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Indole-3-carbinol (I3C), derived from cruciferous vegetables, alters estrogen metabolism. Since lupus is estrogen dependent, we reasoned that I3C might be effective in SLE. I3C significantly thwarted disease progression and prolonged survival in (NZBxNZW) F1 mice. Immunofluorescent and serologic analyses in treated animals indicated a transient blockade in B-cell maturation with increased immature B cells, decreased mature B cells, and a significant reduction of certain autoantibodies. Subsequently, a delay in T-cell maturation occurred in the treated group, manifested by significantly increased naive T cells, decreased mature and memory T cells, and decreased CD4:CD8 T-cell ratios. T cells from the I3C cohort, stimulated in vitro with various mitogens, exhibited enhanced responsiveness. Con A-stimulated T cells from I3C-treated mice produced Th1 cytokines, whereas those from control animals produced Th2 cytokines. Our studies suggest immunological mechanisms by which I3C ameliorates SLE in mice and provide a rationale for its use as an adjunctive therapy for human lupus.

We have identified monoclonal antibodies derived from MRL-lpr/lpr lupus-prone mice that produced nephritis after passive transfer to normal mice. Our present goal was to elucidate the structural and immunochemical features of nephritogenic Ig that facilitate immune deposition. For this purpose the antigen binding properties, capacity to form immune deposits, and nucleotide sequence of a genetically related autoantibody subgroup were compared. The prototype, H147 (an IgG encoded by 7183/81X VH gene), produced glomerular and tubular basement membrane, mesangial immune deposits, and proliferative glomerulonephritis after passive transfer to normal mice. For comparison three other 7183/81X encoded anti-DNA IgG (H257, H171, and H8a) were evaluated (predicted heavy chain aa homology >75%). H257 produced similar types of immune deposits as H147, and this was associated with nephritis; H8a produced predominantly mesangial deposits, whereas H171 did not produce significant deposits. Although their antigen binding profile to a panel of soluble autoantigens was variable, only H147 and H257 bound to both mesangial and aortic endothelial cell surfaces. V gene sequence analysis of the IgG suggests that individual residues, motifs, and conformations influence the autoantigen binding specificities that contributed to the observed differences in immune deposit formation.

The study of T cells in individuals with systemic lupus erythematosus has been limited because a specific marker for the disease has not been identified. To approach this issue, we isolated autoreactive T cell clones from lupus-prone MRL mice, a strain that develops an accelerated form of lupus. These CD4+ T cell clones grew spontaneously from unimmunized mice, and were maintained in culture by intermittent stimulation with syngeneic antigen presenting cells in the absence of exogenous antigen. One autoreactive T cell clone, termed ARTC-1, previously reported to have atypical MHC requirements for activation (both I-Ak and I-Ek were required) and to stimulate B cell proliferation and Ig production in vitro, was found to have an unrestricted pattern of lymphokine secretion. Following stimulation, it produced IL-4, IFN-gamma and IL-2. ARTC-1 induced B cell proliferation both by cell contact and through secretion of soluble lymphokines. B cell proliferation by cell-cell contact was MHC restricted in a manner analogous to ARTC-1 activation by APCs; the B cell response was inhibited by both anti-I-Ak and anti-I-Ek antibodies. The ARTC-1 B cell interaction was also found to result in the production of IgG autoantibodies. These observations suggest that cells such as ARTC-1, if unregulated, could lead to B cell stimulation and autoantibody production in vivo, in the absence of exogenous stimulation. Furthermore, IFN-gamma production by ARTC-1 could also result in enhanced class II expression, leading both to additional T-B cell interactions and to T cell interactions with endogenous cells capable of expressing class II antigens in other organs.

To distinguish the properties of anti-DNA antibodies in patients with lupus from those in normal individuals, we compared the ligand binding, idiotypic and charge properties of serum anti-DNA antibodies derived from: patients with active lupus; normal individuals; and among Ig eluted from the kidneys of two patients with active lupus nephritis (one with mesangial proliferation and the other with membranous nephropathy). The kidney eluate anti-DNA antibodies were the most cross-reactive; they cross-reacted with ssDNA, poly(GdC), poly(dT), poly(dG), poly(dC), ZDNA, SmRNP and the phospholipids cardiolipin and phosphatidyl serine. Lupus serum anti-DNA antibody cross-reacted with polynucleotides but not with phospholipids, whereas anti-DNA antibodies derived from normal serum reacted only with poly(dT). An anti-idiotype (anti-IdD; produced against serum anti-DNA antibodies from one patient) reacted with: anti-DNA antibodies in 8/9 lupus sera; antibodies in both kidney eluates; and anti-DNA antibodies from 5/7 normal sera. Anti-IdD did not react with Ig that did not bind to DNA. Isoelectric focusing of Ig showed that the charge of anti-DNA antibodies from lupus serum and normal serum were similar and unrestricted (pI 5.4-9.0); Ig in kidney eluates varied: membranous lupus pI 4.5-8.6; mesangial lupus pI 8.1-9.1. We conclude that idiotypically related anti-DNA antibodies in tissue lesions, lupus serum and normal serum from different individuals can be distinguished on the basis of their cross-reactive antigen-binding properties. Furthermore the cross-reactive properties of lupus auto-antibodies may influence their capacity to form glomerular immune deposits.

IgG antibodies, eluted from kidneys of rats with Heymann nephritis were separated into cationic and anionic fractions, labeled with 125I and 131I, respectively, mixed in equal amounts, and then injected in incremental doses into 10 rats. Glomerular antibody binding was highly correlated with blood concentration of antibody at 24 hr, however, significantly more cationic antibody bound to glomeruli than did anionic antibody at all blood levels studied. The differences were not due to greater antibody content and/or avidity of the cationic preparation, as measured by binding to isolated glomeruli in vitro. These studies demonstrate the influence of glomerular permselectivity and antibody charge on subepithelial antibody deposition.

Although it has been observed that DNA has a high binding affinity for the glomerular basement membrane (GBM) in vitro, glomerular localization of DNA has not been demonstrated in vivo. To evaluate this possibility, after injection of 125I ssDNA of varying molecular weight (mol. wt.) to normal mice, we measured glomerular levels of DNA in vivo. Following administration of 2 mg of 125I high mol. wt. purified single stranded(ss) DNA (2-6 kilobases; 0.7-2.0 x 10(6)D) to normal mice, DNA was not detected in glomeruli, despite measurable blood levels of DNA for 72 h. In contrast, after injection of 280 micrograms of low mol. wt. 125ssDNA (160-200 bases; mol. wt. = 5.3-6.6 x 10(4)D) to normal mice, glomerular localization was observed throughout the 24-h study period despite relatively low 125IssDNA blood levels. The results of these studies indicate that free circulating DNA can bind to sites within glomeruli in vivo, and that the size of DNA is crucial for this interaction. Since low mol. wt. DNA is present in the plasma of patients with active lupus, these findings raise the possibility that DNA may bind to glomeruli and serve as a planted antigen for in situ immune complex formation with circulating anti-DNA antibodies.