Abstract
S100A8 and S100A9 are members of the S100 family of cytoplasmic EF-hand Ca2+-binding proteins and are abundantly expressed in the cytosol of neutrophils. In addition to their intracellular roles, S100A8/A9 can be secreted in the extracellular environment and are considered as alarmins able to amplify the inflammatory response. The intracellular activity of S100A8/A9 was shown to be regulated by S100A9 phosphorylation, but the importance of this phosphorylation on the extracellular activity of S100A8/A9 has not yet been extensively studied. Our work focuses on the impact of the phosphorylation state of secreted S100A9 on the proinflammatory function of neutrophils. In a first step, we characterized the secretion of S100A8/A9 in different stimulatory conditions and investigated the phosphorylation state of secreted S100A9. Our results on neutrophil-like differentiated HL-60 (dHL-60) cells and purified human neutrophils showed a time-dependent secretion of S100A8/A9 when induced by phorbol 12-myristoyl 13-acetate and this secreted S100A9 was found in a phosphorylated form. Second, we evaluated the impact of this phosphorylation on proinflammatory cytokine expression and secretion in dHL-60 cells. Time course experiments with purified unphosphorylated or phosphorylated S100A8/A9 were performed and the expression and secretion levels of interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor alpha, CCL2, CCL3, CCL4, and CXCL8 were measured by real-time PCR and cytometry bead array, respectively. Our results demonstrate that only the phosphorylated form of the complex induces proinflammatory cytokine expression and secretion. For the first time, we provide evidence that S100A8/PhosphoS100A9 is inducing cytokine secretion through toll-like receptor 4 signaling.
Highlights
Neutrophils are among the first cells to be recruited to inflammatory sites with a crucial role in pathogen killing through the deployment of sophisticated antimicrobial strategies, including production of reactive oxygen species (ROS) by the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), release of cytotoxic products during the degranulation process and formation of neutrophil extracellular traps (NETs)
We determined in a first step the phosphorylation state of S100A8/A9 secreted by neutrophil-like HL-60 cells and purified neutrophils
In order to know under which stimulatory conditions S100A8/A9 are released in the cell culture supernatants, we performed a secretion time-course on differentiated HL-60 (dHL-60) cells or purified neutrophils stimulated with 100 nM fMLF or 100 nM phorbol 12-myristoyl 13-acetate (PMA)
Summary
Neutrophils are among the first cells to be recruited to inflammatory sites with a crucial role in pathogen killing through the deployment of sophisticated antimicrobial strategies, including production of reactive oxygen species (ROS) by the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), release of cytotoxic products during the degranulation process and formation of neutrophil extracellular traps (NETs). Activation of neutrophils was initially recognized as the result of the presence of pathogens, but lately it has become clear that this inflammatory response can be triggered by endogenous ligands known as damage-associated molecular pattern (DAMPs) or alarmins [1]. S100A8 and S100A9 have been characterized as DAMPs, which are released by activated phagocytes such as neutrophils and monocytes [2, 3]. It has been suggested that posttranslational modifications of S100A8 induced by oxidase-producing oxygen derivatives may switch their biological properties from a proinflammatory toward an anti-inflammatory pattern preventing excessive damage to host tissue by scavenging oxidants [14, 15]
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