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- and ß-Chemokine Synthesis in Human Microglial Cells and Astrocytes1
Laboratory of Virus, Neuron and Immunity, Unité de Formation et de Recherche, Kremlin Bicêtre, University of Paris-South, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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- and ß-chemokines (IL-8 >
growth related protein (GRO) >> RANTES >
microphage inflammatory protein (MIP)-1 and MIP-1ß) in
parallel to PGs (PGE2 and PGF2) after
proinflammatory cytokine stimulation: TNF- + IL-1ß induced all
except RANTES, which was induced by TNF- + IFN-
. Purified
cultures of astrocytes and microglia were also induced by the same
combination of cytokines, to produce all these mediators except
MIP-1 and MIP-1ß, which were produced predominantly by astrocytes.
The inhibition of PG production by indomethacin led to a 37–60%
increase in RANTES, MIP-1, and MIP-1ß but not in GRO and IL-8
secretion. In contrast, inhibition of IL-8 and GRO activities using
neutralizing Abs resulted in a specific 6-fold increase in
PGE2 but not in PGF2 production by
stimulated microglial cells and astrocytes, whereas Abs to
ß-chemokines had no effect. Thus, the production of PGs in human
glial cells down-regulates their ß-chemokine secretion, whereas
-chemokine production in these cells controls PG secretion level.
These data suggest that under inflammatory conditions, the
intraparenchymal production of PGs could control chemotactic gradient
of ß-chemokines for an appropriate effector cell recruitment or
activation. Conversely, the elevated intracerebral -chemokine levels
could reduce PG secretion, preventing the exacerbation of inflammation
and neurotoxicity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of chemokines and PGs in several biological effects induced by
proinflammatory cytokines, as well as the presence of common events in
the pathways of synthesis and signal transduction of these two classes
of mediators such as phospholipase A2 activation and intracellular
calcium mobilization (14, 15), prompted us to study a possible
interaction between chemokine and PG pathways of synthesis. In this
report, we demonstrate that human microglia and astrocytes produce -
and ß-chemokines (IL-8, growth related protein (GRO),
RANTES, microphage inflammatory protein (MIP)-1, and MIP-1ß) and
PGs under proinflammatory conditions. The production of ß-chemokines
in these cells is negatively regulated by their PG secretion, whereas
PG production itself is limited by -chemokine secretion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight- to 10-wk-old human embryos were obtained after elective abortion in compliance with the recommendations of the French National Ethics Committee and following approval by the local ethics committee. Primary cultures of spinal cord and prosencephalon were prepared as described (16, 17). Briefly, tissues were dissected, trypsinized, and resuspended in MCDB 153 medium (Polylabo, Paris, France) completed by 5% FCS, 2 mM glutamine, 105 U/liter penicillin, and 0.1 g/liter streptomycin. The cell suspension was seeded at a density of 5 x 105 cells per well (24-well plates, Nunc, Roskilde, Denmark) coated with collagen (100 µg/ml). Cultures were kept at 37°C in a 10% CO2 atmosphere. The medium was changed completely after 48 h and then by half every 3 days. These cultures consisted of neurofilament-positive neuronal clusters lying on a monocellular layer containing 40% glial fibrillary acidic protein (GFAP)-positive astrocytes and 40% CD68-positive microglial cells associated with <10% fibronectin-positive fibroblasts (16, 17, 18).
Purified cultures of human microglial cells and astrocytes
To obtain purified microglial and astrocyte cultures, CNS cells were seeded at a density of 2 x 106 cells per well in 6-well plates. After plating (10–15 days), the microglial cells were released by circular shaking, followed by a 20-min incubation at 37°C to select adherent cells, as described (17). These cells were subsequently grown in the same medium as the primary mixed cultures for 3 wk to 2 mo to obtain a monolayer containing >95% CD68/KiM-7 and EBM11-positive cells (17). The adherent cells remaining after release of microglial cells were trypsinized, plated, and passaged two to four times to obtain purified cultures of astrocytes (>95% GFAP-positive cells) (17). Under the usual experimental conditions, 5 x 105 astrocytes or microglial cells were seeded in 24-well plates and stimulations were performed at confluence.
Cell stimulations and reagents
To stimulate cultures, cells were refed with serum-free MCDB 153
medium containing the tested inducers, inhibitors, or Abs. IL-1ß (200
U/ml) was purchased from Genzyme (Cambridge, MA). TNF- (200 U/ml)
and IFN- (200 U/ml) were purchased from Boehringer Mannheim (Maylan,
France). All are recombinant human cytokines. Human rIL-8, GRO (500
ng/ml), and neutralizing Abs against human RANTES (polyclonal, 200
µg/ml), MIP-1 and MIP-1ß (polyclonal, 100 µg/ml) (19, 20, 21, 22),
IL-8, and GRO (monoclonal, 100 µg/ml) were purchased from R&D Systems
(Minneapolis, MN). The specific inhibitor for cyclooxygenase
(indomethacin, 5–15 µM) was purchased from Sigma (St. Louis, MO).
PGF2 and PGE2 were purchased from Cayman
Chemicals (Ann Arbor, MI; PGF2 and PGE2, 16
ng/ml) (23).
Chemokine and PG titrations
The concentrations of all tested mediators were measured by
enzyme immunoassay (PGF2 and PGE2, Cayman
Chemicals; - and ß-chemokines, R&D Systems). All assays were
performed using standards and instructions supplied by the
manufacturers.
Statistical analysis
Each measurement was performed at least in triplicate.
Student’s t test was used for determination of statistical
significance of two to five independent experiments. Results were
considered significant at p 
0.05.
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Results |
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- and ß-chemokine production by proinflammatory
cytokines in human glial cultures
Spontaneous IL-8 and GRO production was detected in the
supernatant of mixed glial cultures. The effect of proinflammatory
cytokines on this production was tested in the absence of serum:
IL-1ß and, to a lesser extent, TNF-, but not IFN- alone led to
an increase in IL-8 and GRO production by glial cells at day 3
poststimulation (Fig. 1
A).
However, a combination of TNF- + IL-1ß was most efficient at
inducing -chemokine production (IL-8: 290,435 ± 33,483 vs 2,
704 ± 1,590 pg/ml; GRO: 64, 978 ± 10, 884 vs 1, 516
± 977 pg/ml, (TNF- + IL-1ß)-stimulated vs control,
n = 5, p < 0.0001, Fig. 1A). RANTES secretion by mixed glial cultures was also
induced in the presence of proinflammatory cytokines although the
induction profile of the cytokine combinations differed (IFN- +
TNF- > IFN- + IL-1ß > TNF- + IL-1ß, Fig. 1B).
IL-1ß, TNF-, or IFN- alone had no significant effect on RANTES
production by these cells. The level of RANTES production by glial
cells was at least 10-fold lower than that of -chemokines
(4,958 ± 1, 190 vs 7 ± 0.3 pg/ml, (IFN- +
TNF-)-stimulated vs control, day 3, n = 3,
p = 0.001). The same cytokines induced production of
MIP-1 and MIP-1ß in mixed glial cultures (TNF- + IL-1ß >
IFN- + IL-1ß = IFN- + TNF-, Fig. 1C), which
nonetheless remained low compared with RANTES (MIP-1: 881 ± 88
vs 11 ± 1 pg/ml; MIP-1ß: 1, 345 ± 159 vs 36 ± 7
pg/ml, (TNF- + IL-1ß)-stimulated vs control, day 3,
n = 3, p < 0.0001). Kinetics of IL-8,
GRO, RANTES, MIP-1, and MIP-1ß production by
cytokine-stimulated mixed glial cells demonstrated a similar profile of
chemokine induction starting between 6 and 24 h poststimulation
and accumulating over the 3 days tested (Fig. 2). All purified cultures of both
microglia and astrocytes tested secreted high levels of IL-8, GRO,
and RANTES, respectively, after TNF- + IL-1ß and IFN- + TNF-
stimulation (Fig. 3, A and
B). In contrast, only two of six purified cultures of
microglia could be induced by TNF- + IL-1ß to produce MIP-1,
and three of six produced MIP-1ß, whereas four of five purified
cultures of astrocytes were induced to secrete both MIP-1 and
MIP-1ß under the same condition of stimulation (Fig. 3C).
Accordingly, MIP-1 and MIP-1ß secretion was induced more
efficiently in purified cultures of astrocytes compared with microglia.
Nevertheless, the level of MIP-1 and MIP-1ß secretion in purified
cultures of astrocytes remained low in comparison with mixed glial
cultures.
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-chemokines in human glial cells
To investigate the possible involvement of PGs in the regulation
of chemokine production by glial cells, we first tested the induction
of PGF2 and PGE2 in the supernatant of mixed
glial cultures after proinflammatory cytokine stimulation
(PGE2: 19, 814 ± 4, 315 vs 421 ± 230 pg/ml;
PGF2: 23,596 ± 3,739 vs 735 ± 268 pg/ml,
(TNF- + IL-1ß)-stimulated vs control, day 3, n =
5, p < 0.0001, Fig. 1
D). The induction of
PG production was inhibited in the presence of 5 µM indomethacin, a
specific cyclooxygenase inhibitor (PGE2: 12,773 ±
2,098 vs 147 ± 24 pg/ml; PGF2: 13, 983 ± 2,
144 vs 287 ± 25 pg/ml, (TNF- + IL-1ß)-stimulated vs (TNF-
+ IL-1ß + indomethacin)-treated). Both microglial cells and
astrocytes were responsible for PG production as demonstrated
previously (data not shown; see also 17 . The effect of
indomethacin was subsequently tested on the induced production of
chemokines. Treatment of mixed glial cultures with 5–15 µM
indomethacin at the time of stimulation led to a 37–60% increase in
the production of RANTES, MIP-1, and MIP-1ß induced, respectively,
by IFN- + TNF- and TNF- + IL-1ß (Fig. 4, A and B, RANTES:
4,142 ± 597 vs 6, 100 ± 674; MIP-1: 420 ± 56 vs
661 ± 57; MIP-1ß: 602 ± 45 vs 894 ± 52 pg/ml,
cytokine-treated vs (cytokine + 5 µM indomethacin)-treated, day 3,
n 
4, p 0.03). The effect of
indomethacin was dose-independent in the range of the tested
concentrations. This suggested a limitation of ß-chemokine secretion
by human glial cells in the presence of PGs. Addition of exogenous
PGF2 and PGE2 to the culture supernatants
abolished the effect of indomethacin on this chemokine production (Fig. 4, A and B), indicating a direct effect of PGs.
In contrast, inhibition of PG secretion by indomethacin had no effect
on GRO and IL-8 production in (TNF- + IL-1ß)-stimulated glial
cells (Fig. 4C). To investigate the role of microglia and
astrocytes in the regulation of ß-chemokines by PGs, purified
cultures of microglial cells and astrocytes were analyzed under the
same conditions. Although all purified microglial or astrocyte cultures
were induced with IFN- + TNF- to produce RANTES, this production
was not significantly modified after inhibition of PG secretion by
indomethacin (not shown). In contrast, indomethacin treatment of
astrocytes led to a significant increase in MIP-1 and MIP-1ß
production by these cells after TNF- + IL-1ß stimulation (Fig. 5).
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- or ß-chemokines could also regulate PG
secretion by glial cells, we tested the effect of neutralization of
chemokine activities on the induced production of PGs. Neutralization
of GRO and/or IL-8, but not of ß-chemokines, in the presence of
neutralizing Abs led to a strong increase in the production of
PGE2 by mixed glial cultures (Fig. 6A, 23,969 ± 7,500 vs
147,218 ± 14,320 pg/ml, cytokine-treated vs (cytokines +
anti-IL-8 + anti-GRO)-treated, day 3, n = 4,
p < 0.0001), whereas PGF2 secretion
remained unchanged (Fig. 6). The enhancement of PGE2 levels
in mixed glial cultures, in the presence of anti-IL-8 and
anti-GRO Abs, was due to an increase in PGE2 production
in both microglia and astrocytes (Fig. 6). The treatment of
unstimulated glial cells by purified IL-8 and GRO did not
significantly affect their spontaneous production of
PGF2 or PGE2 (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and ß-chemokines in human microglial cells and astrocytes.
This is also the first demonstration of a negative retroregulatory loop
between - and ß-chemokine and PG production in human glial cells.
Studies on chemokine production by CNS resident cells of either human
or rodent origin have focused mainly on a single class of chemokine in
either microglial cells or astrocytes (5, 6, 24, 25, 26, 27, 28). Here we describe
the production of high amounts of IL-8 and GRO by microglia and
astrocytes, either in close interaction or isolated, after stimulation
with TNF- in combination with IL-1ß. Mixed glial cells were also
induced by the same cytokines to secrete MIP-1 and MIP-1ß but to a
lesser extent compared with -chemokines. This production was
probably due to astrocytes rather than microglia because 80% of the
astrocyte cultures tested could be induced to secrete MIP-1 and
MIP-1ß, compared with 20–30% of microglial cultures. However, the
observation of a higher level of MIP-1 production in mixed glial
cultures than in astrocytes suggests that cell interactions between
microglia and astrocytes facilitate MIP-1 and MIP-1ß production.
The difference in the ability of astrocytes and microglia to secrete
MIP-1 and MIP-1ß is reminiscent of previous data describing the
inability of rodent microglial cells to produce monocyte chemotactic
protein-1 (MCP-1) protein, whereas astrocytes from the same origin
secreted MCP-1 under the same condition of stimulation (25). Moreover,
it has been shown that LPS-stimulated murine alveolar and peritoneal
macrophages expressed MIP-1 mRNA, whereas only alveolar
macrophages could produce MIP-1 protein in their supernatant after
stimulation (29). The disparity in MIP-1 secretion between these two
populations of macrophages was attributed to an impairment in MIP-1
protein translation and secretion in peritoneal macrophages (29).
Another explanation for the differentially expressed MIP-1 and
MIP-1ß by astrocytes and microglia could be a difference in
transcription factors between the two cell types such as activating
transcription factor/cAMP response element binding protein
(ATF/CREB)-, activating protein-1 (AP-1)-, NF-
B-,
CCAAT/enhancer binding protein (C/EBP), cellular-E 26-specific
(C-ET)-related proteins, and/or MIP-1 nuclear protein (30, 31, 32).
However, the possibility remains that other stimuli are required for an
efficient induction of these ß-chemokines in microglial cells.
Finally, the production of RANTES, which was less than that of
-chemokines but more than that of MIP-1 and MIP-1ß, was
increased in mixed or isolated glial cultures after stimulation by
TNF- in combination with IFN- rather than IL-1ß. The fact that
the induction of different chemokines did not require the same
combination of cytokines suggests different mechanisms of induction.
The inducing effect of inflammatory cytokines might involve the
induction of different transcription factors such as STAT1, NF-B, or
AP-1, as shown in certain cell types (33, 34, 35, 36).
Numerous in vitro studies have demonstrated the role of - and
ß-chemokines in inducing the migration of specific populations of
leukocytes (6, 25, 27, 37). However, in vivo studies have demonstrated
that the physiological functions of these chemokines are highly
dependent on their level of production. In fact, transgenic mice
overexpressing IL-8 exhibited a marked circulating neutrophilia and a
decreased neutrophilic exudation into body cavities in response to
acute inflammatory stimulants (38). Similarly, transgenic mice
overexpressing MCP-1 displayed no monocyte infiltration into tissues
and showed increased sensitivity to intracellular pathogens (39).
In contrast, mice in which murine GRO or MCP-1 were expressed at
lower levels and in anatomically restricted areas displayed an
appropriate infiltration of neutrophils or monocytes to the site of
chemokine production (40, 41). Thus, overproduction of chemokines in
vivo might lead to a loss of their specific biological effect through
either the inactivation of leukocytes by receptor desensitization or
the abolition of the chemotactic gradient.
We investigated whether chemokine production could be under the control
of other inflammatory mediators such as PGs, secreted in parallel in
proinflammatory cytokine-stimulated glial cells. Inhibition of PG
production in glial cells by the cyclooxygenase inhibitor,
indomethacin, resulted in an increase in glial secretion of RANTES,
MIP-1, and MIP-1ß, but not of IL-8 and GRO. The reversion of
this effect in the presence of exogenous PGs demonstrated that PGs are
directly implicated in the specific limitation of ß-chemokines.
Again, differences were observed between isolated glial cultures and
mixed glial cells. The blockade of PG secretion did not significantly
modify RANTES production by microglia or astrocytes cultured in
isolation. However, it did result in an increase in MIP-1 secretion
(similar to that observed with mixed glial cells) in astrocytes,
which are the main cells producing MIP-1 and MIP-1ß (Fig. 5). This suggested that the feedback regulation of RANTES by PGs might
be dependent on interactions between microglia and astrocytes. Thus,
PGs may control local physiological production of ß-chemokines,
leading to an appropriate activation of monocytes and lymphocytes,
and/or an appropriate recruitment of these cells to the inflammatory
foci (Fig. 7).
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. The addition of Abs to
neutralize RANTES, MIP-1, and MIP-1ß had no effect on
the induced production of PGs by glial cells. Thus, the high levels of
-chemokines produced under proinflammatory conditions might be
responsible, in part, for maintaining intracerebral PG secretion at a
low level. This observation suggests a role for -chemokines in the
protection of cerebral cells from PG toxicity during the inflammatory
response (Fig. 7). The immune surveillance in CNS inflammation has to be tightly controlled so as to mount an efficient defense against pathogens. An imbalance between immunoregulatory factors during infectious or autoimmune diseases may have drastic effects on the integrity of the brain parenchyma. In this respect, studies of anti-inflammatory treatments in different in vivo experimental systems are of great interest. Although in such models, anti-inflammatory drugs generally lead to the inhibition of vasoactive product secretion by effector cells (42, 43, 44), the inconsistency of their effect on the neurological lesions reflects subtle and time-dependent retroregulations, which determine the final biological effect on neuron function and survival.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nazila Janabi, Laboratoire "Virus, Neurone et Immunité," Faculté de Médecine, 63 rue Gabriel Péri, 94276 Le Kremlin Bicêtre Cedex, France.
3 Present address: Laboratory of Molecular Medicine and Neuroscience, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892.
4 Abbreviations used in this paper: CNS, central nervous system; MIP, microphage inflammatory protein; GRO, growth related protein; GFAP, glial fibrillary acidic protein; MCP-1, monocyte chemotactic protein-1.
Received for publication July 21, 1998. Accepted for publication October 16, 1998.
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B, and c-ETs family members and transcriptional regulation of cell-specific and inducible macrophage inflammatory protein 1 immediate-early gene. Mol. Cell. Biol. 13:5276. nuclear protein (MNP), a novel transcription factor expressed in hematopoietic cells that is crucial for transcription of the human MIP-1 gene. Mol. Cell. Biol. 15:3110.[Abstract]
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and interferon- synergistically induce interleukin 8 production in a human gastric cancer cell line through acting concurrently on AP-1 and NF-B-like binding sites of the interleukin 8 gene. J. Biol. Chem. 267:22506.This article has been cited by other articles:
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 K. M. Omari, G. R. John, S. C. Sealfon, and C. S. Raine CXC chemokine receptors on human oligodendrocytes: implications for multiple sclerosis Brain, May 1, 2005; 128(5): 1003 - 1015. [Abstract] [Full Text] [PDF]
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 R. B. Rock, G. Gekker, S. Hu, W. S. Sheng, M. Cheeran, J. R. Lokensgard, and P. K. Peterson Role of Microglia in Central Nervous System Infections Clin. Microbiol. Rev., October 1, 2004; 17(4): 942 - 964. [Abstract] [Full Text] [PDF]
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 K. Arima, K. Nasu, H. Narahara, K. Fujisawa, N. Matsui, and I. Miyakawa Effects of lipopolysaccharide and cytokines on production of RANTES by cultured human endometrial stromal cells Mol. Hum. Reprod., March 1, 2000; 6(3): 246 - 251. [Abstract] [Full Text] [PDF]
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, control; •, TNF-
, IFN-
