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Role of Macrophage Oxidative Burst in the Action of Anthrax Lethal Toxin

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Abstract

Background

Major symptoms and death from systemic Bacillus anthracis infections are mediated by the action of the pathogen’s lethal toxin on host macrophages. High levels of the toxin are cytolytic to macrophages, whereas low levels stimulate these cells to produce cytokines (interleukin-1β and tumor necrosis factor-α), which induce systemic shock and death.

Materials and Methods

Experiments were performed to assess the possibility that the oxidative burst may be involved in one or both of lethal toxin’s effects on macrophages. Toximediated cell lysis, superoxide anion and cytokine production were measured. Effects of antioxidants and macrophage mutations were examined.

Results

RAW264.7 murine macrophages treated with high levels of toxin released large amounts of superoxide anion, beginning at about 1 hr, which correlates with the onset of cytolysis. Cytolysis could be blocked with various exogenous antioxidants or with N-acetyl-L-cysteine and methionine, which promote production of the endogenous antioxidant, glutathione. Mutant murine macrophage lines deficient in production of reactive oxygen intermediates (ROIs) were relatively insensitive to the lytic effects of the toxin, whereas a line with increased oxidative burst potential showed elevated sensitivity. Also, cultured blood monocyte-derived macrophages from a patient with Chronic Granulomatous Disease, a disorder in which the phagocyte’s oxidative burst is disabled, were totally resistant to toxin, in contrast to control monocytes.

Conclusions

These results imply that the cytolytic effect of the toxin is mediated by ROIs. Additionally, cytokine production and consequent pathologies showed partial dependence on macrophage ROIs. Antioxidants moderately inhibited toxin-induced cytokine production in vitro, and BALB/c mice pretreated with N-acetyl-L-cysteine or mepacrine showed partial protection against lethal toxin. Thus ROIs are involved in both the cytolytic action of anthrax lethal toxin and the overall pathologic process in vivo.

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Acknowledgments

The authors would like to thank Maria Steinbeck, Kate Beauregard, and Jill Milne for critical discussion of this study and J.M. also for determination of cAMP values. This work was supported by NIH grants AI-22021, AI-22848 (RJC), AI-08649 (PCH), and AI-07118 (BRB). R.A.B.E. is an Established Investigator of the American Heart Association. B.A.K, is the recipient of a Charles A. Janeway, Child Health Research Scholarship.

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Authors and Affiliations

  1. Department of Microbiology and Molecular Genetics and the Shipley Institute of Medicine, Harvard Medical School, 200 Longwood Avenue, D1-502, Boston, Massachusetts, 02115, USA

    Philip C. Hanna & R. John Collier

  2. Division of Hematology and Infectious Diseases, The Children’s Hospital, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA

    Benjamin A. Kruskal & R. Allen B. Ezekowitz

  3. Howard Hughes Medical Institute and Departments of Microbiology and Immunology, Cell Biology, Molecular Pharmacology, and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York, USA

    Barry R. Bloom

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  1. Philip C. Hanna
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  2. Benjamin A. Kruskal
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  3. R. Allen B. Ezekowitz
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  4. Barry R. Bloom
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  5. R. John Collier
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Hanna, P.C., Kruskal, B.A., Ezekowitz, R.A.B. et al. Role of Macrophage Oxidative Burst in the Action of Anthrax Lethal Toxin. Mol Med 1, 7–18 (1994). https://doi.org/10.1007/BF03403527

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