentage of CD25+ cells was reduced (mean +/- sd: 37 +/- 18 vs 63 +/- 14 in controls), sIL-2R levels were like controls in 7 cases. In sera sIL-2R levels were increased in all cases (mean +/- sd: 1256 +/- 465 U/ml vs 256 +/- 114 U/ml in controls), IL-1alpha was increased in all cases too, while IL-4 levels were increased only in 7 cases. Linear regression analysis generally showed a low relationship between S.I. and IL-2, IL-4 and sIL-2R levels in supernatants of PHA stimulated culture as well as between S.I. and the percentage of CD25+ cells. Taken together these data suggest a state of B- and T-cell hyperactivation in AIHA. The low PBL proliferative response in vitro, explained in previous studies as a temporary functional exhaustion, might be itself a sign of the complete lymphocyte activation occurring in vivo in AIHA.

In autoimmune diseases striking abnormalities of T and B cell activation and of cytokine production are present. In 14 patients with autoimmune hemolytic anemia (AIHA), idiopathic or in the course of: lymphoma, B hepatitis, carcinoma, drug therapy (alpha-methyldopa), systemic lupus erythematosus (SLE), and not yet submitted to immunosuppressive therapy, the PBL proliferative response to PHA and the IL1 alpha, IL2, IL4 and IL2R serum levels have been valued. While the stimulation index of PBL was strongly reduced in 10 cases (64 +/- 56 vs 138 +/- 45 in the control group), IL1 alpha, IL2 and IL2R were greatly increased in all the patients, and IL4 in 5 (IL1 alpha :199 +/- 268 pg/ml in patients vs 0.30 +/- 0.2 in controls; IL2:716 +/- 311 pg/ml vs 16 +/- 4; IL4:29 +/- 13 pg/ml vs 13 +/- 7; IL2R:1233 +/- 471 U/ml vs 256 +/- 114). Cytokine serum levels were not related with the associated disease, with the CD4+ and CD8+ cells absolute number or with PBL blastogenic in vitro response. The high serum levels of cytokines and IL2R suggest that in AIHA there exist a CD4+ lymphocyte hyperactivation (the low proliferative response of PBL might imply a temporary functional exhaustion of T lymphocytes) as in the other autoimmune diseases.