Mechanisms of eosinophil adhesion to endothelial cells under flow conditions

Eosinophils play an important role in allergic inflammatory diseases such as allergic asthma. Infiltrates of these cells are present in the interstitium and the lumen of the bronchi of asthmatic patients. Eosinophils must pass the endothelium to enter this site of inflammation. A widely accepted paradigm for leukocyte extravasation is the multistep model. In this model selectins mediate rolling interactions between leukocytes and endothelium and subsequently, activated integrins facilitate firm adhesion and extravasation of the cells. The interactions between eosinophils and activated endothelial cells have been studied under flow conditions. This has been done in a flow chamber model in which a blood vessel is simulated. In chapter 1 (general introduction) the multistep model of extravasation is explained. Furthermore, eosinophils function and their role in allergic asthma has been described. In chapter 2 data are presented on the function of alpha 4 integrins and E-selectin in the initial attachment of eosinophils to TNF-? activated endothelial cells. In chapter 3 data are presented on the differences in adhesion behavior between eosinophils of healthy blood donors and of allergic asthmatic patients on activated endothelial cells under flow conditions. Remarkably, eosinophils of allergic asthmatic patients bound platelets in contrast to cells of healthy controls. The platelet-eosinophil interactions resulted in increased adhesion to activated endothelial cells via a process that is called secondary tethering. In chapter 4 data are presented on the role of IL-8 on eosinophil arrest. IL-8 is a typical neutrophil activator but is not known to be a eosinophil activator. We showed that IL-8 can induce 1) an alpha 4- and beta 2-integrin dependent arrest of rolling eosinophils on activated endothelial cells and 2) an increase in intracellular Ca2+ concentration of eosinophils binding to fibronectin and activated endothelial cells. Thus, IL-8 is not only a neutrophil activator but also an eosinophil activator. In chapter 5 data are presented on the role of phospholipase C (PLC) in the chemokine-induced integrin activation of eosinophils. Three chemokines, IL-8, eotaxin and C5a were tested on their ability to induce a PLC dependent arrest of eosinophils. Eotaxin mediated a totally PLC-dependent arrest whereas IL-8 and C5a only induced a partial PLC-dependent arrest. In resting eosinophils PLC is also important because inhibition of PLC decreased the functionality of alpha 4 integrins. In chapter 6 data are presented on the migration behavior of eosinophils. Migrating cells display a typical tear-drop shape while migrating. The front is a broad protrusion (lamellipodium) whereas the back of the cell is a small point-like structure (uropod). The small GTP-ase RhoA was found to play an important role in the regulation of the movement of the uropod. In chapter 7 (general discussion) the data of chapter 2-6 are discussed. Special emphasis was give to the validation of the model, the possible working mechanisms of PLC in integrin regulation, the role of platelets in the binding of eosinophils of allergic asthmatic patients to activated endothelial cells and possible target molecules for therapy

Activation of RhoA and ROCK Are Essential for Detachment of Migrating Leukocytesh

Detachment of the rear of the cell from its substratum is an important aspect of locomotion. The signaling routes involved in this adhesive release are largely unknown. One of the few candidate proteins to play a role is RhoA, because activation of RhoA in many cell types leads to contraction, a mechanism probably involved in detachment. To study the role of RhoA in detachment regulation, we analyzed several subsets of expert migratory leukocytes by video microscopy. In contrast to fast-migrating neutrophils, eosinophils do not detach the rear of the cell unless stimulated with serum. When measuring the amount of active RhoA, with the use of a GSTRhotekin pulldown assay, we found that serum is an excellent activator of RhoA in granulocytes. Inhibition of RhoA or one of Rhos target proteins, the kinase ROCK, in neutrophils leads to the phenotype seen in eosinophils: the rear of the cell is firmly attached to the substratum, whereas the cell body is highly motile. ROCK-inhibition leads to impaired migration of granulocytes in filters, on glass, and through endothelial monolayers. Also, the ROCK signaling pathway is involved in changes of integrin-mediated adhesion. Eosinophil transduction by a tat-fusion construct containing active RhoA resulted in detachment stimulation in the presence of chemoattractant. From these results we conclude that activation of the RhoA-ROCK pathway is essential for detachment of migratory leukocytes

Characteristics, causes and treatment of postpartum haemorrhage in first and second pregnancies

Leptin is an adipokine that is thought to be important in many inflammatory diseases, and is known to influence the function of several leukocyte types. However, no clear consensus is present regarding the responsiveness of neutrophils for this adipokine. In this study a 2D DIGE proteomics approach was used as an unbiased approach to identify leptin-induced effects on neutrophils. Additionally chemotaxis and survival experiments were performed to reproduce results from literature showing putative effects of leptin on these neutrophil responses. Leptin did not induce any significant changes in the proteome provided leptin was added at physiologically relevant concentrations (250 ng). Our leptin batches were biologically active as they induced proliferation in LeptinR expressing Ba/F3 cells. At high concentrations (25000 ng) leptin induced a change in neutrophil proteome. Seventeen differently regulated spots were identified of which twelve could be characterized by mass spectrometry. Two of these identified proteins, SerpinB1 and p40 phox, were chosen for further analysis but leptin-induced expression analyzed by western blot were highly variable. Additionally leptin also induced neutrophil survival at these high concentrations. No leptin-induced chemotaxis of human neutrophils was detected at any concentration. In conclusion, physiological concentrations of leptin do not affect neutrophils. High leptin concentrations induced survival and changes in the neutrophils proteome, but this was most likely mediated by an indirect effect. However, it cannot be ruled out that the effects were mediated by a yet not-identified leptin receptor on human neutrophils

Characterization of Eosinophil Adhesion to TNF-a-Activated Endothelium Under Flow Conditions: a4 Integrins Mediate Initial Attachment, and E-Selectin Mediates Rolling

The multistep model of leukocyte adhesion reveals that selectins mediate rolling interactions and that integrins mediate firm adhesion processes. In this study, the interaction between eosinophils and TNF-a-activated HUVEC (second or third passage) was studied under flow conditions (0.8 and 3.2 dynes/cm 2 ). Especially the role of a4 integrins on eosinophils and E-selectin on HUVEC was studied. Inhibition of the integrin a4 chain on eosinophils reduced the number of firmly adhered resting eosinophils to TNF-a-stimulated endothelium by 43% whereas the percentage rolling cells increased 2.2-fold compared with untreated control eosinophils. Blocking of E-selectin on the endothelium reduced the number of adherent eosinophils by only 23% and 16%. In this situation, however, hardly any rolling adhesion was observed, and the few rolling cells showed a low rolling velocity. Blocking both a4 integrin on eosinophils and E-selectin on HUVEC reduced the number of adhered eosinophils by 95%. P-selectin did not significantly participate in eosinophil adhesion to TNF-a-activated HUVEC. Inhibition of both a4 integrins and ß2 integrins on eosinophils resulted in a reduction of adhered cells by 65% and a 3-fold increase in percentage rolling cells. Taken together, these results clearly show that resting eosinophils preferentially use constitutively active a4 integrins ( a4 ß1 , a4 ß7 ) for the first attach-ment to TNF-a-activated HUVEC. In addition, a4 integrins and E-selectin work synergistically in eosinophil adherence to TNF-a-activated HUVEC. Although E-selectin is important for eosinophil rolling under these conditions, P-selectin plays only a minor role

IL-8 Induces a Transient Arrest of Rolling Eosinophils on Human Endothelial Cells

Eosinophils exhibit a rolling interaction with E-selectin-expressing endothelium, and need to be activated by inflammatory me-diators to firmly adhere to this surface. This study shows that IL-8 induces a transient arrest of unprimed eosinophils that roll on E-selectin present on TNF-a-activated HUVEC in an in vitro flow chamber. This process was antagonized by neutralizing Abs directed against IL-8 showing the specificity of the IL-8 effect. Furthermore, blocking Abs against both a4 and ß2 integrins inhibited the IL-8-induced transient arrest while these Abs had no effect when they were added separately. The IL-8-induced arrest was pertussis toxin sensitive. Studying the effect of IL-8 in more detail, we evaluated putative changes in intracellular Ca^(2+) concentration in eosinophils induced by IL-8. We could show that IL-8 induces a transient rise in intracellular Ca^(2+) concentration in ~40% of the cells provided that the eosinophils are interacting with endothelial cells or fibronectin-coated surfaces. Together these data show that resting eosinophils respond to IL-8 provided that the cells adhere on physiological surfaces. The induction of a transient arrest provides a new level of chemokine-induced regulation of leukocyte adhesion under flow conditions

Specificity and effector functions of human RSV-specific IgG from bovine milk

Background Respiratory syncytial virus (RSV) infection is the second most important cause of death in the first year of life, and early RSV infections are associated with the development of asthma. Breastfeeding and serum IgG have been shown to protect against RSV infection. Yet, many infants depend on bovine milk-based nutrition, which at present lacks intact immunoglobulins. Objective To investigate whether IgG purified from bovine milk (bIgG) can modulate immune responses against human RSV. Methods ELISAs were performed to analyse binding of bIgG to human respiratory pathogens. bIgG or hRSV was coated to plates to assess dose-dependent binding of bIgG to human Fc¿ receptors (Fc¿R) or bIgG-mediated binding of myeloid cells to hRSV respectively. S. Epidermidis and RSV were used to test bIgG-mediated binding and internalisation of pathogens by myeloid cells. Finally, the ability of bIgG to neutralise infection of HEp2 cells by hRSV was evaluated. Results bIgG recognised human RSV, influenza haemagglutinin and Haemophilus influenza. bIgG bound to Fc¿RII on neutrophils, monocytes and macrophages, but not to Fc¿RI and Fc¿RIII, and could bind simultaneously to hRSV and human Fc¿RII on neutrophils. In addition, human neutrophils and dendritic cells internalised pathogens that were opsonised with bIgG. Finally, bIgG could prevent infection of HEp2 cells by hRSV. Conclusions The data presented here show that bIgG binds to hRSV and other human respiratory pathogens and induces effector functions through binding to human Fc¿RII on phagocytes. Thus bovine IgG may contribute to immune protection against RSV

GM-CSF and TNFalpha modulate protein expression of human neutrophils visualized by fluorescence two-dimensional difference gel electrophoresis

Item does not contain fulltextIncreased serum levels of TNFalpha and GM-CSF are found in various chronic inflammatory diseases and these cytokines affect the function of circulating and tissue neutrophils. TNFalpha- and GM-CSF-induced protein expression profiles could, therefore, serve as biomarker for the action of these cytokines in vivo. We stimulated human peripheral neutrophils with TNFalpha and GM-CSF in vitro and analyzed changes in their proteome by fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). We report the differential expression of 3 and 18 protein spots following TNFalpha and GM-CSF stimulation, respectively. Differences in protein expression induced by TNFalpha were limited and did not show discriminatory power in a principal component analysis, whereas the profile induced by GM-CSF did. TNFalpha- and GM-CSF-induced both de novo IL-1beta and sIL-1Ra protein expression as detected by Western blot analysis, which confirmed proper neutrophil activation by these cytokines in vitro. Mass spectrometry analysis of cytokine-regulated protein spots resulted in the identification of 8 proteins. Among the identified proteins, enolase 1 and annexin A1 might function as markers for peripheral neutrophil activation. In conclusion, a proteomic analysis of neutrophils by 2D-DIGE provides proof-of-principle that cytokine-induced protein profiles can serve as biomarkers for the action of individual cytokines in vivo

Allele-specific regulation of primary cilia function by the von Hippel-Lindau tumor suppressor.

Contains fulltext : 69666.pdf (publisher's version ) (Closed access)Patients with von Hippel-Lindau (VHL) disease often develop VHL-/- kidney cysts, which possibly progress into clear-cell renal carcinomas (ccRCCs). Recent data link the VHL gene product to formation of the primary cilium, an organelle that extends apically into the renal lumen. Exactly how VHL induces ciliogenesis or function is unknown. Here, we demonstrate that ciliary assembly and mechanotransduction is rapidly restored in VHL-/- ccRCC cells upon ectopic reconstitution of wild-type - but not variant alleles of - VHL. These data support and expand recent studies implicating a role for VHL in the initiation of ciliogenesis. Furthermore, reduction of cellular levels of VHL in this cell system was associated with fewer ciliated cells, suggesting a role for VHL in ciliary maintenance

Steroids induce a disequilibrium of secreted interleukin-1 receptor antagonist and interleukin-1beta synthesis by human neutrophils

Item does not contain fulltextChronic obstructive pulmonary disease (COPD) is characterised by neutrophilic inflammation in the airways and these neutrophils contribute to the production of inflammatory mediators. Dampening the production of proinflammatory mediators might be an important strategy to treat COPD and glucocorticosteroids are known to do so via inhibition of nuclear factor-kappaB. However, this pathway is important for the control of pro- and anti-inflammatory genes. We studied the effects of dexamethasone on production and secretion of pro-inflammatory interleukin (IL)-1beta and anti-inflammatory secreted IL-1 receptor antagonist (sIL-1Ra) by human neutrophils activated with tumor necrosis factor (TNF)-alpha. In vitro, TNF-alpha-stimulated neutrophils produced significant amounts of IL-1beta and sIL-1Ra; this production was inhibited by dexamethasone. However, synthesis and secretion of sIL-1Ra was inhibited at lower concentrations dexamethasone compared to IL-1beta, which changed the IL-1beta:sIL-1Ra ratio significantly. This altered ratio resulted in a more pro-inflammatory condition, as visualised by increased intercellular adhesion molecule-1 expression on human endothelial cells. In vivo, moderate-to-severe COPD patients using inhaled glucocorticosteroids have decreased plasma sIL-Ra levels compared with mild-to-moderate patients not on glucocorticosteroid treatment. In conclusion, dexamethasone induces a pro-inflammatory shift in the IL-1beta:sIL-1Ra cytokine balance in neutrophils in vitro, which might contribute to a lack of endogenous anti-inflammatory signals to dampen inflammation in vivo

Consumption of unprocessed cow's milk protects infants from common respiratory infections

Background: Breast-feeding is protective against respiratory infections in early life. Given the co-evolutionary adaptations of humans and cattle, bovine milk might exert similar anti-infective effects in human infants. Objective: To study effects of consumption of raw and processed cow's milk on common infections in infants. Methods: The PASTURE birth cohort followed 983 infants from rural areas in Austria, Finland, France, Germany, and Switzerland, for the first year of life, covering 37,306 person-weeks. Consumption of different types of cow's milk and occurrence of rhinitis, respiratory tract infections, otitis, and fever were assessed by weekly health diaries. C-reactive protein levels were assessed using blood samples taken at 12 months. Results: When contrasted with ultra-heat treated milk, raw milk consumption was inversely associated with occurrence of rhinitis (adjusted odds ratio from longitudinal models [95% CI]: 0.71 [0.54-0.94]), respiratory tract infections (0.77 [0.59-0.99]), otitis (0.14 [0.05-0.42]), and fever (0.69 [0.47-1.01]). Boiled farm milk showed similar but weaker associations. Industrially processed pasteurized milk was inversely associated with fever. Raw farm milk consumption was inversely associated with C-reactive protein levels at 12 months (geometric means ratio [95% CI]: 0.66 [0.45-0.98]). Conclusions: Early life consumption of raw cow's milk reduced the risk of manifest respiratory infections and fever by about 30%. If the health hazards of raw milk could be overcome, the public health impact of minimally processed but pathogen-free milk might be enormous, given the high prevalence of respiratory infections in the first year of life and the associated direct and indirect costs