A method to study apoptosis in eosinophils by flow cytometry

Sandström, Kristina; Håkansson, Lena; Lukinius, Agneta

doi:10.1016/s0022-1759(00)00176-9

Cited by 24 publications

(23 citation statements)

References 38 publications

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“…Nonapoptotic PMN were separated from apoptotic PMN in 24-h cultures using immunomagnetic techniques, which we modified for this study (9,23). Briefly, cell aliquots were removed from culture and combined to obtain approximately 20 -25 ϫ 10 6 PMN.…”

Section: Methodsmentioning

confidence: 99%

Neonatal Neutrophils with Prolonged Survival Exhibit Enhanced Inflammatory and Cytotoxic Responsiveness

et al. 2005

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Apoptosis is critical to the resolution of inflammation, as it promotes the removal of neutrophils (PMN) by the reticuloendothelial system. In contrast, PMN persistence characterizes the early stages of chronic inflammation. Adult PMN with delayed senescence retain some functionality, although this has not been described for neonatal PMN. We hypothesized that neonatal PMN with prolonged survival retain cytotoxic and inflammatory function. To test one aspect of inflammatory function, we determined surface CD11b expression on 0-h and 24-h PMN after chemotactic formyl-methionine-leucine-phenylalanine (fMLP) stimulation. Although fMLP induced a greater percentage upregulation of CD11b on 0-h adult PMN, this was similar between nonapoptotic cord blood and adult PMN at 24 h. Furthermore, percentage up-regulation of CD11b was more robust for 24-h than for 0-h cord blood PMN. In contrast, there was no difference in responsiveness between 0-h and 24-h adult PMN. In studies of cytotoxic potential, we determined the expression of reactive oxygen intermediates (ROI) in phorbol 12-myristate 13-acetatestimulated cord blood and adult PMN at 0 h and in 24-h nonapoptotic PMN, using the dihydrorhodamine 123 assay.Stimulated cord blood PMN generated more ROI than did adult PMN at both 0 h and 24 h; in addition, ROI levels in 24-h cord blood PMN were similar to those of 0-h adult PMN. We conclude that PMN with prolonged survival retain specific cytotoxic and inflammatory functions, and these are enhanced in cord blood PMN. We speculate that neonatal PMN with prolonged survival have the functional capacity to contribute to the pathogenesis of inflammatory disorders. Abbreviations CLD, chronic lung disease DHR, dihydrorhodamine 123 fMLP, formyl-methionine-leucine-phenylalanine PE, phycoerythrin PMA, phorbol 12-myristate 13-acetate PMN, neutrophil(s) ROI, reactive oxygen intermediates 7-AAD, 7-aminoactinomycin PMN serve as the primary line of host defense during the early stages of sepsis and inflammation (1). After activation by systemic or local factors, primed endothelial cells interact with PMN through adhesion molecules that include selectins and integrins (2). These adhesive interactions initiate a process that allows the extravasation of PMN into inflamed tissue with the goal of neutralizing the inciting stimulus. In the case of inflammation mediated by bacterial or fungal microorganisms, phagocytosis and subsequent killing of these invaders occurs through mechanisms that partly involve ROI and proteases (3,4).The concept that apoptosis of inflammatory PMN and their timely removal by resident macrophages are critical to the resolution of inflammation has been well established (5,6). During the resolution phase, PMN typically undergo the phenotypic changes associated with apoptosis, which promotes their removal by resident macrophages (7). PMN can also undergo necrosis and lysis, a process that induces cytotoxicity in surrounding tissues (8), although apoptosis is the more common and physiologically preferable route. All cells a...

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Section: Methodsmentioning

confidence: 99%

Neonatal Neutrophils with Prolonged Survival Exhibit Enhanced Inflammatory and Cytotoxic Responsiveness

et al. 2005

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“…The cells were washed with PBS and incubated with 50 g/ml ribonuclease A (Sigma-Aldrich) for 30 min at 37°C, then with 50 g/ml of propidium iodide (PI; Sigma-Aldrich) for 10 min. Stained cells were analyzed by flow cytometry (33,34).…”

Section: Apoptosis Assaymentioning

confidence: 99%

Galectin-9 Induces Apoptosis Through the Calcium-Calpain-Caspase-1 Pathway

Kashio

Nakamura

Abedin

et al. 2003

The Journal of Immunology

275

239

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Galectin-9 (Gal-9) induced the apoptosis of not only T cell lines but also of other types of cell lines in a dose- and time-dependent manner. The apoptosis was suppressed by lactose, but not by sucrose, indicating that β-galactoside binding is essential for Gal-9-induced apoptosis. Moreover, Gal-9 required at least 60 min of Gal-9 binding and possibly de novo protein synthesis to mediate the apoptosis. We also assessed the apoptosis of peripheral blood T cells by Gal-9. Apoptosis was induced in both activated CD4+ and CD8+ T cells, but the former were more susceptible than the latter. A pan-caspase inhibitor (Z-VAD-FMK) inhibited Gal-9-induced apoptosis. Furthermore, a caspase-1 inhibitor (Z-YVAD-FMK), but not others such as Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and Z-AEVD-FMK (caspase-10 inhibitor), inhibited Gal-9-induced apoptosis. We also found that a calpain inhibitor (Z-LLY-FMK) suppresses Gal-9-induced apoptosis, that Gal-9 induces calcium (Ca2+) influx, and that either the intracellular Ca2+ chelator BAPTA-AM or an inositol trisphosphate inhibitor 2-aminoethoxydiphenyl borate inhibits Gal-9-induced apoptosis. These results suggest that Gal-9 induces apoptosis via the Ca2+-calpain-caspase-1 pathway, and that Gal-9 plays a role in immunomodulation of T cell-mediated immune responses.

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“…Staining with FDA, PI and Annexin V Thymocytes, untreated or treated for 8 h with CPS at different doses (5, 10 or 25 mM) at 371C, 5% CO 2 , were incubated for 5 min at 371C with 0.125 mg/ml FDA, an agent that is metabolized to fluorescein in living cells, or 0.2 mg PI in 0.2 ml of binding buffer, 67 respectively. After washing in phosphate-buffered saline (PBS), samples were analyzed on a FACScan cytofluorimeter using the CellQuest software.…”

Section: Confocal Laser Scanning Microscopy Analysismentioning

confidence: 99%

Distinct thymocyte subsets express the vanilloid receptor VR1 that mediates capsaicin-induced apoptotic cell death

et al. 2004

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Herein, we provide the first evidence on the capsaicin (CPS) receptor vanilloid receptor type-1 (VR1) by rat thymocytes, and its involvement in CPS-induced apoptosis. VR1 mRNA was identified by quantitative RT-PCR in CD5 þ thymocytes. By immunofluorescence and flow cytometry, we found that a substantial portion of CD5 þ thymocytes, namely CD4 þ and double negative (DN) cell subsets, express VR1 that was present on plasma membrane on discrete spots. By Western blot, VR1 protein was identified as a single band of 95 kDa. We also described that CPS could trigger two distinct pathways of thymocyte death, namely apoptosis and necrosis depending on the dose of CPS exposure. CPS-induced apoptosis involved intracellular free calcium (Ca 2 þ ) influx, phosphatidylserine exposure, mitochondrial permeability transmembrane pore (PTP) opening and mitochondrial transmembrane potential (DW m ) dissipation leading to cytochrome c release, activation of caspase-9 and -3 and oligonucleosomal DNA fragmentation. VR1 was functionally implicated in these events as they were completely abrogated by the VR1 antagonist, capsazepine (CPZ). Finally, we demonstrated that VR1 expression on distinct thymocytes was associated with the selective ability of CPS to trigger DNA fragmentation in VR1 þ CD4 þ and DN thymocytes. Overall, our results suggest that the expression of VR1 on thymocytes may function as a sensor of harmful stimuli present in the thymic environment.

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A method to study apoptosis in eosinophils by flow cytometry

Cited by 24 publications

References 38 publications

Neonatal Neutrophils with Prolonged Survival Exhibit Enhanced Inflammatory and Cytotoxic Responsiveness

Neonatal Neutrophils with Prolonged Survival Exhibit Enhanced Inflammatory and Cytotoxic Responsiveness

Galectin-9 Induces Apoptosis Through the Calcium-Calpain-Caspase-1 Pathway

Distinct thymocyte subsets express the vanilloid receptor VR1 that mediates capsaicin-induced apoptotic cell death

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