Analysis of Signal Transduction Pathways Regulating Cytokine-Mediated Fc Receptor Activation on Human Eosinophils

Igs can be potent stimulants of eosinophil activation since interaction with IgA or IgG-coated particles can lead to eosinophil degranulation. We have investigated the comparative roles of mitogen-activated protein (MAP) kinases (MAPKs; ERK1/2 and p38) and phosphatidylinositol-3 kinase (PI3K) in the priming and regulation of Fc receptor functioning on human eosinophils utilizing a MAPK kinase (MEK) inhibitor (PD98059), a p38 inhibitor SB203580, and the widely used PI3K inhibitors wortmannin and LY294002. We demonstrate that priming of human eosinophils with Th2-derived cytokines, IL-4 and IL-5, differentially activate phosphotyrosine-associated PI3K and ERK and p38 MAP kinases. This activation can be inhibited by pre-incubation with wortmannin or LY294002, PD98059, and SB203580, respectively. Analysis of the effects of the inhibitors on rosette formation between human eosinophils and IgA- or IgG-coated beads revealed that activation of MEK was not required for IgA binding after priming with IL-4 or IL-5. However, inhibition of MEK did inhibit IL-5-primed binding of IgG-beads. The rosette formation of primed eosinophils with IgA-beads could be completely inhibited by wortmannin and LY294002 treatment, demonstrating a critical role for PI3K. Interestingly, inhibition of the p38 pathway also resulted in a complete blockade of IgA rosette formation. This work demonstrates regulatory control by inside-out signaling of Fc receptors by various cytokines on human eosinophils. Thus in vivo the local production of Th2-derived cytokines will regulate the effector functions of Fc receptors

Cytokine-induced inside-out activation of FcaR (CD89) is mediated by a single serine residue (S263) in the intracellular domain of the receptor

Fc receptors play an important role in leukocyte activation and the modulation of ligand binding ("activation") is a criti-cal point of regulation. Previous studies demonstrated that the Fc receptor for IgA (FcaRI/CD89) is regulated by cytokine stimulation, switching it to a high-binding state. To investigate the mechanism by which cytokine-induced signal transduc-tion pathways result in FcaRI activation, cell lines expressing various receptor mu-tants were generated. Binding studies indicated that truncation of the C-termi- nus of the FcaRI resulted in constitutive IgA binding, removing the need for cyto-kine stimulation. Furthermore, mutagen-esis of a single C-terminal serine residue (S263) to alanine (S>A) (single-letter amino acid codes) also resulted in consti-tutive IgA binding, whereas a serine to aspartate (S>D) mutation was no longer functional. The role of S263 might be in regulating the interaction with the cy-toskeleton, because disruption of the cy-toskeleton results in reduced IgA binding to both FcaRwt and FcaR_S>A. In addi- tion, overexpression of a membrane-targeted intracellular domain of FcaR, and the introduction of cell-permeable CD89 fusion proteins blocked IgA bind-ing, implying a competition for endoge-nous proteins. The proposal is made that Fc receptors are activated by cytokines via an inside-out mechanism converging at the cytoplasmic tail of these receptors

Cytokine-mediated cPLA2 phosphorylation is regulated by multiple MAPK family members

Cytosolic phospholipase A2 (cPLA2) plays a critical role in various neutrophil functions including the generation of leukotrienes and platelet-activating factor release. Enzyme activity is regulated both by translocation to the membrane in a Ca^(2+) -dependent manner and serine phosphorylation by members of the mitogen-activated protein kinase (MAPK) family. In this report, we have investigated the role of granulocyte/macrophage colony-stimulating factor (GM-CSF)- mediated signalling pathways in the regulation of cPLA2. GM- CSF-induced cPLA2 phosphorylation was not affected by pharmacological inhibition of p38 MAPK, phosphatidylinositol 3-kinase or Src. However, inhibition of extracellular signal- regulated kinase (ERK) MAPK activation resulted in a partial inhibition of cPLA2 phosphorylation, revealed in a slower onset of phosphorylation. A cell line stably transfected with the GM- CSF receptor was used to further analyze GM-CSF-mediated cPLA2 phosphorylation. Mutation of tyrosine residues 577 and 612 resulted in a delayed cPLA2 phosphorylation similar to the pharmacological ERK inhibition. Furthermore, inhibition of p38 MAPK in cells bearing the double mutant ßc577/612 completely abrogated GM-CSF-induced cPLA2 phosphorylation. We con- clude that GM-CSF can mediate cPLA2 phosphorylation through the redundant activation of both p38 and ERK MAP kinases

Comparison of the roles of mitogen-activated protein kinase kinase and phosphatidylinositol 3-kinase signal transduction in neutrophil effector function

Although it is known that many stimuli can activate mitogen- activated protein kinases (MAPKs) and phosphatidylinositol 3- kinases (PI3K) in human neutrophils, little is known concerning either the mechanisms or function of this activation. We have utilized a selective inhibitor of MAPKkinase (MEK), PD098059, and two inhibitors of PI3K, wortmannin and LY294002, to investigate the roles of these kinases in the regulation of neutrophil effector functions. Granulocyte/macrophage colony- stimulating factor, platelet-activating factor (PAF) and N-for- mylmethionyl-leucyl-phenylalanine are capable of activating both p44^(ERK1) and p42^(ERK2) MAPKs and phosphotyrosine-asso- ciated PI3K in human neutrophils. The activation of extracellular signal-related protein kinases (ERKs) is correlated with the activation of p21^(ras) by both tyrosine kinase and G-protein- coupled receptors as measured by a novel assay for GTP loading. Wortmannin and LY294002 inhibit, to various degrees, super- oxide generation, neutrophil migration and PAF release. In- cubation with PD098059, however, inhibits only the PAF release stimulated by serum-treated zymosan. This demonstrates that, while neither MEK nor ERK kinases are involved in the acti- vation of respiratory burst or neutrophil migration, inhibition of PAF release suggests a potential role in the activation of cytosolic phospholipase A2 . PI3K isoforms, however, seem to have a much wider role in regulating neutrophil functioning

Activation of 12-O-Tetradecanoylphorbol-13-acetate Response Element- and Dyad Symmetry Element-dependent Transcription by Interleukin-5 Is Mediated by Jun N-terminal Kinase/Stress-activated Protein Kinase Kinases

Interleukin-5 (IL-5) is one of the major regulators of eosinophilic granulocytes in vivo. IL-5 exerts its pleiotropic effects by binding to the IL-5 receptor, which is composed of an IL-5-specific a chain and a common bc chain shared with the receptors for IL-3 and granulocyte- macrophage colony-stimulating factor. Previous studies have shown that binding of IL-5 to its receptor triggers the activation of multiple signaling cascades, including the Ras/mitogen-activated protein kinase, the phosphatidyl -3`-kinase, and the Janus kinase/signal transducer and activator of transcription pathways. Here we describe that IL-5 activates the serine/threonine protein kinase Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway. We show that IL-5 activates TPA response element (TRE)-dependent transcription in transfection experiments. TRE activation by IL-5 is mediated by a region of the bc (577- 581) that is also responsible for activation of JNK/SAPK and for activation of dyad symmetry element (DSE)-dependent transcription. Dominant-negative SAPK or ERK kinase-1 was used to demonstrate that JNK/SAPK activation is necessary for induction of DSE- and TREdependent transcription by IL-5, whereas extracellular signal-regulated kinase 2 was not essential for TRE- and DSE-dependent transcription. By contrast, IL-5-induced activation of the tyrosine kinase Janus kinase 2 seems to be a prerequisite for TRE- and DSE-dependent transcription. Taken together, we show for the first time that IL-5 activates kinases of the JNK/SAPK family, and that this activation is linked to IL-5-induced TRE- and DSE-dependent transcription

Intracutaneous and intravesical immunotherapy with keyhole limpet hemocyanin compared with intravesical mitomycin in patients with non-muscle-invasive bladder cancer: results from a prospective randomized phase III trial.

Contains fulltext : 108422.pdf (publisher's version ) (Open Access)PURPOSE: Despite current treatment after transurethral resection of a bladder tumor, recurrences and progression remain a problem. Keyhole limpet hemocyanin (KLH) was beneficial in earlier studies. In this study, safety and efficacy of KLH were compared with that of mitomycin (MM). PATIENTS AND METHODS: Patients with intermediate- and high-risk non-muscle-invasive bladder cancer (NMIBC) without carcinoma in situ were enrolled in a randomized phase III trial. In all, 283 patients were randomly assigned for 16 adjuvant intravesical instillations with KLH after preimmunization, and 270 patients were randomly assigned for 11 adjuvant intravesical instillations with MM. Primary outcome measurement was recurrence-free survival (RFS). Secondary outcome measurements were progression-free survival, adverse events (AEs), and the effect of delayed-type hypersensitivity (DTH) response on clinical outcome. RESULTS: There were significantly more pT1 tumors in the MM group (P = .01). In a log-rank test, univariate and multivariate Cox regression analysis, KLH was less effective than MM regarding RFS (all P < .001). Progression was uncommon (n = 20). In univariate Cox regression analyses, KLH tended to prevent progression more effectively than MM, but in multivariate Cox regression analyses, this could not be shown. AEs were common but mild. Fever, flu-like symptoms, and fatigue occurred significantly more after KLH treatment. Allergic reactions and other skin disorders occurred significantly more after MM treatment. Significantly more DTH-positive patients developed a recurrence than DTH-negative patients. CONCLUSION: KLH had a different safety profile and was inferior to MM in preventing NMIBC recurrences. KLH tended to be more effective than MM in preventing progression. More research is needed to clarify the immunologic effects of KLH and the effects of KLH on progression

The course of post-stroke apathy in relation to cognitive functioning: a prospective longitudinal cohort study

Apathy is common after stroke and has been associated with cognitive impairment. However, causality between post-stroke apathy and cognitive impairment remains unclear. We assessed the course of apathy in relation to changes in cognitive functioning in stroke survivors. Using the Apathy Scale (AS) and cognitive tests on memory, processing speed and executive functioning at six- and 15 months post-stroke we tested for associations between (1) AS-scores and (change in) cognitive scores; (2) apathy course (persistent/incident/resolved) and cognitive change scores. Of 117 included participants, 29% had persistent apathy, 13% apathy resolving over time and 10% apathy emerging between 6-15 months post-stroke. Higher AS-scores were cross-sectionally and longitudinally associated with lower cognitive scores. Relations between apathy and cognitive change scores were ambiguous. These inconsistent relations between apathy and changes in cognition over time suggest that post-stroke apathy does not directly impact cognitive performance. Both these sequelae of stroke require separate attention

Ant-like Traits in Wingless Parasitoids Repel Attack from Wolf Spiders

A recent study showed that a wingless parasitoid, Gelis agilis, exhibits a suite of ant-like traits that repels attack from wolf spiders. When agitated, G. agilis secreted 6-methyl-5-hepten-2-one (sulcatone), which a small number of ant species produce as an alarm/panic pheromone. Here, we tested four Gelis parasitoid species, occurring in the same food chain and microhabitats, for the presence of sulcatone and conducted two-species choice bioassays with wolf spiders to determine their degree of susceptibility to attack. All four Gelis species, including both winged and wingless species, produced sulcatone, whereas a closely related species, Acrolyta nens, and the more distantly related Cotesia glomerata, did not. In two-choice bioassays, spiders overwhelmingly rejected the wingless Gelis species, preferring A. nens and C. glomerata. However, spiders exhibited no preference for either A. nens or G. areator, both of which are winged. Wingless gelines exhibited several ant-like traits, perhaps accounting for the reluctance of spiders to attack them. On the other hand, despite producing sulcatone, the winged G. areator more closely resembles other winged cryptines like A. nens, making it harder for spiders to distinguish between these two species. C. glomerata was also preferred by spiders over A. nens, suggesting that other non-sulcatone producing cryptines nevertheless possess traits that make them less attractive as prey. Phylogenetic reconstruction of the Cryptinae reveals that G. hortensis and G. proximus are ‘sister’species, with G. agilis, and G.areator in particular evolving along more distant trajectories. We discuss the possibility that wingless Gelis species have evolved a suite of ant-like traits as a form, of mimicry to repel predators on the ground.FWN – Publicaties zonder aanstelling Universiteit Leide