The Lymphocyte Dependent Bactericidal Assay of Human Monocyte and Alveolar Macrophage for Mycobacteria

Cheon, Seon Hee; Lee, You Hyun; Lee, Jong Soo; Bae, Ki Sun; Shin, Sue Yeon

Abstract

BACKGROUND: Though mononuclear phagocytes serve as the final effectors in killing intracellular Mycobacterium tuberculosis, the bacilli readily survive in the intracellular environment of resting cells. The mechanisms through which cellular activation results in the intracellular killing is unclear. In this study, we sought to explore an in vitro model of a low-level infection of human mononuclear phagocytes with MAC and H37Ra and determine the extent of the lymphocyte dependent cytotoxicity of human monocytes and alveolar macrophages. METHOD: The peripheral monocytes were prepared using the Ficoll gradient method from PPD positive healthy people and tuberculosis patients. The alveolar macrophages were prepared from PPD positive healthy people via a bronchoalveolar lavage. The human mononuclear phagocytes were infected at a low infection rate (bacilli:phagocyte 1:10) with MAC(Mycobacterium avium) and Mycobacterium tuberculosis H37Ra. Non-adherent cells(lymphocyte) were added at a 10:1 ratio. After 1,4, and 7 days culture in 37degrees C, 5% CO2 incubator, the cells were harvested and inoculated in a 7H10/OADC agar plate for the CFU assay. The bacilli were calculated with the CFU/1 X 10(6) of the cells and the cytotoxicity was expressed as the log killing ratio. RESULTS: The intracellular killing of MAC and H37Ra within the monocyte was greater in patients with tuberculosis compared to the PPD positive controls (p<0.05). Intracellular killing of MAC and H37Ra within the alveolar macrophage appeared to be greater than that within the monocytes of the PPD positive controls. There was significant lymphocyte dependent inhibition of intracellular growth of the mycobacteria within the monocytes in both the controls and tuberculosis patients and within the macrophages in the controls(p<0.05). There was no specific difference in the virulence between the MAC and the H37Ra. CONCLUSION: This study is an in vitro model of a low-level infection with MAC and H37Ra of human mononuclear phagocytes. The intracellular cytotoxicity of the mycobacteria within the phagocytic cells was significantly lymphocyte dependent. During the 7 days culture after the intracellular phagocytosis, the actual confinement of the mycobacteria was observed within the monocytes of tuberculosis patients and the alveolar macrophages of the controls as in the case of adding lymphocytes.