Related Experiment Videos
Bacterial lipopolysaccharide up-regulates platelet-activating factor-stimulated Ca2+ mobilization and eicosanoid
M Aepfelbacher1, H W Ziegler-Heitbrock, I Lux
1Institut für Prophylaxe und Epidemiologie, Kreislaufkrankheiten, München, Germany.
Abstract:
When human monocytic Mono Mac 6 cells were treated with bacterial LPS (10 ng/ml, 72 h), they showed an increase in phagocytic activity, superoxide anion production, and expression of monocyte/macrophage-associated cell surface Ag. In these more mature (LPS-treated) cells but not in untreated cells, platelet-activating factor (PAF) (100 nM) produced a three- to fourfold increase in cytosolic free Ca2+ concentration. The cytosolic free Ca2+ concentration increase was inhibited by the PAF receptor antagonist L-659,989 (10 microM) and by EGTA (2 mM), indicating receptor-dependent Ca2+ influx. Furthermore, L-659,989 (10 microM), as well as PAF (1 microM), inhibited specific [3H]PAF binding in LPS-treated but not in untreated cells. Consistent with these results, PAF (100 nM) stimulated release of arachidonic acid and thromboxane B2 only in LPS-treated cells, and this could be inhibited by L-659,989 (10 microM) and EGTA (2 mM). Our data indicate that LPS up-regulates PAF-induced Ca2+ influx, resulting in arachidonic acid and eicosanoid release in Mono Mac 6 cells.
Insights
Bacterial lipopolysaccharide (LPS) enhances monocyte maturation, increasing their response to platelet-activating factor (PAF). LPS-treated cells show increased calcium influx and eicosanoid release upon PAF stimulation, indicating LPS up-regulates PAF signaling.
Area of Science:
- Immunology
- Cell Biology
Background:
- Human monocytic Mono Mac 6 cells are a model for studying monocyte/macrophage differentiation and function.
- Bacterial lipopolysaccharide (LPS) is known to induce cellular maturation and activate immune responses.
Purpose of the Study:
- To investigate the effect of LPS on Mono Mac 6 cell maturation and their subsequent response to platelet-activating factor (PAF).
- To elucidate the mechanisms underlying PAF-induced signaling in LPS-treated monocytes.
Main Methods:
- Mono Mac 6 cells were treated with bacterial LPS.
- Phagocytic activity, superoxide anion production, and cell surface antigen expression were measured.
- Cytosolic free Ca2+ concentration was assessed using calcium imaging.
- Platelet-activating factor (PAF) receptor binding was evaluated using radioligand assays.
- Release of arachidonic acid and thromboxane B2 was quantified.
Main Results:
- LPS treatment increased phagocytic activity, superoxide production, and monocyte/macrophage marker expression in Mono Mac 6 cells.
- LPS-treated cells exhibited a significant increase in cytosolic free Ca2+ concentration upon PAF stimulation, dependent on PAF receptor and calcium influx.
- Specific [3H]PAF binding and PAF-induced release of arachidonic acid and thromboxane B2 were observed only in LPS-treated cells.
- These PAF-induced responses in LPS-treated cells were inhibited by a PAF receptor antagonist and EGTA.
Conclusions:
- LPS treatment promotes the maturation of Mono Mac 6 cells, enhancing their responsiveness to PAF.
- LPS up-regulates PAF-induced calcium influx, leading to downstream signaling events such as arachidonic acid and eicosanoid release.
- These findings highlight a mechanism by which bacterial components can modulate monocyte/macrophage inflammatory responses.