Background/Purpose: We previously identified a subset of regulatory B cells that express the apoptosis-inducing molecule Fas ligand (FasL), and reported that these B cells were expanded by IL-5. This study tested the effects of IL-4, another type 2 cytokine, on the growth and function of FasL⁺ B cells, and sought to identify pathways downstream of the IL-5 receptor important for FasL⁺B cell function.

Methods: B cells were purified from mouse splenocytes by CD19⁺ MACS beads and cultured with a monolayer of CD40L-expressing fibroblasts in the presence or absence of cytokines. After culture, surface expression of phenotypic markers was assessed by flow cytometry. Cytokine release from stimulated B cells was measured by ELISA. The ability of stimulated B cells to induce apoptosis was measured by culturing B cells with activated T cells from TCR-transgenic mice and assessing apoptosis in CD4⁺cells by Annexin V/propidium iodide staining. Cyclosporine was used to determine the effects of inhibiting the calcineurin signaling pathway on regulatory B cell function.

Results: B cells stimulated with IL-5 displayed regulatory B cell functions, including increased expression of FasL, antigen-specific killing of CD4⁺T cells, and secretion of IL-10. Unexpectedly, the addition of IL-4 to B cell cultures completely abrogated the ability of B cells to induce apoptosis in target T cells and secrete IL-10, and instead induced the secretion of IL-6. Both IL-4 and IL-5 stimulated similar levels of B cell proliferation, and the combination of both cytokines had an additive effect on proliferation. IL-5-stimulated B cells resembled marginal zone B cells, with increased surface expression of CD80, CD86, CD9 and CD5, and reduced CD23 expression. This change in surface phenotype was also inhibited by IL-4. Finally, the calcineurin inhibitor cyclosporine prevented the induction of FasL expression and IL-10 secretion mediated by IL-5.

Conclusion: IL-4 inhibits the regulatory B cell functions induced by IL-5, such as FasL-mediated induction of apoptosis in CD4⁺ T cells and secretion of IL-10. The IL-5 receptor utilizes a calcineurin-dependent pathway for induction of FasL⁺ regulatory B cells. These findings suggest that while IL-4 has regulatory effects on T cells in inflammatory diseases such as rheumatoid arthritis, it may have an opposing role on B cells by antagonizing the IL-5-mediated induction of FasL⁺ regulatory B cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/il-5-induced-fasl-regulatory-b-cells-are-inhibited-by-il-4-and-cyclosporine/