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The Journal of Neuroscience, March 1, 2002, 22(5):1562-1572
Cytokines Regulate Microglial Adhesion to Laminin and Astrocyte
Extracellular Matrix via Protein Kinase C-Dependent Activation of the
 6 1 Integrin
Richard
Milner and
Iain L.
Campbell
Department of Neuropharmacology, The Scripps Research Institute, La
Jolla, California 92037
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ABSTRACT |
Microglia are highly plastic cells that participate in inflammatory
and injury responses within the CNS and that can migrate extensively after activation. Because astrocytes and their
extracellular matrix (ECM) form a large part of the CNS parenchyma, we
undertook to study the adhesive interactions between microglia and
these substrates in vitro. In contrast to
oligodendrocyte precursor cells, microglia formed only weak
interactions with astrocytes and their ECM. On specific ECM substrates
the microglia adhered strongly to fibronectin, vitronectin, and plastic
but only weakly to laminin. Microglial adhesion to laminin was
increased significantly by the proinflammatory cytokines TNF,
IFN- , and IFN- but was decreased by TGF- 1, with the TGF-1
effect being dominant over the other cytokines. Fluorescence-activated
cell sorting (FACS) analysis and immunoprecipitation showed that
microglia constitutively express the 61 integrin, a well
characterized laminin receptor, and that 61 expression levels did
not change after cytokine treatment. Function-blocking studies showed
that microglial adhesion to laminin is mediated entirely by the
61 integrin, strongly suggesting that the cytokine regulation of
adhesion to laminin is mediated by changes in the activation state of
61. Analysis of signaling pathways revealed that activation of
61 is mediated by a PKC-dependent mechanism. In light of the
evidence that laminin expression is upregulated after CNS injury, the
findings suggest that cytokine regulation of microglial adhesion to
laminin may play a fundamental role in determining the extent of
microglial infiltration into and retention at the site of injury.
Key words:
microglia; astrocytes; extracellular matrix; laminin; integrin; cytokine; inflammation; activation; CNS
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INTRODUCTION |
Cell adhesion to the surrounding
extracellular matrix (ECM) or to neighboring cells is a vital function
necessary for cell survival, migration, proliferation, and
differentiation. Ultimately, it helps to determine the complex spatial
cell-cell relationships that are established during development and
that continue to be tightly regulated throughout adult life (Adams and
Watt, 1993 ; Sastry and Horwitz, 1996; Hynes, 1999). In the adult the
importance of regulation of cell adhesion is nowhere more apparent than
in the cells involved in mounting a response to infection or injury, because these cells have to attach and then detach from distinct biological substrates at different stages of the inflammatory process
(Dustin and Springer, 1991; Diamond and Springer, 1994). In the CNS a
principal cell that fulfills this role is the microglial cell.
Microglia behavior is very dynamic and can vary from a quiescent phenotype to an activated, migratory cell that phagocytoses tissue debris and triggers responses involved in the recruitment of other inflammatory cells (Kreutzberg, 1996; Rezaie and Male, 1999).
For microglia and other inflammatory cells to be recruited to a lesion,
the cells have to acquire migratory ability. Adhesive interactions with
the tissue in which they are responding are critical determinants of
migratory behavior (Huttenlocher et al., 1995; Lauffenburger and
Horwitz, 1996). In the CNS the astrocytes and their associated ECM form
a large part of the potential substrate that microglia will engage
during migration; therefore, it is important to understand the
molecular basis of the interaction between microglia and astrocytes and
their ECM. This becomes more relevant considering that brain injury
induces astrocytosis and upregulated expression of many ECM molecules,
including fibronectin (Egan and Vijayan, 1991; Pasinetti et al., 1993),
laminin (Liesi et al., 1984; Frisen et al., 1995), vitronectin (Sobel
et al., 1995; Niquet et al., 1996), and several proteoglycans (Laywell and Steindler, 1991; Bovolenta et al., 1992). In addition, many cytokines also are upregulated after CNS injury, and these molecules can modulate different aspects of glial behavior, including cell adhesion and migration (Feuerstein et al., 1998; Raivich et al., 1999).
Our present understanding of what regulates
microglial-astrocyte adhesive interactions is not well defined.
Previous studies have been contradictory and show that microglia can
display both weak (Gebicke-Haerter et al., 1989; Ben-Hur et al., 1998)
and strong attachment to astrocytes (Tanaka et al., 1999; Toku et al.,
1999). This variation in cell behavior may be explained by the
observation that soluble factors in serum such as lipopolysaccharide (LPS; Gebicke-Haerter et al., 1989) regulate this adhesive interaction (Kloss et al., 2001). The overall objective of our study was to examine
the nature of microglial-astrocyte interactions under serum-free
conditions, thus reducing the potential of influence by extrinsic
factors. Specifically, we wished to (1) examine microglial behavior on
an astrocyte monolayer and on astrocyte ECM, (2) investigate microglial
adhesion to individual ECM molecules produced by astrocytes in
vivo, (3) determine the influence of cytokines on these processes, and (4) elucidate the role of specific signal transduction pathways in
microglial adhesive interactions.
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MATERIALS AND METHODS |
Cell culture. Mixed glial cultures were prepared as
described previously (Milner and ffrench-Constant, 1994) with a
technique modified from McCarthy and de Vellis (1980). Briefly,
forebrains from postnatal mice (days 0-2) were stripped of meninges,
chopped into small chunks, and dissociated in papain before being
cultured for ~10 d on poly-D-lysine-coated (Sigma, St.
Louis, MO) T75 tissue culture flasks (Falcon, Franklin Lakes, NJ) in
DMEM (Sigma) supplemented with 10% fetal calf serum (FCS; Sigma).
After establishment of the astrocyte monolayer, the flasks were shaken
for 1 hr to obtain the loosely attached microglia. Then the microglia
were cultured in serum-free N1 medium (DMEM supplemented with N1;
Sigma) and used in adhesion assays or for analysis of integrin
expression by fluorescence-activated cell sorting (FACS) or immunoprecipitation.
Preparation of astrocyte extracellular matrix. Mixed glial
cultures were prepared in poly-D-lysine-coated 24-well
plates (Nunc, Naperville, IL) as described above and maintained in DMEM
containing 10% FCS. Then 1 week after the astrocytes became confluent,
the cells were lysed in water for 30 min at 37°C. The excess cell debris was removed by washing the substrate several times in PBS; the
remaining ECM substrate was stored in PBS at 4°C and used within
2 d of preparation.
Antibodies and peptides. The following antibodies used in
immunoprecipitations, FACS analysis, and adhesion assays were obtained from PharMingen (San Diego, CA): the monoclonal antibodies specific for
the integrin subunits 1 (Ha2/5), 1 (Ha31/8), 4 (R1-2), 5
(5H10-27), and 6 (GoH3); the isotype control monoclonal antibody (anti-KLH); and the polyclonal donkey anti-rat phycoerythrin-conjugated secondary antibody. The RGD and RGE peptides were obtained from Invitrogen (San Diego, CA).
Cell surface labeling and immunoprecipitation. Cell surface
molecules were labeled with biotin by removing growth media, washing the cell layer twice with PBS, and then incubating the cells
with 0.1 mg/ml NHS-LC-Biotin in PBS (Pierce, Rockford, IL) at 37°C in
5% CO2 for 30 min. Cell monolayers were washed
three times with cell wash buffer (50 mM TRIS-HCl, pH 7.5, 0.15 M NaCl, 1 mM CaCl2,
1 mM MgCl2) and harvested with a cell
scraper before being washed twice more in suspension. Next the cells
were lysed in extraction buffer (cell wash buffer plus 0.5% NP-40, 300 µg/ml PMSF, 1 µg/ml pepstatin A, 2 µg/ml aprotinin, and 4 µg/ml
leupeptin) for 30 min on ice, followed by trituration and
centrifugation at 14,000 rpm at 4°C to remove the insoluble fraction.
The supernatants were precleared in a 1 hr incubation with 30 µl of
protein G-agarose (Amersham Pharmacia Biotech, Piscataway, NJ) per
milliliter of cell lysate. Immunoprecipitations were performed
overnight at 4°C on a rotating platform, using the GoH3 monoclonal
antibody at 1:100 dilution. The immune complexes were collected by
incubation with 30 µl of protein G-agarose beads (Roche Diagnostics,
Mannheim, Germany) for 2 hr, after which time the beads were washed
five times in immunoprecipitation wash buffer [identical to the cell wash buffer except for a higher salt concentration (0.5 M
NaCl) and the addition of 1% NP-40]. Integrins then were separated
from the beads by boiling in nonreducing SDS sample buffer for 5 min before being analyzed by SDS-PAGE on 7.5% resolving gels (Invitrogen) under nonreducing conditions. Proteins were electroblotted for 3 hr
onto nitrocellulose (Invitrogen), blocked for 1 hr with 3% BSA in TBS
(10 mM Tris-HCl and 0.15 NaCl, pH 8.0) containing 0.1% Tween 20 (Sigma), and probed with streptavidin-HRP (Amersham Pharmacia Biotech) for 1 hr. Then the membranes were washed extensively in TBS,
and the proteins were identified with the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech) according to the manufacturer's instructions.
FACS analysis. Microglia were cultured in six-well plates
(Nunc) in serum-free N1 medium in the presence or absence of the following cytokines: interleukin-3 (IL-3; 1 ng/ml; R&D Systems, Minneapolis, MN), IL-6 (0.2 ng/ml; R&D), tumor necrosis factor (TNF; 40 ng/ml; Genentech, San Francisco, CA), transforming growth factor-1
(TGF-1; 2 ng/ml; R&D), interferon- (IFN-;
103 U/ml; Invitrogen), and IFN- (5 U/ml = 1.6 ng/ml; R&D). After a 12 hr incubation with these
cytokines the microglia were scraped off the tissue culture plates and
centrifuged before being blocked in suspension with PBS containing 5%
normal goat serum for 30 min on ice. Then the microglia were
transferred to wells within a round-bottom 96-well plate (Nunc) and
incubated with the primary rat anti-mouse monoclonal antibodies (1:100
dilution) for 1 hr on ice. The cells were washed twice in the blocking
buffer before being labeled with donkey anti-rat-PE conjugate (1:100
dilution; PharMingen) for 1 hr on ice. Next the cells were washed
twice in the blocking buffer before being resuspended in the fixing solution, 2% formaldehyde in PBS. The fluorescence intensity of the
labeled microglia was analyzed with a Becton Dickinson (San Diego, CA)
FACScan machine, with 10,000 events recorded for each condition.
Adhesion assays. All substrates were prepared by coating the
central area of the wells within 24-well tissue culture dishes (Nunc)
with 25 µl of ECM solution (10 µg/ml) for 2 hr at 37°C. Bound
substrates were washed twice with serum-free N1 medium immediately before the addition of the cells. Microglia (prepared as described above) were centrifuged, resuspended in N1 medium, and applied to the
substrates in a 25 µl drop for 1 hr at 37°C. The adhesion assay was
stopped by adding 1 ml of DMEM to each well and washing off loosely
attached cells. The attached cells then were fixed in 4%
paraformaldehyde in PBS for 20 min. Adhesion was quantified by counting
all attached cells under phase microscopy; the results were expressed
as a percentage of the number of cells adhering under control
conditions for each substrate. Within each experiment each condition
was performed in duplicate; the results represent the mean ± SEM
of three experiments. Statistical significance was assessed by using
the Student's paired t test in which p < 0.05 was defined as statistically significant. Murine laminin-1, bovine
fibronectin, and bovine vitronectin (all obtained from Sigma) were
diluted to the 10 µg/ml coating concentration in PBS. Mixed ECM
substrates were prepared by using a coating solution containing 10 µg/ml of each protein for which the level of binding to the
substratum was determined as described below. In antibody or
peptide-blocking experiments the antibody or peptide was added to the
medium that was used to resuspend the cells after their final wash and
therefore was present when the cells were added to the substrate and
throughout the experiment. The RGD or RGE peptides were used at a
concentration of 0.1 mg/ml, and the monoclonal antibodies Ha2/5 and
GoH3 and isotype control were used at a concentration of 5 µg/ml. In
experiments designed to investigate the influence of cytokines on
microglial adhesion to laminin, the individual cytokines were added to
the medium that was used to resuspend the microglia to give the
following final concentrations: IL-3 (1 ng/ml), IL-6 (0.2 ng/ml), TNF
(40 ng/ml), TGF-1 (2 ng/ml), IFN-
(103 U/ml), and IFN- (5 U/ml = 1.6 ng/ml). In the 12 hr experiments involving cytokines, 0.5 ml of N1
medium containing the cytokine was added around the 25 µl drop of
cells that had been allowed to attach for 1 hr. To assess microglial
adhesion to the astrocyte monolayer, we grew astrocytes to
confluence in 24-well plates before they were shaken mechanically for 1 hr to remove microglia. Then fresh microglia were resuspended in N1
medium containing individual cytokines and/or individual integrin
function-blocking reagents, plated on top of the astrocyte monolayer,
and cultured in serum-free N1 medium. After 12 hr the medium was
removed, 1 ml DMEM was added to the wells, and loosely attached
microglia were washed off. Then the cells were fixed in 4%
paraformaldehyde in PBS for 20 min, and microglia adhesion was
quantified by counting all attached microglia under phase microscopy.
In experiments to investigate the role of the signaling molecules,
phorbol-12-myristate 13-acetate (PMA; Sigma) was used at concentrations
of 1, 3, and 10 nM. Calphostin C (Alexis, San
Diego, CA) was used at 10, 30, and 100 nM; H89
(Alexis) was used at 10, 30, and 100 nM. In
experiments aimed at investigating the role of de novo
protein synthesis in response to inflammatory cytokines, cycloheximide
(Sigma) was used at a concentration of 10 µg/ml. Photomicrographs of
microglia were taken on a Nikon Diaphot inverted microscope with phase optics.
Quantification of ECM substrate deposition. To ensure that
equivalent amounts of ECM proteins were deposited on the tissue culture
plastic during the mixed ECM substrate preparation, we quantified
protein deposition by ELISA. ECM substrates were prepared by coating
wells of a 96-well tissue culture plate (Nunc) with 50 µl of ECM
solution for 2 hr at 37°C. The ECM solution contained single ECM
proteins at a range of concentrations (0-30 µg/ml) or, as described
above, a mixture of fibronectin with laminin or vitronectin with
laminin, all at a concentration of 10 µg/ml. Then the bound
substrates were washed twice with PBS before incubation with a 1% BSA
blocking solution in PBS for 30 min at 37°C. The substrates were
incubated with primary rabbit antibodies reactive for fibronectin,
laminin (both from Sigma), or vitronectin (Chemicon, Temecula, CA)
diluted 1:1000 in blocking solution for 1 hr at 37°C. After two
washes with PBS the substrates were incubated with goat anti-rabbit
alkaline phosphatase (Sigma) diluted 1:500 in blocking solution for 1 hr at 37°C before being washed four times in PBS. Next the substrates
were incubated with the alkaline phosphatase substrate
p-nitrophenyl phosphate (Vector Laboratories, Burlingame,
CA), and colorimetric intensity was measured in a microplate reader
(Dynex Technologies, Chantilly, VA). In each experiment standard
concentration curves for the single ECM proteins fibronectin,
vitronectin, and laminin were plotted by using a range of coating
concentrations (0-30 µg/ml) with duplicates for each concentration.
Absorbance values for each protein within the mixed ECM substrate (in
triplicate) then were compared with the absorbance value obtained with
a coating solution of 10 µg/ml of the single protein. Over three
experiments this showed that the presence of 10 µg/ml laminin in the
coating solution did not reduce the deposition of either fibronectin
(122 ± 17.7% of the amount of fibronectin deposited with no
laminin present) or vitronectin (88.7 ± 15.1% of the amount of
vitronectin deposited with no laminin present). In addition, the
deposition of laminin was not reduced by the presence of either
fibronectin (102 ± 25% of the amount of laminin deposited with
no fibronectin present) or the presence of vitronectin (148 ± 32.3% of the amount of laminin deposited with no vitronectin present).
Therefore, these results generally do not support the possibility that
the effect of laminin is to suppress the binding of vitronectin or
fibronectin to the plastic substrate.
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RESULTS |
Microglia adhere weakly to an astrocyte monolayer
To investigate the adhesive interactions between microglia and
astrocytes, we prepared mixed glial cultures from neonatal mouse brain
according to the method of McCarthy and de Vellis, as has been
described previously (McCarthy and de Vellis, 1980; Milner and
ffrench-Constant, 1994). These cultures showed a typical stratification
of cell types, with a base layer of astrocytes and a "top" layer
consisting of oligodendrocyte precursor cells and microglia.
Oligodendrocyte precursor cells and microglia differed fundamentally in
their interactions with the astrocyte monolayer (Fig.
1A). Oligodendrocyte
precursor cells had a phase-dark appearance indicating a firm
attachment to the underlying astrocytes and had several long process
extensions. In contrast, microglia showed a rounded-up,
phase-bright appearance and were either loosely attached or
actually floating around on top of the astrocytes. This differential
adhesion of oligodendrocyte precursors and microglia to the astrocyte
monolayer is well described and actually forms the basis of an
established protocol to separate the two different cell types (Hardy
and Reynolds, 1991; Milner et al., 1996; Ben-Hur et al., 1998). In this
protocol the mechanical shaking of the culture flasks first detaches
microglia from the astrocyte monolayer (1-2 hr) and then much later
detaches oligodendrocyte precursors from the astrocytes (15-24 hr).
When microglia were plated onto an ECM substrate derived from
astrocytes, they showed the same adhesive behavior as on the astrocyte
monolayer and were rounded-up, poorly spread cells (Fig.
1B).
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Figure 1.
Microglial adhesion and morphology on different
substrates. Microglial behavior in established mixed glial cultures
(A) was compared with the behavior of microglia
that had been cultured for 12 hr on astrocyte ECM
(B) and on uncoated plastic
(C). Scale bar, 35 µM. Note that
microglia cultured on the astrocyte monolayer (arrows)
and on astrocyte ECM were only loosely attached, phase-bright,
rounded-up cells, whereas on uncoated plastic the microglia were
strongly adherent and well spread. This is in contrast to the behavior
of oligodendrocyte precursor cells (arrowheads), which
attached very well to the astrocyte monolayer and extended long thin
processes.
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In contrast to adhesion to astrocytes, when microglia were plated onto
uncoated plastic, they strongly attached and showed a fully adherent
and spread morphology within 30 min (Fig. 1C). By
comparison, oligodendrocyte precursors did not attach to plastic and
remained rounded-up and floating in the medium. This raises the
fundamental question as to why microglia, which are such intrinsically adherent cells, attach strongly to a nonspecific plastic substrate but
only weakly to the astrocyte monolayer. Considering that adhesive interactions between microglia and astrocytes are likely to be a major
factor regulating microglial behavior in vivo, it becomes important to understand the molecular basis of the interaction between
these two cell types and to understand how this might be regulated.
Microglia adhere weakly to laminin
After injury to the CNS, astrocytes show upregulated expression of
many ECM molecules, and these molecules also are expressed by
astrocytes grown in culture. These include the glycoproteins fibronectin (Liesi et al., 1986; Oh and Yong, 1996), vitronectin (Oh
and Yong, 1996), and laminin (Liesi et al., 1983; Giotta et al., 1986)
and the proteoglycans (Ard and Bunge, 1988). As a first step to
investigate whether any of these ECM molecules might regulate microglia-astrocyte interactions, we examined microglial adhesion and
spreading characteristics on individual ECM substrates. When 1 hr
adhesion assays were performed, microglia adhered well to the control
plastic substrate and the ECM substrates fibronectin and vitronectin,
but microglial adhesion to laminin was much weaker (see Fig.
3A). In fact, <15% of available microglia adhered to the
laminin substrate (12.9 ± 1.4% compared with 53.7 ± 12.1% adhesion to plastic; p < 0.01), and, as seen in Figure
2, the nature of this adhesion was weak,
with microglia on laminin showing a phase-bright rounded-up appearance
(reminiscent of microglial behavior on astrocytes) in contrast to the
well spread phase-dark microglial morphology on the other substrates.
Therefore, these experiments showed that laminin is a relatively
nonadherent substrate for microglia under these conditions. To
investigate further the nature of microglial interactions with ECM
molecules, we plated microglia into six-well plates in which part of
the surface had been coated with the different ECM molecules
fibronectin, vitronectin, and laminin. After 24 hr of culture we
observed that the areas coated with laminin were almost devoid of any
microglia and represented bare patches on the dish, whereas microglia
adhered and spread well on fibronectin and vitronectin and uncoated
plastic in these longer-term assays. This point is demonstrated in
Figure 3C, which shows an
interface of laminin and uncoated plastic in which microglia are packed
at high density right up to the border of the laminin but do not
transgress onto the laminin substrate
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Figure 2.
Microglial behavior on different ECM substrates.
Microglia were purified from mixed glial cultures, as described in
Materials and Methods, and then plated onto uncoated plastic
(A), laminin (B),
fibronectin (C), or vitronectin
(D). Scale bar, 35 µM. Note that
microglia attached and spread well on all substrates except for
laminin, where they remained rounded-up and phase-bright.
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Figure 3.
A, B, Microglial cell adhesion to
different ECM substrates. Microglia were purified from mixed glial
cultures, as described in Materials and Methods; then 1 hr adhesion
assays were performed, and adhesion was quantified by counting the
cells under phase microscopy. All data points are expressed as a
percentage of cell adhesion to vitronectin and represent the mean ± SEM of three experiments. Note in A that plastic,
fibronectin (Fn), and vitronectin (Vn)
all promoted extensive microglial adhesion, but <15% of microglia
adhered to laminin (Ln). Note in B that
the presence of laminin in a mixed substrate reduced the level of
microglial adhesion to the level of adhesion to laminin alone.
C, Microglia were plated onto a plastic substrate, part
of which had been coated with laminin, and were cultured for 24 hr.
Scale bar, 35 µM. Note that microglia adhered and spread
well on plastic, but not on laminin, and that the cells formed a sharp
interface at the laminin-plastic border.
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The adhesion assays showed that fibronectin and vitronectin are
adhesive substrates for microglia; considering that fibronectin in
addition to laminin is present in the ECM synthesized by astrocytes in vitro (Liesi et al., 1986; Oh and Yong, 1996), it is
surprising that microglia are not strongly adherent to the astrocyte
monolayer and the astrocyte ECM by way of the fibronectin interaction.
One possibility is that laminin exerts a dominant anti-adhesive effect on microglial-astrocyte interactions in the mixed glial cultures. To
investigate this possibility, we performed adhesion assays by using
mixed ECM substrates. To ensure that equivalent amounts of ECM proteins
were deposited on the tissue culture plate during the mixed ECM
substrate preparation, we quantified protein deposition by ELISA. As
described in Materials and Methods, this showed that there was no
significant difference in the final amount of the proteins fibronectin
or vitronectin deposited on the tissue culture plate when prepared by
using either a 10 µg/ml solution of the single protein or a mixed
solution containing 10 µg/ml each of fibronectin and laminin or
vitronectin and laminin. As shown in Figure 3B, the outcome
of this experiment was very clear; although microglia adhered strongly
to fibronectin and vitronectin, the presence of laminin reduced
microglial adhesion to both of these substrates (from 81.9 ± 7.3 to 12.3 ± 1.3%, p < 0.01 on fibronectin; from
100% control to 14.1 ± 1.7%, p < 0.001 on
vitronectin). In other words, laminin exerted a dominant anti-adhesive
effect on microglial adhesion.
Microglia use the 61 integrin to attach to laminin
To investigate whether microglia express any functional cell
surface receptors for laminin, we performed analysis by FACS and
immunoprecipitation by using the monoclonal antibody GoH3, specific for
the well characterized laminin receptor 61 integrin. By FACS
analysis we determined that microglia express high levels of the
61 integrin (Fig.
4A), and this was
confirmed by immunoprecipitation (see Fig. 7A).
Immunoprecipitation experiments also showed that the 11 integrin,
another laminin receptor, was not expressed by microglia (data not
shown).Once a potential laminin receptor on microglia was identified,
the next step was to address the role of this integrin in microglial
adhesion to laminin. Short-term microglial adhesion assays were
performed for 1 hr in the presence of different integrin
function-blocking reagents. As shown in Figure 4B,
microglial adhesion to laminin was reduced to baseline levels, both by
the 1 integrin monoclonal antibody Ha2/5 (12.7 ± 2.5% of
control; p < 0.001) and by the 6 monoclonal
antibody GoH3 (14.4 ± 2.3% of control; p < 0.001), but not by RGD peptides or the 4 or 5 monoclonal
antibodies. This shows that microglial adhesion to laminin is mediated
by 1 integrins and that 61 is the major functional laminin
receptor expressed by microglia.
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Figure 4.
Characterization of functional laminin receptors
on microglia. A, Purified microglial cultures were
analyzed for expression of the 6 integrin subunit by FACS, as
described in Materials and Methods. Note that microglia express the
6 integrin subunit. B, Identification of integrins
involved in microglial adhesion to different ECM substrates. The 1 hr
adhesion assays to fibronectin, vitronectin, or laminin were performed
as described in Materials and Methods in the presence of RGE peptides
(control), RGD peptides, isotype control monoclonal antibody, or
monoclonal antibodies specific for the 1 (Ha2/5), 4 (R1-2), 5
(5H10-27), or 6 (GoH3) integrin subunits. Adhesion is expressed as
a percentage of cell adhesion to each ECM substrate under control
conditions (RGE peptides); all data points represent the mean ± SEM of three experiments. Note that microglial adhesion to laminin was
abolished almost totally by both the anti-1 and anti-6
antibodies, but none of the other reagents had any significant
effect.
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Microglial adhesion to laminin is regulated by cytokines
The results presented so far indicate something of a paradox;
microglia do not attach well to laminin, but they express high levels
of the well recognized laminin receptor 61. Previous work has
shown that the macrophage, the equivalent cell type outside the CNS, is
also weakly adherent to laminin but can be induced to a more adherent
phenotype by the presence of IFN- (Shaw and Mercurio, 1989). On this
basis we set out to determine whether the interaction between microglia
and laminin was modified by cytokines also. Adhesion assays were
performed in the presence of individual cytokines. In the first
instance these assays were performed for 1 hr, and the results showed
no significant differences in microglial adhesion to laminin (data not
shown). However, when the adhesion assays were extended to 12 hr, clear
differences were observed both at the levels of number of cells
adherent and also on the microglial
morphology. As shown in the summary in Figures 5 and
6, we found that over the 12 hr
incubation period only a fraction of the total microglia was
adherent to laminin under control conditions (no cytokines), and the
cells that did attach were rounded-up. Interestingly, TGF-1 reduced
the amount of microglial adhesion to laminin relative to control (no
cytokine) (11.2 ± 2.3% compared with 17.9 ± 1.5%
control; p < 0.05), and the cells were spread even
less than under control conditions. In contrast, the inflammatory
cytokines TNF, IFN-, and IFN- significantly increased the number
of microglia adherent to laminin (to 44.4 ± 5.7%,
p < 0.05; 58.6 ± 7.8%, p < 0.02; and 84.6 ± 5.8%, p < 0.01, respectively,
relative to 17.9 ± 1.5% control) and induced a dramatic
morphological change so that the microglia became well spread. IL-3 and
IL-6 had no noticeable effect. Because these assays showed that
microglial adhesion to laminin could be regulated by cytokines in both
a positive and a negative direction, additional adhesion assays were
performed to determine the effect of combining different cytokines.
Because IFN- and IFN- caused the greatest induction of adhesion
to laminin and TGF-1 had the opposite effect, these cytokines were
used in combination to determine which effect would predominate. As
shown in Figure 6B, TGF-1 reduced microglial adhesion to laminin both in the presence of IFN- (9.0 ± 1.1% compared with 52.5 ± 6.8% for IFN- alone; p < 0.02) and IFN- (9.2 ± 4.1% compared with 71.3 ± 5.9% for IFN- alone; p < 0.01). In fact, in both
of these conditions microglial adhesion to laminin was reduced to the
same level as TGF-1 alone, showing that TGF-1 exerted a dominant
effect over the proinflammatory cytokines. This anti-adhesive effect of
TGF-1 was observed at a range of concentrations from 2.0 to as low
as 0.1 ng/ml.
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Figure 5.
The influence of cytokines on microglial adhesion
to laminin. Microglia were purified from mixed glial cultures, as
described in Materials and Methods, and then plated onto laminin in the
presence of no cytokines (A), TGF-1
(B), IFN- (C), or
IFN- (D). Scale bar, 35 µM. Note
that after 12 hr in culture the microglia under control conditions were
rounded-up but had some small process extensions. In the presence of
TGF-1 the microglia were totally rounded-up and mostly were floating
around the dish. In contrast, in the presence of either IFN- or
IFN- the microglia became more adherent to laminin and adopted a
more flattened and spread morphology.
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Figure 6.
The influence of different cytokines on microglial
adhesion to laminin. Microglia were purified from mixed glial cultures,
as described in Materials and Methods; then 12 hr adhesion assays to a
laminin substrate were performed, and adhesion was quantified by
counting the cells under phase microscopy. All data points are
expressed as a percentage of cell adhesion to plastic and represent the
mean ± SEM of three experiments. A, Microglial
adhesion assays were performed in the presence of IL-3 (1 ng/ml), IL-6
(0.2 ng/ml), TNF (40 ng/ml), TGF-1 (2 ng/ml), IFN-
(103 U/ml), or IFN- (5 U/ml = 1.6 ng/ml).
Note that TGF-1 reduced microglial adhesion to laminin relative to
control, whereas the inflammatory cytokines TNF, IFN-, and IFN-
all increased the adhesion to laminin. B, Microglial
adhesion assays were performed in the presence of TGF-1 (2 ng/ml),
IFN- (103 U/ml), IFN- (5 U/ml = 1.6 ng/ml), or combinations of IFN- + TGF-1 or IFN- + TGF-1
(same concentrations as when used alone). Note that the anti-adhesive
effect of TGF-1 predominated over the proadhesive effect of the
IFNs.
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Having determined that several inflammatory cytokines increase the
adhesion of microglia to laminin, we wanted to investigate the
underlying molecular basis for this change in behavior. There are three
main possibilities: first, that the expression level of the 61
integrin is regulated; second, that the inflammatory cytokines induce
upregulation of another laminin receptor; third, that the activation
state of the 61 integrin is increased by the inflammatory
cytokines, as has been shown before on macrophages (Shaw and Mercurio,
1989). To address the first of these possibilities, we cultured
microglia in the presence of the different cytokines for 12 hr and then
analyzed the 6 integrin expression levels. As shown in Figure
7A, the cytokines produced no
detectable change in 6 expression levels, as seen in
immunoprecipitation, or by FACS analysis. To address the second and
third possibilities together, we performed microglial adhesion assays
over 12 hr in media containing the cytokines shown to promote
microglial adhesion to laminin in the presence or absence of the
different integrin function-blocking antibodies. As shown in Figure
7B, the increased microglial adhesion to laminin induced by
IFN- or IFN- was reduced to background level both by the 1 and
the 6 function-blocking antibodies (anti-1, 2.9 ± 1.2%,
p < 0.01 and anti-6, 2.9 ± 1.4%,
p < 0.01 compared with 47.6 ± 5.8% isotype
control for IFN-; anti-1, 3.2 ± 0.9%, p < 0.01 and anti-6, 2.8 ± 1.0%, p < 0.01 compared with 62.7 ± 4.6% isotype control for IFN-) but was
not affected by RGD peptides or monoclonal antibodies against the 4
or 5 integrin subunits. This strongly suggests that the 61
integrin is solely responsible for mediating microglial adhesion to
laminin and that regulation of the activation of the 61 integrin
is most likely the primary mechanism whereby cytokines regulate
microglial adhesion to laminin.
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Figure 7.
Analysis of the 61 integrin and
its role in microglial adhesion to laminin. A, The
influence of cytokines on 61 integrin expression by microglia.
Purified microglia were cultured in serum-free medium for 12 hr in the
presence of different cytokines. Immunoprecipitations of
biotin-labeled cell surface proteins were performed as described in
Materials and Methods, after which the proteins were separated on
nonreducing gels. Note that the cytokines that were tested did not
alter significantly the level of 61 integrin expression by
microglia. B, Contribution of the 61 integrin to
microglial adhesion to laminin in the presence of IFNs. The 12 hr
adhesion assays to laminin were performed as described in Materials and
Methods in the presence of RGE peptides (control), RGD
peptides, isotype control monoclonal antibody, or monoclonal antibodies
specific for the 1 (Ha2/5), 4 (R1-2), 5 (5H10-27), or 6
(GoH3) integrin subunits and were performed under three different
conditions: control, IFN-, and IFN-. Adhesion is expressed as a
percentage of cell adhesion to plastic under control conditions; all
data points represent the mean ± SEM of three experiments. Note
that, with no cytokine present, microglial adhesion to laminin was
abolished almost totally by both the anti-1 and anti-6 antibodies
and that the increased adhesion induced by IFN- or IFN- also was
reduced to background level by both the anti-1 and anti-6
antibodies. C, Contribution of the 4 and 5
integrins to microglial adhesion to fibronectin. Note that IFN- and
IFN- do not influence microglial adhesion to fibronectin and
that this adhesion is inhibited by RGD peptides and antibodies to the
1, 4, and 5 integrin subunits but is unaffected by the
anti-6 antibody. D, Time course of microglial
adhesion to ECM substrates under the influence of IFN-. Microglial
adhesion assays to fibronectin and laminin were performed in the
presence of IFN- for 1, 2, 4, and 6 hr in the absence or presence of
the protein synthesis inhibitor cycloheximide (10 µg/ml). Adhesion is
expressed as a percentage of cell adhesion to vitronectin after 6 hr;
all data points represent the mean ± SEM of three experiments.
Note that microglial adhesion to laminin increases with time in the
presence of IFN-, and this increased adhesion is blocked effectively
by cycloheximide.
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These results are very similar to those of Shaw et al. (1990), who
showed that macrophage adhesion to laminin increased after exposure to
IFN-, with the response taking up to 8 hr for the maximal effect
(Shaw and Mercurio, 1989). Paradoxically, the activation of cell
surface receptors is normally a fast process taking place within
minutes. One possible explanation for our findings would be that the
IFNs stimulate the synthesis of another factor that is responsible for
directly activating the 61 integrin expressed by microglia. To
test this hypothesis, we performed microglia adhesion assays in the
presence of IFN- or IFN- over a 6 hr time course with or without
the addition of the protein synthesis inhibitor cycloheximide. As shown
in Figure 7D, IFN- increased microglial adhesion to
laminin over the 6 hr time course, and this was blocked effectively by
the presence of cycloheximide (14.2 ± 1.5% in the presence of
cycloheximide compared with 36.7 ± 3.9% cell adhesion with no
cycloheximide present; p < 0.02). Similar results also
were obtained with IFN- (data not shown). To address the possibility
that the cycloheximide treatment over 6 hr may alter integrin
expression by microglia, we quantified integrin expression levels by
FACS analysis. In this experiment the microglia were cultured for 6 hr
with or without IFN-, each in the presence or absence of 10 µg/ml
cycloheximide. In three separate experiments the presence of
cycloheximide had no significant effect on the expression level of
either the 5 or 6 integrin subunits during the 6 hr time course
(data not shown). These results support the hypothesis that the ability
of the IFNs to increase microglial adhesion to laminin is an indirect
effect, involving the synthesis of an intermediate factor that itself
increases the activation state of the 61 integrin.
Activation of the 61 integrin involves a PKC
signaling pathway
Previous work on macrophages has shown that cell adhesion to
laminin is regulated by a change at the level of activation state of
the 61 integrin and that this increased activation also can be
triggered by activators of protein kinase C pathways such as PMA
(Mercurio and Shaw, 1988; Shaw et al., 1990). To determine which
intracellular pathways in microglia are important for mediating the
changes in adhesion to laminin, we performed adhesion assays to
determine the effect of (1) PMA stimulation and (2) IFN- and IFN-
stimulation in the presence of specific inhibitors of PKC-mediated and
PKA-mediated pathways. As shown in
Figures 8 and 9A, microglial adhesion to laminin in 12 hr adhesion assays was enhanced markedly in
the presence of the PKC-activating molecule PMA (1 nM, 55.9 ± 7.5%, p < 0.05; 3 nM, 80.3 ± 9.8%, p < 0.02 relative to 19.6 ± 3.1% for control), and, as shown in
Figure 8B, PMA changed microglial morphology on
laminin from phase-bright, rounded-up cells to phase-dark, fully spread
cells. This proadhesive effect of PMA also was observed in much shorter
1 hr adhesion assays, suggesting that stimulation of the PKC signaling
pathway directly increased the activation state of the 61
integrin. To investigate whether 61 activation in response to
IFN- or IFN- is dependent on PKC-mediated or PKA-mediated
signaling pathways, we used the PKC-specific antagonist Calphostin C
(Jarvis et al., 1994) and the PKA-specific antagonist H89 (Savickiene
et al., 1999) in 12 hr adhesion assays. As shown in Figure
9B, the ability of both IFN- and IFN- to enhance
microglial adhesion to laminin was blocked by the PKC-specific
antagonist Calphostin C (15.9 ± 4.1 compared with 51.2 ± 6.4% control for IFN-, p < 0.05; 18.5 ± 4.0 compared with 63.9 ± 6.1% control for IFN-, p < 0.02), but not by the PKA-specific antagonist H89. In addition, the
PKC-specific antagonist reversed the enhanced spread morphology of
microglia exposed to IFN- (Fig. 8D). These results
make clear two points: first, that activating PKC pathways (by PMA)
leads to activation of the 61 integrin on microglia and, second,
that activation of microglial 61 by IFN- and IFN- involves
signaling via the PKC pathway.
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Figure 8.
Examining the influence of PKC-mediated signaling
on microglial adhesion to laminin. Microglia were purified from mixed
glial cultures, as described in Materials and Methods, and then plated
onto laminin in serum-free medium in the presence of no reagents
(A), 3 nM PMA
(B), 5 U/ml IFN- (C), or
5 U/ml IFN- plus 100 nM Calphostin C
(D). Scale bar, 35 µM. Note that
after 12 hr in culture the microglia under control conditions were
mostly phase-bright and rounded-up. In the presence of PMA the
microglia became more adherent to laminin and adopted a more flattened
and spread morphology. IFN- also increased the level of microglial
adhesion and spreading, whereas the PKC-specific antagonist Calphostin
C reversed this effect.
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Figure 9.
Investigating the role of PKA and PKC-mediated
signaling on microglial adhesion to laminin. Microglia were purified
from mixed glial cultures, as described in Materials and Methods; then
12 hr adhesion assays to a laminin substrate were performed, and
adhesion was quantified by counting the cells under phase microscopy.
All data points are expressed as a percentage of cell adhesion to
plastic and represent the mean ± SEM of three experiments.
A, Microglial adhesion assays were performed in the
presence of different concentrations of PMA (0, 1, and 3 nM). Note that PMA increased microglial adhesion to laminin
in a dose-dependent manner. B, Microglial adhesion
assays were performed in the presence of IFN-
(103 U/ml) or IFN- (5 U/ml = 1.6 ng/ml), and
the effects of specific inhibitors of PKA-mediated (H89; 100 nM) and PKC-mediated (Calphostin C; 100 nM)
signaling pathways were evaluated under each condition. Note that the
increased microglial adhesion to laminin, induced by both IFN- and
IFN-, was inhibited in both cases by the PKC-specific antagonist
Calphostin C, but not by the PKA antagonist H89.
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Microglial adhesion to astrocytes is regulated by cytokines
To investigate whether cytokines also modulate
microglial-astrocyte interactions, we examined mixed glial cultures in
serum-free conditions in the presence of different cytokines.
After 1 d of culture, clear differences in the morphology of
microglia became obvious. Control cultures showed microglia weakly
adherent, with some floating in the media. TGF-1 induced the
microglia to become even less adherent, with far more microglia
floating in the media. In contrast, the inflammatory cytokines TNF,
IFN-, and IFN- induced the microglia to become much more adherent
to the astrocytes and changed the microglial morphology from
rounded-up, phase-bright cells to phase-dark, well spread cells that
have long process extensions (Fig. 10).
This effect was quantified by performing adhesion assays over a 12 hr
time period and assessing microglial adhesion both to astrocytes and to
astrocyte ECM in the presence of the individual cytokines TGF-1,
IFN-, and IFN-. The contribution of the 61 integrin to
microglial adhesion also was assessed within the same experiment by
using the function-blocking antibodies against the 6 and 1
integrin subunits. As shown in Figure
11, the inflammatory cytokines IFN-
and IFN- increased microglial adhesion to astrocytes (to 70.2 ± 6.5%, p < 0.02, and 58.6 ± 4.8%, p < 0.02, respectively, compared with 19.8 ± 3.4% under control conditions) and increased microglial adhesion to
astrocyte ECM (to 58.2 ± 4.0%, p < 0.05, and
68.7 ± 4.7%, p < 0.05, respectively, compared
with 30.4 ± 5.1% under control conditions). In contrast, TGF-1 reduced microglial adhesion to astrocytes (from 19.8 ± 3.4 control to 12.6 ± 0.8%; p < 0.05) and to
astrocyte ECM (from 30.4 ± 4.7 control to 17.4 ± 3.2%;
p < 0.05). The 1 and 6 antibodies significantly
reduced microglial adhesion to astrocytes (from 19.8 ± 3.4%
under control conditions to 11.8 ± 2.1%, p < 0.05, and 10.3 ± 1.4%, p < 0.05, respectively)
and reduced microglial adhesion to astrocyte ECM (from 30.4 ± 4.7% under control conditions to 9.6 ± 1.1%, p < 0.02, and 11.3 ± 2.4%, p < 0.05, respectively). In addition, the 1 and 6 antibodies in large part
abolished the IFN--enhanced microglial adhesion to astrocytes (from
70.2 ± 6.5% under control conditions to 15 ± 2.3%,
p < 0.01, and 16.3 ± 2.8%, p < 0.01, respectively) and reduced microglial adhesion to astrocyte ECM
(from 58.2 ± 4.0% under control conditions to 14.8 ± 3.1%, p < 0.01, and 15.2 ± 2.2%,
p < 0.01, respectively). In a similar manner, the 1
and 6 antibodies also inhibited the IFN- enhanced microglial
adhesion to astrocytes (from 58.6 ± 4.8% under control
conditions to 14.4 ± 3.5%, p < 0.01, and
16.1 ± 2.4%, p < 0.01, respectively) and
reduced adhesion to astrocyte ECM (from 68.7 ± 5.1% under
control conditions to 15.4 ± 1.6%, p < 0.01, and 17.5 ± 2.4%, p < 0.02, respectively). In
these experiments RGD peptides or monoclonal antibodies specific for
the 4 or 5 integrin subunits had no significant effect on
microglial adhesion to either astrocytes or astrocyte ECM. In
conclusion, these experiments show that cytokines influence microglial
adhesion to astrocytes and astrocyte ECM in much the same way that they
regulate microglial adhesion to laminin; furthermore, they show that
the major part of this adhesion and its regulation is mediated by the
61 integrin.
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Figure 10.
The influence of different cytokines on
microglial adhesion to astrocytes. Mixed glial cultures were grown in
serum-free conditions in the presence of no cytokines
(A), TGF-1 (B), IFN-
(C), or IFN- (D). Scale
bar, 35 µM. Note that after 24 hr in culture the
microglia (arrows) under control conditions were
phase-bright and rounded-up. In the presence of TGF-1 the microglia
were also rounded-up and mostly were floating around the dish. In
contrast, in the presence of either IFN- or IFN- the microglia
were more adherent to the astrocyte monolayer and adopted a more spread
and well processed morphology.
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Figure 11.
Analysis of the role of the 61 integrin in
mediating microglial adhesion to astrocytes and astrocyte ECM in
response to cytokines. Microglia were purified from mixed glial
cultures, as described in Materials and Methods; then 12 hr adhesion
assays were performed either on an astrocyte (A)
or astrocyte ECM substrate (B). Within each
substrate, adhesion was performed under control conditions (no
cytokine), +TGF1 (2 ng/ml), +IFN- (10 3 U/ml), or
+IFN- (5 U/ml = 1.6 ng/ml). Each of these conditions contained
the isotype control antibody, the Ha2/5 (anti-1) antibody, or the
GoH3 (anti-6) antibody. Adhesion was quantified by counting
microglial cells under phase microscopy. All data points are expressed
as a percentage of cell adhesion to plastic and represent the mean ± SEM of three experiments. Note that, with no cytokine present,
microglial adhesion to astrocytes and astrocyte ECM is inhibited by
both the anti-1 and anti-6 antibodies and that the increased
adhesion induced by IFN- and IFN- is reduced to a background
level by both the anti-1 and anti-6 antibodies.
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DISCUSSION |
In this study we investigated the nature of adhesive interactions
between microglia and astrocytes and their associated ECM in
vitro. There were four main conclusions. First, microglia attached weakly to an astrocyte monolayer and astrocyte ECM. Second, microglia attached weakly to laminin relative to fibronectin and vitronectin, and
the effect of laminin was dominant, i.e., laminin reduced microglial
adhesion to the other ECM substrates. Third, microglial adhesion to
laminin and astrocytes was dependent on the activation state of the
61 integrin, and this was regulated by cytokines. Fourth, the
change in the activation state of the 61 integrin was shown to be
mediated by PKC-dependent signaling pathways.
Regulation of microglial adhesion to laminin
The observation that microglia adhere only weakly to an astrocyte
monolayer first was described >10 years ago (Gebicke-Haerter et al.,
1989) and represents a surprising finding given that, in the CNS,
astrocytes probably represent the most abundant substrate that
microglia are likely to interact with. The work presented here shows
that microglia have a weak affinity interaction with laminin, one of
the most abundant components of astrocyte ECM in vitro
(Liesi et al., 1983; Giotta et al., 1986). The behavior of microglia on
laminin is in contrast to that of other cells of the CNS, including
neurons, astrocytes, and oligodendroglial cells, all of which display
strong adhesive interactions with laminin (Reichardt and Tomaselli,
1991; Tawil et al., 1993; Letourneau et al., 1994; Milner et al., 1996,
1999). However, the behavior of microglia on laminin is very similar to
that of other inflammatory cells such as neutrophils and macrophages,
which show no adhesion to laminin under resting conditions but, when
activated, dramatically increase their adhesion to laminin. This has
been attributed to a regulation at the level of the activation state of
the 61 integrin (Wei et al., 1997). The evidence presented in
this paper strongly suggests that similar regulation occurs in
microglia within the CNS.
This work takes on more significance considering the observation that
laminin exerted a dominant anti-adhesive effect when presented with
fibronectin or vitronectin. The molecular basis for this is unclear but
would seem not to involve an active anti-adhesive signal via the
61 integrin for two reasons. First, 61 integrin function
provided a proadhesive signal for microglia, which was lost with the
blocking of 61 function on laminin; second, blocking 61
integrin function on mixed laminin/fibronectin substrates did not
result in increased adhesion of microglia (our unpublished observations). In light of the observation that microglia are equally
nonadherent to astrocyte ECM, which contains both fibronectin and
laminin secreted and organized by astrocytes as a physiological matrix
(Liesi et al., 1983; Oh and Yong, 1996), then this interaction takes on
greater biological significance. A dominant anti-adhesive effect of
laminin already has been described in sensory neurons (Calof and
Lander, 1991) and in inflammatory cells (Yanaka et al., 1997). In the
latter study the administration of a laminin peptide was shown to
reduce the degree of brain injury in an animal model of transient focal
cerebral ischemia by reducing leukocyte accumulation and the subsequent
extent of postischemic inflammation.
These studies were performed by using microglia derived from the
neonatal CNS. Although it is possible that age-related differences may
exist, it is accepted widely that neonatal microglia in
vitro display many of the properties of microglia in the adult
CNS, including the ability to switch from a resting to an activated phenotype (Kloss et al., 2001).
Intracellular signaling within microglia
An interesting finding to emerge from our studies was that,
broadly speaking, negative signals appeared to dominate over positive signals in regulating microglial adhesion. First, the anti-adhesive signal from laminin dominated over the proadhesive signal from fibronectin and vitronectin. Second, the anti-adhesive signal from
TGF-1 dominated over the proadhesive signal from the inflammatory cytokines. These observations support the idea that biological damping-down mechanisms have the upper hand and play an important role
in suppressing low-level proinflammatory stimuli, thereby preventing an
excessive inflammatory response. The observation that microglial
adhesion to laminin can be regulated both in a positive and negative
direction provides a useful model in which to investigate the signaling
mechanisms. The intracellular signaling pathways used by these
cytokines are fundamentally different; TGF-1 uses predominantly the
Smad pathway (Baker and Harland, 1997), whereas the IFNs use the
JAK/STAT pathways (Darnell et al., 1994) and other alternative pathways
that include PKC (Jun et al., 1995; Yu and Floyd-Smith, 1997). In this
study we have begun to dissect out the signaling mechanisms used by the
IFNs and have shown that PKC-mediated events are implicated in
enhancing microglial adhesion to laminin. Previous reports have shown
that PKC signaling also mediates other microglial responses to IFN-, including upregulation of nitric oxide synthase (iNOS) and secretion of
neurotoxic agents (Klegeris and McGeer, 2000; Kang et al., 2001). In
the future it will be important to investigate the intracellular signaling events in microglia in more detail, with two specific questions in mind. First, how and at what point of the intracellular signaling cascade does TGF-1 inhibit the IFN-mediated process? Second, is there any connection between the STAT and PKC pathways in
response to IFNs? The observation that the microglial response to IFNs
takes place over several hours and can be blocked by the protein
synthesis inhibitor cycloheximide suggests that IFNs act indirectly, by
inducing synthesis of an intermediate factor that increases the
activation state of the 61 integrin. In ongoing studies we are
investigating the nature of the intermediate factor to determine
whether this is a soluble factor secreted by microglia or a key
intracellular element of the signaling pathway. In addition, we also
are examining the adhesive behavior of microglia obtained from STAT-1
null mice in response to stimulation by IFN-, IFN-, and PMA.
Physiological significance of cytokine regulation of microglial
adhesion to laminin
For inflammatory cells outside the CNS, it has been shown that
cytokines regulate the ability of circulating leukocytes to adhere to
the laminin-rich basal lamina of blood vessels during inflammatory
conditions, thereby promoting extravasation of leukocytes toward the
inflammatory focus (Wei et al., 1997). This raises the important
question, what is the function of enhancing microglial binding to
laminin in the CNS? One possibility is that microglia now could attach
more strongly to the basal lamina around blood vessels and then migrate
along vessels to reach their target site of inflammation. In light of
the extensive evidence that CNS injury is accompanied by the
upregulation of laminin (Liesi et al., 1984; Giftochristos and David,
1988; Yamamoto and Kawana, 1990; Alonso and Privat, 1993; Logan et al.,
1994; Frisen et al., 1995; Stichel et al., 1999), an alternative
possibility is that cytokines influence the ability of microglia to
adhere to and enter the laminin-rich injury site by regulating the
activation state of the 61 integrin expressed by microglia
in vivo (Terpe et al., 1994; Kloss et al., 1999). On the
basis of our data, the ability of microglia to enter the laminin-rich
injury site will be regulated by the balance of cytokines present in
this region. The proinflammatory cytokines TNF, IFN-, and IFN-
will promote increased microglial adhesion to laminin, allowing more
microglia to enter and be retained at the injury site to phagocytose
cell debris and secrete potential regeneration factors. In contrast,
the elevated levels of TGF-1 observed at the injury site (Pasinetti
et al., 1993; Logan et al., 1994) will reduce microglial adhesion to
laminin and thus restrict microglial entry into the laminin-rich area,
leading to increased scar formation because of the persistence of cell debris. In support of this model, a previous study on injured rat brain
showed that function-blocking antibodies against TGF-1 significantly
increased microglial cell number at the site of injury (Logan et al.,
1994) and also attenuated glial scar formation, thus providing an
environment more conducive to neural regeneration.
Several lines of evidence suggest that failure of effective
regeneration in the CNS might be attributable to an inadequate microglial/macrophage response after injury (Perry et al., 1995; Lazarov-Spiegler et al., 1998). First, macrophage recruitment and
activation in the CNS are not just delayed but also spatially restricted when compared with the PNS (Perry et al., 1987; George and
Griffin, 1994). Second, in mice that have a deficient macrophage response the nerve regeneration is delayed (Ludwin and Bisby, 1992).
Third, transplantation of activated microglia and macrophages into
lesioned spinal cord leads to enhanced neurite outgrowth (Prewitt et
al., 1997; Rabchevsky and Streit, 1997). Taken together, this evidence
suggests that one way of promoting more effective regeneration in the
CNS would be to stimulate the macrophage/microglia response after
injury. This could be attained in part by promoting microglial adhesion
and entry into areas of CNS damage. On the basis of the results
described in this paper, microglial adhesion and migration into the
laminin-rich injury site would be promoted by stimulating
activation of the 61 integrin. This could be achieved
either by regulating the balance or action of cytokines, for instance
by blocking TGF-1 activity (as has been shown already), or by
pharmacologically increasing the activation state of the 61
integrin on microglia by specific reagents. Future experiments will
address the value of this approach.
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FOOTNOTES |
Received Sept. 6, 2001; revised Dec. 5, 2001; accepted Dec. 7, 2001.
This work was supported by National Institutes of Health Grants MH50426
and MH47680. R.M. was the recipient of a Wellcome Trust International
Prize Traveling Research Fellowship. This is manuscript 14144-NP from
the Scripps Research Institute.
Correspondence should be addressed to Dr. Iain L. Campbell, Department
of Neuropharmacology, SP315, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail:
icamp{at}scripps.edu.
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ABSTRACT
INTRODUCTION
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