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Efficient central nervous system remyelination requires T cells.

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Efficient Central Nervous
System Remyelination
Requires T Cells
Allan J. Bieber, PhD,1,2 Scott Kerr, BS,1
and Moses Rodriguez, MD1,2,3
General nonspecific immunosuppression often is
used as a treatment for demyelinating disease and spinal cord injury. A better understanding of the role of
the immune system in CNS injury and repair is essential for the ongoing evaluation and development of
these therapies.
Materials and Methods
Animals
We demonstrate a role for immune functions in the
spontaneous remyelination of central nervous system
(CNS) axons after lysolecithin-induced demyelination in
the spinal cord. Rag-1–deficient mice lack both B cells
and T cells and show significantly reduced spontaneous
remyelination compared with control mice of matching
genetic background. Mice lacking or depleted of either
CD4ⴙ T cells or CD8ⴙ T cells also exhibit reduced remyelination. These data indicate that T cells are necessary for efficient CNS remyelination. Thus, general nonspecific immunosuppression as a therapeutic approach
for the treatment of CNS injury and demyelinating disease may have undesirable effects on subsequent tissue
repair.
Ann Neurol 2003;53:680 – 684
Spontaneous remyelination is a normal physiological
response after myelin damage but the factors that control rate and extent of spontaneous myelin repair after
central nervous system (CNS) demyelination are largely
unknown. Several recent reports have suggested that
various immune cells and immune effector molecules
may play a role in myelin repair.1–5
The aim of this study was to establish a role for immune functions in the repair process by examining the
extent of remyelination in mice with genetic deficiencies in immune function or after antibody depletion of
specific sets of immune cells. CNS demyelination was
induced by injection of lysolecithin into the spinal
cord, and remyelination was assessed 35 days later.
Lysolecithin-induced demyelination occurs independently of immune function and therefore is an excellent system in which to assess the contribution that immune functions make to the spontaneous
remyelination process.
From the 1Department of Neurology, 2Program in Molecular Neuroscience, and 3Department of Immunology, Mayo Medical and
Graduate Schools, Rochester, MN.
Received Dec 16, 2002, and in revised form Feb 10, 2003. Accepted for publication Feb 11, 2003.
Address correspondence to Dr Bieber, Department of Neurology,
Guggenheim 418, Mayo Clinic and Foundation, 200 First St. SW,
Rochester, MN 55905. E-mail: bieber.allan@mayo.edu
680
© 2003 Wiley-Liss, Inc.
C57BL/6J, B6-Rag1tm1Mom, B6-CD4tm1Mak, and B6CD8tm1Mak mice were purchased from the Jackson Laboratories (Bar Harbor, ME) or bred in-house. Mice were housed
in plastic cages with food and water provided ad libitum.
Handling of animals conformed to National Institutes of
Health and Mayo Clinic guidelines.
Antibody Depletion
The hybridoma lines Gk1.5 and Lyt2.43, which secrete function neutralizing rat antibodies to CD4 and CD8, respectively, were obtained from the American Type Culture Collection. Hybridomas were adapted to growth in serum and
protein-free medium (Sigma, St. Louis, MO) supplemented
with 1% fetal calf serum. Antibody was precipitated from
culture supernatant with ammonium sulfate and then purified by gel filtration on a Superose-6 column.
Lysolecithin Injection and Quantification
of Remyelination
Lysolecithin injections were performed on 12-week-old mice
as previously described.6 In brief, after dorsal laminectomy a
micropipette was inserted into the dorsolateral part of the
spinal cord and 2.5␮l of 1% lysolecithin in saline was injected. The needle was withdrawn and the wound was
closed. The day of lysolecithin injection was designated day
0. Thirty-five days after lysolecithin injection, mice were
anesthetized and perfused by intracardiac administration of
Trump’s fixative (phosphate-buffered 4% formaldehyde with
1% glutaraldehyde, pH 7.4). Spinal cords were removed and
cut into 1mm sections, postfixed with osmium, and embedded in araldite plastic (Polysciences, Warrington, PA). Onemicrometer-thick cross-sections were cut from each block
and stained with 4% p-phenylenediamine to visualize myelin.
For each animal, the block showing the largest demyelinated lesion was used for quantitative analysis. Blocks showing obvious signs of tissue damage due to injection were excluded from the analysis. The average density of myelinated
axons is much higher in the dorsal columns of the spinal
cord than in the ventral or lateral columns, and mixing
quantitative remyelination data from dorsal versus ventral/
lateral lesions creates added variability in the data. Therefore,
only ventral and lateral lesions were selected for quantification. Productive lesion formation in the ventral and lateral
tracts after lysolecithin injection was observed in 42 of 72
total mice. The total number of animals and the number of
lesions that were analyzed in each experimental group are
listed in the Table. In most animals, only a single ventral/
lateral lesion was present, but in three animals, two separate
ventral/lateral lesions were observed. For statistical analysis,
when two lesions existed in the same animal, the data for
Table. Central Nervous System Remyelination of Lysolecithin:Induced Lesions in Immunodeficient Mice
Experiment
C57BL/6
B6-Rag1tm1Mom
B6-CD4tm1Mak
B6-CD8tm1Mak
B6-CD4tm1Mak ⫻ B6-CD8tm1Mak
C57BL/6 depleted for CD4 T
cells with Gk 1.5 antibody
C57BL/6 depleted for CD8 T
cells with Lyt 2.43 antibody
No. of No. of
Animals Lesions
7
6
5
5
7
7
10
6
5
5
7
7
5
5
Mean (median) Lesion
Areas (mm2) ⫾ SEM
Mean (median)
Remyelinated Axons/
mm2, ⫾ SEM
Remyelination
vs C57BL/6
(P value)
0.0513 ⫾ 0.0070 (0.0546)
0.0468 ⫾ 0.0052 (0.0446)
0.1030 ⫾ 0.0317 (0.0732)
0.0538 ⫾ 0.0155 (0.0418)
0.0384 ⫾ 0.0046 (0.0377)
0.0495 ⫾ 0.0087 (0.0427)
75,447 ⫾ 8,184 (66,833)
26,538 ⫾ 3,893 (29,408)
14,694 ⫾ 3,131 (15,168)
32,772 ⫾ 7,249 (29,235)
63,455 ⫾ 4,976 (62,845)
32,344 ⫾ 6,816 (28,531)
0.001
0.003
0.005
0.259
0.004
0.0683 ⫾ 0.0287 (0.0431) 28,986 ⫾ 4,319 (26,130)
0.003
See Materials and Methods section for additional details. Pair-wise comparison of remyelination to the C57BL/6 control strain was by Mann–
Whitney rank-sum test. Comparison of mean lesion area between C57BL/6 and B6-Rag1tm1Mom by Mann–Whitney rank-sum test showed no
significant difference ( p ⫽ 0.628). Comparison of mean lesion areas between all experimental groups by Kruskal–Wallis one-way analysis of
variance on ranks also showed no significant differences ( p ⫽ 0.564).
lesion area and quantification of remyelination were combined, and the two lesions were considered as one. All analysis was done on coded samples without knowledge of the
experimental group.
Lesions areas were determined using a Zeiss interactive
digital analysis system (ZIDAS; Zeiss, Thornwood, NY) as
described.7,8 Remyelinated axons were identified by their
thin myelin sheaths (Fig) and counted manually on a photographic montage of each individual lesion. Remyelination
is expressed as the number of remyelinated axons per square
millimeter of lesion (see Table). Pair-wise comparison of remyelination to the C57BL/6 control strain was by Mann–
Whitney rank-sum test. Comparison of mean lesion area between C57BL/6 and B6-Rag1tm1Mom was also by Mann–
Whitney rank-sum test, and comparison of mean lesion areas
for all experimental groups was by Kruskal–Wallis one-way
analysis of variance on ranks.
elination after lysolecithin injection is independent of
immune system function.
Morphometric analysis of spinal cord remyelination
showed significantly greater remyelination in normal
controls compared to Rag-1–deficient mice (see Fig;
Table). Control animals showed an average remyelination of 75,447 axons/mm2, whereas B6-Rag1tm1Mom
mice had 26,538 remyelinated axons/mm2. This difference was statistically significant with p ⫽ 0.001. Allowing 60 days for remyelination to occur after lysolecithin injection did not increase the extent of
remyelination in Rag-1 mice, indicating that the observed effect is a true inhibition rather than a delay in
the remyelination process. These data demonstrate that
some aspect of immune system function is necessary
for efficient remyelination of CNS axons.
Results
Remyelination of Lysolecithin-Induced Demyelination
Is Inhibited in B6-Rag1 Mice
To determine whether immune functions play a role in
remyelination after lysolecithin-induced demyelination,
we induced lesions in B6-Rag1tm1Mom mice, which
lack recombination activating gene-1 and therefore
produce no mature B cells or T cells.9 Our previous
experience with this experimental system has shown
that remyelination is well established by 21 days after
lysolecithin injection and complete by 35 days.6,7
Therefore, 35 days after lysolecithin injection, we compared extent of remyelination in B6-Rag1tm1Mom mice
with that in control mice with matching genetic background (C57BL/6).
There were no significant differences in mean lesion
area between any experimental groups in this study ( p
⫽ 0.564, see Table). The mean lesion area in B6Rag1tm1Mom mice was slightly larger than that in
C57BL/6 mice, but this difference was not significant
( p ⫽ 0.628), suggesting that the mechanism of demy-
Remyelination Is Inhibited in Mice Deficient for
Either CD4⫹ or CD8⫹ T Cells
To further explore the role of immune functions in
remyelination, we assessed remyelination in mice deficient for CD4, which is important in MHC class II–
restricted immune responses, or deficient for CD8, important in MHC class I–restricted immune responses.
Remyelination was assessed 35 days after lysolecithin
injection and compared with that in control mice with
matching genetic background (C57BL/6).
Morphometric analysis of remyelination in CD4 and
CD8-deficient mice showed that absence of either
branch of immune function resulted in significantly reduced remyelination (see Fig, Table). B6-CD4tm1Mak
mice had an average of 14,694 remyelinated axons/
mm2, whereas B6-CD8tm1Mak mice had an average of
32,772 remyelinated axons/mm2. These numbers were
significantly different from those of C57BL/6 control
animals ( p ⫽ 0.003 for CD4, p ⫽ 0.005 for CD8).
Reduced remyelination in both CD4 and CD8-
Bieber et al: Remyelination Requires T Cells
681
682
Annals of Neurology
Vol 53
No 5
May 2003
deficient strains was somewhat unexpected. Both
strains were created in the same laboratory; therefore,
we were concerned that an unknown second site mutation that affects remyelination might have existed in
the parent strain. To test this possibility, we crossed
the B6-CD4tm1Mak and B6-CD8tm1Mak strains and assessed remyelination in F1 progeny. These animals
demonstrated an average of 63,455 remyelinated axons/mm2, which was statistically similar to that observed in the C57BL/6 control group ( p ⫽ 0.259).
Antibody-Mediated Depletion of CD4⫹ and CD8⫹
Cells Inhibits Remyelination
Mutations often have pleiotropic effects, and we were
concerned that CD4 and CD8 mutations might have
unknown effects on CNS development that result in
an inability to efficiently remyelinate in adult animals.
To address this concern, we used antibodies to deplete
CD4⫹ or CD8⫹ T cells in adult C57BL/6 mice and
then assessed remyelination.
Mice were injected on three successive days with
0.5mg of either Gk1.5 (anti-CD4) or Lyt2.43 (antiCD8). One week after injection, the peripheral blood
of all animals was assessed for the presence of CD4⫹
and CD8⫹ cells by fluorescence-activated cell sorting.
Average depletion for CD4⫹ cells was 98.53 ⫾ 0.41%
compared with untreated C57BL/6 controls, and
95.78 ⫾ 0.50% for CD8⫹ cells. By 35 days after lysolecithin injection when the animals were prepared for
histological examination, the CD4⫹ or CD8⫹ T-cell
populations in peripheral blood generally had recovered to only 20% of their normal levels.
Mice depleted of either CD4⫹ or CD8⫹ cells displayed levels of remyelination similar to those seem in
animals with a genetic deletion (see Fig, Table). Animals depleted for CD4⫹ cells averaged 32,344 remyelinated axons/mm2, and those depleted of CD8⫹ cells
averaged 28,986 remyelinated axons/mm2. These data
demonstrate that both the CD4 and CD8 branches of
the immune system make independent contributions to
CNS remyelination.
Discussion
The immune response in the adult mouse spinal cord
after lysolecithin injection has been characterized,10,11
and our own immunostaining experiments are consistent with the published observations. Lysolecithin in-
Š
jection induces a rapid but transient influx of T cells
and neutrophils into the CNS. Infiltration and activation of macrophages and microglia begins within hours
after injury but persists for many days. The role that
these cell types play in establishing an environment in
which remyelination can occur is unknown.
A neuroprotective role for T cells after CNS injury has
been suggested.2,12 Systemic injection of myelin-reactive
T cells after spinal cord injury results in enhanced accumulation of T cells, B cells, and macrophages at the site of
injury.13 This T-cell response promotes the expression of
various neurotrophins by macrophages and astrocytes that
may play a role in promoting neuronal survival. Several
growth factors have been identified that show increased
levels of expression during remyelination,14 and it is possible that T cells play a similar role in supporting oligodendrocyte remyelination, either directly or by stimulating
the activity of CNS glia.
Depletion of macrophages impairs oligodendrocyte
remyelination, suggesting that these cells also may be
important for support of the myelin repair process.4
Immunochemical staining of any of the immunodeficient mice used in this study showed normal accumulations of CD45⫹ and CD11b⫹ macrophages at lesion
sites, and the number and morphology of these cells
was not different after remyelination. Whether these
cells were expressing the full array of normal functions
is not clear.
Thus, we demonstrate that the immune system does
provide functions necessary for remyelination and that
CD4⫹ and CD8⫹ T cells are both required for efficient CNS remyelination to occur.
This work was supported by grants from the NIH (NS24180, M.R.,
A.B. and NS40209, M.R.), the National Multiple Sclerosis Society
(RG 3172-A-6, M.R., A.B). We thank Eugene and Marcia Applebaum for their generous support to M.R.
We thank Dr P. C. O’Brien for assistance with the statistical analysis.
References
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Fig. Remyelination of lysolecithin-induced lesions in control and immunodeficient mice and in mice depleted for CD4⫹ or CD8⫹
T cells. C57BL/6 normal white matter is shown in A. Panels B to H show remyelination in lysolecithin lesions: C57BL/6 (B), B6Rag1tm1Mom (C), B6-CD4tm1Mak (D), B6-CD8tm1Mak (E), B6-CD4tm1Mak ⫻ B6-CD8tm1Mak (F), C57BL/6 depleted of CD4⫹ T
cells (G), and C57BL/6 depleted of CD8⫹ T cells (H). Remyelinated axons are identified by their relatively thin myelin sheaths
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decrease in remyelination of approximately two- to fourfold can be seen in the lesions of immunodeficient mice (C–E, G, and H) as
compared with the control animals (B, F).
Bieber et al: Remyelination Requires T Cells
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