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Keywords:

  • Autoimmunity;
  • Astrocytes;
  • EAE;
  • FasL

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

In T-cell-mediated autoimmune diseases of the CNS, apoptosis of Fas+ T cells by FasL contributes to resolution of disease. However, the apoptosis-inducing cell population still remains to be identified. To address the role of astrocytic FasL in the regulation of T-cell apoptosis in experimental autoimmune encephalomyelitis, we immunized C57BL/6 glial fibrillary acid protein (GFAP)-Cre FasLfl/fl mice selectively lacking FasL in astrocytes with MOG35–55 peptide. GFAP-Cre FasLfl/fl mice were unable to resolve EAE and suffered from persisting demyelination and paralysis, while FasLfl/fl control mice recovered. In contrast to FasLfl/fl mice, GFAP-Cre FasLfl/fl mice failed to induce apoptosis of Fas+ activated CD4+ T cells and to increase numbers of Foxp3+ Treg cells beyond day 15 post immunization, the time point of maximal clinical disease in control mice. The persistence of activated and GM-CSF-producing CD4+ T cells in GFAP-Cre FasLfl/fl mice also resulted in an increased IL-17, IFN-γ, TNF, and GM-CSF mRNA expression in the CNS. In vitro, FasL+ but not FasL astrocytes induced caspase-3 expression and apoptosis of activated T cells. In conclusion, FasL expression of astrocytes plays an important role in the control and elimination of autoimmune T cells from the CNS, thereby determining recovery from EAE.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

EAE is a widely used animal model to study MS, an inflammatory demyelinating disease mediated by accumulation of T lymphocytes and macrophages in the CNS [1, 2]. EAE can be induced by either active immunization with myelin Ags including myelin oligodendrocyte glycoprotein (MOG) peptide or passive transfer of myelin-reactive CD4+ T cells, which are both initiators and effectors of EAE. Among CD4+ T lymphocytes, GM-CSF-producing CD4+ T cells, IFN-γ-secreting Th1 cells, and IL-17-secreting Th17 cells have been identified as the most important mediators in the immunopathogenesis of EAE [3-6] and all of them can induce EAE independently, although recent studies point to an essential role of GM-CSF-producing CD4+ T cells, which can induce EAE independent of IFN-γ and IL-17 [7]. Infiltrating T lymphocytes trigger an inflammatory response in the CNS culminating in demyelination and axonal damage clinically resulting in paralysis [8]. Correspondingly, recovery from EAE requires termination of inflammation and the induction of T-cell apoptosis in the CNS [9].

Fas ligand (FasL; CD95L), a cytotoxic cytokine belonging to the TNF superfamily, acts through Fas, a death receptor of the TNFR superfamily, to induce programed cell death via caspase signaling [10]. Local expression of FasL in immunoprivileged organs including eyes, testis, and placenta is essential for deletion of infiltrating inflammatory cells [11-13]. Fas/FasL interaction is of particular importance for homeostasis of the immune system and its dysregulation has been implicated in various autoimmune diseases. Mice carrying autosomal recessive mutations in the Fas (lpr) and FasL (gld) genes develop a spontaneous autoimmune syndrome similar to human systemic lupus erythematosus [14, 15]. Fas and FasL are also involved in the pathogenesis of EAE, as EAE is dramatically ameliorated in lpr and gld mice in terms of disease incidence and mean clinical score [16]. Intrathecal infusion of recombinant FasL induces apoptosis of CNS-infiltrating inflammatory cells, including T cells and macrophages, but does not exert cytotoxicity against CNS-resident cells, resulting in mitigated EAE manifestations [17].

Elimination of infiltrating T cells in the CNS by Fas/FasL-mediated apoptosis is crucial for resolution of EAE [9, 18, 19], since FasL-deficient gld recipients develop prolonged EAE after adoptive transfer of myelin basic protein-reactive WT Fas+ T lymphocytes [20]. The CNS-resident cell population which induces apoptosis of CD4+ T cells in EAE still remains to be identified. We hypothesize that astrocytes, which constitutively express FasL, may play a key role given that FasL-expressing astrocytes are in intimate contact with apoptotic T cells in EAE and can induce apoptosis of activated CD4+ T cells in vitro [21, 22]. Consistently, our previous study also demonstrated that increased apoptosis of gp130-deficient astrocytes exacerbated EAE, partially due to an impaired elimination of CD4+ T cells from the CNS [23]. However, in vivo evidence confirming that astrocytic FasL is involved in the induction of CD4+ T-cell apoptosis in EAE is still lacking.

In order to determine whether FasL+ astrocytes are inducers of CD4+ T-cell apoptosis in EAE, we generated glial fibrillary acid protein (GFAP)-Cre FasLfl/fl mice that are deficient of FasL selectively in astrocytes. We show in the present study that astrocytic FasL is crucial to terminate the autoimmune T-cell response in the CNS, which allows clinical recovery from EAE.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Selective deletion of FasL in astrocytes of GFAP-Cre FasLfl/fl mice

We generated GFAP-Cre FasLfl/fl mice with selective FasL deletion in the CNS (Supporting Information Fig. 1). Further PCR analysis of cultivated cells showed FasL deletion in astrocytes and to a minor extent in neurons (Fig. 1A). In contrast, microglia of GFAP-Cre FasLfl/fl as well as astrocytes, neurons, and microglia of FasLfl/fl control mice did not show deletion of FasL (Fig. 1A). To confirm astrocytic FasL deletion at the protein level, cell surface expression of FasL protein was analyzed by flow cytometry from cultivated astrocytes of GFAP-Cre FasLfl/fl and FasLfl/fl mice. As shown in Figure 1B, FasL expression was reduced on the surface of astrocytes from GFAP-Cre FasLfl/fl as compared to FasLfl/fl mice. Both GFAP-Cre FasLfl/fl mice and FasLfl/fl (control) mice were born in a normal Mendelian ratio and reached adulthood without any CNS defects.

image

Figure 1. Organ and cell-type-specific deletion of FasL in GFAP-Cre FasLfl/fl mice. (A) Deletion of FasL from cultivated and FACS-sorted astrocytes, microglia, and cultivated neurons of GFAP-Cre FasLfl/fl (lanes labeled 1) and FasLfl/fl (lanes labeled 2) mice. Total brain (positive control) was from a GFAP-Cre FasLfl/fl mouse. (B) Cell surface expression of the FasL protein (right) was analyzed by flow cytometry from cultivated astrocytes of GFAP-Cre FasLfl/fl and FasLfl/fl mice. Staining with control mouse Ab is shown (left). Representative data from one of two experiments are shown and the MFI of FasL expression is given in each histogram.

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Collectively, these findings show that astrocyte-specific deletion of FasL was achieved in our newly generated GFAP-Cre FasLfl/fl mice, which did not show abnormalities under physiological conditions, thereby providing a useful tool for studying the function of astrocyte-specific FasL in experimentally induced models of CNS disorders.

Aggravated EAE of GFAP-Cre FasLfl/fl mice with increased inflammation and demyelination

To gain insight into the astrocyte-specific function of FasL in the pathogenesis of EAE, we actively immunized GFAP-Cre FasLfl/fl mice and control mice with MOG35–55 peptide emulsified in complete Freund's adjuvant and assessed clinical disease activity daily. Both GFAP-Cre FasLfl/fl mice and FasLfl/fl control mice developed EAE starting at around day 9 post immunization (p.i.) and reaching peak disease at day 15 p.i.; over this period of time they developed similar clinical symptoms (Fig. 2A). However, beyond the maximum of disease, i.e. day 15 p.i., FasLfl/fl mice recovered gradually while EAE progressed in GFAP-Cre FasLfl/fl mice indicating a significantly more severe course of EAE in the later group of mice (Fig. 2A). Already at day 15 p.i., inflammation of GFAP-Cre FasLfl/fl mice was more severe and more widespread as compared with that in control animals, leading to more severe demyelination. While inflammatory foci consisting of CD3+ T cells and macrophages were confined to the dorsal columns of the spinal cord in FasLfl/fl mice, they also infiltrated the spinocerebellar tracts in GFAP-Cre FasLfl/fl mice. Differences between the two mouse strains were more prominent at day 22 p.i. as compared with those at day 15 p.i. Inflammation and demyelination were mild in FasLfl/fl mice (Fig. 2B and D) as compared with that in GFAP-Cre FasLfl/fl mice, with widespread inflammatory foci consisting of CD3+ T cells and Mac3+ macrophages (Fig. 2C and E). In GFAP-Cre FasLfl/fl mice, demyelination was prominent in the posterior columns as well as in spinocerebellar tracts (Fig. 2C), which also showed evidence of a disturbed axonal transport as evidenced by axonal bulbs. Inflammation was also prominent in the dorsal horn of the spinal cord, where many infiltrates resided (Fig. 2E).

image

Figure 2. Aggravated EAE with increased demyelination and inflammation in GFAP-Cre FasLfl/fl mice. (A) Clinical symptoms of GFAP-Cre FasLfl/fl and FasLfl/fl mice were scored daily after immunization with MOG35–55 peptide in CFA, and data are displayed as the mean ± SEM of 8–10 mice per group, from one experiment representative of four performed. *p < 0.05, **p < 0.01, t-tests. (B) Lack of ongoing demyelination in the posterior column of the spinal cord in a FasLfl/fl mouse, although myelin sheaths are reduced. Inflammatory infiltrates are confined to the leptomeninges (arrows), but do not infiltrate the spinal cord parenchyma. Cresyl violet—luxol fast blue staining. (C) Severe inflammation with leptomeningeal and spinal cord parenchymal infiltrates (arrows) with pronounced demyelination (*) in the posterior column of the spinal cord in a GFAP-Cre FasLfl/fl mouse. Note numerous myelin fragments and pronounced edema. Cresyl violet—luxol fast blue staining. (D) Some CD3+ T cells contributed to leptomeningeal infiltrates (arrowheads) and only single CD3+ T cells are still present in parenchyma of the spinal cord of a FasLfl/fl mouse (small arrows). Anti-CD3 immunostaining, slight counterstaining with hemalum. (E) Many CD3+ T cells form inflammatory cuffs in the posterior columns and around blood vessels in the posterior horn (large arrows) and are also scattered throughout the spinal cord (small arrows) in a GFAP-Cre FasLfl/fl mouse. Anti-CD3 immunostaining, slight counterstaining with hemalum. (B–E) Histological findings of representative mice (day 22 p.i.) from one of two independent experiments with three mice each are shown, bar 50 μm.

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Increased numbers of infiltrating T cells in the spinal cord of GFAP-Cre FasLfl/fl mice

Autoimmune T cells are widely regarded as the key mediator of EAE; therefore, we analyzed T cells infiltrating the spinal cord. At day 15 p.i., flow cytometry revealed that numbers of infiltrating CD4+ and CD8+ T cells were slightly but not significantly increased in the spinal cord of GFAP-Cre FasLfl/fl mice as compared with those in FasLfl/fl mice (Fig. 3A and B), which corresponds to the similar clinical scores at this time point (Fig. 2). At day 22 p.i., significantly more CD4+ and CD8+ T cells were detected in the spinal cord of GFAP-Cre FasLfl/fl mice than in FasLfl/fl mice (Fig. 3A and B; p < 0.01 for CD4+ and CD8+ T cells).

image

Figure 3. Increased numbers of activated and reduced numbers of Treg cells in the CNS of GFAP-Cre FasLfl/fl mice. (A, B) Leukocytes were isolated from the spinal cord of GFAP-Cre FasLfl/fl and FasLfl/fl mice and numbers of (A) CD4+ and (B) CD8+ T cells were determined by flow cytometry. (C) Leukocytes isolated from the spinal cord were stained for extracellular CD4 and CD45 and intracellular GM-CSF. Dot plots (left) show the percentage of CD4+ CD45+ cells (upper left), and GM-CSF+ CD4+ cells (lower left) at day 22 p.i., as well as the gating strategy for CD4+ cells. The relative (upper right) and absolute number (lower right) of GM-CSF+ CD4+ cells is shown. (D, E) The percentage of (D) CD25+ CD4+ T cells and (E) Foxp3+ regulatory CD4+ T cells was analyzed by flow cytometry. (F) Cell surface expression of Fas on CD4+ T cells from both GFAP-Cre FasLfl/fl and FasLfl/fl mice was analyzed by flow cytometry at day 15 p.i. and representative histograms are shown (right). Control staining was performed with hamster IgG (left). The MFI is shown in each histogram. (G) The percentage of 7-AAD+ CD4+ T cells was determined by flow cytometry at day 22 p.i., and the mean + SD is shown. (A–G) Data are shown as representative dot plots as well as the mean + SD of six to nine mice per experimental group from one of two independent experiments. *p < 0.05, **p < 0.01, t-tests.

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As GM-CSF-producing CD4+ T cells are essential for the induction of EAE [7], we determined the percentage and number of GM-CSF-producing CD4+ T cells in the spinal cord of both mouse strains. Flow cytometry revealed that GM-CSF-producing CD4+ T cells accounted for approximately 15% of CD4+ T cells in both mouse strains; however, the absolute number of GM-CSF-producing CD4+ T cells was significantly increased in GFAP-Cre FasLfl/fl mice as compared with that in control animals at day 22 p.i. (Fig. 3C). In addition, we compared the phenotypic composition of CD4+ T cells between the two genotypes to determine whether astrocyte-specific deletion of FasL influenced the activation state of infiltrating CD4+ T cells in EAE. At day 15 p.i., the percentage of Foxp3 CD25+ activated CD4+ T cells (Fig. 3D) and Foxp3+ regulatory CD4+ T cells (Fig. 3E) was similar in both strains of mice, whereas at day 22 p.i., as compared with FasLfl/fl mice, the percentage of Foxp3 CD25+ activated CD4+ T cells was increased while the percentage of Foxp3+ regulatory CD4+ T cells was reduced in GFAP-Cre FasLfl/fl mice, respectively (Fig. 3D and E). Intraspinal CD4+ T cells from both mouse strains expressed Fas, as detected by flow cytometry (Fig. 3F), and, thus, they might be regulated by FasL+ cells. At day 22 p.i., the percentage of 7-aminoactinomycin D (7-AAD)+ CD4+ T cells was significantly reduced in GFAP-Cre FasLfl/fl mice as compared with that in FasLfl/fl mice (Fig. 3G, *p < 0.05) suggesting that elimination of infiltrating T cells by apoptosis was impaired in GFAP-Cre FasLfl/fl mice in late stages of EAE. Annexin V staining was not used to detect CD4+ T-cell apoptosis in vivo because previous reports showed that annexin V did not selectively detect apoptotic T cells, since it also stained activated CD4+ T cells [24].

Increased proinflammatory gene transcription in the spinal cord of GFAP-Cre FasLfl/fl mice

To examine the impact of astrocyte-specific FasL deletion on the expression of proinflammatory genes during EAE, quantitative real-time PCR for cytokines and chemokines was performed on spinal cord tissue at day 15 p.i. and day 22 p.i. of EAE, respectively. At day 15 p.i., IFN-γ and IL-27 mRNA was significantly elevated in GFAP-Cre FasLfl/fl mice as compared to FasLfl/fl mice while mRNA levels of IL-17, TNF, IL-23, and GM-CSF did not differ between the two mouse strains (Fig. 4). In contrast, at day 22 p.i., mRNA levels of all mediators, except for IL-23, were significantly upregulated in GFAP-Cre FasLfl/fl mice as compared with levels in FasLfl/fl mice, indicating an increased proinflammatory response in the spinal cord of GFAP-Cre FasLfl/fl mice at this late time point (Fig. 4). Interestingly, mRNA of IL-17, a main mediator of EAE, persisted at high levels in the spinal cord of GFAP-Cre FasLfl/fl mice up to day 22 p.i. Taken together, these results show that astrocytic deletion of FasL resulted in an increased transcription of important proinflammatory genes in the spinal cord which induce and contribute to severity of EAE.

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Figure 4. Increased proinflammatory gene expression in the spinal cords of GFAP-Cre FasLfl/fl mice. (A–G) Expression of (A) IL-17, (B) IFN-γ, (C) TNF-α, (D) GM-CSF, (E) IL-23, (F) IL-27, and (G) iNOS mRNA was determined by quantitative RT-PCR in GFAP-Cre FasLfl/fl and FasLfl/fl mice at days 15 and 22 p.i. Spinal cords of three mice per group were analyzed. Data represent the mean + SEM as relative increase over nonimmunized mice. *p < 0.05, **p < 0.01, t-tests. One of two independent experiments with three mice each is shown.

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Reduced apoptosis of CD4+ T cells in coculture with FasL-deficient astrocytes

Twenty-four hours after coculture of FasLfl/fl CD4+ T cells with primary astrocytes isolated from the CNS of FasLfl/fl or GFAP-Cre FasLfl/fl mice, T-cell apoptosis induced by FasL-deficient astrocytes was compared to that induced by control astrocytes. In accordance with a previous report of Bechmann et al. [21], significantly lower numbers of T cells cocultured with FasL-deficient astrocytes underwent apoptosis as demonstrated by both annexin V binding and caspase staining (Fig. 5). Based on these findings, we conclude that, during EAE, astrocytic FasL-induced apoptotic elimination of T cells in the CNS of GFAP-Cre FasLfl/fl mice is significantly compromised as compared with that of control animals, resulting in a significantly enhanced disease activity.

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Figure 5. Astrocytes induce apoptosis of activated T cells via FasL in vitro. (A, B) After monoculture or coculture with astrocytes from FasLfl/fl or GFAP-Cre FasLfl/fl mice for 24 h, apoptosis of T cells was detected by (A) caspase 3 and (B) annexin V staining. No difference was observed in the corresponding control stainings with isotype-matched IgG (data not shown). One of two independent experiments is shown.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

The present study demonstrates that elimination of infiltrating encephalitogenic T lymphocytes is a key mechanism for EAE resolution and that FasL+ astrocytes are crucial to induce T-cell apoptosis and elimination from the CNS in this autoimmune disease. In agreement with Bechmann et al. [21], who demonstrated that astrocytes induced apoptosis of activated T cells in a FasL-dependent way in vitro, we observed that FasL+ astrocytes induced apoptosis of activated Fas+ CD4+ T cells in vitro. Since astrocytes are also FasL+ in MS, our data suggest a similar role of astrocytes for the elimination of inflammatory leukocytes in this severe human autoimmune disease.

Astrocytic FasL was protective for mice during EAE, since MOG35–55-immunized mice lacking astrocyte-specific FasL suffered from a clinically more severe EAE compared with their control littermates. The onset of clinical symptoms was similar in both GFAP-Cre FasLfl/fl and control FasLfl/fl mice at day 9 p.i. indicating that homing of myelin-specific leucocytes was not regulated by astrocytic FasL expression. In addition, the clinical score of GFAP-Cre FasLfl/fl and FasLfl/fl mice increased with the same kinetics until the peak of disease in FasLfl/fl mice at day 15 p.i. In accordance, numbers of CD4+ T cells were not significantly increased in GFAP-Cre FasLfl/fl mice at day 15 p.i. However, during the clinical recovery phase of FasLfl/fl mice (day 22 p.i.), numbers of CD4+ T cells were significantly increased in the spinal cord of GFAP-Cre FasLfl/fl mice as shown by both flow cytometry and histology at day 22 p.i. The reduced number of CD4+ T cells positive for 7-AAD, which identifies late apoptotic and dead cells, illustrates the compromised ability of FasL-deficient astrocytes to induce apoptosis and elimination of infiltrating autoimmune T cells. The kinetics of disease and intraspinal CD4+ T-cell numbers indicate that FasL-dependent elimination of CD4+ T cells in EAE plays a particularly protective role in the recovery phase. Noteworthy, the more severe EAE of GFAP-Cre FasLfl/fl mice cannot be attributed to the GFAP-Cre transgene, since C57BL/6 GFAP-Cre mice without a loxP-flanked gene develop the same course of EAE as compared to WT mice [23].

We also observed a significantly higher number of activated CD25+ CD4+ T cells and a significantly reduced number of Foxp3+ regulatory CD4+ T cells in the spinal cord of GFAP-Cre FasLfl/fl as compared with FasLfl/fl mice at day 22 p.i. Lack of astrocytic FasL expression altered the ratio of activated CD25+ versus regulatory Foxp3+ CD4+ T cells in the spinal cord from 5:1 in FasLfl/fl mice to 10:1 in GFAP-Cre FasLfl/fl mice. These data suggest that astrocytic FasL expression predominantly contributes to elimination of activated disease-promoting CD25+ CD4+ T cells but not of protective regulatory Foxp3+ CD4+ T cells in order to recover from EAE and to achieve a restitutio ad integrum. These data are in line with previous reports illustrating that astrocytes induce a regulatory phenotype in autoimmune CD4+ T cells [25], which play an important protective role in EAE [26, 27]. In agreement, the number of GM-CSF-producing CD4+ T cells, i.e. the essential EAE-inducing CD4+ T-cell subset [7], was significantly increased in the spinal cord of GFAP-Cre FasLfl/fl mice at day 22 p.i.

The increased number of infiltrating activated autoreactive CD4+ T cells in GFAP-Cre FasLfl/fl mice was associated with an enhanced production of proinflammatory cytokines. At day 15 p.i., IFN-γ, TNF, GM-CSF, IL-27, and iNOS but not IL17 mRNA was increased in GFAP-Cre FasLfl/fl mice as compared with that in FasLfl/fl mice. IFN-γ, TNF, and GM-CSF have been reported to contribute to disease progression and demyelination in EAE [7, 28]. GM-CSF and IFN-γ are mainly produced by encephalitogenic T cells. GM-CSF sustains neuroinflammation via myeloid cells that infiltrate the spinal cord. In addition to its proinflammatory function, IFN-γ is also a potent stimulator of IL-27 production by astrocytes [29], which might explain the increased production of this immunosuppressive and protective cytokine in the spinal cord of GFAP-Cre FasLfl/fl mice at day 15 p.i. IL-27 can suppress IL-17 production of primed Th17 cells [29], which might explain that GFAP-Cre FasLfl/fl mice did not show elevated IL-17 mRNA expression in the spinal cord as compared to FasLfl/fl mice. IL-17 is an important cytokine contributing to demyelination and progression of EAE [30]. Comparable levels of IL-17 mRNA transcription in GFAP-Cre FasLfl/fl and FasLfl/fl mice at day 15 p.i. might, therefore, explain similar clinical scores in the two mouse strains at this stage of disease. However, at day 22 p.i., when numbers of activated CD25+ and GM-CSF-producing CD4+ T cells were significantly increased and numbers of Foxp3+ regulatory CD4+ T cells decreased in GFAP-Cre FasLfl/fl mice, IL-17 mRNA was very prominently increased in addition to IFN-γ, TNF, GM-CSF, and iNOS mRNA. Thus, aggravation of clinical symptoms in GFAP-Cre FasLfl/fl mice correlated with an increased IL-17 mRNA transcription, indicating that this cytokine was decisive for the more severe and persisting EAE in these mice. IL-23, which drives IL-17 polarization of CD4+ T cells was not increased in the CNS of GFAP-Cre FasLfl/fl mice, which fits to the important role of IL-23 for Th17 polarization in lymphatic organs [31].

Astrocytes play both positive and negative roles in the pathogenesis and development of EAE [32]. As part of the blood-brain barrier, early chemokine release of astrocytes contributes to the recruitment of autoimmune CD4+ T cells to the CNS [33]. At later stages of EAE, astrogliosis develops, which may restrict further invasion of leukocytes into the CNS parenchyma [34]. In fact, genetically induced ablation of reactive astrocytes during EAE led to widespread inflammation and more severe clinical symptoms [35]. In this regard, it is of note that gp130-dependent astrocyte survival and astrogliosis are critical to restrict CNS inflammation in EAE as well as in CNS infections, i.e. Toxoplasma encephalitis [23, 36]. Consistently, blocking NF-κB signaling, which is required for astrocyte activation in EAE, by tissue-specific ablation of key signaling molecules including NEMO, IKK2, and Act1 in the CNS impaired astrocytic production of inflammatory cytokines and chemokines ameliorating EAE as evidenced by decreased leukocyte infiltration and reduced demyelination [5, 37, 38]. Interestingly, in sharp contrast to the proinflammatory function of most astrocyte-derived chemokines, CXCL12, which is upregulated in the CNS of MS patients, particularly produced by astrocytes, suppressed ongoing EAE by redirecting the polarization of effector Th1 cells into IL10-producing Treg cells [39].

Collectively, the present study extends the in vivo function of astrocytes and illustrates that astrocytes also confer protection against EAE by the FasL-dependent apoptotic elimination of activated CD25+ Foxp3 and GM-CSF-producing CD4+ T cells and the concomitant inhibition of proinflammatory cytokine production. Thus, augmentation of astrocytic FasL may provide a favorable strategy for treatment of clinically active MS.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Mice

GFAP-Cre+/− FasLfl/fl mice were generated by crossing C57BL/6 GFAP-Cre transgenic mice [40] with C57BL/6 FasLfl/fl mice [41] and the colony was maintained by breeding of GFAP-Cre+/− FasLfl/fl mice with GFAP-Cre−/− FasLfl/fl mice. Genotyping of offsprings was carried out by PCR of tail DNA with primers targeting GFAP-Cre and FasLfl/fl. Deletion of FasL was analyzed by PCR in various organs and cell types with Del-FasL primers (5′-GTACTTCTTCTGATAAGGACC-3′ and 5′-GGAGTTGAACGAGTAGCCTC-3′). C57BL/6 WT mice were obtained from Harlan (Borchen, Germany). Animal care and experimental procedures were performed according to European regulations and approved by state authorities (Landesverwaltungsamt Halle, Germany; IMMB/G/02–994/10).

Induction and assessment of EAE

MOG35–55 (MEVGWYRSPFSRVVHLYRNGK) was purchased from JPT (Berlin, Germany). Active EAE was induced in 8- to 12-week-old mice by s.c. immunization with 200 μg of MOG35–55 emulsified in complete Freund's adjuvant (Sigma, Taufkirchen, Germany) supplemented with 800 μg of killed Mycobacterium tuberculosis (Sigma). In addition, mice also received two i.p. injections of 200 ng pertussis toxin (Sigma), dissolved in 200 μL PBS, at the time of immunization as well as 48 h thereafter. Clinical signs of EAE were monitored daily and scored according to a scale of severity from 0 to 5 as described previously [23]. Daily clinical scores were calculated as the average of all individual disease scores within each group.

Isolation of intraspinal leukocytes and flow cytometry

Leukocytes were isolated from the spinal cord and stained for CD4+ T cells, CD8+ T cells, and CD45high inflammatory leukocytes as described before [42]. For the detection of regulatory and activated CD4+ T cells, isolated leukocytes were stained with CD25-FITC and CD4-allophycocyanin followed by a Foxp3-PE staining kit as recommended by the manufacturer (eBiosciences, Frankfurt, Germany). For intracellular staining of GM-CSF, isolated leukocytes were incubated with 50 ng/mL PMA, 500 ng/mL ionomycin, Golgi-Plug (1 μL/mL) containing brefeldin A in RPMI-1640 at 37°C for 4 h. Thereafter, cells were stained with rat antimouse CD4-FITC, rat antimouse CD45-V450, fixed and permeabilized with Cytofix/Cytoperm (BD), and stained with rat antimouse GM-CSF-PE (BD). Apoptotic and dead CD4+ T cells were detected by staining with 7-AAD and CD4-allophycocyanin. Fas expression on CD4+ T cells was analyzed by staining with hamster antimouse Fas-PE and CD4-FITC. Controls were stained with isotype-matched control antibodies. All antibodies and reagents were obtained from BD Biosciences (Heidelberg, Germany) unless otherwise mentioned. Flow cytometry was performed on a FACScan (BD Biosciences), and the data were analyzed with WinMDI or Cell Quest software.

In vitro culture, DNA isolation, and flow cytometry of astrocytes, microglia, and neurons

Primary astrocytes were isolated from 1- to 2-day-old newborn mice and cultured as published before [43]. To obtain pure astrocytes, cells were harvested from astrocyte cultures and stained with rat antimouse CD11b-PE. Pure astrocytes (CD11b) were then separated from CD11b+ microglia with a FACSVantage cell sorter (BD). Neuronal cultures were obtained according to Lenz et al. [44] with slight modifications. Briefly, pregnant female mice were sacrificed by cervical dislocation at gestational day 18.5, and dissociated cells of each embryonic brain were cultivated in flasks coated with poly-L-lysine in Neurobasal medium supplemented with B27 (Invitrogen) and 500 μM L-glutamine (Gibco). The purity of cultures for neurons was ≥98%, as determined by immunofluorescence staining for neuron-specific class III β-tubulin. DNA was isolated from sorted astrocytes and microglia, respectively, as well as from cultured neurons using a DNA isolation kit (Qiagen, Germany). For the detection of FasL expressed on the surface of astrocytes, mixed astrocyte/microglia cultures were stained with mouse antimouse FasL-PE and CD11b-FITC. Controls were stained with isotype-matched control antibodies.

Histology

For histology on paraffin sections, mice anesthetized with methoxyflurane were perfused with 0.1 M PBS followed by 4% paraformaldehyde in PBS, spinal cords were processed and stained with hemalum and eosin, cresyl violet, and luxol fast blue. In addition, paraffin sections were used for immunohistochemical demonstration of GFAP, neurofilament, Mac3, and CD3 (Serotec, Düsseldorf, Germany) by an ABC protocol as described [45].

Quantitative RT-PCR

Total mRNA was isolated from the spinal cords of nonimmunized and MOG35–55- immunized mice (RNeasy kit, Qiagen, Germany) at day 15 and day 22 p.i., respectively. SuperScript reverse transcriptase kit with oligo (dT) primers (Invitrogen, Germany) was used to generate cDNA from total mRNA. Quantitative RT-PCR for IL-17, IFN-γ, IL-23, IL-27, TNF, GM-CSF, iNOS, and hypoxanthine phosphoribosyltransferase was performed on the light cycler 480 real-time PCR system (Roche). The ratio between the respective gene and corresponding hypoxanthine phosphoribosyltransferase was calculated per mouse according to the ΔΔ cycle threshold method [46], and data were expressed as the increase of mRNA expression in immunized mice over non immunized controls of the respective mouse strain. All primers and probes were obtained from Applied Biosystems.

Coculture of CD4+ T cells with astrocytes

CD4+ T cells were isolated from spleens and LNs of C57BL/6 mice by MACS (Miltenyi Biotec, Germany) according to the manufacturer’ instructions. Purified CD4+ T cells were activated for 48 h by culturing in anti-CD3 (BD, 5 μg/mL) and anti-CD28 (eBiosciences, 2 μg/mL) coated 96-well plates at 1–2 × 105 cells/well in 200 μL of RPMI-1640 (Gibco) supplemented with 10% FCS (Gibco), 1% L-glutamine (Gibco), 100 U/mL penicillin (Sigma), and 0.1 mg/mL streptomycin (Sigma). For coculture, 1 × 105 activated T cells were inoculated onto the astrocytic monolayers in six-well plates. After 24 h incubation, T cells were collected and apoptosis was detected by staining cells with Annexin-allophycocyanin, Caspase 3-PE, and CD4-Pacific Blue.

Statistics

To test for statistical differences in the clinical scores and cell numbers, the two-tailed Student's t-test was used. p values < 0.05 were accepted as significant. All experiments were performed at least twice.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

This work was supported by grants from the Deutsche Forschungsgemeinschaft (Schl 391 7–1, GRK 1167). The expert technical assistance of Elena Fischer, Nadja Schlüter, and Annette Sohnekind is gratefully acknowledged.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information
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Abbreviations
7-AAD

7-aminoactinomycin D

FasL

Fas ligand

GFAP

glial fibrillary acid protein

MOG

myelin oligodendrocyte glycoprotein

p.i.

post immunization

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

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FilenameFormatSizeDescription
eji2465-sup-0001-FigureS1.pdf445KFigure 1. Deletion of FasL from various organs of nonimmunized GFAP-Cre FasLfl/fl and FasLfl/fl mice was analyzed by PCR. The FasLΔ product has a size of 500 bp. Representative data from one of two experiments with three mice each are shown.

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