munity in ALS, two immune-mediated animal models of mo- toneuron disease have been developed that resemble ALS with respect to the loss of motoneurons, ...
Proc. Natl. Acad. Sci. USA Vol. 88, pp. 647-651, January 1991
Neurobiology
Immunoglobulinp from animal models of motor neuron disease and from human amyotrophic lateral sclerosis patients passively transfer physiological abnormalities to the neuromuscular junction STANLEY H. APPEL*t*, JOZSEF I. ENGELHARDT*, JESTiS GARCIA§, Departments of *Neurology, tBiochemistry, and
AND
ENRICO STEFANI§
§Molecular Physiology and Biophysics, Baylor College of Medicine Houston, TX 77030
Communicated by Salih J. Wakil, October 29, 1990 (received for review August 22, 1990)
Amyotrophic lateral sclerosis (ALS) is a devABSTRACT astating human disease of upper and lower motoneurons of unknown etiology. In support of the potential role of autoimmunity in ALS, two immune-mediated animal models of motoneuron disease have been developed that resemble ALS with respect to the loss of motoneurons, the presence of IgG within motoneurons and at the neuromuscular junction, and with respect to altered physiology of the motor nerve terminal. To provide direct evidence for the primary role of humoral immunity, passive transfer with immunoglobulins from the two animal models and human ALS was carried out. Mice infected with serum or immunoglobulins from the animal disease models and human ALS but not controls demonstrated IgG in motoneurons and at the neuromuscular junction. The mice also demonstrated an increase in miniature end-plate potential (mepp) frequency, with normal amplitude and time course and normal resting membrane potential, indicating an increased resting quantal release of acetylcholine from the nerve terminal. The ability to transfer motoneuron dysfunction with serum immunoglobulins provides evidence for autoimmune mechanisms in the pathogenesis of both the animal models and human ALS.
Amyotrophic lateral sclerosis (ALS) is a devastating neuromuscular disease progressively compromising arms, legs, speech, swallowing, and breathing. Neither the etiology nor the pathogenesis is understood. In an effort to support an autoimmune etiology, two immune-mediated animal models of ALS have recently been developed: one of which, experimental autoimmune motor neuron disease (EAMND), is induced by the inoculation of purified motoneurons and results in lower motoneuron destruction (1); the other, experimental autoimmune gray matter disease (EAGMD), is induced by the inoculation of spinal cord ventral horn homogenate and results in both upper and lower motoneuron destruction (2). Animals with EAMND exhibit electromyographic and morphologic signs of denervation, IgG within lower motoneurons and at the neuromuscular junction, and no evidence of inflammation. Guinea pigs with EAGMD demonstrate loss of upper motoneurons and lower motoneurons, electromyographic and morphologic evidence of denervation, inflammatory foci within gray and white matter in the spinal cord, and IgG in upper and lower motoneurons and at the neuromuscular junction. In both models resting acetylcholine release from the motor nerve terminal is increased at an early stage of the disease (3). ALS resembles the immune-mediated animal models, especially EAGMD, with respect to the loss of motoneurons, the presence of denervation, inflammatory foci within the spinal cord (4, 5), IgG within motoneurons (6), and physiologic abnormalities of the neuromuscular junction (7). The similarities of human ALS to the animal models raise the
question of an autoimmune etiology for these disorders. A major question relevant to an autoimmune etiology is whether serum immunoglobulins can passively transfer the disease. Since the neurological signs of motoneuron dysfunction were presumed to require inoculations over several months, a shorter term goal of passively transferring dysfunction to the neuromuscular junction was established. Similar passive transfer of physiological abnormalities was critical in establishing the role of immune mechanisms in myasthenia gravis (8) and the Eaton-Lambert myasthenic syndrome (9).
MATERIALS AND METHODS Eight- to 10-week-old BALB/c mice were injected intraperitoneally with serum from EAMND (2 animals), EAGMD (4 animals), or control guinea pigs (2 animals) (1 ml/day for 3 days). Three mice were injected with IgG from EAGMD animals with mild signs (20 mg/day for 3 days). Sets of four mice were injected for 3 days with immunoglobulins (50 mg/day) from one of five sporadic ALS patients (20 animals total), from one of two normal patients (8 animals total), and from one of three non-ALS neurologic disease patients (12 animals total). EAMND and EAGMD Induction. Four-month-old male albino outbred Hartley guinea pigs were inoculated with purified bovine motoneurons in complete Freund's adjuvant three times at 4-week intervals with the production of weakness in the hind extremities characteristic of EAMND (1). EAGMD was produced in male albino outbred Hartley white guinea pigs by the inoculation of bovine spinal cord ventral horn homogenate in complete Freund's adjuvant according to established techniques (2). The resulting disease was a more acute onset of hind limb weakness with progression to paresis and often bulbar involvement. The extent of clinical dysfunction varied from mild to extremely severe disease in various animals. Serum was obtained from guinea pigs with EAMND all of whom had mild disease after 3 months of antigen inoculations. Serum was obtained from guinea pigs with EAGMD with either mild or moderate to severe signs after 4 weeks of antigen inoculations. Control animals exhibited no clinical or pathologic evidence of disease after inoculation with complete Freund's adjuvent alone. ALS and Control Patients. The five ALS patients consisted of four males, one of whom had IgG K monoclonal gammopathy, and one female. Their ages were from 44 to 62 years and all had progressive disease with bulbar involvement fulfilling all clinical criteria of ALS. There was no evidence Abbreviations: ALS, amyotrophic lateral sclerosis; mepp, miniature end-plate potential; EAMND, experimental autoimmune motor neuron disease; EAGMD, experimental autoimmune gray matter disease; FITC, fluoroscein isothiocyanate; EDL, extensor digitorum longus. tTo whom reprint requests should be addressed at: Department of Neurology, Baylor College of Medicine, One Baylor Plaza, Hous-
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ton, TX 77030. 647
648
Neurobiology: Appel et al.
of multifocal motor conduction block by electromyography. All patients sera were tested for either IgG or IgM antibodies to GM1 gangliosides, and all were negative. The disease controls consisted of a 44-year-old male with moderately severe generalized myasthenia gravis with bulbar compromise, a 52-year-old female with chronic relapsing inflammatory polyneuropathy, and a 38-year-old male with GuillianBarrd syndrome. The normal controls consisted of two individuals, one male of age 56 and one female of age 67, without evidence of neurologic disease. Immunoglobulin Preparation. Rivanol fractions were obtained by the technique of Horejsi and Smethana (10). After dialysis against distilled water and phosphate-buffered saline, rivanol fractions were employed for most studies of passive transfer with human material. IgG fractions were isolated from guinea pig serum by employing protein A affinity column chromatography. The IgG was isolated from the serum of one ALS patient and one myasthenia gravis patient with protein A chromatography. Immunohistochemical Studies. Twenty-four hours after the last injection, namely on day 4, the inoculated mice underwent ether anesthesia and were perfused with phosphatebuffered saline followed by a solution of 4% (wt/vol) paraformaldehyde, 0.05% glutaraldehyde, and 0.2% picric acid. The extensor digitorum longus (EDL) muscles from the hind limbs as well as 3- to 4-mm blocks of the spinal cord, brain stem, cerebrum, and cerebellum were post-fixed in the same fixative for 3 days at 40C. Muscles were processed as described (1-3). When human immunoglobulins were injected, the fluorescein isothiocyanate (FITC)-conjugated IgG fraction of goat anti-human IgG was employed to detect IgG reactivity whereas the FITC-conjugated IgG fraction of goat anti-guinea pig IgG was employed when guinea pig immunoglobulins were injected into BALB/c mice. Controls consisted of control guinea pig or human immunoglobulininjected mice, pretreatment of the FITC-conjugated goat anti-guinea pig or anti-human IgG with guinea pig or human sera, and incubating sections with rhodamine-conjugated IgG fraction of goat anti-human IgM. Electrophysiological Studies. The animals for electrophysiologic studies were also sacrificed under ether anesthesia and studied 1 day after the last injection but did not undergo perfusion. EDL muscles from the hind extremities were dissected and mounted in the experimental chamber containing normal oxygenated Krebs-Ringer solution [145 mM NaCl/5 mM KCI/2.5 mM CaCl2/1 mM MgSO4/10 mM Hepes/10 mM glucose/neostigmine (0.25 mg/liter); pH was buffered to 7.4 with Hepes-NaOHI. The osmolarity was corrected to 300 milliosmolar. Intracellular electrodes filled with 3 M KCI had a resistance of 20-40 Mfl. The resting membrane potential was measured in reference to an Ag/ AgCl ground electrode placed in the chamber. Spontaneous miniature end-plate potentials (mepps) were measured with an Axoclamp-2A amplifier (Axons Instruments, Burlingame, CA) and stored on magnetic tape for computer analysis. Bath temperature (28°C) was monitored with a thermistor probe placed close to the muscle in the chamber solution.
RESULTS AND DISCUSSION Hind.limb weakness developed in mice (Fig. 1) injected with serum or IgG from guinea pigs with either moderate or severe symptoms of EAGMD, characterized by acute limb weakness with marked muscular atrophy within 4 weeks of inoculation with bovine ventral horn homogenates (2). Mice injected with serum or immunoglobulins from animals with mild EAGMD, mild or moderate EAMND, or from ALS patients did not develop signs of weakness after inoculation. None of the injected mice showed either microscopic or macroscopic morphologic alterations of the central nervous system, peripheral nerve, or the neuromuscular junction.
Proc. Natl. Acad. Sci. USA 88 (1991)
FIG. 1. BALB/c mice after two injections within 24 hr of serum from control guinea pig (on right) and from a guinea pig with EAGMD (on left).
However, by immunohistochemical techniques, reactivity for immunoglobulin approximated the reactivity for a-bungarotoxin in mice injected with serum or immunoglobulins from the two animal models and human ALS (Fig. 2) similar to the colocalization of a-bungarotoxin and IgG in the animal models in vivo (1-3). In mice inoculated with EAGMD serum
FIG. 2. Immunohistochemical reaction of the EDL neuromuscular junction and spinal cord ventral horn cells. (A) End plate from a mouse injected with ALS immunoglobulins. Stained with rhodamineconjugated a-bungarotoxin. (x 1175.) (B) Same end plate stained with FITC-conjugated goat anti-human IgG. (x1175.) (C) Absence of IgG reactivity in ventral horn of a mouse injected with control guinea pig serum. (x525.) (D) Motoneurons from the ventral horn of a mouse injected with EAMND sera demonstrating patchy cytoplasmic IgG
reactivity. (x525.)
Neurobiology: Appel et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
or immunoglobulins, 602 end plates were examined. Sixty percent of the end plates that stained with a-bungarotoxin also displayed fluorescent staining for IgG. In mice injected with EAMND serum, 400 end plates were examined. Twenty-five percent of the neuromuscular junctions stained positively for IgG. Mice injected with immunoglobulins from ALS patients were positive for IgG in 30 ± 5% (mean ± SEM) of the 500 neuromuscular junctions examined. An approximation of IgG and a-bungarotoxin reactivity was seen in 20 mepps per sec). These results indicate that many, but not all, of the nerve terminals were involved in this process. Furthermore, the reported increase in frequency was not due to nonspecific damage of the postsynaptic element since muscle fibers from all injected mice had normal resting potentials and normal mepp amplitude and time course. Thus, the higher frequency in mice injected with serum or IgG from diseased guinea pigs or ALS patients suggests a physiological alteration of the presynaptic terminal. Since the mepp results from opening of the acetylcholine receptor in the postsynaptic membrane, the increase in number of these events reflects larger acetylcholine release from the nerve terminal in the resting state. The fact that immunoglobulins from immune-mediated animal models of motoneuron destruction and human ALS can passively transfer physiologic alterations provides important evidence for the role of immunoglobulins in these syndromes, just as such passive transfer experiments pro-
vided cogent evidence for the role of immunoglobulins in myasthenia gravis (8) and in the Eaton-Lambert myasthenic syndrome (9). The enhanced resting mepp frequency further defines the neuromuscularjunction as a target for the immune attack. However, the question must be raised as to whether the enhanced resting mepp frequency has significance as a physiological sign of early motor neuron disease. In EAMND and EAGMD, enhanced resting mepp frequency was noted early in the course of disease and prior to the onset of weakness and motoneuron destruction (1-3). Studies of anconeus muscle biopsies in ALS have documented changes in spontaneous mepp frequency and quantal content, both of which draw attention to the neuromuscular junction as a site of altered physiology (7), but no studies have been carried out of neuromuscular physiology at an early stage of the disease. Thus it is not clear whether abnormalities of the neuromuscular junction represent the earliest stage of ALS. Acetylcholine is known to be released in quantal form by electrical stimulation of the nerve, whereas at rest acetylcholine is released as molecular leakage as well as quantal release, which is dependent on calcium (13-17). In motoneuron syndromes, the demonstration of enhanced mepp frequency, both in the present passive transfer experiments and in our previous in vitro studies (18), in fact may have more significance as a functional assay of immunoglobulin-induced increase of cytoplasmic calcium, especially since BAYK8644 potentiates calcium currents (19-21) and increases spontaneous and evoked acetylcholine release at the neuromuscular junction (22, 23). The increased cytoplasmic cal-
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FIG. 4. Frequency histograms of mepps in mice. The mean value of mepp frequency for each examined fiber was calculated and all the data of each group of animals were plotted. The data shown, in A correspond to all untreated mice. (B) Pooled data obtained from mice injected with guinea pig serum or IgG from gray matter-immunized guinea pigs. (C and D) Data from mice injected with immunoglobulin from two normal individuals or from three individuals with non-ALS neurological disorders, respectively. (E) Distribution of mepp frequency obtained from mice injected with immunoglobulin from patients with ALS.
Neurobiology: Appel et al. cium may in turn trigger processes that lead to cell death as has been well documented with neuronal cell death in other experimental paradigms (24-26). Thus our data would suggest that EAMND, EAGMD, and ALS immunoglobulins interact with the neuromuscular junction, result in an increased intracellular calcium in the motor nerve terminal and an increased resting acetylcholine release, and trigger processes leading to cell death. The rapidity of immunoglobulin action suggests the possibility of a direct effect on membrane channels or receptors that influence calcium entry. However, the difficulty in carrying out patch-clamp studies on the mammalian motor terminal will necessitate investigation of voltage- and ligand-gated calcium channels in other tissues. Since upper and lower motoneurons may share antigens (27), a definition of the specific target of the immunoglobulins that passively transfer these physiological changes of the neuromuscularjunction could help clarify the specific mechanisms of cell death in motor neuron disease. This study was supported by grants from the Muscular Dystrophy Association through a Muscular Dystrophy Association ALS Center Grant, by the M. H. "Jack" Wagner Memorial Fund and the Robert J. and Helen C. Kleberg Foundation, by a postdoctoral fellowship from the Muscular Dystrophy Association to J.G., by a grant from the National Institutes of Health (RO1-AR 38970), and by a grant from Cephalon, Inc. 1. Engelhardt, J. I., Appel, S. H. & Killian, J. M. (1989) Ann. Neurol. 26, 368-3%. 2. Engelhardt, J. I., Appel, S. H. & Killian, J. M. (1990) J. Neuroimmunol. 27, 21-31. 3. Garcia, J., Engelhardt, J. I., Appel, S. H. & Stefani, E. (1990) Ann. Neurol. 28, 329-334. 4. Troost, D., Van de Oord, J. J., de Jong, J. M. B. V. & Swaab, D. F. (1989) Clin. Neuropathol. 8, 289-294. 5. Appel, S. H., Engelhardt, J. I., Garcia, J. & Stefani, E. (1991) in ALS and Motor Neuron Disease: Research Progress, ed. Rowland, L. P. (Raven, New York), pp. 405-412.
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6. Engelhardt, J. I. & Appel, S. H. (1990) Arch. Neurol. 47, 1210-1216. 7. Maselli, R., Wollman, R., Mass, D., Palanbi, S., SalazarGrueno, E., Roos, R., Nelson, D. & Richman, D. (1989) Neurology 39, Suppl. 1, 400 (abstr.). 8. Toyka, K. V., Drachman, D. B., Pestronk, A. & Kao, I. (1975) Science 190, 397-399. 9. Lang, B., Newsom-Davis, J., Wray, D., Vincent, A. & Murphy, N. (1981) Lancet ii, 224-226. 10. Horejsi, J. & Smetana, R. (1956) Acta Med. Scand. 155, 65-70. 11. Fabian, R. & Ritchie, T. C. (1986) J. Neurol. Sci. 73, 257-267. 12. Fabian, R. (1988) Neurology 38, 1775-1780. 13. Katz, B. & Miledi, R. (1968) J. Physiol. (London) 195, 481-492. 14. Katz, B. & Miledi, R. (1%9) J. Physiol. (London) 203, 689-706. 15. Alnaes, E. & Rahaminoff, R. (1975) J. Physiol. (London) 248, 265-306. 16. Katz, B. & Miledi, R. (1977) Proc. R. Soc. London Ser. B 196, 59-72. 17. Duncan, C. J. & Statham, H. E. (1977) J. Physiol. (London) 268, 319-333. 18. Uchitel, 0. D., Appel, S. H., Crawford, F. & Sczcurpek, L. (1988) Proc. Natl. Acad. Sci. USA 85, 7371-7374. 19. Baeyens, J. M. & Del Pozo, E. (1989) Pharmacol. Toxicol. 65, 398-401. 20. Coronado, R. & Affolter, H. (1986) in Ion Channel Reconstitution, ed. Miller, C. (Plenum, New York), pp. 483-503. 21. Lamb, G. D. & Walsh, T. (1987) J. Physiol. (London) 393, 595-617. 22. Atkinson, W. D. & O'Leary, S. M. (1987) Brain Res. 419, 315-319. 23. Ofiram-Uffenheimer, E., Rahamimoff, R. & Shapira, R. (1990) J. Physiol (London) 430, 65p. 24. Weiss, J. H., Hartley, D. M., Koh, J. & Choi, D. W. (1990) Science 247, 1474-1477. 25. Dreyer, E. G., Kaiser, P. I. C., Offerman, J. T. & Lipton, S. A. (1990) Science 248, 364-367. 26. Rich, K. M. & Hollowell, J. P. (1990) Science 248, 1419-1421. 27. Engelhardt, J. I. & Appel, S. H. (1990) J. Neurol. Sci. 96, 333-352.
