вход по аккаунту


In vitro demyelination by serum antibody from patients with guillain-barr syndrome requires terminal complement complexes.

код для вставкиСкачать
In Vitro Demyelination by Serum Antibody
from Patients with G d a i n - B a d Syndrome
Requires Terminal Complement Complexes
S. Sawant-Mane, PhD," M. B. Clark, PhD,t and C. L. Koski, MD"
Serum from 7 patients who had acute-phase Guillain-Barre syndrome with high anti-peripheral nerve myelin antibody
activity (54 to 210 units/ml) was compared with serum from 3 patients in the recovery phase (0 to 17 unitsiml) and
serum from 7 disease control subjects (0 to 24 unidml) and 7 normal control subjects (0 to 7 unitsiml) for its ability
to demyelinate rodent dorsal root ganglion cultures. The demyelinating capacity of each serum was quantitated by
counting the percent of damaged internodal segments in each of four cultures. All sera from patients in the acute
phase GBS caused 50 to 78% dernyelination, in contrast with 6 to 19% by the sera from all 3 patients in the recovery
phase and all other control subjects. The degree of demyelination correlated with anti-peripheral nerve myelin
antibody activity of the sera and dernyelination was complement-dependent. Further, cultures were treated with an
immunoglobulin M (IgM) fraction of an acute-phase Guillain-Barre syndrome plasma plus normal human serum
depleted of complement component C7. Only those cultures treated with IgM and C7-depleted human serum reconstituted with purified C7 resulted in 50.8% demyelination. which was significantly greater than the 14.2 to 16.2%
demyelination observed in the presence of heat-inactivated, C7-depleted human serum plus purified C7 or in the
absence of C7 or antibody. In summary, our work suggests that anti-peripheral nerve myelin antibody in GuillainBarre syndrome mediated complement dependent-demyelination of rodent dorsal root ganglion cultures. Further,
this in vitro demyelination required generation of activation complexes of the terminal complement cascade.
Sawant-Mane S, Clark MB, Koski CL. In vitro demyelinatlon by serum antibody from patients with
Guillain-Barre syndrome requires terminal complement complexes. Ann Neurol 1991;29:397-404
Guillain-Barre syndrome (GBS) is a monophasic neurological disorder assocked wich an inflammatory demyelination of peripheral nerve in which myelin is the
target of immune attack 11-31. In this disorder peripheral nerves undergo segmental demyelination { 1); infiltration by macrophages, T cells, and B cells {2, 41;
and immunoglobulin (Ig) and complement deposition
15, 61. Although the mechanisms of myelin destruction
are not fully understood, evidence suggests that humoral factors such as antibody (Ah) and complement
are involved [b- 101.
Participation of humord serum factors in demyelination has been tested in both in vivo and in vitro model
systems. Conclusions drawn from these experiments
are conflicting. Controversy exists concerning the ability of GBS and normal human sera to mediate demyelination and whether demyelination is dependent on
complement. Subendoneurial injections of serum from
patients with acute-phase GBS in rat sciatic nerves produced focal demyelination in 41 to 86% of the cases
[I 1- 141 and also induced greater electrophysiological
conduction block when compared with sera from either
patients in the recovery phase of GBS or disease and
normal control subjects {lS, 161. Similar effects were
observed after injections of rabbit anti-galactocerebroside serum [17, 181. Despite the observations in
these smdies, others using this in vivo model could
demonstrate neither histological demyelination nor
electrophysiological conduction block t19, 201. Studies
using organotypic or dissociated dorsal root ganglion
. 1-24] showed qualitatively that
(DRG) cultures 12
acute-phase GBS serum produced myelin damage in
vitro within 16 to 96 hours, but similar damage was
also reported with sera from 30%' of the disease controls and a normal control in one of these studies {24].
Participation of complement in both these model
systems was implicated by the loss of demyelinating
activity following heat inactivation of the test sera { 11,
22, 241. However, in a study 1111 in which intraneural
injections of GBS serum produced focal demyelination
in rodent sciatic nerve, attempted reconstitution of
complement activity by the addition of fresh guinea
From the Departments of 'Neurology and +Anatomy, University of
Maryland at Baltimore, Baltimore, MD.
Address correspondence to Dr Koski, Department of Neurology,
UMH-N4W46, University of Maryland at Baltimore, 22 South
St, Baltimore, MD 21201.
Received May 8, 1990, and in revised form Sep 28. Accepted for
publication Sep 30, 1990.
Copyright 8 1991 by the American Neurological Association 397
pig serum was unsuccessful. Pollard proposed that lymphokines and macrophage-derived, heat-sensitive proteases released in GBS serum could be the effectors
of demyelination [25].
Lack of consistent results in these systems using
acute-phase GBS sera could reflect varying levels of
specific antibodies during the natural course of the disease [7f, lack of an adequate source of serum complement, or activation of complement by the alternative
pathway in the absence of specific antibody [26]. In
the present study, we wanted to determine the relevance of humoral factors in the serum of patients with
GBS to demyelination and, specifically, the possible
role of antibodies to peripheral nerve myelin (antiPNM Ab) in mediating complement-dependent demyelination. We compared a series of sera with known
anti-PNM Ab activity from patients in the acute phase
and patients in the recovery phase of GBS as well as
sera from other disease and normal control subjects for
the ability to mediate demyelination in a tissue culture
system in which dissociated D R G were myelinated by
rodent Schwann cells.
Methods and Materials
Media and Buflers
C-10, CF, N2, and E15-0 media formulations used for culture preparations have been previously described C271. C-10
is minimum essential medium (Earle's salts) with 10% human
placental serum (HPS), 6 gmlliter of glucose, 1.4 mM glutamine, and 50 ng/ml of nerve growth factor (NGF). CF is
C-10 with 2 mM 5-fluoro-deoxyuridine and uridine. E15-0
is MEM with 15% HPS, 1.4 mM glutamine, 2.3 gmiliter of
glucose, 50 pg/ml of ascorbic acid, and 50 ngiml of NGF.
N2 {28} is a serum-free medium modified for Schwann-cell
growth by adding NGF [29]. L-15, Hank's balanced salt solution (calcium, magnesium-free) and Earle's balanced salt solution were used as buffers for washing cells. All the basic
media and buffers were purchased from GIBCO Laboratories (NY); 5-fluoro-deoxyuridine, uridine, bovine insulin,
progesterone, and putrescine dihydrochloride were bought
from Sigma Chemical (St Louis, MO); and rat transferrin was
obtained from Jackson Immunoresearch Laboratories (West
Grove, PA). NGF was a gift of Dr Eugene Johnson (Washington University School of Medicine, St Louis, MO). All
depleted sera and purified complement components were
purchased from Cytotech (San Diego, CA). Aclar (Allied
Chemical, NJ) was used to prepare 12-mm dishes, which
were then coated with rat tail collagen "1.
rats, 15 to 16 days in gestation, were obtained from Zivic
Miller (Allison Park, PA).
Collection of Clinical Material
Serum samples were collected during the acute phase of illness from patients who met the criteria of the Ad Hoc Committee for GBS 1311. From 3 of these patients, serum was
also collected during the recovery phase. Normal and disease
control sera were obtained from laboratory workers without
known disease and from patients with sensory neuropathy,
338 Annals of Neurology Vol 29 No 4 April 1991
diabetic neuropathy, tendinitis, myasthenia gravis, acute
transverse myelitis, or motor neuron disease. Anti-PNM Ab
titers of sera were determined by complement component
C1 fixation and transfer assay against myelin isolated from
human spinal roots obtained at autopsy E7). Anti-PNM Ab
activity for patients in the acute phase and those in che recovery phase of GBS ranged from 54 to 210 unitsiml and 0 to
17 unitsiml, respectively. The sera selected for these studies
reflected the range of anti-PNM Ab titers normally seen (18
to 357 unitsiml) in patients with acute-phase GBS. All sera
were aliquoted and stored at - 70°C. Prior to use as an Ab
source, each serum was dialyzed for 3 hours against barbital
saline solution (pH 7.4)r7) and inactivated by heat at 56°C
for 40 minutes.
Preparation of Alyelinated Cultures of Rodent DRG
Primary cultures of DRG neurons were prepared from 15to 16-day-oldrat embryos as described by Wood 323 except
trypsin-dissociated cultures were used instead of explants.
Cells were plated in C-10 medium at a density equlvalent to
1.5 DRG per Aclar dish. After a 2-week treatment with
alternating pulses of C-10 and CF, pure neuronal cultures
were obtained and wcrc then seeded with Schwann cells obtained from DRG explant cultures [29, 32}. The seeded
Schwann cells were then expanded in N2 for 10 to 14 days.
Cultures were changed to E15-0 to promote myelination
Detection of Dcmyelination
in Dissociated DRG Cultures by GBS Sera
For each experiment in this study, well-myelinated cultures
were matched by microscopic observation for similar cell and
myelin density. Two to three replicates were included in each
group. Before application of test medium, cultures were
washed three times with N2 containing ascorbic acid (50 pg/
ml) to remove complement components of serum in E15-0.
Optimal concentrations of test serum and complement
source were established in preliminary experiments. In all the
experiments using serum as an Ab source, the test medium
consisted of serum diluted tenfold in N2 medium with
ascorbic acid (50 kgiml) and 20% factor B-depleted normal
human serum (FBd-HS) as a source of complement. Cultures
were exposed to each sterile-filtered coded test serum and
complement for a period of 24 to 48 hours, at 35°C in 5%
carbon dioxide. They were examined by phase microscopy
for morphological evidence of demyelination at 6, 18, 24,
and 4 8 hours. After 24 or 4 8 hours, cultures were fixed in
25%glutaraldehyde in 0.1 M phosphate buffer and processed
for Sudan black staining [29}. Each experiment was repeated
at least once.
Quantitation of Demyelination
Demyelination in Sudan black-stained cultures was assessed
by light microscopy. Quantitation of intact and damaged myelin segments was determined in ten regularly placed, highpower ( x 200) fields in each culture. The values are expressed as a mean of percents of damaged myelin segments
per total counted segments in 40 high-power fields for a total
of four to six cultures. The quantitation method was adapted
from that described by Eldridge and colleagues 1291.
Preparation of I g M and Its Depletion of Complement
Component C7
IgM was separated from a single GBS plasma (anti-PNM Ab
titer, 50 unidml) by ion exchange chromatography, on a
diethylaminoethyl (DEAEj-cellulose column (DE-52 Whatman Biosystems Ltd, Kent, England). The column was
packed and loaded with 0.03 M sodium chloride (NaC1) and
0.005 M phosphate (pH 7.5), and IgM containing fractions
were eluted with 0.3 M NaCl and 0.005 M phosphate. Prior
to column application, the GBS plasma was treated with
0.014 M calcium chloride (CaCI,) to precipitate residual fibrinogen and equilibrated by dialysis with the starting buffer.
IgM fractions were pooled and concentrated by ultraiiltration
in Diaflo ultrafilter (XM300, greater than 300,000 molecular
weight; Amicon, MA). IgM from a normal control serum was
prepared in a similar manner.
C7 was absorbed from IgM using the Affi-Gel Hz immunoaffinity kit (Bio-Rad, Richmond, CA) and anti-C7 Ab from
Cytotech. The depleted IgM was tested for anti-PNM Ab
activity [7] and residual C7 activity, inactivated by heat at
56°C for 40 minutes, and dialyzed against barbital saline solution before use in the tissue culture experiments.
Determination of a Requirement for Terminal
Complement Complexes in IgM-Mediated
Demyelination In Vitro
Cultures were divided into five groups of four to six cultures
in each. Each group received one of the following: 15% IgM
plus 20% normal human serum depleted of C7 (C7d-HS),
to prevent formation of terminal complement complexes
(TCCs); 15% IgM plus 205%C7d-HS plus C7, to allow TCC
formation; 15%, IgM plus C7 plus C7d-HS, inactivated by
heat to prevent activation of the early complement cascade;
205% C7d-HS plus C7, to detect demyelination in the absence of Ab; or C7 in N2 medium alone. Cultures were
maintained at 35°C in 5% carbon dioxide for 24 hours and
observed for signs of demyelination. After 20 to 22 hours,
myelin damage was quantitated as previously described.
In a separate series of experiments, demyelination of DRG
cultures by IgM prepared from normal human serum was
assayed in the presence of human serum as a complement
source. Demyelination within groups was reported as mean
SD, and comparisons between groups were performed by
Student's t test.
Demyelination of Dissociated DRG Cultures Sy GBS
Serum and Complement
Groups of myelinated cultures were incubated for 24
to 48 hours at 35"C, with N 2 medium containing 50
Fg/ml of ascorbate and one of the following: 10%
heat-inactivated GBS serum plus 20% FBd-HS (antiPNM Ab titer, 108 unidml), 10% heat-inactivated
GBS serum alone, or 20% FBd-HS alone. Only those
cultures treated with the combination of GBS serum
and FBd-HS showed evidence of demyelination. As
shown in Figure 1A, demyelination was demonstrated
by the swelling of myelin internodes and formation of
myelin blebs. Myelin vesiculation was associated with
Fig 1. Demyelination of dorsal root ganglion (DRG) cultures by
acute-phase Guillain-Barre'syndrome (GBS) serum is complement-dependent. Sets of DRG cultures were incubated for 24 hours
at 35°C with one uf the following: (A) 10% heat-inactivated
GBS serum (anti-peripheral nerue myelin antibody [anti-PNM
Ad) activity, 108 unitsiml) plus 209%factor B-depleted n o m l
h u m n serum (FBd-HS) (A); (B) 10% GBS serum in N2, or
20%' FBd-HS in N2 (not shownj. Pronounced demyelination
(arrowheads) was seen in cultures treated with anti-PNM Ad
in the presence of a complement .rource (A);thir was not evident
with the Ab (B). Bars = 1 mm.
a loss of refractile parallel lines representative of myelinated neurites under phase microscopy. No such
evidence of myelin breakdown was seen with either
heat-inactivated GBS serum alone (see Fig 1B) or
FBd-HS alone (data not shown). These observations
suggest that this in vitro demyelination requires both
a heat-stable component of the GBS serum as well as
the addition of fresh serum.
Kinetics of Demyelination
Observations of cultures incubated with 10% heatinactivated GBS (anti-PNM Ab, 108 unitdml) serum
Sawant-Mane et al: In Vitro Dernyelination by GBS Serum Antibody
Fig 2. Kinetics of dmyelination mediated by anti-peripheral
newe myelin antibody (anti-PNM Ah).Myelinated cultures were
incubated with heat-inactioated, acute-phase Guillain-Bad syndrome semnz (anti-PNM Ab activio, 108 unitslml) in the prcsence 420% factor B-depLeted human serum. Demyelination was
obvious as earb as 6 hours (A) (arrowhead) and Pronounced by
24 hours (B) (arrows).
and 20% FBd-HS in N 2 medium plus ascorbate were
made after 2, 4, 6, 12, 24, and 48 hours. As shown in
Figure 2A, formation of myelin blebs and vesiculation
could be observed as early as 6 hours after application
of the medium containing Ab and complement source
and became pronounced by 24 hours (see Fig 2B).
Longer exposure of up to 48 hours caused severe vesiculation and actual loss of myelin internodes (not
shown). Such samples were not suitable for quantitation since lost segments could not be counted. Therefore, in all further experiments cultures were routinely
fixed after 24 hours of incubation with Ab and complement sources.
Normal sera with lower anti-PNM Ab titers (0 to 7
unitdml), handled in a similar manner, were not associated with significant demyelination (data not shown).
Demyelination of these cultures was not secondary
to primary neuronal damage, since cultures consisting
only of neurons and neurite network were not damaged after exposure to GBS serum with a high antiPNM Ab titer, plus 20% FBd-HS (data not shown).
Correlation of Antz-FNM Ab Activity and Ability of
the Serum t o Mediate Demyelination of Dissociated
DRG Cultures
All 7 acute-phase GBS sera with anti-PNM Ab titers
between 54 and 210 uni dml mediated damage to be400 Annals of Neurology
Vol 29 No 4
April 1991
tween 50.1 and 77.9% of myelinated internodes in the
presence of 20%~FBd-HS (Table 1). Figure 3 represents an example of Sudan black-stained cultures
treated with either low-titer, normal control serum or
high-titer, acute-phase GBS serum. In general, sera
with higher titers were associated with greater percentages of damage to myelin segments (Fig 4). Sera with
Ab titers varying from 0 to 17 unitsiml, obtained during the recovery phase from the 3 patients with GBS,
caused significantly less demyelination than that noted
with the acute-phase serum from the same patients (see
Table 1). The demyelination seen in association with
the 3 recovery-phase sera (11.6, 12.1, and 16.2%) was
not significantly higher than background levels. Background levels of demyelination were defined by incubation of cultures separately in the presence of one
of the following: E15-0, N2 plus ascorbate, N 2 plus
FBd-HS (see Table 1).
Further, demyelination associated with 6 of 7 disease
control (0 to 9 unitsiml) and 7 of 7 normal control sera
(0 to 7 unitsiml) was also comparable with demyelination seen in cultures treated with N 2 plus FBd-HS
(see Table 1). Only one disease control, with an antiPNM Ab titer of 24 unitsiml, produced 26.6% demyelination, which was greater than the background levels
of demyelination seen in this system. Cultures treated
with 3 of 7 acute-phase GBS sera also showed evidence
of Schwann cell damage. In these cultures, Schwann
cells developed cytoplasmic vacuoles and had a
rounded morphology by 24 hours (Fig 5). Changes in
the morphology were more pronounced at the periphery of cultures where many Schwann cells had detached from the dish.
Table 1. Anti-Per$heral Newe Myelin Antibody (Anti-PNM
Ab) Mediation of Complement-DependentDemyelination of
Dissociated Dorsal Root Ganglion Czlltzlres
Ab Titer
GBS, acute phase
% Demyelinationb
13.5 f 2.3
77.9 k 4.8
72.0 k 7.8
66.2 t 1.4
66.9 t 1.6
GBS, recovery
16.2 ? 3.6
12.1 ? 1.3
11.6 2 5.2
Disease control
11.3 2 3.1
13.2 t 4.2
16.9 t 4.6
14.1 t 1.6
12.6 t 4.1
Normal control
N 2 + ascorbate
26.6 2.9
11.2 2 1.2
11.53 -t 5.3
12.1 ? 5.9
11.9 & 5.4
16.2 t 2.8
11.5 4.5
10.0 * 2.7
14.2 * 3.9
18.5 t 1
11.1 ? 1.9
"All serum Ab sources were inactivated by heat at 56°C for 40
minutes and dialyzed against veroneal-buffered s&ne solution containing 0.15 mM calcium chloride and 1.0 mM magnesium chloride.
The final concentration of Ab and FBd-HS in the incubation medium
was 1 0 and 20%, respectively.
bValues represent a mean of percents of damaged myelin segments
per total counted segments in 40 high-power fields for a total of 4
to 6 cultures.
'Same patient from whom acute-phase GBS serum 6 was taken.
dSame patient from whom acute-phase GBS serum 2 was taken.
eSame patient from whom acute-phase GBS serum 3 was taken.
GBS = Guillain-Bard syndrome; FBd-HS
human serum.
factor B-depleted
TCC C5b-9 Is Required for IgM-Mediated
Demyelination In Vitro
As shown in Table 2, only cultures treated with IgM
purified from a patient with acute-phase GBS and
C7d-HS reconstituted with purified C7 showed damage to 50.8% of myelin segments, which was significantly greater than the damage seen in the cultures
incubated in the absence of any one of the three
constituents. Absence of IgM, heat inactivation of
C7d-HS, or lack of reconstitution of C7d-HS with C7
was associated with 14.7, 14.2, and 16.2% demyelination, respectively, which was similar to the background myelin damage seen in the cultures treated
with N2.
Treatment of cultures with similar concentrations of
IgM of a control serum (anti-PNM Ab activity, 15
units/ml) in the presence of human complement did
not result in significant dernyelination (12.8 5 1.2%
damaged internodes).
Anti-PNM Ab in the serum of patients with GBS can
bind to the surface of isolated PNM and fix complement 17, 81. Previous studies, however, were not able
to show consistently that acute-phase GBS serum
could mediate demyelination in either an in vivo or an
in vitro model system. Since these inconclusive findings may have reflected variability in both the Ab activity of the serum and the activity of the complement
source provided, we wanted to know if serum with
high anti-PNM Ab activity could mediate complement-dependent demyelination of a tissue culture
model in which dissociated DRG were myelinated with
rodent Schwann cells.
Our studies suggested that in vitro demyelination
required both a source of antibody and a complement
(see Figs 1 and 2). Results of experiments in which the
complement source was depleted of factor B to prevent alternative pathway activation in the absence of
specific Ab further suggested that myelin damage occurred via the classic pathway and was mediated by
specific Ab binding to antigen on the surface of the
myelin a n d o r rodent Schwann cells.
Myelin disruption was apparent in these cultures as
early as 6 hours and became obvious by 24 hours (see
Figs 2A and 2B). Dernyelination appeared to be primary, occurring along the entire nerve axis in a segmental manner similar to that seen in GBS nerve, and
no neuronal damage was evident. Further, remyelination of cultures following re-exposure to E 15-0 medium began within 3 days and reached their initial myelin density in 15 to 18 days (data not shown). This
suggested that an adequate number of Schwann cells
survived treatment of cultures and could produce new
myelin membrane on existing neurites.
The extent of demyelination in this culture system
correlated with anti-PNM Ab activity in each of the
individual sera (see Fig 4). Serum of GBS patients with
high anti-PNM Ab titers (54 to 210 unitsiml) mediated
significantly greater damage to myelin internodes (50
to 78%) of the DRG cultures than did the sera with
Ab titers varying from 0 to 17 units/rnl (10 to 16.9%
demyelination). The latter was similar to that seen with
cultures treated with N 2 or N2 containing FBd-HS.
Sawant-Mane et al: In Vitro Demyelination by GBS Serum Antibody
Pig 3. Assessment of myelin integrity by use of Sudan blackstuined cultures. Cultures were incubated for 24 hours with heatinactivated control serum or Guillain-Bawe‘syndrome (GAS) serum (A: normal control serum, with anti-peripheral neme myelin
antibody {anti-PNM Ad) activity of I unitlml; B: GBS serum,
with unti-PNM Ab uctivity of 84 unitslml) und complement
sourcei. Cultures were stained with Sudan black as described in
the Methods and Materials section. An intact segment was smooth
with no evidence of disruption. whereas u myelin internode with
blebs (thin arrow) or vesicles (wide arrow) was designated aJ
‘ilamaged.’’ The asterish represent Schwunn cell nuclei.
100 1
The background level of 18.5% damaged segments in
this culture system treated with N 2 medium alone was
somewhat higher than the 11.1% in cultures treated
with N 2 containing 20% FBd-HS. These levels of
damaged internodes in the cultures most likely reflected response to multiple manipulations, repeated
washes, and observation of cultures under phase microscopy outside the carbon dioxide incubator. In
keeping with our hypothesis that measurable levels of
anti-PNM Ab would mediate myelin damage in vitro,
a single serum in our disease control group from a
patient with acute transverse myelitis whose anti-PNM
Ab activity measured 24 unitdm1 mediated damage to
26.6% of myelin segments. The significant quantitative
correlation between the anti-PNM Ab activity of GBS
serum and the capacity of that serum to mediate in
vitro dernyelination (see Fig 4 ) suggests that anti-PNM
Annals of Neurology Vol 29
No 4
April 1991
Anti-PNM Ab Activity (units/ml)
Fig 4. Correlation of unti-peripheral nerve myelin antibody
(anti-PNM Ab) uctitity in experimental serum with rl-emyelination of dorsul root ganglion (DRG) cultures. Sets of four cultures
were incubatedjor 24 hours with each of a series of 24 experimental seru und media (anti-PNM A b artivity, 0 t o 210 unitsiml)
plus 20% fuctor B-depleted human serum. Demyelination was
quantitated as h..ri-ribedin the Methods and Materials section.
Demyelination in cultures increased with increasing anti-PNM
Ab activity in sera.
Table 2. Demyelination Dissociated Dorsal Root Ganglion
Cultures Requires IgM Antibody (Ah) of GBS Serum and
Terminal Complement Complexes"
% Demyelination
+ C7d-HS' + C7
IgM Ab +
IgM Ab +
C7d-HS +
HI-C7d-HSd + C7
16.2 k 5.3
* 3.6
"All Ah and complement sources were dialyzed against veronealbuffered saline solution containing 0.15 mM calcium chloride and
1.0 mM magnesium chloride. Cultures were washed two times in
N2 medium to remove any Ab or complement components from
the 15% human placental serum in E15-0 medium.
'15% IgM purified from an acute-phase GBS piasma by ionexchange chromatography; the ,anti-peripheral nerve myelin Ab titer
was 100 units/ml.
'Medium contained 20% normal human serum depleted of C7 to
inhibit formation of terminal complement complexes.
dC7d-HS was inactivated by heat at 5G"C for 40 minutes.
GBS = Guillain-Barre syndrome; C7d-HS = complement component 7-depleted human serum; HI = heat inactivated.
Fig >. Schuiann cell damage by acute-phase Guillain-Bad ~ y n drome IGBSJ serum and complement. Serum of 3 patients with
acute-phase GBS, in the presence of 20% factor B-depleted human .reTum, produced Schwann cell damuge t5y 24 hours, u6ich
was indicated by rounded morphology and detachment of celh from
the dish. The cytolysis was also accompanied by vesicukztion of
my elin.
Ab is one of the components in GBS and other serum
that may participate in in vivo demyelination once a
damaged blood-nerve barrier allows penetration of the
Ab into the endoneurium.
Morphological evidence of Schwann cell danxage was
evident with 3 of 7 acute-phase GBS sera (see Fig 5).
Ongoing experiments suggest that this phenomenon is
Ah- and complement-mediated, and Schwann cells not
committed to myelin formation are more susceptible.
Schwann cell cytolysis by the serum of some patients
with GBS and not by others could reflect Ab heterogeneity [33} or the degree of Ab-mediated complement
damage. Nucleated cells that are susceptible to cytolysis by insertion of multiple complement channels can
survive a more limited complement attack [34}. Survival of such a sublytic complement attack by oligodcndrocytes results in the production of significant levels
of inflammatory mediators such as leukotriene B,,
which influence migration of macrophages and monocytes 1351. A similar phenomenon is likely in the peripheral nervous system.
Our studies quantitatively demonstrate that both Ab
and complement are required for in vitro demyelination since IgM purified from an acute-phase GBS
plasma and not from a control serum mediated myelin
damage only in the presence of the C7-depleted human serum reconstituted with purified C7. Since C7
depletion of serum would stop the complement cascade prior to C5b-9 formation (at C5b-Q it suggested
that Ab-mediated myelin membrane destruction requires activation of the complement cascade and formation of the TCCs. Similar conclusions were reached
in previous work that studied Ab-mediated damage to
myelinated cerebellar explants [36].
In summary, these studies suggest that complement-fixing Ab against PNM in the serum of patients
with GBS and a disease control can bind a surface
determinant of myelin and Schwann cells and mediate
complement-dependent myelin membrane damage.
The generation of the terminal activation complexes of
the complement cascade is required for demyelination
in this in vitro system.
This work was supported by National Institutes of Health grants
PO1 NS 20022 and PO1 NS 22849 to Dr Koski.
The authors wish to thank Saeid Motevalli for technical assistance
and Dr M. L. Shin for reviewing the manuscript.
1. Asbury AK, Arnason BG, Adams RD. The inflammatory lesion
in idiopathic polyneuritis-its role in pathogenesis. Medicine
2. Dalcanto MC, Wisniewski HM, Johnson AB, et al. Vesicular
disruption of myelin in autoiminuiie demyclination. J Neurol
Sci 1975;24:313-319
3. Koski CL. Guillain-Barr~2syndrome. Neurol Clin 1984;2:355366
4. Cornblath DR, Griffin DE, Chupp M, et a]. Mononuclear cell
typing in inflammatory demyehdtion polyiieuropathy nerve biopsies. Neurology 1987;37 : 25 3 (Abstract)
Sawant-Mane et al: In V i m Demyelination by GBS Serum Antibody
5. Nyland H , Matre R, Mork S. Immunological characterization
21. Birchem K, Mithen FA, Empereur KM, Wessels MM. Ultra-
of sural neme biopsies from patients with Guillain-Barre syndrome. Ann Neurol 1981;9 (suppl):80-86
6. Koski CL, Sanders ME, Swoveland PT,et al. Activation of terminal components of complement in patients with GuillahBarre syndrome and other demyelinating neuropathies. J Clin
Invest 1987;80:1492-1497
7. Koski CL, Humphrey R, Shin ML. Anti-peripheral myelin antibody in patients with demyelinating neuropathy: quantitative
and kinetic determination of serum antibody by complement
coinponeiit 1 fixation. Proc Natl Acad Sci USA 1985;82:
structural effects of Guillain-Barr6 serum in culture rontaining
only rat Schwann cells and dorsal root-ganglion neurons. Brain
Res 1987;42 1:173- 185
Dubois-Dalque M, Buyse M, Buyse G, Gorce F. The action of
Guillain-Barre syndrome serum on myelin: a tissue culture and
electron microscopic analysis. J Neurol Sci 1971;13:67-83
Hirano A, Cook SD, Whitaker JN, et al. Fine structural aspects
of demyelination in vitro. The effects of Guillain-Barre serum.
J Neuropathol Exp Neurol 1931;30:249-265
Cook SD, Dowling PC, Murray MR, Whitaker JN. Circulating
demyelinating factors in acute idiopathic polyneuropathy. Arch
Neurol 1971;24:136-144
Pollard JD. A critical review of therapies in acute and chronic
inflammatory demyelinatiy polyneuropathies. Muscle Nerve
Koski CL, Vanguri P, Shin ML. Activation of the alternative
pathway of complement by human peripheral nerve myelin. J
Immunol 1985;134:1810-18 14
Porter S, Clark MB, Glaser L, Bunge RP. Schwann cells stimulated to proliferate in the absence of neurons retain full functional capability.J Neurosci 1986;6:3070-3078
Bottestein JE, Sat0 GH. Growth of a rat neuroblastoma cell line
in serum free supplemented medium. Proc Natl Acad Sci USA
1979;76:514-5 17
Eldridge CF, Bunge MB, Bunge RP, Wood PM. Differentiation
of axon related Schwann cells in vitro. 1. Ascorbic acid regulates
basal lamina assembly and myelin formation. J Cell Biol
Bornstein MB. Reconstituted rat tail collagen used as substrate
for tissue culture on coverslip in maximow slides and roller
rubes. Lab Invest 1958;7:134-137
Asbury A, Arnason B, Karp H, McFarlin DE. Crireria for the
diagnosis of Guillain-Barre syndrome. Ann Neurol 1978;3:
Wood PM. Separation of functional Schwann cells and neurons
from normal peripheral nerve tissue. Brain Res 1976;115:
Koski CL, Chou KH, Jugalwala FB. Anti-peripheral nerve myelin antibodies in Guillain-Barre syndrome bind a neutral glycolipid of peripheral myelin and cross-react with Forssman antigen.
J CLin Invest 1989;84:2813-287
Koski CL, Ramm LE,Hammer CH, et al. Cytolysis of nucleated
cells by complement: cell death displays multi-hit characteristics.
Proc Natl Acad Sci USA 1983;80:3816-3820
Shirazi Y, Imagawd DK, Shin ML. Release of leukotriene B4
from sublethally injured oligodendrocytes by terminal complement complexes. J Neurochem 1987;48:271-278
Liu WT, Vanguri P, Shin ML. Studies on demyelination in vitro:
the requirement of membrane attack components of complement system. J Immunol 1983;131:778-782
8. Koski CL, Gram E, Sutherland J, Mayer RF. Clinical correlation
with anti-peripheral-nerve myelin antibodies in Guillain-Harr6
syndrome. Ann Neurol 1986;19:573-577
9. Sanders ME, Koski CL, Robbins D, et al. Activated terminal
complement in cerebrospinal Auid in Guillaiti-Bard syndrome
and multiple sclerosis. J Immunol 1986;136:4456-4459
10. The Guillain-Barre syndrome study group. Plasmapheresis and
acute Guillain-Barr6 syndrome. Neurology 1985;35:10961104
11. Feasby TE, Hahn AF, Gilbert JJ. Passive transfer of demyelinating activity in Guillain Barre polyneuropathy. Neurology (NY)
1980;30:363 (Abstract)
12. Feasby TE, Hahn AF, Gilbert JJ. Passive transfer studies in
Guillain-Barrb polyneuropathy. Neurology (NY) 1982;32:
13. Saida T, Saida K, l s a k RP, et al. In vivo demyelinating activity
of sera from patients with Guillain-Barre syndrome. Ann Neurol 1982;11:69-75
14. Brown MJ, Northington JW, Rosen JL, L i d RP. Acute canine
idiopathic polyneuropathy (ACIP) serum deinyelinates peripheral nerve in vivo. J Neuroimmunol 1985;7:239-248
15. Harrison BM, Hansen LA, Pollard JD, McLeod JG. Demyelination induced by serum from patients with Guillain-Barre syndrome. Ann Neurol 1984;15:163-170
16. Sumner AJ, Lisak RP, Brown MJ, Asbury AK. Demyelinating
activity of Guillain-Barre syndrome (GBS) serum. Neurology
1983;33:81 (Abstract)
17. Sumner AJ, Saida K, Saida T, et at. Acute conduction block
associated with experimental antiserum-mediated demyelination
of peripheral nerve. Ann Neurol 1982;11:469-477
18. Saida 7, Saida K, Dorfman SH, et al. Experimental allergic neuritis induced by sensitization with galactocerebroside. Science
1979;204:1103-l106 (Abstract)
19. Tandon DS, Griffin JW, Drachman DB, et al. Studies on the
humoral mechanisms of inflammatory demyelinating neuropathies. Neurology (NY) 1980;30:362
20. Low PA, Schmelzer JD, Dyck PJ. Results of endoneural injection of Guillain-Barre serum in Lewis rats. Mapo Clin Proc
404 Annals of Neurology Vol 29 No 4 April 1991
Без категории
Размер файла
949 Кб
barry, complement, patients, syndrome, terminal, serum, guillain, required, complexes, antibody, demyelination, vitro
Пожаловаться на содержимое документа