Monocyte migration to the synovium in rheumatoid arthritis patients treated with adalimumab.

OBJECTIVES: The mechanism of action of treatment with tumour necrosis factor (TNF) blockers in rheumatoid arthritis (RA) is still not completely understood. The aim of this study was to test if adalimumab treatment could affect the influx of monocytes into the synovium. METHODS: A novel technique was used to analyse the migration of labelled autologous monocytes before and 14 days after initiation of adalimumab treatment using scintigraphy. CD14 monocytes were isolated from patients with RA, using a positive selection procedure with magnetic-activated cell sorting, and labelled with technetium-99m-hexamethylpropylene-amino-oxime. Scintigraphic scans were made 1, 2 and 3 h after re-infusion. RESULTS: As early as 14 days after the start of treatment with adalimumab a significant decrease in disease activity score evaluated in 28 joints was shown. There was no significant decrease in the influx of monocytes into the joint at this time. CONCLUSIONS: This study indicates that adalimumab treatment does not reduce the influx of monocytes into the synovium early after initiation of treatment. As previous studies showed a rapid decrease in macrophage infiltration after TNF-antibody therapy, which could not be explained by increased cell death, this points to an important role for enhanced efflux of inflammatory cells from the synovium.

Labeling monocytes for imaging chronic inflammation.

With growing interest in cell-based scintigraphic diagnosis or therapy monitoring, there is an increasing demand for non-invasive observation and quantification of cell trafficking in the preclinical and clinical setting. Monocytes are members of the human mononuclear phagocyte system originating from a myeloid precursor in the bone. Labeled monocytes are being used for investigation of pathogenesis like atherosclerosis and for monitoring of therapeutic intervention in inflammatory diseases like rheumatoid arthritis. Labeling mononuclear cells at high specific activity without affecting their biological functions allows (delayed) non-invasive imaging with g or PET cameras. Monocytes labeled before their final differentiation into macrophages or dendritic cells may reveal centers of inflammation in a patient and, thereby, contribute to scintigraphic diagnosis. Macrophages or dendritic cells may be in vitro cultured and by means of genetic transformation specified towards specific targets prior to re-injection, an approach with therapeutic potency. This review addresses issues on autologous monocytes, particularly their properties and labeling for non-invasive in vivo radionuclide imaging of chronic inflammation.

Evaluation of 'out-of-specification' CliniMACS CD34-selection procedures of hematopoietic progenitor cell-apheresis products.

BACKGROUND: Immunomagnetic selection of CD34(+) hematopoietic progenitor cells (HPC) using CliniMACS CD34 selection technology is widely used to provide high-purity HPC grafts. However, the number of nucleated cells and CD34+ cells recommended by the manufacturer for processing in a single procedure or with 1 vial of CD34 reagent is limited. METHODS: In this retrospective evaluation of 643 CliniMACS CD34-selection procedures, we validated the capacity of CliniMACS tubing sets and CD34 reagent. Endpoints of this study were the recovery and purity of CD34+ cells, T-cell depletion efficiency and recovery of colony-forming units-granulocyte-macrophage (CFU-GM). RESULTS: Overloading normal or large-scale tubing sets with excess numbers of total nucleated cells, without exceeding the maximum number of CD34+ cells, had no significant effect on the recovery and purity of CD34+ cells. In contrast, overloading normal or large-scale tubing sets with excess numbers of CD34+ cells resulted in a significantly lower recovery of CD34+ cells. Furthermore, the separation capacity of 1 vial of CD34 reagent could be increased safely from 600 x 10(6) CD34+ cells to 1000 x 10(6) CD34+ cells with similar recovery of CD34(+) cells. Finally, T-cell depletion efficiency and the fraction of CD34+ cells that formed CFU-GM colonies were not affected by out-of-specification procedures. DISCUSSION: Our validated increase of the capacity of CliniMACS tubing sets and CD34 reagent will reduce the number of selection procedures and thereby processing time for large HPC products. In addition, it results in a significant cost reduction for these procedures.

Migratory behavior of leukemic cells from acute myeloid leukemia patients.

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 contribute to stem cell homing and may play a role in the trafficking of leukemic cells. Therefore, we analyzed migration across Transwell filters of cells derived from bone marrow (BM) or peripheral blood (PB) from 26 acute myeloid leukemia (AML) patients. The presence of the extracellular matrix protein fibronectin (FN) strongly enhanced the spontaneous and SDF-1-induced migration of leukemic PB and BM cells. No differences in spontaneous, SDF-1-induced migration or CXCR-4 expression were observed between the different AML subtypes. Subsequently, it was determined whether SDF-1 preferentially promoted migration of subsets of leukemic cells. Leukemic cells expressing CD34, CD38 and HLA-DR were preferentially migrating, whereas cells expressing CD14 and CD36 showed diminished migration. Analysis of paired PB and BM samples indicated that significantly higher SDF-1-induced migration was observed in AML for CD34(+) BM-derived cells compared to CD34(+) PB-derived cells, suggesting a role for SDF-1 in the anchoring of leukemic cells in the BM or other organs. The lower percentage of circulating leukemic blasts in patients with a relatively high level of SDF-1-induced migration also supports this hypothesis. In conclusion, we have shown that primary AML cells are migrating towards SDF-1 independent of the AML subtypes.

SDF-1-induced actin polymerization and migration in human hematopoietic progenitor cells.

OBJECTIVE: The capacity of hematopoietic progenitor cells (HPCs; CD34(+) cells) to respond to chemotactic stimulation is essential for their homing efficiency, e.g., during stem cell transplantation. Previous studies established that stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 play an important role in the homing of HPCs. The aim of the present study was to analyze SDF-1-induced actin polymerization and migration of HL-60 cells and primary human CD34(+) cells. MATERIALS AND METHODS: SDF-1-induced migration of CD34(+) cells from cord blood (CB) and peripheral blood (PB) across fibronectin-coated filters was measured in a Transwell assay. Actin polymerization was detected using fluorescent phalloidin and analyzed by confocal microscopy and FACS analysis. RESULTS: SDF-1 induced a rapid and transient increase in actin polymerization and in polarization of the actin cytoskeleton in primary CD34(+) cells and HL-60 cells. SDF-1 was found to induce significantly more actin polymerization in CB CD34(+) cells that show fast migration in vitro compared to slow migrating PB CD34(+) cells. Moreover, CB CD34(+) cells that had migrated toward SDF-1 showed an elevated and prolonged rise in F-actin upon second exposure to SDF-1 compared to nonmigrated cells, although both cell types expressed equal levels of the SDF-1 receptor CXCR-4. CONCLUSIONS: The relatively high migratory capacity of CB-derived human HPCs is not related to cellular polarization or high expression of the SDF-1 receptor but is largely determined by their capacity to efficiently polymerize F-actin in response to SDF-1.

In vitro migratory capacity of CD34+ cells is related to hematopoietic recovery after autologous stem cell transplantation.

To investigate whether the migratory ability of peripheral blood-derived CD34+ cells of patients undergoing autologous peripheral blood stem cell transplantation is related to the homing efficiency of these cells, the migration in vitro of these cells was determined and correlated with in vivo hematopoietic recovery. Large inter-individual differences of the in vitro migratory ability of the CD34+ cells were observed, ranging from 1.1% to 16.4% for spontaneous migration and 6.2% to 40.8% for SDF-1-induced (100 ng/mL) migration. Significantly faster hematologic recovery was observed in those patients who received transplanted CD34+ cells that showed high spontaneous and SDF-1-induced migration in vitro (P <.05). Moreover, CD34+ cells from healthy G-CSF-mobilized donors exhibited significantly higher spontaneous and SDF-1-induced (P <.01) migration than CD34+ cells from patients mobilized with chemotherapy and G-CSF. The lower migratory capacity in vitro of patient-derived CD34+ cells was not due to lower expression of CXCR-4 but probably reflected decreased motogenic behavior of the cells. These results indicate that the migratory capacity of the cells is important for hematopoietic recovery. The data suggest that the engraftment potential of autologous stem cells is more or less impaired by treatment before or during the mobilization procedure and might possibly be restored by in vitro manipulation of the cells. In addition, an exponential relation between CXCR-4 expression and number of CD34+ cells that mobilized to the peripheral blood was found (P <.001), suggesting that CXCR-4 expression plays a role in the mobilization of CD34+ cells.

Homing of human hematopoietic stem and progenitor cells: new insights, new challenges?

In healthy adults, hematopoiesis takes place in the bone marrow, where the majority of hematopoietic progenitor cells (HPC) reside. In patients undergoing chemo- and/or radiotherapy, hematopoiesis is seriously disturbed. Reconstitution of bone-marrow function can be achieved by bone marrow transplantation or peripheral blood stem cell transplantation. The success of stem cell transplantation depends on the ability of intravenously infused stem cells to lodge in the bone marrow, a process referred to as homing. However, the molecular mechanisms that govern this process are poorly understood. It is hypothesized that homing is a multistep process, consisting of adhesion of the HPC to endothelial cells of the marrow sinusoids, followed by transendothelial migration directed by chemoattractants, and finally anchoring within the extravascular bone marrow spaces where proliferation and differentiation will occur. In this review, we discuss the factors that determine the engraftment potential of stem cells, and focus on various aspects of migration and homing of HPC, i.e., the role of the chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4, the involvement of adhesion molecules, and the induction of actin polymerization in the HPC. Defining the role of chemokines and adhesion molecules in human stem cell migration and engraftment will help us uncover the underlying mechanisms that regulate stem cell homing and will eventually advance clinical stem cell transplantation.

Adhesion molecules involved in transendothelial migration of human hematopoietic progenitor cells.

In the process of homing, CD34(+) hematopoietic progenitor cells migrate across the bone marrow endothelium in response to stromal cell-derived factor (SDF)-1. To develop more efficient stem cell transplantation procedures, it is important to define the adhesion molecules involved in the homing process. Here, we identified the adhesion molecules that control the migration of primary human CD34(+) cells across human bone marrow endothelial cells. Migration of CD34(+) cells is enhanced across interleukin 1beta prestimulated bone marrow endothelium, suggesting an important role for the endothelium in adhesion and formation of the chemotactic gradient. Under these conditions, 30-100 ng/ml SDF-1 induced a rapid and efficient migration of CD34(+) cells (+/- 46% migration in 4 h). In contrast, 600-1,000 ng/ml SDF-1 were required for optimal migration across fibronectin-coated filters. Subsequent studies revealed that transendothelial migration of CD34(+) cells is mediated by beta1- and beta2-integrins and PECAM-1 (CD31) but not by CD34 or E-selectin. Whereas these antibodies individually blocked migration for 25%-35%, migration was reduced by 68% when the antibodies were combined. Thus, these adhesion molecules play specific and independent roles in the transmigration process. Finally, O-glycosylated proteins appeared to play a role, since SDF-1-induced migration of CD34(+) cells (treated with a glycoprotease from Pasteurella haemolytica) across endothelial cells was clearly inhibited. In conclusion, we show that efficient SDF-1-induced migration of primary human CD34(+) cells across bone marrow endothelium is mediated by beta1-integrins, beta2-integrins, CD31 and O-glycosylated proteins.

Increased migration of cord blood-derived CD34+ cells, as compared to bone marrow and mobilized peripheral blood CD34+ cells across uncoated or fibronectin-coated filters.

Hematopoietic progenitor cells (CD34+ cells) migrate to the bone marrow after reinfusion into peripheral veins. Stromal cell-derived factor-1 (SDF-1) is a chemokine produced by bone marrow stromal cells that induces migration of CD34+ cells. In this study we compared spontaneous and SDF-1-induced migration of CD34+ cells from bone marrow (BM), peripheral blood (PB), and cord blood (CB) across Transwell filters. Under all circumstances, CB CD34+ cells showed significantly more migration than did BM or PB CD34+ cells. SDF-1 induced migration of BM CD34+ cells was higher than that of PB CD34+ cells, possibly due to differences in sensitivity towards SDF-1. Indeed, PB CD34+ cells showed a significantly lower expression of the receptor for SDF-1 (CXCR-4) than did BM and CB CD34+ cells. The sensitivity to SDF-1, as measured by migration towards different concentrations of SDF-1, was identical for BM and CB-derived CD34+ cells and correlated with their equal CXCR-4 receptor expression. Coating of the filters with the extracellular matrix protein fibronectin (FN) strongly enhanced the SDF-1-induced migration of PB CD34+ cells (2.5 times) and of BM CD34+ cells (1.5 times). SDF-1 induced migration of PB CD34+ cells over FN-coated filters was blocked by antibodies against beta1 integrins. Subsequently, analysis was performed to determine whether SDF-1 preferentially promoted migration of subsets of CD34+ cells. Actively cycling CD34+ cells, which were present in BM (14%) but hardly in PB (2.2%) or CB (1.2%), were found to migrate preferentially towards SDF-1. In the input, 14%+/-2.5% of the BM CD34+ cells were in G2/M and S phase, whereas in the migrated fraction 20%+/-5.7% of the cells were actively cycling (p < 0.05). We did not observe preferential migration of phenotypically recognizable primitive CD34+ subsets, despite the fact that CB CD34+ cells are thought to contain a higher percentage of immature subsets. In conclusion, the relatively lower migration of PB CD34+ cells seems to be due to a lower sensitivity towards SDF-1, and the higher migrational capacity of CB CD34+ cells, in comparison to BM and PB CD34+ cells, seems to have an as yet unknown intrinsic cause. The increased migration of CB CD34+ cells may favor homing of these cells to the bone marrow, which might reduce the number of cells required for hematological reconstitution after transplantation.

Hepatocyte growth factor/scatter factor (HGF/SF) affects proliferation and migration of myeloid leukemic cells.

Hepatocyte growth factor (HGF), also known as scatter factor (SF), is produced by mesenchymal cells, including bone marrow (BM) stromal cells, and has mitogenic and motogenic effects on a variety of cell types. Recently, a role has been assigned to HGF/SF and its receptor, c-MET, in both normal and malignant hemopoiesis. We investigated the function of HGF/SF on hemopoietic mononuclear cells (MNC) from patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) with circulating blasts. In contrast to results with normal MNC, HGF/SF alone stimulated the proliferation and colony formation of MNC from these patients. MNC from some (4/13) of the AML patients also produced HGF/SF (0.1-0.2 ng/ml/day), while we could not detect HGF/SF in cultures from normal MNC. Furthermore, it appeared that HGF/SF induced migration of leukemic cells in Boyden using KG1a cells as a model for leukemic blasts. The membranes dividing the two compartments of the Boyden chambers were coated with fibronectin. HGF/SF significantly promoted migration in 3/5 samples of MDS patients and in 5/7 samples of AML patients. Supernatant of human BM stromal cells, which is chemoattractive for normal human hemopoietic progenitor cells, also promoted migration of MNC from 4/5 MDS patients and 6/7 AML patients. Since HGF/SF is one of the growth factors produced by BM stromal cells, a neutralizing antibody directed against HGF/SF was added to the BM stroma supernatant, which reduced migration significantly in 2/3 MDS and in 3/6 AML responders to BM stroma supernatant. In conclusion, HGF/SF promotes proliferation and migration of hemopoietic cells from AML and MDS patients in vitro and may therefore contribute to the malignant potential of these cells.