Pristane-induced autoimmunity in germ-free mice.

Hypergammaglobulinemia and autoantibodies are reduced in pristane-treated specific pathogen-free mice vs. conventionally housed controls, consistent with the role of microbial stimulation in this model. To determine whether microbial stimulation is required, BALB/c mice housed under germ-free conditions were treated i.p. with sterile PBS or pristane and examined 6 months later. As in conventional mice, pristane-treated germ-free mice developed peritoneal granulomas and hypergammaglobulinemia with increased IgG2a/IgG1 ratios. LPS stimulation induced more IL-6, IL-12, and TNF-alpha, and anti-CD3 induced more IFN-gamma and IL-4 by peritoneal cells from pristane-treated mice vs. control. Anti-nRNP/Sm and -Su autoantibodies were found in 40% and 43%, respectively, of pristane-treated germ-free mice by immunoprecipitation. Thus, bacterial stimulation was not required for lupus autoantibodies, peritoneal granuloma formation, hypergammaglobulinemia, or cytokine overproduction. Although microbial stimulation acts synergistically with pristane, these results clearly indicate that pristane does not act merely by increasing exposure to microbial products such as LPS.

Induction of lupus autoantibodies by adjuvants.

Exposure to the hydrocarbon oil pristane induces lupus specific autoantibodies in non-autoimmune mice. We investigated whether the capacity to induce lupus-like autoimmunity is a unique property of pristane or is shared by other adjuvant oils. Seven groups of 3-month-old female BALB/cJ mice received a single intraperitoneal injection of pristane, squalene (used in the adjuvant MF59), incomplete Freund's adjuvant (IFA), three different medicinal mineral oils, or saline, respectively. Serum autoantibodies and peritoneal cytokine production were measured. In addition to pristane, the mineral oil Bayol F (IFA) and the endogenous hydrocarbon squalene both induced anti-nRNP/Sm and -Su autoantibodies (20% and 25% of mice, respectively). All of these hydrocarbons had prolonged effects on cytokine production by peritoneal APCs. However, high levels of IL-6, IL-12, and TNFalpha production 2-3 months after intraperitoneal injection appeared to be associated with the ability to induce lupus autoantibodies. The ability to induce lupus autoantibodies is shared by several hydrocarbons and is not unique to pristane. It correlates with stimulation of the production of IL-12 and other cytokines, suggesting a relationship with a hydrocarbon's adjuvanticity. The potential to induce autoimmunity may complicate the use of oil adjuvants in human and veterinary vaccines.

Immunoregulatory role of CD1d in the hydrocarbon oil-induced model of lupus nephritis.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that is accompanied by the emergence of autoreactive T cells and a reduction in regulatory T cells. Humans and mice with SLE have reduced numbers of CD1d-restricted NK T cells, suggesting a role for these cells in the regulation of SLE. In this study, we show that CD1d deficiency exacerbates lupus nephritis induced by the hydrocarbon oil pristane. This exacerbation in disease is associated with: 1) reduced TNF-alpha and IL-4 production by T cells, especially during the disease induction phase; and 2) expansion of marginal zone B cells. Strikingly, inoculation of pristane in wild-type mice resulted in reduced numbers and/or functions of NK T cells and CD1d-expressing dendritic cells. These findings suggest that CD1d may play an immunoregulatory role in the development of lupus in the pristane-induced model.

X-linked immunodeficient mice spontaneously produce lupus-related anti-RNA helicase A autoantibodies, but are resistant to pristane-induced lupus.

Murine lupus can occur spontaneously or be induced by hydrocarbons, such as pristane. Spontaneous disease in MRL and NZB/W F1 mice is suppressed by the xid (X-linked immunodeficiency) mutation, which greatly diminishes T cell-independent type 2 responses as well as the number of peritoneal B1 cells. The present study asked whether lupus induced by i.p. injection of pristane likewise is inhibited by the xid defect. Male CBA/N (xid) mice were refractory to the induction of autoantibodies by pristane, whereas 23% of pristane-treated male CBA/CaJ controls produced anti-nRNP/Sm, -Su and/or -OJ (isoleucyl tRNA synthetase) antibodies. Unexpectedly, 43% (12 of 28) of the xid mice spontaneously produced anti-nuclear antibodies that proved highly specific for the lupus antigen RNA helicase A (RHA). Strikingly, this specificity was absent in CBA/CaJ mice (none of 51). Moreover, pristane treatment suppressed the production of anti-RHA antibodies when administered prior to the onset of autoantibody production, but enhanced anti-RHA levels when given after the onset of autoantibody production, suggesting that pristane interferes with anti-RHA production at an early stage. Large amounts of IgG1 anti-RHA autoantibodies were detected in the sera of xid mice, whereas pristane-induced anti-nRNP/Sm and -Su autoantibodies were almost exclusively IgG2a. Cytokine production within the peritoneal cavity reflected the predominant isotypes: IL-12 and IFN-gamma predominated in pristane-treated mice, whereas IL-4 and IL-6 were more predominant in untreated xid mice. The spontaneous production of anti-RHA by xid mice and its suppression by pristane treatment at the level of autoantibody induction supports the idea that lupus autoantibodies may be generated through a variety of mechanisms.

Pivotal role of Stat4 and Stat6 in the pathogenesis of the lupus-like disease in the New Zealand mixed 2328 mice.

We have developed novel genetically lupus-prone (NZB x NZW)F(1)-derived congenic New Zealand mixed (NZM) 2328 lines, which are either Stat4- or Stat6-deficient. Our studies show that the deficiency of Stat4 and Stat6 significantly alters the phenotype of the lupus-like disease in NZM 2328 congenic mice. Specifically, Stat4-deficient NZM mice develop accelerated nephritis and increased mortality in the absence of high levels of autoantibodies including anti-dsDNA Abs, and in the presence of relatively reduced levels of IFN-gamma. In contrast, Stat6-deficient NZM mice display a significant reduction in incidence of kidney disease, with a dramatic increase in survival, despite the presence of high levels of anti-dsDNA Abs. The lack of correlation between levels of these autoantibodies and kidney disease raises the question of the direct cause-effect relationships between the presence of autoantibodies and kidney disease. Furthermore, these results also question the apparent equation of the effect of Stat deficiency with loss of secretion or response to particular cytokines.