Sequential study of vasculitis in MRL mice

The frequency, age of onset and organ distribution of spontaneously occurring vasculitis was examined in a sequential study with 170 MRL mice of both substrains. Necrotizing vasculitis was seen in 55.8% of MRL/Mp-lpr/lpr mice studied, beginning at the age of 3 months. The kidney and urinary bladder were most frequently involved. In MRL/Mp- +/+ mice necrotizing vasculitis was much less frequently present (7.6%), beginning at the age of 18 months, and was seen only in the kidney, stomach and testes. In both substrains mononuclear infiltration of pulmonary vessel walls preceded the occurrence of necrotizing arteritis in other organs. The immunofluorescence study revealed the presence of immune complex components (immunoglobulin G, C3, murine leukaemia virus antigen gp71) in the vessel walls of the renal arteries of six out of 36 lpr/lpr mice with necrotizing arteritis.     

Nucleoside-specific suppression in MRL/MP +/+ mice

The induction of nucleoside-specific nonresponsiveness was further studied in the autoimmune strain MRL/MP +/+ (MRL/n). Experiments were undertaken to determine (i) whether nucleoside-conjugated spleen cells are able to induce specific nonresponsiveness to T-dependent nucleoside antigens in MRL/n mice, and (ii) whether periodic treatment with nucleoside-conjugated spleen cells would retard the development of spontaneous anti-DNA antibodies and associated indicators of autoimmunity. The results show that nonresponsiveness to nucleoside antigens is inducable in male, but not in female, MRL/n mice. Nonresponsiveness in male MRL/n was transferable and mediated by T cells. Treatment of male MRL/n mice with nucleoside-conjugated spleen cells (NSC) appeared to attenuate the progress of autoimmune symptoms in experimental animals. These results are discussed in the context of recent studies exploring the etiology of autoantibody production and the loss of self-tolerance in murine models of autoimmunity.     

Genetic dissection of testis weight in mice: quantitative trait locus analysis using F2 intercrosses between strains with extreme testis weight, and association study using Y-consomic strains

In the present study, dissection of genetic bases of testis weight in mice was performed. Autosomes and the X chromosome were searched using traditional quantitative trait locus (QTL) scans, and the Y chromosome was searched by association studies of Y-consomic strains. QTL analysis was performed in ??DDD?×???CBA F₂ mice; the inbred mouse DDD has the heaviest testes, whereas the inbred mouse CBA has the lightest testes. Two significant testis weight QTLs were identified on chromosomes 1 and X. A DDD allele was associated with increased and decreased testis weight at the locus on chromosomes 1 and X, respectively. In the reciprocal cross ??CBA?×???DDD F₂ mice, QTL on chromosome 1, and not on chromosome X, had a significant effect on testis weight. The DDD allele at the X-linked locus could not sustain testis weight in combination with the Y chromosome of the CBA strain. The Y chromosome per se had a significant effect on testis weight, i.e., DH-Chr Y^DDD had significantly heavier testes than DH-Chr Y^CBA. On the basis of the results of Y-chromosome-wide association studies using 17 Y-consomic strains, variations in Uty, Usp9y, and Sry were significantly associated with testis weight. Thus, testis weight is a complex quantitative phenotype controlled by multiple genes on autosomes and sex chromosomes and their interactions.     

Accuracy of estimation of testis weight from in situ testis measures in ram lambs

At a mean age of 93 +/- 5 days and a mean weight of 29 +/- 5 kg, 44 crossbred ram lambs were castrated to evaluate the accuracy of the estimation of testis weight from in situ testis measures (scrotal circumference and testis diameter). Means and standard deviations for testis weight (both testes), scrotal circumference and average in situ testis diameter were 134 +/- 57 g, 20.2 +/- 3.1 cm and 3.7 +/- .7 cm, respectively. Testis weight (W) was predicted from scrotal circumference (C) and average in situ diameter (D(o)) as W=.131C(1.90) D(o)(.88) (R(2)=.949). When adjusted to the same scrotal circumference and in situ diameter, testes of 3/4-Finnish Landrace rams were heavier (P<.05) than testes of 7/8-Dorset rams. However, the additional accuracy obtained by using equations specific to each crossbred group was small.     

Analyses of acute graft-versus-host-like reaction in [MRL/lpr----MRL/+] chimeric mice using MRL/lpr-Thy-1. 1 congenic mice.

When MRL/Mp(-)+/+(MRL/+) mice are lethally irradiated and then reconstituted with MRL/Mp-lpr/lpr (MRL/lpr) bone marrow and/or spleen cells, these MRL/+ mice develop "lpr-GVHD" which is similar to acute graft-versus-host disease (GVHD). Using a Thy-1 congenic strain of MRL/lpr mice (MRL/lpr-Thy-1.1), we analyzed T cell subpopulations in the thymus and spleen of MRL/+ mice suffering from lpr-GVHD. lpr-GVHD was induced in MRL/+ mice by transplantation of bone marrow cells (BMC) from MRL/lpr-Thy-1.1 mice; severe lymphocyte depletion associated with fibrosis was observed in the spleens after 7 weeks of bone marrow transplantation (BMT). Thymocytes of the host MRL/+ thymus were replaced with donor-derived cells from the early stage of lpr-GVHD, whereas in the spleen, a small number of host T cells (Thy-1.2+) (4-5%) were retained until the late stage of lpr-GVHD. Donor-type (Thy-1.1+) T cell subsets were not different from those of nontreated MRL/+ mice in the thymus, whereas in the spleen. CD8+ T cells (Thy-1.1+) reached a peak at 5 weeks after BMT, and CD4+ T cells (Thy-1.1+), a peak at 6 weeks. The elimination of T cells from MRL/lpr BMC had no evident effect on the prevention of lpr-GVHD. T cell subpopulations showed a similar pattern to GVHD elicited by MHC differences. Analyses of autoreactive T cells expressing V beta 5 or V beta 11 revealed that autoreactive T cells were deleted from the peripheral lymph nodes. Interestingly, the levels of IgG anti-ssDNA antibodies markedly increased, and both IgM and IgG rheumatoid factors slightly increased 5 to 7 weeks after BMT. These findings are discussed in relation to not only GVHD elicited by MHC differences but also autoimmune diseases.     

Anti-Sm autoantibodies in MRL mice: analysis of precursor frequency

Individual MRL-lpr mice vary in their capacity to generate anti-Sm autoantibodies spontaneously. We have compared the frequency of B-cell precursors for this autoantibody in serologically negative and serologically positive MRL-lpr mice, and in normals. Anti-Sm precursors were present in a frequency of approximately 1 per 10-30,000 in spleen cell cultures from anti-Sm positive mice, but were undetectable when spleen cells from serologically negative MRL-lpr mice or from normal mice were examined. Despite LPS stimulation, neither IgM nor IgG precursors could be detected. In parallel cultures, in contrast, anti-DNA autoantibody precursors were readily detected. The results thus indicate that, for the lupus-specific autoantibodies, the absence of antibody in autoimmune mice reflects a deficit in precursor B lymphocytes rather than an active regulatory mechanism. It is suggested that the generation of anti-Sm may reflect a low-probability random event in the generation of B-cell diversity.     

Sexually dimorphic genes regulate healing and regeneration in MRL mice

Abstract The MRL mouse has been shown to display unusual healing properties. In particular, when the ear pinna is hole punched, the hole that is made closes completely without scarring, with reformation of hair follicles and sebaceous glands, and regrowth of cartilage. Initial studies using (MRL/lpr × C57BL/6) F₂ and backcross mice showed that this phenomenon is genetically determined and that multiple loci contribute to this quantitative trait. In the present study, with twice as many animals, we have confirmed many of the original heal loci and identified new ones. We have also found that this phenotype is sexually dimorphic in that female mice heal more quickly and more completely than male mice. To test the cause of this difference, we castrated both males and females. Castration of males led to better healing, although ovariectomy did not lead to worse healing in female mice. Finally, most heal loci were shown to be responsible for regulating healing primarily in male animals more than in females, or vice versa. Thus, sex plays a highly significant role in the closure of wounded tissue in this mammalian model of healing and regeneration.     

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.     

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.     

Abrogation of lupus nephritis in activation-induced deaminase-deficient MRL/lpr mice

We generated MRL/lpr mice deficient in activation-induced deaminase (AID). Because AID is required for Ig hypermutation and class switch recombination, these mice lack hypermutated IgG Abs. Unlike their AID wild-type littermates, AID-deficient MRL/lpr mice not only lacked autoreactive IgG Abs but also experienced a dramatic increase in the levels of autoreactive IgM. This phenotype in AID-deficient mice translated into a significant reduction in glomerulonephritis, minimal mononuclear cell infiltration in the kidney, and a dramatic increase in survival to levels comparable to those previously reported for MRL/lpr mice completely lacking B cells and well below those of mice lacking secreted Abs. Therefore, this study wherein littermates with either high levels of autoreactive IgM or autoreactive IgG were directly examined proves that autoreactive IgM Abs alone are not sufficient to promote kidney disease in MRL/lpr mice. In addition, the substantial decrease in mortality combined with a dramatic increase in autoreactive IgM Abs in AID-deficient MRL/lpr mice suggest that autoreactive IgM Abs might not only fail to promote nephritis but may also provide a protective role in MRL/lpr mice. This novel mouse model containing high levels of autoreactive, unmutated IgM Abs will help delineate the contribution of autoreactive IgM to autoimmunity.     

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.     

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.     

Ig VH gene family repertoire of plasma cells derived from lupus-prone MRL/lpr and MRL/++ mice

VH gene family usage was determined in both spontaneous, in vivo activated plasma cells and LPS-induced plasma cells from individual MRL/lpr mice by using in situ hybridization. It was found that VH gene family expression in spontaneous plasma cells varied from mouse to mouse. Some mice expressed VH families in an apparently random manner similar to that obtained with polyclonal activation. Other mice showed an exaggerated expression of particular VH gene families. VH J558 was overrepresented most frequently, but overrepresentation of VH 7183, Q52, and 36-60 was also observed. Importantly, LPS-induced VH gene family expression in these same mice displaying biased VH family usage in spontaneous plasma cells, appeared normal with no evidence for similar biases in the LPS-induced repertoire. Anti-DNA antibody concentrations and the degree of glomerulonephritis were determined for each mouse to measure the severity of disease. The level of expression of the J558 family was positively correlated with disease severity. The results suggest that the initial autoantibody response is highly diverse but becomes more restricted as the disease progresses.     

Mapping of genetic factors influencing the weight of cystic fibrosis knockout mice

One of the poorly understood clinical manifestations of cystic fibrosis (CF) is low body weight. Mice in which the CF causative gene, cystic fibrosis transmembrane conductance regulator (Cftr), has been knocked out reflect this as they are smaller than age-matched littermates. The variable weight of F2 Cftr -/- (CF) mice derived from a cross between congenic C57BL/6J and BALB/cJ Cftr heterozygotic mice permits the mapping of modifiers of this cystic fibrosis phenotype. In this report, quantitative trait loci (QTL) mapping was used to identify the chromosomal locations of genes that contribute to the body weight of 12-week-old F2 CF mice. Five loci of CF body weight were detected with four of the five acting in a sex-specific manner. Significant linkage of the phenotype to a region of Chromosome (Chr) 13 from D13Mit179 to D13Mit254 (LOD = 4.2) was established in female mice; and suggestive loci on Chrs 7 and 10 were identified. The weights of F2 male CF mice were suggestively linked to regions of Chrs 1 and 6, and to the same locus on Chr 7 as in female mice. The suggestive loci did not influence the weight of the limited set of control mice and thus are presumed to be CF specific in their effects. Further study of these putative CF body weight modifiers may provide insight on the pathogenesis of cystic fibrosis.     

Heat-shock resistance in experimental cryptorchid testis of mice

Cryptorchidism is commonly used for research on spermatogenesis. However, there are few comparative investigations about the strain differences in mice, especially in long-term experiments. In the present study, the authors demonstrate its specific dynamics in the MRL/MpJ mouse strain, and discuss the cause of strain differences. In the mouse strains A/J BALB/c, C3H/He, and C57BL/6, after 2 weeks of experimental cryptorchidism, the ratios of the cryptorchid testis weight against the intact one were 0.38+/-0.05, 0.43+/-0.05, 0.38+/- 0.02, and 0.44+/-0.14, respectively. On the other hand, in the MRL/MpJ strain it was shifted to 0.69+/-0.08. The details of this strain difference were compared by calculation of germ cells with the Sertoli cell index at 2 weeks after operation. The indices of spermatogonia in all strains were not significantly different; however, in MRL/MpJ mice remarkable numbers of late spermatocytes and round spermatids were detected. The decrease of the testis weight ratio was similar until 10 days in the C57BL/6 and MRL/MpJ strains, but continued in C57BL/6 until 21 days, whereas in MRL/MpJ mice it plateaued after 10 days. Northern blot analysis for heat shock protein 70-2 using total RNA prepared from the cryptorchid and intact testes at 2 weeks after operation revealed that the expression was decreased in the cryptorchid testis of C57BL/6, but not MRL/MpJ mice. The results suggested that heat-resistant germ cells were present in MRL/MpJ, originating possibly from the genetic background.     

Activities of dipeptidyl peptidases in BXSB mice and MRL/lpr mice with lupus erythematosus-like syndrome

Activities of peptidases were examined in tissues of male BXSB and male MRL/Mp-lpr/lpr (MRL/lpr) mice which are animal models of human systemic lupus erythematosus. Female BXSB and male MRL/+ + mice without histopathological changes were used as controls. Activity of DPP II in the spleen, kidney, and liver showed an increase at 13 and 20 weeks of age, while that of DPP IV was decreased at 20 weeks of age, as compared to control mice. The ratio of DPP II/DPP IV activities in the tissues was significantly increased and these findings agree with our previous results in the tissues of NZB mice and in the serum of patients with lupus erythematosus, underscoring the importance of hydrolytic enzymes in the pathogenesis of autoimmune diseases.