In vitro and in vivo DC migration assay. (A) In vitro chemoattraction assay of CsA- and LPS-treated murine DCs in response to MIP-1α (upper panel) or MIP-3β (lower panel). (B) Reverse transmigration assay of CsA-treated murine DCs. No chemokines were added to the lower chamber. In panels A-B, if not specifically indicated, the CsA concentration used was 1 μg/mL. (C-D) CsA treatment impairs PKH-26–labeled murine DC migration into spleen and lymph nodes (LN). (C) Immunofluorescence microscopy of PKH-26–positive cells in spleen and LNs. Results here are representative of sections derived from at least 3 samples. Red cells are PKH-26 labeled DCs, and blue cells are stained with Hoechst. Original magnification, × 400. (D) FACS enumeration of PKH-26–positive cells in spleen and LNs. ND indicates not detected. (E) FACS analysis of DCs^GFP+/+ (R2 region) in spleen and LNs following injection into skin transplants. Labels indicate percentage of GFP⁺ cells in cell suspensions. (F) CsA does not affect numbers of FITC⁺CD11c⁺ DCs in skin but decreases the frequency of these cells in spleen and LNs. Mice treated with CsA (20 mg/kg) for 7 days were painted with FITC and then intravenously injected with LPS. Twelve hours later, FITC⁺CD11c⁺ cells in skin, spleen, and LN cell suspensions were counted using FACS. Results are expressed as means ± SD (A-B, D). Results in panel F represent 3 independent experiments. Asterisks in panel F indicate P < .05.