Proliferative response of naive T cells from PPARγ^+/– mice does not differ from wild-type mice. T cells were purified from the spleen of each naive mouse and stimulated with 1 or 10 μg/ml of anti-CD3 Ab in the presence of 1 μg/ml of anti-CD28 Ab for 24 hours. The proliferation was measured by [³H]thymidine incorporation. Bars show the mean ± SD (n = 8 per group). Data from every littermate group of PPARγ^+/– and PPARγ^+/+ mice were statistically analyzed. There was no significant difference in the naive T cell response between the PPARγ^+/– mice (filled circles, +/–) and the littermate ICR (open circles, +/+) wild-type mice.

PPARγ agonistic ligands suppress the proliferation of naive B cells from both PPARγ^+/– and PPARγ^+/+ mice stimulated with LPS or anti–mouse IgM F(ab′)₂. B cells (10⁵) purified from spleens were stimulated with 10 μg/ml of LPS (a) or 20 μg/ml of anti–mouse IgM F(ab′)₂ (b) for 72 hours in the absence or presence of PPARγ agonistic ligands at various concentrations (TZDs: 20, 50 μM; 15d-PGJ₂: 2, 5 μM). Proliferation was measured by [³H]thymidine incorporation. Bars show the mean ± SD (n = 10 per group). All of the tested PPARγ agonistic ligands (15d-PGJ₂, troglitasone, pioglitasone, and rosiglitasone) demonstrated suppressive activity. Data from every littermate group were analyzed statistically using paired t test.

Activation of NF-κB pathway in B cells from PPARγ^+/– mice. (a) Nuclear extracts from B cells were analyzed by EMSA for the ability to bind to an NF-κB consensus probe. Lane 1: No extract, with labeled oligonucleotide; lane 2: resting B cells from wild-type mice (+/+); lane 3: resting B cells from PPARγ^+/– mice (+/–); lane 4: stimulated B cells by LPS for 5 minutes from wild-type mice; lane 5: stimulated B cells from PPARγ^+/– mice; lane 6: lane 5 with excess of cold oligonucleotide; lane 7: lane 5 without labeled oligonucleotide. (b) Phosphorylation of IκBα. Cell lysates from B cells were probed with anti-phosphorylated IκBα (lanes 1–4) and anti- IκBα (lanes 5–8). Lanes 1 and 5 were resting B cells from wild-type mice. Lanes 2 and 6 were resting B cells from PPARγ^+/– mice. Lanes 3 and lane 7 were B cells from wild-type mice, stimulated with LPS for 5 minutes. Lanes 4 and 8 were stimulated B cells from PPARγ^+/– mice.

Antigen-specific immune response was enhanced in PPARγ^+/– mice. (a) Proliferation of splenocytes from wild-type mice (open circles, +/+) and PPARγ^+/– mice (filled circles, +/–), both of which were primed with OVA. Proliferation was measured by [³H]thymidine incorporation in the presence of various concentrations of OVA. Bars show the mean ± SD (n = 10 per group). The difference between the two groups was significant (*P < 0.01). (b) The level of Ab against OVA in sera from mice primed with OVA. Mice were bled 30 days after priming with OVA and assayed for the anti-OVA Ab level by ELISA. The mean Ab titers (± SD) of each group of ten mice are shown. The difference between the two groups was significant (*P < 0.01). (c) Proliferation against mBSA of splenocytes from mBSA-primed wild-type control mice (open circles, +/+) and PPARγ^+/– mice (filled circles, +/–), respectively. Proliferation was measured by [³H]thymidine incorporation in the presence of various concentrations of mBSA. Bars show the mean ± SD (n = 10 per group).The difference between the two groups was significant (*P < 0.001). (d) Mice were bled 30 days after intra-articular challenge with mBSA and assayed for the anti-mBSA Ab level by ELISA. The mean Ab titers (± SD) of each group of the total ten mice are shown. The statistical difference between PPARγ^+/– and PPARγ^+/+ littermates derived from the same litter were significant (*P < 0.01).

Histological examination of ankles from arthritic mice. Hematoxylin and eosin staining of the ankle joint revealed increased infiltration of inflammatory mononuclear cells in PPARγ^+/– mice (a) compared with wild-type mice (b).

Enhanced proliferative response of B cells from PPARγ^+/– mice. Naive B cells (10⁵) purified from the spleen of each mouse were stimulated with either 10 μg/ml of LPS (a) or 20 μg/ml of anti-mouse IgM F(ab′)₂ (b) for 72 hours. The proliferation was measured by [³H]thymidine incorporation. Bars show the mean ± SD (n = 8 per group). B cells from PPARγ^+/– mice (+/–) demonstrated a significantly stronger response to LPS or anti–mouse IgM F(ab′)₂ compared with the cells from wild-type littermates (+/+). Statistical analysis using data from all mice as well as every littermate group gave a significant difference.

The cell viability as well as the proportion of dead/dying cells are different between PPARγ^+/+ and PPARγ^+/– mice, whereas the addition of PPARγ agonists did not alter those parameters, except 5 μM of 15d-PGJ₂. The B cells from PPARγ^+/+ (a) or PPARγ^+/– (b) are cultured in the absence or presence of PPARγ agonists and their viable cell number were counted by trypan blue exclusion. The percentage of dead or dying cells of B cells from PPARγ^+/+ (c) or PPARγ^+/– (d) in the culture were counted by PI staining. B cells were cultured without agonist (closed squares), or with troglitazone 20 μM (filled circles), troglitazone 50 μM (filled triangles), 15d-PGJ₂ 2 μM (filled diamonds), and 15d-PGJ₂ 5 μM (open squares). *P < 0.05 for the cell number or percentage of dead cells compared to those in the absence of the agonist.

Serum IgG and IgM levels were increased in PPARγ^+/– mice. Serum IgG (a) and IgM (b) levels in PPARγ^+/– mice (closed column, +/–) were higher than in wild-type mice (open column, +/+). Both PPARγ^+/– and wild-type 12-week-old mice were bled, and serum IgG and IgM levels were measured by ELISA. The mean Ab titers (± SD) of each group of ten mice are shown. The statistical differences between PPARγ^+/– and PPARγ^+/+ littermates derived from the same litter were significant (*P < 0.01).

PPARγ^+/– mice showed more severe antigen-induced arthritis. Mice primed with mBSA were challenged intra-articularly with the antigen on day 0. The increase in hind paw thickness during the course of the disease was monitored using a dial-gauge calipers. Bars show the mean ± SD (n = 10 per group). The statistical difference (*P < 0.01) between the PPARγ^+/– and PPARγ^+/+ mice from day 1 to day 5 was calculated using data of all mice as well as every littermate group.