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Isolation and Characterization of Neutrophils with Anti-Tumor Properties
Published on: June 19, 2015
Egg white derivatives induce tumor necrosis factor-alpha expression in porcine peripheral blood mononuclear cells
Mhan-Pyo Yang1, In-Kyung Ko, Ji-Houn Kang
1Laboratory of Veterinary Internal Medicine, Department of Veterinary Medicine, College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea. mpyang@cbnu.ac.kr
Abstract:
Porcine PBMC derived phagocytic activity in peripheral blood polymorphonuclear cells (PMN) induced by egg white derivatives (EWD) treatment was analyzed at the protein and mRNA level. EWD alone failed to induce phagocytic activity of PMN measured by flow cytometry. But PMN phagocytosis was enhanced by culture supernatant from PBMC treated with EWD, human (h)rTNF-alpha and porcine (p)rIL-1beta, respectively. To identify this phagocytic inducing factor, the culture supernatant was partially purified by gel filtration. Only fraction 8 revealed the enhanced PMN phagocytic activity. This fraction also had a high cross-reactivity with anti-prTNF-alpha polyclonal (p)Ab but not with anti-prIL-1beta pAb, as measured by ELISA, indicating that the culture supernatant from PBMC treated with EWD was independent from IL-1beta. The enhanced PMN phagocytic activity of fraction 8 was also inhibited by anti-prTNF-alpha pAb. Both fraction 8 and hrTNF-alpha produced a single protein band between 16 and 18kDa upon analysis by sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using anti-prTNF-alpha pAb, suggesting that the promoter of PMN phagocytosis is TNF-alpha, a 16-18 kDa protein produced by EWD-stimulated PBMC. Porcine TNF-alpha mRNA expression in porcine PBMC analyzed by RT-PCR was also increased by addition of EWD. This study strongly suggests that the immunoenhancing effect of EWD on the phagocytic response of porcine PMN is mediated through TNF-alpha produced by PBMC stimulated with EWD. In addition, the expression of porcine TNF-alpha on PBMC is also increased when stimulated with EWD.
Insights
Egg white derivatives (EWD) enhance porcine peripheral blood polymorphonuclear cell (PMN) phagocytosis by stimulating PBMC to produce TNF-alpha. This protein mediates the immunoenhancing effect, increasing PMN
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Phagocytic activity of peripheral blood polymorphonuclear cells (PMN) is crucial for innate immunity.
- Egg white derivatives (EWD) are potential immunomodulators, but their mechanism of action on porcine immune cells requires elucidation.
Purpose of the Study:
- To investigate the effect of EWD on porcine PMN phagocytic activity.
- To identify the specific factor produced by peripheral blood mononuclear cells (PBMC) that enhances PMN phagocytosis upon EWD stimulation.
Main Methods:
- Flow cytometry was used to measure PMN phagocytic activity.
- PBMC culture supernatants were analyzed for phagocytic-enhancing factors.
- Gel filtration, ELISA, SDS-PAGE, Western blotting, and RT-PCR were employed to identify and characterize the active factor.
Main Results:
- EWD alone did not enhance PMN phagocytosis.
- Culture supernatant from EWD-treated PBMC significantly enhanced PMN phagocytosis.
- The active factor was identified as porcine tumor necrosis factor-alpha (TNF-alpha), a 16-18 kDa protein.
- EWD stimulation increased porcine TNF-alpha mRNA expression in PBMC.
Conclusions:
- EWD stimulates porcine PBMC to produce TNF-alpha.
- Porcine TNF-alpha mediates the enhanced phagocytic activity of PMN induced by EWD.
- EWD exhibits an immunoenhancing effect on the porcine innate immune system via TNF-alpha production.
