Neutrophils play an important role in innate immunity by defending the host organism against invading microorganisms. Antimicrobial activity of neutrophils is mediated by release of antimicrobial peptides, phagocytosis as well as formation of neutrophil extracellular traps (NET). These structures are composed of DNA, histones and granular proteins such as neutrophil elastase and myeloperoxidase. This study focused on the influence of NET on the host cell functions, particularly on human alveolar epithelial cells as the major cells responsible for gas exchange in the lung. Upon direct interaction with epithelial and endothelial cells, NET induced cytotoxic effects in a dose-dependent manner, and digestion of DNA in NET did not change NET-mediated cytotoxicity. Pre-incubation of NET with antibodies against histones, with polysialic acid or with myeloperoxidase inhibitor but not with elastase inhibitor reduced NET-mediated cytotoxicity, suggesting that histones and myeloperoxidase are responsible for NET-mediated cytotoxicity. Although activated protein C (APC) did decrease the histone-induced cytotoxicity in a purified system, it did not change NET-induced cytotoxicity, indicating that histone-dependent cytotoxicity of NET is protected against APC degradation. Moreover, in LPS-induced acute lung injury mouse model, NET formation was documented in the lung tissue as well as in the bronchoalveolar lavage fluid. These data reveal the important role of protein components in NET, particularly histones, which may lead to host cell cytotoxicity and may be involved in lung tissue destruction.

Siglecs (sialic acid-binding immunoglobulin-type lectins) are a family of immune regulatory receptors predominantly found on the cells of the hematopoietic system. A V-set Ig-like domain mediates the recognition of different sialylated glycoconjugates, which can lead to the activation or inhibition of the immune response, depending on the involved Siglecs. Siglecs are categorized into two subgroups: one including all CD33-related Siglecs and the other consisting of Siglec-1 (Sialoadhesin), Siglec-2 (CD22), Siglec-4 (myelin-associated glycoprotein, MAG) and Siglec-15. In contrast to the members of the CD33-related Siglecs, which share ∼50-99% sequence identity, Siglecs of the other subgroup show quite low homology (approximately 25-30% sequence identity). Based on the published sequences and functions of Siglecs, we performed phylogenetic analyses and sequence alignments to reveal the conservation of Siglecs throughout evolution. Therefore, we focused on the presence of Siglecs in different classes of vertebrates (fishes, amphibians, birds, reptiles and mammals), offering a bridge between the presence of different Siglecs and the biological situations of the selected animals.

Among the large set of cell surface glycan structures, the carbohydrate polymer polysialic acid (polySia) plays an important role in vertebrate brain development and synaptic plasticity. The main carrier of polySia in the nervous system is the neural cell adhesion molecule NCAM. As polySia with chain lengths of more than 40 sialic acid residues was still observed in brain of newborn Ncam ^−/− mice, we performed a glycoproteomics approach to identify the underlying protein scaffolds. Affinity purification of polysialylated molecules from Ncam ^−/− brain followed by peptide mass fingerprinting led to the identification of the synaptic cell adhesion molecule SynCAM 1 as a so far unknown polySia carrier. SynCAM 1 belongs to the Ig superfamily and is a powerful inducer of synapse formation. Importantly, the appearance of polysialylated SynCAM 1 was not restricted to the Ncam ^−/− background but was found to the same extent in perinatal brain of WT mice. PolySia was located on N-glycans of the first Ig domain, which is known to be involved in homo- and heterophilic SynCAM 1 interactions. Both polysialyltransferases, ST8SiaII and ST8SiaIV, were able to polysialylate SynCAM 1 in vitro, and polysialylation of SynCAM 1 completely abolished homophilic binding. Analysis of serial sections of perinatal Ncam ^−/− brain revealed that polySia-SynCAM 1 is expressed exclusively by NG2 cells, a multifunctional glia population that can receive glutamatergic input via unique neuron-NG2 cell synapses. Our findings sug-gest that polySia may act as a dynamic modulator of SynCAM 1 functions during integration of NG2 cells into neural networks.

Despite optimal therapy, the morbidity and mortality of patients presenting with an acute myocardial infarction (MI) remain significant, and the initial mechanistic trigger of myocardial “ischaemia/reperfusion (I/R) injury” remains greatly unexplained. Here we show that factors released from the damaged cardiac tissue itself, in particular extracellular RNA (eRNA) and tumour-necrosis-factor α (TNF-α), may dictate I/R injury. In an experimental in vivo mouse model of myocardial I/R as well as in the isolated I/R Langendorff-perfused rat heart, cardiomyocyte death was induced by eRNA and TNF-α. Moreover, TNF-α promoted further eRNA release especially under hypoxia, feeding a vicious cell damaging cycle during I/R with the massive production of oxygen radicals, mitochondrial obstruction, decrease in antioxidant enzymes and decline of cardiomyocyte functions. The administration of RNase1 significantly decreased myocardial infarction in both experimental models. This regimen allowed the reduction in cytokine release, normalisation of antioxidant enzymes as well as preservation of cardiac tissue. Thus, RNase1 administration provides a novel therapeutic regimen to interfere with the adverse eRNA-TNF-α interplay and significantly reduces or prevents the pathological outcome of ischaemic heart disease.

Polysialic acid (polySia), a post-translational modification of the neural cell adhesion molecule (NCAM), is the key regulator of NCAM-mediated functions and crucial for normal brain development, postnatal growth, and survival. Two polysialyltransferases, ST8SiaII and ST8SiaIV, mediate polySia biosynthesis. To dissect the impact of each enzyme during postnatal brain development, we monitored the developmental changes in NCAM polysialylation in wild-type, ST8SiaII-, and ST8SiaIV-deficient mice using whole brain lysates obtained at 10 time points from postnatal days 1 to 21 and from adult mice. In wild-type and ST8SiaIV-null brain, polySia biosynthesis kept pace with the rapid increase in brain weight until day 9, and nearly all NCAM was polysialylated. Thereafter, polySia dropped by ∼70% within 1 week, accompanied by the first occurrence of polySia-free NCAM-140 and NCAM-180. In ST8SiaII-null brain, polySia declined immediately after birth, leading to 60% less polySia at day 9 combined with the untimely appearance of polySia-free NCAM. Polysialyltransferase deficiency did not alter NCAM expression level or isoform pattern. In all three genotypes, NCAM-140 and NCAM-180 were expressed at constant levels from days 1 to 21 and provided the major polySia acceptors. By contrast, NCAM-120 first appeared at day 5, followed by a strong up-regulation inverse to the decrease in polySia. Together, we provide a comprehensive quantitative analysis of the developmental changes in polySia level, NCAM polysialylation status, and polysialyltransferase transcript levels and show that the predominant role of ST8SiaII during postnatal brain development is restricted to the first 15 days.