Pharmacological modulation of proinflammatory mediator production by macrophages

The free radical gas nitric oxide is generated in biological systems by a family of oxidoreductases which utilize arginine as the substrate. Elevated levels of nitric oxide have been observed in several inflammatory disorders. Using a novel polarographic self-referencing microsensor, nitric oxide fluxes released from the surface of activated macrophages were measured. With this system, nitric oxide fluxes could be detected at distances from the cells of ∼100–500 μm. In related studies, we found that formamidine, a compound that is structurally similar to arginine, readily inhibited nitric oxide production. Formamidine had no effect on expression of inducible nitric oxide synthase, but did inhibit the activity of the enzyme in a simple competitive manner. Using the nitric oxide microsensor, we found that formamidine required 2–4 hours to inhibit enzyme activity. This may be due to slow uptake of the drug. In the present studies we also observed that heat shock protein 60 (HSP60) acts an effective inducer of NOS2 and cyclooxygenase-2 (COX-2) in macrophages, as well as endothelial cells. In both cell types, HSP60-induced synthesis of COX-2 was coordinate with induction of NOS2. Using promoter constructs in transient transfection assays, optimal expression of COX-2 in macrophages was found to require NFκB, the cAMP response element (CRE), and nuclear factor (NF)-IL-6, but not the E box. Mobility shift assays revealed that HSP60 induced NFκB and CRE binding activity, while C/EBP, which binds to NF-IL-6, was constitutively active in the cells. Both c-JUN and CREB bound to the CRE, while C/EBPβ bound to NF-IL-6. These data indicate that NFκB, C/EBPβ, c-JUN and CREB are important in HSP 60-induced expression of COX-2. The c-JUN-N-terminal kinase (JNK), p44/42 MAP kinase (ERK1/2) and p38 MAP kinase were rapidly activated by HSP60 in the macrophages. PD98059, an inhibitor of phosphorylation of ERK1/2, caused a marked inhibition of HSP60-induced COX-2 and NOS2 expression. SB203580, a selective inhibitor of p38 kinase, inhibited HSP60-induced expression of COX-2, but not NOS2. These data indicate that MAP kinase signaling plays distinct roles in regulating HSP60-induced expression of COX-2 and NOS2.