Neutrophil apoptosis in autoimmune Fas-defective MRL lpr/lpr mice

The apoptosis-defective lpr (fas) mutation in MRL mice causes the early onset of a lupus-like autoimmune disease with concomitant inflammation. In order to analyse the consequences of the impaired Fas-dependent apoptosis on inflammation, the susceptibility to apoptosis of polymorphonuclear leukocytes (PMN), obtained from MRL lpr/lpr mice, has been studied. Peritoneal PMN from lpr/lpr and control (+/+) mice were recruited with a mild inflammatory stimulus. The number of cells collected from the peritoneal cavity of young lpr/lpr mice was comparable to that obtained from age-matched control mice, indicating that PMN homeostasis is maintained regardless of the loss-of-function Fas mutation. Recruited neutrophils were exposed in culture to apoptosis-inducing stimuli. Treatment with agonist anti-Fas antibody increased apoptosis of +/+ PMN, but did not affect lpr/lpr PMN which do not express Fas on their surface. However, lpr/lpr PMN could undergo both spontaneous and stimulus-induced apoptosis in a fashion comparable to or higher than that of control +/+ mice. Analysis of mRNA expression revealed that lpr/lpr PMN have reduced expression of IL-18, whereas IL-1beta, IFNgamma, caspase 1 and caspase 3 are expressed at levels comparable to those of +/+ cells. However, caspase-3-like activity was higher in PMN from lpr/lpr mice than in +/+ cells, and correlated with enhanced apoptosis. It could be concluded that in young, uncompromised lpr/lpr mice, PMN homeostasis is still fully regulated through the involvement of Fas-independent, compensatory, apoptotic mechanisms. This could include an increased participation of caspase 3 in the apoptotic pathway, consequent to enhanced activation of the enzyme and to the decreased production of IL-18, which acts as a competitive caspase 3 substrate.     

Lymphocytes from autoimmune MRL lpr/lpr mice are hyperresponsive to IL-18 and overexpress the IL-18 receptor accessory chain

MRL lpr/lpr mice spontaneously develop a severe autoimmune lupus syndrome characterized by strong autoantibody production and massive lymphoproliferation, in which IFN-gamma plays a major pathogenic effect. The role of the IFN-gamma-inducing cytokine IL-18 in the autoimmune syndrome of lpr/lpr mice has been investigated. In response to IL-18, lymph node cells of lpr/lpr mice produce significant amounts of IFN-gamma and proliferate more potently as compared with cells from +/+ mice. Cells likely responsible for such hyperresponsiveness to IL-18 include NK cells and the CD4(+)/CD8(+) self-reactive T lymphocytes characteristically present in lymph nodes of lpr/lpr mice. Analysis of the expression of IL-18R complex revealed that mRNA for the IL-18R alpha-chain is constitutively expressed at similar level both in +/+ and lpr/lpr lymphocytes. In contrast, the expression of the accessory receptor chain IL-18R beta is low in unstimulated +/+ cells but significantly high in lpr/lpr cells. Thus, the abnormally high expression of the IL-18R chain IL-18R beta could be one of the causes of the hyperresponsiveness of lpr/lpr cells to IL-18 at the basis of consequent enhancement of IFN-gamma production and development of IFN-gamma-dependent autoimmune pathology.     

IL-12 drives IFN-gamma-dependent autoimmune kidney disease in MRL-Fas(lpr) mice

IL-12 is secreted by kidney tubular epithelial cells in autoimmune MRL-Fas(lpr) mice before renal injury and increases with advancing disease. Because IL-12 is a potent inducer of IFN-gamma, the purpose of this study was to determine whether local provision of IL-12 elicits IFN-gamma-secreting T cells within the kidney, which, in turn, incites injury in MRL-Fas(lpr) mice. We used an ex vivo retroviral gene transfer strategy to construct IL-12-secreting MRL-Fas(lpr) tubular epithelial cells (IL-12 "carrier cells"), which were implanted under the kidney capsule of MRL-Fas(lpr) mice before renal disease for a sustained period (28 days). IL-12 "carrier cells" generated intrarenal and systemic IL-12. IL-12 fostered a marked, well-demarcated accumulation of CD4, CD8, and double negative (CD4-CD8- B220+) T cells adjacent to the implant site. We detected more IFN-gamma-producing T cells (CD4 > CD8 > CD4-CD8- B220+) at 28 days (73 +/- 14%) as compared with 7 days (20 +/- 8%) after implanting the IL-12 "carrier cells;" the majority of these cells were proliferating (60-70%). By comparison, an increase in systemic IL-12 resulted in a diffuse acceleration of pathology in the contralateral (unimplanted) kidney. IFN-gamma was required for IL-12-incited renal injury, because IL-12 "carrier cells" failed to elicit injury in MRL-Fas(lpr) kidneys genetically deficient in IFN-gamma receptors. Furthermore, IFN-gamma "carrier cells" elicited kidney injury in wild-type MRL-Fas(lpr) mice. Taken together, IL-12 elicits autoimmune injury by fostering the accumulation of IFN-gamma-secreting CD4, CD8, and CD4-CD8- B220+ T cells within the kidney, which, in turn, promote a cascade of events culminating in autoimmune kidney disease in MRL-Fas(lpr) mice.     

Anti-CD4 monoclonal antibody therapy suppresses autoimmune disease in MRL/Mp-lpr/lpr mice.

MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop systemic autoimmune disease, characterized by vasculitis, lymphadenopathy, glomerulonephritis, and autoantibody formation. The target organ inflammatory lesions are composed largely of CD4+ "helper" T cells, while the massively enlarged lymph nodes are composed primarily of CD3+ CD4- CD8- TCR alpha/beta + "double-negative" T cells. In this study we investigated the effect of treatment of MRL/lpr mice with anti-CD4 monoclonal antibody (mAb); control groups consisted of animals treated with normal saline or rat immunoglobulin (Ig). Anti-CD4 mAb treatment, which was started at 4 weeks and continued through 20 weeks of age, resulted in a dramatic reduction of both the frequency and severity of the autoimmune disease, as demonstrated histologically and serologically. Anti-CD4 mAb therapy markedly reduced the frequency of glomerulonephritis and eliminated vasculitis of the major renal arterial branches. Glomerulonephritis was detected in 9 of 9 saline-treated, 9 of 9 rat Ig-treated, but in only 1 of 9 anti-CD4 mAb-treated mice; vasculitis was detected in 6 of 9 saline-treated, 7 of 9 rat Ig-treated, but in none of 9 anti-CD4 mAb-treated mice. The frequency of antinuclear antibodies, titer of anti-dsDNA antibodies, and total Ig levels were all significantly reduced by anti-CD4 mAb therapy. These data support the hypothesis that CD4+ T cells play a central role in the disease process in this autoimmune strain.     

B cell depletion with anti-CD79 mAbs ameliorates autoimmune disease in MRL/lpr mice

MRL/lpr mice develop a spontaneous systemic lupus erythematosus-like autoimmune syndrome due to a dysfunctional Fas receptor, with contributions from other less well-defined genetic loci. The removal of B cells by genetic manipulation not only prevents autoantibody formation, but it also results in substantially reduced T cell activation and kidney inflammation. To determine whether B cell depletion by administration of Abs is effective in lupus mice with an intact immune system and established disease, we screened several B cell-specific mAbs and found that a combination of anti-CD79alpha and anti-CD79beta Abs was most effective at depleting B cells in vivo. Anti-CD79 therapy started at 4-5 mo of age in MRL/lpr mice significantly decreased B cells (B220(+)CD19(+)) in peripheral blood, bone marrow, and spleens. Treated mice also had a significant increase in the number of both double-negative T cells and naive CD4(+) T cells, and a decreased relative abundance of CD4(+) memory cells. Serum anti-chromatin IgG levels were significantly decreased compared with controls, whereas serum anti-dsDNA IgG, total IgG, or total IgM were unaffected. Overall, survival was improved with lower mean skin scores and significantly fewer focal inflammatory infiltrates in submandibular salivary glands and kidneys. Anti-CD79 mAbs show promise as a potential treatment for systemic lupus erythematosus and as a model for B cell depletion in vivo.     

Combined treatment of autoimmune MRL/Mp-lpr/lpr mice with cholera toxin plus irradiation. Combined treatment of autoimmune MRL/l mice

MRL/Mp-lpr/lpr (MRL/1) mice spontaneously develop autoimmune diseases like systemic lupus erythematosus (SLE) from 2 months of age, accompanied by massive lymphadenopathy. Such mice of 2 months of age were treated with 1 microgram cholera toxin (CT) every 7 days and/or with 400 rad of one-shot 60Co irradiation. CT treatment alone markedly improved nephritis as evaluated by proteinuria and moderately suppressed lymphadenopathy and anti-DNA antibody production, while irradiation alone prominently improved lymphadenopathy but showed little effect on both nephritis and anti-DNA antibody production. On the other hand, when mice were treated with the combination of CT plus irradiation, autoimmune nephritis as well as anti-DNA production and lymphadenopathy were almost completely inhibited. Taken together, each agent exerts the improvement effect at the different points from each other in an abnormal immunological circuit displayed in MRL/1 mice. This kind of combined treatment may be applicable to the clinical use for autoimmune diseases.     

Comparison of antigen-specific T cell responses in autoimmune MRL/Mp-lpr/lpr and MRL/Mp-+/+ mice

The MRL-1 mouse develops severe autoimmune disease characterized by high titers of autoantibodies at an early age (3 to 5 mo). The congeneic MRL-n mouse, which differs only in the lymphoproliferative (lpr) gene, exhibits no such pathologic or serologic abnormalities at the same age. We examined antigen-specific T cell responses in the MRL-1 mouse and compared them to age- and sex-matched MRL-n controls. We found broad defects in these responses in the MRL-1 mouse; an inability to generate primary allospecific and hapten-specific cytolytic T lymphocytes (CTL), secondary hapten- and virus-specific CTL, as well as a deficient proliferative response to hapten and natural antigens and a weak delayed-type hypersensitivity response were demonstrated. Our data furthermore suggest a lack of interleukin 2 (IL 2) acceptor sites in the proliferating T cell, while suggesting no such lack on CTL precursors. In fact, the deficient CTL responses in MRL-1 mice can be restored to levels seen in MRL-n by the in vitro addition of IL 2. The implications of these findings and the possible explanations for the relative deficit in helper function in the MRL-1 mouse are discussed.     

Low-dose targeted complement inhibition protects against renal disease and other manifestations of autoimmune disease in MRL/lpr mice

Complement appears to play a dual role in the progression of systemic lupus erythematosus, serving a beneficial role in enhancing immune complex clearance, while serving a pathogenic role in inducing local inflammation. To investigate these different roles of complement in a therapeutic setting, MRL/lpr mice were treated with the targeted murine C3 complement inhibitor, CR2-Crry, from 16 to 24 wk of age (after the development of proteinuria). The targeting moiety, CR2, binds to C3 breakdown products deposited at sites of complement activation and has the potential to provide complement inhibition locally without causing systemic inhibition. Administration of CR2-Crry i.v., at a dose of 0.25 mg once a week, was associated with a significant survival benefit, improved kidney function, and a significant reduction in glomerulonephritis and renal vasculitis. The presence of skin lesions and lung bronchiolar and vascular inflammation was also dramatically reduced by CR2-Crry treatment. CR2-Crry treatment also resulted in a significant reduction in autoantibody production, as measured by anti-dsDNA Ab levels, and did not cause an increase in circulating immune complex levels. These effects on autoimmunity and circulating immune complexes represent significant potential advantages over the use of Crry-Ig in MRL/lpr mice, a systemic counterpart of CR2-Crry. CR2-Crry localized preferentially to the kidneys in 16-wk MRL/lpr mice with a kidney-localized half-life of approximately 24 h. Thus, targeted complement inhibition at the C3 level is an effective treatment in murine lupus, even beginning after onset of disease.     

Unique surface phenotype of T cells in lymphoproliferative autoimmune MRL/Mp-lpr/lpr mice

MRL/l mice, which carry the mutant gene lpr, develop massive T cell lymphoproliferation and an autoimmune syndrome. The surface antigen profile of MRL/l lymphocytes was analyzed with rat monoclonal antibodies. They included YE1/9.9, anti-transferrin receptor; YE1/7.1, which reacts with a population of proliferating immature T cells; and YE1/19.1, a newly identified antibody reactive with a surface antigen on EL-4 cells, which consists of two disulfide-bonded polypeptide chains, each of 115,000 m.w. Flow cytometric analysis of lymphocytes from the enlarged lymph nodes of MRL/1 mice showed the majority of them to be Lyt-1+, YE1/7.1+, YE1/19.1+, YE1/9.9-. In contrast, YE1/7.1 and YE1/19.1 antibodies did not significantly react with lymphocytes from the congenic MRL/n mice, which lack the lpr gene and do not develop lymphoproliferation. Lymphocytes from younger MRL/l mice, which have almost normal size lymph nodes, were also YE1/7.1-, YE1/19.1-. A small proportion of mitogen-stimulated MRL/n lymph node cells appeared to express these antigens at low densities, but it is not known whether they represent a unique cell population. Among the several lymphoid cell lines tested, only the T lymphoma line EL-4 had the Lyt-1+, YE1/7.1+, YE1/19.1+ phenotype. These results suggest that the lpr mutation induces the expansion of a unique T cell subset.     

Structural changes in the oligosaccharide chains of IgG in autoimmune MRL/Mp-lpr/lpr mice

The structures of the asparagine-linked oligosaccharide chains of IgG from autoimmune arthritic MRL/Mp-lpr/lpr (MRL-lpr/lpr) mice and control MRL/Mp(-)+/+ (MRL(-)+/+) mice were investigated. Two subpopulations of IgG, M1-I and M1-II, were obtained from serum of MRL-lpr/lpr mice by column chromatography on protein A-Sepharose CL-4B. Although M1-I did not bind to the column, its elution was retarded, whereas M1-II was bound and was eluted in acidic buffer. IgG (Mn) from MRL(-)+/+ mice showed the same chromatographic behavior as M1-II. The structures of oligosaccharide chains liberated quantitatively by hydrazinolysis from IgG samples Mn, M1-I, M1-II, and a pooled mixture (M1) of M1-I and M1-II were determined by sequential exoglycosidase digestion, lectin (RCA120) affinity HPLC, and by methylation analysis. Their oligosaccharide structures were the same and shown to be biantennary complex-type chains +/- Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(+/- Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc. The proportion of each oligosaccharide in Mn and M1-II was the same but differed from that in M1-I where the degree of the galactosylation was significantly decreased which caused the change in the oligosaccharide pattern of total serum IgG (M1) of autoimmune MRL-lpr/lpr mice. This phenomenon, which is also found in total serum IgG of patients with rheumatoid arthritis, suggests that alteration of oligosaccharides in IgG may be a common feature in animals which develop arthritis with the production of rheumatoid factor regardless of species.     

Combined treatment of autoimmune MRL/Mp-lpr/lpr mice with cholera toxin plus irradiation

MRL/Mp-lpr/lpr (MRL/1) mice spontaneously develop autoimmune diseases like systemic lupus erythematosus (SLE) from 2 months of age, accompanied by massive lymphadenopathy. Such mice of 2 months of age were treated with 1g cholera toxin (CT) every 7 days and/or with 400 rad of one-shot⁶⁰Co irradiation. CT treatment alone markedly improved nephritis as evaluated by proteinuria and moderately suppressed lymphadenopathy and anti-DNA antibody production, while irradiation alone prominently improved lymphadenopathy but showed little effect on both nephritis and anti-DNA antibody production. On the other hand, when mice were treated with the combination of CT plus irradiation, autoimmune nephritis as well as anti-DNA production and lymphadenopathy were almost completely inhibited. Taken together, each agent exerts the improvement effect at the different points from each other in an abnormal immunological circuit displayed in MRL/1 mice. This kind of combined treatment may be applicable to the clinical use for autoimmune diseases.     

Functional defects of culture-grown bone marrow-derived macrophages from autoimmune MRL/MpJ-lpr/lpr mice

To investigate the primary defects and development of macrophages in MRL/MpJ-/pr/lpr (MRL/l) mice, we used a pure population of macrophages derived from bone marrow precursor cells cultured in the presence of L-cell conditioned medium (LCM) as a source of colony stimulating factor. Bone marrow-derived macrophages (BMM phi) from MRL/l mice had lower antigen presenting activity as detected by the induction of antigen-specific T cell proliferation, than age- and sex-matched control mice (CBA/J). Cell surface antigens (Ia and Mac-1) were determined quantitatively by a cell sorter as markers of macrophage differentiation. The BMM phi from MRL/l contained a much smaller number of Ia antigen-positive macrophages than those from normal mice. Treatment of BMM phi with an Ia-inducing of factor (IFN-gamma) markedly increased the expression of Ia antigens. This increase was significantly greater in BMM phi from MRL/l mice than in BMM phi from control mice. Expression of Mac-1 antigen was not different in BMM phi from the two strains. The Fc-mediated phagocytosis of IgG-coated sheep red blood cells was decreased in BMM phi from MRL/l mice compared with those from control mice. The function of nonspecific phagocytosis as measured by latex-bead incorporation was also impaired in MRL/l mice. The functional defects of MRL/l BMM phi found in these experiments are not secondary defects acquired under the influence of environmental signals during development, but are derived from the primary abnormalities which already exist in myeloid stem cells.     

Cloning of B cells from autoimmune MRL-lpr/lpr and MRL.xid mice

The relationship between colony formation (cloning) of B cells and their activation in murine autoimmunity was investigated in MRL-lpr/lpr and MRL.xid mice. Cells from MRL-lpr/lpr mice showed similar requirements for in vitro growth as normal CBA/J and BALB/c cells, with maximal colony formation in the presence of the supporting factors lipopolysaccharide and sheep red blood cells. The frequency of colony-forming cells from MRL-lpr/lpr spleens or hapten-specific B-cell preparations was slightly greater than the two normal control strains, with this difference significant only for a comparison of BALB/c and MRL-lpr/lpr spleens. In contrast, MRL-lpr/lpr mice bearing the xid gene for B-cell immunodeficiency (MRL.xid) had markedly reduced B-cell colony formation. These mice nevertheless expressed anti-DNA antibodies, although at levels reduced from that of MRL-lpr/lpr controls. These results indicate that enhanced in vitro colony formation need not accompany B-cell hyperactivity in murine autoimmune disease and that autoantibody production can occur in mice with impairment in this growth property.     

Comparison of immunological effects of cholera toxin on autoimmune MRL/lpr and BXSB mice.

MRL/lpr and BXSB mice were treated weekly or biweekly with cholera toxin (CT) in intravenous dose of 2 micrograms/mouse. CT treatment notably alleviated proteinuria in MRL/lpr mice, but did not influence the course of lupus nephritis in BXSB male mice. Flow cytometric analysis showed that anomalous B220+ T cells in spleen and thymus were reduced in CT-treated MRL/lpr mice while no significant change in lymphocyte populations was induced in BXSB male mice by this treatment. The suppressive effect of CT treatment on Con A response and the augmentative action on LPS response were observed in MRL/lpr mice. The latter may reflect increased B cells in relative number in the peripheral lymphoid organs. Mitogenic responses in CT-treated BXSB male mice remained unchanged in comparison with those of untreated group. Increased production of IL-6 by spleen cells was demonstrated in MRL/lpr mice treated with CT while in BXSB mice the level of IL-6 was not changed by the treatment with CT. Production of IFN gamma was suppressed by CT treatment in both strains of mice. This may be attributed to the inhibitory effect of CT on IFN gamma-producing Th1 cells as reported previously (Munoz et al, J. Exp. Med. 172: 95-103, 1990). However, CT treatment did not inhibit anti-DNA antibody production in BXSB mice, whereas the autoantibodies were markedly decreased in MRL/lpr mice treated with CT.     

Estrogen accelerates immune complex glomerulonephritis but ameliorates T cell-mediated vasculitis and sialadenitis in autoimmune MRL lpr/lpr mice.

Estrogen is known to influence immune responses in healthy subjects in a dichotomous fashion. Thus, in number of previous studies we and others have demonstrated that B cell activities are augmented after exposure to estrogen whereas T cell reactivity is suppressed. Furthermore, it has been shown that this hormone has significant impact on the course of certain human and experimental autoimmune diseases. In this study we report that treatment with physiological doses of estradiol exerts dichotomous effects on different manifestations of the lupus disease in MRL/l mice. On one hand immune complex-mediated glomerulonephritis was significantly accelerated. This outcome was due to polyclonal B cell activation with increased production of antibodies to double-stranded DNA and formation of circulating immune complexes. In contrast, T cell-mediated lesions such as focal sialadenitis, renal vasculitis, and periarticular inflammation were all significantly ameliorated in MRL/l mice exposed to estrogen. Thus, we were able to demonstrate that, within one subject and even within one organ, administration of estrogen leads to differential outcome of SLE morbidity. We propose that the differential effect of estrogen on the manifestations of the autoimmune disease of MRL/l mice is due to its dichotomous effects on B and T cell-mediated immune responses.     

Modulation of renal disease in MRL/lpr mice by suberoylanilide hydroxamic acid

Epigenetic regulation of gene expression is involved in the development of many diseases. Histone acetylation is a posttranslational modification of the nucleosomal histone tails that is regulated by the balance of histone deacetylases and histone acetyltransferases. Alterations in the balance of histone acetylation have been shown to cause aberrant expression of genes that are a hallmark of many diseases, including systemic lupus erythematosus. In this study, we determined whether suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor: 1) inhibits inflammatory mediator production in vitro and 2) modulates lupus progression in vivo. Mesangial cells isolated from 10-wk-old MRL/lpr mice were stimulated with LPS/IFN-gamma and incubated with SAHA. TNF-alpha, IL-6, NO, and inducible NO synthase expression were inhibited by SAHA. We then treated MRL/lpr mice with daily injections of SAHA from age 10 to 20 wk. The animals treated with SAHA had decreased spleen size and a concomitant decrease in CD4-CD8- (double-negative) T cells compared with controls. Serum autoantibody levels and glomerular IgG and C3 deposition in SAHA-treated mice were similar to controls. In contrast, proteinuria and pathologic renal disease were significantly inhibited in the mice receiving SAHA. These data indicate that SAHA blocks mesangial cell inflammatory mediator production in vitro and disease progression in vivo in MRL/lpr mice.     

Cryoglobulinemia induced by monoclonal immunoglobulin G rheumatoid factors derived from autoimmune MRL/MpJ-lpr/lpr mice

A MRL strain bearing the autosomal recessive mutant gene, lpr (lymphoproliferation), spontaneously develops, in addition to a lupus-like syndrome, unique serological and pathological manifestations. Production of high titers of IgG rheumatoid factors (RF) may be related to the formation of extremely large amounts of cryoglobulins and the development of tissue lesions such as necrotizing polyarteritis, arthritis, and glomerulonephritis. To analyze more directly the relationship of IgG RF to the development of cryoglobulins and tissue injuries, we have established four monoclonal IgG RF secreting hybridomas from unimmunized MRL-lpr/lpr mice and determined their pathogenic effects in normal strains of mice. All the monoclonal IgG RF obtained in this study were of the IgG3 subclass and generated cryoglobulins. However, the fact that not only IgG3 Rf monoclonals but also four of five non-RF IgG3 monoclonals were able to form cryoglobulins, which were composed exclusively of each IgG3 monoclonal, indicates that the IgG3 molecule has a unique physicochemical property to self-associate via nonimmunological interaction and the ability to form cryoglobulins. When the in vivo pathogenic activities of these IgG3 RF and non-RF monoclonals were examined, three of IgG3 RF monoclonals with the specificity to IgG2a were able to induce extensive pathologic manifestations including peripheral vasculitis and glomerulonephritis characteristic of patients with cryoglobulinemia. Our results indicate that the IgG3 itself, independently of its specificity, could be a potential source of cryoglobulins and IgG3 RF, combined with its activity of cryoglobulin formation, may play a significant role in the development of glomerulonephritis and cutaneous vascular lesions of ears and foot pads observed frequently in aged MRL-lpr/lpr mice.