Role of PPARγ in COX-2 activation in mycobacterial pulmonary inflammation

Abstract

Preliminary studies show that intranasal (i.n.) administration of BCG in mice induces M1 activation of alveolar macrophages (M∅) that increase TNF-α production and cyclooxygenase-2 (COX-2) expression but reduce constitutive peroxisome proliferator-activated receptor gamma (PPARγ) expression. However, COX-2 is catalytically inactive for prostaglandin E(2) release, unlike COX-2 that is active in M1 activation in vitro by BCG. In this study, we determined the role of PPARγ for BCG-induced M1 activation in vivo and in vitro. We found that treatment of mice with GW9662, a PPARγ antagonist, prior to i.n. BCG, partially restored PPARγ expression, and decreased TNF-α production and COX-2 expression. But COX-2 was still inactive. The decreased effects on TNF-α and COX-2 were also observed when alveolar M∅ were treated in vitro with GW9662/BCG, but COX-2 was still active. Our results indicate that PPARγ upregulates M1 activation of alveolar M∅, but inactive COX-2 formation is independent of PPARγ in mycobacterial pulmonary inflammation.

Fig. 1. PPARγ is reduced by BCG treatment in vivo

Alveolar MØ were isolated from groups of mice 24 h after i.n. administration of 0 (saline), 5, 50, and 500 μg BCG. PPARγ, COX-1, COX-2 and β-actin levels were determined by Western blot with 5 γg protein. β-actin bands show equivalent loading of samples. a Representative western blot. b The ratio of each molecule to β-actin obtained by densitometric analysis is expressed as arbitrary units (A.U.). Values are mean ± SEM of three separate experiments. *, p<0.05; **, p<0.005; ***, p<0.0001 compared with control saline group. .

Fig. 2. GW9662 slightly restores PPARγ but reduces COX-1 and COX-2 expression by alveolar MØ activated by i.n. BCG

Mice received 1 mg/kg of a PPARγ antagonist GW9662 (GW) i.p. and 1 mg/kg of GW9662 i.n., 50 min and 20 min, respectively, before 500 μg of i.n. BCG administration. After 24 h, alveolar MØ were isolated from groups of mice. PPARγ, COX-1, COX-2 and β-actin were determined by Western blot with 5 μg protein. β-actin bands show equivalent loading of samples. a Representative western blot. b The ratio of each molecule to β-actin obtained by densitometric analysis is expressed as arbitrary units (A.U.). Values are mean ± SEM of three separate experiments. *, p<0.005; **, p<0.0001 compared with control saline group. †_, p<0.05; ††_, p<0.0001 compared with the BCG group.

Fig. 3. Subcellular localization of COX-1 and COX-2 in alveolar MØ after GW9662/BCG treatments in vivo

Mice were treated as described in Fig. 2. Cells were examined by confocal microscopy following immunefluorescent staining with anti-COX-1 or anti-COX-2 (green) and PI (red) for the nucleus and BCG. Bar = 5 μm. Results are representative of three separate experiments.

Fig. 4. Alveolar MØ cultures with BCG reduce PPARγ and induce COX-2

Alveolar MØ isolated from normal mice were incubated with 0 (saline), 20 or 100 μg/ml BCG for 24 h, and cells were harvested. PPARγ, COX-1, COX-2 and β-actin levels were determined by Western blot with 5 μg protein. β-actin bands show equivalent loading of samples. a Representative western blot. b The ratio of each molecule to β-actin obtained by densitometric analysis is expressed as arbitrary units (A.U.). Values are mean ± SEM of three separate experiments. *, p<0.05; **, p<0.01; ***, p=0.0001 compared with control saline group.

Fig. 5. Effects of GW9662 on PPARγ, COX-1 and COX-2 expression and PGE₂ production in alveolar MØ cultures

Normal alveolar MØ were pre-incubated with 10 μM GW9662 for 30 min and then incubated with 100 μg/ml BCG for 24 h. PPARγ, COX-1, COX-2 and β-actin levels were examined by Western blot with 5 μg protein. β-actin bands show equivalent loading of samples. a Representative western blot. b The ratio of each molecule to β-actin obtained by densitometric analysis is expressed as arbitrary units (A.U.). Values are mean ± SEM of three separate experiments. *, p<0.05 compared with control saline group. †_, p<0.05 compared with the BCG group. ‡_, p<0.005 compared with the GW9662 group. c To measure PGE₂ release, adherent MØ were resuspended in serum-free RPMI 1640 and cultured for 2 h. PGE₂ was assayed by ELISA. Values are mean ± SEM, n=3. *, p<0.005 compared with the saline group. †_, p<0.005 compared with the BCG group. d Subcellular localization of COX-2 in MØ was examined by confocal microscopy following immunofluorescent staining with anti-COX-2 (green) and PI (red) for the nucleus and BCG. Bar = 5 μm. Results are representative of three separate experiments.

Fig. 5. Effects of GW9662 on PPARγ, COX-1 and COX-2 expression and PGE₂ production in alveolar MØ cultures

Normal alveolar MØ were pre-incubated with 10 μM GW9662 for 30 min and then incubated with 100 μg/ml BCG for 24 h. PPARγ, COX-1, COX-2 and β-actin levels were examined by Western blot with 5 μg protein. β-actin bands show equivalent loading of samples. a Representative western blot. b The ratio of each molecule to β-actin obtained by densitometric analysis is expressed as arbitrary units (A.U.). Values are mean ± SEM of three separate experiments. *, p<0.05 compared with control saline group. †_, p<0.05 compared with the BCG group. ‡_, p<0.005 compared with the GW9662 group. c To measure PGE₂ release, adherent MØ were resuspended in serum-free RPMI 1640 and cultured for 2 h. PGE₂ was assayed by ELISA. Values are mean ± SEM, n=3. *, p<0.005 compared with the saline group. †_, p<0.005 compared with the BCG group. d Subcellular localization of COX-2 in MØ was examined by confocal microscopy following immunofluorescent staining with anti-COX-2 (green) and PI (red) for the nucleus and BCG. Bar = 5 μm. Results are representative of three separate experiments.

Fig. 6. TNF-α levels in bronchoalveolar lavage (BAL) from GW9662/BCG-treated mice and supernatants of alveolar MØ cultures with GW9662/BCG

a TNF-α levels were measured by ELISA in BAL obtained by a 1 ml saline-lavage from mice, treated as in Fig. 2. b Normal alveolar MØ were treated as indicated in Fig. 5. TNF-α in the culture supernatants were measured by ELISA. Values are mean ± SEM; n=3. *, p<0.0001 compared with control saline group. †_, p<0.0001 compared with BCG group. Results are representative of three separate experiments.