Characterization of the cytokine secretion profile of highly purified, functional astrocytes

Hui Zhang, Sandy Reiß, Melanie Jungblut, and Andreas BosioMiltenyi Biotec GmbH, Bergisch Gladbach, Germany

IntroductionThe inflammatory response in the central nervous system (CNS) in multiple sclerosis (MS) is a dynamic multifactorial process that involves both the local resident immune cells, such as microglial cells and astrocytes, as well as infiltrating circulating immune cells such as macrophages. These immune cells communicate to each other by secreting different cytokines and chemokines, regulating and initiating their activities. Astrocytes, a population of CNS- resident cells, were found to play multiple roles in neuro-inflammation. However, there are no detail ed and reproducible studies of cytokine and chemokine secretion, partly due to technical restrictions. Most astrocyte populations are not purified effectively and contain contaminating microglial cells, which can lead to erroneous interpretation of the results.

In the past we have set up an automated dissocia tion procedure for neonatal brain using the gentleMACS™ Octo Dissociator with an optimized enzymatic treat ment. For isolation and culture of adult neural cells we have further improved the method and

included a novel protocol for removal of debris and erythrocytes, yielding 2–4×10⁶ living cells per mouse brain. After brain dissociation, astro cytes were isolated to a purity of >98.5% using MACS® MicroBeads coupled to anti bodies against a specific pan astrocyte marker, ACSA-2 (astrocyte cell surface antigen-2).

We also characterized the cytokine secretion profiles of activated neonatal and adult astrocytes. Immu no cytochemistry (ICC) analy-ses showed that neonatal and adult astrocytes expressed a broad range of cytokines and chemokines. When lipo polysaccharide (LPS) or conditioned media from LPS-stimulated microglia were added to neonatal astrocytes, an increased level of GM-CSF was de tected with the MACSPlex Cytokine Kit (Miltenyi Biotec). In case of adult astrocytes distinct cytokine secretion profiles domi nated either by GM-CSF or IL-23 were detected after activation with TNF-α/LPS or IFN-γ/LPS, respect ively. This points to the presence of diverse astrocyte subtypes or to different cellular states, in which the cells are capable of reacting to different stimuli.

• We present a novel technology to purify functional astrocytes from neonatal and adult mouse brain.

• Neonatal and adult astrocytes express a broad range of cytokines and chemokines as shown by ICC.

• A significantly increased level of GM-CSF was de tected in cul tures of neonatal astrocytes when the cells were treated with microglia-conditioned media or with higher concentra tions of LPS. In con trast, there was no significant change in the TNF-α level.

• Distinct cytokine secretion profiles dominated either by GM-CSF or IL-23 were detected following activation of adult astrocytes with TNF-α/LPS or IFN-γ/LPS, respectively. This suggests that diverse astrocyte subtypes are present or that the cells are in different states and therefore capable of react ing to different stimuli.

• Further experiments will focus on the isolation of astrocytes from EAE mouse brain to gain insight into the in vivo cytokine secre tion profile of astrocytes in neuroinflammation.

Highly pure neonatal and adult astrocytes were obtained by MACS® Technology using Anti-ACSA-2 MicroBeads1

Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. gentleMACS, MACS, and MACSQuant are registered trademarks of Miltenyi Biotec GmbH. Copyright © 2015 Miltenyi Biotec GmbH. All rights reserved.

Neonatal (P1–2) and adult (P9–12W) brains were dissect ed from CD1 or C57BL/6 mice, and dissociated with the Neural Tissue Dissociation Kit and Adult Brain Dissociation Kit, respectively. Astro-cytes were isolated with Anti-ACSA-2 MicroBeads. Separated cells were stained with an Anti-ACSA-2-APC anti body for flow cytometry

(MACSQuant® Analyzer) to determine astrocyte purity (fig. 1). High purities were achieved for both neonatal astrocytes (99.17%) and adult astrocytes (98.68%). Iso lated astrocytes were cultivated for 5–14 days, then fixed and subjected to ICC using GLAST- (green) and GFAP (red)–specific antibodies (fig. 2; scale bar: 50 μm).

Results

Figure 1

Magnetic labeling with Anti-ACSA-2

MicroBeads (astrocyte cell

surface antigen)

Magnetic separation Elution of ACSA-2+ cells

Neonatal (P1–2) or adult (P9–12W) brains were dissected from CD1 mice or C57BL/6 mice

Flow cytometry with MACSQuant Analyzer

24-well imaging plate

7-14 DIV Stimuli

Tissue dissociation with Neural Tissue Dissociation Kit or Adult Brain Dissociation Kit and gentleMACS™ Octo Dissociator

Fixation and immunocytochemistry

Supernatant was tested for GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-12, IL-17A, IL-23, and TNF-α with MACSPlex Cytokine Kit

Cells were detached from culture plate for intracellular staining and flow cytometry

Neonatal and adult astrocytes express a broad range of cytokines and chemokines2

Purified neonatal astrocytes were cultured for 5 days before fixation. Cells were stained intracellu larly with antibodies against IL-17F, IL-12, IL-13, CCL-6, MIP-1α (CCL-3), IL-1β, TNF-α, G-CSF, IL-22, and IL-1α. Specific staining was observed for all the markers (fig. 3; scale bar: 50 μm), except IL-1α. Adult astrocytes were cultured for 10 days before fixation. Cells were stained intra cellularly and specific staining was observed for the antibodies against TNF-α, CCL-3, IL-2, G-CSF, and IL-22 (fig. 4; scale bar: 50 μm). ELISA

experiments also show ed a wide range of cytokines expressed by activated adult astrocytes (data not shown). These results need to be confirmed by further experiments. Neonatal and adult astrocytes were cultured in MACS Neuro Medium supplemented with MACS NeuroBrew-21, L-glutamine, and pen/strep. Neonatal astrocytes were cultured on PLL-coated plates, while adult astrocytes were cultured on PLL/Laminin–coated plates.

Figure 4

Activation of neonatal astrocytes3To validate the activation and detection methods for glial cells in vitro, stimulation of microglial cell cultures was first tested as a positive control. Neonatal micro glial cells were first isolated with CD11b (Microglia) MicroBeads (Miltenyi Biotec) and cultured for 4 days. Subsequently, the cells were treated with different concentrations of LPS O127:B8 (LPS1) or LPS O111:B4 (LPS2), with or without addition of IFN-γ, for 24 hours. Supernatants were collect ed and analyzed with a MACSPlex Cytokine Kit (Miltenyi Biotec) to investigate cytokine secre tion. Significantly increased TNF-α and IL-10 levels were detected in the supernatant of LPS-treated neonatal microglia cultures (fig. 5A; one-way ANOVA plus Dunnett‘s multiple comparisons test, SEM, **** p < 0.0001, *** p < 0.001, n = 3).

Neonatal astrocytes were treated with different concentrations of LPS O127:B8 (LPS1) or LPS O111:B4 (LPS2), with or without addi-tion of IFN-γ, for 24 hours. An increased level of GM-CSF was

detected when neonatal astrocytes were treated with very high concentrations of LPS (15 µg/mL) (fig. 5B; one-way ANOVA with Dunnett‘s multiple comparisons test, SEM, ****p < 0.0001, **p < 0.01, *p < 0.05, n = 3). In contrast, no difference in the TNF-α level was detected in the supernatant of LPS-treated neonatal astrocyte cultures.

Neonatal astrocytes were also treated with microglia-conditioned media (MCM), with or with out additional LPS treatment, for 24 hours. Signi ficantly increased GM-CSF levels were observ ed in the supernatants when astrocytes were treated with MCM (fig. 5C; one-way ANOVA with Dunnett‘s multiple comparisons test, SEM, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, n = 3.). However, no significant changes in TNF-α levels were seen (fig. 5D; one-way ANOVA with Dunnett‘s multiple comparisons test, SEM, n = 3).

Figure 5

A

C D

B

Activation of adult astrocytes4Adult astrocytes were cultured for 14 days and then treated with cytokines and LPS for 48 hours. Astrocytes showed a significantly increased GM-CSF level when treated with TNF-α/LPS, and a significantly increased IL-23 level when treated with IFN-γ/LPS. The latter was accompanied by slightly increased GM-CSF, IL-10 and IL-12 (fig. 6A; one-way ANOVA with Dunnett‘s multiple com parisons test, SEM, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, n = 3). Similar to TNF-α, IL-1β treatment also led to a significantly increased level of GM-CSF, but not IL-23 (data not shown).

To investigate the status of astrocytes after culture and stimulation, activated astrocytes were de tached from the culture plates, and stained with Anti-ACSA-2-PE and Anti-GFAP-APC antibodies for flow cytometry analysis (MACSQuant Analyzer). More than 92% of the cells were ACSA-2+GFAP+ (fig. 6B). Astrocytes treated with TNF-α/LPS or IFN-γ/LPS showed a higher GFAP fluorescence intensity (median, mean) compared to untreated astrocytes (fig. 6B), indicating that these astrocytes were in a more active state.

Summary and outlookFigure 2

Isolation and culture of neonatal and adult astrocytes

Figure 3

G-CSF IL-22

IL-17FGFAP IL-12 IL-13 CCL-6

MIP-1α (CCL3) IL-1β TNF-α IL-1α

Figure 6

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Neonatal astrocyte culture: GM-CSF secretion

ControlMCM only (no LPS treatment)LPS (100 ng/mL)

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Anti-GFAP-APC

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92.36% 94.28% 96.07%

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GFAP-APC median

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Control 93.72 7.97 47.63

TNF-α + LPS 95.48 14.31 81.89

IFN-γ + LPS 96.96 16.82 78.71

This work is supported by NeuroKine – Marie Curie Initial Training Networks.

99.17% 98.68%50 μm

CCL-3

IL-2

TNF-α

G-CSF

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GLAST GFAP overlay

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