TLR3 is expressed on all cell types used in this study. Human DCs were generated from peripheral blood monocytes after 5- to 7-day culture in the presence of 10 ng/mL IL-4 and 50 ng/mL GM-CSF. MØs were differentiated from peripheral blood monocytes for 3 to 4 days in the presence of 100 ng/mL M-CSF. ECs were isolated from human umbilical veins and were used at passages 2 to 3. Fibroblasts were isolated from synovial tissue from RA patients undergoing joint replacement. (A) Total RNA was isolated from each cell type and 1 μg was subjected to reverse transcriptase-PCR and analyzed for TLR3, TLR4, MD-2, or GAPDH expression with PCR. − indicates PCR-negative control. (B) DCs, MØs, ECs, and RA-SFs were analyzed for both intracellular and membrane-bound TLR3 by flow cytometry. Cells stained with isotype control (filled histograms) or anti–TLR3-FITC antibody (open black histograms) are shown.