Development of ALS-like disease in SOD-1 mice deficient of B lymphocytes
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- Naor, S., Keren, Z., Bronshtein, T. et al. J Neurol (2009) 256: 1228. doi:10.1007/s00415-009-5097-3
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Several recent studies proposed a role for innate immunity and inflammation in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, possible links, if any, between disease and adaptive immunity are poorly understood. The present study probed for the role of B cells in ALS disease using the G93A-SOD-1 transgenic mouse model. In agreement with other studies, we show here that autoantibodies are detectable in SOD-1 mice. However, SOD-1 B cells did not express any altered phenotype and exhibited indistinguishable responsiveness to immunogenic stimuli relative to wild-type B cells. This was obtained for B cells isolated before, during and after the onset of ALS-like disease. Finally, to obtain an in vivo conclusion, we generated SOD-1 mice that are deficient of B cells, by crossing SOD-1 mice with Igμ-deficient mice (μMT), where B cell development is blocked at the proB stage. The meteoric assays performed on a rota-rod clearly showed the development of ALS-like disease in SOD-1 mice that are deficient of B cells not differently than in control SOD-1 mice. Our results propose that B lymphocytes do not have a major role in the pathogenesis of ALS-like disease in SOD-1 mice.
KeywordsAmyotrophic lateral sclerosis (ALS)B lymphocytesAntibodiesAutoimmunity
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a loss of brain and spinal cord motor neurons with clinical manifestation of progressive muscular weakness, paralysis and death of patients within 3–5 years [27, 35]. The pathogenesis of ALS is incompletely understood, although different hypotheses have been suggested, including mitochondria dysfunction , mutation in the superoxide dismutase gene , defects in neuronal glutamate transports , and autoimmunity . Despite of all this, no effective remedy is available today for these patients. In about 20% of the patients missense mutation (G93A) in the Cu/Zn superoxide dismutase (SOD1) were identified as the primary cause for this fatal disease (familial ALS [2, 3, 14]). These findings have led to the development of a transgenic mouse model, which express the mutant human SOD-1 protein and develop symptoms of familial ALS-like disease. However, most cases of ALS are sporadic and their etiology is unknown .
Inflammatory mechanisms and immune reactivity are hypothesized to play a role in the pathogeneses of central nervous system (CNS) diseases [26, 33]. Numerous studies have shown that autoreactive T and B lymphocytes are responsible for the onset of the disease, and these cells are the target of many drug therapies . Studies now suggest that dysregulation in the immune system may also contribute to the pathogenesis of ALS [6, 41]. The observations proposing a role of the immune system include inflammatory signals [1, 25], activated microglia , infiltration of lymphocytes into the spinal cord , depositions of IgG antibodies , and dysregulation of cytokine expression in the spinal cord of ALS patients . Recently, increased amounts of complement components have been shown in serum, cerebrospinal fluid and neuronal tissue of diseased ALS individuals and in SOD-1 animal models . The findings of autoantibodies to neuronal antigens in patients with lower motor neuron syndromes (LMNs)  may also support a potential involvement of the immune system in ALS. Amyotrophic lateral sclerosis occurs both as a sporadic and a familial disease with a similar pathology of neuronal damage . Thus, although most studies utilize the SOD-1 model for familial ALS, it is hoped that therapies effective in mutant SOD1 models will translate to sporadic ALS.
B lymphocytes are key regulators in the autoimmune process. In addition to antibody secretion, other B cells functions include antigen presentation, T cell regulation and cytokine production [16, 23, 47]. Targeted B cell therapy appears to be very efficient treatment to many autoimmune diseases including rheumatoid arthritis, lupus erythematosus and multiple sclerosis [10, 17]. There are several studies proposing a role of B lymphocytes in the pathogenesis of ALS by showing that autoantibodies to neural antigens are produced in ALS patients. Thus, anti-neural antibodies (GM1-gangliosides, AGM1-gangliosides and sulfatides) were found in serum and cerebrospinal fluid of ALS patients , and antibodies isolated from ALS patients inhibit dopamine release mediated by L-type calcium channels . The present study was undertaken to determine whether or not B lymphocytes contributes to the pathogenesis of ALS-like disease in SOD-1 mouse. We show here that SOD-1 B cells are indistinguishable from wild-type (wt) B cells by both phenotypic marker expression and responsiveness to immunogenic stimulations and that lack of B cells does not prevent, inhibit or reduce ALS-like disease in SOD-1 mice. These results argue against an essential role of B cells in the pathogenesis of ALS.
Materials and methods
The mice used for the experiments were 60–130 days old normal C57Bl6 or deficient of B cells (μMT) . Mice transgenic for the mutant G93A form of human superoxide dismutase-1 gene (SOD-1) were purchased from Jackson Laboratory, Bar Harbor, USA. Typing for SOD-1 G93A transgene was carried out by PCR reaction using a tail DNA template. Primer used for SOD-1 are oIMR0113 (5′-CATCAGCCCTAATCCATCTGA-3′) and oIMR0114 (5′-CGCGACTAACAATCAAAGTGA-3′) . The PCR for the wild type IL-2 gene was carried out for control using primers oIMR0042 (5′-CTAGGCCACAGAATTGAAAGATCT-3′) and oIMR0043 (5′-GTAGGTGGA AATTCTAGCATCATCC-3′). The PCR conditions were 90 s at 94°C followed by 37 cycles as follows: 45 s at 94°C, 60 s at 59°C and 45 s at 72°C. Final elongation at 72°C lasted 10 min. In some experiments, SOD-1 Tg and μMT mice were crossed to generate SOD-1 mice that are deficient of B cells. The mice were housed and bred at the animal facility of the Faculty of Biotechnology and Food Engineering, Technion, and all studies were approved by the committee for the supervision of animal experiments. In our mouse colony, the onset of the ALS-like disease occurs at around 90 days after birth and the sick mice tend to die between days 140–180. In these experiments, the principles of laboratory animal care (NIH publication No. 86-23, revised 1985) were followed, as well as specific national laws as approved by the institutional ethic committee.
Cell culture and stimulation
Mature B cells were purified untouched from spleens after negative selection by magnetic beads (B cell isolation kit, Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultured in supplemented DMEM tissue culture medium (Biological Industries, Kibbutz Beit Haemek, Israel). For stimulation, we used the synthetic ODNs 1826 (CpG-ODN)  (InvivoGen, San Diego, USA) at 100 nM, or with LPS (50 μg/ml) (Sigma). Cells were stimulated for 96 h and proliferation was measured by the MTT assay . Supernatants of stimulated cells were collected and analyzed for antibodies by ELISA (see below).
Flow cytometry and cell sorting
Single cell suspensions from spleens were stained for surface markers using FITC-, PE-, APC and biotin-conjugated antibodies, followed with streptavidin PerCP. Abs used were as follows: B220, RA3-6B2; CD19, 1D3; CD5, 53-7.3 (Pharmingen, Becton Dickinson, San Diego, USA); CD45, 13/2.3; CD69, H1.2F3 [Southern Biotechnology Associates Inc. (SBA), Birmingham, USA]; rat anti mouse IgM (Zymed Laboratories, San Francisco, CA, USA); IgD JA12.5. Data for four-color analysis were collected on a FACSCalibur™ (BD Biosciences, Immunocytometry Systems, Mountain View, CA, USA).and analyzed using FlowJo software.
Tissue array analysis was carried out as described . Briefly, normal mouse tissue samples from healthy mice were homogenized in lysis buffer and after centrifugation, cleared tissue lysates were separated SDS-PAGE gel and transferred to nitrocellulose membrane blots. After blocking, membranes were incubated with sera collected from SOD-1 or from wt mice (diluted 1:500–1:1,500). HRP conjugated goat anti-mouse IgG (Jackson) was used for detection. Visualization of specific bands was performed by ECL reaction.
Analysis of antibodies
Serum samples or supernatants of stimulated cells were analyzed for total amounts of IgM and IgG by sandwich ELISA using specific goat anti-mouse IgM or IgG polyclonal reagents (SBA) as described . Antibody concentrations were calculated using a reference standard curve.
Motor analysis—rota-rod test
Neuromuscular function of SOD1 G93A transgenic mice was assessed using the rota-rod test on an ENV-575 five station mouse USB rota-rod treadmill, MED Associates Inc., St. Albans, USA. Following a 1-week pre-training session on the rotating rod, 70-day-old mice were subjected to gradually increasing rotation speeds from 2.5 to 25 RPM over a 5 min time course and were then subjected to the maximal rotation speed (25 RPM) for an additional 3 min. The time to fall (seconds) and maximal speed achieved (RPM) were recorded twice a week. In each test session the protocol was repeated for 3–5 times and the average value for each time point was assessed. Performance of the mice on the rota-rod was measured as the time the animal holds (seconds)/age(days), or as the maximum speed reached MAX–V(RPM)/age(days). The slopes (dV/dT) of the linear fits were calculated and their analysis of variance was determined. The standard deviation of the slopes was predicted from the upper and lower borders comprising 95% of the data results.
The statistical significance of differences between groups was determined using unpaired Student’s t test, with differences considered significant at P < 0.05.
Tissue array analysis of SOD-1 serum antibodies
Analysis of splenic B cells and serum immunoglobulin
Responsiveness to immunogenic stimulation
Development of ALS-like disease in SOD-1 mice deficient of B cells
The rational for this study stems from several publications proposing that ALS disease is associated with dysregulation in the immune system. In here, we specifically addressed the contribution of B lymphocytes to the pathogenesis of ALS-like disease in SOD-1 mice. The conclusions drawn by this study exclude an essential role of B cells in this disease. However, it is important to mention that the mutated form of SOD-1 gene (G93A) in the mouse model is linked with only 20% of ALS cases and that over 140 mutations are now identified .
While dysfunction of innate immunity has been shown as a pathogenic feature of ALS [6, 19, 22], there are very few studies proposing a pathogenic role of the adaptive immunity [5, 11, 29, 31, 39]. A functional link between the adaptive immune system and neurodegenerative diseases has been shown for Alzheimer’s disease , multiple sclerosis (MS)  and Parkinson’s disease , proposing the existence of a destructive autoimmune process. Recently, the central role of the B lineage in autoimmunity becomes more appreciated as B cell targeting therapies, such as rituximab , yield very promising results even in autoimmune diseases that are traditionally considered as T cell regulated such as MS . The finding that SOD-1 mice that are deficient of B cells develop ALS-like diseases (Fig. 4) appears to exclude a central role of B cells in this disease. However, similar studies using B cell deficient mice proposed that B cells do not play a vital role in experimental autoimmune encephalomyelitis (EAE) , a conclusion that is now reversed in light of the successful use of B cell depletion therapy in MS patients . In addition, the SOD-1 model may only represent 20% of familial ALS cases, providing a rational for more clinical studies using other ALS animal models and patients with a different form of ALS before excluding the role of B cells in the disease.
Despite the fact that autoantibodies are detected in ALS patients and in SOD-1 mice (here and in [29, 31]), it is also possible that B lymphocytes may have a smaller contribution to the pathogenesis of ALS than initially proposed. In autoimmune diseases such as lupus, peripheral B cells are hyperreactive  and express higher levels of activation markers , thereby contributing to the pathogenesis of the diseases. We show here that SOD-1 B cells are not expressing activated phenotype and are not different than wt B cells in responsiveness to LPS and to CpG DNA. Thus, ALS-like disease in the SOD-1 mouse model does not involve polyclonal activation of B lymphocytes or an inherent defect in B cell responsiveness. However, the fact that autoantibodies are produced argues that few, self-reactive B cell clones are activated by specific neuronal tissue. The contribution of these autoantibodies to the pathogenesis of ALS is still questionable although a correlation with the severity of disease course has been shown . In many organ-specific autoimmune diseases autoantibodies can be detected a long time before the clinical onset and during the course of the diseases. In these cases, autoantibodies can be used as predictive markers of an ongoing disease (in healthy individuals) and of disease activity and severity (in patients). So far no biological marker has been described for ALS and extensive research is conducted to identify such a marker for early diagnosis. Several autoantibodies have been identified in cerebrospinal fluid (CSF) and serum of ALS patients including anti-GM-1 ganglioside , neurofilament 68 . However, because not all ALS patients develop autoantibodies and that no pathogenic role has been described for these autoantibodies, it is conceivable that autoantibody production is a secondary immunological consequence of neuronal death. The fact that ALS-like disease develops in B cell-deficient SOD-1 mice strongly supports this hypothesis. Nevertheless, it is possible that the autoantibodies may accelerate the course of neuronal degeneration, as a correlation with the severity of disease course has been shown .
This work was supported by grants provided by IsrA.L.S.—The Association for ALS Research in Israel, and by the Elias Fund for Medical Research.