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. 1998 Aug;57(8):495–499. doi: 10.1136/ard.57.8.495

Ceramide, a mediator of interleukin 1, tumour necrosis factor α, as well as Fas receptor signalling, induces apoptosis of rheumatoid arthritis synovial cells

N Mizushima 1, H Kohsaka 1, N Miyasaka 1
PMCID: PMC1752728  PMID: 9797556

Abstract

OBJECTIVES—To examine the effects of ceramide, which is a lipid second messenger of cell surface receptors, including tumour necrosis factor α (TNFα), interleukin 1 (IL1), and Fas receptors, on rheumatoid arthritis (RA) synovial cells.
METHODS—Synovial cells from RA patients and normal skin fibroblasts were cultured with cell permeable ceramide (C2-ceramide). Apoptosis was assessed by microscopic observation of morphological changes, nuclear staining, and DNA electrophoresis. DNA synthesis was examined by thymidine incorporation.
RESULTS—C2-ceramide induced reversible morphological changes of synovial cells such as cell rounding within four hours. Subsequently, irreversible nuclear changes characteristic to apoptosis were observed at 48 hours. DNA synthesis was not promoted. The addition of ceramide exerted similar effects on cultured dermal fibroblasts.
CONCLUSION—Ceramide induced apoptosis in RA synovial cells. Ceramide could be a second messenger specific for apoptosis of RA synovial cells.

 Keywords: ceramide; apoptosis; rheumatoid arthritis

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Figure 1  .

Figure 1  

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Induction of cell death in RA synovial cells by C2-ceramide. RA synovial cells were incubated with 10 µM of C2-ceramide in serum free RPMI 1640 medium (supplemented with 40 ng/ml PDGF) for 0 hours (A), 4 hours (B), 24 hours (C), and 48 hours (D). Cells were observed by phase contrast microscopy (original magnification × 400).

Figure 2  .

Figure 2  

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Cytotoxicity of C2-ceramide on RA synovial cells and dermal fibroblasts. RA synovial cells (A) and normal dermal fibroblasts (B) were treated with C2-ceramide, C2-dihydroceramide, and dioctanoylglycerol in serum free RPMI 1640 medium (supplemented with 40 ng/ml PDGF), C2-ceramide in RPMI 1640 containing 5% FCS for 48 hours. Cell viability was determined by crystal violet assay. Values are the mean (SD) of triplicate cultures. The data were representative of synovial cells from eight RA patients and dermal fibroblasts from normal skin of three controls.

Figure 3  .

Figure 3  

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Inhibitory effect of ceramide on synovial cell DNA synthesis. RA synovial cells were treated with C2-ceramide in serum free RPMI 1640 medium (supplemented with 40 ng/ml PDGF, open symbols) or in RPMI 1640 containing 5% FCS (closed symbols) for 48 hours. DNA synthesis was determined by 3H-thymidine incorporation assay. Values are the mean (SD) of triplicate cultures. The data were representative of synovial cell samples from eight RA patients.

Figure 4  .

Figure 4  

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Nuclear condensation and fragmentation of RA synovial cells induced by C2-ceramide. RA synovial cells were treated with 0.1% ethanol, as a control (A) or 10 µM C2-ceramide (B) for 48 hours. Nuclei were stained with Hoechst 33258 and observed by fluorescence microscopy (original magnification × 400).

Figure 5  .

Figure 5  

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DNA fragmentation of ceramide treated RA synovial cells. RA synovial cells were treated with 10 µM C2-ceramide for the indicated periods. Extracted DNA was electrophoresed on a 2% agarose gel, and stained with ethidium bromide. Lane 1:1kb ladder DNA molecular weight marker, lane 2:0.1% ethanol, lane 3:C2-ceramide for 24 hours, lane 4:C2-ceramide for 48 hours.

Figure 6  .

Figure 6  

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Reversibility of early morphological changes induced by ceramide. RA synovial cells were incubated with 10 µM of C2-ceramide in serum free RPMI 1640 medium (supplemented with 40 ng/ml PDGF) for 24 hours. The medium was changed to ceramide free medium, and the culture was continued for additional 24 hours. The cells were observed by phase contrast microscopy (original magnification × 400).

Selected References

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