Gabriele Seitz

The Role of Sphingosine 1‐Phosphate Receptors in the Trafficking of Hematopoietic Progenitor Cells

Abstract: Sphingosine 1‐phosphate (S1P) is an ubiquitously present extracellular lipid mediator that is released by several cell types, particularly by activated platelets. The effects of S1P are mediated by a specific family of G protein‐coupled sphingosine 1‐phosphate receptors (S1P1‐S1P5). We demonstrate that S1P acts on hematopoietic progenitor cells as a chemotactic factor, attracting peripheral blood CD34+ cells in vitro. Furthermore, constant activation of S1P receptors augments CXCR4‐mediated signal transduction induced by stromal cell‐derived factor 1 (SDF‐1). These effects are most likely mediated by the S1P1 receptor consistently expressed in both primitive and committed CD34+ hematopoietic progenitor cells (HPCs). In vivo, sustained activation of S1P1 by a receptor agonist during the homing process resulted in increased engraftment. Given the fact that activated platelets represent a major source of extracellular S1P, SDF‐1‐mediated stem cell homing may occur at sites of tissue injury in addition to the bone marrow. This could explain the previously observed contribution of primary hematopoietic stem cells to tissue repair in myocardial infarction and other diseases.

Ascorbic acid stimulates DOPA synthesis and tyrosine hydroxylase gene expression in the human neuroblastoma cell line SK-N-SH

Ascorbic acid is well known to induce noradrenaline synthesis in sympathetic nervous cells. In a series of experiments we found that incubation of the neuroblastoma cell line SK-N-SH with ascorbic acid (100-500 microM) for 2 h results in a significantly enhanced synthesis of 3,4-dihydroxyphenylalanine (DOPA) and dopamine. Additionally, cDNA-polymerase chain reaction (cDNA-PCR) analysis of relative mRNA levels corresponding to the enzymes involved in catecholamine synthesis revealed a 3-fold increase of tyrosine hydroxylase gene expression after 5 days of incubation with ascorbic acid (200 microM), whereas expression of dopamine-beta-hydroxylase was found to be unaltered. In summary the data give evidence that ascorbic acid leads to enhanced DOPA production in SK-N-SH cells by two different mechanisms: at the metabolic level after short-term incubation and by increasing the tyrosine hydroxylase gene expression after long-term incubation. Based on these data we suppose that enhancement of DOPA synthesis by ascorbic acid may be useful in the treatment of early Parkinsons disease.

Efficient in vitro generation of adult multipotent cells from mobilized peripheral blood CD133+ cells

Abstract. Objectives: To generate non‐haematopoietic tissues from mobilized haematopoietic CD133+ stem cells. Materials and methods: Mobilized peripheral blood CD133+ cells from adult healthy donors were used. In vitro ability of highly enriched CD133+ cells from mobilized peripheral blood to generate multipotent cells, and their potential to give rise to cells with characteristics of neuroectoderm, endoderm and mesoderm layers was investigated. Results: We found that a recently identified population of CD45+ adherent cells generated in vitro after culture of highly purified CD133+ cells for 3–5 weeks with Flt3/Flk2 ligand and interleukin‐6 can, in presence of the appropriate microenvironmental cues, differentiate into neural progenitor‐like cells (NPLCs), hepatocyte‐like cells and skeletal muscle‐like cells. We have termed them to be adult multipotent haematopoietic cells (AMHCs). AMHC‐derived NPLCs expressed morphological, phenotypic and molecular markers associated with primary neural progenitor cells. They can differentiate into astrocyte‐like cells, neuronal‐like cells and oligodendrocyte‐like cells. Moreover, AMHC‐derived NPLCs produced 3,4‐dihydrophenylalanine and dopamine and expressed voltage‐activated ion channels, suggesting their functional maturation. In addition, AMHC‐derived hepatocyte‐like cells and skeletal muscle‐like cells, showed typical morphological features and expressed primary tissue‐associated proteins. Conclusion: Our data demonstrate that AMHCs may therefore serve as a novel source of adult multipotent cells for autologous replacement cell therapies.

Modulation of CXC chemokine receptor expression and function in human neutrophils during aging in vitro suggests a role in their clearance from circulation.

In mice, differential regulation of CXC chemokine receptor expression in circulating polymorphonuclear neutrophils (PMNs) undergoing senescence results in homing to the bone marrow. However, the role of this compartment and of the chemokine receptor CXCR4 is still under discussion, and only scarce data exist about CXCR4 function in human PMN. In our study, we provide evidence that also in human neutrophils, expression (cell surface and mRNA), chemotactic and signaling functions of the homing-related chemokine receptor CXCR4 are upregulated during aging in vitro, independent of addition of stimulatory cytokines (TNF, IL-1, IL-8, G-CSF). In contrast, interleukin-8 receptors are downmodulated (CXCR2) or remain unchanged (CXCR1), suggesting that human PMNs undergoing senescence acquire a phenotype that impairs inflammatory extravasation and favors homing to the bone marrow or other tissues involved in sequestration. Partially retained responsiveness to interleukin-8 may be important for neutrophil function when senescence occurs after extravasation in inflamed tissues.

The CysLT1 Ligand Leukotriene D4 Supports α4β1- and α5β1-Mediated Adhesion and Proliferation of CD34+ Hematopoietic Progenitor Cells

Cytokines and chemokines control hematopoietic stem and progenitor cell (HPC) proliferation and trafficking. However, the role of nonpeptide mediators in the bone marrow microenvironment has remained elusive. Particularly CysLT1, a G protein-coupled receptor recognizing inflammatory mediators of the cysteinyl leukotriene family, is highly expressed in HPCs. We therefore analyzed the effects of its ligands on human CD34+ HPCs. The most potent CysLT1 ligand, LTD4, rapidly and significantly up-regulated α4β1 and α5β1 integrin-dependent adhesion of both primitive and committed HPC. LTD4-triggered adhesion was inhibited by specific CysLT1 antagonists. The effects of other CysLT1 ligands were weak (LTC4) or absent (LTE4). In serum-free liquid cultures supplemented with various hematopoietic cytokines including IL-3, only LTD4 significantly augmented the expansion of HPCs in a dose-dependent manner comparable to that of peptide growth factors. LTC4 and LTE4 were less effective. In CD34+ cell lines and primary HPCs, LTD4 induced phosphorylation of p44/42 ERK/MAPK and focal adhesion kinase-related tyrosine kinase Pyk2, which is linked to integrin activation. Bone marrow stromal cells produced biologically significant amounts of cysteinyl leukotrienes only when hematopoietic cells were absent, suggesting a regulatory feedback mechanism in the hematopoietic microenvironment. In contrast to antagonists of the homing-related G protein-coupled receptor CXCR4, administration of a CysLT1 antagonist failed to induce human CD34+ HPC mobilization in vivo. Our results suggest that cysteinyl leukotriene may contribute to HPC retention and proliferation only when cysteinyl leukotriene levels are increased either systemically during inflammation or locally during marrow aplasia.

Differential Effects of G Protein–Coupled Receptors on Hematopoietic Progenitor Cell Growth Depend on their Signaling Capacities

Abstract:  We have shown that CD34+ hematopoietic progenitor and stem cells (HPCs) consistently express several G protein–coupled receptors (GPCRs): the chemokine receptor CXCR4, the cysteinyl‐leukotriene receptor cysLT1, and receptors for sphingosine 1‐phosphate (S1P), particularly S1P1. These GPCRs differentially mediate chemotactic, adhesive, and proliferative responses in HPCs. To elucidate the diversity of the responses observed, we compared their signaling capacities in CD34+ cells. In primary CD34+ progenitors, the strongest effects on calcium signaling (intracellular calcium fluxes) were mediated by cysLT1. Analyses in CD34+ cell lines revealed that calcium signaling induced by cysLT1 was only partially inhibited by pertussis toxin (PTX), while responses induced by CXCR4 and S1P receptors were completely blocked. These findings indicate that cysLT1 signals via Gi and Gq proteins, while CXCR4 and also S1P receptors (e.g., S1P1) only induce Gi protein‐mediated effects. By analysis of downstream signaling, we could provide further evidence that combined activation of PTX‐insensitive (Gq‐mediated) and PTX‐sensitive (Gi‐mediated) pathways by cysLT1 may explain the strong and broad effects of cysteinyl‐leukotrienes in early hematopoietic cells, while signaling of CXCR4 and S1P1 solely depends on Gi proteins, resulting in effects mainly restricted to migration and adhesion.

The G protein-coupled receptor CysLT1 mediates chemokine-like effects and prolongs survival in chronic lymphocytic leukemia

Abstract The G protein-coupled receptor (GPCR) CXCR4 is involved in bone marrow tropism and survival of chronic lymphocytic leukemia (CLL) cells. The function of the GPCRs cysteinyl leukotriene receptor 1 (CysLT1) and CysLT2 remains elusive. Here we demonstrate that in CLL and normal B lymphocytes, CysLT1 mRNA is consistently expressed, in contrast to low CysLT2 levels. Similar to the CXCR4 ligand CXCL12, the cysteinyl leukotriene (cysLT) LTD4 induces calcium fluxes, actin polymerization, and chemotaxis. These effects are blocked by specific CysLT1 antagonists. Their inhibition by pertussis toxin suggests Giα/o protein involvement. Furthermore, CysLT1 mediates MAP-kinase phosphorylation, which implicates contribution of cysLT to survival. Indeed, CysLT1 antagonists induce apoptosis and reduce viability independent of Gαi/o protein signaling. Considering the production of cysLTs in the bone marrow, our data suggest that CysLT1 induces chemokine-like effects, supports accumulation and survival of CLL cells in the bone marrow and thus represents a potential treatment target.

6-fluorodopamine selectively destroys neuroblastoma cells expressing the noradrenaline transporter.

BACKGROUND 6-Hydroxydopamine (6-OHDA) was used for ex vivo purging of bone marrow from neuroblastoma cells before autologous transplantation. However, this concept failed because of the rapid autoxidation of 6-OHDA, which leads to the generation of cytotoxic reactive oxygen species (ROS), mainly in the incubation medium before 6-OHDA can be incorporated by neuroblastoma cells. PROCEDURE We based our experiments on the theory that, in contrast, 6-fluorodopamine (6-FDA), which is slowly converted to 6-OHDA at neutral pH, is able to enter neuroblastoma cells via the noradrenaline transporter (NA-T). Therefore, most ROS are generated inside the target cells. RESULTS Small amounts of ascorbate prevent the extracellular conversion of 6-FDA to 6-OHDA without affecting its cytotoxicity, leading to an even more selective effect of 6-FDA. CONCLUSIONS We conclude that 6-FDA is a promising substance for selective destruction of NA-T-positive neuroblastoma cells.

Detection of minimal neuroblastoma cell contamination using tyrosine hydroxylase nested RT-PCR depends on basal tyrosine hydroxylase gene expression

Improved detecion methods of minimal residual disease in patients with neuroblastoma who are prepared for autologous transplantation by harvesting bone marrow or peripheral stem cells would allow their more accurate therapy. Gene expression of tyrosine bydroxylase, the first step enzyme in catecholamine biosynthesis, is characteristic for catecholaminergic neuroblastoma cells. A very specific method of neuroblastoma cell identification among hematopoetie cells was established by RT-PCR with 2 nested primers (nRT-PCR). The study shows that in every neuroblastoma cell line investigated (SK-N-SH, IMR 32, Kelly and SiMa) tyrosine hydroxylase gene expression occured (nRT-PCR). In reconstitution experiments with neuroblastoma cells in separated bone marrow of healthy donors or buffy coat 1 SK-N-SH per 1,000; 1 IMR 32 cell per 10,000, 1 Kelly per 100,000 and 1 SiMa cell per 1,000,000 bone marrow cells bone marrow cells could be detected. The differences in sensitivity correlated with basal tyrosine hydroxylese gene expression levels in cells used for reconstitution experiments (semiquantitative RT-PCR). Based on these resuks, this technique may be of value in the detection of minimal residuals in neuroblastoma. However, a wide range of sensitivity depending on heterogenous basal tyrosine hydroxylase gene expression levels of patients tumor cells is expected. Since an inverse correlation of tyrosine hydroxylasr and noradrenaline transportergene expression was described a combination of different methods of minimal residual disease detection including e.g. noradrenaline transporter nRT-PCR, FACS-analysis using FITC-anti-GD2-antibody or immunohistoehemical methods is thought to be more edvantagous.