MRL Lpr Research Articles

Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells

Global DNA hypomethylation in CD4+ cells in systemic lupus erythematosus (SLE) was suggested to play a key role in the pathogenesis. To identify new methylation-sensitive genes, we integrated genome-wide DNA methylation and mRNA profiling data in CD4+ cells of MRL/lpr (MRL) and C57BL6/J (B6) mice. We identified Cathepsin E (Ctse), in which 13 methyl-CpGs within 583 bp region of intron 1 were hypomethylated, and Ctse mRNA upregulated in MRL compared with B6 mice. One of methyl-CpGs, mCGCG was 93.3 ± 2.05% methylated in B6 mice, while 80.0 ± 6.2% methylated and mutated to CGGG in MRL mice. Kaiso is known to bind to mCGCG and we hypothesized that it represses expression of Ctse in B6 mice. The binding of Kaiso to mCGCG site in B6 mice was reduced in MRL mice revealed by ChIP-PCR. EL4 cells treated with 5-azaC and/or Trichostatin A showed the suppression of binding of Kaiso to mCGCG motif by ChIP-PCR and the overexpression of Ctse was demonstrated by qPCR. Ctse gene silencing by siRNA in EL4 cells resulted in reduction of IL-10 secretion. The hypomethylation of mCGCG motif, reduced recruitment of Kaiso, and increased expression of Ctse and Il-10 in CD4+ cells may be involved in the pathogenesis of SLE.

Therapeutic efficacy and mechanism of total lymphoid irradiation with electron beam in MRL/lpr mouse model of lupus

Objective To evaluate the therapeutic efficacy of total lymphoid irradiation with electronic beam in MRL/lpr mouse model of lupus,and to assess the relationship between total lymphoid irradiation and regulatory T (Treg) cells.Methods Eighteen four-month-old MRL/lpr lupus mice were randomly divided into three groups with three female mice and three male mice in each group:negative control group treated with intraperitoneal sodium chloride physiological solution 0.5 ml once daily,radiotherapy group receiving total lymphoid irradiation with the electronic beam (2 Gy per day) five times a week,and dexamethasone group treated with intraperitoneal dexamethasone 1 mg/kg once daily for two consecutive weeks.Then,Coomassie brilliant blue method was used to quantify the 24-hour urinary protein,and enzyme-linked immunosorbent assay (ELISA) to measure the serum levels of antinuelear antibody (ANA) and anti-dsDNA antibody.All the mice were sacrificed,renal tissue was obtained and subjected to direct immunofluorescence assay for the observation of IgG deposits as well as periodic acid-Schiff staining for the evaluation of renal injury,and spleen tissue was isolated and subjected to flow cytometry for the determination of proportion of Treg cells in spleen cells.Data were processed using the SPSS 11.5 software.Statistical analysis was done with one-way analysis of variance.Results Significant differences were observed between the radiotherapy group and negative control group in 24-hour urinary protein level ((3.33 ± 0.17) vs.(3.97 ± 0.33) mg,P＜ 0.05),serum ANA antibody level ((275.12 ± 30.18) vs.(434.82 ± 36.63) U/ml,P＜ 0.05) and anti-dsDNA antibody level ((455.74 ± 43.38) vs.(956.78 t 37.69) IU/ml,P ＜ 0.05).The glomerular deposition of IgG antibody was obviously decreased with attenuated renal injury in the radiotherapy group and dexamethasone group compared with the negative control group,but no significant difference was noted between the radiotherapy group and dexamethasone group.The proportion of Treg cells in the radiotherapy group and dexamethasone group was significantly higher than that in the negative control group ((1.08 ± 0.52)％ and (1.18 ± 0.60)％ vs.(0.38 ± 0.14)％,both P ＜ 0.05).Conclusions Total lymphoid irradiation with electronic beam can effectively reduce urine protein level,serum ANA and anti-dsDNA antibody level in MRL/lpr lupus mice,alleviate renal injury,likely by up-regulating the proportion of Treg cells in vivo. Key words: Mice, inbred MRL lpr; Lupus erythematosus, systemic; T-lymphocytes, regulatory; Lymphatic irradiation

Toll-Like Receptor 2 or Toll-Like Receptor 4 Deficiency Does Not Modify Lupus in MRLlpr Mice

Systemic lupus erythematosus is an autoimmune disease with a high morbidity and nephritis is a common manifestation. Previous studies in murine lupus models have suggest a role for Toll-like receptor 2 and 4. We examined the role of these molecules in MRL lpr mice which is one of the most established and robust murine models. We compared disease parameters in Toll-like receptor 2 or Toll-like receptor 4 deficient mice with their littermate controls. We found no difference in the severity of glomerulonephritis as assessed by histology, serum creatinine and albuminuria when Toll-like receptor 2 or Toll-like receptor 4 deficient MRLlpr mice were compared with Toll-like receptor sufficient controls. We also found similar levels of anti-dsDNA and anti-ssDNA antibodies. These results show that Toll-like receptor 2 and Toll-like receptor 4 do not play a significant role in MRLlpr mice, and therefore they may not be important in human lupus.

Roles of regulation of plasma cell function by Blimp1-B cell maturation antigen in the pathogenesis of systemic lupus erythematosus

Objective To detect the expression of Blimpl gene in MRL/lpr mice, so as to provide new ideas for plasma cell-targeting therapy of systemic lupus erythematosus （SLE）. Methods Electrophoretic mobility shift assay （EMSA） and chromatin immunoprecipitation analysis （CHIP） were performed to evaluate the regulatory effect of Blimpl on B cell maturation antigen （BCMA）, and fluorescence-based quantitative PCR was carried out to detect the expression of Blimp1 mRNA in spleen and lymph node tissue of MRL/lpr mice and normal control mice. The intergroup difference in Blimpl mRNA expression was assessed by rank sum test. Results Blimpl could bind to the BCMA gene promoter. Increased Blimpl mRNA expression was observed in both the spleen and lymph node tissue of MRL/Ipr mice compared with the normal control mice （Z = 2.609, 3.402, respectively, both P 〈 0.01 ）. Conclusions BCMA appears to be the target gene of Blimpl, and Blimpl may plays a certain role in the maintenance of plasma cell survival and antibody production via directly regulating BCMA gene expression. Key words: Lupus erythematosus, systemic; Blimpl; B-cell maturation antigen; Plasma cell; Mice, inbred MRL lpr

4SC-101, A Novel Small Molecule Dihydroorotate Dehydrogenase Inhibitor, Suppresses Systemic Lupus Erythematosus in MRL-(Fas)lpr Mice

Immunosuppressive treatments of systemic lupus (SLE) remain associated with significant toxicities; hence, compounds with better toxicity profiles are needed. Dihydroorotate dehydrogenase (DHODH) inhibition with leflunomide has proven to be effective in autoimmune diseases including SLE, but leflunomide can cause a variety of side effects. We hypothesized that 4SC-101, a novel DHODH inhibitor with a more favorable toxicity profile, would be as effective as high-dose cyclophosphamide (CYC) in controlling experimental SLE of female MRL(Fas)lpr mice. Daily oral gavage of 30, 100, and 300 mg/kg 4SC-101 from 12 to 22 weeks of age was compared with either vehicle or CYC treatment (30 mg/kg/week, i.p.) in terms of efficacy and toxicity. Three hundred milligrams per kilogram 4SC-101 was as effective as CYC in depleting spleen autoreactive T cells, B cells, and plasma cells as well as the respective DNA and RNA serum autoantibodies. This was associated with a comparable amelioration of the renal, dermal, and pulmonary SLE manifestations of MRL(Fas)lpr mice. However, even the highest dose of 4SC-101 had no effect on bone marrow neutrophil counts, which were significantly reduced in CYC-treated mice. Together, the novel DHODH inhibitor 4SC-101 is as effective as high dose CYC in controlling SLE without causing myelosuppression. Hence, DHODH inhibition with 4SC-101 might be suitable to treat active SLE with fewer side effects than CYC.

Detection of glomerular-binding immune elements in murine lupus using a tissue-based ELISA.

The glomerulonephritis in systemic lupus erythematosus (SLE) is presumably triggered by the binding of circulating immune elements (autoantibodies and immune complexes) to the glomerulus; however, the nature of these elements is unclear. In order to detect and characterize such elements, we developed an ELISA using whole intact glomeruli as the substrate. With this assay, glomerular binding activity (GBA) was detected in the serum of MRL lpr, NZB x W, and B x SB mice, but not in non-autoimmune BALB/c mice. Less activity was present in the serum of C3H lpr, C57Bl/6J lpr and AKR lpr animals which develop signs of autoimmunity but only modest renal disease. The GBA in MRL lpr mice contained IgG (subclasses 1, 2a and 2b), but not IgG3, IgM, IgA, or C3. GBA was not significantly decreased by preadsorption of MRL lpr serum by DNA-agarose (although anti-DNA antibodies were). Binding activity in serum, however, was diminished by DNAase treatment. Fractionation of MRL lpr serum over a molecular sizing column showed that GBA eluted in a broad peak. GBA bound to the glomerulus ex vivo in a tissue-specific fashion and was enriched in renal eluates relative to serum in vivo. In sum, the binding activity detected by this assay appeared to be a heterogeneous entity (possibly in part immune complexes containing DNA) which bound specifically to the glomerulus and which appeared to parallel the presence of renal disease. This novel assay system may help elucidate the pathogenesis of SLE nephritis and have utility as a disease marker.

Leukocytes and the kidney contribute to interstitial inflammation in lupus nephritis

Interstitial leucocyte infiltration, a prominent feature of lupus nephritis, predicts deterioration of renal function. We used two models of lupus nephritis in mice, one with chronic spontaneous disease and the other with acute interferon-alpha (IFN alpha)-mediated disease. The latter is characterized by the virtual absence of interstitial infiltration. In vivo migration assays showed that splenic leukocytes from spontaneously nephritic mice tended to migrate into non-inflamed syngeneic kidneys. This was enhanced if the recipient kidneys were already inflamed. Kidneys from both chronically and acutely nephritic mice showed similar ability to recruit splenic leukocytes from chronically diseased mice. Leukocytes from acutely diseased mice, however, failed to migrate into chronically inflamed kidney. Compared with those with chronic nephritis, the kidneys of acute nephritic mice expressed less of the inflammatory chemokine CXCL13/BLC. Moreover, leukocytes from acute nephritic mice displayed impaired migration, in vitro, to T-cell chemokine attractants. This study links leukocyte infiltration to both kidney chemokine expression, and leukocyte chemotaxis to kidney-expressed chemokines.

C5a Receptor Deficiency Attenuates T Cell Function and Renal Disease in MRLlpr Mice

The development and progression of systemic lupus erythematosus (SLE) is strongly associated with complement activation and deposition. To characterize the role of C5a and its receptor (C5aR) in SLE, C5aR-deficient mice were backcrossed nine generations onto the lupus-like MRL(lpr) genetic background. Evidence is presented that C5aR modulates both renal injury and T cell responses in MRL(lpr) mouse. C5aR-deficient MRL(lpr) mice had prolonged viability, with a mean survival time of 33.0 wk compared with 22.6 wk in control mice. Renal injury was also attenuated in the C5aR-/- MRL(lpr) mice. At 20 wk of age C5aR-/- MRL(lpr) mice had a complete absence of glomerular crescents and marked reductions in glomerular hypercellularity. There was no difference in the degree of glomerular C3 deposition; however, IgG deposits were reduced in the C5aR-/- MRL(lpr) mice. The reduction in glomerular injury was also associated with a four-fold decrease in renal CD4+ T cell infiltrates. Whereas there were modest differences in total IgG anti-dsDNA antibody titers, C5aR-deficient mice had 3.5-fold higher levels of IgG1 and 15-fold lower levels of IgG2a anti-dsDNA antibody titers compared to controls. The differences in anti-dsDNA IgG subclasses were associated with reduced CD4+ Th-1 responses in the C5aR-/- MRL(lpr) mice, including diminished production of IL-12p70, IFN-gamma, and increased expression of the Th-2 transcription factor GATA-3. These findings indicate that the C5aR plays a major role in modulating complement-dependent renal injury and T helper cell Th-1 responses in the MRL(lpr) mouse.

Mycophenolic acid inhibits inosine 5 ′-monophosphate dehydrogenase and suppresses production of pro-inflammatory cytokines, nitric oxide, and LDH in macrophages

Mycophenolic acid (MPA) inhibits reversibly inosine 5 ′-monophosphate dehydrogenase, an enzyme involved in the de novo synthesis of guanine nucleotides. Previously, mycophenolate mofetil (MMF), the pro-drug of MPA, was shown to exert beneficial effects on the systemic lupus erythematosus (SLE)-like disease in MRL lpr/ lpr mice. In this study MPA’s immunomodulating effects in vitro on the murine macrophage cell line IC-21 were investigated. The cells were exposed to MPA together with lipopolysaccharide and IFN-γ. Cytokine, NO 2 −, and lactate dehydrogenase levels in supernatants and cell lysates were analysed as well as the proliferation of IC-21 cells. MPA exposure reduced the total levels of all molecules investigated and suppressed the proliferation. All MPA-induced effects were reversed by the addition of guanosine to the cultures. Since macrophages play a role in lupus nephritis, our results indicate that modulation of macrophages may be involved in the ameliorating effects of MMF in SLE.

CTLA4IgG gene delivery prevents autoantibody production and lupus nephritis in MRL/ lpr mice

MRL MP - lpr lpr ( MRL lpr ) mice spontaneously develop an autoimmune syndrome closely resembling systemic lupus erythematosus (SLE) in humans, characterized by hypergammaglobulinemia, various autoantibody production, and the development of fatal glomerulonephritis. We have previously demonstrated that systemic administration of soluble form of CTLA4IgG prevented autoantibody-related diseases in MRL lpr mice. To test the potential protective effects of CTLA4IgG gene delivery on the development of lupus nephritis, we injected MRL lpr mice with a recombinant adenovirus vector containing CTLA4IgG gene, AdexlCACTLA4IgG (AdCTLA4IgG). It was demonstrated that a single administration of intravenous injection of AdCTLA4IgG into MRL lpr mice resulted in almost complete amelioration of lupus nephritis.

Dietary vitamin A deficiency and the immune system in a murine model of systemic lupus erythematosus

The purpose of this study was to investigate the effects of dietary vitamin A deficiency on the onset and progression of systemic lupus erythematosus (SLE), a severe autoimmune disorder, using the murine SLE model, MRL MPJ - lpr lpr ( MRL l ) mice. Sixty weanling female 4-week old MRL lpr mice were randomly assigned to either the vitamin A-deficient (A −), A-sufficient (A +), and A-sufficient diet/feed restricted (DR) groups. The weight losses due to the onset of SLE for both the A+ and the DR groups were greater than that for the A- group (P<0.01 at 16 weeks of treatment or 24 weeks of age). Both thymus and spleen weights as the percentage of body weights for the A- group were lower than those for the A + and the DR groups within 12 weeks of diet treatment (or 20 weeks of age). The degree of the abnormality in splenocyte composition, viz. an increased ratio of Thy 1.2 (total T cells) and a relatively decreased ratio of SmIg positive splenocytes (total B cells), was more severe in both the A + and the DR groups than in the A − group. This change suggests that vitamin A deficiency may suppress the proliferation of abnormal T splenocytes in MRL l mice. The results of T and B splenocyte proliferation stimulated by mitogens, either concanavalin A (Con A) or lipopolysaccharide (LPS) in vitro, indicated that vitamin A deficiency exerted a suppressive effect on the activities of abnormal T and B splenocytes of MRL l mice. The deficiency of vitamin A in the diet also suppressed serum anti-DNA autoantibody production in MRL l mice. In summary, it caused a suppression in autoimmunity as expressed by a decreased hyperactivity in both cellular and humoral immune functions in MRL l mice. The overall results suggest that dietary vitamin A deficiency retarded the progression of SLE and resulted in a better survival of MRL l mice.

Blood clearance of (1 → 3)-β- d-glucan in MRL [formula omitted] mice

(1 → 3)-β- d-Glucans have a variety of biological and immunopharmacological properties, and they are used clinically as biological response modifiers (BRMs). Clinically, these glucans have often been used for long periods by multiple dosing. During studies on the clearance and metabolism of the glucans in mice, we have found that, in the case of a single dose, the glucan was cleared from blood eventually, and remained constant in the organs for at least one month. Here, we investigated the clearance of glucans from the blood following multiple dosing using MRL lpr lpr mice with an autoimmune disease. Two kinds of glucans, GRN from Grifola frondosa and SSG from Sclerotinia sclerotiorum, were administered to the mice once a week for more than 35 weeks (250 μg/week/mouse by the intraperitoneal route). Examination of the blood clearance of the glucans in these mice revealed that the glucan concentrations were always high (about 20 μg/ml for GRN and 200 μg/ml for SSG). It is also shown that the glucans were significantly deposited in the liver and spleen of these mice. These findings suggest that administration of a large quantity of the glucan saturated the reticuloendothelial system, resulting in circulation of the glucan in the blood.

Two kappa immunoglobulin light chains are secreted by an anti-DNA hybridoma: Implications for isotypic exclusion

An anti-DNA hybridoma derived from an MRL lpr mouse secretes two different kappa light chains in combination with a single heavy chain. Multiple single cell clones express and secrete immunoglobulin containing both kappa light chains. The N-terminal protein sequences of the light chains correspond to sequences predicted from functionally rearranged mRNAs subjected to reverse transcription and amplified by polymerase chain reaction (PCR). Karyotype analysis of the hybridoma indicates a clonal line derived from the fusion of two cells. By amino acid sequence comparison and PCR analysis, both functional kappa light chains are derived from the MRL lpr spleen. The two functional light chain cDNAs were cloned and co-transfected into COS-7 cells with the heavy chain cDNA. Only one of the light chains in combination with mAb 3E10 heavy chain confers anti-DNA reactivity. The presence of two separate kappa light chains and, therefore, two separate antigen receptors on a single B cell may have ramifications for both polyclonal activation and toleration of lupus B cells.

Calorie restriction modulates lymphocyte subset phenotype and increases apoptosis in [formula omitted] mice

Defective expression of the Fas apoptotic gene may account for overproduction of CD4 −CD8 −B220 + cells (double-negative) in MRL/MpJ-lpr/lpr (lpr) mice. Previous studies have shown that calorie restriction (CR) inhibits the development of autoimmune disease and extends life span in these animals. The present studies describe the effects of CR on the distribution of lymphocyte phenotypes, lymphocyte proliferative response, and cytokine release. The effects of CR on dexamethasone (DEX)-induced apoptosis were also studied using propidium iodide (PI) uptake and DNA fragmentation in splenocytes and lymph node (LN) cells. Weanling female mice were fed a nutritionally adequate semipurified diet either ad libitum (AL) or with 40% fewer calories than AL (CR), and killed at 5 months of age. CR mice had fewer palpable lymph nodes, and decreased serum anti-dsDNA antibodies. Mitogen (ConA, anti-CD3, and LPS) and superantigen (SEB)-induced proliferative response was significantly lower in lymphoid cells from AL fed animals. FACS analysis of cells from CR animals showed decreased CD4 −CD8 − cells in spleen (1.7-fold, P < 0.025) and LN (1.6-fold, P < 0.01) and significantly higher CD4 + (spleen, 1.7-fold, P < 0.0001; LN, 2.6-fold, P < 0.025) and CD8 + (spleen, 1.6-fold, P < 0.001; LN, 5.2-fold, P < 0.005) cells. ConA-stimulated IL-2 release was increased in CR animals (splenocytes, 7.5-fold, P < 0.001; LN cells, 6.1-fold, P < 0.01). Finally, apoptosis in response to Dex was increased in CR animals as indicated by the presence of more PI-positive cells (spleen, 15.8%; LN, 10.7%; P < 0.01) and increased DNA fragmentation. In summary, the amelioration of autoimmune disease in MRL lpr mice by CR is accompanied by prevention of the rise in ‘double-negative’ T cells and by maintenance of lymphocyte responsiveness to mitogens and DEX-induced apoptosis at higher levels.

Glomerular binding activity in MRL lpr serum consists of antibodies that bind to a DNA/histone/type IV collagen complex.

To clarify the pathogenesis of lupus nephritis, we developed an assay that defines a glomerular binding activity (GBA) in both murine and human lupus sera highly correlated with nephritis. In the current study, we used a cross-adsorption strategy to establish that the GBA in MRL lpr serum binds to the glomerular basement membrane (GBM). We subsequently observed that this binding to the GBM was competitively inhibited by either exogenous DNA or histone, abrogated by pretreatment of the GBM with DNAse, and restored after DNase treatment with DNA/histone in a synergistic fashion. GBM binding was also completely inhibited by pretreatment of GBM with collagenase but not heparatinase. The effect of collagenase was not reversed by the subsequent addition of DNA, but was restored by the sequential re-addition of type IV collagen and DNA. By using purified basement membrane components, we found that MRL lpr serum bound avidly to DNA coated on type I collagen but less well (or not at all) to DNA coated on type IV collagen, laminin, or fibronectin. Histone pretreatment of type IV collagen before DNA addition, however, synergistically enhanced binding in a fashion similar to that seen with native GBM. Thus, the GBA in MRL lpr serum seems to be comprised of autoantibodies that bind to histones and/or DNA that adhere to type IV collagen within the GBM. These data support the planted Ag hypothesis as the principal pathogenic mechanism in lupus nephritis and suggest that multiple autoantibodies may contribute to this disorder.

Comparison of wasting syndrome in [ [formula omitted]] chimera and graft versus host disease in [ [formula omitted]] chimera and an attempt to transfer the wasting syndrome in [ [formula omitted]] to MRL +/+ mice

We compared the findings in the wasting syndrome seen in [ MRL lpr lpr → MRL +/+ ] chimeras with those of chronic graft versus host disease (GVHD) in [ B10.D2 → BALB c ] chimeras. BALB c mice were lethally irradiated and administered B10.D2 spleen and bone marrow cells. These mice are identical to MHC and Mls but differ as to genetic background. As a result of chronic GVHD, these [ B10.D2 → BALB c ] chimeras showed hair loss, weight loss and atrophy of lymph nodes and spleen beginning 5 weeks after the transplantation. MRL lpr lpr mice carry the lpr gene and spontaneously develop generalized lymph node swelling and lupus-like autoimmune disease, while congenic MRL +/+ mice lack the lpr gene. The [ MRL lpr lpr → MRL +/+ ] chimeras showed wasting and the same symptoms as in [ B10.D2- BALB c ] chimeras beginnng 16 weeks after cell transfer. Skin biopsy from both chimeras showed very similar changes on HE staining and on immunoperoxidase staining for Ia and Thy-1. Our data suggest that very small differences in minor histocompatibility may induce GVHD which produces severe wasting with lethal consequences. Finally, we succeeded in transferring the wasting syndrome seen in the [ MRL lpr lpr → MRL +/+ ] chimera to other MRL +/+ mice by transplanting spleen cells from the [ MRL lpr lpr → MRL +/+ ] chimera to lethally irradiated MRL +/+ mice.

Injections of Complexes made of dsDNA and Specific Polyclonal Antibodies Extend MRL LPR Mouse Survival: a Pilot Study

Antibodies towards double-strain (ds) DNA are responsible for the development of lupus nephritis both in human and animal models. A method by which one would suppress the production of pathogenic idiotypes could therefore prevent the development of nephritis. To this end, we prepared polyclonal anti-dsDNA antibodies by immunoaffinity from a serum pool of MRL/MpJ-lpr mice, a strain that develops an early form of nephritis identical to its human counterpart. Antigen-antibody complexes were prepared by addition of dsDNA. Such complexes have the potential of altering the anti-DNA antibody response and boosting the production of specific anti-idiotypic antibodies. Two groups of 14 MRL lpr mice were treated by regular intraperitoneal injections of 10 micrograms dsDNA-anti-dsDNA complexes or carrier buffer, starting at the age of 4 weeks, namely, prior to the appearance of nephritogenic anti-dsDNA IgG antibodies. We show here that such a treatment significantly extended the survival of treated mice compared with the control group. Five treated mice were still alive at month 11 compared with two in the control group. In addition, microscopic kidney examination at the time of death showed less lesions in the treated group compared with controls. This study indicates that complexes made of dsDNA and anti-dsDNA can delay the development of nephritis in the MRL lpr mouse strain.

Composition of peritoneal macrophage membranes in autoimmune MRL lpr/lpr mice

Adult MRL lpr / lpr mice display phenotypic features that are consistent with both rheumatoid arthritis and systematic lupus erythematosus. Previous studies have reported that peritoneal macrophages harvested from this model have an increased propensity for both spontaneous and elicited release of prostaglandins and leukotrienes relative to immunologically normal control mice. To investigate whether one aspect of the differences in secretory potential between autoimmune and normal mice was at the level of increased substrate availability, gas chromatographic analysis of peritoneal macrophage membranes from autoimmune MRL lpr / lpr , young lpr, wild type ±/±, and immunologically normal mice was done. The results demonstrate enrichment of arachidonate in adult lpr macrophage membranes in all major phospholipid classes relative to young lpr, ±/± and immunologically normal C3H/HeN mice. Similarly, there was an increased mole % of arachidonic acid in lpr mice relative to controls. Elevated membrane arachidonate may contribute to the increased propensity of autoimmune strains to participate in the inflammatory process.

Comparison of wasting syndrome in [ [formula omitted]] chimera and graft versus host disease in [ [formula omitted]] chimera and an attempt to transfer the wasting syndrome in [ [formula omitted]] chimera to MRL+/+ mice

Abstract We compared the findings in the wasting syndrome seen in [ MRL lpr lpr → MRL +/+ ] chimeras with those of chronic graft versus host disease (GVHD) in [ B10.D2 → BALB c ] chimeras. BALB c mice were lethally irradiated and administered B10.D2 spleen and bone marrow cells. These mice are identical to MHC and Mls but differ as to genetic background. As a result of chronic GVHD, these [ B10.D2 → BALB c ] chimeras showed hair loss, weight loss and atrophy of lymph nodes and spleen beginning 5 weeks after the transplantation. MRL lpr lpr mice carry the lpr gene and spontaneously develop generalized lymph node swelling and lupus-like autoimmune disease, while congenic MRL +/+ mice lack the lpr gene. The [ MRL lpr lpr → MRL +/+ ] chimeras showed wasting and the same symptoms as in [ B10.D2- BALB c ] chimeras beginnng 16 weeks after cell transfer. Skin biopsy from both chimeras showed very similar changes on HE staining and on immunoperoxidase staining for Ia and Thy-1. Our data suggest that very small differences in minor histocompatibility may induce GVHD which produces severe wasting with lethal consequences. Finally, we succeeded in transferring the wasting syndrome seen in the [ MRL lpr lpr → MRL +/+ ] chimera to other MRL +/+ mice by transplanting spleen cells from the [ MRL lpr lpr → MRL +/+ ] chimera to lethally irradiated MRL +/+ mice.

CD2-CD4-CD8- lymph node T lymphocytes in MRL lpr/lpr mice are derived from a CD2+CD4+CD8+ thymic precursor.

MRL lpr/lpr (lymphoproliferative, lpr) mice demonstrate an age-dependent lymphoproliferation and development of autoimmunity. Characteristic of the lymphoproliferation in these mice is the accumulation of large numbers of CD4-CD8-(CD4-8-),CD3+ T lymphocytes in their lymph nodes. The development of the CD4-8- cells, which also aberrantly express B220 and CD44 (Pgp-1) but are CD2-, has been shown to be thymus dependent. An unusual feature of lpr CD4-8-T lymphocytes is that although they appear unresponsive to stimulation, as defined by proliferation and IL-2 production, they have undergone thymic negative selection. As thymic deletion normally occurs at the CD4+CD8+ (CD4+8+) stage, this raises the dilemma that lpr CD4-8- T lymphocytes have either previously been CD4+8+, or they are able to undergo thymic selection as CD4-8- cells. We have addressed this question by examining the methylation status of the CD8 gene in MRL lpr CD4-8- lymph node cells. Demethylation of the CD8 gene has been shown to be an indicator of previous CD8 expression. We find that the CD8 gene in lpr CD4-8- lymph node cells, as well as in the abnormal B220+ CD4-8- lpr thymocytes, is demethylated, suggesting that these cells have previously expressed CD8. In addition, we find that the lpr CD4+8+ thymocyte population contains an increased percentage of atypical B220+, CD44+ cells that are virtually all CD2+. Taken together, these data are consistent with the lpr CD2-CD4-8- population of LNC having arisen from a CD2+ CD4+8+ thymic stage of differentiation.