Immunofluorescence and Confocal Microscopy of Neutrophils

Allen, Lee‐Ann H.

doi:10.1007/978-1-62703-845-4_16

Cited by 19 publications

(14 citation statements)

References 22 publications

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“…For immunofluorescence by confocal microscopy, neutrophils were seeded on acid washed coverslips that were treated with Coating Buffer and placed in 24 well plates as described (26). Neutrophils were infected with S. aureus as described above and incubated at 37°C for 1 minute, supernatant was removed and cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes.…”

Section: Methodsmentioning

confidence: 99%

IL-20 Signaling in Activated Human Neutrophils Inhibits Neutrophil Migration and Function

Gough

Ganesan

Datta

2017

The Journal of Immunology

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Neutrophils possess multiple antimicrobial mechanisms that are critical for protection of the host against infection with extracellular microbes, such as the bacterial pathogen Staphylococcus aureus. Recruitment and activation of neutrophils at sites of infection is driven by cytokine and chemokine signals that directly target neutrophils via specific cell surface receptors. The IL-20 subfamily of cytokines has been reported to act at epithelial sites and contribute to psoriasis, wound healing, and anti-inflammatory effects during S. aureus infection. However, the ability of these cytokines to directly affect neutrophil function remains incompletely understood. Here, we show that human neutrophils altered their expression of IL-20 receptor chains upon migration and activation in vivo and in vitro. Such activation of neutrophils under conditions mimicking infection with S. aureus conferred responsiveness to IL-20 that manifested as modification of actin polymerization and inhibition of a broad range of actin-dependent functions, including phagocytosis, granule exocytosis, and migration. Consistent with the previously described homeostatic and anti-inflammatory properties of IL-20 on epithelial cells, the current studies provide evidence that IL-20 directly targets and inhibits key inflammatory functions of neutrophils during infection with S. aureus.

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Section: Methodsmentioning

confidence: 99%

IL-20 Signaling in Activated Human Neutrophils Inhibits Neutrophil Migration and Function

Gough

Ganesan

Datta

2017

The Journal of Immunology

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Section: Image Analysismentioning

confidence: 99%

“…Particle binding was synchronized by centrifugation (15°C, 2 min at 800 g), followed by incubation at 37°C to initiate phagocytosis as described previously [21,25,26]. At the indicated times, samples were fixed and processed for indirect immunofluorescence and confocal microscopy using our established methods [21,25,26]. NADPH oxidase subunits p22 phox and gp91 phox were detected using mAb 44.1 [27] and 54.1 [16], and mAb 7D5 was used to detect an epitope of gp91 phox that is present in primate flavocytochrome b 558 [17].…”

Section: Image Analysismentioning

confidence: 99%

Effects of IFN-γ on intracellular trafficking and activity of macrophage NADPH oxidase flavocytochrome b558

Casbon

Long

Dunn

et al. 2012

Journal of Leukocyte Biology

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IFNγ regulates trafficking and synthesis of flavocytochrome b558, suggesting a role to control superoxide production in macrophages. Flavocytochrome b558, the catalytic core of the phagocyte NADPH oxidase (NOX2), mediates electron transfer from NADPH to molecular oxygen to generate superoxide, the precursor of highly ROS for host defense. Flavocytochrome b558 is an integral membrane heterodimer consisting of a large glycosylated subunit, gp91phox, and a smaller subunit, p22phox. We recently showed in murine macrophages that flavocytochrome b558 localizes to the PM and Rab11-positive recycling endosomes, whereas in primary hMDMs, gp91phox and p22phox reside in the PM and the ER. The antimicrobial activity of macrophages, including ROS production, is greatly enhanced by IFN-γ, but how this is achieved is incompletely understood. To further define the mechanisms by which IFN-γ enhances macrophage NADPH oxidase activity, we evaluated changes in flavocytochrome b558 expression and localization, along with NADPH oxidase activity, in IFN-γ stimulated RAW 264.7 cells and primary murine BMDMs and hMDMs. We found that enhanced capacity for ROS production is, in part, a result of increased protein expression of gp91phox and p22phox but also demonstrate that IFN-γ induced a shift in the predominant localization of gp91phox and p22phox from intracellular membrane compartments to the PM. Our results are the first to show that a cytokine can change the distribution of macrophage flavocytochrome b558 and provide a potential, new mechanism by which IFN-γ modulates macrophage antimicrobial activity. Altogether, our data suggest that the mechanisms by which IFN-γ regulates antimicrobial activity of macrophages are more complex than previously appreciated.

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“…To analyze bacterial binding and uptake at the level of single cells, PMNs in suspension were infected with opsonized or unopsonized sGFP-expressing F. novicida for 60 min at 37°C and then cytocentrifuged onto acid-washed coverslips or plated onto coverslips that had been precoated with polylysine. Attached PMNs were fixed with 10% formalin, permeabilized with cold acetone:methanol (1:1), washed once with PBS, and blocked in PBS containing 0.5 mg/ml NaN 3 , 5 mg/ml BSA, and 10% horse serum, as we described previously [36,41]. Fixed and permeabilized cells were stained with DAPI (University of Iowa Central Microscopy Research Facility, Iowa City, IA, USA) to label PMN nuclei or with mAb specific for CD16 (Clone 3G8) at 1:100 dilution (BioLegend, San Diego, CA, USA) and Dylight 549-conjugated goat anti-mouse F(ab9) 2 secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at 1:500 dilution to label PMN plasma membranes.…”

Section: Quantitation Of Bacterial Binding and Uptake By Fluorescencementioning

confidence: 99%

“…For synchronized phagocytosis assays, PMNs were plated onto acid-washed glass coverslips in 35 mm tissue-culture dishes that had been precoated with 10% serum [41]. Opsonized or unopsonized U112 were added to each dish at an MOI of 25:1 in 2 ml serum-free RPMI 1640.…”

Section: Quantitation Of Bacterial Binding and Uptake By Fluorescencementioning

confidence: 99%

Francisella novicida inhibits spontaneous apoptosis and extends human neutrophil lifespan

Kinkead

Fayram

Allen

2017

Journal of Leukocyte Biology

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is a Gram-negative bacterium that is closely related to the highly virulent facultative intracellular pathogen, Data published by us and others demonstrate that virulence correlates directly with its ability to impair constitutive apoptosis and extend human neutrophil lifespan. In contrast, is attenuated in humans, and the mechanisms that account for this are incompletely defined. Our published data demonstrate that binds natural IgG that is present in normal human serum, which in turn, elicits NADPH oxidase activation that does not occur in response to As it is established that phagocytosis and oxidant production markedly accelerate neutrophil death, we predicted that may influence the neutrophil lifespan in an opsonin-dependent manner. To test this hypothesis, we quantified bacterial uptake, phosphatidylserine (PS) externalization, and changes in nuclear morphology, as well as the kinetics of procaspase-3, -8, and -9 processing and activation. To our surprise, we discovered that not only failed to accelerate neutrophil death but also diminished and delayed apoptosis in a dose-dependent, but opsonin-independent, manner. In keeping with this, studies of conditioned media (CM) showed that neutrophil longevity could be uncoupled from phagocytosis and that stimulated neutrophil secretion of CXCL8. Taken together, the results of this study reveal shared and unique aspects of the mechanisms used by species to manipulate neutrophil lifespan and as such, advance understanding of cell death regulation during infection.

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Immunofluorescence and Confocal Microscopy of Neutrophils

Cited by 19 publications

References 22 publications

IL-20 Signaling in Activated Human Neutrophils Inhibits Neutrophil Migration and Function

IL-20 Signaling in Activated Human Neutrophils Inhibits Neutrophil Migration and Function

Effects of IFN-γ on intracellular trafficking and activity of macrophage NADPH oxidase flavocytochrome b558

Francisella novicida inhibits spontaneous apoptosis and extends human neutrophil lifespan

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