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Abstract
Bacillus anthracis is a gram-positive, aerobic, spore forming, and rod-shaped bacterium. Anthrax spores are introduced into macrophage by phagocytosis and multiply after germination. The anthrax spores infected in macrophage produce lethal toxin eventually caused cell death. In this study, we analyzed apoptosis and cytokine TNF-α and IL-12 secretion after the infection of spores of B. anthracis Sterne in the murine macrophage RAW264.7 cells and in the primary human macrophages. In murine macrophage RAW264.7 cells infected by spore of B. anthracis Sterne, the cells were markedly changed in secretion of TNF-α (482∼6,213 pg/ml) by lethal toxin, and induced apoptosis. In case of RAW264.7 cells infected by formalin-inactivated spores of B. anthracis, the cells were not able to produce lethal toxin, which released lower level concentration of TNF-α (7.7∼97.2 pg/ml), and rarely induced apoptosis. When primary human macrophage cells infected with spores of B. anthracis Sterne, they secreted TNF-α (5∼16 pg/ml), and induced apoptosis about 1% of total cells. We presented that inducing apoptosis by spores of B. anthracis Sterne capable of expressing lethal toxin is related with the secretion of TNF-α in murine macrophage RAW264.7 cells. These studies revealed that human and murine macrophages has affected differently by anthrax lethal toxin produced by spores of B. anthracis Sterne.
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Figure 1.
Cytokine analysis in murine macrophage RAW264.7 cells infected with spores of B. anthracis Sterne at 1, 3, 5, and 7 hours using ELISA method. Control was murine macrophage RAW264.7 cells treated with PBS buffer. The concentration of 2 cytokines in the supernatants collected from murine macrophage RAW264.7 cells infected with formalin-inactivated spores (white bars), spores (gray bars), and vegetative cells (black bars) of B. anthracis Sterne at the indicated time points are shown for TNF-α (A), and IL-12 (B). Three experiments were performed, and the results of a representative experiment are shown. Each value reported is the average of three samples. Error bars represent 1 standard deviation. Statistical significance was determined by Student's t test analysis. In all cases, means were compared to control group only treated with PBS buffer. ∗p<0.05, ∗∗p<0.01
Figure 2.
Cytokine analysis in primary human macrophage infected with spores of B. anthracis Sterne at 1, 3, 5, and 7 hours using ELISA method. Control was murine macrophage RAW264.7 cells treated with PBS buffer. The concentration of 3 cytokines in the supernatants collected from primary human macrophage infected with formalin-inactivated spores (white bars), spores (gray bars), and vegetative cells (black bars) of B. anthracis Sterne at the indicated time points are shown for TNF-β (A), IL-1β (B), and IL-12 (C). Three experiments were performed, and the results of a representative experiment are shown. Each value reported is the average of three samples. Error bars represent 1 standard deviation. Statistical significance was determined by Student's t test analysis. In all cases, means were compared to control group only treated with PBS buffer. ∗p<0.05, ∗∗p<0.01
Figure 3.
Apoptosis analysis in murine macrophage RAW264.7 cells infected with spores of B. anthracis Sterne at 1, 3, 5, and 7 hours by flow cytometry with annexin-PI double-labeled cells. The annexin-positive population corresponding to apoptotic cells, PI-positive population corresponds to necrotic cells, and the double-positive position corresponds to necrotic and/or late apoptotic cells. Murine macrophage RAW264.7 cells were treated with PBS buffer (A), formalin-inactivated spores (B), spores (C), and vegetative cells (D) of B. anthracis Sterne.
Figure 4.
Apoptosis analysis in primary human macrophages infected with spores of B. anthracis Sterne at 1, 3, 5, 7 hours by flow cytometry with annexin-PI double-labeled cells. The annexin-positive population corresponding to apoptotic cells, PI-positive population corresponds to necrotic cells, and the double-positive position corresponds to necrotic and/or late apoptotic cells. Primary human macrophages were treated with PBS buffer (A), formalin-inactivated spores (B), spores (C), and vegetative cells (D) of B. anthracis Sterne.
Table 1.
Cytokine expression by anthrax lethal toxin on different cells of immune system
| Cell Lines | Toxin | Time (hr) | Cytokines | References |
|---|---|---|---|---|
| RAW264.7 | LeTx∗ | 2∼4 | TNF-α ↑ | 11 |
| RAW264.7 | LeTx∗ | 24 | IL-1β, IL-18 ↑ | 5 |
| J774A.1 | LeTx∗ | 2 | TNF-α ↓ | 7 |
| J774A.1 | spore | 5∼7.5 | TNF-α, IL-12 ↑ | 23 |
| Human macrophage | LeTx∗ | 48 | TNF-α ↓, IL-1β, IL-6 ↑ | 25 |
| Mouse macrophage | spore | 5∼7.5 | TNF-α, IL-6 ↑ | 24 |
Table 2.
The amounts of TNF-α or IL-12 secreted from murine macrophage RAW264.7 cells were infected with spores of B. anthracis Sterne
| TNF-α (pg/ml) | IL-12 (pg/ml) | |||||||
|---|---|---|---|---|---|---|---|---|
| 1 hr | 3 hr | 5 hr | 7 hr | 1 hr | 3 hr | 5 hr | 7 hr | |
| Inactivated spores | ND∗ | ND∗ | ND∗ | ND∗ | 215 | 254 | 210 | 234 |
| Live spores | 482 | 2,433 | 6,022 | 6,213 | 110 | 246 | 210 | 234 |
| Vegetative cells | 692 | 2,844 | 5,128 | 6,045 | 151 | 175 | 318 | 340 |
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