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Background and objectives Monosodium urate (MSU) is one of the endogenous danger molecules generated during apoptosis. Precipitated MSU crystals in tissues and joints induce an inflammatory process with interleukin 1 β (IL-1β) induction after NALP3 inflammasome (NALP3i) activation resulting in gouty arthritis. However, the exact mechanism of NALP3i activation is still elusive. The goal is the identification of the mechanism of NALP3i idependent IL-1β production induced by MSU crystals. Materials and methods Uptake of MSU was analysed by time lapse microscopy and cytofluorometry. Via REM-EDX the morphological and chemical structure of urate crystals was identified. The relative intracellular sodium concentration was analysed by Sodium Green fluorescence. The human IL-1β induction in presence of inhibitors of lysosomal acidification and of aquaporins in culture supernatants was quantified by ELISA. Additionally, mice were treated i.p. with chloroquine following injection of MSU in generated air pouches. IL-1β production in the pouch fluid was quantified. Results Blood borne phagocytes ingest MSU crystals and substantially increase their side scatter reflecting increased ‘granularity’. After the uptake of the crystals the cells swell shown by augmented forward scatter and time-lapse microscopy. Most of the phagocytes show an intact plasma membrane after ingestion of crystals. Incubation of phagocytes with MSU results in an induction of IL-1β production. Needle-shaped MSU crystals release sodium after treatment with acidic milieu and switch into barrel-shaped crystals. In swollen cells the intracellular sodium concentration of phagocytes after the uptake of MSU displays a markedly increased intracellular sodium concentration. Furthermore, the inhibition of lysosomal acidification and of aquaporins results in reduction of IL-1β production in in vitro and in vivo. Conclusions We suggest a novel model of NALP3i activation after phagocytosis of MSU crystals: the phagocytes ingest crystals into endosomes resulting in fusion with acidic lysosomes. That induces a massive release of sodium from the MSU crystal and enhances the intracellular osmolarity. The hyperosmolarity of the cell is compensated by passive water influx through aquaporins following cell swelling. The latter reduces the intracellular potassium concentration below the threshold of NALP3i activation. Finally, the decreased IL-1β production by treatment with inhibitors of lysosomal acidification or inhibitors of aquaporins supports our new model of inflammasome activation by MSU. [ABSTRACT FROM PUBLISHER]

Background and objectives C-reactive protein (CRP) is an acute-phase protein whose serum concentration increases massively in response to tissue injury or inflammation. Beside membranes of bacteria CRP is known to bind calcium dependent to altered mammalian membranes (as occurring in cell death) via phosphocholine, the polar headgroup of phosphatidylcholine and its derivatives. CRP also opsonises intracellular components leaking from necrotic cells. This study was performed to analyse whether CRP differentially binds to primary and secondary necrotic cells. Primary necrosis is defined as immediate cell death appearing after severe death stimuli, whereas secondary necrosis follows after an apoptotic stadium. Materials and methods We prepared directly Fitc-labelled CRP to determine the binding of CRP to primary and secondary necrotic cells by flow cytometry and confocal microscopy. The release of a CRP target from dying cells was detected in the supernatant by inhibition assays. The released CRP antagonist was identified by enzymatic cleavage and by mass spectrometry. Results The binding of CRP to primary necrotic cells was significantly stronger compared to secondary necrotic ones. We detected a CRP antagonistic activity in supernatants of secondary necrotic cells, which was identified as glycerophosphocholine (GlyceroPC). Actually, CRP binding to necrotic cells was blocked by synthetic GlyceroPC. The release of the CRP antagonist was significantly reduced by inhibitors of Phospholipase A2 and Caspases. Conclusions Secondary necrotic cells show a weaker binding of CRP resulting from the release of the CRP target during late apoptosis. That may have implications for opsonisation with CRP of dying and dead cells and for CRP-mediated effects in clearance. Secondary necrotic cells, for example in SLE patients, may release high amounts of the CRP antagonist with the following consequences (1) missing CRP binding targets on the dead cells and (2) neutralising of circulating CRP, both possibly contributing to clearance failure. [ABSTRACT FROM PUBLISHER]

Background and objectives Analysis of gouty tophi initiated by injection of MSU (monosodium urate) crystals intraperitoneally in mice and patients suffering from gout. Materials and methods Human PMN (polymorphonuclear cells) were incubated with MSU crystals and analysed by fluorescence microscopy (DAPI). Moreover, mice were treated intraperitoneally with MSU crystals resulting in generation of gouty tophi. Paraffin sections of the tophi were analysed by nuclear staining and immunhistology. Furthermore, synovial fluids and tophi of patients with acute gouty attacks were analysed. Results In the in vitro assays we found the formation of extracellular DNA induced by MSU crystals. Next we analysed the gouty tophi generated intraperitoneally in mice and again detected extranuclear DNA and histones. Last we investigated material of gouty patients and observed the formation of extranuclear DNA in the synovial fluid and in tophous material. Conclusions We conclude that the extranuclear DNA has been ejected by the neutrophils in order to trap the inflammation inducing crystals. A similar mechanism is employed by neutrophils in the defense against massive bacterial or fungal infection, where the extranuclear DNA helps for immobilisation and inactivation of pathogeneic microoragnisms. Further investigations are required to elucidate whether the extranuclear DNA is ameliorating or precipitating inflammation and pathology in gout. [ABSTRACT FROM PUBLISHER]

Background and objectives Systemic lupus erythematosus (SLE) is a multifactorial genetically predisposed disease resulting from inflammatory responses of the immune system against several autoantigens. Considering actual evidence, the onset of autoimmune manifestations is triggered by the interplay between environmental factors and several major complex intrinsic defects. Deficiencies in the recognition and engulfment of apoptotic cells have been reported in patients with SLE. If dying cells are not promptly cleared, accumulate, undergo secondary necrosis, and expose nuclear autoantigens. The goal of this work is to analyse whether the remnants of apoptotic cells are involved in the induction of inflammatory responses in SLE. Materials and methods Secondarily necrotic cell (SNEC) were generated by treatment of dead human lymphocytes with serum DNase-I and C1q. We employed fluorescence microscopy, FACS, and an ex vivo phagocytosis assays to demonstrate the availability of autoantigens to cells of the innate immune system. Results SNEC binds several dead and dying cells ligands, and are readily recognised by lupus autoantibodies. Autoantibodies recognising DNA in the surface of nuclear remnants are able to opsonise them and to foster its uptake by non-professional blood borne phagocytes. We observed a significantly secretion of inflammatory cytokines by the phagocytes which were cultured with SLE plasma containing AAb. Phagocytosis of SNEC and the subsequent production of inflammatory cytokines were strongly influenced by the presence of DNA in the target particle. Conclusions Lupus autoantigens are exposed on the surface of SNEC and available to assemble immune complexes. The clearance of cellular remnants that are not properly removed from the organism is targeted to innate phagocytes by lupus autoatibodies causing systemic inflammation in some patients with SLE. [ABSTRACT FROM PUBLISHER]

Background and objectives During inflammation and tissue damage, pathogens and dying cells are ingested by different phagocytes such as macrophages and dendritic cells. Both the nature and the activation state of the respective phagocyte determine the resulting immune response, which ranges from specific immunity to tolerance. In this context, the coordinated removal of apoptotic cells is essential to prevent autoimmunity and imposes a special task on the innate immune system. Materials and methods The authors both studied the removal of apoptotic thymocytes in vitro and the resulting immunological consequences in the pristine model of murine lupus in vivo. Results During peritonitis the removal of apoptotic cells is performed by a distinct population of alternatively activated and 12/15-LO-expressing resident macrophages (resMΦ). In the absence of 12/15-LO, this pattern was deranged and inflammatory macrophages (infMΦ) started to engulf apoptotic cells. The authors hypothesised a 12/15-LO-dependent block of phagocytosis in infMΦ and consequently identified 12/15-LO-derived oxidation products of phosphatidylethanolamine (OxPE) as the corresponding mediators, which are generated by resMΦ to selectively block ingestion of apoptotic cells by infMΦ. In contrast, phagocytosis of bacteria, micropinocytosis or the ingestion of apoptotic cells by resMΦ was unaffected. OxPE, which is exposed on the outer membrane of resMΦ in a 12/15-LO-dependent manner, bound to soluble milk fat globule-epidermal growth factor 8 protein and consequently blocked the major pathway responsible for the uptake of apoptotic cells into infMΦ. In consistence, the authors observed a break in self-tolerance in aged 12/15-LO-deficient mice including spontaneous production of autoantibodies and occurrence of glomerulonephritis, which both exacerbated after apoptotic challenge in the pristane-induced model of experimental murine lupus. Conclusions This data point towards a so far unrecognised role for lipid oxidation during the maintenance of self-tolerance and identify a mechanism, which orchestrates the cell- and context-specific uptake of antigens by different subsets of phagocytes, imposing a new paradigm in their understanding of the clearance of apoptotic cells. [ABSTRACT FROM PUBLISHER]