the role of autoantibodies in lambert-eaton myasthenic syndromeaa

The Role of Autoantibodies in Lambert-EatonMyasthenic Syndromea

BETHAN LANG,b SALLY WATERMAN,b ASHWIN PINTO,b

DOMINIC JONES,b FRASER MOSS,c JOHN BOOT,c PAUL BRUST,d

MARK WILLIAMS,d KENNETH STAUDERMAN,d MICHAEL HARPOLD,d

MASAKATSU MOTOMURA,e J. WIBE MOLL,f ANGELA VINCENT,b

AND JOHN NEWSOM-DAVISb

bNeurosciences GroupInstitute of Molecular Medicine

University of OxfordOxford OX3 9DU, United Kingdom

cLilly Research Center LimitedWindlesham, Surrey, United Kingdom

dSIBIA Neurosciences IncorporatedLa Jolla, California 92037

eFirst Department of Internal MedicineNagasaki University School of Medicine

Nagasaki 852, Japan

fDepartment of Neuro-OncologyDr. Daniel den Hoed Cancer Center3075 EA Rotterdam, the Netherlands

The Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder ofneuromuscular transmission that causes fatigable muscle weakness, loss of tendonreflexes, and autonomic dysfunction.1–3 LEMS antibodies bind to and induce adownregulation of voltage-gated channels (VGCCs),4,5 resulting in a reduction in thenerve-evoked, Ca2+-dependent release of acetylcholine from motor nerve terminals.Approximately 60% of patients have an associated small cell lung carcinoma(SCLC),6 a tumor that is thought to be neuroendocrine in origin. This strong associa-tion with cancer makes LEMS a member of the group of paraneoplastic disorders.SCLC cells have been shown to express functional VGCCs,7,8 and preincubation inLEMS sera or IgG reduces the K+-stimulated 45Ca2+ flux into these cells.8–10

596

aThis work was supported by the Medical Research Council (United Kingdom), the Myas-thenia Gravis Association (United Kingdom), the Queen’s Trust of Australia, the LeopoldMuller Estate, the Sir Jules Thorne Charitable Trust, the Nuffield Foundation, and Jesus Col-lege, Oxford. A. Pinto is a recipient of a Wellcome Trust Research Fellowship and M. Motomu-ra held a Wellcome Trust Research Travelling Fellowship.

DETECTION OF ANTIBODIES TO THE N- AND P-/Q-SUBTYPES OFVGCCS USING RADIOIMMUNOASSAYS

Neurons have been shown to express multiple types of VGCCs, which may becharacterized by their electrophysiological and pharmacological profile.11–13 The L-type VGCC, characterized by its long-lasting conductance, is found in excitable cellsand in the cardiovascular system and is sensitive to dihydropyridines. By contrast, theN-, P-, and Q-type VGCCs appear to be confined to neuronal/neuroendocrine cells.The N-type VGCCs are involved in neurotransmitter release in amphibian neuromus-cular junctions and mammalian brain, but the P-/Q-type VGCCs mediate transmitterrelease at the mammalian neuromuscular junction.14,15 N-, P-, and Q-type VGCCshave been demonstrated in SCLC cells by biochemical, electrophysiological, andmolecular biological techniques.16–18

The presence of serum antibodies to the VGCC subtypes have been detected inLEMS patients using specific neurotoxins. Initially, �-conotoxin GVIA (�-CgTx)derived from the fish-eating snail, Conus geographus, which binds specifically andessentially irreversibly to N-type VGCCs, was used as a basis for a radioimmunoas-say (see FIGURE 1). Although initially a high number of patients were reported as hav-ing antibodies against 125I-�-CgTx-labeled (N-type) VGCCs,19 subsequent reports inthe literature have put this figure at 44–52%.20,21

Greater positivity has been obtained using a different Conus snail toxin, �-cono-toxin MVIIC (�-CmTx), which binds to both P- and Q-type VGCCs.22–24 Using125I-�-CmTx to label digitonin extracts of human cerebellum, we have detected anti-bodies in greater than 92% of clinically definite LEMS patients (n = 72).23 In our ini-tial study, we also looked for the presence of these antibodies in control patients (seeFIGURE 1). Only 1 of 66 was positive, an amyotrophic lateral sclerosis (ALS) patientwith a very low titer. We have subsequently investigated larger numbers of controls(TABLE 1). In 90 patients with SCLC, but no neurological symptoms, we found anti-125I-�-CmTx-labeled VGCC antibodies in 3.3%. Also, 15% of patients (n = 88) withparaneoplastic encephalomyelitis or subacute sensory neuronopathy, who were posi-tive for antineuronal nuclear antibody (anti-Hu or ANNA-1), had anti-P-/Q-typeVGCC antibodies. Interestingly, a higher association was seen in patients with para-neoplastic encephalomyelitis or subacute sensory neuronopathy without associatedantineuronal antibodies.25

Immunohistochemical staining has demonstrated that some typical LEMS pa-tients, with or without cerebellar symptoms, do have antineuronal antibodies of asyet unknown specificity in their sera (see Polizzi et al. elsewhere in this volume).

ROLE OF AUTOANTIBODIES IN THE AUTONOMICDYSFUNCTION OF LEMS PATIENTS

In addition to muscle weakness, around 80% of LEMS patients have some auto-nomic dysfunction such as dry mouth, constipation, impaired sweating, poor bladdercontrol, and (in men) sexual impotence.6 The neuronal basis of the autonomic symp-toms has not been previously investigated. N-, P-, and Q-type VGCCs subserve trans-

LANG et al.: AUTOANTIBODIES 597

ANNALS NEW YORK ACADEMY OF SCIENCES598

FIGURE 1. Serum antibodies in LEMS and control patients measured by immunoprecipita-tion of solubilized human cerebellar VGCCs labeled with 125I-�-CgTx GVIA (A) or 125I-�-CmTx MVIIC (B). Antibody titers were considered positive if greater than 10 pM (A) or 18pM (B) (mean titer + 3 SD above mean for healthy controls). Terms: LEMS, Lambert-Eatonmyasthenic syndrome; SCLC, small cell lung carcinoma; NCD, no cancer detected; SU, statusuncertain; HC, healthy control; ALS, amyotrophic lateral sclerosis; MG, myasthenia gravis;RA/SLE, rheumatoid arthritis/systemic lupus erythematosus.


mitter release from autonomic neurons,26,27 and we present evidence here that anti-bodies to P- and Q-type VGCCs are responsible for the autonomic dysfunction.

We used the passive transfer model of LEMS, previously developed to investigateskeletal neuromuscular function, to study the effects on transmitter release frompostganglionic parasympathetic neurons in the bladder and from postganglionic sym-pathetic neurons in the vas deferens.28 Plasma was obtained from patients undergo-ing therapeutic plasmapheresis. IgG was prepared by the rivanol-ammonium sulfatemethod as previously described.28 Control IgG was prepared from patients with otherneurological disorders [Guillain-Barré syndrome (GBS), myasthenia gravis (MG),polymyositis (PM), and acquired neuromyotonia (NMT)], from patients with SCLC,but without neurological signs, and also from healthy volunteers.

Male albino mice were injected ip with IgG (10 mg in 1 mL per day) for eightdays. On day 9, the mice were killed. The urinary bladder and vas deferentia were ex-cised and mounted in organ baths in Krebs solution (see references 26 and 27). Elec-trically evoked contractions were recorded as described previously. Capsaicin (10�M), guanethidine (3 �M), and hexamethonium (500 �M) were added to bladderpreparations to inactivate functionally sensory neurons, block transmitter releasefrom sympathetic neurons, and block nicotinic ganglionic transmission, respectively.Calcium-dependent, tetrodotoxin-sensitive contractions could thus be attributed totransmitter release from postganglionic parasympathetic neurons. Similarly, postgan-glionic sympathetic responses in vas deferens preparations were pharmacologicallyisolated using capsaicin and hexamethonium. Yohimbine (0.3 �M) was also added tovas deferens preparations to block presynaptic inhibition mediated by �2 adrenocep-tors. Preparations were stimulated at frequencies of 1–50 Hz in the absence and pres-ence of calcium-channel neurotoxins. �-CgTx GVIA (30 nM) was used to block N-type VGCCs. In the continued presence of �-CgTx, �-agatoxin IVA (�-AgaTx) (300

TABLE 1. Anti-�-CmTx-labeled (P-/Q-type) VGCC Antibodies in Control Sera

No. of PositivesGroup n (>20 pM) Positive (%)

SCLC (without neurological signs) 90 3 3.3

Paraneoplastic (subacute sensory 94 13 14neuronopathy, paraneoplastic encephalomyelitis, paraneoplasticcerebellar degeneration)

ANNA1+ (SCLC) 88 13 15ANNA2+ (breast) 2 0 0PCA+ (breast/gynecol.) 4 0 0

Other autoimmune diseasesa 36 0 0

Healthy controls 40 0 0

aOther autoimmune disease controls: Guillain-Barré syndrome (3), myasthenia gravis (15),acquired neuromyotonia (3), polymyositis (1), systemic lupus erythematosus (10), and insulin-dependent diabetes mellitus (4).


nM), a toxin that specifically blocks P-type VGCCs, was added. �-CmTx MVIIC(300 nM) was then added to block Q-type VGCCs.

FIGURE 2 shows the VGCC subtypes required for transmitter release from theparasympathetic neurons of the bladder of mice injected with control IgG. �-CgTxGVIA reduced the contraction amplitude by approximately 50%, demonstrating theimportant role of N-type VGCCs in transmitter release at this site. Subsequent addi-tion of �-AgaTx IVA and �-CmTx MVIIC also produced significant inhibition, indi-cating roles for P- and Q-type channels, respectively, in transmitter release. The re-maining contraction was abolished by tetrodotoxin. Similar neurotoxin profiles wereconstructed for the vas deferens.

Mice passively transferred with LEMS IgG for eight days showed a marked dif-ference (FIGURE 3) in both preparations. �-CgTx GVIA abolished transmitter re-lease, but �-AgaTx IVA and �-CmTx MVIIC had no further effect. Thus, we con-clude that P- and Q-type VGCCs had already been downregulated by the LEMS anti-bodies and that transmitter release was solely dependent on calcium influx throughN-type channels. Similar results were obtained in postganglionic sympathetic neu-rons in the vas deferens of mice injected with LEMS IgG. Injection of IgG from 9 pa-tients caused a functional inhibition of P-type VGCCs. Eight of the 9 caused an inhi-bition of Q-type VGCCs and only 1 patient caused an inhibition of the N-typeVGCCs. None of the controls produced any effect (TABLE 2).

FIGURE 2. Subtypes of VGCCs required for transmitter release from parasympathetic neu-rons in the mouse bladder. Mice were injected with IgG from pooled healthy control serum (n =4). �-CgTx GVIA (30 nM) reduced contraction amplitude by approximately 50%, demonstrat-ing the important role of N-type channels in transmitter release. Subsequent addition of�-AgaTx IVA (300 nM) and �-CmTx MVIIC (300 nM) also produced significant inhibition,indicating roles for P- and Q-type channels, respectively. The remaining contraction was abol-ished by tetrodotoxin (0.3 �M).


EFFECT OF LEMS IgG ON CLONED NEURONAL VGCCS

Neuronal VGCCs are multimeric proteins that consist of an �1 subunit (which isthought to possess both channel function and drug binding sites), a � subunit (whichis thought to be intracellular), and disulfide bridge–linked �2/� subunits. Six genesencoding the �1 subunit have been reported (�1A, �1B, �1C, �1D, �1E, and �1S). �1C,�1D, and �1S encode for L-type (dihydropyridine-sensitive) VGCCs, while the chan-nel phenotype encoded for by the �1E is as yet unknown. The �1B gene has beenshown to code for the �-CgTx GVIA–labeled (N-type) VGCCs. The exact nature of

FIGURE 3. Effect of LEMS IgG on transmitter release from parasympathetic neurons in thebladder. �-CgTx GVIA abolished transmitter release, but �-AgaTx IVA and �-CmTx MVIIChad no effect. Thus, P- and Q-type VGCCs had been downregulated by the LEMS antibodiesand transmitter release was solely dependent on calcium influx through N-type channels (n =4).

TABLE 2. Effect of LEMS IgG on Transmitter Release in Autonomic Neurons

Functional Functional Functional Inhibition Inhibition Inhibition of P-type of Q-type of N-type Antibody AutonomicChannels Channels Channels Titer Symptoms

LEMS 9/9 8/9 1/9 9/9 P/Q+ 9/92/9 N+

Disease controlsa 0/6 0/6 0/6 0/6 1/6 (GBS)

Healthy controls 0/2 0/2 0/2 0/2 0/2

aDisease controls: Guillain-Barré syndrome (GBS) (2), myasthenia gravis (1), acquired neu-romyotonia (1), polymyositis (1), and small cell lung carcinoma without neurological symp-toms (1).


the channel encoded for by the �1A gene is uncertain and its expression with differentisoforms of the other subunits appears to determine whether the channel exhibits P-or Q-type characteristics.

Human neuronal VGCC subunits have now been cloned and transfected into hu-man embryonic kidney (HEK) cells. Four lines were studied: 10-13 (�1A-2, �4a, �2/�),which is potently blocked by �-AgaTx IVA; G1A1 (�1B-1, �1b, �2/�) blocked by �-CgTx GVIA; 5D12-20 (�1D, �2/�, �3a) blocked by dihydropyridines; and the resistantcell line E52-3 (�1E-3, �1b, �2/�) (for further methodology, see Pinto et al. elsewherein this volume).

Transfected cells were incubated overnight in IgG prepared from patients withLEMS or from controls. The cells were loaded the following day with the fluorescentcalcium-sensitive dye Fluo-3AM for one hour and then depolarized by exposure to ahigh concentration of KC1. The fluorescence change, which relates to the influx ofcalcium into the cells, was measured in real time using the Ascent Fluoroscan II. FIG-URE 4 shows the effect on calcium flux of preincubation of the cell lines in LEMS orcontrol IgG as compared to cells grown in media alone. LEMS IgG causes a reduc-tion in the K+-stimulated calcium flux into cell line 10-13 by 75% (p < 0.01) as com-pared to control IgG. In the three other cell lines (G1A1, 5D12-20, E52-3), preincu-

FIGURE 4. Effect of LEMS and control IgG on calcium flux in transfected HEK cell lines.Results are expressed as the percentage change in K+-stimulated calcium flux as compared tocells grown in media alone. Incubation with LEMS IgG leads to a reduction in calcium flux inthe �1A-2 (10-13) cell line only.

bation in both LEMS and control IgG causes a rise in calcium flux. Further experi-ments have shown that, within each of these three cell lines, there is no significantdifference in K+-stimulated calcium influx between LEMS and control treated cells(data not shown). These results show that, in an intact cell system, LEMS antibodiesaffect the function of P-/Q-type VGCCs, but not N-, L-, or R-type VGCCs.

CONCLUSIONS

We have used three different methods to investigate the target of autoantibodiesfrom patients with LEMS. Using a standard radioimmunoassay, we have shown thatmore than 90% of patients have antibodies against the 125I-�-CmTx MVIIC–labeled(P-/Q-type) VGCCs, but less than 40% have them against the 125I-�-CgTx GVIA–labeled (N-type) VGCCs. We have investigated the role of these antibodies in the au-tonomic system using the mouse passive transfer model. These results demonstratedthat 9/9 and 8/9 patients’ IgG respectively block transmission through P-type and Q-type VGCCs, whereas only 1 of the 9 has any effect on N-type VGCCs. Finally, usingHEK cells transfected with the genes for subunits of human VGCC subtypes, calciuminflux through the 10-13 line that expresses the �1A gene was affected by LEMS IgG,while the other VGCC subtypes were not affected. Thus, antibodies against the P-/Q-type VGCCs are the main pathogenic target in the majority of LEMS patients.

ACKNOWLEDGMENTS

We are grateful to P. Busby and H. McMath for technical assistance and to Pfizerfor the kind gift of the �-AgaTx IVA.

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the role of autoantibodies in lambert-eaton myasthenic syndromeaa

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