Role of S100A8/A9 for Cytokine Secretion, Revealed in Neutrophils Derived from ER-Hoxb8 Progenitors.

S100A9, a Ca2+-binding protein, is tightly associated to neutrophil pro-inflammatory functions when forming a heterodimer with its S100A8 partner. Upon secretion into the extracellular environment, these proteins behave like damage-associated molecular pattern molecules, which actively participate in the amplification of the inflammation process by recruitment and activation of pro-inflammatory cells. Intracellular functions have also been attributed to the S100A8/A9 complex, notably its ability to regulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. However, the complete functional spectrum of S100A8/A9 at the intracellular level is far from being understood. In this context, we here investigated the possibility that the absence of intracellular S100A8/A9 is involved in cytokine secretion. To overcome the difficulty of genetically modifying neutrophils, we used murine neutrophils derived from wild-type and S100A9-/- Hoxb8 immortalized myeloid progenitors. After confirming that differentiated Hoxb8 neutrophil-like cells are a suitable model to study neutrophil functions, our data show that absence of S100A8/A9 led to a dysregulation of cytokine secretion after lipopolysaccharide (LPS) stimulation. Furthermore, we demonstrate that S100A8/A9-induced cytokine secretion was regulated by the nuclear factor kappa B (NF-κB) pathway. These results were confirmed in human differentiated HL-60 cells, in which S100A9 was inhibited by shRNAs. Finally, our results indicate that the degranulation process could be involved in the regulation of cytokine secretion by S100A8/A9.

Secretion of the Phosphorylated Form of S100A9 from Neutrophils Is Essential for the Proinflammatory Functions of Extracellular S100A8/A9.

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-1ß, 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.

New insights into the regulation of neutrophil NADPH oxidase activity in the phagosome: a focus on the role of lipid and Ca(2+) signaling.

SIGNIFICANCE: Reactive oxygen species, produced by the phagosomal NADPH oxidase of neutrophils, play a significant physiological role during normal defense. Their role is not only to kill invading pathogens, but also to act as modulators of global physiological functions of phagosomes. Given the importance of NADPH oxidase in the immune system, its activity has to be decisively controlled by distinctive mechanisms to ensure appropriate regulation at the phagosome. RECENT ADVANCES: Here, we describe the signal transduction pathways that regulate phagosomal NADPH oxidase in neutrophils, with an emphasis on the role of lipid metabolism and intracellular Ca(2+) mobilization. CRITICAL ISSUES: The potential involvement of Ca(2+)-binding S100A8 and S100A9 proteins, known to interact with the plasma membrane NADPH oxidase, is also considered. FUTURE DIRECTIONS: Recent technical progress in advanced live imaging microscopy will permit to focus more accurately on phagosomal rather than plasma membrane NADPH oxidase regulation during neutrophil phagocytosis.

iPLA2, a novel determinant in Ca2+- and phosphorylation-dependent S100A8/A9 regulated NOX2 activity.

The neutrophil NADPH oxidase (NOX2) is a key enzyme responsible for host defense against invading pathogens, via the production of reactive oxygen species. Dysfunction of NOX2 can contribute to inflammatory processes, which could lead to the development of diseases such as atherosclerosis. In this paper, we characterize a pathway leading to NOX2 activation in which iPLA(2)-regulated p38 MAPK activity is a key regulator of S100A8/A9 translocation via S100A9 phosphorylation. Studies in cell-free or recombinant systems involved two Ca2+-binding proteins of the S100 family, namely S100A8 and S100A9, in NOX2 activation dependent on intracellular Ca2+ concentration ([Ca2+](i)) elevation. Using differentiated HL-60 cells as a model of neutrophils, we provide evidence that [Ca2+](i)-regulated S100A8/A9 translocation is mediated by an increase in [Ca2+](i) through intracellular Ca2+ store depletion. Moreover, we confirm that p38 MAPK induces S100A9 phosphorylation, a mandatory precondition for S100 translocation. Based on a pharmacological approach and an siRNA strategy, we identify iPLA(2) as a new molecular player aiding S100 translocation and NOX2 activity. Inhibition of p38 MAPK activity and S100A9 phosphorylation by bromoenol lactone, a selective inhibitor of iPLA(2), indicated that p38 MAPK-mediated S100A9 phosphorylation is dependent on iPLA(2). In conclusion, we have characterized a pathway leading to NOX2 activation in which iPLA(2)-regulated p38 MAPK activity is a key regulator of S100A8/A9 translocation via S100A9 phosphorylation.

ILK as a potential marker gene to ascertain specific adenocarcinoma cell mRNA isolation from frozen prostate biopsy tissue sections.

A major technical challenge related to gene expression profiling of tissue samples is the difficulty of procuring selected cell populations from tissues that by nature are heterogeneous, such as prostate tissue. In this study we have examined the expression of integrin-linked kinase (ILK) mRNA in prostate adenocarcinoma cells versus normal prostate epithelial cells in order to determine whether ILK could be used as a reference marker gene for prostate adenocarcinoma cell mRNA isolation. Using laser microdissection (LMD) technology and real-time PCR, together with immunohistochemistry, we have analyzed ILK mRNA expression in epithelial cells isolated from frozen prostate biopsy specimens as well as 4 prostate cell lines (RWPE-1, LNCaP, PC-3 and DU 145) and correlated ILK mRNA expression with ILK protein expression. We demonstrate that quantitative upregulation of ILK mRNA expression in prostate epithelial cells derived from prostate tissue correlated with ILK protein expression and with the histopathology diagnosis of prostate adenocarcinoma. We further show that the level of ILK overexpression was directly influenced by the method used to isolate prostate adenocarcinoma cells (bulk tissue versus LMD dissected cells). These data provide evidence that ILK mRNA is quantitatively upregulated in prostate adenocarcinoma cells versus normal epithelial cells and is therefore a useful internal reference gene marker to evaluate the quality of prostate adenocarcinoma cell derived mRNA used for large scale prostate cancer cDNA gene profiling.