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INFLAMMATION AND IMMUNOPHARMACOLOGY

The Phase 2 Enzyme Inducers Ethacrynic Acid, DL-Sulforaphane, and Oltipraz Inhibit Lipopolysaccharide-Induced High-Mobility Group Box 1 Secretion by RAW 264.7 Cells

Departments of Critical Care Medicine (J.A.E., M.E.K., M.S., Y.H., R.L.D., J.A.K., M.P.F.), Cell Biology and Physiology (D.B.S.), Pathology (R.L.D.), Medicine (J.A.K.), and Surgery (M.P.F.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

The diuretic ethacrynic acid (EA) has been shown to inhibit signaling by the proinflammatory transcription factor nuclear factor-B (NF-B). Accordingly, we sought to determine whether this compound is capable of inhibiting the release of cytokines [interleukin (IL)-6 and IL-10] and NO from RAW 264.7 murine macrophage-like cells stimulated with lipopolysaccharide (LPS). Additionally, we sought to determine whether EA can inhibit secretion of high-mobility group box 1 (HMGB1), a nuclear protein that is secreted by immunostimulated macrophages and functions in the extracellular milieu as a proinflammatory mediator. In a concentration-dependent manner, EA inhibited secretion of IL-6, IL-10, nitric oxide, and HMGB1. As expected, EA inhibited NF-B DNA binding in LPS-stimulated RAW 264.7 cells. Treating these cells with pyrrolidine dithiocarbamate, SN50 (amino acid sequence AAVALLPAVLLALLAPVQRKRQKLMP) or 5-(thien-3-yl)-3-aminothiophene-2-carboxamide (SC-514) also inhibited LPS-induced NF-B DNA binding, but these compounds failed to inhibit LPS-induced HMGB1 secretion. These findings suggested that inhibition of HMGB1 secretion by EA might occur via a mechanism unrelated to the NF-B signaling pathway. Because EA is an electrophilic compound that is known to be capable of inducing expression of so-called phase 2 proteins, we sought to determine whether two other phase 2 enzyme inducers, oltipraz and DL-sulforaphane, also are capable of inhibiting HMGB1 release from immunostimulated macrophages. Incubating RAW 264.7 cells with either oltipraz or DL-sulforaphane inhibited LPS-induced HMGB1 secretion. Moreover, both EA and DL-sulforaphane inhibited relocalization of nuclear HMGB1 into the cytoplasm of LPS-stimulated RAW 264.7 cells. These data suggest that phase 2 inducers may exert anti-inflammatory effects by inhibiting secretion of the cytokine-like nuclear protein HMGB1.

Address correspondence to: Dr. Mitchell P. Fink, Department of Critical Care Medicine, 615 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. E-mail: finkmp{at}ccm.upmc.edu

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