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Geldanamycin treatment ameliorates the response to LPS in murine macrophages by decreasing CD14 surface expression
Virginia L Vega1, Antonio De Maio
1Division of Pediatric Surgery and Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Abstract:
Geldanamycin (GA) is an antibiotic produced by Actinomyces, which specifically inhibits the function of the heat shock protein 90 family. Treatment of a murine macrophage cell line (J774) with GA resulted in a reduced response to Escherichia coli lipopolysaccharide (LPS) as visualized by a decrease of NF-kappaB translocation into the nucleus and secretion of tumor necrosis factor alpha (TNF-alpha). To elucidate the mechanism of this effect, the expression of CD14, the formal LPS receptor, was analyzed. Cells treated with GA showed a reduced level of surface CD14 detected by immunostaining, whereas the expression of other surface receptors, such as FC-gamma receptor and tumor necrosis factor receptors (TNF-R1 and TNF-R2), was unaffected. The reduced surface level of CD14 was not due to a reduction in its expression because CD14 steady state mRNA levels or the total cellular pool of CD14 was not altered by GA treatment. Surface CD14 was more rapidly internalized after GA treatment (2-3 h) than after incubation with cycloheximide. Immunostaining of permeabilized cells after GA treatment revealed a higher intracellular content of CD14 colocalizing with calnexin, an endoplasmic reticulum (ER) protein. These results suggest that the decrease in CD14 surface expression after GA treatment is due to rapid internalization without new replacement. These effects may be due to the inhibition of Hsp90 and Grp94 by GA in macrophages.
Insights
Geldanamycin antibiotic treatment reduces macrophage response to lipopolysaccharide (LPS) by decreasing surface CD14 levels. This occurs through accelerated CD14 internalization, not altered gene expression.
Area of Science:
- Immunology
- Cell Biology
- Pharmacology
Background:
- Geldanamycin (GA) is an antibiotic that inhibits heat shock protein 90 (Hsp90) family proteins.
- Macrophage activation by lipopolysaccharide (LPS) involves NF-kappaB translocation and TNF-alpha secretion.
- CD14 is the primary receptor for LPS on macrophages.
Purpose of the Study:
- To investigate the mechanism by which Geldanamycin (GA) affects macrophage response to LPS.
- To determine the impact of GA on CD14 expression and function in macrophages.
Main Methods:
- Murine macrophage cell line (J774) treated with Geldanamycin (GA).
- Analysis of NF-kappaB translocation and TNF-alpha secretion.
- Immunostaining to assess surface and intracellular CD14 levels, and other surface receptors.
- Measurement of CD14 mRNA levels and total cellular CD14.
- Internalization assays and co-localization studies with ER protein calnexin.
Main Results:
- GA treatment reduced LPS-induced NF-kappaB translocation and TNF-alpha secretion.
- Surface CD14 levels were significantly decreased by GA, while other receptors remained unaffected.
- GA did not alter CD14 mRNA or total cellular CD14 levels, indicating no change in expression.
- GA accelerated CD14 internalization and increased intracellular CD14 co-localizing with calnexin.
- The reduction in surface CD14 is attributed to rapid internalization without compensatory new synthesis.
Conclusions:
- Geldanamycin (GA) inhibits LPS-induced inflammatory responses in macrophages.
- GA reduces surface CD14 expression by promoting its rapid internalization from the cell surface.
- These effects are likely mediated by GA's inhibition of Hsp90 and Grp94 in macrophages.