 |
 Previous Article | Next Article 
The Journal of Neuroscience, January 15, 2000, 20(2):729-735
Immune Deficiency in Mouse Models for Inherited Peripheral
Neuropathies Leads to Improved Myelin Maintenance
Christoph D.
Schmid1, 2,
Martina
Stienekemeier1,
Stephan
Oehen3,
Frank
Bootz4,
Jürgen
Zielasek1,
Ralf
Gold1,
Klaus V.
Toyka1,
Melitta
Schachner5, and
Rudolf
Martini1, 2
1 Department of Neurology, University of
Würzburg, D-97080 Würzburg, Germany,
2 Department of Neurobiology, Swiss Federal Institute of
Technology, CH-8093 Zürich, Switzerland, Institutes of
3 Experimental Immunology and 4 Laboratory
Animal Science, University of Zürich, CH-8092 Zürich,
Switzerland, and 5 Zentrum für Molekulare
Neurobiologie, University of Hamburg, D-20246 Hamburg, Germany
 |
ABSTRACT |
The adhesive cell surface molecule P0 is the most
abundant glycoprotein in peripheral nerve myelin and fulfills pivotal
functions during myelin formation and maintenance. Mutations in the
corresponding gene cause hereditary demyelinating neuropathies. In mice
heterozygously deficient in P0
(P0+/ mice), an established animal
model for a subtype of hereditary neuropathies, T-lymphocytes are
present in the demyelinating nerves. To monitor the possible
involvement of the immune system in myelin pathology, we cross-bred
P0+/ mice with null mutants for the
recombination activating gene 1 (RAG-1) or with mice deficient in the
T-cell receptor  -subunit. We found that in
P0+/ mice myelin degeneration and
impairment of nerve conduction properties is less severe when the
immune system is deficient. Moreover, isolated T-lymphocytes from
P0+/ mice show enhanced reactivity to
myelin components of the peripheral nerve, such as
P0, P2, and myelin basic protein.
We hypothesize that autoreactive immune cells can significantly foster
the demyelinating phenotype of mice with a primarily genetically based
peripheral neuropathy.
Key words:
Charcot-Marie-Tooth disease; myelin degeneration; Schwann
cell; P0; myelin protein zero; immune system; immune
deficiency; T-lymphocytes; macrophages; electron microscopy; electrophysiology
| |
INTRODUCTION |
Myelin protein zero (MPZ or
P0), the most abundant glycoprotein of the myelin
sheath of peripheral nerves, is a transmembrane adhesion molecule of
the Ig superfamily that fulfills multiple functions during myelin
development and maintenance (for review, see Martini and Schachner,
1997 ). Its pivotal role in the peripheral nervous system is reflected
by the fact that mutations in the corresponding gene cause inherited
neuropathies, such as Charcot-Marie-Tooth (CMT) disorder, type 1B, the
Déjérine-Sottas syndrome, and congenital hypomyelination
(for review, see De Jonghe et al., 1997; Martini et al., 1998; Nelis et
al., 1999). These disorders are typically associated with muscular
weakness and atrophy, sensory dysfunction, and skeletal deformities.
Biopsies from CMT1B patients revealed that, dependent on the mutation
in the P0 gene, divergent pathological hallmarks can be found. Such hallmarks comprise either formation of
myelin tomacula, i.e., focally thickened myelin sheaths of reduced
stability or, alternatively, myelin decompaction and demyelination (Thomas et al., 1994; Gabreëls-Festen et al., 1996; Tachi et al.,
1997). Recently, we have shown that heterozygous
P0 null mutant mice
(P0+/) are appropriate
models for mild CMT1B forms with an initially normal myelin formation
followed by myelin decompaction and demyelination starting at 4 months
of life (Martini et al., 1995a; for review, see Martini, 1997).
Although it is well accepted that various heterozygous mutations or the
reduction in gene dosage of P0 lead to
demyelination (for review, see Martini et al., 1998), the molecular and
cellular mechanisms that lead to myelin degeneration are not yet
understood. Based on the observation that nerves of patients suffering
from inherited neuropathies (Schmidt et al., 1996) or nerves of
P0+/ mice contain
endoneurial T-lymphocytes (Schmid et al., 1996; Shy et al., 1997), we
tested the hypothesis that the immune system might be involved in the
development of the demyelinating phenotype. We, therefore, cross-bred
P0+/ mice with
homozygous null mutants for the recombination activating gene 1 (RAG-1)
or with mice deficient in the T-cell receptor -subunit (TCR),
i.e., with mutants that are deficient in mature T- and B-lymphocytes or
only T-lymphocytes, respectively. We found that, in the absence of an
intact immune system, myelin degeneration in
P0+/ mice is
significantly reduced in comparison to
P0+/ mice with normal
immune cells. Moreover, isolated T-lymphocytes from
P0+/ mice show enhanced
reactivity to myelin components of the peripheral nerve, such as
P0, P2, and myelin basic
protein (MBP). We conclude that the immune system may be functionally
involved in the demyelinating phenotype of mice with a primarily
genetically mediated peripheral neuropathy.
| |
MATERIALS AND METHODS |
Animals and determination of genotypes. Mice
deficient in the gene for protein zero
(P0/; Giese et al.,
1992) or the recombination activating gene 1 (RAG-1/; Mombaerts et al., 1992) were
generated as described previously and backcrossed to the C57BL/6 strain
for six to eight generations. Double mutants were generated by
cross-breeding heterozygous P0-mutants (P0+/ mice) with
RAG-1/ mice under specific
pathogen-free conditions (Institute of Laboratory Animal
Science, University of Zürich) in analogy to the breeding protocol described previously (Martini et al., 1995b; Carenini et al.,
1997). Mice deficient for the T-cell receptor subunit (TCR/; Philpott et al., 1992) were
bred on a mixed background (129/Ola/Hsd, BALB/c, C57Bl/6) and
cross-bred with P0+/
mice as described above. Genotypes of P0-mutants
were determined by conventional PCR using oligonucleotides
5'-TCAGTTCCTTGTCCCCCGCTCTC-3', 5'-GGCTGCAGGGTCGCTCGGTGTTC-3', and
5'-ACTTGTCTCTTCTGGGTAATCAA-3' leading to 334 or 500 bp products for the
P0 null mutation or wild-type allele,
respectively. The phenotype of the RAG-1 or the TCR null
mutation was determined by fluorescence activated cell sorting (FACS)
analysis of peripheral blood cells stained with phycoerythrin
(PE)-coupled anti-CD4 and fluorescein-isothiocyanate-coupled anti-CD8
antibodies (PharMingen, Hamburg, Germany) using a FACScan instrument
(Becton Dickinson, Heidelberg, Germany). To rule out the possibility
that the effects observed were a consequence of differences in the
genetic background, all pathological alterations were investigated in
littermates. For all pathological studies, the femoral quadriceps nerve
was chosen that contains ~500 myelinated axons in wild-type mice
(Lindberg et al., 1999), thus being an ideal paradigm for quantitative
analyses. All experiments described here on animals were approved by
the Veterinary Administration of Zürich, Switzerland and by the
Regierung von Unterfranken (Würzburg, Germany).
Immunohistochemistry. Fourteen-micrometer-thick serial
sections from fresh frozen femoral nerve were immunostained as
previously described (Guénard et al., 1999). For detection of
T-lymphocytes, anti-mouse CD8 (1:50 of supernatants of hybridoma cell
lines TS169; Cobbold et al., 1984) were used. For detection of
macrophages, anti-mouse F4/80 (1:500; Serotec, Oxford, UK) was used.
Sections of spleen of wild-type mice were used as positive controls,
and for negative control the primary antibody was omitted. Positive profiles were counted on cryosections using a Zeiss Axiophot
microscope, and the mean number of positive profiles per
mm2 section was calculated. Statistical
analysis of the number of immune cells in cryosections was performed by
a two-way ANOVA test.
Electrophysiology. Nerve conduction properties of sciatic
nerves from 13-month-old myelin mutants homozygously deficient in RAG-1
(P0+//RAG-1/)
and myelin mutants heterozygously deficient in RAG-1
(P0+//RAG-1+/)
as well as from myelin wild-type mice either homozygously or heterozygously deficient in RAG-1
(P0+/+/RAG-1/,
P0+/+/RAG-1+/)
were determined by established electrophysiological methods as
previously described in detail (Zielasek et al., 1996). In brief,
anesthetized mice (Hypnorm, Janssen, Belgium) received subcutaneous
stimulating electrodes at the sciatic notch (proximal stimulation) and
above the ankle (distal stimulation). Recording electrodes were placed
subcutaneously close to the hallux and between digits 2 and 3. Studies
were performed with a current 50% higher than that needed to elicit
maximal stimulation. In all experiments, the investigator (J.Z.) was
not aware of the genotype of the mice. For motor nerve conduction
studies, the following parameters were measured: proximal and distal
M-response latencies, proximal and distal F-wave latencies, and
amplitudes of compound muscle action potentials. Nerve conduction
velocities were calculated from the latency measurements and the
distance of the stimulation sites. In analogy to the different
P0/RAG-1 mutants, nerve conduction properties from
13-month-old
P0+//TCR/,
P0+//TCR+/,
P0+/+/TCR/
and
P0+/+/TCR+/
mice were determined. Statistical analysis was performed using a
one-sided t test.
Tissue preservation for electron microscopy. Femoral nerves
of mice were processed for light and electron microscopy as previously described (Martini et al., 1995b; Lindberg et al., 1999). Briefly, mice
were transcardially perfused with 0.1 M cacodylate buffer containing 4% paraformaldehyde and 2% glutaraldehyde, and nerves were
post-fixed in the same fixative overnight. After osmification and
dehydration, nerves were embedded in Spurr's medium.
Morphometry. For light microscopy, semithin sections
(0.5-µm-thick) were stained with alkaline methylene blue. For
electron microscopy, ultrathin sections (80 nm) were contrasted with
lead citrate. Quantification of total nuclei and myelin competent axons was performed on cross sections of the quadriceps branch of the femoral
nerve. For this purpose, all nuclei were counted on semithin sections
at a final magnification of 600×. The number of myelin competent axons
(>2 µm in diameter) that were not or only thinly myelinated (less
than five turns) was determined by electron microscopy. Analysis of
morphometric parameters such as the g-ratio (myelin thickness; Friede,
1972), the diameter of axons (Friede, 1972), and the endoneurial space
was done by electron microscopy on randomly selected parts of ultrathin
cross sections of the quadriceps branch of the femoral nerve. The
endoneurial space was determined as the fraction of total nerve area
not occupied by fibers. Electron micrographs of two randomly selected
parts per nerve cross section were digitized with a scanner (HP 4C).
Morphometric parameters were evaluated at a final magnification of
8000× using the Image Tool Application, version 1.28 program
(University of Texas UTHSCSA). Statistical significance of
differences between mean values of P0+//RAG1/
versus
P0+//RAG1+/
mice was determined by double-sided Student's t test using
Microsoft Excel software.
Culture of splenocytes and proliferation assay. Splenocytes
from four myelin wild-type
(P0+/+) and four
P0+/ mice at the age of
8 months were seeded into 96-well microtiter plates at a density of
1.5 × 106/ml (Nunc, Wiesbaden,
Germany). Splenocytes were exposed to recombinant human (rh)
P0, rhP2 (Weishaupt et al.,
1995), rat MBP (10 µg/ml each), or concanavalin A (Con A; 2.5 µg/ml), as described (Pette et al., 1990; Stienekemeier et al., 1999)
except that we used 1% autologous serum and added 5 × 105 M 2-mercaptoethanol.
After 3 d, murine interleukin (IL)-2 (2.5 ng/ml; R & D Systems,
Wiesbaden, Germany) was added. On day 8, the nonadherent cells from
each plate were split into two new microtiter plates. Cells were
reactivated on day 14 by addition of irradiated, syngeneic splenocytes
to both subcultures, whereas the respective antigens were added to only
one as described for the split well technique (Pette et al., 1990).
After 48 hr, cells were pulsed with tritiated thymidine and harvested
16 hr later (Stienekemeier et al., 1999). Corresponding cultures were
compared for specific proliferation. Wells showing a thymidine
incorporation of >1000 counts per minute and a stimulation index of
>3 in comparison to control wells without antigen were considered
positive. For each antigen, at least 96 primary wells were screened.
Statistical analysis was performed with the Mann-Whitney U
test using the Prism computer program (Graph Pad, San Diego, CA).
p values < 0.05 were considered statistically significant.
| |
RESULTS |
Quantification of immune cells in peripheral nerves
In an attempt to confirm previous observations of the presence of
immune system derived cells within the nerves of adult
P0+/ mice (Schmid et al.,
1996), markers for lymphocytes (CD8; Brideau et al., 1980) or for mouse
macrophages (F4/80; Austin and Gordon, 1981) were used. At the age of 4 and 6 months, three P0+/+ and three
P0+/ mice were considered. At older ages (8 and 21 months), two mice of each genotype were included, with the exception of
three P0+/ mice investigated at 21 months. In cross
sections of femoral nerves of 4- to 21-month-old mice, CD8-positive
cells were always rounded, whereas F4/80-positive cells were usually
ramified or showed at least a few slender processes. During the
analysis, the investigator (C.D.S.) was not aware of the genotype of
the nerve sections. In general, CD8-positive cells as well as
F4/80-positive cells were predominantly detectable in the endoneurium
of nerves. When comparing nerves from
P0+/ versus
P0+/+ mice, there was no
quantitative difference in the number of CD8-positive cells at 4 months
of age (Fig. 1, top left
diagram), a stage where the very first pathological changes are
detectable in P0+/ mice
(Martini et al., 1995a). However, at 6 months and older, the number of
CD8-positive profiles was always higher in nerves from
P0+/ versus
P0+/+ mice, although
there was considerable heterogeneity. This heterogeneity was possibly
caused by the previously reported nonuniform distribution of
lymphocytes along the course of the nerve (Shy et al., 1997; Fig. 1,
top left diagram). F4/80-positive endoneurial profiles outnumbered CD8-positive cells by a factor of ~20. In contrast to
CD8-positive profiles, the numbers of F4/80-positive cells were already
elevated in 4-month-old
P0+/ mice when compared
to age-matched P0+/+ mice
(Fig. 1, top right diagram).
View larger version (61K):
[in this window]
[in a new window]
|
Figure 1.
Quantification of CD8- and F4/80-positive cells in
cryosections of quadriceps nerves from
P0+/+ (top diagrams, filled
bars) and P0+/ mice
(top diagrams, open bars) and quantification of
F4/80-positive cells in quadriceps nerves from
P0+//TCR+/
(bottom diagram, stippled bars) and
P0+//TCR/ mice
(bottom diagram, hatched bars). Top
diagrams, Note elevated levels of immune-derived cells in
P0+/ mice. Statistical significance
was achieved for F4/80-positive cells, but not for CD8-positive cells,
which might be related to the heterogeneous distribution of the
lymphocytes along the nerve. Error bars indicate SD. Bottom
diagram, Note reduced levels of F4/80-positive cells in
quadriceps nerves of
P0+/ /TCR/
versus P0+//TCR+/
mice at 8 months of age. Error bars indicate SD. p < 0.05 (Student's t test).
|
|
Statistical analysis comparing the numbers of immune cells in
P0+/+ versus
P0+/ mice by a two-way
ANOVA test considering all time points investigated resulted in
p values of 0.0035 and 0.064 for F4/80- and CD8-positive cells, respectively. Thus, the difference of F4/80-positive cells in
P0+/+ versus
P0+/ mice was
statistically significant, whereas the difference of the CD8-positive
cells did not reach statistical significance, which might be related to
the considerable heterogeneity in the distribution of the cells (see above).
Generation of double mutants
By cross-breeding
P0+/ mice with
RAG-deficient (RAG-1/) mice and
intercrossing the individuals of the F1 generation, we obtained an F2
generation with myelin mutants homozygously deficient in RAG-1
(P0+//RAG-1/)
and myelin mutants heterozygously deficient in RAG-1
(P0+//RAG-1+/).
In addition, P0+/+ mice
either homozygously or heterozygously deficient in RAG-1 (P0+/+/RAG-1/,
P0+/+/RAG-1+/)
were obtained. RAG/ mice are reported
to be deficient for mature T- and B-lymphocytes (Mombaerts et al.,
1992). Based on FACS and in agreement with previous reports (Mombaerts
et al., 1992), CD4- and CD8-single positive T-lymphocytes in peripheral
blood were not detectable in RAG-1/
genotypes. In RAG-1+/ mice, the number
of CD4- and CD8-positive T-lymphocytes was indistinguishable from
values from RAG-1+/+ genotypes reflecting
normal immune cell development (Mombaerts et al., 1992). A
corresponding pattern of cross-breeding of
P0+/ mice was performed
with mice deficient in the TCR-subunit
(TCR/ mice) that lack mature
TCR -lymphocytes, but contain B-lymphocytes and small populations
of TCR- and TCR  -T-lymphocytes (Philpott et al., 1992; Neuhaus
et al., 1996; see Materials and Methods).
Deficiency in the RAG-1 gene leads to milder pathological changes
in P0+/ mice
We first investigated semithin sections of femoral quadriceps
nerves of
P0+/+/RAG-1+/
and
P0+/+/RAG-1/
mice at the age of 13 months. We found that, independent of the RAG-1
genotype, these nerves were of normal phenotype with thick myelin,
overall compact appearance with small endoneurial spaces between the
nerve fibers, and absence of features indicative of demyelination
(Table 1, Fig.
2a,b).
View larger version (152K):
[in this window]
[in a new window]
|
Figure 2.
Representative semithin cross sections of femoral
quadriceps nerves from
P0+/+/RAG-1+/
(a),
P0+/+/RAG-1/
(b),
P0+//RAG-1+/
(c), and
P0+//RAG-1/
mice (d) and ultrathin cross sections of femoral
quadriceps nerves from
P0+//RAG-1+/
(e) and
P0+//RAG-1/
mice (f) at the age of 13 months. In
P0+//RAG-1+/ mice
(c, e), numerous thinly myelinated and demyelinated
axons are visible as well as extended endoneurial spaces with
supernumerary Schwann cells (e, f, arrows). In
P0+//RAG-1/
mice (d, f), these pathological characteristics
are less pronounced. Note normal appearance of axon-Schwann cell units
in P0+/+/RAG-1+/
(a) and
P0+/+/RAG-1/ mice
(b). Scale bars: a-d, 20 µm (in
d); e, f, 5 µm (in
f).
|
|
Next, we compared quadriceps femoral nerves from 13-month-old
P0+//RAG-1+/
mice with nerves from
P0+//RAG-1/
littermates. In semithin sections, all mice of
P0+/ genotype showed
pathological alterations indicative of compromised myelin maintenance
(Fig. 2c,d). However, two principle categories of
pathological severity could be distinguished by an investigator who was
not aware of the genotype (R.M.). In all cases, the more severely
affected nerves were from
P0+//RAG-1+/
mice, whereas the nerves with less pronounced pathological alterations were derived from immune-deficient
P0+//RAG-1/
mice (Fig. 2c,d). The nerves of the latter mutants were
characterized by thicker myelin sheaths and significantly lower numbers
of cell nuclei (Table 1) that mostly belonged to typical Schwann cell onion bulbs. Such supernumerary Schwann cells are a well established indicator of demyelination in mice and humans (for review, see Martini,
1997) so that their reduced numbers in the
P0+//RAG-1/
mutants reflect a lower degree of myelin degeneration. As a consequence of the lower number of supernumerary Schwann cells, the nerves of the
P0+//RAG-1/
mice appeared more compact in that the myelinated fibers were separated
by smaller endoneurial spaces. The number of myelin-competent, i.e.,
thick caliber axons was similar in both
P0+//RAG-1+/
and
P0+//RAG-1/
mutants (data not shown).
Next,
we quantified the pathological alterations in the different
mutants by electron microscopy. As exemplified in Figure 2,
e and f, and in agreement with the
light-microscopic findings, pathological features were less severe in
P0+//RAG-1/
versus
P0+//RAG-1+/
mice (Table 1). Particularly striking were the thicker myelin sheaths
in the
P0+//RAG-1/
mice, as indicated by their significantly lower g-ratio values (see
Materials and Methods) when compared to the
P0+//RAG-1+/
littermates. Supporting the impression by light microscopy, the endoneurial space was significantly less expanded in
P0+//RAG-1/
mice in comparison to the
P0+//RAG-1+/
littermates when quantified on electron micrographs (Table 1).
We also investigated the number of F4/80-positive macrophages in
P0+//RAG-1/
versus
P0+//RAG-1+/
mice. Most interestingly, macrophages were reduced in numbers in one
P0+//RAG-1/
mouse (140 per mm2) versus a
P0+//RAG-1+/
littermate (346 per mm2) at 13 months of
age. Although this observation refers to only one individual of each
genotype, we consider the reduced number of macrophages in the
P0+//RAG-1/
mouse as relevant, because analogous investigations in other immune-deficient mice
(P0+//TCR+/
and
P0+//TCR/
mice) with higher numbers of individuals investigated
(n = 3) revealed similar observations (see below; Fig.
1, bottom diagram).
Deficiency in the TCR gene leads to milder pathological changes
in P0+/ mice
We also compared quadriceps nerves from 8-month-old
P0+//TCR+/
(n = 4) versus
P0+//TCR/
(n = 3) mice both at the light- and
electron-microscopic level. Similar to the findings in
P0+//RAG-1+/
and
P0+//RAG-1/
mice, the pathological alterations in the
P0+//TCR/
mice were less pronounced than in the
P0+//TCR+/
littermates. For instance, in
P0+//TCR/
mice, the number of Schwann cell nuclei was significantly reduced when
compared to values from
P0+//TCR+/
littermates (26.7 ± 11.2 vs 53.8 ± 13.3; p < 0.05). Furthermore, the endoneurial space was less expanded in
P0+//TCR/
mice in comparison to the
P0+//TCR+/
littermates (42.1 ± 6.3% vs 65.0 ± 11.3% of total nerve
area; p < 0.05). Also, myelin was thicker in
P0+//TCR/
mice than in
P0+//TCR+/
mice, although the differences between g-ratios were not statistically significant (0.75 ± 0.027 vs 0.78 ± 0.015). We also
investigated the number of F4/80-positive macrophages in quadriceps
nerves of
P0+//TCR/
mice in comparison to the
P0+//TCR+/
littermates at 8 months of age. Similar to the findings in
P0+//RAG-1/
versus
P0+//RAG-1+/
mice, numbers of macrophages were reduced in the immune-deficient myelin mutants (Fig. 1, bottom diagram).
Deficiency in the RAG-1 gene leads to less impaired conduction
properties in P0+/ mice
To test whether the immune system has an impact on conduction
properties of peripheral nerves of
P0+/ mutants, mice were
subjected to an electrophysiological investigation by an examiner not
aware of the genotypes.
In sciatic nerves of 13-month-old
P0+/+/RAG-1+/
and
P0+/+ /RAG-1/
mice, conduction properties were comparable to those previously
described for P0+/+ mice
(Zielasek et al., 1996; Fig. 3.). In both
P0+//RAG-1+/
and
P0+//RAG-1/
mice, conduction properties were impaired when compared with littermates of P0+/+
genotype. However, when comparing
P0+//RAG-1+/
and
P0+//RAG-1/
mice, the latter mutants showed better conduction properties. This was
reflected by significantly shorter latencies of M-responses and of
F-waves after distal or proximal stimulations (Fig. 3). Amplitudes of
compound muscle action potentials were not significantly different in
P0+//RAG-1+/
versus
P0+//RAG-1/
mutants (13.9 ± 4.1 mV vs 11.8 ± 3.7 mV after distal
stimulations; n = 4 for each genotype).
View larger version (29K):
[in this window]
[in a new window]
|
Figure 3.
Schematic representation of parameters of
conduction properties of sciatic nerves of
P0+/+/RAG-1+/,
P0+/+/RAG-1/,
P0+//RAG-1+/, and
P0+//RAG-1/
mice (asterisks). Note that in
P0+//RAG-1/
mice, nerve conduction properties are significantly improved when
compared to values from
P0+//RAG-1+/
mice. Each black dot represents the value of one sciatic
nerve from one single mouse investigated; short horizontal
bars represent mean values ± SDs.
|
|
Deficiency in the TCR gene leads to less impaired nerve
conduction properties in P0+/ mice
In addition, we investigated conduction properties in
P0+/+/TCR+/,
P0+/+/TCR/,
P0+//TCR+/,
and
P0+//TCR/
mice at 13 months of age. Similar to
P0/RAG-1-mice, conduction properties in
P0+//TCR/
mice were less affected than in
P0+//TCR+/
littermates. Motor nerve conduction velocities were significantly higher in the
P0+//TCR/
mice (37 ± 7 m/sec vs 26 ± 4 m/sec; p < 0.05; n = 3 for each genotype). Other parameters, such
as latencies of M-responses and of F-waves tended to show less impaired
nerve conduction without reaching statistical significance, probably
because of the low number of mice available (data not shown).
Increased frequency of myelin-reactive splenocytes in
P0+/ mice
Based on the observation that the immune system appears to be
involved in demyelination in
P0+/ mice, we tested
the hypothesis that in
P0+/ mice an increased
proportion of T-lymphocytes might recognize components of peripheral
nerve myelin as autoantigens. We used the split-well technique (Pette
et al., 1994) and compared the specific proliferative responses of
isolated splenocytes from 8-month-old
P0+/+ and
P0+/ mice, i.e., at an
age of advanced myelin degeneration in
P0+/ mice.
Interestingly, the frequencies of cells proliferating in response to
different myelin components were significantly higher in
P0+/ versus
P0+/+ mice (Fig.
4). By contrast, stimulation with
concanavalin A caused comparable proliferative responses in both
P0+/+ and
P0+/ mice (Fig. 4).
View larger version (25K):
[in this window]
[in a new window]
|
Figure 4.
Schematic representation of the proliferative
response of splenocytes from 8-month-old
P0+/+ (filled
bars) and P0+/ mice
(open bars) to myelin components and to concanavalin A. Stimulation of isolated splenocytes from
P0+/+ or
P0+/ mice by different myelin antigens
was performed by split-well technique. Each column
represents the mean of the percentage of positive wells of four
mice ± SEM. All myelin components tested elicited a higher
proliferative response in P0+/ versus
P0+/+ mice, and statistical significance
(p < 0.05) was achieved when the
proliferative responses against the three myelin components were summed
up. Stimulation with concanavalin A (Con A) was taken as
positive control for the proliferative capacity of splenocytes from
P0+/+ and
P0+/ mice.
|
|
| |
DISCUSSION |
In the present study, we demonstrate that in a mouse model for
hereditary neuropathies, demyelination is less severe when the immune
system is impaired. Removal of mature T-lymphocytes and amelioration of
the pathological phenotype was achieved by two different genetic immune
defects (RAG-1/ and
TCR/) thus supporting a role of the
immune system in the demyelinating phenotype of
P0+/ mice. Another and
independent argument in favor of this view is the observation that in
splenocyte preparations from
P0+/ mice the
frequencies of leukocytes proliferating in response to myelin
components was higher than in preparations from
P0+/+ mice. Although we
did not investigate the cytokine release of these myelin-specific
T-lymphocytes, we suggest that the elevated frequency of proliferating
T-lymphocytes in P0+/
mice reflects the involvement of the immune system in the myelin pathology of P0+/ mice.
The highest frequency of proliferating cells was observed when
P0 was offered to leukocytes from
P0+/ mice. Why
P0 appears to be a main immunogenic component in
P0+/ mice is presently
not known. In experimental autoimmune neuritis (EAN) in rat,
P2 is a much stronger neuritogen than
P0 (Linington et al., 1992), but there might be
differences among species. On the other hand, even in heterozygous
mutants, P0 is still the most abundant peripheral
myelin protein (Giese et al., 1992) which could explain a substantial
role of P0 as an immunogenic component. This does
not preclude the possibility that other myelin components that have not
been tested could elicit even stronger immune responses.
The mechanism leading to an involvement of the immune system during
demyelination in P0+/
mice is presently not known. It is possible that the reduction of
P0 may result in an intrinsic instability of
myelin leading to an initial attraction of macrophages by Schwann
cell-derived cytokines followed by a macrophage-mediated attraction of
T-lymphocytes. An argument in favor of this model is our observation
that the number of macrophages is increased already at 4 months of age in P0+/ mice, whereas
the number of T cells is not yet elevated at this age. The activation
of autoimmune T-lymphocytes by antigen-presenting macrophages could
eventually result in a local cascade of cellular and humoral immune
reactions leading to demyelination (Gold et al., 1999) and/or to a
continued attraction and activation of macrophages by
T-lymphocyte-derived cytokines. The reduced numbers of macrophages in
the less impaired nerves of immune-deficient myelin mutants could
indicate a devastating role of macrophages during genetically induced
demyelination, as is the case in primarily immune-mediated disorders of
the nervous system, such as EAE and EAN (Huitinga et al., 1990; Jung et
al., 1993). However, it is still possible that the lower number of
macrophages is rather the consequence of than the cause for a less
severe pathological status of the immune deficient myelin mutants.
Thus, further studies are needed to characterize the roles of
macrophages in genetically mediated demyelination.
In contrast to acute and primarily immune-mediated disorders, such as
EAN, we observed only few T-lymphocytes within the nerve. As mentioned
above, activated T-lymphocytes can lead to a significant damage by
direct cytotoxicity or by T-cell-derived signals attracting other
immune cells, such as macrophages. In addition, chronic instability of
myelin induced by a gene defect may favor the perpetuation of immune
reactions to self by repeated exposure of antigen or antigens toward
patrolling T-lymphocytes. Furthermore, Schwann cells of
P0+/ mice with an
intrinsically reduced stability of myelin might react very sensitive to
a mild inflammatory attack carried by low numbers of cells. An
alternative interpretation could be that the relatively few lymphocytes
in the diseased nerve are not the only culprits leading to an
aggravation of the pathology. It is possible that mice deficient in the
immune system may generally express lower levels of gliotoxic cytokines
than mice with a wild-type immune system and that the intrinsically
instable Schwann cells of
P0+/ mice react
particularly sensitive on such cytokines.
A relevant question concerns the clinical implications of our findings.
Most forms of P0-related hereditary neuropathies
are caused by heterozygous mutations that only seldom result in a pure
reduction of gene dosis (Scherer and Chance, 1995; Warner et al.,
1996). Rather, toxic gain-of-function and dominant-negative effects
might be the leading detrimental mechanisms in most of the
P0-mutation-mediated disorders (Warner et al.,
1996; Wong and Filbin, 1996; Scherer, 1997; Zhang and Filbin, 1998).
This, however, does not argue against the possibility that in patients suffering from such detrimental processes immune-mediated mechanisms are involved as a secondary cause as we have shown for the
P0+/ mice. Rather, it
is plausible to assume that the involvement of the immune system as an
aggravating component is not confined to hereditary neuropathies caused
by P0-mutations, but might be widespread among
the different types of hereditary neuropathies. Interestingly, in a
variety of CMT patients of unknown genotype, elevated levels of
activated T-lymphocytes have been detected in the peripheral blood
(Williams et al., 1987, 1992). Furthermore, some patients are reported
to develop a sudden worsening of their clinical neuropathy and show
inflammatory infiltrates in nerve biopsies (Malandrini et al., 1999)
and clinically respond to corticosteroids (Dyck et al., 1993). Taking
into consideration that immune reactions can be superimposed on the
clinical and pathological phenotype of CMT, it might be worthwhile to
investigate whether among patients with chronic inflammatory
demyelinating polyradiculopathy some might carry CMT mutations that
would lead to a more pronounced immune reaction. In analogy, it is
intriguing to speculate that mutations in myelin and other genes of the
CNS could elicit similar immune reactions that could manifest
themselves in multiple sclerosis and modulate the disease course.
Our finding that in
P0+/ mice, the immune
system contributes to demyelination might offer the possibility to
understand the pathological mechanisms of demyelination in at least
some forms of inherited neuropathies. It might, therefore, be
worthwhile to reconsider treatment strategies with appropriate
immunomodulators or suppressors to ameliorate the immune-mediated
pathological components of the still untreatable monogenic disorders.
In addition, the knowledge about the participation of the immune system
in primarily genetically based neuropathies might extend our
understanding of the pathomechanisms occurring in neurodegenerative
disorders in general.
| |
FOOTNOTES |
Received June 14, 1999; revised Sept. 10, 1999; accepted Oct. 22, 1999.
This study was supported by the Swiss National Science Foundation (NF
31-45890.95 to R.M.), Gemeinnützige Hertie-Stiftung (GHS
2/3378/96 to R.M. and M. Sch.), the Deutsche
Forschungsgemeinschaft (Priority Program "Microglia," MA1053/3-1,
to R.M.), Research Funds of the University of Würzburg European
Union (Clinical, genetic and functional analysis of peripheral
neuropathies), and by the German Ministry for Education and Research.
We are grateful to Professor Rolf Zinkernagel (University of
Zürich) for valuable advice and discussions, Heinrich Blazyca and
Kathrin Rohner for excellent technical assistance, and Dr. Stefano
Carenini for discussions. We are indebted to Dr. Imme Haubitz
(Department of Mathematics and Statistics, University of
Würzburg) for performing the statistical analysis of the
quantification of immune cells in peripheral nerves and of the
splenocyte proliferation assay. We also thank Dr. M. Koltzenburg for
the possibility to use electronic equipment.
Correspondence should be addressed to Rudolf Martini, Department of
Neurology, Section of Developmental Neurobiology, University of
Würzburg, D-97080 Würzburg, Germany. E-mail:
neuk176{at}rzkli.uni-wuerzburg.de.
| |
REFERENCES |
-
Austin JM,
Gordon S
(1981)
A monoclonal antibody directed specifically against the mouse macrophage.
Eur J Immunol
10:805-815.
-
Brideau RJ,
Carter PB,
McMaster WR,
Mason DW,
Williams AF
(1980)
Two subsets of rat T lymphocytes defined with monoclonal antibodies.
Eur J Immunol
10:609-615[Web of Science][Medline].
-
Carenini S,
Montag D,
Cremer H,
Schachner M,
Martini R
(1997)
Absence of the myelin-associated glycoprotein (MAG) and the neural cell adhesion molecule (N-CAM) interferes with the maintenance, but not with the formation of peripheral myelin.
Cell Tissue Res
287:3-9[Web of Science][Medline].
-
Cobbold SP,
Jayasuriya A,
Nash A,
Prospero TD,
Waldmann H
(1984)
Therapy with monoclonal antibodies by elimination of T-cell subsets in vivo.
Nature
312:548-551[Medline].
-
De Jonghe P,
Timmerman V,
Nelis E,
Martin JJ,
Van Broeckhoven C
(1997)
Charcot-Marie-Tooth disease and related peripheral neuropathies.
J Periph Nerv Syst
2:370-387[Medline].
-
Dyck PJ,
Chance P,
Lebo R,
Carney JA
(1993)
Hereditary motor and sensory neuropathies.
In: Peripheral Neuropathy (Dyck PJ,
Thomas PK,
Griffin JW,
Low PA,
Poduslo JF,
eds), pp 1094-1136. Philadelphia: WB Saunders.
-
Friede RL (1972) Control of myelin formation by axon caliber
(with a model of the control mechanism). J Comp Neurol
233-252.
-
Gabreëls-Festen AA,
Hoogendijk JE,
Meijerink PH,
Gabreëls FJ,
Bolhuis PA,
van Beersum S,
Kulkens T,
Nelis E,
Jennekens FG,
de Visser M,
van Engelen BG,
Van Broeckhoven C,
Mariman EC
(1996)
Two divergent types of nerve pathology in patients with different P0 mutations in Charcot-Marie-Tooth disease.
Neurol
47:761-765[Abstract/Free Full Text].
-
Giese KP,
Martini R,
Lemke G,
Soriano P,
Schachner M
(1992)
Mouse P0 gene disruption leads to hypomyelination, abnormal expression of recognition molecules, and degeneration of myelin and axons.
Cell
71:565-576[Web of Science][Medline].
-
Gold R,
Archelos JJ,
Hartung H-P
(1999)
Mechanisms of immune regulation in the peripheral nervous system.
Brain Pathol
9:343-360[Web of Science][Medline].
-
Guénard V,
Schweitzer B,
Flechsig E,
Hemmi S,
Martini R,
Suter U,
Schachner M
(1999)
Effective gene transfer of lacZ and P0 into Schwann cells of P0-deficient mice.
Glia
25:165-178[Web of Science][Medline].
-
Huitinga I,
v Rooijen N,
Groot CJA,
Uitdehaag BMJ,
Dijkstra CD
(1990)
Suppression of experimental allergic encephalomyelitis in Lewis rats after elimination of macrophages.
J Exp Med
172:1025-1033[Abstract/Free Full Text].
-
Jung S,
Huitinga I,
Schmidt B,
Zielasek J,
Dijkstra CD,
Toyka KV,
Hartung HP
(1993)
Selective elimination of macrophages by dichlormethylene diphosphonate-containing liposomes suppresses experimental autoimmune neuritis.
J Neurol Sci
119:195-202[Web of Science][Medline].
-
Lindberg RLP,
Martini R,
Baumgartner M,
Erne B,
Borg J,
Zielasek J,
Ricker K,
Steck A,
Toyka KV,
Meyer UA
(1999)
Motor neuropathy in porphobilinogen deaminase-deficient mice imitates the peripheral neuropathy of human acute porphyria.
J Clin Invest
103:1127-1134[Web of Science][Medline].
-
Linington C,
Lassmann H,
Ozawa K,
Kosin S,
Mongan L
(1992)
Cell adhesion molecules of the immunoglobulin supergene family as tissue-specific autoantigens: induction of experimental allergic neuritis (EAN) by PO protein-specific T cell lines.
Eur J Immunol
22:1813-1817[Web of Science][Medline].
-
Malandrini A,
Villanova M,
Dotti MT,
Frederico A
(1999)
Acute inflammatory neuropathy in Charcot-Marie-Tooth disease.
Neurology
52:859-861[Abstract/Free Full Text].
-
Martini R
(1997)
Animal models for inherited peripheral neuropathies.
J Anat
191:321-336.
-
Martini R,
Schachner M
(1997)
Molecular bases of myelin formation as revealed by investigations on mice deficient in glial cell surface molecules.
Glia
19:298-310[Web of Science][Medline].
-
Martini R,
Zielasek J,
Toyka KV,
Giese KP,
Schachner M
(1995a)
Protein zero (P0)-deficient mice show myelin degeneration in peripheral nerves characteristic of inherited human neuropathies.
Nat Genet
11:281-286[Web of Science][Medline].
-
Martini R,
Mohajeri MH,
Kasper S,
Giese KP,
Schachner M
(1995b)
Mice doubly deficient in the genes for P0 and myelin basic protein show that both proteins contribute to the formation of the major dense line in peripheral nerve myelin.
J Neurosci
15:4488-4495[Abstract].
-
Martini R,
Zielasek J,
Toyka KV
(1998)
Inherited demyelinating neuropathies: from gene to disease.
Curr Opin Neurol
11:545-556[Web of Science][Medline].
-
Mombaerts P,
Iacomini J,
Johnson RS,
Herrup K,
Tonegawa S,
Papaioannou VE
(1992)
RAG-1-deficient mice have no mature B and T lymphocytes.
Cell
68:869-877[Web of Science][Medline].
-
Nelis E,
Haites N,
Van Broeckhoven C
(1999)
Mutations in the peripheral myelin genes and associated genes in inherited peripheral neuropathies.
Hum Genet
13:11-28.
-
Neuhaus O,
Emoto M,
Kaufmann SH
(1996)
Constitutive biological activity of thymus-independent TCR-alpha-beta+ intestinal intraepithelial lymphocytes in TCR-alpha
 / gene disruption mice.
Immunol Lett
54:53-57[Web of Science][Medline]. -
Pette M,
Fujita K,
Wilkinson D,
Altmann DM,
Trowsdale J,
Giegerich G,
Hinkkanen A,
Epplen JT,
Kappos L,
Wekerle H
(1990)
Myelin autoreactivity in multiple sclerosis: recognition of myelin basic protein in the context of HLA-DR2 products by T lymphocytes of multiple-sclerosis patients and healthy donors.
Proc Natl Acad Sci USA
87:7968-7972[Abstract/Free Full Text].
-
Pette M,
Linington C,
Gengaroli C,
Grosse-Wilde H,
Toyka KV,
Hartung HP
(1994)
T lymphocyte recognition sites on peripheral nerve myelin P0 protein.
J Neuroimmunol
54:29-34[Web of Science][Medline].
-
Philpott KL,
Viney JL,
Kay G,
Rastan S,
Gardiner EM,
Chae S,
Hayday AC,
Owen MJ
(1992)
Lymphoid development in mice congenitally lacking T cell receptor alpha beta-expressing cells.
Science
256:1448-1452[Abstract/Free Full Text].
-
Scherer SS
(1997)
Molecular genetics of demyelination: new wrinkles on an old membrane.
Neuron
18:13-16[Web of Science][Medline].
-
Scherer SS,
Chance PF
(1995)
Myelin genes: getting the dosage right.
Nat Genet
11:226-228[Web of Science][Medline].
-
Schmid CD,
Schnell L,
Schachner M,
Martini R
(1996)
Peripheral nerves of a mouse model for Charcot-Marie-Tooth neuropathy 1B show infiltration of macrophages and CD4- and CD8-positive lymphocytes.
Soc Neurosci Abstr
22:835.819.
-
Schmidt B,
Toyka KV,
Kiefer R,
Full J,
Hartung HP,
Pollard J
(1996)
Inflammatory infiltrates in sural nerve biopsies in Guillain-Barre syndrome and chronic inflammatory demyelinating neuropathy.
Muscle Nerve
19:474-487[Web of Science][Medline].
-
Shy ME,
Arroyo E,
Sladky J,
Menichella D,
Jiang H,
Xu W,
Kamholz J,
Scherer SS
(1997)
Heterozygous P0 knockout mice develop a peripheral neuropathy that resembles chronic inflammatory demyelinating polyneuropathy (CIDP).
J Neuropathol Exp Neurol
56:811-821[Web of Science][Medline].
-
Stienekemeier M,
Herrmann T,
Kruse N,
Weishaupt A,
Weilbach FX,
Giegerich G,
Theofilopoulos A,
Jung S,
Gold R
(1999)
Heterogeneity of T-cell receptor usage in experimental autoimmune neuritis in the Lewis rat.
Brain
122:523-535[Abstract/Free Full Text].
-
Tachi N,
Kozuka N,
Ohya K,
Chiba S,
Sasaki K
(1997)
Tomaculous neuropathy in Charcot-Marie-Tooth disease with myelin protein zero gene mutation.
J Neurol Sci
153:106-109[Web of Science][Medline].
-
Thomas FP,
Lebo RV,
Rosoklija G,
Ding XS,
Lovelace RE,
Latov N,
Hays AP
(1994)
Tomaculous neuropathy in chromosome 1 Charcot-Marie-Tooth syndrome.
Acta Neuropathol (Berl)
87:91-97[Medline].
-
Warner LE,
Hilz MJ,
Appel SH,
Killian JM,
Kolodny EH,
Karpati G,
Carpenter S,
Watters GV,
Wheeler C,
Witt D,
Bodell A,
Nelis E,
Van Broeckhoven C,
Lupski JR
(1996)
Clinical phenotypes of different MPZ (P0) mutations may include Charcot Marie Tooth type 1B, Dejerine Sottas, and congenital hypomyelination.
Neuron
17:451-460[Web of Science][Medline].
-
Weishaupt A,
Giegerich G,
Jung S,
Gold R,
Enders U,
Pette M,
Hayasaka K,
Hartung HP,
Toyka KV
(1995)
T cell antigenic and neuritogenic activity of recombinant human peripheral myelin P2 protein.
J Neuroimmunol
63:149-156[Web of Science][Medline].
-
Williams LL,
Shannon BT,
O'Dougherty M,
Wright FS
(1987)
Activated T cells in type I Charcot-Marie-Tooth disease: evidence for immunologic heterogeneity.
J Neuroimmunol
16:317-330[Web of Science][Medline].
-
Williams LL,
Kissel JT,
Shannon BT,
Wright FS,
Mendell JR
(1992)
Expression of Schwann cell and peripheral T-cell activation epitopes in hereditary motor and sensory neuropathy.
J Neuroimmunol
36:147-155[Web of Science][Medline].
-
Wong MH,
Filbin MT
(1996)
Dominant-negative effect on adhesion by myelin P0 protein truncated in its cytoplasmic domain.
J Cell Biol
134:1531-1541[Abstract/Free Full Text].
-
Zhang K,
Filbin MT
(1998)
Myelin Po protein mutated at Cys21 has a dominant-negative effect on adhesion of wild type Po.
J Neurosci Res
53:1-6[Web of Science][Medline].
-
Zielasek J,
Martini R,
Toyka KV
(1996)
Functional abnormalities in P0-deficient mice resemble human hereditary neuropathies linked to P0 gene mutations.
Muscle Nerve
19:946-952[Web of Science][Medline].
Copyright © 2000 Society for Neuroscience 0270-6474/00/202729-07$05.00/0
This article has been cited by other articles:
|
|
|
|
 
C. W. Ip, A. Kroner, M. Bendszus, C. Leder, I. Kobsar, S. Fischer, H. Wiendl, K.-A. Nave, and R. Martini
Immune Cells Contribute to Myelin Degeneration and Axonopathic Changes in Mice Overexpressing Proteolipid Protein in Oligodendrocytes
J. Neurosci.,
August 2, 2006;
26(31):
8206 - 8216.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Visan, I. A. Visan, A. Weishaupt, H. H. Hofstetter, K. V. Toyka, T. Hunig, and R. Gold
Tolerance Induction by Intrathymic Expression of P0
J. Immunol.,
February 1, 2004;
172(3):
1364 - 1370.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Ginsberg, O. Malik, A. R. Kenton, D. Sharp, J. R. Muddle, M. B. Davis, J. B. Winer, R. W. Orrell, and R. H. M. King
Coexistent hereditary and inflammatory neuropathy
Brain,
January 1, 2004;
127(1):
193 - 202.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. V. Toyka and R. Gold
The pathogenesis of CIDP: Rationale for treatment with immunomodulatory agents
Neurology,
April 22, 2003;
60(90083):
S2 - 7.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
I. Kobsar, M. Berghoff, M. Samsam, C. Wessig, M. Maurer, K. V. Toyka, and R. Martini
Preserved myelin integrity and reduced axonopathy in connexin32-deficient mice lacking the recombination activating gene-1
Brain,
April 1, 2003;
126(4):
804 - 813.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Samsam, W. Mi, C. Wessig, J. Zielasek, K. V. Toyka, M. P. Coleman, and R. Martini
The Wlds Mutation Delays Robust Loss of Motor and Sensory Axons in a Genetic Model for Myelin-Related Axonopathy
J. Neurosci.,
April 1, 2003;
23(7):
2833 - 2839.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C J Klein, P J B Dyck, S M Friedenberg, T M Burns, A J Windebank, and P J Dyck
Inflammation and neuropathic attacks in hereditary brachial plexus neuropathy
J. Neurol. Neurosurg. Psychiatry,
July 1, 2002;
73(1):
45 - 50.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C M Gabriel, N A Gregson, N W Wood, and R A C Hughes
Immunological study of hereditary motor and sensory neuropathy type 1a (HMSN1a)
J. Neurol. Neurosurg. Psychiatry,
February 1, 2002;
72(2):
230 - 235.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Carenini, M. Maurer, A. Werner, H. Blazyca, K. V. Toyka, C. D. Schmid, G. Raivich, and R. Martini
The Role of Macrophages in Demyelinating Peripheral Nervous System of Mice Heterozygously Deficient in P0
J. Cell Biol.,
January 22, 2001;
152(2):
301 - 308.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Tohyama, M. T. Vanier, K. Suzuki, T. Ezoe, J. Matsuda, and K. Suzuki
Paradoxical influence of acid {beta}-galactosidase gene dosage on phenotype of the twitcher mouse (genetic galactosylceramidase deficiency)
Hum. Mol. Genet.,
July 1, 2000;
9(11):
1699 - 1707.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
