Regulation of Neutrophil Degranulation and Cytokine Secretion: A Novel Model Approach Based on Linear Fitting

Neutrophils participate in the maintenance of host integrity by releasing various cytotoxic proteins during degranulation. Due to recent advances, a major role has been attributed to neutrophil-derived cytokine secretion in the initiation, exacerbation, and resolution of inflammatory responses. Because the release of neutrophil-derived products orchestrates the action of other immune cells at the infection site and, thus, can contribute to the development of chronic inflammatory diseases, we aimed to investigate in more detail the spatiotemporal regulation of neutrophil-mediated release mechanisms of proinflammatory mediators. Purified human neutrophils were stimulated for different time points with lipopolysaccharide. Cells and supernatants were analyzed by flow cytometry techniques and used to establish secretion profiles of granules and cytokines. To analyze the link between cytokine release and degranulation time series, we propose an original strategy based on linear fitting, which may be used as a guideline, to (i) define the relationship of granule proteins and cytokines secreted to the inflammatory site and (ii) investigate the spatial regulation of neutrophil cytokine release. The model approach presented here aims to predict the correlation between neutrophil-derived cytokine secretion and degranulation and may easily be extrapolated to investigate the relationship between other types of time series of functional processes

Regulation of neutrophil cytokine release and degranulation during inflammation: Role of SNARE fusion proteins

Neutrophil granulocytes are the first effector cells to be recruited to sites of infection. They rapidly release granule proteins and pro-inflammatory cytokines to efficiently kill intruding pathogens and recruit other immune cells. In exocytotic events, specific interactions of so-called soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins lead to the formation of complexes in order to mediate membrane fusion. Because of the excessive release of neutrophil-derived mediators leading to the development of chronic inflammatory disorders, we aimed in the present scientific work to investigate in more details the regulatory processes of mediator release in neutrophils during inflammation, with emphasis on SNARE proteins. The main objectives were i) to characterize the release of pro-inflammatory mediators, ii) to profile SNARE expression, iii) to determine the functional role of SNAREs and SNARE complexes in cytokine release and degranulation, and iv) to identify the intracellular localization of SNAREs. To characterize the pro-inflammatory response in neutrophils in regard to exocytosis, extensive kinetic studies were performed in a first step on LPS-stimulated primary neutrophils. A novel linear fitting approach was created to correlate the relationship between granule proteins and cytokines secreted to the inflammatory site. In a second step, SNARE expression levels were determined by whole-transcript analysis and the similar profiles in primary neutrophils as well as DMSO-differentiated HL-60 cells (dHL-60 cells), a neutrophil cell model, were underlined. Using an RNAi strategy, the SNARE syntaxin 3 (STX3) was identified as an essential actor in the release of the cytokines IL-1α, IL-1β, IL-12b, and CCL4. It was also involved in MMP-9 exocytosis from gelatinase granules where it could partly be localized. The SNARE SNAP29, which shares common localization with STX3, functionally affects the release of IL-12b, CCL2 and IL-8 as well as MMP-9, and represents a potential candidate to form cognate complexes with STX3. The knockdown of VAMP3, another SNARE candidate, showed deregulated secretion of IL-12b, CCL4, IL-8 as well as MMP-9. However, VAMP3 was located at the plasma membrane and was thus excluded as being part of the STX3-SNAP29 complex. Our findings provide first evidence that SNARE fusion proteins are involved in the release of IL- 12b, IL-1α, IL-1β, CCL4, IL-8, and CCL2 in a neutrophil-like cell model. The impact of SNAREs on gelatinase degranulation led us to hypothesize that cytokines might be packaged in these granules before subsequent exocytosis.

An essential role of syntaxin 3 protein for granule exocytosis and secretion of IL-1α, IL-1β, IL-12b, and CCL4 from differentiated HL-60 cells

Besides their roles in the killing of pathogens, neutrophils have the capacity to package a variety of cytokines into cytoplasmic granules for subsequent release upon inflammatory conditions. Because the rapid secretion of cytokines orchestrates the action of other immune cells at the infection site and thus, can contribute to the development and chronicity of inflammatory diseases, we aimed to determine the intracellular SNARE machinery responsible for the regulation of cytokine secretion and degranulation. From a constructed gene-expression network, we first selected relevant cytokines for functional validation by the CBA approach. We established a cytokine-secretion profile for human neutrophils and dHL-60 cells, underlining their similar ability to secrete a broad variety of cytokines within proinflammatory conditions mimicked by LPS stimulation. Secondly, after screening of SNARE genes by microarray experiments, we selected STX3 for further functional studies. With the use of a siRNA strategy, we show that STX3 is clearly required for the maximal release of IL-1α, IL-1β, IL-12b, and CCL4 without alteration of other cytokine secretion in dHL-60 cells. In addition, we demonstrate that STX3 is involved in MMP-9 exocytosis from gelatinase granules, where STX3 is partly localized. Our results suggest that the secretion of IL-1α, IL-1β, IL-12b, and CCL4 occurs during gelatinase degranulation, a process controlled by STX3. In summary, these findings provide first evidence that STX3 has an essential role in trafficking pathways of cytokines in neutrophil granulocytes

Fibroblast growth factor receptor 4: a putative key driver for the aggressive phenotype of hepatocellular carcinoma

Recently, we found upregulation of fibroblast growth factor receptor 4 (FGFR4) in a subset of hepatocellular carcinoma (HCC). Here, we provide mechanistic insight into the role of FGFR4-mediated signalling for the aggressive behaviour of HCC cells. To overexpress FGFR4, hepatoma/hepatocarcinoma cells were transfected with a construct coding for FGFR4. For downmodulation of endogenous FGFR4, we used small interfering RNA or adenoviral infection with dominant-negative FGFR4 constructs being either kinase dead (kdFGFR4) or coding for the autoinhibitory soluble domain (solFGFR4). FGFR4 overexpression in non-tumourigenic hepatocarcinoma cells significantly reduced cell-matrix adhesion, enabled cells to grow anchorage-independently in soft agar, to disintegrate the lymph-/blood-endothelial barrier for intra-/extravasation of tumour cells and to form tumours in SCID mice. Transcriptome analysis revealed altered expression of genes involved in cell-matrix interactions. Conversely, in highly tumourigenic cell lines, kdFGFR4 or solFGFR4 lowered the proportion of cells in S phase of the cell cycle, enhanced the G0/G1 and G2/M-phase proportions, reduced anchorage-independent growth in vitro and attenuated disintegration of the lymph-/blood-endothelium and tumour formation in vivo. These findings were confirmed by altered expression profiles of genes being important for late stages of cell division. Deregulated FGFR4 expression appears to be one of the key drivers of the malignant phenotype of HCC cells. Accordingly, blockade of FGFR4-mediated signalling by soluble dominant-negative constructs, like solFGFR4, may be a feasible and promising therapeutic approach to antagonize aggressive behaviour of hepatoma/hepatocarcinoma cells. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]