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.     

Prednisone effects on blood-brain barrier permeability and CNS IgG synthesis in healthy humans

Corticosteroids reportedly decrease blood-brain barrier (BBB) permeability and/or IgG synthesis in patients with multiple sclerosis or brain tumors. However, these effects have not been studied in healthy humans. We investigated the effects of prednisone, 80 mg/day for five days, on the ratio of cerebrospinal fluid (CSF) albumin/serum albumin, a measure of blood-brain barrier (BBB) permeability, and on CSF and serum IgG levels in six healthy, normal volunteers. We found significant steroid-induced decreases in serum and CSF albumin levels and in serum IgG levels. However, we found only a nonsignificant decrease in BBB permeability and no significant change in CNS IgG synthesis. These findings, based on a small number of volunteers, suggest that it may be difficult to further decrease BBB permeability and CNS IgG synthesis in medically healthy subjects.     

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.     

Intrathecal antibodies and brain damage in autoimmune MRL mice

Neuropsychiatric (NP) manifestations and brain pathology are poorly understood and potentially fatal concomitants of systemic lupus erythematosus (SLE). For many years, autoantibodies to brain tissue (i.e., brain-reactive antibodies, BRA) were proposed as a key factor in pathogenesis of CNS manifestations. Recent evidence suggests that intrathecal BRA, rather than serum autoantibodies, are a better predictor of disturbed brain morphology and function. We presently test this hypothesis by examining the relationship among BRA in cerebrospinal fluid (CSF), behavioral deficits, and brain pathology in a well-established animal model of CNS lupus. We showed earlier that significant diversity in disease manifestations within genetically homogenous MRL-lpr mice allows for constructive and informative correlational analysis. Therefore, levels of CSF antibodies were presently correlated with behavioral, neuropathological and immune measures in a cohort of diseased MRL-lpr males (N = 40). ELISA, Western Blotting, standardized behavioral battery, digital planimetry, HE staining, and immunohistochemistry were employed in overall data collection. The IgG antibodies from CSF were binding to different regions of brain parenchyma, with dentate gyrus, amygdale, and subventricular zones showing enhanced immunoreactivity. High levels of CSF antibodies correlated with increased immobility in the forced-swim test and density of HE⁺ cells in the paraventricular nucleus. Peripheral measures of autoimmunity were associated with other deficits in behavior and neuropathology. This correlation pattern suggests that etiology of brain damage in lupus-prone mice is multifactorial. Intrathecal BRA may be important in altering motivated responses and activity of major neuroendocrine axes at the onset of SLE-like disease.     

Neurotoxic properties of cerebrospinal fluid from behaviorally impaired autoimmune mice

The chronic, lupus-like autoimmune disease in MRL-lpr mice is associated with leucocyte infiltration into the choroid plexus, brain cell death, and deficits in motivated behavior. The presence of lymphoid cells in the ventricular lumen and the increased number of TUNEL-positive cells in periventricular areas led to the hypothesis that immune cells enter into the cerebrospinal fluid (CSF) and induce primary neuronal damage in regions bordering the cerebral ventricles. Using an in vitro approach, we presently examine the possibility that CSF from autoimmune mice is neurotoxic and/or gliotoxic. The CSF and serum from diseased MRL-lpr mice, less symptomatic MRL +/+ controls, and healthy Swiss/Webster mice (non-autoimmune controls) were frozen until their effects on the viability of pyramidal neurons and astrocytes were assessed in a two-color fluorescence assay. Significant reduction in neuronal viability (in some cases as low as 67%) was observed in the co-cultures of hippocampal neurons and astrocytes incubated for 24 h with CSF from autoimmune MRL-lpr mice. The viability of astrocytes did not differ among the groups, and the CSF from autoimmune mice appeared more toxic than the serum. The behavior of MRL-lpr mice differed significantly from the control groups, as indicated by impaired exploration, reduced intake of palatable food, and excessive immobility in the forced swim test. The present results suggest that CSF from the behaviorally impaired lupus-prone mice is neurotoxic and are consistent with the hypothesis that neuroactive metabolites are produced intrathecally in neuropsychiatric lupus erythematosus.     

Abnormal cerebrospinal fluid protein indices in schizophrenia

Determinations of albumin and immunoglobulin G (IgG) were performed in paired cerebrospinal fluid (CSF) and serum samples from 24 subjects with schizophrenia. These determinations allowed calculation of two indices, one that is an indicator of integrity of the blood-brain barrier and the other a measure of selective IgG production within the central nervous system (CNS). In comparison with previously determined reference values, 7 of 24 (29%) subjects showed increased blood-brain barrier permeability, and 8 of 24 (33%) demonstrated elevated endogenous CNS IgG production. One of these eight also demonstrated oligoclonal banding on high-resolution protein electrophoresis of the CSF.     

Altered neuroendocrine status at the onset of CNS lupus-like disease

Neuropsychiatric (NP) manifestations and brain atrophy are common, etiologically unexplained complications of the systemic autoimmune disease lupus erythematosus (SLE). Similar to patients with NP SLE, behavioral deficits and neurodegeneration occur in aged, lupus-prone MRL/lpr mice. In order to gain a better understanding of the time course and nature of CNS involvement, we compare the neuro-immuno-endocrine profiles of two lupus-prone MRL/lpr stocks, which differ in disease onset and severity. Mice from stock 485 (characterized by early lupus-like manifestations) display blunted responsiveness to palatable solutions and impaired nocturnal activity as early as 7 weeks of age. They also have increased IgG in cerebrospinal fluid (CSF) before high serum autoantibody levels and splenomegaly are detected. Moreover, when compared to age-matched 6825 controls, 485 mice exhibit elevated serum corticosterone, enlarged left adrenal gland, and enhanced haematoxylin/eosin staining in the hypothalamic paraventricular nucleus. Swimming speed and novel object exploration become impaired only when more severe peripheral manifestations are documented in 17 week-old 485 mice. The obtained results suggest that performance deficits during the prodromal phase of NP SLE-like disease are associated with autoantibodies in CSF and asymmetric activation of the hypothalamus–pituitary–adrenal axis. Subsequent deterioration in behavioral performance evolves alongside systemic autoimmunity and inflammation. Although a leaky blood–CSF barrier is a possible explanation, one may hypothesize that, similar to neonatal lupus, maternal antibodies to brain antigens cross blood–placental barrier during embryogenesis and induce early endocrine and behavioral deficits in offspring.     

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.     

Distribution and prevalence of leukocyte phenotypes in brains of lupus-prone mice

Autoantibody-mediated compromise of central neurotransmission is a pathogenic mechanism proposed in etiology of neuropsychiatric lupus (NP-SLE). Recent experimental data support the hypothesis that intrathecally-synthesized antibodies play a key role in brain damage and behavioral dysfunction. However, autoantibody-producing plasma cells have not yet been detected in brain tissue. We presently use contemporary immunohistochemical markers and flow cytometry to assess distribution and prevalence of plasma cells and other phenotypes, which infiltrate brains of lupus-prone MRL-lpr mice. The functional status of infiltrates was confirmed by in situ hybridization for TNF-alpha mRNA. Consistent with the notion of breached blood-CSF and blood-brain barriers, CD3+ T-cells (approximately 20% of the mononuclear cell infiltrate) were plentiful in choroid plexuses and commonly seen around blood vessels. The CD138+ plasma cells were restricted to the choroid plexus and stria medullaris of diseased MRL-lpr mice. Although accounting for less than 1% of the total cell infiltrate, CD19+IgM+ B-cells increased with age in brains of MRL-lpr mice. Severe mononuclear cell infiltration was accompanied by splenomegaly and retarded brain growth. The results obtained support the hypothesis of progressive neurodegeneration as a consequence of leukocyte infiltration and intrathecal autoantibody synthesis. Further characterization of neuroactive antibodies and their targets may contribute to a better understanding of brain atrophy and behavioral dysfunction in the MRL model, and potentially in NP-SLE.     

Failure of intravenous and intrathecal cytarabine to modify central nervous system IgG synthesis in multiple sclerosis

To study the nature of the multiple sclerosis (MS) central nervous system (CNS) immune reaction, cytarabine (ara-C) was administered intravenously to three patients and intrathecally to another seven. Although intravenous administration severely suppressed circulating leukocytes derived fom the bone marrow, there was no change in de novo CNS IgG synthesis (rate or presence of CSF IgG oligoclones). Therefore, the MS CNS immune reaction can persist in the presence of severe granulocytopenia and severe monocytopenia of one to two weeks' duration. In four patients who were given ara-C intrathecally, cytotoxic levels (> 10 micrograms/ml) were present for a least 24 hours. A brief elevation in CSF albumin indicated transient damage to the blood-brain barrier. Leukopenia did not result in the seven patients treated intrathecally, and de novo CNS IgG synthesis did not change or was transiently increased. Assuming that cytotoxic levels in CSF diffused to the CNS sites responsible for synthesizing IgG, results indicate that CNS IgG synthesis does not depend on in situ rapid turnover of immune cells. None of the ten patients demonstrated neurological improvement or persistent adverse effects.     

益肾达络饮与变应性脑脊髓炎小鼠血脑屏障的完整性

为了验证益肾达络饮对实验性变应性脑脊髓炎小鼠血脑屏障完整性的保护作用,应用实时荧光定量RT-PCR和分光光度计检测实验性变应性脑脊髓炎小鼠脑和脊髓内T淋巴细胞表面标记物CD4、CD8以及单核细胞表面标记物CD11b mRNA的表达变化以及伊文思蓝含量。结果提示实验性变应性脑脊髓炎小鼠血脑屏障通透性增高与脑内CD4、CD8、CD11b mRNA表达的升高相关,应用益肾达络饮可以明显减轻实验性变应性脑脊髓炎小鼠大脑和脊髓炎性细胞的聚集。     

The evaluation of humoral immunity response in subacute sclerosing panencephalitis (SSPE)]

The purpose of the study was the assessment of humoral response markers in 20 patients with SSPE in phase I, II and III of the disease during immunomodulating treatment. In the CSF IgG levels were determined by nephalometric method, with its share in the total protein level, and with determination of the local IgG synthesis in CNS by Reiber method, 24-hour IgG synthesis by Tourtellotte-Boce method, IgG index by Link-Tibbling method and albumin index. Oligoclonal IgG was determined by isoelectrofocusing. All mathematical tests indicating IgG synthesis in CSF were abnormal in all cases, and also in all cases oligoclonal IgG was found (type 3 of electrophoresis separation pattern). The results point to far reaching changes of the humoral immune response in SSPE persisting also during immunomodulating treatment independently of disease phase and duration. In only 10% of cases abnormal values of the albumin index suggested damage to the blood-brain barrier.     

Increased TUNEL staining in brains of autoimmune Fas-deficient mice

Profound changes in brain morphology and behavior coincide with the spontaneous development of systemic autoimmune/inflammatory disease in Fas-deficient MRL-lpr mice. The dendrites atrophy, the density of hippocampal and cortical neurons decreases, and an anxious/depressive-like behavior emerges while lymphoid cells infiltrate into the choroid plexus of MRL-lpr mice. We hypothesized that the inherited lack of the Fas-dependent anti-inflammatory mechanism would lead to unsuppressed immune activity, characterized by reduced apoptosis in the MRL-lpr brain. Using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeled (TUNEL) method as an indicator of apoptosis, a surprisingly high incidence of TUNEL-positive cells was observed in the hippocampus, choroid plexus and periventricular regions of MRL-lpr mice, 5-10-fold higher than that found in the MRL +/+ control brain. Immunostaining with anti-CD3, CD4 and CD8 monoclonal antibodies showed limited overlap between CD-positive and TUNEL-positive cells, suggesting that the dying cells are for the most part (approximately 70%) not T-lymphocytes. Although further characterization of the phenotype of the dying cells and the mechanism of cell death are required, the present results suggest the involvement of a Fas-independent apoptotic process in neurodegeneration induced by systemic autoimmune disease.     

Autoantibody responses in the cerebrospinal fluid of guinea pigs with chronic relapsing experimental allergic encephalomyelitis

We report the use of the ELISA technique to measure IgG specific for whole cord, myelin, myelin basic protein and Mycobacterium tuberculosis in the cerebrospinal fluid (CSF) of Strain 13 guinea pigs in different stages of chronic relapsing experimental allergic encephalomyelitis (CR-EAE). Specific antibody levels to all 4 antigen preparations were related to the severity of clinical signs, with the highest levels of IgG in the CSF of guinea pigs in relapse or in stable chronic disease. Total IgG levels in the CSF, though elevated throughout the course of CR-EAE, did not show any association with the category of disease. Control animals inoculated with complete Freund's adjuvant (CFA) alone showed CSF IgG levels specific for M. tuberculosis that were not significantly different from those in animals with chronic EAE, indicating that CFA may itself induce a late-acting increase in blood-brain barrier permeability.     

The mouse blood-brain barrier and blood-nerve barrier for IgG: A tracer study by use of the avidin-biotin system

Summary To study the permeability of the blood-brain barrier (BBB) and the blood-nerve barrier (BNB) for immunoglobulin G (IgG) we adapted the avidin-biotin system for postembedding demonstration of the tracer IgG in the central and peripheral nervous system (CNS, PNS). Normal mouse and human IgG were biotinylated and injected daily into the intraperitoneal (i.p.) space of adult BDF₁ mice. After 24 h, IgG was detected in blood vessels and in the interstitium of various organs, but staining was restricted to the dura mater in the CNS, to the spinal ganglia, and to the perineurium of peripheral nerves. After 4 days, IgG was also present in the endoneurial connective tissue of peripheral nerves, while the brain, spinal cord, and spinal roots remained free of IgG. Our results show a partial permeability of the normal mouse BNB for homologous and heterologous IgG.Supported by the Deutsche Forschungsgemeinschaft SFB 200, B 5 and C 1, and by the Ministerium MWF (North-Rhine Westphalia)     

Characterization of brain lesions in a mouse model of progressive multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system characterized by damage to the neuronal myelin sheath, which results in different levels of muscle paralysis that can lead to neuronal death. In most MS mouse models, the neurologic damage mostly affects the spinal cord with limited damage to the brain, which cannot be monitored by magnetic resonance imaging (MRI) as used for humans. We show that immunization of non-obese diabetic (NOD) mice with myelin oligodendrocyte glycoprotein peptide 35-55 leads to the development of relapsing-remitting stages, evident from days 20 to 70, which then develops into a chronic progressive stage. This cycle is similar to MS stages found in humans. Brain MRI gadolinium-enhanced T1-weighted image analysis showed an increased blood-brain barrier permeability in brain gray and white matter specific to the corpus callosum, fimbria, and internal capsule as found in humans. MRI fractional anisotropy analysis showed demyelination and axonal damage in identical regions. Immunohistologic analysis supported the MRI data. No evidence of brain lesions was found in a common model of MS using C57BL/6 mice. We suggest that an increase in astrocyte toxicity in experimental autoimmune encephalomyelitis-induced NOD mice may be linked to brain lesion development. We suggest using NOD mice as a suitable model for studying MS using MRI methods toward future diagnostic and drug development.     

Blood-brain barrier in chronic relapsing experimental allergic encephalomyelitis: A correlative study between cerebrospinal fluid protein concentrations and tracer leakage in the central nervous system

Summary Blood-brain barrier (BBB) permeability in chronic relapsing experimental allergic encephalomyelitis was studied morphologically in tracer studies with horseradish peroxidase (HRP) as well as by quantitative determination of HRP, albumin, and IgG in serum and cerebrospinal fluid (CSF), BBB damage was found to be localized in demyelinating plaques and in blood vessels with vasculitis. Actively demyelinating lesions showed massive increase in BBB permeability, whereas in inactive or remyelinated lesions BBB damage was either minimal or absent. Determination of serum proteins in the CSF of animals with severe disease and a high incidence of actively demyelinating lesions showed evidence of BBB damge (reduction of Q-albumin) and an IgG-index in the normal range. In animals with only inactive lesions the Q-albumin was normal, the IgG index, however, was elevated. This finding indicates intrathecal IgG synthesis.A correlation between morphologically visualized tracer leakage in the central nervous system (CNS) with serum protein concentrations in the CSF revealed that elevated CSF albumin is a reliable indicator for BBB damage in lesions, located near the inner or outer surface of the brain and spinal cord. However, singular focal lesions with BBB damage located in the depth of the CNS parenchyma may not be accompanied by CSF protein alterations. The invariable presence of BBB damage in active inflammatory demyelinating lesions and its absence in inactive plaques or in the unaffected nervous tissue may be important in therapy, not only in experimental allergic encephalomyelitis but also in multiple sclerosis (MS).Supported by Fonds zur Förderung der wissenschaftlichen Forschung, Austria, Project no. S25/07     

Altered neurotransmission in brains of autoimmune mice: pharmacological and neurochemical evidence

Depressive-like behavior is the most profound manifestation of autoimmunity-associated behavioral syndrome in lupus-prone MRL-lpr mice. This led to the hypothesis that chronic autoimmunity and inflammation alter the activity of central serotonergic and dopaminergic systems. Three drugs with a selective mode of action were used to probe the functional status of these two systems in vivo. The behavioral effects of single and repeated intraperitoneal (i.p.) injections of sertraline, quinpirole (QNP) and risperidone were measured in the forced swim and brief sucrose preference tests. In comparison to MRL +/+ controls, autoimmune MRL-lpr mice did not show a reduction in sucrose intake after the administration of sertraline. Acute injection of quinpirole increased floating more in the MRL-lpr than in the control group, while intermittent administration induced self-injurious behavior in both groups. Acute injection of risperidone significantly increased floating in MRL-lpr mice, while repeated administration abolished the difference between the substrains in sucrose intake. These discrepancies in responsiveness implied that the central neurotransmitter activity is dissimilar in the two MRL substrains. This notion was confirmed in a cohort of untreated MRL-lpr and MRL +/+ mice by comparing their neurotransmitter/metabolite levels in several brain regions. In particular, MRL-lpr brains showed increased dopamine (DA) levels in the paraventricular nucleus (PVN) and median eminence (ME), decreased concentrations of serotonin in the PVN and enhanced levels in the hippocampus, as well as decreased norepinephrine (NE) levels in the prefrontal cortex. Behavioral deficits correlated with the changes in PVN and median eminence. These results are consistent with the hypothesis that imbalanced neurotransmitter regulation of the hypothalamus-pituitary axis plays an important role in the etiology of behavioral dysfunction induced by systemic autoimmune disease.     

Serum and cerebrospinal fluid proteins and the blood-brain barrier in Alzheimer's disease and multi-infarct dementia

Serum concentrations of IgG, IgA, IgM, haptoglobin, transferrin, prealbumin and albumin quantitated nephelometrically in 22 patients with Alzheimer's disease (AD), 29 patients with multi-infarct dementia (MID) and their age-matched controls were normal. Cerebrospinal fluid (CSF) albumin and CSF/serum ratio for albumin were higher in AD and MID patients compared to controls, but no significant differences were found between AD and MID. Patients with MID had elevated CSF IgG, IgA, IgM and prealbumin levels compared to controls and to AD. An increased CSF IgG index was found in 5 MID patients but none of the AD patients. Thus, the blood-brain barrier permeability is often increased in MID as well as in AD. There is no increased intrathecal IgG synthesis in AD but it may occur in MID.