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. 1995 May;63(5):1876–1886. doi: 10.1128/iai.63.5.1876-1886.1995

Intracellular killing of Listeria monocytogenes in the J774.1 macrophage-like cell line and the lipopolysaccharide (LPS)-resistant mutant LPS1916 cell line defective in the generation of reactive oxygen intermediates after LPS treatment.

S Inoue 1, S Itagaki 1, F Amano 1
PMCID: PMC173238  PMID: 7729897

Abstract

Listeria monocytogenes is a facultative intracellular pathogen and survives within phagocytic cells by escaping from phagosomes into the cytoplasm. It has been reported that, in vivo, L. monocytogenes is effectively eliminated through cell-mediated immunity, especially by macrophages which have been immunologically activated by cytokines such as gamma interferon (IFN-gamma). However, this killing mechanism for L. monocytogenes and the role of macrophage activation in this bacterial killing are unclear. We demonstrated the listericidal effect of oxidative radicals induced by lipopolysaccharide (LPS) and IFN-gamma, using a macrophage-like cell line, J774.1, and a mutant cell line, LPS1916. LPS1916 cells do not exhibit normal generation of O2- and H2O2 after treatment with 0.1 microgram of LPS per ml, although J774.1 cells generate 100 times the normal level of oxidative radicals with the same LPS treatment. The growth of L. monocytogenes was strongly inhibited in J774.1 cells pretreated with 0.1 microgram of LPS per ml or the combination of 0.1 microgram of LPS per ml and 10 U of IFN-gamma per ml. On the other hand, in LPS1916 cells, the growth of L. monocytogenes was not inhibited by treatment with LPS only, although LPS1916 cells pretreated with the combination of LPS and IFN-gamma showed moderate inhibition of listerial growth. This killing was not influenced by treatment with NG-monomethyl-L-arginine, which is a strong inhibitor of nitrite oxide generation. Interestingly, J774.1 cells treated with LPS did not show enhanced intraphagosomal killing of a nonhemolytic strain of avirulent L. monocytogenes that lacks the ability to escape from phagosomes, and this killing was not influenced by treatment with NG-monomethyl-L-arginine either. These results suggest that the reactive oxygen radicals are more important than nitric oxide in the mechanism underlying the intracellular killing of virulent L. monocytogenes and that there seem to be different killing mechanisms for virulent and avirulent strains of L. monocytogenes in activated-macrophage cell lines.

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Selected References

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  1. Adams L. B., Hibbs J. B., Jr, Taintor R. R., Krahenbuhl J. L. Microbiostatic effect of murine-activated macrophages for Toxoplasma gondii. Role for synthesis of inorganic nitrogen oxides from L-arginine. J Immunol. 1990 Apr 1;144(7):2725–2729. [PubMed] [Google Scholar]
  2. Amano F., Akamatsu Y. A lipopolysaccharide (LPS)-resistant mutant isolated from a macrophagelike cell line, J774.1, exhibits an altered activated-macrophage phenotype in response to LPS. Infect Immun. 1991 Jun;59(6):2166–2174. doi: 10.1128/iai.59.6.2166-2174.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anthony L. S., Morrissey P. J., Nano F. E. Growth inhibition of Francisella tularensis live vaccine strain by IFN-gamma-activated macrophages is mediated by reactive nitrogen intermediates derived from L-arginine metabolism. J Immunol. 1992 Mar 15;148(6):1829–1834. [PubMed] [Google Scholar]
  4. Beattie I. A., Swaminathan B., Ziegler H. K. Cloning and characterization of T-cell-reactive protein antigens from Listeria monocytogenes. Infect Immun. 1990 Sep;58(9):2792–2803. doi: 10.1128/iai.58.9.2792-2803.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beckerman K. P., Rogers H. W., Corbett J. A., Schreiber R. D., McDaniel M. L., Unanue E. R. Release of nitric oxide during the T cell-independent pathway of macrophage activation. Its role in resistance to Listeria monocytogenes. J Immunol. 1993 Feb 1;150(3):888–895. [PubMed] [Google Scholar]
  6. 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]
  7. Campbell P. A., Canono B. P., Cook J. L. Mouse macrophages stimulated by recombinant gamma interferon to kill tumor cells are not bactericidal for the facultative intracellular bacterium Listeria monocytogenes. Infect Immun. 1988 May;56(5):1371–1375. doi: 10.1128/iai.56.5.1371-1375.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cossart P., Vicente M. F., Mengaud J., Baquero F., Perez-Diaz J. C., Berche P. Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation. Infect Immun. 1989 Nov;57(11):3629–3636. doi: 10.1128/iai.57.11.3629-3636.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Czuprynski C. J., Campbell P. A., Henson P. M. Killing of Listeria monocytogenes by human neutrophils and monocytes, but not by monocyte-derived macrophages. J Reticuloendothel Soc. 1983 Jul;34(1):29–44. [PubMed] [Google Scholar]
  10. Czuprynski C. J., Henson P. M., Campbell P. A. Enhanced accumulation of inflammatory neutrophils and macrophages mediated by transfer of T cells from mice immunized with Listeria monocytogenes. J Immunol. 1985 May;134(5):3449–3454. [PubMed] [Google Scholar]
  11. Dabiri G. A., Sanger J. M., Portnoy D. A., Southwick F. S. Listeria monocytogenes moves rapidly through the host-cell cytoplasm by inducing directional actin assembly. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6068–6072. doi: 10.1073/pnas.87.16.6068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Davies W. A. Kinetics of killing Listeria monocytogenes by macrophages: correlation of 3H-DNA release from labeled bacteria and changes in numbers of viable organisms by mathematical model. J Reticuloendothel Soc. 1982 Dec;32(6):461–476. [PubMed] [Google Scholar]
  13. Flesch I. E., Kaufmann S. H. Mechanisms involved in mycobacterial growth inhibition by gamma interferon-activated bone marrow macrophages: role of reactive nitrogen intermediates. Infect Immun. 1991 Sep;59(9):3213–3218. doi: 10.1128/iai.59.9.3213-3218.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gaillard J. L., Berche P., Frehel C., Gouin E., Cossart P. Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci. Cell. 1991 Jun 28;65(7):1127–1141. doi: 10.1016/0092-8674(91)90009-n. [DOI] [PubMed] [Google Scholar]
  15. Gaillard J. L., Berche P., Mounier J., Richard S., Sansonetti P. In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2. Infect Immun. 1987 Nov;55(11):2822–2829. doi: 10.1128/iai.55.11.2822-2829.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Godfrey R. W., Wilder M. S. Relationships between oxidative metabolism, macrophage activation, and antilisterial activity. J Leukoc Biol. 1984 Oct;36(4):533–543. doi: 10.1002/jlb.36.4.533. [DOI] [PubMed] [Google Scholar]
  17. Granger D. L., Hibbs J. B., Jr, Perfect J. R., Durack D. T. Specific amino acid (L-arginine) requirement for the microbiostatic activity of murine macrophages. J Clin Invest. 1988 Apr;81(4):1129–1136. doi: 10.1172/JCI113427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gregory S. H., Wing E. J., Hoffman R. A., Simmons R. L. Reactive nitrogen intermediates suppress the primary immunologic response to Listeria. J Immunol. 1993 Apr 1;150(7):2901–2909. [PubMed] [Google Scholar]
  19. Hage-Chahine C. M., Del Giudice G., Lambert P. H., Pechere J. C. Hemolysin-producing Listeria monocytogenes affects the immune response to T-cell-dependent and T-cell-independent antigens. Infect Immun. 1992 Apr;60(4):1415–1421. doi: 10.1128/iai.60.4.1415-1421.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Havell E. A. Synthesis and secretion of interferon by murine fibroblasts in response to intracellular Listeria monocytogenes. Infect Immun. 1986 Dec;54(3):787–792. doi: 10.1128/iai.54.3.787-792.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hiemstra P. S., Eisenhauer P. B., Harwig S. S., van den Barselaar M. T., van Furth R., Lehrer R. I. Antimicrobial proteins of murine macrophages. Infect Immun. 1993 Jul;61(7):3038–3046. doi: 10.1128/iai.61.7.3038-3046.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Higginbotham J. N., Lin T. L., Pruett S. B. Effect of macrophage activation on killing of Listeria monocytogenes. Roles of reactive oxygen or nitrogen intermediates, rate of phagocytosis, and retention of bacteria in endosomes. Clin Exp Immunol. 1992 Jun;88(3):492–498. doi: 10.1111/j.1365-2249.1992.tb06477.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Igarashi K., Mitsuyama M., Muramori K., Tsukada H., Nomoto K. Interleukin-1-induced promotion of T-cell differentiation in mice immunized with killed Listeria monocytogenes. Infect Immun. 1990 Dec;58(12):3973–3979. doi: 10.1128/iai.58.12.3973-3979.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. James S. L., Glaven J. Macrophage cytotoxicity against schistosomula of Schistosoma mansoni involves arginine-dependent production of reactive nitrogen intermediates. J Immunol. 1989 Dec 15;143(12):4208–4212. [PubMed] [Google Scholar]
  25. Karunasagar I., Krohne G., Goebel W. Listeria ivanovii is capable of cell-to-cell spread involving actin polymerization. Infect Immun. 1993 Jan;61(1):162–169. doi: 10.1128/iai.61.1.162-169.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kathariou S., Pine L., George V., Carlone G. M., Holloway B. P. Nonhemolytic Listeria monocytogenes mutants that are also noninvasive for mammalian cells in culture: evidence for coordinate regulation of virulence. Infect Immun. 1990 Dec;58(12):3988–3995. doi: 10.1128/iai.58.12.3988-3995.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kaufmann S. H. Acquired resistance to facultative intracellular bacteria: relationship between persistence, cross-reactivity at the T-cell level, and capacity to stimulate cellular immunity of different Listeria strains. Infect Immun. 1984 Jul;45(1):234–241. doi: 10.1128/iai.45.1.234-241.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kingdon G. C., Sword C. P. Effects of Listeria monocytogenes Hemolysin on Phagocytic Cells and Lysosomes. Infect Immun. 1970 Apr;1(4):356–362. doi: 10.1128/iai.1.4.356-362.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kuhn M., Goebel W. Identification of an extracellular protein of Listeria monocytogenes possibly involved in intracellular uptake by mammalian cells. Infect Immun. 1989 Jan;57(1):55–61. doi: 10.1128/iai.57.1.55-61.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kuhn M., Kathariou S., Goebel W. Hemolysin supports survival but not entry of the intracellular bacterium Listeria monocytogenes. Infect Immun. 1988 Jan;56(1):79–82. doi: 10.1128/iai.56.1.79-82.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kuhn M., Prévost M. C., Mounier J., Sansonetti P. J. A nonvirulent mutant of Listeria monocytogenes does not move intracellularly but still induces polymerization of actin. Infect Immun. 1990 Nov;58(11):3477–3486. doi: 10.1128/iai.58.11.3477-3486.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kura F., Suzuki K., Watanabe H., Akamatsu Y., Amano F. Difference in Legionella pneumophila growth permissiveness between J774.1 murine macrophage-like JA-4 cells and lipopolysaccharide (LPS)-resistant mutant cells, LPS1916, after stimulation with LPS. Infect Immun. 1994 Dec;62(12):5419–5423. doi: 10.1128/iai.62.12.5419-5423.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Köhler S., Bubert A., Vogel M., Goebel W. Expression of the iap gene coding for protein p60 of Listeria monocytogenes is controlled on the posttranscriptional level. J Bacteriol. 1991 Aug;173(15):4668–4674. doi: 10.1128/jb.173.15.4668-4674.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  35. Leijh P. C., Nathan C. F., van den Barselaar M. T., van Furth R. Relationship between extracellular stimulation of intracellular killing and oxygen-dependent microbicidal systems of monocytes. Infect Immun. 1985 Feb;47(2):502–507. doi: 10.1128/iai.47.2.502-507.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Liew F. Y., Millott S., Parkinson C., Palmer R. M., Moncada S. Macrophage killing of Leishmania parasite in vivo is mediated by nitric oxide from L-arginine. J Immunol. 1990 Jun 15;144(12):4794–4797. [PubMed] [Google Scholar]
  38. MACKANESS G. B. Cellular resistance to infection. J Exp Med. 1962 Sep 1;116:381–406. doi: 10.1084/jem.116.3.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mandel T. E., Cheers C. Resistance and susceptibility of mice to bacterial infection: histopathology of listeriosis in resistant and susceptible strains. Infect Immun. 1980 Dec;30(3):851–861. doi: 10.1128/iai.30.3.851-861.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Marshall N. E., Ziegler H. K. Role of bacterial hemolysin production in induction of macrophage Ia expression during infection with Listeria monocytogenes. J Immunol. 1991 Oct 1;147(7):2324–2332. [PubMed] [Google Scholar]
  41. Mitsuyama M., Igarashi K., Kawamura I., Ohmori T., Nomoto K. Difference in the induction of macrophage interleukin-1 production between viable and killed cells of Listeria monocytogenes. Infect Immun. 1990 May;58(5):1254–1260. doi: 10.1128/iai.58.5.1254-1260.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. North R. J. The relative importance of blood monocytes and fixed macrophages to the expression of cell-mediated immunity to infection. J Exp Med. 1970 Sep 1;132(3):521–534. doi: 10.1084/jem.132.3.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Peck R. Gamma interferon induces monocyte killing of Listeria monocytogenes by an oxygen-dependent pathway; alpha- or beta-interferons by oxygen-independent pathways. J Leukoc Biol. 1989 Nov;46(5):434–440. doi: 10.1002/jlb.46.5.434. [DOI] [PubMed] [Google Scholar]
  44. Portnoy D. A., Jacks P. S., Hinrichs D. J. Role of hemolysin for the intracellular growth of Listeria monocytogenes. J Exp Med. 1988 Apr 1;167(4):1459–1471. doi: 10.1084/jem.167.4.1459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Portnoy D. A., Schreiber R. D., Connelly P., Tilney L. G. Gamma interferon limits access of Listeria monocytogenes to the macrophage cytoplasm. J Exp Med. 1989 Dec 1;170(6):2141–2146. doi: 10.1084/jem.170.6.2141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rosen H., Gordon S., North R. J. Exacerbation of murine listeriosis by a monoclonal antibody specific for the type 3 complement receptor of myelomonocytic cells. Absence of monocytes at infective foci allows Listeria to multiply in nonphagocytic cells. J Exp Med. 1989 Jul 1;170(1):27–37. doi: 10.1084/jem.170.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rácz P., Tenner K., Mérö E. Experimental Listeria enteritis. I. An electron microscopic study of the epithelial phase in experimental listeria infection. Lab Invest. 1972 Jun;26(6):694–700. [PubMed] [Google Scholar]
  48. Stevenson M. M., Kongshavn P. A., Skamene E. Genetic linkage of resistance to Listeria monocytogenes with macrophage inflammatory responses. J Immunol. 1981 Aug;127(2):402–407. [PubMed] [Google Scholar]
  49. Vincendeau P., Daulouède S. Macrophage cytostatic effect on Trypanosoma musculi involves an L-arginine-dependent mechanism. J Immunol. 1991 Jun 15;146(12):4338–4343. [PubMed] [Google Scholar]
  50. Welch D. F., Sword C. P., Brehm S., Dusanic D. Relationship between superoxide dismutase and pathogenic mechanisms of Listeria monocytogenes. Infect Immun. 1979 Mar;23(3):863–872. doi: 10.1128/iai.23.3.863-872.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yoshikawa H., Kawamura I., Fujita M., Tsukada H., Arakawa M., Mitsuyama M. Membrane damage and interleukin-1 production in murine macrophages exposed to listeriolysin O. Infect Immun. 1993 Apr;61(4):1334–1339. doi: 10.1128/iai.61.4.1334-1339.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. van Dissel J. T., Stikkelbroeck J. J., Michel B. C., van den Barselaar M. T., Leijh P. C., van Furth R. Inability of recombinant interferon-gamma to activate the antibacterial activity of mouse peritoneal macrophages against Listeria monocytogenes and Salmonella typhimurium. J Immunol. 1987 Sep 1;139(5):1673–1678. [PubMed] [Google Scholar]

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