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Recruitment of neutrophils from the intravascular compartment into injured tissue is an essential component of the inflammatory response. It involves intracellular trafficking of vesicles within neutrophils and endothelial cells, both containing numerous proteins that have to be distributed in a tightly controlled and precise spatiotemporal fashion during the recruitment process. Rab proteins, a family of small GTPases, together with their effectors, are the key players in guiding and regulating the intracellular vesicle trafficking machinery during neutrophil recruitment. This review will provide a short overview on this process and highlight new findings as well as current controversies in the field. [ABSTRACT FROM AUTHOR]

Hypothesizing that therapeutic use of DEL-1 might be an interesting and effective way to attenuate hypertension-induced cardiovascular remodeling, Failer and colleagues performed an excellent study to address a potential beneficial effect of therapeutic DEL-1 in hypertension-induced cardiovascular disease [[7]]. Accordingly, recruitment of neutrophils and monocytes as well as T-cells into the arterial wall has been well-documented in patients with hypertension as well as in various animal models, including angiotensin II-induced hypertension [[2]]. [Extracted from the article]

Talin is an integrin adaptor, which controls integrin activity in all hematopoietic cells. How intracellular signals promote talin binding to the integrin tail leading to integrin activation is still poorly understood, especially in leukocytes. In vitro studies identified an integrin activation complex whose formation is initiated by the interaction of active, guanosine triphosphate (GTP)-bound Ras-related protein 1 (Rap1) with the adapter protein Rap1-GTP-interacting adapter molecule (RIAM) followed by the recruitment of talin to the plasma membrane. Unexpectedly, loss-of-function studies in mice have shown that the talin-activating role of RIAM is neither required for development nor for integrin activation in platelets. In this study, we show that leukocyte integrin activation critically depends on RIAM both in vitro and in vivo. RIAM deficiency results in a loss of β2 integrin activation in multiple leukocyte populations, impaired leukocyte adhesion to inflamed vessels, and accumulation in the circulation. Surprisingly, however, the major leukocyte β1 integrin family member, α4β1, was only partially affected by RIAM deficiency in leukocytes. Thus, although talin is an essential, shared regulator of all integrin classes expressed by leukocytes, we report that β2 and α4 integrins use different RIAM-dependent and -independent pathways to undergo activation by talin. [ABSTRACT FROM AUTHOR]

The ability of leukocytes to navigate through the different body compartments is an essential component for functioning immune defense and surveillance systems. In order to exit the blood circulation, leukocytes follow distinct recruitment steps, including capture of free-flowing leukocytes to, and rolling along, the vessel wall; firm leukocyte arrest on the endothelial lining; and postarrest modifications (spreading and crawling), which prepare the leukocyte for transmigration through the vascular wall. Post-translational glycosylation (including sialylation) has been known for many years to be functionally relevant for selectin ligands and, hence, selectin-mediated capture and rolling. Recently, sialylation by the α2-3 sialyltransferase ST3Gal-IV was identified to significantly influence chemokine-triggered firm leukocyte arrest, expanding the role of α2-3 sialylation from leukocyte rolling to subsequent chemokine-triggered leukocyte arrest. These findings make ST3Gal-IV an interesting drug target for modulating leukocyte trafficking in human disorders, including autoimmune diseases and cancer. [ABSTRACT FROM AUTHOR]

Abstract: Recruitment of innate immune cells from the vasculature into infected tissue is a key event in primary host defense against invading pathogens. This highly regulated process requires a functional interplay of specialized adhesion molecules and involves a series of steps leading from rolling of leukocytes along the endothelium to firm adhesion and finally transmigration. In the developing fetus, innate immune functions are ontogenetically regulated and show increasing maturation throughout gestation. Developmental differences in the innate immune response leave the neonate and especially the premature newborn at high risk of severe infections. Understanding the ontogeny of immune functions in the fetus and newborn is therefore essential for the prevention and treatment of neonatal infections. In this review, an overview will be given of the developmental aspects of innate immune cell recruitment including a discussion of controversial findings and open questions. [Copyright &y& Elsevier]

The transmittance of force from the actin cytoskeleton via integrins to extracellular ligands is essential for multiple aspects of leukocyte function. In addition, integrins must be efficiently linked to the cytoskeleton in order to resist external forces that act on the cell. Recently, the mammalian actin-binding protein 1 (mAbp1) was identified as a novel adaptor involved in linking adhesion molecules of the β2 integrin family to the actin cytoskeleton, indicating that this protein might have a fundamental impact on leukocyte functions that are associated explicitly with force transmittance; namely, intraluminal adhesion and phagocytosis. Here, we review the current understanding of the molecular and cellular functions of mAbp1 with a focus on its impact in leukocyte biology. [ABSTRACT FROM AUTHOR]

Leukocyte recruitment encompasses cell adhesion and activation steps that enable circulating leukocytes to roll, arrest, and firmly adhere on the endothelial surface before they extravasate into distinct tissue locations. This complex sequence of events relies on adhesive interactions between surface structures on leukocytes and endothelial cells and also on signals generated during the cell–cell contacts. Cell surface glycans play a crucial role in leukocyte recruitment. Several glycosyltransferases such as α1,3 fucosyltransferases, α2,3 sialyltransferases, core 2 N-acetylglucosaminlytransferases, β1,4 galactosyltransferases, and polypeptide N-acetylgalactosaminyltransferases have been implicated in the generation of functional selectin ligands that mediate leukocyte rolling via binding to selectins. Recent evidence also suggests a role of α2,3 sialylated carbohydrate determinants in triggering chemokine-mediated leukocyte arrest and influencing β1 integrin function. The recent discovery of galectin- and siglec-dependent processes further emphasizes the significant role of glycans for the successful recruitment of leukocytes into tissues. Advancing the knowledge on glycan function into appropriate pathology models is likely to suggest interesting new therapeutic strategies in the treatment of immune- and inflammation-mediated diseases. [ABSTRACT FROM AUTHOR]

Modification of glycoproteins by the attachment of fucose residues is widely distributed in nature. The importance of fucosylation has recently been underlined by identification of the monogenetic inherited human disease "congenital disorder of glycosylation IIc," also termed "leukocyte adhesion deficiency II." Due to defective Golgi GDP-fucose transporter (SLC35C1) activity, patients show a hypofucosylation of glycoproteins and present clinically with mental and growth retardation, persistent leukocytosis, and severe infections. To investigate effects induced by the loss of fucosylated structures in different organs, we generated a mouse model for the disease by inactivating the Golgi GDP-transporter gene (Slc35c1). Lectin binding studies revealed a tremendous reduction of fucosylated glycoconjugates in tissues and isolated cells from Slc35c1-/- mice. Fucose treatment of cells from different organs led to partial normalization of the fucosylation state of glycoproteins, thereby indicating an alternative GDP-fucose transport mechanism. Slc35c1-deficient mice presented with severe growth retardation, elevated postnatal mortality rate, dilatation of lung alveoles, and hypocellular lymph nodes. In vitro and in vivo leukocyte adhesion and rolling assays revealed a severe impairment of P-, E-, and L-selectin ligand function. The diversity of these phenotypic aspects demonstrates the broad general impact of fucosylation in the mammalian organism. [ABSTRACT FROM AUTHOR]

Leukocyte rolling is an important step for the successful recruitment of leukocytes into tissue and occurs predominantly in inflamed microvessels and in high endothelial venules of secondary lymphoid organs. Leukocyte rolling is mediated by a group of C-type lectins, termed selectins. Three different selectins have been identified – P-, E- and L-selectin – which recognize and bind to crucial carbohydrate determinants on selectin ligands. Among selectin ligands, P-selectin glycoprotein ligand-1 is the main inflammatory selectin ligand, showing binding to all three selectins under in vivo conditions. Functional relevant selectin ligands expressed on high endothelial venules of lymphoid tissue are less clearly defined at the protein level. However, high endothelial venule-expressed selectin ligands were instrumental in uncovering the crucial role of post-translational modifications for selectin ligand activity. Several glycosyltransferases, such as core 2 β1,6- N-acetylglucosaminyltransferase-I, β1,4-galactosyltransferases, α1,3-fucosyltransferases and α2,3-sialyltransferases have been described to participate in the synthesis of core 2 decorated O-glycan structures carrying the tetrasaccharide sialyl Lewis X, a carbohydrate determinant on selectin ligands with binding activity to all three selectins. In addition, modifications, such as carbohydrate or tyrosine sulfation, were also found to contribute to the synthesis of functional selectin ligands. [ABSTRACT FROM AUTHOR]

Objective. Symptomatic patent ductus arteriosus (sPDA) is a common problem in premature infants and can be treated effectively with intravenous indomethacin, leading to permanent ductal closure in 70% to 80% of infants. Infants who do not respond to pharmacologic closure of the duct ultimately have to undergo surgical or interventional closure of the PDA. Optimizing the pharmacologic treatment could offer an interesting approach to reduce the number of infants who need surgical closure of the duct. Methods. We conducted a retrospective analysis in infants who were <33 weeks' gestation, had sPDA, and were treated with high-dose intravenous indomethacin. From 1993 to 2002, 129 infants with sPDA received indomethacin after diagnosis of sPDA was confirmed by echocardiography. Treatment was started in all infants with intravenous indomethacin (0.2 mg/kg given 5 times at 0 hours, 12 hours, 24 hours, 48 hours, and 72 hours). When the ductus was still open at 36 hours, indomethacin every 12 hours was continued and single doses increased up to 1 mg/kg until ductal closure was achieved. Results. in 68 (53%) of 129 infants who were treated with indomethacin, ductal closure occurred during intermediate-dose indomethacin therapy (up to 1.5 mg/kg total dose). In the 61 initial nonresponders, the continuation of indomethacin led to ductal closure in 59 infants. When infants who were treated with an intermediate dose were compared with the initial nonresponders, no differences in the incidences of renal or electrolyte abnormalities, gastrointestinal bleeding, intraventricular hemorrhage, or periventricular leukomalacia were found. Conclusions. High-dose indomethacin after intermediate-dose therapy resulted in an overall closure rate of 98.5% (127 of 129). Although single indomethacin doses of up to 1 mg/kg were given, high-dose indomethacin was safe. [ABSTRACT FROM AUTHOR]

P-selectin glycoprotein ligand-1 (PSGL-1) mediates rolling of leukocytes on P-selectin under flow. The glycoproteins that enable leukocyte tethering to or rolling on E-selectin are not known. We used gene targeting to prepare PSGL-1-deficient (PSGL-1-/-) mice, which were healthy but had moderately elevated total blood leukocytes. Fluid-phase E-selectin bound to approximately 70% fewer sites on PSGL-1-/- than PSGL-1+/+ neutrophils. Compared with PSGL-1+/+ leukocytes, significantly fewer PSGL-1-/- leukocytes rolled on E-selectin in vitro, because their initial tethering to E-selectin was impaired. The residual cells that tethered rolled with the same shear resistance and velocities as PSGL-1+/+ leukocytes. Compared with PSGL-1+/+ mice, significantly fewer PSGL-1-/- leukocytes rolled on E-selectin in TNF-alpha-treated venules of cremaster muscle in which P-selectin function was blocked by an mAb. The residual PSGL-1-/- leukocytes that tethered rolled with slow velocities equivalent to those of PSGL-1+/+ leukocytes. These results reveal a novel function for PSGL-1 in tethering leukocytes to E-selectin under flow. [ABSTRACT FROM AUTHOR]

Eosinophil recruitment is a pathological hallmark of many allergic and helminthic diseases. Here, we investigated chemokine receptor CCR3-induced eosinophil recruitment in sialyltransferase St3gal4-/- mice. We found a marked decrease in eosinophil extravasation into CCL11-stimulated cremaster muscles and into the inflamed peritoneal cavity of St3gal4-/- mice. Ex vivo flow chamber assays uncovered reduced adhesion of St3gal4-/- compared to wild type eosinophils. Using flow cytometry, we show reduced binding of CCL11 to St3gal4-/- eosinophils. Further, we noted reduced binding of CCL11 to its chemokine receptor CCR3 isolated from St3gal4-/- eosinophils. This was accompanied by almost absent CCR3 internalization of CCL11-stimulated St3gal4-/- eosinophils. Applying an ovalbumin-induced allergic airway disease model, we found a dramatic reduction in eosinophil numbers in bronchoalveolar lavage fluid following intratracheal challenge with ovalbumin in St3gal4-deficient mice. Finally, we also investigated tissue-resident eosinophils under homeostatic conditions and found reduced resident eosinophil numbers in the thymus and adipose tissue in the absence of ST3Gal-IV. Taken together, our results demonstrate an important role of ST3Gal-IV in CCR3-induced eosinophil recruitment in vivo rendering this enzyme an attractive target in reducing unwanted eosinophil infiltration in various disorders including allergic diseases. [ABSTRACT FROM AUTHOR]

The use of cell-based reporter systems has provided valuable insights into the molecular mechanisms of integrin activation. However, current models have significant drawbacks because their artificially expressed integrins cannot be regulated by either physiological stimuli or endogenous signaling pathways. Here, we report the generation of a Hoxb8 cell line expressing human β2 integrin that functionally replaced the deleted mouse ortholog. Hoxb8 cells are murine hematopoietic progenitor cells that can be efficiently differentiated into neutrophils and macrophages resembling their primary counterparts. Importantly, these cells can be stimulated by physiological stimuli triggering classical integrin inside-out signaling pathways, ultimately leading to β2 integrin conformational changes that can be recorded by the conformation-specific antibodies KIM127 and mAb24. Moreover, these cells can be efficiently manipulated via the CRISPR/Cas9 technique or retroviral vector systems. Deletion of the key integrin regulators talin1 and kindlin3 or expression of β2 integrins with mutations in their binding sites abolished both integrin extension and full activation regardless of whether only one or both activators no longer bind to the integrin. Moreover, humanized β2 integrin Hoxb8 cells represent a valuable new model for rapidly testing the role of putative integrin regulators in controlling β2 integrin activity in a physiological context. [ABSTRACT FROM AUTHOR]

A commetary on an article on the synthesis and structural characterization of carboxyethylpyrrole (CEP) is presented.

Aims Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA. Methods and results Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager −/− mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager −/− mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager −/− mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-κB activation. Conclusion We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA–RAGE interaction thus represents a novel mechanism in thrombo-inflammation. [ABSTRACT FROM AUTHOR]

The mammalian sterile 20-like (MST) kinases are central constituents of the evolutionary ancient canonical Hippo pathway regulating cell proliferation and survival. However, perhaps surprisingly, MST1 deficiency in human patients leads to a severe combined immunodeficiency syndrome with features of autoimmune disease. In line with this, Mst1 -deficient mice exhibit severe defects in lymphocyte and neutrophil functions as well as disturbed intracellular vesicle transport. These findings spurred research on the noncanonical functions of MST1 in leukocytes. Here, we summarise the latest findings on this topic and discuss MST1 as a critical regulator of various leukocyte functions. [ABSTRACT FROM AUTHOR]

A response by Markus Sperandi and colleagues to a letter to the editor about their article on ductal closure with the use of high-dose indomethacin in pre-term infants is presented.

The article announces that Luca Scorrano, one of the experts in the field of apoptosis and mitochondrial pathophysiology, has won the 2013 ESCI Award for Excellence in Basic/Translational Research.

The article announces that professor Triantafyllos Chavakis has received the 2012 European Society for Clinical Investigation (ESCI) Award for Excellence in Biomedical Investigation.

Aims Neutrophil trafficking within the vasculature strongly relies on intracellular calcium signalling. Sustained Ca2+ influx into the cell requires a compensatory efflux of potassium to maintain membrane potential. Here, we aimed to investigate whether the voltage-gated potassium channel KV1.3 regulates neutrophil function during the acute inflammatory process by affecting sustained Ca2+ signalling. Methods and results Using in vitro assays and electrophysiological techniques, we show that KV1.3 is functionally expressed in human neutrophils regulating sustained store-operated Ca2+ entry through membrane potential stabilizing K+ efflux. Inhibition of KV1.3 on neutrophils by the specific inhibitor 5-(4-Phenoxybutoxy)psoralen (PAP-1) impaired intracellular Ca2+ signalling, thereby preventing cellular spreading, adhesion strengthening, and appropriate crawling under flow conditions in vitro. Using intravital microscopy, we show that pharmacological blockade or genetic deletion of KV1.3 in mice decreased neutrophil adhesion in a blood flow dependent fashion in inflamed cremaster muscle venules. Furthermore, we identified KV1.3 as a critical component for neutrophil extravasation into the inflamed peritoneal cavity. Finally, we also revealed impaired phagocytosis of Escherichia coli particles by neutrophils in the absence of KV1.3. Conclusion We show that the voltage-gated potassium channel KV1.3 is critical for Ca2+ signalling and neutrophil trafficking during acute inflammatory processes. Our findings do not only provide evidence for a role of KV1.3 for sustained calcium signalling in neutrophils affecting key functions of these cells, they also open up new therapeutic approaches to treat inflammatory disorders characterized by overwhelming neutrophil infiltration. [ABSTRACT FROM AUTHOR]

Abstract: The innate immune system responds to inflammation, infection and injury by recruiting neutrophils and other leukocytes. These cells are able to leave the intravascular compartment in a process called leukocyte recruitment. This process involves several distinct steps: selectin-mediated rolling, firm adhesion via integrins, postarrest modifications including adhesion strengthening and leukocyte crawling and finally transmigration into tissue. Genetic defects affecting the different steps of the cascade can result in severe impairment in leukocyte recruitment. So far, three leukocyte adhesion deficiencies (LAD I-III) have been described in humans. These LADs are rare autosomal recessive inherited disorders and, although clinically distinct, exhibit several common features including recurrent bacterial infections and leukocytosis. In LAD-I, mutations within the β2-integrin gene result in a severe defect in β2 integrin-mediated firm leukocyte adhesion. Defects in the posttranslational fucosylation of selectin ligands dramatically reduce leukocyte rolling and lead to LAD-II. Finally, LAD-III, also known as LAD-I variant, is caused by impaired integrin activation due to mutations within the kindlin-3 gene. This review provides an overview on the molecular basis of leukocyte adhesion and its deficiencies. [Copyright &y& Elsevier]

Background: Pathological cardiac hypertrophy is a result of afterload-increasing pathologies including untreated hypertension and aortic stenosis. It features progressive adverse cardiac remodeling, myocardial dysfunction, capillary rarefaction, and interstitial fibrosis often leading to heart failure.Objectives: This study aimed to establish a novel porcine model of pressure-overload-induced heart failure and to determine the effect of inhibition of microribonucleic acid 132 (miR-132) on heart failure development in this model.Methods: This study developed a novel porcine model of percutaneous aortic constriction by implantation of a percutaneous reduction stent in the thoracic aorta, inducing progressive remodeling at day 56 (d56) after pressure-overload induction. In this study, an antisense oligonucleotide specifically inhibiting miR-132 (antimiR-132), was regionally applied via intracoronary injection at d0 (percutaneous transverse aortic constriction induction) and d28.Results: At d56, antimiR-132 treatment diminished cardiomyocyte cross-sectional area (188.9 ± 2.8 vs. 258.4 ± 9.0 μm2 in untreated hypertrophic hearts) and improved global cardiac function (ejection fraction 48.9 ± 1.0% vs. 36.1 ± 1.7% in control hearts). Moreover, at d56 antimiR-132-treated hearts displayed less increase of interstitial fibrosis compared with sham-operated hearts (Δsham 1.8 ± 0.5%) than control hearts (Δsham 10.8 ± 0.6%). Of note, cardiac platelet and endothelial cell adhesion molecule 1+ capillary density was higher in the antimiR-132-treated hearts (647 ± 20 cells/mm2) compared with in the control group (485 ± 23 cells/mm2).Conclusions: The inhibition of miR-132 is a valid strategy in prevention of heart failure progression in hypertrophic heart disease and may be developed as a treatment for heart failure of nonischemic origin. [ABSTRACT FROM AUTHOR]

At the transition from intrauterine to postnatal life, drastic alterations are mirrored by changes in cellular immunity. These changes are in part immune cell intrinsic, originate in the replacement of fetal cells, or result from global regulatory mechanisms and adaptation to changes in the tissue microenvironment. Overall, longer developmental trajectories are intersected by events related to mother-infant separation, birth cues, acquisition of microbiota and metabolic factors. Perinatal alterations particularly affect immune niches, where structures with discrete functions meet, the intestinal mucosa, epidermis and lung. Accordingly, the following questions will be addressed in this review: How does the preprogrammed development supported by endogenous cues, steer innate immune cell differentiation, adaptation to tissue structures, and immunity to infection? How does the transition at birth impact on tissue immune make-up including its topology? How do postnatal cues guide innate immune cell differentiation and function at immunological niches? [ABSTRACT FROM AUTHOR]

Leukocytes are rapidly recruited to sites of inflammation via interactions with the vascular endothelium. The steroid hormone dehydroepiandrosterone (DHEA) exerts anti-inflammatory properties; however, the underlying mechanisms are poorly understood. In this study, we show that an anti-inflammatory mechanism of DHEA involves the regulation of developmental endothelial locus 1 (DEL-1) expression. DEL-1 is a secreted homeostatic factor that inhibits b2-integrin-dependent leukocyte adhesion, and the subsequent leukocyte recruitment and its expression is downregulated upon inflammation. Similarly, DHEA inhibited leukocyte adhesion to the endothelium in venules of the inflamed mouse cremaster muscle. Importantly, in a model of lung inflammation, DHEA limited neutrophil recruitment in a DEL-1-dependent manner. Mechanistically, DHEA counteracted the inhibitory effect of inflammation on DEL-1 expression. Indeed, whereas TNF reduced DEL-1 expression and secretion in endothelial cells by diminishing C/EBPb binding to the DEL-1 gene promoter, DHEA counteracted the inhibitory effect of TNF via activation of tropomyosin receptor kinase A (TRKA) and downstream PI3K/AKT signaling that restored C/EBPb binding to the DEL-1 promoter. In conclusion, DHEA restrains neutrophil recruitment by reversing inflammation-induced downregulation of DEL-1 expression. Therefore, the anti-inflammatory DHEA/DEL-1 axis could be harnessed therapeutically in the context of inflammatory diseases. [ABSTRACT FROM AUTHOR]

The recruitment of neutrophils to sites of inflammation, their battle against invading microorganisms through phagocytosis and the release of antimicrobial agents is a highly coordinated and tightly regulated process that involves the interplay of many different receptors, ion channels and signalling pathways. Changes in intracellular calcium levels, caused by cytosolic Ca2+ store depletion and the influx of extracellular Ca2+ via ion channels, play a critical role in synchronizing neutrophil activation and function. In this review, we provide an overview of how Ca2+ signalling is initiated in neutrophils and how changes in intracellular Ca2+ levels modulate neutrophil function. [ABSTRACT FROM AUTHOR]

Background: The development of models that incorporate intact microvascular networks enables the investigation of multicellular dynamics during angiogenesis. Our laboratory introduced the rat mesentery culture model as such a tool, which would be enhanced with mouse tissue. Since mouse mesentery is avascular, an alternative is mouse mesometrium, the connective tissue of uterine horns. The study’s objective was to demonstrate that mouse mesometrium contains microvascular networks that can be cultured to investigate multicellular dynamics during angiogenesis. Methods: Harvested mesometrium tissues from C57Bl/6 female mice were cultured in media with serum for up to 7 days. PECAM, NG2, αSMA, and LYVE-1 labeling identified endothelial cells, pericytes, smooth muscle cells, and lymphatic endothelial cells, respectively. Results: These cells comprised microvascular networks with arterioles, venules, and capillaries. Compared to day 0, capillary sprouts per vascular length were increased by 3 and 5 days in culture (day 0, 0.08 ± 0.01; day 3, 3.19 ± 0.78; day 5, 2.49 ± 0.05 sprouts/mm; p < 0.05). Time-lapse imaging of cultured tissues from FlkEGFP mice showcases the use of the model for lineage studies. The impact is supported by the identification of endothelial cell jumping from one sprout to another. Conclusion: These results introduce a novel culture model for investigating multicellular dynamics during angiogenesis in real-time ex vivo microvascular networks. [ABSTRACT FROM AUTHOR]

Neutrophil extravasation and interstitial migration are important steps during the recruitment of neutrophils to sites of inflammation. In the present study, we addressed the functional importance of the unconventional class I myosin 1f (Myo1f) for neutrophil trafficking during acute inflammation. In contrast to leukocyte rolling and adhesion, the genetic absence of Myo1f severely compromised neutrophil extravasation into the inflamed mouse cremaster tissue when compared with Myo1f1/1 mice as studied by intravital microscopy. Similar results were obtained in experimental models of acute peritonitis and acute lung injury. In contrast to 2-dimensional migration, which occurred independently of Myo1f, Myo1f was indispensable for neutrophil migration in 3-dimensional (3D) environments, that is, transmigration and migration in collagen networks as it regulated squeezing and dynamic deformation of the neutrophil nucleus during migration through physical barriers. Thus, we provide evidence for an important role of Myo1f in neutrophil trafficking during inflammation by specifically regulating neutrophil extravasation and migration in 3D environments. [ABSTRACT FROM AUTHOR]

Trafficking of polymorphonuclear neutrophils (PMNs) during inflammation critically depends on the β2 integrins lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18) and macrophage-1 antigen (CD11b/CD18). Here, we identify coronin 1A (Coro1A) as a novel regulator of β2 integrins that interacts with the cytoplasmic tail of CD18 and is crucial for induction of PMN adhesion and postadhesion events, including adhesion strengthening, spreading, and migration under flow conditions. Transition of PMN rolling to firm adhesion critically depends on Coro1A by regulating the accumulation of highaffinity LFA-1 in focal zones of adherent cells. Defective integrin affinity regulation in the genetic absence of Coro1A impairs leukocyte adhesion and extravasation in inflamed cremaster muscle venules in comparison with control animals. In a Helicobacter pylori mouse infection model, PMN infiltration into the gastric mucosa is dramatically reduced in Coro1A-/- mice, resulting in an attenuated gastric inflammation. Thus, Coro1A represents an important novel player in integrin biology, with key functions in PMN trafficking during innate immunity. [ABSTRACT FROM AUTHOR]

Neutrophils and monocytes belong to the first line of immune defence cells and are recruited to sites of inflammation during infection or sterile injury. Both cells contain huge amounts of the heterodimeric protein S100A8/A9 in their cytoplasm. S100A8/A9 belongs to the Ca 2 + binding S100 protein family and has recently gained a lot of interest as a critical alarmin modulating the inflammatory response after its release (extracellular S100A8/A9) from neutrophils and monocytes. Extracellular S100A8/A9 interacts with the pattern recognition receptors Toll-like receptor 4 (TLR4) and Receptor for Advanced Glycation Endproducts (RAGE) promoting cell activation and recruitment. Besides its biological function, S100A8/A9 (also known as myeloid related protein 8/14, MRP8/14) was identified as interesting biomarker to monitor disease activity in chronic inflammatory disorders including inflammatory bowel disease and rheumatoid arthritis. Furthermore, S100A8/A9 has been tested successfully in pre-clinical imaging studies to localize sites of infection or sterile injury. Finally, recent evidence using small molecule inhibitors for S100A8/A9 also suggests that blocking S100A8/A9 activity exerts beneficial effects on disease activity in animal models of autoimmune diseases including multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis and inflammatory bowel disease. This review will provide a comprehensive and detailed overview into the structure and biological function of S100A8/A9 and also will give an outlook in terms of diagnostic and therapeutic applications targeting S100A8/A9. [ABSTRACT FROM AUTHOR]

Neutrophils need to penetrate the perivascular basement membrane for successful extravasation into inflamed tissue, but this process is incompletely understood. Recent findings have associated mammalian sterile 20-like kinase 1 (MST1) loss of function with a human primary immunodeficiency disorder, suggesting that MST1 may be involved in immune cell migration. Here, we have shown that MST1 is a critical regulator of neutrophil extravasation during inflammation. Mst1-deficient (Mst1-/-) neutrophils were unable to migrate into inflamed murine cremaster muscle venules, instead persisting between the endothelium and the basement membrane. Mst1-/- neutrophils also failed to extravasate from gastric submucosal vessels in a murine model of Helicobacter pylori infection. Mechanistically, we observed defective translocation of VLA-3, VLA-6, and neutrophil elastase from intracellular vesicles to the surface of Mst1-/- neutrophils, indicating that MST1 is required for this crucial step in neutrophil transmigration. Furthermore, we found that MST1 associates with the Rab27 effector protein synaptotagmin-like protein 1 (JFC1, encoded by Sytl1 in mice), but not Munc13-4, thereby regulating the trafficking of Rab27-positive vesicles to the cellular membrane. Together, these findings highlight a role for MST1 in vesicle trafficking and extravasation in neutrophils, providing an additional mechanistic explanation for the severe immune defect observed in patients with MST1 deficiency. [ABSTRACT FROM AUTHOR]

ARHGAP25 is a Rac-specific GTPase-activating protein that is expressed primarily in hematopoietic cells. The involvement of ARHGAP25 in regulating the recruitment of leukocytes to inflammatory sites was investigated in genetically modified mice. Using intravital microscopy, we show that Arhgap25 deficiency affects all steps of leukocyte recruitment with a predominant enhancement of transendothelial migration of neutrophilic granulocytes. Increased transmigration of Arhgap25-deficient leukocytes is demonstrated in inflamed cremaster muscle venules, in a peritonitis model, and in an in vitro chemotaxis assay. Using bone marrow chimeric mice lacking ARHGAP25 in the hematopoietic compartment, we show that enhanced migration in the absence of ARHGAP25 is due to defective leukocyte function. In search for potential mechanisms of ARHGAP25-regulated migration of neutrophils, we detected an increase in the amount of active, GTP-bound Rac and Rac-dependent cytoskeletal changes in the absence of ARHGAP25, suggesting a critical role of ARHGAP25 in counterbalancing the Rac-activating effect of nucleotide exchange factors. Taken together, using Arhgap25-deficient mice, we identified ARHGAP25 as a relevant negative regulator of leukocyte transendothelial migration. [ABSTRACT FROM AUTHOR]

ARHGAP25 is a Rac-specific GTPase-activating protein that is expressed primarily in hematopoietic cells. The involvement of ARHGAP25 in regulating the recruitment of leukocytes to inflammatory sites was investigated in genetically modified mice. Using intravital microscopy, we show that Arhgap25 deficiency affects all steps of leukocyte recruitment with a predominant enhancement of transendothelial migration of neutrophilic granulocytes. Increased transmigration of Arhgap25-deficient leukocytes is demonstrated in inflamed cremaster muscle venules, in a peritonitis model, and in an in vitro chemotaxis assay. Using bone marrow chimeric mice lacking ARHGAP25 in the hematopoietic compartment, we show that enhanced migration in the absence of ARHGAP25 is due to defective leukocyte function. In search for potential mechanisms of ARHGAP25-regulated migration of neutrophils, we detected an increase in the amount of active, GTP-bound Rac and Rac-dependent cytoskeletal changes in the absence of ARHGAP25, suggesting a critical role of ARHGAP25 in counterbalancing the Rac-activating effect of nucleotide exchange factors. Taken together, using Arligap25-deficient mice, we identified ARHGAP25 as a relevant negative regulator of leukocyte transendothelial migration. [ABSTRACT FROM AUTHOR]

P-selectin ligands (P-ligs) support the recruitment of lymphocytes into inflamed tissues. Binding to P-selectin is mediated by oligosaccharide groups synthesized by means of several glycosyltransferases including core 2 ß1,6- N -acetylglucosaminyltransferase-I (C2GlcNAcT-I), encoded by the gene Gcnt1. Using Gcnt1 −/− Th1 cells, we show that C2GlcNAcT-I is crucial for inflammatory T cell homing in vivo. To understand the molecular regulation of Gcnt1 in CD4 + T helper cells, we performed ChIP-on-chip experiments across the Gcnt1 locus assessing the chromatin structure in P-lig-expressing versus non-expressing CD4 + T cells. This identified a distal region about 20 kb upstream of the promoter where the presence of a H3K27me3 mark correlated with Gcnt1 repression. This region possessed IL-12-dependent enhancer activity in reporter assays, in accordance with preferential IL-12-dependent induction of Gcnt1 in vitro . STAT4 and T-bet cooperated in control of the enhancer activity. Deficiency in either one resulted in drastically reduced Gcnt1 mRNA expression in differentiated Th1 cells. While both STAT4 and T-bet were bound to the enhancer early after activation only T-bet binding persisted throughout the expansion phase after TCR signal cessation. This suggests sequential action of STAT4 and T-bet at the enhancer. In summary, we show that Gcnt1 transcription and subsequent P-lig induction in Th1 cells is governed by binding of STAT4 and T-bet to a distal enhancer and further regulated by epigenetic marks such as H3K27me3. [ABSTRACT FROM AUTHOR]

Hematopoietic cells dependonintegrin-mediated adhesion and signaling,whichis induced by kindlin-3 and talin-1. To determine whether platelet and polymorphonuclear neutrophil (PMN) functions require specific thresholds of kindlin-3, we generated mouse strains expressing 50%, 10%, or5%of normal kindlin-3 levels.We report that in contrast to kindlin-3-null mice, which die perinatally of severe bleeding and leukocyte adhesion deficiency, mice expressing as little as 5% of kindlin-3 were viable and protected from spontaneous bleeding and infections. However, platelet adhesion and aggregation were reduced in vitro and bleeding times extended. Similarly, leukocyte adhesion, extravasation, and bacterial clearancewerediminished.Quantificationofproteincopynumbers revealedstoichiometric quantities of kindlin-3 and talin-1 in platelets and neutrophils, indicating that reduction of kindlin-3 in our mouse strains progressively impairs the cooperation with talin-1. Our findings show that very low levels of kindlin-3 enable basal platelet and neutrophil functions, whereas in stress situations such as injury and infection, platelets and neutrophils require a maximum of functional integrins that is achieved with high and stoichiometric quantities of kindlin-3 and talin-1. [ABSTRACT FROM AUTHOR]

Fetal haematopoiesis is a highly regulated process in terms of time and location. It is characterized by the emergence of specific cell populations at different extra- and intraembryonic anatomical sites. Trafficking of haematopoietic stem cells (HSCs) between these supportive niches is regulated by a set of molecules, i.e. integrins and chemokine receptors, which are also described for the recruitment of differentiated innate immune cells. In this review, an overview will be given on fetal haematopoiesis as well as trafficking of HSCs during fetal life. In addition, we will focus on the appearance of the first differentiated neutrophils and monocytes in the fetal circulation and describe how they acquire the ability to roll, adhere, and transmigrate into inflamed fetal tissue. Furthermore, we will discuss other effector functions of innate immune cells evolving during fetal ontogeny. [ABSTRACT FROM AUTHOR]

Background: Neutrophilic inflammation often persists for days despite effective antibiotic treatment and contributes to brain damage in bacterial meningitis. We propose here that myeloid-related protein 14 (MRP14), an abundant cytosolic protein in myeloid cells, acts as an endogenous danger signal, driving inflammation and aggravating tissue injury. Methods: The release pattern of MRP14 was analyzed in human and murine cerebrospinal fluid (CSF), as well as in isolated neutrophils. Its functional role was assessed in a mouse meningitis model, using MRP14-deficient mice. Results: We detected large quantities of MRP14 in CSF specimens from patients and mice with pneumococcal meningitis. Immunohistochemical analyses and a cell-depletion approach indicated neutrophils as the major source of MRP14. In a meningitis model, MRP14-deficient mice showed a better resolution of inflammation during antibiotic therapy, which was accompanied by reduced disease severity. Intrathecal administration of MRP14 before infection reverted the phenotype of MRP14-deficient mice back to wild type. Moreover, intrathecal injection of MRP14 alone was sufficient to induce meningitis in a Toll-like receptor 4 (TLR4)-CXCL2-dependent manner. Finally, treatment with the MRP14 antagonist paquinimod reduced inflammation and disease severity significantly, reaching levels comparable to those achieved after genetic depletion of MRP14. Conclusions: The present study implicates MRP14 as an essential propagator of inflammation and potential therapeutic target in pneumococcal meningitis. [ABSTRACT FROM AUTHOR]

Gut microbiota encompasses a wide variety of commensal microorganisms consisting of trillions of bacteria, fungi, and viruses. This microbial population coexists in symbiosis with the host, and related metabolites have profound effects on human health. In this respect, gut microbiota plays a pivotal role in the regulation of metabolic, endocrine, and immune functions. Bacterial metabolites include the short chain fatty acids (SCFAs) acetate (C2), propionate (C3), and butyrate (C4), which are the most abundant SCFAs in the human body and the most abundant anions in the colon. SCFAs are made from fermentation of dietary fiber and resistant starch in the gut. They modulate several metabolic pathways and are involved in obesity, insulin resistance, and type 2 diabetes. Thus, diet might influence gut microbiota composition and activity, SCFAs production, and metabolic effects. In this narrative review, we discuss the relevant research focusing on the relationship between gut microbiota, SCFAs, and glucose metabolism. [ABSTRACT FROM AUTHOR]

Emerging evidence suggests a role of the cytokine midkine (MK) in inflammation. In this study, its functional relevance for recruitment of polymorphonuclear neutrophils (PMNs) during acute inflammation was investigated. Intravital microscopy and histologic analysis of tumor necrosis factor-α-stimulated cremaster muscle venules revealed severely compromised leukocyte adhesion and extravasation in MK-/- mice compared with MK+/+animals. Systemic administration of recombinant MK completely rescued the adhesion defect in MK-/- mice. In a hind limb ischemia model, leukocyte accumulation in MK-/- mice was significantly diminished compared with MK+/+animals. However, MK did not lead to an inflammatory activation of PMNs or endothelial cells suggesting that it does not serve as classical proinflammatory cytokine. Unexpectedly, immobilized MK mediated PMN adhesion under static and flow conditions, whereas PMN-derived MK was dispensable for the induction of adhesion. Furthermore, adhesion strengthening remained unaffected by MK. Flow cytometry revealed that immobilized, but not soluble MK, significantly promoted the high affinity conformation of β2 integrins of PMNs. Blocking studies of low-density lipoprotein receptor-related protein 1 (LRP1) suggested that LRP1 may act as a receptor for MK on PMNs. Thus, MK seems to support PMN adhesion by promoting the high affinity conformation of β2 integrins, thereby facilitating PMN trafficking during acute inflammation. [ABSTRACT FROM AUTHOR]

ChemR23 is a chemotactic receptor expressed by APCs, such as dendritic cells, macrophages, and NK cells. Chemerin, the ChemR23 ligand, was detected by immunohistochemistry, to be associated with inflamed endothelial cells in autoimmune diseases, such as lupus erythematosus, psoriasis, and rheumatoid arthritis. This study reports that blood and lymphatic murine endothelial cells produce chemerin following retinoic acid stimulation. Conversely, proinflammatory cytokines, such as TNF-α, IFN-γ, and LPS, or calcitriol, are not effective. Retinoic acid-stimulated endothelial cells promoted dendritic cell adhesion under shear stress conditions and transmigration in a ChemR23-dependent manner. Activated endothelial cells upregulated the expression of the atypical chemotactic receptor CCRL2/ACKR5, a nonsignaling receptor able to bind and present chemerin to ChemR23+ dendritic cells. Accordingly, activated endothelial cells expressed chemerin on the plasma membrane and promoted in a more efficient manner chemerin-dependent transmigration of dendritic cells. Finally, chemerin stimulation of myeloid dendritic cells induced the high-affinity binding of VCAM-1/CD106 Fc chimeric protein and promoted VCAM-1-dependent arrest to immobilized ligands under shear stress conditions. In conclusion, this study reports that retinoic acid-activated endothelial cells can promote myeloid and plasmacytoid dendritic cell transmigration across endothelial cell monolayers through the endogenous production of chemerin, the upregulation of CCRL2, and the activation of dendritic cell β1 integrin affinity. [ABSTRACT FROM AUTHOR]

Septic shock is characterized by increased vascular permeability and hypotension despite increased cardiac output. Numerous vasoactive cytokines are upregulated during sepsis, including angiopoietin 2 (ANG2), which increases vascular permeability. Here we report that mice engineered to inducibly overexpress ANG2 in the endothelium developed sepsis-like hemodynamic alterations, including systemic hypotension, increased cardiac output, and dilatory cardiomyopathy. Conversely, mice with cardiomyocyte-restricted ANG2 overexpression failed to develop hemodynamic alterations. Interestingly, the hemodynamic alterations associated with endothelial-specific overexpression of ANG2 and the loss of capillary-associated pericytes were reversed by intravenous injections of adeno-associated viruses (AAVs) transducing cDNA for angiopoietin 1, a TIE2 ligand that antagonizes ANG2, or AAVs encoding PDGFB, a chemoattractant for pericytes. To confirm the role of ANG2 in sepsis, we i.p. injected LPS into C57BL/6J mice, which rapidly developed hypotension, acute pericyte loss, and increased vascular permeability. Importantly, ANG2 antibody treatment attenuated LPS-induced hemodynamic alterations and reduced the mortality rate at 36 hours from 95% to 61%. These data indicate that ANG2-mediated microvascular disintegration contributes to septic shock and that inhibition of the ANG2/TIE2 interaction during sepsis is a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Neutrophilic inflammation, which often persists over days despite appropriate antibiotic therapy, contributes substantially to brain damage in bacterial meningitis. We hypothesized that persistent inflammation is the consequence of a vicious cycle in which inflammation-induced cell injury leads to the release of endogenous danger molecules (e.g. high mobility group box 1) that drive the inflammatory response, causing further damage. The present study aimed to assess the mechanisms of high mobility group box 1 protein release and its functional relevance for the development and progression of pneumococcal meningitis. High mobility group box 1 was found in large quantities in cerebrospinal fluid samples of patients and mice with pneumococcal meningitis (predominantly in advanced stages of the disease). By using macrophages, we demonstrated that the release of high mobility group box 1 from macrophages following pneumococcal challenge is passive in nature and probably not connected with inflammasome- and oxidative stress-dependent inflammatory cell death forms. In a mouse meningitis model, treatment with the high mobility group box 1 antagonists ethyl pyruvate or Box A protein had no effect on the development of meningitis, but led to better resolution of inflammation during antibiotic therapy, which was accompanied by reduced brain pathology and better disease outcome. Additional experiments using gene-deficient mice and murine neutrophils provided evidence that high mobility group box 1 acts as a chemoattractant for neutrophils in a receptor for advanced glycosylation end products-dependent fashion. In conclusion, the present study implicated high mobility group box 1, likely released from dying cells, as a central propagator of inflammation in pneumococcal meningitis. Because persistent inflammation contributes to meningitis-associated brain damage, high mobility group box 1 may represent a promising target for adjunctive therapy of this disease. [ABSTRACT FROM AUTHOR]

Recruitment of polymorphonuclear neutrophils (PMNs) to sites of acute inflammation critically depends on β2 integrins (CD11/CD18). Recently, the mammalian actin-binding protein 1 (mAbpl) was identified as an important adaptor protein regulating PMN trafficking downstream of β2 integrins. Here, we show that mAbpl constitutively co-immunoprecipitated with hematopoietic progenitor kinase 1 (HPK1) in neutrophil-like differentiated HL-60 (dHL-60) cells. HPK1 was enriched at the lamellipodium of polarized dHL-60 cells, where it colocalized with mAbpl and actin. Functional analysis of PMNs from HPK1-deficient mice showed that HPK1 was critical for CXCL1-induced lymphocyte function-associated antigen 1 (LFA-l)-mediated PMN adhesion to ICAM-1 under flow conditions. Accordingly, CXCL1-mediated induction of high-affinity LFA-1 required HPK1, but macrophage antigen 1 (Mac-1) affinity regulation was independent of HPK1. Intravital microscopy of the mouse cremaster muscle confirmed the defect of CXCL1-induced leukocyte adhesion in HPK1-deficient mice. Furthermore, β2 integrin-mediated post-adhesion processes--adhesion strengthening, spreading, and directed mechanotactic crawling of PMNs under flow conditions--involved HPK1 in vitro and in vivo. Upon intrascrotal administration of tumor necrosis factor α(TNF-)α, PMN adhesion and extravasation were severely compromised in HPK1 -deficient mice. In summary, our results indicate that HPK1 is critically involved in LFA-1-mediated PMN trafficking during acute inflammation. [ABSTRACT FROM AUTHOR]

Binding of selectins to their glycan ligands is a prerequisite for successful leukocyte trafficking. During synthesis and transport through the secretory pathway, selectin ligands are constructed with the participation of one or more sialyl-transferases of the ST3Gal subfamily. Previous studies established that ST3Gal-IV only partially contributes to selectin li-gand formation, indicating that other ST3Gal-sialyltransferases are involved. By generating and analyzing St3gal6-nu\\ mice and St3gal4/St3gal6 double-deficient mice, in the present study, we found that binding of E- and P-selectin to neutrophils and L-selectin binding to lymph node high endothelial venules is reduced in the absence of ST3Gal-VI and to a greater extent in double-deficient mice. In an ex vivo flow chamber assay, P- and E-selectin-dependent leukocyte rolling was mildly reduced in Sf3ga/6-null mice and more severely in double-deficient mice. In inflamed cremaster muscle venules of St3gal6-nu\\ mice, we found impaired P-selectin-dependent, but not E-selectin-dependent leukocyte rolling, whereas in double-deficient mice, E-selectin-dependent rolling was almost completely absent. Furthermore, neutrophil recruitment into the inflamed peritoneal cavity and lymphocyte homing to secondary lymphoid organs were impaired in Sf3ga/6-null mice and more severely in double-deficient mice. The results of the present study demonstrate the coordinated participation of both ST3Gal-VI and ST3Gal-IV in the synthesis of functional selectin ligands. (Blood. 2012;120(5):1015-1026) [ABSTRACT FROM AUTHOR]

Recently, the mammalian actin-binding protein 1 (mAbpl; Hip-55, SH3P7, debrin-like protein) was identified as a novel component of the β2 integrin-mediated signaling cascade during complement-mediated phagocytosis and firm adhesion of polymorphonuclear neutrophils (PMN) under physiological shear stress conditions. In this study, we found that the genetic ablation of mAbpl severely compromised not only the induction of adhesion, but also subsequent spreading of leukocytes to the endothelium as assessed by intravital microscopy of inflamed vessels of the cremaster muscle of mice. In vitro studies using murine PMN confirmed that mAbpl was required for β2 integrin-mediated spreading under shear stress conditions, whereas mAbpl was dispensable for spreading under static conditions. Upon β2 integrin-mediated adhesion and chemotactic migration of human neutrophil-Iike differentiated HL-60 cells, mAbpl was enriched at the leading edge of the polarized cell. Total internal reflection fluorescence microscopy revealed that mAbpl formed propagating waves toward the front of the lamellipodium, which are characteristic for dynamic reorganization of the cytoskeleton. Accordingly, binding of mAbpl to actin was increased upon β2 integrin-mediated adhesion, as shown by coimmunoprecipitation experiments. However, chemotactic migration under static conditions was unaf-fected in the absence of mAbpl. In contrast, the downregulation of mAbpl by RNA interference technique in neutrophil-Iike differentiated HL-60 cells or the genetic ablation of mAbpl in leukocytes led to defective migration under flow conditions in vitro and in inflamed cremaster muscle venules in the situation in vivo. In conclusion, mAbpl is of fundamental importance for spreading and migration under shear stress conditions, which are critical prerequisites for efficient PMN extravasation during inflammation. [ABSTRACT FROM AUTHOR]

Neutrophils form a vital part of the innate immune response, but at the same time their inappropriate activation contributes to autoimmune diseases. Many molecular components are involved in fine-tuning neutrophil function. We report here the first characterization of the role of ARAP3, a PI3K and Rap-regulated GTPase-activating protein for RhoA and Arf6 in murine neutrophils. We show that neutrophils lacking ARAP3 are preactivated in vitro and in vivo, exhibiting increased β2 integrin affinity and avidity. ARAP3-deficient neutrophils are hyperresponsive in several adhesion-dependent situations in vitro, including the formation of reactive oxygen species, adhesion, spreading, and granule release. ARAP3-deficient cells adhere more firmly under flow conditions in vitro and to the vessel wall in vivo. Finally, loss of ARAP3 interferes with integrin-dependent neutrophil chemotaxis. The results of the present study suggest an important function of ARAP3 downstream of Rap. By modulating β2 integrin activity, ARAP3 guards neutrophils in their quiescent state unless activated. [ABSTRACT FROM AUTHOR]

Despite Imatinib's remarkable success in chronic myelogenous leukemia treatment, monotherapy frequently causes resistance, underlining the rationale for combination chemotherapy. A potential approach would be interrupting the SDF-1/CXCR4 axis using the selective CXCR4 antagonist Plerixafor (previously AMD3100), as this axis has been reported to provide survival-enhancing effects to myeloid progenitor cells. By efficient CXCR4 blocking in the CXCR4+/BCR-ABL+ cell line BV-173, plerixafor (1-100 μM) significantly inhibits SDF-1α-mediated chemotaxis and cell migration toward the murine stroma cell line FBMD-1. Furthermore, plerixafor also significantly (10-100 μM) increased the detachment rate of SDF-1-mediated/VCAM-1-associated cell adherence under shear stress. Using a stroma-dependent coculture assay, plerixafor sensitized BCR-ABL+ cells toward tyrosine kinase inhibitor therapy. Because the level of cell killing nearly reached that of samples cultured without stroma, a cell-cell interaction disruption seems to improve the efficacy of BCR-ABL-targeting drugs. In addition, we could show that exposure of BCR-ABL+ cells to Imatinib or Nilotinib induced an increase in surface CXCR4 expression. Our data suggest that for BCR-ABL+ leukemia, the selective blocking of the SDF-1/CXCR4 axis by plerixafor is a potential mechanism to overcome the protective effect of the bone marrow environment, thereby increasing the therapeutic potency of anti-BCR-ABL drugs and the therapeutic window. [ABSTRACT FROM AUTHOR]