Prostaglandin E<sub>2</sub>Inhibits the Phospholipase D Pathway Stimulated by Formyl-methionyl-leucyl-phenylalanine in Human Neutrophils. Involvement of EP<sub>2</sub>Receptors and Phosphatidylinositol 3-kinase γ

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Polymorphonuclear neutrophil (PMN) stimulation with fMLP stimulates small G proteins such as ADP-ribosylation factors (Arfs) Arf1 and Arf6, leading to phospholipase D (PLD) activation and functions such as degranulation and the oxidative burst. However, the molecular links between fMLF receptors and PLD remain unclear. PMNs express cytohesin-1, an Arf-guanine exchange factor that activates Arfs, and its expression is strongly induced during the acquisition of the neutrophilic phenotype by neutrophil-like cells. The role of cytohesin-1 in the activation of the fMLF-Arf-PLD signaling axis, and the accomplishment of superoxide anion production, and degranulation was investigated in PMNs using the selective inhibitor of cytohesin, Sec 7 inhibitor H3 (secinH3). Cytohesin-1 inhibition with secinH3 leads to Arf6 but not Arf1 inhibition, demonstrating the specificity for Arf6, and fMLF-mediated activation of PLD and of the oxidative burst as well. We observed a decrease in fMLF-mediated protein secretion and expression of cell surface markers corresponding to primary (CD63/myeloperoxidase), secondary (CD66/lactoferrin), and tertiary (matrix metalloproteinase-9) granules in PMNs incubated with secinH3. Similarly, silencing cytohesin-1 or Arf6 in PLB-985 cells negatively affected fMLF-induced activation of PLD, superoxide production, and expression of granule markers on the cell surface. In contrast, stable overexpression of cytohesin-1 in PLB-985 cells enhanced fMLF-induced activation of Arf6, PLD, and NADPH oxidase. The results of this study provide evidence for an involvement of cytohesin-1 in the regulation of the functional responses of human PMNs and link these events, in part at least, to the activation of Arf6.

Section: Discussionmentioning

confidence: 77%

Section: Discussionmentioning

confidence: 99%

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Cytohesin-1 Regulates the Arf6-Phospholipase D Signaling Axis in Human Neutrophils: Impact on Superoxide Anion Production and Secretion

Azreq

Garceau

Harbour

et al. 2009

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“…To analyze further the role of calcium and distinguish it from other fMLF receptor-induced transduction pathways, we evaluated the effect of ionomycin, a calcium ionophore that bypasses the receptor activation step, on the subcellular distribution of Munc13-4 in neutrophils. As described previously (38), fMLF induced a rapid and transient increase in [Ca 2ϩ ] c , whereas ionomycin induced a sustained and long-lasting increase in [Ca 2ϩ ] c (Fig. 3C).…”

Section: Calcium-dependent Recruitment Of Munc13-4 To Membranesmentioning

confidence: 71%

Munc13-4 Regulates Granule Secretion in Human Neutrophils

Pivôt-Pajot

Varoqueaux

Basile

et al. 2008

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The neutrophil plays a central role in the innate host immune defense. Regulated exocytosis of its granules and release of antimicrobial and cytotoxic substances are key events to limit the spread of pathogens. However, the molecular mechanisms that control exocytosis of neutrophil granules are ill-defined. Recently, it was shown that Munc13-4 is essential for the priming of granules in several hematopoietic cells. In this study, we show that Munc13-4 is expressed in human neutrophils, and that its expression is increased during granulocytic differentiation of HL-60 and PLB-985 cells. Cell fractionation analysis reveals that Munc13-4 is mainly cytosolic and is recruited rapidly to membranes following stimulation with fMLF (N-formyl-methionyl-leucyl-phenylalanine). Moreover, a pool of Munc13-4 associated with mobilizable secondary and tertiary granules is relocalized to the plasma membrane after stimulation with fMLF. The fMLF-induced translocation of Munc13-4 is strictly dependent on calcium in neutrophils. C2 domains of Munc13-4 are essential for binding to phospholipid vesicles in a Ca2+-independent manner. Finally, down-regulation of Munc13-4 using small interfering RNA decreases exocytosis of tertiary granules in PLB-985 cells, whereas overexpression of Munc13-4 enhances secretion of MMP-9 (matrix metalloproteinase-9) from tertiary granules. Our findings suggest a role for Munc13-4 as a component of the secretory machinery in neutrophils.

“…Apart from increasing cAMP biosynthesis, a downstream event following stimulation of EP2 receptors is activation of PI3K (45,46). Moreover, activation of PI3K can eventually lead to PKC activation (47,48).…”

Section: Discussionmentioning

confidence: 99%

Prostaglandin E2 Inhibits Eosinophil Trafficking through E-Prostanoid 2 Receptors

Sturm

Schratl

Schuligoi

et al. 2008

108

110

The accumulation of eosinophils in lung tissue is a hallmark of asthma, and it is believed that eosinophils play a crucial pathogenic role in allergic inflammation. Prostaglandin (PG) E2 exerts anti-inflammatory and bronchoprotective mechanisms in asthma, but the underlying mechanisms have remained unclear. In this study we show that PGE2 potently inhibits the chemotaxis of purified human eosinophils toward eotaxin, PGD2, and C5a. Activated monocytes similarly attenuated eosinophil migration, and this was reversed after pretreatment of the monocytes with a cyclooxygenase inhibitor. The selective E-prostanoid (EP) 2 receptor agonist butaprost mimicked the inhibitory effect of PGE2 on eosinophil migration, whereas an EP2 antagonist completely prevented this effect. Butaprost, and also PGE2, inhibited the C5a-induced degranulation of eosinophils. Moreover, selective kinase inhibitors revealed that the inhibitory effect of PGE2 on eosinophil migration depended upon activation of PI3K and protein kinase C, but not cAMP. In animal models, the EP2 agonist butaprost inhibited the rapid mobilization of eosinophils from bone marrow of the in situ perfused guinea pig hind limb and prevented the allergen-induced bronchial accumulation of eosinophils in OVA-sensitized mice. Immunostaining showed that human eosinophils express EP2 receptors and that EP2 receptor expression in the murine lungs is prominent in airway epithelium and, after allergen challenge, in peribronchial infiltrating leukocytes. In summary, these data show that EP2 receptor agonists potently inhibit eosinophil trafficking and activation and might hence be a useful therapeutic option in eosinophilic diseases.