Expression of 25(OH)D₃-1α-hydroxylase and production of 1,25(OH)₂D₃ by different types of immature and mature DCs. (A) RNA of monocyte-derived DCs (GM/IL-4, GM/IFNα) and MACs (AB, GM) cultured for 5 days was used for Northern blot analysis. Terminal differentiation of DCs was induced by LPS (DC/LPS). The expression of 25(OH)D₃-1α-hydroxylase protein in DCs matured by stimulation with LPS (DC/LPS) was determined by immunohistochemistry. As negative control, staining without the primary antibody is shown (B, left panel). (C) 1,25(OH)₂D₃ synthesis was measured by ELISA. Freshly isolated monocytes or monocytes differentiated along the DC pathway for 3 or 6 days were harvested and cultured for another 24 hours under serum-free conditions with 25(OH)D₃ in the absence or presence of 100 ng/mL LPS. The values represent mean ± SEM of at least 3 experiments. (D) The maturation of DCs was induced on day 4 of culture for 72 hours with tumor necrosis factor (DC/TNF-α) or LPS (DC/LPS) or a mixture containing TNF-α, IL-1β, IL-6, and prostaglandin E₂ (DC/mix). The values represent mean ± SEM of 4 experiments. (E) Blood DCs were isolated from mononuclear cells,¹⁵  and the expression of 25(OH)D₃-1α-hydroxylase was investigated by immunohistochemistry. Original magnification of panels B and E, × 400. In B and E, left panels show negative control (without primary antibody) and right panels show the positive sample (with primary antibody).