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

Abstract

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.

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

“…Since recent reports have implicated neutrophils in the development of chronic inflammatory disorders, we wanted to characterize the regulatory mechanisms in the release of neutrophil-derived products. In a first step, cytokine candidates found secreted by highly purified (≥98%) neutrophils upon LPS stimulation [ 17 ] were selected for integration into our mathematical model. These cytokines have a particular relevance since they have been reported to contribute to the development of different chronic inflammatory diseases through the recruitment of diverse immune cells to the inflammatory site ( Table 1 ).…”

“…In accordance with the literature, the most relevant markers have been selected for degranulation analysis [ 17 ]. Degranulation was determined by measuring the expression of CD markers characteristic for azurophil granules (CD63-PE), specific granules (CD15-FITC, CD66b-FITC), gelatinase granules (CD11b-PE), and secretory vesicles (CD13-APC, CD14-APC, CD18-FITC, and CD45-APC) at the plasma membrane by flow cytometry (all antibodies are from BD Biosciences except CD14-APC from Immunotools).…”

Myeloperoxidase targets oxidative host attacks to Salmonella and prevents collateral tissue damage

“…1b, 1e), as expected. ABAH and DPI did not affect neutrophil degranulation as measured by three different assays 26 ( Supplementary Fig. 1f).…”

The Ontogeny of a Neutrophil: Mechanisms of Granulopoiesis and Homeostasis

“…Moreover, recently identified variations in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes explain these differences: whereas all neutrophil granules contain syntaxin 4 and SNARE complexes, specific and gelatinase granules have SNARE complexes with high concentrations of VAMP1, VAMP2, and 23-kDa synaptosome-associated protein (SNAP-23), while azurophilic granules have increased levels of VAMP1 and VAMP7 (186,187). Additionally, gelatinase granules are also regulated by syntaxin 3, which controls the degranulation of cytokines, including IL-1␣, IL-1␤, IL-12b, and CCL4, during early inflammatory responses (188,189). SNARE-interacting Sec1/Munc18 (SM) family members, particularly MUNC18-2 and MUNC18-3, also play essential roles in selective vesicular trafficking in neutrophils (189).…”