Neutrophils play a pivotal role in the defense against bacterial, viral and fungal infections and are important mediators in the acute inflammatory response. At the same time, neutrophils are also involved in sterile inflammatory responses that are triggered by endogenous ligands. A series of immediate effector functions and the expression of proinflammatory genes enable neutrophils to initiate the immune response against the injurious agent. Among these, interleukin-1b (IL-1b) plays a key role in the orchestration of the inflammatory response. Induction of IL-1b expression leads to production of cytosolic pro-IL-1b, which requires further processing by a proteolytic cleavage event. Caspase-1 was initially identified as the main IL-1b-converting enzyme, and the upstream events leading to caspase-1 activation were identified as so-called inflammasome complexes. Up to now, the inflammasome system has mainly been studied in macrophages, whereas the inflammasome was thought to play a redundant or no role in the cell intrinsic processing of pro-IL-1b in neutrophils. Here, we identify the expression of the components of the NLRP3 inflammasome complex in neutrophils and show that the NLRP3 inflammasome pathway is indeed operational in neutrophils. Our findings establish the NLRP3 inflammasome as a key step in the secretion of matured IL-1b by neutrophils.

We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software xiSEARCH. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12‐fraction protocol for crosslinked multi‐protein complexes and cell lysates, quantitative analysis, and high‐density crosslinking, without requiring specific crosslinker features. This overall approach reveals dynamic protein–protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.

Neutrophil granulocytes are important mediators of innate immunity, but also participate in the pathogenesis of (auto)inflammatory diseases. Neutrophils express a specific set of proteolytic enzymes, the neutrophil serine proteases (NSPs), which are stored in cytoplasmic granules and can be secreted into the extra- and pericellular space upon cellular activation. These NSPs, namely cathepsin G (CG), neutrophil elastase (NE), and proteinase 3 (PR3), have early been implicated in bacterial defense. However, NSPs also regulate the inflammatory response by specifically altering the function of cytokines and chemokines. For instance, PR3 and NE both inactivate the anti-inflammatory mediator progranulin, which may play a role in chronic inflammation. Here, we provide a concise update on NSPs as modulators of inflammation and discuss the biological and pathological significance of this novel function of NSPs. Mounting evidence support an important proinflammatory function for PR3, which may have been underestimated in the past.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-010-0677-3) contains supplementary material, which is available to authorized users.

We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software Xi. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12-fraction protocol for crosslinked multi-protein complexes and cell lysates, quantitative analysis, and high-density crosslinking, without requiring specific crosslinker features. This overall approach reveals dynamic protein-protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.Crosslinking mass spectrometry (CLMS) has become a standard tool for the topological analysis of multi-protein complexes and has begun delivering high-density information on protein structures, insights into structural changes and the wiring of interaction networks in situ 1 .The technological development currently focuses on enrichment strategies for crosslinked peptides and mass spectrometric data acquisition 2-4 , including newly designed crosslinkers 5 .