Demonstration of specific receptors for fluoresceinated casein on human neutrophils and monocytes using flow cytometry

Sharon L. Lewis; Dennis E. Van Epps

doi:10.1007/bf00916301

Mouse IgG3 binding to macrophage-like cells is prevented by deglycosylation of the antibody or by Accutase treatment of the cells

Scientific Reports ◽

10.1038/s41598-021-89705-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alicja Karabasz ◽

Monika Bzowska ◽

Joanna Bereta ◽

Maria Czarnek ◽

Maja Sochalska ◽

...

Keyword(s):

Flow Cytometry ◽

Complex Network ◽

Immune Cells ◽

Specific Receptor ◽

Fcγ Receptors ◽

Specific Receptors ◽

First Time

AbstractThe binding of mouse IgG3 to Fcγ receptors (FcγR) and the existence of a mouse IgG3-specific receptor have been discussed for 40 years. Recently, integrin beta-1 (ITGB1) was proposed to be a part of an IgG3 receptor involved in the phagocytosis of IgG3-coated pathogens. We investigated the interaction of mouse IgG3 with macrophage-like J774A.1 and P388D1 cells. The existence of an IgG3-specific receptor was verified using flow cytometry and a rosetting assay, in which erythrocytes clustered around the macrophage-like cells coated with an erythrocyte-specific IgG3. Our findings confirmed that receptors binding antigen-free IgG3 are present on J774A.1 and P388D1 cells. We demonstrated for the first time that the removal of N-glycans from IgG3 completely abolished its binding to the cells. Moreover, we discovered that the cells treated with Accutase did not bind IgG3, indicating that IgG3-specific receptors are substrates of this enzyme. The results of antibody-mediated blocking of putative IgG3 receptors suggested that apart from previously proposed ITGB1, FcγRII, FcγRIII, also additional, still unknown, receptor is involved in IgG3 binding. These findings indicate that there is a complex network of glycan-dependent interactions between mouse IgG3 and the surface of effector immune cells.

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Role of Mitogen-Activated Protein Kinases in Activation of Human Neutrophils by Antineutrophil Cytoplasmic Antibodies

Journal of the American Society of Nephrology ◽

10.1681/asn.v12137 ◽

2001 ◽

Vol 12 (1) ◽

pp. 37-46

Author(s):

RALPH KETTRITZ ◽

ADRIAN SCHREIBER ◽

FRIEDRICH C. LUFT ◽

HERMANN HALLER

Keyword(s):

Flow Cytometry ◽

Cell Surface ◽

P38 Mapk ◽

Respiratory Burst ◽

Antineutrophil Cytoplasmic Antibodies ◽

Human Neutrophils ◽

Tnf Α ◽

Mitogen Activated Protein ◽

P38 Mapk Inhibitor ◽

Mapk Inhibitor

Abstract. Antineutrophil cytoplasmic antibodies (ANCA) may be important in the pathophysiology of necrotizing vasculitis. ANCA activate human neutrophils primed with tumor necrosis factor-α (TNF-α) in vitro. TNF-α priming results in translocation of ANCA antigens to the cell surface, where they are recognized by the antibodies. The signaling mechanisms involved in TNF-α priming and subsequent ANCA-induced activation were investigated. TNF-α-primed neutrophils were stimulated with monoclonal antibodies (MAb) to human myeloperoxidase (MPO) and proteinase 3 (PR3), and with preparations of human ANCA (three patients with PR3-ANCA and two patients with MPO-ANCA). Respiratory burst was measured with superoxide dismutase-inhibitable ferricytochrome C reduction and using dihydro-rhodamine-1,2,3. Phosphorylation of p38 mitogen-activated protein kinase (p38-MAPK) and the extracellular signal-regulated kinase (ERK) were assessed by immunoblotting. ANCA-antigen translocation was studied by flow cytometry. The tyrosine phosphorylation inhibitor genistein, but not calphostin or staurosporin, resulted in a significant dose-dependent superoxide generation inhibition (11.6 ± 1.7 nmol to 2.1 ± 0.5 for PR3-ANCA, and 16.0 ± 2.8 to 3.3 ± 1.3 for MPO-ANCA). The p38-MAPK inhibitor (SB202190) and the ERK inhibitor (PD98059) diminished PR3-ANCA-mediated superoxide production dose dependently (11.6 ± 1.7 nmol O2- to 1.9 ± 0.6 with 50 μM SB202190 and 4.0 ± 0.6 with 50 μM PD098059, respectively). For MPO-ANCA, the results were similar (16.0 ± 2.8 nmol to 0.9 ± 1.0 nmol with SB202190 and 6.4 ± 2.4 nmol with PD98059, respectively). Western blot showed phosphorylation of both p38-MAPK and ERK during TNF-α priming. The p38-MAPK inhibitor and the ERK inhibitor showed the strongest effect on respiratory burst when added before TNF-α priming, further supporting an important role for both signaling pathways in the priming process. Flow cytometry showed that p38-MAPK inhibition decreased the translocation of PR3 (by 93 ± 2%) and of MPO (by 64 ± 2%). In contrast, no such effect was seen when ERK was inhibited. Thus, p38-MAPK and ERK are important for the TNF-α-mediated priming of neutrophils enabling subsequent ANCA-induced respiratory burst. However, both pathways show differential effects, whereby p38-MAPK controls the translocation of ANCA antigens to the cell surface.

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Flow cytometry reveals different lag times in rapid cytoplasmic calcium elevations in human neutrophils in response to N-formyl peptide

Journal of Cellular Physiology ◽

10.1002/jcp.1041570325 ◽

1993 ◽

Vol 157 (3) ◽

pp. 637-643 ◽

Cited By ~ 8

Author(s):

Jörn Elsner ◽

Johannes Norgauer ◽

Gustav J. Dobos ◽

Andreas Emmendörffer ◽

Erwin Schöpf ◽

...

Keyword(s):

Flow Cytometry ◽

Human Neutrophils ◽

Cytoplasmic Calcium ◽

Formyl Peptide ◽

Lag Times

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Endocytosis of ß2 integrins by stimulated human neutrophils analyzed by flow cytometry

Journal of Leukocyte Biology ◽

10.1002/jlb.53.4.462 ◽

1993 ◽

Vol 53 (4) ◽

pp. 462-469 ◽

Cited By ~ 23

Author(s):

J. David Chambers ◽

Scott I. Simon ◽

Elaine M. Berger ◽

Larry A. Sklar ◽

Karl-E. Arfors

Keyword(s):

Flow Cytometry ◽

Human Neutrophils

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A method for high-purity isolation of neutrophil granulocytes for functional cell migration assays

Turkish Journal of Biochemistry ◽

10.1515/tjb-2019-0089 ◽

2019 ◽

Vol 44 (6) ◽

pp. 810-821

Author(s):

Edibe Avci ◽

Yeliz Z. Akkaya-Ulum ◽

Digdem Yoyen-Ermis ◽

Gunes Esendagli ◽

Banu Balci-Peynircioglu

Keyword(s):

Flow Cytometry ◽

Cell Migration ◽

Neutrophil Migration ◽

Cost Effective ◽

In Vitro Assays ◽

Human Neutrophils ◽

Neutrophil Granulocytes ◽

Inflammatory Conditions ◽

Migration Analysis

Abstract Background Neutrophil-mediated killing of pathogens is one of the most significant functions of the primary defense of the host. Neutrophil activity and migration play a key role in inflammatory conditions. To gain insights into the interactions between neutrophils and neutrophil migration-related disorders, a large number of sophisticated methods have been developed. The technical limitations of isolating highly purified neutrophil populations, minimizing both cell death and activation during the isolation process, and the short lifespan of neutrophils present challenges for studying specific functions of neutrophils in vitro. In this study, we aimed to evaluate a separation medium-based density gradient method to obtain highly purified neutrophil populations and combined this protocol with a model for studying neutrophil migration in-vitro. Materials and methods Human granulocytes were isolated using Lympholyte-poly solution. The purity and viability of isolated neutrophils were assessed by flow cytometry and morphological analysis. Neutrophil activation was confirmed by immunocytochemistry. Lastly, filter assay was performed to measure neutrophil chemotaxis. Results and discussion All validation experiments revealed that this method was capable of generating a highly purified neutrophil population for further functional in-vitro assays. Consequently, this study demonstrates a quick, cost effective, and easy-to-follow model, and may be a significant alternative to isolation methods that need extra subsequent steps such as flow cytometry-based cell sorting for reaching highly purified neutrophil population. Conclusion The suggested combination of methods for the isolation and cell migration analysis of human neutrophils is highly recommended to use for disease models involving neutrophil migration such as autoinflammatory disorders.

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Control of the Oxidative Burst of Human Neutrophils by Staphylococcal Leukotoxins

Infection and Immunity ◽

10.1128/iai.71.7.3724-3729.2003 ◽

2003 ◽

Vol 71 (7) ◽

pp. 3724-3729 ◽

Cited By ~ 31

Author(s):

Didier A. Colin ◽

Henri Monteil

Keyword(s):

Flow Cytometry ◽

Oxidative Burst ◽

Pore Formation ◽

Human Neutrophils ◽

Polymorphonuclear Cells ◽

Phosphatidylinositol 3 Kinase ◽

Intracellular Release ◽

Two Component ◽

Phosphatidylinositol 3

ABSTRACT The ability of staphylococcal two-component leukotoxins to induce an oxidative burst and/or to prime human polymorphonuclear cells (PMNs) was studied by using spectrofluorometry or flow cytometry. At sublytic concentrations, the HlgA-HlgB, HlgA-LukF-PV, LukS-PV-LukF-PV, and HlgC-LukF-PV combinations of leukotoxins, but not the LukS-PV-HlgB and HlgC-HlgB combinations, were able to induce H2O2 production similar to the H2O2 production induced by 1 μM N-formyl-Met-Leu-Phe (fMLP). In addition, when added at sublytic concentrations, all of the leukotoxin combinations primed PMNs for H2O2 production induced by fMLP. Leukotoxin activation was dependent on the presence of Ca2+ and was inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, but not by N-methyl-l-arginine, an inhibitor of NO generation, which eliminates the possibility that NO plays a role in the action of leukotoxins. At higher concentrations, all leukotoxins inhibited H2O2 production by PMNs activated by fMLP, phorbol 12-myristate 13-acetate (PMA), or the leukotoxins themselves. This inhibition was not related to the pore formation induced by leukotoxins. Intracellular release of H2O2 induced by fMLP and PMA was not primed by leukotoxins but was inhibited. It seems that leukotoxin inhibition of H2O2 release is independent of pore formation but secondary to an intracellular event, as yet unknown, triggered by leukotoxins.

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Graded responses of human neutrophils induced by serum-treated zymosan

Blood ◽

10.1182/blood.v66.5.1182.1182 ◽

1985 ◽

Vol 66 (5) ◽

pp. 1182-1188 ◽

Cited By ~ 3

Author(s):

JC Whitin ◽

DH Ryan ◽

HJ Cohen

Keyword(s):

Flow Cytometry ◽

Membrane Potential ◽

Fluorescent Probe ◽

Dose Response ◽

Dose Dependence ◽

Human Neutrophils ◽

Graded Responses ◽

Dose Dependent ◽

O2 Production ◽

Stimulation Of

Abstract A modified zymosan preparation was used to probe the interaction of particulate stimuli with human neutrophils (PMNs). After extraction with alkali and detergent, the zymosan particles retained their ability to be opsonized in serum and to stimulate PMNs. Serum-treated zymosan (STZ) induced dose-dependent superoxide (O2-) production and membrane potential depolarization in the range of 1 to 10 mg/mL of STZ. The rate and extent of secretion of lysozyme and beta-glucuronidase were also dose-dependent in the range of 1 to 10 mg/mL of STZ. Cytochemical studies using nitroblue tetrazolium, however, showed that 92% of PMNs were stimulated to produce O2- at 0.1 mg/mL of STZ. The dose response of O2- production induced by STZ is therefore due to increasing O2- production by individual PMNs and not to the stimulation of more PMNs to produce O2-. Evidence for O2- production was found only in the area of PMN-zymosan contact, suggesting a mechanism for the graded responses of PMNs treated with particulate stimuli. In order to determine the nature of the dose dependence of depolarization (a measure of PMN activation), PMNs equilibrated with the fluorescent probe 3,3′- dipentyloxacarbocyanine were analyzed by flow cytometry. The results demonstrate that STZ induces a dose-dependent depolarization of the membrane potential of individual PMNs. These results also demonstrate that increasing concentrations of STZ can induce increasing PMN responses even when all of the PMNs have been activated. These results are consistent with the hypothesis that receptor-mediated particulate stimulation of PMNs is a phenomenon that results in graded PMN responses.

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Identification and characterization of specific receptors for monocyte-derived neutrophil chemotactic factor (MDNCF) on human neutrophils.

Journal of Experimental Medicine ◽

10.1084/jem.169.3.1185 ◽

1989 ◽

Vol 169 (3) ◽

pp. 1185-1189 ◽

Cited By ~ 88

Author(s):

A K Samanta ◽

J J Oppenheim ◽

K Matsushima

Keyword(s):

Human Peripheral Blood ◽

Platelet Activating Factor ◽

Leukotriene B4 ◽

Chemotactic Factor ◽

Single Type ◽

Human Neutrophils ◽

Scatchard Analysis ◽

Specific Receptors ◽

Neutrophil Chemotactic Factor

Specific receptors for a recently purified and cloned monocyte-derived neutrophil chemotactic factor (MDNCF) have been identified on the surface of normal human peripheral blood neutrophils using 125I-labeled recombinant human MDNCF (125I-MDNCF). Competitive binding of 125I-MDNCF to human neutrophils reached a maximal level at 1-3 h at 4 degrees C. The Scatchard analysis showed that there are approximately 20,000 receptors per cell with a single type of high affinity binding (Kd, 8 x 10(-10) M). The receptors for MDNCF are clearly distinct from the receptors for other cytokines and chemotactic agents, e.g., IL-1 alpha, TNF-alpha, and FMLP, C5a, leukotriene B4, and platelet activating factor. Based on the SDS-PAGE analysis of chemically crosslinked 125I-MDNCF receptor complex, there are two polypeptides that bind MDNCF; the molecular weight of these two MDNCF receptors were estimated to be 67,000 and 59,000. Treatment of a promyelocytic cell line, HL60, with 1.25% DMSO for 5 d in vitro increased the number of receptors up to 7,000 receptors/cell with a Kd of 1.2 x 10(-9) M.

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