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The Journal of Neuroscience, May 1, 2002, 22(9):3352-3358
Tissue Plasminogen Activator Mediates Microglial Activation via
Its Finger Domain through Annexin II
Chia-Jen
Siao and
Stella E.
Tsirka
Department of Pharmacological Sciences and Program in Molecular and
Cellular Pharmacology, University Medical Center at Stony Brook, Stony
Brook, New York 11794
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ABSTRACT |
Microglia are the immunocompetent cells of the CNS, and
their activation is thought to play an important neurotoxic role in many diseases modeled by glutamate-induced excitotoxicity. One molecule
whose expression is upregulated after excitotoxic injury is tissue
plasminogen activator (tPA), a serine protease with dual roles in the
CNS. The catalytic activity of tPA, which converts plasminogen into
plasmin, leads to neuronal death during excitotoxicity. Via a
nonproteolytic mechanism, tPA also mediates microglial activation. We
show here in culture studies that stimulated wild-type neurons and
microglia can release the tPA that elicits the activation, and that
tPA acts in combination with other factors. We also show that
the finger domain of tPA is necessary to trigger the activation and
identify annexin II as its probable binding partner-receptor. Together, these findings suggest that tPA released by either neurons or
microglia can act as a neural cytokine, signaling through annexin II to
activate microglia in settings of disease and injury. Developing methods to inhibit the interaction of tPA with annexin II would offer a
new and selective approach to interfere with microglial activation for
therapeutic purposes.
Key words:
nonproteolytic; cell signaling; cytokine; receptor; domain deletions; F4/80 glycoprotein
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INTRODUCTION |
Microglia, derived from the
monocyte/macrophage lineage, are considered the immune cells of the
brain (Kreutzberg, 1996 ). After injury, they become activated and
acquire a stereotypical phenotype (Streit et al., 1999) that includes
proliferation and increased expression of specific markers [e.g.,
nitric oxide (NO), interleukin-1 (IL-1), tumor necrosis factor
(TNF)- , proteases, and the surface glycoprotein F4/80], changes in
morphology (from ramified to amoeboid), migration toward injured cells,
and acquisition of antigen presentation and phagocytosis properties.
Activated microglia accumulate in many CNS diseases. They cluster
around amyloid plaques in Alzheimer's disease (Giulian, 1999), are
found in the brains of animals with scrapie (Russelakis-Carneiro et
al., 1999), and have been implicated in the progression of multiple
sclerosis and human immunodeficiency virus dementia (Gonzalez-Scarano and Baltuch, 1999). Ischemic events induce vigorous and prolonged microglial activation (for review, see Stoll et al., 1998), which increases the ensuing neurotoxicity by exacerbating the initial insult
into the surrounding region (Yenari and Giffard, 2001).
Microglial activation in part regulates the neuronal death pathway
known as excitotoxicity (Rogove and Tsirka, 1998). Excitotoxicity, which is observed after overstimulation of neurons with excitatory neurotransmitters, is a component in several neuropathologies (Dickson
et al., 1993; DiPatre and Gelman, 1997) and is modeled in animals by
injection of glutamate analogs such as kainate (KA) (Olney,
1986). Pharmacological delay of microglial activation protects neurons
from KA-induced excitotoxicity (Rogove and Tsirka, 1998).
Tissue plasminogen activator (tPA) is a serine protease; its activation
of plasmin is critical for the progression of excitotoxicity (Tsirka et
al., 1995, 1997). Plasmin degrades laminin (Chen and Strickland, 1997)
and possibly other extracellular matrix (ECM) molecules, and the loss
of this substratum promotes neuronal death.
Microglia from tPA / mice show
attenuated activation after KA injection. Infusion of either
catalytically active or catalytically inactive tPA into these mice
before KA injection restores microglial activation (Rogove et al.,
1999). These data indicate that contrary to its proteolytic role in
neurotoxicity, tPA activates microglia via a nonproteolytic mechanism.
In addition to its catalytic domain, tPA also contains a fibronectin
type-3 finger domain (F), an epidermal growth factor-like (GF) domain,
and two kringle domains (K1 and K2), which are thought to mediate
protein-protein interactions. The finger domain binds fibrin (van
Zonneveld et al., 1986) and annexin II (Hajjar et al., 1994). The GF
domain, well characterized in other proteins, is necessary for
urokinase plasminogen activator, the other mammalian plasminogen
activator, to bind to and activate its receptor (Rabbani et al., 1992).
The kringle domains bind fibrin (van Zonneveld et al., 1986), and the
second kringle also binds lysine (Gething et al., 1988). Moreover, in
addition to mediating the proteolytic function of tPA, the catalytic
domain binds to vascular smooth muscle cells (Werner et al., 1999). The catalytic domain also mediates binding to inhibitors such as the plasminogen activator inhibitors (van Zonneveld et al., 1986).
In this report, we use deletion analysis of tPA to define the mechanism
through which tPA activates microglia. We find that tPA released from
neurons or microglia activates microglia via its finger domain, which
most likely interacts with annexin II, a cell-surface receptor, to
initiate an intracellular signaling cascade.
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MATERIALS AND METHODS |
Recombinant human tPA and mutants lacking the finger ( F),
epidermal growth factor (GF), kringle 1 (K1), or kringle 2 (K2) domains were a generous gift from Genentech Inc. (San
Francisco, CA). The tPA proteins were used at a concentration of 500 ng/ml unless otherwise noted. Bacterial lipopolysaccharide (LPS; strain O55:B5; Sigma, St. Louis, MO) was used at 100 ng/ml.
Animal studies and cell culture
C57BL/6 (wild-type) and tPA/
(Carmeliet et al., 1994) mice were obtained from The Jackson Laboratory
(Bar Harbor, ME) and maintained in the Stony Brook Department of
Laboratory Animal Research with access to food and water ad
libitum. The tPA/ animals were
further backcrossed to C57BL/6 mice for nine generations, and progeny
of those mice were used for the experiments. All procedures followed
guidelines set by the National Institutes of Health and were approved
by Stony Brook University. Cultured cells were incubated at 37°C with
5% CO2. All tissue culture media and additives
were from Invitrogen (San Diego, CA) except as noted.
Neuronal cultures were made as described previously (Rogove and Tsirka,
1998). Briefly, the hippocampi from embryonic day 17.5 mouse embryos
were dissected and gently trypsinized (0.25% trypsin in HBSS) at
37°C for 15 min and then triturated to form single-cell suspensions.
The cells were plated onto poly-L-lysine precoated
coverslips at a density of 100,000 cells/cm2 in neurobasal medium with B27
supplements, 25 µM glutamate, 0.5 mM
L-glutamine, and 10 gm/l gentamycin sulfate. The medium was changed 4 hr after the initial plating, and the glutamate was removed
after 4 d in culture. Neurons were used after at least 7 d
in vitro. Neuronal conditioned medium (CM) was obtained from cells stimulated with 25 µM
L-glutamate for 4 hr.
Microglial cultures were made from mixed cortical cultures as described
previously (Rogove and Tsirka, 1998) except that single cells, obtained
by passage through a 70 µm nylon mesh, were plated into
poly-L-lysine-precoated 75 cm2
tissue culture flasks at a density of two brains per flask. The mixed
cortical cultures were used to isolate microglia after 14 d
in vitro, when the cells were selectively released from the flasks by incubation with 15 mM lidocaine for 7 min at room temperature, followed by gentle shaking for 3 min by hand.
After low-speed centrifugation, the cell pellet was resuspended in
complete medium and plated onto
poly-L-lysine-precoated 48 well plates (0.25 ml culture volume per well). The microglia were used at least 2 d after subculture, by which time their morphology resembled that of
resting microglia. To obtain microglial CM with the LPS removed, microglia were primed with 100 ng/nl LPS overnight, and then the medium
was collected, passed over a polymyxin B-agarose column as directed by
the manufacturer (Sigma), and sterilized through a 0.22 µm
syringe filter. Endotoxin removal was assayed using the Pyrogent
Plus kit (BioWhittaker, Walkersville, MD). Ten endotoxin units (E.U.)
of residual LPS were detected, compared with the 25 E.U.
originally added. Blocking molecules (see below), when used, were added
to the cell cultures 1 hr before addition of tPA. After the addition of
tPA, the cultures were continued for 1-24 hr before adding LPS for a
final overnight culture period. We found no difference in microglial
activation regardless of whether the tPA was added for short (1 hr) or
long (24 hr) periods before the addition of LPS. For some experiments,
tPA at a 500 ng/ml final concentration was added to the
tPA/ CM, or -tPA antibody (clone
N-14, 600 ng/ml; Santa Cruz Biotechnology, Santa Cruz, CA) was added to
the wild-type CM, before the addition of these CM to the microglial
cultures (Fig. 1).
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Figure 1.
tPA/ microglia in culture
respond to signals released from injured neurons and microglia by
becoming activated and changing morphology. Cells were treated as
described in Results, and the expression of F4/80 was examined.
a, Control, untreated tPA/
microglia (400× magnification). b, 100 ng/ml LPS (400×
magnification). c, CM prepared from quiescent
tPA/ neurons (400× magnification).
d, CM prepared from glutamate
(Glu)-stimulated tPA/ neurons
(400× magnification). e, Same as d
(100× magnification). f, CM prepared from
glutamate-stimulated tPA/ neurons additionally
containing tPA at 500 ng/ml (100× magnification). g, CM
prepared from quiescent tPA/ microglia (400×
magnification). h, CM prepared from LPS-stimulated
tPA/ microglia. The LPS was partially removed
using a polymyxin B column before addition of the CM to the responding
tPA/ microglia (400× magnification).
i, Same as h (100×
magnification). j, CM prepared from LPS-stimulated
tPA/ microglia additionally containing tPA at
500 ng/ml. The LPS was partially removed using a polymyxin B column
before addition of the CM to the responding tPA/
microglia (100× magnification). k, CM prepared from
quiescent wild-type neurons (400× magnification). l, CM
prepared from glutamate-stimulated wild-type neurons (400×
magnification). m, Same as l (100×
magnification). n, CM prepared from -tPA antibody of
glutamate-stimulated wild-type neurons (100× magnification).
o, CM prepared from quiescent wild-type microglia (400×
magnification). p, CM prepared from LPS-stimulated
wild-type microglia. The LPS was partially removed using a polymyxin B
column before addition of the CM to the responding
tPA/ microglia (400× magnification).
q, Same as p (100× magnification).
r, CM prepared from -tPA antibody of LPS-stimulated
wild-type microglia. The LPS was partially removed using a polymyxin B
column before addition of the CM to the responding
tPA/ microglia (100× magnification).
Arrowheads, Membrane ruffles or pseudopods.
Arrows, Phagocytic vacuoles.
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Microglial activation
Immunocytochemistry. Microglia were fixed on their
coverslips with 4% paraformaldehyde-20% sucrose and then
permeabilized using 0.1% Triton X-100. After blocking with goat serum
(10% in PBS), the rat anti-F4/80 antibody (Serotec, Indianapolis, IN) was used at a 1:200 dilution, followed by incubation with biotinylated anti-rat IgG (1:1000; Vector Laboratories, Burlingame, CA). The signal
was amplified using the Vector Laboratories avidin-biotin complex
Elite kit (avidin coupled with horseradish peroxidase) and detected
using diaminobenzidine-peroxide (Sigma), after which the coverslips
were successively dehydrated in ethanol, delipidated in xylenes, and
mounted using Permount (Fisher, Houston, TX).
Quantitative Western blot. Cultured microglia were lysed
in 0.25% Triton X-100 in PBS. After centrifuging to remove cell
debris, the total protein concentration was measured using the Bio-Rad (Hercules, CA) Bradford detergent-compatible (Dc) assay. Equal amounts (20 µg) of protein from each sample were separated on 10%
SDS-PAGE, transferred onto polyvinylidene difluoride membrane, blocked
with 5% nonfat dry milk in PBS/0.5% Tween 20 (PBS-T), and incubated
with F4/80 antibody (1:200 dilution) overnight in milk/PBS-T.
Biotinylated secondary anti-rat antibodies were used (1:500 dilution)
and were detected using FITC-labeled ExtrAvidin (1:200; Sigma).
Fluorescence was detected by a FluorImager (Molecular Devices, Menlo
Park, CA) and quantified using the ImageQuant (Amersham, Sunnyvale, CA) software.
Nitrite detection. The production of nitric oxide from
activated cells was measured in the form of nitrites according to the method of Si et al. (1997). CM from treated microglia was collected and
refrigerated before detection, after which 20 µl of 0.05 mg/ml diaminonaphthalene (Molecular Probes, Eugene, OR) in 0.28N HCl was
added to 100 µl of CM per well in a 96 well black plate (Nunc, Naperville, IL), which was then incubated at room temperature for 10 min. The reactions were terminated by the addition of 100 µl of 0.28N
NaOH followed by incubation for 10 min. The generation of nitrites was
measured using a microplate fluorescence reader (Titertek, Huntsville,
AL) with 365 nm excitation and 450 nm emission filters.
Annexin II analysis
Reverse transcription-PCR. Total RNA was isolated
from cultured microglia and neurons using Trizol (Invitrogen) according to the manufacturer's directions. Reverse transcription (RT) was performed using poly-T primers and Moloney murine leukemia virus (Invitrogen) according to the manufacturer's directions. PCR for annexin II was performed using the following primers:
5'-ATGTCTACTGTCCACGAAATC-3' (forward) and 5'-CAGGTAGAGCCACTTCTGGG-3'
(reverse). For amyloid precursor protein (APP), the primers used were
5'-CGATGGGGGATGCTTCTTGTG-3' (forward) and
5'-GCTATCATGGCATAAGCAATG-3' (reverse).
Western blot analysis. Total protein from cultured
neurons or microglia was lysed in 0.25% Triton X-100 and clarified by
centrifugation. Total protein (5 µg) from the supernatant, quantified
using the Bio-Rad Bradford Dc assay, was separated on a 10%
SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride
membrane. -Annexin II was detected using a monoclonal antibody
(1:1000 final dilution; Transduction Laboratories, Lexington, KY). To
verify cell-specific blotting, -microtubule-associated
protein-2 (a neuronal marker; 1:700; Sigma) antisera and F4/80
(a macrophage/microglial marker) were used. To show equal loading of
proteins, an -actin antibody (1:250; Sigma; a generous gift from Dr.
M. Berrios, State University of New York at Stony Brook, Stony Brook,
NY) was used.
Antibody blocking assays. Primary
tPA/ microglia were plated as above. Antibodies
were added in the following amounts (in µg): 1 -annexin II,
1 mouse IgG1 (Sigma), 5 -low-density lipoprotein receptor-related
protein (LRP) (rabbit anti-human antiserum; a generous gift from Dr. G. Bu, Washington University, St. Louis, MO). After incubation for
1 hr, tPA was added for another hour, and finally LPS was added and
cells were incubated overnight before analysis.
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RESULTS |
Both neurons and microglia can activate microglia in a paracrine
and tPA-dependent manner
Purified wild-type microglia stimulated with LPS become activated,
whereas tPA/ microglia do not unless
exogenous tPA is added to the culture (Rogove et al., 1999). However,
addition of exogenous tPA in the absence of LPS does not result in
activation. These findings suggest that LPS priming stimulates several
events required for activation, one of which could be the release of
tPA, which would then further stimulate the microglia in an
autocrine-paracrine manner. Alternatively, we have proposed that
neuronal release of tPA could also activate the primed microglia at
this step. To address these hypotheses, we developed a system to assess
whether defective tPA/ microglial
activation can be rescued using CM from glutamate-stimulated purified
neurons and LPS-primed microglia.
In the absence of manipulation, the cultured
tPA/ microglia used for the
experiments displayed primarily a resting phenotype, which is
characterized by small cell bodies with long, thin processes (Fig.
1a). With LPS stimulation, these cells began the process of
activation in that their cell bodies and processes became slightly increased in volume (Fig. 1b).
In the presence of CM prepared from unstimulated wild-type or
tPA/ neurons or microglia, the
tPA/ microglia similarly exhibited
very limited signs of activation (Fig. 1c,g,k,o). However,
dramatic activation was observed in the presence of CM prepared from
wild-type neurons subjected to simulated injury (66.5% of the
microglia were activated, as judged by altered morphology) [Fig. 1,
l (higher magnification) and m (lower
magnification)]. The cell membranes became ruffled (Fig. 1,
arrowheads), and phagocytic vacuoles were observed in the
cell bodies (Fig. 1, arrows). In contrast, CM prepared from
injured tPA/ neurons did not trigger
activation (6.8%) (Fig. 1d,e). CM prepared from
LPS-stimulated wild-type microglia similarly triggered activation (92.6%) (Fig. 1p,q), whereas CM prepared from
LPS-stimulated tPA/ microglia did not
(10.6%) (Fig. 1h,i). However, the
tPA/-stimulated neuronal or
microglial CM could be made functional with the addition of tPA (Fig.
1f,j). Finally, addition of an -tPA antibody to the
stimulated wild-type neuronal or microglial CM impaired the ability of
the CM to fully activate the responding tPA/ microglia (Fig.
1n,r).
Together, these results show that wild-type but not
tPA/ microglia and neurons, once
primed or injured, release a factor(s) in the medium that activates
tPA/ microglia. Because this factor
can be replaced by purified tPA and can be blocked by anti-tPA
antiserum, it is most likely tPA. The results demonstrate that both
neurons and microglia are capable of mediating microglial activation
through the release of tPA.
tPA activates microglia in synergy with other stimuli
Cultured microglia often adopt a slightly activated morphology
[probably attributable to the culture preparation and
conditions (Fig. 2a) (Nakajima
et al., 1989)]. Stimulation of tPA/
microglia with tPA does not induce significant morphological changes
(Fig. 2, compare a with b and c). LPS
treatment alone also induces a rapid (within 30 min) but quite limited
change in morphology in many cells (Fig. 2d). However, when
the cells are treated with 500 ng/ml tPA plus LPS, the microglia become highly activated (Fig. 2f). This finding indicates
that tPA alone does not suffice to activate microglia; instead, a
second signal is required, which can be provided by LPS priming or by
factors other than tPA released by stimulated microglia and neurons
(Fig. 1).
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Figure 2.
tPA and LPS are required to activate
tPA/ microglia. Microglia were cultured as
indicated and then fixed and immunostained for F4/80 expression.
a, Not treated; b, 5 ng/ml tPA;
c, 1 µg/ml tPA; d, 100 ng/ml LPS;
e, 5 ng/ml tPA, LPS; f, 500 ng/ml tPA,
LPS. All panels are shown at 100× magnification.
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Microglial activation as a function of tPA and LPS addition was
quantified by Western blot analysis. The expression of the glycoprotein
F4/80 is upregulated when cells of the monocyte-macrophage lineage are
activated (Lawson et al., 1990). We detected an increase in F4/80
immunoreactivity after culturing tPA/
microglia with LPS and increasing concentrations of tPA. From baseline
unstimulated levels, we found a 0.8-fold increase with the lowest dose
of tPA added (5 ng/ml), which increased to 2-fold with 50 ng/ml tPA and
to 2.4-fold with 500 ng/ml tPA, indicating that the level of activation
is dependent on the concentration of tPA (data not shown). Microglia
secrete many other molecules during activation, including NO and
TNF- (Meda et al., 1995). The activated microglia were also found to
secrete higher amounts of nitrites (2.5 ± 0.2 µM at
500 ng/ml tPA, compared with 0.9 µM from control cells),
consistent with the immunochemical data shown in Figure 2 (data not shown).
The finger domain of tPA mediates microglial activation
tPA mediates microglial activation via a nonproteolytic mechanism,
possibly through interactions with protein(s) on the microglial cell
surface. We used tPA mutants lacking the finger (F), growth factor
(GF), and kringle (K1 or K2) domains to determine whether the
absence of one or more of these domains abolished microglial activation. The GF, K1, and K2 proteins activated
tPA/ microglia as well as or better
than full-length tPA, as judged by F4/80 upregulation (Fig.
3a), NO release (Fig.
3b), and morphological change (Fig. 3c). However,
the F mutant did not elicit significant activation (see also Fig.
5a). The F mutant did not act as a dominant-negative or
toxic factor, because wild-type levels of activation were observed when
full-length tPA and F were used together in the activation assay
(data not shown). Thus, the presence of the tPA finger domain is
critical for mediating microglial activation.
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Figure 3.
The finger domain of tPA mediates microglial
activation. tPA/ microglia were cultured with
LPS in combination with full-length tPA (tPA LPS), tPA
lacking the finger domain (F LPS) or the growth
factor domain (GF LPS), or either of the kringle
domains (K1 LPS and K2 LPS).
a, Quantification of F4/80 expression by Western blot.
b, Detection of NO release by its product, nitrite
(NO2). *p < 0.01;
**p < 0.05. Data expressed are averages ± SEM of triplicate experiments; all analyses were conducted using
Student's t test relative to the control cells that did
not receive additions to the culture. c,
tPA/ microglia were cultured on coverslips under
the indicated conditions and then immunostained for F4/80 expression;
panels are shown at 200× magnification.
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Microglial activation by the finger domain of tPA is
receptor-mediated through annexin II
One candidate for a microglial tPA receptor is annexin II (Hajjar
et al., 1994). The Ca2+- and
phospholipid-dependent annexin II is a known receptor for tPA in
endothelial cells (Hajjar et al., 1994). We found by RT-PCR and Western
blot analysis that annexin II is expressed by microglial cells but not
neurons (Fig. 4), suggesting that there
could be a microglial-specific tPA signaling pathway.
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Figure 4.
Primary microglia, but not neurons, express
annexin II, a potential tPA receptor. a, RT-PCR results
from microglia (mg) or neurons
(n), amplified using either annexin II
(ann II) or APP primers. The quality of the
neuronal cDNA was established using the APP as a positive control
(lane 4). b, Western blot
confirming that primary microglia but not neurons express annexin II.
The cell lysates used for the Western blot were also probed with the
neuronal marker microtubule-associated protein 2 and the microglial
marker F4/80 to confirm that the respective cell types had been highly
enriched as intended and indicated (data not shown). c,
An anti-actin antibody was used to ascertain equal protein loading for
the lysates analyzed on the Western blot.
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To assess whether this pathway was functional, we preincubated
tPA/ microglia with a monoclonal
-annexin II antibody for 1 hr before tPA/LPS activation (Fig.
5a). Addition of the antibody
alone (Fig. 5A, column 4) did not
affect unstimulated cells (Fig. 5A, column 1).
However, the antibody did block (Fig. 5A, column
5) the activation normally elicited by tPA and LPS (Fig.
5A, column 2), as assessed by F4/80 upregulation.
Similar results were observed by morphology (Fig. 5b-d) but
not in the presence of an IgG1 isotype control (Fig. 5e).
Finally, incubation with an antibody directed against LRP, which is a
known receptor for tPA in liver cells (Bu et al., 1992), did not
prevent microglial activation (Fig. 5f). These results suggest that the finger domain of tPA mediates microglial activation by signaling through the cell surface protein annexin II.
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Figure 5.
tPA-mediated microglial activation is blocked by
-annexin II antibodies. a, Measurement of F4/80
expression by Western blot. tPA/ microglia were
cultured with medium only [not treated (n.t.)]; LPS
and 500 ng/ml tPA (tPA, LPS); LPS and 500 ng/ml F
mutant (F, LPS); 1 µg/ml anti-annexin II
(-annII); or LPS, 1 µg/ml anti-annexin II,
and 500 ng/ml tPA (-annII, tPA,LPS). Data from two
independent experiments were pooled and plotted as fold activation over
the control cells, which received no additions. Error bars indicate
range of data points. b-e, tPA/
microglia were cultured alone or with -annexin II or -LRP
antibodies for 1 hr, 500 ng/ml tPA was added for another hour, and then
LPS was added and the cells were incubated overnight. Activation was
examined using F4/80 immunocytochemistry on fixed cells as described in
Materials and Methods. The panels are shown at 400×
magnification.
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DISCUSSION |
tPA mediates neuronal death and microglial activation during
excitotoxic injury. We have reported previously that the proteolytic activation of plasmin by tPA is required for neurodegeneration to
progress (Tsirka et al., 1997). The role of plasmin has been proposed
to involve subsequent degradation of ECM molecules, such as laminin
(Chen and Strickland, 1997), resulting in loss of neuronal contact with
the ECM, which may lead to apoptosis (Frisch and Ruoslahti, 1997;
Frisch, 2000). However, the proteolytic activity of tPA is not
necessary to activate microglia, suggesting an additional, cell-signaling function for tPA in the CNS (Rogove et al., 1999). Because tPA is secreted by both neurons and microglia, we addressed first whether either cell type released enough tPA into CM under conditions of stimulation to activate microglia. We found that stimulated tPA/ neurons or microglia
failed to induce activation in tPA/
microglia but that this result was reversed by addition of tPA into the
cultures (Fig. 1). Finally, incubation with an antibody that sequesters
the released tPA present in the stimulated wild-type CM partially
(neuronal CM) to fully (microglial CM) inhibits the ability of the CM
to mediate activation (Fig. 1; also see below for further discussion).
These experiments confirm that one of the factors responsible for
microglial activation after stimulation in our system is soluble tPA.
The finding that glutamate-injured neurons can release sufficient tPA
to activate tPA/ microglia suggests a
paracrine mechanism of microglial activation in vivo, in
which the injured neurons would signal the immune cells of the CNS
(i.e., the microglia) to scavenge damaging molecules secreted from the
dying cells. In addition, we show that microglial activation can
further stimulate release of microglial factors, such as tPA, that can
in turn activate neighboring microglia in an autocrine or paracrine
manner. This signal amplification ultimately leads to recruitment of
microglia to the site of injury in the brain and can promote both a
timely resolution of cellular injury and an overly sensitive
inflammatory response. The data presented here support the role of tPA
as a cytokine in this setting.
Our dose-response results indicate that there is a threshold for
microglial reactivity to injury. It is widely accepted that microglia
are the sensors of injury in the brain (Kreutzberg, 1996), and we
propose that tPA acts as a microglial sensor molecule. Normally, the
level of tPA released into the extracellular space is tightly
regulated. tPA is stored in intracellular vesicles and is released only
by Ca2+ influx during neuronal
depolarization (Gualandris et al., 1996). Another level of control is
afforded by the presence of several serine protease inhibitors in many
regions of the brain [e.g., plasminogen activator inhibitors-1 and -2 and neuroserpin (Akiyama et al., 1995; Krueger et al., 1997)]. We and
others have shown that tPA plays an important role in physiological
processes such as neurite outgrowth (Seeds et al., 1999; Wu et al.,
2000). This highly localized secretion of tPA, and hence localized
activation of plasmin, is probably necessary for neurites to degrade
ECM structures so that they can reach their targets. Therefore, neurons must be able to tolerate significant local increases in tPA activity surrounding them without sending out injury/death and inflammatory signals. To this end, we have shown that tPA alone is not neurotoxic (Tsirka et al., 1996) (C.-J. Siao and S. E. Tsirka, unpublished observations). Like neurons, microglia must also be able to
differentiate between high levels of tPA alone (which may be
physiological) and high levels of tPA and some other factor, the
combination of which is now an injury signal for microglia to become
activated. In most of our experimental conditions, that other factor is
bacterial LPS, whose presence is uncommon in the normal CNS.
Physiological candidates would include proinflammatory molecules such
as TNF- and IL-1 that play important roles in activating
microglia in culture (Meda et al., 1995). We believe that this
cell-culture approach will help to define the identity of this other
factor or factors. Indeed, our data using antibodies to block tPA
binding to microglia and subsequent activation support this hypothesis (Fig. 1). Figure 1n (wild-type neuronal CM plus -tPA)
shows more activated microglia than those in Figure 1r
(wild-type microglial CM plus -tPA). Because the same concentration
of antibody was used and the same amount of tPA was released from each
cell type (data not shown), an additional factor other than soluble tPA presumably is responsible for the advanced activation using neuronal CM. Recent data from our laboratory suggest that excitotoxic
stimulation of hippocampal neurons results in the upregulation and
secretion of active monocyte chemoattractant protein-1, which promotes
microglial activation-migration (A. D. Rogove, J. Z. Sheehan, Y.-P.
Wu, Tsirka, unpublished observations).
The use of CM in a cell-culture setting is artificial, because of,
among other reasons, the absence of cell types such as astrocytes,
which can contribute to both amplification of tissue injury and
containment of damage (Cui et al., 2001; Mandell et al., 2001). In
addition, although LPS is widely used to prime cells of the
monocyte/macrophage lineage for activation in vitro, this
priming step would presumably be mediated in vivo by unknown endogenous factors. Finally, our findings show that microglia and
neurons can release sufficient tPA in vitro, but not that they take up this role in vivo. All of these issues will be
resolved by developing methods to address the questions using
transgenic animal models, and these experiments are currently in progress.
To explore the mechanism of receptor-mediated microglial activation by
tPA, we undertook a domain-deletion approach. Our results suggest that
the finger domain of tPA plays a key role in activating microglia
during excitotoxic injury. The slight activation of microglia in the
cells treated with the F mutant and LPS is probably attributable to
LPS priming. We would not predict that addition of the finger domain
alone would completely reconstitute microglial activation; evidence
suggests that the finger-growth factor domains interact with the
catalytic domain (Novokhatny et al., 1991). Thus, the protease domain
could also be involved (noncatalytically) in physically mediating
activation. Recent data indicate that the catalytic domain may mediate
tPA binding to human vascular smooth muscle cells (Werner et al.,
1999). Mutants with catalytic domain deletions could be used to answer
this question.
Annexin II, a Ca2+- and
phospholipid-binding protein, belongs to a family of proteins
characterized by a highly conserved set of -helical repeats in the C
terminus that mediate membrane binding (Mollenhauer, 1997). Annexin II
is usually found as a heterotetramer consisting of two annexin II (p36)
and two S100A10 (p11) subunits. A hexapeptide sequence in the N
terminus of annexin II has been shown to bind to the finger domain of
tPA and to increase tPA activity on endothelial cells (Hajjar et al.,
1998). Because annexin II is a membrane-associated protein to which tPA
and plasminogen presumably only dock, it is unclear how it may
transduce inside the cells the activation signal resulting from the
binding of extracellular tPA. Members of the annexin family are
secreted to the cell surface via an unknown mechanism (Mollenhauer,
1997). Annexin II binds phosphatidylserine, so it could be
cotranslocated across the membrane during early stages of apoptosis,
although how and why this might occur during microglial activation is
not at all clear.
Once inside the cell, the tPA/annexin II complex could signal in
various ways. For example, the site at which tPA binds to annexin II is
also where S100A10/p11 binds annexin II. One could envision a
displacement and release of S100A10/p11 to function as an intracellular
signal molecule. Alternatively, another as yet unidentified molecule
associated with annexin II may mediate signaling cascades to activate
microglia when tPA binds to annexin II. Annexin II is also a prominent
intracellular signaling protein found in caveolas and lipid rafts, and
it is phosphorylated by several kinases at key residues. Finally,
annexin II is known to mediate interaction between cholesterol-rich
membrane domains and the actin cytoskeleton (Filipenko and Waisman,
2001). This observation may underlie the changes in morphology during
microglial activation. We are currently characterizing biochemically
the interactions of the finger domain with annexin II and examining how
this interaction mediates signal transduction.
tPA is used clinically as a thrombolytic agent during acute stroke.
However, its neurotoxic effects in a mouse ischemia model suggest
caution (Wang et al., 1998), because it can also activate microglia and
lead to an exacerbation of neuronal death (Rogove and Tsirka, 1998;
Rogove et al., 1999). Identification and further characterization of
the tPA domain that activates microglia may aid in reducing
inflammation and neurotoxicity. Mutating this binding region could lead
to a clinically useful form of tPA that retains its thrombolytic
properties yet does not trigger microglial activation.
A large body of research suggests that microglia can undertake either
neurotoxic or neuroprotective roles in different disease or model
settings (Dickson et al., 1993; Gehrmann et al., 1995; Benveniste,
1997; DiPatre and Gelman, 1997). It is highly likely that both roles
exist. In addition to cleaning up debris from dead and dying cells,
phagocytes are also required to contain damage so that other cells in
the regions surrounding the injury are not damaged. However,
overzealous reactive microglia can secrete excessive proinflammatory
molecules, thus worsening the initial injury. Identification of annexin
II as a microglial cell surface tPA receptor and characterization of
the detailed interaction between the finger domain of tPA and annexin
II would allow a potentially powerful method of interfering with
microglial activation when such activation may be detrimental to the
system as a whole.
| |
FOOTNOTES |
Received Sept. 19, 2001; revised Jan. 24, 2002; accepted Jan. 29, 2002.
This work was supported by a Neurotoxin Research Program grant from the
United States Army Medical Research to S.E.T. We thank Drs. Michael
Frohman, Lincoln Stein, and Chuck O'Neal for critically reviewing this
manuscript. We are also grateful to Genentech Inc. for providing the
deletion domain mutant tPA proteins.
Correspondence should be addressed to Dr. Stella E. Tsirka, Department
of Pharmacological Sciences, BST-7, Room 183, University Medical Center
at Stony Brook, Stony Brook, NY 11794-8651. E-mail: stella{at}pharm.sunysb.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
