Cholera toxin induces a shift from inactive to active cyclooxygenase 2 in alveolar macrophages activated by Mycobacterium bovis BCG

Abstract

Intranasal vaccination stimulates formation of cyclooxygenases (COX) and release of prostaglandin E(2) (PGE(2)) by lung cells, including alveolar macrophages. PGE(2) plays complex pro- or anti-inflammatory roles in facilitating mucosal immune responses, but the relative contributions of COX-1 and COX-2 remain unclear. Previously, we found that Mycobacterium bovis BCG, a human tuberculosis vaccine, stimulated increased release of PGE(2) by macrophages activated in vitro; in contrast, intranasal BCG activated no PGE(2) release in the lungs, because COX-1 and COX-2 in alveolar macrophages were subcellularly dissociated from the nuclear envelope (NE) and catalytically inactive. This study tested the hypothesis that intranasal administration of BCG with cholera toxin (CT), a mucosal vaccine component, would shift the inactive, NE-dissociated COX-1/COX-2 to active, NE-associated forms. The results showed increased PGE(2) release in the lungs and NE-associated COX-2 in the majority of COX-2(+) macrophages. These COX-2(+) macrophages were the primary source of PGE(2) release in the lungs, since there was only slight enhancement of NE-associated COX-1 and there was no change in COX-1/COX-2 levels in alveolar epithelial cells following treatment with CT and/or BCG. To further understand the effect of CT, we investigated the timing of BCG versus CT administration for in vivo and in vitro macrophage activations. When CT followed BCG treatment, macrophages in vitro had elevated COX-2-mediated PGE(2) release, but macrophages in vivo exhibited less activation of NE-associated COX-2. Our results indicate that inclusion of CT in the intranasal BCG vaccination enhances COX-2-mediated PGE(2) release by alveolar macrophages and further suggest that the effect of CT in vivo is mediated by other lung cells.

Fig 1

COX-1 and COX-2 expression by alveolar Mϕ isolated from mice given i.n. BCG and/or CT. Mice were given 500 μg of BCG and/or 1 μg of CT intranasally. After 24 h, alveolar Mϕ were isolated from groups of mice. COX-1, COX-2, and PPARγ levels were determined by Western blotting 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 means ± SEM (n = 3). *, P < 0.05 for comparisons between control saline and BCG groups. ns, not significant for comparisons between the BCG and BCT/CT groups.

Fig 2

Subcellular localization of COX-1 and COX-2 in alveolar Mϕ by i.n. BCG and/or CT. (A) Mice were treated as indicated in Fig. 1. Alveolar Mϕ were examined by confocal microscopy following immunofluorescent staining with anti-COX-1 (green) or anti-COX-2 (green) and PI (red) for the nucleus and BCG. Bars, 5 μm. Results are representative of three separate experiments. (B) The percentages of COX-1⁺ Mϕ and COX-2⁺ Mϕ that expressed NE-associated (white bars) and NE-dissociated (black bars) forms were calculated. Values are means ± SEM (n = 3). ns, not significant. *, P < 0.001, for comparisons of NE association between the BCG and BCG/CT groups.

Fig 3

PGE₂ levels in BAL specimens. Mice were treated as indicated in Fig. 1. After 24 h, levels of PGE₂ in BAL specimens were measured by ELISA. Values are means ± SEM (n = 3). *, P < 0.05 for comparisons between the BCG and BCG/CT groups.

Fig 4

COX-1 and COX-2 expression by alveolar epithelial cells. Shown is a representative Western blot showing protein levels in alveolar epithelial cells isolated 24 h after mice were given 500 μg of BCG and/or 1 μg of CT intranasally. COX-1 and COX-2 levels were determined by Western blotting with 5 μg protein, as indicated in Materials and Methods; the β-actin bands show equivalent loading of samples. Unlike alveolar Mϕ, PPARγ was not detected in all 4 groups (data not shown).

Fig 5

Effects of post- or pretreatment with CT on COX-2 expression and PGE₂ release in BCG-treated RAW 264.7 cells. For CT pretreatment (CT-BCG), RAW 264.7 cells were incubated with CT at the indicated doses, and 18 h after CT treatment, BCG was added at the indicated doses. For CT posttreatment (BCG-CT), RAW 264.7 cells were incubated with BCG, and 18 h after BCG treatment, CT was added. Controls received saline. Cells were harvested at 24 h. Results are representative of three separate experiments. (A) Levels of COX-2 following CT-BCG and BCG-CT treatments, respectively, were measured by Western blotting after loading of 1.5 μg protein. (B) The ratio of COX-2 to β-actin obtained by densitometric analysis is expressed as arbitrary units (A.U.). Values are means ± SEM of three separate experiments. *, P < 0.005, and **, P < 0.0001, compared with the saline group; †, P < 0.05, and ††, P < 0.0001, compared with Mϕ treated with BCG alone. (C) To measure PGE₂ release, adherent RAW 264.7 cells were resuspended in serum-free RPMI 1640 and further cultured for 2 h. PGE₂ levels in the culture fluids were measured by ELISA. Values are means ± SEM (n = 3). *, P < 0.01 for comparisons between the BCG groups and the corresponding groups containing CT and BCG.

Fig 6

Effects of CTB on COX-2 expression in BCG-treated RAW 264.7 cells. For CT pretreatment (CTB-BCG), RAW 264.7 cells were incubated with CTB or CT at the indicated doses (μg/ml), and 18 h after the treatment, 5 μg/ml BCG was added. For CTB posttreatment (BCG-CTB), RAW 264.7 cells were incubated with BCG, and 18 h later, CTB or CT was added. Controls received saline. Cells were harvested at 24 h. (A) Levels of COX-2 following CTB-BCG and BCG-CTB treatments, respectively, were measured by Western blotting with 5 μg protein; β-actin bands show equivalent loading of samples. (B) The ratio of COX-2 to β-actin obtained by densitometric analysis is expressed as arbitrary units (A.U.). Values are means ± SEM (n = 3). *, P < 0.0001 compared with the saline group; †, P < 0.0001 compared with Mϕ treated with BCG alone.

Fig 7

Subcellular localization of COX-2 in alveolar Mϕ by i.n. CT-BCG and BCG-CT. Mice were given i.n. 500 μg of BCG and/or 1 μg of CT on the following schedules: CT at 0 h followed by BCG at 18 h (CT-BCG), a mixture of BCG and CT at 0 h (BCG/CT), and BCG at 0 h followed by CT at 18 h (BCG-CT). BCG alone and CT alone were given at 18 h. At 24 h, alveolar Mϕ were isolated in all groups. (A) Alveolar Mϕ were examined by confocal microscopy following immunofluorescent staining with anti-COX-2 and PI for the nucleus and BCG. The percentages of COX-2⁺ Mϕ that expressed NE-associated (white bars) and NE-dissociated (black bars) forms were calculated. Values are means ± SEM (n = 3). *, P < 0.05, and **, P < 0.001, compared with NE association of BCG alone. (B) Alveolar Mϕ were cultured in serum-free RPMI 1640 for 2 h. PGE₂ in the supernatant was measured by ELISA. Values are means ± SEM (n = 3). *, P < 0.05, and **, P < 0.01, compared with BCG alone.