Fig. 2. Neutralization of the TNFR1 and TNFR2 receptors suppresses TNF-α-induced CCL4 expression. THP-1 monocytic cells were pre-treated with 2μg/mL of anti-TNFR1/2 neutralizing antibodies or isotype-matched IgG1 control antibody and incubated at 37°C for 40 min. Later, cells were stimulated with TNF-α (10ng/ml) and incubated for 24 hrs. Cells and culture media were collected and CCL4 mRNA in cell lysates and protein expression in cell supernatants were determined using real-time RT-PCR and ELISA, respectively. The data show that CCL4 (A) gene (TNFR1 Ab: P=0.0004; TNFR2 Ab: P=0.0005) and (B) secreted protein (TNFR1 Ab: P=0.0002; TNFR2 Ab: P=0.0002) expression was significantly reduced in monocytic cells that were pre-treated with either TNFR1 or TNFR2 specific neutralizing antibodies compared to isotype control antibody treated cells.