Figure 4

GCSF modulates inflammatory responses in vitro and ex vivo. Cells were treated in vitro with 10 ng/ml LPS for 24 h and TNFα release was quantified with ELISA. GCSF decreased the production of TNFα in primary microglia (A, p <0.001, n = 3). GCSF also decreased the TNFα production in BM monocytes obtained from wt and mutant SOD1 mice (B, p <0.001, n = 6-7). TNFα production was also decreased in BM Ly6C monocytes isolated from 20 week-old, long-term pegfilgrastim-treated mutant SOD1 mice, as compared to their vehicle-treated littermates (C, p <0.001, n = 3): TNFα production was analyzed with intracellular cytokine staining with flow cytometry after 4 h incubation of 1 μg/ml LPS ex vivo. NO production was detected with determination of NO metabolites NO₂ and NO₃ from the medium after 24 h of incubation with 10 ng/ml LPS. NO production was increased after LPS stimulation in wt (D, p <0.001, n = 4-8) and mutant SOD1 BM monocytes in vitro (D, p <0.05, n = 7-8). GCSF further increased the NO production in mutant SOD1 monocytes up to the level of wt monocytes (D, p <0.05, n = 7-8). NO production was also increased in BM (E, p <0.001, n = 3) and spleen (F, p <0.001, n = 3) leukocytes obtained from long-term pegfilgrastim-treated mutant SOD1 mice, as compared to their vehicle-treated littermates: NO production was analyzed after 24 h of incubation with 100 ng/ml LPS.