26 research outputs found
Der Einfluss von Dipyridamol auf die Gap Junction-abhängige Kommunikation in Zellen des vaskulären Systems : physiologische und molekulare Analysen
No full text[no abstract
Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca<sup>2+</sup> influx through cyclic nucleotide-gated channels.
No full textThe human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood–brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the gap junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of gap junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+, suppressed the 2-PAA-related enhancement of gap junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood–brain barrier
Nuclear Deformation During Neutrophil Migration at Sites of Inflammation
No full textCell migration is indispensable for various biological processes including angiogenesis, wound healing, and immunity. In general, there are two different migration modes described, the mesenchymal migration mode and the amoeboid migration mode. Neutrophils rapidly migrate toward the sites of injury, infection, and inflammation using the amoeboid migration mode which is characterized by cell polarization and a high migration velocity. During site-directed trafficking of neutrophils from the blood stream into the inflamed tissue, neutrophils must first withstand shear stress while migrating on the 2-dimensional endothelial surface. Subsequently, they have to cross different physical barriers during the extravasation process including the squeezing through the compact endothelial monolayer that comprises the blood vessel, the underlining basement membrane and then the 3-dimensional meshwork of extracellular matrix (ECM) proteins in the tissue. Therefore, neutrophils have to rapidly switch between distinct migration modes such as intraluminal crawling, transmigration, and interstitial migration to pass these different confinements and mechanical barriers. The nucleus is the largest and stiffest organelle in every cell and is therefore the key cellular element involved in cellular migration through variable confinements. This review highlights the importance of nuclear deformation during neutrophil crossing of such confinements, with a focus on transendothelial migration and interstitial migration. We discuss the key molecular components involved in the nuclear shape changes that underlie neutrophil motility and squeezing through cellular and ECM barriers. Understanding the precise molecular mechanisms that orchestrate these distinct neutrophil migration modes introduces an opportunity to develop new therapeutic concepts for controlling pathological neutrophil-driven inflammation
Intracellular β<sub>2</sub>integrin (CD11/CD18) interacting partners in neutrophil trafficking
No full textHow neutrophils resist shear stress at blood vessel walls: molecular mechanisms, subcellular structures, and cell–cell interactions
No full textAbstract
Neutrophils are the first cells arriving at sites of tissue injury or infection to combat invading pathogens. Successful neutrophil recruitment to sites of inflammation highly depends on specific molecular mechanisms, fine-tuning the received information into signaling pathways and converting them into well-described recruitment steps. This review highlights the impact of vascular flow conditions on neutrophil recruitment and the multitude of mechanisms developed to enable this sophisticated process under wall shear stress conditions. The recruitment process underlies a complex interplay between adhesion and signaling molecules, as well as chemokines, in which neutrophils developed specific mechanisms to travel to sites of lesion in low and high shear stress conditions. Rolling, as the first step in the recruitment process, highly depends on endothelial selectins and their ligands on neutrophils, inducting of intracellular signaling and subsequently activating β2 integrins, enabling adhesion and postadhesion events. In addition, subcellular structures, such as microvilli, tethers, and slings allow the cell to arrest, even under high wall shear stress. Thereby, microvilli that are pulled out from the cell body form tethers that develop into slings upon their detachment from the substrate. In addition to the above-described primary capture, secondary capture of neutrophils via neutrophil–neutrophil or neutrophil–platelet interaction promotes the process of neutrophil recruitment to sites of lesion. Thus, precise mechanisms based on a complex molecular interplay, subcellular structures, and cell–cell interactions turn the delicate process of neutrophil trafficking during flow into a robust response allowing effective neutrophil accumulation at sites of injury.</jats:p
Small interfering RNA-mediated connexin gene knockdown in vascular endothelial and smooth muscle cells
No full textGlobal knockout of vascular connexins can result in premature/neonatal death, severe developmental complications, or compensatory up-regulation of different connexin isoforms. Thus, specific connexin gene knockdown using RNAi-mediated technologies is a technique that allows investigators to efficiently monitor silencing effects of single or multiple connexin gene products. The present chapter describes the transient knockdown of connexins in vitro and ex vivo for cells of the blood vessel wall. In detail, different transfection methods for primary endothelial cells and ex vivo thoracodorsal arteries are described. Essential controls for validating transfection efficiency as well as targeted gene knockdown are explained. These protocols provide researchers with the ability to modify connexin gene expression levels in a multitude of experimental setups
Decoding the signaling profile of hematopoietic progenitor kinase 1 (HPK1) in innate immunity: A proteomic approach
No full textSignaling via beta(2) integrins (CD11/CD18) as well as TCRs and BCRs involves similar pathways. However, the activation of the same signaling molecule can result in opposing effects. One such example is the hematopoietic progenitor kinase 1 (HPK1), which negatively regulates T and B cell activation but enforces neutrophil adhesion via beta(2) integrins. This difference may be defined by specific HPK1 interacting networks in different leukocyte subsets which have already been described in the adaptive immune system. Here, we set out to identify interacting proteins of HPK1 in neutrophil-like differentiated HL-60 cells exposed to immobilized fibrinogen and left nonactivated or Mn2+-activated to allow beta(2) integrin-dependent adhesion. Co-IP experiments followed by mass spectrometry led to the identification of 115 HPK1-interacting proteins. A total of 58 proteins were found only in nonactivated cells and 39 proteins only in Mn2+-activated adherent cells. From these results, we decoded a pre-existing signaling cluster of HPK1 in nonactivated cells encompassing proteins essential for beta(2) integrin-mediated signaling during neutrophil trafficking, namely DNAX-activation protein 12 (DAP12), spleen tyrosine kinase (Syk), and Rac1. Thus, our study provides novel insights into the complex architecture of the signaling processes during neutrophil activation and the complex signaling profile of HPK1 in leukocytes
Molecular Insights Into Neutrophil Biology From the Zebrafish Perspective: Lessons From CD18 Deficiency
No full textNeutrophils are key players in innate immunity and originate from the bone marrow of the adult mammalian organism. In mammals, mature neutrophils are released from the bone marrow into the peripheral blood where they circulate until their recruitment to sites of inflammation in a multistep adhesion cascade. Here, adhesion molecules of the beta(2) integrin family (CD11/CD18) are critically required for the initial neutrophil adhesion to the inflamed endothelium and several post-adhesion steps allowing their extravasation into the inflamed tissue. Within the mammalian tissue, interstitial neutrophil migration can occur widely independent of beta(2) integrins. This is in sharp contrast to neutrophil recruitment in zebrafish larvae (Danio rerio) where neutrophils originate from the caudal hematopoietic tissue and mainly migrate interstitially to sites of lesion upon the early onset of inflammation. However, neutrophils extravasate from the circulation to the inflamed tissue in zebrafish larvae at later-time points. Although zebrafish larvae are a widely accepted model system to analyze neutrophil trafficking in vivo, the functional impact of beta(2) integrins for neutrophil trafficking during acute inflammation is completely unknown in this model. In this study, we generated zebrafish with a genetic deletion of CD18, the beta subunit of beta(2) integrins, using CRISPR/Cas9 technology. Sequence alignments demonstrated a high similarity of the amino acid sequences between zebrafish and human CD18 especially in the functionally relevant I-like domain. In addition, the cytoplasmic domain of CD18 harbors two highly conserved NXXF motifs suggesting that zebrafish CD18 may share functional properties of human CD18. Accordingly, CD18 knock-out (KO) zebrafish larvae displayed the key symptoms of patients suffering from leukocyte adhesion deficiency (LAD) type I due to defects in ITGB2, the gene for CD18. Importantly, CD18 KO zebrafish larvae showed reduced neutrophil trafficking to sites of sterile inflammation despite the fact that an increased number of neutrophils was detectable in the circulation. By demonstrating the functional importance of CD18 for neutrophil trafficking in zebrafish larvae, our findings shed new light on neutrophil biology in vertebrates and introduce a new model organism for studying LAD type I
Structure and emerging functions of LRCH proteins in leukocyte biology
No full textActin-dependent leukocyte trafficking and activation are critical for immune surveillance under steady state conditions and during disease states. Proper immune surveillance is of utmost importance in mammalian homeostasis and it ensures the defense against pathogen intruders, but it also guarantees tissue integrity through the continuous removal of dying cells or the elimination of tumor cells. On the cellular level, these processes depend on the precise reorganization of the actin cytoskeleton orchestrating, e.g., cell polarization, migration, and vesicular dynamics in leukocytes. The fine-tuning of the actin cytoskeleton is achieved by a multiplicity of actin-binding proteins inducing, e.g., the organization of the actin cytoskeleton or linking the cytoskeleton to membranes and their receptors. More than a decade ago, the family of leucine-rich repeat (LRR) and calponin homology (CH) domain-containing (LRCH) proteins has been identified as cytoskeletal regulators. The LRR domains are important for protein-protein interactions and the CH domains mediate actin binding. LRR and CH domains are frequently found in many proteins, but strikingly the simultaneous expression of both domains in one protein only occurs in the LRCH protein family. To date, one LRCH protein has been described in drosophila and four LRCH proteins have been identified in the murine and the human system. The function of LRCH proteins is still under investigation. Recently, LRCH proteins have emerged as novel players in leukocyte function. In this review, we summarize our current understanding of LRCH proteins with a special emphasis on their function in leukocyte biology
