Proinflammatory cytokine response of macrophages derived from GM-CSF–stimulated LKS+ or Lin− progenitors. (A) C57BL/6 mice were IV injected with PBS or 1.5 μg per mouse of GM-CSF and 24 hours later Lin− or LKS+ cells were isolated from the bone marrow and differentiated into macrophages with M-CSF (50 ng/mL) for 6 or 8 days, respectively; TNF-α and IL-6 production was measured by enzyme-linked immunosorbent assay (ELISA) in supernatants upon Pam3CSK4 (Pam3) or LPS stimulation (B) and by intracellular flow cytometry upon LPS stimulation (C). (D) Lin− or LKS+ cells isolated from the bone marrow of mice were cultured in vitro with M-CSF (50 ng/mL) + SCF (20 ng/mL) and stimulated with or without GM-CSF (50 ng/mL). After 24 hours, cells were washed and differentiated into macrophages with M-CSF for 6 or 8 days, respectively; TNF-α and IL-6 production was measured by ELISA in supernatants upon Pam3 or LPS stimulation (E) and by intracellular flow cytometry upon LPS stimulation (F). For the ELISA measurements triplicate samples were analyzed and expressed as means + standard deviation (SD) and for intracellular cytokine measurements mean fluorescence intensity (MFI) is indicated in the histograms; data presented are 1 representative experiment of at least 3 independent experiments. (G) Total bone marrow or LKS+ cells were isolated from 24 hours PBS- or GM-CSF–treated DsRed mice, transplanted into irradiated C57BL/6 recipients, and analyzed 16 weeks after their hematopoietic reconstitution. (H) Bone marrow from 16-week reconstituted mice was obtained, stimulated with LPS for 4 hours and with brefeldin A for the final 3 hours, and TNF-α production was measured in DsRed+ Ly6Chi monocytes (Ly6G− CD11b+ CD115+ Ly6Chi) by intracellular flow cytometry (3 representative mice are shown). (I) Bar graphs for MFIs and box plots for cell percentages are expressed as means + SD (n = 5-6 mice per condition). Statistical significance was assessed by the Student t test (∗P < .05; ∗∗P < .01; ∗∗∗P < .001). mφ, macrophage; unstim., unstimulated.

Proinflammatory cytokine response of macrophages derived from GM-CSF–stimulated LKS+ or Lin− progenitors. (A) C57BL/6 mice were IV injected with PBS or 1.5 μg per mouse of GM-CSF and 24 hours later Lin− or LKS+ cells were isolated from the bone marrow and differentiated into macrophages with M-CSF (50 ng/mL) for 6 or 8 days, respectively; TNF-α and IL-6 production was measured by enzyme-linked immunosorbent assay (ELISA) in supernatants upon Pam3CSK4 (Pam3) or LPS stimulation (B) and by intracellular flow cytometry upon LPS stimulation (C). (D) Lin− or LKS+ cells isolated from the bone marrow of mice were cultured in vitro with M-CSF (50 ng/mL) + SCF (20 ng/mL) and stimulated with or without GM-CSF (50 ng/mL). After 24 hours, cells were washed and differentiated into macrophages with M-CSF for 6 or 8 days, respectively; TNF-α and IL-6 production was measured by ELISA in supernatants upon Pam3 or LPS stimulation (E) and by intracellular flow cytometry upon LPS stimulation (F). For the ELISA measurements triplicate samples were analyzed and expressed as means + standard deviation (SD) and for intracellular cytokine measurements mean fluorescence intensity (MFI) is indicated in the histograms; data presented are 1 representative experiment of at least 3 independent experiments. (G) Total bone marrow or LKS+ cells were isolated from 24 hours PBS- or GM-CSF–treated DsRed mice, transplanted into irradiated C57BL/6 recipients, and analyzed 16 weeks after their hematopoietic reconstitution. (H) Bone marrow from 16-week reconstituted mice was obtained, stimulated with LPS for 4 hours and with brefeldin A for the final 3 hours, and TNF-α production was measured in DsRed+ Ly6Chi monocytes (Ly6G− CD11b+ CD115+ Ly6Chi) by intracellular flow cytometry (3 representative mice are shown). (I) Bar graphs for MFIs and box plots for cell percentages are expressed as means + SD (n = 5-6 mice per condition). Statistical significance was assessed by the Student t test (∗P < .05; ∗∗P < .01; ∗∗∗P < .001). mφ, macrophage; unstim., unstimulated.

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