Mechanisms of immunological tolerance loss versus erythrocyte self-antigens and autoimmune hemolytic anemia.

Recent studies on animal and human autoimmune hemolytic anemia (AIHA) suggest that immunological tolerance loss toward red blood cells (RBC) self-antigens may be originate by different, non-mutually exclusive, mechanisms. According to now available data the identified mechanisms may be: ignorance against RBC self-antigens; molecular mimicry; polyclonal T and/or B cells activation; errors in central or peripheral tolerance; immunoregulatory disorders including cytokine network alteration. In some patients with AIHA, stimulation of PMBC by synthetic Rh peptides indicate that ignorant T and/or B cell clones may recognize cryptic RBC self-antigens. AIHA associated with bacterial or viral infections seems to be produced by polyclonal T and/or B cells activation against foreign antigens which mimic protein or carbohydrate epitopes on RBC. Polyclonal activation of host B cell clones by donor alloreactive T cells causes the AIHA in chronic GVHD. As the tolerance loss is concerned, experiments on mouse lines expressing a transgene with autoantibody activity against murine RBC have shown that non-deleted peripheral B cell clones may produce RBC autoantibodies. In humans a genetic defect of Fas/FasL autoreactive lymphocytes apoptosis may be associated to AIHA. Immunoregulatory disorders due to depletion of CD4+ CD25+ T cells or Th1/Th2 cytokines imbalance may induce autoimmune diseases. In mice AIHA may be induced or improved by cytokines or anticytokine antibodies administration. In NZB/W mice and human AIHA there is an increased production of Th2 cytokines as IL4 and IL10 but INF-gamma reduced production. In addition in human AIHA has been shown a downregulation of IL12 and therefore, an IL10/IL12 immunoregulatory circuit imbalance which might facilitate the RBC autoantibodies production.

Cytokine network in autoimmune haemolytic anaemia: new probable targets for therapy.

AIMS: Several immunological mechanisms seems to be similar in cancer and autoimmune disease. Studying interleukins production and proliferative response in autoimmune haemolytic anaemia (AIHA), it is possible to observe that manipulation of IL-10/IL-12 balance can have profound effect on the incidence of autoimmune diseases and this might be useful for the control of AIHA. METHODS: Respective role of IL-2, IL-4, IFN-gamma, IL-10 and IL-12 in non-cancer associated AIHA were investigated by examining the spontaneous and mitogen-induced (OKT3 or LPS) synthesis of these cytokines in PBMC cultures by ELISA methods. RESULTS: Our results affirmed that AIHA is a disease which exhibited an increased basal synthesis of IL-4 and decreased levels of IFN-gamma by AIHA PBMC compared with controls and then there is a basal increase of Th2 cytokines. Th1-type cytokine decrease in basal state occurred in parallel with an increase of constitutive IL-10 production and a IL-12 decrease. CONCLUSIONS: 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 then the neutralisation 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 over B cell activation associated with AIHA and sometimes with cancer.

Th1 and Th2 cytokine modulation by IL-10/IL-12 imbalance in autoimmune haemolytic anaemia (AIHA).

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.

Th2 cytokine role in autoimmune haemolytic anaemia (AIHA) pathogenesis.

BACKGROUND: AIHA is characterised by the destruction of antibody-coated red blood cells, but the mechanism that initiates the production of autoantibodies remains unclear. We have studied the proliferative response and the spontaneous and mitogen-induced (PHA and OKT3) synthesis of IFN-g, IL-2 and IL-4 by peripheral blood mononuclear cells from patients with AIHA before any treatment to investigate the activation of Th1 and Th2 subsets. METHODS: Thirteen AIHA patients, both idiopathic and associated with other diseases, were studied by ELISA methods and H3 thymidine incorporation to determine in vitro cytokine production and T cell proliferative response, respectively. RESULTS: Under basal conditions the proliferative response induced in peripheral blood mononuclear cells was enhanced in AIHA patients suggesting a basal state of hyperactivation. This increase in proliferative response can be related to the basal enhanced levels of IL-2 (p<0.04), a key cytokine, that regulates the growth, differentiation and function of lymphocytes. High IL-2 levels in AIHA patients supernatants, with or without OKT3 stimulation, justify the high basal activation of T lymphocytes. Under basal conditions levels of IFN-g are decreased and IL-4 levels are increased. CONCLUSIONS: AIHA is characterised by the destruction of antibody-coated red blood cells, but the mechanism that initiates the production of autoantibodies remains unclear. But looking to this data it is possible to suppose that there is a prevalent contribution of Th2 lymphocytes to the pathogenesis of AIHA.

Enhanced IL-10 production in vitro by monocytes in autoimmune haemolytic anaemia.

Our previous studies on autoimmune haemolytic anaemia (AIHA) have shown a hyperactivation concerning cytokine production in T and B lymphocytes obtained from AIHA patients. In this study the production of interleukin (IL)-10, basal and stimulated by lipopolysaccharide (LPS), was determined in cultured monocytes obtained from patients affected by AIHA, either idiopathic or associated with other autoimmune diseases, e.g. idiopathic thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE), myastenia gravis (MG). In cultured AIHA monocytes the IL-10 basal production (mean+/-SD) was increased in all but one patient, compared to the controls, 125.96+/-76.10 pg/mL and 19.18+/-15.80 pg/mL respectively. Specifically, LPS stimulation was able to induce a higher IL-10 production by monocytes in two AIHA cases, whereas in five patients there was no difference and in two cases the LPS stimulated IL-10 production was even decreased compared to the levels observed in the controls. Interestingly in the latter two cases AIHA was associated with other autoimmune diseases (ITP, SLE). These results indicate a constitutively higher basal IL-10 production in monocytes from AIHA patients. The increased level of IL-10 could play an important role in the modified pathways of the immune response in AIHA.

Lymphocyte activation and cytokine production in autoimmune hemolytic anaemia (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.

High cytokine serum levels in patients with autoimmune hemolytic anemia (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.

Peroxidative events in stored platelet concentrates.

Changes in metabolic and functional activity of platelets stored as platelet concentrates in plastic bags highly permeable to gases were investigated. The following parameters were measured daily: pH, pO2, pCO2, HCO3, glucose, lactic acid, lactic dehydrogenase, cellular ATP and platelet aggregation induced by different agents (collagen and ADP). As indexes of lipid peroxidative damage, the cellular levels of conjugated dienes, malonyldialdehyde and some antioxidant molecules such as coenzyme Q10 and vitamin E were determined. A marked increase in pO2, conjugated dienes, malonyldialdehyde, lactic acid and lactic dehydrogenase activity was observed during the preservation. Platelet ATP content was unmodified and a remarkable decrease in platelet aggregability was found. pCO2, cyclooxygenase activity, vitamin E, coenzyme Q10, bicarbonate and glucose showed a rapid fall. Our data seem to indicate a preservation of platelet metabolic activity and a correlation between increased lipid peroxidation and functional impairement.

Biochemistry of packed red blood cell concentrates stored in PAGGS-sorbitol solution for 42 days.

We investigated metabolic, blood gas and acid-base balance modifications of erythrocyte concentrates resuspended in PAGGS-sorbitol solution during 6 weeks of storage. Glucose utilization was impaired during the last 2 weeks, while the intraerythrocytic ATP level decreased only 50% from the 1st to the 6th week. The K+ and Hb concentration in the medium showed a progressive increase which was more pronounced during the last 2 weeks. The decrease in pH progressed to reach 6.27 at the 6th week of storage. Intraerythrocytic 2,3-DPG dropped rapidly in the course of the 1st week and the percentage of oxyhemoglobin showed a remarkable increase after the 1st week (90%). Our data suggest that erythrocyte concentrates in PAGGS-sorbitol should be transfused, for optimal efficacy, within 4 weeks.

Biochemical and functional changes of platelet stored for transfusional use.

A protocol for the biochemical study of platelet stored for transfusional use at 22 degrees C and under continuous shaking in a plastic bag highly permeable to gases and with a suitable area/volume ratio, is described. Plasmatic dextrose, lactic acid, lactic dehydrogenase activity, cellular ATP and malonyldialdehyde were monitored during the storage, as well as some acid-base indexes namely: pH, pCO2, HCO3-, pO2. The platelet functional status was checked as aggregating power induced by ADP and collagen and by beta-thromboglobulin release. The results obtained are indicative of a discrete maintenance of aerobic metabolism by platelets which are able to give up CO2 and take up O2 so that the plasmatic pH is constant during the storage. However, the malonyldialdehyde increase suggests that platelets become increasingly susceptible to peroxidative attacks. The aggregating response was dramatically reduced even on the third day of storage. The data obtained point out that, under the conditions reported, platelets can be transfused up to the third day of storage.

Biochemical parameters to assess viability of blood stored for transfusional use.

Blood collected in citrate-phosphate-dextrose-adenine (CPDA-1) containing transfusional bags, was weekly tested throughout a 35 day period. Biochemical assays included plasmatic glucose, electrolytes, free Hb, acid-base balance and hemogasanalysis. Intraerythrocytic ATP and 2,3 DPG were also determined. Results show that an almost total 2,3 DPG depletion occurs during the first three weeks, whereas intracellular ATP are about 50% of the initial values, at the same time. Lowering of pH is also maximal at the third week. pCO2 variation pattern is biphasic: an early increase due to HCO3- titration by lactic acid arising from red cells glycolysis, followed by a decrease probably due to plastic bag permeability to CO2 itself. The percentage of O2Hb also rises during blood storage: this might be the combined result of increase in pO2 and decrease in 2,3 DPG content. A rise of free Hb was obtained; extracellular K+ levels underwent a sixfold increase in 35 days. The mechanism of relative variations of these parameters and the gas transport are discussed. Some of these parameters might be used as routine controls to asses viability and functional status of stored red cells for transfusional use.

T lymphocyte subpopulations and lymphocytotoxic antibodies in patients with autoimmune haemolytic anaemia.

Evaluation of T lymphocyte subpopulations was performed on peripheral blood of patients affected by idiopathic or associated autoimmune haemolytic anaemia. A marked reduction of absolute number of T gamma and T mu cells was observed in 11 of 16 patients; a decrease of both OKT4+ and OKT8+ cells was found in 8 of 10 patients. Circulating cytotoxic antibodies against autologous and allogenic T lymphocytes and/or thymocytes were found in almost all the cases. T lymphocyte subsets depletion, probably connected to antibodies against T lymphocytes and their thymic precursors, could play a role in autoimmunity because of T3+/T4+ cell depletion.

IgM plasmacytoma. Report of a case and review of the literature.

IgM paraproteinaemia is described in a patient with diffuse plasmacytoma involving the pleura, lymph nodes, and kidneys. Sixteen cases of pure plasmacellular tumor (diffuse or solitary plasmacytoma and plasmacellular leukaemia) associated with IgM paraproteinaemia have only been reported. These cases and experimental data indicate that neoplastic plasma cells can synthetize IgM as well as other immunoglobulins and that this particular M component is not associated solely with Waldenström's macroglobulinaemia.

Bone marrow amyloid in patients with M-component.

Amyloid substance was looked for in bone marrow aspirates of 28 patients presenting an M-component, in association with different illnesses (plasmocytoma, chronic lymphatic leukemia, lymphocytic lymphoma, "benign monoclonal gammapathy" and liver cirrhosis). Amyloid was detected in 12 out of 20 patients with plasmocytoma (60%) and in 3 out of 8 patients with other illnesses (37.5%); it was found in 9 out of 13 cases (69.2%) presenting exclusive light chain production and in 6 out of 15 cases (40%) with a monoclonal complete Ig. Lambda type light chain, either free or Ig-bound, was slightly more often (6 cases out of 10, 60%) associated with amyloidosis than was K (9 cases out of 17, 52.9%). Bone marrow appeared to be more frequently involved than other sites considered elective such as rectal mucosa. Amyloid appeared as an intensively fluorescent substance present intercellularly in areas of variable number and size.

Immune haemolytic anaemia after exchange transfusion.

Two cases of immune haemolytic anaemia in newborn infants who had received exchange transfusion on the first day of life because of hyperbilirubinaemia probably due to a conjugation defect, are reported. The first baby, born in the 30th week of gestation presented erythrocyte-fixed IgM antibodies + C, neutropenia, and circulating leucocytotoxic antibodies. The second baby, born at term to a diabetic mother showed erythrocyte-fixed IgG + IgM antibodies + C and a selective IgA deficit. In both cases the anaemia improved and the Coombs test became negative spontaneously in the 4th and 5th months of life, respectively. These haematologic alterations are similar to a graft versus host (GVH) reaction due to the persistence in the neonate blood of the donor's immunocompetent cells.