G-CSF is an endothelial survival factor in LPS-induced inflammatory conditions

Background

Granulocyte-colony stimulating factor (G-CSF) may induce stem cell mobilization and stimulates myelopoiesis. Functionally, G-CSF upregulates myeloid cell survival and effector function (e.g. phagocytosis, Fc-mediated killing, etc.). G-CSF has been discussed to play a role in the protection of endothelial cells and anti-apoptosis via bcl-2 family protein, caspase 9 and cIAPs. We were therefore interested to test the effect of G-CSF on endothelial cell viability in the presence and absence of granulocytes (PMN) with and without activation by bacterial endotoxin (LPS).

Methods

Endothelial monolayers were established from bone marrow and layers were preincubated with rhG-CSF (filgrastim, 3000 U/ml) for 24 hours. PMN isolated from patients with septic shock or healthy donors were incubated with or without LPS (E. coli 0111:B4, 100 ng/ml); or were preactivated with LPS, then washed before being cocultured with the endothelial layer for another 24 hours. As a measure for cell viability, we quantified ATP contents by luciferin-based chemiluminescence. The inflammatory response by endothelial cells and/or PMN was studied by measuring IL-8, IL-6, IL-1β and TNF-α released into the culture supernatant.

Results

In G-CSF treated endothelial/PMN co-cultures as well as in G-CSF substituted cultures of PMN alone, the ATP content in cell lysates was upregulated. This G-CSF effect was independent from the addition of exogenous LPS. PMN as well as LPS stimulated the cytokine release by endothelial cells. We found IL-6 levels ranging between 1 and 1054 pg/ml being exclusively secreted by endothelial cells, and IL-8 levels originating from PMN and endothelial cells between 55 and 3278 pg/ml, respectively. G-CSF had no effect on IL-6 levels but downmodulated IL-8 in all endothelial/PMN co-cultures as well as PMN cultures alone. All IL-1β levels were close to detection levels and TNF-β levels were in the range of 5–20 pg/ml. This indicates that PMN preparations were not significantly contaminated by monocytes or macrophages. This was confirmed by morphological examination.

Conclusion

G-CSF increases the ATP content in both endothelial/PMN co-cultures and PMN cultures alone even if LPS or LPS-treated PMN are added, to mirror an inflammatory response. Moreover, G-CSF attenuates levels of the inflammatory cytokine IL-8, but does not modulate IL-6.