Factors in the cerebrospinal fluid of multiple sclerosis patients interfering with voltage-dependent sodium channels

Neuroscience Letter~s, 150 (1993) i 72 i ',© 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/03/$ 06011

172

NSL 09629

Letter to the Editor

Factors in the cerebrospinal fluid of multiple sclerosis patients interfering with voltage-dependent sodium channels H. Brinkmeier a, K . H . Wollinsky b, M.J. Seewald", P.-J. Hiilser c, H.-H. M e h r k e n s b, H . H . K o r n h u b e r c a n d R. Rfidel a '~Abteilungfiir Allgemeine Physiologie der Universitgit UIm, bAbteilungfiir Aniisthesiologie und lntensivmedizin, Rehabilitationskrankenhaus UIm and 'Abteilung fi'ir Neurologie, Rehabilitationskrankenhaus Ulm, UIm (FRG) (Received 2 March 1993; Revised version received 5 April 1993; Accepted 5 April 19933

Key words: Sodium channel; Multiple sclerosis: Autoimmune disorder; Human myoball; Whole-cell recording; Cerebrospinal fluid The effect of cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) on voltage-dependent Na + channels in human myoballs was studied. The transient Na + currents, elicited by whole-cell depolarization from -85 to - 2 0 mV, were decreased to 75--25% the control value in the presence of CSF from all 7 MS patients investigated. The effect was complele in about 5 s and was fully reversible on admission of standard external fluid. Such decrease was not or only to a minor extent observed with 10 out of 11 control CSFs from patients without inflammatory neurological disease. The origin of the factors interfering with the Na * channels is unknown. It is suggested that, in addition to demyelination, impaired Na* channel function might cause the symptoms in MS.

Multiple sclerosis (MS) is the most common chronic inflammatory disease of the nervous system in man. A pathologic reaction of the immune system directed against structures of the nervous system, in particular against components of myelin, is commonly regarded as underlying the disease [1]. The destruction of the myelin sheath impairs impulse conduction of the affected neurons. Both reduced and increased nerve activity can result, so that the clinical picture of MS is characterized by pareses and spasticity. The autoimmune reaction against myelin is assumed to be an inflammatory process [1, 14] started by an activation of autoantigenic T lymphocytes which then penetrate into the brain [13, 14]. Macrophages and antibodyproducing B lymphocytes would follow [9, 12] through an impaired blood-brain barrier [11]. Increased amounts of immunoglobulins [12] and increased levels of cytokines [8, 10] are observed in the cerebrospinal fluid (CSF) of the patients. The common explanation of the characteristic impairments of impulse conduction in MS as caused by the demyelination of the neurons is based on evidence produced by electrophysiology [6] and histology [16]. HowCorrespondence: R. Rtidel, Abteilung fiir Allgemeine Physiologie der Universit/it Ulm, Albert-Einstein-Allee 11, D-7900 Ulm, FRG. Fax: (49) 731-502-3242.

ever, doubts have been raised whether demyelination is the only reason for the neuronal defects in MS [15]. In a recent editorial for 'Muscle and Nerve', it was postulated that 'systemic factors (such as antibodies) might interact with Na + channels in this disease' [17], In fact, for another autoimmune disorder, polyradiculoneuritis (Guillain Barr6 syndrome, GBS), the existence of such Na + channel-blocking factors in the CSF from patients has recently been demonstrated [4]. In the present study, we have searched for the postulated factors in the CSF from MS patients that can interfere with the Na + channels responsible for the excitatory process. Samples of CSF were obtained from MS patients being in the active phase. All patients were in a late stage of the disease and had ceased to respond to the usual treatments after many exacerbations. Spinal puncturing was performed for individual therapeutic trials [18], and the present study was a by-product of these tests. Control CSFs were from patients having no inflammatory neurological disease. All patients gave informed consent to the tests, according to the regulations of the Ethics Committee of the University of UIm. Cells for assaying the effects on the Na + channels were taken from primary human skeletal muscle cultures grown routinely in our laboratory [3]. The cells were in the differentiation stage of myotubes when they were converted into myoballs, floating spherical muscle cells

173

suited for electrophysiological experiments. For recordings of the Na + current, the myoballs were transferred to a hydrophobic experimental dish filled with standard 'external fluid' containing (in mM); NaC1 140, KC1 3.5, CaC1z 1.0, MgC12 1.0, 2,4-(2-hydroxyethyl)-l-piperazineethane sulphonic acid (HEPES) 2, yielding pH 7.4. While being inspected with an inverted microscope, the cells were sucked to patch pipettes filled with 'internal solution' containing (in raM): CsCI 140, MgC12 1.4, [ethylenebis(oxononitrilo)]tetraacetate (EGTA) 10, HEPES 10 (tip resistances 300 to 500 kf~). Recording of Na + currents according to the whole-cell technique and the evaluating of data were as described [2, 4]. For an extensive characterization of the Na ÷ channels in human myoballs see ref. 7. CSF and control fluid were applied by quickly shifting the pipette with the attached myoball between two parallel streams ejected into the experimental chamber [7]. Electrophysiological parameters were measured with every CSF probe in 3-5 myoballs, and for each pa-

tient means were calculated + S.D. Grand means were calculated for the results obtained with MS patients and controls, taking into account the individual S.D. values. When CSF from an MS patient was flushed against a myoball that was repetitively stimulated at 1 Hz with depolarizing pulses going from -85 to -20 mV, the Na + currents were always decreased, in the most extreme case to 0.25 the original value (mean value for 7 patients 0.51 + 0.22; see Table 1). Fig. 1A shows two original current traces measured in the same myoball before and after CSF application. The effect occurred within a few seconds (Fig. 1B) and was completely reversible upon washing with external fluid. Much less effect, or none, was seen in 11 control CSFs, except control 7 (Fig. 1C,D; Table 1). We have no explanation for the effect exerted by control 7, at any rate, even with the abnormal result included, the difference between the effect of MS and control CSFs was highly significant (P < 0.01, Wilcoxon rank sum test).

TABLE I E L E C T R O P H Y S I O L O G I C A L P A R A M E T E R S O F T H E S O D I U M C U R R E N T S IN M Y O B A L L S B E F O R E , D U R I N G A N D A F T E R T H E A P P L I C A T I O N OF C E R E B R O S P I N A L F L U I D (CSF) F R O M PATIENTS W I T H M U L T I P L E SCLEROSIS (ABOVE) A N D F R O M C O N T R O L S (BELOW) C o l u m n 1: inhibition of the peak Na t currents, Im~x,elicited by 1-Hz stimulation after application of CSF (conditions as in Fig. 1). Column 2: changes of Im,x (rel.), the amplitude of the m a x i m u m sodium current (related to the previous value in standard external fluid). Column 3: A Vh, the shift (with respect to value in standard external fluid) of the membrane potential (in mV) at which the h= curve has its point of inflexion. Columns 4 and 5: same as columns 2 and 3, after readmission of standard external fluid (values related to those of the first exposure to standard external fluid, n, number of myoballs tested. All data are means ± S.D, Standard external fluid

Cerebrospinal fluid

MS Patients

Controls

Im,x at 1 Hz

Im~x (rel.)

AVh (mY)

Im,x (rel.)

zlVh (mV)

0.45+--0.17 0.53 ± 0.06 0.38 -2_0.13 0.77 _+ 0.18 0.73 -+ 0.05 0.60 + 0.14 0.25 + 0.18

1.14__-0.06 1.07 _+ 0.08 0.93 _+ 0.03 1.42 _+ 0.09 1.33 _+ 0.10 1.19 _+ 0.05 1.28 -+ 0.10

- 7.67+2.40 -12.34 + 1.83 -13.23 _+ 2.08 -11.68 + 2.93 -11.26 + 2.60 -10.27 +- 4.14 -19.78 -+ 1.78

0.97_+0.09 0.89 +- 0.10 0.85 + 0.04 1.03 + 0.07 1.03 + 0.11 0.98 -+ 0.04 0.90 + 0.08

-2.11_+2.29 -2.97 + 0,49 -1.86 + 2,41 -2.01 + 2.96 -3.92 _+ 4.06 -0.82 + 2.81 -1.18 + 0.92

(3) (3) (3) (3) (3) (3) (5)

0.51-+0.22"

1.20-+0.17

-12.99_+4.55"

0.95"+0.09

-2.04_+2.07

(23)

1 2 3 4 5 6 7 8 9 10 11

0.74_+0.17 1.07 -+ 0.02 1.21 ±'0.21 0.94_+0.04 0.90 ± 0.06 0.66 _+ 0.08 0.40-+0.18 1.08 -+ 0.30 0.81 -+ 0.08 0.94 -+ 0.08 1.00_+0.18

1.19_+0.01 1.14 i 0.03 1.27 + 0.12 1.31 _+0.06 1.27 -+ 0.10 0.93 _+ 0.07 1.11-+0.14 1.49 + 0.12 1.15 _+ 0.12 1.16 -+ 0.02 1.15_+0.04

- 7.89_+2.71 - 0.98 -+ 1.03 - 3.25 -+ 1.98 - 5.01 _+ 1.56 - 8.66 + 0.78 - 9.02 + 1.45 -13.69-+4.11 - 3.60 + 4.87 - 7.52 -+ 2.04 - 5.35 _+ 3.35 - 0.83_+1.52

0.92-+0.11 1.00 ± 0.04 1.04 + 0.06 0.98_+0.11 1.00 _+ 0.04 0.84 ± 0.04 0.96-+0.12 0.93 ± 0.07 0.93 ± 0.10 0.95 ± 0.06 0,83_+0.08

-2.43+0.92 0.90 + 0.27 0.14 + 1.18 -0.74_+0.52 -1.15 _+ 0.40 -2.13 + 1.60 -1.94-+3.54 -0.93 -+ 3.12 -1.00 -+ 2.29 -1.21 + 2.36 -0.96+1.79

(3) (3) (3) (3) (3) (4) (4) (4) (4) (4) (3)

"x

0.88+0.26

1.19+_0.16

- 6.21-+444

0.94+0.08

-1.09-+2.00

(38)

1 2 3 4 5 6 7

* significantly different from controls, P < 0.01, Wilcoxon rank sum test.

(n)

174

A 0

8

I[nA] lO

-5 -10

l/'-~ e×,t. ,. L/

5

~ solution

-15

2 ms

0 1~

i

i

20

4-0 time [s]

I[nA]

-20 -~

C

I[nA]

i

20

ext, solution

-40

10 0

2

ms

D

I

1

20

40 time Is]

Fig. 1. Effect of cerebrospinal fluid (CSF) from MS patient 3 on wholecell Na + currents. A: current transients induced by square voltage pulses from - 8 5 to - 2 0 mV before (external solution) and after the application of CSF. B: time course of interference with Na ÷ currents elicited by l-Hz pulses (CSF application indicated by arrow). The maxima of the currents transients - - two of them shown in A - - are plotted against time. C,D: identical recordings as in A and B, but with CSF from control patient 4.

of control CSF (Table 1). Uh, the point ofinflexion of the h~ curve was left-shifted by 13 mV (Fig. 2C). The potential at which the Na* current had its maximum was leftshifted by the same amount (Fig. 2A). Again, these changes were almost fully reversible on re-admission of the standard external fluid (open squares in Fig. 2A,C). In contrast, CSF from controls produced only minor shifts, if at all (Fig. 2B,D and Table 1). These results show that factors can exist in human CSF that reversibly interfere with the opening of the Na + channels responsible for the excitatory inward current producing the action potential. It is not excluded that small amounts of these factors may prevail in normal CSF at small amounts. At any rate, their presence during acute phases of multiple sclerosis is high enough to be readily demonstrated. Na + channel blocking factors have been found in the CSF from GBS patients [4]. The results with CSF from GBS patients were different in two respects: (i) the Na + currents were on average more de-

MS CSF

Control

CSF

A

B test potential [mY]

test potential [mV] -90

-60

-30

0

,30

-90

-60

-30

50

~o

In an attempt to characterize the mode of interference exerted by the unknown substances in the MS CSFs, we have recorded current-voltage relations (I-V curves) and the voltage dependence of steady-state inactivation (h= curves). For each CSF sample from both patients and controls, we determined these curves and compared them with the corresponding curves measured in standard external fluid before and after the exposure to the CSF. MS CSF caused, on average, a 1.2-fold increase of the maximum Na ÷ current, /max, which was reversed to 0.95 the original control value on return to standard external fluid (Table 1). This increase in Ira,× is not specific for multiple sclerosis, as the mean values obtained for the changes with control CSF were virtually the same. We do not exactly know the reason for the increase of Im,x during the application of CSF, but most probably it is caused by the fact that the ionic compositions of human CSF and our external fluid do not perfectly match. The value of 0.95 for /max after return to standard fluid is probably caused by some rundown of the channels. In contrast to the maximum amplitude, the parameters describing the voltage dependence of the Na ÷ currents were altered by the presence of MS CSF to an extent that was significantly different from the much smaller effect

Ii

I[nA]

~,,o;

-70

-70

D

C

1.0

I[nA]

hoo

boo 0.5

0.0

I

I

-120

-90

I --~--'r

-60

....

-30

prepulse potential [mV]

-120

-90

-60

-30

prepulse potential rmV]

Fig. 2. Electrophysiological analysis of Na ÷ channel inhibition by cerebrospinal fluid (CSF). A,B: current-voltage curves recorded first in standard external fluid (©), then in CSF from MS patient 7 (A) and control patient 4 (B) (e), and finally again in external fluid (m). Pulse program: holding potential - 8 5 inV, conditioning pulse to - 135 mV for 100 ms, test potential varying between - 6 5 and +31 mV in 4-mV steps. C,D: voltage dependence of inactivation of Na + currents (h_ curves fitted using Boltzmann equation), recorded in external fluid, CSF, and again external fluid. Same symbols, cells and CSFs as in A and B. Pulse program: holding potential - 8 5 mV, conditioning pulse to - 135 mV for 100 ms, prepotential of 32 ms duration variable between - 135 and - 19 mV in 4-mV steps, test potential - 2 0 mV. Each of the curves shown in A and C is averaged from 3 measurements with 3 different cells.

175 creased, a n d (ii) for m o s t o f the C S F s tested the c u r r e n t v o l t a g e r e l a t i o n s h i p was n o t shifted as in M S . S i m u l t a n e o u s shifts o f the c u r r e n t - v o l t a g e curve a n d the h= curve o f the N a ÷ currents, as o b s e r v e d with M S C S F s , also occur w h e n the external free c o n c e n t r a t i o n s o f H ÷ or C a 2+ are lowered, b o t h ions influencing the m e m b r a n e surface charge. T h e M S C S F s used in this s t u d y were on a v e r a g e ( m e a n s + S.D.; n=7) indeed m o r e alkaline ( p H 7.8 + 0.43) a n d h a d a lower [Ca2+]e (0.73 + 0.08 m M ) t h a n o u r external fluid, b u t these devia t i o n s were to small to explain the effects on the N a + c u r r e n t s b y the C S F s . Testing the effect o f p H 8.5 a n d o f 0.33 m M [Ca2+]c on o u r m y o b a l l s , we f o u n d left-shifts o f the h= curves o f 1-3 m V u n d e r b o t h c o n d i t i o n s . A n increase in p H f r o m 7.4 to 10.4 shifted the ha curve b y only 5-6 m V in frog p e r i p h e r a l nerve [5]. C o m p a r i s o n o f the effects o f the C S F s with the largest ( p a t i e n t 4) a n d the smallest ( p a t i e n t 2) d e v i a t i o n s o f p H a n d [Ca 2+] (Table 1) does n o t suggest t h a t the d e s c r i b e d effects on N a ÷ currents seen in the presence o f M S C S F s are c a u s e d by a m o r e alkaline p H a n d / o r r e d u c e d [Ca2+]e. O t h e r molecules r e d u c i n g the positive o r increasing the negative surface charge at the N a + channels c o u l d be responsible for the d e s c r i b e d effects. F u r t h e r b i o c h e m i c a l analyses o f M S C S F s c o m b i n e d with e l e c t r o p h y s i o l o g i cal studies m a y reveal the identity o f these factors. T h e e n d o g e n o u s factors m a y o c c u r in high c o n c e n t r a t i o n s within the M S lesions. T h e y m a y i m p a i r impulse c o n d u c tion a n d cause n e u r o l o g i c a l deficits in a d d i t i o n to the well-established effects o f d e m y e l i n a t i o n . We are grateful to Ms, S. Sch/ifer a n d Ms. M. R u d o l f for c u l t u r i n g the cells, a n d Drs. Ch. F a h l k e a n d H. L o r k o v i c for c o m m e n t s on the m a n u s c r i p t . 1 Bradley, J., Recent advances in the clinical investigation of immune dysfunction relevant to multiple sclerosis, Curr. Opinion Neurol. Neurosurg., 3 (1990) 191-198. 2 Brinkmeier, H., Kaspar, A., Wieth61ter, H. and Riidel R., Interleukin-2 inhibits sodium currents in human muscle cells, Pfliigers Arch., 420 (1992) 621-623. 3 Brinkmeier, H., Mutz, J.V., Seewald, M.J., Melzner, I. and Rfidel, R., Specific modifications of the membrane fatty acid composition of human myotubes and their effect on muscular sodium channels, Biochim. Biophys. Acta, 1145 (1993) 8-14.

4 Brinkmeier, H., Wollinsky, K.H., Hiilser, P.-J., Seewald, M.J., Mehrkens, H.-H., Kornhuber, H.H. and Riidel, R., The acute paralysis in Guillain-Barr6 syndrome is related to a Na ÷ channel blocking factor in the cerebrospinal fluid, Pfliigers Arch., 421 (1992) 552-557. 5 Chernoff, D.M. and Strichartz, G.R., Kinetics of local anaesthetic inhibition of neuronal sodium currents - - pH and hydrophobicity dependence, Biophys. J., 58 (1990) 69-81. 6 Davis, F.A. and Schauf, C.L., Approaches to the development of pharmacological interventions in multiple sclerosis. In S.G. Waxman and J.M. Ritchie (Eds.), Demyelinating Disease: Basic and Clinical Electrophysiology, Raven, New York, 1981, pp. 505510. 7 Fakler, B., Ruppersberg, J.P., Spittelmeister, W. and Rfidel R., Inactivation of human sodium channels and the effect of tocainide, Pflfigers Arch., 415 (1990) 693-700. 8 Hartung, H.E, Jung, S., Stoll, G., Zielasek, J., Schmidt, B., Archelos, J.J. and Toyka, K.V., Inflammatory mediators in demyelinating disorders of the CNS and PNS, J. Neuroimmunol., 40 (1992) 197210. 9 Hauser, S,L., Bhan, A.K., Gillis, F., Kemp, M., Kerr, C. and Weiner, H.L., Immunohistochemical analysis of the cellular infiltrate in multiple sclerosis lesions, Ann. Neurol., 19 (1986) 578--587. 10 Hauser, S.L., Doolittle, T.H., Lincoln, R., Brown, R.H. and Dinarello, C.A., Cytokine accumulations in CSF of multiple sclerosis patients, Neurology, 40 (1990) 1735-1739. 11 Hawkins, C.E, Mackenzie, F., Tofts, P., Duboulay, E.P.G.H. and McDonald, W.I., Patterns of blood/brain barrier breakdown in inflammatory demyelination, Brain, 114 (1991) 801-810. 12 Kenneth, G., Catz, W. and Catz, I., Purification of autoantibodies to myelin basic protein by antigen specific affinity chromatography from cerebrospinal fluid IgG of multiple sclerosis patients, J. Neurol. Sci., 103 (1991) 90-96. 13 Martin, R., McFarland, H.F. and McFarlin, D.E., Immunological aspects of demyelinating diseases, Annu. Rev. Immunol., 10 (1992) 153 187. 14 Rodriguez, M., Multiple Sclerosis: Basic concepts and hypothesis, Mayo Clin. Proc., 64 (1989) 570-576. 15 Schauf, C.L. and Davis, F.A., Circulating toxic factors in multiple sclerosis: A perspective. In S.G. Waxman and J.M. Ritchie (Eds.), Demyelinating Disease: Basic and Clinical Electrophysiology, Raven, New York, 1981, pp. 267-280. 16 Selmaj, K., Raine, C.S., Cannella B. and Brosnan C.F., Identification of lymphotoxin and tumor necrosis factor in multiple sclerosis lesions, J. Clin. Invest., 87 (1991) 949-954. 17 Waxman, S.G., Editorial: peripheral nerve abnormalities in multiple sclerosis, Muscle Nerve, 16 (1993) 1-5. 18 Wollinsky, K.H., Hiilser P.-J., Westarp, M.E., Mehrkens, H.H. and Kornhuber H.H., Liquorpherese bei 10 Patienten mit Multipler Sklerose, Verh. Dtsch. Ges. Neurol., in press.