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Reactive nitrogen intermediates (RNI) derived from L-arginine have been implicated as important anti-bacterial agents in the control of Listeria monocytogenes by murine macrophages. However, not all evidence is consistent with this conclusion. In the present study, this issue was examined using a simple experimental system to assess the correlation between macrophages Listericidal activity and production of nitrite (a stable end product of RNI) in culture. Various levels of nitrite production were achieved by activating macrophages with interferon-gamma (IFN-gamma) (20 or 500 U/ml) with or without lipopolysaccharide (LPS) (10 ng/ml) for 20 h before the Listericidal assay, and by using normal and arginine-free culture medium during the Listericidal assay. Nitrite concentration was measured for the same wells used to assess Listericidal activity. There was essentially no correlation between initial or final nitrite concentration and Listericidal activity in resident peritoneal macrophages. Significant correlations were noted between initial and final nitrite concentration and Listericidal activity in proteose peptone-elicited peritoneal macrophages. However, the correlation coefficients (0.34 and 0.52) suggested marginal biological relevance. In addition, no correlation was noted when LPS-activated macrophages were omitted from analysis. A previous study suggested that the enhanced Listericidal activity of LPS-treated macrophages could be accounted for by an enhanced rate of phagocytosis during the initial phase of the assay. These results suggest RNI are probably not the predominant bactericidal agents used by macrophages from female CD-1 mice to kill L. monocytogenes. However, it remains possible that RNI are important anti-bacterial agents in highly activated (LPS-treated) macrophages, and that there are other mechanisms whereby RNI contribute to host resistance to L. monocytogenes.

The role of macrophage activation in the killing of L. monocytogenes is unclear. Some studies suggest that activation for enhanced production of reactive oxygen and nitrogen intermediates may not be of central importance. Recent data have indicated an important role for interferon-gamma (IFN-gamma) induced retention of L. monocytogenes in endosomes. Data from the present study indicate that proteose peptone-elicited macrophages from DBA2/J, CD-1, and C3H/HeN mice are listericidal. Activation of these cells in vitro for 20 h by IFN-gamma (20 or 500 U/ml) increased H2O2 or nitrite production, but did not increase the number of L. monocytogenes killed during a subsequent 6-h or 7-h culture. Incubation of macrophages with IFN-gamma plus lipopolysaccharide (LPS) caused greater activation and increased the number of Listeria killed during a 6-h or 7-h culture. However, this seems primarily attributable to enhanced phagocytosis. Proteose peptone-elicited macrophages were significantly more effective than resident macrophages in preventing the escape of L. monocytogenes from endosomes into the cytoplasm. This capability was not significantly enhanced by IFN-gamma in vitro, but was enhanced by IFN-gamma plus LPS. This correlates well with the effects of these activation stimuli on killing of L. monocytogenes by proteose peptone-elicited macrophages. These results indicate that enhanced retention of L. monocytogenes in endosomes is induced by proteose peptone elicitation and that further macrophage activation in vitro by IFN-gamma does not improve listericidal activity.

A number of investigators have reported that human serum inhibits the proliferation of IL-2-dependent mouse cells in IL-2 bioassays, but the mechanism of inhibition has not been carefully examined. We noticed that IL-2-dependent mouse cells (CTLL-2) are killed within 30 min in the presence of a 1/10 dilution of human serum. However, CTLL-2 cells totally deprived of IL-2 did not begin to die until at least 6 h in culture. Thus, even complete inhibition of IL-2 by human serum could not account for the rapid cytotoxicity caused by human serum. Since humans have 'natural' antibodies which react with mouse cells, it seemed possible that the cytotoxicity was due to antibody/complement-mediated cell lysis. This was supported by the observation that EDTA (at a concentration sufficient to inhibit complement) protected CTLL-2 cells from the cytotoxic effects of human sera from four normal donors. In addition, preincubation of CTLL-2 cells with heat-inactivated human sera at 4 degrees C rendered them much more susceptible to lysis with rabbit complement than cells which were preincubated with complete culture medium. The cytotoxicity of human serum is not limited to IL-2-dependent mouse cells but was also observed with EL4 and Ag8.653 cells as well as normal splenocytes. The cytotoxic effect of human serum was lost upon removal of IgM, but not upon removal of IgG. These results strongly suggest that the inhibition of proliferation of IL-2-dependent mouse cells by human serum is due to antibody/complement-mediated lysis of those cells. In addition, non-heat-inactivated human serum did not inhibit the IL-2-mediated proliferation of human PHA blasts, indicating that there is no inherent inhibitory activity in human serum apart from the cytotoxic effect on xenogeneic cells. Thus the reported IL-2 inhibitory activity of whole human serum is probably not biologically relevant.