Induction of WNT5A expression in human monocyte-derived macrophages, but not lymphocytes. Human cells were stimulated as indicated. Total RNA was isolated, reverse transcribed, and used for quantitative (A) or conventional RT-PCR (B,C). Representative results of 1 of at least 3 independent experiments are shown. (A) Dose response of Wnt5a expression in human macrophages after 4 hours of stimulation with M avium, M tuberculosis, synthetic lipopeptide (Pam₃CSK₄), or LPS, monitored by quantitative RT-PCR normalized to B2M mRNA levels; mean ± SD of duplicate determinations of 1 of 3 experiments are shown. (B) Kinetics of WNT5A and B2M mRNA expression in human macrophages induced by M avium (multiplicity of infection [MOI] 3) or LPS (10 ng/mL) analyzed by RT-PCR. Lane 1, 0 hours; lane 2, 2 hours; lane 3, 4 hours; lane 4, 6 hours; lane 5, 8 hours; lane 6, 10 hours; lane 7, 24 hours; lane 8, 48 hours; lane 9, 72 hours; RNA, RNA without reverse transcription; +, cDNA from human macrophages stimulated with M tuberculosis for 4 hours; and -, no cDNA. (C) Isolated human lymphocytes were left unstimulated (lane 1) or were stimulated with anti-CD3 (lane 2), anti-CD3/anti-CD28 (lane 3), 1 μg/mL (lane 4), or 5 μg/mL (lane 5) PHA or 10 ng/mL PMA/A23187 (lane 6). Cells were stimulated in duplicates as indicated for 4 hours; total RNA was isolated and reverse transcribed. Expression of WNT5A, IL-2, and beta-2-microglobulin (b2m) were analyzed by RT-PCR. One of 3 experiments is shown. +, cDNA from human macrophages stimulated with M tuberculosis for 4 hours (WNT5A); cDNA from PBMC stimulated with anti-CD3 for 4 hours (IL-2); -, no cDNA.