Astrocyte-Derived ATP Induces Vesicle Shedding and IL-1β Release from Microglia

Bianco, Fabio; Pravettoni, Elena; Colombo, Alessio; Schenk, Ursula; Möller, Thomas; Matteoli, Michela; Verderio, Claudia

doi:10.4049/jimmunol.174.11.7268

Cited by 519 publications

(526 citation statements)

References 45 publications

Supporting

Mentioning

499

Contrasting

Unclassified

Order By: Relevance

“…27 Prolonged activation of P2X7 is known to induce the formation of an aspecific pore, measured by dye uptake, which is known to induce sustained intracellular calcium levels in microglia leading to the release of neuroinflammatory mediators. 28 In our experiment, we show that ATP conveying in the second chamber as a consequence of astrocyte stimulation with glutamate leads to an uptake of PI by microglia. The micrographs in Figure 5b-g show images (10× objective) of microglia after three independent experiments.…”

Section: Resultssupporting

confidence: 49%

“…We currently are scaling the concept of oMFNs to having three or more interconnected cell chambers, in view of depositing different cell populations in each chamber for dissecting the intercellular flows of information occurring among brain cells. 27,28 This approach might help identify the mediators and the cellular sources of molecules involved in neuroinflammation, a process that plays a very active role in the pathophysiology of progressive neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases, and synaptopathies in general.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Overflow Microfluidic Networks for Open and Closed Cell Cultures on Chip

et al. 2010

Self Cite

View full text Add to dashboard Cite

Microfluidics have a huge potential in biomedical research, in particular for studying interactions among cell populations that are involved in complex diseases. Here, we present "overflow" microfluidic networks (oMFNs) for depositing, culturing, and studying cell populations, which are plated in a few microliters of cell suspensions in one or several open cell chambers inside the chip and subsequently cultured for several days in vitro (DIV). After the cells have developed their phenotype, the oMFN is closed with a lid bearing microfluidic connections. The salient features of the chips are (1) overflow zones around the cell chambers for drawing excess liquid by capillarity from the chamber during sealing the oMFN with the lid, (2) flow paths from peripheral pumps to cell chambers and between cell chambers for interactive flow control, (3) transparent cell chambers coated with cell adhesion molecules, and (4) the possibility to remove the lid for staining and visualizing the cells after, for example, fixation. Here, we use a two-chamber oMFN to show the activation of purinergic receptors in microglia grown in one chamber, upon release of adenosine triphosphate (ATP) from astrocytes that are grown in another chamber and challenged with glutamate. These data validate oMFNs as being particularly relevant for studying primary cells and dissecting the specific intercellular pathways involved in neurodegenerative and neuroinflammatory brain diseases.Microfluidics are emerging as invaluable tools for research on cells for a number of reasons. 1-3 First, they enable experiments on cells to be performed with well-defined chemical and topographical environments. 4-9 Second, they can reduce the number of cells needed for experiments. 1 Third, they can be used for experiments in which a precise number of cells can be stimulated and observed accurately. 10-12 Last, they permit experiments to be performed in parallel and/or with high throughput. 13 Despite these possibilities, microfluidics are not well established for daily research work on cells, as they lack user friendliness and require specific and often cumbersome methods for depositing and culturing cells. This is further complicated when taking into consideration more relevant cell systems such as primary cells, 14,15 which may require longer culturing times before being ready for specific assays (i.e., primary neurons need to be cultured several days in vitro (DIV) before establishing mature synaptic contacts). Although progress on sealing a Petri dish with a hybrid microfluidic-vacuum platform was recently demonstrated by Chung et al., 9 interfacing "open" cell cultures with microfluidics for cell cultures that are only a few microliters in volume remains a great challenge. Similarly, depositing cells on a treated glass slide and gluing a lid having ports onto the slide provides a method for enclosing living cells inside a microfluidic flow path. 16 Such a method would be hard to implement with multiple types of cells and can be simplified if the gluing step coul...

show abstract

Section: Resultssupporting

confidence: 49%

Section: Discussionmentioning

confidence: 99%

Overflow Microfluidic Networks for Open and Closed Cell Cultures on Chip

et al. 2010

Self Cite

View full text Add to dashboard Cite

show abstract

“…Triggering of P2 receptors has been reported to induce shedding of plasma membrane MVs from HEK293 expressing the P2X7 subtype, the human acute monocytic leukemia cell line THP-1, mouse primary microglia, and monocytederived human DCs [6,24,31].…”

Section: Atp and Bzatp Induce Rapid Release Of Mvsmentioning

confidence: 99%

IL‐18 associates to microvesicles shed from human macrophages by a LPS/TLR‐4 independent mechanism in response to P2X receptor stimulation

et al. 2012

View full text Add to dashboard Cite

Extracellular ATP, released upon microbial infection, cell damage, or inflammation, acts as an alert signal toward immune cells by activating P2 receptors. The nucleotide causes microvesicle (MV) shedding from immune and nonimmune cells. Here, we show that IL-18 associates with MVs shed by human ex vivo macrophages upon P2X receptor stimulation.MV shedding was potently induced by ATP and by the P2X7 agonist 3 -benzoylbenzoyl adenosine 5 -triphosphate, while it was greatly reduced by P2X irreversible inhibitoroxidized ATP and by the specific P2X7 inhibitors KN-62, A-740003, and A-438079. Peculiarly, the P2X7 subtype was highly present in the MVs, while on the contrary the P2X3 and P2X4 subtypes were almost absent. The Ca 2+ ionophore A23187 mimicked the effect of 3 -benzoylbenzoyl adenosine 5 -triphosphate suggesting that an intracellular Ca 2+ increase was sufficient to evoke MV shedding. Caspase inhibitors Ac-YVAD-CMK or Z-YVAD-CMK did not block the cleavage of MV-associated pro-IL-18. Pro-IL-18 formation in macrophages did not require pretreatment of cells with LPS, as the procytokine was already present in unprimed macrophages and did not decrease by incubating cells with the LPS-binding antibiotic polymyxin B nor with the TLR-4 intracellular inhibitor CLI-095. These data reveal a nucleotide-based mechanism responsible for the shedding of MV to which IL-18 is associated.Keywords: Extracellular ATP r Human macrophages r IL-18 r P2 receptors r P2X7 Supporting Information available online IntroductionMicrovesicles (MVs) are small vesicles derived from the plasma membrane of eukaryotic cells [1,2]. MVs are gaining momenCorrespondence: Dr. Davide Ferrari e-mail: dfr@unife.it tum as they have been isolated in tumors, infectious and autoimmune diseases, and a role in delivery different messages to the cells has also been demonstrated [2][3][4]. They can transport and deliver proteins, enzymes, mRNA, or microRNA. Recent * These authors contributed equally to this work.C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2012. 42: 3334-3345 Innate immunity 3335 observations have shown that MVs shed by mesenchymal stem cells play a role in inducing peripheral tolerance and modulation of the immune response [5]. Proinflammatory cytokines such as IL-1β and IL-18 are leaderless secreted proteins. It has been shown that IL-1β is efficiently released from immune cells in association to MVs containing the cytokine. Shedding of MV requires stimulation of membrane receptors for the extracellular nucleotides [6]. IL-18, previously named IFN-γ-inducing factor for its ability to stimulate IFN-γ production in T lymphocytes and natural killer cells, is one of the main cytokines contributing to the pathogenesis of autoimmune and inflammatory diseases [7]. Besides lymphocytes, IL-18 is secreted by a variety of cell types including epithelial cells, keratinocytes, synovial fibroblasts, monocytes, macrophages, and DCs [8,9], thus inducing the synthesis of other proinflammatory cytokines (IFN-γ, IL-1β...

show abstract

“…However, compared with the control group, BBG not only reversed the inhibition of rotenone, but even promoted the secretion. An explanation for this phenomenon may be that in the control group, astrocytes could secrete ATP without cell damage [25,26]. Such ATP would inhibit TNF-α secretion from astrocytes themselves through native P2X7 receptors.…”

Section: Discussionmentioning

confidence: 99%

Astroglial P2X7 receptor current density increased following long-term exposure to rotenone

Gao

Wang

et al. 2011

Purinergic Signalling

View full text Add to dashboard Cite

The role of the interaction between neurons and glial cells in the pathogenesis of neurodegenerative diseases is gaining more attention. Neuroinflammation participates in the progressive nature of diverse neurologic diseases including Parkinson's disease, Alzheimer's disease and multiple sclerosis. Activated microglia release neurotoxic molecules, which take part in the neuroinflammatory responses. Astrocytes are also key players in these responses. Reactive astrocytes secrete inflammatory factors, including tumor necrosis factor-α (TNF-α). This secretion can be regulated by extracellular ATP mediated through P2X7 receptors. However, whether the activity of astrocytic P2X7 receptors changes in Parkinson's disease and whether these changes would influence the secretion of inflammatory factors in astrocytes are still unclear. In our study, through immunocytochemistry, whole-cell patch clamp and ELISA assay, we found that P2X7 receptors were expressed in midbrain astrocytes, and that, rotenone, a Parkinson's disease model used at a low concentration (2-20 nM) for 48 h increased the P2X7 receptor current density and thereby inhibited the secretion of TNF-α. Our research suggests that rotenone can regulate cytokine secretion of astrocytes through elevated P2X7 channel current density and, in turn, take part in the neuroinflammatory process in the rotenone Parkinson's disease model.

show abstract

Astrocyte-Derived ATP Induces Vesicle Shedding and IL-1β Release from Microglia

Abstract: Material

Cited by 519 publications

References 45 publications

Overflow Microfluidic Networks for Open and Closed Cell Cultures on Chip

Overflow Microfluidic Networks for Open and Closed Cell Cultures on Chip

IL‐18 associates to microvesicles shed from human macrophages by a LPS/TLR‐4 independent mechanism in response to P2X receptor stimulation

Astroglial P2X7 receptor current density increased following long-term exposure to rotenone

Contact Info

Product

Resources

About