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The Journal of Neuroscience, December 15, 1998, 18(24):10457-10463
The Chemokine Growth-Regulated Oncogene- Promotes Spinal Cord
Oligodendrocyte Precursor Proliferation
Shenandoah
Robinson1, 2,
Marie
Tani3,
Robert M.
Strieter4,
Richard M.
Ransohoff3, and
Robert H.
Miller2
Departments of 1 Neurosurgery and
2 Neurosciences, School of Medicine, Case Western Reserve
University, Cleveland, Ohio 44106, 3 Departments of
Neurology and Neurosciences, Lerner Research Institute, Cleveland
Clinic Foundation, Cleveland, Ohio 44195, and 4 The
University of Michigan Medical Center, Department of Internal
Medicine, Ann Arbor, Michigan 48109
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ABSTRACT |
Chemokines, (chemotactic cytokines) are a family of regulatory
molecules involved in modulating inflammatory responses. Here we
demonstrate that the chemokine growth-regulated oncogene- (GRO-)
is a potent promoter of oligodendrocyte precursor proliferation. The
proliferative response of immature spinal cord oligodendrocyte precursors to their major mitogen, platelet derived growth factor (PDGF), is dramatically enhanced by GRO- present in spinal cord conditioned medium. One source of GRO- is a subset of spinal cord
astrocytes. Cultures of astrocytes contain GRO- mRNA and protein and
secrete biologically active concentrations of GRO-. In postnatal
spinal cord white matter the location of GRO--immunoreactive cells
is developmentally regulated: GRO-+ cells first appear in ventral
and later in dorsal spinal cord white matter. These results suggest
that localized proliferation of oligodendrocytes is mediated by synergy
between PDGF and GRO-.
Key words:
oligodendrocytes; chemokines; GRO-; cell
proliferation; development; spinal cord
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INTRODUCTION |
The local control of oligodendrocyte
precursor proliferation is critical to ensure that all axons in CNS
white matter tracts are myelinated effectively. In the spinal cord,
oligodendrocytes, the myelinating cells of the CNS, develop from
precursors that arise in specific regions (Warf et al., 1991 ; Pringle
and Richardson, 1993) and undergo extensive migration (Noll and Miller,
1993; Warrington et al., 1993) and proliferation (Gilmore, 1971; Miller et al., 1997) before differentiation. The mitogenic response of oligodendrocyte precursors depends on their level of maturity. Immature
rodent oligodendrocyte precursors identified by labeling with
monoclonal antibody (mAb) A2B5 (Raff et al., 1983) proliferate in
response to platelet-derived growth factor (PDGF-AA) (Noble et al.,
1988; Richardson et al., 1988) and are the predominant cell population
expressing the PDGF- receptor in the developing CNS (Pringle et al.,
1992; Pringle and Richardson, 1993). More mature precursors identified
by the binding of mAb O4 proliferate primarily in response to basic
fibroblast growth factor (bFGF) and not PDGF (Gard and Pfeiffer, 1990,
1993; Fok-Seang and Miller, 1994) suggesting that the sequential action
of multiple factors regulate oligodendrocyte development.
In the spinal cord of postnatal animals, the majority of
oligodendrocyte precursor proliferation occurs in presumptive white matter (Gilmore, 1971; Miller et al., 1997), where it is spatially and
temporally regulated (Gilmore, 1971; Schwab and Schnell, 1989). In
ventral regions most oligodendrocyte precursor proliferation is
complete, and myelination begins in the first postnatal week. By
contrast, in dorsal regions such as the cortical spinal tract, maximal
oligodendrocyte proliferation and myelination do not occur until the
second or third postnatal week (Schwab and Schnell, 1989, 1991),
implying an influence of the local environment on the generation of
oligodendrocytes. Likewise, a transient rapid proliferation of very
immature spinal cord oligodendrocyte precursors has recently been
demonstrated (Calver et al., 1998). In addition, elevated local
proliferation of oligodendrocyte precursors occurs in a number of
pathological conditions in the adult CNS (Raine et al., 1981; Amat et
al., 1991; Prineas et al., 1993), suggesting that environmental factors
contribute to spatially localized oligodendrocyte precursor
proliferation. The localization of oligodendrocyte proliferation is not
simply a reflection of the distribution of the mitogen PDGF.
Platelet-derived growth factor is synthesized by neurons (Yeh et al.,
1991) and astrocytes (Pringle et al., 1989) and is ubiquitously
distributed in the developing spinal cord (Calver et al., 1998).
Environmental signals influence oligodendrocyte precursor
responses to PDGF (Robinson and Miller, 1996). For example, PDGF induces high levels of proliferation in A2B5+ cells in mixed spinal cord but not optic nerve cultures or purified cultures of A2B5+ cells
(Robinson and Miller, 1996), suggesting that spinal cord contains an
activity that enhances the response of oligodendrocyte precursors to
PDGF. Oligodendrocyte precursor proliferative responses to PDGF can be
influenced by bFGF, which promotes extended proliferation (Bogler et
al., 1990; McKinnon et al., 1990), and the proteoglycan NG2 (Stallcup
and Beasley, 1987), which enhances the PDGF-driven proliferative
response (Nishiyama et al., 96). Neither of these two factors, nor
ciliary neurotrophic factor or leukemia inhibitory factor, has
properties consistent with the spinal cord activity (Robinson and
Miller, 1996).
An alternate candidate for the spinal cord biological activity is
a member of the chemokine family (Rollins, 1997). The chemokine growth-regulated oncogene- (GRO-) was initially cloned as KC, a
gene strongly induced in quiescent murine NIH 3T3 fibroblasts by PDGF
(Cochran et al., 1983). Human, rat, and hamster orthologs have since
been identified, and their characterization has provided insights into
the functions of GRO peptides. Anisowicz et al. (1987) isolated
a gene that was overexpressed in human diploid fibroblasts, rendered
tumorigenic by cDNAs from Chinese hamster ovary cells. Nontumorigenic
sibling cell lines expressed lower levels of this gene, which was
designated "gro." Independently, Richmond et al. (1988) described a
peptide, designated melanocyte growth stimulatory activity (MGSA),
secreted from human melanoma cells that possessed autocrine
growth-promoting properties. The molecular identity of GRO- and MGSA
and a homologous relationship to KC were described early on (Oquendo et
al., 1989), and a family of closely related human genes was
characterized (Haskill et al., 1990). An avian homolog of GRO-,
9E3/CEF-4, was isolated from chick embryo fibroblasts during
exponential growth and shown to be expressed in developing wings and in
tissues undergoing neovascularization (Martins-Green and Bissell,
1990). These investigators proposed that CEF-4 might be involved in
regulating angiogenesis; an extension of this hypothesis to mammalian
CXC chemokines [interleukin 8 (IL-8) and GRO-] has gained
substantial recent support (Strieter et al., 1995). Growth regulation
by GRO peptides has now been described for cells of diverse lineages,
including fibroblasts, melanocytes, and hemopoietic progenitors
(Broxmeyer et al., 1993). In parallel, a substantial literature
describing the roles of these molecules in inflammatory responses has
appeared (Baggiolini et al., 1997; Rollins, 1997; Baggiolini,
1998).
Little is known about the role of CXC chemokines in the CNS.
Elevated levels of CXC chemokine expression in the CNS are found in
infection (Spanaus, 1997), tumors (Van Meir et al., 1992), ischemia
(Liu, 1993), and demyelination (Glabinski et al., 1997). Overexpression
of GRO- in oligodendrocytes induced neutrophil invasion and
astrogliosis (Tani, 1996). Similarly, leukocyte infiltration and
blood-brain barrier breakdown occurred after overexpression of a CXC
chemokine by astrocytes (Bell, 1996).
Here we demonstrate that GRO- secreted by spinal cord
astrocytes regulates oligodendrocyte precursor proliferation. In
purified cultures of oligodendrocyte precursors, GRO- markedly
increased PDGF-induced proliferation in a narrow dose range. The
presence of GRO-+ astrocytes in spinal cord white matter correlated
spatially and temporally with elevated levels of oligodendrocyte
precursor proliferation, suggesting that a combination of PDGF and
GRO- locally regulates oligodendrocyte precursor proliferation.
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MATERIALS AND METHODS |
Cell culture. Highly enriched populations of immature
oligodendrocyte precursors were prepared by immunopanning with mAb A2B5 as previously described (Robinson and Miller, 1996). Cultures were
grown in serum-free basal defined medium. To quantify cellular proliferation, a bromodeoxyuridine (BrdU) incorporation assay was used
and quantitated (Robinson and Miller, 1996). Briefly, cells were grown
for the last 18 hr in the presence of 10 µM BrdU, and the
number of BrdU-labeled cells was determined as described below. In
experimental cultures, rat GRO-/KC (Peprotech), PDGF-AA (Boehringer
Mannheim, Indianapolis, IN), and 2 µg/ml goat anti-GRO- (Santa
Cruz Biotechnology, Santa Cruz, CA) were added as described. The
anti-GRO- antibody was specific for GRO- and non-cross-reactive with GRO- and GRO- . Purified O4+ pro-oligodendroblasts were prepared using similar protocols with mAb O4 substituted for mAb A2B5.
To determine the molecular mass of the biological activity, spinal cord
conditioned medium (CM) was fractionated using Centriprep concentrators
(Amicon, Beverly, MA) according to the manufacturer's directions.
Fractions with maximal molecular weights of 10, 30, and 50 kDa were
prepared from postnatal day 0 (P0) rat spinal cord cultures. The data
represent the mean ± SD of BrdU+/A2B5+ cells from at least six
coverslips from three independent preparations.
Immunofluorescent labeling. Spinal cord cells were
labeled with mAbs A2B5, O4, anti-GFAP, and anti-BrdU according to
standard protocols (Robinson and Miller, 1996). For GRO- labeling,
mixed spinal cord cells were cultured in basal defined medium with 10 ng/ml PDGF, fixed with 1% formalin, incubated sequentially with mAb
anti-GRO- (Sigma, St. Louis, MO), biotin-conjugated anti-mouse IgG
(ICN Pharmaceuticals, Costa Mesa, CA), and streptavidin Cy3 (Jackson
ImmunoResearch, West Grove, PA). Cultures were then fixed with
methanol-acetic acid and double-labeled with anti-GFAP antibodies (Robinson and Miller, 1996). Vibratome sections (25 µm) of spinal cord previously fixed in Bouin's fixative were sequentially incubated with 0.3% hydrogen peroxide, mAb anti-GRO-, and biotin-conjugated anti-mouse IgG, and labeling was visualized by DAB using a Vectastain kit (Vector Laboratories, Burlingame, CA). Sections were dehydrated through graded alcohol, cleared in Hemo-de (Fisher Scientific, Houston,
TX), and mounted in Permount. In control cultures the primary antibody
was deleted, and no specific labeling was seen. ELISAs for GRO-,
GRO-, and IL-8 were performed as previously described (Keane, 1997)
from coded samples of CM from two distinct cell cultures of both whole
spinal cord and purified astrocytes. The purity of astrocyte cultures
was determined by double labeling with mAbs A2B5, ED1 (Chemicon,
Temecula, CA), and anti-GFAP to be >85% type 1 astrocytes.
RT-PCR. Cells of the oligodendrocyte lineage were
selectively removed from neonatal spinal cord cultures through
complement-mediated cell lysis (Fok-Seang and Miller, 1994). The
remaining cells (>85% GFAP+ astrocytes) were collected. RNA was
prepared by the acid-guanidinium method using TRIzol. GRO- mRNA was
analyzed by RT-PCR, using gene-specific primers (forward,
5'-TCGCTTCTCTGTGCAGCGCT-3'; backward, 5'-GTGGTTGACACTTAGTGGTCTC-3') as
previously described (Glabinski et al., 1997). PCR products were
amplified for 20 cycles at 94°C for 2 min, 60°C for 2 min, and
72°C for 1 min. The PCR products were analyzed by Southern transfer
and hybridization with a radiolabeled full-length murine GRO- probe.
After high-stringency washes, a discrete band of expected size was
detected by autoradiography in each of the two samples derived from two
different cultures.
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RESULTS |
Spinal cord CM enhances the proliferative response of
oligodendrocyte precursors to PDGF
To determine whether the enhanced proliferation of oligodendrocyte
precursors to PDGF in spinal cord cultures reflected the influence of a
soluble factor, the effect of spinal cord CM on PDGF-driven
proliferation was assayed in purified cultures of A2B5+ cells. The
proliferative response of purified immature oligodendrocyte precursors
to PDGF is enhanced by spinal cord CM (Fig.
1). In control cultures in the absence of
PDGF, <5% of A2B5+ spinal cord cells proliferate. Likewise, in the
presence of spinal cord CM or in whole spinal cord cultures, low levels
of BrdU incorporation were seen. Addition of PDGF resulted in a small
increase in purified A2B5+ cell proliferation in defined medium such
that ~16% of cells incorporated BrdU (Fig. 1). By contrast, when
PDGF was added to whole spinal cord cultures containing all classes of
neural cells, BrdU incorporation in the endogenous A2B5+ cells
increased to >65%. Spinal cord CM in combination with PDGF increased
proliferation 4.5-fold in purified cultures of A2B5+ spinal cord cells,
and these levels were indistinguishable from those in whole spinal cord
cultures (Fig. 1). These data suggest that spinal cord CM contains an
activity, that while not an independent mitogen acts in synergy with
PDGF to enhance the proliferation of oligodendrocyte precursors.
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Figure 1.
Oligodendrocyte precursor proliferation is
regulated by soluble factors in addition to PDGF. To determine the
influence of CM on PDGF-induced proliferation of oligodendrocyte
precursors, purified A2B5+ cells were grown in combinations of PDGF and
CM. A2B5+ oligodendrocyte precursors have low levels of BrdU
incorporation in basal defined medium (BDM), in
whole spinal cord cultures (WSC), and in the presence of
spinal cord CM. PDGF-AA (10 ng/ml) alone had a limited effect on cell
proliferation. By contrast, WSC or CM in combination with PDGF resulted
in a 4.5-fold increase in cell proliferation. Denatured CM partially
reduces the BrdU incorporation. The data represent the proportion of
A2B5+ cells that incorporated BrdU. In all cases P0 rat spinal cord
cells were cultured for 3 d, with BrdU added during the last 18 hr. The data represent the mean ± SD taken from two separate
coverslips from three independent experiments.
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To test whether increasing levels of PDGF could replicate the
effect of the spinal cord CM, concentrations of PDGF up to 30 ng/ml in
the absence of CM were added to purified cultures of A2B5+ cells, but
no significant enhancement of proliferation was seen compared with 10 ng/ml PDGF (data not shown). Denaturing the CM reduced the synergistic
effect significantly but did not totally abolish it (Fig. 1),
suggesting the activity was a protein. To determine the molecular mass
of the biological activity, spinal cord CM was size-fractionated, and
the fractions were tested for synergy with PDGF. Addition of complete
CM in the presence of PDGF resulted in 65% BrdU incorporation in
purified A2B5+ oligodendrocyte precursors. Addition of the fractions
with molecular weights <10 kDa resulted in similar levels (67%) of
BrdU incorporation as complete CM (Fig.
2). Addition of larger molecular weight
fractions resulted in a slight increase in BrdU incorporation (~30%)
over that seen with PDGF alone but was not comparable with that seen with the smaller fraction (Fig. 2), suggesting that although there may
be multiple activities, the majority of the synergistic activity was
present in the small-size fraction.
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Figure 2.
The active factor in spinal cord CM was soluble
with an Mr of <10 kDa. CM was separated
into fractions depending on size, and equivalent quantities were added
to cultures of purified oligodendrocyte precursors. The fraction with
an Mr <10 kDa had a level of activity similar to that of whole CM,
whereas fractions >10 kDa had little proliferation-enhancing activity.
Data represent the mean ± SD for duplicate preparations in three
independent experiments.
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Spinal cord CM activity can be replaced by the
chemokine GRO-
The relatively small apparent molecular weight associated
with the biological activity suggested a member of the chemokine family. Because the chemokine GRO- had established mitogenic activity (Anisowicz et al., 1987; Richmond et al., 1987; Richmond and
Thomas, 1988), the effect of GRO- on PDGF-driven oligodendrocyte precursor proliferation was assayed. In cultures of purified
oligodendrocyte precursors, addition of 0.5 ng/ml GRO- resulted in a
fourfold increase in BrdU incorporation over that seen with PDGF alone (Figs. 3, 4). The level of precursor
proliferation seen with a combination of GRO- and PDGF (60%) was
similar to that seen with a combination of CM and PDGF (Figs. 3, 4).
The synergistic effect between GRO- and PDGF was totally abolished
by addition of 2 µg/ml neutralizing anti-GRO antibodies (Fig.
4), and the neutralizing effects of the
anti-GRO- antibodies were reversed by addition of 100 ng/ml GRO-
(data not shown).
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Figure 3.
Neutralizing antibodies to GRO- block the
enhancement of oligodendrocyte precursor proliferation by spinal cord
CM. A2B5 (A, C, E) and BrdU (B, D,
F) immunofluorescent staining of purified A2B5+
oligodendrocyte precursors cultured with PDGF and GRO- (A,
B), PDGF and spinal cord astrocyte CM (C, D),
and PDGF, CM, and anti-GRO- antibody (E, F) is
shown. The anti-GRO- antibody neutralizes the marked increase in
proliferation induced by CM, suggesting that the neonatal spinal cord
factor is closely homologous to GRO-. Scale bar, 20 µm.
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Figure 4.
Quantification of the effect of GRO- on the
proliferation of spinal cord oligodendrocyte precursors. Addition of
GRO- (0.5 ng/ml) enhances PDGF (10 ng/ml)-driven proliferation of
oligodendrocyte precursors to the same extent as spinal cord astrocyte
CM. The amplification of PDGF-driven proliferation by both GRO- and
CM is blocked by addition of anti-GRO- antibody (Ab),
suggesting that the active factor in CM is GRO-. Purified A2B5+ P0
rat spinal cord cells were cultured 2 d with BrdU added during the
last 16 hr. The data represent the mean ± SD of the proportion of
cells that have incorporated BrdU taken from duplicate cultures in
three independent experiments.
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To determine whether the biological activity in CM was related to
GRO-, neutralizing GRO- antibody was added to CM. Addition of
anti-GRO- essentially abolished the synergistic effect of spinal
cord CM on PDGF-mediated proliferation of oligodendrocyte precursors
(Figs. 3, 4), suggesting that the active factor in spinal cord CM is a
GRO- chemokine-like protein.
The proliferative response to PDGF and GRO- is
concentration-dependent and a characteristic of A2B5+ precursor
cells
Like spinal cord CM, purified GRO- was not an independent
mitogen for oligodendrocyte precursors (Fig.
5). In the absence of PDGF no increase in
BrdU incorporation was seen with GRO- concentrations ranging from
0.01 to 50 ng/ml. Thus, PDGF appears essential for A2B5+ cell
proliferation. In the presence of 5 ng/ml PDGF, GRO- had little
effect on cell proliferation at concentrations <1.0 ng/ml. At higher
concentrations (10-50 ng/ml) the proliferative response increased
until ~25% of cells incorporated BrdU (Fig. 5). In the presence of
10 ng/ml PDGF, a peak of proliferative response was seen with 0.5 ng/ml
GRO- (Fig. 5). This relatively narrow concentration dependence
coupled with the threshold requirement for PDGF suggests that GRO-
enhancement of PDGF-driven oligodendrocyte precursor proliferation is
precisely regulated in a dose-dependent manner.
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Figure 5.
The GRO- enhancement of proliferation of
oligodendrocyte precursors requires a threshold concentration of PDGF
and is optimal at a concentration of 0.5 ng/ml GRO-. In the absence
of PDGF, GRO- is not mitogenic. At 5 ng/ml PDGF a limited
enhancement of precursor proliferation is seen at concentrations of
GRO- >1 ng/ml. By contrast, at 10 ng/ml an optimal 4.5-fold
increase in BrdU incorporation is seen at a concentration of 0.5 ng/ml.
This restricted optimal range of GRO- concentrations is
characteristic of chemokine responses. Purified P0 A2B5+ cells were
cultured for 2 d with BrdU added during the last 16 hr.
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The response of oligodendrocyte precursors to GRO- is a
characteristic of immature (A2B5+) precursors not shared by more mature
O4+ oligodendrocyte precursors. In parallel studies, purified O4+
pro-oligodendrocytes were examined for responses to combinations of
PDGF and GRO-. No increase in proliferation was seen with any
combination of factor concentrations tested (data not shown), consistent with previous studies indicating that PDGF is not a major
mitogen for these cells (Gard and Pfeiffer, 1990; Fok-Seang and Miller,
1994).
Astrocytes contain GRO- mRNA and protein and release
biologically active quantities
The major cell type in the spinal cord cultures used to generate
CM was astrocytes. To independently confirm the identity of the CM
biological activity as GRO- and to demonstrate astrocytes as a
cellular source, cultures enriched in spinal cord astrocytes (>85%
GFAP+ cells) were assayed by RT-PCR using GRO--specific primers.
Analysis of RT-PCR products by Southern blotting revealed that a single
band at the expected size was detected (Fig.
6), indicating that cultured spinal cord
type 1 astrocytes contained GRO- mRNA. To determine whether all
spinal cord astrocytes expressed GRO- immunoreactivity, cultures
were double-labeled with antibodies to GFAP and GRO-. Although
>85% of the cells were GFAP+, only a subpopulation (~25%) of the
astrocytes cells were GRO--immunoreactive (Fig.
7). There were no clearly distinctive
morphological characteristics of the GRO-+ cells. The GRO-
immunoreactivity was localized in the cytoplasm and abundant in the
perinuclear region and under high magnification appeared to be
predominantly vesicular. Some non-GFAP+ cells also expressed GRO-.
Some of these cells were likely ED1+ microglia, which constituted <1%
of cells in the cultures, whereas others were immature astrocytes,
residual neurons, or oligodendrocyte lineage cells.
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Figure 6.
Neonatal rat spinal cord astrocytes contain
GRO- mRNA in vitro. Two independent cultures of
purified spinal cord astrocytes were subjected to RT-PCR analysis with
GRO--specific probes. Both samples (S) had a strong band
of the appropriate size that comigrated with the major band detected in
extracts of lipopolysaccharide-treated murine macrophages (+).
No band was detected in control ( ) samples from unstimulated
macrophages.
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Figure 7.
A subpopulation of astrocytes contains GRO-
protein. Cultures of spinal cord astrocytes were double-labeled with
anti-GFAP (B, E) and anti-GRO- (C,
F) antibodies. Approximately 23% of the GFAP+
astrocytes in these cultures labeled with anti-GRO- antibodies.
A, D, Corresponding phase-contrast
micrographs. Scale bar, 30 µm.
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To determine whether the chemokines produced by cultured astrocytes
were released in detectable quantities into the culture medium,
chemokine-specific ELISAs (Keane, 1997) were performed on CM derived
from both complete spinal cord and purified astrocyte cultures. Both
complete spinal cord and astrocyte CM contained biologically active
levels of GRO- (Table 1). The
concentrations of GRO- (0.5 ng/ml) were similar to that which
induced maximal increase in BrdU incorporation in the in
vitro dose-response assay (Table 1, compare with Fig. 4). By
contrast, conditioned media from both cultures contained only low
levels of GRO-, and the closely related chemokine IL-8 was not
detectable (Table 1). Taken together, these data provide strong
evidence that the biological activity in CM that synergizes with PDGF
to enhance the proliferation of oligodendrocyte precursors is the
chemokine GRO-.
The distribution of GRO-+ cells in the spinal cord is
developmentally regulated
In neonatal rat spinal cord the proliferation of oligodendrocyte
precursors occurs primarily in the developing white matter (Miller et
al., 1997). To determine whether GRO- was expressed in the
developing spinal cord in a pattern that correlated with oligodendrocyte precursor proliferation, transverse sections of spinal
cord were labeled with an anti-GRO- antibody. In cervical spinal
cord of postnatal day 1 animals the majority of GRO-+ cells were
seen in ventral and ventrolateral white matter. These GRO-+ cells
had morphological characteristics of spinal cord white matter
astrocytes (Liuzzi and Miller, 1987). The cells were mainly radially
oriented and frequently had endfeet that terminated at the pial surface
(Fig. 8). Fewer GRO- cells were
present in dorsal white matter; they were less intensely
GRO--immunoreactive and not obviously radially oriented. In gray
matter, GRO-+ cells were present both ventrally and dorsally.
Although some of these cells were most likely astrocytes, others were
clearly neurons. In cervical spinal cord of postnatal day 8 animals the
pattern of GRO- expression was clearly different. Far fewer GRO-
cells were present in gray matter and in ventral white matter compared with P1 animals; however, many more GRO-+ cells were detectable in
dorsal spinal cord white matter (Fig. 8). This pattern of GRO- expression in the developing spinal cord is consistent with the pattern
of emergence of oligodendrocytes (Gilmore, 1971; Schwab and Schnell,
1989) and suggests that local control of proliferation of spinal cord
oligodendrocyte precursors may be, in part, regulated by the local
expression of GRO-.
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Figure 8.
The expression of GRO- in spinal cord white
matter astrocytes is developmentally regulated. In ventral spinal cord
white matter (A, C) radially oriented cells with feet on
the pial surface express relatively high levels of GRO- at P1
(A, arrows) but low levels at P8
(C). By contrast, in dorsal spinal cord
(B, D) there are relatively low levels of GRO-
expression at P1 (B) and higher levels of
expression in some cells at P8 (arrows). Scale bar, 30 µm.
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DISCUSSION |
Oligodendrocyte precursor proliferation is spatially and
temporally regulated in the vertebrate CNS. How such local control of
proliferation is mediated is currently unclear. Here we show that the
chemokine GRO- synergizes with PDGF to enhance rat oligodendrocyte precursor proliferation. GRO- is produced by a subset of astrocytes and neurons in a developmentally regulated pattern. In
vitro, cultured astrocytes secrete biologically active GRO-,
whereas in vivo, the location of GRO-+ cells in spinal
cord white matter is developmentally regulated. The proliferative
response of oligodendrocyte precursors to GRO- and PDGF is dependent
on the concentration of both ligands and the maturational state of the
cells. Below a threshold concentration of PDGF, GRO- has no effect,
whereas the maximal proliferative response occurs in a clearly defined GRO- concentration range.
The interaction between GRO- and PDGF suggests a novel mechanism for
the precise local control of oligodendrocyte precursor proliferation.
As an immediate response gene, GRO- facilitates transient local
bursts of oligodendrocyte precursor proliferation in the presence of
threshold levels of PDGF. Thus, stimuli that induce the expression of
GRO- would promote the local proliferation of oligodendrocyte
precursors. This hypothesis is consistent with the known requirements
of PDGF for spinal cord oligodendrogenesis (Calver et al., 1998) and
may explain the elevation of oligodendrocyte precursor proliferation
seen during specific stages of early development (Calver et al.,
1998).
The inducers of GRO- expression in the CNS remain to be identified.
In an astrocyte cell line, IL-1 and tumor necrosis factor- (TNF-) induce expression of the related CXC chemokine IL-8
(Anisowicz et al., 1991; Kasahara, 1991), and TNF- is known to be
expressed in the CNS under specific conditions (Liu, 1993). One well
characterized inducer of GRO- is PDGF (Cochran et al., 1983).
Because both PDGF and GRO- are required for maximal oligodendrocyte
precursor proliferation, it is an attractive hypothesis that PDGF
stimulates astrocyte production of GRO-. Focal elevations in PDGF as
a result of astrocyte or neuronal stimulation would therefore provide
both threshold levels of the mitogen and induction of GRO-, leading to a localized amplification of oligodendrocyte proliferation.
Expression of GRO- may also regulate local elevated levels of
oligodendrocyte precursor proliferation in the adult CNS. Enhanced oligodendrocyte precursor proliferation is seen adjacent to
demyelinating lesions (Raine et al., 1988; Prineas et al., 1993).
Although the expression of GRO- adjacent to multiple
sclerosis plaques has yet to be addressed, GRO- is locally
elevated in experimental autoimmune encephalitis, a model for multiple
sclerosis (Glabinski et al., 1997). Several other pathological
conditions result in elevated expression of GRO-, including ischemia
(Liu, 1993), infection (Spanaus, 1997), and autoimmune demyelination
(Glabinski et al., 1997), although the concomitant proliferation of
oligodendrocyte precursors has not been assayed. Chemokines of the CXC
family may play other roles in the nervous system in addition to
enhancing oligodendrocyte precursor proliferation. Properties
consistent with directing astrocyte migration (Heesen et al., 1996) and
supporting the survival of distinct neuronal populations (Araujo and
Cotman, 1993) have been described.
The finding that GRO- enhances oligodendrocyte precursor
proliferation suggests novel therapies in which GRO- functions are
enhanced to boost oligodendroglial progenitor proliferation and to
improve neurological recovery after demyelination. Alternatively, dysregulated chemokine expression might contribute to abnormal glial
proliferation that occurs in gliosis and tumors. In conclusion, the
regulation of oligodendrocyte precursor proliferation by synergy between GRO- and PDGF provides a novel mechanism for the precise regulation of glial proliferation in the developing vertebrate CNS.
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FOOTNOTES |
Received Aug. 3, 1998; revised Oct. 5, 1998; accepted Oct. 7, 1998.
This work was supported by National Institutes of Health Grants NS
36674 (to R.H.M.), 32151 (to R.M.R.), and RG2838 from the National
Multiple Sclerosis Society. M.T. was supported by a Multiple Sclerosis fellowship. We thank Kim Dyer for outstanding assistance.
Correspondence should be addressed to Robert H. Miller, Department of
Neurosciences, Case Western Reserve University School of Medicine,
10900 Euclid Avenue, Cleveland, OH 44106.
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Top
Abstract
Introduction
