Direct Interaction of G-CSF-Stimulated-Neutrophils and HUVECs Are Necessary for Production of IL-8 in Endothelial Cells.

Background: We previously reported that interleukin (IL)-8 increased up to 207 pg/ml in vivo after administration of granulocyte colony-stimulating factor (G-CSF) at a dose 5μg/kg for five days to mobilize peripheral blood stem cells (PBSC) in hematopoietic stem cell transplantation in cancer patients or healthy donors. Since pre-apheresis serum IL-8 levels correlated with the yield of CD34-positive cells, IL-8 production may be critical to G-CSF induced stem cell mobilization. IL-8 is known to be surged from granules of endothelial cells (EC) by stimulation of GM-CSF, however, precise mechanisms of increased production of IL-8 by G-CSF in vivo is unclear. First, in this study we examined the relationship between G-CSF and IL-8 in vitro.

Method: Peripheral blood was drawn from healthy donor with consent. Neutrophils were separated by density centrifugation method. Human umbilical vein endothelial cells (HUVECs) and G-CSF were purchased. HUVECs were cultured under serum-containing medium and stimulated by G-CSF or HUVECs were co-cultured with neutrophils. IL-8 in culture media was measured by ELISA.

Results: G-CSF alone does not increase IL-8 production in the culture medium either of neutrophils or HUVECs alone, in either short time (6 hours) or long time (up to 4 days) stimulation. However, when HUVECs were co-cultured with neutrophils, IL-8 started to increase 10 hours after starting co-culture and gradually increased up to 3000pg/ml after 4 days culture. IL-8 in co-culture media increased five fold compared to that in the single culture of HUVECs. This increased production of IL-8 was dependent on the number of neutrophils. Further, when neutrophils were pre-incubated by G-CSF at a dose 50ng/ml, IL-8 production in co-culture media increased 70 percent compared to pre-incubation without G-CSF. This increased production of IL-8 was dependent on G-CSF dose. IL-8 production remarkably decreased to 15% when HUVECs and neutrophils were cultured separately by mesh to avoid direct contact.

Conclusion: These data indicates that direct contact between HUVECs and neutrophils is necessary for production of IL-8. Some molecule(s) induced by G-CSF might play an important role in IL-8 production. The effect of stimulation by G-CSF seems less important than that of direct interaction between neutrophils and HUVECs. This correlates well to the fact that high WBC count after G-CSF administration in vivo correlates with the yield of CD34-positive cells in peripheral blood stem cells mobilization. Molecular mechanisms of interaction between HUVECs and neutrophils need to be clarified further.