Calcium changes in immune complex-stimulated human neutrophils. Simultaneous measurement of receptor occupancy and activation reveals full population stimulus binding but subpopulation activation

Brunkhorst, Beatrice; Lazzari, K G; Strohmeier, G; Weil, Gary J.; Simons, Elizabeth R.

doi:10.1016/s0021-9258(18)98799-5

Cited by 35 publications

(1 citation statement)

References 61 publications

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“…Immediately after purification, neutrophils were suspended at 5 ϫ 10 6 /ml in RPMI-1640 medium supplemented with 10% low endotoxin fetal bovine serum [Ͻ0.06 EU/ml by Limulus amoebocyte lysate (LAL) assay, BioWhittaker, Verviers, Belgium], treated with or without 1000 U/ml IFN-␣ (Roferon, Roche Laboratories, Nutley, NJ), 1000 U/ml IFN-␤ (Betaferon, Schering AG, Berlin, Germany), or 200 U/ml IFN-␥ (R&D Systems, Minneapolis, MN), unless otherwise specified, and then, usually plated in 24-well tissue-culture wells (Orange, Trasadingen, Switzerland). After 20 h of incubation, cultures were usually stimulated for an additional 4 h (unless otherwise indicated) with the following substances: TNF-␣ (0.5-5 ng/ml; Peprotech, Rocky Hill, NJ), LPS (0.1-100 ng/ml, from Escherichia coli, serotype 026:B6, Sigma Chemical Co., St. Louis, MO), fMLP (10 -1000 nM, Sigma Chemical Co.), 60 g/ml insoluble IC prepared as described previously [32], and hsp Gp96 (1-50 g/ml, Immatics Biotechnologies GmbH, Tubingen, Germany). The levels of contaminating endotoxin in the Gp96 preparations (declared by Immatics Biotechnologies GmbH as Ͻ0.026 EU/g by LAL assay and equivalent to Ͻ0.1 ng/ml LPS) had no effect on TRAIL release.…”

Section: Cell Purification and Culturementioning

confidence: 99%

Interferon-activated neutrophils store a TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand) intracellular pool that is readily mobilizable following exposure to proinflammatory mediators

Cassatella

Huber

Calzetti

et al. 2005

Journal of Leukocyte Biology

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Neutrophils are versatile cells, which play a role, not only in inflammatory processes but also in immune and antitumoral responses. Recently, we have reported that interferon (IFN)-activated neutrophils are able to release biologically active tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2 ligand), a molecule exerting selective, apoptotic activities toward tumor and virus-infected cells, as well as immunoregulatory functions on activated T lymphocytes. Herein, we show that only a minor fraction of the total TRAIL, newly synthesized by IFN-activated neutrophils within 24 h, is released outside, the rest being retained intracellularly, mainly in secretory vesicles and light membrane fractions. We demonstrate that the intracellular pool of TRAIL present in IFN-pretreated neutrophils is rapidly mobilizable to the cell surface and can be secreted following exposure to proinflammatory mediators such as TNF-alpha, lipopolysaccharide, formyl-methionyl-leucyl-phenylalanine, CXC chemokine ligand 8/interleukin-8, insoluble immunocomplexes, and heat shock protein Gp96. These various proinflammatory agonists functioned as effective secretagogue molecules only, in that they failed to augment TRAIL mRNA expression or TRAIL de novo synthesis in freshly isolated neutrophils or cultured with or without IFN. In addition, supernatants from IFN-treated neutrophils stimulated with proinflammatory mediators induced the apoptosis of target cells more effectively than supernatants from neutrophils activated with IFNs alone. Collectively, our results uncover a novel mechanism, whereby the release of soluble TRAIL by neutrophils can be greatly amplified and further reinforce the notion that neutrophils are important cells in tumor surveillance and immunomodulation.

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Section: Cell Purification and Culturementioning

confidence: 99%

Interferon-activated neutrophils store a TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand) intracellular pool that is readily mobilizable following exposure to proinflammatory mediators

Cassatella

Huber

Calzetti

et al. 2005

Journal of Leukocyte Biology

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Measurement of Phagocytosis and of the Phagosomal Environment in Polymorphonuclear Phagocytes by Flow Cytometry

Simons

2010

CP Cytometry

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Phagocytes are the most important early components of the immune response, programmed to recognize, engulf, and destroy immune complexes (formed when antibodies recognize their specific antigens), foreign particles, bacteria, mycobacteria, apoptotic cells, etc. Neutrophils, monocytes, macrophages, and dendritic cells all participate in this process. Flow cytometry permits observation of phagocytes that have responded and, with the appropriate fluorescent probes, of the environment in the phagosome that has enclosed the foreign matter. This unit gives the background and the protocols for performing such studies. Curr. Protoc. Cytom. 51:9.31.1‐9.31.10. © 2010 by John Wiley & Sons, Inc.

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Fcγ receptor activation of neutrophils in cryoglobulin‐induced leukocytoclastic vasculitis

Hundt

Zielinska‐Skowronek

Schmidt

1993

Arthritis & Rheumatism

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Objective. The role of Fcγ receptors (FcγR) in type I cryoglobulinemia was investigated to characterize novel mechanisms of neutrophil activation in the pathogenesis of leukocytoclastic vasculitis. Methods. Neutrophils from healthy donors were incubated with purified monoclonal IgG1k cryoglobulin complexes in vitro. Changes in surface antigen expression and mechanisms of intracellular hydrogen peroxide production and calcium release were measured by flow cytometry. Results. After incubation for 2 hours, surface expression of FcγRI (CD64), CD66, and CD67 was up‐regulated; FcγRII (CDw32), FcγRIII (CD16), and LAM‐1 were down‐regulated. Using solubilized and complexed cryoglobulins, it was demonstrated that complex formation is necessary to induce intracellular H2O2 production and calcium release from intracellular stores. Both H2O2 generation and calcium mobilization could be inhibited by pretreatment with F(ab')2 fragments of monoclonal antibodies (MAb) against FcγRIII. In contrast, Fab fragments of anti‐FcγRII MAb failed to block these activations. Neither the cryoglobulin complex–induced production of H2O2 nor the increase in cytoplasmic calcium was affected by treatment with pertussis toxin, which suggests that pertussis toxin–sensitive G proteins are not involved in signal transduction. Conclusion. These results indicate that FcγRIII plays a major role in the pathogenesis of leukocytoclastic vasculitis.

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Calcium changes in immune complex-stimulated human neutrophils. Simultaneous measurement of receptor occupancy and activation reveals full population stimulus binding but subpopulation activation

Cited by 35 publications

References 61 publications

Interferon-activated neutrophils store a TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand) intracellular pool that is readily mobilizable following exposure to proinflammatory mediators

Interferon-activated neutrophils store a TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand) intracellular pool that is readily mobilizable following exposure to proinflammatory mediators

Measurement of Phagocytosis and of the Phagosomal Environment in Polymorphonuclear Phagocytes by Flow Cytometry

Fcγ receptor activation of neutrophils in cryoglobulin‐induced leukocytoclastic vasculitis

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