Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1996 Feb;64(2):428–433. doi: 10.1128/iai.64.2.428-433.1996

Nonadherent cultures of human monocytes kill Mycobacterium smegmatis, but adherent cultures do not.

K Barker 1, H Fan 1, C Carroll 1, G Kaplan 1, J Barker 1, W Hellmann 1, Z A Cohn 1
PMCID: PMC173781  PMID: 8550187

Abstract

Human peripheral blood monocytes are permissive for the growth of Mycobacterium tuberculosis, but the fate of nonpathogenic Mycobacterium smegmatis in these cells is not known. Since M. smegmatis may be used as a host with which to express and screen for M. tuberculosis genes needed for survival in monocytes, we determined whether human peripheral blood monocytes could restrict the growth of Mycobacterium smegmatis. Adherent human peripheral blood monocytes were permissive for the growth of M. smegmatis, as measured by ex vivo [3H]uracil uptake. However, human peripheral blood monocytes which were cultured nonadherently in Teflon wells were able to restrict the growth of M. smegmatis while remaining permissive for the growth of M. tuberculosis H37Ra. The loss of viability of M. smegmatis in nonadherent cells was correlated with an increase in nonspacious phagocytic vacuoles. The killing of M. smegmatis was not blocked by NG-monomethyl-L-arginine, suggesting that it was not due to the production of reactive nitrogen intermediates. Incubation of the monocytes for 1 to 7 days before infection had no effect on the fate of M. smegmatis, suggesting that adherence versus nonadherence, and not differentiation, was the key determinant for the difference in functional ability. Nonadherent human peripheral blood monocytes may be a more appropriate model than adherent cells for the study of factors employed by bacterial to survive within monocytes and for selection screening of bacterial genes needed for intracellular survival.

Full Text

The Full Text of this article is available as a PDF (645.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. ALLEN J. M., BRIEGER E. M., REES R. J. ELECTRON MICROSCOPY OF THE HOST-CELL PARASITE RELATION IN MURINE LEPROSY. J Pathol Bacteriol. 1965 Jan;89:301–306. doi: 10.1002/path.1700890131. [DOI] [PubMed] [Google Scholar]
  2. Armstrong J. A., Hart P. D. Phagosome-lysosome interactions in cultured macrophages infected with virulent tubercle bacilli. Reversal of the usual nonfusion pattern and observations on bacterial survival. J Exp Med. 1975 Jul 1;142(1):1–16. doi: 10.1084/jem.142.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Armstrong J. A., Hart P. D. Response of cultured macrophages to Mycobacterium tuberculosis, with observations on fusion of lysosomes with phagosomes. J Exp Med. 1971 Sep 1;134(3 Pt 1):713–740. doi: 10.1084/jem.134.3.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arruda S., Bomfim G., Knights R., Huima-Byron T., Riley L. W. Cloning of an M. tuberculosis DNA fragment associated with entry and survival inside cells. Science. 1993 Sep 10;261(5127):1454–1457. doi: 10.1126/science.8367727. [DOI] [PubMed] [Google Scholar]
  5. Belisle J. T., Pascopella L., Inamine J. M., Brennan P. J., Jacobs W. R., Jr Isolation and expression of a gene cluster responsible for biosynthesis of the glycopeptidolipid antigens of Mycobacterium avium. J Bacteriol. 1991 Nov;173(21):6991–6997. doi: 10.1128/jb.173.21.6991-6997.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Denis M., Forget A., Pelletier M., Gervais F., Skamene E. Killing of Mycobacterium smegmatis by macrophages from genetically susceptible and resistant mice. J Leukoc Biol. 1990 Jan;47(1):25–30. doi: 10.1002/jlb.47.1.25. [DOI] [PubMed] [Google Scholar]
  7. Denis M. Growth of Mycobacterium avium in human monocytes: identification of cytokines which reduce and enhance intracellular microbial growth. Eur J Immunol. 1991 Feb;21(2):391–395. doi: 10.1002/eji.1830210221. [DOI] [PubMed] [Google Scholar]
  8. Denis M. Tumor necrosis factor and granulocyte macrophage-colony stimulating factor stimulate human macrophages to restrict growth of virulent Mycobacterium avium and to kill avirulent M. avium: killing effector mechanism depends on the generation of reactive nitrogen intermediates. J Leukoc Biol. 1991 Apr;49(4):380–387. doi: 10.1002/jlb.49.4.380. [DOI] [PubMed] [Google Scholar]
  9. Douvas G. S., Berger E. M., Repine J. E., Crowle A. J. Natural mycobacteriostatic activity in human monocyte-derived adherent cells. Am Rev Respir Dis. 1986 Jul;134(1):44–48. doi: 10.1164/arrd.1986.134.1.44. [DOI] [PubMed] [Google Scholar]
  10. Dumarey C. H., Labrousse V., Rastogi N., Vargaftig B. B., Bachelet M. Selective Mycobacterium avium-induced production of nitric oxide by human monocyte-derived macrophages. J Leukoc Biol. 1994 Jul;56(1):36–40. doi: 10.1002/jlb.56.1.36. [DOI] [PubMed] [Google Scholar]
  11. Dumont A., Sheldon H. Changes in the fine structure of macrophages in experimentally produced tuberculous granulomas in hamsters. Lab Invest. 1965 Nov;14(11):2034–2055. [PubMed] [Google Scholar]
  12. Flesch I. E., Kaufmann S. H. Attempts to characterize the mechanisms involved in mycobacterial growth inhibition by gamma-interferon-activated bone marrow macrophages. Infect Immun. 1988 Jun;56(6):1464–1469. doi: 10.1128/iai.56.6.1464-1469.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Garbe T., Harris D., Vordermeier M., Lathigra R., Ivanyi J., Young D. Expression of the Mycobacterium tuberculosis 19-kilodalton antigen in Mycobacterium smegmatis: immunological analysis and evidence of glycosylation. Infect Immun. 1993 Jan;61(1):260–267. doi: 10.1128/iai.61.1.260-267.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hart P. D., Young M. R. Interference with normal phagosome-lysosome fusion in macrophages, using ingested yeast cells and suramin. Nature. 1975 Jul 3;256(5512):47–49. doi: 10.1038/256047a0. [DOI] [PubMed] [Google Scholar]
  16. Husson R. N., James B. E., Young R. A. Gene replacement and expression of foreign DNA in mycobacteria. J Bacteriol. 1990 Feb;172(2):519–524. doi: 10.1128/jb.172.2.519-524.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kaplan G. In vitro differentiation of human monocytes. Monocytes cultured on glass are cytotoxic to tumor cells but monocytes cultured on collagen are not. J Exp Med. 1983 Jun 1;157(6):2061–2072. doi: 10.1084/jem.157.6.2061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Leake E. S., Myrvik Q. N. Digestive vacuole formation in alveolar macrophages after phagocytosis of Mycobacterium smegmatis in vivo. J Reticuloendothel Soc. 1966 May;3(1):83–100. [PubMed] [Google Scholar]
  19. Leake E. S., Ockers J. R., Myrvik Q. N. In vitro interactions of the BCG and Ravenel strains of Mycobacterium bovis with rabbit macrophages: adherence of the phagosomal membrane to the bacterial cell wall and the problem of the peribacillary space. J Reticuloendothel Soc. 1977 Aug;22(2):129–147. [PubMed] [Google Scholar]
  20. Mautino G., Paul-Eugène N., Chanez P., Vignola A. M., Kolb J. P., Bousquet J., Dugas B. Heterogeneous spontaneous and interleukin-4-induced nitric oxide production by human monocytes. J Leukoc Biol. 1994 Jul;56(1):15–20. doi: 10.1002/jlb.56.1.15. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Nathan C. F. Neutrophil activation on biological surfaces. Massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes. J Clin Invest. 1987 Dec;80(6):1550–1560. doi: 10.1172/JCI113241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pascopella L., Collins F. M., Martin J. M., Lee M. H., Hatfull G. F., Stover C. K., Bloom B. R., Jacobs W. R., Jr Use of in vivo complementation in Mycobacterium tuberculosis to identify a genomic fragment associated with virulence. Infect Immun. 1994 Apr;62(4):1313–1319. doi: 10.1128/iai.62.4.1313-1319.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pawlowski N. A., Abraham E. L., Pontier S., Scott W. A., Cohn Z. A. Human monocyte-endothelial cell interaction in vitro. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8208–8212. doi: 10.1073/pnas.82.23.8208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pawlowski N. A., Kaplan G., Abraham E., Cohn Z. A. The selective binding and transmigration of monocytes through the junctional complexes of human endothelium. J Exp Med. 1988 Nov 1;168(5):1865–1882. doi: 10.1084/jem.168.5.1865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pawlowski N. A., Kaplan G., Hamill A. L., Cohn Z. A., Scott W. A. Arachidonic acid metabolism by human monocytes. Studies with platelet-depleted cultures. J Exp Med. 1983 Aug 1;158(2):393–412. doi: 10.1084/jem.158.2.393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rook G. A., Steele J., Ainsworth M., Champion B. R. Activation of macrophages to inhibit proliferation of Mycobacterium tuberculosis: comparison of the effects of recombinant gamma-interferon on human monocytes and murine peritoneal macrophages. Immunology. 1986 Nov;59(3):333–338. [PMC free article] [PubMed] [Google Scholar]
  28. Schweizer A., Rohrer J., Hauri H. P., Kornfeld S. Retention of p63 in an ER-Golgi intermediate compartment depends on the presence of all three of its domains and on its ability to form oligomers. J Cell Biol. 1994 Jul;126(1):25–39. doi: 10.1083/jcb.126.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Snapper S. B., Lugosi L., Jekkel A., Melton R. E., Kieser T., Bloom B. R., Jacobs W. R., Jr Lysogeny and transformation in mycobacteria: stable expression of foreign genes. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6987–6991. doi: 10.1073/pnas.85.18.6987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Snapper S. B., Melton R. E., Mustafa S., Kieser T., Jacobs W. R., Jr Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol. 1990 Nov;4(11):1911–1919. doi: 10.1111/j.1365-2958.1990.tb02040.x. [DOI] [PubMed] [Google Scholar]
  31. Stach J. L., Gros P., Forget A., Skamene E. Phenotypic expression of genetically-controlled natural resistance to Mycobacterium bovis (BCG). J Immunol. 1984 Feb;132(2):888–892. [PubMed] [Google Scholar]
  32. Stevenson H. C., Schlick E., Griffith R., Chirigos M. A., Brown R., Conlon J., Kanapa D. J., Oldham R. K., Miller P. Characterization of biological response modifier release by human monocytes cultured in suspension in serum-free medium. J Immunol Methods. 1984 May 25;70(2):245–255. doi: 10.1016/0022-1759(84)90189-3. [DOI] [PubMed] [Google Scholar]
  33. Sturgill-Koszycki S., Schlesinger P. H., Chakraborty P., Haddix P. L., Collins H. L., Fok A. K., Allen R. D., Gluck S. L., Heuser J., Russell D. G. Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science. 1994 Feb 4;263(5147):678–681. doi: 10.1126/science.8303277. [DOI] [PubMed] [Google Scholar]
  34. Tsai V., Firestein G. S., Arend W., Zvaifler N. J. Cytokine-induced differentiation of cultured nonadherent macrophages. Cell Immunol. 1992 Oct 1;144(1):203–216. doi: 10.1016/0008-8749(92)90237-j. [DOI] [PubMed] [Google Scholar]
  35. Vidal S. M., Malo D., Vogan K., Skamene E., Gros P. Natural resistance to infection with intracellular parasites: isolation of a candidate for Bcg. Cell. 1993 May 7;73(3):469–485. doi: 10.1016/0092-8674(93)90135-d. [DOI] [PubMed] [Google Scholar]
  36. Wright S. D., Silverstein S. C. Tumor-promoting phorbol esters stimulate C3b and C3b' receptor-mediated phagocytosis in cultured human monocytes. J Exp Med. 1982 Oct 1;156(4):1149–1164. doi: 10.1084/jem.156.4.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zembala M., Siedlar M., Marcinkiewicz J., Pryjma J. Human monocytes are stimulated for nitric oxide release in vitro by some tumor cells but not by cytokines and lipopolysaccharide. Eur J Immunol. 1994 Feb;24(2):435–439. doi: 10.1002/eji.1830240225. [DOI] [PubMed] [Google Scholar]
  38. Zhang Y., Heym B., Allen B., Young D., Cole S. The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature. 1992 Aug 13;358(6387):591–593. doi: 10.1038/358591a0. [DOI] [PubMed] [Google Scholar]
  39. Zhang Y., Lathigra R., Garbe T., Catty D., Young D. Genetic analysis of superoxide dismutase, the 23 kilodalton antigen of Mycobacterium tuberculosis. Mol Microbiol. 1991 Feb;5(2):381–391. doi: 10.1111/j.1365-2958.1991.tb02120.x. [DOI] [PubMed] [Google Scholar]
  40. van der Meer J. W., Bulterman D., van Zwet T. L., Elzenga-Claasen I., van Furth R. Culture of mononuclear phagocytes on a teflon surface to prevent adherence. J Exp Med. 1978 Jan 1;147(1):271–276. doi: 10.1084/jem.147.1.271. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES