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Oligodendrocyte – type-2 astrocyte progenitor cells (O-2A/OPCs) populate the CNS and generate oligodendrocytes and astrocytes in vitro and in vivo. Understanding how O-2A/OPCs respond to their environment is crucial to understanding how these cells function in the CNS and how to best promote their therapeutic proliferation and differentiation. We show that IFN-γ was not toxic to highly purified perinatal or adult rat O-2A/OPCs. IFN-γ treatment led to downregulation of PDGFR-α and Ki-67 and decreased self-renewal in clonal populations. IFN-γ also significantly increased the proportion of cells in the G0/G1 phase of the cell cycle, decreased BrdU incorporation, and led to increased expression of the cell cycle inhibitors Rb and p27kip1. While p27kip1 expression was not necessary for IFN-γ – mediated quiescence, its upstream regulator IRF-1 was required. The quiescent state of O-2A/OPCs caused by INF-γ was reversible as the withdrawal of IFN-γ allowed O-2A/OPCs to appropriately respond to both proliferation and differentiation signals. Differentiation into oligodendrocytes induced by either thyroid hormone or CNTF was also abrogated by IFN-γ. This inhibition was specific to the oligodendrocyte pathway, as O-2A/OPC differentiation into astrocytes was not inhibited. IFN-γ alone also led to the generation of GFAP positive astrocytes in a subset of O-2A/OPCs. Taken together these results demonstrate a reversible inhibitory effect of IFN-γ on O-2A/OPC proliferation with a concomitant generation of astrocytes. We propose that neuroinflammation involving increased IFN-γ can reduce progenitor numbers and inhibit differentiation, which has significant clinical relevance for injury repair, but may also contribute to the generation of astrocytes.
A number of CNS disorders exhibit dysfunction of differentiated oligodendrocytes and/or of the progenitor cells that give rise to oligodendrocytes (Franklin and ffrench-Constant, 2008). The oligodendrocyte-type-2 astrocyte progenitor cell (O-2A/OPC) that gives rise to oligodendrocytes (Raff et al., 1983; Barres et al., 1992; Levine et al., 2001; Miller, 2002; Noble et al., 2004) proliferate and migrate extensively during the first 4 postnatal weeks and persist throughout adulthood (Dawson et al., 2003; Cayre et al., 2009). O-2A/OPCs also have the potential to generate GFAP+ astrocytes with stellate morphology (also referred to as type-2 astrocytes) (Raff et al., 1983; Lillien et al., 1990; Mayer et al., 1994; Aguirre and Gallo, 2004; Windrem et al., 2004; Guo et al., 2009). In multiple sclerosis (MS) it is suggested that the repair function of O-2A/OPCs is impaired as determined by their failure to proliferate and effectively replace dying oligodendrocytes (Franklin and ffrench-Constant, 2008). In MS brains O-2A/OPCs have been detected at the periphery of active lesions and within chronic inactive lesions (Chang et al., 2000) but appear to be quiescent (Wolswijk, 1998a).
The reasons for O-2A/OPC failure to consistently facilitate remyelination are largely unknown and may occur at one or all of the requisite steps: recruitment, migration, differentiation, and remyelination (Wolswijk, 1998b; Dawson et al., 2000; Levine et al., 2001). Of the many differences between developmental myelination and conditions requiring remyelination, one of the most striking is the change in the extracellular environment, which is often associated with neuroinflammation and conditions of myelin breakdown (Franklin, 2002). The pro-inflammatory cytokine interferon-γ (IFN-γ) is present during episodes of neuroinflammation, and is thought to play a role in the demyelination that occurs in MS (Beck et al., 1988; Popko et al., 1997; Imitola et al., 2005). IFN-γ expression precedes clinical symptoms of MS and exacerbates disease (Panitch et al., 1987; Beck et al., 1988), although it can be both protective and enhancing of pathology in EAE (Popko et al., 1997; Imitola et al., 2005; Lin and Popko, 2009).
As oligodendrocyte cell death is a major hallmark of MS, many studies have been focused on the mechanisms of IFN-γ induced cell death. We were, however, intrigued by studies that describe quiescent oligodendrocyte progenitors surrounding MS lesion sites (Wolswijk, 1998a; Chang et al., 2000). Our specific interest was to identify mechanisms that could contribute to the failure of remyelination in the absence of cell death. We show that highly purified O-2A/OPCs dissected from the postnatal corpus callosum, and also from the adult CNS, treated with IFN-γ (10 ng/ml) exit the cell cycle. Upon withdrawal of IFN-γ the cells can resume self-renewal and differentiation. Surprisingly, IFN-γ-arrested O-2A/OPCs were inhibited from generating oligodendrocytes but showed an increased tendency to generate GFAP+ astrocytes. These results reveal that O-2A/OPCs are highly plastic cells that can withstand neuroinflammation by exiting the cell cycle and suggest that a subset of O-2A/OPCs may respond to the neuroinflammatory insult by differentiating into astrocytes.
All experiments except for those on adult cells, were performed on primary corpus callosum O-2A/OPCs isolated from post-natal day 7 rat pups by GalC negative and A2B5 positive magnetic cell sorting (MACS; Miltenyi Biotec, Auburn, CA, USA) and differential adherence on tissue culture plastic as described previously (Ibarrola et al., 1996; Power et al., 2002; Li et al., 2007). All chemicals were purchased from Sigma (St. Louis, MO, USA) unless otherwise indicated. Cells for clonal analyses were grown in proliferation media consisting of DMEM/F12 containing insulin (1 mg/ml), transferrin (100 mg/ml), putrescine (0.1 mM), selenium (0.224 mM), progesterone (0.2 mM), and pathocyte-4 BSA (0.0286% v/v; ICN Biochemicals, Costa Mesa, CA, USA) roughly according to Bottenstein and Sato (Bottenstein and Sato, 1979), with the addition of 10 ng/ml PDGF-AA (PeproTech, Rocky Hill, NJ, USA) on poly(L) lysine coated tissue culture plastic (Nunc, Rochester, NY, USA). Adult O-2A/OPCs were isolated from female adult optic nerve essentially as described before (Wolswijk and Noble, 1989), with the exception that between trituration and A2B5 magnetic sorting cells were suspended in a sucrose gradient and centrifuged to separate progenitor cells from myelin debris.
For clonal analyses 200 cells were seeded per well in a 24-well plate immediately after sorting (within 4 hours of dissection) in 10 ng/ml PDGF-AA for 24 hours. After for 24 hours of recovery cells were treated as described in figure legends.
All other experiments involved first expanding O-2A/OPCs through one passage in PDGF-AA and bFGF (10 ng/ml; PeproTech). At passage, cells were plated for experiments and allowed 24 hours for recovery in 10 ng/ml PDGF-AA before treatment. Mass culture analyses consisted of plating O-2A/OPCs at a density of 1000 cells per well of a 24 well plate or 100,000 cells per 10-centimeter dish for western blot. Proliferation experiments consisted of exposure to PDGF-AA (10 ng/ml) unless otherwise indicated. Differentiation conditions consisted of 1 ng/ml PDGF-AA plus 40 nM thyroid hormone (TH; 30 ng/ml thyroxine and 36 ng/ml tri-iodothrionine) or 1 ng/ml PDGF-AA plus 10 ng/ml CNTF (PeproTech) as indicated in figure legends. To induce differentiation into astrocytes O-2A/OPCs were treated with either 1 ng/ml PDGF-AA plus 10 ng/ml BMP-4 (PeproTech) or 1 ng/ml PDGF-AA plus 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) for indicated times.
Cells were plated as indicated. Cells were permeablized in 0.5% triton x-100 and probed overnight at 4° C with the following antibodies, all diluted in Hanks Balanced Salt Solution (Gibco) plus 5% fetal calf serum with 0.5% sodium azide and 0.1% triton x-100: A2B5 (mouse IgM, hybridoma supernatant; 1:5 dilution), anti-GalC (mouse IgG3, hybridoma supernatant; 1:10 dilution), Ki-67 (mouse IgG1; 1:1000 dilution; BD Biosciences, San Diego, CA, USA), anti-PDGFR-α (rabbit IgG; 1:1000 dilution; Santa Cruz, Santa Cruz, CA, USA), anti-GFAP (mouse IgG2b; 1:1000 dilution; BD Biosciences). Secondary antibodies were Alexa-conjugated 405, 488, or 568 (all used at 1:2000 dilution). Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride; Molecular Probes, Eugene, OR, USA). Thymidine analogs BrdU (5-bromo-2′-deoxyuridine) and EdU (5-ethynyl-2′-deoxyuridine) were from Invitrogen. Cells were rinsed in wells with PBS and dH2O, dried, mounted with fluoromount-G (Southern Biotech, Birmingham, AL, USA) and coverslipped with glass. Cells were imaged on an inverted fluorescence Nikon Eclipse TE300 microscope and pictures were taken with a Spot camera using Spot software (Diagnostic Instruments Inc., Sterling Hts, MI, USA).
Cells were plated on 10-cm tissue culture dishes at 105 cells per dish. Cells were harvested in modified RIPA buffer containing protease and phosphatase inhibitors. O-2A/OPC lysates were subjected to SDS-PAGE, transferred to PVDF, and probed with antibodies form Santa Cruz specific against the following substrates: phospho-Rb (goat IgG; 1:1000 dilution), total-Rb (mouse IgG1; 1:1000 dilution), Actin-HRP (mouse IgG1; 1:1000 dilution), Cyclin E (rabbit IgG; 1:1000), p27kip1 (mouse IgG1; 1:1000 dilution), and β-tubulin (rabbit IgG; 1:2500 dilution).
O-2A/OPCs were treated as described. RNA was isolated from cells and converted to cDNA following standard protocols. Taqman assays (Applied Biosystems, Carlsbad, CA) for p27 and IRF-1 were run multiplexed with GAPDH and values were calculated using the delta-delta CT method. Three independent experiments were performed in triplicate.
Constructs containing shRNA sequences for p27, IRF-1, and scrambled control were purchased from Open Biosystems (Huntsville, AL) as inserts into the pLKO.1 vector. HEK-293T cells were transfected with 2 μg pLKO.1, and the packaging vectors PAX2 (3 μg) and VSV (1.5 μg). Cells were fed 24 hrs after transfection with DMEM containing 1% FetalClone II (HyClone, Thermo Fisher Scientific, Rockford, IL). At 48 hours after feeding, viral supernatant was sterile filtered and added to O-2A/OPCs at a 1:5 dilution. Infection was allowed to proceed 4–18 hours, after which cells recovered for 24 hours prior to treatment.
For DAPI cell cycle analysis, O-2A/OPCs were treated as indicated for 5 days, detached with trypsin, and stained with DAPI. 20,000 cells from each treatment were counted and DNA content determined on a BD-LSRII (Becton-Dickinson, CA) and data was imported into FlowJo 8 (V 8.8.6; Tree Star, Inc., OR) and analyzed using Modfit LT (Verity Software House, ME). Cells from a given dissection and treatment were split to yield triplicate scans and the entire experiment was repeated 3 times. Cells were processed similarly for EdU incorporation and DNA content except that EdU was added 24 hours before staining and cells were permiabilized with saponin. EdU was detected with Click-It EdU buffer (Invitrogen) at 488m.
Clonal analyses presented as scatter plots indicate the value of each data point from an individual experiment, with the horizontal bar indicating the mean. Summary graphs of clonal data are presented as bar charts with the means of at least 3 independent experiments grouped to give mean +/− SEM. All experiments were performed at least three independent times with the exception of the adult O-2A/OPC EdU assay and the EdU incorporation flow cytometry experiment, which were both performed two independent times. Student’s t-test and one-way ANOVA with Bonferoni’s multiple comparison post hoc test were performed as indicated using Prism 4.0 (GraphPad Software, Inc, La Jolla, CA).
In order to examine the response of O-2A/OPCs to IFN-γ we first exposed highly purified progenitors to this cytokine to define concentrations in which we observed no cell death (Figure 1A) and used a sub-lethal concentration throughout the rest of this work (10ng/ml, which represents ≈ 50 Units/ml (Chew et al., 2005)) O-2A/OPCs were grown at mass culture density (see Methods and Materials) in proliferation media (10 ng/ml PDGF-AA, the primary mitogen for O-2A/OPCs (Noble et al., 1988; Richardson et al., 1988)) in the presence or absence of IFN-γ. The number of cells expressing PDGFR-α was significantly decreased with IFN-γ compared to controls (Figure 1C, D and quantified in 1E), as was total protein expression (Figure 1B). Treatment with IFN-γ also decreased the number of O-2A/OPCs expressing Ki-67 compared to controls (Figure 1C, D and quantified in 1F), which is a marker for all active phases of the cell cycle but is specifically absent from cells in the G0 phase (Scholzen and Gerdes, 2000). Mass culture analysis of O-2A/OPCs showed that continuous treatment with IFN-γ for up to 10 days led to a decrease in the number of cells that expressed either Ki-67 or PDGFR-α (Figure 5).
Given the robust effect of IFN-γ on expression of Ki-67, we next utilized clonal analyses to examine whether IFN-γ suppresses O-2A/OPC division. We performed clonal analyses by plating O-2A/OPCs at very low density (see Methods and Materials) and counted clonally derived A2B5 positive, DAPI positive, and GalC negative cells generated from individual O-2A/OPCs in the presence or absence of IFN-γ. Average clonal size in proliferation media containing 10 ng/ml PDGF-AA alone or in combination with bFGF, a cooperating mitogen that keeps the O-2A/OPC in a prolonged self-renewal state (Bogler et al., 1990), shows clonal expansion at 3 days (Figure 2A,D,E). The addition of IFN-γ to parallel cultures significantly decreased self-renewal of O-2A/OPCs. We confirmed that the decreased number of cells was not due to cell death, as IFN-γ had no effect on the number of clones (Figure 2D) or on TUNEL and activated caspase-3 staining at each time point (data not shown). Average clone sizes in cultures exposed to IFN-γ for 5 or 7 days were reduced by approximately 60% and 75% compared to cultures exposed to PDGF or PDGF + bFGF, (Figure 2B,C, D, E). To test whether this decrease in O-2A/OPC self-renewal was dependent on the loss of PDGFR-α, we pharmacologically inhibited PDGFR-α degradation using the lysosomal inhibitor NH4Cl. NH4Cl had little effect on PDGFR-α levels in control treated cells (Figure 2F). When O-2A/OPCs were treated with NH4Cl and IFN-γ we found that lysosomal inhibition significantly increased PDGFR-α expression compared to INF-γ treatment alone. Clonal analysis revealed, however, that enhanced PDGFR-α expression did not increase O-2A/OPC self-renewal (Figure 2G), suggesting that the effect on self-renewal is not regulated at the level of PDGFR-α. Thus, these results demonstrate that IFN-γ is a potent inhibitor of O-2A/OPC self-renewal.
In order to test whether the loss of proliferation occurs due to cell cycle exit, O-2A/OPCs were grown at mass culture density in proliferation media treated with or without IFN-γ in the presence of BrdU (Figure 3A). IFN-γ significantly decreased BrdU incorporation and PDGFR-α expression in O-2A/OPCs (Figure 3A–C). We next measured cell cycle status by flow cytometry using the nucleic acid dye DAPI and found a significant increase in the proportion of O-2A/OPCs in G0/G1 and subsequently fewer in G2/M and S phase (Figure 3D; with cell cycle profiles for Ctl (D′) and IFN-γ (D″) with representative scatter plots in inset) after exposure to IFN-γ. Considered in conjunction with the Ki-67 staining (Figure 1D, F) these data indicate that the majority of the O-2A/OPCs exposed to IFN-γ are in the G0 phase, and therefore have exited the cell cycle. We next measured EdU incorporation and DNA content together by flow cytometry and found that there was a significant decrease in EdU incorporation and a lower average DNA content in O-2A/OPCs treated with IFN-γ (Figure 3E, F).
O-2A/OPCs are found in the adult CNS but have different properties from perinatal O-2A/OPCs. Cell cycle length for adult progenitors is ~65 hours, compared to ~16 hours for perinatal cells and the cells are less abundant (Wolswijk and Noble, 1989). While recent studies suggest that remyelination in adults originates from cells that behave similar to perinatal O-2A/OPCs, whether they be from the adult SVZ or adult-derived oligospheres (Zhang et al., 1999; Menn et al., 2006) we wanted to address whether adult O-2A/OPCs exit the cell cycle in response to IFN-γ. Adult O-2A/OPCs were cultured for 4 or 9 days in the presence or absence of IFN-γ. EdU incorporation was significantly decreased in cells treated for 4 days with IFN-γ (Figure 3G). Consistent with this data and that from perinatal progenitors, we found the number of clones with more than one cell was significantly decreased in the presence of IFN-γ (Figure 3H). Finally, we determined that IFN-γ had no significant effect on the percentage of GalC positive cells in culture (Figure 3I), although we found that all GalC+ cells were also A2B5+ and appeared immature (data not shown). Collectively, these data demonstrate that perinatal and adult O-2A/OPCs exit the cell cycle in response to IFN-γ.
Having established that IFN-γ significantly decreases O-2A/OPC self-renewal, we next sought to investigate the mechanism of these actions. We assayed levels of critical cell cycle protein expression at all time points examined. We detected a significant increase in the product of the retinoblastoma gene Rb while there was no change in the proportion of Rb that was phosphorylated (Figure 4A). Rb is a tumor suppressor which prevents cell cycle progression when hypophosphorylated prior to the G1 to S phase transition (Weinberg, 1991) and it has been shown that Rb levels also increase during normal cell cycle exit of CNS progenitor cells (Burkhart and Sage, 2008). These results, in addition to the data demonstrating that these cells down-regulate Ki-67 (Figure 1D and F), strongly suggest that O-2A/OPCs are not progressing through the G1 restriction point and remain in the G0 phase of the cell cycle. We next measured protein levels of p27kip1, a member of the cyclin-dependant kinase inhibitor family that negatively regulates the interaction between cyclin E and cdk2. In order for O-2A/OPCs to differentiate into oligodendrocytes it has been demonstrated that they need to first exit the cell cycle and then initiate their differentiation gene expression program. p27kip1 loss of function experiments results in massive expansion of O-2A/OPCs in vivo (Casaccia-Bonnefil et al., 1999), while gain of function experiments generated O-2A/OPCs that never acquired typical oligodendrocyte markers (Tikoo et al., 1998; Tang et al., 2000). In our experiments, p27kip1 was significantly upregulated by three days with IFN-γ treatment (Figure 4B) consistent with cell cycle exit. We also measured levels of Cyclin E, which is upregulated upon transition from G1 to S phase, and found no change between control and IFN-γ treatment. Next, we measured p27 gene expression p27 over time after IFN-γ treatment (Figure 4C) and found that p27 transcription increased within 1 hour and was significantly greater than expression levels in controls by 3 days.
To investigate the necessity of p27 in IFN-γ – mediated quiescence, we knocked down p27 expression using lentiviral based shRNA. This knockdown significantly increased proliferation in PDGF-AA – exposed O-2A/OPCs, as predicted, compared to control cells treated with lentivirus expressing scrambled shRNA (Figure 4E). Surprisingly, however, we observed that O-2A/OPC proliferation was still significantly decreased in p27 knockdown cells upon the addition of IFN-γ, indicating that while p27 is an important component of cell cycle progression, it is not necessary for IFN-γ – mediated quiescence (Figure 4E, F).
We next asked whether IFN-γ signaling through the IFN-γ response factor, IRF-1, which is immediately upstream of IRF-1 (Lee et al., 2005), might be required for IFN-γ – induced quiescence. We found that within 15 minutes, IFN-γ treatment highly upregulated IRF-1 gene expression (Figure 4D). To test whether this upregulation was required for IFN-γ – meditated quiescence we infected cells with lentivirus expressing shRNA against IRF-1 and found that there was no decrease in O-2A/OPC self-renewal in the presence of IFN-γ, indicating that IRF-1 is critical for IFN-γ – mediated quiescence (Figure 4E, F). Collectively, these data demonstrate that while the loss of individual cell cycle inhibitors is important for O-2A/OPC cell cycle control, the robust cell cycle exit induced by IFN-γ likely involves multiple factors that are downstream of the transcription factor IRF-1.
Data presented thus far demonstrate that a large proportion of O-2A/OPCs exit the cell cycle upon exposure to IFN-γ. Since IFN-γ is associated with many types of neuroinflammation we wanted to determine whether transient exposure to IFN-γ would permanently inhibit O-2A/OPC proliferation and differentiation into oligodendrocytes. To test the potential of O-2A/OPCs after IFN-γ exposure, cells were treated for 3 days with IFN-γ in proliferation media. At this time, IFN-γ was either withdrawn to allow for 1, 2, 3, 5, or 7 days of recovery or IFN-γ remained in the medium. O-2A/OPCs were plated at clonal density and the number of A2B5-positive cells per clone was counted (Figure 5A, B). Within three days after IFN-γ withdrawal there was a significant increase in O-2A/OPC proliferation (Figure 5A, B), which continued through 7 days (Figure 5A, B). While cultures in which IFN- γ was withdrawn showed increased self-renewal, there was no increase in the number of O-2A/OPCs per clone during continuous IFN-γ treatment. Critically, however, there was still the same number of clones in both treatments, indicating that these cells were still alive for up to 10 days with IFN-γ exposure. Moreover, these cells still labeled with the A2B5 monoclonal antibody, suggesting that they had not lost their progenitor cell identity. O-2A/OPCs also retained the ability to differentiate into GalC positive oligodendrocytes after IFN-γ withdrawal (Figure 5C). As early as two days after IFN- γ withdrawal there was a significant increase in the number of clones containing at least one GalC positive oligodendrocyte, thus indicating that IFN-γ treated O-2A/OPCs can re-engage in both proliferation and differentiation.
We next confirmed the clonal data in mass culture experiments. O-2A/OPCs arrested in the cell cycle by IFN-γ showed a robust increase in the percentage of cells expressing Ki-67 or PDGFR-α and an increase in the number of O-2A/OPCs after the withdrawal of IFN-γ (Figure 5D). The increase in Ki-67 labeling indicates that a greater proportion of O-2A/OPCs are entering the active phases of the cell cycle (Figure 5D). The increase in PDGFR-α-positive cells suggests that O-2A/OPCs regain the ability to respond to this mitogen after exiting the cell cycle (Figure 5D). Taken together, these data demonstrate that O-2A/OPCs maintain their proliferative potential and regain their responsiveness to PDGF. In order to determine if these cells were able to re-enter the cell cycle after a longer exposure to IFN-γ, we treated cells for 7 days before withdrawal. O-2A/OPCs rapidly increased expression of both PDGFR-a and Ki-67 upon IFN-γ withdrawal (Figure 6A, B) and incorporated BrdU (Figure 6C, D). At 7 days after withdrawal there were significantly more BrdU and PDGFR-α positive cells compared to continuous IFN-γ treatment (Figure 6D). Collectively, these data indicate that O-2A/OPCs can remain in cell cycle phase G0 for long periods of time while still retaining the ability to reenter the cell cycle, proliferate, and differentiate.
As cessation of division is associated with differentiation of O-2A/OPCs into oligodendrocytes (Noble et al., 1988), it was surprising that our results indicate that there was far less spontaneous differentiation in the presence of IFN-γ (Figure 5C), as these cells were grown in proliferation medium, versus that of control-treated cells. We therefore asked whether IFN-γ altered differentiation into oligodendrocytes. We tested the differentiation capacity of O-2A/OPCs by growing cells at clonal density in medium supplemented with a low concentration of PDGF-AA (1ng/ml) and thyroid hormone (40 nM) in the presence or absence of IFN-γ. After 3, 5, or 7 days cells were labeled with A2B5 and anti-GalC antibodies and clones were counted. The conditions we used that promote differentiation still allowed for a small degree of proliferation, but there was no significant proliferation in either condition (Figure 7A, B). There were, however, significantly fewer GalC positive oligodendrocytes in the presence of IFN-γ suggesting that O-2A/OPCs exposed to IFN-γ do not differentiate into oligodendrocytes (Figure 7C). At day 5 there were over 4-fold fewer clones with GalC positive oligodendrocytes in the presence of IFN-γ, and by day 7 there were over 6-fold fewer oligodendrocyte-containing clones with IFN-γ treatment compared to control cultures that demonstrated robust differentiation (Figure 7C). We also performed similar experiments with O-2A/OPCs grown in proliferation media as in Figure 1 and found that there was no change in the proportion of cells that became GalC positive (fewer than 3% of clones contained at least one oligodendrocyte; data not shown).
While thyroid hormone is a particularly well-characterized pro-oligodendrocyte differentiation factor we performed similar experiments with ciliary neurotrophic factor (CNTF), which has also been shown to promote oligodendrocyte differentiation from postnatal O-2A/OPCs (Barres et al., 1993; Mayer et al., 1994). While CNTF treatment alone leads to an increase in O-2A/OPC proliferation followed by differentiation (Figure 7D, E, F), this proliferation and differentiation was significantly inhibited by IFN-γ (Figure 7D, E, F). The percentage of clones containing at least one GalC positive oligodendrocyte was reduced significantly at 5 and 7 days with an ~5-fold reduction and ~7-fold reduction, respectively, in the number of clones containing at least one GalC positive oligodendrocyte with IFN-γ treatment (Figure 7E). These results demonstrate that IFN-γ treatment abrogates the pro-differentiation effects of at least two distinct pro-oligodendrocyte differentiation factors.
O-2A/OPCs were originally noted for their ability to generate type-2 astrocytes, in addition to oligodendrocytes, with the addition of serum (Raff et al., 1983), certain signaling factors in combination with extracellular matrix molecules (Lillien et al., 1990; Mayer et al., 1994), or bone morphogenic proteins (BMPs; (Mabie et al., 1997), endogenous signaling factors of the TGF-β family. We were therefore interested in determining whether IFN-γ would inhibit the induced differentiation into type-2 astrocytes as it inhibited induced differentiation into oligodendrocytes. We found that IFN-γ had no inhibitory effect on O-2A/OPC differentiation into type-2 astrocytes as virtually all of the cells in culture expressed GFAP after treatment with BMP-4 alone or in combination with IFN-γ, respectively at 3, 5, and 7 days (Figure 7G, H). All astrocytes were GFAP/A2B5 double positive and GalC negative, consistent with the type-2 astrocyte antigenic phenotype. Similarly, IFN-γ did not inhibit differentiation into astrocytes induced by 10% fetal bovine serum (data not shown).
We next determined whether IFN-γ had any effect on astrocyte generation in the absence of differentiation factors or in the presence of pro-oligodendrocyte differentiation conditions. O-2A/OPCs grown in 1 ng/ml PDGF-AA in the presence of IFN-γ gave rise to significantly more GFAP positive astrocytes compared to the absence of IFN-γ (Figure 8A, B). The percentage of astrocytes was, however, never greater than 10%, unlike that observed in BMP-4 or serum. Finally, O-2A/OPC differentiation into was promoted by IFN-γ even in the presence of the pro-oligodendrocyte differentiation factors TH (Figure 8C) and CNTF (data not shown) while the absence of IFN-γ allowed for no astrocyte generation in the presence of TH at any time point examined (Figure 8C).
Based on our results that IFN-γ treatment led to an increase in GFAP positive cells we became concerned with the formal possibility of having a small number of contaminating GFAP+/A2B5− type-1 astrocytes in our O-2A/OPC cultures that might have been converted by IFN-γ into the GFAP+/A2B5+ positive cell population we observed. To test whether IFN-γ could lead to hypertrophy and/or an up regulation of both A2B5 and GFAP in type-1 astrocytes, cultures of type-1 astrocytes generated from the postnatal day-1 rat brain were grown and treated in the presence of TH or BMP-4 with and without IFN-γ and stained with A2B5 antibody, anti-GFAP, and DAPI (data not shown). There was no up regulation of either GFAP or A2B5 staining in cortical derived type-1 astrocytes upon treatment with IFN-γ (data not shown). This finding makes it highly unlikely that the GFAP/A2B5 double positive cells we observed in previous experiments were due to type-1 astrocyte contamination in our cultures.
While this data suggests that O-2A/OPCs are differentiating into astrocytes, it does not rule out the possibility that astrocyte progenitor cells or a subset O-2A/OPCs is responsible for the astrocyte generation. In order to test this possibility we performed clonal analyses in the presence of BMP-4 alone or in combination with IFN-γ. As shown in Figure 8D, the average clone had fewer than five A2B5 positive cells at 3, 5, and 7 days indicating that these cells were not proliferating (Figure 8D, E). There were similarly fewer than five GFAP positive cells, on average, per clone at each time point examined indicating that there is no subset of GFAP positive cells that is proliferating (Figure 8F, G). Collectively, these data indicate that virtually all cells from a single O-2A/OPC clone differentiated into GFAP/A2B5 double positive astrocytes and that there is no other population of cells giving rise to the GFAP positive astrocytes generated by IFN-γ treatment.
Our goal in these studies was to understand the sub-lethal response of O-2A/OPCs to IFN-γ, which has confounded investigators since it was tested as therapy in MS patients where it exacerbated MS relapse (Panitch et al., 1987). IFN-γ expression precedes the clinical symptoms of MS (Beck et al., 1988) and its expression correlates with EAE severity (Renno et al., 1994). Paradoxically, IFN-γ has been shown to reduce the severity of EAE by intra-ventricular injection (Voorthuis et al., 1990), while depletion of IFN-γ worsens EAE (Billiau et al., 1988; Duong et al., 1992), and IFN-γ gene knockout confers susceptibility to EAE in mice that are normally resistant (Krakowski and Owens, 1996). Recently, IFN-γ signaling was found to decrease neuroinflammation and pathogenesis in the cerebellum and brainstem (Lees et al., 2008) and activate a cytoprotective stress response in EAE mice (Lin et al., 2007; Lin et al., 2008).
In vitro analyses of IFN-γ (up to 200ng/ml) have been described by others to induce apoptosis in oligodendrocyte progenitors generated by the shake-off method (McCarthy and de Vellis, 1980), but these cultures were exposed to serum (Vartanian et al., 1995; Andrews et al., 1998; Baerwald and Popko, 1998; Chew et al., 2005; Horiuchi et al., 2006; Wang et al., 2009) and contain in a mixture of O-2A/OPCs, microglia, and astrocytes (Miller et al., 2007). In contrast another group reported that IFN-γ had no adverse effect on viability but inhibits proliferation (Agresti et al., 1996). Based upon the contradictory results from various groups using mixed glial cell preparations, we set out to characterize the O-2A/OPC response to IFN-γ in the absence of other cells. The perinatal progenitor cells used in our studies were highly purified from corpus callosum, while adult progenitors were isolated from the optic nerve, grown in culture for up to only one passage, and were never exposed to serum.
Our data demonstrate that IFN-γ inhibits O-2A/OPC proliferation and induces differentiation of a subset of O-2A/OPCs into astrocytes. Our conclusion that IFN-γ induces cell cycle exit as opposed to cell death or differentiation into oligodendrocytes is supported by several lines of evidence: First, IFN-γ did not alter the number of clones, although there was a decrease in the amount of proliferation within each given clone at all time points examined (Figure 2). Exposure also decreased BrdU incorporation (Figure 3), and did not change the number of dead or dying cells as detected by either TUNEL (Figure 1A) or activated caspase-3 staining (data not shown). Similarly, in differentiation conditions, with both TH and CNTF, there was no change in clone numbers with IFN-γ but a decrease in the number of clones containing GalC positive oligodendrocytes (Figure 8). We also found that this response was robust in that adult O-2A/OPCs from the optic nerve also showed decreased self-renewal (Figure 4). Second, IFN-γ exposure had no effect on O-2A/OPC viability (Figure 1). In long-term cultures O-2A/OPCs treated with IFN- γ for over 40 days showed no difference in viability compared to control cells (DCT, JDC, and MM-P, unpublished observations). Third, the proliferation marker Ki-67 was down regulated upon IFN-γ treatment (Figure 1), strongly indicating that a majority of cells were in phase G0. Furthermore, the cell cycle inhibitor proteins Rb and p27kip1 were both upregulated in response to IFN-γ (Figure 5). A recent report demonstrates that IFN-γ upregulates IRF-1 in O-2A/OPCs (Wang et al., 2009), which has been shown to up-regulate p27kip1 in cancer cells (Lee et al., 2005), and supports our conclusions with the important exception that, unlike Wang et al., we do not observe cell death. This discrepancy could be due to different cell isolation methods and/or the higher concentration of IFN-γ they used. An alternative explanation of their viability data is that the decrease in MTT labeling in their cultures exposed to IFN-γ is not an indication of cell death, as was concluded, but rather a lack of proliferation and/or metabolic activity. Fourth, DNA content, as analyzed by flow cytometry robustly demonstrates that the vast majority of O-2A/OPCs were neither in S phase nor G2/M phase of the cell cycle (Figure 3) but rather in G0/G1. This, coupled with the decrease in Ki-67 immunostaining strongly suggests that the majority of these cells were in G0. This observation is in contrast with a previous report (Chew et al., 2005) but these authors were studying the effect of higher concentrations of IFN-γ on differentiating precursors making it difficult to directly compare results. Fifth, the cell cycle exit induced by IFN-γ was reversible (Figures 6 and and77).
While IFN-γ exposure decreased PDGFR-α expression, our data suggest that IFN-γ – mediated cell cycle exit is not primarily due to loss of PDGFR-α. Inhibiting PDGFR-α degradation did not abrogate the effect of IFN-γ on O-2A/OPC self-renewal (Figure 2F, G). Moreover, O-2A/OPCs treated with both PDGF-AA and FGF-2 undergo similar cell cycle exit (Figure 2A – C).
Exposure to IFN-γ decreased differentiation of O-2A/OPCs into oligodendrocytes (Figure 8). Interestingly, differentiation is not inhibited per se as cells exposed to IFN-γ were still able to differentiation into astrocytes (Figures 8 and 9). O-2A/OPC differentiation into type-2 astrocytes in culture has been shown in the presence of CNTF and extracellular matrix (Lillien et al., 1990; Mayer et al., 1994), and after exposure to BMPs (Mabie et al., 1997; Grinspan et al., 2000; Gomes et al., 2003; Samanta and Kessler, 2004; Hampton et al., 2007; Weible and Chan-Ling, 2007). This astrocyte differentiation is not restricted to in vitro conditions, but also has been shown to occur in vivo. BMP-4 overexpression and conditional BMP receptor mutants both demonstrate that BMP signaling induces astrocyte generation at the expense of oligodendrocyte number in vivo (Gomes et al., 2003; Samanta et al., 2007). Rodent NG2+ progenitors can also give rise to astrocytes in the absence of ectopic or disrupted signaling (Aguirre and Gallo, 2004; Zhu et al., 2008; Guo et al., 2009) and human progenitors transplanted into mice differentiate into oligodendrocytes in the white matter and astrocytes when they migrate into gray matter (Windrem et al., 2004). O-2A/OPC differentiation into astrocytes is also observed in vivo in response to stab wounds or cryo-injury (Alonso, 2005; Buffo et al., 2005; Tatsumi et al., 2005; Hampton et al., 2007; Magnus et al., 2007). Our results also demonstrate that IFN-γ can induce O-2A/OPC differentiation into astrocytes, even in the presence of pro-oligodendrocyte signaling factors such as TH and CNTF (Figure 8 and data not shown). This observation is intriguing as mice over-expressing IFN-γ under control of the MBP promoter have been shown to exhibit an increase in GFAP immunoreactivity as well as reactive gliosis in white matter tracts (Corbin et al., 1996). Our data suggests that IFN-γ is associated with the generation of GFAP positive astrocytes from O-2A/OPCs and not from type-1 astrocytes as IFN-γ led to neither increased proliferation nor to upregulation of A2B5 or GFAP in cortical A2B5-negative type-1 astrocytes (data not shown). Type-2 astrocytes could formally also be progeny of a subpopulation of astrocyte progenitor cells (APCs), but it appears unlikely that our cultures contain APCs, however, as it has been shown that the survival of APCs is dependent on glial growth factor (GGF) (Mi and Barres, 1999), which is not present in our conditions. In addition, our clonal data show that virtually every cell in clones generated in the presence of BMP and IFN-γ was immunoreactive for both A2B5 and GFAP and no clones contained cells that labeled with anti-GFAP only and could therefore be progeny of a putative APC. Taken together, our conclusions most strongly support the idea that the GFAP+/A2B5+ positive astrocytes in our cultures differentiated from O-2A/OPCs. These data suggest that endogenous O-2A/OPCs can directly contribute to IFN-γ induced astrogliosis, a possibility that has not yet been examined.
These data demonstrate the potential for IFN-γ to influence cell fate in CNS injury and inflammation in previously unrecognized ways. While the environment in vivo is much more complex, high focal concentrations of IFN- γ might initially lead to a decrease of O-2A/OPC numbers via apoptosis. After acute inflammation ends and the levels of IFN- γ decrease (Franklin, 2002) O-2A/OPCs could then differentiate into astrocytes. While this could reduce the number of progenitors available to generate oligodendrocytes, astrocyte generation might also be associated with local production of beneficial factors, as seen for type-1 astrocyte production of interleukin-11 (Zhang et al., 2006), nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 (Schwartz et al., 1993). Were this so, differentiation of O-2A/OPCs into astrocytes could represent a protective measure that helps to safeguard the remaining progenitor cells. Such an effect might help to explain the contradictory effects of IFN- γ in vivo, and would suggest that a positive outcome of inhibiting inflammation through therapeutic interventions is highly dependent on the time point of the intervention and local concentration of inflammatory cytokines like IFN- γ. Finally, more in vitro and in vivo investigation of IFN-γ in combination with other inflammatory cytokines will advance our understanding of O-2A/OPC behavior and hopefully expose new therapeutic avenues.
This work was supported in part by the NIH (grant numbers ES012708 and NS39511). DCT was supported in part by a Kirschstein NRSA fellowship (NINDS; T32NS051152) and a postdoctoral fellowship from the National Multiple Sclerosis Society (FG1812A1). The authors are grateful for insight and comments from Drs. Fred Strathmann, Ibro Ambeskovic, Christoph Proschel, Mark Noble, and Kerry O’Bannion.
Author contributions: D.C.T. and J.D.C. performed the experiments. D.C.T. and J.D.C. analyzed the data. D.C.T. and M.M-P. conceived the project and designed the experiments and wrote the manuscript.