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Human monocytes lose 5-lipoxygenase and FLAP as they mature into monocyte-derived macrophages in vitro
W L Ring1, C A Riddick, J R Baker
1Department of Medicine, University of California, San Diego, USA.
Abstract:
Previous studies in mononuclear phagocytes have shown that macrophages have substantially greater 5-lipoxygenase activity than monocytes and that this is associated with greater amounts of 5-lipoxygenase and its activating protein (FLAP). The aim of this study was to examine the effect of mononuclear phagocyte maturation in vitro on 5-lipoxygenase expression. At baseline, monocytes had significant 5-lipoxygenase activity, but then lost all detectable 5-lipoxygenase activity over 7 days. Immunoblot and Northern blot analysis revealed that immunoreactive protein and mRNA for both 5-lipoxygenase and FLAP were significantly decreased over time. These studies demonstrate that in vitro differentiation of monocytes into a macrophage phenotype is not accompanied by the enhanced expression of 5-lipoxygenase and FLAP seen in macrophages derived from in vivo sources. In fact, baseline expression of 5-lipoxygenase and FLAP by monocytes is lost in vitro. These studies have clear implications for the use of cultured monocytes as a model of macrophages, and they also further our understanding of the regulation of the 5-lipoxygenase pathway.
Insights
Monocytes lose 5-lipoxygenase activity and expression of 5-lipoxygenase (LO) and FLAP during in vitro maturation into macrophages. This contradicts in vivo findings and impacts using cultured monocytes as a macrophage model.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Macrophages exhibit higher 5-lipoxygenase (LO) activity than monocytes.
- This activity is linked to increased levels of 5-LO and 5-LO-activating protein (FLAP).
Purpose of the Study:
- To investigate how in vitro maturation of mononuclear phagocytes affects 5-lipoxygenase expression.
- To compare in vitro monocyte-derived macrophages with in vivo macrophages regarding 5-LO and FLAP expression.
Main Methods:
- Monocytes were cultured in vitro for 7 days to induce differentiation into macrophages.
- 5-lipoxygenase activity was measured.
- Immunoblot and Northern blot analyses were performed to quantify 5-LO and FLAP protein and mRNA levels.
Main Results:
- Monocytes exhibited significant 5-lipoxygenase activity at baseline.
- This activity was completely lost during the 7-day in vitro culture period.
- Both 5-LO and FLAP protein and mRNA levels significantly decreased over time in vitro.
Conclusions:
- In vitro differentiation of monocytes into macrophages does not enhance 5-lipoxygenase and FLAP expression, unlike in vivo-derived macrophages.
- Monocytes lose their baseline 5-LO and FLAP expression during in vitro culture.
- These findings have implications for using cultured monocytes as a macrophage model and understanding 5-LO pathway regulation.