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Serum amyloid A (SAA) is one of the acute‐phase reactants, a group of plasma proteins that increases immensely in concentration during microbial infections and inflammatory conditions, and a close relationship between SAA levels and disease activity in rheumatoid arthritis (RA) has been observed. RA is an inflammatory disease, where neutrophils play important roles, and SAA is thought to participate in the inflammatory reaction by being a neutrophil chemoattractant and inducer of proinflammatory cytokines. The biological effects of SAA are reportedly mediated mainly through formyl peptide receptor like‐1 (FPRL1), a G protein‐coupled receptor (GPCR) belonging to the formyl peptide receptor family. Here, we confirmed the affinity of SAA for FPRL1 by showing that stably transfected HL‐60 cells expressing FPRL1 were activated by SAA and that the response was inhibited by the use of the FPRL1‐specific antagonist WRWWWW (WRW4). We also show that SAA activates the neutrophil NADPH‐oxidase and that a reserve pool of receptors is present in storage organelles mobilized by priming agents such as TNF‐α and LPS from Gram‐negative bacteria. The induced activity was inhibited by pertussis toxin, indicating the involvement of a GPCR. However, based on FPRL1‐specific desensitization and use of FPRL1 antagonist WRW4, we found the SAA‐mediated effects in neutrophils to be independent of FPRL1. Based on these findings, we conclude that SAA signaling in neutrophils is mediated through a GPCR, distinct from FPRL1. Future identification and characterization of the SAA receptor could lead to development of novel, therapeutic targets for treatment of RA.

Uric acid, the naturally occurring degradation product of purine metabolism, is a danger signal, driving maturation of dendritic cells. It is well known that uric acid crystals display potent proinflammatory properties—the cause of gout—whereas the biological properties of soluble uric acid are less well documented. We have demonstrated previously that nucleic acids of endogenous and exogenous origin display proinflammatory properties. The aim of the present study was to assess the impact of soluble uric acid on in vivo inflammatory responses. Mice were administered with uric acid suspension in saline or saline alone prior to induction of neutrophil‐mediated inflammation, delayed‐type hypersensitivity, histamin‐induced edema (measure of vasodilation capacity), as well as double‐stranded (ds)RNA‐triggered arthritis. Frequency and severity of arthritis were decreased significantly in mice exposed to dsRNA and simultaneously treated with uric acid as compared with saline‐treated controls. Also, granulocyte‐mediated inflammatory response and vasodilation capacity were reduced significantly in mice treated with uric acid as compared with their control group. The data suggest that down‐regulation of inflammation was mediated by skewing the inflammatory response from the peripheral sites to the peritoneal cavity and down‐regulating vasodilatatory capacity and thereby affecting leukocyte migration. In contrast, the T cell‐mediated delayed‐type hypersensitivity reaction was not affected significantly in mice exposed to uric acid. These findings demonstrate that uric acid displays a potent, distant anti‐inflammatory effect in vivo. This property seems to be mediated by down‐regulation of neutrophil influx to the site of inflammatory insult.

Mature human neutrophils contain small amounts of interleukin‐8 [CXC chemokine ligand 8 (CXCL‐8)], which upon proinflammatory activation, increases significantly. It has been suggested that the CXCL‐8 content of resting human neutrophils is stored in the secretory vesicles. Here, we have used a fractionation technique, which allows isolation of these vesicles, and we find that CXCL‐8 neither colocalizes with the secretory vesicles nor with markers of any of the classical neutrophil granules. To increase resolution in the system, we induced CXCL‐8 production by lipopolysaccharide. After 8 h of stimulation, CXCL‐8 was visualized within the cell using immunoelectron microscopy. The images revealed CXCL‐8‐containing stuctures resembling neutrophil granules, and these were distinct from all known neutrophil organelles, as shown by double immunostaining. Further, the CXCL‐8 organelle was present in nonstimulated neutrophil cytoplasts, entities lacking all other known granules and secretory vesicles. Upon fractionation of the cytoplasts, CXCL‐8 was found to partly cofractionate with calnexin, a marker for endoplasmic reticulum (ER). Thus, part of CXCL‐8 may be localized to the ER or ER‐like structures in the neutrophil.

The neutrophil NADPH‐oxidase may be activated in the plasma membrane, resulting in release of oxygen metabolites extracellularly, or in the granule or phagosomal membranes, giving intracellular production of oxidants. An increase in [Ca2+]imediated through binding of fMLF to its receptor is part of a signaling cascade that activates the plasma membrane‐localized oxidase. In contrast, a rise in [Ca2+]iinduced by a Ca2+ionophore results in activation of the intracellular pool of oxidase. We mimicked fMLF‐induced emptying of intracellular Ca2+stores with thapsigargin. This induced a pronounced intracellular oxidase activity but no extracellular release of oxidants. The thapsigargin‐induced effect was dependent on capacitative Ca2+influx, because the effect was inhibited dose‐dependently by EGTA and the Ca2+channel blocker La3+. At La3+concentrations between 200 and 400 μM, thapsigargin also induced a massive extracellular production of superoxide anion. No other channel blockers tested induced a similar effect. We conclude that elevation in [Ca2+]iby capacitative Ca2+influx induces NADPH‐oxidase activation at an intracellular site. Further, activation of the plasma membrane‐localized NADPH‐oxidase is regulated by a more complex Ca2+signaling, involving capacitative Ca2+influx and possibly the specific action of La3+‐sensitive Ca2+channels.