Fas and Fas Ligand Gene

The identification of defects in Fas mapped to chromosome 19, which is involved in apoptosis, in lupus-prone MRL mice with the lpr phenotype, represented an important contribution to our understanding of the genetic basis of SLE.⁴ This mutation produces a massive enlargement of lymph nodes with the accumulation of a particular subset of T cells that are phenotypically TCRαβ⁺, CD4⁻, CD8⁻, but express the B220 molecule characteristic of B cells.³ Notably, the gld (generalized lymphoproliferative disease) mutation, discovered in a colony of the C3H/HeJ strain, induces marked lymphadenopathy phenotypically indistinguishable from that induced by the lpr mutation.¹³ As a matter of fact, gld was identified as a mutation of the gene encoding the Fas ligand (FasL), present in chromosome 1.¹⁴ These Fas^lpr and Fasl^gld mutations not only accelerate the progression of autoimmune disease in lupus-prone MRL mice, but also induce the production of a broad spectrum of autoantibodies in various strains of mice, including those not predisposed to SLE.^2,15,16 Fas is highly expressed in activated B and T cells, while the expression of FasL is limited to activated T cells.³ However, the Fas apoptosis pathway does not appear to be essential for negative selection during T and B cell development in thymus and bone marrow, respectively.^17,18 Therefore, it has been speculated that the abnormal regulation of the Fas apoptotic pathway could result in prevention of antigen-induced apoptotic death of autoreactive lymphocytes in the periphery, thereby promoting the development of lupus-like autoimmune responses. However, it should be stressed that the mutation of Fas or FasL alone is not sufficient to induce severe autoimmune disease in mice that are not predisposed to SLE,^13,15,16 underlining the importance of other still undefined lupus susceptibility background genes in the development of full-blown SLE.

Although Fas mutations have been identified in children with a rare autoimmune lymphoproliferative syndrome (ALPS),^19,20 this was not really the case in SLE patients.²¹ Nevertheless, studies in mice bearing the lpr or gld mutation clearly indicate the importance of the genes regulating apoptosis in the development of SLE. It can be speculated that one of the defects in lupus-prone mice may be the failure to efficiently eliminate autoreactive B cells upon interaction with autoantigens. In fact, the resistance of mature B cells to B-cell receptor (BCR)-mediated apoptosis has been reported in lupus-prone (NZB × NZW)F1 mice.²² This concept is consistent with the findings that transgenic overexpression of the antiapoptotic proto-oncogene bcl-2 in B cells led to spontaneous development of an SLE-like autoimmune syndrome in certain strains of mice,²³ and that the constitutive expression of the bcl-2 gene is able to counteract the apoptotic death of autoreactive B cells upon interaction with autoantigens in the periphery.²⁴

Yaa Mutation

In contrast to the accelerated development of SLE in (NZB × NZW)F1 female mice, male BXSB mice develop disease much more rapidly than their female counterparts.² This striking sexual dimorphism is not hormonally mediated, but results from a mutant gene, Yaa, present in the Y chromosome of the BXSB strain.^25–27 The contribution of the Yaa mutation to lupus susceptibility remains limited without other background genes, since nonautoimmune strains, such as CBA/J and B6, were largely unaffected by the Yaa mutation. Notably, when B6.Yaa consomic males are mated with NZW females, which are phenotypically normal but have a genetic potential to develop SLE, F1 hybrid males bearing the Yaa mutation develop typical SLE.²⁶ In addition, studies in B6 or C57BL/10 (B10) mice carrying different lupus susceptibility loci derived from either NZB, NZW or BXSB mice have shown that the combination of a single lupus susceptibility locus with Yaa can be sufficient to induce the development of lupus-like autoimmune syndrome, although the severity of the disease was variable, depending on the individual lupus susceptibility locus studied.^28–30 These results indicate that the Yaa mutation by itself is unable to promote SLE in mice which are not predisposed to autoimmune diseases, but in combination with autosomal susceptibility alleles present in different lupus-prone strains, it can induce or accelerate the development of SLE.

It is clear that the molecular characterization of Yaa would give valuable information on the general mechanisms implicated in the development of SLE. Unfortunately, it is impossible to map this locus by conventional genetics because of the lack of homologous recombination of the Y chromosome. In an attempt to identify the cell types which express the Yaa mutation, we have produced Yaa plus non-Yaa double-bone-marrow chimeric mice, and analyzed the origin of autoantibodies produced in these mice. This analysis revealed that only B cells of Yaa origin participated in the production of IgG anti-DNA autoantibodies, and that they maintained this production even after selective depletion of T cells of Yaa origin.^31,32 Thus, the Yaa abnormality is functionally expressed in B cells, but not in T cells. Based on this finding, it has been hypothesized that the action of Yaa may be to decrease the threshold for BCR-dependent stimulation, thereby promoting the activation of autoreactive B cells.³³ This hypothesis is in agreement with the selective autoimmune enhancing effect of the Yaa mutation: the Yaa effect appears to be essential for the promotion of autoimmune responses in mice having only a limited activity of T-helper cells specific for a given autoantigen.³⁴ It has been reported that lupus-like autoimmunity could be detected in genetically engineered mice lacking or overexpressing molecules implicated in the regulation of BCR signaling, which indicates that the deregulation of BCR signaling may be a critical element in the triggering and activation of potentially autoreactive B cells. According to this model, the Yaa mutation would have a direct enhancing effect on BCR signaling and thereby trigger the cascade of events initiating SLE.

A unique cellular abnormality associated with the Yaa mutation is monocytosis.³⁵ At 8 months of age, monocytes reached a frequency of more than 50% of peripheral blood mononuclear cells in BXSB Yaa male mice. The development of monocytosis was closely associated with the progression of SLE, since monocytosis was observed in (NZB × B6.Yaa)F1 male mice developing SLE, but not in B6.Yaa males, which fail to develop a lupus-like autoimmune syndrome.³⁶ Furthermore, recent analysis of B6 × (NZB × B6.Yaa)F1 back-cross males bearing the Yaa mutation revealed a remarkable correlation of monocytosis with autoantibody production and subsequent development of lupus nephritis,³⁷ indicating that monocytosis is a useful and predictive marker for severe SLE. Significantly, Yaa-mediated monocytosis resulted in selective expansion of a monocyte subset expressing the CD11c dendritic cell marker,³⁶ which is therefore considered to be a potential source of tissue-resident dendritic cells. Thus, the association of monocytosis with the development of SLE could be explained by a possible expansion of dendritic cells, which would further promote the production of pathogenic autoantibodies. In addition, given the considerable role of infiltrating macrophages in the progression of glomerular lesions, and given the implication of stimulatory IgG Fc receptor (FcγR) in murine lupus nephritis,³⁸ monocytosis could promote glomerular inflammation and injury through increased secretion of proinflammatory cytokines, reactive oxygen species, and matrix-specific proteases, possibly as a result of IC-mediated, FcγR-dependent activation of infiltrating macrophages. Although the precise cellular and molecular basis of the Yaa gene-linked development of monocytosis has not been defined, the analysis of Yaa plus non-Yaa mixed-bone-marrow chimeras showed no overrepresentation of monocytes of Yaa origin over those of non-Yaa origin, indicating that the development of monocytosis is not due to an intrinsic abnormality in the growth potential of monocyte lineage cells from Yaa mice.³⁶ Rather, Yaa-mediated monocytosis may result from an excessive production of monocyte-specific growth factor(s)—for example, by activated macrophages—possibly due to hyper-responsiveness of FcγR to autoimmune IC that arise during the course of the disease.

Association of H2^d/z Heterozygosity with Murine SLE

Extensive studies in New Zealand mice have demonstrated a strong association of H2^d/z heterozygosity (vs. H2^d/d or H2^z/z) with the development of SLE, indicating a co-dominant contribution from each strain, i.e., H2^d from NZB and H2^z from NZW.^39,40 However, it is still unknown how this H2 heterozygosity mechanistically contributes to murine SLE. It has been proposed that mixed haplotype class-II molecules produced by heterozygous pairing of an α-chain from one haplotype with a β-chain from the other haplotype might play a critical role in the development of SLE. However, the lack of disease enhancement by an Ab^z transgene introduced into H2^d homozygous (NZB × NZW.H2^d)F1 mice⁴¹ and by E^z or A^z transgene in (B6 × NZB)F1 × NZB back-cross mice^42,43 argue against this possibility. Significantly, a comparative serologic analysis of two different nephritogenic anti-DNA and anti-gp70 autoantibody productions in (NZB × NZW)F1 × NZW and (NZB × NZW)F1 × NZB back-cross mice revealed that in the F1 × NZW back-cross, H2^d/z (compared with H2^z/z) was associated preferentially with the production of anti-gp70 rather than anti-DNA autoantibodies, whereas the opposite influence was noted for H2^d/z (compared with H2^d/d) in the F1 × NZB back-cross.⁴⁴ These results suggest that enhancement of disease by H2^d/z heterozygosity is related to separate contributions from H2^d and H2^z, thus providing one explanation as to why H2^d/z heterozygosity is required for full expression of disease in (NZB × NZW)F1 mice.

Association of H2^b with Murine SLE

Another contribution of the MHC to the regulation of murine SLE, which is different from that seen in (NZB × NZW)F1 hybrid mice, has been well documented in BXSB and (NZB × BXSB)F1 mice, in which lupus susceptibility was more closely linked with the H2^b haplotype than with the H2^d and H2^k haplotypes.^45–47 However, this MHC effect was limited, as it was markedly influenced by other factors in the genetic background of individual lupus-prone mice. In the context of the BXSB background, in which the development of SLE is dependent on the Yaa mutation, the H2^d or H2^k haplotype almost completely prevented the development of autoimmune responses occurring in H2^b-bearing conventional BXSB mice. In contrast, (NZB × BXSB)F1 female hybrids homozygous for H2^d still developed typical SLE, although its development was markedly delayed as compared with mice homozygous for H2^b. This indicates that the genetic complementation of NZB and BXSB genomes allows the development of spontaneous autoimmune responses in the context of H2^d, even without the Yaa mutation. More strikingly, the Yaa mutation dramatically accelerated the progression of SLE in (NZB × BXSB)F1 H2^d mice to an extent comparable with that observed in F1 H2^b mice. Thus, no more MHC association was evident in these F1 hybrid males when they expressed the Yaa gene. Notably, similar results were observed in mice bearing the Fas^lpr mutation; the production of autoantibodies in B6 mice bearing the Fas^lpr mutation was highly dependent on H2^b,⁴⁸ while lupus-like disease was developed equally well in both H2^k and H2^b lupus-prone MRL-Fas^lpr mice.⁴⁹ All these experiments indicate that the MHC class-II genes likely provide at least some of the genetic requirements for the predisposition to SLE, and that conventional MHC class-II molecules are sufficient in mice with an appropriate autoimmune genetic background. Most significantly, the MHC-linked autoimmune promoting effect is no longer apparent in mice which are highly predisposed to SLE, for example by powerful autoimmune accelerating genes, such as Yaa or Fas^lpr.

The autoimmune inhibitory effect of the H2^d and H2^k haplotypes, as compared with H2^b, can be related at least in part to the expression of I-E molecules, since mice bearing the H2^b haplotype do not express I-E because of the deletion of the promoter region of the Ea gene. The development of SLE was almost completely prevented in BXSB (H2^b) mice expressing two copies of an Ea transgene encoding I-E α-chains, which is the case of H2^d and H2^k BXSB mice. In addition, the expression of two functional Ea (one transgenic and the other endogenous) genes in either H2^d/b (NZB × BXSB)F1 or H2^k/b (MRL × BXSB)F1 mice provided protection from SLE at levels comparable to those conferred by the H2^d/d or H2^k/k haplotype.⁴⁷ These results suggest that the reduced susceptibility associated with the I-E⁺ H2^d and H2^k haplotypes (vs. the I-E⁻ H2^b haplotype) is largely, if not exclusively, contributed by the Ea gene. This idea is further supported by the recent demonstration that (NZB × NZW)F1 mice expressing I-A^d but lacking I-E molecules developed SLE as severe as that of wild-type H2^d/z heterozygous (NZB × NZW)F1 mice.⁵⁰ However, it should be stressed that since H2^d/z (NZB × NZW)F1 mice express I-E, the unique autoimmune-promoting effect conferred by the H2^d/z heterozygosity apparently overcomes the protective effect of I-E in this genetic background, as in the case of (NZB × BXSB)F1 mice expressing the Yaa mutation and MRL mice bearing the Fas^lpr mutation.

The precise mechanism(s) responsible for the Ea gene-mediated protection from SLE remains to be elucidated. Studies of Ea transgenic and nontransgenic mixed-bone-marrow chimeras revealed that these chimeric mice developed a typical lupus-like autoimmune syndrome, in which anti-DNA autoantibody production was preferentially induced by nontransgenic B cells.^51,52 These results suggested that B cells are the major target of Ea-mediated suppression of autoimmune responses, and that Ea gene expression may interfere with an efficient interaction between autoreactive T and B cells, possibly by modulating the presentation of pathogenic self-peptides by MHC class II molecules. This could occur as a result of increased formation of peptides derived from the I-E α-chains. In fact, one of the peptides, Eα52-68 peptide, has been identified as one of the major self-peptides presented by I-A molecules.^53,54 The hypothesis that the protective action of the Ea gene is mediated through binding of its degradation products by MHC class-II molecules, thereby competing with potentially pathogenic self-peptides, was further supported by the demonstration that the protective effect of the Ea transgene was highly dependent on the host H2 haplotype.⁵⁵ This idea was consistent with recent in vitro demonstration that the capacity of Ea transgenic B cells to activate T-helper cells by presenting I-A—restricted peptides of foreign antigens was substantially diminished, compared with that of nontransgenic B cells.⁵⁶

The MHC class-II Ea gene apparently contributes to the reduced susceptibility to SLE by suppressing autoimmune responses in mice, but its protective effect is influenced by the host H2 haplotype. However, the Ea gene is not the only gene encoded within the MHC region that determines the genetic susceptibility to murine SLE, and the MHC region likely encodes additional lupus-associated genes, which can potentiate or suppress the development of SLE by acting at various levels of the disease process. Studies have claimed the presence of a lupus suppressor gene, Sles1 (SLE suppressor 1), within the H2 region of the NZW strain,⁵⁷ although it remains to be confirmed whether the observed suppressive effect was due to this novel suppressor gene and not to MHC region polymorphisms. In addition, the Tnfa allele of the NZW strain, which is associated with down-regulated TNF-α synthesis, has been previously proposed as a candidate gene that may underlie the H2^z contribution to lupus in (NZB × NZW)F1 mice.^58,59