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Figure 3 CRP Induction of NF- B Activation in Circulating Monocytes as Assessed by EMSA (Protocol B)
In 8 healthy volunteers, aliquots of 5 x 105 cells were incubated at 37°C in PBS and stimulated with increasing doses of recombinant human (rh) CRP (5 to 10 to 25 µg/ml) for 30 min to assess the effects on NF-B activation. A cell aliquot was incubated with the NF-B inhibitor pyrrolidine dithiocarbamate (PDTC) (50 mmol/l) for 1 h before CRP stimulation (25 µg/ml), and another cell aliquot was stimulated with CRP (25 µg/ml) denatured by incubation in boiling water bath for 1 h (25den). As a positive control (Pos) nuclear extracts were generated from 5 x 105 monocytes incubated for 30 min with 10 ng/ml LPS, and as a negative control (Neg) nuclear extracts were generated from 5 x 105 monocytes incubated for 30 min with PBS only. As shown in this representative EMSA, NF-B activation in circulating monocytes was induced by stimulation with increasing doses of rhCRP and inhibited by preincubation with PDTC (50 mmol/l) and by the use of denatured rhCRP (A); coincubation of both rhCRP and LPS had a greater-than-additive effect (B). Gel supershift experiments with monoclonal antibodies to the p50 subunit indicated that complexes consisted of heterodimers containing the p50 subunit. Other abbreviations as in Figures 1 and 2.
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