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Annals of the Rheumatic Diseases logoLink to Annals of the Rheumatic Diseases
. 2004 Oct 21;64(5):708–714. doi: 10.1136/ard.2004.025577

Expression and regulation of cryopyrin and related proteins in rheumatoid arthritis synovium

S Rosengren 1, H Hoffman 1, W Bugbee 1, D Boyle 1
PMCID: PMC1755498  PMID: 15498798

Abstract

Background: Rheumatoid arthritis (RA) synovium is characterised by enhanced NF-κB activity and proinflammatory cytokines. Cryopyrin (CIAS-1, NALP-3, PYPAF-1) has been shown to regulate NF-κB and caspase-1 activation.

Objective: To study the expression of cryopyrin, its effector molecule ASC, and its putative antagonist pyrin in RA and osteoarthritis (OA) synovium, and the main two cellular constituents of synovial lining, cultured fibroblast-like synoviocytes (FLS) and macrophages.

Methods: FLS and macrophages were cultured in the presence of inflammatory mediators. Real time polymerase chain reaction was used to quantify message levels in synovial biopsy specimens and cells. In situ hybridisation was employed to localise expression of cryopyrin mRNA.

Results: Cryopyrin mRNA was raised in RA synovium and detected in both lining and sublining regions. FLS from RA and OA tissue expressed low baseline levels of cryopyrin transcripts that were induced by tumour necrosis factor α (TNFα). In contrast, macrophages differentiated in vitro expressed relatively high cryopyrin levels, which were further induced by TNFα, but not by interleukin 1ß. ASC mRNA levels were comparable in RA and OA tissue, FLS, and macrophages, and were depressed by TNFα in macrophages. Pyrin expression was higher in RA synovium than in OA tissue, and virtually undetectable in FLS but high in macrophages where it was unchanged by TNFα treatment.

Conclusion: These results suggest that enhanced cryopyrin levels in RA synovium are due to a greater numbers of tissue macrophages, and demonstrate transcriptional regulation of cryopyrin in a chronic inflammatory disease.

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Figure 1.

Figure 1

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 Expression of cryopyrin, ASC, and pyrin mRNA in synovial tissue from patients with RA and OA undergoing joint replacement surgery, as determined by real time qPCR. REU (relative expression units) data are standardised to known dilutions of PBMC cDNA and normalised by GAPDH to control for cellularity. Results are from 14 patients for each group. *p<0.05 by Student's t test on log transformed data. Both the cryopyrin and pyrin message was significantly raised in RA.

Figure 2.

Figure 2

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 Detection of cryopyrin mRNA in synovial tissue by in situ hybridisation. (A) CHO cells transfected with a cryopyrin-GFP fusion construct and hybridised with cryopyrin antisense probe to demonstrate its specificity. No signal was obtained with cryopyrin sense probe (not shown). (B) RA synovium hybridised with MMP-3 antisense probe shows typical lining distribution. MMP-3 sense probe did not hybridise (not shown). (C) Cryopyrin mRNA is expressed in both lining and sublining in RA synovium as shown by the antisense probe. (D) The cryopyrin sense probe did not hybridise to RA synovium (serial section, same area as in C).

Figure 3.

Figure 3

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 Expression of cryopyrin (A), ASC (B), and pyrin (C) mRNA in FLS after stimulation with TNFα (50 ng/ml) at time zero. Results are from real time qPCR studies of three cell lines each and are expressed as GAPDH normalised REU as in fig 1 legend. * p<0.05 from time zero and †p<0.05 between RA and OA, by repeated measures ANOVA. (A) Expression of cryopyrin was transiently raised in both RA and OA FLS. (B) ASC was up regulated in RA but not OA FLS. (C) Pyrin was not detectable at baseline but was induced. (D) As a control, continuous MMP-1 expression demonstrates that FLS were fully activated.

Figure 4.

Figure 4

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 Expression of (A) cryopyrin, (B) ASC and pyrin mRNA in macrophage-like cells differentiated from healthy donor monocytes by 5 day culture in M-CSF and serum. Cells were treated with LPS (10 ng/ml), TNFα (50 ng/ml), or IL1ß (2 ng/ml) for 18 hours before real time qPCR analysis. Results are from three donors and expressed as GAPDH normalised REU as in the legend to fig 1. *p<0.05 compared with control, untreated cells by single ANOVA, and Dunnett's post hoc test. The cryopyrin message was induced by LPS and TNFα, whereas ASC message levels decreased under the same conditions. Pyrin was only raised by LPS but not by either cytokine. The IL1ß message was significantly up regulated by LPS and TNFα (A), whereas IL1ß itself up regulated MMP-9 expression (see text), indicating macrophage activation in all cases.

Figure 5.

Figure 5

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 Time course of mRNA expression in monocyte derived, macrophage-like cells in response to treatment with TNFα (50 ng/ml). Results are from three donors and expressed as GAPDH normalised REU as in the legend to fig.1. *p<0.05 from time zero by repeated measures ANOVA. (A) Cryopyrin message was induced in two phases, whereas (B) ASC levels fell. (C) Pyrin remained unaltered. (D) Cells were fully activated as indicated by continuous expression of IL1ß.

Selected References

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