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A5.23 Multiparameter Phospho-Flow Analysis of B Cells from Patients with Rheumatoid Arthritis
  1. Daniel Szili1,
  2. Judit Pozsgay1,
  3. Zsuzsanna Banko1,
  4. Tamas Gati2,
  5. Gyorgy Nagy2,3,
  6. Bernadett Rojkovich2,
  7. Gabriella Sarmay1
  1. 1Department of Immunology, Eötvos Lorand University, Budapest
  2. 2Buda Hospital of Hospitaller Brothers of St. John, Budapest
  3. 3Department of Rheumatology, Semmelweis University, Budapest, Hungary

Abstract

Background and Objectives Rheumatoid arthritis (RA) is a common, relapsing autoimmune disease, which affects approximately 1% of the population worldwide. While the specific molecular events that lead to initiation and onset of RA are not known, an uncontrolled activation of the immune system is considered to be a critical component of the disease. B lymphocytes undoubtedly play a critical role in disease aetiology. Antigen binding to B-cell receptor (BCR) triggers B cell activation, although the threshold of activation can be influenced by other receptors, such as TLR9. TLR9 has received substantial attention as a pathogenic co-stimulator of autoreactive B cell responses. Our aim was to compare basal activity and induced phosphorylation of AKT, ERK, p38 MAPK and CREB after stimulation of B cells from RA patients and healthy individuals via BCR and/or TLR9.

Materials and Methods Blood samples were collected from healthy donors and RA patients having moderate (DAS28 3.2 < 5.1) and active (DAS28 > 5.1) disease. B cells were stimulated with anti-Ig (Fab’)2 and/or CpG ODN. Naive and memory B cells were identified by anti-CD20-A647 and anti-CD27-PE Phosphorylation level of AKT, ERK, p38 and CREB was detected before and after the stimuli by specific phospho-antibodies using multiparameter phospho-flow analysis. Results were evaluated by the FlowJo software.

Results The basal level of phosphorylation of signalling molecules was significantly higher in RA patients as compared to healthy donors. The induced level of phosphorylation was also higher in RA samples in most of cases, CpG stimulated memory B cells from patients with active disease have shown the highest values. In anti-Ig plus CpG ODN stimulated samples phosphorylation of all molecules was significantly higher in both naïve and memory RA B cells as compared to healthy controls. However, when compared to unstimulated cells, the increment of phosphorylation in the stimulated cells was the same or lower in RA samples.

Conclusions We have shown differences in the activation state of AKT, ERK, p38 and CREB in B cells from healthy individuals and RA patients. The higher basal phosphorylation level indicates the activated state of RA B cells. The lower capability of activation-induced phosphorylation may be a result of lower responsiveness of RA B cells. The analysis of phosphorylation signature in RA B-cells may provide new information to a better understanding of the disease.

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