A critical role for IL-21 receptor signaling in the pathogenesis of systemic lupus erythematosus in BXSB-Yaa mice

Abstract

Interleukin 21 (IL-21) is a pleiotropic cytokine produced by CD4 T cells that affects the differentiation and function of T, B, and NK cells by binding to a receptor consisting of the common cytokine receptor gamma chain and the IL-21 receptor (IL-21R). IL-21, a product associated with IL-17-producing CD4 T cells (T(H)17) and follicular CD4 T helper cells (T(FH)), has been implicated in autoimmune disorders including the severe systemic lupus erythematosus (SLE)-like disease characteristic of BXSB-Yaa mice. To determine whether IL-21 plays a significant role in this disease, we compared IL-21R-deficient and -competent BXSB-Yaa mice for multiple parameters of SLE. The deficient mice showed none of the abnormalities characteristic of SLE in IL-21R-competent Yaa mice, including hypergammaglobulinemia, autoantibody production, reduced frequencies of marginal zone B cells and monocytosis, renal disease, and premature morbidity. IL-21 production associated with this autoimmune disease was not a product of T(H)17 cells and was not limited to conventional CXCR5(+) T(FH) but instead was produced broadly by ICOS(+) CD4(+) splenic T cells. IL-21 arising from an abnormal population of CD4 T cells is thus central to the development of this lethal disease, and, more generally, could play an important role in human SLE and related autoimmune disorders.

Fig. 1.

IL-21 is the product of T cells. Fold change computations were based on the mean C_T (cycle threshold) values normalized to 18S RNA from spleen cells pooled from 2 21-week-old BXSB-Yaa and 2 BXSB-wt mice, depleted of both CD4⁺ and CD8⁺ T cells or left intact.

Fig. 2.

IL-21R deficiency prevents humoral components of the BXSB-Yaa autoimmune disease. (A) ELISA analysis of Ig subclass levels in plasma from 10 Yaa/Il21r^+/− (gray bars) and 10 Yaa/Il21^−/− (black bars) 16-week-old mice. (B) Anti-nuclear antibodies (ANA) from Yaa/Il21r^+/− mice compared with Yaa/Il21^−/− mice detected by staining of Hep-2 cell slides (Right) with quantitation by ImageJ (Left). *, P < 0.05; **, P < 0.002.

Fig. 3.

IL-21R deficiency prevents the abnormal leukocyte populations characteristic of BXSB-Yaa mice. (A) Spleens of Yaa/Il21^−/− mice exhibit greatly reduced cellularity compared with spleens of Yaa/Il21r^+/− mice. Yaa/Il21r^+/− mice have high numbers of total B cells, and immature and mature B cells; this phenotype was abolished in Yaa/Il21^−/− mice. (B) The frequencies of splenic MZ B cells (CD19⁺ CD21^hi CD23^lo) typically depleted in Yaa/Il21r^+/− mice are restored in Yaa/Il21^−/− mice. (C) Yaa/Il21r^+/− but not Yaa/Il21^−/− spleens lack an obvious MZ and show expansion in red pulp with monocytosis and accumulations of plasma cells resulting in compression of the white pulp. Central arteriole (CA), follicle (F) of H&E-stained sections. (D) Yaa/Il21^−/− mice do not develop spleen cell monocytosis as measured by CD11b expression and have fewer CD11c-positive dendritic cells compared with Yaa/Il21r^+/− littermate controls. Data are representative of spleens from 16-week-old mice. Data from BXSB female mice thus lacking Yaa of the same age are included for comparison.

Fig. 4.

IL-21R deficiency prevents renal disease and mortality characteristic of BXSB-Yaa mice. (A and B) Kidney sections from 16-week-old Yaa/Il21r^+/− and Yaa/Il21^−/− mice were stained with H&E, periodic acid/Schiff reagent (PAS), or Masson's trichrome. (A) Representative sections. (B) Kidneys were graded for severity of changes. N.S., not significant at P ≤ 0.05. (C) Kaplan–Meier lifespan analysis indicated a significant Wilcoxon P value of 0.0017 for survival differences.

Fig. 5.

IL-21R deficiency decreases numbers of T_FH cells. Splenic CD4 T cells from 16-week-old Yaa/Il21^−/−, Yaa, and Yaa/Il21r^+/− mice were analyzed for ICOS and CXCR5 expression by flow cytometry analysis. (A) Representative data. (B) Mean fluorescence intensity (MFI) and percent ICOS^hi splenic CD4 T cells; there were 10 mice per group. (C) Correlation of total mononuclear spleen cell numbers with MFI of ICOS (Upper) or CXCR5 (Lower) on CD4 T cells of individual 16-week-old Yaa/Il21r^+/− and Yaa/Il21^−/− mice. (D) Gene expression comparison of flow cytometry-sorted ICOS^hi and ICOS^lo splenic CD4 T cells (Upper) and ICOS^hi CXCR5^hi and CXCR5^lo splenic CD4 T cells (Lower) from BXSB-Yaa mice. See Fig. S4 for flow cytometric gates.