Functional activity of individual abscess neutrophils from mice

In the absence of antibiotic therapy, viable bacteria can persist within intra-abdominal abscesses in mice for at least 10 weeks. The mechanisms contributing to this survival are unknown, but abscess-derived neutrophils have impaired abilities to kill, in vitro, organisms engulfed in vivo. In order to determine whether subpopulations of abscess neutrophils might be discernible on the basis of phenotypic or functional criteria, cells from murine intra-abdominal abscesses were examined for phagocytic activity, CR3 expression, and H₂O₂ production in response to soluble and particulate stimuli. With respect to phagocytosis of Proteus mirabilis, abscess cells were no less efficient than peritoneal exudate neutrophils; no significant subpopulation of cells was incapable of phagocytosis in the presence of normal mouse serum. Using flow cytometry to examine abscess neutrophils for CR3 expression, we found that no subpopulations of cells were observed with unstimulated cells or with cells incubated with either phorbol 12-myristate 13-acetate or bacteria and serum. Intracellular H₂O₂ levels were measured by using the probe 2',7'-dichlorofluorescein diacetate. In general, incubation with phorbol 12-myristate 13-acetate resulted in similar increases in H₂O₂ production in all cells of the population. However, stimulation with bacteria and serum revealed a variable but consistent, poorly responsive subpopulation of neutrophils in abscess cell populations. Cell-sorting experiments showed that cells from the poorly responsive section of the FACS profile contained significantly higher numbers of abscess-derived bacteria, suggesting the presence of a subpopulation of viable abscess neutrophils harboring persisting viable bacteria.