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Membrane potential modulates release of tumor necrosis factor in lipopolysaccharide-stimulated mouse macrophages
A Haslberger1, C Romanin, R Koerber
1Sandoz Forschungsinstitut, Vienna, Austria.
Abstract:
Lipopolysaccharide (LPS)-mediated synthesis of macrophage gene products such as tumor necrosis factor (TNF) is controlled by different signaling pathways. We investigated intracellular free Ca2+ (Ca2+ic) and the membrane potential as early cellular responses to LPS and their role in the synthesis and release of TNF. In peritoneal macrophages and in the RAW 269 mouse macrophage cell line, LPS and its biologically active moiety lipid A stimulated TNF synthesis but exerted no significant effects on these early cellular responses using Fura-2/Indo-1 to measure Ca2+ic and bis-oxonol, as well as the patch-clamp technique to monitor membrane potential. In contrast, the platelet-activating factor transiently induced both an increase in Ca2+ic and cell membrane depolarization but no significant TNF release. Increased extracellular K+ concentrations or K(+)-channel blockers, such as quinine, tetraethylammonium, or barium chloride, inhibited the LPS-stimulated release of TNF alpha, as well as the accumulation of cell-associated TNF alpha as found by enzyme-linked immunosorbent assay analysis, but did not inhibit TNF alpha mRNA accumulation. Concentrations of quinine (greater than 125 microM) or of enhanced extracellular K+ (25-85 mM) required to inhibit TNF production both significantly depolarized macrophages. These results indicate a lack of ion transport activities as early cellular responses of macrophages to LPS but suggest an important regulatory role of the membrane potential on the posttranscriptional synthesis and release of TNF in macrophages.
Insights
Lipopolysaccharide (LPS) does not alter early cellular responses in macrophages. However, membrane potential regulates the posttranscriptional synthesis and release of tumor necrosis factor (TNF).
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Lipopolysaccharide (LPS) triggers macrophage responses, including tumor necrosis factor (TNF) synthesis, via complex signaling pathways.
- Early cellular responses like intracellular calcium (Ca2+ic) and membrane potential are investigated for their role in LPS-mediated TNF production.
Purpose of the Study:
- To investigate the role of intracellular free Ca2+ (Ca2+ic) and membrane potential as early cellular responses to LPS.
- To determine the involvement of these early responses in the synthesis and release of TNF by macrophages.
Main Methods:
- Measurement of Ca2+ic using Fura-2/Indo-1 and membrane potential using bis-oxonol and patch-clamp techniques.
- Stimulation of macrophages with LPS, lipid A, and platelet-activating factor.
- Enzyme-linked immunosorbent assay (ELISA) analysis for TNF alpha and TNF alpha mRNA accumulation.
- Pharmacological manipulation of membrane potential using extracellular K+ and K+-channel blockers (quinine, tetraethylammonium, barium chloride).
Main Results:
- LPS and lipid A stimulated TNF synthesis but did not affect Ca2+ic or membrane potential.
- Platelet-activating factor induced Ca2+ic increase and depolarization but no TNF release.
- K+ channel blockers and high extracellular K+ inhibited LPS-stimulated TNF release and accumulation, but not mRNA levels.
- Inhibitory concentrations of quinine or high K+ significantly depolarized macrophages.
Conclusions:
- Macrophage responses to LPS do not involve early changes in ion transport (Ca2+ic or membrane potential).
- Membrane potential plays a crucial regulatory role in the posttranscriptional synthesis and release of TNF in macrophages.