Bronchial Mucus in Health and Disease

Lopez-Vidriero, M T; Reid, Lynne

doi:10.1093/oxfordjournals.bmb.a071461

Cited by 88 publications

(42 citation statements)

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“…The present study was undertaken to elucidate the potential effect of DTT in sputum analysis. Sputum cells are embedded in a matrix of airway secretions produced by mucosal epithelial cells and submucosal glands [21]. The major component of this are mucins or mucus glycoproteins, which comprise up to 1±3% of sputum wet weight [22,23].…”

Section: Discussionmentioning

confidence: 99%

Flow cytometric analysis of the effect of dithiothreitol on leukocyte surface markers

Loppow¹,

Böttcher²,

Gercken³

et al. 2000

Eur Respir J

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Pretreatment with dithiothreitol (DTT) is necessary to dissolve mucus in samples of induced sputum prior to analysis. However, DTT may affect cell surface markers which are essential for lymphocyte subtyping. Therefore, the aim of this study was to evaluate the effect of DTT on an appropriate panel of surface markers. Peripheral blood leukocytes were used because these cells, in contrast to sputum cells, could be obtained without DTT treatment.Peripheral blood from healthy donors was incubated with either DTT according to standard sputum procedures or phosphate-buffered saline (PBS), washed and incubated with fluorochrome-labelled antibodies. After lysis of erythrocytes, analysis was performed using a calibrated flow cytometer. Leukocyte populations were identified by their light scattering properties. For analysis, fluorescence intensity was compared between DTT-and PBS-treated samples.After treatment with DTT, fluorescence intensity was significantly increased in CD16-positive granulocytes; it was reduced in CD2-positive lymphocytes, CD45-positive lymphocytes and CD14-positive monocytes (p#0.001). These changes occurred in all samples. The fluorescence intensity of CD3-, CD4-, CD8-, CD19-, CD56-and histocompatibility leukocyte antigen DR-positive lymphocytes was not altered by DTT. However, there were statistically significant (p<0.001), although small, changes in the percentages of leukocytes.The present data demonstrate that, although dithiothreitol as used in sputum analysis affects some surface markers of peripheral blood leukocytes, comparability between samples concerning lymphocyte surface markers is preserved. Therefore, it is suggested that treatment of sputum samples with dithiothreitol does not invalidate the immunocytochemical analysis of lymphocytes. Eur Respir J 2000; 16: 324±329. The method of induced sputum is widely used as a noninvasive procedure for obtaining biological samples from the airways [1,2] in order to determine their cellular and biochemical composition in relation to airway diseases [3,4]. As sputum cells are embedded in airway secretions, the material has to be liquified in order to prepare singlecell suspensions for flow cytometry. This can be achieved by incubation with the potent reducing agent dithiothreitol (DTT, 2,3-dihydroxybutane-1,4-dithiol) [5]. Therefore, DTT is widely used in sputum processing [6±9]. However, owing to its reducing properties, DTT could affect the three-dimensional structure of proteins, which is maintained by disulphide bonds, thereby leading to changes in the availability and integrity of epitopes which could hamper immunological detection procedures [10].Indeed, the data which are available indicate effects of DTT or dithioerythritol (DTE) on surface markers assessed by flow cytometry on eosinophils or neutrophils even if cell viability remains unchanged [11±13]. Data regarding markers that are essential for lymphocyte subtyping have been published in abstract form [14]. Recent data have shown improved cell and sputum supernatant inflamma...

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Section: Discussionmentioning

confidence: 99%

Flow cytometric analysis of the effect of dithiothreitol on leukocyte surface markers

Loppow¹,

Böttcher²,

Gercken³

et al. 2000

Eur Respir J

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“…However, we estimated the percent dry weight (percentage of solids) of normal mucus at ϳ1.5-2.5% and of thick CF-like mucus at ϳ6 -15%, based on data reported from cell culture (26), a mouse model (4), and human studies (27)(28)(29)(30)(31)(32)(33). Mucus of 1.5% solids may be less than the normal optimal concentration because the network of mucin fibers and transport were heterogeneous (Fig.…”

Section: Figurementioning

confidence: 99%

Reduced Three-Dimensional Motility in Dehydrated Airway Mucus Prevents Neutrophil Capture and Killing Bacteria on Airway Epithelial Surfaces

Matsui¹,

Verghese²,

Kesımer

et al. 2005

The Journal of Immunology

128

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Cystic fibrosis (CF) lung disease is characterized by persistent lung infection. Thickened (concentrated) mucus in the CF lung impairs airway mucus clearance, which initiates bacterial infection. However, airways have other mechanisms to prevent bacterial infection, including neutrophil-mediated killing. Therefore, we examined whether neutrophil motility and bacterial capture and killing functions are impaired in thickened mucus. Mucus of three concentrations, representative of the range of normal (1.5 and 2.5% dry weight) and CF-like thickened (6.5%) mucus, was obtained from well-differentiated human bronchial epithelial cultures and prepared for three-dimensional studies of neutrophil migration. Neutrophil chemotaxis in the direction of gravity was optimal in 1.5% mucus, whereas 2.5% mucus best supported neutrophil chemotaxis against gravity. Lateral chemokinetic movement was fastest on airway epithelial surfaces covered with 1.5% mucus. In contrast, neutrophils exhibited little motility in any direction in thickened (6.5%) mucus. In in vivo models of airway mucus plugs, neutrophil migration was inhibited by thickened mucus (CF model) but not by normal concentrations of mucus (“normal” model). Paralleling the decreased neutrophil motility in thickened mucus, bacterial capture and killing capacity were decreased in CF-like thickened mucus. Similar results with each mucus concentration were obtained with mucus from CF cultures, indicating that inhibition of neutrophil functions was mucus concentration dependent not CF source dependent. We conclude that concentrated (“thick”) mucus inhibits neutrophil migration and killing and is a key component in the failure of defense against chronic airways infection in CF.

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“…In an early study on the composition of mucus from pulmonary secretions, Potter et al [7] showed a wide spectrum of values for DNA, protein, lipid and carbohydrate in 'normal' (laryngectomised patients), bronchiectatic, and CF patients; the highest values were found in the CF group. Later, LopezVidreiro and Reid [8] concluded that the quantitative changes in mucus composition were secondary to infection rather than a primary abnormality of the disease. Abnormalities were also described in the ionic composition of CF sputum by Kilbourn [9] who correlated an increased calcium concentration with colonisation by Staphylococcus aureus, and an increased magnesium content with the presence of mucoid P aeruginosa [lo].…”

Section: Introductionmentioning

confidence: 99%

The high amino-acid content of sputum from cystic fibrosis patients promotes growth of auxotrophic Pseudomonas aeruginosa

Barth

Pitt

1996

Journal of Medical Microbiology

126

116

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Bronchial Mucus in Health and Disease

Cited by 88 publications

References 0 publications

Flow cytometric analysis of the effect of dithiothreitol on leukocyte surface markers

Flow cytometric analysis of the effect of dithiothreitol on leukocyte surface markers

Reduced Three-Dimensional Motility in Dehydrated Airway Mucus Prevents Neutrophil Capture and Killing Bacteria on Airway Epithelial Surfaces

The high amino-acid content of sputum from cystic fibrosis patients promotes growth of auxotrophic Pseudomonas aeruginosa

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