Epratuzumab inhibits the production of the proinflammatory cytokines IL-6 and TNF-α, but not the regulatory cytokine IL-10, by B cells from healthy donors and SLE patients

Abstract

Introduction: Cytokines produced by B cells are believed to play important roles in autoimmune diseases. CD22 targeting by epratuzumab has been demonstrated to inhibit phosphorylation of B cell receptor (BCR) downstream signaling in B cells. It has been shown that other sialoadhesin molecules related to CD22 have immunoregulatory functions; therefore, in the present study, we addressed the role of epratuzumab on the production of key cytokines by B cells of patients with systemic lupus erythematosus (SLE) and of healthy donors (HD).

Methods: Peripheral blood B cells were purified and activated by BCR with or without Toll-like receptor 9 (TLR9) stimulation in the presence or absence of epratuzumab. Cytokine production by B cells (interleukin [IL]-6, tumor necrosis factor [TNF]-α and IL-10) in the supernatant and the induction of IL-10(+) B cells from patients with SLE and HD were analyzed.

Results: The secretion of the proinflammatory cytokines TNF-α and IL-6 by anti-BCR and BCR- and/or TLR9-activated B cells from HD and patients with SLE was inhibited by epratuzumab. In contrast, the production of IL-10 by B cells was not affected by epratuzumab under either stimulation condition. Consistently, the induction of IL-10-producing B cells in culture was not affected by epratuzumab.

Conclusions: Epratuzumab, by targeting CD22, was able to inhibit the production of the proinflammatory cytokines IL-6 and TNF-α by B cells, in contrast to IL-10, in vitro. These data suggest that targeting CD22 alters the balance between proinflammatory cytokines (TNF-α, IL-6) and the regulatory cytokine IL-10 as another B cell effector mechanism.

Fig. 1

Targeting of CD22 by epratuzumab influences the secretion of the proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6, but not IL-10, by stimulated B cells. Peripheral blood B cells were purified from patients with systemic lupus erythematosus (SLE) (left) and healthy donors (HD) (right), pretreated with (gray squares) or without F(ab′)₂ epratuzumab (open squares) and cultured with media alone (RPMI) or stimulated with anti–B cell receptor (anti-BCR) or anti-BCR + CpG. After 2 days of culture, the supernatants were harvested and tested for TNF-α, IL-6 and IL-10 protein production using a Bio-Plex Pro™ Human Th17 Cytokine Panel assay. Combined data from 13 patients with SLE and 9 HD are shown for TNF-α (a), IL-6 (b) and IL-10 (c) (Mann–Whitney U test; ns = not significant, *p < 0.05, **p < 0.01)

Fig. 2

Epratuzumab does not influence the generation of intracellular interleukin (IL)-10-positive B cells. (a) Frequency of intracellular IL-10⁺ B cells after 2 days of peripheral blood mononuclear cell culture of representative healthy donors (HD) or patients with systemic lupus erythematosus, respectively, without stimulation (RPMI 1640 medium) or stimulated by anti–B cell receptor (anti-BCR) or anti-BCR + CpG using flow cytometry. (b) Comparison of the frequency of IL-10–producing B cells from 6 patients with SLE and 10 HD without (open squares) and with F(ab′)₂ epratuzumab (gray squares) incubation in response to the indicated stimulations (Mann–Whitney U test)

Fig. 3

Epratuzumab influences the balance between interleukin (IL)-10 and the proinflammatory cytokines tumor necrosis factor (TNF)-α and IL-6 secreted by B cells activated by anti–B cell receptor (anti-BCR) + CpG in patients with systemic lupus erythematosus (SLE). The balance between IL-10 and proinflammatory cytokines (IL-6 and TNF-α) produced by B cells from healthy donors (HD) and patients with SLE was evaluated based on the ratio of IL-10 to IL-6 (a) or IL-10 to TNF-α (b) (Mann–Whitney U test; **p < 0.01)