Volume 3 Supplement 2

21st European Workshop for Rheumatology Research

Open Access

Analysis of VH and VL mRNA in single synovial and peripheral B/plasma cells of patients with rheumatoid arthritis

  • S Ruzickova1, 3,
  • J Vencovsky1,
  • O Krystufkova1,
  • Z Cimburek1,
  • J Sinkora2,
  • O Horvath3 and
  • T Doerner4
Arthritis Research & Therapy20013(Suppl 2):P107

DOI: 10.1186/ar276

Received: 15 January 2001

Published: 26 January 2001

Introduction

Synovial tissue in rheumatoid arthritis displays a complex infiltration of many cell types like T and B lymphocytes, plasma cells, folicular dendritic cells, macrophages etc. Presence of B and plasma cells results in secretion of large amounts of multiple pathologic autoantibodies.

Aim

To analyze immunoglobulin VH and VL gene usage on the level of mRNA from a single B or plasma cell isolated from synovium and peripheral blood of patients with RA, in order to determine a clonality and a molecular structure of produced antibodies.

Materials and methods

RA synovial tissue obtained during synovectomy was enzymatically digested and single synovial B/plasma cells were sorted using immunofluorescent staining with anti-CD19 or anti-CD138. Peripheral B cells were isolated in the same way without enzyme treatment. cDNA library from each single B and plasma cell was generated. Two stage polymerase chain reaction to analyse VH and VL genes was performed. VH, DH and JH or VL and JL gene segments were assigned and somatic mutations determined by comparison with germline sequences on the V BASE/Genbank data base. As a control peripheral B lymphocytes from healthy donors were screened.

Results

Analysis of RA synovial mRNA transcripts revealed prevalence of C gamma recombinants that contained rearranged VH1, VH3, VH4, VH5 and VH6 genes. Utilization of VH segments was similar between RA patients and normal subjects, but the accumulation of somatic mutations was elevated in RA synovial and peripheral B cells. We also found preferential utilization of a limited number of VH and DH gene segments. There was increased frequency of kappa light chains containing unusually long CDR3 when compared to normal peripheral B cells.

Conclusions

Our findings are consistent with hypothesis that B cell response RA synovium is probably antigen driven and oligoclonal.

The project was supported by grant VS96129 from Ministry of Education, Youth and Sport in the Czech Republic.

Authors’ Affiliations

(1)
Institute of Rheumatology and Laboratory of Gene Expression, Institute of Microbiology CAS
(2)
Division of Immunology and Gnotobiology, Institute of Microbiology CAS
(3)
Department of Immunology, Institute of Microbiology CAS
(4)
Department of Rheumatology and Clinical Immunology, Charite University Hospital

Copyright

© BioMed Central Ltd 2001

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