Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear receptor transcription factor that regulates cell growth, differentiation, and homeostasis. PPARγ agonists are potent therapeutic agents for type 2 diabetes, obesity, and inflammation. Experimental allergic encephalomyelitis (EAE) is a Th1 cell-mediated inflammatory demyelinating autoimmune disease model of multiple sclerosis. We have shown recently that PPARγ agonists inhibit EAE by blocking IL-12 production, IL-12 signaling, and neural Ag-induced Th1 differentiation. In this study, we show that the PPARγ-deficient heterozygous mice develop an exacerbated EAE with prolonged clinical symptoms than the wild-type littermates, following immunization with myelin oligodendrocyte glycoprotein (MOG) p35–55 peptide. The exacerbation of EAE in PPARγ+/− mice associates with an increased expansion of CD4+ and CD8+ T cells and expression of CD40 and MHC class II molecules in response to MOGp35–55 Ag. The PPARγ+/− mice also showed an increase in T cell proliferation and Th1 response to MOGp35–55 Ag than the wild-type littermates. These findings suggest that PPARγ be a critical physiological regulator of CNS inflammation and demyelination in EAE and perhaps multiple sclerosis and other Th1 cell-mediated autoimmune diseases.

Multiple sclerosis (MS) 3 is an inflammatory demyelinating autoimmune disease of the CNS that afflicts about one million people worldwide (1, 2, 3). Approximately 30% of MS patients develop clinical paralysis and become wheelchair bound for the rest of their lives (4). The disease usually affects young adults and women more frequently than men (5, 6). Although the etiology of MS is not known, it is generally viewed as an autoimmune inflammatory disease, mediated by myelin-reactive T cells in the CNS (7). Activation of immune cells, secretion of inflammatory cytokines, and differentiation of encephalitogenic Th1 cells are key processes associated with the pathogenesis of MS (3, 4, 5, 6, 7). Although destruction of oligodendrocyte myelin sheath in the CNS is the pathological hallmark of MS, axonal degeneration contributes to irreversible long-term disability (7, 8, 9). Experimental allergic encephalomyelitis (EAE) is an inflammatory demyelinating autoimmune disease of the CNS (10, 11). EAE can be induced by immunization with neural Ags such as myelin basic protein, myelin oligodendrocyte glycoprotein (MOG), or proteolipid protein and by adoptive transfer of neural Ag-sensitized T cells in susceptible rodents and primates. The clinical and pathological features of EAE show close similarity to human MS, and therefore have been commonly used as a model system to study the mechanism of MS pathogenesis and to test the efficacy of potential therapeutic agents for the treatment of MS (10, 11, 12, 13).

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent nuclear hormone receptor transcription factors that play critical roles in cell growth, differentiation, and homeostasis (14, 15). PPARα, δ, and γ are the three PPAR subtypes, encoded by distinct genes (16). PPARγ1, PPARγ2, and PPARγ3 are three isoforms of PPARγ that arise as products of different promoter usage and alternate splicing. Although PPARγ2 is expressed predominantly in adipose tissue, PPARγ1 and PPARγ3 are expressed in adipose tissue, heart, kidney, pancreas, spleen, intestine, colon epithelial cells, and skeletal muscle (17). The targeted disruption of PPARγ is embryonically lethal, and the mice die by day E10 due to defects in the development of placental, cardiac, and adipose tissue (18). Several fatty acids and eicosanoids including 9-hydroxyoctadecadienoic acid and 13-hydroxyoctadecadienoic acid function as physiological ligands for PPARγ (19, 20). The 15-deoxy Δ12,14 PGJ2 (15d-PGJ2) is a natural ligand for PPARγ, and thiazolidinediones are high affinity synthetic agonists for PPARγ (21, 22). PPARγ is an important regulator of adipogenesis, and PPARγ agonists prevent obesity (22, 23). PPARγ regulates glucose metabolism, and PPARγ agonists modulate insulin sensitivity, thereby reducing plasma glucose and insulin levels in human and animal models of type 2 diabetes (24, 25). The PPARγ agonists, troglitazone (rezulin), pioglitazone (ACTOS), and rosiglitazone (avandia), have been used as therapeutic agents for type 2 diabetes. PPARγ agonists inhibit malignant growth of liposarcoma (26), breast cancer (27), myeloid leukemia (28), lung cancer (29), and colon cancer (30, 31), suggesting their use in the treatment of cancer. Interestingly, recent studies have also shown that PPARγ is an important regulator of immune and inflammatory responses (32, 33). In vivo treatment with PPARγ agonists inhibits rheumatoid arthritis (34), atherosclerosis (35, 36), colitis (37), and psoriasis (38) in animal models, indicating their use for the treatment of human inflammatory diseases (39, 40).

We and others have shown recently that PPARγ agonists inhibit CNS inflammation and demyelination in EAE, implying their use in the treatment of MS (41, 42, 43, 44). Our studies further showed that the inhibition of EAE by PPARγ agonists was associated with the blockade of IL-12 production, IL-12 signaling, and neural Ag-specific Th1 differentiation (40). However, the physiological role of PPARγ in the regulation of inflammation and demyelination in EAE/MS was not known. In this study, we show that the PPARγ-deficient heterozygous (PPARγ+/−) mice develop an exacerbated EAE in association with an augmented autoimmune Th1 response than the wild-type littermates, suggesting a critical physiological role for PPARγ in the regulation of EAE and perhaps MS and other Th1 cell-mediated autoimmune diseases.

The generation of PPARγ+/− mice has been described elsewhere (18). Genotypes were determined by PCR of tail DNA (18). The age-matched female wild-type (PPARγ+/+) and PPARγ+/− offsprings were used in the experiments. The mice were maintained in the animal care facility at Vanderbilt University Medical Center. All the animal use and experimental protocols were approved by the Vanderbilt University Institutional Animal Care and Use Committee.

Recombinant murine IL-12, IFN-γ, and anti-IL-12 mAb419 (anti-IL-12p70) were purchased from R&D Systems (Minneapolis, MN). Anti-IL-12 mAb C15.6 (anti-IL-12p40) was prepared from hybridoma cells kindly provided by G. Trinchieri (Wistar Institute, Philadelphia, PA) and conjugated with biotin according to standard protocol. The anti-IFN-γ mAb, R4-6A2, was purified from ascitic fluid collected from nude mice following transplantation of R4-6A2 hybridoma cells (ATCC HB 170; American Type Culture Collection, Manassas, VA). The anti-IFN-γ mAb, MM700, was obtained from Endogen (Woburn, MA) and conjugated with biotin according to standard protocol. Anti-CD40 and anti-MHC class II mAbs were obtained from BioScience (San Diego, CA). The 21-aa peptide corresponding to the published sequence of mouse MOGp35–55 (MEVGWYRSPFSRVVHLYRNGK) was obtained from ResGen (Invitrogen, Huntsville, AL).

EAE was induced in 4- to 6-wk-old female PPARγ+/− or wild-type (C57BL/6) mice by immunization (s.c.) with 200 μg MOGp35–55 in a 150-μl emulsion of IFA containing 50 μg/ml Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI) in the lower dorsum on days 0 and 7. The mice received (i.p.) 100 ng of pertussis toxin (Sigma-Aldrich, St. Louis, MO) on days 0 and 2. The clinical symptoms were scored on every day from day 0 to 32 in a blinded manner, as follows; 0, normal; 0.5, stiff tail; 1, limp tail; 1.5, limp tail with inability to right; 2, paralysis of one limb; 2.5, paralysis of one limb and weakness of one other limb; 3, complete paralysis of both hind limbs; 4, moribund; 5, death (41). Mean clinical score (MCS) was calculated by adding everyday clinical score for all mice in a group and then dividing by total number of mice. Mean maximum clinical score (MMCS) is the MCS at the peak of disease. Average MCS (AMCS) was calculated by adding the MCS for all days (from days 0 to 32) and then dividing by 32, the total number of days. AMCS provides valuable information on experiments like this in which the animals develop an exacerbated and prolonged disease. The mean clinical score more than 1 (MCS >1) was obtained by counting the number of days with MCS more than 1.

The degrees of inflammation and demyelination in the CNS of mice with EAE were determined by histological analysis. The wild-type and PPARγ+/− mice induced to develop EAE were euthanized on day 25 (at the peak of disease) by CO2 asphyxiation and perfused by intracardiac infusion of 4% paraformaldehyde and 1% glutaraldehyde in PBS. Brain and spinal cord samples were removed and fixed in zinc-buffered formalin. Tissues were processed, and transverse sections from cervical, upper thoracic, lower thoracic, and lumbar regions of the spinal cord were stained with Luxol Fast Blue or H&E. Inflammation and demyelination in the CNS were assessed under microscope in a blinded manner. The spinal cord sections were viewed as anterior, posterior, and two lateral columns (four quadrants). Each quadrant displaying the infiltration of mononuclear cells or loss of myelin was assigned a score of one inflammation or one demyelination, respectively. Thus, each animal has a potential maximum score of 16, and this study represents the analysis of spinal cord from 10 mice per group. The pathological score from each group is expressed as percentage of positive over total number of quadrants examined (41).

The spleen cells from PPARγ+/− and wild-type mice were analyzed for the presence of CD4-, CD8-, MHC class II-, or CD40-positive cells by immunostaining and flow cytometry. The spleen cells were isolated from PPARγ+/− and wild-type mice before and after immunization with MOGp35–55 Ag. The freshly isolated spleen cells and those cultured with 25 μg/ml MOGp35–55 Ag for 4 days were washed in cold PBS and incubated with PBS/1% BSA containing Abs specific to CD4, CD8, MHC class II, or CD40, conjugated with FITC or PE at 4°C for 1 h. The cells were then washed in cold PBS and analyzed by flow cytometry. The isotype-matched secondary Abs conjugated with FITC or PE were used as negative controls.

The proliferative response of splenic T cells to neural Ags was determined by [3H]thymidine incorporation assay. The spleen cells were isolated from PPARγ+/− and wild-type mice on days 0, 7, 14, 21, 28, and 35 after immunization with MOGp35–55 Ag. The cells were cultured in 96-well tissue culture plates in RPMI 1640 medium containing 10% FBS (2 × 105/200 μl/well) in the presence of 0, 10, 25, or 50 μg/ml MOGp35–55 Ag. [3H]Thymidine (0.5 μCi/ml) was added at 72 h, and the uptake of radiolabel was measured after 96 h by Wallac beta plate scintillation counter (41).

The spleen cells from MOGp35–55-sensitized PPARγ+/− and wild-type mice were cultured in 24-well plates in RPMI 1640 medium (5 × 105/ml) in the absence or presence of 25 μg/ml MOGp35–55 Ag. The culture supernatants were collected after 48 h, and the levels of IL-12 and IFN-γ were measured by ELISA, as described earlier (41). Briefly, 96-well ELISA plates were coated with 2 μg/ml anti-IL-12 mAb, 419, anti-IFN-γ mAb, or R4-6A2 capture Abs in 100 μl/well bicarbonate buffer, pH 9.3. After overnight incubation at 4°C, the excess Abs were washed off, and the residual binding sites were blocked by the addition of 3% BSA in PBS for 1 h. The test samples (culture supernatants) and standards (rIL-12 or rIFN-γ) were added and incubated overnight at 4°C. Plates were washed with PBS containing 0.05% Tween 20 and 0.2 μg/ml biotin-conjugated anti-IL-12 mAb, C15.6, or anti-IFN-γ mAb, MM700, added as detection Ab. After incubation at room temperature for 1 h, the plates were washed three times, and avidin-alkaline phosphatase was added, followed by 1 mg/ml of p-nitrophenyl phosphate. After a 30-min incubation at room temperature, the absorbance was read at 405 nm and the concentration of IL-12 or IFN-γ in the culture supernatants was calculated from standard curve.

All experiments were repeated at least three times, and the values are expressed as mean ± SD. The differences in clinical and pathological scores between wild-type and PPARγ+/− mice were analyzed using nonparametric Mann-Whitney testing. The differences in immune responses between wild-type and PPARγ+/− mice were analyzed by parametric Student’s t test. The values of p < 0.05 were considered significant.

To define the role of PPARγ in the regulation of CNS inflammation and demyelination, we first examined the pathogenesis of EAE in PPARγ-deficient heterozygous mice. Four- to 6-wk-old female PPARγ+/− and wild-type mice were induced to develop EAE by active immunization with MOGp35–55. As shown in Fig. 1, the PPARγ+/− mice developed a more exacerbated EAE with prolonged clinical paralysis than the wild-type littermates. In the early phase of EAE, the wild-type and heterozygous mice showed only a marginal difference in the day of onset and MCS of the disease. However, in the later phase of EAE, while the wild-type mice recovered, the PPARγ+/− mice continued to progress to a state of more severe clinical paralysis. Further analyses showed that the wild-type mice developed a MMCS of 1.5 with an AMCS of 0.59 and MCS >1 for 10 days with an ending MCS of 0.2 on day 32. Conversely, the PPARγ+/− mice displayed an MMCS of 2.4 (1.6-fold increase, p < 0.05) with an AMCS of 1.26 (2.14-fold increase, p < 0.01) and MCS >1 for 22 days (2.2-fold increase, p < 0.01) with an ending MCS of 1.85 on day 32 (9.25-fold increase, p < 0.001). These results suggested that the PPARγ+/− mice develop an exacerbated EAE with prolonged clinical paralysis and an impaired remission than the wild-type littermates.

FIGURE 1.

Development of exacerbated EAE in PPARγ+/− mice than the wild-type littermates. PPARγ+/− mice and the wild-type littermates were induced to develop EAE by immunization with MOGp35–55, as described in Materials and Methods, and the clinical symptoms were scored everyday in a blinded manner. The MCS of all 10 mice per group from one experiment is shown. The figure is a representative of four independent experiments.

FIGURE 1.

Development of exacerbated EAE in PPARγ+/− mice than the wild-type littermates. PPARγ+/− mice and the wild-type littermates were induced to develop EAE by immunization with MOGp35–55, as described in Materials and Methods, and the clinical symptoms were scored everyday in a blinded manner. The MCS of all 10 mice per group from one experiment is shown. The figure is a representative of four independent experiments.

Close modal

We then examined whether the clinical symptoms of EAE in PPARγ+/− mice associate with an increased pathology of inflammation and demyelination in the CNS. Spinal cord sections from PPARγ+/− mice and wild-type littermates induced to develop EAE were analyzed for the myelin loss (demyelination) and infiltration of mononuclear cells (inflammation) in the CNS. As shown in Fig. 2, A and B, the PPARγ+/− mice showed an extensive demyelination and inflammation in the CNS than the wild-type littermates. Although the wild-type mice showed 45.5% demyelination, the PPARγ+/− mice showed 76% (1.67-fold increase, p < 0.01) demyelination in the CNS (Fig. 2, A and C). Similarly, while the wild-type mice showed 38.75% inflammation, the PPARγ+/− mice showed 67.25% (1.74-fold increase, p < 0.01) inflammation in the CNS (Fig. 2, B and C). No inflammation or demyelination was observed in the CNS of wild-type or PPARγ+/− mice that were not induced to develop EAE. These results suggest that the pathology of CNS inflammation and demyelination correlates with the clinical severity of EAE in wild-type and PPARγ+/− mice.

FIGURE 2.

Pathology of spinal cord sections from wild-type and PPARγ+/− mice induced to develop EAE. The spinal cord sections (four sections per mouse) were prepared from wild-type and PPARγ+/− control mice not induced to develop EAE (WT/CONT and KO/CONT) or mice with EAE (WT/EAE and KO/EAE) at the peak of disease. The representative pictures of demyelination (A) and inflammation (B) were taken under phase-contrast microscope (×20). The spinal cord sections were scored for the presence of demyelination or inflammation in a blinded manner, and the pathological scores are expressed as percentage of positive spinal cord quadrants over total number of quadrants examined (C). The average number of quadrants examined per mouse was 16, and the number of mice per group analyzed was 10; therefore, this study included the analysis of 160 spinal cord quadrants per group.

FIGURE 2.

Pathology of spinal cord sections from wild-type and PPARγ+/− mice induced to develop EAE. The spinal cord sections (four sections per mouse) were prepared from wild-type and PPARγ+/− control mice not induced to develop EAE (WT/CONT and KO/CONT) or mice with EAE (WT/EAE and KO/EAE) at the peak of disease. The representative pictures of demyelination (A) and inflammation (B) were taken under phase-contrast microscope (×20). The spinal cord sections were scored for the presence of demyelination or inflammation in a blinded manner, and the pathological scores are expressed as percentage of positive spinal cord quadrants over total number of quadrants examined (C). The average number of quadrants examined per mouse was 16, and the number of mice per group analyzed was 10; therefore, this study included the analysis of 160 spinal cord quadrants per group.

Close modal

To define the mechanisms involved in the exacerbation of EAE in PPARγ+/− mice, we first analyzed the spleen cell population by flow cytometry. As shown in Fig. 3,A, the naive wild-type mice displayed 23% CD4+ T cells in the spleen that decreased to 22% after immunization with MOGp35–55 and increased to 29% (1.26-fold increase) following in vitro culture of immune spleen cells with 25 μg/ml MOGp35–55 Ag for 4 days, whereas the naive PPARγ+/− mice showed only 17% CD4+ T cells that increased to 22% following immunization with MOGp35–55 and further increased to 37% (2.18-fold increase) after in vitro culture of immune spleen cells with 25 μg/ml MOGp35–55 Ag for 4 days. These results suggested that despite a 26% reduction in naive PPARγ+/− mice, the CD4+ T cells increased to the level of 27.6% (p < 0.01) higher than wild-type littermates following in vivo and in vitro sensitization with MOGp35–55 Ag. We have further examined whether such a difference was also present in CD8+ T cell population. As shown in Fig. 3 B, the naive wild-type mice showed 14% CD8+ T cells that reduced to 12% following immunization and increased to 18% (1.29-fold increase) after in vitro culture of immune spleen cells with 25 μg/ml MOGp35–55 Ag for 4 days, whereas the naive PPARγ+/− mice showed only 9% CD8+ T cells that increased to 19% following immunization and 47% (5.22-fold increase) after in vitro culture of immune cells with 25 μg/ml MOGp35–55 Ag for 4 days. These results suggested that despite a 35.7% reduction in naive PPARγ+/− mice, the CD8+ T cells expanded to the level of 161% (p < 0.001) higher than wild-type littermates following in vivo and in vitro sensitization with MOGp35–55 Ag.

FIGURE 3.

Flow cytometry analyses of CD4- and CD8-positive spleen cells in PPARγ+/− and wild-type mice with EAE. Spleen cells were isolated from wild-type and PPARγ+/− mice before (day 0) or after induction of EAE by immunization with MOGp35–55 Ag (day 14) and cultured in the presence of 25 μg/ml MOGp35–55 Ag for 4 days. The freshly isolated and cultured spleen cells were stained with FITC-conjugated anti-CD4 (A) or anti-CD8 (B) Ab and analyzed by flow cytometry. The viable cells with log fluorescence intensities >102 were gated as CD4- or CD8-positive T cells. The figures are representatives of three independent experiments.

FIGURE 3.

Flow cytometry analyses of CD4- and CD8-positive spleen cells in PPARγ+/− and wild-type mice with EAE. Spleen cells were isolated from wild-type and PPARγ+/− mice before (day 0) or after induction of EAE by immunization with MOGp35–55 Ag (day 14) and cultured in the presence of 25 μg/ml MOGp35–55 Ag for 4 days. The freshly isolated and cultured spleen cells were stained with FITC-conjugated anti-CD4 (A) or anti-CD8 (B) Ab and analyzed by flow cytometry. The viable cells with log fluorescence intensities >102 were gated as CD4- or CD8-positive T cells. The figures are representatives of three independent experiments.

Close modal

To further understand the mechanisms in the development of exacerbated EAE in PPARγ+/− mice, we examined the expression of MHC class II in spleen cells. As shown in Fig. 4,A, the naive wild-type mice showed 62% MHC class II-positive cells in the spleen that increased to 74% (1.19-fold increase) following immunization with MOGp35–55 Ag, as described in Materials and Methods, whereas the naive PPARγ+/− mice showed 78% MHC class II-positive cells in the spleen that increased to 85% (1.15-fold increase) following immunization with MOGp35–55 Ag. However, when compared with wild-type littermates, the naive and MOGp35–55-immune PPARγ+/− mice respectively showed 25.8% (p < 0.01) and 14.9% (p < 0.01) more MHC class II+ spleen cells than the wild-type littermates. We have also examined the expression of CD40 in spleen cells by flow cytometry. As shown in Fig. 4 B, the naive wild-type mice showed 62% spleen cells positive for CD40 that decreased to 52% following immunization with MOGp35–55 Ag, whereas the naive PPARγ+/− mice showed 69% CD40+ spleen cells that increased to 75% following immunization with MOGp35–55 Ag. When compared with wild-type littermates, the naive and MOGp35–55-immune PPARγ+/− mice respectively showed 11.3% (p < 0.05) and 44.2% (p < 0.01) more CD40+ spleen cells than the wild-type mice. These results suggested that the increased expression of MHC class II and CD40 molecules contributes to the development of exacerbated EAE in PPARγ+/− mice.

FIGURE 4.

Flow cytometry analyses of MHC class II and CD40 expression in spleen cells from wild-type and PPARγ+/− mice with EAE. Spleen cells were isolated from wild-type and PPARγ+/− mice before (day 0) or after induction of EAE by immunization with MOGp35–55 (day 14), stained with PE-conjugated anti-MHC class II (A) or anti-CD40 (B) Ab, and analyzed by flow cytometry. The viable cells with log fluorescence intensities >102 were gated as MHC class II-positive cells, and those with fluorescence intensities 101 were gated as CD40-positive cells. The figures are representatives of three independent experiments.

FIGURE 4.

Flow cytometry analyses of MHC class II and CD40 expression in spleen cells from wild-type and PPARγ+/− mice with EAE. Spleen cells were isolated from wild-type and PPARγ+/− mice before (day 0) or after induction of EAE by immunization with MOGp35–55 (day 14), stained with PE-conjugated anti-MHC class II (A) or anti-CD40 (B) Ab, and analyzed by flow cytometry. The viable cells with log fluorescence intensities >102 were gated as MHC class II-positive cells, and those with fluorescence intensities 101 were gated as CD40-positive cells. The figures are representatives of three independent experiments.

Close modal

To further define the mechanisms involved in the exacerbation of EAE in PPARγ+/− mice, we examined the neural Ag-induced proliferation of splenic T cells isolated from wild-type and PPARγ+/− mice on different days following immunization with MOGp35–55. As shown in Fig. 5, MOGp35–55-sensitized T cells isolated from PPARγ+/− and wild-type mice on days 7, 14, 21, 28, and 35 displayed a dose-dependent increase in proliferative response to MOGp35–55 Ag. When compared with wild-type mice, the spleen cells from PPARγ+/− mice showed a significant increase in proliferation at 10, 25, and 50 μg/ml doses of MOGp35–55 Ag. At 50 μg/ml MOGp35–55, the spleen cells from wild-type mice showed 1.2-, 12.1-, 8.8-, 3.8-, 1.9-, and 1.3-fold increase in proliferation from background on days 0, 7, 14, 21, 28, and 35, respectively, whereas stimulation of spleen cells from PPARγ+/− mice with 50 μg/ml MOGp35–55 resulted in 1.2-, 28.5-, 17.7-, 4.7-, 2.5-, and 2.0-fold increase in proliferation from the background on days 0, 7, 14, 21, 28, and 35, respectively. When compared with wild-type mice, the increase in proliferation of splenic T cells from PPARγ+/− mice was 0-, 16.4-, 8.9-, 0.9-, 0.6-, and 0.7-fold higher than the wild-type mice on respective days. This increase in T cell proliferation was statistically significant (p < 0.01) on days 7, 14, 21, 28, and 35. The naive PPARγ+/− mice (day 0) showed no significant increase in MOGp35–55-induced T cell proliferation than the wild-type littermates.

FIGURE 5.

Neural Ag-specific T cell proliferative response in wild-type and PPARγ+/− mice. Spleen cells were isolated from PPARγ+/− and wild-type mice on days 0, 7, 14, 21, 28, and 35 following immunization with MOGp35–55 Ag, as described in Materials and Methods. The cells were stimulated in vitro with 0, 10, 25, or 50 μg/ml MOGp35–55 Ag, and the proliferative response was measured by [3H]thymidine uptake assay. The values are mean of triplicates at each point, and the error bars represent SD. The figures are representatives of three independent experiments.

FIGURE 5.

Neural Ag-specific T cell proliferative response in wild-type and PPARγ+/− mice. Spleen cells were isolated from PPARγ+/− and wild-type mice on days 0, 7, 14, 21, 28, and 35 following immunization with MOGp35–55 Ag, as described in Materials and Methods. The cells were stimulated in vitro with 0, 10, 25, or 50 μg/ml MOGp35–55 Ag, and the proliferative response was measured by [3H]thymidine uptake assay. The values are mean of triplicates at each point, and the error bars represent SD. The figures are representatives of three independent experiments.

Close modal

Finally, we have examined the secretion of Th1 cytokines, IL-12 and IFN-γ, in spleen cells from wild-type and PPARγ+/− mice in culture. As shown in Fig. 6,A, stimulation of MOGp35–55-sensitized spleen cells with MOGp35–55 Ag induced a dose-dependent increase in the secretion of IL-12 in culture. In PPARγ+/+ mice, the secretion of IL-12 increased from 38.6 ± 2.1 pg/ml in the background control to 785 ± 59 pg/ml after stimulation with 25 μg/ml MOGp35–55, whereas in PPARγ+/− mice, the levels of IL-12 increased from 42 ± 23 pg/ml in the background control to 1033 ± 67 pg/ml after stimulation with 25 μg/ml MOGp35–55. When compared with wild-type mice, the PPARγ+/− mice produced 31.6% more IL-12 in response to 25 μg/ml Ag (p < 0.01) than the wild-type littermates. Further analyses showed that stimulation with MOGp35–55 also resulted in a dose-dependent increase in the secretion of IFN-γ in both wild-type and PPARγ+/− mice (Fig. 6 B). The levels of IFN-γ in wild-type mice increased from 0.65 ± .03 ng/ml in the control background to 4.71 ± 0.31 ng/ml after stimulation with 25 μg/ml MOGp35–55 Ag, whereas the levels of IFN-γ in PPARγ+/− mice increased from 0.67 ± .02 ng/ml in the control to 7.14 ± 0.53 ng/ml in response to 25 μg/ml MOGp35–55. We found that the PPARγ+/− mice produced 50.7% more IFN-γ (p < 0.01) in response to 25 μg/ml neural Ag than the wild-type littermates. These results suggested that the PPARγ+/− mice develop an augmented Th1 immune response to neural Ags that contributes to the development of an exacerbated EAE than the wild-type littermates.

FIGURE 6.

Neural Ag-induced Th1 response in wild-type and PPARγ+/− mice. Spleen cells were isolated from PPARγ+/− and wild-type mice on day 14 following immunization with MOGp35–55 and stimulated in vitro with 0, 10, or 25 μg/ml MOGp35–55 Ag. The culture supernatants were collected after 48 h, and the levels of IL-12 (A) and IFN-γ (B) were measured by ELISA. The values are mean of triplicates, and the error bars represent SD. The figures are representatives of three independent experiments.

FIGURE 6.

Neural Ag-induced Th1 response in wild-type and PPARγ+/− mice. Spleen cells were isolated from PPARγ+/− and wild-type mice on day 14 following immunization with MOGp35–55 and stimulated in vitro with 0, 10, or 25 μg/ml MOGp35–55 Ag. The culture supernatants were collected after 48 h, and the levels of IL-12 (A) and IFN-γ (B) were measured by ELISA. The values are mean of triplicates, and the error bars represent SD. The figures are representatives of three independent experiments.

Close modal

In this study, we have shown that the PPARγ-deficient heterozygous mice develop an exacerbated EAE with severe clinical and pathological symptoms than the wild-type littermates. The exacerbation of EAE in PPARγ+/− mice following immunization with MOGp35–55 Ag was associated with a significant increase in CD4+, CD8+, MHC class II+, and CD40+ cells than the wild-type littermates. PPARγ+/− mice also showed a significant increase in neural Ag-induced T cell proliferation, IL-12/IFN-γ secretion, and Th1 differentiation than the wild-type littermates. Our results suggested a critical physiological role for PPARγ in the regulation of CNS inflammation and demyelination in EAE.

The pathogenesis of EAE/MS is a complex process involving activation of macrophage/microglial cells and differentiation of neural Ag-specific Th1 cells in which proinflammatory cytokines determine the outcome of the disease (7, 8, 9, 10, 11, 12, 13). Using the EAE model of MS, we have shown earlier that in vivo treatment with PPARγ agonists, ciglitazone or 15d-PGJ2, inhibits the clinical and pathological symptoms of MS in SJL/J mice (41). In this study, using PPARγ+/− mice, we have examined the physiological role of PPARγ in the regulation of inflammation and demyelination in EAE/MS. The development of an exacerbated EAE in PPARγ+/− mice following immunization with MOGp35–55 Ag than the wild-type littermates is evident to conclude that PPARγ is a physiological regulator of inflammation and demyelination in EAE/MS. The clinical severity of paralysis correlates well with increased inflammation and demyelination in the CNS. Although the exact mechanisms involved in the regulation of EAE by PPARγ are not known, it is likely that the physiological ligands produced systemically in mice with EAE activate the PPARγ system in autoimmune cells, thereby regulating EAE. There was no difference in the day of onset of EAE, but the exacerbated disease profile in PPARγ+/− mice than the wild-type littermates was more apparent in the latter phase of EAE. Although the wild-type mice recover to normal, the PPARγ+/− mice progress to severe clinical conditions. This prolonged clinical disease has further suggested that PPARγ and its physiological ligands play crucial roles in the spontaneous remission of EAE and perhaps MS and other autoimmune diseases. Although it is likely that the PPARγ+/− mice systemically produce adequate physiological ligands for PPARγ, the deficiency in PPARγ receptor itself is sufficient to contribute a more prolonged EAE or impaired recovery from the disease in PPARγ+/− mice than the wild-type littermates.

To help understand the mechanisms involved in the exacerbation of EAE in PPARγ+/− mice, we analyzed the immune responses of spleen cells that are critical in the pathogenesis of EAE/MS. Flow cytometric analyses showed a significant increase in the number of CD4+ and CD8+ spleen cells in both wild-type and PPARγ+/− mice, following immunization and in vitro culture with MOGp35–55. However, the expansion of CD4+ and CD8+ T cells in PPARγ+/− mice was more profound than the wild-type littermates. We have also observed an increase in the number of MHC class II+ and CD40+ spleen cells in PPARγ+/− mice following immunization with MOGp35–55. The increase of MHC class II+ and CD40+ spleen cells in PPARγ+/− mice was higher than the wild-type mice. Moreover, the T cells from PPARγ+/− mice showed a significantly higher proliferative response to MOGp35–55 than the wild-type littermates. IL-12 and IFN-γ are two important proinflammatory Th1 cytokines that play crucial roles in the pathogenesis of EAE and MS. In this study, we found that the PPARγ+/− mice produce elevated levels of IL-12 and IFN-γ in response to neural Ag than the wild-type mice. This is consistent with our previous studies showing the inhibition of EAE in SJL mice by blocking IL-12 production, IL-12 signaling, and Th1 differentiation, following treatment with ciglitazone or 15d-PGJ2 (41). Although the exact mechanisms are not known, we believe that the impaired regulation of APCs and neural Ag-specific Th1 cells by systemically produced physiological ligands through PPARγ-dependent mechanisms contributes to the development of an exacerbated EAE in PPARγ+/− mice.

The intracellular mechanisms involved in the anti-inflammatory actions of PPARγ agonists are controversial. Although some effects have been attributed to PPARγ, the involvement of PPARγ-independent mechanisms in the anti-inflammatory actions of PPARγ agonists is evident (45). We have shown earlier that the natural and synthetic PPARγ agonists inhibit the development of neural Ag-specific Th1 response and pathogenesis of EAE in SJL/J mice. In this study, we have demonstrated that the PPARγ+/− mice develop an augmented neural Ag-induced Th1 response and exacerbated EAE with prolonged clinical symptoms than the wild-type mice. Although the physiological ligands for PPARγ responsible for the regulation of EAE are yet to be identified, our findings highlight the fact that PPARγ is a critical physiological regulator of CNS inflammation and demyelination in EAE and perhaps MS. This study further suggests the use of PPARγ agonists in the treatment of MS and other Th1 cell-mediated autoimmune inflammatory diseases.

1

This work was supported by National Institutes of Health Grant R01 NS42257-01A1 (to J.J.B.).

3

Abbreviations used in this paper: MS, multiple sclerosis; AMCS, average MCS; 15d-PGJ2, 15-deoxy Δ12,14 PGJ2; EAE, experimental allergic encephalomyelitis; MCS, mean clinical score; MMCS, mean maximum clinical score; MOG, myelin oligodendrocyte glycoprotein; PPAR, peroxisome proliferator-activated receptor.

1
Hemmer, B., J. J. Archelos, H. Hartung.
2002
. New concepts in the immunopathogenesis of multiple sclerosis.
Nat. Rev. Neurosci.
3
:
204
.
2
Dean, G..
1994
. How many people in the world have MS?.
Neuroepidemiology
13
:
1
.
3
Noseworthy, J. H., C. Lucchinetti, M. Rodriguez, B. G. Weinshenker.
2000
. Multiple sclerosis.
N. Engl. J. Med.
343
:
938
.
4
Bitsch, A., W. Bruck.
2002
. Differentiation of multiple sclerosis subtypes: implications for treatment.
CNS Drugs
16
:
405
.
5
Wingerchuk, D. M., C. F. Lucchinetti, J. H. Noseworthy.
2001
. Multiple sclerosis: current pathophysiological concepts.
Lab. Invest.
81
:
263
.
6
Whitacre, C. C., S. C. Reingold, P. A. O’Looney.
1999
. A gender gap in autoimmunity.
Science
283
:
1277
.
7
Steinman, L., R. Martin, C. Bernard, P. Conlon, J. R. Oksenberg.
2002
. Multiple sclerosis: deeper understanding of its pathogenesis reveals new targets for therapy.
Annu. Rev. Neurosci.
25
:
491
.
8
Franklin, R. J..
2002
. Why does remyelination fail in multiple sclerosis?.
Nat. Rev. Neurosci.
3
:
705
.
9
Coleman, M. P., V. H. Perry.
2002
. Axon pathology in neurological disease: a neglected therapeutic target.
Trends Neurosci.
25
:
532
.
10
Gold, R., H. P. Hartung, K. V. Toyka.
2000
. Animal models for autoimmune demyelinating disorders of the nervous system.
Mol. Med. Today
6
:
88
.
11
Owens, T., S. Sriram.
1995
. The immunology of multiple sclerosis and its animal model, experimental allergic encephalomyelitis.
Neurol. Clin.
13
:
51
.
12
Bright, J. J., B. F. Musuro, C. Du, S. Sriram.
1998
. Expression of IL-12 in CNS and lymphoid organs of mice with experimental allergic encephalomyelitis.
J. Neuroimmunol.
82
:
22
.
13
Bright, J. J., M. Rodriguez, S. Sriram.
1999
. Differential influence of interleukin-12 in the pathogenesis of autoimmune and virus-induced CNS demyelination.
J. Virol.
73
:
1637
.
14
Evans, R. M..
1988
. The steroid and thyroid hormone receptor superfamily.
Science
240
:
889
.
15
Blumberg, B., R. M. Evans.
1998
. Orphan nuclear receptors: new ligands and new possibilities.
Genes Dev.
12
:
3149
.
16
Mukherjee, R., L. Jow, G. E. Croston, J. R. Paterniti.
1997
. Identification, characterization and tissue distribution of human peroxisome proliferator activated receptor isoforms 1 and 2 and activation with retinoid X receptor agonists and antagonists.
J. Biol. Chem.
272
:
18779
.
17
Elbrecht, A., Y. Chen, C. A. Cullinan, N. Hayes, M. Leibowitz, D. E. Moller, J. Berger.
1996
. Molecular cloning, expression and characterization of human peroxisome proliferator activated receptors γ1 and γ2.
Biochem. Biophys. Res. Commun.
224
:
431
.
18
Barak, Y., M. C. Nelson, E. S. Ong, Y. Z. Jones, P. Ruiz-Lozano, K. R. Chien, A. Koder, R. M. Evans.
1999
. PPARγ is required for placental, cardiac, and adipose tissue development.
Mol. Cell
4
:
585
.
19
Willson, T. M., W. Wahli.
1997
. Peroxisome proliferator activated receptor agonists.
Curr. Opin. Chem. Biol.
1
:
235
.
20
Krey, G., O. Braissant, F. L’Horset, E. Kalkhoven, M. Perroud, M. G. Parker, W. Wahli.
1997
. Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay.
Mol. Endocrinol.
11
:
779
.
21
Forman, B. M., P. Tontonoz, J. Chen, R. P. Brun, B. Spiegelman, R. M. Evans.
1995
. 15-Deoxy-12, 14 prostaglandin J2 a ligand for the adipocyte determination factor PPAR.
Cell
83
:
803
.
22
Lehmann, J. M., L. B. Moore, T. A. Smith-Oliver, W. O. Wilkison, T. M. S. A. Willson, S. A. Kliewer.
1995
. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ).
J. Biol. Chem.
270
:
12953
.
23
Kliewer, S. A., T. M. Willson.
1998
. The nuclear receptor PPAR: bigger than fat.
Curr. Opin. Genet. Dev.
8
:
576
.
24
Schwartz, S., P. Raskin, V. Fonseca, J. F. Graveline.
1998
. Effect of troglitazone in insulin-treated patients with type II diabetes mellitus: Troglitazone and Exogenous Insulin Study Group.
N. Engl. J. Med.
338
:
861
.
25
Barroso, I., M. Gurnell, V. E. Crowley, M. Agostini, J. W. Schwabe, M. A. Soos, G. L. Maslen, T. D. Williams, H. Lewis, A. J. Schafer, et al
1999
. Dominant negative mutations in human PPARγ associated with severe insulin resistance, diabetes mellitus and hypertension.
Nature
402
:
880
.
26
Demetri, G. D., C. D. Fletcher, E. Mueller, G. D. Demetri, C. D. Fletcher, E. Mueller, P. Sarraf, R. Naujoks, N. Campbell, B. M. Spiegelman, S. Singer.
1999
. Induction of solid tumor differentiation by the peroxisome proliferator-activated receptor-γ ligand troglitazone in patients with liposarcoma.
Proc. Natl. Acad. Sci. USA
196
:
3951
.
27
Elstner, E., C. Muller, K. Koshizuka, E. A. Williamson, D. Park, H. Asou, P. Shintaku, J. W. Said, D. Heber, H. P. Koeffler.
1998
. Ligands for peroxisome proliferator-activated receptor γ and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice.
Proc. Natl. Acad. Sci. USA
95
:
8806
.
28
Asou, H., W. Verbeek, E. Williamson, E. Elstner, T. Kubota, N. Kamada, H. P. Koeffler.
1999
. Growth inhibition of myeloid leukemia cells by troglitazone, a ligand for peroxisome proliferator activated receptor γ, and retinoids.
Int. J. Oncol.
15
:
1027
.
29
Tsubouchi, Y., H. Sano, Y. Kawahito, S. Mukai, R. Yamada, M. Kohno, K. Inoue, T. Hla, M. Kondo.
2000
. Inhibition of human lung cancer cell growth by the PPARγ agonists through induction of apoptosis.
Biochem. Biophys. Res. Commun.
270
:
400
.
30
Sarraf, P., E. Mueller, W. M. Smith, H. M. Wright, J. B. Kum, L. A. Aaltonen, A. de la Chapelle, B. M. Spiegelman, C. Eng.
1999
. Loss-of-function mutations in PPARγ associated with human colon cancer.
Mol. Cell
3
:
799
.
31
Sarraf, P., E. Mueller, D. Jones, F. J. King, D. J. DeAngelo, J. B. Partridge, S. A. Holden, L. B. Chen, S. Singer, C. Fletcher, B. M. Spiegelman.
1998
. Differentiation and reversal of malignant changes in colon cancer through PPARγ.
Nat. Med.
4
:
1046
.
32
Ricote, M., A. C. Li, T. M. Willson, C. J. Kelly, C. K. Glass.
1998
. The peroxisome proliferator-activated receptor-γ is a negative regulator of macrophage activation.
Nature
391
:
79
.
33
Jiang, C., A. T. Ting, B. Seed.
1998
. PPARγ agonists inhibit production of monocyte inflammatory cytokine.
Nature
391
:
82
.
34
Kawahito, Y., M. Kondo, Y. Tsubouchi, A. Hashiramoto, D. Bishop-Bailey, K. Inoue, M. Kohno, R. Yamada, T. Hla, H. Sano.
2000
. 15-Deoxy-δ(12, 14)-PGJ2 induces synoviocyte apoptosis and suppresses adjuvant-induced arthritis in rats.
J. Clin. Invest.
106
:
189
.
35
Neve, B. P., J. C. Fruchart, B. Staels.
2000
. Role of the peroxisome proliferator-activated receptors (PPAR) in atherosclerosis.
Biochem. Pharmacol.
60
:
1245
.
36
Chen, Z., S. Ishibashi, S. Perrey, J. Osuga, T. Gotoda, T. Kitamine, Y. Tamura, H. Okazaki, N. Yahagi, Y. Iizuka, et al
2001
. Troglitazone inhibits atherosclerosis in apolipoprotein E-knockout mice: pleiotropic effects on CD36 expression and HDL.
Arterioscler. Thromb. Vasc. Biol.
21
:
372
.
37
Su, C. G., X. Wen, S. T. Bailey, W. Jiang, S. M. Rangwala, S. A. Keilbaugh, A. Flanigan, S. Murthy, M. A. Lazar, G. D. Wu.
1999
. A novel therapy for colitis utilizing PPAR-γ ligands to inhibit the epithelial inflammatory response.
J. Clin. Invest.
104
:
383
.
38
Pershadsingh, H. A., J. A. Sproul, E. Benjamin, J. Finnegan, N. M. Amin.
1998
. Treatment of psoriasis with troglitazone therapy.
Arch. Dermatol.
134
:
1304
.
39
Nagy, L., P. Tontonoz, J. G. Alvarez, H. Chen, R. M. Evans.
1998
. Oxidized LDL regulates macrophage gene expression through ligand activation of PPARγ.
Cell
93
:
229
.
40
Spiegelman, B. M..
1998
. PPARγ in monocytes: less pain, any gain?.
Cell
93
:
153
.
41
Natarajan, C., J. J. Bright.
2002
. Peroxisome proliferator-activated receptor-γ agonists inhibit experimental allergic encephalomyelitis by blocking IL-12 production, IL-12 signaling and Th1 differentiation.
Genes Immun.
3
:
59
.
42
Niino, M., K. Iwabuchi, S. Kikuchi, M. Ato, T. Morohashi, A. Ogata, K. Tashiro, K. Onoe.
2001
. Amelioration of experimental autoimmune encephalomyelitis in C57BL/6 mice by an agonist of PPARγ.
J. Neuroimmunol.
116
:
40
.
43
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-γ agonist 15-deoxy-δ(12, 14)-prostaglandin J2 ameliorates experimental autoimmune encephalomyelitis.
J. Immunol.
168
:
2508
.
44
Feinstein, D. L., E. Galea, V. Gavrilyuk, C. F. Brosnan, C. C. Whitacre, L. Dumitrescu-Ozimek, G. E. Landreth, H. A. Pershadsingh, G. Weinberg, M. T. Heneka.
2002
. Peroxisome proliferator-activated receptor-γ agonists prevent experimental autoimmune encephalomyelitis.
Ann. Neurol.
51
:
694
.
45
Chawla, A., Y. Barak, L. Nagy, D. Liao, P. Tontonoz, R. M. Evans.
2001
. PPARγ dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation.
Nat. Med.
7
:
48
.