Interleukin-6 receptor blockade selectively reduces IL-21 production by CD4 T cells and IgG4 autoantibodies in rheumatoid arthritis

Abstract

Interleukin-6 (IL-6) levels are known to be increased in patients with rheumatoid arthritis (RA). Tocilizumab, a monoclonal antibody to the IL-6 receptor (IL-6R), reduces disease activity in RA, although its mechanisms of action remain unclear. Since IL-6 regulates cytokine production by CD4 T cells during activation, we investigated whether treatment with tocilizumab altered the phenotype and cytokine production by CD4 T cells in patients with rheumatoid arthritis. We show here that tocilizumab treatment does not change the production of cytokines by naïve CD4 T cells. However, tocilizumab treatment causes a selective decrease of IL-21 production by memory/activated CD4 T cells. Since IL-21 is known to promote plasma cell differentiation, we examined the effect of tocilizumab on the production of autoantibodies. We show that there is a decrease in the levels of IgG4 anti-CCP antibodies, but there is no effect on IgG1 anti-CCP antibodies. In addition, we show that IL-21 is a powerful inducer of IgG4 production by B cells. Thus, IL-6 contributes to the presence of IgG4-specific anti-CCP autoantibodies in RA patients, likely through its effect on IL-21 production by CD4 T cells, and IL-6R blockade down-regulates this pathway.

Keywords: CD4 T; IL-21; IL-6; IgG4; Interleukin-6; auti-CCP; rheumatoid arthritis; tocilizumab.

Figure 1

Tocilizumab treatment reduces production of IL-21 by CD4 CD45RO T cells in RA patients. (A) CD4 CD45RO cells were isolated from each patient at the indicated period of time and activated for 24 h with anti-CD3 and anti-CD28 Abs. IL-21 levels in the supernatant were determined by Multiplex analysis. (B) Relative IL-21 mRNA levels in freshly isolated CD4 CD45RO T cells from patients were determined by real time RT-PCR using HPRT as house keeping gene, using the delta delta CT analysis. IL-21 mRNA levels for each patients were relative to the levels at time 0 (prior to the first treatment). Levels of IL-21 mRNA at 6 months were statistically significantly reduced relative to the levels prior to the treatment (p=0.03) using Wilcoxon signed rank test.

Figure 2

Effect of tocilizumab on IgG isotypes and IgG4 autoantibodies. (A) and (B) Serum levels of total IgG2 (A) and IgG4 (B) prior to (0 months) and 6 months after the treatment with tocilizumab in the eight patients. (C) IgG4-specific anti-CCP Ab levels in serum. Six patients (Patients #1, 2, 4, 5, 6 and 7) had detectable levels of IgG4-anti-CCP Abs in serum prior to the initiation of tocilizumab treatment (0 months). (D) IgG1-specific anti-CCP Ab levels in serum. Five patients (Patients #1, 2, 4, 6 and 7) showed detectable levels of IgG1-anti-CCP Abs prior to the treatment. (E) Fold reduction in the serum levels of IgG1-specific anti-CCP Abs and IgG4-specific anti-CCP Abs between 0 months (prior to the treatment) and 6 months after the initiation of the treatment. Results of the repeated measures analysis of variance suggest that the fold reduction in IgG1 differs from that observed in IgG4 (p=0.011 for the interaction effect). Follow-up tests of simple effects shows a statistically significant (p =0.011) fold-reduction in IgG4 anti-CCP Abs (denoted by *) while there was no reduction in IgG1 anti-CCP Abs levels (p=0.185). Note that a fold-reduction equal to 1 between 0 months and 6 months means no effect on IgG levels with treatment. (F) Purified B cells from healthy volunteers (n=4) were activated in vitro with CD40L-expressing cells in the presence of medium, IL-4 or IL-21. The levels of IgG4 in the supernatants were determined after 6 days. Fold induction for each subject in the levels of IgG4 produced by B cells activated with IL-4 or IL-21 relative to the levels by B cells activated with just medium is shown. The statistically significant difference between fold-induction obtained with IL-21 relative to IL-4 Abs was determined by paired t test analysis, p =0.0206.