Legionnaires' disease bacterium (Legionella pneumophila) multiples intracellularly in human monocytes

We have studied the interaction between virulent egg yolk-grown Legionella pneumophila Philadelphia 1 and human blood monocytes in vitro. The leukocytes were cultured in antibiotic-free tissue culture medium supplemented with 15% autologous human serum.L. pneumophila multiplied several logs, as measured by colony-forming units, when incubated with monocytes or mononuclear cells; the mid-log phase doubling time was 2 h. The level to which L. pneumophila multiplied was proportional to the number of mononuclear cells in the culture. L. pneumophila multiplied only in the adherent fraction of the mononuclear cell population indicating that monocytes but not lymphocytes support growth of the bacteria. Peak growth of L. pneumophila was correlated with destruction of the monocyte monolayer. By fluorescence microscopy using fluorescein conjugated rabbit anti-L. pneumophila antiserum, the number of monocytes containing L. pneumophila increased in parallel with bacterial growth in the culture. At the peak of infection, monocytes were packed full with organisms. By electron microscopy, L. pneumophila in such monocytes were found in membrane-bound cytoplasmic vacuoles studded with structures resembling host cell ribosomes. Several lines of evidence indicate that L. pneumophila grows within monocytes. (a) In the absence of leukocytes, L. pneumophila did not grow in tissue culture medium with or without serum even if the medium was conditioned by monocytes. (b) L. pneumophila did not grow in sonicated mononuclear cells. Lysis of these cells at various times during logarithmic growth of L. pneumophila was followed by cessation of bacterial multiplication. Growth resumed when intact mononuclear cells were added back to the culture. (3) In parabiotic chambers separated by 0.1-mum Nuclepore filters, L. pneumophila multiplied only when placed on the same side of the filter as mononuclear cells. These findings indicate that L. pneumophila falls into a select category of bacterial pathogens that evade host defenses by parasitizing monocytes. It remains to be determined whether cell-mediated immunity plays a dominant role in host defense against L. pneumophila as it does against other intracellular pathogens.