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Differential inhibition of IL-1 and TNF-alpha mRNA expression by agents which block second messenger pathways in
E J Kovacs1, D Radzioch, H A Young
1Department of Anatomy, Loyola University Stritch School of Medicine, Maywood, IL 60153.
Abstract:
IL-1 and TNF-alpha are induced in macrophages by LPS; however, it is unclear whether similar mechanisms control the expression of both genes. Here, we report on the detection of differential regulation of LPS induced IL-1 and TNF-alpha mRNA expression and protein production in murine macrophages based on the use of inhibitors of second messenger pathways. Northern blot analysis was performed with total RNA obtained from murine (C57Bl/6) peritoneal macrophages stimulated in vitro with LPS with or without an inhibitor of protein kinase C (PKc)(1-(5-isoquinolinesulfonyl)-2-methylpiperazine hydrochloride; H7) or an inhibitor of calmodulin (CaM)-dependent kinase (N-(6-amino-hexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride; W7). Northerns were analyzed with probes for IL-1 alpha and IL-1 beta and TNF-alpha. The expression of the three cytokine mRNA by LPS was inhibited in a dose response manner by H7. In contrast, the expression of IL-1 mRNA, but not TNF-alpha mRNA, was blocked by treatment with W7. Parallel studies monitoring biologic activities of these two cytokines confirm the mRNA data. PKc inhibitors, H7 and retinal, block both IL-1 and TNF-alpha protein production and inhibitors of CaM kinase, W7, N-(6-aminobutyl)-5-chloro-2-naphthalenesulfonamide, calmidazolum, and trifluoperazine dichloride inhibit only IL-1 production. These data suggest that both PKc and CaM kinase dependent pathways are involved in the induction of IL-1 mRNA by LPS. In contrast, TNF-alpha expression appears to be PKc dependent but not CaM kinase dependent.
Insights
Lipopolysaccharide (LPS) induces interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) in macrophages. Protein kinase C (PKc) and calmodulin (CaM)-dependent kinase pathways differentially regulate IL-1 and TNF-alpha expression.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- Interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) are key inflammatory cytokines produced by macrophages.
- Lipopolysaccharide (LPS) is a potent inducer of these cytokines, but the precise signaling pathways involved remain incompletely understood.
- Investigating differential regulation provides insight into macrophage activation and cytokine production.
Purpose of the Study:
- To elucidate the distinct roles of protein kinase C (PKc) and calmodulin (CaM)-dependent kinase pathways in regulating LPS-induced IL-1 and TNF-alpha expression.
- To compare the mRNA and protein level regulation of IL-1 and TNF-alpha.
Main Methods:
- Murine peritoneal macrophages were stimulated with LPS in the presence or absence of specific inhibitors of PKc (H7) and CaM kinase (W7).
- Northern blot analysis was used to quantify mRNA levels of IL-1 alpha, IL-1 beta, and TNF-alpha.
- Cytokine bioactivity assays were performed to assess protein production.
Main Results:
- Both PKc and CaM kinase inhibitors significantly reduced LPS-induced IL-1 mRNA and protein production in a dose-dependent manner.
- PKc inhibition (H7) reduced TNF-alpha mRNA and protein production.
- CaM kinase inhibition (W7) did not affect TNF-alpha mRNA or protein production, indicating differential pathway dependency.
Conclusions:
- LPS-induced IL-1 production in macrophages is regulated by both PKc and CaM kinase-dependent signaling pathways.
- TNF-alpha production is primarily dependent on PKc signaling, but not CaM kinase signaling.
- These findings highlight distinct molecular mechanisms controlling the expression of key inflammatory cytokines.