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Fucose-activated killer cells. I. Enhanced TNF-alpha mRNA accumulation and protein production
1Immunology Division, Faculty of Medicine, University of Sherbrooke, Québec, Canada.
Abstract:
Our previous studies have shown that the monosaccharide alpha-L-fucose significantly enhances the cytolytic capacity of peripheral blood mononuclear leukocytes (PBMLs). To examine possible mechanisms through which fucose affects cytolytic activity, we studied the production of cytokines after alpha-L-fucose stimulation. In this report, we show that fucose induced a minor but significant augmentation of production of interleukin-2 (IL-2), but anti-IL-2 antibodies did not completely inhibit fucose-activated cytolysis. Fucose induced significantly higher secretion of TNF-alpha by both lymphocytes and monocytes. The nature of the lytic molecule detected in the TNF bioassay was verified with specific neutralizing antibodies. In addition, fucose induced the accumulation of TNF-alpha mRNA in a time-dependent manner with a peak at 8 h and a return to baseline values at 20 h after stimulation. In vitro nuclear transcription assays determined that fucose augmented the rate of transcription of the TNF-alpha gene, and inhibition of de novo transcription with actinomycin D indicated that the turnover rate of the TNF-alpha mRNA was not affected by fucose stimulation. We also determined that fucose did not modulate the mRNA expression of the pore-forming protein, a major lytic protein involved in lymphocyte cytotoxicity. Specific neutralizing antibodies indicated that TNF-alpha was not an effector molecule in fucose-activated killing of K562 or Raji target cells but that this cytokine had an essential role in the induction of the augmented killing by alpha-L-fucose.
Insights
Alpha-L-fucose enhances immune cell killing by increasing tumor necrosis factor-alpha (TNF-alpha) production and gene transcription. While not directly causing cell death, TNF-alpha is crucial for fucose-mediated immune cell activation.
Area of Science:
- Immunology
- Molecular Biology
- Cellular Biology
Background:
- Peripheral blood mononuclear leukocytes (PBMLs) exhibit enhanced cytolytic capacity with alpha-L-fucose.
- Understanding the molecular mechanisms behind fucose-enhanced cytotoxicity is crucial for immune modulation.
Purpose of the Study:
- To investigate the role of cytokines, specifically interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha), in alpha-L-fucose-mediated enhancement of PBML cytolytic activity.
- To elucidate the transcriptional and post-transcriptional regulation of TNF-alpha production induced by alpha-L-fucose.
Main Methods:
- Stimulation of PBMLs with alpha-L-fucose and measurement of cytokine production (IL-2, TNF-alpha).
- Use of neutralizing antibodies to assess the role of IL-2 and TNF-alpha in fucose-activated cytolysis.
- Analysis of TNF-alpha mRNA accumulation and gene transcription rates.
- Assessment of pore-forming protein mRNA expression.
Main Results:
- Alpha-L-fucose significantly increased TNF-alpha secretion and mRNA levels in PBMLs in a time-dependent manner.
- Fucose enhanced TNF-alpha gene transcription, but not mRNA turnover.
- While IL-2 production was slightly increased, it was not the primary mediator of fucose-enhanced cytolysis.
- TNF-alpha was essential for the induction of fucose-augmented killing, though not a direct effector molecule.
Conclusions:
- Alpha-L-fucose enhances PBML cytolytic activity primarily through the induction of TNF-alpha.
- The mechanism involves increased TNF-alpha gene transcription, leading to augmented immune cell killing.
- TNF-alpha plays a critical inductive role in fucose-mediated cytotoxicity, highlighting its importance in immune responses.