Kinetics of IL-6 gene expression and protein production after CD44 ligation on normal peripheral blood PMNs.
Freshly purified PMNs were isolated from a buffy coat from a normal donor and stimulated in complete medium. At the indicated times after stimulation, PMNs were harvested as source of total RNA, and the supernatants were collected for ELISA IL-6 protein assays. (A) This panel shows that, using sense and antisense specific primers, RT-PCR analyzed IL-6 and β-actin gene expression: lanes 1, 5, 9, 13, and 17, unstimulated PMNs; lanes 2, 6, 10, 14, and 18, NIH44.1 F(ab)2; lanes 3, 7, 11, 15, and 19, HA 100 μg/mL; and lanes 4, 8, 12, 16, and 20, LPS 12.5 ng/mL. (B) Left side shows IL-6 production measured in the supernatants collected at the indicated times: unstimulated PMNs (open squares), PMNs stimulated with NIH44.1 F(ab)2 (closed circles), HA 100 μg/mL (open circles), and LPS 12.5 ng/mL (closed triangles). Right side shows the IL-6 production measured in the supernatants of human PMNs stimulated overnight with immobilized or soluble HA at the indicated doses.
