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Antisera to an axolemma-enriched fraction inhibit neurite outgrowth and destroy axons in vitro
Brain Research, 366 (1986) 333-337 Elsevier
333
BRE21365
Antisera to an axolemma-enriched fraction inhibit neurite outgrowth and destroy axons in vitro DENNIS N. BOURDETYEt, FREDRICK J. SEILl, JOHN W. BIGBEE2, GEORGE H. DeVRIES 2, MARGARET M. GARWOOD3and HARISH C. AGRAWAL3
1Neurology Research, VeteransAdministration Medical Centerand Departmentof Neurology, OregonHealth Sciences University, Portland, OR; 2Departmentof Biochemistry, MedicalCollegeof Virginia, Richmond, VA; 3Departmentsof Pediatricsand Neurology, Washington University School of Medicine, and Division of PediatricNeurology Research, St. Louis Children's Hospital, St. Louis, MO (U.S.A.) (Accepted October 15th, 1985)
Key words: anti-axolemma antibody - - anti-axon antibody-- axolemma - - neural tissue culture
Antisera prepared to an axolemma-enriched fraction derived from rat brain inhibited neurite outgrowth and destroyed mature axons in spinal cord-dorsal root ganglia cultures. Similar antibody-mediated anti-axon effects may be important in some diseases of the human nervous system. Antibodies to components of axons occur in patients with some inflammatory and degenerative diseases of the nervous systeml-g,lt,24. Antibodies to axons might participate in the pathogenesis of these diseases by destroying axons or preventing regeneration of damaged axons. There are few published studies to support these speculationsi6,25, 26 and little is currently known about the biologic activity of anti-axon antibodies. Explant cultures of spinal cord (SC) and dorsal root ganglia ( D R G ) provide an in vitro system for studying the effects of antibodies to axons. Pieces of 14 day fetal mouse SC with attached D R G can be cultured in nutrient medium on collagen-coated glass coverslips in Maximow chambers by established methods6A 5. After explantation, neurites, fibrocytes and glia grow out from the SC and D R G explants and form an outgrowth zone (Fig. 1A). The outgrowth zone contains axons from D R G neurons and anterior horn cells in the SC explant as well as axons from SC neurons with presumably central targets which loop into the outgrowth zone and return to the explant12,23. Abundant central axons are found within and restricted to the SC explant. These cultures pro-
vide an in vitro system for assessing the effects of antibodies on both developing and mature axons. Techniques for preparation of axolemma-enriched fractions (AEF) from mammalian central nervous system (CNS) have been developedS-10. A E F contain fragments of CNS axolemma and are relatively free of myelin and axoplasmic components. Two antisera (G and T in Table I) were produced by immunizing rabbits to rat brain AEF, as previously describers. Two additional antisera (001 and 002) were made by injecting two rabbits in multiple sites with an emulsion containing 1 mg rat brain A E F (dry weight), 1.5 ml complete Freund's adjuvant, 5 mg M. butyricum and 1.5 ml phosphate-buffered saline. These animals were injected 4 times at weekly intervals. Serum was obtained 10 days after the last injection and stored at - 7 0 °C until tested. The antisera had antibodies which bound several proteins contained in CNS and peripheral nervous system (PNS) AEF as demonstrated by immunoblot staining. Using immunohistochemical techniques, the antisera specifically stained both nodal and internodal axolemma in the PNS and appeared to stain synaptic terminals in the CNS 5.
Correspondence: D.N. Bourdette, Neurology Research (151N), Veterans Administration Medical Center, Portland, OR 97201, U.S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
334 We assessed the effects of the antisera to A E F on
posed for 4 days to m e d i u m containing 2 0 - 2 5 % anti-
fetal mouse S C - D R G explant cultures. To test for ef-
serum to A E F and 10% guinea pig serum. For both
fects on axonal d e v e l o p m e n t in the outgrowth zone,
sets of experiments, controls consisted of cultures
S C - D R G cultures were grown in m e d i u m containing
maintained in normal nutrient medium and those ex-
I 0 - 2 0 % antiserum to A E F with 10% pooled guinea
posed to medium containing rabbit preimmune serum
pig serum as a source of complement• Cultures were
and pooled guinea pig serum. Assessment of axons
exposed to antiserum to A E F either from the time of explantation or after 4 days of initial growth in normal
was made after cultures were fixed as whole mount
medium containing 2.5S mouse salivary gland nerve
preparations and stained by a H o l m e s silver impregnation technique modified for tissue culture 27.
growth factor (100 ng/ml) to increase the n u m b e r of
S C - D R G cultures maintained for 15-18 days in
surviving D R G neurons 7. Cultures were fixed after
vitro in medium containing antiserum to rat A E F dis-
15-18 days in m e d i u m containing antiserum to A E F .
played marked inhibition of axonal outgrowth from
To assess for effects on mature axons, S C - D R G cul-
both the SC and the D R G explants (Fig. 1B). Both
tures were initially grown in normal nutrient m e d i u m
the area of neurite outgrowth, which is primarily a
for 18 days. These mature cultures were then ex-
function of average axon length, and the average
Fig. 1. Cultures were fixed as whole mount preparations, impregnated with silver and photographed with darkfield optics. A: normal SC (c) and DRG (g) explants (18 days ill vitro (DIV)). The outgrowth zone (o) contains abundant axons. B: SC (c) and DRG (g) cxplants ( 18 D1V) after 14 days of exposure to antiserum to AEF. There are few axons in the outgrowth zone (o). C: outgrowth zone of a control SC-DRG culture (18 DIV) demonstrating normal axons. D: outgrowth zone of a culture (18 DIV) after 4 days of exposure to antiserum to AEF. There are many axon fragments and no intact axons. Magnification: 37× (A, B): 58× (C, D).
335 TABLE I
10% of that of controls, and the average axon density
Inhibition of axonal outgrowth
was reduced by 5 0 - 7 5 % . Growth of axons into the outgrowth zone from both D R G and SC n e u r o n s appeared equally inhibited. There were no significant differences between the effects of the 4 antisera, and
Camera lucida tracings of the zone of neurite outgrowth from spinal cord-dorsal root ganglia explants (15-22 days in vitro) were made with darkfield microscopy at 25 x. The areas of the tracings were measured with a Zeiss MOP3 image analyzer. Average axon densities 100~m from the edge of the spinal cord explants were determined by counting silver-stained axons with brightfield microscopy at 500x within a 0.001 mm2 area. Measurements were taken from 6 standard locations and an average axon density was calculated for each culture. Values expressed are means of 6-24 cultures + S.E.M. The results of cultures exposed to preimmune sera are presented as pooled data since there were no significant differences among cultures exposed to the 4 preimmune serums. Statistical analysis was performed using the Mann-WhitneyU-test.
Medium
Area of neurite outgrowth (mm 2)
Axon density (axons/ O.001 mm 2)
Normal + Preimmune Sera + Antiserum to AEF: G T 001 002
32.3 + 29.2 + 0.2 + 2.1 + 0.7 + 0.6 +
27 + 26 + 15 + 11 + 6+ 7+
3.6 2.6 0.1" 1.6" 0.3* 0.4*
1.5 1.7 1.8" 2.7* 2.5* 1.7"
* Significant at the 0.001 level.
initial growth in normal m e d i u m containing nerve growth factor did not alter the inhibitory effects. All cultures initially m a i n t a i n e d in normal m e d i u m for 18 days and then exposed to antiserum to A E F for 4 days had markedly reduced n u m b e r s of neurites and many axon fragments in the outgrowth zone (Fig. 1C, D). Distal portions of axons were more severely affected than the proximal portions, although axon destruction occurred throughout the outgrowth zone. There was no appreciable destruction of central axons restricted to the SC explant, but axons in the outgrowth zone originating from SC n e u r o n s were destroyed as were axons from D R G neurons. In some cultures the axon destruction was nearly complete with few axons remaining in the outgrowth zone. All 4 antisera to A E F destroyed outgrowth axons while p r e i m m u n e sera did not. D R G explants exposed to antiserum to A E F con-
neurite density in the outgrowth zone were diminished in antiserum-treated cultures (Table I). The average area of neurite outgrowth from S C - D R G explants exposed to antiserum to A E F was less than
tained many surviving n e u r o n s which were morphologically altered (Fig. 2). In silver-stained cultures, D R G n e u r o n a l cell bodies were smaller than normal, contained densely clumped neurofibrils and had eccentrically displaced nuclei. These changes were
Fig. 2. A: DRG neurons in normal culture (18 DIV) have centrally placed nuclei and a delicate cytoplasmic neurofibriUary pattern; 950x. B: DRG neurons in an explant (18 DIV) after 14 days of exposure to antiserum to AEF are smaller than normal, contain clumped neurofibrils and have eccentrically placed nuclei; 1100x.
336 most prominent in cultures in which neurite outgrowth had been inhibited by p r o l o n g e d exposure to antiserum to A E F but were also present in mature cultures which had been exposed to antiserum for only 4 days prior to fixation. The light microscopic appearance of the neurofibrillary changes was similar to those occurring in D R G neurons exposed to aluminum sulfate in vitro 2~. Antisera to CNS A E F inhibit axonal outgrowth from S C - D R G neurons, destroy m a t u r e outgrowth axons from both SC and D R G neurons and induce neurofibrillary changes in D R G neurons. The identity of the antigen(s) responsible for these effects is not known, but it is likely that these effects are m e d i a t e d by antibodies to c o m p o n e n t s of axolemma. This is the first r e p o r t e d evidence of axon destruction by antibodies to axolemma. Similar anti-axon effects have not been seen during in vitro studies on antisera to whole CNS tissue, whole PNS tissue, purified myelin and components of myelin, including basic protein, proteolipid protein, myelin-associated glycoprotein,
c h l o r o f o r m - m e t h a n o l insoluble proteins of myelin and galactocerebroside~3-'4.~7-20.2:. These results d e m o n s t r a t e that antisera to A E F can produce a relatively selective axotomy and provide a means of investigating the response of neurons to i m m u n e - m e d i a t e d axotomy. The neurofibrillary changes induced by antisera to A E F are of interest and may represent an accumulation of neurofilaments as a consequence of a combination of antibody-induced axotomy and inhibition of axon regeneration. The p r o f o u n d effect of antisera to A E F on axons in vitro suggests a possible role for anti-axon antibodies in inflammatory and degenerative dis-
1 Abrams, G.M., Latov, N., Hays, A.P., Sherman, W. and Zimmerman, E.A., Immunocytochemical studies of human peripheral nerve with serum from patients with polyneuropathy and paraproteinemia, Neurology, 32 (1982) 821-826. 2 Bahmanyar, S., Gajdusek, D.C., Sotelo, J. and Gibbs, C.J., Jr., Longitudinal spinal cord sections as substratum for anti-neurofilament antibody detection, J. Neurol. Sci., 53 (1982) 85-90. 3 Babmanyar, S., Liem, R.K.H., Griffin, J.W. and Gajdusek, D.C., Characterization of antineurofilament autoantibodies in Creutzfeldt-Jakob disease, J. Neuropathol. Exp. Neurol., 43 (1984) 369-375. 4 Bahmanyar, S., Moreau-Dubois, M.-C., Brown, P., Cathala, F. and Gajdusek, D.C., Serum antibodies to neurofilament antigens in patients with neurological and other diseases and in healthy controls, J. Neuroimmunol., 5 (1983) 191-196. 5 Bigbee, J.W., Calabrese, V.P. and DeVries, G.H., Characterization of an antiserum against an axolemma-enriched fraction, J. Neuroimmunol,, 7 (1985) 221-229. 6 Bornstein, M.B., Organotypic mammalian central and peripheral nerve tissue. In P.F. Kruse, Jr. and M.K. Patterson, Jr. (Eds.), Tissue Culture Methods and Applications, Academic Press, New York, 1973, pp. 86-92. 7 Crain, SM. and Peterson, E.R., Enhanced afferent synaptic functions in fetal mouse spinal cord-sensory ganglion explants foUowing NGF-induced ganglion hypertrophy, Brain Research, 79 (1974) 145-152. 8 DeVries, G.H., Isolation of axolemma-enriched fractions from mammalian CNS. In N. Marks and R. Rodnight
(Eds.), Research Methods in Neurochemistry, Plenum Press, New York, 1981, pp. 3-38. DeVries, G.H.. Anderson. M.G. and Johnson. D., Fractionation of isolated rat CNS myelinated axon by sucrose density gradient centrifugation in a zonal rotor, J. Neurochem., 40 (1983) 1709-1717. DeVries, G.H.. Matthieu, J.-M., Beny, M., Chicheportiche, R.. Lazdunski. M. and Dotivo, M., Isolation and partial characterization of rat CNS axolemma enriched fractions, Brain Research, 147 f1978) 339-352. Eiizan, T.S., Casals, J. and Yahr, M.D.. Antineurofilament antibodies in postencephalitic and idiopathic Parkinson's disease, J. Neurol. Sci. 59 (1983) 341-347. GuiUery, R.W., Sobkowicz, H.M. and Scott, G.L., Light and electron microscopical observations of the ventral horn and ventral root in Ions term cultures of the spinal cord of the fetal mouse, Y. Comp. Neurol., 134 (!968) 433-476. Hruby, S., Alvord, E.C., Jr. and Seil, F.J., Synthetic galactocercbrosides evoke myelination-inhibiting antibodies. Science, 195 (1977) 173-175. Kies, M.W., Dri.seoll, B.F., Seil, F.J. and Alvord, E.C.. Jr., Myelination inlaibition factor: dissociation from induction of experimental allergic encephalomyelitis, Science, 179 (1973) 689-690. Peterson, E.R., Crain, S.M. and Murray, M.R., Differentiation and prolon~d m m n t e m c e of bioelectriesfly active spinal cord cultures (rat, chick and human), Z. Ze~[orsch.. 66 (1965) t30-154. Schwartz, M. and Spirman, N., Sprouting from chicken embryo dorsal root ganglia induced by nerve growth factor is specifically inhibited by affinity-purified antiganglioside an-
eases of the nervous system. This study provides impetus to investigate with a similar in vitro system the role of antibodies to axons in human diseases. This work was s u p p o r t e d by the ,Veterans A d m i n istration ( D . N . B . and F.J.S.), N I H - N S 10821 and 15408 ( G . H . D . ) and N I H - N S 13464 and 19414 (H.C.A.).
9
10
11 12
13 14
15
16
337 tibodies, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 6080-6083. 17 Seil, F.J. and Agrawal, H.C., Myelin-proteolipid protein does not induce demyelinating or myelination-inhibiting antibodies, Brain Research, 194 (1980) 273-277. 18 Seil, F.J., Falk, G.A., Kies, M.W. and Alvord, E.C., Jr., The in vitro demyelinating activity of sera from guinea pigs sensitized with whole CNS and with purified encephalitogen, Exp. Neurol., 22 (1968) 545-555. 19 Seil, F.J., Garwood, M.M., Clark, H.B. and Agrawal, H.C., Demyelinating and myelination-inhibitingfactors induced by chloroform-methanol insoluble proteins of myelin, Brain Research, 288 (1983) 384-388. 20 Seil, F.J., Kies, M.W. and Bacon, M.L, A comparison of demyelinating and myelination-inhibiting factor induction by whole peripheral nerve tissue and P2 protein, Brain Research, 210 (1981) 441-448. 21 Seil, F.J., Lampert, P.W. and Klatzo, I., Neurofibrillary spheroids induced by aluminum phosphate in dorsal root ganglia neurons in vitro, J. Neuropathol. Exp. Neurol., 28 (1969) 74-85. 22 Seil, F.J., Quarles, R.H., Johnson, D. and Brady, R.O.,
23
24
25
26 27
Immunization with purified myelin-associated glycoprotein does not evoke myelination-inhibitingor demyelinating antibodies, Brain Research, 209 (1981) 470-475. Sobkowicz, H.M., Guillery, R.W. and Bornstein, M.B., Neuronal organization in long term cultures of the spinal cord of the fetal mouse, J. Comp. Neurol., 132 (1968) 365-396. Sotelo, J., Gibbs, C.J., Jr. and Gajdusek, D.C., Autoantibodies against axonal neurofilaments in patients with kuru and Creutzfeldt-Jakob disease, Science, 210 (1980) 190-193. Spirman, N., Sela, B.-A., Gitler, C., Calef, E. and Schwartz, M., Regeneration capacity of the goldfish visual system is affected by antibodies specific to gangliosides injected intraocularly, J. Neuroimmunol., 6 (1984) 197-207. Spirman, N., Sela, B.-A. and Schwartz, M., Anti-ganglioside antibodies inhibit outgrowth from regenerating goldfish retinal explants, J. Neurochem., 39 (1982) 874-877. Wolf, M.K., Differentiation of neuronal types and synapses in myelinating cultures of mouse cerebellum, J. Cell Biol., 22 (1964) 259-279.
333
BRE21365
Antisera to an axolemma-enriched fraction inhibit neurite outgrowth and destroy axons in vitro DENNIS N. BOURDETYEt, FREDRICK J. SEILl, JOHN W. BIGBEE2, GEORGE H. DeVRIES 2, MARGARET M. GARWOOD3and HARISH C. AGRAWAL3
1Neurology Research, VeteransAdministration Medical Centerand Departmentof Neurology, OregonHealth Sciences University, Portland, OR; 2Departmentof Biochemistry, MedicalCollegeof Virginia, Richmond, VA; 3Departmentsof Pediatricsand Neurology, Washington University School of Medicine, and Division of PediatricNeurology Research, St. Louis Children's Hospital, St. Louis, MO (U.S.A.) (Accepted October 15th, 1985)
Key words: anti-axolemma antibody - - anti-axon antibody-- axolemma - - neural tissue culture
Antisera prepared to an axolemma-enriched fraction derived from rat brain inhibited neurite outgrowth and destroyed mature axons in spinal cord-dorsal root ganglia cultures. Similar antibody-mediated anti-axon effects may be important in some diseases of the human nervous system. Antibodies to components of axons occur in patients with some inflammatory and degenerative diseases of the nervous systeml-g,lt,24. Antibodies to axons might participate in the pathogenesis of these diseases by destroying axons or preventing regeneration of damaged axons. There are few published studies to support these speculationsi6,25, 26 and little is currently known about the biologic activity of anti-axon antibodies. Explant cultures of spinal cord (SC) and dorsal root ganglia ( D R G ) provide an in vitro system for studying the effects of antibodies to axons. Pieces of 14 day fetal mouse SC with attached D R G can be cultured in nutrient medium on collagen-coated glass coverslips in Maximow chambers by established methods6A 5. After explantation, neurites, fibrocytes and glia grow out from the SC and D R G explants and form an outgrowth zone (Fig. 1A). The outgrowth zone contains axons from D R G neurons and anterior horn cells in the SC explant as well as axons from SC neurons with presumably central targets which loop into the outgrowth zone and return to the explant12,23. Abundant central axons are found within and restricted to the SC explant. These cultures pro-
vide an in vitro system for assessing the effects of antibodies on both developing and mature axons. Techniques for preparation of axolemma-enriched fractions (AEF) from mammalian central nervous system (CNS) have been developedS-10. A E F contain fragments of CNS axolemma and are relatively free of myelin and axoplasmic components. Two antisera (G and T in Table I) were produced by immunizing rabbits to rat brain AEF, as previously describers. Two additional antisera (001 and 002) were made by injecting two rabbits in multiple sites with an emulsion containing 1 mg rat brain A E F (dry weight), 1.5 ml complete Freund's adjuvant, 5 mg M. butyricum and 1.5 ml phosphate-buffered saline. These animals were injected 4 times at weekly intervals. Serum was obtained 10 days after the last injection and stored at - 7 0 °C until tested. The antisera had antibodies which bound several proteins contained in CNS and peripheral nervous system (PNS) AEF as demonstrated by immunoblot staining. Using immunohistochemical techniques, the antisera specifically stained both nodal and internodal axolemma in the PNS and appeared to stain synaptic terminals in the CNS 5.
Correspondence: D.N. Bourdette, Neurology Research (151N), Veterans Administration Medical Center, Portland, OR 97201, U.S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
334 We assessed the effects of the antisera to A E F on
posed for 4 days to m e d i u m containing 2 0 - 2 5 % anti-
fetal mouse S C - D R G explant cultures. To test for ef-
serum to A E F and 10% guinea pig serum. For both
fects on axonal d e v e l o p m e n t in the outgrowth zone,
sets of experiments, controls consisted of cultures
S C - D R G cultures were grown in m e d i u m containing
maintained in normal nutrient medium and those ex-
I 0 - 2 0 % antiserum to A E F with 10% pooled guinea
posed to medium containing rabbit preimmune serum
pig serum as a source of complement• Cultures were
and pooled guinea pig serum. Assessment of axons
exposed to antiserum to A E F either from the time of explantation or after 4 days of initial growth in normal
was made after cultures were fixed as whole mount
medium containing 2.5S mouse salivary gland nerve
preparations and stained by a H o l m e s silver impregnation technique modified for tissue culture 27.
growth factor (100 ng/ml) to increase the n u m b e r of
S C - D R G cultures maintained for 15-18 days in
surviving D R G neurons 7. Cultures were fixed after
vitro in medium containing antiserum to rat A E F dis-
15-18 days in m e d i u m containing antiserum to A E F .
played marked inhibition of axonal outgrowth from
To assess for effects on mature axons, S C - D R G cul-
both the SC and the D R G explants (Fig. 1B). Both
tures were initially grown in normal nutrient m e d i u m
the area of neurite outgrowth, which is primarily a
for 18 days. These mature cultures were then ex-
function of average axon length, and the average
Fig. 1. Cultures were fixed as whole mount preparations, impregnated with silver and photographed with darkfield optics. A: normal SC (c) and DRG (g) explants (18 days ill vitro (DIV)). The outgrowth zone (o) contains abundant axons. B: SC (c) and DRG (g) cxplants ( 18 D1V) after 14 days of exposure to antiserum to AEF. There are few axons in the outgrowth zone (o). C: outgrowth zone of a control SC-DRG culture (18 DIV) demonstrating normal axons. D: outgrowth zone of a culture (18 DIV) after 4 days of exposure to antiserum to AEF. There are many axon fragments and no intact axons. Magnification: 37× (A, B): 58× (C, D).
335 TABLE I
10% of that of controls, and the average axon density
Inhibition of axonal outgrowth
was reduced by 5 0 - 7 5 % . Growth of axons into the outgrowth zone from both D R G and SC n e u r o n s appeared equally inhibited. There were no significant differences between the effects of the 4 antisera, and
Camera lucida tracings of the zone of neurite outgrowth from spinal cord-dorsal root ganglia explants (15-22 days in vitro) were made with darkfield microscopy at 25 x. The areas of the tracings were measured with a Zeiss MOP3 image analyzer. Average axon densities 100~m from the edge of the spinal cord explants were determined by counting silver-stained axons with brightfield microscopy at 500x within a 0.001 mm2 area. Measurements were taken from 6 standard locations and an average axon density was calculated for each culture. Values expressed are means of 6-24 cultures + S.E.M. The results of cultures exposed to preimmune sera are presented as pooled data since there were no significant differences among cultures exposed to the 4 preimmune serums. Statistical analysis was performed using the Mann-WhitneyU-test.
Medium
Area of neurite outgrowth (mm 2)
Axon density (axons/ O.001 mm 2)
Normal + Preimmune Sera + Antiserum to AEF: G T 001 002
32.3 + 29.2 + 0.2 + 2.1 + 0.7 + 0.6 +
27 + 26 + 15 + 11 + 6+ 7+
3.6 2.6 0.1" 1.6" 0.3* 0.4*
1.5 1.7 1.8" 2.7* 2.5* 1.7"
* Significant at the 0.001 level.
initial growth in normal m e d i u m containing nerve growth factor did not alter the inhibitory effects. All cultures initially m a i n t a i n e d in normal m e d i u m for 18 days and then exposed to antiserum to A E F for 4 days had markedly reduced n u m b e r s of neurites and many axon fragments in the outgrowth zone (Fig. 1C, D). Distal portions of axons were more severely affected than the proximal portions, although axon destruction occurred throughout the outgrowth zone. There was no appreciable destruction of central axons restricted to the SC explant, but axons in the outgrowth zone originating from SC n e u r o n s were destroyed as were axons from D R G neurons. In some cultures the axon destruction was nearly complete with few axons remaining in the outgrowth zone. All 4 antisera to A E F destroyed outgrowth axons while p r e i m m u n e sera did not. D R G explants exposed to antiserum to A E F con-
neurite density in the outgrowth zone were diminished in antiserum-treated cultures (Table I). The average area of neurite outgrowth from S C - D R G explants exposed to antiserum to A E F was less than
tained many surviving n e u r o n s which were morphologically altered (Fig. 2). In silver-stained cultures, D R G n e u r o n a l cell bodies were smaller than normal, contained densely clumped neurofibrils and had eccentrically displaced nuclei. These changes were
Fig. 2. A: DRG neurons in normal culture (18 DIV) have centrally placed nuclei and a delicate cytoplasmic neurofibriUary pattern; 950x. B: DRG neurons in an explant (18 DIV) after 14 days of exposure to antiserum to AEF are smaller than normal, contain clumped neurofibrils and have eccentrically placed nuclei; 1100x.
336 most prominent in cultures in which neurite outgrowth had been inhibited by p r o l o n g e d exposure to antiserum to A E F but were also present in mature cultures which had been exposed to antiserum for only 4 days prior to fixation. The light microscopic appearance of the neurofibrillary changes was similar to those occurring in D R G neurons exposed to aluminum sulfate in vitro 2~. Antisera to CNS A E F inhibit axonal outgrowth from S C - D R G neurons, destroy m a t u r e outgrowth axons from both SC and D R G neurons and induce neurofibrillary changes in D R G neurons. The identity of the antigen(s) responsible for these effects is not known, but it is likely that these effects are m e d i a t e d by antibodies to c o m p o n e n t s of axolemma. This is the first r e p o r t e d evidence of axon destruction by antibodies to axolemma. Similar anti-axon effects have not been seen during in vitro studies on antisera to whole CNS tissue, whole PNS tissue, purified myelin and components of myelin, including basic protein, proteolipid protein, myelin-associated glycoprotein,
c h l o r o f o r m - m e t h a n o l insoluble proteins of myelin and galactocerebroside~3-'4.~7-20.2:. These results d e m o n s t r a t e that antisera to A E F can produce a relatively selective axotomy and provide a means of investigating the response of neurons to i m m u n e - m e d i a t e d axotomy. The neurofibrillary changes induced by antisera to A E F are of interest and may represent an accumulation of neurofilaments as a consequence of a combination of antibody-induced axotomy and inhibition of axon regeneration. The p r o f o u n d effect of antisera to A E F on axons in vitro suggests a possible role for anti-axon antibodies in inflammatory and degenerative dis-
1 Abrams, G.M., Latov, N., Hays, A.P., Sherman, W. and Zimmerman, E.A., Immunocytochemical studies of human peripheral nerve with serum from patients with polyneuropathy and paraproteinemia, Neurology, 32 (1982) 821-826. 2 Bahmanyar, S., Gajdusek, D.C., Sotelo, J. and Gibbs, C.J., Jr., Longitudinal spinal cord sections as substratum for anti-neurofilament antibody detection, J. Neurol. Sci., 53 (1982) 85-90. 3 Babmanyar, S., Liem, R.K.H., Griffin, J.W. and Gajdusek, D.C., Characterization of antineurofilament autoantibodies in Creutzfeldt-Jakob disease, J. Neuropathol. Exp. Neurol., 43 (1984) 369-375. 4 Bahmanyar, S., Moreau-Dubois, M.-C., Brown, P., Cathala, F. and Gajdusek, D.C., Serum antibodies to neurofilament antigens in patients with neurological and other diseases and in healthy controls, J. Neuroimmunol., 5 (1983) 191-196. 5 Bigbee, J.W., Calabrese, V.P. and DeVries, G.H., Characterization of an antiserum against an axolemma-enriched fraction, J. Neuroimmunol,, 7 (1985) 221-229. 6 Bornstein, M.B., Organotypic mammalian central and peripheral nerve tissue. In P.F. Kruse, Jr. and M.K. Patterson, Jr. (Eds.), Tissue Culture Methods and Applications, Academic Press, New York, 1973, pp. 86-92. 7 Crain, SM. and Peterson, E.R., Enhanced afferent synaptic functions in fetal mouse spinal cord-sensory ganglion explants foUowing NGF-induced ganglion hypertrophy, Brain Research, 79 (1974) 145-152. 8 DeVries, G.H., Isolation of axolemma-enriched fractions from mammalian CNS. In N. Marks and R. Rodnight
(Eds.), Research Methods in Neurochemistry, Plenum Press, New York, 1981, pp. 3-38. DeVries, G.H.. Anderson. M.G. and Johnson. D., Fractionation of isolated rat CNS myelinated axon by sucrose density gradient centrifugation in a zonal rotor, J. Neurochem., 40 (1983) 1709-1717. DeVries, G.H.. Matthieu, J.-M., Beny, M., Chicheportiche, R.. Lazdunski. M. and Dotivo, M., Isolation and partial characterization of rat CNS axolemma enriched fractions, Brain Research, 147 f1978) 339-352. Eiizan, T.S., Casals, J. and Yahr, M.D.. Antineurofilament antibodies in postencephalitic and idiopathic Parkinson's disease, J. Neurol. Sci. 59 (1983) 341-347. GuiUery, R.W., Sobkowicz, H.M. and Scott, G.L., Light and electron microscopical observations of the ventral horn and ventral root in Ions term cultures of the spinal cord of the fetal mouse, Y. Comp. Neurol., 134 (!968) 433-476. Hruby, S., Alvord, E.C., Jr. and Seil, F.J., Synthetic galactocercbrosides evoke myelination-inhibiting antibodies. Science, 195 (1977) 173-175. Kies, M.W., Dri.seoll, B.F., Seil, F.J. and Alvord, E.C.. Jr., Myelination inlaibition factor: dissociation from induction of experimental allergic encephalomyelitis, Science, 179 (1973) 689-690. Peterson, E.R., Crain, S.M. and Murray, M.R., Differentiation and prolon~d m m n t e m c e of bioelectriesfly active spinal cord cultures (rat, chick and human), Z. Ze~[orsch.. 66 (1965) t30-154. Schwartz, M. and Spirman, N., Sprouting from chicken embryo dorsal root ganglia induced by nerve growth factor is specifically inhibited by affinity-purified antiganglioside an-
eases of the nervous system. This study provides impetus to investigate with a similar in vitro system the role of antibodies to axons in human diseases. This work was s u p p o r t e d by the ,Veterans A d m i n istration ( D . N . B . and F.J.S.), N I H - N S 10821 and 15408 ( G . H . D . ) and N I H - N S 13464 and 19414 (H.C.A.).
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337 tibodies, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 6080-6083. 17 Seil, F.J. and Agrawal, H.C., Myelin-proteolipid protein does not induce demyelinating or myelination-inhibiting antibodies, Brain Research, 194 (1980) 273-277. 18 Seil, F.J., Falk, G.A., Kies, M.W. and Alvord, E.C., Jr., The in vitro demyelinating activity of sera from guinea pigs sensitized with whole CNS and with purified encephalitogen, Exp. Neurol., 22 (1968) 545-555. 19 Seil, F.J., Garwood, M.M., Clark, H.B. and Agrawal, H.C., Demyelinating and myelination-inhibitingfactors induced by chloroform-methanol insoluble proteins of myelin, Brain Research, 288 (1983) 384-388. 20 Seil, F.J., Kies, M.W. and Bacon, M.L, A comparison of demyelinating and myelination-inhibiting factor induction by whole peripheral nerve tissue and P2 protein, Brain Research, 210 (1981) 441-448. 21 Seil, F.J., Lampert, P.W. and Klatzo, I., Neurofibrillary spheroids induced by aluminum phosphate in dorsal root ganglia neurons in vitro, J. Neuropathol. Exp. Neurol., 28 (1969) 74-85. 22 Seil, F.J., Quarles, R.H., Johnson, D. and Brady, R.O.,
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Immunization with purified myelin-associated glycoprotein does not evoke myelination-inhibitingor demyelinating antibodies, Brain Research, 209 (1981) 470-475. Sobkowicz, H.M., Guillery, R.W. and Bornstein, M.B., Neuronal organization in long term cultures of the spinal cord of the fetal mouse, J. Comp. Neurol., 132 (1968) 365-396. Sotelo, J., Gibbs, C.J., Jr. and Gajdusek, D.C., Autoantibodies against axonal neurofilaments in patients with kuru and Creutzfeldt-Jakob disease, Science, 210 (1980) 190-193. Spirman, N., Sela, B.-A., Gitler, C., Calef, E. and Schwartz, M., Regeneration capacity of the goldfish visual system is affected by antibodies specific to gangliosides injected intraocularly, J. Neuroimmunol., 6 (1984) 197-207. Spirman, N., Sela, B.-A. and Schwartz, M., Anti-ganglioside antibodies inhibit outgrowth from regenerating goldfish retinal explants, J. Neurochem., 39 (1982) 874-877. Wolf, M.K., Differentiation of neuronal types and synapses in myelinating cultures of mouse cerebellum, J. Cell Biol., 22 (1964) 259-279.