K2.1 is a regulator of inflammatory cell responses in idiopathic inflammatory myopathies.

K2.1 (TREK1), a two-pore domain potassium channel, has emerged as regulator of leukocyte transmigration into the central nervous system. In the context of skeletal muscle, immune cell infiltration constitutes the pathogenic hallmark of idiopathic inflammatory myopathies (IIMs).

Nuclease activity and protein A release of clinical isolates determine the virulence in a murine model of acute lung infection.

is a common cause of hospital-acquired pneumonia associated with high mortality. Adequate clinical treatment is impeded by increasing occurrence of antibiotic resistances. Understanding the underlying mechanisms of its virulence during infections is a prerequisite to finding alternative treatments.

The Fatal Circle of NETs and NET-Associated DAMPs Contributing to Organ Dysfunction.

The innate immune system is the first line of defense against invading pathogens or sterile injuries. Pattern recognition receptors (PRR) sense molecules released from inflamed or damaged cells, or foreign molecules resulting from invading pathogens. PRRs can in turn induce inflammatory responses, comprising the generation of cytokines or chemokines, which further induce immune cell recruitment.

Resolving immune cells with patrolling behaviour by magnetic resonance time-lapse single cell tracking.

Background: Immune cells show distinct motion patterns that change upon inflammatory stimuli. Monocytes patrol the vasculature to screen for pathogens, thereby exerting an early task of innate immunity. Here, we aimed to non-invasively analyse single patrolling monocyte behaviour upon inflammatory stimuli.

A Fragile Balance: Does Neutrophil Extracellular Trap Formation Drive Pulmonary Disease Progression?

Neutrophils act as the first line of defense during infection and inflammation. Once activated, they are able to fulfil numerous tasks to fight inflammatory insults while keeping a balanced immune response. Besides well-known functions, such as phagocytosis and degranulation, neutrophils are also able to release "neutrophil extracellular traps" (NETs).

The integrin-linked kinase is required for chemokine-triggered high-affinity conformation of the neutrophil β2-integrin LFA-1.

Neutrophil adhesion and extravasation into tissue at sites of injury or infection depend on binding of the integrin lymphocyte function-associated antigen 1 (LFA-1) to ICAM-1 expressed on activated endothelial cells. The activation-dependent conformational change of LFA-1 to the high-affinity conformation (H+) requires kindlin-3 binding to the β2-integrin cytoplasmic domain. Here we show that genetic deletion of the known kindlin interactor integrin-linked kinase (ILK) impaired neutrophil adhesion and extravasation in the cremaster muscle and in a clinically relevant model of renal ischemia reperfusion injury.

The ITIM Domain-Containing NK Receptor Ly49Q Impacts Pulmonary Infection by Mediating Neutrophil Functions.

Pulmonary infection is a frequent pathology associated with excessive neutrophil infiltration. Ly49Q, an ITIM domain-bearing receptor expressed on different leukocytes, has been recently reported to impact neutrophil migration and polarization. Utilizing a murine model of induced pulmonary infection in combination with additional in vivo and in vitro assays, we show that Ly49Q is critically involved in different steps of the leukocyte adhesion cascade.

Skap2 is required for β integrin-mediated neutrophil recruitment and functions.

Integrin activation is required for neutrophil functions. Impaired integrin activation on neutrophils is the hallmark of leukocyte adhesion deficiency (LAD) syndrome in humans, characterized by impaired leukocyte recruitment and recurrent infections. The Src kinase-associated phosphoprotein 2 (Skap2) is involved in integrin functions in different leukocyte subtypes.

Endothelial cell-derived CD95 ligand serves as a chemokine in induction of neutrophil slow rolling and adhesion.

Integrin activation is crucial for the regulation of leukocyte rolling, adhesion and trans-vessel migration during inflammation and occurs by engagement of myeloid cells through factors presented by inflamed vessels. However, endothelial-dependent mechanisms of myeloid cell recruitment are not fully understood. Here we show using an autoperfused flow chamber assay of whole blood neutrophils and intravital microscopy of the inflamed cremaster muscle that CD95 mediates leukocyte slow rolling, adhesion and transmigration upon binding of CD95-ligand (CD95L) that is presented by endothelial cells.

The Neutrophil Btk Signalosome Regulates Integrin Activation during Sterile Inflammation.

Neutrophils are recruited from the blood to sites of sterile inflammation, where they are involved in wound healing but can also cause tissue damage. During sterile inflammation, necrotic cells release pro-inflammatory molecules including formylated peptides. However, the signaling pathway triggered by formylated peptides to integrin activation and leukocyte recruitment is unknown.

Gnb isoforms control a signaling pathway comprising Rac1, Plcβ2, and Plcβ3 leading to LFA-1 activation and neutrophil arrest in vivo.

Chemokines are required for leukocyte recruitment and appropriate host defense and act through G protein-coupled receptors (GPCRs), which induce downstream signaling leading to integrin activation. Although the α and β subunits of the GPCRs are the first intracellular molecules that transduce signals after ligand binding and are therefore indispensable for downstream signaling, relatively little is known about their contribution to lymphocyte function-associated antigen 1 (LFA-1) activation and leukocyte recruitment. We used knockout mice and short hairpin RNA to knock down guanine nucleotide binding protein (GNB) isoforms (GNB1, GNB2, GNB4, and GNB5) in HL60 cells and primary murine hematopoietic cells.

Cross-Talk between Shp1 and PIPKIγ Controls Leukocyte Recruitment.

Neutrophil recruitment to the site of inflammation plays a pivotal role in host defense. However, overwhelming activation and accumulation of neutrophils in the tissue may cause tissue damage and autoimmunity due to the release of cytokines, oxidants, and proteases. Neutrophil adhesion in acute inflammation is initiated by activation of αLβ2 (LFA-1), which can be induced by rolling on E-selectin (slowly) or by exposure to the chemokine CXCL1 (rapidly).

Purification of GST-Tagged Proteins.

This protocol describes the purification of recombinant proteins fused to glutathione S-transferase (GST, GST-tagged proteins) by Glutathione Affinity purification. The GST tag frequently increases the solubility of the fused protein of interest and thus enables its purification and subsequent functional characterization. The GST-tagged protein specifically binds to glutathione immobilized to a matrix (e.

Proteolytic Affinity Tag Cleavage.

Here, we present protocols describing the use of the dipeptidyl-aminopeptidase-1 (DPP1, DAPase) exoprotease-based TAGZyme system and the endoprotease, Factor Xa. Both enable the recovery of proteins free of any amino acids encoded by the vector and/or protease recognition site. They also provide the possibility of removing the proteases from the preparation of the target protein by a simple subtractive chromatography step.

Purification of His-Tagged Proteins.

Ni-NTA affinity purification of His-tagged proteins is a bind-wash-elute procedure that can be performed under native or denaturing conditions. Here, protocols for purification of His-tagged proteins under native, as well as under denaturing conditions, are given. The choice whether to purify the target protein under native or denaturing conditions depends on protein location and solubility, the accessibility of the His tag, and the desired downstream application.

Expression and purification of membrane proteins.

Approximately 30% of a genome encodes for membrane proteins. They are one of the most important classes of proteins in that they can receive, differentiate, and transmit intra- and intercellular signals. Some examples of classes of membrane proteins include cell-adhesion molecules, translocases, and receptors in signaling pathways.

The PSGL-1-L-selectin signaling complex regulates neutrophil adhesion under flow.

Neutrophils are recruited from the blood to sites of inflammation, where they contribute to immune defense but may also cause tissue damage. During inflammation, neutrophils roll along the microvascular endothelium before arresting and transmigrating. Arrest requires conformational activation of the integrin lymphocyte function-associated antigen 1 (LFA-1), which can be induced by selectin engagement.

Integrin activation by P-Rex1 is required for selectin-mediated slow leukocyte rolling and intravascular crawling.

Integrin activation is essential for the function of leukocytes. Impaired integrin activation on leukocytes is the hallmark of the leukocyte adhesion deficiency syndrome in humans, characterized by impaired leukocyte recruitment and recurrent infections. In inflammation, leukocytes collect different signals during the contact with the microvasculature, which activate signaling pathways leading to integrin activation and leukocyte recruitment.

Regulation of PTEN activity by p38δ-PKD1 signaling in neutrophils confers inflammatory responses in the lung.

Despite their role in resolving inflammatory insults, neutrophils trigger inflammation-induced acute lung injury (ALI), culminating in acute respiratory distress syndrome (ARDS), a frequent complication with high mortality in humans. Molecular mechanisms underlying recruitment of neutrophils to sites of inflammation remain poorly understood. Here, we show that p38 MAP kinase p38δ is required for recruitment of neutrophils into inflammatory sites.

Severe impairment of leukocyte recruitment in ppGalNAcT-1-deficient mice.

P-selectin glycoprotein ligand-1 plays an important role in leukocyte recruitment. Its binding affinity to selectins is modulated by posttranslational modifications. The polypeptide N-acetylgalactosamine transferase-1 (ppGalNAcT-1) initiates core-type protein O-glycosylation.

The role of the tec kinase Bruton's tyrosine kinase (Btk) in leukocyte recruitment.

Recruitment of leukocytes into inflamed tissue is a key component of the immune system. The activation of integrins on leukocytes is required for their recruitment into the inflamed tissue. Btk is a cytoplasmic nonreceptor tyrosine kinase belonging to the Tec-kinase family.

Crucial role of SLP-76 and ADAP for neutrophil recruitment in mouse kidney ischemia-reperfusion injury.

Neutrophils trigger inflammation-induced acute kidney injury (AKI), a frequent and potentially lethal occurrence in humans. Molecular mechanisms underlying neutrophil recruitment to sites of inflammation have proved elusive. In this study, we demonstrate that SLP-76 (SH2 domain-containing leukocyte phosphoprotein of 76 kD) and ADAP (adhesion and degranulation promoting adaptor protein) are involved in E-selectin-mediated integrin activation and slow leukocyte rolling, which promotes ischemia-reperfusion-induced AKI in mice.

Dissociation of VE-PTP from VE-cadherin is required for leukocyte extravasation and for VEGF-induced vascular permeability in vivo.

We have recently shown that vascular endothelial protein tyrosine phosphatase (VE-PTP), an endothelial membrane protein, associates with VE-cadherin and is required for optimal VE-cadherin function and endothelial cell contact integrity. The dissociation of VE-PTP from VE-cadherin is triggered by vascular endothelial growth factor (VEGF) and by the binding of leukocytes to endothelial cells in vitro, suggesting that this dissociation is a prerequisite for the destabilization of endothelial cell contacts. Here, we show that VE-cadherin/VE-PTP dissociation also occurs in vivo in response to LPS stimulation of the lung or systemic VEGF stimulation.

Reprint of: Immobilized-Metal Affinity Chromatography (IMAC): A Review.

This article reviews the development of immobilized-metal affinity chromatography (IMAC) and describes its most important applications. We provide an overview on the use of IMAC in protein fractionation and proteomics, in protein immobilization and detection, and on some special applications such as purification of immunoglobulins and the Chelex method. The most relevant application- purification of histidine-tagged recombinant proteins-will be reviewed in greater detail with focus of state-of-the-art materials, methods, and protocols, and the limitations of IMAC and recent advances to improve the technology and the methods will be described.

Immobilized-metal affinity chromatography (IMAC): a review.

This article reviews the development of immobilized-metal affinity chromatography (IMAC) and describes its most important applications. We provide an overview on the use of IMAC in protein fractionation and proteomics, in protein immobilization and detection, and on some special applications such as purification of immunoglobulins and the Chelex method. The most relevant application-purification of histidine-tagged recombinant proteins-will be reviewed in greater detail with focus of state-of-the-art materials, methods, and protocols, and the limitations of IMAC and recent advances to improve the technology and the methods will be described.