Optimized protocol for the isolation of spleen‐resident murine neutrophils

Coquery, Christine M.; Loo, William; Buszko, Maja; Lannigan, Joanne; Erickson, Loren D.

doi:10.1002/cyto.a.22096

Cited by 21 publications

(27 citation statements)

References 30 publications

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“…Under homeostatic conditions, neutrophils are present in SLO tissues at a frequency of 1–2% of total splenocytes, although their numbers can increase dramatically during an immune response (25). In order to characterize neutrophils in lupus and their impact on adaptive immunity, we first quantitated neutrophil infiltration of the spleen at several points over the course of lupus progression by FACS analysis of splenocytes from female NZB/W mice at 11, 23, 25, and 30 weeks of age.…”

Section: Resultsmentioning

confidence: 99%

Neutrophils Slow Disease Progression in Murine Lupus via Modulation of Autoreactive Germinal Centers

Bird

Chang

Barnard

et al. 2017

The Journal of Immunology

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Neutrophils are well characterized as mediators of peripheral tissue damage in lupus, but it remains unclear whether they influence loss of self-tolerance in the adaptive immune compartment. Lupus neutrophils produce elevated levels of factors known to fuel autoantibody production, including interleukin-6 and B cell survival factors, but also reactive oxygen intermediates, which can suppress lymphocyte proliferation. In order to assess whether neutrophils directly influence the progression of auto-reactivity in secondary lymphoid organs (SLO), we characterized the localization and cell-cell contacts of splenic neutrophils at several stages in the progression of disease in the NZB/W murine model of lupus. Neutrophils accumulate in SLO over the course of lupus progression, preferentially localizing near T lymphocytes early in disease and B cells with advanced disease. RNA sequencing reveals that the splenic neutrophil transcriptional program changes significantly over the course of disease, with neutrophil expression of anti-inflammatory mediators peaking during early- and mid-stage disease, and evidence of neutrophil activation with advanced disease. To assess whether neutrophils exert predominantly protective or deleterious effects on loss of B cell self-tolerance in vivo, we depleted neutrophils at different stages of disease. Neutrophil depletion early in lupus resulted in a striking acceleration in the onset of renal disease, SLO germinal center formation, and auto-reactive plasma cell production. In contrast, neutrophil depletion with more advanced disease did not alter SLE progression. These results demonstrate a surprising temporal and context dependent role for neutrophils in restraining auto-reactive B cell activation in lupus.

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Section: Resultsmentioning

confidence: 99%

Neutrophils Slow Disease Progression in Murine Lupus via Modulation of Autoreactive Germinal Centers

Bird

Chang

Barnard

et al. 2017

The Journal of Immunology

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“…We chose this method since detection of BCMA protein on the cell surface of murine cells is unreliable due to poor reagents [33]. Using a protocol developed by our laboratory to isolate spleen-resident murine neutrophils [26], we found that BCMA was not expressed in neutrophils from B6.Fas lpr /J mice compared to WT, Tnfrsf17 −/− , and B6.Fas lpr /J Tnfrsf17 −/− mice (Fig. 2F).…”

Section: Resultsmentioning

confidence: 99%

Neutrophils Contribute to Excess Serum BAFF Levels and Promote CD4+ T Cell and B Cell Responses in Lupus-Prone Mice

et al. 2014

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Despite increased frequencies of neutrophils found in autoimmune diseases such as systemic lupus erythematosus (SLE), how they contribute to disease pathogenesis and the mechanisms that affect the accumulation of neutrophils are poorly understood. The aim of this study was to identify factors in autoantibody-mediated autoimmunity that controls the accumulation of spleen resident neutrophils and to determine whether neutrophils contribute to abnormal B cell responses. Increased levels of the cytokine BAFF have been linked to loss of B cell tolerance in autoimmunity, but the cellular source responsible for excess BAFF is unknown. B cell maturation antigen (BCMA) is a receptor for BAFF and is critical for the survival of bone marrow plasma cells. Paradoxically, BCMA deficiency exacerbates the formation of autoantibody-secreting plasma cells in spleens of lupus-prone mice and the reasons for this effect are not understood. Here we analyzed the phenotype, localization and function of neutrophils in spleens of healthy mice and congenic lupus-prone mice, and compared mice sufficient or deficient in BCMA expression. Neutrophils were found to be significantly increased in frequency and activation status in spleens of lupus-prone mice when BCMA was absent. Furthermore, neutrophils localized within T cell zones and enhanced CD4+ T cell proliferation and IFNγ production through the production of BAFF. Reduced BAFF and IFNγ serum levels, decreased frequencies of IFNγ-producing T cells, germinal center B cells, and autoantibody production after neutrophil depletion indicated the involvement of neutrophils in these autoimmune traits. Thus, we have identified a novel role for BCMA to control excess BAFF production in murine lupus through restraining the accumulation of BAFF-producing neutrophils. Our data suggests that devising therapeutic strategies to reduce neutrophils in autoimmunity may decrease BAFF levels and ameliorate disease.

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“…Further detail characterization would be needed to elucidate the origin of these events, which was outside the focus in this study. Performing a ficoll density gradient centrifugation may lead to further enrichment of intact immune cells from debris as we and others have observed for other tissues [42-44]. However, since cell suspensions collected from muscle biopsies are rather small we preferred not to risk losing cells by performing additional procedures as well as losing specific immune cells such as neutrophils that are discarded by such centrifugation.…”

Section: Discussionmentioning

confidence: 99%

Dissociation of skeletal muscle for flow cytometric characterization of immune cells in macaques

Liang

Ploquin²,

Hernández³

et al. 2015

Journal of Immunological Methods

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The majority of vaccines and several treatments are administered by intramuscular injection. The aim is to engage and activate immune cells, although they are rare in normal skeletal muscle. The phenotype and function of resident as well as infiltrating immune cells in the muscle after injection are largely unknown. While methods for obtaining and characterizing murine muscle cell suspensions have been reported, protocols for nonhuman primates (NHPs) have not been well defined. NHPs comprise important in vivo models for studies of immune cell function due to their high degree of resemblance with humans. In this study, we developed and systematically compared methods to collect vaccine-injected muscle tissue to be processed into single cell suspensions for flow cytometric characterization of immune cells. We found that muscle tissue processed by mechanical disruption alone resulted in significantly lower immune cell yields compared to enzymatic digestion using Liberase. Dendritic cell subsets, monocytes, macrophages, neutrophils, B cells, T cells and NK cells were readily detected in the muscle by the classical human markers. The methods for obtaining skeletal muscle cell suspension established here offer opportunities to increase the understanding of immune responses in the muscle, and provides a basis for defining immediate post-injection vaccine responses in primates.

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Optimized protocol for the isolation of spleen‐resident murine neutrophils

Cited by 21 publications

References 30 publications

Neutrophils Slow Disease Progression in Murine Lupus via Modulation of Autoreactive Germinal Centers

Neutrophils Slow Disease Progression in Murine Lupus via Modulation of Autoreactive Germinal Centers

Neutrophils Contribute to Excess Serum BAFF Levels and Promote CD4+ T Cell and B Cell Responses in Lupus-Prone Mice

Dissociation of skeletal muscle for flow cytometric characterization of immune cells in macaques

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