Joel Schiffenbauer

Polygenic control of susceptibility to murine systemic lupus erythematosus

Susceptibility to glomerulonephritis (GN) and anti-dsDNA autoantibody production was analyzed in crosses with a newly developed systemic lupus erythematosus-susceptible inbred strain, NZM/Aeg2410. The mode of inheritance and the number and location of systemic lupus erythematosus-associated susceptibility loci were analyzed by interval mapping in a backcross with C57BL/6. Three chromosomal intervals containing strong recessive GN susceptibility alleles were identified on chromosomes 1, 4, and 7, each containing several potentially interesting candidate genes. Heterozygosity at H-2 was also found to correlate strongly with GN susceptibility, consistent with previous findings in the NZB/NZW parental strain model. Logistic regression analysis indicated that each of these susceptibility alleles independently accounted for a component of GN susceptibility, and that susceptibility was inherited as a threshold genetic liability in this model system.

MULTIPLEX INHERITANCE OF COMPONENT PHENOTYPES IN A MURINE MODEL OF LUPUS

Abstract. We analyzed the linkage of GN and a wide spectrum of serological phenotypes associated with systemic lupus erythematosus in a (NZM2410 × C57BL/6)F2 cross. Some phenotypes, such as glomerulonephritis (GN) and anti-chromatin IgG antibody production, were more penetrant in females, but others, such as anti-dsDNA antibody production, did not show a gender bias. These results suggest that gender bias affects only a subset of SLE-component phenotypes, and that NZM2410 can be used to dissect the genetic basis of this phenomenon. Genome scanning linked six chromosomal intervals with the expression of one or more component phenotypes. These loci included two Sle loci previously identified in an (NZM2410 × B6)F1× NZM2410 backcross, loci identified by others in the NZB/W model. Our analysis also suggested two new intervals on chromosomes (Chrs.) 10 and 11. Detailed analysis of the segregation of different phenotypes within these intervals suggests that they encompass more than one susceptibility locus. This clustering has been a common finding in several murine polygenic traits. Each of NZM2410 susceptibility loci can be aligned with a specific genetic pathways contributing to SLE pathogenesis on the basis of the spectrum of component phenotypes expressed.

Acceleration of Autoimmunity by Organochlorine Pesticides in (NZB × NZW)F1 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disorder that affects women more frequently than men. In the (NZB × NZW)F1 mouse, a murine SLE model, the presence or absence of estrogen markedly influences the rate of progression of disease. Three organochlorine pesticides with estrogenic effects were administered chronically to ovariectomized female (NZB × NZW)F1 mice, and we measured the time to development of renal disease, the principal clinical manifestation of lupus in this model. Treatment with chlordecone, methoxychlor, or o,p′-dichlorodiphenyl-trichloroethane (o,p′-DDT) significantly decreased the time to onset of renal impairment, as did treatment with 17β-estradiol used as a positive control. In an expanded study of chlordecone, we found a dose-related early appearance of elevated anti–double-strand DNA autoantibody titers that corresponded with subsequent development of glomerulonephritis. Immunohistofluorescence confirmed early deposition of immune complexes in kidneys of mice treated with chlordecone. These observations are consistent with an effect of these organochlorine pesticides to accelerate the natural course of SLE in the (NZB × NZW)F1 mouse. Although we originally hypothesized that the effect on progression of autoimmunity was due to estrogenic properties of the pesticides, autoimmune effects and estrogenicity, assessed through measurement of uterine hypertrophy, were not well correlated. This may indicate that uterine hypertrophy is a poor indicator of comparative estrogenic effects of organochlorine pesticides on the immune system, or that the pesticides are influencing autoimmunity through a mode of action unrelated to their estrogenicity.

Genetic Dissection of Systemic Lupus Erythematosus Pathogenesis: Evidence for Functional Expression of Sle3/5 by Non-T Cells

On the non-autoimmune C57BL/6 (B6) background, the chromosome 7-derived lupus susceptibility loci Sle3 and Sle5 have been shown to mediate an elevated CD4:CD8 ratio with an increase in activated CD4+ T cells, decreased susceptibility to apoptosis, and a break in humoral tolerance. Development of subcongenic strains has subsequently shown that the elevated CD4:CD8 ratio is due to Sle3 but that both loci contribute to the development of autoantibodies. To elucidate the functional expression patterns of these loci, adoptive transfer experiments were conducted. All possible combinations of bone marrow reconstitution, including syngenic, were conducted between the congenic B6 and B6.Sle3/5 strains. It was found that the Sle3/5 locus was functionally expressed by bone marrow-derived cells, but not by host cells, and that the elevated CD4:CD8 phenotype could be reconstituted in radiation chimeras. Using Ly5-marked congenic strains and B6 host mice, additional experiments surprisingly demonstrated that the elevated CD4:CD8 ratio was neither an intrinsic property of the T cells nor of single positive thymocytes. Allotype-marked chimeras indicated that autoantibody production by B cells was also an extrinsic property, as shown by the fact that B cells without the Sle3/5 interval contributed to autoantibody production. These experiments strongly suggest that a gene within the B6.Sle3/5 interval was expressed by a bone marrow-derived, nonlymphocyte population in the thymus and periphery and was affecting T cell selection and/or survival.

The possible role of bacterial superantigens in the pathogenesis of autoimmune disorders

The ability of superantigens (SAgs) to activate the immune system suggests that they may play a role in the course of autoimmune disorders. Here, Joel Schiffenbauer and colleagues review evidence from animal models of autoimmunity, as well as human data that support this hypothesis, and propose a model for SAg involvement in autoimmune disorders.

Treatment of PL/J mice with the superantigen, staphylococcal enterotoxin B, prevents development of experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE), an antigen induced autoimmune disease, is mediated by V beta 8+ CD4+ T cells in PL/J mice after injection with the autoantigen, myelin basic protein (MBP). Recently the superantigen, staphylococcal enterotoxin B (SEB), has been shown to peripherally anergize and delete T cells in a V beta specific manner. By treatment of PL/J mice with SEB, we have been able to protect PL/J mice from the development of EAE. Two-color FACS analysis of the spleens of SEB treated mice showed depletion of V beta 8+ CD4+ T cells. Consistent with this observation, spleen cells of SEB treated mice that did not show signs of EAE could not be stimulated in vitro with SEB but did respond to SEA. Thus, V beta specific superantigens may prove to be a preventative therapy for autoimmune diseases mediated by V beta specific T lymphocytes.

Interleukin-2 gene deletion produces a robust reduction in susceptibility to experimental autoimmune encephalomyelitis in C57BL/6 mice

Dysregulation of interleukin-2 (IL-2), the prototypical T cell growth factor and immunoregulatory cytokine, may modify self-tolerance and predisposition to autoimmunity. The available literature suggested that IL-2 could be hypothesized to either propagate or inhibit the development autoimmune demyelinating disorders of the central nervous system such as multiple sclerosis. Thus, the present study sought to test these competing hypotheses by examining whether disrupting one or both IL-2 gene alleles would render mice more or less vulnerable to experimental autoimmune encephalomyelitis (EAE). Myelin oligodendrocyte glycoprotein was used to induce EAE in C57BL/6-IL-2(-/-) knockout, C57BL/6-IL-2(+/-) heterozygote and C57BL/6-IL-2(+/+) wild-type mice. All of the wild-type and heterozygote mice developed signs of EAE compared with only 23% of the IL-2 knockout mice. Histopathological examination of lumbar spinal cord sections confirmed that subpial perivascular inflammatory infiltrates found in wild-type and heterozygote mice were absent in the unaffected IL-2 knockout mice. These data demonstrate that vulnerability to EAE is markedly reduced in C57BL/6 mice lacking IL-2, and suggest that this cytokine may play a critical role in autoimmune processes of the central nervous system.

Intramolecular epitope spreading induced by staphylococcal enterotoxin superantigen reactivation of experimental allergic encephalomyelitis

The staphylococcal enterotoxin superantigens are among the most potent T cell stimulators known. They have been shown to alter the course of disease in experimental allergic encephalomyelitis, an animal model for multiple sclerosis (MS). We have previously demonstrated that two of the staphylococcal enterotoxins, SEA and SEB, are able to reactivate paralysis in PL/J mice which had been immunized with myelin basic protein (MBP) and resolved an initial episode of paralysis. In PL/J mice, Ac1-11 is the dominant encephalitogenic determinant of MBP. We hypothesized that superantigen reactivation of experimental allergic encephalomyelitis (EAE) may result in the spreading of T cell specificities for other epitopes of MBP. PL/J mice which had resolved an initial episode of EAE were treated with SEA and developed a second episode of paralysis. At the onset of symptoms, mice were sacrificed and splenocytes were stimulated in vitro with a panel of MBP peptides. EAE reactivation by SEA resulted in the spreading of T cell specificites to residues 100 to 120 of MBP. While intramolecular spreading did occur, spreading to other antigens did not, as evidenced by the lack of response to a proteolipid protein (PLP) peptide and heat shock protein 60 (hsp 60). To further characterize the epitope MBP 100-120, PL/J mice were immunized with MBP 100-120. No initial development of disease was observed. However, administration of SEA 2 weeks after MBP 100-120 immunization resulted in the onset of paralysis. In addition to a proliferative response to MBP 100-120, these mice also exhibited a proliferative response to the flanking MBP peptides 81-100 and 120-140. Thus, SEA is able to induce intramolecular epitope spreading in PL/J mice after reactivation of EAE.

Superantigens as virulence factors in autoimmunity and immunodeficiency diseases

Virulence factors are microbial products that are known to be harmful to the host and may assist in the pathogenesis of the micro-organism. Superantigens, including those produced by bacteria and viruses, clearly act as virulence factors. The clinical effects of superantigens can be not only acute but also chronic and complex. Recent evidence suggests that superantigens may play a central role in the pathogenesis of autoimmune and immunodeficiency disorders. It is our contention that superantigens, as environmental factors, can change a controllable disease into one that becomes relentless for susceptible individuals. To illustrate the detrimental effects of superantigens on disease outcome, modulation of experimental allergic encephalomyelitis by superantigen, as well as the potential role of superantigens in human immunodeficiency virus pathogenesis will be discussed. The information presented may provide valuable insight into the role of superantigens in autoimmunity and human immunodeficiency virus infection.

Background genes mediate the development of autoimmunity in (NZB × PLJ)F1 or (NZB × BIO.PL)F1 mice

The (NZB x NZW)F1 mouse develops a lupus-like disease including anti-double-stranded DNA antibodies and renal disease. It has been demonstrated that genes linked to the H-2 locus contributed by NZW correlate with development of this disease. We investigated whether mice with identical class II molecules but different background genes could contribute to autoimmunity when crossed with NZB. We report that two strains, PL/J and BIO.PL, when crossed to NZB, do not result in F1 with autoimmunity. Therefore, background genes present in NZW but not in PL/J or BIO.PL contribute to the development of disease.