The fact that SAA is a chemoattractant for neutrophils20 and stimulates the secretion of IL-8,22 an important chemokine for neutrophils, suggests that SAA produced at the sites of inflammation may be involved in neutrophil infiltration, but whether it plays a role in the systemic increase in neutrophils has never been investigated. results in the activation of nuclear factorCB and binding activity to the CK-1 element of the G-CSF promoter region. In vitro reconstitution experiments also support that TLR2 mediates SAA-induced G-CSF expression. In addition, SAA-induced secretion of N-Desethyl Sunitinib G-CSF was IDH2 sensitive to heat and proteinase K treatment, yet insensitive to polymyxin B treatment, indicating that the induction is a direct effect of SAA. Finally, our in vivo studies confirmed that SAA treatment results in a significant increase in plasma G-CSF and neutrophilia, whereas these responses are ablated in G-CSFC or TLR2-deficient mice. Introduction Serum amyloid A (SAA) is one of the major acute-phase proteins. Its plasma concentration can increase 1000-fold, reaching as high as 80 M or 1 mg/mL during the acute-phase response.1,2 Many studies have shown that SAA plasma levels are also significantly elevated in patients with a broad spectrum of chronic inflammatory diseases, such as atherosclerosis,3,4 rheumatoid arthritis,5 Crohn disease,6 diabetes,7,8 and ankylosing spondylitis.9 However, the precise role of SAA in inflammation remains unclear. In humans, the acute-phase or inducible SAA is encoded by the and alleles.1 Bacterial products, such as lipopolysaccharide (LPS-del), and inflammatory cytokines, including interleukin-1 (IL-1), IL-6, and tumor necrosis factor- (TNF-), induce acute-phase SAA expression in hepatocytes as well as in tissue macrophages and synoviocytes.10C13 In circulation, the newly synthesized SAA is incorporated into high-density lipoprotein (HDL).14 Increased SAA can displace apolipoprotein ACI (ApoA-I) and change HDL composition. This change may be associated with a loss of HDL’s atheroprotective properties and fundamentally alter the function of HDL.15 At elevated concentrations, SAA may also dissociate from HDL, generating lipoprotein fractions that contain primarily lipid-poor ApoA-I and SAA.16,17 At sites of inflammation, SAA produced by inflammatory macrophages and synoviocytes is in the lipid-poor form.11,12 This form of SAA has numerous proinflammatory actions: it is chemotactic to neutrophils, monocytes, and T lymphocytes, causing leukocyte infiltration and promoting neutrophil adhesion to endothelial cells,18C20 and it stimulates neutrophils and monocytes to release cytokines,21,22 tissue factor,23 and matrix metalloproteinases.24 These findings suggest a key role for SAA in the establishment and maintenance of inflammation. There are 3 receptors involved in SAA’s proinflammatory effects: formyl peptide receptor likeC1 (FPRL1/ALX), which was shown to be responsible for SAA-induced chemotaxis, IL-8 secretion, and matrix metalloproteinaseC9 production20,22,24; receptor for advanced glycation end products, which was reported to mediate SAA-induced tissue factor expression23; and CLA-1 (CD36 and LIMPII analogousC1, a human ortholog of rodent scavenger receptor BI), which was found to facilitate SAA-triggered proinflammatory downstream signaling pathways, such as extracellular signal-regulated kinase (ERK) and p38 activation.25 In addition, there are several proteins and molecules to which SAA binds: Tanis, heparin, heparan sulfate, and certain glycoproteins, although whether these interactions lead to transmembrane signaling remains to be tested.26C28 The Toll-like receptors (TLRs) are key players of the innate immune system, functioning as pattern recognition receptors that recognize a wide range of microbial pathogens. In addition to microbial products, there are several endogenous TLR ligands that have been identified.29 For instance, high-mobility group box 1 is a ubiquitous, host-derived protein that interacts with multiple TLRs and plays a role in inflammation.30 The presence of endogenous TLR ligands supports the notion that TLRs play an important role in the detection of danger signals.31,32 The acute-phase proteins, such as SAA, could be danger-signaling molecules31 which, when recognized N-Desethyl Sunitinib by the host, may initiate tissue-controlled immune response.32 In this study, we explore the role of TLRs in inflammatory responses to SAA. Neutrophils are emerging as key players in the pathogenesis of several inflammatory diseases.33 They N-Desethyl Sunitinib are an essential component of the acute-phase response and a major contributor to inflammation. Granulocytosis or neutrophilia often results from infection and inflammation and is a feature of several autoimmune diseases, such as rheumatoid arthritis. One.