Autofluorescence of Eosinophils: a Bone Marrow Study

Fuerst, Donald E.; Jannach, Joseph R.

doi:10.1038/2051333a0

Cited by 29 publications

(15 citation statements)

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“…Endobronchial biopsy specimens were obtained 48 hours after antigen (and normal saline) challenge. Specific autofluorescence imaging of eosinophil peroxidase in endobronchial biopsies was performed as described elsewhere (38)(39)(40). EPO-specific in situ peroxidase staining of tissues was performed after treatment of slides with 0.01 M KCN to inhibit MPO (41,42).…”

Section: Methodsmentioning

confidence: 99%

“…First, endobronchial biopsy specimens were examined by fluorescence microscopy using excitation and emission wavelengths specific for the autofluorescence of the porphyrin group of EPO (38)(39)(40). Dramatic increases in fluorescence intensity were observed in allergen-challenged, but not normal saline-challenged, specimens from allergic asthmatic subjects ( Figure 3, c and d).…”

Section: Figurementioning

confidence: 99%

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Eosinophils generate brominating oxidants in allergen-induced asthma

Wu¹,

Samoszuk²,

Comhair³

et al. 2000

J. Clin. Invest.

274

280

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Section: Methodsmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Eosinophils generate brominating oxidants in allergen-induced asthma

Wu¹,

Samoszuk²,

Comhair³

et al. 2000

J. Clin. Invest.

274

280

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“…All biopsies were stained on the same day. Specific autofluorescence imaging of EPO in biopsies was performed as described (47)(48)(49). EPO-specific in situ peroxidase staining of tissues was performed on anti-nitrotyrosine-stained tissue sections following treatment of slides with 0.01 M KCN to inhibit MPO (50,51).…”

Section: Histological Analysis Of Lung and Bronchial Tissuesmentioning

confidence: 99%

Eosinophils Are a Major Source of Nitric Oxide-Derived Oxidants in Severe Asthma: Characterization of Pathways Available to Eosinophils for Generating Reactive Nitrogen Species

MacPherson

Comhair²,

Erzurum

et al. 2001

The Journal of Immunology

261

225

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Eosinophil recruitment and enhanced production of NO are characteristic features of asthma. However, neither the ability of eosinophils to generate NO-derived oxidants nor their role in nitration of targets during asthma is established. Using gas chromatography-mass spectrometry we demonstrate a 10-fold increase in 3-nitrotyrosine (NO2Y) content, a global marker of protein modification by reactive nitrogen species, in proteins recovered from bronchoalveolar lavage of severe asthmatic patients (480 ± 198 μmol/mol tyrosine; n = 11) compared with nonasthmatic subjects (52.5 ± 40.7 μmol/mol tyrosine; n = 12). Parallel gas chromatography-mass spectrometry analyses of bronchoalveolar lavage proteins for 3-bromotyrosine (BrY) and 3-chlorotyrosine (ClY), selective markers of eosinophil peroxidase (EPO)- and myeloperoxidase-catalyzed oxidation, respectively, demonstrated a dramatic preferential formation of BrY in asthmatic (1093 ± 457 μmol BrY/mol tyrosine; 161 ± 88 μmol ClY/mol tyrosine; n = 11 each) compared with nonasthmatic subjects (13 ± 14.5 μmol BrY/mol tyrosine; 65 ± 69 μmol ClY/mol tyrosine; n = 12 each). Bronchial tissue from individuals who died of asthma demonstrated the most intense anti-NO2Y immunostaining in epitopes that colocalized with eosinophils. Although eosinophils from normal subjects failed to generate detectable levels of NO, NO2−, NO3−, or NO2Y, tyrosine nitration was promoted by eosinophils activated either in the presence of physiological levels of NO2− or an exogenous NO source. At low, but not high (e.g., >2 μM/min), rates of NO flux, EPO inhibitors and catalase markedly attenuated aromatic nitration. These results identify eosinophils as a major source of oxidants during asthma. They also demonstrate that eosinophils use distinct mechanisms for generating NO-derived oxidants and identify EPO as an enzymatic source of nitrating intermediates in eosinophils.

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“…The fluorophoreeosinophil granule complexes described in this study were clearly photoreactive, increasing their quantum emission and then stabilizing with continuous photoexcitation, whereas background Biebrich scarlet staining photobleached. This characteristic is particularly useful in eosinophils, which possess diffuse autofluorescence (520 nm) due to a granule-associated flavin adenine dinucleotide (Fuerst and Jannach 1965;Mayeno et al 1992). The masking of eosinophil autofluorescence by Biebrich scarlet not only provides definitive specific granule profiles not seen by autofluorescence but also allows dual labels with fluorescent markers that emit at this wavelength.…”

Section: Discussionmentioning

confidence: 99%

A Photoreactive Fluorescent Marker for Identifying Eosinophils and Their Cytoplasmic Granules in Tissues

Eversole

Mackenzie

Beuving

2003

J Histochem Cytochem.

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S U M M A R YHere we describe a simple histochemical technique that provides an improved approach to identifying eosinophil components in tissues through the formation of photoreactive complexes that produce stable fluorescent emissions. This method worked readily with histological tissue sections 6-60 m thick, which were fixed in neutral buffered formalin (NBF), and with cell suspensions similarly fixed and unfixed. Deep red ( Ͼ 605 nm) fluorescent emissions were produced by eosinophil-specific granules when exposed to broadband excitation spectra from a 100-W mercury lamp source (510-590 nm), as well as single-wavelength excitations from both an argon laser (488 nm) and a UV-visible laser (514 nm). The fluorophore-granule complex emissions increased in intensity during the first minute of continuous photoexcitation, then remained stable ( Ͼ 10 min). All nonspecific autofluorescence phenomena associated with these tissues were photobleached in the first minute, including areas of background Biebrich scarlet binding where photoreactive complexes were not formed (i.e., collagen), indicating environmental influences on the fluorophore. This technique allows the visualization of eosinophil granules over a greater period of time than is usually permissible with standard fluorescent markers. Therefore, techniques such as confocal microscopy can be utilized to their fullest extent, providing much more detailed information on the location and distribution of the cytoplasmic contents of eosinophils.

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Autofluorescence of Eosinophils: a Bone Marrow Study

Cited by 29 publications

References 1 publication

Eosinophils generate brominating oxidants in allergen-induced asthma

Eosinophils generate brominating oxidants in allergen-induced asthma

Eosinophils Are a Major Source of Nitric Oxide-Derived Oxidants in Severe Asthma: Characterization of Pathways Available to Eosinophils for Generating Reactive Nitrogen Species

A Photoreactive Fluorescent Marker for Identifying Eosinophils and Their Cytoplasmic Granules in Tissues

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