Further serological and RFLP analysis of the MRL-+/+ and MRL-lpr/lpr mice

During their ageing, MRL-lpr/lpr mice (H-2k) suffer from progressive lymph node enlargement, associated with the development of several acute autoimmune lesions. The primary effect and location of the lpr mutation is unknown. However, a minority of the lymphoid cells of some MRL-+/+, as well as MRL-lpr/lpr mice, express 'alien' H-2d antigenic specificities. In the course of the investigation of the origin of the latter, we have carried out the genotyping of the MHC of several MRL-+/+ and MRL-lpr/lpr mice using the Southern blotting technique and employing a variety of MHC class I and class II probes, and restriction enzymes known to discriminate between the d and the k haplotypes. None of the results obtained so far suggests the contribution of genuine H-2d genes to the MHC of the MRL-+/+ and MRL-lpr/lpr mouse strains. Alternative hypotheses for the generation of the 'alien' epitopes are discussed.     

Hormone control of autoantibodies to calf thymus nuclear extract (CTE) and DNA in MRL-lpr and MRL-+/+ mice

Because hormonal influences on autoimmune disease in MRL-lpr and MRL-+/+ mice have not been defined completely, we examined animals which had been castrated and implanted with the opposite sex hormone. Antibodies directed at non-DNA antigens in a calf thymus nuclear extract (designated CTE) and specific anti-DNA antibodies were increased in estrogen-treated males, testosterone-treated females, and sham-operated female controls compared to sham-operated males. Analysis by sucrose gradient ultracentrifugation revealed that gonadal hormones exerted marked differences in the distribution and nature of circulating IgM anti-CTE antibodies. Although 19 S IgM was the predominant form of anti-CTE antibodies in experimental groups showing elevated anti-CTE responses, estrogen-treated male MRL-lpr mice expressed a large additional population of anti-CTE IgM antibody released by acid dissociation of apparently cryptic complexes. An unexpected additional finding was the presence of cryptic anti-CTE IgG (7 S) in all groups of MRL-lpr and MRL-+/+ mice, revealed only in sucrose gradient analysis under acid conditions. It is suggested that sex-related factors may account, in part, for apparent differences in levels of circulating autoantibodies observed in MRL mice by influencing the degree to which autoantibody populations exist in circulating complexes.     

Genetic association between natural autoantibody responses to histones and DNA in murine lupus.

Genetic regulation of the spontaneous anti-histone antibody production in systemic lupus erythematosus (SLE) was studied using the H-2-congenic and T cell receptor beta chain gene complex (TCR beta)-congenic NZB and NZW strains and their crosses. We found that the original, parental H-2d/d NZB mice produced significantly higher titers of serum IgM class anti-histone antibodies than did the congenic H-2d/z or H-2z/z NZB mice. However, none of these three NZB strains produced IgG antibodies. The NZW strain of any H-2 haplotype did not produce IgM and IgG anti-histone antibodies. The IgG anti-histone antibodies were produced only by H-2d/z heterozygous NZB x NZW F1, but not by homozygous H-2z/z or H-2d/d NZB x NZW F1 mice. In studies using (NZB x NZW) F1 x NZB backcross mice, only the progeny having both H-2d/z and NZW-type TCR beta genotypes produced high amounts of IgG antibodies. There was a tight linkage between the NZW-type TCR beta and the production of IgG anti-histone antibodies in TCR beta-congenic NZB x NZW F1 mice. All these findings were in keeping with our preceding observations on the genetic regulation of anti-DNA antibodies in these mice and suggest that certain common mechanisms such as super-antigen-mediated or common idiotope-mediated regulations may underlie the production of these two distinct autoantibodies in NZB x NZW F1 mice.     

Lymphocyte migration patterns in autoimmune MRL-lpr/lpr mice: relationship to age, disease manifestations and lymphocyte homing receptor expression

We have previously reported that lymphoid cells from systemic lupus erythematosus (SLE) mice with established disease migrate aberrantly. This study evaluates the abnormal lymphocyte migration patterns found in MRL-lpr/lpr (MRL/l) mice in relation to age, disease manifestations and the expression of lymphocyte homing receptors. 51chromium-labelled lymph node cells from MRL/l and from normal histocompatible CBA mice of different ages were injected i.v. into age and sex-matched CBA recipients. Diminished lymph node and increased hepatic uptake of MRL/l compared to CBA cells was evident as early as 6 weeks of age. Abnormalities in lymphocyte migration antedated the appearance of elevated antihistone antibody (AHA) levels but not the development of lymphadenopathy. Using the monoclonal antibody MEL-14, no differences in the expression of lymphocyte homing receptors between MRL/l and CBA lymph node cells were found at any age. Thus abnormalities in lymphocyte migration in MRL/l mice appear as early as six weeks and are not related to changes in homing receptor expression.     

MRL-lpr/lpr mice show an impairment of IgG aggregate removal which relates to parameters of disease activity.

MRL-lpr/lpr mice show an age-related impairment in the removal of heat-aggregated IgG (HAGG) from the circulation. The female mice, which have an earlier mortality than their male counterparts, clear HAGG more slowly than the male animals. Delay in clearance of this probe of mononuclear phagocytic system (MPS) function relates directly to high levels of circulating immune complexes (CIC) and to significant renal damage. In addition it relates indirectly to hepatic and splenic uptake of HAGG. MPS saturation plays a significant role in the pathogenesis of the disease seen in MRL-lpr/lpr mice.     

Autoreactive T cells from MRL-lpr/lpr mice secrete multiple lymphokines and induce the production of IgG anti-DNA antibodies.

The study of T cells in individuals with systemic lupus erythematosus has been limited because a specific marker for the disease has not been identified. To approach this issue, we isolated autoreactive T cell clones from lupus-prone MRL mice, a strain that develops an accelerated form of lupus. These CD4+ T cell clones grew spontaneously from unimmunized mice, and were maintained in culture by intermittent stimulation with syngeneic antigen presenting cells in the absence of exogenous antigen. One autoreactive T cell clone, termed ARTC-1, previously reported to have atypical MHC requirements for activation (both I-Ak and I-Ek were required) and to stimulate B cell proliferation and Ig production in vitro, was found to have an unrestricted pattern of lymphokine secretion. Following stimulation, it produced IL-4, IFN-gamma and IL-2. ARTC-1 induced B cell proliferation both by cell contact and through secretion of soluble lymphokines. B cell proliferation by cell-cell contact was MHC restricted in a manner analogous to ARTC-1 activation by APCs; the B cell response was inhibited by both anti-I-Ak and anti-I-Ek antibodies. The ARTC-1 B cell interaction was also found to result in the production of IgG autoantibodies. These observations suggest that cells such as ARTC-1, if unregulated, could lead to B cell stimulation and autoantibody production in vivo, in the absence of exogenous stimulation. Furthermore, IFN-gamma production by ARTC-1 could also result in enhanced class II expression, leading both to additional T-B cell interactions and to T cell interactions with endogenous cells capable of expressing class II antigens in other organs.     

Murine monoclonal antibodies to DNA. A comparison of MRL/lpr NZB/W and chronically graft-versus-host-diseased mice

Hybridomas producing monoclonal antibodies to DNA were prepared from NZB/W F1 (n = 20), MRL/lpr (n = 13), mice with a chronical graft versus-host-disease (GVHD) (n = 8) and polyclonally stimulated mice (n = 9). Screening was performed by means of an anti-DNA ELISA. Reaction patterns in four different anti-DNA assays (anti-DNA ELISA, indirect immunofluorescence on Crithidia luciliae, PEG assay and Farr assay) as well as avidity and cross-reactivity of these monoclonals were studied in relation to anti-DNA (sub)class and murine origin of the clones. It was found that monoclonal anti-DNA derived from mice with chronic GVHD did not differ from monoclonal anti-DNA derived from NZB/W F1 or MRL/lpr mice, with respect to isotype distribution, avidity towards DNA, cross-reactivity and assay behaviour in the anti-DNA assays mentioned before. In contrast, monoclonal anti-DNA obtained from polyclonally stimulated mice were all of the IgM isotype and displayed a stronger cross-reactive behaviour than the other three models. Altogether, these results exclude the possibility that anti-DNA in the GVHD mice originates from the non-specific pool of natural autoantibodies and further emphasize the relevance of chronic GVHD as a murine model of systemic lupus erythematosus.     

Kupffer cell activation and hematopoiesis in the liver of autoimmune MRL-lpr/lpr mice

Hematopoiesis can be induced in the adult murine liver by the administration of macrophage activators. The proliferation of macrophages and extrathymic T cells is spontaneously induced in the liver of autoimmune MRL-lpr/lpr mice, and deeply involved in the development of disease. To study the role of Kupffer cell activation in the induction of hematopoiesis and lymphocyte proliferation in the liver, we histologically analysed the kinetic and spatial relationship between Kupffer cells and hematopoietic cells or lymphocytes. At 5 weeks of age before the onset of disease, there were no appreciable histological changes in the liver. At 7 weeks, Kupffer cells had slightly increased in number, while hematopoietic islands were not yet detected. When disease had fully developed at 14 weeks, Kupffer cells were considerably increased in number and size, and exhibited numerous lysosomes. Hematopoietic cells of erythroid and myeloid series frequently appeared in the sinusoid, and lay in close apposition to Kupffer cells. Promyelocytes further migrated into the space of Disse to cluster there, being surrounded by the stellate cells (or fat-storing cells) and hepatocytes. After maturation, metamyelocytes and mature granulocytes were released into the sinusoidal circulation. Mitotic figures were detected in the cells of both erythroid and myeloid series. Lymphocytes proliferated in various sites such as in the sinusoid lumen, the space of Disse, and interlobular connective tissue, whether associated or not with Kupffer cells. The present results indicate that erythropoiesis, granulopoiesis, and lymphocyte proliferation are induced in the liver of MRL-lpr/lpr mice and are closely associated with Kupffer cell activation.     

Rapid B cell apoptosis induced by antigen receptor ligation does not require Fas (CD95/APO-1), the adaptor protein FADD/MORT1 or CrmA-sensitive caspases but is defective in both MRL-+/+ and MRL-lpr/lpr mice

Antigen receptor ligation-induced apoptosis is thought to play a role in self-tolerance by deleting autoreactive lymphocytes. Antigen receptor ligation-induced apoptosis of mature T cells and T cell lines requires autocrine or paracrine activation of Fas (CD95/APO-1). Whether B cell antigen receptor (BCR)-mediated apoptosis requires Fas or related molecules is unclear. Here we demonstrate that expression of either CrmA, the cowpox virus serpin, or an inhibitor of the adapter protein FADD/MORT1 blocks Fas-mediated apoptosis but has no effect on BCR ligation-induced apoptosis of the B cell line WEHI-231. In contrast, expression of Bcl-2 blocks BCR-mediated but not Fas-induced apoptosis in WEHI-231 cells. These results indicate that BCR ligation activates an apoptotic signaling pathway distinct from Fas-mediated apoptosis in WEHI-231 cells, and that BCR-mediated apoptosis of WEHI-231 cells does not require Fas or related molecules such as DR3, DR4 and DR5, as all of these death receptors require FADD/MORT1 and/or CrmA-sensitive caspases for induction of apoptosis. Moreover, extensive BCR ligation induces death of mature B cells from C57BL/6-lpr/lpr mice as efficiently as those from C57BL/6 mice, indicating that Fas is not essential for BCR-mediated apoptosis of mature B cells. In contrast, BCR ligation-induced apoptosis is reduced in mature B cells from MRL mice and this is not affected by the lpr mutation. Since MRL-lpr/lpr mice but not C57BL/6-lpr/lpr mice develop severe autoimmune disease, defects in BCR-mediated apoptosis in the MRL background, together with lpr mutation, may contribute to the development of severe autoimmune disease in MRL-lpr/lpr mice by allowing survival of self-reactive B cells.     

Administration of monoclonal anti-Sm antibody prolongs the survival and renal function of MRL-lpr/lpr mice.

The repeated administration of a monoclonal anti-Sm antibody (KSml) resulted in a significant prolongation of life in MRL-lpr/lpr lupus mice with a 50% mortality of 36 weeks compared with 18-24 weeks in the control groups. Control animals injected with APC11 (a myeloma protein of the same isotype) lived no longer than an untreated group. In addition the renal function as assessed by blood urea levels was less impaired in the KSml-injected mice than in the controls. All KSml-injected mice showed the presence of circulating anti-Sm antibodies which had a different Sm polypeptide binding specificity from that of the injected monoclonal antibody; the increased prevalence of these antibodies compared to the control mice (10-30%) suggested that the anti-Sm antibody response had been induced. The increased longevity in the KSml-treated animals was not associated with alterations in the anti-dsDNA antibody response. The data suggest that administration of anti-Sm antibodies modifies the course of murine lupus.     

Lack of association of Vβ8+ T cells with lupus-like syndrome in MRL-lpr/lpr mice

To evaluate the role of Vβ8⁺ T cells in the development of lupus-like autoimmune syndrome in MRL-lpr/lpr mice, we treated them with the F23.1 anti-Vβ8 monoclonal antibody (mAb) from birth to 4 months of age. Here we report that almost complete depletion of Vβ8⁺ T cells by the F23.1 mAb treatment neither inhibited nor delayed the development of hypergammaglobulinemia, autoantibody production and autoimmune glomerulonephritis in MRL-lpr/lpr mice. In addition, the F23.1 mAb treatment did not prevent the development of lymphadenopathy and the generation of a CD4^?CD8^? double-negative T cell subset, characteristically accumulating in lpr lymph nodes. Our results strongly argue against the idea that the Vβ8⁺ T cells play a critical role in the development of lupus-like autoimmune syndrome in MRL-lpr/lpr mice.     

霉酚酸酯对MRL-lpr/lpr小鼠狼疮性肾炎的治疗作用

目的　采用MRL lpr/lpr自发狼疮小鼠观察霉酚酸酯 (MMF)对狼疮性肾炎的治疗作用 ,探讨其作用机制。方法　 12周龄雌性MRL lpr/lpr自发狼疮小鼠分别予以MMF(90mg·kg-1·d-1,po)、甲基强的松龙 (MPS) (2 5mg·kg-1·d-1,ip)、环磷酰胺 (CTX) (2 5mg·kg-1·w-1,ip)治疗 2 4周。分别测定3种不同药物对小鼠尿蛋白排泄量、动物死亡率的影响 ;放免法测定血清抗dsDNA抗体的变化 ;运用RT PCR和免疫组化方法观察治疗药物对小鼠肾组织表达ICAM 1、PAI 1的影响。结果　治疗 2 4周后 ,各治疗组与对照组相比 ,体重均有不同程度增长 (P <0 .0 5 ) ,尿蛋白排泄量明显下降 (P <0 .0 1) [治疗9个月时 ,对照组为 (11.9± 2 .8)mg/d ,MMF组 (5 .1± 1.2 )mg/d ,MPS组 (3.4± 0 .8)mg/d ,CTX组 (2 .9±0 .3)mg/d],动物累积死亡率明显降低 ,血清中抗dsDNA抗体水平均有一定程度的降低 (P <0 .0 1) [治疗 9个月后 ,对照组为 (6 1.6± 7.1) % ,MMF组 (2 8.7± 4.6 ) % ,MPS组 (37.6± 4.3) % ,CTX组 (4 0 .1± 5 .8) % ],肾间质炎细胞浸润、肾小球硬化及间质纤维化程度较对照组减轻 ;肾组织免疫组化、RT PCR结果表明 ,3种药物在基因转录、蛋白质表达水平可不同程度地降低肾组织内ICAM 1、PAI 1表达。结论　MMF对MRL lpr/lpr自发     

Dietary fat influences the expression of autoimmune disease in MRL/lpr/lpr mice.

Near-isocaloric diets with qualitative and quantitative differences in fat content have a profound influence on the manifestation and progression of the autoimmune syndrome that occurs in female MRL/lpr mice. In these animals, a high (9%) lipid intake resulted in a significantly higher mortality rate: 60% (saturated fat) and 75% (unsaturated fat) compared to 35% at 1 year for a group fed a diet low in fat. Furthermore, beginning at 7 months of age mice from both of the high fat diet groups exhibited a significantly higher incidence of proteinuria than mice in the low fat group. Immunologically, the group fed the high unsaturated fat diet had the highest incidence of anti-dsDNA autoantibodies, and the high saturated fat group had the poorest macrophage phagocytic function. The low fat diet preserved near 'normal' immune function in general, particularly IL-2 production. No significant differences were noted in either the production of rheumatoid factor or natural killer cell activity, irrespective of age or diet.     

An allogeneic microenvironment influences the phenotype of intermediate T-cell receptor cells expanding in MRL-lpr/lpr mice.

MRL-lpr/lpr (lpr) mice fall victim to autoimmune disease owing to a lymphoproliferative disorder mainly of double-negative (DN) CD4- CD8- alpha beta T cells expressing a low density of interleukin-2 receptor beta-chain (IL-2R beta). It was previously revealed that the lpr gene is a defective Fas gene, into which an early transposon (ETn) of retrovirus is transfected. As a result of the failure of apoptosis, intermediate T-cell receptor (TCR) cells (i.e. TCRint cells) with DN phenotype abnormally accumulate in the periphery of lpr mice. We investigated herein how these TCRint cells are selected in terms of CD4, CD8 and TCR in lpr mice. When a whole fraction of mononuclear cells (MNC) in various immune organs of lpr mice was injected into scid mice (allogeneic circumstance), CD8+ TCRint cells mainly expanded. They had a high density of IL-2R beta. This was true when bone marrow cells of lpr mice were injected into scid mice. On the other hand, when MNC of the spleen and bone marrow in lpr mice were injected into irradiated (9 Gy) lpr mice (syngeneic circumstance), the major expanding cells were DN TCRint cells expressing a low density of IL-2R beta. A cell-sorting experiment for purified fractions demonstrated that only CD8- cells reconstituted TCRint cells in scid mice. Namely, DN CD4- CD8- cells as well as CD4+ cells which once acquired the mature phenotype, no longer switched their phenotype. These results suggest that the phenotype of TCRint cells is influenced by the surrounding microenvironment.     

Relationship of macrophages to defective delayed-type hypersensitivity in B6/lpr mice.

These experiments were undertaken to clarify whether or not suppressor macrophages contribute to deficiency of delayed-type hypersensitivity induced by BCG CW immunization in B6/lpr mice. Addition of indomethacin in vitro did not increase MIF production by immunized lpr lymph node cells. Adherent cells in lymph node cell suspensions from lpr mice did not interfere with MIF production by nonadherent lymph node cells from immunized B6 mice. On the other hand, MIF production by nonadherent lymph node cells from immunized B6/lpr mice was not restored even if they were cultured with adherent lymph node cells from immunized B6 mice. In addition, macrophages from nonimmunized B6/lpr mice showed normal reactivity to MIF when lymphocytes coexisting with the macrophages in large proportions were depleted from PEC. These results suggest that macrophages make no contribution to the deficiency of DTH expression of B6/lpr mice.     

Lack of connectivity between the induced and autoimmune repertoires of lpr/lpr mice.

It has been proposed that the autoantibody-secreting cells active during autoimmune diseases are derived from B cells initially responding to environmental antigens. In order to test the relationship between the antigen-induced and autoimmune repertoires, we monitored the fate of antigen-activated idiotypically defined B cells present in mice that developed the systemic lupus erythematosus (SLE)-like syndrome associated with the lpr mutation. Mice homozygous for both the A/J-derived Igh and Ig kappa region haplotypes and the lpr mutation were bred. Immunization of these mice with p-azophenylarsonate (Ars)-protein conjugates elicited the idiotypic components (IdCR) characteristic of the A/J anti-Ars response and did not interfere with the spontaneous development of the lpr-mediated autoimmune disease. These Id/lpr mice provided an ideal system for studying the relationship between the exogenously and endogenously induced responses because: (1) VHIdCR antibodies have been shown to bind autoantigens in vitro; and (2) serological and molecular reagents exist which can identify and monitor VHIdCR antibody production as disease progresses. Serum samples and hybridoma cell lines derived from non-immune as well as Ars-keyhole limpet haemocyanin (KLH)-immunized Id/lpr mice were monitored for idiotype expression as well as Ars and ssDNA reactivity at various stages of disease progression. We found that antibodies utilizing the VHIdCR gene segment did not preferentially contribute to the autoantibody pool. Moreover, even when IdCR B-cell clones were expanded by deliberate immunization with Ars-KLH, Ars non-binding variants were only rarely detected among the activated B-cell populations of diseased mice. These results indicate that there is only minimal overlap between the VHIdCR conventional and autoimmune repertoires.     

Sequential autoantigenic determinants of the small nuclear ribonucleoprotein Sm D shared by human lupus autoantibodies and MRL lpr/lpr antibodies.

Autoantibodies directed against the Sm proteins of the spliceosome complex are found in approximately 25% of systemic lupus erythematosus (SLE) patients sera. To determine which regions of the Sm D polypeptide are involved in the lupus autoimmune response, binding to overlapping octapeptides of Sm D has been evaluated with sera from nine Sm D-positive patients, six patients with other autoimmune serology, and five normal human sera. Lupus patient sera which are Sm precipitin-positive bind various combinations of five regions of the peptide. The major antigenic region, Epitope 5 (REAVA(GR)10GGPRR), is bound by eight of nine Sm precipitin-positive sera tested. This region of Sm D shows significant sequence homology with Epstein-Barr nuclear antigen-1. To determine the fine specificity of the murine Sm response, four unique Sm D MoAbs derived from MRL lpr/lpr mice and three adult anti-Sm-positive MRL lpr/lpr mouse sera have been analysed. Two of these monoclonals, KSm 4 and Y12, as well as the MRL lpr/lpr sera tested, show binding with Epitope 5. Another of these monoclonals, KSm 2, binds octapeptides 84-91, DVEPKVKSKKREAVAG, which corresponds to Epitope 4 of this study. Antibodies from SLE patients with autoimmune serology other than anti-Sm bind the carboxyl glycine-arginine repeat (GR)10 peptides of Sm D. However, none of the antibodies tested from patients who do not have lupus and who have different autoimmune serology binds any of the Sm D octapeptides. Normal controls did not significantly bind any of the Sm D octapeptides. These results describe two major regions of shared antigenicity of Sm D between sera from SLE patients and MRL lpr/lpr mice, thereby establishing a basis for the cross-species similarity of autoimmunity to the Sm autoantigen in SLE.     

Naturally occurring antibodies to poly(ADP-ribose) in autoimmune MRL/Mp-lpr/lpr mice.

The presence of antibodies to poly(ADP-ribose) was demonstrated in the sera of MRL/Mp-lpr/lpr (MRL/l) mice by an enzyme linked immunosorbent assay (ELISA). Antibody to poly(ADP-ribose) was strongly inhibited by single stranded DNA (ssDNA) and poly(ADP-ribose), and less by double stranded DNA (dsDNA). Affinity purified anti-poly(ADP-ribose) antibodies bound more with immobilized ssDNA than with poly(ADP-ribose), and were significantly inhibited by soluble ssDNA, although poly(ADP-ribose) was the best soluble inhibitor. On the contrary, affinity purified anti-ssDNA antibodies bound best with ssDNA and significantly but less with poly(ADP-ribose); however, they were scarcely inhibited by poly(ADP-ribose). These results suggest that similar antigenic determinants exist in poly(ADP-ribose) and ssDNA. It is conceivable, however, at the present moment that 'naturally occurring antibodies to poly(ADP-ribose)' in MRL/l mice are subpopulations of anti-ssDNA antibodies that react equally well with poly(ADP-ribose) and ssDNA.     

Hepatic reticuloendothelial system activation in autoimmune mice: differences between (NZB X NZW)F1 and MRL-lpr/lpr strains

5'-Nucleotidase (5'N) activity and Ia expression of hepatic nonparenchymal cells (NPCs) of the autoimmune (NZB X NZW)F1 and MRL-lpr/lpr and nonautoimmune C3H/FeJ, DBA/2J, and A/J strains were assayed to determine endogenous activation of the hepatic reticuloendothelial system (RES). Pretreatment of the nonautoimmune strains with Corynebacterium parvum resulted in decreases in Fc receptor expressor and 5'N and an increase in Ia expression of NPCs. Endogenous hepatic RES activation, as measured by low 5'N and high Ia expression, was observed in both the autoimmune MRL-lpr/lpr and (NZB X NZW)F1 strains even without C. parvum pretreatment. However, in the MRL-lpr/lpr strain, age is a dependent variable in activation and correlates with delayed clearance from the circulation of soluble IgG immune complexes. In the (NZB X NZW)F1 strain, the observation of decreased 5'-nucleotidase activity and increased percentage of Ia-positive cells at all ages suggests a primary abnormality. Therefore, different genetic or exogenous factors may be responsible for the activation of the hepatic RES in these autoimmune strains.