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Expression of interleukin-3 and tumor necrosis factor-beta mRNAs in cultured microglia
K Appel1, P Honegger, P J Gebicke-Haerter
1Department of Psychiatry, University of Freiburg Medical School, Psychiatr. Universitätsklinik, Germany.
Abstract:
The function of interleukin-3 (or multi-CSF) in the hemopoietic system has been studied in great detail. Although its growth promoting activity on brain microglial cells has been confirmed both in vitro and in vivo, its presence in the brain and even in cultured brain cells has repeatedly been questioned. We have shown recently that isolated rat microglia express mRNA(IL-3) and synthesize IL-3 polypeptide. It is shown here by use of the PCR method, that mRNA(IL-3) is found also in C6 glioblastoma, in rat aggregate cultures, and in newborn and adult rat brain. Quantitation of amplified cDNA(IL-3) was achieved by non-competitive RT-PCR using an elongated internal standard. IL-3 messenger RNA was almost undetectable in vivo and low in (serum-free) aggregate cultures. In isolated microglia, mRNA(IL-3) was increased upon treatment with LPS, PHA, with the cytokines IL-1 or TNF-alpha, with retinoic acid, dbcAMP or the phorbol ester TPA. Effects of LPS were inhibited by dexamethasone, while the glucocorticoid by itself had no effect on basal IL-3 expression. LPS increased mRNA(IL-3) in a concentration-dependent manner beginning with 10 pg/ml and reaching plateau levels at 10 ng/ml. LPS also increased mRNAs of TNF-alpha and TNF-beta. TNF-alpha mRNA was already detectable in untreated microglia and LPS-increased levels were sustained for a few days. In contrast, TNF-beta mRNA was observed only between 4 and 16 h of LPS incubation. It was absent in LPS-free microglia, and after 24 h of LPS-treatment or later.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Interleukin-3 (IL-3) is present in the brain, with its messenger RNA (mRNA) detected in microglia and brain tissue. Microglial IL-3 mRNA expression is upregulated by inflammatory stimuli like LPS and cytokines.
Area of Science:
- Neuroimmunology
- Molecular Biology
- Cellular Biology
Background:
- Interleukin-3 (IL-3), also known as multi-CSF, is crucial for hematopoiesis.
- While IL-3's growth-promoting effects on brain microglia are known, its presence in the brain has been debated.
- Previous work confirmed IL-3 synthesis by isolated rat microglia.
Purpose of the Study:
- To investigate the presence and regulation of IL-3 mRNA in various brain cells and tissues.
- To quantify IL-3 mRNA levels in vivo and in vitro.
- To determine the effects of inflammatory stimuli and other factors on microglial IL-3 mRNA expression.
Main Methods:
- Polymerase Chain Reaction (PCR) to detect mRNA(IL-3) in C6 glioblastoma, aggregate cultures, and rat brain.
- Non-competitive Reverse Transcription PCR (RT-PCR) for quantitative analysis of cDNA(IL-3).
- Treatment of isolated microglia with lipopolysaccharide (LPS), phytohemagglutinin (PHA), cytokines (IL-1, TNF-alpha), retinoic acid, dbcAMP, and TPA.
Main Results:
- mRNA(IL-3) was detected in C6 glioblastoma, aggregate cultures, and both newborn and adult rat brain.
- In vivo and in serum-free aggregate cultures, mRNA(IL-3) was nearly undetectable or low, respectively.
- Isolated microglia showed increased mRNA(IL-3) upon stimulation with LPS, PHA, IL-1, TNF-alpha, retinoic acid, dbcAMP, or TPA.
- LPS treatment upregulated mRNA(IL-3) in a dose-dependent manner and also increased TNF-alpha and TNF-beta mRNA levels.
- Dexamethasone inhibited LPS-induced mRNA(IL-3) increases, but did not affect basal expression.
Conclusions:
- This study provides evidence for the presence of IL-3 mRNA in the rat brain and cultured brain cells, including microglia.
- Microglial IL-3 expression is significantly modulated by inflammatory and other signaling pathways.
- These findings contribute to understanding the role of IL-3 in neuroinflammation and brain function.