Figure 1.
Microarray analysis of neutrophils subsets during human experimental endotoxemia.
(A) Flow cytometry dot plot of neutrophils prior to, and 4 hours after in vivo LPS administration. The fluorescence signal for CD16 is displayed on the x-axis and the fluorescence signal for CD62L is displayed on the y-axis. At t = 0 hours (upper panel) a large population of CD16bright/CD62Lbright neutrophils, a small population of CD16high/CD62Ldim neutrophils and a population of CD16negative cells representing eosinophils is present. At t = 4 hours after LPS (lower panel), neutrophil with CD16dim/CD62Lbright and CD16dim/CD62Ldim subsets appeared and these were FACS sorted for microarray analysis. (B) Absolute cell numbers of different neutrophil subsets in the blood at 0 and 4 hours after LPS challenge (n = 6). Data are expressed as means ± SEM. (C) Overrepresented functional categories in CD16high/CD62Ldim neutrophils based on the total list of differentially expressed genes relative to prior to LPS. A minimum of 5 genes and a p value of 0,01 were taken as cutoff. All significantly overrepresented categories are shown. The 4 highest parent levels of the Gene ontology tree were excluded for this graph since various general processes are involved in these. (D) Network of several interferon-induced genes that are upregulated in CD16high/CD62Ldim neutrophils after intravenous administration of LPS. The color intensity of the nodes indicates the level of upregulation. (E) Expression of CD274 on isolated neutrophil subsets from volunteers intravenous administered LPS at 4 and 6 hours after LPS. *P<0.05. Data are expressed as means ± SEM (n = 4).
Figure 2.
PD-L1 expression on IFN-γ treated neutrophils.
(A) Neutrophils were stimulated 18–20 hours with different cytokines and growth factors and CD274 mean fluorescence intensity (MFI) was measured. (B) Neutrophil survival after 18–20 hours stimulation with different cytokines and growth factors shown on the x-axis and the percentage of cells that were positive for either Annexin-V, 7-AAD or both on the y-axis. (C) Freshly isolated neutrophils were stimulated 18–20 hours with IFN-γ or GM-CSF and CD274 mean fluorescence intensity (MFI) was measured on annexin-V negative and annexin-V positive neutrophils (D) Neutrophils were stimulated 18–20 hours with different concentrations of IFN-γ and CD274 mean fluorescence intensity (MFI) was measured. (E) Neutrophils were stimulated with 100 ng/ml IFN-γ and incubated for different periods before washing and further incubation till 18–20 hours and CD274 mean fluorescence intensity (MFI) was measured. (F) Gene expression of CD274 in IFN-γ stimulated neutrophils. Expression is shown in time with the use of GAPDH as reference gene. Surface expression of (G) CD274, (H) CD273 and (I) CD279 after 0, 2, 4, 6, 8 and 20 hours stimulation with IFN-γ. *P<0.05, **P<0.01, ***P<0.001. Data are expressed as means ± SEM (n = 4).
Figure 3.
IFN-γ stimulated neutrophils suppress T-cell proliferation.
(A) Neutrophils stimulated 18–20 hours with either GM-CSF or IFN-γ or left untreated and inhibition of PHA-induced PBMC proliferation was measured after 3 days. (B) Neutrophils stimulated 18–20 hours with IFN-α, IFN-β, IFN-γ or left untreated and inhibition of PHA-induced PBMC proliferation was measured after 3 days. (C) Neutrophils stimulated 18–20 hours with IFN-γ or left untreated and inhibition of CD3/CD28-induced PBMC proliferation was measured after 3 days. (D) Neutrophils stimulated 18–20 hours with IFN-γ or left untreated and inhibition of Candida albicans-induced proliferation was measured after 7 days. (E) CD16positive CD14negative CD3negative sorted neutrophils were stimulated 18–20 hours with IFN-γ or left untreated and inhibition of PHA-induced CD3positive CD14negative CD16negative sorted lymphocyte proliferation was measured after 3 days. (F) Percentage of CD4 and CD8 lymphocytes after 3 days PHA-induced PBMC proliferation in the presence of neutrophils stimulated 18–20 hours with IFN-γ or left untreated.
Figure 4.
Suppression of T-cell proliferation by IFN-γ stimulated neutrophils is dependent on cell-cell contact and PD-L1.
(A) Neutrophils stimulated with IFN-γ or left untreated for 18–20 hours and PHA-stimulated PBMCs were co-cultured for 3 days in separate compartments by the use of cell culture inserts. Percentage of inhibition of PHA-induced proliferation was calculated. (B) Neutrophils stimulated 18–20 hours with IFN-γ or left untreated and inhibition of PHA-induced PBMC proliferation in the presence of αCD11b, αCD274 or αPAFr was measured after 3 days. (C) Neutrophils stimulated 18–20 hours with either GM-CSF or IFN-γ or left untreated and interactions between Calcein-blue labeled neutrophils with Calcein-AM labeled PBMCs after 120 minutes of co-culture measured by flow cytometer. Ratios indicate neutrophils: lymphocytes (Figure A–E). *P<0.05, **P<0.01, ***P<0.001. Data are expressed as means ± SEM (n = 4).