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Proinflammatory cytokines released from microglia inhibit gap junctions in astrocytes: potentiation by ß-amyloid

INSERM U587, Collège de France, Paris, France

³Correspondence: INSERM U587, Collège de France, 11 Place Marcelin Berthelot, Paris 75005, France. E-mail: christian.giaume{at}college-de-france.fr

SPECIFIC AIMS

In astrocytes, gap junctions provide an important mode of intercellular communication involved in several glial functions, including neuroprotection. We have previously reported that nonactivated microglia (MG) decreases the expression of connexin 43 (Cx43), the molecular constituent of gap junctions in astrocytes, and inhibits their function. The purpose of this study was to determine how and to what extent activated MG, a main actor in brain inflammation, affects astrocyte gap junctions. To address the functional status of gap junctions after activated MG application we have assessed: 1) the level of gap junction communication by dye coupling experiments; and 2) the level of Cx43 expression by Western blot and immunofluorescence analyses. An inflammatory response characterized by an activation of both MG and astrocytes occurs at site of ß-amyloid (Aß) deposits in Alzheimer’s disease. Thus, the present study has also investigated whether this peptide interferes with two proinflammatory cytokines produced by activated MG that control astrocyte gap junctions.

PRINCIPAL FINDINGS

1. Activated microglial cells inhibit gap junctional communication in astrocytes through the release of soluble factors
The level of astrocyte gap junctional communication (AGJC) was measured using the scrape-loading/dye transfer (SL/DY) technique on confluent astrocyte cultures where MG or conditioned medium (CM) harvested from cultured MG were added for 24 h. A significant decrease (up to 30%) in AGJC was observed after plating untreated MG on confluent astrocytes, while plating with CM was without effect (Fig. 1 A). In contrast, when activated by the bacterial endotoxin lipopolysaccharide (LPS) (10 ng /mL, 6 h), plating of activated MG produced a strong inhibition (74%) of AGJC (Fig. 1A ). The inhibitory effect of LPS-activated MG is mainly due to the release of soluble factors because: 1) an inhibition was still observed when physical contact between the two cell types was prevented by seeding activated MG on cell inserts; and 2) the incubation with conditioned medium harvested from LPS-activated MG (CM*) resulted in a strong inhibition (70%) of AGJC (Fig. 1A ).

View larger version (39K): [in this window] [in a new window]   Figure 1. LPS-activated microglial cells inhibit gap junctional communication and Cx43 expression in astrocytes. AGJC was evaluated by SL/DT in primary cultures of astrocytes where untreated (+MG) or LPS-activated MG (+MG*) were seeded overnight. A) Inhibitory effect of MG on AGJC requires physical contact between the two cell types: the direct addition of MG reduced the level of AGJC while MG addition into cell inserts (+MG insert) had not significant effect. In contrast, seeding MG* to astrocyte culture (+MG*) or into cell insert (+MG*, insert) induced a strong inhibition. Finally, the addition of conditioned media (1:2 dilution) harvested from either MG (CM) or activated MG (CM*) indicated that only the later was efficient in reducing AGJC. Data were obtained from 3–14 independent experiments. Differences were considered significant at **P < 0.01 and ***P < 0.001. B, C) Western blot analysis of Cx43 expression in protein extracts from various cell treatment. B) Down-regulation of Cx43 in astrocytes when MG were stimulated with LPS at 10 ng/mL (+MG*) or 1 µg/mL (+MG**) prior to seeding on astrocyte cultures. In contrast, no Cx43 was detected in purified MG exposed to similar concentrations of LPS (MG* and MG**). C) Compared with untreated astrocyte cultures, the level of Cx43 expression was weakly altered by the direct addition of MG (+MG) and strongly reduced by that of LPS-activated MG (+MG*) seeded into cell insert (MG*, insert) or by conditioned medium (1:2 dilution) harvested from activated MG (CM*). In this experiment, membranes were also hybridized with anti--tubulin antibody to visualize the amount of protein loaded for each sample.

2. Activated microglial cells down-regulate Cx43 expression in astrocytes
To define the molecular mechanisms involved in the MG-induced inhibition of AGJC, the level of Cx43 expression was investigated by immunoblot analysis. Treatment carried out with LPS (10 ng/mL) had no effect on Cx43 expression in astrocyte cultures, but the amount of Cx43 was significantly reduced when activated MG were added (Fig. 1B ). Evidence that Cx43 is solely expressed in astrocytes was demonstrated by the lack of Cx43 detection in MG alone activated or not by LPS (10 ng/mL and 1 µg/mL) for 6 or 24 h. In agreement with dye-coupling data, application of CM from MG had no effect on Cx43 expression while CM* from LPS-activated MG strongly reduced the amount of Cx43 protein. In addition, MG cultured on insert did not affect Cx43 expression while a marked reduction was obtained with activated MG (Fig. 1C ). Finally, the activated MG-induced astrocyte decrease in Cx43 expression was confirmed by an immunofluorescent analysis.

3. Co-application of IL-1ß and TNF- inhibits intercellular communication and Cx43 expression in astrocytes
Activated MG are known to release a number of immunoregulatory mediators among which is the proinflammatory cytokine IL1-ß previously shown to affect astrocyte gap junctions. Accordingly, the effect of four compounds potentially released by activated MG was tested on AGJC. IL-6, INF, and TNF- (10 ng/mL, 24 h) applied alone did not result in a significant change in AGJC (even at concentration up to 100 ng/mL) while IL-1ß produced a weak but significant inhibition. Alternatively, co-application of these compounds was performed, and among all possible combinations tested, only the co-application of TNF- and IL-1ß (10 ng/mL, 24 h) strongly reduced AGJC with a more than additive effect (Fig. 2 A). These observations were reinforced by immunoblot and immunofluorescence analysis that demonstrated a strong down-regulation of Cx43 expression in astrocytes treated with both TNF- and IL-1ß.

View larger version (21K): [in this window] [in a new window]   Figure 2. ß-Amyloid potentiates the inhibitory effect of proinflammatory cytokines on AGJC. A) The presence of Aß25–35, which by itself has no effect on AGJC, when co-applied with either IL1 or TNF enhances the blocking effect of IL1 and TNF. A similar potentiation was observed when Aß25–35 was co-applied with diluted CM* (Dil.CM*). The dilution of the CM* varied from 1:40 to 1:64 depending on the selected sample and was chosen at the threshold dilution found to have no significant effect on AGJC. This potentiation was blocked in the presence of IL-1ra and sTNFR1 (Aß+Dil.CM*+2antag). Data were obtained from 3–18 independent experiments. Differences were considered significant at **P < 0.01 and ***P < 0.001.

4. TNF- and IL-1ß are involved in the inhibitory effect of the conditioned medium harvested from activated microglial cells
To evaluate the involvement of these cytokines in the CM*-induced inhibition of AGJC, blockers of each cytokine activity were used. Treatment of astrocytes with CM*, used at 1:4 dilution, in the presence of IL-1ra or sTNFR1 (the recombinant receptor antagonist for IL-1ß and the soluble receptor for TNF-, respectively) (100 ng/mL) resulted in partial reduction of the inhibition of AGJC (27 and 23% inhibition, respectively). In the presence of both agents, the inhibition induced by 1:4 diluted CM* was almost abolished (only 15% inhibition remained). These observations demonstrate that these two proinflammatory cytokines contribute to the CM*-induced inhibitory effect on AGJC.

5. ß-Amyloid potentiates the inhibitory effect of conditioned medium from activated microglia on astrocyte gap junctions: role of IL-1ß and TNF
Since Aß deposits are observed at sites where proinflammatory cytokines are potentially released, co-application of Aß_25–35, the active fragment of the Aß peptide, with either of the two cytokines active on AGJC was tested. While IL-1ß, TNF (10 ng/mL, 24 h) or Aß_25–35 (20 µM, 24 h) applied alone had only a slight inhibitory effect on their own, the co-application of each cytokine with Aß_25–35 resulted in a significant inhibition of AGJC and the inhibitory effect of these cytokines applied together was also enhanced (Fig. 2B ).

While the effect of CM* diluted 1/40 to 1/64 was not significant, the addition of Aß_25–35 resulted in a marked AGJC inhibition (Fig. 2B ). Such inhibitory effect was correlated with a decrease in the level of Cx43 expression analyzed by immunoblot as well as immunofluorescence.

Finally, the inhibition induced by the co-application of diluted CM* and Aß_25–35 was prevented by the presence of IL-1ra or sTNFR1 used at concentrations (100 ng/mL) that were shown to block the IL-1ß plus TNF inhibitory effect.

CONCLUSIONS AND SIGNIFICANCE

These studies demonstrated that activated MG control gap junctions by down-regulating Cx43 expression in astrocytes, mainly through the release of two proinflammatory cytokines, IL-1ß and TNF-. Moreover, Aß_25–35 potentiates the inhibitory effect of these MG products on astrocyte gap junctions. As the inhibition of AGJC and Cx43 expression is observed when diluted CM* and Aß are co-applied on astrocyte cultures (i.e., in the absence of MG) this potentiation may directly operate at the level of astrocytes. These observations are of particular interest because a critical step in AD is apparently reached when pathological glial activation is not restricted to MG but includes astrocytes. As a whole, they indicated that the vicinity of reactive MG with sites of Aß deposits may result in a lack of intercellular communication between astrocytes involved in the glial inflammatory response.

Although some evidence suggests that functional gap junctions in astrocytes could be deleterious for neurons, several reports indicate that they rather play a neuroprotective role. The inhibition of AGJC enhances the neurotoxicity induced by oxidative agents or glutamate. After a stroke lesion, the volume of brain infarct is larger and an increased neuronal apoptosis is observed several days after focal ischemia in heterozygote Cx43 knockout mice. Thus, AGJC may contribute to the neuroprotective role of astrocytes and its inhibition may affect neuronal fate in brain injury and pathologies. Accordingly, the strong inhibition of AGJC induced by Aß in the presence of proinflammatory agents secreted by activated MG could account for the proposed glial contribution to neurodegenerative processes in AD.

Finally, such interaction between astrocytes and MG resulting in the inhibition of AGJC can be considered in the more general context of neurodegenerative disease progression. This inhibitory effect may play a role in an amplification phenomenon leading to the extension of neuronal damages. Because neuronal death has also been reported to down-regulate Cx43 expression and AGJC, both effects may converge to diminish the neuroprotective role of astrocytes. As neuronal death is associated with a glial reaction, a repetition of the above steps could take place by establishing a "vicious circle." It is critical to determine whether the inhibition of astrocyte gap junctions reported here affects neuronal survival and disrupts neuron-glia interaction, an important contributing factor to neurodegeneration.

View larger version (31K): [in this window] [in a new window]   Figure 3. Summary diagram indicating that the inhibition of astrocyte gap junctions by microglia depends on their activation status. This interaction, which targets the expression of Cx43 in astrocytes, is mediated by the release of proinflammatory cytokines and potentiated in the presence of Aß25–35.

FOOTNOTES

¹ These authors contributed equally to this work.

² Present address: Laboratoire de Neurobiologie, Université d’Orléans, BP 6759, Orléans 45067, France

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4297fje;

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