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THU0109 The destructive process of rheumatoid arthritis synovial fibroblasts (ra-sf) is independent of p53
  1. CA Seemayer1,
  2. S Kuchen1,
  3. M Neihart1,
  4. P Kuenzler1,
  5. E Neumann2,
  6. M Pruschy3,
  7. T Pap1,
  8. WK Aicher4,
  9. U Muller-Ladner1,
  10. BA Michel1,
  11. RE Gay1,
  12. S Gay1
  1. 1Center Exp Rheumat
  2. 2Int Medicine, University, Regensburg
  3. 3Rad Onc, University Hospital, Zürich, Switzerland
  4. 4Bas Sci Lab, Orth Surg, Tübingen, Germany

Abstract

Objectives The aim of this study was to investigate, whether the destructive process of synovial fibroblasts in rheumatoid arthritis is dependent on the expression of p53. Moreover, we searched for the expression of p53 in RA-SF in vitro and in vivo. In addition, we examined the inducibility of p53 in RA-SF by X-rays in vitro.

Methods Paraffin embedded synovial tissues from 14 RA, 3 osteoarthritis (OA), 3 normal synovia and a neurological p53 positive tumour were investigated with DO7 anti-p53 antibodies by immunohistochemistry. RA-SF from passages 2 to 12 (n = 10), OA-SF (n = 2), normal synoviocytes (N-SF, n = 1) and foreskin fibroblasts (FSFB, n = 1)) from passages 4–8 were grown on chamber slides and analysed with the same primary antibodies utilising immunofluorescence. SV40 transformed RA-SF (HSE) and SV40 transformed N-SF (K4IM) as well as the SV40 transformed bronchial epithelial cell line BEAS 2B served as positive controls. The percentage of positive cells was evaluated by a scoring system. RA-SF and HSE cultured in vitro were co-implanted with human cartilage under the renal capsule or the under the skin of SCID mice and kept there for 60 days. Paraffin sections from these SCID mice experiments were stained for p53. In addition, sections from former SCID mouse experiments including retroviral and adenoviral gene transfer studies revealing a strong invasion of synovial cells into the cartilage were investigated for the expression of p53. Moreover, RA-SF (n = 2) and controls (FSFB, n = 1) were irradiated with 10 Gy (X-rays) and analysed time dependently for the expression of p53 by immunofluorescence.

Results In vivo, p53 was not abundantly expressed in RA synovial tissue and in particular not at sites of invasion. In general, less than 5% of the cells in RA and OA synovial tissues stained positively for p53, while no staining in the normal synovia occurred. In contrast, the positive control revealed a strong nuclear p53 signal in almost 100% of the tumour cells. In vitro, the expression rate of p53 in RA-SF was comparable low, while the SV40 transformed cells demonstrated a strong and specific nuclear staining. Furthermore, RA-SF invading the human cartilage in the SCID mouse co-implantation model did not show any expression of p53 at sites of invasion, whereas the implanted SV40 transformed HSE revealed a strong p53 staining indicating that those cells survived at least for 60 days in SCID mice. RA-SF and FSFB cultured in vitro responded equally to the irradiation with X-rays by producing transiently p53, demonstrating an adequate and functional response of p53.

Conclusion We suggest that the expression of p53 plays only a limited role in the pathogenesis of RA and particularly not in the invasive process of RA-SF.

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