MRL-lpr Mice Research Articles

Isolation and Characterization of A T-Suppressor Factor Specific for the Lupus-Associated Autoantigen RNP-Sm

We have previously described a monoclonal antibody, B16G, which has been found to be specific for T-cell derived suppressor factors (TsF). B16G has been shown to react with T-suppressor cells, TsF in the spleen of normal or tumor-bearing mice, the TsF produced by tumour-specific, or hapten-specific T-cell hybridomas, and with polyclonal whole human TsF isolated from tonsillar tissue. This pan-reactivity inherent to the B16G antibody suggests that it recognizes some common, shared epitope of the TsF molecule. In this study, we have used B16G as a probe to isolate a TsF-producing T-cell hybridoma, S-50, from CBA mice (H-2k) that is specific for the Lupus-associated antigen, RNP-Sm. The TsF bound specifically to RNP-Sm and inhibited the production of anti-RNP-Sm antibody cell cultures from MRL-lpr mice. SDS-PAGE analysis of purified S-50 TsF revealed a B16G-reactive band with a molecular weight of 43 kd.

Novel and enhanced IL-1 gene expression in autoimmune mice with lupus.

IL-1 is a pleiotropic factor encoded for by at least two genes, alpha and beta, and capable of eliciting a broad set of immunologic and inflammatory events. MRL/MP-lpr (MRL-lpr) mice are an appealing model for studies of renal injury inasmuch as disease in this strain is spontaneous, rapid, predictable, and regulated by the lpr gene. Infiltration of macrophages and the proliferation of the glomerular mesangial cells are prominent features of renal disease. Because both mesangial cells and macrophages can synthesize IL-1, the purpose of this study was to determine whether enhanced IL-1 gene expression is associated with lupus nephritis in the MRL-lpr mouse model. Glomerular macrophages, abundant in the kidneys of MRL-lpr mice but rarely present in the kidney of congenic MRL/MP-++(MRL-++) mice, were isolated and cultured and found to express a 10-fold increase in both IL-1 alpha and IL-1 beta mRNA transcripts as compared with MRL-++ and MRL-lpr mesangial cells. IL-1 alpha was not detected in the total RNA extracted from freshly excised kidney, whereas IL-1 beta transcripts were detected in both the renal cortex of MRL-lpr as well as MRL-++ animals. A previously undetected truncated 1200 nucleotide IL-1 beta transcript together with the conventional 1600 nucleotide IL-1 beta transcript was found in kidneys from MRL-lpr and was abundantly expressed in glomeruli of MRL-lpr mice with lupus nephritis. Isolated glomeruli from MRL-lpr mice with nephritis produce IL-1, whereas in normal glomeruli from MRL-++ and C3H/FeJ mice this cytokine was not detected. Glomerular macrophages and mesangial cells cultured from MRL-lpr mice with nephritis both secrete IL-1. These studies indicate that IL-1 beta gene expression and IL-1 protein are increased in the kidneys of autoimmune mice with lupus nephritis and is generated, at least in part, by glomerular macrophages. We speculate that an alteration in IL-1 beta gene expression may be responsible for causing a cascade of events leading to acute and chronic renal injury.

Effect of in vivo injection of recombinant human Interleukin-2 on peritoneal macrophages from [formula omitted] mice

Different characteristics of peritoneal macrophages have been studied, to assess the role of macrophages in the pathogenesis of MRL-lpr lpr mice which develop a lupus-like syndrome. Resident peritoneal macrophages from MRL-lpr lpr mice (> 10 weeks old) displayed characteristics of activation, while thioglycollate-elicited or resident macrophages from normal mice ( Balb c or MRL-+/+) did not. In addition to Ia antigens, macrophages spontaneously expressed Interleukin-2 receptors (IL2-R) whereas resident macrophages from normal mice did not. Injection of recombinant human Interleukin-2 (rHu-IL2) by the i.p. route to normal mice did not modify the cellular composition of the resident peritoneal population. On the contrary, rHu-IL2 treatment of MRL-lpr lpr mice induced an enhancement in cell number in the peritoneal cavity. At the same time, macrophages harvested from treated MRL-lpr lpr mice showed enhanced chemiluminescence triggered by phorbol-12-myristate-13-acetate (PMA) whereas peritoneal macrophages from treated normal mice did not. These results indicate that MRL-lpr lpr peritoneal macrophages display features of selective ‘activation’ and suggest that the expression of IL2-R could be involved in the pathogenesis of inflammatory disorders seen in MRL-lpr lpr autoimmunity.

Quantitation and IgG subclass distribution of antichromatin autoantibodies in SLE mice

Antibodies to a native chromatin preparation were found in most mice suffering from spontaneous SLE. All MRL Mp - lpr lpr ( MRL lpr ) sera tested (more than 500) contained antibodies to chromatin and antichromatin levels increased with age. Approximately 50% of the IgG antichromatin antibody in the MRL lpr sera was of the IgG2a subclass, 30% IgG2b, 10% IgG1, and 10% IgG3. Interestingly, the relative restriction of antichromatin autoantibodies to the IgG2a subclass was apparent in MRL lpr mice as young as 1 month, well before the onset of lymphadenopathy. Antichromatin autoantibodies were also detectable in sera from MRL Mp -+/+ (MR//+), NZB, (NZB × NZW)F1 (B × W), and BXSB mice, but were not found in sera from normal mice. A similar subclass distribution skewed toward IgG2a was seen for MRL/+, B × W, and NZB mice. These results indicate that the spontaneous autoantibody directed against chromatin is a good marker for murine SLE, and is predominantly of the IgG2a subclass.

Phenotypes of immunocompetent cells and Ia antigen expression in oral mucosa and skin of autoimmune mouse strains.

The phenotypic characteristics of resident and infiltrating cells in skin and oral mucosa of MRL/Mp-lpr/lpr (MRL-lpr), MRL/Mp+/+ (MRL-(+)), NZB x NZW F1 (NZB/W) and Balb/c mice have been studied using monoclonal antibodies (Mabs) and immunoperoxidase staining with the Avidin-Biotin Complex method. Macroscopically evident skin lesions appeared spontaneously exclusively in aging MRL-lpr mice. Infiltration of lymphocytes was detected within the mucosa and the skin of MRL-lpr-mice. These lymphocytes expressed predominantly L3T4 and to a lesser extent Lyt-2 phenotype (T helper/inducer and T suppressor/cytotoxic subset, respectively). Class II major histocompatibility complex antigen (Ia) expression was detected on macrophages, dendritic cells and few lymphoid cells in normal oral mucosa and skin of all strains examined. However, in the oral mucosa and skin lesions of the 4 months old MRL-lpr mouse also keratinocytes expressed Ia antigens. Keratinocytic Ia expression was also detected in oral mucosa of 8 months old MRL-(+) mice. The described immunopathological findings in skin and oral mucosa of old MRL-lpr mice show a number of important similarities to human SLE disease.

Rôle du placenta dans le maintien de l'allogreffe fétale

Present knowledge of the alloantigenic status of the placenta, which makes it a natural semi-allogeneic allograft is briefly surveyed. The systemic immunoregulatory mechanisms operating during normal allopregnancy which are not a prerequisite for a successful pregnancy are recalled. The placenta--dependent local mechanisms, e.g. trophoblast dependent decidual suppressor cells, factor mediated and factor independent resistance to cell mediated lysis, are surveyed as well as some of the mechanisms of action of trophoblast regulatory factors, namely suppressor cell induction and inhibition of IL-2 dependent cell growth/activation. The main feature of the CBA x DBA/2 model of spontaneous abortions in mice, and its prevention by anti Balb/c leukocyte immunisation are described. It was shown that anti-T cell depletion prevents the anti-abortive effects of immunisation. Such a treatment is also able to restore normal placental weight in auto-immune MRL lpr mice, which are known to display excess seric CSF beta-like activity (CSFs being in vitro efficient growth factors for trophoblasts). Transfer of such cells into the MHC compatible CBA/J prevents resorbtions upon a subsequent mating with DBA/2. Thus, direct effects of CSFs beta 1 and beta 2 as anti abortifacient (IL3 and GM CSF) are described together with the abortificacient effects of TNFs and NKs activators.

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.

Functional alterations of macrophages in autoimmune MRL-lpr/lpr mice.

To assess the role of macrophages (MAC) in the pathogenesis of systemic lupus erythematosus, we investigated functional aspects of peritoneal MAC obtained from autoimmune MRL/MpJ-lpr/lpr (MRL-lpr) mice. MRL-lpr and control C3H/HeN MAC were obtained from untreated mice or mice injected i.p. with 1 ml of 10% sterile peptone 3 days before cell harvest. MRL-lpr mice had significantly more peritoneal cells (MAC and lymphocytes) than did control mice. In endotoxin-free conditions, MRL-lpr MAC were similar to C3H/HeN MAC in their baseline, and IFN-gamma and/or LPS enhanced cytolysis of 3T12 fibrosarcoma tumor cells. Compared with C3H/HeN MAC, MRL-lpr MAC had a significant increase in antibody-dependent cellular cytotoxicity activity against sheep erythrocytes. This enhanced activity was not accompanied by a similar increase in adherence and/or phagocytosis of the same targets. Finally, in response to phorbol myristate acetate stimulation, both resident and peptone-induced MAC from MRL-lpr mice produced significantly more hydrogen peroxide than did those from control mice. These results indicate that MAC from MRL-lpr mice display features of selective "activation", and suggest that MAC or their products may play a role in the pathogenesis of inflammatory disorders seen in autoimmune diseases.

Correction of defective IL 3 responses of T lymphocytes from autoimmune mice.

MRL-+ and MRL-lpr congenic mice differ by the presence and expression of the homozygous recessive lymphoproliferation (lpr) gene. One manifestation of this gene is a massive T cell proliferation that results in a generalized lymphadenopathy in older animals. Interleukin 3 (IL 3), a recently described lymphokine, has been shown to influence lymphocyte differentiation. It was possible that abberrant IL 3 production was the mechanism responsible for the lpr controlled lymphadenopathy. Consequently, in this paper we tested the MRL congenic mice for their ability to produce IL 3. We report that the T lymphocytes from MRL-+ and MRL-lpr could neither respond to pokeweed mitogen in the induction of proliferation nor produce IL 3. Moreover, IL 3 was not produced constitutively nor could be induced at any time period up to 5 days in vitro. This hyporesponsiveness was shown to be controlled at the accessory cell level. Addition of T cell-depleted peritoneal exudate cells from normal major histocompatibility complex (MHC) compatible mice was able to restore the ability to secrete IL 3 in response to pokeweed mitogen in MRL-+ and young MRL-lpr mice. The defect in the accessory cells could be overridden by two means: the incorporation of phorbol myristate acetate in the induction system and preincubation of the cells in tissue culture.

Isolation of functional polyclonal TsF from MRL-lpr spleens using monoclonal antibody absorbance

We have previously described a monoclonal antibody, B16G, which has been found to be specific for T-cell derived suppressor factors (TsF). B16G has been shown to react with T-suppressor cells, TsF in the spleens of normal or tumor-bearing mice, the TsF produced by a tumorspecific T-cell hybridoma, and with polyclonal whole human TsF isolated from tonsilar tissue. This panreactivity inherent to the B16G MAb has made it clear that it recognizes some common, shared epitope of the TsF molecule. In this study we have used B16G as a probe to isolate TsF from the spleens of MRL-lpr mice and compare the activity with these factors isolated from the spleens of an MHC compatible nonautoimmune strain, CBA. We find that equivalent quantities of functional TsF are isolable from both strains and thus, it can be concluded that the associated oligoclonal B-cell activation characteristic of MRL-lpr mice is not due to a polyclonal T-suppressor cell deficit, nor to the ability of TsC in these mice to produce soluble, functional TsFs. The molecular and biochemical characteristics of these TsFs are discussed.

Interstitial pneumonitis in autoimmune [formula omitted] mice and its treatment with cyclosporin A

Age-associated changes of anti-double-stranded (ds) DNA antibodies, anti-single-stranded (ss) DNA antibodies, and serum immune complex concentrations were studied in MRL lpr mice. All anti-ds DNA antibodies, anti-ss DNA antibodies, and immune complexes began to be detected in the sera of MRL lpr mice aged 8 to 13 weeks and increased remarkably after 17 weeks of age. Almost no pathological findings were observed histologically in the lungs of MRL lpr mice aged 8 weeks but interstitial pneumonitis became evident at 14 weeks of age. Peribronchial and perivascular lymphocyte infiltrations were seen in the lungs of 14-week-old MRL lpr mice and became more severe at 21 weeks of age. Oral administration of cyclosporin A to 15-week-old MRL lpr mice markedly prolonged their life span. The lungs of 44-week-old MRL lpr mice given cyclosporin A showed few pathological findings except for minimal perivascular lymphocyte infiltration.

Strain-dependence and cellular aspects of the acceleration of age-dependent shift in class-specific helper and suppressor activity in the thymus of MRL/Mp mice by the LPR gene

Previous studies of the immunoregulatory activity of thymocytes from SJL/J mice have shown loss of suppressor activity for the antibody response by 24 weeks of age with appearance of helper activity. At the same time, suppressor cells developed which inhibit the generation of cytotoxic T lymphocytes (CTL). We now show a similar pattern of helper and suppressor activity in MRL/Mp mice. Presence of the lpr/lpr genotype significantly accelerated the onset of these changes in thymocyte activity. A similar pattern of thymocyte activity was not detected in C57Bl/6 mice. In aged MRL-lpr mice, evidence of increased suppressor cell activity for the CTL response could be demonstrated in spleen, and the suppressor was sensitive to treatment with anti-thy 1.2 + complement. The magnitude of the deficiency in the CTL response in MRL-lpr mice was greater than could be accounted for by suppressor cell activity alone. Measurement of the frequency of CTL precursors (CTLP), the yield of CTL per CTLP, and the ability to produce and to respond to interleukin 2 (IL-2) indicated that a drop in CTLP frequency, subnormal generation of IL-2, and probably an intrinsic defect in the responsiveness of MRL-lpr CTLP to IL-2 was contributing to the defective CTL response. We were not able to link suppressor T cells with reduced responsiveness to IL-2. Ageing involves different patterns of change in immunoregulatory T-cell subsets in different strains of mice, depending on their genetic constitution. The general implications of this conclusion for prediction of immune dysfunction with age in genetically distinct members of an outbred population are discussed.

A fish oil diet rich in eicosapentaenoic acid reduces cyclooxygenase metabolites, and suppresses lupus in MRL-lpr mice.

Dietary supplementation of fish oil as the exclusive source of lipid suppresses autoimmune lupus in MRL-lpr mice. This marine oil diet decreases the lymphoid hyperplasia regulated by the lpr gene, prevents an increase in macrophage surface Ia expression, reduces the formation of circulating retroviral gp70 immune complexes, delays the onset of renal disease, and prolongs survival. We show that a fatty acid component uniquely present in fish oil but not in vegetable oil decreases the quantity of dienoic prostaglandin E, thromboxane B, and prostacyclin normally synthesized by multiple tissues, including kidney, lung, and macrophages, and promotes the synthesis of small amounts of trienoic prostaglandin in autoimmune mice. We suggest that this change in endogenous cyclooxygenase metabolite synthesis directly suppresses immunologic and/or inflammatory mediators of murine lupus.

The lymphoproliferating cells of MRL-lpr/lpr mice are a polyclonal population that bear the T lymphocyte receptor for antigen.

Mice bearing the recessive gene lpr develop an age-dependent, massive lymphoproliferation, primarily in the lymph nodes (LN), with associated autoimmunity. LN cells from these mice express T cell receptor protein on the cell surface at 50-70% of normal levels. Normal levels of T cell receptor alpha, beta and gamma mRNA were found in these cells as compared to normal LN cells. Southern blot analysis of MRL-lpr/lpr LN DNA showed rearrangements in 80-90% of the chromosomes at the beta gene loci. The pattern of rearrangement indicated that a polyclonal rather than monoclonal expansion of T cells occurred. These data support a lymphokine-induction model of lymphoproliferation in MRL-lpr mice.

Dissociation of severe lupus-like disease from polyclonal B cell activation and IL 2 deficiency in C3H-lpr/lpr mice.

The lymphoproliferation characteristic of all strains of mice homozygous for the gene lpr results from the expansion of an unusual subset of cells that express reduced levels of Ly-1 and Thy-1 antigens and high levels of Ly-5(B220), an antigen that is normally only detected on cells of the B lineage. In the present study, C3H-lpr mice were studied to determine when this population of cells first appears in lymph node (LN) and spleen and whether its appearance relates to the development of B cell activation and deficiencies in interleukin 2 (IL 2) production. The results showed that Ly-5(B220)+, sIg- cells were first detected in LN at 4 wk of age; thereafter their numbers increased exponentially until at 16 wk of age they represented more than 80% of LN cells. Two subpopulations of Ly-5(B220)+, sIg- cells were present in LN; one Ly-1+, Thy-1+ and the other Ly-1+, Thy-1-. Ly-5(B220)+, sIg- cells were not detected in C3H-lpr spleen until 6 to 10 wks after their appearance in LN, and their proportions never reached those in LN. Polyclonal B cell activation in C3H-lpr spleens was not observed until Ly-5(B220)+, sIg- cells were present, suggesting that this population may play a role in B cell stimulation. IL 2 production by C3H-lpr spleen and LN cells was normal up to 6 wk of age and was significantly impaired thereafter, with LN being more severely affected than spleen. The IL 2 defect could be significantly repaired by the addition of PMA to the cultures. Although defective IL 2 production coincided with the appearance of Ly-5(B220)+, sIg- cells in LN, it preceded the appearance of these cells in spleen. In spite of the impaired ability to produce IL 2 in vitro, CTL responses to alloantigens were normal. Although C3H-lpr mice share many of the lymphoid abnormalities observed in MRL-lpr mice, they do not develop severe, early-onset SLE-like disease characteristic of the latter strain. This suggests that factors other than defective IL 2 production and polyclonal B cell activation are required for the development of fulminant autoimmune disease.

Limiting dilution analysis of interleukin 2 and colony-stimulating factor producer cells in normal and autoimmune mice.

MRL/MP lpr-lpr (MRL-lpr) mice spontaneously develop an age-related disease characteristic of human systemic lupus erythematosus (SLE). Old MRL-lpr mice (4 mo of age) develop antibodies to nucleic acids, display immune complex glomerulonephritis, and have a massive T cell-associated lymphadenopathy. In concert with disease development is data showing an age-related loss of interleukin 2 (IL 2) production by mitogen-stimulated lymphoid cells from these mice. The loss of IL 2 production has been suggested to be involved in the onset and/or development of autoimmune disease seen in these animals. In this report, we examined the frequency of both IL 2 and colony-stimulating factor (CSF) producer T cells in the MRL-lpr mouse by using a limiting dilution analysis assay. Our results show that the number of IL 2 and CSF producer cells present in autoimmune animals is similar to the number found in normal control mice. In addition, IL 2 and CSF producer T cells from autoimmune MRL-lpr mice make similar levels of lymphokine activity, as do producer T cells from normal mice. Our data argue against the previously hypothesized role that a paucity of IL 2 production may be involved in the etiology of autoimmune disease.

A monoclonal antibody (100C5) to the Lyt-2-T cell population expanding in MRL/Mp-lpr/lpr mice detects a surface antigen normally expressed on Lyt-2+ cells and B cells.

MRL/Mp-lpr/lpr (MRL-lpr) mice develop a generalized lymphadenopathy reflecting the expansion of a Lyt-2- T cell population. The present report describes the pattern of reactivity of a xenogeneic monoclonal antibody (mAb 100C5) directed against this T cell population. By single- and two-color flow cytofluorometry analysis this mAb was found to stain brightly 80-90% of T cells in enlarged MRL-lpr lymph nodes. In contrast lymph node T cells from congenic MRL-MP-+/+ (MRL-+) mice were either unstained (70%) or weakly stained (30%), these latter cells being mostly Lyt-2+. Unexpectedly, all lymph node B cells from MRL-+ or MRL-lpr mice were as strongly 100C5+ as MRL-lpr T cells. Similar observations were made in C57BL/6-lpr/lpr and C57BL/6-+/+ mice. Molecular weight determination suggested that the 100C5 mAb binds to the same molecule (Mr = 220 000) on MRL-lpr T cells and normal B cells.

In vitro correction of the interleukin 2 defect of autoimmune mice.

The effect of concanavalin A (Con A) and/or phorbol myristate acetate (PMA) on interleukin 2 (IL 2) production and tritiated thymidine incorporation was measured in young (6 weeks) and old (16-24 weeks) autoimmune mice by pulsing 5 X 10(6) unfractionated spleen cells with 5 micrograms of Con A and/or 5 ng of PMA for variable periods of time. The apparent deficiency in Con A-stimulated IL 2 production manifested by mice prone to the development of autoimmune disease was repaired by the addition of PMA. PMA did not enhance interleukin 1 (IL 1) secretion in autoimmune MRL-lpr mice either alone or in combination with Con A. The addition of purified IL 1 to Con A-pulsed autoimmune cells did not increase IL 2 production. Freeze-thaw experiments suggested that PMA does not promote IL 2 synthesis. Con A-pulsed cells plus PMA-pulsed cells do not syngergize. These data indicate that autoimmune-prone mice are capable of producing IL 2 and of proliferating in response to Con A provided the comitogen PMA is present. It can be argued that the failure to produce IL 2 or to respond to proliferative signals is not fundamental to the development of the disease sustained by autoimmune-prone mice.

Thymic influences on autoimmunity in MRL-lpr mice.

We have examined the role of the thymus in the development of autoimmunity in MRL/Mp-lpr/lpr (MRL-lpr) mice. MRL-lpr mice develop a lymphoproliferative disorder characterized by features of systemic lupus erythematosus and by massive proliferation of a subpopulation of Lyt-1+23- T cells. Using fluorescein-conjugated monoclonal antibodies and the fluorescence-activated cell sorter, we have found an abnormal pattern of differentiation within the MRL-lpr thymus characterized by a loss of Lyt-123+ thymocytes and an increased frequency of Lyt-1+23- thymocytes. Neonatal thymectomy retarded lymphoproliferation, reduced autoantibody concentrations, improved renal function, and prolonged life. Furthermore, neonatal thymectomy resulted in a relatively specific elimination of the subset of T cells involved in the lymphoproliferative process. These findings suggest that thymic maturation of T cells with alloantigenic characteristics of a helper subpopulation may contribute to the marked lymphoproliferation and severe autoimmunity of MRL-lpr mice. Neonatal thymectomy may protect against autoimmunity by preventing the maturation of this helper subpopulation.

Loss of resistance to listeria infection in autoimmune [formula omitted] mice: Protection by prostaglandin E 1

MRL lpr mice develop a severe SLE-like disease associated with a massive T-cell proliferation. Our studies show that this strain becomes incapable of resisting listeria infection only after there is an increase in their T cells. Age-matched congenic animals which lack the lpr gene never lose this resistance. In addition, treatment with PGE 1, which inhibits this T-cell expansion, prevents the loss of bacterial resistance in the MRL lpr mice. The discovery of this defect in MRL lpr mice, its relation to T-cell proliferation, and its protection by prostaglandin E provide a valuable system for investigating cell-mediated mechanisms operative in autoimmune disease.