CD34+ Marrow Cells from MDS Patients Express Homing-Associated Cell Surface Molecules but Impaired In Vitro Migration.

Homing and retention of hematopoietic stem cells in the marrow microenvironment are multi-step processes mainly controlled by interactions between chemokines, adhesion receptors and proteases. In the case of MDS, whether these interactions are perturbed and how they are implicated in the pathogenesis of the disease or progression to AML is not known. In this study we evaluated the cell surface expression of homing-associated adhesion molecules, CD11a, CD26, CD45, CD49d, CD49e and CXCR4 by flow cytometric analysis in BM mononuclear (MN) and CD34⁺ cells from 19 MDS patients [7 RA, 1RARS, 11RAEB(t)] and age-matched healthy individuals (n=9). CD34⁺ and MN cells from RAEB(t) samples expressed higher levels of CD11a and CD49e (2–3 fold increase) and 1,5–3 fold lower levels of CD45 compared with the cells from the healthy control group, as measured by the mean fluorescent intensity. None of the remaining molecules was found to have altered expression in the RAEB(t) subtype. A similar expression pattern of all homing-associated molecules was seen in the cells from the RA/RARS and normal control samples. However, when CD34⁺ cells from MDS patients were allowed to migrate in vitro toward a gradient of SDF-1α (125ng/ml), they displayed an impaired chemotactic response [mean values of % input: 9% (0,5–30%)], whereas those from healthy individuals actively migrated [mean values of % input: 20% (10–50%)]. Both migrated and non migrated CD34⁺ cells from MDS patients displayed little hematopoietic activity in vitro (CFC and LTC-IC). The addition of neutralizing monoclonal antibody anti-CXCR4 decreased in vitro chemotactic response of normal and MDS derived CD34⁺ cells by 90% and 80% respectively indicating that migration was dependent on SDF1/CXCR4 interactions. To further investigate the differences observed in response to SDF1a, immunofluorescence detection of intracellular expression of CXCR4 was performed. MDS derived CD34⁺ and MN cells from all the subtypes studied significantly expressed high levels of intracellular CXCR4 and were induced to cell surface CXCR4 expression after 48 hours of SCF (100ng/ml) and IL-6 (20ng/ml) stimulation, as was seen with normal cells.

In conclusion marrow CD34⁺ cells from MDS patients showed reduced capacity to migrate in vitro, although no significant perturbations of adhesion molecules expression were detected. We are currently investigating the signaling events and pathways, which control homing and adhesion of hematopoietic cells in the marrow.