Interleukin-6 is responsible for aberrant B-cell receptor-mediated regulation of RAG expression in systemic lupus erythematosus

Abstract

Defective regulation of secondary immunoglobulin V(D)J gene rearrangement promotes the production of autoantibodies in systemic lupus erythematosus (SLE). It remains unclear, however, whether the regulation of the recombination-activating genes RAG1 and RAG2 is effective in SLE. RAG1 and RAG2 messenger RNA expression was analysed before and after in vitro activation of sorted CD19(+) CD5(-) B cells with anti-immunoglobulin M antibodies, in 20 SLE patients and 17 healthy controls. The expression of CDK2 and p27(Kip1) regulators of the RAG2 protein, were examined. The levels of interleukin-6 (IL-6) and its influence on RAG regulation were also evaluated in vitro. SLE patients had increased frequency of RAG-positive B cells. B-cell receptor (BCR) engagement induced a shift in the frequency of kappa- and lambda-positive cells, associated with a persistence of RAG messenger RNA and the maintenance of RAG2 protein within the nucleus. While expression of the RAG2-negative regulator CDK2 was normal, the positive regulator p27(Kip1) was up-regulated and enhanced by BCR engagement. This effect was the result of the aberrant production of IL-6 by SLE B cells. Furthermore, IL-6 receptor blockade led to a reduction in p27(Kip1) expression, and allowed the translocation of RAG2 from the nucleus to the cytoplasm. Our study indicates that aberrant production of IL-6 contributes to the inability of SLE B cells to terminate RAG protein production. Therefore, we hypothesize that because of constitutive IL-6 signalling in association with BCR engagement, SLE B cells would become prone to secondary immunoglobulin gene rearrangements and autoantibody production.

Figure 1

RAG1 and RAG2 expression in peripheral blood B cells. (a) Peripheral cells from healthy controls (C) and systemic lupus erythematosus patients (SLE) were stained with anti-CD19 and anti-CD5 mAb, and expression of IgD and CD38 was assessed on CD19⁺ CD5⁺ and CD19⁺ CD5^– B-cell subpopulations. A representative example is shown. IgD^low CD38^high, including transitional type 1, B cells are indicated. (b) RAG1 and RAG2 mRNA were amplified by nested RT-PCR in fluorescence-activated sorted CD19⁺ CD5^– B cells from SLE patients and healthy controls. (c) Individual B cells were sorted with the Autoclone® single-cell deposition unit of the flow cytometer. Among 40 cells sorted in each sample, only GAPDH mRNA-positive cells were analysed for expression of RAG1 and RAG2 mRNA by nested RT-PCR. A representative example of three SLE patients is shown (SLE2) where crosses indicate cells excluded from the RAG1 and RAG2 analyses because of the absence of cDNA or contamination with genomic DNA. (d) The frequencies of single RAG1-positive and/or RAG2-positive cells are shown. Mean ±SD of three SLE patients and the two healthy controls.

Figure 2

Expression of κ and λ light chains, and RAG mRNA in IgM cross-linked B cells. The CD19⁺ CD5^– B cells from patients with SLE and healthy controls were sorted and stimulated with anti-IgM-coated beads at 1 μg/ml for 24 hr. (a) FITC-anti-κ and PE-anti-λ antibodies were used to determine the proportion of κ- and λ-positive cells by flow cytometry. A representative example of 11 healthy controls and 14 SLE patients is shown (C2 and SLE8, respectively). (b) Ratios of κ⁺/λ⁺ B cells were determined before (T0) and after anti-IgM-stimulation (anti-IgM). (c) RAG1 and RAG2 mRNA expression after anti-IgM stimulation was analysed by nested RT-PCR and that of GAPDH by RT-PCR. (d) Sorted κ⁺ B cells were stimulated with anti-IgM antibody, and the expression of λ light chain was determined by flow cytometry. A representative example of three healthy controls and three SLE patients is shown (C9 and SLE20, respectively).

Figure 3

CDK2 and p27^Kip1 expression in peripheral blood B cells. (a) Fluorescence-activated sorted CD19⁺ CD5^– B cells from patients with SLE and from healthy controls were stained intracellularly with anti-CDK2 or anti-p27^Kip1 antibodies and analysed. A representative example of the healthy controls and SLE patients (C1 and SLE7, respectively) is shown. (b) Frequency of CDK2- and p27^Kip1-positive B cells (filled symbols) and mean fluorescence intensity (MFI: open symbols) determined by flow cytometry. (c) The expression of p27^Kip1 was analysed by Western blotting: four representative examples of the SLE patients and the healthy controls are shown. For all the samples tested, the level of the p27^Kip1 protein was normalized relative to that of the β-actin by densitometry. (d) The expression of p27^Kip1 mRNA was determined by quantitative RT-PCR and the level was established following normalization to that of the 18S. Medians are indicated.

Figure 4

Variations in p27^Kip1 protein and mRNA expression in anti-IgM-stimulated B cells. The CD19⁺ CD5^– B cells from patients with SLE and from healthy controls were sorted and stimulated with anti-IgM-coated beads at 1 μg/ml for 24 hr. (a) Following Western blotting, the level of p27^Kip1 protein was measured by densitometry analysis relative to that of the β-actin. Fold increases induced by anti-IgM stimulation for all the samples tested are shown. (b) The expression of p27^Kip1 mRNA was determined by quantitative RT-PCR, and the level was normalized relative to that of the 18S. Fold increases induced by anti-IgM stimulation for all the samples tested is shown.

Figure 5

IL-6-dependent regulation of p27^Kip1 and RAG2. (a) The CD19⁺ CD5^– B cells from patients with SLE and healthy controls were sorted and stimulated with anti-IgM-coated beads at 1 μg/ml for 24 hr; the concentration of IL-6 in the supernatant was then determined by ELISA and expressed as pg for 10⁶ cultured B cells. (b) Some SLE B cells were costimulated with anti-IgM antibody at 1 μg/ml and anti-IL-6 receptor (IL-6R) antibody at 40 ng/ml for 24 hr. p27^Kip1 mRNA level was estimated by quantitative RT-PCR following normalization relative to the 18S mRNA. (c) Following costimulation with anti-IgM and anti-IL-6R antibodies, p27^Kip1 protein level was calculated by Western blotting and densitometry relative to that of β-actin. (d) Anti-IgM-stimulated SLE B cells were costimulated or not with anti-IL-6R antibody for 24 hr, permeabilized and stained with propidium iodide and anti-RAG2 antibody revealed by FITC-conjugated anti-rabbit IgG. The localization of RAG2 was determined by confocal microscopy. A representative example of three experiments is shown. (e) Healthy and SLE B cells were sorted, stimulated with anti-IgM antibody in the presence of a range of rIL-6 concentrations (0, 500 or 1000 pg/ml) with or without anti-IL-6R antibody for 24 hr, and RAG1 and RAG2 mRNA was amplified by nested RT-PCR. A representative example of three experiments is shown.