Tumor necrosis factor is increased in the spinal cord of an animal model of motor neuron degeneration

ARTICLE

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive death of upper and lower motor neurons. The two main pathogenic hypothesis include: autoimmunity and oxidative/excitotoxic damage. The autoimmune hypothesis is supported mainly by the presence of anti-neural and anti-ganglioside antibodies in ALS patients [1] and by the similarities between ALS and an inducible experimental autoimmune motor neuron disease in the guinea pig [2, 3]. The involvement of a glutamate-mediated, excitotoxic damage is suggested by papers reporting defects in glutamate metabolism [4] or reuptake [5], and strong support to this hypothesis comes from clinical studies showing that riluzole, a drug which reduces glutamate release, increases survival [6].

TNF is a proinflammatory cytokine implicated in the pathogenesis of multiple sclerosis [7]. Its role in animal models of experimental autoimmune encephalomyelitis and of stroke was demonstrated by the protective effect of anti-TNF antibodies [8-10]. Interestingly, TNF is induced by excitotoxins [11] and potentiates the neurotoxicity of glutamate [12].

The aim of this work was to obtain evidence of the possible presence of TNF in mnd mouse, originally proposed as a model of motor neuron degeneration [13, 14], and more recently characterized as a model of ceroid lipofuscinosis [15]. This is a late-onset mutation that shows progressive deterioration of motor functions accompanied by degeneration of motor neurons in the spinal cord and of upper motor neurons in brain regions [16]. In spite of the diffuse lipofuscin accumulation [15], mnd mice share with human ALS an increase in blood levels of glutamic acid and a decrease of the spinal GLT-1 glutamate transporter [17, 18]. In addition, a slightly increased expression of IL-1 mRNA has been reported in peri-pheral macrophages from mnd mice [19].

We studied these mice when the pathology was evident (7 months) and at earlier time points, when mice showed no symptoms (3 months). We measured the levels of TNF in the serum, in the brain and in the spinal cord in these mice using a bioassay. TNF in the spinal cord was also evaluated by immunohistochemistry, using an antibody specific for TNF-alpha. We also studied in vitro TNF production by splenocytes or by blood cells from control or mnd mice, as a previous report had shown that TNF production by blood cells in vitro was increased in multiple sclerosis patients in parallel with the recurrence of the disease [20, 21]. The results indicate that TNF is increased in the spinal cord of mnd mice when symptoms of the disease develop, suggesting the possible involvement of this cytokine as a novel pathogenetic mediator.

MATERIALS AND METHODS

Animals

Homozygous mutants mnd/mnd male mice (B6KB2) were obtained from Jackson Laboratories (Bar Harbor, ME, USA) at the age of 10 or 27 weeks. Control mice with the same background (C57Bl6J) were obtained from IFFA Credo (Lyon, France), since in previous studies we found no differences between C57Bl6J imported directly from Jackson Laboratories and those obtained from IFFA Credo [18]. On arrival, the animals were housed, five per cage and acclimatized for at least one week. Temperature and relative humidity in the animal room were set at 21 ± 1° C and 55 ± 10%. Room illumination was: 12 hours of light (7 a.m.-7 p.m.) and 12 hours of dark (7 p.m.-7 a.m.). Food and water were supplied ad libitum.

Mice were sacrified by decapitation and blood was collected in heparinized tubes. Spleens were removed and immediatley processed for splenocyte preparation as described below. Brains and spinal cords were homogenized in 10 volumes (w/v) of ice-cold saline, clarified by centrifugation (9,000 rpm for 15 min, at 4° C) and the supernatants used for TNF measurement as described below.

For immunohistochemistry, animals were anesthetized with Equithesin and perfused through the ascending aorta with 20 ml phosphate buffered saline (PBS, 50 mM, pH 7.4) followed by 50 ml of a 4% formaldehyde solution in PBS, then the brain and spinal cord were removed and processed for immunohistochemistry analysis as described below.

Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national and international laws and policies (EEC Council Directive 86/609, OJ L 358, 1, December 12, 1987; Italian Legislative Decree 116/92, Gazzetta Ufficiale della Repubblica Italiana No. 40, February 18, 1992; NIH Guide for the Care and Use of Laboratory Animals, US National Research Council, 1996).

Splenocytes and whole blood cultures

Spleens were disaggregated by washing with RPMI 1640 medium using a 2.5 ml syringe. Splenocytes were then counted and plated at 5 x 10⁶/ml in 96-well culture plates (100 µl/well). Cells were cultured in RPMI 1640 medium with 10% FCS in the presence of 1 µg/ml LPS with and without IL-6 or CNTF (0.2, 2 or 20 µg/ml) in a final volume of 200 µl/well. Four hours later, supernatants were harvested and used for TNF determination.

For whole blood cultures, heparinized (14 U/ml; Liquemin, Roche, Milan, Italy) whole blood, freshly obtained, was plated in 96-well tissue culture plates (100 µl/well) and incubated for 4 hours at 37° C, 5% CO₂ with the indicated LPS concentration. Then, plates were centrifuged and supernatant collected for TNF determination.

TNF measurement

TNF was measured in sera or homogenates using a bioassay based on L929 cell cytotoxicity as previously described [22] using recombinant murine TNF as standard (kind gift of W. Fiers, Gent, Belgium).

Immunohistochemistry

All chemicals were purchased from Sigma Chemicals Co. (Milano, Italy), unless otherwise specified. All reagents for immunohistochemistry, including secondary antibodies, were purchased from Vector Laboratories Inc. (Burlingame, USA). Rat monoclonal anti-mouse TNF-alpha antibody was purchased from Keystone Laboratories, Inc. (Menlo Park CA, USA).

After sacrifice, spinal cords were rapidly removed on a chilled plate, postfixed for at least two days in 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4), and immersed for a further 2-3 days in a hypertonic sucrose solution (30% in PB). Forty µm thick sections were then cut by means of a freezing microtome (Leitz), collected and stored in PB at 4° C until immunostaining. Free-floating sections were rinsed in Tris-buffered saline (TBS, pH 7.6) and then incubated for 30 min with 10% normal rabbit serum (NRS) in TBS, and incubated overnight with the primary anti-TNF-alpha antibody, diluted to 1: 4,000 in TBS containing 1% NRS. In control sections, the primary antibody was replaced with normal rat serum. The following day, sections were rinsed in TBS and then incubated for 30 min with 10% NRS in TBS, 60 min with biotinylated rabbit anti-rat serum diluted to 1: 200 in TBS containing 1% NRS, 60 min with the avidin-biotinylated peroxidase complex (ABC), diluted to 1: 100 in TBS, and, finally, 4-8 min with a solution containing 3-3' diaminobenzidine tetrahydrochloride in 0.05 M Tris buffer with hydrogen peroxide to visualize peroxidase activity. Sections were then mounted, air dried and coverslipped, after the addition of an appropriate amount of DPX mountant for microscopy (BDH Chemicals, Poole, UK).

RESULTS

Table 1 shows the levels of bioactive TNF detectable in brains (including motor neurons of cranial nerves) and spinal cords from 3 and 7 month-old mnd or control mice. Elevated levels of TNF were detected at 7 months in mnd mice. No TNF was detectable in the sera from any of these mice (data not shown).

The in vitro TNF production by whole blood or splenocytes from 7-month-old control or mnd mice is shown in Table 2. There was no significant difference between the two groups.

Figure 1 shows the immunohistochemical analysis for TNF-alpha in normal, wild type mice, 3-month-old mnd mice and 7-month-old mnd mice with obvious disease. In normal, wild type mice (7 month-old), specific immunostaining was not detectable in cell bodies located in the gray matter of the spinal cord (upper panel). Weak immunostaining was observed in some cell bodies of motor neurons in the ventral horn of the spinal cord of 3-month-old mnd mice (Figure 1, middle panel). In 7-month-old mnd mice, intense immunostaining for TNF-alpha was observed in numerous cell bodies of motor neurons located in the ventral horn of the spinal cord (Figure 1, lower panel). Immunostaining was occasionally found in small-diameter cell bodies in the grey matter as well as in the white matter of the spinal cord sections. These cells, clearly distinguishable from motor neurons, probably belonged to the glial population and were more frequently observed in mnd mice. No immunostaining was obtained in control experiments in which the primary anti-serum was omitted, or when normal rat serum was used instead of the primary anti-serum. Similarly, no immunostaining occurred in sections in which either the biotinylated anti-serum, the avidin-biotin complex, or any component of the chromogen solution were omitted (not shown).

DISCUSSION

The data reported here show that TNF is produced in the brain and the spinal cord of mnd mice when the disease is evident, thus suggesting, for the first time, the possibility that TNF might be a pathogenetic mediator in this disease. In fact, in spite of diffuse lipofuscin accumulation in the central nervous system and in many somatic organs of mnd mice [15], we did not observe increased TNF production by splenocytes or blood cells from these mice, thus indicating that TNF increases with the specific localization of the disease. The immunohistochemical analysis of the spinal cord showed a localization of TNF-alpha that seems to be associated with motor neurons, which is evident in 3-month-old mnd mice, although to a lesser extent than in 7-month-old mnd mice. This selective expression in motor neurons could not be related to lipofuscin accumulation, since stored material has also been found in other neurons and glial cells [15], which do not express TNF-alpha immunoreactivity. Thus, the expression of TNF-alpha in motor neurons seems to be related to a specific localization of the pathology in these cells.

It is of interest to note that the TNF-producing cells in the CNS may differ according to the disease. While in experimental autoimmune encephalomyelitis, which has a strong inflammatory component, TNF is produced mainly by glial cells [23], in excitotoxic and ischemic brain injury neuronal TNF production was reported [24, 25]. In 7-month-old mnd mice there is an increase in astroglial proliferation [18], but these glial cells do not seem to be involved in TNF-alpha production in these animals.

Interestingly, the immunohistochemistry also showed a detectable TNF-alpha expression in 3-month-old mnd mice, that, although asymptomatic, are regarded as disease-prone. However, the observation that TNF is produced locally in the CNS of mnd mice does not necessarily mean that it plays a possible pathogenetic role in this animal model. Increased serum concentrations of TNF-alpha, sTNF-RI and sTNF-RII have been reported in ALS patients when compared to healthy controls, with no apparent relation with disease severity, duration or weight loss (Facchetti D, Bachetti T, Mai R, Micheli A, Agnoletti L, Francolini G, Mora G, Camana C, Mazzini L and Poloni M). Tumor necrosis factor in amyotrophic lateral sclerosis. submitted for publication). Our study suggests that serum concentrations of TNF (apart from the sensitivity of the technical assays) do not reflect levels of the cytokine in the central nervous system, therefore experimental studies of inhibition of TNF in vivo will be required to support its pathogenetic role in ALS. Unfortunately, this being a late-onset disease, the administration of anti-TNF antibodies is problematic and the use of knock-out mice lacking TNF or its receptors might help to answer the question of the role of TNF in ALS.

On the other hand, these findings might suggest a new target for pharmacological manipulation in ALS therapy. In fact, while administration of antibodies may not be an easy approach for the treatment of a chronic, progressive degenerative disease, one may consider the possibility of inhibiting TNF with low molecular weight drugs. In fact, the phosphodiesterase inhibitor rolipram and the guanylhydrazone CNI-1463, which inhibit TNF production, are active in murine experimental autoimmune encephalomyelitis [26, 27], whereas CNI-1463 also inhibits cerebral TNF production and is protective in a rat model of stroke [10].

It is of interest to note that ciliary neurotrophic factor (CNTF) is protective in other animal models of motor neuron diseases [28, 29], and knock-out mice lacking the CNTF receptor show a motor neuron deficit [30]. Recently, we have observed that CNTF administration inhibits LPS-induced TNF production in the brain and in the periphery [31]. Although the background for the therapeutic use of CNTF in ALS is based on its neurotrophic activity, inhibition of TNF production may contribute to its in vivo action in motor neuron diseases. Interestingly, it has been reported that IL-6 administration is protective in the wobbler mouse motor neuron disease model [32], and IL-6, like CNTF and other cytokines whose receptors share the gp130 signal transducer, also inhibits TNF production [33]. It should be noted, however, that recent studies using TNF receptor-deficient mice [25] have shown that TNF might have a protective effect in excitotoxic and ischemic brain injury. Thus, it cannot be excluded that the increase in TNF reported here has not a pathogenic role in the disease but may represent, on the contrary, a neuroprotective response.

Another pathogenetic hypothesis for ALS is that of oxidative injury, which is also implicated in the neurotoxic effect of glutamate [34]. Oxidative injury could occur directly as suggested by the studies of familial ALS (FALS), as 20-25% of FALS cases are associated with dominantly inherited mutations of the gene encoding for the anti-oxidant enzyme superoxide dismutase-1 (SOD-1) that might convert it into an enzyme capable of generating reactive intermediates by a peroxidation reaction [35], and transgenic mice with mutated SOD undergo motor neuron degeneration [36]. The expression of the anti-apoptotic gene bcl-2, which acts through an anti-oxidant pathway at the mitochondrial level is decreased in spinal cord motor neurons from ALS patients, and the anti-oxidants N-acetylcysteine and vitamin E are protective in animal models [37, 38]. With respect to this pathogenetic hypothesis, it should be noted that TNF toxicity in vitro is mediated by free radical production at the mitochondrial level [39], while anti-oxidants inhibit TNF synthesis [40].

CONCLUSION

The hypothesis of a possible role of TNF in ALS is compatible with all the pathogenic mechanisms actually proposed for ALS, although further studies will be required to clarify whether TNF behaves as a pathogenic mediator or a neuroprotective factor in the mnd mouse and other models of ALS.

Acknowledgments

This work was supported by the CNR (National Research Council, Rome, Italy), contract no. 96.00789.CT04, and by Telethon (grant no.1004).

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