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Infection and Immunity, November 2002, p. 6456-6459, Vol. 70, No. 11
0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.11.6456-6459.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Departments of Medicine,1 Pathology, University Hospitals of Cleveland and Case Western Reserve University,3 Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 441092
Received 19 February 2002/
Returned for modification 24 April 2002/
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ATP-induced lysis in monocytes.
First, the concentration of extracellular ATP required to induce P2X7 signaling was determined by measuring ATP-induced cytolysis of blood monocytes (MN). Extracellular ATP induces apoptosis and lysis through P2X7 signaling in macrophages (3, 21). For all experiments, MN from normal donors were purified with immunomagnetic beads by negative selection (monocyte isolation kit; Miltenyi) and pretreated with gamma interferon (IFN-
) (100 U/ml) for 2 days to upregulate P2X7 receptor expression, thereby increasing sensitivity to ATP. The degree of ATP-induced lysis was determined by release of 51Cr from MN 4 h after ATP exposure (Fig. 1A). Both donors responded maximally to 3 mM ATP, while exhibiting intermediate responses to 1 mM ATP and no response to 0.3 mM ATP. Thus, an ATP concentration range of 1 to 3 mM was found to be optimal for the killing of both Mycobacterium bovis BCG and M. tuberculosis in human macrophages (15, 17). ATP antagonists were found to completely inhibit the lysis of macrophages (Fig. 1B).
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These data do not exclude the possibility that vectorial ATP release into the small volume of an "immunologic synapse" can activate macrophages through P2X7 to kill mycobacteria. However, the low ATP concentrations are unlikely to be adequate for signaling the P2X7 receptor long enough, given the high ecto-ATPase activity on the surfaces of activated T cells. ATP antagonists, KN-62 and oxidized ATP, were found to have nonspecific inhibitory effects in 51Cr macrophage T-cell lysis assays and growth restriction assays. Thus, they could not be used to determine whether the small amounts of ATP after T-cell degranulation reached a concentration sufficient to signal the P2X7 receptor.
Bystander ATP does not inhibit BCG growth. T cells may also generate extracellular ATP by lysing cells, which precipitates release of cytosolic ATP in the immediate vicinity of infected macrophages. As cytoplasmic ATP concentrations are 3 to 5 mM, bystander cells may be a significant source of ATP for signaling through purinergic receptors such as P2X7 (7, 11, 14).
Two experimental systems were established to determine if bystander ATP, which is released when T cells lyse target cells, affects BCG growth in MN. They were designed to generate extracellular ATP by lysis of bystander cells. The acute myeloid leukemia cell line KG-1 (American Type Culture Collection, Manassas, Va.), served as an ATP donor cell line because it has low ecto-ATPase activity (6). In the first experimental system, pretreatment of KG-1 cells with UV irradiation for 30 min induced lysis over the course of several hours. In the second, KG-1 cells were pretreated with the superantigen staphylococcal enterotoxin B (SEB), which binds their major histocompatibility complex class II molecules to label them as selective targets for activated T-cell lines. The maximum number of KG-1 cells that could be supported by the tissue medium was used. In each experimental system, KG-1 cells were in intimate contact with BCG-infected MN at the time of lysis to create a microenvironment where ATP concentrations would be highest locally.
Table 2 presents the concentrations of ATP and total concentrations of ATP plus ADP plus AMP detected in supernatants by these two experimental systems. Nucleotide levels were measured by rephosphorylation assays. By 3 h, UV-treated KG-1 cells were lysed by 50%, as measured by 51Cr release, and concentrations of total nucleotide (ATP plus ADP plus AMP) reached 2.6 µM. After incubation with T cells, 24 to 50% of SEB-treated KG-1 cells, measured by a 51Cr release assay, were lysed after 4 h. The lower nucleotide concentrations in cultures of T cells with SEB-treated KG-1 cells compared to those with KG-1 cells alone may have been due to the high ecto-ATPase activity of activated T cells.
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-treated MN were infected with BCG at a 1:1 multiplicity of infection. After the cells were washed extensively, KG-1 cells, T cells, or ATP was added and the solution was briefly centrifuged to maximize cell contact. After overnight culture, cells were lysed with saponin (0.2%) and the number of CFU/well was measured. Figure 2A demonstrates that there were no reductions in the numbers of BCG CFU in experimental groups when lysed KG-1 cells served as an extracellular source of ATP. Figure 2B demonstrates the mean changes in the numbers of CFU in the experimental groups expressed as percentages relative to the number of CFU in infected MN alone. For the three donors, there was no statistically significant difference in numbers of CFU by paired t test between UV-treated and nontreated wells or between SEB-KG-1 cell-treated and nontreated wells. To constitute a positive control, BCG-infected MN were treated with extracellular ATP, which resulted in a significant reduction in growth.
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| ACKNOWLEDGMENTS |
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This work is funded by National Institutes of Health grants K08 AI 01581 (D.H.C.) and AI 27243 (W.H.B.) and Tuberculosis Research Unit grants AI 95383 and GM 36387 (G.R.D.) and HL 59858 (R.F.S.). This work was also supported in part by the Center for AIDS Research at Case Western Reserve/University Hospitals of Cleveland (grant AI-36219).
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