Figure 5.

ERK is involved in production and activity of soluble survival factor(s). (a) Plated or unplated N99 PMNs were stimulated with 100 ng/ml lipid A for the indicated times (5 min through 60 min); or with fMLF (300 nM, 5 min) as positive control for plated N99. Cell lysates were analyzed by immunoblot using Abs specific for phosphorylated or total ERK1/2 and p38 MAPK. Representative blots are shown; see Supplemental Figure S3 for quantitation of all blots. (b and c) Plated N90 PMNs were incubated with 20 μM ERK inhibitor or diluted vehicle (DMSO) for 30 min before they were cultured with 100 ng/ml lipid A for 24 h. GM-CSF was used as positive control. (d) N99 PMNs pre-treated with ERK inhibitor or vehicle for 30 min were cultured with supernatants from lipid A-stimulated (Supnt Lipid A 100) or untreated (Supnt UT) N90 PMNs for 24 h under adherent culture conditions. (e) N99 PMNs were cultured with supernatants collected from ERK inhibitor or vehicle pre-treated N90 PMNs that were subsequently stimulated with 100 ng/ml of lipid A. Cell viability was analyzed by Annexin V-APC/7-AAD staining and flow cytometry. Each symbol represents one donor; means are shown as black (b) or red (d and e) bars. ****P ≤ 0.0001, ***P ≤ 0.001, *P ≤ 0.05, and ns P > 0.05 when compared with the corresponding vehicle group. (c) Bars show mean percentage of viability ± SEM from five individuals; ****P ≤ 0.0001 when compared with untreated neutrophils. Each donor (n) represents an independent experiment.