Cytotoxic tumor necrosis factor activity produced by equine alveolar macrophages: preliminary characterization.
Abstract: Blood monocytes and alveolar macrophages (AM) were harvested from foals (aged 46 days to 6 months) and cultured in either medium alone or medium containing 10 micrograms/ml bacterial lipopolysaccharide (LPS). After 24 h, culture supernates were collected and analyzed for cytotoxic activity on sensitized L929 cells. Both monocytes and AM that had been treated with LPS produced significantly more cytotoxic activity than the same cell type exposed to medium lacking LPS. LPS-treated macrophages secreted significantly more cytotoxic activity (120 +/- 17.8 U/ml) than did LPS-treated monocytes (47.3 +/- 17.0 U/ml); however, constitutive production of cytotoxin by monocytes was higher (16.7 +/- 4.1 versus 1.2 +/- 1.2 U/ml). The identification of the cytotoxin as tumor necrosis factor (TNF) was strongly suggested by its reactivity with a rabbit antiserum directed against the N-terminal 15 amino acids of human TNF. TNF secretion by AM increased in a dose-dependent manner between LPS concentrations of 0.0001 and 1 microgram/ml, then leveled off. Most of the cytotoxic TNF activity produced by AM was secreted within the first 8 h after initial contact with LPS. Macrophage supernatant TNF was stable over a pH range of 6-11, but lost activity when kept at a pH less than 6. Equine TNF also was destroyed by exposure for 1 h to temperatures more than 60 degrees C. TNF bioactivity was recovered as a single peak after crude macrophage supernate was subjected to analysis by either anion exchange or gel filtration chromatography (molecular weight approximately 56,000).
Publication Date: 1991-08-01 PubMed ID: 1949576DOI: 10.1016/0165-2427(91)90049-iGoogle Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research paper explores how bacterial lipopolysaccharide (LPS) enhances the production of tumor necrosis factor (TNF), a cytotoxic (cell killing) substance, in monocytes and alveolar macrophages (AM) of young horses, and discusses the characteristics and stability of the resulting TNF.
Experimental Procedures
- The researchers collected blood monocytes and AM from foals (horses aged between 46 days and 6 months) and cultured them in a medium with or without bacterial LPS.
- After 24 hours, the supernates (liquid part) from cultures were collected and their cytotoxic activity on sensitized L929 cells was observed.
- The level of cytotoxic activity in the samples from cells exposed to LPS was compared with that from cells cultured in medium without LPS.
Findings
- Monocytes, as well as AM that were treated with LPS, produced significantly more cytotoxic activity than non-LPS treated cells.
- LPS-treated AM produced more cytotoxic activity than LPS-treated monocytes, but untreated monocytes naturally produced more cytotoxins than untreated AM.
- The cytotoxin was identified as TNF due to its reactivity with a rabbit antiserum that responds specifically to the initial 15 amino acids of human TNF.
TNF Characteristics
- After making initial contact with LPS, the AM produced and secreted most of the cytotoxic TNF within the first 8 hours.
- The produced TNF was stable in a pH level range of 6-11, meaning it maintained its cytotoxic activity in these conditions. However, under acidic conditions (pH less than 6), TNF lost its activity.
- Exposure to high temperature (over 60 degrees Celsius) also destroyed equine TNF.
- When the crude AM supernate was analyzed using either anion exchange or gel filtration chromatography, TNF bioactivity was recovered as a single peak, indicating the presence of a dominant, active form of TNF with a molecular weight of approximately 56,000.
Cite This Article
APA
MacKay RJ, King RR, Dankert JR, Reis KJ, Skelley LA.
(1991).
Cytotoxic tumor necrosis factor activity produced by equine alveolar macrophages: preliminary characterization.
Vet Immunol Immunopathol, 29(1-2), 15-30.
https://doi.org/10.1016/0165-2427(91)90049-i Publication
Researcher Affiliations
- Department of Large Animal Clinical Sciences, University of Florida, Gainesville 32610.
MeSH Terms
- Animals
- Bronchoalveolar Lavage Fluid / cytology
- Cells, Cultured
- Chromatography, Gel
- Cytotoxicity, Immunologic
- Dose-Response Relationship, Immunologic
- Horses / immunology
- Lipopolysaccharides
- Macrophages / immunology
- Monocytes / immunology
- Protein Denaturation
- Tumor Necrosis Factor-alpha / biosynthesis
Citations
This article has been cited 1 times.- Rütten S, Schusser GF, Abraham G, Schrödl W. Release kinetics of tumor necrosis factor-α and interleukin-1 receptor antagonist in the equine whole blood.. BMC Vet Res 2016 Jun 17;12(1):117.
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