Proc. Natl. Acad. Sci. USAVol. 88, pp. 647-651, January 1991Neurobiology

Immunoglobulinp from animal models of motor neuron disease andfrom human amyotrophic lateral sclerosis patients passivelytransfer physiological abnormalities to the neuromuscular junctionSTANLEY H. APPEL*t*, JOZSEF I. ENGELHARDT*, JESTiS GARCIA§, AND ENRICO STEFANI§Departments of *Neurology, tBiochemistry, and §Molecular Physiology and Biophysics, Baylor College of Medicine Houston, TX 77030

Communicated by Salih J. Wakil, October 29, 1990 (receivedfor review August 22, 1990)

ABSTRACT Amyotrophic lateral sclerosis (ALS) is a dev-astating human disease of upper and lower motoneurons ofunknown etiology. In support of the potential role of autoim-munity in ALS, two immune-mediated animal models of mo-toneuron disease have been developed that resemble ALS withrespect to the loss of motoneurons, the presence of IgG withinmotoneurons and at the neuromuscular junction, and withrespect to altered physiology of the motor nerve terminal. Toprovide direct evidence for the primary role of humoral immu-nity, passive transfer with immunoglobulins from the two ani-mal models and human ALS was carried out. Mice infected withserum or immunoglobulins from the animal disease models andhuman ALS but not controls demonstrated IgG in motoneuronsand at the neuromuscular junction. The mice also demonstratedan increase in miniature end-plate potential (mepp) frequency,with normal amplitude and time course and normal restingmembrane potential, indicating an increased resting quantalrelease of acetylcholine from the nerve terminal. The ability totransfer motoneuron dysfunction with serum immunoglobulinsprovides evidence for autoimmune mechanisms in the patho-genesis of both the animal models and human ALS.

Amyotrophic lateral sclerosis (ALS) is a devastating neuro-muscular disease progressively compromising arms, legs,speech, swallowing, and breathing. Neither the etiology northe pathogenesis is understood. In an effort to support anautoimmune etiology, two immune-mediated animal modelsof ALS have recently been developed: one of which, exper-imental autoimmune motor neuron disease (EAMND), isinduced by the inoculation of purified motoneurons andresults in lower motoneuron destruction (1); the other, ex-perimental autoimmune gray matter disease (EAGMD), isinduced by the inoculation of spinal cord ventral horn ho-mogenate and results in both upper and lower motoneurondestruction (2). Animals with EAMND exhibit electromyo-graphic and morphologic signs of denervation, IgG withinlower motoneurons and at the neuromuscular junction, andno evidence of inflammation. Guinea pigs with EAGMDdemonstrate loss of upper motoneurons and lower motoneu-rons, electromyographic and morphologic evidence of de-nervation, inflammatory foci within gray and white matter inthe spinal cord, and IgG in upper and lower motoneurons andat the neuromuscular junction. In both models resting ace-tylcholine release from the motor nerve terminal is increasedat an early stage of the disease (3).ALS resembles the immune-mediated animal models, es-

pecially EAGMD, with respect to the loss of motoneurons,the presence of denervation, inflammatory foci within thespinal cord (4, 5), IgG within motoneurons (6), and physio-logic abnormalities of the neuromuscular junction (7). Thesimilarities of human ALS to the animal models raise the

question of an autoimmune etiology for these disorders. Amajor question relevant to an autoimmune etiology iswhether serum immunoglobulins can passively transfer thedisease. Since the neurological signs of motoneuron dysfunc-tion were presumed to require inoculations over severalmonths, a shorter term goal of passively transferring dys-function to the neuromuscular junction was established.Similar passive transfer of physiological abnormalities wascritical in establishing the role of immune mechanisms inmyasthenia gravis (8) and the Eaton-Lambert myasthenicsyndrome (9).

MATERIALS AND METHODSEight- to 10-week-old BALB/c mice were injected intraperi-toneally with serum from EAMND (2 animals), EAGMD (4animals), or control guinea pigs (2 animals) (1 ml/day for 3days). Three mice were injected with IgG from EAGMDanimals with mild signs (20 mg/day for 3 days). Sets of fourmice were injected for 3 days with immunoglobulins (50mg/day) from one of five sporadic ALS patients (20 animalstotal), from one of two normal patients (8 animals total), andfrom one of three non-ALS neurologic disease patients (12animals total).EAMND and EAGMD Induction. Four-month-old male

albino outbred Hartley guinea pigs were inoculated withpurified bovine motoneurons in complete Freund's adjuvantthree times at 4-week intervals with the production of weak-ness in the hind extremities characteristic of EAMND (1).EAGMD was produced in male albino outbred Hartley whiteguinea pigs by the inoculation of bovine spinal cord ventralhorn homogenate in complete Freund's adjuvant according toestablished techniques (2). The resulting disease was a moreacute onset ofhind limb weakness with progression to paresisand often bulbar involvement. The extent of clinical dysfunc-tion varied from mild to extremely severe disease in variousanimals. Serum was obtained from guinea pigs with EAMNDall of whom had mild disease after 3 months of antigeninoculations. Serum was obtained from guinea pigs withEAGMD with either mild or moderate to severe signs after 4weeks of antigen inoculations. Control animals exhibited noclinical or pathologic evidence of disease after inoculationwith complete Freund's adjuvent alone.ALS and Control Patients. The five ALS patients consisted

of four males, one of whom had IgG K monoclonal gam-mopathy, and one female. Their ages were from 44 to 62 yearsand all had progressive disease with bulbar involvementfulfilling all clinical criteria of ALS. There was no evidence

Abbreviations: ALS, amyotrophic lateral sclerosis; mepp, miniatureend-plate potential; EAMND, experimental autoimmune motor neu-ron disease; EAGMD, experimental autoimmune gray matter dis-ease; FITC, fluoroscein isothiocyanate; EDL, extensor digitorumlongus.tTo whom reprint requests should be addressed at: Department ofNeurology, Baylor College of Medicine, One Baylor Plaza, Hous-ton, TX 77030.

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Proc. Natl. Acad. Sci. USA 88 (1991)

of multifocal motor conduction block by electromyography.All patients sera were tested for either IgG or IgM antibodiesto GM1 gangliosides, and all were negative. The diseasecontrols consisted of a 44-year-old male with moderatelysevere generalized myasthenia gravis with bulbar compro-mise, a 52-year-old female with chronic relapsing inflamma-tory polyneuropathy, and a 38-year-old male with Guillian-Barrd syndrome. The normal controls consisted of twoindividuals, one male of age 56 and one female of age 67,without evidence of neurologic disease.Immunoglobulin Preparation. Rivanol fractions were ob-

tained by the technique of Horejsi and Smethana (10). Afterdialysis against distilled water and phosphate-buffered saline,rivanol fractions were employed for most studies of passivetransfer with human material. IgG fractions were isolatedfrom guinea pig serum by employing protein A affinitycolumn chromatography. The IgG was isolated from theserum of one ALS patient and one myasthenia gravis patientwith protein A chromatography.

Immunohistochemical Studies. Twenty-four hours after thelast injection, namely on day 4, the inoculated mice under-went ether anesthesia and were perfused with phosphate-buffered saline followed by a solution of 4% (wt/vol) para-formaldehyde, 0.05% glutaraldehyde, and 0.2% picric acid.The extensor digitorum longus (EDL) muscles from the hindlimbs as well as 3- to 4-mm blocks of the spinal cord, brainstem, cerebrum, and cerebellum were post-fixed in the samefixative for 3 days at 40C. Muscles were processed asdescribed (1-3). When human immunoglobulins were in-jected, the fluorescein isothiocyanate (FITC)-conjugated IgGfraction of goat anti-human IgG was employed to detect IgGreactivity whereas the FITC-conjugated IgG fraction of goatanti-guinea pig IgG was employed when guinea pig immuno-globulins were injected into BALB/c mice. Controls con-sisted of control guinea pig or human immunoglobulin-injected mice, pretreatment of the FITC-conjugated goatanti-guinea pig or anti-human IgG with guinea pig or humansera, and incubating sections with rhodamine-conjugated IgGfraction of goat anti-human IgM.

Electrophysiological Studies. The animals for electrophys-iologic studies were also sacrificed under ether anesthesiaand studied 1 day after the last injection but did not undergoperfusion. EDL muscles from the hind extremities weredissected and mounted in the experimental chamber contain-ing normal oxygenated Krebs-Ringer solution [145 mMNaCl/5 mM KCI/2.5 mM CaCl2/1 mM MgSO4/10 mMHepes/10 mM glucose/neostigmine (0.25 mg/liter); pH wasbuffered to 7.4 with Hepes-NaOHI. The osmolarity wascorrected to 300 milliosmolar. Intracellular electrodes filledwith 3 M KCI had a resistance of 20-40 Mfl. The restingmembrane potential was measured in reference to an Ag/AgCl ground electrode placed in the chamber. Spontaneousminiature end-plate potentials (mepps) were measured withan Axoclamp-2A amplifier (Axons Instruments, Burlingame,CA) and stored on magnetic tape for computer analysis. Bathtemperature (28°C) was monitored with a thermistor probeplaced close to the muscle in the chamber solution.

RESULTS AND DISCUSSIONHind.limb weakness developed in mice (Fig. 1) injected withserum or IgG from guinea pigs with either moderate or severesymptoms of EAGMD, characterized by acute limb weak-ness with marked muscular atrophy within 4 weeks of inoc-ulation with bovine ventral horn homogenates (2). Miceinjected with serum or immunoglobulins from animals withmild EAGMD, mild or moderate EAMND, or from ALSpatients did not develop signs of weakness after inoculation.None of the injected mice showed either microscopic or

macroscopic morphologic alterations of the central nervoussystem, peripheral nerve, or the neuromuscular junction.

FIG. 1. BALB/c mice after two injections within 24 hr of serumfrom control guinea pig (on right) and from a guinea pig with EAGMD(on left).

However, by immunohistochemical techniques, reactivityfor immunoglobulin approximated the reactivity for a-bun-garotoxin in mice injected with serum or immunoglobulinsfrom the two animal models and human ALS (Fig. 2) similarto the colocalization of a-bungarotoxin and IgG in the animalmodels in vivo (1-3). In mice inoculated with EAGMD serum

FIG. 2. Immunohistochemical reaction of the EDL neuromuscu-lar junction and spinal cord ventral horn cells. (A) End plate from amouse injected withALS immunoglobulins. Stained with rhodamine-conjugated a-bungarotoxin. (x 1175.) (B) Sameend plate stained withFITC-conjugated goat anti-human IgG. (x1175.) (C) Absence of IgGreactivity in ventral horn of a mouse injected with control guinea pigserum. (x525.) (D) Motoneurons from the ventral horn of a mouseinjected with EAMND sera demonstrating patchy cytoplasmic IgGreactivity. (x525.)

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Proc. Natl. Acad. Sci. USA 88 (1991) 649

or immunoglobulins, 602 end plates were examined. Sixtypercent of the end plates that stained with a-bungarotoxinalso displayed fluorescent staining for IgG. In mice injectedwith EAMND serum, 400 end plates were examined. Twen-ty-five percent of the neuromuscular junctions stained pos-itively for IgG. Mice injected with immunoglobulins fromALS patients were positive for IgG in 30 ± 5% (mean ± SEM)of the 500 neuromuscular junctions examined. An approxi-mation of IgG and a-bungarotoxin reactivity was seen in<5% of =600 neuromuscular junctions stained with a-bun-garotoxin in control conditions in mice injected with immu-noglobulins from normal or disease control patients or fromcontrol guinea pigs.

After 3 days of inoculation with EAMND serum, slightreactivity for IgG was detected within lower motoneurons, ina granular cytoplasmic distribution (Fig. 2). The cytoplasm ofpyramidal cells was also positively stained, while the nucleuswas unstained. Analogous uptake of anti-synaptosomal IgGand transport to the cell body has been documented (11, 12).The staining of cortical cells was mainly in the corticalrepresentation of the hind legs, the frontal horns of the lateralventricles, the choroid plexus, and to a lesser extent Purkinjecells. In mice injected with serum of EAGMD animals,staining was also observed in lower motoneurons and brainstem motoneurons, with more intense staining of pyramidalcells than noted with EAMND sera. In addition, Purkinjecells and small cerebellar neurons were stained. Mice inoc-ulated for 3 days with ALS IgG exhibited staining ofthe lowermotoneurons and pyramidal cells with a similar intensity anddistribution as animals injected with EAMND serum. Only atrace of IgG activity was noted in motoneurons and other

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cells in mice inoculated with control human IgG or controlguinea pig serum or immunoglobulins. No IgM reactivity wasdetected within the central nervous system after injection ofguinea pig or ALS globulins.The EDL muscle was used to monitor physiologic alter-

ations of the neuromuscular junction. Fig. 3 shows repre-sentative records of mepps from EDL muscles from normalmice and mice injected with EAMND and EAGMD guineapig serum and with immunoglobulins from a normal patientand an ALS patient. These records clearly demonstrate thatthe frequency of mepps in mice injected with serum fromEAMND and EAGMD and immunoglobulin from ALS pa-tients is significantly increased. The postsynaptic element(muscle fibers) was not affected since the resting membranepotential, the amplitude, and time course of mepp in controland injected mice was identical, regardless of whether themepp frequency was increased or not. The values of meppamplitude and decay time constant were, respectively (mean± SEM): (i) control conditions, 0.56 ± 0.01 mV and 4.36 ±0.03 msec (22 fibers, 11 animals); (ii) mice injected withEAMND and EAGMD serum, 0.54 ± 0.01 mV and 4.47 ±0.25 msec (24 fibers, 9 animals); (iii) mice injected with ALSimmunogloublin, 0.56 + 0.01 mV and 5.01 ± 0.21 msec (48fibers, 12 animals). The resting membrane potential of themuscle fibers had similar values in mice injected with normalhuman globulins (-76.4 ± 1.7 mV; n = 62), disease controlglobulins (-74.7 ± 0.9 mV; n = 62), and ALS globulins(-73.1 ± 1.4 mV; n = 73).

All muscles from mice injected with EAMND and EAGMDserum and globulins exhibited an increase in mepp frequency,although individual muscle fibers were affected to a different

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FIG. 3. Continuous records of mepps from normal mice, mice injected with guinea pig serum with EAGMD, EAMND, or humanimmunoglobulins from control patients orALS patients. Each mouse received daily injections intraperitoneally for 3 days. Control animals wereuntreated mice or mice injected with normal guinea pig serum, normal human immunoglobulin, or immunoglobulin from patients with myastheniagravis, Guillian-Barr6 syndrome, or chronic relapsing inflammatory polyneuropathy. (A) Records correspond to a normal (untreated) mouse.(B and C) Records correspond to mice injected with serum obtained from guinea pigs immunized with gray matter or motoneurons, respectively.(D and E) Mepps obtained from a control mouse injected with normal human immunoglobulin and from a mouse injected with immunoglobulinfrom a patient with ALS, respectively.

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extent. To assess the degree of involvement of differentmuscle fibers, histograms of mepp frequencies were con-structed (Fig. 4). In control conditions, mepp frequencieswere similar. Normal mice had a mean frequency of 4 ± 0.3mepps per sec (n = 44); mice injected with immunoglobulinfrom normal patients had a mean frequency of 4.5 ± 0.3mepps per sec (n = 55); and mice injected with immunoglob-ulin from non-ALS neurologic controls had a mean frequencyof 3.9 ± 0.3 mepps per sec (n = 55). On the other hand, inmice injected with EAMND, EAGMD, orALS immunoglob-ulins, -50%o of fibers had very high mepp frequencies (>20mepps per sec). These results indicate that many, but not all,of the nerve terminals were involved in this process. Fur-thermore, the reported increase in frequency was not due tononspecific damage of the postsynaptic element since musclefibers from all injected mice had normal resting potentials andnormal mepp amplitude and time course. Thus, the higherfrequency in mice injected with serum or IgG from diseasedguinea pigs or ALS patients suggests a physiological alter-ation ofthe presynaptic terminal. Since the mepp results fromopening of the acetylcholine receptor in the postsynapticmembrane, the increase in number of these events reflectslarger acetylcholine release from the nerve terminal in theresting state.The fact that immunoglobulins from immune-mediated

animal models of motoneuron destruction and human ALScan passively transfer physiologic alterations provides im-portant evidence for the role of immunoglobulins in thesesyndromes, just as such passive transfer experiments pro-

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vided cogent evidence for the role of immunoglobulins inmyasthenia gravis (8) and in the Eaton-Lambert myasthenicsyndrome (9). The enhanced resting mepp frequency furtherdefines the neuromuscularjunction as a target for the immuneattack. However, the question must be raised as to whetherthe enhanced resting mepp frequency has significance as aphysiological sign ofearly motor neuron disease. In EAMNDand EAGMD, enhanced resting mepp frequency was notedearly in the course of disease and prior to the onset ofweakness and motoneuron destruction (1-3). Studies of an-coneus muscle biopsies in ALS have documented changes inspontaneous mepp frequency and quantal content, both ofwhich draw attention to the neuromuscular junction as a siteof altered physiology (7), but no studies have been carried outof neuromuscular physiology at an early stage of the disease.Thus it is not clear whether abnormalities of the neuromus-cular junction represent the earliest stage of ALS.

Acetylcholine is known to be released in quantal form byelectrical stimulation of the nerve, whereas at rest acetyl-choline is released as molecular leakage as well as quantalrelease, which is dependent on calcium (13-17). In motoneu-ron syndromes, the demonstration of enhanced mepp fre-quency, both in the present passive transfer experiments andin our previous in vitro studies (18), in fact may have moresignificance as a functional assay ofimmunoglobulin-inducedincrease of cytoplasmic calcium, especially since BAY-K8644 potentiates calcium currents (19-21) and increasesspontaneous and evoked acetylcholine release at the neuro-muscular 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 dataof each group of animals were plotted. The data shown, in A correspond to all untreated mice. (B) Pooled data obtained from mice injected withguinea pig serum or IgG from gray matter-immunized guinea pigs. (C and D) Data from mice injected with immunoglobulin from two normalindividuals or from three individuals with non-ALS neurological disorders, respectively. (E) Distribution ofmepp frequency obtained from miceinjected with immunoglobulin from patients with ALS.

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cium may in turn trigger processes that lead to cell death ashas been well documented with neuronal cell death in otherexperimental paradigms (24-26). Thus our data would sug-gest that EAMND, EAGMD, and ALS immunoglobulinsinteract with the neuromuscular junction, result in an in-creased intracellular calcium in the motor nerve terminal andan increased resting acetylcholine release, and trigger pro-cesses leading to cell death. The rapidity of immunoglobulinaction suggests the possibility of a direct effect on membranechannels or receptors that influence calcium entry. However,the difficulty in carrying out patch-clamp studies on themammalian motor terminal will necessitate investigation ofvoltage- 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 thatpassively transfer these physiological changes of the neuro-muscularjunction could help clarify the specific mechanismsof cell death in motor neuron disease.

This study was supported by grants from the Muscular DystrophyAssociation through a Muscular Dystrophy Association ALS CenterGrant, by the M. H. "Jack" Wagner Memorial Fund and the RobertJ. and Helen C. Kleberg Foundation, by a postdoctoral fellowshipfrom the Muscular Dystrophy Association to J.G., by a grant fromthe National Institutes of Health (RO1-AR 38970), and by a grant fromCephalon, Inc.

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