Autoantibodies reactive with the snRNP complex are often found in lupus patients and those reactive with the Sm proteins are an important diagnostic marker for the disease (1
). Recent studies have indicated a role for IFN-α in the generation of the anti-snRNP antibody response (8
). A reduced incidence of anti-Sm/RNP antibodies is observed in MRL.lpr
mice deficient in TLR7 (6
). The RNA moiety associated with the snRNP complex can activate TLR7 and induce type I IFN production (9
). These studies implicate RNA within the snRNP particle engages TLR7 segregated in endosomal compartment resulting in the production of IFN-α, which in turn plays a role in the production of anti-snRNP antibodies. Recent findings with respect to TLR9, which may be pivotal in production of anti-dsDNA antibodies, suggest that formation of autophagosome brings together TLR9 and B cell receptor bound ligand to the endosomal location (24
). Whether a similar mechanism is operative for TLR7 is not known. The RNA moiety within the snRNP particle can gain access to TLR7 through the uptake and internalization of immune complexes containing snRNP particle and induce type I IFN production (10
). However, it should be noted that despite the presence of high type I IFN, not all lupus patients develop anti-Sm/RNP autoantibodies. Thus, additional genetic factors contribute towards the generation anti-snRNP autoantibodies. Our results from the present investigation add support to this hypothesis.
In this study, we have shown genetic complementation in generation of the anti-snRNP antibody response. This conclusion is drawn from the observation that NZM/NOD F1 mice have very high titers and high incidence of anti-snRNP antibodies. The parental lines, NOD mice have low titers and low incidence of anti-snRNP antibodies, whereas NZM2328 mice fail to produce such antibodies. It is of significant interest that NZM2328 strain is high in type 1 IFN expression, NOD is low in type 1 IFN expression and the F1 mice have intermediate level of expression. Although there is a general correlation between type 1 IFN expression and anti-snRNP antibody levels, the type 1 IFN expression in F1 mice does not exceed that in NZM2328. The F1 mice also have much higher ANA and anti-dsDNA antibody titers than the NZM2328 mice. These data indicate type I IFN responses might be better regulated by genetic predisposition than just by levels of anti-snRNP and anti-dsDNA autoantibodies. These observations provide new insights in the relationship of IFN expression and anti-snRNP antibody production. At 23 weeks of age, the mean serum IgM levels between the F1 mice and NZM2328 mice were not significantly different and only a modest elevation in mean serum IgG level (1.52 times, p=0.014) was seen in the F1 mice in comparison with the NZM2328 mice. In contrast, the titers of ANA or anti-dsDNA antibodies in F1 mice were a log scale higher than the parental strains. In addition, in younger mice (10-12 weeks of age) we did not see any evidence for splenomegaly and the total spleen cell numbers in the F1 and NZM2328 mice were very similar (data not shown). These data suggest that augmented autoantibody responses in F1 mice are not a result of generalized polyclonal activation of B cells. However, whether B cells from F1 mice respond better to signals from helper T cells needs to be determined.
A similar observation of genetic complementation has been reported in the SWR × SJL F1 hybrid mice (26
). Sera from parental strains were negative for the presence of anti-snRNP autoantibodies. However, by 40 weeks of age, 70% of the F1 mice were positive for anti-snRNP antibodies. Genetic complementation between the SWR and SJL strains of mice was considered as a mechanism for the observed phenotype. The role of type I IFN in this model was not investigated.
Genes contributing towards the development of anti-snRNP antibodies have not been clearly defined. From the aforementioned discussion, TLR7 is obviously one of the contenders. Studies from BXSB mice have demonstrated that increased expression of TLR7 due to gene duplication contributes towards the generation of antibodies reactive with ribonucleoproteins (5
). In our study, comparison of TLR7 expression between the F1 mice and parental strains did not show any differences. Similarly we did not see any differences in TLR9 expression between these strains (data not shown). These findings are important as a recent study failed to demonstrate any significant association between relative TLR7 gene copy number and autoantibody profiles in lupus patients (27
). Also, there was considerable variation in the relative gene copy number of TLR7 in both healthy controls and SLE patients, without any significant increase in the patients. Thus, expression level of TLR7 does not seem to be a major determining factor for generation of anti-snRNP autoantibodies in our model system. However, in the NZM/NOD F1 mice the level of autoantibodies correlated with the gene expression levels type I IFN responsive genes PRKR and M×1. These data suggest that both TLR7 and TLR9 must contribute towards the amplification of type I IFN responses in the F1 mice.
Genetic manipulation of NOD mice was shown to induce production of anti-snRNP antibodies (16
). Presence of B10 mouse derived Idd9.3
locus on the NOD background (NOD.B10Idd9.3
) was sufficient to increase the penetrance of anti-Sm antibodies (17
). It was proposed that CD137 might be one of the candidate genes playing an important role in this autoantibody production through modulation of costimulatory signals. However, analysis of data from different crosses involving the NOD congenic mice shows multigenic regulation of anti-snRNP autoantibody responses. Our study also supports the notion of polygenic control of autoantibody production as heterozygosity with NZM genes was sufficient to induce a very high incidence of anti-snRNP antibodies. Whether in addition to genetic complementation, suppression of resistance genes plays a role in the F1 mice remains to be determined. The increased IL-6 transcript levels in F1 mice suggest that genes within this pathway might also be important for anti-snRNP, ANA and anti-dsDNA autoantibody generation. Clearly, the NZM/NOD F1 mouse model will be a valuable tool to identify these genes as well as to investigate the pathogenic potential of anti-Sm/RNP autoantibodies in lupus-nephritis.