Autoimmunity to antigens of the central nervous system is usually considered
detrimental. T cells specific to a central nervous system self antigen, such
as myelin basic protein, can indeed induce experimental autoimmune encephalomyelitis,
but such T cells may nevertheless appear in the blood of healthy individuals.
We show here that autoimmune T cells specific to myelin basic protein can
protect injured central nervous system neurons from secondary degeneration.
After a partial crush injury of the optic nerve, rats injected with activated
anti−myelin basic protein T cells retained approximately 300% more retinal
ganglion cells with functionally intact axons than did rats injected with
activated T cells specific for other antigens. Electrophysiological analysis
confirmed this finding and suggested that the neuroprotection could result
from a transient reduction in energy requirements owing to a transient reduction
in nerve activity. These findings indicate that T−cell autoimmunity
in the central nervous system, under certain circumstances, can exert a beneficial
effect by protecting injured neurons from the spread of damage.
Maintenance of central nervous system (CNS) integrity is a complex balancing
act in which compromises are struck with the immune system. In most tissues,
the immune system plays an essential part in protection, repair and healing.
In the CNS, because of its unique immune privilege, immunological reactions
are relatively limited1,
2. A growing body of evidence indicates
that the failure of the mammalian CNS to achieve functional recovery after
injury is a reflection of an ineffective 'dialog' between the damaged tissue
and the immune system. For example, the restricted communication between the
CNS and blood−borne macrophages affects the capacity of axotomized axons
to regrow, and transplantation of activated macrophages can promote CNS regrowth3,
4. Here we have extended the study of CNS maintenance to T cells.
Activated T cells have been shown to enter the CNS parenchyma, irrespective
of their antigen specificity, but only T cells capable of reacting with a
CNS antigen seem to persist there5. T cells reactive to antigens
of CNS white matter, such as myelin basic protein (MBP), can induce the paralytic
disease experimental autoimmune encephalomyelitis (EAE) within several days
of their inoculation into naive recipient rats6. Anti−MBP
T cells may also be involved in the human disease multiple sclerosis7,
8. However, despite their pathogenic potential, anti−MBP
T−cell clones are present in the immune systems of healthy subjects9,
10,
11,
12. Activated T cells, which normally patrol the intact
CNS, transiently accumulate at sites of CNS white matter lesions13.
These observations prompted us to study whether the T cells that accumulate
after axonal injury exert a beneficial or a deleterious effect on the damaged
CNS.
A catastrophic consequence of CNS injury is that the primary damage is
often compounded by the gradual secondary loss of adjacent neurons that apparently
were undamaged, or only marginally damaged, by the initial injury14,
15,
16.
The primary lesion causes changes in extracellular ion concentrations, elevation
of amounts of free radicals, release of neurotransmitters, depletion of growth
factors, and local inflammation. These changes trigger a cascade of destructive
events in the adjacent neurons that initially escaped the primary injury17,
18,
19. This secondary damage is mediated by activation of voltage−dependent
or agonist−gated channels, ion leaks, activation of calcium−dependent
enzymes such as proteases, lipases and nucleases, mitochondrial dysfunction
and energy depletion, culminating in neuronal cell death20,
21,
22,
23.
The widespread loss of neurons beyond the loss caused directly by the primary
injury has been called 'secondary degeneration'.
We have developed a model for studying secondary degeneration, based on
a partial crush injury of the rat optic nerve23,
24,
25. Here
we demonstrate morphologically that activated T cells specific to the CNS
self antigen MBP can reduce the secondary degeneration of neurons after a
primary crush injury. T cells specific to an epitope of a different self antigen,
the 60−kDa heat shock protein (hsp60), or to the foreign antigen ovalbumin
(OVA) did not protect neurons against secondary degeneration, although these
T cells did 'home' to the site of optic nerve injury. Electrophysiological
analysis confirmed the neuroprotective effect of the anti−MBP T cells.
The observed neuroprotection was preceded by a transient reduction in nerve
conduction. These findings indicate that the anti−MBP T cells might
reduce injury−induced secondary damage by inducing a resting state in
the damaged nerve, thereby reducing its energy demands and enhancing its ability
to cope with the stress resulting from the injury.
Presence of T cells in the injured optic nerve Passively transferred, activated T cells accumulate at a site of CNS injury,
independent of their antigen specificity13. Therefore, we analyzed
crush−injured optic nerves for the presence of T cells after systemic
injection of various T−cell lines. Autoimmune T cells specific to MBP,
T cells specific to the non−self antigen OVA, or T cells specific to
peptide 277 (p277) of hsp60 (a self antigen not restricted to the CNS) were
activated with their respective antigens for 3 days, and then injected (1
107 cells) intraperitoneally into rats immediately
after unilateral optic nerve injury. Control rats were injected intraperitoneally
with phosphate−buffered saline (PBS). Seven days after injury, the optic
nerves were excised, cryosectioned and analyzed immunohistochemically for
the presence of T cells. No T cells could be detected in the uninjured optic
nerves of PBS−injected rats (Fig. 1). Anti−MBP
T cells are known to 'home' to intact CNS white matter26, and
small numbers of T cells were indeed observed in the uninjured optic nerves
of rats injected with anti−MBP T cells, but not in rats injected with
anti−OVA or anti−p277 T cells. Crush injury of the optic nerve
in PBS−injected control rats was accompanied by the presence of a small
number of endogenous T cells at the injury site, possibly reflecting a response
to self antigens triggered by the injury27. The number of T
cells in the injured optic nerve in rats injected with PBS was 16% to 25%
of the number in rats injected with anti−OVA, anti−p277 or anti−MBP
T cells. These observations confirmed our previous finding that axonal injury
in the CNS is accompanied by the accumulation of endogenous T cells, and that
this accumulation is significantly augmented by systemic injection of activated
T cells13. 'Homing' of exogenous T cells to the site of optic
nerve injury can be demonstrated by prelabeling the injected T cells13.
Figure 1. T−cell presence in injured optic nerve 1 week after injury.
Adult Lewis rats were injected with activated T cells of the anti−MBP
(TMBP), anti−OVA (TOVA), or anti−p277 (T
p277) lines, or with PBS, immediately after unilateral crush injury
of the optic nerve. Seven days later, both the injured and uninjured optic
nerves were removed, cryosectioned and analyzed immunohistochemically for
the presence of immunolabeled T cells. T cells were counted at the site of
injury and at randomly selected areas in the uninjured optic nerves. The histogram
shows the mean number of T cells per mm2 s.e.m., counted
in two to three sections of each nerve. Each group contained three to four
rats. The number of T cells was considerably higher in injured nerves of rats
injected with anti−MBP, anti−OVA or anti−p277 T cells; statistical
analysis (one−way ANOVA) showed significant differences between T cell
numbers of injured optic nerves of rats injected with anti−MBP, anti−OVA,
or anti−p277 T cells and injured optic nerves of rats injected with
PBS (P < 0.001); and between injured optic nerves and uninjured
optic nerves of rats injected with anti−MBP, anti−OVA, or anti−p277
T cells (P < 0.001).
Neuroprotection by autoimmune anti−MBP T cells Morphological analyses were done to assess the effect of the T cells on
the response of the nerve to injury, and specifically on secondary degeneration.
Immediately after optic nerve injury, rats were injected intraperitoneally
with PBS or with 1 107 activated T cells of the various
cell lines. The degree of primary damage to the optic nerve axons and their
attached retinal ganglion cells (RGCs) was measured by injecting the dye 4−Di−10−Asp
distal to the site of the lesion immediately after the injury. A time lapse
of 2 weeks between a moderate crush injury and dye application is optimal
for demonstrating the number of still−viable labeled neurons as a measure
of secondary degeneration, and as the response of secondary degeneration to
treatment. Therefore, secondary degeneration was quantified here by injecting
the dye immediately or 2 weeks after the primary injury, and calculating the
additional loss of RGCs between the first and the second injections of the
dye. The percentage of RGCs that had survived secondary degeneration was then
calculated. The percentage of labeled RGCs (reflecting still−viable
axons) was significantly greater in the retinas of the rats injected with
anti−MBP T cells than in the retinas of the PBS−injected control
rats (Fig. 2). In contrast, the percentage of labeled
RGCs in the retinas of the rats injected with anti−OVA or anti−p277
T cells was not significantly greater than those in the control retinas. Thus,
although the three T−cell lines accumulated at the site of injury, only
the MBP−specific autoimmune T cells had a substantial effect in limiting
the extent of secondary degeneration. Photomicrographs show the labeled RGCs
of injured optic nerves of rats injected with PBS, anti−p277 T cells
or anti−MBP T cells (Fig. 3).
Figure 2. T cells specific to MBP, but not to OVA or p277 of hsp60, protect neurons
from secondary degeneration.
Immediately after optic nerve injury, rats were injected with anti−MBP,
anti−OVA or anti−p277 T cells, or with PBS. The neurotracer dye
4−Di−10−Asp was applied to optic nerves distal to the site
of the injury, immediately after injury (for assessment of primary damage)
or 2 weeks later (for assessment of secondary degeneration). Five days after
dye application, the retinas were excised and flat−mounted. Labeled
retinal ganglion cells (RGCs) from three to five randomly selected fields
in each retina (all located at approximately the same distance from the optic
disk) were counted by fluorescence microscopy. RGC survival in each group
of injured nerves was expressed as the percentage of the total number of neurons
spared after the primary injury (42% of axons remained undamaged after the
primary injury). The neuroprotective effect of anti−MBP T cells compared
with that of PBS was significant (P < 0.001, one−way ANOVA).
Anti−OVA T cells or anti−p277 T cells did not differ significantly
from PBS in their effect on the protection of neurons that had escaped the
primary injury (P > 0.05, one−way ANOVA). The results are a summary
of five experiments. Each group contained five to ten rats.
Figure 3. Photomicrographs of retrogradely labeled retinas of injured optic nerves
of rats.
Immediately after unilateral crush injury of their optic nerves, rats were
injected with PBS (a) or with activated anti−p277 T cells
(b) or activated anti−MBP T cells (c). Two weeks
later, the neurotracer dye 4−Di−10−Asp was applied to the
optic nerves, distal to the site of injury. After 5 days, the retinas were
excised and flat−mounted. Labeled (surviving) RGCs, located at approximately
the same distance from the optic disk in each retina, were photographed.
The functional autoimmunity of the injected anti−MBP T cells was
demonstrated by the development of transient EAE in the recipients of these
cells. The course and severity of the EAE was not affected by the presence
of the optic nerve crush injury (Fig. 4a). Moreover,
the administration of anti−MBP T cells in rats that had not suffered
an optic nerve crush did not affect the numbers of RGCs (Fig.
4b). Thus, contracting EAE in the absence of a nerve crush did
not cause any change in the numbers of viable RGCs or optic nerve axons.
Figure 4.a, Clinical severity of EAE is not influenced by an optic nerve
crush injury.
Lewis rats, either uninjured () or immediately after optic nerve
crush injury (), were injected with activated anti−MBP T cells.
EAE was evaluated according to a neurological paralysis scale. Data points
represent means s.e.m. These results represent a summary of three
experiments. Each group contained five to nine rats. b,
The number of RGCs in the uninjured optic nerve is not influenced by injection
of anti−MBP T cells. Two weeks after the injection of anti−MBP
T cells or PBS, 4−Di−10−Asp was applied to the optic nerves.
After 5 days the retinas were excised and flat−mounted. Labeled RGCs
from five fields (located at approximately the same distance from the optic
disk) in each retina were counted and their average number per mm2
was calculated. There was no difference in the number of labeled
RGCs between rats injected with anti−MBP T cells (TMBP)and
PBS−injected control rats.
To determine whether the neuroprotective effect of the anti−MBP T
cells is correlated with their virulence, we examined the effect of T cells
reactive to a 'cryptic' epitope of MBP, the peptide 51−70 (p51−70).
'Cryptic' epitopes activate specific T cells after an animal is immunized
with the particular peptide, but not with the whole antigen28.
The T−cell line reactive to the whole MBP and the T−cell line
reactive to the cryptic epitope p51−70 were compared for the severity
of the EAE they induced, and for their effects on secondary degeneration.
In rats injected with the T−cell line reactive to the cryptic epitope,
disease severity (as manifested by the maximal EAE score) was significantly
lower than that in rats injected with the T−cell line reactive to the
whole protein (Table 1). Whereas anti−MBP
T cells caused clinical paralysis of the limbs, rats injected with the anti−p51−70
T cells developed only tail atony, not hind limb paralysis, and almost none
showed weakness of the hind limbs. Despite this difference in EAE severity,
the neuroprotective effect of the less virulent (anti−p51−70)
T cells was similar to that of the more virulent (anti−MBP) T cells
(Fig. 5). The percentage of RGCs surviving secondary
degeneration in the retinas of rats injected with either of the lines was
significantly higher than in the retinas of the PBS−injected rats. Thus,
there was no correlation between the neuroprotective effect of the autoimmune
T cells and their virulence. It is possible that the anti−p51−70
T cells encounter little antigen in the intact CNS, and therefore cause only
mild EAE. Their target antigen may however become more available after injury,
enabling these T cells to exert a neuroprotective effect.
Figure 5. T cells specific to p51−70 of MBP protect neurons from secondary
degeneration.
Immediately after optic nerve injury, rats were injected with anti−MBP
T cells, anti−p51−70 T cells or PBS. The neurotracer dye 4−Di−10−Asp
was applied to optic nerves distal to the site of the injury, immediately
after injury (for assessment of primary damage) or 2 weeks later (for assessment
of secondary degeneration). Five days after dye application, the retinas were
excised and flat−mounted. Labeled retinal ganglion cells (RGCs) from
three to five randomly selected fields in each retina (all located at approximately
the same distance from the optic disk) were counted by fluorescence microscopy.
RGC survival in each group of injured nerves was expressed as the percentage
of the total number of neurons spared after the primary injury. Compared with
that of PBS treatment, the neuroprotective effects of anti−MBP and anti−p51−70
T cells were significant (P < 0.001, one−way ANOVA).
To confirm the neuroprotective effect of the anti−MBP T cells, we
did electrophysiological studies. Immediately after optic nerve injury, the
rats were injected intraperitoneally with PBS or with 1 10
7 activated anti−MBP or anti−OVA T cells. The optic nerves
were excised 7, 11 or 14 days later and the compound action potentials (CAPs),
a measure of nerve conduction, were recorded from the uninjured nerves and
from the distal segments of the injured nerves. On day 14, the mean CAP amplitude
of the distal segments recorded from the injured nerves obtained from from
rats injected with the anti−MBP T cells was about 250% that of recorded
from the PBS−injected contrtol rats (Fig. 6a
and Table 2). As the distal segment of the injured
nerve contains both axons that escaped the primary insult and injured axons
that have not yet degenerated, the observed neuroprotective effect could reflect
the rescue of spared neurons, or a delay of Wallerian degeneration of the
injured neurons (which normally occurs in the distal stump), or both. No effect
of the injected anti−MBP T cells on the mean CAP amplitudes of uninjured
nerves was observed (Fig. 6b,
Table 2). It is unlikely that the neuroprotective effect observed
on day 14 could have been due to the regrowth of nerve fibers, as the time
period was too short for this.
Figure 6. Anti−MBP T cells increase the CAP amplitude of injured optic
nerves.
Immediately after optic nerve injury, rats were injected with either PBS
or activated anti−MBP T cells (TMBP). Two weeks later, the
CAPs of injured (a) and uninjured (b) nerves were recorded.
There were no significant differences in mean CAP amplitudes between uninjured
nerves obtained from PBS−injected and T cell−injected rats (
n = 8; P = 0.8, Student's t−test). The neuroprotective
effect of anti−MBP T cells (relative to PBS) on the injured nerve on
day 14 after injury was significant (n = 8; P < 0.01, Student's
t−test).
Table 2. Transient reduction in electrophysiological activity of the injured
optic nerve induced by anti−MBP T cells, followed by a neuroprotective
effect.
The strong neuroprotective effect of the anti−MBP T cells seen on
day 14 was associated with a significantly decreased CAP amplitude recorded
on day 7 (Table 2). The anti−MBP T cells manifested
no substantial effect on the uninjured nerve on day 7, indicating that the
reduction in electrophysiological activity observed in the injured nerve on
day 7 might reflect the larger number of T cells present at the injury site
relative to the uninjured nerve (Fig. 1). The observed
reduction in CAP amplitude in the injured nerve on day 7 reflected a transient
reduction in conduction, which may have imposed a transient resting state
in the injured nerve. This transient effect had not only disappeared, but
was even reversed by day 14 (Table 2). Early signs
of the neuroprotective effect could already be detected on day 11 in the rats
injected with anti−MBP T cells (data not shown). In rats injected with
anti−OVA T cells, no reduction in CAP amplitude on day 7 could be detected
in either the injured or the uninjured nerves, and no neuroprotective effect
was observed on day 14 (Table 2). Thus, it seems
that the early reduction in CAP and the late neuroprotection shown specifically
by the anti−MBP T cells are related.
Discussion The CNS is an immune−privileged site in which local immune responses
are restricted1,
2 and in which the capacity for repair and
recovery after injury is poor29. The highly specialized CNS
must avoid the possibly destructive consequences of severe inflammation and
autoimmune disease. However, immune reactions are required for healing and
recovery after injury in the CNS, as in other tissues30. An
accumulating body of evidence indicates that the failure of the CNS to recover
from injury is related to its immune−privileged status. In particular,
the resistance of the CNS to regeneration might be closely related to the
restriction in the numbers of macrophages recruited and activated by the injured
CNS (3). Here we have demonstrated that the
administration of autoimmune anti−MBP T cells to rats with injured optic
nerves, rather than aggravating the damage, can lead to a significant degree
of protection from secondary degeneration. Thus, enhancement of the T−cell
autoimmune response to a component of CNS myelin seemed to be beneficial in
limiting the spread of secondary damage after partial injury of CNS axons.
Autoimmune responses directed against antigens of the CNS are usually regarded
as detrimental. As an example, passive transfer of T−cell lines specific
for MBP or proteolipid protein induces a paralytic disease, EAE6,
31.
We have shown here, however, using both morphological and electrophysiological
techniques, that T−cell autoimmunity can mediate significant neuroprotection
after CNS injury. The slight difference in therapeutic index obtained by these
two techniques can be attributed to the fact that the former detects only
axons that are still morphologically intact, whereas the latter, in which
CAPs are recorded from the nerve segment distal to the injury site, might
reflect the activities of both intact axons and injured axons that are still
functioning.
CNS damage can activate latent autoimmunity to MBP: myelin−reactive
antibodies are elevated after CNS injury32,
33, and T cells
isolated from rats with spinal cord injury are capable of causing EAE in naive
rats27. Therefore, it will be important to determine whether
the endogenous T cells accumulating at the site of damaged optic nerve include
anti−MBP clones. The results reported here using anti−MBP T cell
lines indicate that endogenous anti−MBP T cells, if sufficiently numerous,
might function in a similar way to protect damaged CNS tissue. The lack of
correlation between the clinical pathogenicity of the autoimmune T cells and
their neuroprotective effect indicates that a benign autoimmunity, achieved
by non−encephalitogenic T cells specific to a cryptic antigen, might
serve as an effective mechanism for neuroprotection. In the uninjured CNS,
cryptic epitopes might not be readily accessible and, therefore, T cells to
such epitopes might cause only mild, if any, autoimmune disease. After injury,
however, cryptic epitopes might become available and the specific T cells
could then be activated at the site of injury to exert their neuroprotective
effect.
At present, it seems that neuroprotection is exerted only by the CNS−specific
autoimmune T cells. The inefficiency of the anti−p277 T cells is noteworthy,
given that the hsp60 molecule is expressed in injured tissues34
and anti−hsp60 T cells have been isolated from EAE lesions35.
Thus, not all autoimmune T cells can inhibit secondary degeneration, even
if the target antigen is present in the CNS lesion. It is possible, however,
that the p277 epitope of hsp60 is not strongly expressed in the injured optic
nerve. We are now investigating the neuroprotective potential of T cells specific
for other self antigens.
In addition to the question of specificity, the molecular mechanisms by
which anti−MBP T cells protect the injured nerve from secondary degeneration
of neurons need to be explored further. Secondary degeneration results from
metabolic insult, among other factors20,
21,
23,
36. Given our
electrophysiological findings here, the anti−MBP T cells might exert
neuroprotection by causing a transient reduction in the nerve's electrophysiological
activity. Putting the damaged nerve to rest, even transiently, could reduce
the nerve's metabolic oxygen and glucose requirements and prevent energy depletion,
thus helping to preserve neuronal viability. The mechanism proposed here is
reminiscent of the neuroprotection obtained by therapeutic hypothermia37. Our finding here agrees with earlier work showing that anti−MBP
T cells could reversibly block signal conduction in the isolated rat optic
nerve in vitro38, and indicating that no invading cells
other than T cells are required for the observed reduction in electrophysiological
activity of the nerve. The exposure of myelin at the site of the crush might
activate MBP−specific T cells to secrete molecules that put the injured
nerve to rest, whereas T cells specific for other antigens might not be activated
because of inadequate antigen recognition. The anti−MBP T cells may
act directly on neurons or on glial cells (for example, astrocytes), which
can indirectly mediate neuronal dysfunction after immunological activation39.
There is evidence that cytokines can directly affect the electrophysiological
functions of neurons and glial cells. Cytokines can induce a reduction in
neuronal excitability, for example by increasing inactivation of the Na
+ current39. Inhibition of neuronal excitability by
T−cell cytokines may contribute to the CAP reduction we observed in
the optic nerves of the rats injected with the anti−MBP T cells. It
is unlikely that this reduction in electrophysiological activity is due to
demyelination, as EAE adoptively transferred to Lewis rats by anti−MBP
T cells is typically a brief and monophasic disease with sparse demyelination40
Although the mechanism proposed here seems most promising, alternative
explanations cannot be excluded. Growth factors secreted by anti−MBP
T cells in the tissue might protect the injured CNS. CD4+ T
cells were indeed found to synthesize and release biologically active nerve
growth factor41,
42,
43. Growth factors can attenuate the elevation
of levels of Ca++ and free radicals otherwise 'triggered' in
neurons by damage. Thus, the benefit provided by the anti−MBP T cells
could be mediated by several actions, working together.
Our findings here indicate that the activation of specific autoimmunity
in the CNS might not always be detrimental, but could, under certain circumstances,
have a physiological role in protecting the damaged CNS. Beneficial autoimmunity
is functionally distinguishable from autoimmune disease. If autoimmunity to
a particular protein or to a selected epitope can be advantageous, we might
have an explanation for the wide prevalence of well−regulated autoimmunity
naturally directed to a particular set of self antigens, a phenomenon that
has been called the 'immunological homunculus'44. The immunological
dominance of the main self antigens constituting the homunculus is encoded
by naturally autoimmune T cells and B cells and their anti−idiotypic
regulatory cells45. Indeed, the idea that the immune system's
primary goal is to discriminate between self and non−self was recently
called into question. Our results are consistent with the idea that a primary
function of the immune system is to maintain the body by receiving signals
from an extended network of body tissues, without necessarily ignoring self
antigens. Thus, the immune system can be activated to deal with tissue damage,
rather than exclusively with the danger associated with pathogens46.
The fact that endogenous autoimmune T cells apparently do not function optimally
in protecting neurons after CNS injury may reflect evolutionary constraints
derived from the special needs of the highly specialized CNS, which restrict
immune reactions. If, however, autoimmunity to particular injury−exposed
epitopes can be selectively augmented, it might be possible to achieve neuroprotection
without the threat of autoimmune disease.
Our findings here not only demonstrate the role that T cells might play
in nerve recovery, but also further substantiate the idea that natural autoimmunity
can be benign44 and may even function as a protective mechanism.
Methods Animals. Inbred female adult Lewis rats (8−12
weeks old) were supplied by the Animal Breeding Center of The Weizmann Institute
of Science. The rats were housed in a light− and temperature−controlled
room and matched for age in each experiment.
Antigens. MBP from the spinal cords of guinea pigs
was prepared as described47. OVA was purchased from Sigma. The
p51−70 of the rat 18.5−kDa isoform of MBP (sequence APKRGSGKDSH—TRTTHYG)
and the p277 of the human hsp60 (sequence VLGGGCALLRCPALDSLTPANED)(48) were synthesized using the 9−fluorenylmethoxycarbonyl
technique with an automatic multiple peptide synthesizer (AMS 422; ABIMED,
Langenfeld, Germany). The purity of the peptides was analyzed by HPLC and
amino−acid composition.
T−cell lines. T−cell lines were generated
from draining lymph node cells obtained from Lewis rats immunized with the
antigens above49. The antigen was dissolved in PBS (1 mg/ml)
and emulsified with an equal volume of incomplete Freund's adjuvant (Difco
Laboratories, Detroit, Michigan) supplemented with 4 mg/ml Mycobacterium
tuberculosis (Difco Laboratories, Detroit, Michigan). Ten days after the
antigen was injected into the rats' hind foot pads in 0.1 ml of the emulsion,
the rats were killed and draining lymph nodes were surgically removed and
dissociated. The cells were washed and activated with the antigen (10 g/ml)
in proliferation medium containing Dulbecco's modified Eagle's medium (DMEM)
supplemented with L−glutamine (2 mM), 2−mercaptoethanol (5
10−5 M), sodium pyruvate (1 mM), penicillin (100 IU/ml),
streptomycin (100 g/ml), nonessential amino acids (1 ml/100 ml) and autologous
serum 1% (volume/volume). After incubation for 72 h at 37 °C, 90% relative
humidity and 7% CO2, the cells were transferred to propagation
medium consisting of DMEM, L−glutamine, 2−mercaptoethanol, sodium
pyruvate, nonessential amino acids and antibiotics in the same concentrations
as above, with the addition of 10% fetal calf serum (FCS) (volume/volume)
and 10% T−cell growth factor derived from the supernatant of concanavalin
A−stimulated spleen cells50. Cells were grown in propagation
medium for 4−10 days before being re−stimulated with their antigen
(10 g/ml) in the presence of irradiated (2000 rad) thymus cells (10
7 cells/ml) in proliferation medium. The T−cell lines were expanded
by repeated stimulation and propagation.
Crush injury of optic nerve. Crush injury of the optic
nerve was done as described51. Rats were deeply anesthetized
by intraperitoneal injection of xylazine, (10 mg/kg; Vitamed, Bat−Yam,
Israel) and ketamine (50 mg/kg; Fort Dodge Laboratories, Fort Dodge, Iowa).
Using a binocular operating microscope, a lateral canthotomy was done in the
right eye and the conjunctiva was incised lateral to the cornea. After separation
of the refractor bulbi muscles, the optic nerve was exposed intraorbitally
by blunt dissection. Using calibrated cross−action forceps, the optic
nerve was subjected to a moderate crush injury 1−2 mm from the eye.
The uninjured contralateral nerve was left undisturbed.
Immunocytochemistry of T cells. Longitudinal cryosections
of the excised nerves (20 m in thickness) were picked up onto gelatin−coated
glass slides and frozen until preparation for fluorescence staining. Sections
were fixed in ethanol for 10 min at room temperature, washed twice in double−distilled
water and incubated for 3 min in PBS containing 0.05% polyoxyethylene−sorbitan
monolaurate (Tween−20). Sections were then incubated for 1 h at room
temperature with mouse monoclonal antibody to rat T−cell receptor52 (donated by B. Reizis) diluted in PBS containing 3% FCS and 2%
bovine serum albumin. The sections were washed three times with PBS containing
0.05% Tween−20 and incubated with fluorescein isothiocyanate−conjugated
goat anti−mouse IgG (with minimal cross−reaction to rat, human,
bovine and horse serum proteins)(Jackson ImmunoResearch, West Grove, Pennsylvania),
for 1 h at room temperature. The sections were washed with PBS containing
Tween−20 and treated with glycerol containing 1,4−diazobicyclo−(2,2,2)
octane to inhibit quenching of fluorescence. Sections were viewed with a Zeiss
microscope and cells were counted. Staining in the absence of first antibody
was negative.
Retrograde labeling and measurement of primary damage and secondary
degeneration. Primary damage of the optic nerve axons and their
attached RGCs was measured after the immediate post−injury application
of the fluorescent lipophilic dye 4−(4−(didecylamino)styryl)−n−methylpyridinium
iodide (4−Di−10−Asp) (Molecular Probes Europe BV, Leiden,
Netherland) distal to the site of injury. Only axons that are intact are capable
of transporting the dye back to their cell bodies; therefore, the number of
labeled cell bodies is a measure of the number of axons that survived the
primary damage. Secondary degeneration was also measured by application of
the dye distal to the injury site, but 2 weeks after the primary lesion was
inflicted. Application of the neurotracer dye distal to the site of the primary
crush after 2 weeks ensures that only axons that survived both the primary
damage and the secondary degeneration will be counted. This approach makes
it possible to differentiate between neurons that are still functionally intact
and neurons in which the axons are injured but the cell bodies are still viable,
as only those neurons whose fibers are morphologically intact can take up
dye applied distally to the site of injury and transport it to their cell
bodies. Using this method, the number of labeled ganglion cells reliably reflects
the number of still−functioning neurons. Labeling and measurement were
done by exposing the right optic nerve for a second time, again without damaging
the retinal blood supply. Complete axotomy was done 1−2 mm from the
distal border of the injury site and solid crystals (0.2−0.4 mm in diameter)
of 4−Di−10−Asp were deposited at the site of the newly formed
axotomy. Uninjured optic nerves were similarly labeled at approximately the
same distance from the globe. Five days after dye application, the rats were
killed. The retina was detached from the eye, prepared as a flattened whole
mount in 4% paraformaldehyde solution and examined for labeled ganglion cells
by fluorescence microscopy. The percentage of RGCs surviving secondary degeneration
was calculated using the following formula: (Number of spared neurons after
secondary degeneration)/(Number of spared neurons after primary damage)
100.
Electrophysiological recordings. Nerves were excised
and their CAPs were recorded in vitro using the suction electrode experimental
set−up described53. At different times after injury and
injection of T cells or PBS, rats were killed by intraperitoneal injection
of pentobarbitone (170 mg/kg)(CTS Chemical Industries, Tel Aviv, Israel).
Both optic nerves were removed while still attached to the optic chiasma,
and were immediately transferred to a vial containing a fresh salt solution
consisting of 126 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4,
26 mM NaHCO3, 2 mM MgSO4, 2 mM CaCl2 and
10 mM D−glucose, aerated with 95% O2 and 5% CO2
at room temperature. After 1 hour, electrophysiological recordings were made.
In the injured nerve, recordings were made in a segment distal to the injury
site. This segment contains axons of viable retinal ganglion cells that have
escaped both primary and secondary damage, as well as the distal stumps of
non−viable retinal ganglion cells that have not yet undergone Wallerian
degeneration. The nerve ends were connected to two suction Ag−AgCl electrodes
immersed in the bathing solution at 37 °C. A stimulating pulse was applied
through the electrode, and the CAP was recorded by the distal electrode. A
stimulator (SD9; Grass Medical Instruments, Quincy, Massachusetts) was used
for supramaximal electrical stimulation at a rate of 1 Hz to ensure stimulation
of all propagating axons in the nerve. The measured signal was transmitted
to a microelectrode AC amplifier (model 1800; A−M Systems, Everett,
Washington). The data were processed using the LabView 2.1.1 data acquisition
and management system (National Instruments, Austin, Texas). For each nerve,
the difference between the peak amplitude and the mean plateau of eight CAPs
was computed and was considered as proportional to the number of propagating
axons in the optic nerve. The experiments were done by experimenters 'blinded'
to sample identity. In each experiment the data were normalized relative to
the mean CAP of the uninjured nerves from PBS−injected rats.
Clinical evaluation of EAE. Animals were scored every
1−2 days according to the following neurological scale: 0, no abnormality;
1, tail atony; 2, hind limb paralysis; 3, paralysis extending to thoracic
spine; 4, front limb paralysis; 5, moribund state.
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Acknowledgments We thank S. Smith and P. Taylor for editorial assistance, and I. Friedmann
for help with graphics. I.R. Cohen is the incumbent of the Mauerberger Chair
in Immunology, the director of the Robert Koch−Minerva Center for Research
in Autoimmune Disease and the director of the Center for the Study of Emerging
Diseases. M.S. holds the Maurice and Ilse Katz Professorial Chair in Neuroimmunology.
107 cells) intraperitoneally into rats immediately
after unilateral optic nerve injury. Control rats were injected intraperitoneally
with phosphate−buffered saline (PBS). Seven days after injury, the optic
nerves were excised, cryosectioned and analyzed immunohistochemically for
the presence of T cells. No T cells could be detected in the uninjured optic
nerves of PBS−injected rats (
s.e.m., counted
in two to three sections of each nerve. Each group contained three to four
rats. The number of T cells was considerably higher in injured nerves of rats
injected with anti−MBP, anti−OVA or anti−p277 T cells; statistical
analysis (one−way ANOVA) showed significant differences between T cell
numbers of injured optic nerves of rats injected with anti−MBP, anti−OVA,
or anti−p277 T cells and injured optic nerves of rats injected with
PBS (P < 0.001); and between injured optic nerves and uninjured
optic nerves of rats injected with anti−MBP, anti−OVA, or anti−p277
T cells (P < 0.001).
) or immediately after optic nerve
crush injury (
), were injected with activated anti−MBP T cells.
EAE was evaluated according to a neurological paralysis scale. Data points
represent means 
g/ml)
in proliferation medium containing Dulbecco's modified Eagle's medium (DMEM)
supplemented with L−glutamine (2 mM), 2−mercaptoethanol (5 
