Activation of H-mDCs by TREM-1 cross-linking. (A) mDCs were generated under hypoxic conditions from 2 different donors, and total cell lysates were subjected to immunoprecipitation with anti-TREM-1 mAb or control IgG1. The precipitates were resolved on a 12% SDS-PAGE and immunoblotted with anti-DAP12 mAb, as described in “Western blot analysis and immunoprecipitation.” DAP12 and Ig light chain (IgL) are indicated by arrows. Molecular weight markers are given at the side. (B-C) H-mDCs were seeded onto plates precoated with agonist anti-TREM-1 mAb or control IgG1 at 10 μg/mL, after extensive washing to remove cytokines, and cultured for 20 minutes (B) or 24 hours (C) under hypoxic conditions. (B) Protein phosphorylation. Cell lysates (40 μg) were resolved on 10% SDS-PAGE and immunoblotted with Abs antiphospho (p)-ERK, Akt, and IκBα. Abs against the nonphosphorylated forms (tot) were used as loading controls. Representative experiments performed with cells from 2 different donors are shown. T0 indicates H-mDCs from one of the donors not subjected to Ab cross-linking, used as a negative control of phosphorylation. The figure was arranged by combining the lanes containing not-crosslinked samples (T0) with those containing samples subjected to cross-linking from different parts of the same gel. (C) Cytokine production. CM was assayed for TNF-α, IL-6, IL-12p70, IL-10, CXCL8, CCL4, and CCL5 content by specific ELISA. Results are expressed as picograms or nanograms/8 × 105 cells/mL and are represented as the mean ± SE of 3 different experiments. Values significantly different from those of H-mDCs cross-linked with IgG1: * P < .05; **P < .001; ***P < .0001.
