Interleukin 6 dependence of anti-DNA antibody production: evidence for two pathways of autoantibody formation in pristane-induced lupus

Abstract

Pristane induces a lupus-like syndrome in nonautoimmune mice characterized by the development of glomerulonephritis and lupus-associated autoantibodies. This is accompanied by overproduction of interleukin (IL)-6, a cytokine linked with autoimmune phenomena. The goal of this study was to evaluate the role of IL-6 in autoantibody production in pristane-induced lupus. BALB/cAn IL-6-deficient (-/-) and -intact (+/+) mice were treated with pristane or phosphate-buffered saline, and autoantibody production was evaluated. Pristane induced high levels of immunoglobulin (Ig)G anti-single-stranded DNA, -double-stranded (ds)DNA, and -chromatin antibodies in IL-6(+/+), but not IL-6(-/-) mice by enzyme-linked immunosorbent assay. High titer IgG anti-dsDNA antibodies also were detected in sera from +/+, but not -/-, mice by Crithidia luciliae kinetoplast staining. The onset of IgG anti-dsDNA antibody production in +/+ mice occurred >5 mo after pristane treatment, well after the onset of nephritis, suggesting that these antibodies are not directly responsible for inducing renal disease. In contrast to anti-DNA, the frequencies of anti-nRNP/Sm and anti-Su antibodies were similar in pristane-treated IL-6(-/-) and IL-6(+/+) mice. However, levels were higher in the +/+ group. These results suggest that IgG anti-DNA and chromatin antibodies in pristane-treated mice are strictly IL-6 dependent, whereas induction of anti-nRNP/Sm and Su autoantibodies is IL-6 independent. The IL-6 dependence of anti-DNA, but not anti-nRNP/Sm, may have implications for understanding the patterns of autoantibody production in lupus. Anti-DNA antibodies are produced transiently, mainly during periods of disease activity, whereas anti-nRNP/Sm antibody levels are relatively insensitive to disease activity. This may reflect the differential IL-6 dependence of the two responses.

Figure 1

Anti-ssDNA and anti-dsDNA autoantibody levels. (left) Sera from BALB/cAn IL-6^−/− or IL-6^+/+ mice 3 wk after pristane or PBS treatment were tested by ELISA for IgM anti-ssDNA antibodies at a dilution of 1:500. Sera from the same mice 8 mo after treatment were tested by ELISA for IgG anti-ssDNA antibodies. Symbols represent individual animals and horizontal lines represent the mean A_{405 nm} of each group (*P < 0.005 in the IL-6^+/+ pristane versus IL-6^−/− pristane by Mann Whitney test). (right) Sera from BALB/cAn mice 8 mo after pristane or PBS treatment, 5-mo-old MRL/lpr mice, and mAb 6/O2 576-2 (IgG anti-ssDNA) were analyzed by ELISA for IgG anti-dsDNA antibodies at a 1:160 dilution. Samples were negative (•) or positive (□) positive by Crithidia assay at a 1:40 dilution.

Figure 1

Anti-ssDNA and anti-dsDNA autoantibody levels. (left) Sera from BALB/cAn IL-6^−/− or IL-6^+/+ mice 3 wk after pristane or PBS treatment were tested by ELISA for IgM anti-ssDNA antibodies at a dilution of 1:500. Sera from the same mice 8 mo after treatment were tested by ELISA for IgG anti-ssDNA antibodies. Symbols represent individual animals and horizontal lines represent the mean A_{405 nm} of each group (*P < 0.005 in the IL-6^+/+ pristane versus IL-6^−/− pristane by Mann Whitney test). (right) Sera from BALB/cAn mice 8 mo after pristane or PBS treatment, 5-mo-old MRL/lpr mice, and mAb 6/O2 576-2 (IgG anti-ssDNA) were analyzed by ELISA for IgG anti-dsDNA antibodies at a 1:160 dilution. Samples were negative (•) or positive (□) positive by Crithidia assay at a 1:40 dilution.

Figure 3

Anti-nRNP/Sm and anti-chromatin antibody ELISAs.Sera from pristane treated IL-6^−/− (n = 28, •) or IL-6^+/+ (n = 26, ▪) mice and from PBS-treated IL-6^−/− (n = 10, ○) or IL-6^+/+ (n = 10, □) mice 0–8 mo after treatment were tested for anti-nRNP/Sm (A and B) and antichromatin (C and D) antibodies by ELISA. (Top) Frequencies (%) of sera containing IgG anti-nRNP/Sm (A) or antichromatin (B) autoantibodies (defined as >3 SDs above the mean of negative controls). (Bottom) Levels of anti-nRNP/Sm (B) or anti-chromatin (D) autoantibodies (in units). Data represent the mean of all animals in each group.

Figure 2

IgG antichromatin antibody ELISA. Serial serum samples from all pristane- or PBS-treated mice were analyzed at a dilution of 1:500 for IgG anti-chromatin antibodies by ELISA. Levels of IgG antichromatin are expressed in units × 10⁶ (see Materials and Methods).