Rheumatoid arthritis is associated with signaling alterations in naturally occurring autoreactive B-lymphocytes

Abstract

Immune tolerance established during the development of B lymphocytes can be subverted in mature cells and lead to autoimmunity. This study focuses on the recently discovered subset of CD19(+)CD27(-)IgD(+)IgM(low/-) B cells that recognize self-antigens and have the capacity to produce autoantibodies, but under normal conditions do not generate autoimmune response due to intrinsic signaling inhibition (a condition known as clonal anergy and characterized by impaired antigen receptor signaling). Phosphorylation of intracellular signaling proteins and Ca(2+) responses in anergic B cells were measured by multicolor flow cytometry. Our results demonstrate a distinct phosphorylation pattern for major signal transduction proteins, which distinguishes anergic B cells. Comparison of B cell signaling properties in Rheumatoid Arthritis patients and healthy controls revealed a reversal of pTyr and Ca(2+) anergic signaling features in patients, accompanied by phosphorylation decreases of Blnk, Syk, SHP2, CD19. We identified BCR signaling pathway alterations associated with the loss of anergic B cell tolerance in Rheumatoid Arthritis.

Conflict of interest statement

Authors have no conflict of interest to declare.

Published by Elsevier Ltd.

Figures

Figure 1

Groups of 20 RA patients (mean age 55.9; male/female ratio 0.54) and 32 healthy control subjects (mean age 35.7; male/female ratio 0.68) recruited for the study. All RA patients are RF and anti-CCP positive and have not undergone B cell targeted treatments.

Figure 2

(A) Gating strategy to identify a subset of CD19+CD27−IgD+IgM− B cells (Ban) in healthy controls. (B) The majority of Ban lymphocytes gated in (A) are CD27-. Minor CD27+ subpopulations also present in the gated CD19+IgD+ subsets consist of IgM+ memory cells and IgM− Cδ class switched B cells. (C) Ca2+ responses to anti-BCR stimulation in IgM+ and CD19+IgD+IgMlow/− B cells. (D) total pTyr responses to anti-BCR stimulation in CD19+ cells (fluorescence intensity shift after the stimulation; duplicate experiments).

Figure 3

(A) Phosphorylation levels of intracellular signaling proteins (baseline and after stimulation with anti-BCR Ab) in IgM+ and Ban subsets of B cells isolated from healthy controls; mean fluorescence intensity (MFI); p values of paired two-tailed t-test. (B) Phosphorylation levels in B cells from patients with active RA.

Figure 4

(A) Intracellular Ca2+ response amplitude (% increase over baseline after stimulation with anti-BCR) in IgM+ and Ban B cells isolated from healthy controls or RA patients. (B) Comparison of baseline protein phosphorylation levels in Ban cells from healthy controls and RA patients; mean fluorescence intensity (MFI); p values of unpaired two-tailed t-test; statistically significant differences marked with α.

Figure 5

(A) Comparison of Ban phospho-response amplitudes (% increase over baseline after anti-BCR stimulation normalized to IgM+ response within each group) demonstrates relative signaling differences between Ban cells from healthy controls and RA patients; each bar shows that Ban response is Y % higher/lower than IgM+ in the same donor group; parameters YBan(RA) and YBan(control) are shown. (B) Color map representation of the numerical data in Figure 5A (parameters YBan(RA) and YBan(control)). (C) Qualitative phospho-protein signaling differences between RA and control Ban cells; YBan(RA) increase (↑) or decrease (↓) relative to YBan(control).

Figure 6

Schematic representation of signaling pathways alterations in Ban cells as compared the bulk of CD19+ cells in healthy controls (left) and RA patients (right); normalized phosphorylation change over baseline (YBan(control)).

Figure 7

(A) Correlations between phosphoprotein responses to anti-BCR in Ban cells from healthy controls and RA patients (nonparametric Spearman’s multivariate analysis). Statistically significant pairwise correlations (|ρ|<0.05) highlighted in grey. Bottom panels display scatterplot correlation matrices for each group (each dot represents individual sample). Alterations in pairwise relationships observed in the RA group are highlighted in red. (B) Graphical representation of pairwise relationships among phospho-protein responses to anti-BCR in Ban cells from healthy controls and RA patients.