Host response to injury and infection is IntroductionSerum amyloid A (SAA) is a major acute-phase protein released to circulation in response to infection and injury. Within the first 24 to 36 hours after infection or injury, the blood concentration of SAA can increase by as much as 1000-fold over basal level, reaching a concentration of 80 M or 1 mg/mL. 1,2 The liver is a major source of acute-phase SAA, but extrahepatic expression of SAA has also been documented and is known to involve cells of atherosclerotic lesions, that is, smooth muscle cells, endothelial cells, and monocytes/macrophages. 3,4 Inflammatory cytokines such as interleukin 1␤ (IL-1␤), tumor necrosis factor ␣ (TNF-␣), and IL-6 are potent inducers of SAA expression by hepatocytes and, to various degrees, by macrophages and synoviocytes. 2,[5][6][7] In circulation, SAA is associated with high-density lipoproteins (HDLs) at lower concentrations, but it dissociates from HDLs at higher concentrations. 8,9 Free SAA is also found in the inflammatory sites, 3,4 suggesting a role of SAA in local inflammation.The marked increase of SAA has been used as an important indicator for diagnosis and prognosis of inflammatory diseases. 7,10 In addition, SAA is implicated as both a beneficial and harmful factor in the inflammatory process. Potential beneficial roles include reverse transport of cholesterol at sites of inflammation, through its ability to displace cholesterol from HDL. 11,12 With respect to being a harmful factor, SAA is the precursor of amyloid A, the deposit of which causes amyloidosis. 7,13 The findings that SAA is produced locally in atherosclerotic lesions and in arthritic joints suggest a potential role of this acute-phase protein in chronic inflammatory diseases such as atherosclerotic and rheumatoid arthritis. [13][14][15] Despite these important findings, a precise function of SAA in acute inflammation has not been defined. It is notable that a number of studies suggest a link between SAA and leukocyte infiltration. SAA is chemotactic to leukocytes including monocytes, mast cells, and T lymphocytes at concentrations attained in the blood during an acute-phase response. [16][17][18] These early observations have led to the recent identification of a cell surface receptor that mediates SAA-stimulated chemotaxis in monocytes. 19 There is also accumulating evidence suggesting that SAA possesses cytokinelike activities and is able to induce the production of matrix metalloproteinases (MMPs), 20 cytokines, and cytokine receptors including IL-1␤, interleukin-1 receptor antagonist (IL-1ra), and soluble TNF-␣ type II receptor (sTNFr-II). 21 Neutrophils that are stimulated by SAA for 24 hours release TNF-␣, IL-1␤, and IL-8 into culture medium. 22 However, it is not clear whether this is a primary response to SAA or a secondary response to other secreted cytokines because of the long incubation time. The receptor that mediates this function of SAA has not been identified.In this study, we investigated whether SAA induces primary cytokine responses i...

Although arsenic trioxide (As 2 O 3 ) induces apoptosis in a relatively wide spectrum of tumors, the sensitivity of different cell types to this treatment varies to a great extent. Because reactive oxygen species (ROS) are critically involved in As 2 O 3 -induced apoptosis, we attempted to explore the possibility that elevating the cellular ROS level might be an approach to facilitate As 2 O 3 -induced apoptosis. Emodin, a natural anthraquinone derivative, was selected because its semiquinone structure is likely to increase the generation of intracellular ROS. Its independent and synergistic effects with As 2 O 3 in cytotoxicity were studied, and the plausible signaling mechanism was investigated in HeLa cells. Cell Proliferation Assay and flow cytometry were used to assess cell viability and apoptosis. Electrophoretic mobility shift assay, luciferase reporter assay, and Western blotting were performed to analyze signaling alteration. The results demonstrated that coadministration of emodin, at low doses of 0.5-10 M, with As 2 O 3 enhanced As 2 O 3 -rendered cytotoxicity on tumor cells, whereas these treatments caused no detectable proproliferative or proapoptotic effects on nontumor cells. ROS generation was increased, and activation of nuclear factor B and activator protein 1 was suppressed by coadministration. All enhancements by emodin could be abolished by the antioxidant N-acetyl-L-cysteine. Therefore, we concluded that emodin sensitized HeLa cells to As 2 O 3 via generation of ROS and ROS-mediated inhibition on two major prosurvival transcription factors, nuclear factor B and activator protein 1. This result allows us to propose a novel strategy in chemotherapy that uses mild ROS generators to facilitate apoptosisinducing drugs whose efficacy depends on ROS.

Formyl peptide receptor-like 1 (FPRL1) is a G protein-coupled receptor that binds natural and synthetic peptides as well as lipoxin A 4 and mediates important biological functions. To facilitate its pharmacological characterization, we screened a compound library and identified a substituted quinazolinone (Quin-C1, 4-butoxy-N-[2-(4-methoxy-phenyl)-4-oxo-1,4-dihydro-2H-quinazolin-3-yl]-benzamide) as a ligand for FPRL1. Quin-C1 induces chemotaxis and secretion of ␤-glucuronidase in peripheral blood neutrophils with a potency of approximately 1/1000 of that of the peptide agonist WKYMVm. In studies using transfected rat basophilic leukemia (RBL) cell lines expressing either formyl peptide receptor or FPRL1, Quin-C1 induced enzyme release from RBL-FPRL1 but not RBL-FPR cells. Likewise, Quin-C1 selectively stimulates calcium mobilization in RBL-FPRL1 cells, a response that was markedly inhibited by pertussis toxin. Quin-C1 also stimulates phosphorylation of extracellular signal-regulated protein kinases 1 and 2 and induces internalization of an FPRL1 fused to green fluorescent protein. In degranulation assays, both the FPRL1-selective peptide agonist MMK1 and Quin-C1 exhibited lower efficacy and potency than WKYMVm, with EC 50 values of 7.17 ϫ 10 Ϫ8

The guanine nucleotide-binding regulatory protein α-subunit, Gα16, is primarily expressed in hemopoietic cells, and interacts with a large number of seven-membrane span receptors including chemoattractant receptors. We investigated the biological functions resulting from Gα16 coupling of chemoattractant receptors in a transfected cell model system. HeLa cells expressing a κB-driven luciferase reporter, Gα16, and the formyl peptide receptor responded to fMLP with a ∼7- to 10-fold increase in luciferase activity. This response was accompanied by phosphorylation of IκBα and elevation of nuclear κB-DNA binding activity, indicating activation of NF-κB. In contrast to Gα16, expression of Gαq, Gα13, and Gαi2 resulted in a marginal increase in κB luciferase activity. A GTPase-deficient, constitutively active Gα16 mutant (Q212L) could replace agonist stimulation for activation of NF-κB. Furthermore, expression of Gα16 (Q212L) markedly enhanced TNF-α-induced κB reporter activity. The Gα16-mediated NF-κB activation was paralleled by an increase in phospholipase C-β activity, and was blocked by pharmacological inhibitors of protein kinase C (PKC) and by buffering of intracellular Ca2+. The involvement of a conventional PKC isoform was confirmed by the finding that expression of PKCα enhanced the effect of Gα16, and a dominant negative PKCα partially blocked Gα16-mediated NF-κB activation. In addition to formyl peptide receptor, Gα16 also enhanced NF-κB activation by the C5a and C3a receptors, and by CXC chemokine receptor 2 and CCR8. These results suggest a potential role of Gα16 in transcriptional regulation downstream of chemoattractant receptors.