IL-18 cDNA vaccination protects mice from spontaneous lupus-like autoimmune disease

Abstract

The lupus-like autoimmune syndrome of MRL/Mp-Tnfrsf6lpr (lpr) mice is characterized by progressive lymphadenopathy and autoantibody production, leading to early death from renal failure. Activation of T helper lymphocytes is one of the events in the pathogenesis of the disease in these mice and likely in human systemic lupus erythematosus. Among T helper lymphocyte-dependent cytokines, IFN-gamma plays a pivotal role in the abnormal cell activation and the fatal development of the lpr disease. IL-18, an inducer of IFN-gamma in T lymphocytes and natural killer cells, may contribute to the disease because cells from lpr mice are hypersensitive to IL-18 and express high levels of IL-18. To assess the contribution of IL-18 to the pathogenesis in the animal model, in vivo inhibition of IL-18 was attempted. Young lpr mice were vaccinated against autologous IL-18 by repeated administration of a cDNA coding for the murine IL-18 precursor. Vaccinated mice produced autoantibodies to murine IL-18 and exhibited a significant reduction in spontaneous lymphoproliferation and IFN-gamma production as well as less glomerulonephritis and renal damage. Moreover, mortality was significantly delayed in anti-IL-18-vaccinated mice. These studies support the concept that IL-18 plays a major role in the pathogenesis of the autoimmune syndrome of lpr mice and that a reduction in IL-18 activity could be a therapeutic strategy in autoimmune diseases.

Fig. 1.

Spontaneous IL-18 expression in MRL lpr mice. (Upper) Steady-state IL-18 mRNA expression was evaluated by semiquantitative RT-PCR in LN cells from young (4–7 wk) and adult (>12 wk) MRL/Mp +^lpr mice (open columns) and lpr mice (solid columns). Data are the mean ± SEM of results from 5–11 mice per group.**, P < 0.01 vs. +^lpr. (Lower) IL-18 protein was measured by ELISA in homogenates of different organs of 12-week-old MRL/Mp +^lpr mice (open columns) and lpr mice (solid columns). Data are the mean ± SEM of results from four mice per group within a single experiment representative of two performed. *, P < 0.05 vs. + ^lpr; **, P < 0.01 vs. + ^lpr.

Fig. 2.

Serum IL-18 and anti-IL-18 autoantibodies in vaccinated lpr mice. (a) IL-18 was measured by ELISA in sera of 8–10 single MRL/Mp lpr mice, 1–2 weeks after completion of the vaccination protocol with the pEF2 plasmid (control; open symbols) or the pEF2-IL-18 plasmid (vaccinated; solid symbols). Horizontal bars represent mean values. IL-18 in normal mouse serum was <200 pg/ml (data not shown). (b) Anti-IL-18 antibodies were evaluated as percentage IL-18 concentration in acid-treated vs. crude serum samples. Untreated sera (U1, U2, U3; open columns) are the preimmune sera of the same animals subjected to vaccination (V1, V2, V3; solid columns). Controls are serum from a pEF2-treated mouse (C) and normal mouse serum (NMS; striped column) spiked with 1 ng/ml recombinant murine IL-18. Antibody titers were as follows: NMS (spiked with 1 ng of IL-18), 0 units/ml; untreated controls (preimmune sera), 0, 0, and 22 units/ml; control with pEF2, 0 units/ml; vaccinated with pEF2-IL-18, 136, 282, and 452 units/ml. Data are from a single experiment representative of three performed. (c) IL-18-neutralizing activity of anti-IL-18 antibodies was assessed in an assay of IL-18-dependent proliferation of autoreactive T cells, in the presence of processed serum from vaccinated (pEF2-IL-18; V, solid columns) lpr mice or pEF2 control animals (C, open columns). Serum from untreated mice (diluted 1/100; U, striped column) was used as additional control. Data are expressed as percentage of IL-18-induced proliferation (in the absence of serum samples) and reported as the mean ± SEM of results from 3–14 animals in five experiments. *, P < 0.05 vs. IL-18-induced proliferation; **, P < 0.01 vs. IL-18-induced proliferation.

Fig. 3.

IL-18 vaccination inhibits IFN-γ production in lpr LN cells. LN cells from control mice (BALB/c, C57BL/6, MRL/Mp + ^lpr) and 16-week-old MRL/Mp lpr mice (untreated, treated with pEF2, vaccinated with pEF2-IL-18) were analyzed cytofluorimetrically after staining with phycoerythrin-labeled anti-CD4 antibodies followed by intracellular staining with a FITC-labeled anti-murine IFN-γ antibody. Results are from gated CD4⁺ LN cells from individual mice (representative of three to eight animals tested in each group) in a single experiment. Dark areas: control without antibody. Clear areas: with anti-IFN-γ antibody.

Fig. 4.

Anti-IL-18 vaccination inhibits proteinuria in lpr mice. Protein levels in urine of MRL/Mp lpr control mice (treated with pEF2; ○) and mice vaccinated against IL-18 (pEF2-IL-18; ) were assessed at weekly intervals starting at 10 weeks of age. Data are from 15–24 mice, tested in three experiments. The difference between control and vaccinated mice was highly significant (P < 0.0001).

Fig. 5.

IL-18 vaccination inhibits glomerulonephritis in lpr mice. Silver-based reticulin-stained (a and b) and IgG immunofluorescence-stained (c and d) kidney sections in 17-week-old MRL/Mp lpr control mice (vaccinated with pEF2; a and c) and IL-18 vaccinated mice (b and d). (Magnifications: ×100, a and b; ×200, c and d.)

Fig. 6.

Anti-IL-18 vaccination prolongs survival of lpr mice. Cumulative survival data of MRL/Mp lpr control mice (treated with pEF2; ○) and mice vaccinated against IL-18 (pEF2-IL-18; ) are reported (17–18 mice per group in four separate experiments). Survival of IL-18-vaccinated mice was significantly higher than that of control mice (P < 0.0299). Survival of untreated MRL/Mp lpr mice was fully superimposable to that of pEF2-treated mice (data not shown).