Peroxisome proliferator-activated receptor-gamma haploinsufficiency enhances B cell proliferative responses and exacerbates experimentally induced arthritis

Abstract

Peroxisome proliferator-activated receptor-gamma (PPAR gamma) controls adipogenesis and glucose metabolism. It was reported recently that PPAR gamma activation by its agonistic ligands modifies lymphocyte function. Since synthetic ligands are known to exert their effect via PPAR gamma-dependent and -independent pathways, we examined the physiological role of PPAR gamma in lymphocytes by using heterozygote mutant mice in which one allele of PPAR gamma is deleted (PPAR gamma(+/-)). In contrast to T cells, which did not exhibit a significant difference, B cells from PPAR gamma(+/-) showed an enhanced proliferative response to stimulation by either lipopolysaccharide or cross-linking of antigen receptors. Dysregulation of the NF-kappa B pathway in B cells from PPAR gamma(+/-) was indicated by spontaneous NF-kappa B activation, as well as increased I kappa B alpha phosphorylation and gel-shift activity following LPS stimulation. Mice primed with either ovalbumin or methylated BSA also showed enhanced antigen-specific immune response of both T and B cells, an immunological abnormality that exacerbated antigen-induced arthritis. These findings indicate that PPAR gamma plays a critical role in the control of B cell response and imply a role in diseases in which B cell hyperreactivity is involved, such as arthritis and autoimmunity.

Figures

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.

Figure 6

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).

Figure 7

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).

Figure 8

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.

Figure 9

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).