Technical Advance: Autofluorescence-based sorting: rapid and nonperturbing isolation of ultrapure neutrophils to determine cytokine production

Dorward, David A.; Lucas, Christopher D.; Alessandri, Ana L.; Marwick, John A.; Rossi, Fiona; Dransfield, Ian; Haslett, Christopher; Dhaliwal, Kevin; Rossi, Adriano G.

doi:10.1189/jlb.0113040

Cited by 53 publications

(44 citation statements)

References 33 publications

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“…PMNs [37]. To prove further that the H 4 receptor mRNA is present in PMNs and not in contaminating cells (i.e., mononuclear cells and eosinophils), we used an autofluorescencebased sorting method [26] to obtain ultrapure (99.95%) PMNs. Forward-and side-scatter profiles separated granulocytes from contaminating mononuclear cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

The histamine H4 receptor is a potent inhibitor of adhesion-dependent degranulation in human neutrophils

Dib

Perečko

Jenei

et al. 2014

Journal of Leukocyte Biology

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The histamine H4 receptor regulates the inflammatory response. However, it is not known whether this receptor has a functional role in human neutrophils. We found that fMLP (1 μM), but not histamine (0.1-1 μM), induced Mac-1-dependent adhesion, polarization, and degranulation (release of lactoferrin). A pretreatment of neutrophils with histamine (0.001-1 μM) or JNJ 28610244 (0.1-10 μM), a specific H4 receptor agonist, led to inhibition of degranulation. Total inhibition of degranulation was obtained with 0.1 μM histamine and 10 μM JNJ 28610244. Furthermore, such inhibition by histamine of degranulation was reversed by JNJ 7777120 and JNJ 28307474, two selective H4 receptor antagonists. However, neither histamine nor the H4 receptor agonist JNJ 28610244 prevented fMLP-induced, Mac-1-dependent adhesion, indicating that the H4 receptor may block signals emanating from Mac-1-controlling degranulation. Likewise, engagement of the H4 receptor by the selective agonist JNJ 28610244 blocked Mac-1-dependent activation of p38 MAPK, the kinase that controls neutrophil degranulation. We also show expression of the H4 receptor at the mRNA level in ultrapure human neutrophils and myeloid leukemia PLB-985 cells. We concluded that engagement of this receptor by selective H4 receptor agonists may represent a good, therapeutic approach to accelerate resolution of inflammation.

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Section: Resultsmentioning

confidence: 99%

The histamine H4 receptor is a potent inhibitor of adhesion-dependent degranulation in human neutrophils

Dib

Perečko

Jenei

et al. 2014

Journal of Leukocyte Biology

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“…Cell sorting with an antibody against CD11b, CD16 and CD66b allowed for the enrichment of highly pure mature neutrophils in the blood Density gradients used to isolate neutrophils result in neutrophil cell purity of Z 95%, with contaminating cells being identified as 1-5% for eosinophils, basophils, mast cells and 1-2% for other mononuclear cells [23]. Even a 5% cellular contamination can alter phenotypical and functional assays, which is why researchers are constantly striving for methods that yield increased purity.…”

Section: Oral Neutrophils In Cp Patients Are Characterized By An Actimentioning

confidence: 99%

Identification of neutrophil surface marker changes in health and inflammation using high-throughput screening flow cytometry

Lakschevitz

Hassanpour

Rubin

et al. 2016

Experimental Cell Research

134

117

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“…Within neutrophils, these include chemotaxis, ROS production, degranulation, cytokine expression, phagocytosis, and changes in cell surface marker expression. 23,45,46 FPR1 Desensitization As discussed, regulation of GPCR function is essential in vivo for controlling neutrophil functions, including chemotaxis and the exact localization of cells to the inflammatory site. 9,23,47 After FPR1-ligand binding, the cell rapidly becomes unresponsive to subsequent stimulation by the same ligand (homologous desensitization).…”

Section: Fpr1 Binding Propertiesmentioning

confidence: 98%

The Role of Formylated Peptides and Formyl Peptide Receptor 1 in Governing Neutrophil Function during Acute Inflammation

Dorward

Lucas

Chapman

et al. 2015

The American Journal of Pathology

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202

200

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Neutrophil migration to sites of inflammation and the subsequent execution of multiple functions are designed to contain and kill invading pathogens. These highly regulated and orchestrated processes are controlled by interactions between numerous receptors and their cognate ligands. Unraveling and identifying those that are central to inflammatory processes may represent novel therapeutic targets for the treatment of neutrophil-dominant inflammatory disorders in which dysregulated neutrophil recruitment, function, and elimination serve to potentiate rather than resolve an initial inflammatory insult. The first G protein-coupled receptor to be described on human neutrophils, formyl peptide receptor 1 (FPR1), is one such receptor that plays a significant role in the execution of these functions through multiple intracellular signaling pathways. Recent work has highlighted important observations with regard to both receptor function and the importance and functional relevance of FPR1 in the pathogenesis of a range of both sterile and infective inflammatory conditions. In this review, we explore the multiple components of neutrophil migration and function in both health and disease, with a focus on the role of FPR1 in these processes. The current understanding of FPR1 structure, function, and signaling is examined, alongside discussion of the potential importance of FPR1 in inflammatory diseases suggesting that FPR1 is a key regulator of the inflammatory environment.

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Technical Advance: Autofluorescence-based sorting: rapid and nonperturbing isolation of ultrapure neutrophils to determine cytokine production

Abstract: A novel method to ultra-purify unstimulated neutrophils by flow sorting and subsequent demonstration of the neutrophils' ability to release IL-1β following inflammatory stimulus.

Cited by 53 publications

References 33 publications

The histamine H4 receptor is a potent inhibitor of adhesion-dependent degranulation in human neutrophils

The histamine H4 receptor is a potent inhibitor of adhesion-dependent degranulation in human neutrophils

Identification of neutrophil surface marker changes in health and inflammation using high-throughput screening flow cytometry

The Role of Formylated Peptides and Formyl Peptide Receptor 1 in Governing Neutrophil Function during Acute Inflammation

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