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. Author manuscript; available in PMC: 2009 Jan 30.
Published in final edited form as: Vaccine. 2007 Dec 26;26(5):601–613. doi: 10.1016/j.vaccine.2007.11.084

Figure 1. The activation-induced upregulation of 1α-hydroxylase in murine dendritic cells alters their trafficking properties in vivo and their chemotaxis in vitro.

Figure 1

(A) CD11c+ BMDCs were stimulated with LPS (10ng/ml) for 24 hours at 37°C or left untreated. Cell lysates were then prepared and analyzed for the presence of 1α-hydroxylase by Western Blot. Proteins were visualized by ECL Plus Western Blotting Detection System. The blot was later stripped and reprobed with antibodies against β-actin to ensure an equal loading of protein. Results are representative of five experiments. (B) CD11c+ BMDCs were matured in vitro for 24 hours in the presence of LPS (10ng/ml) with or without 1α25(OH)2D3 (VD3, 10−8 M) or 25(OH)D3 (10−7 M). The BMDCs were then washed and evaluated for chemotactic responses toward CCL21 (100ng/ml) and S1P (1000nM) using Transwells (5μm pores). After a 3-hour incubation at 37°C, cells that migrated to the bottom chamber were collected and counted. Results are presented as mean of triplicates ± SD. *- difference between numbers of LPS matured DCs in the presence of LPS in the presence of 25(OH)D3 or 1α25(OH)2D3 that migrated into the bottom chamber and numbers of LPS only treated BMDCs that migrated to the bottom chamber was statistically significant (p<0.02-0.009). This experiment was repeated twice with similar results. (C) CD11c+ BMDCs were treated with 10ng/ml of LPS in the presence or absence of 10 8M 1α25(OH)2D3 or 10−7M 25(OH)D3. After a 24-hour incubation at 37°C, the BMDCs were stained with CFSE and subcutaneously injected into naive recipients (three mice per group). Forty-eight hours later, mice were sacrificed and single cell suspensions from individual lymphoid organs (popliteal (PLN), axillary (ALN) lymph nodes, spleens (SPL) and Peyer’s patches (PP)) were prepared and analyzed by FACScan. A total of 400,000 events were collected. Results are presented as mean ± SD. * - difference between numbers of CFSE+ DCs detected in various secondary lymphoid organs of C3H/HeN mice that received a subcutaneous injection of BMDCs exposed to the influences of LPS in the presence of 25(OH)D3 or 1α25(OH)2D3 and numbers of CFSE+ DCs detected in various secondary lymphoid organs of mice that received a subcutaneous injection of BMDCs treated LPS alone was statistically different (p<0.05-0.01).