Cytotoxic tumor necrosis factor activity produced by equine alveolar macrophages: preliminary characterization.

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.