Purine receptor antagonist modulates serology and affective behaviors in lupus-prone mice: evidence of autoimmune-induced pain?

Neurologic and psychiatric (NP) manifestations are severe complications of systemic lupus erythematosus (SLE). As commonly seen in patients, spontaneous disease onset in the MRL/MpJ-Fas(lpr)/J (MRL-lpr) mouse model of NP-SLE is accompanied by increased autoantibodies, pro-inflammatory cytokines and behavioral dysfunction which precede neuroinflammation and structural brain lesions. The role of purinergic receptors in the regulation of immunity and behavior remains largely unexplored in the field of neuropsychiatry. To examine the possibility that purinoception is involved in the development of affective behaviors, the P2X purinoceptor antagonist, suramin, was administered to lupus-prone mice from 5 to 14 weeks of age. In addition to food and water measures, novel object and sucrose preference tests were performed to assess neophobic anxiety- and anhedonic-like behaviors. Enzyme-linked immunosorbant assays for anti-nuclear antibodies (ANA) and pro-inflammatory cytokines were employed in immunopathological analyses. Changes in dendritic morphology in the hippocampal CA1 region were examined by a Golgi impregnation method. Suramin significantly lowered serum ANA and prevented behavioral deficits, but did not prevent neuronal atrophy in MRL-lpr animals. In a new batch of asymptomatic mice, systemic administration of corticosterone was found to induce aberrations in CA1 dendrites, comparable to the "stress" of chronic disease. The precise mechanism(s) through which purine receptor inhibition exerted beneficial effects is not known. The present data supports the hypothesis that activation of the peripheral immune system induces nociceptive-related behavioral symptomatology which is attenuated by the analgesic effects of suramin. Hypercortisolemia may also initiate neuronal damage, and metabolic perturbations may underlie neuro-immuno-endocrine imbalances in MRL-lpr mice.

Neuroimmunopathology in a murine model of neuropsychiatric lupus.

Animal models are extremely useful tools in defining pathogenesis and treatment of human disease. For many years researchers believed that structural damage to the brain of neuropsychiatric (NP) patients lead to abnormal mental function, but this possibility was not extensively explored until recently. Imaging studies of NP-systemic lupus erythematosus (SLE) support the notion that brain cell death accounts for the emergence of neurologic and psychiatric symptoms, and evidence suggests that it is an autoimmunity-induced brain disorder characterized by profound metabolic alterations and progressive neuronal loss. While there are a number of murine models of SLE, this article reviews recent literature on the immunological connections to neurodegeneration and behavioral dysfunction in the Fas-deficient MRL model of NP-SLE. Probable links between spontaneous peripheral immune activation, the subsequent central autoimmune/inflammatory responses in MRL/MpJ-Tnfrsf6(lpr) (MRL-lpr) mice and the sequential mode of events leading to Fas-independent neurodegenerative autoimmune-induced encephalitis will be reviewed. The role of hormones, alternative mechanisms of cell death, the impact of central dopaminergic degeneration on behavior, and germinal layer lesions on developmental/regenerative capacity of MRL-lpr brains will also be explored. This model can provide direction for future therapeutic interventions in patients with this complex neuroimmunological syndrome.

Impaired response to amphetamine and neuronal degeneration in the nucleus accumbens of autoimmune MRL-lpr mice.

Spontaneous development of lupus-like disease in MRL-lpr mice is accompanied by a constellation of behavioral deficits, including blunted responsiveness to sucrose. Although autoimmunity-induced damage of limbic areas is proposed to underlie this deficit, the systemic nature of the disease precludes inference of a causal relationship between CNS damage and functional loss. Based on the stimulatory effects of d-amphetamine sulfate (AMPH) on sucrose intake, the present study pharmacologically probes the functional status of central dopaminergic circuits involved in control of behavioral reward. The response rates were compared between diseased MRL-lpr mice and congenic MRL +/+ controls tested in the sucrose preference paradigm. Neuronal loss was assessed by Fluoro Jade B (FJB) staining of nucleus accumbens and the CA2/CA3 region. While control mice significantly increased intake of sucrose solutions 60 min after administration of AMPH (i.p., 0.5 mg/kg), the intake in drugged MRL-lpr mice was comparable to those given saline injection. Increased FJB staining was detected in the nucleus accumbens and hippocampus of diseased mice, and AMPH treatment neither altered this nor other measures of organ pathology. The results obtained are consistent with previously observed changes in the mesolimbic dopamine system of MRL-lpr mice and suggest that the lesion in the nucleus accumbens and deficits in dopamine release underlie impaired responsiveness to palatable stimulation during the progress of systemic autoimmune disease. As such, they point to a neurotransmitter-specific regional brain damage which may account for depressive behaviors in neuropsychiatric lupus erythematosus.

Elevated immunoglobulin levels in the cerebrospinal fluid from lupus-prone mice.

The systemic autoimmune disease lupus erythematosus (SLE) is frequently accompanied by neuropsychiatric manifestations and brain lesions of unknown etiology. The MRL-lpr mice show behavioral dysfunction concurrent with progression of a lupus-like disease, thus providing a valuable model in understanding the pathogenesis of autoimmunity-induced CNS damage. Profound neurodegeneration in the limbic system of MRL-lpr mice is associated with cytotoxicity of their cerebrospinal fluid (CSF) to mature and immature neurons. We have recently shown that IgG-rich CSF fraction largely accounts for this effect. The present study examines IgG levels in serum and CSF, as well as the permeability of the blood-brain barrier in mice that differ in immune status, age, and brain morphology. In comparison to young MRL-lpr mice and age-matched congenic controls, a significant elevation of IgG and albumin levels were detected in the CSF of aged autoimmune MRL-lpr mice. Two-dimensional gel electrophoresis and MALDI-TOF MS confirmed elevation in IgG heavy and Ig light chain isoforms in the CSF. Increased permeability of the blood-brain barrier correlated with neurodegeneration (as revealed by Fluoro Jade B staining) in periventricular areas. Although the source and specificity of neuropathogenic antibodies remain to be determined, these results support the hypothesis that a breached blood-brain barrier and IgG molecules are involved in the etiology of CNS damage during SLE-like disease.

Proliferating brain cells are a target of neurotoxic CSF in systemic autoimmune disease.

Brain atrophy, neurologic and psychiatric (NP) manifestations are common complications in the systemic autoimmune disease, lupus erythematosus (SLE). Here we show that the cerebrospinal fluid (CSF) from autoimmune MRL-lpr mice and a deceased NP-SLE patient reduce the viability of brain cells which proliferate in vitro. This detrimental effect was accompanied by periventricular neurodegeneration in the brains of autoimmune mice and profound in vivo neurotoxicity when their CSF was administered to the CNS of a rat. Multiple ionic responses with microfluorometry and protein peaks on electropherograms suggest more than one mechanism of cellular demise. Similar to the CSF from diseased MRL-lpr mice, the CSF from a deceased SLE patient with a history of psychosis, memory impairment, and seizures, reduced viability of the C17.2 neural stem cell line. Proposed mechanisms of cytotoxicity involve binding of intrathecally synthesized IgG autoantibodies to target(s) common to different mammalian species and neuronal populations. More importantly, these results indicate that the viability of proliferative neural cells can be compromised in systemic autoimmune disease. Antibody-mediated lesions of germinal layers may impair the regenerative capacity of the brain in NP-SLE and possibly, brain development and function in some forms of CNS disorders in which autoimmune phenomena have been documented.

Autoimmune-induced damage of the midbrain dopaminergic system in lupus-prone mice.

Spontaneous development of lupus-like disease is accompanied by impaired dopamine catabolism and degenerating axon terminals in the mesencephalon of MRL-lpr mice. We presently examine the hypothesis that systemic autoimmunity affects the central dopaminergic system in behaviorally impaired animals. The functional damage of the nigrostriatal pathway was assessed from rotational behavior after a single injection of the D1/D2-receptor agonist apomorphine. Neurodegeneration in the midbrain was estimated by Fluoro Jade B (FJB) staining. The causal role of autoimmunity was tested by comparing asymptomatic and diseased MRL-lpr mice, and by employing the immunosuppressive drug cyclophosphamide. Damage of dopaminergic neurons was assessed by tyrosine-hydroxylase (TH) staining of the midbrain. Apomorphine induced significant asymmetry in limb use, which lead to increased circling in the diseased MRL-lpr group. While FJB-positive somas were not seen in the striatum, increased staining in the substantia nigra (SN) and ventral tegmental area (VTA) were detected in behaviorally impaired MRL-lpr mice, but not in age-matched controls. Reduced brain mass and increased levels of TNF-alpha in their cerebrospinal fluid (CSF) suggested cerebral atrophy and inflammation. In addition, CSF was neurotoxic to a dopaminergic progenitor cell line. Immunosuppression attenuated CSF cytotoxicity, TNF-alpha levels, and midbrain neurodegeneration. Supportive of the notion that dying neurons were dopaminergic, the SN of autoimmune mice showed approximately a 35% reduction in the number of TH-positive cells. A three-fold increase in serum brain-reactive antibodies accompanied this loss. Although the source of toxic mediator(s) remains unknown, present results are consistent with the hypothesis that autoimmunity-induced destruction of mesonigral and mesolimbic dopaminergic pathways contributes to the etiology of aberrant behavior in an animal model of neuropsychiatric lupus.

Taste responsiveness and diet preference in autoimmune MRL mice.

One of the most profound behavioural deficits in lupus-prone MRL-lpr mice is blunted responsiveness to sweet solutions. Given the systemic nature of autoimmune/inflammatory disease, it was not clear whether impaired taste sensitivity or motivated response to palatable food underlie this deficit. The present study compares response rates of MRL-lpr mice (which develop disease early), congenic MRL +/+ mice (which develop disease later in life) and non-autoimmune Swiss Webster (SW) mice to different tastes and diets. Healthy SW mice showed the highest responsiveness to palatable stimulation throughout the study. Conversely, the preference for palatable solutions progressively declined in MRL-lpr mice as the disease developed. No differences between the two MRL substrains were seen in responsiveness to quinine or saline, suggesting that blunted responsiveness to palatable solutions cannot be accounted for by reduced taste sensory function (hypogeusia). In addition, changes in response rates to palatable solutions were associated with systemic upregulation of pro-inflammatory cytokines. With a new cohort of mice fed on carbohydrate-rich and fat-rich diets, we also examined whether reduced sucrose intake in MRL-lpr mice can be accounted for by a reduced craving for carbohydrates. Contrary to this expectation, diseased MRL-lpr mice preferred carbohydrate-rich food while consuming a food mass comparable to controls. These results further support the hypothesis that the onset of lupus-like disease alters motivated behaviour, independent of changes in neurologic function and food metabolism.

Neurodegeneration in autoimmune MRL-lpr mice as revealed by Fluoro Jade B staining.

As in many humans suffering from lupus erythematosus, the development of systemic autoimmunity and inflammation in Fas-deficient MRL-lpr mice is accompanied by CNS dysfunction of unknown etiology. Experimental studies revealed infiltration of lymphoid cells into the choroid plexus, reduced neuronal complexity, retarded brain growth, and enlargement of cerebral ventricles. Moreover, an increased presence of cells with nicked-DNA (TUNEL+ cells) in the periventricular areas suggested accelerated apoptosis in brain cells of MRL-lpr mice. However, direct evidence that the dying cells were neurons was lacking. For this purpose, we presently use Fluoro-Jade B (FJB), a novel fluorescent dye which has high affinity for dying neurons (both apoptotic and necrotic). As expected, in comparison to the control groups, the brains of diseased, 5-month-old MRL-lpr mice showed increased numbers of FJB-positive (+) cells in cortical and periventricular regions. The FJB+ cells were significantly more numerous than TUNEL+ cells, and only approximately 7% co-localized with TUNEL. Immunostaining for CD4 and CD8 markers did not correlate with the number of FJB+ cells, suggesting that T-lymphocyte infiltration into the brain tissue is not a reliable predictor of neuronal demise. Conversely, indices of systemic autoimmunity (splenomegaly and high serum anti-nuclear antibody levels) were associated with increased FJB+ cell numbers in brains of autoimmune MRL-lpr mice, supporting the causal link between autoimmunity and neurodegeneration. Taken together, the above results suggest that factors other than T-cell infiltration and cell death mechanisms other than Fas-mediated apoptosis dominate neuronal degeneration in lupus-prone MRL-lpr mice.

Ibuprofen fails to prevent brain pathology in a model of neuropsychiatric lupus.

OBJECTIVE: Neurologic and psychiatric manifestations are severe complications of systemic lupus erythematosus (SLE). As commonly seen in patients, spontaneous development of lupus-like disease in MRL-lpr mice is accompanied by brain atrophy and behavioral dysfunction. We examined inflammatory and ultrastructural aspects of central nervous system (CNS) involvement using a nonselective cyclooxygenase-2 (COX-2) inhibitor and measuring effects on behavior, microglial activation, and neuronal morphology. METHODS: Ibuprofen (IBU) was provided in a rodent chow (375 ppm) for animals 5-19 weeks of age. Exploration of a novel environment and performance in the forced swim test assessed effects on behavior. Immunohistochemistry, fluoro-Jade B (FJB) staining, and flow cytometry were employed in neuropathological analysis. Transmission electron microscopy was used to examine ultrastructural morphology of cortical, hippocampal, hypothalamic, nigral, and cerebellar cells. RESULTS: Chronic IBU treatment failed to normalize immune status, behavior, and brain mass in lupus-prone MRL-lpr mice. It also did not reduce density of CD3+ lymphocytes in the choroid plexus, or FJB+ neurons in the hypothalamus. Activated F4/80+ microglia increased with age, but IBU treatment was not effective in reducing their numbers. Although numerous dark cells were seen in functionally critical brain regions (e.g., paraventricular nucleus and subgranular zone), ultrastructural morphologies of classical apoptosis or necrosis were not detected. CONCLUSION: The COX-dependent pathway does not seem to be critical in the etiology of CNS disease in this model of neuropsychiatric lupus. Reduced brain mass, increased microglial activation, and condensation of cytoplasm point to a metabolic perturbation (e.g., excitotoxic damage) that compromises function and survival of central neurons during lupus-like disease.

Hippocampal damage in mouse and human forms of systemic autoimmune disease.

Systemic lupus erythematosus (SLE) is frequently accompanied by neuropsychiatric (NP) and cognitive deficits of unknown etiology. By using autoimmune MRL-lpr mice as an animal model of NP-SLE, we examine the relationship between autoimmunity, hippocampal damage, and behavioral dysfunction. Fluoro Jade B (FJB) staining and anti-ubiquitin (anti-Ub) immunocytochemistry were used to assess neuronal damage in young (asymptomatic) and aged (diseased) mice, while spontaneous alternation behavior (SAB) was used to estimate the severity of hippocampal dysfunction. The causal relationship between autoimmunity and neuropathology was tested by prolonged administration of the immunosuppressive drug cyclophosphamide (CY). In comparison to congenic MRL +/+ controls, SAB acquisition rates and performance in the "reversal" trial were impaired in diseased MRL-lpr mice, suggesting limited use of the spatial learning strategy. FJB-positive neurons and anti-Ub particles were frequent in the CA3 region. Conversely, CY treatment attenuated the SAB deficit and overall FJB staining. Similarly to mouse brain, the hippocampus from a patient who died from NP-SLE showed reduced neuronal density in the CA3 region and dentate gyrus, as well as increased FJB positivity in these regions. Gliosis and neuronal loss were observed in the gray matter, and T lymphocytes and stromal calcifications were common in the choroid plexus. Taken together, these results suggest that systemic autoimmunity induces significant hippocampal damage, which may underlie affective and cognitive deficits in NP-SLE.