FIGURE 4.

Extracellular adenosine rescues the ability of PMNs from old mice to kill Streptococcus pneumoniae. (a) PMNs were isolated from the bone marrow of young and old C57BL/6 mice as well as CD73^−/− young mice and incubated in assay buffer for 5 min. The amount of adenosine in the supernatants was then measured. *Significant differences calculated by Student's t‐test. (b) Bone marrow PMNs isolated from young and old C57BL/6 mice were infected with S. pneumoniae pre‐opsonized with sera from the same mouse for 45 min at 37°C. The amount of adenosine in the supernatants was then measured and the fold change in extracellular adenosine production was calculated by dividing the values of infected reactions by uninfected controls for each condition. *Significantly different from 1 by one‐sample t‐test. (a, b) are representative data from one of three separate experiments, where each condition was tested in quadruplicate. (c) PMNs were isolated from the bone marrow of young and old C57BL/6 mice and treated with 1 μM Adenosine (+ADE) or PBS (VC) for 30 min at 37°C. The reactions were then infected with pre‐opsonized S. pneumoniae for 45 min at 37°C. Reactions were stopped on ice, and viable CFU were determined after serial dilution and plating. The percentage of bacteria killed upon incubation with PMNs was determined by comparing surviving CFU to a no PMN control. Data shown are pooled from three separate experiments (n = 3 biological replicates or mice per strain) where each condition was tested in triplicate (n = 3 technical replicates) per experiment. *Significant differences calculated by one‐way ANOVA followed by Tukey's test