In Vitro Differentiation of Mouse Granulocytes

Reinhart, Ramona; Wicki, Simone; Kaufmann, Thomas

doi:10.1007/978-1-4939-3581-9_8

Cited by 8 publications

(13 citation statements)

References 23 publications

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“…We performed qPCR array analysis in IL-3 cond Hoxb8 basophils originating from conditionally immortalized mouse bone marrow progenitors 27 , 28 (further referred to as in vitro -differentiated mouse basophils) to study the impact of IL-3 on genes regulating apoptosis. As shown in Figure 1 , IL-3 increased pro-survival genes such as Bcl-2 , Bcl-x L and Mcl-1 in mouse basophils, while pro-apoptotic genes like caspase-2, -3, -9 and Bim were downregulated (see Supplementary Table S1 for full array list).…”

Section: Resultsmentioning

confidence: 99%

“…Using in vitro -differentiated mouse basophils, 27 , 28 we revealed that BCL-2 and BCL-X L prevent apoptotic cell death at steady state and identified both BCL-2 and BCL-X L as potential targets to counteract the IL-3-mediated pro-survival effects. Especially the combined inhibition of both proteins by ABT-263 was able to fully neutralize the IL-3 induced survival advantage.…”

Section: Discussionmentioning

confidence: 99%

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BH3 mimetics efficiently induce apoptosis in mouse basophils and mast cells

et al. 2017

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Basophil granulocytes and mast cells are recognized for their roles in immunity and are central effectors of diverse immunological disorders. Despite their similarities, there is emerging evidence for non-redundant roles of the circulating yet scarce basophils and tissue-resident mast cells, respectively. Because of their importance in allergic pathogenesis, specific induction of apoptosis in basophils and mast cells may represent an interesting novel treatment strategy. The pro-inflammatory cytokine interleukin-3 serves as a key factor for basophil and mouse mast cell survival. Interleukin-3 increases the expression of anti-apoptotic BCL-2 family members, such as BCL-2, BCL-XL or MCL-1; however, little is known how strongly these individual proteins contribute to basophil survival. Here, we were applying small molecule inhibitors called BH3 mimetics, some of which show remarkable success in cancer treatments, to neutralize the function of anti-apoptotic BCL-2 family members. We observed that expression levels of anti-apoptotic BCL-2 proteins do not necessarily correlate with their respective importance for basophil survival. Whereas naive in vitro-differentiated mouse basophils efficiently died upon BCL-2 or BCL-XL inhibition, interleukin-3 priming rendered the cells highly resistant toward apoptosis, and this could only be overcome upon combined targeting of BCL-2 and BCL-XL. Of note, human basophils differed from mouse basophils as they depended on BCL-2 and MCL-1, but not on BCL-XL, for their survival at steady state. On the other hand, and in contrast to mouse basophils, MCL-1 proved critical in mediating survival of interleukin-3 stimulated mouse mast cells, whereas BCL-XL seemed dispensable. Taken together, our results indicate that by choosing the right combination of BH3 mimetic compounds, basophils and mast cells can be efficiently killed, even after stimulation with potent pro-survival cytokines such as interleukin-3. Because of the tolerable side effects of BH3 mimetics, targeting basophils or mast cells for apoptosis opens interesting possibilities for novel treatment approaches.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

BH3 mimetics efficiently induce apoptosis in mouse basophils and mast cells

et al. 2017

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“…45, 56 The cells were cultivated in RPMI-1640 AQmedia supplemented with 10% FCS, 1% penicillin/streptomycin, 5% SCF and 0.1 μ M 4-OHT. Mature neutrophils were obtained within 5 days upon removal of 4-OHT from the medium.…”

Section: Methodsmentioning

confidence: 99%

Loss of XIAP facilitates switch to TNFα-induced necroptosis in mouse neutrophils

et al. 2016

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Neutrophils are essential players in the first-line defense against invading bacteria and fungi. Besides its antiapoptotic role, the inhibitor of apoptosis protein (IAP) family member X-linked IAP (XIAP) has been shown to regulate innate immune signaling. Whereas the role of XIAP in innate signaling pathways is derived mostly from work in macrophages and dendritic cells, it is not known if and how XIAP contributes to these pathways in neutrophils. Here we show that in response to bacterial lipopolysaccharides (LPS), mouse neutrophils secreted considerable amounts of tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) and, in accordance with earlier reports, XIAP prevented LPS-induced hypersecretion of IL-1β also in neutrophils. Interestingly, and in contrast to macrophages or dendritic cells, Xiap-deficient neutrophils were insensitive to LPS-induced cell death. However, combined loss of function of XIAP and cIAP1/-2 resulted in rapid neutrophil cell death in response to LPS. This cell death occurred by classical apoptosis initiated by a TNFα- and RIPK1-dependent, but RIPK3- and MLKL-independent, pathway. Inhibition of caspases under the same experimental conditions caused a shift to RIPK3-dependent cell death. Accordingly, we demonstrate that treatment of neutrophils with high concentrations of TNFα induced apoptotic cell death, which was fully blockable by pancaspase inhibition in wild-type neutrophils. However, in the absence of XIAP, caspase inhibition resulted in a shift from apoptosis to RIPK3- and MLKL-dependent necroptosis. Loss of XIAP further sensitized granulocyte–macrophage colony-stimulating factor (GM-CSF)-primed neutrophils to TNFα-induced killing. These data suggest that XIAP antagonizes the switch from TNFα-induced apoptosis to necroptosis in mouse neutrophils. Moreover, our data may implicate an important role of neutrophils in the development of hyperinflammation and disease progression of patients diagnosed with X-linked lymphoproliferative syndrome type 2, which are deficient in XIAP.

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“…; Reinhart et al . ), no studies have been published so far using these cells to investigate SOCE. Whether these cells can serve as a reliable experimental tool to support or replace animal studies will depend on whether they can fully recapitulate the functions of terminally differentiated neutrophils.…”

Section: Ca2+ Signalling In Neutrophil Functionmentioning

confidence: 99%

“…The technique offers advantages over other myeloid cell lines due to the non-cancerous origin of cells and their physiological differentiation programme. Although the technique has been further developed by other groups (Gurzeler et al 2013;Reinhart et al 2016), no studies have been published so far using these cells to investigate SOCE. Whether these cells can serve as a reliable experimental tool to support or replace animal studies will depend on whether they can fully recapitulate the functions of terminally differentiated neutrophils.…”

Section: Considerations In Studying and Interpreting The Role Of Stimmentioning

confidence: 99%

The role of STIM proteins in neutrophil functions

Demaurex

Saul

2018

The Journal of Physiology

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Stromal interaction molecule (STIM) proteins regulate store‐operated Ca2+ entry (SOCE) in innate and adaptive immune cells and participate in the Ca2+ signals that control the functions of neutrophils, the first line of host defence against bacterial and fungal infections. Loss‐of‐function experiments in animal and cellular models indicate that both STIM1 and STIM2 regulate neutrophil functions, but the complexity of the SOCE machinery and the versatility of neutrophils complicate the evaluation of the results. This review aims to summarize the latest progress in the field, with special attention to the details of the experimental designs. Future study design should aim to improve the standardization of experimental procedures and to provide a more holistic understanding of the role of STIM proteins in neutrophils function.

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In Vitro Differentiation of Mouse Granulocytes

Cited by 8 publications

References 23 publications

BH3 mimetics efficiently induce apoptosis in mouse basophils and mast cells

BH3 mimetics efficiently induce apoptosis in mouse basophils and mast cells

Loss of XIAP facilitates switch to TNFα-induced necroptosis in mouse neutrophils

The role of STIM proteins in neutrophil functions

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