Hematopoietic Stem Cells Mobilization by G-CSF and LTB4 Was Significantly Suppressed by an Oxygen Radical Scavenger or an Inhibitor of NADPH Oxidase.

Introduction: We previously had reported that LTB4 was able to mobilize HSC within 4 hours without significant side effects and that LTB4 receptor was involved in both of the pathways of mobilization induced by G-CSF and LTB4 in the murine model. Though the roles of protease (ie. NE, MMPs) seem to be important, the precise mechanisms are still unknown. ROS (reactive oxygen species) have cell signaling roles that are involved in signal transduction cascades of numerous growth factor-, cytokine-, and hormone-mediated pathways, and regulate many biological systems. Because a variety of inflammatory cytokines and chemokines, including G-CSF and LTB4, induce a rapid increase of intracellular ROS, we hypothesized that the role of ROS on HSC mobilization induced by G-CSF and LTB4 might be important.

Methods: MS5, murine stromal cell line cells, or bEnd3, murine microvascular cell line cells, were grown to confluence on the microporous transwell membrane. Murine marrow cells were placed on top of the prepared transwell membrane. The transwells were then seated in wells containing media and G-CSF or LTB4 with or without pretreatment of NAC, an oxygen free radical scavenger, or DPI, an inhibitor of NADPH oxidase-like flavoproteins. Cells that migrated through the stromal or endothelial layer into the wells were assayed for mobilization. NAC and DPI were given to C57BL/6 mice followed by rhG-CSF (5μg, IV) or LTB4 (1μg, IV) after a period of 2 hours. 24 hours after the rhG-CSF injection or 4 hours after the LTB4 injection, peripheral blood samples were obtained via cardiac puncture. The samples were analyzed for TNC using a trypan blue stain and FACS analysis were performed using Sca-1 and lineage markers, including CD45R (B220), CD116, Gr-1 and TER119.

Results: The numbers of migrated cell through the MS5 or bEnd3 were increased by treatment of G-CSF or LTB4. However, increasing effects of G-CSF or LTB4 to the transmigration through the MS5 or bEnd3 were inhibited by pretreatment of NAC or DPI. Comparing the control arm, the numbers of WBC and HSC (Sca-1+Lin−) in blood were decreased in the G-CSF or LTB4 mobilized mice (N=4), in which NAC or DPI were pretreated (Sca-1+Lin− fraction in peripheral blood: 1.91±0.80% in G-CSF alone, 0.55±0.37% in G-CSF with NAC, 0.36±0.09% in G-CSF with DPI, P<0.05; 1.92±0.49% in LTB4 alone, 0.55±0.29% in LTB4 with NAC, 0.67±0.52% in LTB4 with DPI, P<0.05).

Conclusions: Through our data, it is suggested that ROS are involved in the HSC mobilization induced by G-CSF and LTB4 in the murine model. It would be very interesting to test the effect of G-CSF or LTB4 on ROS K/O mice in the future. As for now, we will try to find the critical signal transduction pathway, through which we may find more effective and efficient molecules for HSC mobilization.