The measured decrease in TNFα production is not due to alterations in mRNA production but to retention of TNFα within monocytes. (A) Cytokine mRNA levels were assessed by TaqMan RT-PCR. The results are expressed as the relative change in mRNA compared with a calibrator. (B,C) Intracellular cytokine staining by FACS revealed that TNFα, but not IL-6, was being retained within monocytes after culture in the presence of LPS and anti-MyD-1. The results are expressed as the percentage TNFα-positive CD14⁺ cells, and these data are representative of 3 experiments. (D) Cell lysates were prepared from human PBMCs stimulated with either LPS (100 ng/mL) plus anti-MyD-1 (10 μg/mL) or LPS plus IgG1 (10 μg/mL) for 24 hours at 37°C. The cell lysates were then subjected to Western blot analysis of TACE expression as described in “Materials and methods.” These lysates were prepared from a single donor and were representative of those from 2 other independent donors.