Direct stimulation of the oxidative activity of isolated equine neutrophils by TNF-alpha and IL-1beta.
Abstract: The capacity of the two cytokines TNF-alpha and IL-1beta to directly stimulate the oxidative activity of polymorphonuclear neutrophils remains debated. The purpose of this study was to verify if a direct stimulation of equine neutrophils by TNF-alpha and IL-1beta was possible. Equine neutrophils were isolated from blood by discontinuous density gradient centrifugation. The cell viability after isolation was >98%. The neutrophils were used at 1.25 x 10(6) cells by assay, immediately after isolation. The oxidative activity of neutrophils was measured by luminol- or lucigenin-enhanced chemiluminescence (CL), and the CL was recorded for 60 min. TNF-alpha and IL-1beta were used at concentrations ranging from 0.001 to 100 ng (0.0017-167 ng ml(-1)) for 1.25 x 10(6) neutrophils, and added to the cells just before the CL measurement. Both cytokines highly stimulated the lucigenin-enhanced CL of equine neutrophils in a dose-dependent manner. TNF-alpha was already active at 0.001 ng and IL-1beta at 0.01 ng. The CL response obtained with TNF-alpha was maximal after 5 min and more pronounced with luminol than with lucigenin. With IL-1beta, the luminol-enhanced CL response of neutrophils was short-lived and inversely proportional to the cytokine concentration: the CL response returned to baseline after 12 min, and became even lower than the baseline value for 10 and 100 ng IL-1beta. As luminol (but not lucigenin) enters the cell, we hypothesized that a rapid intracellular consumption of the luminol molecules occurred, explaining the rapid and intense CL response. The choice of the CL enhancer used in previous CL studies of neutrophils stimulation by cytokines could perhaps explain that controversial results were reported. In conclusion, we demonstrated a direct activation of the oxidative activity of equine neutrophils by TNF-alpha and IL-1beta, which was dose-dependent and obtained with very low doses equivalent to the plasma concentrations measured for both cytokines in equine septic shock. TNF-alpha and IL-1beta can thus aggravate neutrophils oxidative activity during septic shock in horses.
Publication Date: 2007-10-02 PubMed ID: 18006077DOI: 10.1016/j.vetimm.2007.09.006Google Scholar: Lookup
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- Journal Article
Summary
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The research is a study on how cytokines TNF-alpha and IL-1beta can stimulate the oxidative activity in equine neutrophils directly. This study is significant as it allows a better understanding of septic shock in horses and highlights the influence of TNF-alpha and IL-1beta in the inflammation process.
Research Methodology
- The study began with the isolation of equine neutrophils from blood through a process called discontinuous density gradient centrifugation. This ensured that the neutrophils used in the study were alive and active, with a viability rate of over 98%.
- The isolated neutrophils were used immediately in the assays at a concentration of 1.25 x 10⁶ cells.
- The oxidative activity of the neutrophils was then measured using luminol- or lucigenin-enhanced chemiluminescence (CL) over 60 minutes.
- The cytokines, TNF-alpha, and IL-1beta, were introduced at concentrations ranging from 0.001 to 100 ng for 1.25 x 10⁶ neutrophils. This was added to the neutrophils before measuring chemiluminescence.
Research Findings
- The two cytokines powerfully stimulated the lucigenin-enhanced chemiluminescence of equine neutrophils in a dose-dependent manner.
- Even at extremely low concentrations, TNF-alpha (0.001 ng) and IL-1beta (0.01 ng) were active in enhancing oxidative activities in neutrophils.
- The chemiluminescent response with TNF-alpha was noticeable within 5 minutes and more pronounced using a luminol enhancer compared to lucigenin.
- With IL-1beta, the luminol-enhanced CL response was short-lived and inversely proportional to the cytokine’s concentration. It returned to baseline after 12 minutes and even dropped below the baseline value at concentrations of 10 and 100 ng IL-1beta.
Interpretation and Conclusion
- The pattern of luminol-enhanced CL response led to a hypothesis that there was a rapid intracellular consumption of luminol molecules, which explained the intense CL response.
- The choice of chemiluminescent enhancer, either luminol or lucigenin, used in previous studies on neutrophil stimulation by cytokines could explain the different outcomes reported in the past.
- The study concluded that there was a direct activation of the oxidative activity in equine neutrophils by both TNF-alpha and IL-1beta. This activation was dose-dependent and could be triggered by extremely low doses.
- These findings suggest that TNF-alpha and IL-1beta may cause an intensified oxidative activity in neutrophils during septic shock in horses, thereby worsening the inflammation and damage to the body’s tissue.
Cite This Article
APA
Benbarek H, Deby-Dupont G, Deby C, Serteyn D.
(2007).
Direct stimulation of the oxidative activity of isolated equine neutrophils by TNF-alpha and IL-1beta.
Vet Immunol Immunopathol, 121(1-2), 101-106.
https://doi.org/10.1016/j.vetimm.2007.09.006 Publication
Researcher Affiliations
- Department of Clinical Sciences, Large Animal Surgery, Faculty of Veterinary Medicine, B41, University of Liège, Sart Tilman, 4000 Liège, Belgium. benbarekh@yahoo.com
MeSH Terms
- Acridines / chemistry
- Animals
- Horses / blood
- Interleukin-1beta / pharmacology
- Luminescent Measurements
- Luminol / chemistry
- Neutrophil Activation / drug effects
- Neutrophil Activation / immunology
- Neutrophils / drug effects
- Neutrophils / immunology
- Respiratory Burst / drug effects
- Respiratory Burst / immunology
- Tumor Necrosis Factor-alpha / pharmacology
Citations
This article has been cited 1 times.- Bahadır Acikara Ö, Hošek J, Babula P, Cvačka J, Budešínský M, Dračinský M, Saltan İşcan G, Kadlecová D, Ballová L, Šmejkal K. Turkish Scorzonera Species Extracts Attenuate Cytokine Secretion via Inhibition of NF-κB Activation, Showing Anti-Inflammatory Effect in Vitro. Molecules 2015 Dec 30;21(1):E43.
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