Anti-SAA1 monoclonal antibody, clone C333A

Serum amyloid A (SAA) is a major acute-phase protein and a precursor of amyloid A, the deposit of which leads to amyloidosis. Different alleles exist in SAA1, a predominant form of the human SAA gene family. Emerging evidence has shown correlations between these alleles and diseases including familiar Mediterranean fever and amyloidosis. However, it remains unclear how the proteins encoded by these SAA1 alleles act differently. Here we report the characterization of proteins encoded by SAA1.1, SAA1.3, and SAA1.5, in comparison to that encoded by SAA2.2, for their preference of the SAA receptors including formyl peptide receptor 2 (FPR2) and Toll-like receptor 2 (TLR2). SAA1.1 was more efficacious than SAA1.3 and SAA1.5 but equally efficacious to SAA2.2 in calcium mobilization and chemotaxis assays, which measure the activation of the G protein-coupled FPR2. In agreement with this, SAA1.1 and SAA2.2 induced more robust phosphorylation of ERK than SAA1.3 and SAA1.5. Only small differences were observed between the SAA1 proteins tested and SAA2.2 in TLR2-dependent NF-kappa B luciferase reporter assay. In comparison, SAA1.3 was most effective in stimulating ERK and p38 MAPK phosphorylation. Using bone marrow-derived macrophages from C57BL/10ScN (Tlr4lps-del) mice, we examined the SAA isoforms for their induction of selected pro- and anti-inflammatory cytokines. SAA1.3 was most potent in the induction of TNE alpha and IL-1rn, whereas SAA1.5 induced robust IL-10 expression. These results show differences of the SAA1 isoforms in their selectivity for the SAA receptors, which may affect their roles in modulating inflammation and immunity. (C) 2014 Elsevier GmbH. All rights reserved.

Background: Acute-phase response is a systemic reaction to environmental/inflammatory insults and involves production of acute-phase proteins, including serum amyloid A (SAA). Interleukin-1 beta (IL-1 beta), a master regulator of neuroinflammation produced by activated inflammatory cells of the myeloid lineage, in particular microglia, plays a key role in the pathogenesis of acute and chronic diseases of the peripheral nervous system and CNS. IL-1 beta release is promoted by ATP acting at the purinergic P2X(7) receptor (P2X(7)R) in cells primed with toll-like receptor (TLR) ligands. Methods: Purified (> 99%) microglia cultured from neonatal rat cortex and cerebellum were first primed with the putative TLR4/TLR2 agonist SAA (recombinant human Apo-SAA) or the established TLR4 agonist lipopolysaccharide (LPS) followed by addition of ATP. Expression of genes for the NLRP3 inflammasome, IL-1 beta, tumor necrosis factor-a (TNF-alpha), and SAA1 was measured by quantitative real-time polymerase chain reaction (q-PCR). Intracellular and extracellular amounts of IL-1 beta were determined by ELISA. Results: Apo-SAA stimulated, in a time-dependent manner, the expression of NLRP3, IL-1 beta, and TNF-alpha in cortical microglia, and produced a concentration-dependent increase in the intracellular content of IL-1 beta in these cells. A 2-h 'priming' of the microglia with Apo-SAA followed by addition of ATP for 1 h, resulting in a robust release of IL-1 beta into the culture medium, with a concomitant reduction in its intracellular content. The selective P2X(7)R antagonist A740003 blocked ATP-dependent release of IL-1 beta. Microglia prepared from rat cerebellum displayed similar behaviors. As with LPS, Apo-SAA upregulated SAA1 and TLR2 mRNA, and downregulated that of TLR4. LPS was less efficacious than Apo-SAA, perhaps reflecting an action of the latter at TLR4 and TLR2. The TLR4 antagonist CLI-095 fully blocked the action of LPS, but only partially that of Apo-SAA. Although the TLR2 antagonist CU-CPT22 was inactive against Apo-SAA, it also failed to block the TLR2 agonist Pam(3)CSK(4). Conclusions: Microglia are central to the inflammatory process and a major source of IL-1 beta when activated. P2X(7)Rtriggered IL-1 beta maturation and export is thus likely to represent an important contributor to this cytokine pool. Given that SAA is detected in Alzheimer disease and multiple sclerosis brain, together with IL-1 beta-immunopositive microglia, these findings propose a link between P2X(7)R, SAA, and IL-1 beta in CNS pathophysiology.