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IL-4-Induced Peroxisome Proliferator-Activated Receptor Activation Inhibits NF-B Trans Activation in Central Nervous System (CNS) Glial Cells and Protects Oligodendrocyte Progenitors under Neuroinflammatory Disease Conditions: Implication for CNS-Demyelinating Diseases¹

Ajaib S. Paintlia^2,*, Manjeet K. Paintlia^2,*, Inderjit Singh^* and Avtar K. Singh^3,

^* Department of Pediatrics and Department of Pathology and Laboratory Medicine, Medical University of South Carolina and Ralph H. Johnson Veterans Administration Medical Center, Charleston, SC 29425

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Th2 phenotype cytokine, IL-4, plays an important role in the regulation of Th1 cell responses and spontaneous remission of inflammatory CNS demyelinating diseases such as multiple sclerosis (MS). In this study we demonstrate IL-4-induced down-regulation of inducible NO synthase (iNOS) expression and survival of differentiating oligodendrocyte progenitors (OPs) in proinflammatory cytokine (Cyt-Mix)-treated CNS glial cells, which is a condition similar to that observed in the brain of a patient with MS. IL-4 treatment of Cyt-Mix-treated CNS glial cells significantly decreased iNOS expression/NO release with a parallel increase in survival of differentiating OPs. IL-4 effects were concentration-dependent and could be reversed by anti-IL-4R Abs. The use of inhibitors for Akt, p38 MAPK, and peroxisome proliferator-activated receptor (PPAR-) antagonist revealed that inhibition of Cyt-Mix-induced iNOS expression and survival of differentiating OPs by IL-4 is via PPAR- activation. There was a coordinate increase in the expression of both PPAR- and its natural ligand-producing enzyme 12/15-lipoxygenase (12/15-LOX) in IL-4-treated cells. Next, EMSA, immunoblots, and transient cotransfection studies with reporter plasmids (pNF-B-Luc and pTK-PPREx3-Luc) and 12/15-LOX small interfering RNA revealed that IL-4-induced PPAR- activation antagonizes NF-B transactivation in Cyt-Mix-treated astrocytes. In support of this finding, similarly treated 12/15-LOX^–/– CNS glial cells further corroborated the result. Furthermore, there was reversal of IL-4 inductive effects in the brain of LPS-challenged 12/15-LOX^–/– mice when compared with LPS-challenged wild-type mice. Together, these data for the first time demonstrate the inhibition of Cyt-Mix-induced NF-B transactivation in CNS glial cells by IL-4 via PPAR- activation, hence its implication for the protection of differentiating OPs during MS and other CNS demyelinating diseases.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Multiple sclerosis (MS)⁴ is the leading cause of neurological deficits in young adults (1). Currently, animals with experimental autoimmune encephalomyelitis (EAE) serve as a disease model, which mimics many aspects of MS. Autoreactive T and B cells responding to myelin proteins are implicated in the pathogenesis of MS and EAE (2, 3). The presence of primary T cell-driven aberrant immune response to various myelin Ags, including myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) (4) as well as high titers of myelin-specific Abs (5, 6), suggests that severe pathological changes in MS and EAE are probably a consequence of synergy between autoimmune T and B cells. There was an extensive CNS demyelination in adoptively transferred MBP-induced EAE when subsequently injected with a mAb to MOG (7) or during recovery phase in SJL/J mice (8). Indirect evidences for an involvement of anti-myelin Abs for the disease comes from the finding of Ig deposits in MS lesions, the presence of Ig together with complement components at the edge of demyelinating lesion (9, 10), and deposition of anti-MBP and anti-MOG Abs in demyelinating MS and EAE lesions (5, 6), which suggests that B cell-mediated responses are also important in CNS demyelination.

The infiltration of T cell subtype Th1 cells (secrete IL-2, IFN-, and TNF-) into the CNS triggers the activation of resident glial cells resulting in NF-B-dependent expression of proinflammatory cytokines (i.e., TNF-, IL-1, and IFN-) and cerebrovascular disturbances, i.e., breaching of the blood-brain barrier and secondary CNS inflammatory injury (11). Local production of TNF- in the CNS has been implicated in oligodendrocyte apoptosis (12). In addition, a greater loss of oligodendrocytes by apoptosis resulting in severe demyelination has been reported in the CNS of transgenic mice engineered for TNF- expression (13). Likewise, IL-1 has been reported to cause oligodendrocyte death in coculture with microglia and astrocytes, an event likely attributable to impaired uptake and metabolism of glutamate by astrocyte-glutamate receptors (14). Reversible damage of CNS integrity and severe demyelination has been documented in the brain of mice engineered for chronic expression of IL-1 (15). Recent studies have indicated that acute LPS-induced white matter injury and oligodendrocyte progenitor (OP) loss are mediated by increased expression of TNF- and IL-1 (16). However, both in vivo and in vitro studies have suggested the beneficial effects of IL-1, as it is essential for the differentiation of oligodendrocytes (17, 18). IFN- and LPS have been shown to induce apoptosis of mature oligodendrocytes and OPs via endogenous NO production in vitro (19), and in vivo studies have documented that IFN- is involved in the progression of chronic demyelination and neurological deficits in EAE (20). Predominantly, the excessive release of NO and reactive oxygen species during neuroinflammatory diseases lead to the production of peroxynitrite, which is responsible for degeneration of both oligodendrocytes and neuronal axons thereby resulting in subsequent demyelination and permanent neurological deficits in affected individuals (21, 22).

IL-4 secreted by Th2 cells blocks the production of IL-1 and TNF- by CNS glial cells and inhibits the differentiation of Th0 cells into Th1 cells but induces their differentiation into Th2 cells thereby helping develop resistance to EAE (23, 24). The exogenous administration of IL-4 after adoptive transfer of MBP-reactive cells attenuated the disease severity in EAE animals (25). However, transgenic expression of IL-4 in T cells did not reduce EAE disease severity (26), whereas encephalitogenic T cells transduced with a retroviral gene construct expressing IL-4 delayed the onset and reduced the disease severity of EAE when adoptively transferred into MBP-immunized mice (27). IL-4-deficient mice (PL/J and C57BL/6) exhibited a similar incidence and disease severity of EAE as that observed in wild-type mice, with a prolongation of disease in IL-4-deficient PL/J mice, suggesting a role for IL-4 in EAE disease termination (26, 28). Based upon these evidences, current therapeutic strategies are essentially targeted to treat inflammatory demyelinating diseases that favor shifting the Th1 phenotype immune response toward Th2 phenotype response. For instance, the FDA approved MS drug Copaxone induces a shift from Th1 to Th2 and preferentially produces IgG1 over IgG2 Abs in patients treated for MS (29).

Previously, IL-4 has been shown to down-regulate the expressions of inducible NO synthase (iNOS) and COX-2 via inhibition of NF-B transactivation in Theiler’s virus-infected brain astrocytes (30, 31). Although it is known that IL-4 inhibits NF-B-mediated iNOS expression in activated astrocytes, we have yet to elucidate the mechanism by which IL-4 modulates NF-B transactivation in activated CNS glial cells and its subsequent effect on the survival of differentiating OPs during neuroinflammatory diseases. Using both in vitro and in vivo approaches, we explored the molecular mechanisms of IL-4-induced NF-B transactivation in activated glial cells and its effect on the fate of differentiating OPs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Chemicals and Abs

DMEM (4.5 g/L glucose) and FBS were obtained from Invitrogen Life Technologies. Recombinant (rat, mouse, and human) IFN-, TNF-, IL-1, and IL-4 proteins and anti-IL-4R Abs were purchased from R&D Systems. GW9662 and protease inhibitor mixture were purchased from Sigma-Aldrich. Akt inhibitor (1L-6-hydroxymethyl-chloro-inositol-2-(R)-2-O-methyl-3-O-octadecylcarbonate) and p38 MAPK inhibitor (SB203580) were purchased from Alexis Biochemicals. [-³²P]dCTP (3000 Ci/mmol), ECL detecting reagents, and nitrocellulose membranes were purchased from Amersham Biosciences. Anti-mouse platelet-derived growth factor- receptor (PDGF-R, rat anti-mouse CD140A) Abs were purchased from Research Diagnostics. Rabbit anti-NG2 chondroitin sulfate proteoglycan, mouse anti-O4, mouse anti-A2B5, anti-isolectin B4, and rabbit anti--actin Abs were purchased from Chemicon International. The murine anti-mouse MBP (clone 1: 129–138) Abs were acquired from Serotec. Anti-glial fibrillary acidic protein (GFAP) was purchased from DakoCytomation. Mouse IgG and rabbit polyclonal IgG (control primary Abs) and secondary Abs such as Texas Red X-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG were from Vector Laboratories. Anti-iNOS polyclonal rabbit Abs purchased from Advanced ImmunoChemical. Abs for peroxisome proliferator-activated receptor (PPAR)-, IB, p65, p50, and -actin were purchased from Santa Cruz Biotechnology.

Plasmids, primers, and small interfering RNA (siRNA) oligonucleotides

The source of peroxisome proliferators-response element-containing reporter plasmids (pTK-PPREx3-Luc) and pNF-B luciferase plasmids (pNF-B-Luc) used in the study are same described earlier (32). The primers for real-time PCR analysis and 12/15-LOX siRNA and a siRNA of scramble sequence (scrRNA) used as control were designed by using free software (available at www.idt.com) and synthesized from Integrated DNA Technologies. Five different siRNAs with antisense strands targeting 12/15-lipoxygenase (12/15-LOX) at nucleotides 350–370, 377–397, 458–478, 1461–1481, and 1485–1505 (GenBank accession no. NM_031010) were screened. 12/15-LOX siRNA, targeting nucleotides 1461–1481 (antisense, 5'-ttcuggcuguuucgaguu-3' and sense, 5'-gaccgacaaagcugugcaatt-3'), revealed maximum knockdown activity (60%; data not shown). The scrRNA sequence (antisense, 5'-uccucauaagcuucauaggcg-3' and sense, 5'-ccuaugaagcuuaugaggatt-3') was used as control. The oligonucleotides were 21-mer double-stranded with a 19-bp oligoribonucleotide region and dinucleotide overhangs (TT) on the 3' end of each strand consisting of deoxynucleotide residues linked by means of a phosphodiesterase group. Antisense and sense strands were annealed in isotonic buffer (100 mM potassium acetate, 30 mM HEPES-KOH, 2 mM magnesium acetate, 26 mM NaCl (pH 7.4)) at 37°C followed by mixing with FuGENE 6 (Roche Molecular Systems) for transient transfection studies.

Mixed glial cells, microglia, and astrocyte cultures

CNS mixed glial cell cultures were generated from both rats (SD; Charles River Breeding Laboratories) and mice (12/15-LOX^–/–), or wild-type (C57BL/6; The Jackson Laboratory) P1–P2 brains as previously described (33). Cells were cultured in poly-D-lysine-coated T75 flasks in DMEM containing 10% FBS (heat-inactivated) and antibiotic mixture (100 U/ml/100 µg/ml, penicillin/streptomycin, respectively) at 37°C in room air and 5% CO₂. The cells were fed every 2–3 days with fresh medium until they reached confluence and then used for experimental studies. For purification of astrocytes and microglia, cultures were shaken for 30 min at 200 x g at 37°C to collect microglia, followed by further shaking for 18 h to remove OPs and remaining microglia. The astrocyte monolayer was then trypsinized and cultured in six-well plates and 100-mm culture plates coated with poly-D-lysine (10 µg/ml). The cultures were immunocytochemically characterized by specific markers for different cell types. For instance, >95% of the cells stained positively with a mAb against GFAP (specific for astrocytes) in purified astrocytes containing <5% of both A2B5-positive (OPs) and isolectin B4-positive (microglia). Cultures of astrocytes containing >3% of A2B5-positive or isolectin B4-positive cells were discarded. Likewise, purified microglia cells were >97% positive for isolectin B4 containing 3% of A2B5-positive and GFAP-positive cells.

Mixed glial cells and purified astrocytes or microglia were cultured on glass-chamber slides (Lab Tek II) or 100-mm plates at a density of 1 x 10⁴ cells/ml. After 24 h, fresh DMEM without FBS was changed, and cells were treated with rIL-4 protein for 30 min before addition of mixture of recombinant cytokine (TNF-, IL-1, and IFN-; each 10 ng/ml) proteins. For oligodendrocyte immunostaining, mixed glial cells were grown in the absence of FBS and treated similarly as described and immunostained with anti-MBP Abs after 6 days posttreatment. Cells were harvested after 6 and 24 h of posttreatment for mRNA and protein expression analysis, respectively. Likewise, cells were treated in the presence or absence of inhibitors (their optimal inhibitory concentrations were used per manufacturer’s instructions) and processed as described for chamber slides and plates. Culture supernatants were collected for analysis of nitrite levels after 24 h posttreatment. For FACS analysis, mixed glial cells cultured in 100-mm plates were treated similarly and harvested after 4 days posttreatment. Human oligodendroglial cell line MO3.13 cells (1 x 10⁴ cells/ml) were cultured similarly as described for DMEM for mixed glial cells. After 24 h, cells were treated with rIL-4 protein and a mixture of proinflammatory cytokine proteins in 100-mm plates and harvested after 24 h posttreatment.

NO release determination

Release of NO was determined by assay of culture supernatants for nitrite, a stable product of NO produced by iNOS activity using Griess reagent as reported earlier (33).

Immunocytochemistry

For single-label immunocytochemistry, standard methodology was used. Briefly, slides were blocked with a serum-PBS solution and incubated with appropriately diluted primary Abs (1/100) at 4°C overnight followed by washing and incubation with secondary Abs. For double-labeling immunocytochemistry, slides were incubated simultaneously with both types of primary Abs after blocking with a serum-PBS solution at 4°C overnight as earlier described. Then, secondary Abs for the appropriate marker (anti-IgG conjugated with FITC or anti-IgM conjugated with Texas Red) were used. Slides were also incubated with Texas Red-conjugated IgM and FITC-conjugated IgG without primary Abs as negative controls, and an appropriate mouse IgG and rabbit polyclonal IgG were used as isotype controls. After thorough washings, slides were mounted with aqueous mounting media (Vectashield; Vector Laboratories). Slides were analyzed by immunofluorescence microscopy (Olympus BX-60) with an Olympus digital camera (Optronics) using a dual-band pass filter. Images were captured and processed using Adobe Photoshop 7.0 and were adjusted using the brightness and contrast level to enhance image clarity. Total numbers of MBP⁺ cells per field were determined by manual counting in 10 fields/slide from three to five experiments in a blinded fashion. Mean numbers of MBP⁺ cells per field were computed for statistical analysis among groups and plotted.

Quantification of immunofluorescence intensity

The immunofluorescence intensity of slides immunostained with anti-MBP, anti-GFAP, and anti-iNOS Abs was quantified with Image-Pro Plus (Media Cybernetics) as previously described (33). A distance scale of 20 µm was chosen for measurement in all groups. The intensity for MBP, GFAP, and iNOS was computed for statistical analysis among groups and plotted.

Total RNA extraction, ss cDNA synthesis, and real-time PCR analysis

Total RNA from treated cells was purified using TRIzol Reagent (Invitrogen Life Technologies) as described (34). ss cDNA was synthesized from total RNA/sample using the Superscript Preamplification system for first-strand cDNA synthesis (Invitrogen Life Technologies) using methodology and conditions explained earlier (34). Real-time PCR was performed using Bio-Rad iCycler IQ real-time PCR. The primer sequences were as follows: GAPDH, forward 5'-cctacccccaatgtatccgttgtg-3' and reverse 5'-ggaggaatgggagttgctgttgaa-3'; iNOS, forward 5'-ggaagaggaacaactactgctggt-3' and reverse 5'-gaactgagggtacatgctggagc-3'; GFAP, forward 5'-ccaagccagacctcacagc-3' and reverse 5'-ccgataccactcttctgtttcttg-3'; PPAR-, forward 5'-gaagacaaaatcaagttcaaacat-3' and reverse 5'-atacttgagcagagtcacttggtc-3'; 12/15-LOX, forward 5'-agataaatgtcgtttggctcctgg-3' and reverse 5'-ttacgaatctcaatttccttatcc-3'; PDGF-R, forward 5'-cagacattgaccctgttccagagg-3' and reverse 5'-gaatctatgccaatatcatccatc-3'; SOX10, forward 5'-tctacacggccatctctgacc-3' and reverse 5'-gtcgtatatactggctgttcccagtg-3'; proteolipid protein (PLP), forward 5'-gccttccctagcaagacctctgag-3' and reverse 5'-gaacttggtgcctcggcccatgag-3'; and MyT1-L, forward 5'-ggtgcccaagagcaaagaa-3' and reverse 5'-atcacagccaggtaccgga-3'. IQ SYBR Green Supermix was purchased from Bio-Rad. Thermal cycling conditions were as follows: activation of iTaq DNA polymerase at 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 30 s and 55–58°C for 30–60 s. Then, normalized expression data were generated by dividing the amount of target gene concentration with the amount of reference gene (GAPDH). The detection threshold was set above the mean baseline fluorescence determined by the first 20 cycles. Amplification reactions in which the fluorescence increased above the threshold were defined as positive. A standard curve for each template was generated using a serial dilution of the template (cDNA). The quantities of target gene expression were normalized to the corresponding GAPDH mRNA quantities in test samples. Similar results were obtained when normalized with reference genes such as -actin or 18 S RNA (data not shown).

Preparation of cytosolic extract and nuclear extract

Cells grown and treated on 100-mm plates were washed with ice-cold PBS, scraped off, and collected by centrifugation. Cell pellets were homogenized in 200 µl of buffer A (10 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 50 µg/ml leupeptin, 10 µg/ml aprotinin, 5 mM benzamidine, 1 mM sodium orthovanadate, 2 mM NaF, and 1 mM DTT). After 10 min at 4°C, Nonidet P-40 was added to a final concentration of 0.5%. The tubes were vigorously vortexed for 10 s and the nuclei were collected by centrifugation at 13,000 x g for 30 s. The supernatants were stored at –80°C and the nuclear pellet was resuspended by gentle shaking for 30 min at 4°C in 100 µl of buffer A supplemented with 20% glycerol and 0.4 M NaCl. Nuclear proteins were obtained by centrifugation at 13,000 x g for 5 min and the supernatants were stored at –80°C. Protein concentration was quantified using Bradford protein assay (Bio-Rad).

Immunoblotting

Cells were processed for immunoblotting as described (35). Briefly, cells were lysed in ice-cold lysis buffer (50 mm Tris-HCl, (pH 7.4), containing 50 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% glycerol, and protease inhibitors mixture). Protein (20 of µg protein/lane) was separated by 10% SDS-PAGE and blotted to nitrocellulose (Amersham Biosciences). Immunoblots were incubated with primary Abs (1/1000) followed by incubation with secondary peroxidase-conjugated Abs (1/10,000; Sigma-Aldrich). Immunoreactivity was detected using the ECL detection method according to the manufacturer’s instructions with subsequent exposure of immunoblot to x-ray films (Amersham Biosciences), followed by autoradiography. Autoradiograph was scanned and analyzed by using densitometry (Imaging Densitometer; Bio-Rad).

EMSA

An oligonucleotide sequence 5'-tgctagggggattttccctctctctgt-3' corresponding to the consensus B site (nucleotides –978 to –952) of the murine iNOS promoter was used. The DNA probes were end-labeled using Klenow enzyme and 50 µCi of [-³²P]dCTP. The DNA probe (5 x 10⁴ dpm) was incubated for 15 min at 4°C with 5 µg of nuclear protein, 2 µg of poly(dT:dC), 100 mM KCl, 5% glycerol, 1 mM DTT, 5 mM MgCl₂, 10 mM Tris-HCl (pH 7.9), in a final volume of 20 µl. The DNA-protein complexes were resolved on nondenaturing 5% polyacrylamide gels in 0.5% Tris borate-EDTA running buffer as previously described. For super shift assays, nuclear extracts were incubated for 30 min at 4°C with 20 µg/ml Abs (anti-p50 and anti-p65) followed by EMSA. The gels were vacuum-dried and exposed to x-ray films followed by autoradiography.

Transient cotransfection studies

Transient cotransfection was performed according to the manufacturer’s instructions using a FuGENE 6 transfection kit (Roche Molecular Systems). Briefly, FuGENE 6 was mixed with the plasmid DNA and with annealed siRNA or scrRNA oligonucleotides at a ratio of 2:1. The mixture was incubated for 20 min at room temperature and then added to 70–80% confluence cells in six-well plates. After 6 h, cells were washed twice with RPMI 1640 and replaced in normal medium. After 24 h, cells were treated with or without Cyt-Mix and IL-4 for the next 6 h and the luciferase reporter assay was performed.

Luciferase reporter assays

Cells were lysed in lysis buffer and centrifuged at 12,000 x g in a microcentrifuge for 2 min at 4°C. The supernatant was transferred into a new tube and 20 µl of lysate was mixed with 100 µl of luciferase assay reagent in cuvettes for the illuminometer. The luciferase assay measurement was normalized by the protein amounts. The protein concentration was determined by Bradford protein assay (Bio-Rad) and used to normalize the luciferase enzyme activity in each sample.

Flow cytometry (FACS) analysis

Mixed glial cells were harvested after treatment by incubation in trypsin-EDTA (1x) solution (Invitrogen Life Technologies). Cells were washed and resuspended in PBS containing 3% BSA and incubated with 10 µg/ml nonimmune mouse IgG for 15 min. After washing, cells were incubated with 2 µg/ml mouse anti-PDGF-R IgG or rabbit anti-NG2 IgG or anti-O4 IgM diluted 1/100 in PBS containing 3% BSA at 4°C for 30 min. After washing, cells were incubated at 4°C for 30 min with PE-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) for PDGF-R, PE-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) for NG2, and FITC-conjugated goat anti-mouse IgM (Sigma-Aldrich) for O4 diluted at 1/200 and measured in a FL-1 channel (530 ± 15 nm bandpass filter). Cells were washed before analysis on a FACSCalibur flow cytometer (BD Biosciences) operating with the CellQuest software. Dead cells and debris were excluded from the analysis by gating living cells from size/structure density plots. Data were displayed on a logarithmic scale with increasing fluorescence intensity (data not shown). Each histogram plot was recorded for at least 10,000 gated events. The percentage of positive cells was plotted in each group.

Cytotoxicity assay

Cytotoxicity was evaluated by measuring the release of lactate dehydrogenase in the culture medium, using a commercial available kit (Roche Diagnostics).

Measurement of caspase-3 activity

For measurement of caspase-3 activity, treated cells were washed twice with ice-cold PBS, resuspended in DMEM (100 µl), and treated with the fluorometric caspase-3 assay system (Promega). In brief, caspase-3 substrate and homogeneous caspase buffer (100 µl) were mixed and added to the cells (100 µl) in 96-well plates. The fluorescence intensity was measured with a Bio-Tek FL600 microplate reader (Bio-Tek Instruments) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm every 15 min for 3 h.

Animals and LPS treatment

The use of animals was in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication number 86-23) and the protocol was approved by Medical University of South Carolina Institutional Animal Care and Use Committee. Female pregnant SD rats, and 12/15-LOX^–/– and wild-type (C57BL/6) mice (30–40 gm) were group housed at room temperature under 12 h light/dark conditions with food and water available ad labitum. Mice were injected with IL-4 (50 mg/kg, i.p.) 30 min before LPS administration (0.5 mg/kg, i.p.) dissolved in 0.9% saline or 0.9% sterile saline alone. After 10 h, the brain (cerebral-cortex) was isolated and frozen in liquid nitrogen and stored at –70°C until later use.

Statistical analysis

Using the Student’s unpaired t test and ANOVA (Student-Newman-Keuls, comparison of all pairs of columns), p-values were determined for real-time PCR analyzed data, luciferase reporter assay data, FACS analysis data, densitometer analysis data, and immunofluorescence intensity data in triplicate from three independent experiments using GraphPad software. A value of p < 0.05 was used as the criterion for statistical significance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
IL-4 attenuates the release of NO and protects differentiating OPs in activated CNS-mixed glial cell cultures

CNS-mixed glial cell cultures were treated with a mixture of proinflammatory cytokine (Cyt-Mix; TNF-, IFN-, and IL-1; 10 ng/ml each) proteins in the presence/absence of rIL-4 protein. Cyt-Mix-treated mixed glial cells produced NO, whereas pretreatment with IL-4 (15 ng/ml), 30 min before addition of Cyt-Mix, significantly (p < 0.001) reduced the levels of nitrite (a stable product of NO) in culture supernatants (Fig. 1A). This attenuation of NO release by IL-4 was concentration-dependent in Cyt-Mix-treated mixed glial cells and was reversed by anti-IL-4R Abs (Fig. 1B). Likewise, IL-4 significantly decreased nitrite in both Cyt-Mix-treated primary astrocytes (Fig. 1C) and microglia (Fig. 1D) compared with those treated with Cyt-Mix only. Parallel immunocytochemistry studies showed a significant increase in number of MBP⁺ oligodendrocytes by IL-4 in Cyt-Mix-treated mixed glial cells or controls compared with those treated with Cyt-Mix only (Fig. 1, E and F). Interestingly, IL-4 alone demonstrated a significant (p < 0.05) increase in MBP⁺ cells as compared with controls. Together, these data suggest that IL-4 treatment attenuates the release of NO in Cyt-Mix-treated CNS glial cells thereby protecting differentiating OPs.

View larger version (49K): [in this window] [in a new window]   FIGURE 1. IL-4 attenuates nitrite release and protects against degeneration of differentiating OPs. A, Mixed glial cells were treated with IL-4 (15 ng/ml) and cytokine mixture (Cyt-Mix) for 24 h, and nitrite levels were determined in cultured supernatants. Anti-IL-4R Abs were used to reverse the IL-4-mediated effects (B). The effect of increased IL-4 was determined by nitrite release in culture supernatants of mixed glial cells treated with Cyt-Mix. Purified primary astrocytes (C) and microglia (D) cells were also treated similarly with IL-4 and Cyt-Mix. E, Plot represents MBP+ cell counts per field in similarly treated mixed glial cells determined by manual counting as described in Materials and Methods. F, Representative slides shown are control (CTL) (a and b), IL-4 (c and d), Cyt-Mix (e and f), and IL-4 plus Cyt-Mix (g and h) show immunocytochemical staining for MBP (oligodendrocytes; red) and GFAP (astrocytes; green) in similarly treated mixed glial cells for 6 days posttreatment. Arrowhead indicates differentiating oligodendrocytes in similarly treated mixed glial cells (magnification, x400). Results are expressed as the mean ± SD from three independent experiments. Statistical significance is *, p < 0.05; **, p < 0.01; ***; p < 0.001 vs Cyt-Mix and #, p < 0.05; ###; p < 0.001 vs control.

To further elucidate the fate of differentiating OPs in IL-4 and Cyt-Mix-treated glial cell cultures, the cell population of oligodendrocyte lineages, i.e., NG2⁺ and O4⁺ was determined by FACS analysis, considering these lineage cells were previously reported to be more vulnerable to apoptotic cell death under hypoxic-ischemic conditions (36). FACS analysis revealed a significant increase in the percentage of NG2⁺ and O4⁺ cells by IL-4 in Cyt-Mix-treated mixed glial cells when compared with those treated with Cyt-Mix or controls (Fig. 2A). Notably, mixed glial cells treated with IL-4 alone had a significant increase in the percentage of O4⁺ cells as compared with untreated-controls; however, there was no significant change in the NG2⁺ cell percentage. There was a parallel increase in mRNA expression for PDGF-R (Fig. 2B) and SOX10 (Fig. 2C), as markers of proliferating OPs as well as PLP (Fig. 2D) and MyT1-L (Fig. 2E), as markers of mature oligodendrocytes by IL-4 in Cyt-Mix-treated mixed glial cells compared with those treated with Cyt-Mix only. Corresponding with the FACS analysis data, real-time PCR analysis revealed a significant increase in the expression of both PDGF-R and PLP mRNA in IL-4 alone treated mixed glial cells as compared with controls. Furthermore, IL-4 treatment significantly attenuated the induction of GFAP expression in Cyt-Mix-treated mixed glial cells as compared with those treated with Cyt-Mix only (Fig. 2F). Altogether, these data suggest that IL-4 protects differentiating OPs against Cyt-Mix-induced cytotoxic effects by attenuation of reactive astrogliosis and iNOS expression in activated CNS glial cells.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. IL-4 protects and increases the proliferation and maturation of OPs in activated CNS glial cells. Mixed glial cells were treated with IL-4 (15 ng/ml) and Cyt-Mix as described in Materials and Methods followed by FACS analysis after day 4 posttreatment. A, Both NG2+ and O4+ cell counts were significantly less in Cyt-Mix-treated mixed glial cells compared with those treated with IL-4 plus Cyt-Mix or controls. Real-time PCR analysis demonstrated mRNA expression for PDGF-R (B), SOX10 (C), PLP (D), MyT1-L (E), and GFAP (F) in similarly treated mixed glial cells after 48 h posttreatment. Plots demonstrate nitrite release (G), LDH release (H), and caspase-3 activity (I) in oligodendroglial MO3.13 cells treated similarly with IL-4 and Cyt-Mix. Results are expressed as the mean ± SD from three independent experiments. Statistical significance shows *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs Cyt-Mix and @, p < 0.05; @@, p < 0.01; @@@, p < 0.001 vs control or NS vs control.

IL-4 modulates Cyt-Mix-induced PI3K-Akt and PI3K-p38 MAPK pathways and iNOS expression in CNS glial cells

Previously, proinflammatory cytokine-induced increase in expression of iNOS has been shown to be mediated via activation of both PI3K-Akt-NF-B and PI3K-p38 MAPK-NF-B pathways in primary astrocytes (32, 38), next we determined the effect of IL-4 on the activation of these pathways in Cyt-Mix-treated mixed glial cells. First, we examined the activation of Akt and p38 MAPK proteins in IL-4 and Cyt-Mix-treated mixed glial cells. As expected, Cyt-Mix treatment of mixed glial cells demonstrated that the phosphorylation of Akt (i.e., phosphorylation of Akt protein at the serine residue) peaked after 30 min of Cyt-Mix treatment and p38 MAPK (i.e., phosphorylation of p38 MAPK protein) peaked after 60 min of Cyt-Mix treatment (Fig. 3A). Interestingly, the activation of these proteins induced by Cyt-Mix was not altered by IL-4 treatment of mixed glial cells. Next, we used specific inhibitors for Akt and p38 MAPK activation to determine at what stage IL-4 has an effect on the attenuation of iNOS expression. The presence of inhibitors for both Akt and p38 MAPK demonstrated abolition of NO release (Fig. 3B) and iNOS expression (Fig. 3C) in IL-4 plus Cyt-Mix-treated or Cyt-Mix-treated mixed glial cells compared with those treated with IL-4 plus Cyt-Mix or Cyt-Mix only. Similar to what was observed in IL-4 plus Cyt-Mix-treated mixed glial cells, MBP⁺ cell counts were significantly increased in IL-4 plus Cyt-Mix-treated or Cyt-Mix-treated mixed glial cells in the presence of both Akt and p38 MAPK inhibitors compared with those cells treated with Cyt-Mix alone (Fig. 3, D and E). There was a relative increase in reactive astrogliosis in Cyt-Mix-treated mixed glial cell cultures as revealed by GFAP immunostaining. There was also the characteristic stellate appearance of hypertrophic (anisomorphic) astrocytes as compared with those treated with IL-4 and Cyt-Mix in the presence of inhibitors of Akt or p38 MAPK, wherein GFAP⁺ astrocytes had more expanded and characteristically normal shape (isomorphic) (Fig. 3D). In addition, the quantification of immunofluorescence intensity for MBP and GFAP also corroborated these data (Fig. 3F). Evidently, GFAP intensity was significantly elevated with a parallel decrease in MBP intensity in Cyt-Mix-treated mixed glial cells. Conversely, IL-4 plus Cyt-Mix-treated cells in the presence or absence of Akt or p38 MAPK inhibitors demonstrated a significant increase in intensity for MBP with a parallel decrease in intensity for GFAP (Fig. 3F). Together, these data indicate that IL-4 inductive effects on the Cyt-Mix-induced iNOS expression may be downstream of the PI3K-Akt and PI3K-p38 MAPK pathways in activated primary CNS glial cells.

View larger version (74K): [in this window] [in a new window]   FIGURE 3. IL-4 modulates PI3K-Akt and MAPK pathways induced iNOS expression. Mixed glial cells were treated with IL-4 and Cyt-Mix in the presence or absence of inhibitors for Akt and p38 MAPK. A, Representative immunoblot demonstrates both phosphorylated (activated) and total proteins of Akt and p38 MAPK at different time points in similarly treated mixed glial cells. B, Plot depicts nitrite release in cultured supernatants of treated cells after 24 h posttreatment. C, Plot depicts iNOS mRNA expression in similarly treated mixed glial cells after 6 h posttreatment. D, Plot represents MBP+ cell counts per field in similarly treated mixed glial cells after day 6 posttreatment, determined by manual counting as described in Materials and Methods. E, Representative slides depict control (CTL) (a and b), Cyt-Mix (c and d), IL-4 (e and f), IL-4 plus Cyt-Mix (g and h), IL-4 plus Akt-inh (i and j), IL-4 plus Cyt-Mix+Akt-inh (k and l), Cyt-Mix plus Akt-inh (m and n), and Cyt-Mix plus p38 MAPK-inh (o and p) show immunocytochemical staining of similarly treated mixed glial cells after day 6 posttreatment for MBP (oligodendrocytes; red) and GFAP (astrocytes; green). Arrowhead indicates mature oligodendrocytes in treated and untreated mixed glial cells (magnification, x400). F, Plot depicts the quantification of immunofluorescence intensity for MBP and GFAP expressions in similarly treated mixed glial cells by Image-Pro Plus analysis as described in Materials and Methods. Results are expressed as the mean ± SD in three independent experiments. Statistical significance is *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs Cyt-Mix or NS vs Cyt-Mix and #, p < 0.05; ###, p < 0.001 vs control; and @@@, p < 0.001 vs other groups.

Activation of PPAR- mediates IL-4-induced effects in activated CNS glial cells

Next, we examined the possible involvement of PPAR- in IL-4-induced effects in activated mixed glial cells. For this, we used PPAR--specific antagonist, GW9662, to determine the involvement of PPAR- on the attenuation of iNOS expression. Interestingly, the presence of GW9662 abolished IL-4 inductive effects as indicated by the increase in levels of nitrite and expression of iNOS (mRNA and protein) in IL-4 plus Cyt-Mix-treated mixed glial cells (Fig. 4, A–C). Anti-IL-4R Abs reversed IL-4-mediated inhibition of iNOS expression and nitrite release in IL-4 plus Cyt-Mix-treated mixed glial cells, documenting the specificity of IL-4 effects (Fig. 4, A–C). The presence of GW9662 in Cyt-Mix-treated glial cells had a similar profile of iNOS expression and NO release, which was observed in mixed glial cells treated with Cyt-Mix only (Fig. 4, A–C). Double-immunostaining for expression of GFAP and iNOS proteins in treated primary astrocytes showed an increase in colocalization of iNOS and GFAP in Cyt-Mix-treated astrocytes compared with those treated with IL-4 plus Cyt-Mix (Fig. 4D). As expected, GW9662 reversed the IL-4-mediated attenuation of expression of GFAP and iNOS in IL-4 plus Cyt-Mix-treated astrocytes. Furthermore, the quantification of immunofluorescence intensity demonstrates a significant increase in GFAP and iNOS expression in Cyt-Mix-treated astrocytes as compared with IL-4 plus Cyt-Mix-treated cells or controls (Fig. 4E). Similar to what was observed in Cyt-Mix-treated astrocytes, the presence of GW9662 in IL-4 plus Cyt-Mix-treated astrocytes produced a significant increase in intensity for iNOS and GFAP. Furthermore, immunostaining of similarly treated mixed glial cells revealed weak MBP immunostaining and a significant decrease in the number of MBP⁺ oligodendrocytes in IL-4 plus Cyt-Mix-treated mixed glial cells in the presence of GW9662 compared with those treated with IL-4 plus Cyt-Mix or controls (Fig. 5, A and B). Next, the quantification of immunofluorescence intensity for MBP and GFAP revealed reversal of the IL-4-induced increase in MBP expression with parallel decrease in GFAP expression in IL-4 plus Cyt-Mix-treated mixed glial cells in the presence of GW9662 (Fig. 5C). Altogether, these data suggest that the down-regulation of Cyt-Mix-induced iNOS expression by IL-4 is mediated by PPAR- activation in CNS glial cells.

View larger version (66K): [in this window] [in a new window]   FIGURE 4. IL-4 inductive effects are mediated through activation of PPAR-. Mixed glial cells were treated with IL-4 and Cyt-Mix in the presence or absence of GW9662. A, Plot depicts nitrite levels in culture supernatants of treated mixed glial cells after 24 h posttreatment. B, Plot depicts iNOS mRNA expression in similarly treated mixed glial cells after 6 h posttreatment. Anti-IL-4R Abs were used to reverse IL-4 induced effects. C, Representative immunoblot shows the expression of iNOS protein in similarly treated astrocytes after 24 h posttreatment. D, Representative slides of similarly treated astrocytes after 24 h posttreatment demonstrate immunostaining for iNOS (red) and GFAP (green) expression as described in Materials and Methods. Arrowhead indicates double positive iNOS+/GFAP+ activated astrocytes. E, Plot shows the quantification of immunofluorescence intensity for iNOS and GFAP expression in similarly treated astrocytes by Image-Pro Plus analysis as described in Materials and Methods. The results are expressed in plots as the mean ± SD in three independent experiments. Statistical significance is *, p < 0.01; ***, p < 0.001 or NS vs Cyt-Mix.

View larger version (69K): [in this window] [in a new window]   FIGURE 5. Antagonist of PPAR- reverses IL-4-mediated survival of differentiating OPs. Mixed glial cells were treated with IL-4 and Cyt-Mix in the presence or absence of GW9662 and immunostained with anti-MBP and anti-GFAP Abs. A, Representative slides demonstrate both MBP+ (oligodendrocytes; red) and GFAP+ (astrocytes; green) cells in treated mixed glial cells after 6 days posttreatment. Arrowhead indicates mature oligodendrocytes in treated or untreated mixed glial cells. B, Plot represents MBP+ cell counts per field determined by manual counting as described in Materials and Methods in similarly treated mixed glial cells. C, Representative plot shows immunofluorescence intensity for GFAP and MBP expression in similarly treated mixed glial cells by Image-Pro Plus analysis as described in Materials and Methods. The results are expressed in plots as the mean ± SD in three independent experiments. Statistical significance is *, p < 0.05; **, p < 0.01; ***, p < 0.001 or NS vs Cyt-Mix and #, p < 0.05; ###, p < 0.001 vs control.

IL-4 induces a coordinate increase in the expression of PPAR- and 12/15-LOX in CNS glial cells

Next, we determined the status of mRNA expression for PPAR- and arachidonate 12/15-LOX, a key enzyme involved in the biosynthesis of PPAR- ligands (13-HODE and 15-HETE) in IL-4-treated glial cells. The expression of PPAR- mRNA was significantly elevated in both IL-4 and IL-4 plus Cyt-Mix-treated mixed glial cells as compared with those cells treated with Cyt-Mix or controls (Fig. 6A). Notably, this increase in expression of PPAR- in IL-4-treated cells was reversed by anti-IL-4R Abs, which is indicative of IL-4-induced effects in glial cells (Fig. 6A). However, no significant difference was observed in PPAR- expression between glial cells treated with Cyt-Mix or controls. Interestingly, IL-4-induced increase in expression of PPAR- was concentration-dependent (peaked at IL-4 concentration 20 ng/ml) in treated mixed glial cells (Fig. 6B). Similar to what was observed in mixed glial cells, the expression of PPAR- (mRNA and protein) was significantly elevated in IL-4-treated and IL-4 plus Cyt-Mix-treated primary microglia compared with Cyt-Mix-treated microglia or controls (Fig. 6, C and D). Likewise, PPAR- (mRNA and protein) expression was also elevated significantly (p < 0.001) in IL-4-treated and IL-4 plus Cyt-Mix-treated astrocytes compared with Cyt-Mix-treated astrocytes or controls (Fig. 6, E and F). The expression of PPAR- was significantly less in Cyt-Mix-treated primary microglia (Fig. 6, C and D) or astrocytes (Fig. 6, E and F) as compared with untreated microglia and astrocytes. Interestingly, parallel to PPAR- expression in mixed glial cells, the expression of 12/15-LOX mRNA was also increased significantly in IL-4-treated and IL-4 plus Cyt-Mix-treated astrocytes as compared with Cyt-Mix-treated astrocytes or controls (Fig. 6G). Together, these data show that IL-4 induces a coordinate increase in expression of both PPAR- and 12/15-LOX in activated CNS glial cells.

View larger version (29K): [in this window] [in a new window]   FIGURE 6. IL-4 increases the expression of PPAR- and 12/15-LOX expression in CNS glial cells. A, Mixed glial cells were treated with IL-4, anti-IL-4R Abs, and Cyt-Mix for 6 h and determined the expression of PPAR- mRNA by real-time PCR analysis. B, PPAR- mRNA expression in IL-4 treated cells with increasing concentrations in the presence of Cyt-Mix. Plots depict PPAR- mRNA expression (C) and densitometric analysis (D) of PPAR- protein in a representative immunoblot in similarly treated primary microglia. Likewise, plots depict PPAR- mRNA expression (E) and densitometric analysis (F) of PPAR- protein levels in a representative immunoblot in similarly treated astrocytes. G, Plot depicts mRNA expression of 12/15-LOX in treated or untreated astrocytes after 6 h posttreatment. Results are expressed as the mean ± SD in three independent experiments. D and F, Results are mean of two independent experiments. Significant difference is *, p < 0.05; **, p < 0.01; and ***, p < 0.001 vs control and ###, p < 0.001 vs Cyt-Mix.

IL-4-induced PPAR- activation inhibits the nuclear translocation of NF-B

To delineate the molecular mechanism of IL-4-mediated down-regulation of iNOS expression in activated CNS glial cells, the nuclear extracts of primary astrocytes treated with IL-4 and Cyt-Mix in the presence or absence of GW9662 were analyzed for NF-B transactivation by EMSA. As expected, a gel shift for NF-B subunit proteins, i.e., p65 and p50, was observed in Cyt-Mix-treated astrocytes, whereas IL-4 plus Cyt-Mix treatment of astrocytes showed weak intensity bands (Fig. 7, A and B). In contrast, the presence of GW9662 reversed these effects of IL-4 in Cyt-Mix-treated astrocytes to the levels similar to ones observed in astrocytes treated with Cyt-Mix alone, as indicative of IL-4-induced PPAR- activation and inhibition of NF-B transactivation. Immunoblots further supported the EMSA data and revealed an increase in nuclear translocation of NF-B (p50) protein with a parallel decrease in PPAR- protein in Cyt-Mix-treated primary astrocytes (Fig. 7C). In contrast, there was an increase in nuclear translocation of PPAR- protein with a corresponding decrease in NF-B (p50) protein in IL-4 plus Cyt-Mix-treated astrocytes (Fig. 7C). These data reveal that the reversal of IL-4-induced effects by GW9662 resulted in an increase in nuclear translocation of NF-B (p50) protein and decreased nuclear translocation of PPAR- in IL-4 plus Cyt-Mix-treated astrocytes. Next, double immunostaining of similarly treated astrocytes for PPAR- and GFAP was performed, which indicated an increase in nuclear translocation of PPAR- protein in both IL-4-treated and IL-4 plus Cyt-Mix-treated astrocytes (Fig. 7D). Absolutely none or weak PPAR- immunostaining was detected in both the cytoplasm and nucleus of astrocytes treated with Cyt-Mix and in controls. The cytoplasmic distribution of IB was decreased in Cyt-Mix-treated astrocytes as compared with IL-4 plus Cyt-Mix-treated astrocytes or controls (Fig. 7E). This distribution of IB protein was absent in the cytoplasm of astrocytes treated with Cyt-Mix indicating the subsequent degradation of IB possibly by phosphorylation and ubiquitination pathways. Taken together, these data show that IL-4-induced activation of PPAR- antagonizes the nuclear translocation of NF-B in activated CNS glial cells.

View larger version (44K): [in this window] [in a new window]   FIGURE 7. IL-4 inhibits the nuclear translocation of NF-B through PPAR- activation. A, Astrocytes were treated with IL-4 and Cyt-Mix for 2 h in the presence or absence of GW9662 followed by EMSA to NF-B proteins in nuclear extracts as described in Materials and Methods. B, The super gel shift assay was performed using anti-p50 and anti-p65 Abs. C, A representative immunoblot demonstrates the expression of p50 and PPAR- protein in nuclear extracts of similarly treated astrocytes. D, Representative slides demonstrate double immunostaining of IL-4 and Cyt-Mix treated astrocytes with anti-GFAP (green) and anti-PPAR- (red) Abs after 2 h posttreatment. Arrowhead indicates the distribution of PPAR- in nucleus of IL-4-treated astrocytes. E, Likewise, representative slides demonstrate immunostaining for IB in similarly treated astrocytes after 2 h posttreatment. Arrowhead indicates IB protein distribution in the cytoplasm of astrocytes.

Transient cotransfection studies corroborate PPAR--mediated effects of IL-4

Next, transient cotransfection of astrocytes with plasmids (pNFB-Luc and pTK-PPREx3-Luc), and siRNA/scrRNA for 12/15-LOX was performed to evaluate the role of IL-4-induced PPAR- activation and nuclear translocation of NF-B. Similar to what was observed in Cyt-Mix-treated astrocytes transiently transfected with pNF-B-Luc, the relative luciferase activity was significantly increased in IL-4 plus Cyt-Mix-treated primary astrocytes transiently cotransfected with pNF-B-Luc and siRNA for 12/15-LOX compared with those transfected with pNF-B-Luc only (Fig. 8A). In contrast, IL-4 plus Cyt-Mix-treated astrocytes transiently cotransfected with pNF-B-Luc and scrRNA 12/15-LOX (control oligonucleotides) demonstrated a similar profile of relative luciferase activity, which was observed in IL-4 plus Cyt-Mix-treated astrocytes transiently transfected with pNF-B-Luc only (Fig. 8A). These observations suggest a possible role of 12/15-LOX products (e.g., agonist of PPAR-) on the IL-4-induced modulation of NF-B transactivation in activated astrocytes. Furthermore, the relative luciferase activity was significantly decreased in IL-4 plus Cyt-Mix-treated astrocytes transiently cotransfected with pTK-PPREx3-Luc and siRNA for 12/15-LOX compared with similarly treated astrocytes transiently transfected with pTK-PPREx3-Luc only (Fig. 8B). In contrast, no significant difference was observed between IL-4 plus Cyt-Mix-treated astrocytes and controls transiently cotransfected with pTK-PPREx3-Luc and scrRNA 12/15-LOX or transfected with pTK-PPREx3-Luc only (Fig. 8B). The effect of 12/15-LOX siRNA was also evident in IL-4-treated astrocytes transiently cotransfected with pTK-PPREx3-Luc and siRNA for 12/15-LOX (Fig. 8B). Collectively, these data further validate our observations that IL-4-dependent regulation of iNOS expression is mediated through induction of PPAR- activation and inhibition of NF-B transactivation in activated CNS glial cells.

View larger version (44K): [in this window] [in a new window]   FIGURE 8. Transient cotransfection studies demonstrate PPAR--mediated effects of IL-4 in activated glial cells. A, Plot depicts relative luciferase activity in transiently cotransfected primary astrocytes with pNF-kB-Luc plasmid along with siRNA or scrRNA for 12/15-LOX followed by treatment with IL-4 and Cyt-Mix for 6 h as described in Materials and Methods. B, Likewise, plot depicts relative luciferase activity in transiently cotransfected primary astrocytes with pTK-PPREx3-Luc plasmid along with siRNA or scrRNA for 12/15-LOX followed by treatment with IL-4 and Cyt-Mix for 6 h as described in Materials and Methods. C, Plot demonstrates nitrite levels in 12/15-LOX–/– mixed glial cells after 24 h posttreatment with IL-4 and Cyt-Mix. IL-4 concentration was ranging from 0 to 50 ng/ml. D, Plot demonstrates PDGF-R+ and O4+ cell counts per field in similarly treated 12/15-LOX–/– mixed glial cells after 4 days posttreatment, as determined by FACS analysis described in Materials and Methods. Results are expressed as the mean ± SD in three independent experiments. Significant difference is *, p < 0.05; ***, p < 0.001 or NS vs Cyt-Mix and @@@, p < 0.001 vs control.

IL-4-induced, PPAR--mediated effects were evident in 12/15-LOX^–/– CNS glial cells

Next, to validate IL-4-induced, PPAR- mediated inhibition of iNOS expression and its effect on the survival of OPs, we used 12/15-LOX^–/– mixed glial cell cultures. In contrast to what was observed in rat mixed glial cells as well as in wild-type mice mixed glial cells (data not shown), similarly treated 12/15-LOX^–/– mice mixed glial cells demonstrated no attenuation of nitrite release in culture supernatants by IL-4 treatment in Cyt-Mix-treated cells (Fig. 8C). Even the dose-dependent effect of IL-4 observed in wild-type mice or rat mixed glial cells was absent in 12/15-LOX^–/– mice mixed glial cells. Furthermore, in contrast to the observed survival of oligodendrocyte lineage cells in IL-4 and Cyt-Mix-treated rat mixed glial cells or wild-type mixed glial cells (data not shown), no significant increase in survival of PDGF-R⁺ or O4⁺ cells was observed by IL-4 in Cyt-Mix-treated 12/15-LOX^–/– mice mixed glial cells compared with those treated with Cyt-Mix only (Fig. 8D). However, PDGF-R⁺ cell counts were still significantly increased at higher concentrations of IL-4 (15–20 ng/ml) compared with Cyt-Mix-treated cells, but without any significant change in O4⁺ cells. Altogether, these data corroborate that IL-4 induced increased synthesis of PPAR- ligands by increased expression of 12/15-LOX, resulting in activation of PPAR- in activated CNS glial cells and, hence, protection of differentiating OPs.

IL-4 inductive effects were evident in the brain of LPS-challenged C57BL/6 mice, but not in LPS-challenged 12/15-LOX^–/– mice

Because IL-4 exhibited anti-inflammatory properties in CNS glial cells (by PPAR--mediated inhibition of NF-B transactivation), it was of interest to examine the same effect of IL-4 in vivo. It is well established that expression of proinflammatory cytokines, such as TNF-, IL-1, and IFN-, can be induced by the i.p. injection of LPS in vivo (39). Although the LPS-induced neuroinflammatory response does not precisely resemble EAE pathology, it tends to induce similar inflammatory mediators in the brain used in in vitro system. Moreover, more severe EAE (worse clinical score) has been reported previously in 12/15-LOX^–/– mice compared with wild-type mice (40). Therefore, we examined the effect of IL-4 on the expression of TNF-, iNOS, and PPAR- in the brain of LPS-challenged wild-type (C57BL/6) and 12/15-LOX^–/– mice. As shown in Fig. 9, A and B, LPS administration significantly induced the expression of TNF- and iNOS in the brain of C57BL/6 mice, whereas IL-4 pretreatment attenuated the LPS-induced expression of these mediators in brain. Conversely, IL-4 had no effect on the attenuation of these mediators in the brain of LPS challenged 12/15-LOX^–/– mice (Fig. 9, A and B). IL-4 induced a significant increase in PPAR- expression in LPS-challenged C57BL/6 mice compared with untreated LPS-challenged C57BL/6 mice (Fig. 9C). On the contrary, no significant increase in PPAR- expression was observed in the brain of similarly treated 12/15-LOX^–/– mice (Fig. 9C). Notably, a significant increase in the expression of TNF- mRNA was observed in the brain of both C57BL/6 (wild-type) and 12/15-LOX^–/– mice treated with IL-4 alone compared with controls. Altogether these in vivo studies corroborated our in vitro observations, i.e., inhibition of proinflammatory cytokine-induced NF-B transactivation by IL-4 induced PPAR- activation is mediated by 12/15-LOX in CNS glial cells.

View larger version (20K): [in this window] [in a new window]   FIGURE 9. IL-4 inductive effects in the brain of LPS-challenged C57BL/6 and 12/15-LOX–/– mice. C57BL/6 and 12/15-LOX–/– mice were challenged with LPS and treated with IL-4 as described in Materials and Methods. Plots demonstrate mRNA expression of TNF- (A), iNOS (B), and PPAR- (C) in the brain of IL-4-treated and LPS-challenged mice after 10 h posttreatment. Results are expressed as the mean ± SD in three of five independent experiments. Significant difference is ***, p < 0.001 or NS vs LPS and #, p < 0.05; ##, p < 0.01; and ###, p < 0.001 or nonsignificant (ns) vs control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

The present study revealed the molecular mechanisms whereby IL-4 down-regulates proinflammatory cytokine-induced NF-B transactivation and iNOS expression in primary glial cells and thereby protects differentiating OPs under neuroinflammatory disease conditions. The observed increase in survival of differentiating OPs with parallel decrease in iNOS expression by IL-4 in proinflammatory cytokine-treated CNS glial cells are indicative of IL-4-induced anti-inflammatory effects and possible induction of a promyelinating environment in CNS glial cell cultures. Conditions used in this study revealed that IL-4-induced activation of PPAR- inhibits proinflammatory cytokine-induced iNOS expression mediated by both PI3K-Akt-NF-B and PI3K-p38 MAPK-NF-B pathways in CNS glial cells. Furthermore, IL-4 inductive effect was on downstream of both Akt and p38 MAPK signaling proteins in these pathways through inhibition of NF-B transactivation by activated PPAR- (Fig. 10). The administration of 15-deoxy-^12,14-PGJ₂, a PPAR- agonist before and at the onset of EAE, demonstrated a significant reduction of EAE severity (48), whereas in vitro studies revealed that 15-deoxy-^12,14-PGJ₂ inductive effects on the modulation of PI3K-Akt-NF-B pathway mediated expression of iNOS/NO release in primary astrocytes are independent of PPAR- activation (32). In contrast, our findings suggest that IL-4-induced activation of PPAR- by its natural ligands, e.g., 13-HODE and 15-HETE, synthesized by 12/15-LOX in activated glial cells is important to exert its effect. In accordance with such findings, there was coordinate induction of both PPAR- and 12/15-LOX expression in IL-4-treated CNS glial cells. Furthermore, IL-4 inductive effects were absent in similarly treated 12/15-LOX^–/– CNS glial cells, which supports the notion that IL-4-induced activation of PPAR- mediates its effect in activated primary glial cells. Earlier, IL-4 has been shown to regulate the expression of CD36 gene in macrophages by coordinate induction of PPAR- and 12/15-LOX (49, 50). Accordingly, our data substantiate that IL-4 inductive effects are mediated by PPAR- activation perhaps by increased synthesis of 13-HODE and 15-HETE with subsequent inhibition of NF-B transactivation in CNS glial cells under neuroinflammatory disease conditions.

View larger version (24K): [in this window] [in a new window]   FIGURE 10. Mechanism of action of IL-4-induced effects in glial cells. Proinflammatory cytokines induce iNOS expression or inflammatory mediator release by transactivation of NF-B via PI3K-Akt or MAPK pathways in glial cells. IL-4 induces the increase in expression of 12/15-LOX leading to increased synthesis of natural ligands (13-HODE and 15-HETE) for PPAR- activation, which in turn antagonize the transactivation of NF-B in activated glial cells.

Additionally, the observed down-regulation of GFAP (mRNA and protein) expression in proinflammatory cytokine-treated glial cells by IL-4 treatment indicates that IL-4 has the potential to attenuate reactive astrogliosis in the CNS. In reactive astrogliosis, astrocytes transform into a characteristic hypertrophic stellate shape (anisomorphic) and secrete inflammatory mediators including inflammatory cytokines, iNOS, and reactive oxidative species leading to tissue injury and development of a scar (55). In contrast, the exposure of astrocytes to mild levels of inflammatory cytokines transforms them into a pronounced stellate shape (isomorphic) and increases the production of many cytosolic enzymes, antioxidants, soluble trophic, and growth factors that enhance the survival of adjacent neurons and glial cells as well as coordinate tissue remodeling (56). The attenuation of reactive astrogliosis is important for induction of a promyelinating environment in the CNS of EAE animals during remission, as observed in lovastatin-treated EAE animals (33). IL-4 has been shown to inhibit the activation of astrocytes and induce secretion of neurotrophic growth factor in bacterial endotoxin-treated astrocyte cultures (57). In accordance with these findings, the present study indicates that IL-4 has a potential to attenuate reactive astrogliosis and induction of a promyelinating environment to augment the differentiation of OPs under neuroinflammatory disease conditions. Furthermore, IL-4 treatment also inhibited the degeneration of OLs against the cytotoxic effects of proinflammatory cytokines (Fig. 2, H and I). There was a significant increase in the expression of neurotrophic factors in similarly treated CNS glial cells as an indicator of IL-4-induced promyelinating environment in the CNS, which is important for proliferation and differentiation of OPs (data not shown). Interestingly, IL-4 alone demonstrated the enhanced differentiation of oligodendrocytes (MBP⁺) as compared with untreated CNS glial cells as well as the expression of TNF- in the brain of IL-4 treated 12/15-LOX^–/– or wild-type mice. However, the relationship between these two events is not understood at present.

These pleiotropic effects of IL-4 on CNS glial cells are in addition to its effect on the differentiation of Th0 cells to Th2 phenotype against Th1 phenotype responses during EAE recovery or remissions (23, 24). Th1 responses predominantly elicit IgG2a Abs, whereas Th2 responses produce higher levels of IgG1 in mice (58). IL-4 has been shown to promote switching to IgG1 production by B lymphocytes and inhibits the differentiation of Th1 phenotype immune cells (59). In addition, the treatment with immunomodulatory drugs such as Copaxone induced a shift toward Th2 phenotype response in MS patients and higher titers of IgG1 over IgG2 isotype Abs in patient sera thereby limit CNS demyelination (60). These studies indicate that IL-4 plays an important role in the attenuation of EAE/MS via modulation of both autoreactive T and B cells. However, the possible role of IL-4 on the secretion of anti-myelin/anti-MOG Abs, critical for aggravating CNS demyelination in an Ab-dependent form of EAE/MS (6, 7, 61), is presently not known.

In conclusion, the present study reveals the molecular mechanisms of anti-inflammatory effects of IL-4, i.e., activated PPAR- mediated inhibition of NF-B transactivation and iNOS expression in activated primary glial cells and thus survival of differentiating OPs under CNS neuroinflammatory disease conditions. These pleiotropic effects of IL-4 are mediated by 1) coordinate induction of PPAR- and 12/15-LOX expressions, 2) activated PPAR--mediated inhibition of NF-B transactivation, and 3) the attenuation of reactive astrogliosis thereby enhancing the survival of differentiating OPs in activated CNS glial cells (Fig. 10). Understanding the molecular mechanisms of IL-4-induced PPAR- activation and inhibition of NF-B transactivation and iNOS expression in CNS glial cells will be useful in designing additional therapeutic targets to treat MS and other CNS demyelinating diseases.

	Acknowledgments

 
We thank all members of our laboratory for valuable comments and help during the course of this study. We also thank Dr. Kamesh Aysolla, Joyce Brian, and Hope Terry for technical assistance. We especially thank Dr. Jennifer G. Schnellmann for critical reading of this manuscript.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported in part by the National Institutes of Health Grants NS-22576, NS-34741, NS-37766, AG-025307, NS-40810, C06-RR015455, and C06-RR018823.

² A.S.P. and M.K.P. contributed equally to this study.

³ Address correspondence and reprint requests to Dr. Avtar K. Singh, Department of Pediatrics, 504-D, Children Research Institute, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425. E-mail address: singhi{at}musc.edu

⁴ Abbreviations used in this paper: MS, multiple sclerosis; PPAR, peroxisome proliferator-activated receptor; EAE, experimental autoimmune encephalomyelitis; iNOS, inducible NO synthase; Cyt-Mix, proinflammatory cytokine mixture; 12/15-LOX, 12/15-lipoxygenase; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; PLP, proteolipid protein; PDGF-R, platelet-derived growth factor- receptor; OP, oligodendrocyte progenitor; GFAP, glial fibrillary acidic protein.

Received for publication July 15, 2005. Accepted for publication January 10, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Noseworthy, J. H., C. Lucchinetti, M. Rodriguez, B. G. Weinshenker. 2000. Multiple sclerosis. N. Engl. J. Med. 343: 938-952. [Free Full Text]
2. Lindert, R.-B., C. G. Haase, U. Brehm, C. Linington, H. Wekerle, R. Hohlfeld. 1999. Multiple sclerosis: B- and T-cell responses to the extracellular domain of the myelin oligodendrocyte glycoprotein. Brain 122: (Pt. 11):2089-2100. [Abstract/Free Full Text]
3. Sun, J. B., T. Olsson, W. Z. Wang, B. G. Xiao, V. Kostulas, S. Fredrikson, H. P. Ekre, H. Link. 1991. Autoreactive T and B cells responding to myelin proteolipid protein in multiple sclerosis and controls. Eur. J. Immunol. 21: 1461-1468. [Medline]
4. Kerlero de Rosbo, N., M. Hoffman, I. Mendel, I. Yust, J. Kaye, R. Bakimer, S. Flechter, O. Abramsky, R. Milo, A. Karni, A. Ben-Nun. 1997. Predominance of the autoimmune response to myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis: reactivity to the extracellular domain of MOG is directed against three main regions. Eur. J. Immunol. 27: 3059-3069. [Medline]
5. Genain, C. P., B. Cannella, S. L. Hauser, C. S. Raine. 1999. Identification of autoantibodies associated with myelin damage in multiple sclerosis. Nat. Med. 5: 170-175. [Medline]
6. Raine, C. S., B. Cannella, S. L. Hauser, C. P. Genain. 1999. Demyelination in primate autoimmune encephalomyelitis and acute multiple sclerosis lesions: a case for antigen-specific antibody mediation. Ann. Neurol. 46: 144-160. [Medline]
7. Linington, C., M. Bradl, H. Lassmann, C. Brunner, K. Vass. 1988. Augmentation of demyelination in rat acute allergic encephalomyelitis by circulating mouse monoclonal antibodies directed against a myelin/oligodendrocyte glycoprotein. Am. J. Pathol. 130: 443-454. [Abstract]
8. Schluesener, H. J., R. A. Sobel, C. Linington, H. L. Weiner. 1987. A monoclonal antibody against a myelin oligodendrocyte glycoprotein induces relapses and demyelination in central nervous system autoimmune disease. J. Immunol. 139: 4016-4021. [Abstract]
9. Prineas, J. W., J. S. Graham. 1981. Multiple sclerosis: capping of surface immunoglobulin G on macrophages engaged in myelin breakdown. Ann. Neurol. 10: 149-158. [Medline]
10. Storch, M. K., S. Piddlesden, M. Haltia, M. Iivanainen, P. Morgan, H. Lassmann. 1998. Multiple sclerosis: in situ evidence for antibody- and complement-mediated demyelination. Ann. Neurol. 43: 465-471. [Medline]
11. Chu, C. Q., S. Wittmer, D. K. Dalton. 2000. Failure to suppress the expansion of the activated CD4 T cell population in interferon -deficient mice leads to exacerbation of experimental autoimmune encephalomyelitis. J. Exp. Med. 192: 123-128. [Abstract/Free Full Text]
12. Selmaj, K., C. S. Raine. 1988. Tumor necrosis factor mediates myelin damage in organotypic cultures of nervous tissue. Ann. NY Acad. Sci. 540: 568-570. [Medline]
13. Akassoglou, K., J. Bauer, G. Kassiotis, M. Pasparakis, H. Lassmann, G. Kollias, L. Probert. 1998. Oligodendrocyte apoptosis and primary demyelination induced by local TNF/p55TNF receptor signaling in the central nervous system of transgenic mice: models for multiple sclerosis with primary oligodendrogliopathy. Am. J. Pathol. 153: 801-813. [Abstract/Free Full Text]
14. Takahashi, J. L., F. Giuliani, C. Power, Y. Imai, V. W. Yong. 2003. Interleukin-1 promotes oligodendrocyte death through glutamate excitotoxicity. Ann. Neurol. 53: 588-595. [Medline]
15. Ferrari, C. C., A. M. Depino, F. Prada, N. Muraro, S. Campbell, O. Podhajcer, V. H. Perry, D. C. Anthony, F. J. Pitossi. 2004. Reversible demyelination, blood-brain barrier breakdown, and pronounced neutrophil recruitment induced by chronic IL-1 expression in the brain. Am. J. Pathol. 165: 1827-1837. [Abstract/Free Full Text]
16. Sherwin, C., R. Fern. 2005. Acute lipopolysaccharide-mediated injury in neonatal white matter glia: role of TNF-, IL-1, and calcium. J. Immunol. 175: 155-161. [Abstract/Free Full Text]
17. Mason, J. L., K. Suzuki, D. D. Chaplin, G. K. Matsushima. 2001. Interleukin-1 promotes repair of the CNS. J. Neurosci. 21: 7046-7052. [Abstract/Free Full Text]
18. Vela, J. M., E. Molina-Holgado, A. Arévalo-Martín, G. Almazán, C. Guaza. 2002. Interleukin-1 regulates proliferation and differentiation of oligodendrocyte progenitor cells. Mol. Cell. Neurosci. 20: 489-502. [Medline]
19. Molina-Holgado, E., J. M. Vela, A. Arévalo-Martín, C. Guaza. 2001. LPS/IFN- cytotoxicity in oligodendroglial cells: role of nitric oxide and protection by the anti-inflammatory cytokine IL-10. Eur. J. Neurosci. 13: 493-502. [Medline]
20. Renno, T., V. Taupin, L. Bourbonnière, G. Verge, E. Tran, R. De Simone, M. Krakowski, M. Rodriguez, A. Peterson, T. Owens. 1998. Interferon- in progression to chronic demyelination and neurological deficit following acute EAE. Mol. Cell. Neurosci. 12: 376-389. [Medline]
21. Tran, E. H., H. Hardin-Pouzet, G. Verge, T. Owens. 1997. Astrocytes and microglia express inducible nitric oxide synthase in mice with experimental allergic encephalomyelitis. J. Neuroimmunol. 74: 121-129. [Medline]
22. Danilov, A. I., M. Andersson, N. Bavand, N. P. Wiklund, T. Olsson, L. Brundin. 2003. Nitric oxide metabolite determinations reveal continuous inflammation in multiple sclerosis. J. Neuroimmunol. 136: 112-118. [Medline]
23. Cua, D. J., D. R. Hinton, S. A. Stohlman. 1995. Self-antigen-induced Th2 responses in experimental allergic encephalomyelitis (EAE)-resistant mice: Th2-mediated suppression of autoimmune disease. J. Immunol. 155: 4052-4059. [Abstract]
24. Karpus, W. J., K. E. Gould, R. H. Swanborg. 1992. CD4⁺ suppressor cells of autoimmune encephalomyelitis respond to T cell receptor-associated determinants on effector cells by interleukin-4 secretion. Eur. J. Immunol. 22: 1757-1763. [Medline]
25. Racke, M. K., A. Bonomo, D. E. Scott, B. Cannella, A. Levine, C. S. Raine, E. M. Shevach, M. Rocken. 1994. Cytokine-induced immune deviation as a therapy for inflammatory autoimmune disease. J. Exp. Med. 180: 1961-1966. [Abstract/Free Full Text]
26. Bettelli, E., M. P. Das, E. D. Howard, H. L. Weiner, R. A. Sobel, V. K. Kuchroo. 1998. IL-10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of IL-10- and IL-4-deficient and transgenic mice. J. Immunol. 161: 3299-3306. [Abstract/Free Full Text]
27. Shaw, M. K., J. B. Lorens, A. Dhawan, R. DalCanto, H. Y. Tse, A. B. Tran, C. Bonpane, S. L. Eswaran, S. Brocke, N. Sarvetnick, et al 1997. Local delivery of interleukin 4 by retrovirus-transduced T lymphocytes ameliorates experimental autoimmune encephalomyelitis. J. Exp. Med. 185: 1711-1714. [Abstract/Free Full Text]
28. Liblau, R., L. Steinman, S. Brocke. 1997. Experimental autoimmune encephalomyelitis in IL-4-deficient mice. Int. Immunol. 9: 799-803. [Abstract/Free Full Text]
29. Miller, A., S. Shapiro, R. Gershtein, A. Kinarty, H. Rawashdeh, S. Honigman, N. Lahat. 1998. Treatment of multiple sclerosis with copolymer-1 (Copaxone): implicating mechanisms of Th1 to Th2/Th3 immune-deviation. J. Neuroimmunol. 92: 113-121. [Medline]
30. Molina-Holgado, E., A. Arévalo-Martín, S. Ortiz, J. M. Vela, C. Guaza. 2002. Theiler’s virus infection induces the expression of cyclooxygenase-2 in murine astrocytes: inhibition by the anti-inflammatory cytokines interleukin-4 and interleukin-10. Neurosci. Lett. 324: 237-241. [Medline]
31. Molina-Holgado, E., A. Arévalo-Martín, A. Castrillo, L. Boscá, J. M. Vela, C. Guaza. 2002. Interleukin-4 and interleukin-10 modulate nuclear factor B activity and nitric oxide synthase-2 expression in Theiler’s virus-infected brain astrocytes. J. Neurochem. 81: 1242-1252. [Medline]
32. Giri, S., R. Rattan, A. K. Singh, I. Singh. 2004. The 15-deoxy-12,14-prostaglandin J₂ inhibits the inflammatory response in primary rat astrocytes via down-regulating multiple steps in phosphatidylinositol 3-kinase-Akt-NF-B-p300 pathway independent of peroxisome proliferator-activated receptor . J. Immunol. 173: 5196-5208. [Abstract/Free Full Text]
33. Paintlia, A. S., M. K. Paintlia, M. Khan, T. Vollmer, A. K. Singh, I. Singh. 2005. HMG-CoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis. FASEB J. 19: 1407-1421. [Abstract/Free Full Text]
34. Paintlia, A. S., A. G. Gilg, M. Khan, A. K. Singh, E. Barbosa, I. Singh. 2003. Correlation of very long chain fatty acid accumulation and inflammatory disease progression in childhood X-ALD: implications for potential therapies. Neurobiol. Dis. 14: 425-439. [Medline]
35. Paintlia, A. S., M. K. Paintlia, A. K. Singh, R. Stanislaus, A. G. Gilg, E. Barbosa, I. Singh. 2004. Regulation of gene expression associated with acute experimental autoimmune encephalomyelitis by Lovastatin. J. Neurosci. Res. 77: 63-81. [Medline]
36. Back, S. A., B. H. Han, N. L. Luo, C. A. Chricton, S. Xanthoudakis, J. Tam, K. L. Arvin, D. M. Holtzman. 2002. Selective vulnerability of late oligodendrocyte progenitors to hypoxia-ischemia. J. Neurosci. 22: 455-463. [Abstract/Free Full Text]
37. Buntinx, M., M. Moreels, F. Vandenabeele, I. Lambrichts, J. Raus, P. Steels, P. Stinissen, M. Ameloot. 2004. Cytokine-induced cell death in human oligodendroglial cell lines. I. Synergistic effects of IFN- and TNF- on apoptosis. J. Neurosci. Res. 76: 834-845. [Medline]
38. Dukic-Stefanovic, S., J. Gasic-Milenkovic, W. Deuther-Conrad, G. Münch. 2003. Signal transduction pathways in mouse microglia N-11 cells activated by advanced glycation endproducts (AGEs). J. Neurochem. 87: 44-55. [Medline]
39. Giri, S., N. Nath, B. Smith, B. Viollet, A. K. Singh, I. Singh. 2004. 5-aminoimidazole-4-carboxamide-1--4-ribofuranoside inhibits proinflammatory response in glial cells: a possible role of AMP-activated protein kinase. J. Neurosci. 24: 479-487. [Abstract/Free Full Text]
40. Emerson, M. R., S. M. LeVine. 2004. Experimental allergic encephalomyelitis is exacerbated in mice deficient for 12/15-lipoxygenase or 5-lipoxygenase. Brain Res. 1021: 140-145. [Medline]
41. Wujek, J. R., C. Bjartmar, E. Richer, R. M. Ransohoff, M. Yu, V. K. Tuohy, B. D. Trapp. 2002. Axon loss in the spinal cord determines permanent neurological disability in an animal model of multiple sclerosis. J. Neuropathol. Exp. Neurol. 61: 23-32. [Medline]
42. Bjartmar, C., B. D. Trapp. 2003. Axonal degeneration and progressive neurologic disability in multiple sclerosis. Neurotox. Res. 5: 157-164. [Medline]
43. Lassmann, H., W. Brück, C. Lucchinetti. 2001. Heterogeneity of multiple sclerosis pathogenesis: implications for diagnosis and therapy. Trends Mol. Med. 7: 115-121. [Medline]
44. Ding, M., M. Zhang, J. L. Wong, N. E. Rogers, L. J. Ignarro, R. R. Voskuhl. 1998. Antisense knockdown of inducible nitric oxide synthase inhibits induction of experimental autoimmune encephalomyelitis in SJL/J mice. J. Immunol. 160: 2560-2564. [Abstract/Free Full Text]
45. Fenyk-Melody, J. E., A. E. Garrison, S. R. Brunnert, J. R. Weidner, F. Shen, B. A. Shelton, J. S. Mudgett. 1998. Experimental autoimmune encephalomyelitis is exacerbated in mice lacking the NOS2 gene. J. Immunol. 160: 2940-2946. [Abstract/Free Full Text]
46. Dalton, D. K., S. Wittmer. 2005. Nitric-oxide-dependent and independent mechanisms of protection from CNS inflammation during Th1-mediated autoimmunity: evidence from EAE in iNOS KO mice. J. Neuroimmunol. 160: 110-121. [Medline]
47. Barnett, M. H., J. W. Prineas. 2004. Relapsing and remitting multiple sclerosis: pathology of the newly forming lesion. Ann. Neurol. 55: 458-468. [Medline]
48. Diab, A., C. Deng, J. D. Smith, R. Z. Hussain, B. Phanavanh, A. E. Lovett-Racke, P. D. Drew, M. K. Racke. 2002. Peroxisome proliferator-activated receptor- agonist 15-deoxy-^12,1412,14-prostaglandin J₂ ameliorates experimental autoimmune encephalomyelitis. J. Immunol. 168: 2508-2515. [Abstract/Free Full Text]
49. Huang, J. T., J. S. Welch, M. Ricote, C. J. Binder, T. M. Willson, C. Kelly, J. L. Witztum, C. D. Funk, D. Conrad, C. K. Glass. 1999. Interleukin-4-dependent production of PPAR- ligands in macrophages by 12/15-lipoxygenase. Nature 400: 378-382. [Medline]
50. Ricote, M., J. S. Welch, C. K. Glass. 2000. Regulation of macrophage gene expression by the peroxisome proliferator-activated receptor-. Horm. Res. 54: 275-280. [Medline]
51. Delerive, P., P. Gervois, J. C. Fruchart, B. Staels. 2000. Induction of IB expression as a mechanism contributing to the anti-inflammatory activities of peroxisome proliferator-activated receptor- activators. J. Biol. Chem. 275: 36703-36707. [Abstract/Free Full Text]
52. Ruan, H., H. J. Pownall, H. F. Lodish. 2003. Troglitazone antagonizes tumor necrosis factor--induced reprogramming of adipocyte gene expression by inhibiting the transcriptional regulatory functions of NF-B. J. Biol. Chem. 278: 28181-28192. [Abstract/Free Full Text]
53. Bennett, B. L., R. Cruz, R. G. Lacson, A. M. Manning. 1997. Interleukin-4 suppression of tumor necrosis factor -stimulated E-selectin gene transcription is mediated by STAT6 antagonism of NF-B. J. Biol. Chem. 272: 10212-10219. [Abstract/Free Full Text]
54. Ohmori, Y., T. A. Hamilton. 2000. Interleukin-4/STAT6 represses STAT1 and NF-B-dependent transcription through distinct mechanisms. J. Biol. Chem. 275: 38095-38103. [Abstract/Free Full Text]
55. Norenberg, M. D.. 1994. Astrocyte responses to CNS injury. J. Neuropathol. Exp. Neurol. 53: 213-220. [Medline]
56. Liberto, C. M., P. J. Albrecht, L. M. Herx, V. W. Yong, S. W. Levison. 2004. Pro-regenerative properties of cytokine-activated astrocytes. J. Neurochem. 89: 1092-1100. [Medline]
57. Brodie, C., N. Goldreich, T. Haiman, G. Kazimirsky. 1998. Functional IL-4 receptors on mouse astrocytes: IL-4 inhibits astrocyte activation and induces NGF secretion. J. Neuroimmunol. 81: 20-30. [Medline]
58. Hooper, D. C., K. Morimoto, M. Bette, E. Weihe, H. Koprowski, B. Dietzschold. 1998. Collaboration of antibody and inflammation in clearance of rabies virus from the central nervous system. J. Virol. 72: 3711-3719. [Abstract/Free Full Text]
59. Karulin, A. Y., M. D. Hesse, H. C. Yip, P. V. Lehmann. 2002. Indirect IL-4 pathway in type 1 immunity. J. Immunol. 168: 545-553. [Abstract/Free Full Text]
60. Brenner, T., R. Arnon, M. Sela, O. Abramsky, Z. Meiner, R. Riven-Kreitman, N. Tarcik, D. Teitelbaum. 2001. Humoral and cellular immune responses to Copolymer 1 in multiple sclerosis patients treated with Copaxone. J. Neuroimmunol. 115: 152-160. [Medline]
61. von Büdingen, H.-C., S. L. Hauser, J.-C. Ouallet, N. Tanuma, T. Menge, C. P. Genain. 2004. Frontline: epitope recognition on the myelin/oligodendrocyte glycoprotein differentially influences disease phenotype and antibody effector functions in autoimmune demyelination. Eur. J. Immunol. 34: 2072-2083. [Medline]

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