Volume 5 Supplement 1

23rd European Workshop for Rheumatology Research

Open Access

Differential expression of IgVH mRNAs in human RA synovium detected by single-cell RT-PCR

  • S Ruzickova1, 3,
  • Z Cimburek1,
  • J Niederlova1,
  • O Horvath2,
  • O Krystufkova1,
  • T Dörner2 and
  • J Vencovsky1
Arthritis Res Ther20035(Suppl 1):23

DOI: 10.1186/ar653

Received: 14 January 2003

Published: 24 February 2003

Introduction

In rheumatoid arthritis (RA), the synovial membranes contain lymphocytic infiltrates sometimes resembling germinal centres. The production of clonally related immunoglobulin (Ig) transcripts, the presence of plasma cells within RA synovial tissue, somatic mutations and isotype switching in RF-specific synovial B lymphocytes have been observed. In addition, the expression of recombination-activating genes 1 and 2 (Rag1 and 2) has been detected in RA synovial B cells.

Objective

Analysis of the presence of plasma cells, mutational frequencies of their Ig heavy-chain transcripts, the signs of isotype switching and expression of Rag1 and 2 genes in inflamed RA synovial tissue.

Methods

Synovial tissue sections were labeled with anti-CD19, anti-CD138 and anti-CD38 antibodies and visualized using the alkaline phosphatase technique. Individual CD19+ CD38+ plasma cells were isolated from digested synovium of two caucasian RA patients using single-cell deposition. The cDNA from each single B cell was generated, and nested polymerase chain reaction specific for VH genes and Rag1 and Rag2 genes was performed. After sequencing, the VBASE database was used to assign VH, DH and JH gene segments and somatic mutations.

Results

Three different subsets of CD19+ CD38+ plasma cells were detected. The first subset represents cells expressing only IgM transcripts (IgM+, 13.5%), the second expressed only IgG transcripts (IgG+, 48.7%) and the third produced both IgM and IgG mRNAs (IgM+ IgG+, 37.8%). All of these detected IgVH mRNAs contained mutated sequences, indicating their memory cell origin. However, the differences of mutational frequencies between these subsets were statistically significant (IgM+ plasma cells 3.8%, IgG+ plasma cells 11.2% and IgM+ IgG+ cells 6.3%). Interestingly, either Rag1 or Rag2 mRNA was observed in 83.3% of all analyzed CD19+CD38+ plasma cells, with the highest frequency in the IgM+ IgG+ subset of plasma cells (71.4%).

Conclusion

The population of CD19+ CD38+ plasma cells differentially expressing mutated IgVH mRNAs and reinducing Rag 1 and 2 genes was observed in RA synovium. The IgM+ IgG+ cells might represent cells switching from IgM to IgG isotype and IgG+ plasma cells might correspond to post-switched cells producing high-affinity (auto)antibodies.

Authors’ Affiliations

(1)
Institute of Rheumatology and Laboratory of Gene Expression, Czech Academy of Sciences
(2)
Institute of Microbiology, Czech Academy of Sciences
(3)
Department of Rheumatology/Immunology, Medical Faculty, Charité, Humboldt University

Copyright

© The Author(s) 2003

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