Expression of BAFF and APRIL in myeloid lineage cells. (A) Myeloid lineage cells express comparable levels of BAFF and APRIL mRNAs. First-strand cDNA was synthesized from RNase-free DNase-digested total RNA using oligo(dT) primers and Superscript II reverse transcriptase. BAFF, APRIL, and GAPDH were amplified by PCR using gene-specific primers. PCR products were resolved on NuSieve 3:1 agarose. DNA molecular size markers are indicated in the far left lanes. (B-C) Macrophages constitutively release BAFF; cytokines and bacterial cell wall components induce BAFF release from macrophages. Anti-human BAFF mAb-coated ELISA plates were incubated with PBS/BSA for 2 hours at room temperature. After blocking, culture supernatants or soluble BAFF as a standard was incubated overnight at 4°C with anti-BAFF—coated plates. Samples were incubated for 2 hours at room temperature with rabbit anti-human BAFF polyclonal serum and subsequently with goat anti-rabbit HRP for 1 hour at room temperature. HRP enzyme activity was quantified using TMB as a substrate at 450 nm. All values are the mean ± SD of triplicate samples. (D-E) Macrophages but not mono-DCs or monocytes constitutively express high relative levels of APRIL protein. APRIL is induced by LPS and interferon-γ in mono-DCs. Cell lysates (50 μg total protein) from B cells, macrophages, mono-DCs, and monocytes were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with APRIL antiserum (upper panel) or reprobed with p38 MAPK antiserum (lower panel) as a control for protein loading.