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Department of Neurobiology, The Weizmann Institute of Science, Rehovot, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We were interested in investigating whether the T cells recruited in the specific environments of different injury sites for the purpose of coping with the local stressful situation have the same or different antigenic specificities. Our previous work indicated that although passive transfer of anti-myelin autoimmune T cells (11) or vaccination with myelin Ags (16) can protect retinal ganglion cells (RGCs)3 after an insult to the optic nerve axons, these procedures are not protective after a direct insult to the RGCs themselves (17). This finding led us to consider the possibility that each tissue has its own specific self-Ags that signal the immune system when the tissue needs help. In the case of axotomy, since the Ags that send signals summoning the immune system to the aid of the stressed neurons are myelin proteins associated not with neurons but with oligodendrocytes, we considered the possibility that the relevant Ags are not necessarily expressed on the cells that require assistance but on other cells in the vicinity. In addition, if an autoimmune disease is indeed the outcome of failure to control an autoimmune response whose original purpose was beneficial, it seems reasonable to postulate that the protection (beneficial response) and the disease (destructive response) share the same antigenic specificity.
Experimental autoimmune uveoretinitis (EAU) is an experimental model for uveitis, a T cell-mediated autoimmune disease of the eye (18, 19, 20, 21). In the present study we tested our working hypothesis (namely, that the protective and the destructive autoimmune response share the same antigenic specificity) by investigating whether a pathogenic, uveitis-related, retinal self-Ag can protect against direct and indirect insults to the RGCs. The results showed that RGCs exposed to a glutamate insult or suffering the secondary consequences of an optic nerve crush injury could be protected by vaccination with a uveitis-associated peptide. In the case of glutamate insult, protection was observed under conditions where no such protective effect could be obtained by vaccination with myelin Ags.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adult male Sprague Dawley (SPD), Fisher (F344), and Lewis rats (8–12 wk old) and adult female Lewis rats (16–18 wk old) were supplied by the Animal Breeding Center of the Weizmann Institute of Science under germfree conditions. The rats were housed in a light- and temperature-controlled room and were matched for age in each species for each experiment. Animals were handled according to the regulations formulated by the institutional animal care and use committee.
Antigens
The peptides R16 (sequence 1177–1191 of bovine interphotoreceptor retinoid-binding protein (IRBP), ADGSSWEGVGVVPDV), G-8 (sequence 347–354 of human retinal soluble Ag (S-Ag), TSSEVATE), a G-8 analog (TSSEAATE), M-8 (sequence 307–314 of human retinal S-Ag, DTNLASST), and an M-8 analog (DTALASST) were prepared in the Synthesis Unit at The Weizmann Institute. G-8 and M-8 are uveitogenic, while their analogs are immunogenic, but not immunopathogenic (22).
Partial crush injury of the rat optic nerve
The optic nerve was subjected to a well-calibrated crush injury as previously described (3). Briefly, rats were deeply anesthetized by i.p. injection of Rompun (xylazine, 10 mg/kg; VMD, Arendonk, Belgium) and Vetalar (ketamine, 50 mg/kg; Fort Dodge Laboratories, Fort Dodge, IA). Using a binocular operating microscope, lateral canthotomy was performed in the right eye, and the conjunctiva was incised laterally to the cornea. After separation of the retractor bulbi muscles, the optic nerve was exposed intraorbitally by blunt dissection. Using calibrated cross-action forceps, the optic nerve was subjected to a severe crush injury 1–2 mm from the eye. The contralateral nerve was left undisturbed.
Glutamate injection
The right eye of the anesthetized rat was punctured with a 27-gauge needle through the conjunctiva and sclera, anterior to the pars plana so that the retina was untouched, and a 10-µl Hamilton syringe (Reno, NV) with a 30-gauge needle was inserted as far as the vitreal body. Rats were injected with 2 µl (400 nmol) of L-glutamate.
Active immunization
Rats were subjected to optic nerve crush injury and then immediately immunized by s.c. injection at the base of the tail of R16 (30 µg), G-8, G-8 analog, M-8, or M-8 analog (200 or 500 µg) emulsified in CFA supplemented with 2.5 mg/ml Mycobacterium tuberculosis (Difco, Detroit, MI) in a total volume of 0.1 ml. Rats in another group were exposed to a glutamate insult (by intravitreal glutamate injection) and then immediately immunized s.c. at the base of the tail with 30 µg of R16 emulsified in CFA supplemented with 2.5 or 0.5 mg/ml of M. tuberculosis in a total volume of 0.1 ml. Control rats were injected with PBS in CFA. In another set of experiments rats were actively immunized with 30 µg of R16 emulsified in CFA supplemented with 2.5 mg/ml M. tuberculosis 1 wk before the crush injury and given a booster of 30 µg of R16 emulsified in IFA (Difco) immediately after the injury. Control rats were injected with PBS in CFA and boosted with PBS in IFA.
Passive immunization
Male Lewis rats were unilaterally injected in the hind footpads with 30 µg of R16 emulsified in CFA supplemented with 2.5 mg/ml of M. tuberculosis in a total volume of 0.1 ml. Seven days after immunization, spleens from immunized and naive rats were removed and pooled in ice-cold PBS. A single-cell suspension was prepared, and the cells (2 x 106 cells/ml) were cultured with naive thymocytes (2 x 106 cells/ml) in the presence of R16 (20 µg/ml) in proliferation medium containing DMEM supplemented with 2 mM glutamine, 2-ME (5 x 10-5M), sodium pyruvate (1 mM), nonessential amino acids (1 ml/100 ml), 1% fresh autologous rat serum, 100 U/ml of penicillin, and 0.1 mg/ml of streptomycin. After incubation for 72 h the cultures were collected and washed, and the lymphoblasts (1.3 x 107 cells in 3 ml PBS) or PBS alone (3 ml) were injected i.p. into Lewis rats immediately after optic nerve crush injury.
Assessment of secondary degeneration in the rat optic nerve by retrograde labeling of retinal ganglion cells
Secondary degeneration of optic nerve axons and their corresponding RGCs was evaluated after application, 2 wk after injury, of the fluorescent lipophilic dye 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide (4-Di-10-Asp; Molecular Probes Europe, Leiden, The Netherlands), distal to the lesion site. The right optic nerve was exposed for the second time, again without damaging the retinal blood supply. Complete axotomy was performed 1–2 mm distal to 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 new axotomy. Five days after dye application the rats were killed. Retinas were detached from the eyes, prepared as flattened whole mounts in 4% paraformaldehyde solution, and examined for labeled RGCs by fluorescence microscopy and confocal microscopy. Since only intact axons can transport the dye back to their cell bodies, application of the dye distal to the lesion site 2 wk after injury ensures that only axons that survived both primary damage and secondary degeneration will be counted. This approach enables us to differentiate between neurons that are still functional and neurons in which the axons are injured but the cell bodies are still viable.
Spinal cord contusion
Female Lewis rats were anesthetized by i.p. injection of Rompun and Vetalar, and their spinal cords were exposed by laminectomy at the level of T8. One hour after induction of anesthesia, a 10-g rod was dropped onto the laminectomized cord from a height of 50 mm using the NYU impactor, a device shown to inflict a well-calibrated contusive injury of the spinal cord (10, 23, 24, 25).
Active immunization
Rats were immunized s.c. on a random basis with 100 µg of R16 or injected with PBS, each emulsified in CFA supplemented with 0.5 mg/ml M. tuberculosis, in a total volume of 0.1 ml. Rats were immunized within 1 h after contusion.
Animal care
In contused rats bladder expression was assisted by massage at least twice a day (particularly during the first 48 h after injury, when it was performed three times a day) throughout the experiment. All rats were carefully monitored for evidence of urinary tract infection or any other sign of systemic disease. During the first week after contusion and in any case of hematuria after that period, they received a course of sulfamethoxazole (400 mg/ml) and trimethoprim (8 mg/ml; Resprim; Teva Pharmaceutical Industries, Ashdod, Israel), administered orally with a tuberculin syringe (0.3 ml solution/day). Daily inspections included examination of the laminectomy site for evidence of infection and assessment of the hind limbs for signs of autophagia or pressure.
Assessment of recovery from spinal cord contusion
Behavioral recovery was scored in an open field using the locomotor rating scale of Basso, Beattie, and Bresnahan, where a score of 0 registers complete paralysis and a score of 21 indicates complete mobility (23, 26, 27). Blind scoring ensured that observers were not aware of the treatment received by individual rats. Approximately once a week the locomotor activities of the trunk, tail, and hind limbs were evaluated in an open field by placing each rat for 4 min in the center of a circular enclosure (90 cm in diameter, 7-cm wall height) made of molded plastic with a smooth, non-slip floor. Before each evaluation the rats were examined carefully for perineal infection, wounds in the hind limbs, and tail and foot autophagia (24, 28).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test our working hypothesis, we first investigated whether RGCs
can be protected by vaccination with a self-peptide associated with
uveitis, an autoimmune disease affecting the eye. The peptide selected
for this experiment was R16 (29, 30), an immunodominant
sequence within IRBP known to cause uveitis. First we examined whether
vaccination with R16 could protect the RGCs of Lewis rats (a strain
susceptible to autoimmune disease induction) from glutamate toxicity
under conditions where immunization with myelin peptides is not
effective (17). Vaccination of Lewis rats with R16 after a
glutamate insult indeed resulted in a reduced loss of RGCs (Fig. 1
). Relative to normal retinas, the
percentage of RGC loss (mean ± SEM) was 14 ± 2% in rats
vaccinated with R16 emulsified in CFA compared with 28 ± 4% in
rats treated with PBS in CFA (p < 0.04). This finding
substantiates our contention that immune protection requires the
activity of T cells specific to Ag present within the injured tissue.
Since R16 is known to cause uveitis in Lewis rats (but not in SPD or
Fisher rats), it was interesting to discover that the RGCs received
protection despite massive infiltration of lymphocytes into the eyes of
these rats. It is important to mention, however, that the
neuroprotective effect of R16 in this model was detected only when the
disease in these rats was mild (i.e., when the amount of bacteria in
the adjuvant was 0.5 mg/ml). Immunization of glutamate-injected rats
with R16 emulsified in CFA at a concentration of 2.5 mg/ml was not
protective and even caused additional neuronal loss compared with that
seen in rats immunized with PBS in CFA (data not shown). These findings
constitute further evidence of the delicate balance between the
processes of destruction and protection attributable to these specific
autoimmune T cells.
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Next we examined the effectiveness of R16 vaccination in
protecting RGCs from secondary degeneration after optic nerve crush, an
insult known to trigger secondary degeneration in the cell bodies or
axons of neurons that escaped direct injury (31). This
examination was conducted in the two resistant rat strains (SPD and
Fisher) and in the susceptible strain (Lewis). In all three strains
vaccination with R16 emulsified in CFA (with the high bacterial content
of 2.5 mg/ml) on the day of injury significantly reduced the
injury-induced loss of RGCs (Figs. 2 and 3). In SPD rats the number of surviving RGCs per square millimeter
(mean ± SEM) was 150 ± 13 in rats immunized with R16 in CFA
and 60 ± 14 in rats injected with PBS in CFA (p
< 0.01; Fig. 2). The corresponding results were 183 ± 16 and
114 ± 9, respectively, in Fisher rats (p < 0.01;
Fig. 2), and 192 ± 8 and 73 ± 10, respectively, in Lewis
rats (p < 0.0001; Fig. 3, A–C). Based on the
antigenic specificity found in the case of glutamate toxicity, we
attributed the dramatic protection of RGCs observed after R16
vaccination in the crush model to protection by T cells that had
migrated to the retina and become activated there, rather than to
protection adjacent to the lesion site.
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D).
It is interesting to note that the clinical onset of EAU in Lewis rats
occurred on day 10 after immunization, and inflammation peaked on day
14. Thus, at the time of assessment of neuronal survival in the crush
injury model the EAU disease in Lewis rats was still severe. We were
therefore interested in knowing whether under such conditions
immunization by itself would have an adverse effect on RGC survival in
Lewis rats despite the overall protection. We therefore examined
whether R16 immunization in the absence of insult causes any RGC loss
in Lewis rats. The percentage of RGC survival (mean ± SEM) was
significantly lower in the R16-immunized Lewis rats than in their
matched PBS-injected controls (83 ± 5%; p =
0.02, by one-tailed t test). Immunization with R16 did not
affect RGC survival in Fisher rats (97 ± 8% survival). Thus,
some loss of RGCs was evident 2 wk after R16 vaccination in Lewis, but
not in Fisher rats, suggesting that uncontrolled autoimmunity leading
to autoimmune disease can indeed be destructive in a susceptible
strain, but that even in this strain the beneficial effect of
autoimmunity on neuronal survival exceeds its destructive effect, so
that the net outcome is favorable. In resistant rats controlled
autoimmunity allows the beneficial effect of autoimmunity to be
expressed under a wider range of conditions. Support for this
suggestion comes from the finding that in the resistant Fisher rats,
unlike in the susceptible Lewis rats, vaccination 1 wk before injury
resulted in significant protection (Fig. 4).
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To gain further support for the idea that the protective response
is Ag specific we used two additional uveitogenic epitopes (G-8 and
M-8) of another retinal autoantigen, S-Ag, and their immunogenic (but
not immunopathogenic) analogs. As with R16, vaccination with the
uveitogenic peptides G-8 and M-8 or their immunogenic analogs
immediately after optic nerve crush injury resulted in a significant
increase in RGC survival in Fisher rats. The numbers of surviving RGCs
per square millimeter (mean ± SEM) were 159 ± 5, 153
± 10, and 159 ± 19 in rats immunized with 200 µg G-8, M-8, or
M-8 analog in CFA and 109 ± 12 in rats injected with PBS in CFA
(p < 0.01, p < 0.03, and
p < 0.04, respectively; Fig. 5A). In the case of the G-8
analog, immunization with 500 µg (but not with 200 µg) of the
peptide resulted a significant increase in RGC survival compared with
that in rats injected with PBS in CFA (175 ± 15 and 90 ±
11, respectively; p < 0.01; Fig. 5B).
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The above results suggest that when a neuronal insult affects the
retinal cell bodies directly, immune neuroprotection is restricted to
Ags expressed within the retina. This suggests that vaccination with
R16 should not protect against injury to the spinal cord, for example,
even though spinal cord tissue can benefit from autoimmunity directed
to myelin Ags. To examine whether R16 can protect against incomplete
spinal cord injury, we subjected Lewis rats to severe spinal cord
contusion and then either vaccinated them with R16 in CFA
or injected them with PBS in CFA. Recovery was
assessed by experimenters who were blinded to the treatment received.
At no time were any differences observed in the recovery of motor
activity by the two groups (Fig. 6).
Under the same experimental conditions in this model, vaccination with
a pathogenic peptide derived from myelin basic protein led to better
recovery than that seen in nonvaccinated rats (10, 24, 32).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our research group recently demonstrated that after injury to myelinated CNS axons (optic nerve or spinal cord), passive transfer of T cells reactive to myelin-associated self-Ags reduces the injury-induced loss of tissue (9, 10, 11, 35). A substantial body of evidence indicates that any fibers that were directly injured will inevitably degenerate, but that neighboring neurons that escaped the primary injury can be protected (via immune system activity) from the toxicity of injury-evoked self-destructive compounds. On the basis of the experimental evidence, our group suggested that the protective immune response to injury of myelinated axons is manifested in the activity of Th1 cells directed against myelin-associated Ags (16). Moreover, the injury itself initiates a signal to the immune system, recruiting its activity at the injury site to help the tissue cope with the stressful conditions (13, 15). The ability to spontaneously manifest an autoimmune response with a beneficial outcome was found to be correlated with the ability to resist autoimmune disease development (13). It was found in our laboratory that the ability to spontaneously protect neuronal tissue does not correspond to a lack of autoimmunity, but to the operation of an autoimmune response whose times of onset, duration, and intensity, are tightly controlled (I. Shaked, Z. Porat, J. Kipnis, and M. Schwartz, unpublished observations).
The results of this study show that the self-Ag associated with uveitis protects RGCs from both glutamate toxicity and death induced as a consequence of axonal injury. This protective potential is not restricted to the R16 peptide, as two uveitogenic peptides derived from another retinal Ag, S-Ag, exerted a similar protective effect in the rat optic nerve injury model. In addition, analogs of those peptides, designed to evoke an immune response without causing disease, enhanced RGC survival after optic nerve injury, suggesting that retinal Ags can be used to protect RGCs without the risk of developing autoimmune disease. It is important to emphasize that the protection is Ag specific, as the protection of RGCs from death caused by a direct insult (such as glutamate toxicity) is conferred by vaccination with R16, but not with myelin Ags, while the opposite is true for injury to the spinal cord. In the case of injury to the optic nerve, however, vaccination with either myelin Ags (16, 36) or retinal Ags improved RGC survival, presumably by attenuating secondary degeneration at the injury site or in the retina, respectively (37).
Also similar to the case of myelin Ags was the finding that individuals
with a limited ability to regulate the autoimmune response are often
unable to benefit from the immune response to this peptide if the
disease is too severe (24, 36). Protection is obtained,
however, if the intensity of autoimmunity is reduced,
resulting in a milder disease. Resistant strains benefit from the
evoked autoimmune response under a wide range of experimental
conditions. An interesting finding of this study was that EAU, an
autoimmune disease that affects both the anterior and posterior parts
of the eye, can cause loss of RGCs. This loss, however, is minor when
weighed against the potential benefit of the autoimmune response. Loss
of RGCs recorded when the disease resolved itself showed that the
maximal loss measured 2 wk after vaccination in noninjured Lewis rats
was 
17%, whereas the maximal benefit after a neuronal insult was as
high as 263% (192 ± 8 surviving RGCs/mm2
in rats immunized with R16 compared with 73 ± 10 in rats injected
with PBS). These results show that even if the autoimmune response to
the uveitogenic Ag causes some loss of RGCs, this cost is outweighed by
the benefit that the neurons derive under injurious conditions.
In our view any tissue uses certain safeguards in its front line of self-defense. We suggest that the Ag that operates evokes an immune response that, in the event of malfunction, induces disease, but not necessarily in the cells that conveyed the stress signal. It thus appears that the tissue endangers some cells for the purpose of saving others. The cells at risk by the disease are neither the RGCs in uveitis nor the myelinated CNS neurons in EAE. Nevertheless, in the absence of appropriate regulation, the intensive autoimmune response against myelin Ags in EAE or against IRBP in uveitis, might eventually lead to neuronal loss as well. We showed here that an anti-IRBP response in uninjured Lewis rats can indeed lead to some RGC loss.
Studies in vitro have shown that activated T cells secrete neurotropins
(NTs) such as nerve growth factor, brain-derived neurotropic factor,
NT-3, and NT-4/5, suggesting that the neuroprotective effect of
autoimmune T cells requires the secretion of these neuronal
survival-promoting factors upon Ag-specific reactivation of the T cells
at the injury site (38). Other studies have demonstrated
in vivo up-regulation of the costimulatory molecule B7-2 on
microglia/macrophages and T cells after spinal cord injury in rats
treated with T cells reactive to myelin basic protein and suggest that
such T cells help prevent cyst formation after spinal cord injury
(35). Among the T cells that home to the lesion site, the
ones encountering their relevant Ag will be activated. As a consequence
they will produce a high level of cytokines. There is evidence to
indicate that IFN-
, being a product of Th1 (the phenotype of the
protective T cells) affects glutamate uptake by astrocytes (39, 40). In addition, the T cells and their secreted cytokines can
amplify the microglial activity of glutamate uptake (I. Shaked, O.
Butovsky, R. Gersner, and M. Schwartz, unpublished observations)
and remove the threat as rapidly and as effectively as possible, even
at the cost of eliminating some healthy cells (U. Nevo, J. Kipnis, I.
Golding, I. Shaked, A. Newmann, S. Akselrod, and M. Schwartz,
unpublished observations). Thus, autoimmune T cells may exert their
protective effect directly on neurons (41), indirectly
(for example, by affecting the state of activation of
microglia/macrophages), or both. Further studies are needed to
determine whether harnessing of autoimmune T cells as part of a
self-defense mechanism also occurs in non-neural tissues and in tissues
that, unlike the eye or the CNS, are not immune-privileged.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michal Schwartz, Department of Neurobiology, The Weizmann Institute of Science, 76100 Rehovot, Israel. E-mail address: michal.schwartz{at}weizmann.ac.il
3 Abbreviations used in this paper: RGC, retinal ganglion cell; 4-Di-10-Asp, 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide; EAU, experimental autoimmune uveoretinitis; IRBP, interphotoreceptor retinoid-binding protein; NT, neurotropin; SPD, Sprague Dawley; S-Ag, soluble Ag.
Received for publication May 9, 2002. Accepted for publication September 3, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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