A map of gene expression in neutrophil-like cell lines

Rincón, Esther; Rocha-Gregg, Briana L; Collins, Sean R.

doi:10.1101/204875

Cited by 3 publications

(3 citation statements)

References 42 publications

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“…The differentiated HL60 cell also highly expresses CAPRI (Fig. S2 B ), consistent with a previous report (37), and provides a suitable cell system in which to study CAPRI’s function in human neutrophils. We found that chemoattractant fMLP (N-formyl-L-methionyl-L-leucyl-phenylalanine) stimulation promoted the association between CAPRI and N-Ras/Rap1(Fig 1 A ), suggesting a role of CAPRI in the regulation of chemoattractant-induced Ras and Rap1 signaling.…”

Section: Resultssupporting

confidence: 90%

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Ras Inhibitor CAPRI Enables Neutrophils to Chemotax Through a Higher-Concentration Range of Gradients

Wen

moosa

et al. 2020

Preprint

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Neutrophils sense and migrate through an enormous range of chemoattractant gradients through adaptation. Here, we reveal that, in human neutrophils, Calcium-promoted Ras inactivator (CAPRI) locally controls the GPCR-stimulated Ras adaptation. Human neutrophils lacking CAPRI (caprikd) exhibit chemoattractant-induced non-adaptive Ras activation; significantly increased phosphorylation of AKT, GSK3α/3β, and cofilin; and excessive actin polymerization. caprikd cells display defective chemotaxis in response to high-concentration gradients but exhibit improved chemotaxis in low- or subsensitive-concentration gradients of various chemoattractants as a result of their enhanced sensitivity. Taken together, our data reveal that CAPRI controls GPCR activation-mediated Ras adaptation and lowers the sensitivity of human neutrophils so that they are able to chemotax through a higher concentration range of chemoattractant gradients.Significance StatementNeutrophils provide first-line host defense by migrating through chemoattractant gradients to the sites of inflammation. Inappropriate recruitment and mis-regulated activation of neutrophils contribute to tissue damage and cause autoimmune and inflammatory disease. One fascinating feature of chemotactic neutrophils is their ability to migrate through an enormous concentration range of chemoattractant gradients (10−9 ∼ 10−5 M) through “adaptation,” in which cells no longer respond to the present stimuli, but remain sensitive to stronger stimuli. The inhibitory mechanism largely remains elusive, although many molecules of the excitatory signaling pathway have been identified. Our study reveals, for the first time, that the inhibitory component, CAPRI, is essential for both the sensitivity and the GPCR-mediated adaptation of human neutrophils.

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Section: Resultssupporting

confidence: 90%

“…HL60 cells also expressed RASAL1 and RASAL2 (Fig. S2)(37), which also deactivate Ras (34, 39). In mouse and human neutrophils, there are other RasGAP proteins expressed in addition to CAPRI (Fig.…”

Section: Discussionmentioning

confidence: 99%

Ras Inhibitor CAPRI Enables Neutrophils to Chemotax Through a Higher-Concentration Range of Gradients

Wen

moosa

et al. 2020

Preprint

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“…Given that neutrophils are relatively onerous to work with, due to their short half-life and the likelihood of overactivation and exhaustion, the use of a cell-line representative of neutrophil activity would be ideal. The human HL-60 cell line can be differentiated into neutrophil-like cells after culture with dimethyl sulfoxide (DMSO); however, FcR transcription and cell-surface levels are far lower and the profile quite different from those of true neutrophils [58,59]. Recently, ADCP assays using HIV-positive sera with both DMSO-treated HL-60 and primary neutrophils were compared, and ADCP scores were found to correlate well [59].…”

Section: Discussionmentioning

confidence: 99%

Primary Human Neutrophils Exhibit a Unique HIV-Directed Antibody-Dependent Phagocytosis Profile

et al. 2018

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The only clinical HIV vaccine trial to demonstrate efficacy, RV144, correlated protection with the antibodies (Abs) mediating function via the “constant” immunoglobulin region, the crystallizable fragment (Fc). These data have supported a focus on the induction of Abs by vaccines that trigger antiviral activities by relevant leukocytes via Fc receptors (FcRs). Neutrophils are phagocytes that comprise > 50% of leukocytes and display unique FcRs. We sought to compare the Ab-dependent cellular phagocytosis (ADCP) activity of human neutrophils to the commonly assayed THP-1 cell line. HIV-specific Abs were employed to elicit ADCP of beads coated with HIV envelope protein. Overall, trends were noted among neutrophil donors and the ADCP profile was different from that of THP-1 cells. mAb ELISA titers correlated with ADCP by THP-1 cells but not neutrophils. Monoclonal (m)Abs were also tested with primary monocytes. Donor-to-donor variation was high, and hindered the analysis of this dataset, but it was, in itself, an important finding. This study illustrates the concept that the assessment of FcR-mediated Ab activity with a frequently used cell line such as THP-1 is not necessarily indicative of relevant Ab functionality in vivo, and this calls for in-depth study of the properties of the HIV antibodies best-suited to eliciting antiviral activities by primary cells.

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Parallel High-Resolution Imaging of Leukocyte Chemotaxis Under Agarose with Rho-Family GTPase Biosensors

Bell

Natwick

Collins

2018

Methods in Molecular Biology

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Neutrophils are key early responders in the innate immune response that use chemotaxis, the directed migration along chemical gradients, to reach sites of infection or inflammation. This process requires integrating inputs from cell surface receptors with the cell's polarity and motility signaling network, in which highly dynamic and interconnected signaling by Rho-family GTPases plays a central role. To understand this fundamentally important behavior, we describe a high-resolution, under-agarose chemotaxis assay for use with neutrophil-like cell lines (HL-60 or PLB-985) or with primary neutrophils. We also describe how to use optical uncaging of chemoattractants to stimulate cells in this assay. These techniques are compatible with epifluorescence, total internal reflection fluorescence (TIRF), and confocal microscopy. Additionally, we cover how to measure the activities of Rho-family GTPases in this context using Förster resonance energy transfer (FRET)-based biosensors. The specific experimental steps outlined in this chapter include how to (1) set up the under-agarose assay, (2) optically pattern chemoattractant gradients, (3) image cells, and (4) conduct basic image analysis for FRET biosensors.

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A map of gene expression in neutrophil-like cell lines

Abstract: 23Background 24

Cited by 3 publications

References 42 publications

Ras Inhibitor CAPRI Enables Neutrophils to Chemotax Through a Higher-Concentration Range of Gradients

Ras Inhibitor CAPRI Enables Neutrophils to Chemotax Through a Higher-Concentration Range of Gradients

Primary Human Neutrophils Exhibit a Unique HIV-Directed Antibody-Dependent Phagocytosis Profile

Parallel High-Resolution Imaging of Leukocyte Chemotaxis Under Agarose with Rho-Family GTPase Biosensors

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