Anti-erythroblast autoimmunity in early myelodysplastic syndromes: enhancement of EPO/EPO-R system and PRO-apoptotic effect

Background: Anti-erythroblast autoimmunity has been preliminary demonstrated in a small group of patients with early myelodysplatic syndromes (MDS), i.e. refractory anemia (RA) and RA with ringed sideroblasts (RARS), by mitogen-stimulated-direct antiglobulin test (MS-DAT) performed on marrow samples. This autoantibodies, rather than being cytopathic, were found to induce an hyperplastic and diserythropoietic growth in vitro of bone marrow (BM) progenitors. Aims: To confirm the presence of anti-erythroblast autoantibodies in a larger cohort of patients with early MDS, and further characterise their biologic properties and target BM cell. Methods: We evaluated 70 consecutive MDS patients (median age 78 years, range 41-93; 27 women and 43 men). Fourty-eight out of 70 patients were classified as RA and 22 as RARS, IPSS index was Low for 55/70 (78%) patients and Int-1 for 15/70 (22%). MS-DAT was performed by stimulating BM with PMA and PHA; antibodies were detected in culture supernatants by competitive solid phase ELISA. To further identify the target population of the autoimmune reaction, BM-MS-DAT positive and negative supernatants were tested on normal bone marrow and cells were labeled with Fab’anti-human IgG FITC, anti-glycophorin-A PE, anti-CD71 PerCP, anti-CD34 PECy7 and anti-CD45 APC antibodies, at 4°C in the dark for cytometric analysis. Acquisition and analysis were performed on a FACSCanto II flow cytometer, using FACSDiva 6.0 software. Results: Thirty-eight out of 70 patients (54%) showed BM-MS-DAT positive values (cut-off value of 150 ng/mL); gender, MDS type, IPSS index, blood counts and therapy were comparable between the two groups. BM-MS-DAT positive patients showed an absolute number of reticulocytes (68±5.5 vs 43±4.2, mean±SE, p=0.018), unconjugated bilirubin and LDH levels higher and haptoglobin levels lower (63±6 vs 102.5±16.7, mean±SE, p=0.05) compared with negative patients. Moreover, the total marrow erythroblasts, as well as proerythroblasts, basophilic, polychromatic, and orthochromatic erythroblasts were higher in positive versus negative cases (p=0.029). Flowcytometric experiments aimed at further characterize the target BM cell of anti-erythroblast autoimmunity, showed a definite binding (20%) of BM-MS-DAT positive supernatants on CD45dimGly-AdimCD71bright, cells (erythrocyte precursors at different maturation stage), and no staining on CD45-GlybrightCD71- cells (erythrocytes); moreover we found an intermediate population CD45-GlyAdimCD71dim, with a lower IgG binding (6%). BM-MS-DAT negative supernatants gave no binding on both populations. EPO levels were reduced (13.8±3.2 versus 33.9±7.8 mUI/ml, mean±SE, p=0.033) and EPO-R expression increased (45.6±8.0versus 19.6±3.2 pg/ml, p=0.03) in bone marrow culture supernatants from BM-MS-DAT-positive patients compared with negative ones. Finally, the level of the pro-apoptotic protein Bax was higher in BM-MSDAT positive versus negative patients (66.9±14.1 versus 21.3±3.3 pg/ml, p=0.03) and, consistently, Bcl2 level was lower in the former (10.9±2.4 vs 15±3.2 pg/ml), although not significantly. Summary and Conclusions: Our results confirm the presence of autoimmunity directed against erythroblast precursors in a larger cohort of RA and RARS patients. We suggest that these autoantibodies may amplify the EPO/EPO-R system leading to a pro-apoptotic rather than a cytopathic effect.