Proclivity to self-injurious behavior in MRL-lpr mice: implications for autoimmunity-induced damage in the dopaminergic system.

Systemic lupus erythematosus is frequently accompanied by psychiatric manifestations of unknown origin. Although damage of central neurons had been documented, little is known about neurotransmitter systems affected by the autoimmune/inflammatory process. Recent studies on lupus-prone MRL-lpr mice point to imbalanced dopamine function and neurodegeneration in dopamine-rich brain regions. We follow up on anecdotal observations of singly housed mice developing chest wounds. Compulsive grooming and/or skin biting accounted for open lesions, lending itself to the operational term 'self-injurious behavior' (SIB). Low incidence of spontaneous SIB increased significantly after repeated injections of dopamine-2/3 receptor (D2/D3R) agonist quinpirole (QNP). To further probe the dopaminergic circuitry and examine whether SIB is associated with development of lupus-like disease, we compared behavioral responses among cohorts that differed in the immune status. Two-week treatment with QNP (intraperitoneal, 0.5 mg kg(-1) body weight per day) induced SIB in 60% of diseased MRL-lpr mice, and exacerbated their splenomegaly. Although increased grooming and stereotypy were observed in less symptomatic MRL+/+ controls, only one mouse (10%) developed SIB. Similarly, SIB was not seen in young, asymptomatic groups despite dissimilar ambulatory responses to QNP. In situ hybridization revealed treatment-independent upregulation of D2R mRNA in substantia nigra of diseased MRL-lpr mice. The above results suggest that development of systemic autoimmunity alters sensitivity of the dopaminergic system and renders MRL-lpr mice prone to SIB. Although pathogenic factors were not examined, we hypothesize that immune and endocrine mechanisms jointly contribute to early neuronal damage, which underlies behavioral deficiency in the adulthood.

Social isolation stress exacerbates autoimmune disease in MRL/lpr mice.

The potential physiological mechanisms explaining an influence of psychosocial stress on autoimmune diseases remain undetermined. Exposure of chronic social isolation stress to MRL/lpr mice significantly enhanced the degree of proteinuria after 20 weeks of age and reduced the survival rate. The serum anti-dsDNA IgG2a levels were increased significantly by stress at 19 weeks of age, which was simultaneously accompanied by inhibition of the serum corticosterone elevation. Furthermore, stress caused increased IFN-gamma production from anti-CD3-stimulated splenic mononuclear cells, whereas IL-4 and IL-10 production decreased. These results indicated that isolation stress exacerbated autoimmune disease in MRL/lpr mice, the possible mechanism for which might be related to stress-induced dysregulation of Th1/Th2 balance and inhibition of the blood corticosterone response to inflammatory stimuli.

Altered CNS response to injury in the MRL/MpJ mouse.

The MRL/MpJ mouse has a greatly enhanced healing response and an absence of scarring compared with other mouse strains. Following lesions to the CNS mammals show a scarring response known as reactive gliosis, and this CNS scar tissue blocks regeneration of cut axons. We have therefore compared reactive gliosis in the MRL/MpJ mouse and the Swiss Webster mouse, which exhibits normal scarring in the periphery. The lesion model was a stab lesion to the cortex, in which reactive gliosis has previously been quantified. Axon regeneration was examined following a cut lesion to the dopaminergic projection from the substantia nigra to the striatum used in previous regeneration experiments. In the MRL/MpJ following the lesion compared with Swiss Webster mice there was greater cell loss around the lesion followed by greater and more widespread and more prolonged cellular proliferation. Early after the lesion there was a greater loss of glial fibrillary acidic protein (GFAP)-positive astrocytes around the injury site in the MRL/MpJ, and an enhancement and prolongation of the microglial inflammatory response. This was accompanied by greater and more widespread blood-brain barrier leakage following injury. RNA levels for the matrix metalloproteinases (MMP)-2 and MMP-9 as well as for the thrombin receptors PAR-1 and PAR-4 were also greater at the MRL/MpJ injury site. All of these differences were transient and by 14 days post-injury there were no differences observed between MRL/MpJ and control mice. No axonal regeneration was observed following axotomy to the nigrostriatal pathway of the MRL/MpJ or the Swiss Webster mice at any time point.

Spallanzani's mouse: a model of restoration and regeneration.

The ability to regenerate is thought to be a lost phenotype in mammals, though there are certainly sporadic examples of mammalian regeneration. Our laboratory has identified a strain of mouse, the MRL mouse, which has a unique capacity to heal complex tissue in an epimorphic fashion, i.e., to restore a damaged limb or organ to its normal structure and function. Initial studies using through-and-through ear punches showed rapid full closure of the ear holes with cartilage growth, new hair follicles, and normal tissue architecture reminiscent of regeneration seen in amphibians as opposed to the scarring usually seen in mammals. Since the ear hole closure phenotype is a quantitative trait, this has been used to show-through extensive breeding and backcrossing--that the trait is heritable. Such analysis reveals that there is a complex genetic basis for this trait with multiple loci. One of the major phenotypes of the MRL mouse is a potent remodeling response with the absence or a reduced level of scarring. MRL healing is associated with the upregulation of the metalloproteinases MMP-2 and MMP-9 and the downregulation of their inhibitors TIMP-2 and TIMP-3, both present in inflammatory cells such as neutrophils and macrophages. This model has more recently been extended to the heart. In this case, a cryoinjury to the right ventricle leads to near complete scarless healing in the MRL mouse whereas scarring is seen in the control mouse. In the MRL heart, bromodeoxyuridine uptake by cardiomyocytes filling the wound site can be seen 60 days after injury. This does not occur in the control mouse. Function in the MRL heart, as measured by echocardiography, returns to normal.

Relationship between the development of autoimmunity and sensorimotor gating in MRL-lpr mice with reduced IL-2 production.

MRL-lpr mice develop systemic lupus-like autoimmune disease associated with changes in emotional reactivity and spatial learning and memory. Although the major immunological deficit in MRL-lpr mice is uncontrolled lymphoproliferation associated with a Fas gene mutation, these mice have a marked deficit in interleukin-2 (IL-2) production which, when treated, can prevent the development of autoimmune disease. Moreover, both MRL-lpr and IL-2 knockout mice manifest alterations in hippocampal cytoarchitecture and cognitive behavior. We found previously that IL-2 knockout mice have alterations in prepulse inhibition (PPI), a measure of sensorimotor gating. Thus, the present study sought to test the hypothesis that that PPI would be altered in MRL-lpr mice. Compared to MRL(+/+) control mice, MRL-lpr mice exhibited different patterns of PPI during development. Whereas 7 and 12-week MRL-lpr mice with evidence of autoimmune disease (the onset and early stages, respectively) showed increased PPI, 5 week predisease MRL-lpr mice did not. MRL-lpr mice also exhibited increased acoustic startle reactivity that was independent of autoimmune disease. These behavioral changes were not associated with increased brain expression of the proinflammatory cytokines genes, IL-1alpha and IL-6, CD3, or c-myc-associated apoptosis.

Modulation of renal disease in MRL/lpr mice by pharmacologic inhibition of inducible nitric oxide synthase.

BACKGROUND: MRL-MPJFaslpr (MRL/lpr) mice spontaneously develop lupus-like disease characterized by immune complex glomerulonephritis and overproduction of nitric oxide (NO). Blocking NO production pharmacologically by a non-specific nitric oxide synthase (NOS) inhibitor ameliorated renal disease in MRL/lpr mice while genetically deficient inducible NOS (iNOS) mice developed proliferative glomerulonephritis similar to wild-type controls. METHODS: To clarify the role of iNOS in the pathogenesis of nephritis in MRL/lpr mice, we treated mice with two different NOS inhibitors. Either NG-monomethyl-l-arginine (L-NMMA), a nonspecific NOS inhibitor, or l-N6-(1-iminoethyl)lysine (L-NIL), an iNOS specific inhibitor, was administered in the drinking water from 10 through 22 weeks of age with disease progression monitored over time. Control mice received water alone. RESULTS: Both L-NMMA and L-NIL blocked NO production effectively in MRL/lpr mice. As expected, neither L-NNMA nor L-NIL had an effect on antibody production, immune complex deposition or complement activation. Although both NOS inhibitors decreased protein excretion, L-NMMA was more effective than L-NIL. Pathologic renal disease was significantly decreased at 19 weeks in both treatment groups. At 22 weeks the L-NIL treated mice, but not the L-NMMA mice, had significantly reduced renal disease scores compared to controls. CONCLUSION: These results indicate that specific inhibition of iNOS blocks the development of pathologic renal disease in MRL/lpr mice.

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.

Heart regeneration in adult MRL mice.

The reaction of cardiac tissue to acute injury involves interacting cascades of cellular and molecular responses that encompass inflammation, hormonal signaling, extracellular matrix remodeling, and compensatory adaptation of myocytes. Myocardial regeneration is observed in amphibians, whereas scar formation characterizes cardiac ventricular wound healing in a variety of mammalian injury models. We have previously shown that the MRL mouse strain has an extraordinary capacity to heal surgical wounds, a complex trait that maps to at least seven genetic loci. Here, we extend these studies to cardiac wounds and demonstrate that a severe transmural, cryogenically induced infarction of the right ventricle heals extensively within 60 days, with the restoration of normal myocardium and function. Scarring is markedly reduced in MRL mice compared with C57BL/6 mice, consistent with both the reduced hydroxyproline levels seen after injury and an elevated cardiomyocyte mitotic index of 10-20% for the MRL compared with 1-3% for the C57BL/6. The myocardial response to injury observed in these mice resembles the regenerative process seen in amphibians.

Nephritogenic cytokines and disease in MRL-Fas(lpr) kidneys are dependent on multiple T-cell subsets.

BACKGROUND: Renal parenchymal cells produce cytokines, colony-stimulating factor-1 (CSF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha), which recruit autoreactive T cells and, in turn, elicit renal injury in MRL-Fas(lpr) mice. METHODS: To determine whether select T-cell populations regulate intrarenal nephritogenic cytokines (CSF-1, GM-CSF, and TNF-alpha) and renal disease, we compared MRL-Fas(lpr) mice that are genetically deficient in T-cell receptor (TCR) alpha beta T cells, CD4 T cells, and major histocompatibility complex class I (MHC class I), lacking CD8 and double negative (DN) T cells, with wild-type mice. To identify the T cells instrumental in downstream (effector) events, we delivered CSF-1 or GM-CSF into the kidney via gene transfer in these select T-cell-deficient and wild-type strains. RESULTS: Intrarenal CSF-1, GM-CSF, and TNF-alpha were absent or dramatically reduced in TCR alpha beta, CD4, and class I-deficient MRL-Fas(lpr) strains as compared with wild-type mice. In addition, the decrease in CSF-1, GM-CSF, and TNF-alpha was associated with a reduced kidney leukocytic infiltrates and spontaneous autoimmune nephritis. Intrarenal ex vivo retroviral gene transfer of CSF-1 and GM-CSF failed to elicit nephritis in these T-cell-deficient MRL strains (TCR alpha beta, CD4, CD8/DN) as compared with wild-type mice. CONCLUSIONS: Multiple T-cell populations initiate renal disease by increasing intrarenal nephritogenic cytokines, CSF-1, GM-CSF, and TNF-alpha. CSF-1 and GM-CSF recruit additional CD4 and CD8 and DN T cells, which augment downstream events, resulting in progressive autoimmune renal disease. We suggest that MRL-Fas(lpr) kidney disease is driven by a T-cell amplification feedback loop dependent on multiple T-cell populations.

The Fas ligand/Fas system in renal injury.

The FasL-Fas system regulates renal cell apoptosis, as well as the immune and inflammatory responses. Evidence that FasL and Fas participate in renal injury may be summarized along modified Koch's postulates (Table 1): (i) FasL is expressed by renal cells and during renal injury, (ii) activation of the Fas receptor promotes apoptosis of cultured renal cells, (iii) Fas agonists induce glomerular injury but they may also decrease renal injury by limiting injurious immunological responses, (iv) mice with disrupted FasL/Fas systems are protected from tubular cell injury during ischaemia reperfusion, although they develop autoimmune glomerulonephritis if other genetic predisposing factors are present. FasL/Fas must be considered a new target for therapeutic intervention in renal injury. Therapeutic modulation of Fas should aim not only at protecting intrinsic glomerular or tubular epithelial cells from death, but also at modulating the immune, inflammatory, and fibrogenic responses. Possible therapeutic interventions include Fas agonists, soluble Fas receptors, or other antagonists, and targetting of Fas to undesired cells, such as fibroblasts, in order to decrease their numbers in a physiological manner through apoptosis. Any therapeutic attempt should carefully take into account the possible effects of interference with Fas in other 1833 cell systems. Given the complexities of the FasL/Fas system, further studies are warranted.

Ultrastructural pathology in the stria vascularis of the MRL-Fasl(lpr) mouse.

The MRL-Fas(lpr) mouse, a model of multisystemic, organ nonspecific autoimmune disease, has been proposed as a model of immune-mediated inner ear disease. A preliminary study employing light microscopy indicated that it develops cochlear pathology that appeared most striking in the stria vascularis, where cells underwent edema and degeneration. However, other structures, including the inner and outer hair cells and the supporting cells, also appeared to display pathology. The current study analyzed cochlear ultrastructure using transmission electron microscopy to better delineate the cochlear lesions found in these animals. MRL-Fas(lpr) animals were allowed to develop systemic disease (20 weeks old) and then had auditory brainstem response (ABR) thresholds determined. Animals were then killed and their cochleas prepared for electron microscopy. Age-matched MRL-+/+ and BALB/c mice served as controls. Results indicated that MRL-Fas(lpr) mice demonstrated elevated ABR thresholds. In contrast to a preliminary report, the cochlear pathology was observed exclusively in the stria vascularis, where cells demonstrated hydropic degeneration. Strial capillary structure was normal as were the rest of the cellular cochlear constituents. No inflammatory infiltrate was noted. These studies confirm that the MRL-Fas(lpr) mouse develops cochlear abnormalities focused in the stria vascularis. Whether the mechanism of the cellular degeneration involves autoimmune, genetic, or uremic processes has yet to be determined.

Blockade of CD28/CTLA4-B7 pathway prevented autoantibody-related diseases but not lung disease in MRL/lpr mice.

We studied the role of CD28/CTLA4 costimulatory T-cell activation pathway on the pathogenesis of MRL/lpr mice. Administration of CTLA4IgG from day 0 significantly inhibited autoantibody production such as anti-double-stranded DNA antibody and rheumatoid factor. In addition, end-organ diseases in kidney, salivary gland, and liver were significantly improved. Improvement of pathologic findings coincided with a significant improvement in survival. At 350 days of age, 90% of mice treated with CTLA4IgG from day 0 were still alive, compared with none of mice treated with hIgG. As expected, activation of conventional T cells was significantly inhibited after CTLA4IgG treatment. However, lung disease that was characterized by perivascular accumulation and interstitial infiltration of lymphocytes and macrophages was not inhibited. Even after CTLA4IgG treatment from day 0, pathologic findings of lung disease were not improved. Additionally, the expression of activation markers such as B7-1, B7-2, CD71, ICAM1, and LFA1 on Mac1+ fraction in both spleen and lung and the concentration of TNFalpha in bronchoalveolar lavage fluid were not significantly suppressed. These results demonstrated that lung disease was independent of the CD28/CTLA4-B7 pathway. Thus, this study emphasizes the differential dependence of the CD28/CTLA4-B7 pathway in development of diseases in MRL/lpr mice.

Rheumatic diseases in an MRL strain of mice with a deficit in the functional Fas ligand.

OBJECTIVE: To characterize Fas antigen expression on the cell surface, and to determine the effect of this expression in rheumatic diseases using a newly established gld-congenic MRL strain of mice (MRL/gld), which is defective in its functional Fas ligand (Fas-L). METHODS: Flow cytometric analyses of lymphoid cells and macrophages were performed using anti-Fas and other cell surface markers. Histopathologic manifestations were examined using immunochemistry and light and electron microscopy. Serum levels of IgG and anti-DNA antibodies were measured by single radial immunodiffusion and enzyme-linked immunosorbent assay, respectively. RESULTS: MRL/gld mice developed systemic lymphadenopathy with an accumulation of Thy1.2+, B220+ and CD4-, CD8- T cells, which both express the Fas antigen. Splenic B cells positive for surface IgM and/or surface IgD, and resident peritoneal macrophages exhibited up-regulated expression of the Fas antigen, at much higher levels than those observed in MRL/MpJ-+/+ (MRL/+) mice. Forms of rheumatic disease were observed in these mice, although not in C3H/HeJ-gld/gld mice. These forms included diffuse glomerulonephritis, granulomatous arteritis, and arthritis, and were associated with the infiltration of mononuclear cells expressing the Fas antigen. Serum levels of IgG and anti-DNA antibodies were significantly increased in MRL/gld mice compared with MRL/+ mice. CONCLUSION: Rheumatic disease was generated by the gld gene in mice with an MRL background, as it is by the lpr gene, which is a Fas deletion mutant, associated with autoimmune traits. Rheumatic disease in this MRL strain was initiated by an incapacity for Fas/Fas-L-induced apoptosis, resulting in the development of autoimmunity and allowing for a persistent immune response in the affected lesions.

Dysregulated transforming growth factor-beta in neonatal and adult autoimmune MRL-lpr mice.

Transforming growth factor- beta (TGF- beta) is a cytokine that promotes inflammatory processes and prevents tissue injury. Autoimmune destruction of the kidney in MRL-lpr mice is spontaneous, rapid, fatal and consists of glomerular damage and an influx of lymphocytes surrounding vessels and in the interstitium. In MRL-lpr mice, cytokine dysregulation is apparent in neonates and continues throughout the life span. Circulating levels of tumour necrosis factor (TNF- alpha) and colony stimulating factor-1 (CSF-1) are detected in neonatal mice and progressively increase in proportion to the loss of renal function. We now report elevated intracellular expression of distinct isoforms of TGF- beta (TGF- beta 3, TGF- beta 2, and TGF- beta 1) detected immunohistochemically in MRL-lpr kidneys and other tissues including the liver and thymus. Enhanced TGF- beta 3 and TGF- beta 2 isoforms are detectable in neonatal mice within the renal tubular epithelial cells (TEC) and vascular smooth muscle cells (VSMC). In MRL-lpr mice 4-6 months of age, TGF- beta 2 and TGF- beta 1 are detected in TEC, VSMC, glomerular epithelial cells (GEC) and in perivascular infiltrating cells. By comparison, TGF- beta is minimally detectable in the normal kidneys of age and sex matched MRL(-)+2 or C3H/Fej mice. Paradoxically, in vitro cultured TEC and VSMC from MRL-lpr mice secrete less TGF- beta than TEC and VSMC isolated from MRL(-)+2 or C3H/FeJ mice. TNF- alpha, but not IL-6, CSF-1, or IFN- gamma stimulated the secretion of TGF- beta in TEC and VSMC. Our data demonstrate the dysregulation of TGF- beta isoforms in neonatal and adult MRL-lpr mice prior to and after the onset of autoimmune renal disease. We suggest that TNF- alpha and/or other molecules increase TGF- beta expression in MRL-lpr mice. We speculate that enhanced expression of TGF- beta promotes autoinmune renal injury in MRL-lpr mice.