Figure 3.

M1 macrophages activated with IFN‐γ + LPS show the commonly accepted marker profile for these cells. Histamine up‐regulates OSM mRNA expression in a dose‐dependent manner in fully differentiated M1 macrophages. Primary human monocytes were obtained from peripheral blood mononuclear cells after 2‐hr adherence. M1 macrophages were differentiated from primary human monocytes in the presence of GM‐CSF (10 ng·ml⁻¹) for 10 days. One part was activated with IFN‐γ (200 ng·ml⁻¹) and LPS (50 ng·ml⁻¹) for additional 24 hr. (a) The M1 marker profile expression of CD68 and CD80 and expression of the M2 marker CD163 were determined by flow cytometry in fully differentiated non‐stimulated and IFN‐γ + LPS‐activated cells to prove that the cells are M1 and not M2 polarized (n = 6 independent donors and experiments). Mean fluorescence intensities were calculated. One representative histogram out of six for (b) CD68, (c) CD80, and (d) CD163 is shown for fully differentiated non‐stimulated M1 macrophages, IFN‐γ + LPS‐activated M1 macrophages and the isotype control. (e) OSM mRNA expression in fully differentiated M1 macrophages after treatment with different concentrations of histamine as indicated for 2 hr is depicted (n = 9 independent donors and experiments). (f) OSM mRNA expression in fully differentiated M1 macrophages after treatment with different concentrations of histamine as indicated for 2 hr and activated with IFN‐γ (200 ng·ml⁻¹) and LPS (50 ng·ml⁻¹) for additional 24 hr is depicted (n = 10 independent donors and experiments). The amount of the target mRNA relative to the amount of the reference gene, rps 20 mRNA in each stimulated sample was normalized to the amount of the target mRNA relative to the amount of the reference gene in the non‐stimulated sample (calibrator) and expressed as normalized ratio. This was calculated using the comparative Ct method also known as the ΔΔCt method provided by the Software LC 480 (Roche Molecular Biochemicals). Data shown are individual values with medians. In ( e) *P < .05; significantly different as indicated; Friedman Dunn's Multiple Comparisons test selected pairs. In (a) and (f), *P < .05; significantly different as indicated; Wilcoxon matched‐pairs signed rank test. Hist, histamine; NS, non‐stimulated; OSM, oncostatin M