Gene dosage determines the negative effects of polymorphic alleles of the P2X7 receptor on adenosine triphosphate-mediated killing of mycobacteria by human macrophages

Background. Stimulation of the P2X(7) purinergic receptor (P2X(7)) in bacille Calmette- Guerin (BCG)-infected human macrophages with extracellular adenosine triphosphate (ATP) leads to pore formation and killing of mycobacteria. We examined the effect of polymorphisms in the P2X(7) gene (P2X7) on the capacity of macrophages to kill mycobacteria. Methods. Polymorphisms and mutations in P2X7 were identified by both DNA sequence analysis and determination of uptake of ethidium by time-resolved flow cytometry. Macrophages from affected subjects were infected with Mycobacterium bovis BCG. Apoptosis was determined by use of Annexin V staining, and BCG growth was determined by use of quantitative mycobacterial cultures. Results. Three new mutations were identified. Macrophages from subjects heterozygous for a polymorphism in P2X7 had a 50% reduction in uptake of ethidium and a 75% reduction in the number of apoptotic cells, compared with macrophages from wild-type (wt) subjects, after stimulation with interferon (IFN)-gamma and ATP. Furthermore, after stimulation with IFN-gamma and ATP, there was a reduction in BCG growth of up to similar to 0.5 log(10) in macrophages from single-heterozygous subjects, compared with a reduction of 1.0 log(10) in macrophages from wt subjects. Interestingly, BCG-infected macrophages from compound-heterozygous subjects, for different combinations of polymorphisms in P2X7, had no uptake of ethidium, failed to undergo apoptosis, and were unable to kill mycobacteria after stimulation with IFN-g and ATP. Conclusions. Various polymorphisms in P2X7 abrogate IFN-gamma/ATP-induced killing of mycobacteria by human macrophages and, thus, may contribute to variability in susceptibility to mycobacterial infections.