Abstract
Background/AimsSerum amyloid A (SAA) is an acute phase reactant with significant immunological activities, including effects on cytokine synthesis and neutrophil chemotaxis. Neutrophils can also release cytokines with proinflammatory properties. IL-1β is a key proinflammatory cytokine, the secretion of which is controlled by inflammasome. We investigated the proinflammatory effects of SAA in vitro in relation to the NLRP3 inflammasome in neutrophils.Methodology/Principal FindingsHuman neutrophils isolated form healthy subjects were stimulated with serum amyloid A (SAA). The cellular supernatants were analyzed by western blot using anti-IL-1β or anti-caspase-1 antibodies. IL-1β or Nod-like receptor family, pyrin domain containing 3 (NLRP3) mRNA expressions were analyzed by real-time PCR or reverse transcription-PCR (RT-PCR) method. SAA stimulation induced pro-IL-1β mRNA expression in neutrophils. Furthermore, SAA engaged the caspase-1-activating inflammasome, resulting in the production of active IL-1β. SAA-induced pro-IL-1β expression was marginally suppressed by the Syk specific inhibitor, R406, and SAA-induced pro-IL-1β processing in neutrophils was prevented by R406. Furthermore, SAA-induced NLRP3 mRNA expression was completely blocked by R406. Analysis of intracellular signaling revealed that SAA stimulation activated the tyrosine kinase Syk and mitogen-activated protein kinase (MAPK).Conclusions/SignificanceThese results demonstrate that the innate neutrophil immune response against SAA involves a two-step activation process: an initial signal promoting expression of pro-IL-1β and a second signal involving Syk-dependent activation of the NLRP3 inflammasome and caspase-1, allowing processing of pro-IL-1β and secretion of mature IL-1β.
Highlights
Serum amyloid A (SAA) is a major acute phase protein produced mainly in the liver, as a result of stimulation by proinflammatory cytokines
We examined the proinflammatory cytokine response in human neutrophils stimulated with SAA for different periods
To confirm whether SAA-induced mature IL-1b secretion was dependent on caspase-1-mediated processing of pro-IL-1b, IL-1b secretion was measured in neutrophils activated by SAA in the presence of the specific caspase-1 inhibitor, Z-YVAD
Summary
Serum amyloid A (SAA) is a major acute phase protein produced mainly in the liver, as a result of stimulation by proinflammatory cytokines. SAA possesses proinflammatory properties that induce the release of cytokines from different cell types, including monocytes [1,2]. Recent studies showed that SAA induced the expression of pro-IL-1b and activated the NRLP3 inflammasome, resulting in the secretion of mature IL-1b [3,4,5]. IL-1b is a key proinflammatory cytokine with a central role in the damaging inflammatory processes that accompany sterile disease [6]. Caspase-1 is a key protease required for the processing of proIL-1b, and its activation is regulated through recruitment to multimolecular scaffolds called inflammasomes [7]. Inflammasomes are composed of a cytosolic pattern-recognition receptor, pro-caspase-
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