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. 1997 Nov;65(11):4850–4857. doi: 10.1128/iai.65.11.4850-4857.1997

Mycobacterial growth and sensitivity to H2O2 killing in human monocytes in vitro.

P Laochumroonvorapong 1, S Paul 1, C Manca 1, V H Freedman 1, G Kaplan 1
PMCID: PMC175696  PMID: 9353075

Abstract

The intracellular growth and susceptibilities to killing by H2O2 in cultured human monocytes of a number of mycobacterial species including laboratory strains and clinical isolates of Mycobacterium tuberculosis, and Mycobacterium bovis bacillus Calmette-Guerin (BCG) and a clinical isolate of Mycobacterium avium-M. intracellulare were examined. The clinical isolate of M. avium-M. intracellulare did not replicate in freshly explanted monocytes (generation time of >400 h); BCG replicated with a generation time of 95 h, and M. tuberculosis strains CDC551, H37Rv, and H37Ra replicated with generation times of 24, 35, and 37 h, respectively, during the 4-day growth assay. When cultured in monocytes for 4 days, the mycobacteria were variably sensitive to H2O2-induced killing. A positive correlation between the generation time and percent killing of intracellular bacilli was observed. By comparison, mycobacterial strains were similarly sensitive to H2O2 treatment in cell-free culture media and in sonicated cell suspensions. Using a number of inhibitors of reactive oxygen intermediates we determined that other than catalase the inhibitors tested did not affect H2O2-induced killing of intracellular mycobacteria. Our studies suggest that the killing of mycobacteria growing in human monocytes in vitro by the addition of exogenous H2O2 is dependent on the susceptibility to a peroxide-induced killing pathway as well as on the intracellular growth rate of the mycobacteria.

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Selected References

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  1. Bortolussi R., Vandenbroucke-Grauls C. M., van Asbeck B. S., Verhoef J. Relationship of bacterial growth phase to killing of Listeria monocytogenes by oxidative agents generated by neutrophils and enzyme systems. Infect Immun. 1987 Dec;55(12):3197–3203. doi: 10.1128/iai.55.12.3197-3203.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Byler R. M., Sherman N. A., Wallner J. S., Horwitz L. D. Hydrogen peroxide cytotoxicity in cultured cardiac myocytes is iron dependent. Am J Physiol. 1994 Jan;266(1 Pt 2):H121–H127. doi: 10.1152/ajpheart.1994.266.1.H121. [DOI] [PubMed] [Google Scholar]
  3. Crowle A. J., Tsang A. Y., Vatter A. E., May M. H. Comparison of 15 laboratory and patient-derived strains of Mycobacterium avium for ability to infect and multiply in cultured human macrophages. J Clin Microbiol. 1986 Nov;24(5):812–821. doi: 10.1128/jcm.24.5.812-821.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Curtis W. E., Muldrow M. E., Parker N. B., Barkley R., Linas S. L., Repine J. E. N,N'-dimethylthiourea dioxide formation from N,N'-dimethylthiourea reflects hydrogen peroxide concentrations in simple biological systems. Proc Natl Acad Sci U S A. 1988 May;85(10):3422–3425. doi: 10.1073/pnas.85.10.3422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Denis M. Interferon-gamma-treated murine macrophages inhibit growth of tubercle bacilli via the generation of reactive nitrogen intermediates. Cell Immunol. 1991 Jan;132(1):150–157. doi: 10.1016/0008-8749(91)90014-3. [DOI] [PubMed] [Google Scholar]
  6. Denis M. Killing of Mycobacterium tuberculosis within human monocytes: activation by cytokines and calcitriol. Clin Exp Immunol. 1991 May;84(2):200–206. doi: 10.1111/j.1365-2249.1991.tb08149.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Flesch I. E., Hess J. H., Oswald I. P., Kaufmann S. H. Growth inhibition of Mycobacterium bovis by IFN-gamma stimulated macrophages: regulation by endogenous tumor necrosis factor-alpha and by IL-10. Int Immunol. 1994 May;6(5):693–700. doi: 10.1093/intimm/6.5.693. [DOI] [PubMed] [Google Scholar]
  8. Flesch I., Kaufmann S. H. Mycobacterial growth inhibition by interferon-gamma-activated bone marrow macrophages and differential susceptibility among strains of Mycobacterium tuberculosis. J Immunol. 1987 Jun 15;138(12):4408–4413. [PubMed] [Google Scholar]
  9. GOTTLIEB S. F., ROSE N. R., MAURIZI J., LANPHIER E. H. OXYGEN INHIBITION OF GROWTH OF MYCOBACTERIUM TUBERCULOSIS. J Bacteriol. 1964 Apr;87:838–843. doi: 10.1128/jb.87.4.838-843.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gordon A. H., Hart P. D. Stimulation or inhibition of the respiratory burst in cultured macrophages in a mycobacterium model: initial stimulation is followed by inhibition after phagocytosis. Infect Immun. 1994 Oct;62(10):4650–4651. doi: 10.1128/iai.62.10.4650-4651.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hawkins C. C., Gold J. W., Whimbey E., Kiehn T. E., Brannon P., Cammarata R., Brown A. E., Armstrong D. Mycobacterium avium complex infections in patients with the acquired immunodeficiency syndrome. Ann Intern Med. 1986 Aug;105(2):184–188. doi: 10.7326/0003-4819-105-2-184. [DOI] [PubMed] [Google Scholar]
  12. Jackett P. S., Aber V. R., Lowrie D. B. Virulence and resistance to superoxide, low pH and hydrogen peroxide among strains of Mycobacterium tuberculosis. J Gen Microbiol. 1978 Jan;104(1):37–45. doi: 10.1099/00221287-104-1-37. [DOI] [PubMed] [Google Scholar]
  13. Jackett P. S., Aber V. R., Lowrie D. B. Virulence of Mycobacterium tuberculosis and susceptibility to peroxidative killing systems. J Gen Microbiol. 1978 Aug;107(2):273–278. doi: 10.1099/00221287-107-2-273. [DOI] [PubMed] [Google Scholar]
  14. Klebanoff S. J., Shepard C. C. Toxic effect of the peroxidase-hydrogen peroxide-halide antimicrobial system on Mycobacterium leprae. Infect Immun. 1984 May;44(2):534–536. doi: 10.1128/iai.44.2.534-536.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kochan I., Pellis N. R., Pfohl D. G. Effects of normal and activated cell fractions on the growth of tubercle bacilli. Infect Immun. 1972 Aug;6(2):142–148. doi: 10.1128/iai.6.2.142-148.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Korchak H. M., Garty B. Z., Stanley C. A., Baker L., Douglas S. D., Kilpatrick L. Impairment of calcium mobilization in phagocytic cells in glycogen storage disease type 1b. Eur J Pediatr. 1993;152 (Suppl 1):S39–S43. doi: 10.1007/BF02072086. [DOI] [PubMed] [Google Scholar]
  17. Kouassi E., Hmama Z., Lina G., Vial J., Faure-Barba F., Normier G., Binz H., Revillard J. P. Activation of human monocyte chemiluminescence response by acylpoly(1,3)galactosides derived from Klebsiella pneumoniae. J Leukoc Biol. 1992 Nov;52(5):529–536. doi: 10.1002/jlb.52.5.529. [DOI] [PubMed] [Google Scholar]
  18. Laochumroonvorapong P., Paul S., Elkon K. B., Kaplan G. H2O2 induces monocyte apoptosis and reduces viability of Mycobacterium avium-M. intracellulare within cultured human monocytes. Infect Immun. 1996 Feb;64(2):452–459. doi: 10.1128/iai.64.2.452-459.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lepay D. A., Steinman R. M., Nathan C. F., Murray H. W., Cohn Z. A. Liver macrophages in murine listeriosis. Cell-mediated immunity is correlated with an influx of macrophages capable of generating reactive oxygen intermediates. J Exp Med. 1985 Jun 1;161(6):1503–1512. doi: 10.1084/jem.161.6.1503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Locksley R. M., Klebanoff S. J. Oxygen-dependent microbicidal systems of phagocytes and host defense against intracellular protozoa. J Cell Biochem. 1983;22(3):173–185. doi: 10.1002/jcb.240220306. [DOI] [PubMed] [Google Scholar]
  21. MITCHISON D. A., SELKON J. B., LLOYD J. VIRULENCE IN THE GUINEA-PIG, SUSCEPTIBILITY TO HYDROGEN PEROXIDE, AND CATALASE ACTIVITY OF ISONIAZID-SENSITIVE TUBERCLE BACILLI FROM SOUTH INDIAN AND BRITISH PATIENTS. J Pathol Bacteriol. 1963 Oct;86:377–386. doi: 10.1002/path.1700860213. [DOI] [PubMed] [Google Scholar]
  22. Marolia J., Mahadevan P. R. Reactive oxygen intermediates inactivate Mycobacterium leprae in the phagocytes from human peripheral blood. Int J Lepr Other Mycobact Dis. 1989 Jun;57(2):483–491. [PubMed] [Google Scholar]
  23. Molloy A., Laochumroonvorapong P., Kaplan G. Apoptosis, but not necrosis, of infected monocytes is coupled with killing of intracellular bacillus Calmette-Guérin. J Exp Med. 1994 Oct 1;180(4):1499–1509. doi: 10.1084/jem.180.4.1499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Molloy A., Meyn P. A., Smith K. D., Kaplan G. Recognition and destruction of Bacillus Calmette-Guerin-infected human monocytes. J Exp Med. 1993 Jun 1;177(6):1691–1698. doi: 10.1084/jem.177.6.1691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Morel D. W., Hessler J. R., Chisolm G. M. Low density lipoprotein cytotoxicity induced by free radical peroxidation of lipid. J Lipid Res. 1983 Aug;24(8):1070–1076. [PubMed] [Google Scholar]
  26. Murray H. W., Cohn Z. A. Macrophage oxygen-dependent antimicrobial activity. III. Enhanced oxidative metabolism as an expression of macrophage activation. J Exp Med. 1980 Dec 1;152(6):1596–1609. doi: 10.1084/jem.152.6.1596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Murray H. W. Interaction of Leishmania with a macrophage cell line. Correlation between intracellular killing and the generation of oxygen intermediates. J Exp Med. 1981 Jun 1;153(6):1690–1695. doi: 10.1084/jem.153.6.1690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Murray H. W., Juangbhanich C. W., Nathan C. F., Cohn Z. A. Macrophage oxygen-dependent antimicrobial activity. II. The role of oxygen intermediates. J Exp Med. 1979 Oct 1;150(4):950–964. doi: 10.1084/jem.150.4.950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Murray H. W., Rubin B. Y., Rothermel C. D. Killing of intracellular Leishmania donovani by lymphokine-stimulated human mononuclear phagocytes. Evidence that interferon-gamma is the activating lymphokine. J Clin Invest. 1983 Oct;72(4):1506–1510. doi: 10.1172/JCI111107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nathan C. F., Murray H. W., Wiebe M. E., Rubin B. Y. Identification of interferon-gamma as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J Exp Med. 1983 Sep 1;158(3):670–689. doi: 10.1084/jem.158.3.670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. North R. J., Izzo A. A. Mycobacterial virulence. Virulent strains of Mycobacteria tuberculosis have faster in vivo doubling times and are better equipped to resist growth-inhibiting functions of macrophages in the presence and absence of specific immunity. J Exp Med. 1993 Jun 1;177(6):1723–1733. doi: 10.1084/jem.177.6.1723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Paul S., Laochumroonvorapong P., Kaplan G. Comparable growth of virulent and avirulent Mycobacterium tuberculosis in human macrophages in vitro. J Infect Dis. 1996 Jul;174(1):105–112. doi: 10.1093/infdis/174.1.105. [DOI] [PubMed] [Google Scholar]
  33. Remick D. G., Villarete L. Regulation of cytokine gene expression by reactive oxygen and reactive nitrogen intermediates. J Leukoc Biol. 1996 Apr;59(4):471–475. doi: 10.1002/jlb.59.4.471. [DOI] [PubMed] [Google Scholar]
  34. Sasada M., Johnston R. B., Jr Macrophage microbicidal activity. Correlation between phagocytosis-associated oxidative metabolism and the killing of Candida by macrophages. J Exp Med. 1980 Jul 1;152(1):85–98. doi: 10.1084/jem.152.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Scully S. P., Segel G. B., Lichtman M. A. Relationship of superoxide production to cytoplasmic free calcium in human monocytes. J Clin Invest. 1986 Apr;77(4):1349–1356. doi: 10.1172/JCI112440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sherman D. R., Mdluli K., Hickey M. J., Arain T. M., Morris S. L., Barry C. E., 3rd, Stover C. K. Compensatory ahpC gene expression in isoniazid-resistant Mycobacterium tuberculosis. Science. 1996 Jun 14;272(5268):1641–1643. doi: 10.1126/science.272.5268.1641. [DOI] [PubMed] [Google Scholar]
  37. SivaSai K. S., Prasad H. K., Misra R. S., Ramesh V., Wilfred D., Nath I. Effect of recombinant interferon gamma administration on lesional monocytes/macrophages in lepromatous leprosy patients. Int J Lepr Other Mycobact Dis. 1993 Jun;61(2):259–269. [PubMed] [Google Scholar]
  38. Styblo K. Overview and epidemiologic assessment of the current global tuberculosis situation with an emphasis on control in developing countries. Rev Infect Dis. 1989 Mar-Apr;11 (Suppl 2):S339–S346. doi: 10.1093/clinids/11.supplement_2.s339. [DOI] [PubMed] [Google Scholar]
  39. Tomioka H., Yamada Y., Saito H., Jidoi J. Susceptibilities of Mycobacterium leprae and M. avium complex to the H2O2-Fe-mediated halogenation system supplemented with antimicrobial agents. Int J Lepr Other Mycobact Dis. 1989 Sep;57(3):628–632. [PubMed] [Google Scholar]
  40. Wilson T. M., de Lisle G. W., Collins D. M. Effect of inhA and katG on isoniazid resistance and virulence of Mycobacterium bovis. Mol Microbiol. 1995 Mar;15(6):1009–1015. doi: 10.1111/j.1365-2958.1995.tb02276.x. [DOI] [PubMed] [Google Scholar]
  41. Young L. S., Inderlied C. B., Berlin O. G., Gottlieb M. S. Mycobacterial infections in AIDS patients, with an emphasis on the Mycobacterium avium complex. Rev Infect Dis. 1986 Nov-Dec;8(6):1024–1033. doi: 10.1093/clinids/8.6.1024. [DOI] [PubMed] [Google Scholar]

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