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Consiglio Nazionale delle Ricerche Cellular and Molecular Pharmacology and "B. Ceccarelli" Centers, Department of Medical Pharmacology, Milan, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. The intracellular calcium increases in astrocytes,
occurring spontaneously or as a result of mechanical or bradykinin
stimulation, induced the release of ATP, which, in turn, was
responsible for triggering a delayed calcium response in microglial
cells. Repeated stimulations of microglial cells by astrocyte-released
ATP activated P2X7 purinergic receptor on microglial cells
and greatly increased membrane permeability, eventually leading to
microglial apoptosis. IFN- increased ATP release and potentiated the
P2X7-mediated cytolytic effect. This is the first study
showing that ATP mediates a form of calcium signaling between
astrocytes and microglia. This mechanism of intercellular communication
may be involved in controlling the number and function of microglial
cells under pathophysiologic CNS conditions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ATP is a major factor mediating intercellular communication in the immune and nervous systems and triggers a variety of strikingly different biological effects (4, 5, 6, 7). In the brain it is considered to be the dominant extracellular messenger for astrocyte-to-astrocyte calcium-mediated communication. Astrocytes release ATP upon mechanical stimulation (8) or glutamatergic receptor activation (9) and respond to ATP with a propagating wave of intracellular calcium ([Ca2+]i)3 increases (8), a process that is thought to serve as a long-range signaling system in the CNS (10, 11).
Functionally active purinergic receptors have been detected in cultured
and in situ microglial cells (12, 13, 14, 15, 16, 17), thus suggesting the
possibility that ATP may also act in astrocyte-to-microglia
communication. In this study we demonstrate the existence of an
ATP-mediated calcium signaling mechanism between astrocytes and
microglial cells that is increased in the presence of the inflammatory
cytokine IFN- and eventually leads to microglial apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hippocampal mixed glia cultures from embryonic rat pups (embryonic day 18) were obtained using previously described methods (18). The cultures were grown in MEM supplemented with 20% FCS and 5.5 g/L glucose. The astrocytic and microglial components of the cultures were determined by means of immunostaining for glial fibrillar acidic protein (GFAP) and CSF-1R. The purified microglia were harvested by shaking 3-wk-old cultures, seeded on glass coverslips, and cultured in the same medium.
Fura-2 videomicroscopy
The cultures were loaded with 5 µM fura-2 pentacetoxy-methylester in Krebs-Ringer solution buffered with HEPES (KRH; 150 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 2 mM CaCl2, 10 mM glucose, and 10 mM HEPES/NaOH, pH 7.4) for 1 h at 37°C, washed in the same solution to allow de-esterification of the dye, and transferred to the recording chamber of an inverted microscope (Axiovert 100; Zeiss, New York, NY) equipped with a calcium-imaging unit. A modified CAM-230 dual wavelength microfluorometer (Jasco, Tokyo, Japan) was used as a light source for the assays. The experiments were performed at room temperature (24–25°C) using an Axon Imaging Workbench 2.2 equipped with a PCO Super VGA SensiCam (Axon Instruments, Foster City, CA). A single astrocyte in the field was gently mechanical stimulated with a glass microelectrode to evoke intercellular calcium waves.
ATP measurements
Bioluminescence assay. ATP levels in the extracellular saline incubated 30 min with dishes containing a pure hippocampal astrocytic monolayer were measured using a luciferin/luciferase assay (Molecular Probes, Leiden, The Netherlands) and a luminometer (Lumat LB9501; Berthold, Nashua, NH). The experimental samples were compared with a standard ATP curve created on the basis of saline samples containing known concentrations of ATP. Each sample was run in duplicate. Most of the samples were assayed within 5–10 min of collection; the others were frozen for subsequent ATP determination.
Extracellular ATP bioassay.
One milliliter of extracellular saline incubated for 30 min with dishes
containing a pure hippocampal astrocytic monolayer in the presence or
in the absence of IFN- was split into two aliquots before testing on
fura-2-loaded astrocytes. One aliquot was pretreated with apyrase (30
U/ml) for 15 min before testing. Five hundred microliters of test
solution or saline containing known concentrations of ATP were applied
to astrocytes as ATP sensor cells during image acquisition, and the
amplitude of the [Ca2+]i
response was measured.
Immunocytochemistry
At the end of the recording session, cultures were fixed at room temperature with 4% paraformaldehyde in 0.1 M phosphate buffer containing 0.12 M sucrose for 25 min. Fixed cells were permeabilized with detergent and labeled with anti-GFAP monoclonal Abs (Sigma, Milan, Italy), and anti-CSF-R1 polyclonal Abs (Santa Cruz Biotechnology, Santa Cruz, CA), followed by fluorescein-anti-mouse Abs (Jackson ImmunoResearch, West Grove, PA) and rhodamine-conjugated anti-rabbit Abs (Jackson ImmunoResearch). The coverslips were mounted in 70% glycerol in phosphate buffer containing 1 mg/ml phenylenediamine. The images were acquired using a Bio-Rad MRC-1024 confocal microscope (Bio-Rad, Hercules, CA) equipped with LaserSharp 3.2 software. The morphological features of apoptosis were monitored by means of phase contrast microscopy and labeling with propidium iodide in PBS on cultures fixed in methanol precooled at -20°C.
Chemicals
Most of the chemicals
(pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid, tetrasodium
(PPADS), ATP, bradykinin, apyrase, and 18-
-glycyrrhetinic acid)
were obtained from Sigma. Fura-2-acetoxymethyl ester was
purchased from Calbiochem (La Jolla, CA), and periodate-oxidized ATP
was a gift from Prof. F. Di Virgilio (University of Ferrara, Ferrara,
Italy).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary cultures of embryonic hippocampal glial cells
predominantly contain astrocytes, with a variable percentage of
microglial cells. Microglial cells are characterized by their amoeboid
shape and can be unambiguously distinguished from astrocytes because
they are stained by Abs against the CSF receptor (CSF-1R; Fig. 1
, A and B, red),
but not by Abs against the GFAP cytoskeletal glial protein (green). The
mixed cultures were loaded with the cell-permeant fura-2-acetoxymethyl
ester fluorescent calcium dye and imaged using digital imaging
techniques in a static bath. Gentle mechanical contact between a patch
pipette and the surface of a single astrocyte (pseudocolor images in
Fig. 1C, arrow) induced a propagating calcium wave in
neighboring cells (Fig. 1C) that were identified as
astrocytes or microglia on the basis of morphological criteria or
CSF-1R and GFAP immunostaining at the end of the recording session
(Fig. 1C, inset). Analysis of the kinetics of
[Ca2+]i changes in the
stimulated astrocytes and in two adjacent microglia cells revealed a
transient, delayed
[Ca2+]i response in the
microglial cells (Fig. 1D).
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E, bottom panel), but a
large increase in [Ca2+]i
was induced in the majority of cocultured astrocytes and microglial
cells (Fig. 1E, top panel). Whereas bradykinin
application induced a prompt
[Ca2+]i response in
astrocytes, [Ca2+]i
increases occurred several seconds later in microglia (50 ±
20 s; mean ± SE; n = 12). Efficient
propagation of calcium signaling between astrocytes and microglia was
also observed in cultures characterized by spontaneous
[Ca2+]i oscillations
(Fig. 1F), a phenomenon that may depend on cycles of calcium
release and uptake from intracellular stores (10, 11, 20, 21). Analysis of several fields containing both astrocytes and
microglial cells (n = 31) revealed that spontaneous
astrocytic [Ca2+]i
oscillations were coupled to a delayed response in adjacent microglial
cells (Fig. 1F). Delayed [Ca2+]i responses in microglial cells are mediated by ATP
The delayed response of microglia triggered by spontaneous or
evoked [Ca2+]i increases
in astrocytes is consistent with a secondary response to a bioactive
compound released by astrocytes upon
[Ca2+]i elevations.
Glutamate and ATP have recently been identified as diffusable factors,
capable of inducing calcium waves among astrocytes (8, 22). As microglial cells from hippocampus lack calcium-permeable
glutamate receptors (our unpublished observation) and express
purinergic receptors permeable to calcium ions (15), we
investigated whether ATP might be involved in intercellular calcium
signaling between astrocytes and microglial cells. We found different
lines of evidence indicating that this was the case. First, ATP was
detected in the extracellular medium of bradykinin-stimulated and
spontaneously oscillating astrocytes by means of the sensitive
luciferase bioluminescence assay (not shown) and was also detected in
the medium collected from mechanically stimulated astrocytes
(8). Second, samples of extracellular medium taken from
spontaneously oscillating astrocytes and gently added to fura-2-loaded
astrocytes as ATP-sensor cells induced a
[Ca2+]i response that was
prevented by pretreatment of the conditioned medium with the
ATP-degrading enzyme apyrase (Fig. 5C, right
panel). Third, when the astrocytes in astrocyte-microglia
cocultures were stimulated mechanically or with bradykinin, a potent
inhibition of the propagation of the
[Ca2+]i signal to
microglial cells was recorded in the presence of either the P2X/P2Y
purinergic receptor antagonist PPADS (50 µM) or apyrase (Fig. 2). The series of pseudocolor images in
Fig. 2A shows an example of a mechanically induced calcium
wave (Fig. 2A, patch pipette in position 1) that did not
propagate to microglia in the presence of PPADS (Fig. 2A,
arrows, see inset in image 64 for immunocytochemical
identification) despite the efficient transmission of the calcium
signal to adjacent astrocytes (Fig. 2A, images from 0–64
s). After extensive washing of PPADS, the calcium wave induced by
mechanical stimulation of another astrocyte in the field (Fig. 2A, patch pipette shifted to position 2) did propagate to
microglia according to the diffusion of the wave front (Fig. 2B, images from 0 to 31 s). The kinetics of
[Ca2+]i changes in the
two stimulated astrocytes (green traces) and in one microglial cell
(red trace, positioned between 1 and 2) are shown in Fig. 2C. The calcium signal propagation from astrocytes to
microglial cells was also potently inhibited in the presence of the
ATP-degrading enzyme apyrase (Fig. 2D). Fig. 2E
shows the quantitative analysis of PPADS and apyrase inhibition of the
delayed response of microglia (81% inhibition in the presence of
PPADS, n = 8; 66.5% inhibition in the presence of
apyrase, with a 420% increase in the time to peak response,
n = 13; controls, n = 14); a similar
reduction in calcium signal propagation was produced by PPADS when
increased astrocyte
[Ca2+]i levels were
evoked by bradykinin (Fig. 2E; 83.9% inhibition in the
presence of PPADS, n = 9; controls, n =
12). Efficient propagation of the calcium signal between astrocytes and
microglia was detected in the presence of a cocktail of glutamate
receptor antagonists (100 µM 2-amino-5-phosphonovaleric acid, 20 µM
6-cyano-7-nitroquinoxanine-2,3-dione, and 1 mM
(S)--methyl-carboxyphenylglycine) or after 5- to
10-min incubation in the presence of the gap junction blocker
18-glycyrrhetinic acid (10 µM; data not shown). These results
indicate that [Ca2+]i
increases in astrocytes induce the release of ATP, which, in turn,
activates purinergic receptors in microglial cells.
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Different concentrations of exogenous ATP were applied to purified
microglia shaken from mixed glial cultures and plated onto glass
coverslips. As shown in Fig. 3A (left
panel), ATP (10 µM) triggered a
[Ca2+]i spike, followed,
or not, by a [Ca2+]i
plateau, and returned to basal levels upon ATP removal. As previously
described (15, 17), the increase in
[Ca2+]i presumably
reflects two different mechanisms: the release of stored calcium (first
spike) and calcium influx from the extracellular medium (second
shoulder). The biphasic
[Ca2+]i response was
completely prevented by 50 µM PPADS (data not shown), thus suggesting
that the [Ca2+]i
response, triggered by micromolar amounts of ATP, is linked to the
activation of PPADS-sensitive P2X/Y receptors. Addition of 1 mM ATP
(Fig. 3A, left panel) greatly enhanced the
duration of the spike and the plateau, with the latter lasting several
minutes after ATP removal. The persistent increase in
[Ca2+]i was followed by a
rapid decrease in the 340:380 fluorescence ratio, thus indicating a
progressive increase in membrane permeability that led to the complete
efflux of FURA2 from the cell (Fig. 3A, left
panel, light blue trace). This decrease in the fluorescence signal
was paralleled by the disappearance of the microglial cell profiles in
the pseudocolor images, although the cells were still attached to the
coverslips (data not shown).
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1000 Da in size (23), and, upon activation, allow the
efflux of cytoplasmic water-soluble molecules such as fura-2-free acid
into the extracellular medium (25). Oxidized ATP (oATP) is
a rather selective blocker of P2X7 receptors.
Preincubation of purified microglial cells for 1–2 h with 300 µM
oATP did not significantly affect the
[Ca2+]i response induced
by 10–20 µM ATP (Fig. 3A, right panel), but
abolished the long-lasting increase in calcium and membrane
permeability in 66.6% of the cells exposed to 1 mM ATP (Fig. 3A, right panel; n = 15). This
indicates that microglia express P2X7 receptors
that mediate fura-2 efflux once activated by millimolar amounts or
repeated applications of micromolar amounts of ATP.
Interestingly, repetitive challenges with 10–20 µM ATP (two or
three pulses of 1–3 min) resulted, after a several minute delay, in a
sustained calcium plateau followed by an increase in membrane
permeability that led to the complete efflux of fura-2 from the cell
(Fig. 3B).
Activation of P2X7 receptors in microglial cells cocultured with astrocytes
In microglial cells cocultured with astrocytes,
long-lasting P2X7-mediated
[Ca2+]i increases
occurred in the absence of exogenously applied ATP, but were triggered
by the [Ca2+]i
oscillations that spontaneously occur in astrocytes (Fig. 3G). The persistent increases in
[Ca2+]i were followed by
fura-2 leakage (Fig. 3G, blue trace, and Fig. 3, D and F). As in the case of the purified
microglia stimulated with 1 mM ATP, the sharp decrease in the
fluorescence signal occurring in microglia cocultured with astrocytes
was paralleled by the disappearance of all microglial cell profiles in
the pseudocolor images within 180 min (Fig. 3, C and
E, pseudocolor images; Fig. 3, D and
F, temporal plots). Brightfield analysis revealed that the
cells were still attached to the coverslips (Fig. 3, C and
E, asterisks). No changes in
[Ca2+]i were detected in
the purified microglial cultures imaged for up to 6 h in control
medium and in the absence of astrocytes (data not shown), which further
confirms that the persistent
[Ca2+]i increase recorded
in cocultures is specifically attributable to the presence of
astrocytes.
The sustained [Ca2+]i
elevations followed by the complete loss of fura-2 in microglia
cocultured with astrocytes were greatly delayed when the cultures were
pretreated with the P2X7 antagonist oATP (see the
different kinetics of microglial cell lysis in Fig. 3H). On
the contrary, the presence of the P2Y/P2X antagonist PPADS (added at
the beginning of recording to the extracellular solution) was much less
effective in delaying the increase in membrane permeability (Fig. 3H). These results indicate that the large increase in
calcium and membrane permeability of microglial cells may be mediated
by the paracrine activation of microglial P2X7
receptors caused by locally high ATP concentrations or lower ATP
concentrations repeatedly released from astrocytes.
Astrocytes-released ATP mediates cytotoxicity in microglial cells
It has been reported that activation of P2X7
receptors by exogenous ATP induces microglial cell death
(24), because pore opening radically disrupts ionic
homeostasis and causes the depletion of intracellular low weight
metabolites. Fig. 4 shows that microglial
cell death is produced by pharmacological treatments that increase
astrocyte [Ca2+]i and
enhance ATP release; 24-h treatment of astrocyte-microglia cocultures
with repeated challenges of 1 µM bradykinin in KRH led to massive
microglial death accompanied by chromatin condensation as revealed by
the intercalation of propidium iodide in the DNA (Fig. 4B).
The astrocytes present in the mixed glial cultures did not show any
morphological changes typical of apoptosis (Fig. 4B) and had
intact nuclei. The percentage of condensed nuclei in microglia
associated with astrocytes was 73.5% in bradykinin-treated cultures;
however, such degenerative changes occurred to a much lesser extent
(18.4%) in the microglia present in the same coverslip but not
directly in contact with astrocytes (Fig. 4A). The
percentage of condensed nuclei in microglia associated with astrocytes
in the absence of pharmacological treatments was 51.5%. Thus, ATP
locally released by astrocytes, either spontaneously or upon
stimulation, activates P2X7 receptors, leading to
the apoptotic death of microglial cells.
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potentiates ATP-mediated calcium signaling between
astrocytes and microglia
Application of IFN- to pure astrocyte cultures caused an
average increase in the frequency of spontaneously occurring
[Ca2+]i oscillations of
520% (n = 37 control; n = 42
IFN--treated cells), with a preonset latency of 5–10 min (Fig. 5, A and B). The
mean frequency of [Ca2+]i
oscillations was derived from 30-min recordings made before and after
application of IFN-. No significant changes in the frequency of
[Ca2+]i oscillations were
observed between the two 30-min recordings made in the absence of
IFN- (data not shown).
The increased frequency of astrocytic
[Ca2+]i oscillations was
associated with a 4- to 5-fold increase in the released ATP (Fig. 5C), which was detected in samples of extracellular medium
incubated with astrocytes for 30 min before and after the addition of
IFN- using the sensitive luciferase bioluminescence assay (Fig. 5C, left panel). The amount of ATP released by
astrocytes was also determined using a biological assay
(8) in which samples of extracellular medium taken before
and after treatment with IFN- were gently added to fura-2-loaded
astrocytes as ATP sensor cells. Although both samples of extracellular
medium evoked a measurable
[Ca2+]i response, the
[Ca2+]i transients
induced by the extracellular saline incubated with IFN--treated
astrocytes were significantly higher (see quantitation in Fig. 5C, right panel). Based on a standard
dose-response curve of the calcium response amplitude vs different ATP
concentrations, the actual ATP level in the extracellular medium was
estimated to be 140 ± 16 nM in basal conditions and 650 ±
45 nM after IFN- treatment. The local concentration of ATP at the
releasing astrocyte was probably higher, as microglial cells do not
respond to exogenous application of ATP <10 µM. The
[Ca2+]i responses were
prevented when the conditioned medium was treated with apyrase (30 U/ml
for 10–15 min), thus indicating that ATP is the bioactive compound
released in the medium and is responsible for the observed effects.
IFN- potentiates P2X7 receptor activation and
microglial apoptosis
In line with the enhanced stimulation of ATP release from
astrocytes, IFN- considerably shortened the time of activation of
P2X7 receptors in microglial cells cocultured
with astrocytes (Fig. 6A). The
treatment of mixed glial cells cultures with IFN- for 24 h
exacerbated the apoptotic death of microglial cells, as revealed by
chromatin condensation of the nuclei stained with propidium iodide. The
quantitative results are shown in Fig. 6B; 51.5% of the
microglial cells lying in strict contact with astrocytes underwent
apoptosis under control conditions, but a significantly higher
proportion of microglial cells (88.8%) showed typically apoptotic
morphological changes after IFN- treatment. On the contrary, no
significant apoptotic changes were detected in the microglial cells
present in the same coverslip but not directly in contact with
astrocytes, indicating a local cytolytic effect of astrocyte-released
ATP in the cultures.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results indicate that ATP is involved in the communication between astrocytes and microglial cells. As revealed by a biological assay and the luciferase bioluminescence assay, spontaneously oscillating and stimulated astrocytes release ATP in the extracellular medium. Furthermore, the purinergic antagonist PPADS (but not glutamate antagonists) potently inhibits the [Ca2+]i transient induced in microglial cells as a result of mechanical stimulation of single astrocytes or bradykinin application. Finally, the ATP-degrading enzyme apyrase blocks the biological activity of the released mediator in the collected medium and inhibits propagation of the calcium wave to microglial cells.
This blocking action of purinergic antagonists also suggests that the
transfer of calcium signaling between astrocytes and microglial cells
is entirely due to an extracellular messenger. Astrocyte-microglia
intercellular communication mediated by gap junctions is unlikely
because microglial cells are not dye-coupled with astrocytes
(28). Furthermore, we observed that astrocytic calcium
signaling efficiently propagates to microglial cells in the presence of
the gap junction blocker 18-glycyrrhetinic acid. Given that
18-glycyrrhetinic acid as well as other gap junction blockers may
also block the release of ATP controlled by connexins
(32), our data support the existence of a
calcium-dependent, gap junction blocker-insensitive release of ATP from
astrocytes. How ATP crosses the membrane of astrocytes is still
unknown. The existence in astrocytes of typical secretory granules
undergoing regulated secretion has been recently reported
(33), and a number of synaptic proteins for regulated
secretion in neuronal cells have been identified in glial cells
(34, 35, 36). The association of these proteins with
ATP-storing organelles in astrocytes (36), along with the
partial sensitivity of ATP release to clostridial toxins (S. Coco, M.
Matteoli, and C. Verderio, unpublished observations) suggest that ATP
in astrocytes is stored in intracellular vesicles that undergo
regulated secretion.
The main finding of this study is that astrocyte-released ATP mediates a paracrine activation of microglial P2X7 receptors that triggers a perturbation of calcium homeostasis and finally leads to microglial cell death. This is consistent with previously reported data showing that P2X7 activation by exogenously administered ATP mediates cytolysis in mouse as well as in human macrophages and microglial cells (27, 37, 38, 39, 40). Although other P2X receptor subtypes can form a pore permeable to ethidium dyes (41, 42), uniquely for the P2X7 receptors this pore dilation progresses to cell lysis (41).
The physiological source of the massive amounts of ATP needed to trigger P2X7 activation in macrophages and microglia in situ is still not defined, although it has been proposed to be neighboring dying cells. Our data identify astrocytes as the major source of endogenous ATP. The purine is released in the absence of cell injury as a consequence of increased [Ca2+]i in astrocytes. Furthermore, our data, by showing that repetitive challenges with low doses of ATP result in microglial cell lysis, provide clues for understanding how P2X7 receptors, which are characterized by a low affinity for ATP, might be activated in situ. ATP, released by astrocytes following [Ca2+]i increases, may reach local concentrations sufficient to activate high affinity purinergic receptors in microglial cells. This repetitive activation could, in turn, lead to either calcium-dependent P2X7 recruitment to the plasma membrane or induction of an autocrine/paracrine loop resulting in an even higher extracellular ATP concentration, able to activate P2X7 receptors.
[Ca2+]i changes, which
occur spontaneously (20) or in response to different kinds
of stimuli (8, 9) in cultured astrocytes, have been shown
to occur in brain as a consequence of neuronal activation and glutamate
release (26). Alterations of calcium homeostasis in
astrocytes have been suggested to occur in specific pathophysiological
conditions of the CNS (43). Interestingly, we have found
that the inflammatory cytokine IFN- increases the frequency of
spontaneous calcium oscillations in glial cells. IFN- receptor
stimulation has been previously associated with elevation of cAMP,
elevations of inositol trisphosphate that cause release of
calcium from stores, and protein kinase C activation in cells of the
immune system and astrocytes (44). The effect of IFN-
on astrocyte calcium homeostasis potentiates astrocyte-to-microglia
communication, with the enhanced ATP release from astrocytes presumably
accounting for the increased apoptosis of microglial cells. This
finding adds to the reported up-regulation of
P2X7 receptors caused by prolonged IFN-
treatment in human macrophages and mouse microglial cells (38, 25).
The activation of P2X7 receptors induces IL-1
release from microglia (15, 45). It has recently been
reported that IL-1 significantly down-regulates gap junction
connectivity among astrocytes and also potentiates interastrocyte
calcium signaling mediated by the extracellular messenger ATP
(46). Microglial cells activated by ATP released from
astrocytes upon IFN- treatment can therefore feed back to glial
cells via IL-1 and thus activate a paracrine loop, sustaining
further ATP release.
The repeated secretion of ATP from astrocytes and the apoptotic
response in microglial cells may represent a homeostatic mechanism for
controlling the number of microglial cells in pathophysiological
conditions of the CNS. It is interesting to note that an increased
number of microglial cells undergoing apoptosis has been reported in
inflammatory demyelinating diseases, including experimental autoimmune
encephalomyelitis in rats (47, 48). In acute multiple
sclerosis (MS) plaques, in particular, microglia and macrophages
represent a relevant percentage (60%) of the TUNEL-positive cells
(49, 50). It is noteworthy that IFN- has been
selectively detected in active plaques, where it is predominantly found
on astrocytes (51). As the interaction between microglia
and T cells is important in the development of CNS inflammation, the
apoptosis of microglia could be related to the development and
progression of MS, as it has been shown that Ag-stimulated T cells
undergo apoptosis in the presence of microglial cells
(52, 53, 54). By acting locally to kill activated T cells,
microglia seem to be a component of the so-called immunological brain
barrier (3) and may contribute to the down-regulation of
immunopathologic processes in the brain. Our data showing that in the
presence of the inflammatory cytokine IFN-, astrocytes induce
extensive microglia apoptosis due to the release of ATP suggest that
this mechanism may operate during MS inflammation and may thus
represent a potential target for therapeutic strategies.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Claudia Verderio, Consiglio Nazionale delle Ricerche, Cellular and Molecular Pharmacology and "B. Ceccarelli" Centers, Department of Medical Pharmacology, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy.
3 Abbreviations used in this paper: [Ca2+]i, intracellular calcium; KRH, Krebs-Ringer solution with HEPES; CSF-1R, CSF receptor; PPADS, pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid, tetrasodium; oATP, oxidized ATP; MS, multiple sclerosis; GFAP, glial fibrillar acidic protein.
Received for publication October 16, 2000. Accepted for publication March 7, 2001.
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 L. K. Parvathenani, S. Tertyshnikova, C. R. Greco, S. B. Roberts, B. Robertson, and R. Posmantur P2X7 Mediates Superoxide Production in Primary Microglia and Is Up-regulated in a Transgenic Mouse Model of Alzheimer's Disease J. Biol. Chem., April 4, 2003; 278(15): 13309 - 13317. [Abstract] [Full Text] [PDF]
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 W. S. Nesbitt, S. Giuliano, S. Kulkarni, S. M. Dopheide, I. S. Harper, and S. P. Jackson Intercellular calcium communication regulates platelet aggregation and thrombus growth J. Cell Biol., March 31, 2003; 160(7): 1151 - 1161. [Abstract] [Full Text] [PDF]
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 E. HANSSON and L. RONNBACK Glial neuronal signaling in the central nervous system FASEB J, March 1, 2003; 17(3): 341 - 348. [Abstract] [Full Text] [PDF]
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J. Aleu, M. Martin-Satue, P. Navarro, I. P. de Lara, L. Bahima, J. Marsal, and C. Solsona Release of ATP induced by hypertonic solutions in Xenopus oocytes J. Physiol., February 15, 2003; 547(1): 209 - 219. [Abstract] [Full Text] [PDF]
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S. Coco, F. Calegari, E. Pravettoni, D. Pozzi, E. Taverna, P. Rosa, M. Matteoli, and C. Verderio Storage and Release of ATP from Astrocytes in Culture J. Biol. Chem., January 3, 2003; 278(2): 1354 - 1362. [Abstract] [Full Text] [PDF]
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 H. Sauer, R. Stanelle, J. Hescheler, and M. Wartenberg The DC electrical-field-induced Ca2+ response and growth stimulation of multicellular tumor spheroids are mediated by ATP release and purinergic receptor stimulation J. Cell Sci., August 15, 2002; 115(16): 3265 - 3273. [Abstract] [Full Text] [PDF]
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J. M. Labasi, N. Petrushova, C. Donovan, S. McCurdy, P. Lira, M. M. Payette, W. Brissette, J. R. Wicks, L. Audoly, and C. A. Gabel Absence of the P2X7 Receptor Alters Leukocyte Function and Attenuates an Inflammatory Response J. Immunol., June 15, 2002; 168(12): 6436 - 6445. [Abstract] [Full Text] [PDF]
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, 
, 
) and in the presence of IFN-
, 
). Note that the cytokine significantly shortens the time
required to induce microglial cell lysis. B,
Quantitative analysis of the percentages of apoptotic microglial cells
in mixed cultures after 15-h incubation in control KRH or in the
presence of IFN-