The role of platelet/endothelial cell adhesion molecule 1 (CD31) and CD38 antigens in marrow microenvironmental retention of acute myelogenous leukemia cells.

In acute myelogenous leukemia (AML), leukemic cell-microenvironment interactions within various niches (stromal/osteoblastic or sinusoidal endothelial cell niches) have a role in leukemia cell survival and drug resistance. The AML leukemic cells express platelet/endothelial cell adhesion molecule-1 (CD31) and CD38, two adhesion molecules that could interact with microenvironmental elements, i.e., CD31 on the surface of marrow endothelial cells (CD31/CD31 and CD38/CD31 interactions) and hyaluronate (CD38/hyaluronate interactions). We report a physical association of these two antigens on the plasma membrane of myeloid leukemic cells. In this context, in vitro experiments done using interaction-blocking anti-CD31 and anti-CD38 monoclonal antibodies (CLB-HEC75 and OKT10, respectively) indicate that an excess of CD31 on the cell membrane of leukemic cells (CD31/CD38 MFI ratio >1) promotes a homotypic interaction with marrow endothelial cells, resulting in higher transendothelial migration. Conversely, an excess of CD38 (CD31/CD38 MFI ratio <1) allows leukemic cells to be entrapped within the bone marrow microenvironment through hyaluronate adhesion. The results obtained in vitro using fluorescence resonance energy transfer, co-capping, and co-immunoprecipitation experiments, and hyaluronate adhesion and transendothelial migration assays, are supported by immunophenotypic characterization of marrow leukemic cells from 78 AML patients on which CD38 expression levels were found to be positively correlated with those of CD31. Importantly, the excess of CD31 in those samples was associated with a higher peripheral WBC count. These findings indicate that bone marrow retention of AML cells depends on CD31 and CD38 coexpression levels.

Stromal-derived factor 1 and matrix metalloproteinase 9 levels in bone marrow and peripheral blood of patients mobilized by granulocyte colony-stimulating factor and chemotherapy. Relationship with mobilizing capacity of haematopoietic progenitor cells.

The roles of the chemokine stromal-derived factor 1 (SDF-1) and the matrix metalloproteinase 9 (MMP-9) in haematopoietic progenitor cell (HPC) mobilization are still unclear, particularly when patients are mobilized by granulocyte colony-stimulating factor (G-CSF) plus chemotherapy. We determined bone marrow (BM) and peripheral blood (PB) plasma levels of SDF-1, together with CXC-chemokine receptor 4 (CXCR-4) expression on CD34+ cells, and interleukin 8 (IL-8) and MMP-9 in 55 patients mobilized for autologous PB transplantation compared with 10 normal BM and PB samples. Plasma samples were tested at steady state (SS-) and after mobilization by cyclophosphamide and G-CSF administration (M-). SDF-1, CXCR-4, IL-8 and MMP-9 levels were significantly lower in SS- and M-PB than in SS-BM. Differences in SDF-1 levels between SS-PB and SS-BM were also observed after mobilization. We showed for the first time a clear relationship between the levels of circulating HPC, both at steady state and after mobilization, and those of secreted MMP-9 but not of SDF-1 or IL-8. However, a negative correlation was observed between mobilizing capacity and CXCR-4 expression on CD34+ cells. These findings suggest that G-CSF-induced mobilization of HPC from BM involves MMP-9, without reversing the positive gradient of SDF-1 between BM and PB.

Long-term marrow reconstitutive ability of autologous grafts in lymphoma patients using peripheral blood mobilized with granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor compared to bone marrow.

OBJECTIVES: The aim of this study was designed to compare the in vivo long-term hematopoietic potential of bone marrow and peripheral blood grafts. MATERIALS AND METHODS: Marrow progenitor cell recovery was assessed for up to 4 years in 227 patients. One hundred patients were treated for malignant lymphomas by autologous bone marrow transplantation (BMT) and 127 by peripheral blood progenitor cell transplantation (PBPCT). RESULTS: Marrow progenitor cell counts were decreased for several years with both bone marrow and peripheral blood grafts. They were not different according to the origin of the graft, despite the reduced duration of peripheral blood cell recovery observed after PBPCT. Granulocyte colony-stimulating factor (G-CSF) used for PB graft mobilization and after transplantation resulted in faster neutrophil recovery compared to granulocyte-macrophage colony-stimulating factor (GM-CSF) with no evidence of decreased marrow progenitor cell recoveries. On the other hand, postgraft administration of GM-CSF enhanced long-term colony-forming unit granulocyte-macrophage reconstitution only after BMT. Factors that influenced marrow progenitor cell reconstitution have been identified by univariate and multivariate analysis: age, gender, type of lymphoma, and postgraft administration of hematopoietic growth factors (HGF) for the whole patient group; gender, graft progenitor cell yields, and type of HGF (G-CSF vs GM-CSF) for the PBPCT group; and only type of HGF for the BMT group. Despite faster peripheral blood cell recovery, persistent deficiency of marrow progenitor cells was found several years after PBPCT, as observed after BMT. G-CSF-mobilized PBPCT resulted in faster neutrophil recovery compared to GM-CSF mobilization, with no difference in long-term hematopoietic reconstitution.

Decreased stroma adhesion capacity of CD34+ progenitor cells from mobilized peripheral blood is not lineage- or stage-specific and is associated with low beta 1 and beta 2 integrin expression.

Molecular mechanisms leading to mobilization of hematopoietic cells from bone marrow (BM) to peripheral blood (PB) involve modulation of adhesion molecule expression on these cells that probably result in changes in adhesion capacity to the microenvironment. However, it is not clear whether these changes involve different stages or lineages of progenitor cells. In this study, we compared the capacity of mature and immature clonogenic progenitor cells from granulocyte colony-stimulating factor (G-CSF)-mobilized PB and normal BM CD34+ cells to adhere to complete marrow stroma. This functional capacity was assessed concurrently with molecular expression on CD34+ cells of integrins VLA-4 (alpha 4/beta 1), VLA-5 (alpha 5/beta 1), and LFA-1 (alpha L/beta 2) by interindividual (between mobilized PB and normal BM) and intraindividual (between mobilized PB and steady-state BM and PB in the same patient) analysis. The proportion of adherent clonogenic progenitor cells was significantly lower in PB than in BM, not only for total progenitor cells but also for mature and immature progenitor cells, and the difference was found for granulocytic and particularly for erythroid lineages. The lower adhesion capacity of PB CD34+ cells to stroma was associated with decreased expression (signal/noise MFI ratio) of integrin alpha 4, beta 1, alpha L, and beta 2 chains whereas that of alpha 5 chain did not differ from BM cells with the lowest expression level. Similar differences in integrin expression levels were also found between mobilized PB and steady-state BM CD34+ cells in the same patient except for the alpha L chain. Moreover, we demonstrated for the first time a strong positive correlation between mobilizing capacity and expression levels on mobilized CD34+ cells for the LFA-1 alpha L chain but not for VLA-4 or VLA-5. In conclusion, the decreased adhesion capacity of mobilized PB progenitor cells to stroma involves different maturation stages and different lineages. This is associated with down-regulation of integrins VLA-4 and LFA-1, but mobilizing capacity appears positively correlated with LFA-1 levels.

Phosphatidylinositol 3-kinases are involved in the all-trans retinoic acid-induced upregulation of CD38 antigen on human haematopoietic cells.

All-trans retinoic acid (ATRA) is a specific inducer of CD38 antigen on marrow CD34+ cells as well as on blast cells in acute promyelocytic and myeloblastic leukaemia. The CD38 antigen contributes to the control of blast cell proliferation, and the upregulation of CD38 might constitute an element in the pathogenesis of retinoic acid syndrome. The aim of this study was to determine whether phosphoinositide 3-kinase (PI3-K) is involved in the modification of CD38 antigen expression on myeloid cells, as PI3-K plays a major role in the ATRA-induced granulocytic differentiation of HL-60 cells. We evaluated the effects of PI3-K inhibitors (wortmannin and LY294002) on the levels of CD38 antigen and mRNA in HL-60 and normal marrow CD34+ cells exposed to ATRA (1 micromol/l). The inhibitors prevented increase in CD38 mRNA expression and the overexpression of membrane CD38 antigen, without modification of the cytoplasmic level of this antigen. Interestingly, PI3-K activity was also necessary for CD38 expression on normal marrow CD34+ cells and for the ATRA-induced upregulation of CD157, a CD38-related antigen. In conclusion, PI3-K activity plays an essential role in the regulation of CD38 expression on human haematopoietic cells, and might constitute an interesting therapeutic target in haematological disorders involving CD38 overexpression.

All-trans retinoic acid prevents apoptosis of human marrow CD34+ cells deprived of haematopoietic growth factors.

The regulation of apoptosis plays a key role in haematopoiesis. It has been demonstrated that haematopoietic progenitor cells progressively undergo apoptotic cell death in the absence of appropriate growth factors. We studied the effects of pharmacological doses of all-transretinoic acid (ATRA) on the apoptosis of human adult marrow CD34+ progenitor cells cultured for 7 d in a serum-free medium. We quantified CD34+ cells, clonogenic progenitors and 5 week colony-forming cells (CFC) before and after ATRA exposure. Moreover, we defined the apoptotic status of the CD34+ cell fraction by analysis of phosphatidylserine externalization (using annexin V), the relative membrane permeability to 7-aminoactinomycin D (7AAD) and the mitochondrial membrane potential [using 3,3'-dihexyloxacarbocyanine iodide, DiOC6(3)]. In the drastic experimental conditions used, a decrease in viable CD34+ cells, granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E) and 5 week CFC were observed. Exposure to ATRA partially prevented the decrease in viable CD34+, without a concomitant effect on the clonogenic and more immature progenitors. ATRA-treated CD34+ cells displayed changes in apoptotic status compared with control cultures, particularly in lower annexin V-binding. These results were confirmed using 7AAD and DiOC6(3). Our results demonstrate that ATRA exerts a protective effect on CD34+ cells exposed to such apoptotic stress.

Quantitative and qualitative analysis of the human primitive progenitor cell compartment after autologous stem cell transplantation.

The aim of this study was to examine whether the severe prolonged deficiency in marrow clonogenic progenitor cells reported after autologous stem cell transplantation (ASCT) is associated with impairment of the primitive progenitor cell compartment. We performed Dexter-type marrow cultures and limiting dilution assays with CD34(+) cells from patients 1 year and/or later after autografting with peripheral blood stem cells for non-Hodgkin's lymphoma (NHL). Flow cytometric analysis was used to assess the CD38 antigen expression and apoptotic state (7-ADD(-)/annexin-V(+) cells) of the CD34(+) cell population. We found a dramatic decrease in both clonogenic progenitor cell production and frequency of long term culture-initiating cells (LTC-IC) in all the patients tested at 1 year, even in those displaying normal progenitor cell frequency. Surprisingly, the clonogenic capacity of each LTC-IC was not increased. Flow cytometric analysis of the CD34(+) cell population confirmed this quantitative defect, with a reduction in the CD38(dim/neg) cell population but no increase in apoptosis. This defect did not improve over time up to 4 years after transplantation. In addition, qualitative abnormalities were revealed, demonstrated by decreased CD34 antigen expression, together with impaired differentiating properties of LTC-IC toward erythroid lineage at 1 year. This study indicates that both quantitative and qualitative abnormalities of the primitive progenitor cell compartment are a constant feature up to 4 years after autologous stem cell transplantation.

Frequency and differentiation capacity of circulating LTC-IC mobilized by G-CSF or GM-CSF following chemotherapy: a comparison with steady-state bone marrow and peripheral blood.

OBJECTIVE: The present study was designed to compare directly the frequency of circulating LTC-IC and E-LTC-IC mobilized in peripheral blood (PB) after chemotherapy supported by either G-CSF (PB-G) or GM-CSF (PB-GM) in comparison to steady-state bone marrow (BM) and PB (PB-ST) values in the same patients. MATERIALS AND METHODS: Long-term cultures (LTC) were performed from 20 patients with malignant lymphoma at saturating cell concentrations to assess bulk progenitor cell production and by limiting dilution assay (LDA) to measure both frequency of LTC-IC and their proliferative and differentiation capacities. RESULTS: While CFC production in bulk LTC was higher at weeks 3-5 with PB-G than with PB-GM samples, week-5 LTC-IC and week-10 LTC-IC (E-LTC-IC) frequencies were not different using a LDA. However, the number of CFC derived from a single LTC-IC was higher in PB-G patients than in PB-GM patients (p = 0.01). Interestingly, the frequency of LTC-IC per 1 x 10(5) MNC in mobilized PB positively correlated with one-year marrow progenitor cell recovery, in contrast to the number of autografted CD34(+) cells and CFU-GM per kg. CONCLUSION: Both G-CSF and GM-CSF resulted in similar increases in LTC-IC and E-LTC-IC in PB at comparable levels to those present in BM. However, the differentiation capacity of LTC-IC was higher after mobilization with G-CSF than with GM-CSF, suggesting qualitative differences in LTC-IC mobilized with these growth factors.