IL-6 gene expression on peripheral blood PMNs on in vitro stimulation with HA.

(A) Normal freshly isolated PMNs were stained without (dash line) or with FITC-conjugated HA (solid line). (B) PMNs were stained with a PE-conjugated anti-CD44 in the absence (thick, solid, line) or in the presence of 25 μg (dotted line) and 50 μg (thin, solid line) unlabeled HA. The dashed line represents the red fluorescence of PMNs stained with a PE-conjugated, matching, isotype mAb. (C) PMNs were isolated from a buffy coat from a normal donor and stimulated for 18 hours in the absence or presence of logarithmic dose concentrations of soluble HA: 0 μg/mL (lane 1), 1 μg/mL (lane 2), 10 μg/mL (lane 3), and 100 μg/mL (lane 4), as well as linear dose concentrations of immobilized HA: 0 μg/mL (lane 5), 250 μg/mL (lane 6), and 1000 μg/mL (lane 7). Using specific sense and antisense primers, RT-PCR analyzed IL-6 or β-actin gene expression. The figure shows 1 of 3 significant experiments performed with similar results. FL1-H and FL2-H indicate the logarithmic numbers of green and red fluorescence, respectively.