SUMMARYCR3 and FccRs are the main receptors involved in the phagocytic process leading to engulfment and killing of microbes by production of reactive oxygen intermediates (ROI) and degranulation. Various in¯ammatory mediators, such as tumour necrosis factor-a (TNF-a) and lipopolysaccharide (LPS), are known to prime neutrophils leading to increased bactericidal responses, but the underlying mechanism of priming has only been partially elucidated. The purpose of this study was to investigate how TNF-a primes neutrophils for subsequent stimuli via either CR3 or FccR. The receptors were speci®cally activated with pansorbins (protein-A-positive Staphylococcus aureus) coated with anti-CR3, anti-FccRIIa, or anti-FccRIIIb monoclonal antibody. Activation of neutrophils with these particles resulted in ROI production as measured by chemiluminescence. Anti-CR3 pansorbins induced the most prominent ROI production in neutrophils. TNF-a potentiated the CR3-mediated respiratory burst but had little effect on that mediated by FccRs. The priming effect of TNF-a on CR3-mediated ROI production is associated with an increased activation of p38 MAPK as well as tyrosine phosphorylation of p72 syk . Pretreatment of neutrophils with the inhibitors for p38 MAPK and p72 syk markedly suppressed the respiratory burst induced by CR3. Furthermore, TNF-a induced about a three-fold increase in the expression of CR3 in neutrophils, an effect which is blocked by the p38 MAPK inhibitor. Taken together, these results showed that TNF-a potentiates the CR3-mediated respiratory burst in neutrophils not only by triggering a p38 MAPK-dependent up-regulation of CD11b/CD18 but also by modulating the signalling pathways.
Human neutrophils express two different types of phagocytic receptors, complement receptors (CR) and Fc receptors. In order to characterize the different signaling properties of each receptor we have used non-adherent human neutrophils and investigated CR3, FcgammaRIIA and FcgammaRIIIB for their signaling capacity. Selective activation of each receptor was achieved by coupling specific antibodies to heat-killed Staphylococcus aureus particles, Pansorbins, through their Fc moiety. Despite the fact that these particles are not phagocytosed, we show that addition of Pansorbins with anti-CD18 antibodies recognizing CR3 induced prominent signals leading to a respiratory burst. Stimulation with anti-FcgammaRIIIB Pansorbins induced about half of the response induced by anti-CR3 Pansorbins, whereas anti-FcgammaRIIA Pansorbins induced an even weaker signal. However, FcgammaRIIA induced strong phosphorylation of p72(syk) whereas FcgammaRIIIB induced only a very weak p72(syk) phosphorylation. During CR3 stimulation no tyrosine phosphorylation of p72(syk) was seen. Both phospholipase D and NADPH oxidase activities were dependent on intracellular calcium. This is in contrast to tyrosine phosphorylation of p72(syk) that occurred even in calcium-depleted cells, indicating that oxygen metabolism does not affect p72(syk) phosphorylation. Inhibitors of tyrosine phosphorylation blocked the respiratory burst induced by both FcgammaRIIA and FcgammaRIIIB as well as CR3. This shows that tyrosine phosphorylation of p72(syk) is an early signal in the cascade induced by FcgammaRIIA but not by CR3.
Abstract. /$2 integrins are involved in the adhesion of leukocytes to other cells and surfaces. Although adhesion is required for cell locomotion, little is known regarding the way/32 integrin-receptors affect the actin network in leukocytes. In the present study filamentous actin (F-actin) levels in non-adherent human neutrophils have been measured by phalloidin staining after antibody cross-linking of/$2 integrins. Antibody engagement of/$2 integrins resulted in a rapid and sustained (146 and 131% after 30 and 300 s, respectively) increase in the neutrophil F-actin content. This is in contrast to stimulation with N-formyl-l-methionyl-lleucyl-/-phenylalanine (fMLP), which causes a prompt and pronounced but rapidly declining rise in F-actin (214 and 127% after 15 and 300 s, respectively). Priming neutrophils with 1 nM PMA, a low concentration that did not influence the F-actin content per se, increased the magnitude of the/$2 integrin-induced response but had no effect on the kinetics (199% after 30 s and 169% after 300 s). Removal of extracellular Ca 2÷ only marginally affected the/$~ integrin-induced F-actin response for cells that were pretreated with PMA whereas the response for nonprimed cells was reduced by half. This suggests that even though extracellular Ca 2÷ has a modulatory effect it is not an absolute requirement for/$2 integrin-induced actin polymerization. /$2 integrin engagement did not affect the resting cellular level of cAMP arguing against a role of cAMP in/$2 integrin-induced actin assembly. The lack of a cAMP signal might instead explain, at least in part, the prolonged F-actin response triggered by/$2 integrins, since addition of cAMP and 1-isobutylmethylxanthine (IBMX) caused a prompt reversal of the/32 integrin-induced F-actin elevation in electropermeabilized neutrophils. Engagement of/32 integrins, as previously shown for activation of the chemotactic peptide receptor, resulted in a significant formation of PtdInsP3. The capacity of/$2 integrins and chemotactic peptide receptors to induce phosphatidylinositol trisphosphate (PtdlnsP3) formation correlated with their ability to induce actin polymerization.
This article revolves around the educational policy introduced in Swedish schools that has extended national testing to younger pupils. The policy is intended to support equal assessment and grading. With the exception of short-term preparations for the tests focused on here, the testing routines are regulated by the state. The paper aims to examine how the policy of national testing in grade six is enacted in different school contexts from a pupil’s point of view, and how this affects equivalence in school. A narrative analysis was conducted of pupils’ ( n = 150) stories about preparing for national tests in 11 schools. Three forms of enactments were distinguished according to how responsibility for test preparations was allocated in each school. In some schools, teachers invited the pupils systematically to the translation process. In other schools, pupils were given most of the responsibility for preparation and were left alone as actors vis-a-vis the policy. Finally, in schools that applied ad hoc preparations, the pupils’ position as actors became less secure and more multifaceted. This variety regarding the pupils’ test preparations in school stress that the different enactments of this policy of national testing have implications for the interpretation of equivalence in school.
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