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The Journal of Neuroscience, July 1, 2001, 21(13):4649-4656
MIP-1 , MCP-1, GM-CSF, and TNF- Control the Immune Cell
Response That Mediates Rapid Phagocytosis of Myelin from the Adult
Mouse Spinal Cord
Shalina S.
Ousman and
Samuel
David
Centre for Research in Neuroscience, Montreal General Hospital
Research Institute and McGill University, Montréal, Québec,
Canada, H3G 1A4
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ABSTRACT |
The slow immune response in the adult mammalian CNS results
in slow myelin phagocytosis along degenerating white matter after injury. This has important consequences for axon regeneration because
of the presence of axon growth inhibitors in myelin. In addition,
abnormal immune cell responses in the CNS lead to demyelinating disease. Lysophosphatidylcholine (LPC) can induce an inflammatory response in the CNS, producing rapid demyelination without much damage
to adjacent cells. In this study, we searched for the molecular switches that turn on this immune cell response. Using reverse transcription PCR analysis, we show that mRNA expression of macrophage inflammatory protein-1 (MIP-1), macrophage chemotactic protein-1 (MCP-1), granulocyte macrophage-colony stimulating factor (GM-CSF), and
tumor necrosis factor- (TNF-) in the spinal cord is
rapidly and transiently upregulated after intraspinal injection of LPC. Neutralizing these signaling molecules with function-blocking antibodies suppresses recruitment of T-cells, neutrophils, and monocytes into the spinal cord, as well as significantly reduces the
number of phagocytic macrophages and the demyelination induced by LPC.
These findings will have important implications for CNS regeneration
and demyelinating disease.
Key words:
lysophosphatidylcholine; myelin; cytokine; chemokine; macrophage; T-cells
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INTRODUCTION |
The immune cell response in the
adult mammalian CNS generally occurs at a slower rate than in non-CNS
tissues (Perry et al., 1987 ; Stoll et al., 1989a,b). This inadequate
immune reaction underlies the slow removal of myelin and axonal debris
distal to the site of injury during Wallerian degeneration in the CNS. Wallerian degeneration takes several weeks to months to complete in the
CNS (Bignami and Ralston, 1969; Perry et al., 1987; George and Griffin,
1994) but occurs within 7-14 d in injured peripheral nerves (Griffin
et al., 1992; George and Griffin, 1994). Slow myelin clearance has
important implications for axon regeneration in the injured CNS because
of the presence of axon growth inhibitors in myelin (David, 1998;
Bandtlow and Schwab, 2000). The robust regeneration of peripheral
nerves, despite the presence of inhibitors in peripheral nerve myelin
(Bahr and Przyrembel, 1995; David et al., 1995; Shen et al., 1998), is
likely attributable to the rapid clearance of myelin debris by
immune cells after injury (Beuche and Friede, 1984; Griffin et al.,
1992; George and Griffin, 1994). However, a rapid immune cell response
leading to myelin phagocytosis can be provoked in the CNS under certain
experimental conditions, such as after lysophosphatidylcholine (LPC)
injections (Ousman and David, 2000).
LPC triggers a rapid and highly reproducible form of demyelination in
the CNS without producing much damage to adjacent cells and axons
(Hall, 1972; Jeffrey and Blakemore, 1993). It is therefore an ideal
model to study the control of the immune response underlying rapid
myelin phagocytosis. We have shown previously that LPC induces rapid
macrophage recruitment (6-12 hr) and activation in the adult mouse
spinal cord (Ousman and David, 2000). These macrophage responses were
preceded and accompanied by recruitment of T-cells and neutrophils. Furthermore, the rapid activation of macrophages to the phagocytic state led to the removal of myelin debris within 4 d of LPC
injection into the spinal cord. We have now performed experiments to
identify the molecular triggers, i.e., chemokines and cytokines, that
signal these immune cell responses.
Chemokines mediate chemotaxis, extravasation, and activation of
leukocytes (Asensio and Campbell, 1999). These molecules induce leukocytes to migrate along concentration gradients and are cell type-selective chemoattractants, e.g., macrophage inflammatory protein-1 (MIP-1) and macrophage chemotactic protein-1 (MCP-1) promote chemotaxis of monocytes and T-cells (Karpus and Ransohoff, 1998). In addition to chemokines, an immune response represents the
differential actions of multiple cytokines. Besides immune cells,
astrocytes and microglia are capable of producing many proinflammatory
[e.g., interleukin-1 (IL-1) and tumor necrosis factor-
(TNF-)], immunosuppressive [e.g., IL-10 and transforming growth
factor- (TGF-)], and hematopoetic [e.g., macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-colony
stimulating factor (GM-CSF)] (Campbell, 1998) cytokines. Regulation of
cytokine and chemokine expression is complex, and understanding their
interactions that ensue in an immune response is important,
particularly in terms of enhancing myelin clearance during Wallerian
degeneration in the injured CNS.
In this study, the expression and role of 11 chemokines and cytokines
was examined after intraspinal injections of LPC. We provide evidence
for the involvement of MIP-1, MCP-1, GM-CSF, and TNF- in the
rapid recruitment of immune cells, activation of macrophages, and
clearance of myelin from the adult mouse spinal cord.
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MATERIALS AND METHODS |
Microinjections and tissue preparation for
reverse transcription-PCR
Female BALB/c mice (8-12 weeks old) were deeply
anesthetized with ketamine-xylazine (150 and 10 mg/kg,
respectively), and spinal cord segments T12-L1 were exposed.
One microliter of LPC (L1381; Sigma, St. Louis, MO) at a concentration
of 2 µg/µl or 1 µl of sterile PBS was injected into the left half
of the spinal cord immediately lateral to the midline dorsal artery
using a 50- to 75-µm-diameter-tipped glass micropipette. Animals were killed after survival times of 0.5, 1, 3, 6, 12, 24, 48, and 96 hr by decapitation. A 5-mm-long portion of the spinal cord
containing the injection site was immediately removed and placed in 1 ml of TRIZOL reagent (Life Technologies, Frederick, MD) at
4°C. For each experiment at every time point, three mice were
injected with either LPC or PBS. Experiments were repeated with another set of mice. The tissues from each time point were pooled, and RNA was
isolated according to the protocol of the manufacturer (Life Technologies).
Reverse transcription-PCR and Southern blotting
The appropriate 5' and 3' primers for PCR and the internal
probes for Southern blotting were designed using Gene Runner,
and the complete cDNA sequences were obtained from the NIH GenBank Entrez program. For reverse transcription, 1 µg/µl RNA from each sample was transcribed at 37°C using murine Moloney leukemia virus reverse transcriptase. For PCR, 10 µl of cDNA from each sample was
amplified for various cytokines and chemokines using ampliTaq polymerase (N801-0060; PerkinElmer Life Sciences, Branchburg, NJ). The
appropriate conditions [e.g., annealing temperatures (MgCl2)] for each cytokine and chemokine were
first established (Table 1) using a
positive control consisting of lung tissue of BALB/c mice infected with
Pseudomonas or spleen from malaria-infected mice before
their expression in the experimental samples was determined. PCR for
each cytokine and chemokine, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was performed within the linear range of amplification. GAPDH was amplified at 25 cycles and the chemokines and
cytokines at 30 cycles. For Southern blotting, internal probes were
labeled with [32P]dCTP and hybridized
with the appropriate cDNA oligonucleotide for 18 hr at 42°C. The
results are expressed as a proportion of the optical density of GAPDH
as scanned from the autoradiographs after Southern blotting. For each
experiment, reverse transcription (RT)-PCR was performed two to three
times with the same RNA sample.
Neutralizing antibody experiments
Microinjection and tissue preparation. Female BALB/c
mice were anesthetized as described above. On the basis of the RT-PCR results, neutralizing antibodies against MCP-1 (18240D, hamster monoclonal; PharMingen, San Diego, CA), MIP-1 (AB-450-NA, goat polyclonal; R & D Systems, Minneapolis, MN), GM-CSF (1723-01, rat
monoclonal; Genzyme, Cambridge, MA), and TNF- (IP-400, rabbit polyclonal; Genzyme) were used for microinjections into the spinal cord. One microliter of a cocktail containing LPC (2 µg/µl) and the
neutralizing antibodies individually or together (0.4 µg/µl each)
was injected into the left side of the mouse cord between T12 and L1.
Control animals received a 1 µl injection containing LPC (2 µg/µl) and the appropriate species and isotype-specific control
Ig: hamster IgG (HM00; Cedarlane, Burlingame, CA), rat IgG
(sc-2026; Santa Cruz Biotechnology, Santa Cruz, CA), goat IgG (sc-2028;
Santa Cruz Biotechnology), and rabbit IgG (sc-2027; Santa Cruz
Biotechnology). Six hours and 4 d after injection, the mice were
killed by perfusion with 0.1 M phosphate buffer, followed by 4% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.5. Longitudinal cryostat sections of the spinal
cord containing the injection site were used for immunohistochemistry.
Immunohistochemistry. This was performed to detect the
following cell types: monocytes and microglia (rat monoclonal
antibody, Mac-1); CD4+ T-cells (goat polyclonal; Santa
Cruz Biotechnology); CD8+ T-cells (goat polyclonal; Santa Cruz
Biotechnology), and neutrophils (rat monoclonal, Clone 7/4; Serotec,
Oxford, UK). Immunohistochemistry was performed as described
previously. Binding of the primary antibodies was revealed using the
chromogen diaminobenzidine (D5905; Sigma) enhanced with nickel ammonium
sulfate (Ousman and David, 2000). Sections were counterstained with 1%
Neutral Red.
Quantification
The RT-PCR results for the cytokines and chemokines at each time
point are expressed as a proportion of the corresponding optical
density value of GAPDH as scanned from the Southern blot autoradiographs. As a consequence, the scales of the y-axes
in Figure 1 are different because the intensity of the autoradiographic bands for each cytokine-chemokine varied. A total of 48 mice injected with LPC and 48 mice injected with PBS were used for RT-PCR. RT-PCR for
MIP-1, MCP-1, TNF-, GM-CSF, RANTES (regulated upon activation, normal T expressed, and secreted), IL-1, and TGF- was performed two to three times with each of the two batches of mice, i.e., a total
of four to five times. For each batch of mice, RNA from three mice were
pooled for each of the eight survival times. RT-PCR for IL-4, IL-10,
IL-1, and interferon- (IFN-) were done two or more
times using RNA from one batch of mice.
Counts of the various cell types in the white and gray matter were made
from longitudinal sections of the spinal cord at 25× magnification
using an ocular grid. Only cells containing a cell nucleus were
counted. These estimates were obtained from three tissue sections,
which were 45 µm apart and contained the injection site. Cell counts
were obtained from regions that extended for 500 µm on either side of
the injection site. Graphs depict the number of cells per square
millimeter. Statistically significant differences between
various experimental and control groups was determined using the
Student's t test.
Epon embedding
Some of the mice used for the antibody blocking experiments were
perfused with 0.5% paraformaldehyde and 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.5. One-millimeter-thick
cross-sections of the spinal cord containing the injection site were
post-fixed in 2% osmium tetroxide for 2 hr at room temperature and
then processed for embedding in Epon. One-µm-thick cross-sections of
the spinal cord were stained with 1% toluidine blue for light microscopy.
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RESULTS |
MIP-1, MCP-1, GM-CSF, and TNF- are expressed rapidly after
intraspinal injection of LPC
We have shown previously that rapid demyelination in the adult
mouse spinal cord induced by LPC is accompanied by recruitment of
monocytes, T-cells, and neutrophils and activation of macrophages. These immune cell changes lead to myelin phagocytosis and
demyelination. To identify the molecules that mediate these immune cell
responses, we examined by RT-PCR the mRNA expression of 10 chemokines
and cytokines after LPC or PBS injections into the adult mouse spinal cord. Of the 11 chemokines and cytokines examined, LPC induced rapid
expression of MIP-1, MCP-1, GM-CSF, and TNF- in the spinal cord
compared with PBS-injected controls (Fig.
1). In the normal, uninjured spinal cord,
the mRNA of three of these molecules (MIP-1, MCP-1, and GM-CSF) was
not detectable by RT-PCR, whereas TNF- was expressed at very low
levels (Fig. 1).
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Figure 1.
Time course of changes in cytokine and
chemokine mRNA levels in the adult mouse spinal cord after injection of
LPC and PBS. MCP-1, MIP-1, TNF-, and GM-CSF mRNA levels increased
above that in PBS controls within 0.5-3 hr after LPC ( ) injection
compared with animals injected with PBS ( ). Levels reached a peak
between 0.5 and 6 hr. Upregulation of RANTES, IL-1, and TGF- mRNA
was seen later between 12 and 96 hr. An upregulation of IL-10 was seen
only in the LPC group at one time point, 12 hr. IL-1, IFN-, and
IL-4 mRNA was not detected at any time point in either the LPC- or
PBS-injected mice. Graphs represent densitometric values that were
normalized to GAPDH. N indicates normal uninjured spinal
cord. Mean ± SEM.
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The expression of MCP-1, GM-CSF, and TNF- increased as early as 30 min after LPC injection compared with mice injected with PBS, whereas
the level of MIP-1 mRNA increased by 3 hr (Fig. 1). Interestingly,
the peak level of expression of GM-CSF was reached at 30 min, TNF-
at 1 hr, MCP-1 at 3 hr, and MIP-1 at 6-24 hr after LPC injection
(Fig. 1), which precedes the LPC-induced recruitment of monocytes,
neutrophils, and T-cells (6 hr) and activation of macrophages (12-96
hr) into mouse spinal cord (Ousman and David, 2000). At these times,
MCP-1 and TNF- levels were increased approximately fivefold after
LPC compared with PBS injections, whereas MIP-1 mRNA was ~40-fold
greater in the LPC versus the PBS group. At the 30 min time point and
at 12 and 24 hr, GM-CSF mRNA expression was only detected in the LPC
group. However, at 1-6 hr, GM-CSF mRNA level in the LPC group was not
significantly different from PBS-injected controls (Fig. 1). The high
expression of this cytokine at 1-6 hr in both groups of mice is
possibly attributable to the mechanical injury induced by the
microinjection pipette.
A delayed increase in mRNA expression was observed for other cytokines
and chemokines. RANTES mRNA levels increased between 12 and 96 hr after
injection of LPC and were at least twofold higher than in mice injected
with PBS between 12 and 48 hr (Fig. 1). A threefold to fourfold higher
expression of IL-1 was also detected in the LPC group between 12 and
96 hr. High levels of IL-1 mRNA were detected early after both LPC
and PBS injections, at 3 and 6 hr, arguing for a mechanical
injury-induced response at these time points. Increased expression of
TGF- mRNA was evident in the LPC group between 24 and 96 hr.
Expression of IL-10 mRNA was detected only at 12 hr after LPC injection
but not at any other times points or in the PBS group (Fig. 1). The
mRNA for IFN-, IL-4, and IL-1 was not detected at any time points
in either group of mice, although strong expression was detected in the
positive control consisting of lung tissue infected with Pseudomonas. These data indicate that upregulation of the
mRNA for MCP-1, MIP-1, GM-CSF, and TNF- after LPC injection
occurs before and during the period of immune cell recruitment and
activation (Ousman and David, 2000). These molecules are,
therefore, good candidates to be involved in triggering the immune cell
responses induced by LPC.
Blocking the activity of MCP-1, MIP-1, GM-CSF, or TNF-
decreases LPC-induced activation of macrophages and immune cell
recruitment
Effects on macrophage activation
We next used function-blocking antibodies to obtain direct
evidence whether MCP-1, MIP-1, GM-CSF, and TNF-, which are
rapidly expressed after intraspinal LPC injection, mediate the
activation of macrophages in the spinal cord induced by LPC. We have
shown previously that large, rounded,
Mac-1+ cells with clear unstained areas in
the cytoplasm are activated macrophages that have phagocytosed myelin
debris (Ousman and David, 2000). These
Mac-1+ cells are seen in the spinal cord
within 4 d after LPC injection. Neutralizing antibodies against
each of the four chemokines and cytokines were tested individually by
injecting 1 µl of solution containing the antibody and LPC into the
spinal cord. All four antibodies significantly decreased the number of
large, round Mac-1+ macrophages at 4 d after LPC injection compared with the LPC plus control Ig injections
(Fig. 2). The largest effect was seen after blocking MIP-1, which showed a 10-fold reduction of the number
of activated Mac-1+ macrophages (Fig. 2).
Inhibition of MCP-1, GM-CSF, or TNF- also induced a threefold to
fourfold decrease in the number of activated macrophages (Fig. 2).
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Figure 2.
Neutralizing MIP-1, MCP-1, GM-CSF, and TNF-
individually with function-blocking antibodies reduces the number of
LPC-induced phagocytic macrophages. Graphs show the number of
Mac-1+ phagocytic macrophages in the white matter
4 d after injection of LPC along with blocking antibodies () or
LPC plus control antibodies (). Blocking each of these molecules
resulted in a statistically significant reduction in the number of
phagocytic macrophages compared with controls. Anti-MIP-1 injection
produced the greatest reduction, ~10-fold. Mean ± SEM
(*p < 0.003, MCP-1; p < 0.003, MIP-1; p < 0.02, TNF-;
p < 0.01, GM-CSF); n = 4 animals.
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Because blocking the activity of MCP-1, MIP-1, GM-CSF, and TNF-
individually resulted in only a partial reduction in the number of
activated macrophages induced by LPC, we assessed whether inhibiting
all four molecules together would lead to a more pronounced decrease.
Few if any Mac-1+-activated macrophages
were detected in the spinal cord after neutralizing all four molecules
together (Fig. 3A-C). The
Mac-1 staining of the few large, round cells that were present was
weaker than that seen in mice injected with LPC plus control Ig.
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Figure 3.
Neutralizing MIP-1, MCP-1, GM-CSF, and TNF-
together results in marked reduction in the number of LPC-induced
phagocytic macrophages. A, B, Mac-1
immunohistochemistry of longitudinal sections of the adult mouse spinal
cord 4 d after injection of LPC along with either control
antibodies (A) or all four neutralizing
antibodies (B). In control animals, large, round
Mac-1+ macrophages that have clear areas in the
cytoplasm are seen (A). In contrast, very few of
these Mac-1+ cells are seen in animals injected with
LPC plus blocking antibodies (B). Sections were
counterstained with Neutral Red. Scale bar, 80 µm. C,
Quantification of Mac-1+ macrophages in the spinal
cord 4 d after injections of LPC along with either control
antibodies () or blocking antibodies (). Blocking MCP-1,
MIP-1, TNF-, and GM-CSF together almost completely reduced the
number of Mac-1+ macrophages induced by LPC.
Mean ± SEM (*p < 0.001);
n = 4 animals. Ab, Antibodies.
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Effects on recruitment of monocytes, T-cells, and neutrophils
We have shown previously that activated macrophages seen after LPC
injection into the spinal cord are likely to arise in part from
monocytes recruited to the area within 6 hr (Ousman and David, 2000).
Monocytes were identified on the basis of their size, shape, and Mac-1
immunoreactivity. We therefore examined whether the absence of
LPC-induced Mac-1+ phagocytic macrophages
4 d after neutralizing MCP-1, MIP-1, GM-CSF, and TNF- may be
attributable to a lack of recruitment of monocytes from the peripheral
circulation. Neutralizing all four molecules with blocking antibodies
almost completely inhibited LPC-induced recruitment of monocytes into
the spinal cord at 6 hr (Table 2). In
contrast, the spinal cord of control mice injected with LPC plus
control Ig contained a large number of monocytes. In addition to this
effect on macrophage recruitment, only a few, weakly labeled
Mac-1+ ramified microglia were seen ~500
µm away from the injection site compared with controls (data not
shown).
In addition to monocytes, LPC also induces an early and transient
recruitment (6-12 hr) of T-cells and neutrophils into the white and
gray matter of the adult mouse spinal cord (Ousman and David, 2000).
Neutralizing MCP-1, MIP-1, GM-CSF, and TNF- together completely
blocked the LPC-induced recruitment of CD4+ T-cells, CD8+ T-cells, and
neutrophils into the spinal cord (Table 2).
Neutralizing MCP-1, MIP-1, GM-CSF, and TNF- prevents
LPC-induced myelin phagocytosis and demyelination
To further assess whether neutralizing the activity of MCP-1,
MIP-1, GM-CSF, and TNF- also results in impairment of myelin clearance, i.e., demyelination, we examined cross-sections of Epon-embedded spinal cord sections 4 d after intraspinal injection of all four blocking antibodies together with LPC. The area of demyelination in function-blocking antibody-injected animals was limited to a very narrow region immediately adjacent to the needle tract (Fig. 4B). In
contrast, sections from mice injected with LPC and control Ig displayed
a large area of demyelination (Fig. 4A). Neutralizing
antibody treatment reduced the area of demyelination approximately
eightfold (Fig. 4C). Furthermore, a threefold greater amount
of myelin was preserved within areas showing demyelination in
neutralizing antibody-treated mice compared with controls (Fig. 4D). These results clearly show that the MCP-1,
MIP-1, GM-CSF, and TNF- induce immune cell responses that control
myelin phagocytosis leading to demyelination.
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Figure 4.
Neutralizing MCP-1, MIP-1, TNF-, and GM-CSF
together reduces LPC-induced demyelination in the mouse spinal cord.
A, B, Light micrographs of toluidine
blue-stained Epon-embedded cross-sections of the spinal cord through
the region of the dorsal columns, 4 d after injection of LPC plus
either control antibodies (A) or all four
blocking antibodies (B). In the control
antibody-treated animal, LPC produces a wide area of demyelination
(A). The size of this area is substantially
reduced in mice treated with blocking antibodies
(B). Arrows point to the injection
sites. Scale bar, 25 µm. C, D,
Quantification of the area of demyelination (C)
and the amount of myelin present in a 15,000 µm2
area on either side of the injection site (D)
4 d after injections of LPC plus either neutralizing antibodies
() or control antibodies (). Measurements were obtained from
Epon-embedded sections of the mouse dorsal column. Neutralizing antibodies reduced the area of
demyelination induced by LPC by approximately sixfold
(C). In addition, myelin clearance within this
area was reduced approximately four times in the antibody-treated mice
compared with controls (D). Mean ± SEM
(*p < 0.008, C;
p < 0.002, D);
n = 3 animals. Ab, Antibodies.
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DISCUSSION |
In this study, we provide evidence that MCP-1, MIP-1, GM-CSF,
and TNF- mediate rapid recruitment of monocytes, T-cells and neutrophils, and activation of macrophages in the adult mammalian CNS
in response to the demyelinating agent LPC. In addition, we show that
blocking the activity of all four of these molecules suppresses the
rapid demyelination characteristically induced by LPC. We therefore
present the first clear evidence that these four chemokines and
cytokines play a key role in initiating the immune cell responses that
lead to rapid phagocytosis and clearance of myelin from the adult
mammalian CNS. These findings have important implications for
stimulating rapid myelin clearance during Wallerian degeneration in the
CNS, as well as provide additional insights into the role of chemokines
and cytokines in the pathogenesis of demyelinating diseases, such as
multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE).
Chemokines and cytokines that mediate immune cell responses leading
to rapid clearance of CNS myelin
Immune cell responses generally occur very slowly in the CNS.
However, during LPC-induced demyelination T-cells, neutrophils and
macrophages are recruited into the CNS within hours (Ousman and David,
2000). We now show that these cellular responses are accompanied by a
rapid upregulation in the expression of MCP-1, MIP-1, GM-CSF, and
TNF- mRNA as early as 30 min to 3 hr after injection of LPC into the
adult mouse spinal cord. In addition, this high level of expression is
maintained for 12-24 hr.
MCP-1 is a potent chemoattractant for monocytes, whereas MIP-1
induces chemotaxis of both T-cells and monocytes (Karpus and Ransohoff,
1998). These chemokines are therefore likely to be involved in
promoting the migration of T-cells and monocytes into the CNS after LPC
injection (Ousman and David, 2000). Activated T-cells, astrocytes,
microglia, and monocytes secrete MCP-1 and MIP-1 (Ransohoff and
Tani, 1998; Asensio and Campbell, 1999), and all are possible sources
of these two chemokines after LPC injection. Furthermore, TNF- can
induce MCP-1 expression in astrocytes in vitro (Hurwitz et
al., 1995; Guo et al., 1998), which hints at a complex interplay
between cell types and their secreted molecular signals in generating
the cytokine-chemokine profile seen after LPC injection.
The activation of macrophages to phagocytose the damaged myelin in the
LPC-demyelination model may be facilitated by the increased presence
of TNF- and GM-CSF. GM-CSF stimulates proliferation of microglial
cells in vitro and the phagocytic activity of CNS macrophages in vivo (Giulian and Ingeman, 1988). In
degenerating peripheral nerves, this cytokine induces activation of
peripheral macrophages (Saada et al., 1996). The rapid upregulation of
GM-CSF in the LPC-injected mice argues for a local CNS source, likely astrocytes. These glial cells are capable of expressing GM-CSF in
vitro (Ohno et al., 1990) and are suggested to secrete a
GM-CSF-like activity that was detected in injured CNS (Giulian et al.,
1990). In addition to its macrophage activation role (Philip and
Epstein, 1986), TNF- is implicated in promoting the demyelination
seen in MS and EAE because of its damaging effects on myelin and
oligodendrocytes in vitro (Robbins et al., 1987; Selmaj and
Raine, 1988). However, its brief upregulation in the spinal cord after
LPC injection likely prevents any substantial injury to adjacent cells.
In fact, axons that are stripped of their myelin sheaths after
injection of LPC appear morphologically intact (Hall, 1972; Jeffrey and Blakemore, 1993).
With respect to the other cytokines and chemokines we
investigated, several interesting observations were noted.
IL-1 expression displayed a biphasic response (at 6 and 96 hr)
after LPC and PBS injections. This response was much more pronounced
and significantly higher in the LPC group at 96 hr. A biphasic (1 and 6 hr) increase in IL-1 mRNA has also been reported after spinal cord
hemisection (Bartholdi and Schwab, 1997). The initial expression was
attributed to a CNS cellular source, likely microglia, and resurged by
the later inflammatory cell influx. Streit et al. (1998) have also suggested microglia to be the source of rapidly expressed IL-1 (within 1 hr) in contused rat spinal cord. The functional importance for the higher levels in the LPC group at the later time points is not
known at present.
Interestingly, the temporal pattern of upregulation of MCP-1, MIP-1,
GM-CSF, and TNF- mRNA precedes and/or accompanies LPC-induced recruitment of immune cells (6-12 hr) into the spinal cord and activation of macrophages (12-48 hr) (Ousman and David, 2000). Antibody neutralization of each of these four molecules significantly reduced macrophage activation. Furthermore, neutralizing all four together led to almost complete blocking of T-cells, neutrophils and
monocytes, and activation of macrophages, suggesting that two or more
of these four molecules are involved in or sufficient to promote the
immune response seen after LPC injection. Blocking the function of
these molecules with antibodies also ensued in a marked reduction in
LPC-induced demyelination. These data therefore provide strong evidence
for the contribution of these immunoregulatory molecules in inducing
rapid immune cell changes and myelin phagocytosis in the adult
mammalian CNS.
Can chemokines stimulate a safe and effective immune
cell response?
Increased expression of several chemokines and cytokines,
including MCP-1, MIP-1, TNF-, and IL-1, is also seen at the
immediate site of CNS injury (Bartholdi and Schwab, 1997; McTigue et
al., 1998; Streit et al., 1998; Lee et al., 2000) and have been
implicated in the immune cell response seen at this site (Perry et al.,
1987; Dusart and Schwab, 1994; Popovich et al., 1997; Schnell et al., 1999). Infiltrating immune cells are also implicated in mediating oligodendrocyte and myelin damage, leading to demyelination after CNS
trauma (Blight, 1994; Popovich et al., 1999), and in MS and EAE
(Huitinga et al., 1990; Tran et al., 1998). These immune cells, as well
as CNS resident cells, can release toxic molecules that can cause
tissue damage (Selmaj and Raine, 1988; Merrill et al., 1993). However,
the LPC-induced immune response produces demyelination without much
damage to axons or cells. What then may account for the rapid and safe
immune response seen in the LPC model compared with the tissue damage
seen in EAE or after spinal cord injury? The time course of expression
of cytokines and chemokines may provide some answers. MCP-1, MIP-1,
TNF-, and GM-CSF were expressed within 0.5-3 hr after LPC injection
and returned to control levels by 24 hr. In EAE, however, MIP-1,
MCP-1, and TNF- expression is seen later, between 10 and 18 d
(Hulkower et al., 1993; Godiska et al., 1995; Karpus et al., 1995;
Glabinski et al., 1997). This may account for the later and prolonged
appearance of immune cells in EAE (Fritz et al., 1983; Hickey et al.,
1983). Therefore, after LPC injection, any damaging effects that these
chemokines and cytokines could mediate would be aborted rapidly because
of the limited period during which they are expressed. Although
proinflammatory cytokines (IL-1 and TNF-) and chemokines (MCP-1
and MIP-1) are upregulated rapidly (within 1-6 hr) after spinal
cord injury (Bartholdi and Schwab, 1997; McTigue et al., 1998; Streit
et al., 1998; Lee et al., 2000), massive tissue damage still ensues
compared with the LPC-induced demyelination model. One possible
explanation for this difference is the slightly later appearance (72 hr) of immunosuppressive TGF- expression in contused spinal cord
(Streit et al., 1998; McTigue et al., 2000) and the expression of IL-10 after LPC injection. Second, a contusion or hemisection injury creates
a very large lesion, leading to widespread breakdown of the
blood-brain barrier and massive influx of inflammatory cells (Popovich
et al., 1997; Schnell et al., 1999) secreting a plethora of cytotoxic
mediators that can gain wider access to the CNS parenchyma. In this
study, the mechanical injury to the cord was minimal, being sustained
with a single injection with a 50-µm-diameter glass micropipette. It
is also possible that the histological differences in the two models
may be attributable to differences in the levels of these
chemokines-cytokines, lower levels of which may serve to sculpt a more
subtle and controlled response that limits tissue damage.
In addition to the downregulation of the proinflammatory chemokines and
cytokines, TGF- and IL-10 mRNA are upregulated at later times after
LPC injection. Because these two cytokines are known to be capable of
reducing the immune response (Tsunawaki et al., 1988; Lodge and Sriram,
1996), they can serve to control and dampen the continued progression
of the inflammatory response after LPC injection. The overlapping
expression of IL-10 with the appearance of "irregular-shaped"
macrophages at 12 hr after LPC injection (Ousman and David, 2000)
suggests that these cells and/or astrocytes (Cannella and Raine, 1995;
Renno et al., 1995) are the most likely source of this
immunosuppressive cytokine during LPC-induced demyelination. The
profile of different chemokines and cytokines expressed in different
experimental models are thus likely to influence whether one type of
response is cytotoxic, whereas another can safely and effectively
remove myelin or other debris from the adult mammalian CNS.
Possible chemokine-cytokine network involved in rapid myelin
phagocytosis in the CNS
In our previous work, we showed that monocytes are recruited to
the CNS at the site of LPC injection within 6-12 hr. T-cells are also
seen at this time, which coincides with the expression of MIP-1 that
has been shown to mediate T-cell recruitment via a very late antigen-1
(VLA-1)-mediated adhesion mechanism (Carr et al., 1996). VLA-1
is expressed within 3-6 hr of LPC injection (Ousman and David, 2000).
The recruited T-cells may then be activated by perivascular macrophages
to secrete TNF- that could induce astroglial expression of MCP-1
(Hurwitz et al., 1995; Guo et al., 1998), a potent chemoattractant of
monocytes. LPC has been shown in vitro to have chemotactic
effects on monocytes (Quinn et al., 1988) and may also contribute to
influx of monocytes after LPC injection (Ousman and David, 2000).
Cytokines, such as TNF- and GM-CSF, released by T-cells, astrocytes,
or other cells could then lead to the activation of macrophages of both
monocytic and microglial origin. Although we do not yet know which cell
types are expressing which chemokine or cytokine, we know from our
blocking experiments that MCP-1, MIP-1, GM-CSF, and TNF- are
indeed involved in mediating rapid phagocytosis of CNS myelin damaged
by LPC.
This work will have important implications for developing strategies to
speed the rate of Wallerian degeneration in the CNS after trauma, as
well as provide additional insights into our understanding of the
pathogenesis of autoimmune demyelinating diseases, such as EAE and MS.
| |
FOOTNOTES |
Received Dec. 8, 2000; revised March 30, 2001; accepted April 5, 2001.
This work was funded by Canadian Institutes of Health Research Grant
14828. S.S.O. was supported by a studentship from the Multiple
Sclerosis Society of Canada.
Correspondence should be addressed to Dr. Samuel David, Centre for
Research in Neuroscience, Montreal General Hospital Research Institute,
1650 Cedar Avenue, Montréal, Québec, Canada, H3G 1A4.
E-mail: sdavid11{at}po-box.mcgill.ca.
| |
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ABSTRACT
INTRODUCTION
