Tag: neutrophils

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Neutrophil Chemotactic Activity of Sputum From Patients With COPD

Neutrophilic inflammation is a major feature of COPD.  Several factors in bronchial secretions have been identified as chemoattractants for neutrophils. The present study was designed to assess the contribution of interleukin (IL)-8 and leukotriene B4 (LTB4) to neutrophil chemotaxis evoked by sputum obtained from patients with established COPD.
Design: Sputum supernatant of 20 patients with COPD was used as chemoattractant in a 96-well
chemotaxis chamber, with subsequent quantification of migrated cells by a luminescence assay.
The contribution of IL-8 and LTB4 to chemotaxis was determined by addition of a neutralizing
antibody and a selective receptor antagonist, respectively.
Measurements and results: COPD sputum caused neutrophil chemotaxis in a concentration dependent manner, with a maximum response evoked with a 10-fold dilution of the original
sample. Pretreatment of sputum or neutrophils with either an anti–IL-8 antibody or the LTB4
antagonist, SB 201146, led to a concentration-dependent inhibition of sputum-induced neutrophil chemotaxis, with a maximum suppression (mean  SEM) of 29.2  4.9% (p < 0.001) from baseline by 100 ng/mL of anti–IL-8 antibody, and 45.6  7% (p < 0.02) by 10 mol/L of SB
201146. The combination of the anti–IL-8 antibody and SB 201146 inhibited neutrophil
chemotaxis, but this was not significantly greater than the effect of SB 201146 or anti–IL-8 alone.
Conclusions: These data confirm the importance of IL-8 and LTB4 as chemoattractants for
neutrophils in bronchial secretions from patients with COPD, and suggest that specific inhibitors
may have therapeutic potential in COPD.

http://journal.publications.chestnet.org/article.aspx?articleid=1081469

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A Bovine Whey Protein Extract Can Enhance Innate Immunity by Priming Normal Human Blood Neutrophils

Bovine milk-derived products, in particular whey proteins, exhibit beneficial properties for human health, including the acquired immune response. However, their effects on innate immunity have received little attention. Neutrophils are key cells of innate defenses through their primary functions of chemotaxis, phagocytosis, oxidative burst, and degranulation.
A whey protein extract (WPE) purified from bovine lactoserum was evaluated for its direct and indirect effects on these primary functions of normal human blood neutrophils in vitro. Although WPE had no direct effects on primary functions, a 24-h pretreatment of neutrophils with WPE was associated with a significant and dose-dependent increase of their chemotaxis, superoxide production, and degranulation in response to N-formyl-methionine-leucine-phenylalanine, as well as of their phagocytosis of bioparticles. The pretreatment increased the surface expression of CD11b, CD16B, and CD32A receptors. The major WPE protein components b-lactoglobulin (b-LG) and a-lactalbumin (a-LA) were the main active fractions having an additive effect on human neutrophils that became more responsive to a subsequent stimulation. This effect on NADPH oxidase activity was associated with translocation of p47phox to plasma membrane. Glycomacropeptide, a peptide present in measurable amounts in WPE products, was able to enhance the individual effect of b-LG or a-LA on neutrophils. The present data suggest that WPE, through b-LG and a-LA, has the capacity to enhance or ‘‘prime’’ human neutrophil responses to a subsequent stimulation, an effect that could be associated with increased innate defenses in vivo.

jn.nutrition.org/content/early/2008/12/23/jn.108.098459.full.pdf

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Rapid fluorescence-based measurement of neutrophil migration in vitro

We have standardized a new chemotaxis chamber that uses fluorescence as the cellular marker for the measurement of leukocyte migration in vitro in disposable 96-well microplates. This new fluorescence-based assay is a robust assay because filter pore size, cell density, filter composition, and filter thickness do not affect PMN migration towards interleukin-8 or the complement fragment, C5a. When compared to two separate chemotaxis assays in which the migrated cells are counted
visually, the fluorescence-based assay was more rapid, less labor intensive, and more sensitive. This new assay is a significant advance in the measurement of leukocyte migration in vitro.

Frevert, Wong, Goodman, Goodwin, and Martin. “Rapid Fluorescence-based Measurement of Neutrophil Migration in Vitro.” 1998, Journal of Immunological Methods, 213, 41-52.

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Rapid, multiwell colorimetric assay for measuring neutrophil chemoattractant activity in bronchoalveolar lavage fluid of horses with recurrent airway obstruction

The criteria used to diagnose recurrent airway obstruction (RAO) in affected horses include demonstration of reversible lower airway obstruction and greater than 25% neutrophils in bronchoalveolar lavage fluid (BALF). Additional objective laboratory tests are needed to improve diagnostic accuracy and to monitor response to treatment. The goal of this study was to determine if neutrophil chemoattractant activity of BALF could be measured by using a previously described, rapid, multiwell colorimetric assay for chemotaxis. In this assay, neutrophils that have migrated through a membrane filter are collected into the bottom well of a disposable chemotaxis–cell migration chamber. The number of viable cells collected in the
bottom well is quantified by measurement of the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT), which is reduced by dehydrogenase in mitochondria of live cells. The number of migrating cells corresponds to the amount of MTT reduced, which is measured with an enzyme-linked immunosorbent assay plate reader. Fourteen adult horses were enrolled in this study, 7 of which had owner
histories consistent with RAO. Each horse was sedated, a bronchoalveolar lavage tube was passed, and saline was infused and immediately aspirated. An aliquot of BALF was used for differential cell count, and BALF supernatant was harvested to assess neutrophil chemoattractant activity. Normal control horses and RAOaffected
horses were distinguished according to clinical signs and percent neutrophils in BALF. Neutrophil chemoattractant activity of BALF was significantly greater in RAO-affected horses (P 5 0.001) compared with control horses. This assay may be useful in future studies for monitoring response to therapy in RAO affected horses.

http://vdi.sagepub.com/content/18/3/257.abstract (link to pdf)

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Neutrophils exhibit distinct phenotypes toward chitosans with different degrees of deacetylation: implications for cartilage repair

Osteoarthritis is characterized by the progressive destruction of cartilage in the articular joints. Novel therapies that promote resurfacing of exposed bone in focal areas are of
interest in osteoarthritis because they may delay the progression of this disabling disease in patients who develop focal lesions. Recently, the addition of 80% deacetylated chitosan to cartilage microfractures was shown to promote the regeneration of hyaline cartilage. The molecular mechanisms by which chitosan promotes cartilage regeneration remain unknown. Because neutrophils are transiently recruited to the microfracture site, the effect of 80% deacetylated chitosan on the function of neutrophils was investigated. Most studies on neutrophils use preparations of chitosan with an uncertain degree of deacetylation. For therapeutic purposes, it is of interest to determine whether the degree of deacetylation influences the response of neutrophils to chitosan. The effect of 95% deacetylated chitosan on the function of neutrophils was therefore also investigated and compared with that of 80%
deacetylated chitosan.

www.biomedcentral.com/content/pdf/ar2703.pdf