Inhibition of nonspecific binding of fluorescent-labelled antibodies to human eosinophils

Mahmudi‐Azer, Salahaddin; Lacy, Paige; Bablitz, Ben; Moqbel, Redwan

doi:10.1016/s0022-1759(98)00105-7

Cited by 27 publications

(18 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Boiling of this preparation improved the visualization of the nuclear positive signal as shown in this work. High nonspecific binding of IgG to the cytoplasm in eosinophils is also observed by others (32). Nuclear antigen masking in neutrophils and eosinophils and cytoplasmic high nonspecific binding of IgG in eosinophils probably made it difficult to distinguish a difference in signals between nonimmune IgG and immune IgG in the previous work.…”

Section: Discussionmentioning

confidence: 52%

Nuclear Localization of Peptidylarginine Deiminase V and Histone Deimination in Granulocytes

Nakashima

Hagiwara

Yamada

2002

Journal of Biological Chemistry

340

296

View full text Add to dashboard Cite

Peptidylarginine deiminase (PAD) deiminates arginine residues in proteins to citrulline residues Ca 2؉ dependently. There are four types of PADs, I, II, III, and V, in humans. We studied the subcellular distribution of PAD V in HL-60 granulocytes and peripheral blood granulocytes. Expression of green fluorescent proteintagged PADs in HeLa cells revealed that PAD V is localized in the nucleus, whereas PAD I, II, and III are localized in the cytoplasm. PAD V deletion mutants indicated that the sequence residues 45-74 have a nuclear localization signal (NLS). A sequence feature of this NLS is a three-lysine residue cluster preceded by a proline residue and is not found in the three other PADs. Substitution of the lysine cluster by an alanine cluster abrogated the nuclear import activity. These results suggested that the NLS is a classical monopartite NLS. HL-60 granulocytes, neutrophils, and eosinophils stained with antibody specific for PAD V exhibited distinct positive signals in the nucleus. Subcellular fractionation of HL-60 granulocytes also showed the nuclear localization of the enzyme. When neutrophils were stimulated with calcium ionophore A23187, protein deimination occurred in the nucleus. The major deiminated proteins were identified as histones H2A, H3, and H4. The implication of PAD V in histone modifications is discussed.A family of peptidylarginine deiminases (PAD) 1 (EC 3.5.3.15) catalyzes the conversion of arginine residues in proteins into citrulline residues in the presence of calcium ion. Four types of rodent PADs, I, II, III, and IV, and of human PADs, I, II, III, and V, are known (1-8). Rodent PAD IV is most closely related to human PAD V. The structures of these PADs are relatively conserved in the C-terminal two-thirds of the sequence and diverge more distantly in the N-terminal one-third of the sequence. Rodent enzymes have different substrate specificities toward synthetic substrates and different tissue distributions (9, 10). Biochemical and immunocytochemical studies have suggested the involvement of PAD I in the terminal differentiation of epidermis (11)(12)(13), that of PAD II in myelination and demyelination of central nerve axons (14,15), and that of PAD III in the keratinization of hair follicles (8,16,17). Information on the biological functions of mouse PAD IV and human PAD V is rather limited. In addition, increased amounts of deiminated myelin basic proteins are in the afflicted area of brains of patients with multiple sclerosis, and prevalent autoantibodies recognizing citrulline residues as an epitope of autoantigen in patients with rheumatoid arthritis have suggested the pathological involvement of PAD in diseases (15, 18 -21).PAD V was first found in HL-60 cells when cells were induced to differentiate into granulocytes (HL-60 granulocytes) by all-trans-retinoic acid (RA) and differentiate into monocytes by 1␣,25-dihydroxyvitamin D 3 (7). PAD V was also found in peripheral blood granulocytes (22). PAD V in HL-60 cells can be activated to deiminate nuclear proteins of nucleop...

show abstract

Section: Discussionmentioning

confidence: 52%

Nuclear Localization of Peptidylarginine Deiminase V and Histone Deimination in Granulocytes

Nakashima

Hagiwara

Yamada

2002

Journal of Biological Chemistry

340

296

View full text Add to dashboard Cite

show abstract

“…Granulocyte cytospins (50 l of 0.8 ϫ 10 6 cells/ml in RPMI 1640 supplemented with 20% FCS) were prepared by Cytospin 2 centrifugation (Shandon, Astmoor, Runcorn, U.K.) as previously optimized (39). Primary labeling was conducted with 20 g/ml mouse monoclonal anti-human Rac, specific for both Rac1 and Rac2 (mouse IgG2b; Upstate Biotechnology).…”

Section: Double Labeling and Confocal Laser Scanning Microscopymentioning

confidence: 99%

Divergence of Mechanisms Regulating Respiratory Burst in Blood and Sputum Eosinophils and Neutrophils from Atopic Subjects

Lacy

Latif

Steward

et al. 2003

The Journal of Immunology

View full text Add to dashboard Cite

Eosinophil respiratory burst is an important event in asthma and related inflammatory disorders. However, little is known concerning activation of the respiratory burst NADPH oxidase in human eosinophils. Conversely, neutrophils are known to assemble NADPH oxidase in intracellular and plasma membranes. We hypothesized that eosinophils and neutrophils translocate NADPH oxidase to distinct intracellular locations, consistent with their respective functions in O2−-mediated cytotoxicity. PMA-induced O2– release assayed by cytochrome c was 3.4-fold higher in atopic human eosinophils than in neutrophils, although membrane-permeable dihydrorhodamine-123 showed similar amounts of release. Eosinophil O2– release was dependent on Rac, in that it was 54% inhibited by Clostridium difficile toxin B (400–800 ng/ml). In eosinophils stimulated with PMA, a pronounced shift of cytosolic Rac to p22phox-positive plasma membrane was observed by confocal microscopy, whereas neutrophils directed Rac2 mainly to intracellular sites coexpressing p22phox. Similarly, ex vivo sputum eosinophils from asthmatic subjects exhibited predominantly plasma membrane-associated immunoreactivity for Rac, whereas sputum neutrophils exhibited cytoplasmic Rac2 staining. Thus, activated sputum eosinophils, rather than neutrophils, may contribute significantly to the pathogenesis of asthma by extracellular release of tissue-damaging O2–. Our findings suggest that the differential modes of NADPH oxidase assembly in these cells may have important implications for oxidant-mediated tissue injury.

show abstract

“…10,11,30 Briefly, samples of peripheral blood (100 mL) were collected in heparin-containing tubes. Erythrocytes were sedimented for 45 minutes at room temperature with 5% dextran (100 000-200 000 kd; Sigma, Oakville, ON, Canada).…”

Section: Preparation Of Eosinophilsmentioning

confidence: 99%

Translocation of the tetraspanin CD63 in association with human eosinophil mediator release

Mahmudi‐Azer

Downey

Moqbel

2002

Blood

View full text Add to dashboard Cite

The tetraspanin CD63 (also known as LAMP-3) has been implicated in phagocytic and intracellular lysosome-phagosome fusion events. It is also present in eosinophils, with predominant expression on crystalloid granule membrane. However, its role in eosinophil function is obscure. We hypothesized that CD63 is associated with intracellular events involved in eosinophil activation and mediator release. We used a combination of confocal immunofluorescence microscopy, flow cytometry, and secretion assays, including ␤-hexosaminidase, eosinophil peroxidase, and RANTES, to examine CD63 expression, intracellular localization, and its association with cell activation and mediator release. In resting eosinophils, CD63 immunoreactivity was localized to plasma and crystalloid granule membranes. In interferon-␥ (IFN-␥)-or C5a/CB-stimulated cells (10 minutes), intracellular CD63 appeared to shift to the cell periphery and plasma membrane, while stimulation with a cocktail of interleukin-3 (IL-3)/IL-5/granulocyte-macrophage colony-stimulating factor induced the appearance of discrete intracellular clusters of CD63 immunoreactivity. IFN-␥ induced mobilization of CD63 to the cell periphery, which coincided with selective mobilization of RANTES prior to its release, implying CD63 association with piecemeal degranulation. Agonist-induced CD63 mobilization and cell surface upregulation was associated with ␤-hexosaminidase, eosinophil peroxidase, and RANTES release. Dexamethasone as well as genistein (a broad-spectrum inhibitor of tyrosine kinases) inhibited agonistinduced intracellular mobilization of CD63 and RANTES together with cell surface up-regulation of CD63 and mediator release. This is the first report of an association between CD63 mobilization and agonist-induced selective mediator release, which may imply the involvement of CD63 in eosinophil activation and piecemeal degranulation. IntroductionEosinophils are major effector cells in allergic inflammation and asthma. 1-3 They synthesize, store, and release a wide range of proinflammatory mediators, including at least 4 cationic proteins 1,2 and up to 23 cytokines and growth factors. 4,5 Eosinophils contain different populations of mediator-storage organelles, including small secretory vesicles as well as crystalloid granules. The latter secretory granules are the site of storage of cytotoxic cationic proteins as well as a number of cytokines, chemokines, and growth factors. 6,7 The membrane-bound crystalloid granule comprises 2 compartments: an electron-dense crystalline core (internum) where major basic protein (MBP) 8,9 is stored and an electron-lucent matrix 6 where 3 cationic proteins-namely, eosinophil cationic protein, eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin 1,8 -together with a number of other cytokines, including interleukin-6 (IL-6) 10 and RANTES (regulated on activation normal T cells expressed and secreted), 11 are stored. Among eosinophilderived mediators, ␤-hexosaminidase (␤-hex) has been shown to localize to both small secretory vesicle...

show abstract

Inhibition of nonspecific binding of fluorescent-labelled antibodies to human eosinophils

Cited by 27 publications

References 20 publications

Nuclear Localization of Peptidylarginine Deiminase V and Histone Deimination in Granulocytes

Nuclear Localization of Peptidylarginine Deiminase V and Histone Deimination in Granulocytes

Divergence of Mechanisms Regulating Respiratory Burst in Blood and Sputum Eosinophils and Neutrophils from Atopic Subjects

Translocation of the tetraspanin CD63 in association with human eosinophil mediator release

Contact Info

Product

Resources

About