Sputum processing for evaluation of inflammatory mediators

Kim, Jungsoo; Hackley, Grant H.; Okamoto, Kosuke; Rubin, Bruce K.

doi:10.1002/ppul.1101.abs

Cited by 14 publications

(21 citation statements)

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“…Studies showed that large polyanionic DNA polymers present in the purulent secretions in CF patients electrostatically bind and sequester cationic granule proteins, such as neutrophil elastase and MPO (57,58). In line with these findings, we found that MPO released by neutrophils became immobilized to matrix extracellular DNA.…”

Section: Discussionsupporting

confidence: 86%

Extracellular DNA: A Major Proinflammatory Component of Pseudomonas aeruginosa Biofilms

Bass

Russo

Gabelloni

et al. 2010

The Journal of Immunology

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We previously demonstrated that extracellular bacterial DNA activates neutrophils through a CpG- and TLR9-independent mechanism. Biofilms are microbial communities enclosed in a polymeric matrix that play a critical role in the pathogenesis of many infectious diseases. Because extracellular DNA is a key component of biofilms of different bacterial species, the aim of this study was to determine whether it plays a role in the ability of biofilms to induce human neutrophil activation. We found that degradation of matrix extracellular DNA with DNase I markedly reduced the capacity of Pseudomonas aeruginosa biofilms to induce the release of the neutrophil proinflammatory cytokines IL-8 and IL-1β (>75%); reduced the upregulation of neutrophil activation markers CD18, CD11b, and CD66b (p < 0.001); reduced the number of bacteria phagocytosed per neutrophil contacting the biofilm; and reduced the production of neutrophil extracellular traps. Consistent with these findings, we found that biofilms formed by the lasI rhlI P. aeruginosa mutant strain, exhibiting a very low content of matrix extracellular DNA, displayed a lower capacity to stimulate the release of proinflammatory cytokines by neutrophils, which was not decreased further by DNase I treatment. Together, our findings support that matrix extracellular DNA is a major proinflammatory component of P. aeruginosa biofilms.

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Section: Discussionsupporting

confidence: 86%

Extracellular DNA: A Major Proinflammatory Component of Pseudomonas aeruginosa Biofilms

Bass

Russo

Gabelloni

et al. 2010

The Journal of Immunology

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“…Compared to other mediators, MPO concentration is greatly affected by the method used for its detection or for sputum sample processing [24][25][26], and sputum MPO concentrations have been reported to be extremely variable in different studies, with a large standard deviation [27]. In the samples in this study, an acceptable interassay coefficient of variation and a fairly good recovery of MPO were found, which, however, was lower in sputum induced by HS than in sputum induced by IS inhalation.…”

Section: Discussionmentioning

confidence: 54%

Granulocyte markers in hypertonic and isotonic saline-induced sputum of asthmatic subjects

Cianchetti

Bacci²,

Ruocco³

et al. 2004

Eur Respir J

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The aim of this study was to assess whether hyperosmolarity affects granulocyte mediator levels in induced sputum of asthmatic subjects.A total of 32 mild-to-moderate asthmatics, who inhaled either hypertonic (HS; 4.5% NaCl) or isotonic (IS; 0.9% NaCl) solutions for 15 min, were studied. Selected sputum was used for analysis. Eosinophil cationic protein (ECP), eosinophil protein X (EPX), myeloperoxidase (MPO) and free neutrophil elastase (NE) were measured in sputum supernatant.Sample weight, total and differential cell counts, as well as viability and squamous cell percentage were no different after the two tests. No significant differences in ECP, EPX, MPO or NE levels were observed between HS-and IS-induced sputum. Repeatability of the two tests was good for macrophages, neutrophils, eosinophils, ECP, EPX and NE, but not for lymphocytes and MPO.In conclusion, hyperosmolarity does not affect sputum cell counts and the levels of most granulocyte degranulation markers examined in this study, confirming that both hypertonic and isotonic solutions can be reliably used to induce sputum in asthmatics. Hypertonic saline (HS) inhalation is currently used to collect sputum from the airways of asthmatic subjects who do not produce sputum spontaneously. However, in high-risk subjects, inhalation with isotonic saline (IS) is recommended, at least at the beginning of sputum induction [1]. Thus, it is important that sputum samples collected after either induction method have a similar composition. Several studies have compared the cell composition of sputum obtained after the two different methods of induction and showed good concordance for inflammatory cell percentages [2-4]; however, few studies have compared the concentrations of soluble mediators in the sputum supernatant [5].A large number of in vivo and in vitro studies have shown that hypertonic solutions modify mediator release from inflammatory cells by acting as a strong stimulus for cell activation [6-8], or, conversely, by inhibiting leukocyte degranulation [5,9]. In sputum induction, the hypertonic solution might increase osmolarity of the airway lining fluid, thus influencing mediator release from granulocytes during inhalation or sputum processing, or both.Therefore, the aim of this study was to compare preformed soluble mediator levels derived from eosinophils and neutrophils, such as eosinophil cationic protein (ECP), eosinophil protein X (EPX), myeloperoxidase (MPO) and neutrophil elastase (NE), in induced sputum obtained by means of either HS or IS inhalation in mild-to-moderate asthmatic subjects. In addition, the total and differential cell counts were also evaluated in the same sputum samples. Subjects and methods SubjectsA total of 44 mild-to-moderate asthmatic subjects, who were recruited from the authors9 asthma clinic, were examined. Asthma was diagnosed at the time of the first examination, according to internationally accepted criteria [10], after assessing reversible airway obstruction and/or nonspecific bronchial hyperresponsiveness ...

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“…DNA was measured by microfluorimetry and compared with a calf thymus DNA standard (Sigma, St. Louis, MO) at 0.25 to 10.0 g/ml (22). The supernatant was diluted 1:50 for CF and 1:10 for ETT specimens with SSC solution (0.0154 M NaCl-0.015 M Na 3 -citrate, pH 7.0).…”

Section: Methodsmentioning

confidence: 99%

“…The supernatant was diluted 1:50 for CF and 1:10 for ETT specimens with SSC solution (0.0154 M NaCl-0.015 M Na 3 -citrate, pH 7.0). One microliter of 33258 Hoechst (1.5ϫ 10 Ϫ6 M; (Calbiochem, La Jolla, CA) was added to the sample, and fluorescence was measured by spectrophotofluorimetry with an excitation wavelength at 360 nm and emission at 450 nm (22). Mucin degradation in vitro and by proteases with and without protease inhibitors.…”

Section: Methodsmentioning

confidence: 99%

Serine Proteases Degrade Airway Mucins in Cystic Fibrosis

et al. 2011

Self Cite

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Airway mucins are the major molecular constituents of mucus. Mucus forms the first barrier to invading organisms in the airways and is an important defense mechanism of the lung. We confirm that mucin concentrations are significantly decreased in airway secretions of subjects with cystic fibrosis (CF) who have chronic Pseudomonas aeruginosa infection. In sputum from CF subjects without a history of P. aeruginosa, we found no significant difference in the mucin concentration compared to mucus from normal controls. We demonstrate that mucins can be degraded by synthetic human neutrophil elastase (HNE) and P. aeruginosa elastase B (pseudolysin) and that degradation was inhibited by serine proteases inhibitors (diisopropyl fluorophosphates [DFP], phenylmethylsulfonyl fluoride [PMSF], and 1-chloro-3-tosylamido-7-amino-2-heptanone HCl [TLCK]). The mucin concentration in airway secretions from CF subjects is similar to that for normal subjects until there is infection by P. aeruginosa, and after that, the mucin concentration decreases dramatically. This is most likely due to degradation by serine proteases. The loss of this mucin barrier may contribute to chronic airway infection in the CF airway.

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Sputum processing for evaluation of inflammatory mediators

Cited by 14 publications

References 0 publications

Extracellular DNA: A Major Proinflammatory Component of Pseudomonas aeruginosa Biofilms

Extracellular DNA: A Major Proinflammatory Component of Pseudomonas aeruginosa Biofilms

Granulocyte markers in hypertonic and isotonic saline-induced sputum of asthmatic subjects

Serine Proteases Degrade Airway Mucins in Cystic Fibrosis

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