No abstract

IntroductionMyeloid-related protein 8 (MRP8) and MRP14, both S100 proteins, are the major calcium-binding proteins expressed in phagocytes during specific stages of differentiation. 1,2 They form stable complexes and are present in circulating neutrophils and monocytes, representing the first cells invading inflammatory lesions. 3 The protein complex is found in inflammatory fluids in distinct inflammatory conditions, including rheumatoid arthritis, allograft rejection, inflammatory bowel disease, and lung disease. [4][5][6][7][8][9] Prerequisite for its secretion is the contact of phagocytes with extracellular matrix proteins or inflamed endothelium, resulting in elevated intracellular calcium levels and activated protein kinase C. 10,11 MRP8/MRP14 is thereby released specifically at inflammatory sites and leads to increased serum levels in correlation with the degree of inflammation, indicating an extracellular role of these molecules in inflammatory processes. However, little is known about the extracellular functions of MRP8/MRP14. The protein complex is deposited on endothelia for which different mechanisms are proposed. MRP14 has been shown to bind specifically to human microvascular endothelial cells (HMECs) by way of heparan sulphate proteoglycans. 12 Another group 13 reported that MRP8/MRP14 binds to novel carboxylated N-glycans expressed on inflammatory activated endothelial cells (ECs). Blocking these N-glycans with specific antibodies inhibited leukocyte extravasation in a murine model. 13 The hypothesis of a prominent role of MRP8/MRP14 for leukocyte recruitment is further supported by the finding that MRP8/MRP14 increases the binding capacity of CD11b-CD18 on leukocytes to intracellular adhesion molecule-1 (ICAM-1) on endothelium. 14 A recently identified inflammatory disorder, with the hallmark of an extraordinarily high abundance of MRP8 and MRP14, finally underscores a direct pathogenetic role for these 2 molecules in inflammation in vivo. 15 Thus, multiple findings indicate important interactions between MRP8/MRP14 and ECs, whereas the functional consequences and the underlying molecular mechanisms are completely unknown.In our study, oligonucleotide microarray analysis of HMECs demonstrated that MRP8/MRP14 directly induces a distinct inflammatory, thrombogenic response in microvascular ECs. The inflammatory response is characterized by the induction of proinflammatory chemokines and adhesion molecules and by increased vascular permeability. Patients, materials, and methods Purification of MRP8 and MRP14MRP8 and MRP14 were purified from human granulocytes as described previously. 16 The purity of the protein was greater than 98%, as verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MALDI-MS) ( Figure 1A), as described elsewhere. 17 MRP8/MRP14-containing stock solutions (1.5 mg/mL) were essentially free of endotoxin, as tested by a limulus lysate assay (E-Toxate Reagent Kit, sensitive to 0.05-0.1 endotoxin U/mL; Sigma, Deisenhofen, Germany). ...

The type I interferon (IFN) system is a first line of defense against viral infections. Viruses have developed various mechanisms to counteract this response. So far, the interferon antagonistic activity of influenza A viruses was mainly observed on the level of IFNβ gene induction via action of the viral non-structural protein 1 (NS1). Here we present data indicating that influenza A viruses not only suppress IFNβ gene induction but also inhibit type I IFN signaling through a mechanism involving induction of the suppressor of cytokine signaling-3 (SOCS-3) protein. Our study was based on the observation that in cells that were infected with influenza A virus and subsequently stimulated with IFNα/β, phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1) was strongly reduced. This impaired STAT1 activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral 5′ triphosphate RNA in the cell. SOCS proteins are potent endogenous inhibitors of Janus kinase (JAK)/STAT signaling. Closer examination revealed that SOCS-3 but not SOCS-1 mRNA levels increase in an RNA- and nuclear factor kappa B (NF-κB)-dependent but type I IFN-independent manner early in the viral replication cycle. This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes. As a consequence, progeny virus titers were reduced in SOCS-3 deficient cells or in cells were SOCS-3 expression was knocked-down by siRNA. These data provide the first evidence that influenza A viruses suppress type I IFN signaling on the level of JAK/STAT activation. The inhibitory effect is at least in part due to the induction of SOCS-3 gene expression, which results in an impaired antiviral response.

S. aureus intracellular persistence via the development of an adapted subpopulation of SCVs most likely represents an important strategy of S. aureus to hide within the host cells, which could be a reservoir for chronic infections.

No abstract

The high risk of neonatal death from sepsis is thought to result from impaired responses by innate immune cells; however, the clinical observation of hyperinflammatory courses of neonatal sepsis contradicts this concept. Using transcriptomic, epigenetic and immunological approaches, we demonstrated that high amounts of the perinatal alarmins S100A8 and S100A9 specifically altered MyD88-dependent proinflammatory gene programs. S100 programming prevented hyperinflammatory responses without impairing pathogen defense. TRIF-adaptor-dependent regulatory genes remained unaffected by perinatal S100 programming and responded strongly to lipopolysaccharide, but were barely expressed. Steady-state expression of TRIF-dependent genes increased only gradually during the first year of life in human neonates, shifting immune regulation toward the adult phenotype. Disruption of this critical sequence of transient alarmin programming and subsequent reprogramming of regulatory pathways increased the risk of hyperinflammation and sepsis. Collectively these data suggest that neonates are characterized by a selective, transient microbial unresponsiveness that prevents harmful hyperinflammation in the delicate neonate while allowing for sufficient immunological protection.

The MEK5/Erk5 MAPK cascade has recently been implicated in the regulation of endothelial integrity and represents a candidate pathway mediating the beneficial effects of laminar flow, a major factor preventing vascular dysfunction and disease. Here we expressed a constitutively active mutant of MEK5 (MEK5D) to study the transcriptional and functional responses to Erk5 activation in human primary endothelial cells. We provide evidence that constitutive Erk5 activation elicits an overall protective phenotype characterized by increased apoptosis resistance and a decreased angiogenic, migratory, and inflammatory potential. This is supported by bioinformatic microarray analysis, which uncovered a statistical overrepresentation of corresponding functional clusters as well as a significant induction of anti-thrombotic, hemostatic, and vasodilatory genes. We identify KLF4 as a novel Erk5 target and demonstrate a critical role of this transcription factor downstream of Erk5. We show that KLF4 expression largely reproduces the protective phenotype in endothelial cells, whereas KLF4 siRNA suppresses expression of various Erk5 targets. Additionally, we show that vasoprotective statins potently induce KLF4 and KLF4-dependent gene expression via activation of Erk5. Our data underscore a major protective function of the MEK5/Erk5/KLF4 module in ECs and implicate agonistic Erk5 activation as potential strategy for treatment of vascular diseases.The vascular endothelium, located at the interface between blood and tissue, fulfills a plethora of important functions, including the supply of nutrients and oxygen to the surrounding tissues as well as regulation of hemostasis and inflammatory responses. Endothelial dysfunction contributes to several diseases including chronic inflammation, hemophilia, thrombosis, and atherosclerosis. Thus, elucidation of the factors and molecular mechanisms that influence endothelial function is essential for the development of novel prophylactic and therapeutic strategies against diseases involving the vascular system. A major determinant influencing endothelial integrity is the hemodynamic force exerted by steady pulsatile blood flow. This force generates a continuous shear stress on the endothelial cells (ECs) 3 in the vessel wall, resulting in gene expression changes that protect the vessel from excessive inflammatory responses and thrombosis and provides an essential survival and quiescence signal for the vascular endothelium (1).Various signaling pathways and transcription factors are involved in perception and mediation of shear stress responses. These include the MEK5/Erk5 mitogen-activated protein kinase (MAPK) pathway (2), which is activated by laminar shear stress in ECs (3). In analogy to the related Erk1/2 MAPK, Erk5 activation is triggered by dual phosphorylation at a TEY consensus motif by a mitogen-activated protein kinase kinase (MAPKK or MEK), which in turn is activated via phosphorylation by a MEK kinase (MEKK or MAP3K) (4). In the case of Erk5, the activating phosphorylation is e...

S100A12 and its receptor RAGE are found at high concentrations in pulmonary tissue and bronchoalveolar lavage fluid in acute lung injury. S100A12 expression may reflect neutrophil activation during lung inflammation and contribute to pulmonary inflammation and endothelial activation via binding to RAGE.