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IL-1-alpha and TNF-alpha differentially regulate CD4 and Mac-1 expression in mouse microglia
N Yu1, X Zhang, P J Magistretti
1Department of Neuropharmacology, The Scripps Research Institute, La Jolla, Calif 92037, USA.
Abstract:
The regulatory effects of the proinflammatory cytokines, interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) were investigated on CD4 and Mac-1 expression in mouse microglial cultures. The identity of the microglia in cultures was confirmed by multiple indices including morphology, uptake of acetylated low-density lipoprotein and lectin RCA 120 staining. Microglia growing on a monolayer of astrocytes (astrocyte-supported microglia) were both CD4- and Mac-1 positive (out of 94.5 % Mac-1-positive cells, 85.3% were also CD4 positive). When astrocyte-supported microglia were replated directly onto culture dishes (plate-supported microglia), the percentage of CD4- and Mac-1-positive cells decreased to 12-29 and 20-25% respectively. The addition of IL-1alpha or TNF-alpha to plate-supported microglia led to an upregulation of Mac-1 expression in a time- and dose-dependent manner with different EC50s (0.5 ng/ml for IL-1alpha and 2 ng/ml for TNF-alpha) but exhibited similar time-to-peak responses (over 12 h). The addition of IL-1alpha, but not TNF-alpha, also led to an increase in CD4 expression on plate-supported microglia with a similar dose response and time course. IL-1alpha treatment gave rise to an increase in the level of CD4 mRNA as assessed by RT-PCR. The possibility that cell proliferation was responsible for the observed effects on microglia was excluded by an analysis of 3H-thymidine incorporation. Our results suggest that cultured mouse microglia express CD4 molecules which can be upregulated by IL-1alpha while Mac-1 can be upregulated by both IL-1alpha and TNF-alpha.
Insights
Proinflammatory cytokines interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) upregulate Mac-1 expression in mouse microglia. IL-1alpha also increases CD4 expression, suggesting a role in microglial immune responses.
Area of Science:
- Neuroimmunology
- Cellular and Molecular Neuroscience
- Cytokine Signaling
Background:
- Microglia are the primary immune cells of the central nervous system.
- Proinflammatory cytokines play crucial roles in neuroinflammation.
- The expression of cell surface markers like CD4 and Mac-1 on microglia is dynamic and can be modulated by inflammatory signals.
Purpose of the Study:
- To investigate the regulatory effects of interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) on CD4 and Mac-1 expression in mouse microglial cultures.
- To determine if these cytokines influence microglial immune marker expression in a dose- and time-dependent manner.
- To explore the molecular mechanisms underlying these regulatory effects, including mRNA level changes.
Main Methods:
- Primary mouse microglial cultures were established and characterized.
- Microglia were cultured with or without astrocytes, and cell surface marker expression (CD4, Mac-1) was assessed.
- The effects of IL-1alpha and TNF-alpha on CD4 and Mac-1 expression were analyzed using dose-response and time-course studies, with mRNA levels measured by RT-PCR and proliferation assessed by 3H-thymidine incorporation.
Main Results:
- Astrocyte-supported microglia exhibited high co-expression of CD4 and Mac-1.
- Replating microglia onto culture dishes (plate-supported) significantly reduced CD4 and Mac-1 expression.
- Both IL-1alpha and TNF-alpha upregulated Mac-1 expression in a time- and dose-dependent manner in plate-supported microglia.
- IL-1alpha, but not TNF-alpha, increased CD4 expression and CD4 mRNA levels in plate-supported microglia.
- Cell proliferation was ruled out as the cause of observed marker changes.
Conclusions:
- Cultured mouse microglia express CD4 molecules that can be upregulated by IL-1alpha.
- Mac-1 expression on microglia can be upregulated by both IL-1alpha and TNF-alpha.
- These findings highlight the regulatory role of specific proinflammatory cytokines on microglial immune marker expression, contributing to our understanding of neuroinflammatory processes.