Measurement of bacterial ingestion and killing by macrophages.

This unit presents fairly simple assays for measuring the binding of bacteria to macrophages, internalization of bacteria (also called ingestion or phagocytosis), and bacterial killing by macrophages. The first basic protocol describes how to measure the ability of macrophages to ingest bacteria. Because it is critical to remove residual extracellular organisms, the protocol presents two alternative steps to accomplish this: a washing procedure and a more stringent method in which cells are sedimented through sucrose. In addition, it is important to distinguish those bacteria truly ingested by a macrophage from those that are bound to, but not internalized by, the cell. A simple but effective way to do this is described in an alternate protocol. The unit also presents two ways to measure the ability of a macrophage to kill bacteria it has internalized. The first is a straightforward assay in which bacterial colonies are enumerated before and after a killing period; a subsequent colony count will indicate whether the bacteria grew within or were killed by the macrophage. The second protocol describes a way to measure bacterial viability based on bacterial metabolism, in which the ability of bacterial dehydrogenases to mediate the reduction of a tetrazolium salt to purple formazan is monitored by measuring absorbance spectrophotometrically.

TNF-alpha and IFN-gamma stimulate a macrophage precursor cell line to kill Listeria monocytogenes in a nitric oxide-independent manner.

Macrophages are important effector cells for resolving infection with the facultative intracellular bacterium Listeria monocytogenes. However, not all macrophages have the ability to kill this organism. Certain factors, such as cytokines, are apparently required for induction of macrophage bactericidal activity. In vivo studies have shown that both TNF-alpha and IFN-gamma play important roles in resistance against Listeria. Yet whether they act directly on macrophages has been difficult to determine, because homogeneous populations of cells that can be induced to express microbicidal activity have not been available. Instead, bactericidal macrophages are typically found in heterogeneous exudates, such as those elicited by inflammatory agents. In this study we show that sequential stimulation with TNF-alpha and IFN-gamma induces the nonphagocytic, nonbactericidal mouse macrophage precursor hybrid cell line W1C3 to phagocytose and kill Listeria efficiently. This provides the first direct evidence that TNF-alpha and IFN-gamma are both necessary and sufficient to induce macrophages to kill Listeria, and that they act directly on macrophages. Data presented here also show that TNF-alpha and IFN-gamma induced the macrophages to produce large amounts of reactive nitrogen intermediates (RNI), but complete inhibition of RNI generation did not decrease bactericidal activity. This indicates that induction of listericidal activity in these cells does not require generation of RNI. Taken together, these findings suggest that TNF-alpha and IFN-gamma act in synergy directly on at least some macrophages to induce them to express listericidal activity in a RNI-independent manner.

Gentamicin kills intracellular Listeria monocytogenes.

The purpose of the experiments described here was to test whether membrane-impermeant antibiotics present in the extracellular milieu could kill bacteria within macrophages. For this, mouse macrophage hybrids and elicited mouse peritoneal macrophages first were allowed to phagocytose the facultative intracellular bacterium Listeria monocytogenes. The cells were incubated with or without gentamicin, and their bactericidal activity was measured. The results show that gentamicin caused normally nonbactericidal macrophages to kill L. monocytogenes. In addition, gentamicin caused listericidal cells to kill significantly more bacteria. To determine whether gentamicin accumulated within macrophages during culture, we tested whether lysates of macrophage hybrids cultured for 72 h in gentamicin-containing medium and then washed could kill Listeria cells. When cultured with 50 to 100 micrograms of gentamicin per ml, but not when cultured with 0 to 5 micrograms of gentamicin per ml, cell lysates were extremely listericidal, demonstrating the presence of intracellular gentamicin. Because gentamicin does not penetrate cell membranes, we hypothesized that it can be internalized by the cell through pinocytosis and can enter the same intracellular compartment as does phagocytosed L. monocytogenes. To test this, macrophages which had phagocytosed L. monocytogenes were incubated with the fluorochrome lucifer yellow to trace pinocytosed medium. About half of the Listeria cells within the macrophages were surrounded by lucifer yellow, indicating delivery of pinocytosed fluid, which could contain antibiotics, to phagosomes containing bacteria. The experiments described here indicate that membrane-impermeant antibiotics can enter macrophages and kill intracellular bacteria. Thus, the use of gentamicin in macrophage bactericidal assays can interfere with the results and interpretation of experiments designed to study macrophage bactericidal activity.

Listericidal and nonlistericidal mouse macrophages differ in complement receptor type 3-mediated phagocytosis of L. monocytogenes and in preventing escape of the bacteria into the cytoplasm.

Listeria monocytogenes is a facultative intracellular bacterium that escapes phagocytic vesicles and replicates in the cytoplasm, where it becomes coated with F-actin. Macrophages, important anti-Listeria effector cells, are heterogeneous in their ability to kill Listeria. Complement receptor type 3 (CR3) mediates most phagocytosis of Listeria by listericidal macrophages. Experiments described here tested whether nonlistericidal macrophages also phagocytosed Listeria through CR3 and whether the ability of Listeria to escape into the cytoplasm correlated with lack of listericidal activity. We show here that CR3 mediated an average of 66% of the phagocytosis of serum-opsonized Listeria by listericidal peptone-elicited macrophages but only 35% by nonlistericidal thioglycolate-elicited macrophages. In thioglycolate-elicited macrophages, most Listeria were cytoplasmic and actin coated, whereas in peptone-elicited macrophages most were retained in the phagosome. These results indicate that listericidal and nonlistericidal macrophages phagocytose Listeria through different receptors and that nonlistericidal macrophages allow Listeria to escape into the cytoplasm.

Recombinant mouse interferon-gamma is not chemotactic for macrophages or neutrophils.

Recent evidence implicates interferon-gamma (IFN-gamma) in resistance to infection with the facultative intracellular bacterium, Listeria monocytogenes. Reports showing that inflammatory macrophages and neutrophils are highly bactericidal for listeria suggest that IFN-gamma might act by directly or indirectly causing recruitment of these cells. The purpose of the experiments described here was to test whether recombinant mouse IFN-gamma is chemotactic for macrophages or neutrophils in vivo or in vitro. In vivo experiments showed rIFN-gamma to have no ability to cause recruitment of inflammatory neutrophils or macrophages into the peritoneal cavities of mice. When tested in vitro, rIFN-gamma also did not induce migration of inflammatory neutrophils or macrophages through cellulose nitrate filters in a chemotaxis assay. The data indicate that rIFN-gamma has no direct or indirect chemotactic activity in vivo or in vitro for mouse macrophages or neutrophils.

Mouse macrophages stimulated by recombinant gamma interferon to kill tumor cells are not bactericidal for the facultative intracellular bacterium Listeria monocytogenes.

Data presented here demonstrate that recombinant gamma interferon (rIFN-gamma) activated a single population of 10% fetal calf serum-elicited mouse peritoneal exudate cells to express tumoricidal activity but not bactericidal activity for the facultative intracellular bacterium Listeria monocytogenes. Fetal calf serum-elicited cells incubated with rIFN-gamma phagocytosed listeriae normally, suggesting that their inability to kill this bacterium is not because they cannot phagocytose it. Data also show that proteose peptone-elicited peritoneal exudate cells, which are bactericidal but not tumoricidal, acquired tumoricidal activity but lost bactericidal activity following incubation overnight with rIFN-gamma. These experiments show that under conditions sufficient for rIFN-gamma to induce macrophages to express tumoricidal activity, the same cell population does not express bactericidal activity for the facultative intracellular bacterium L. monocytogenes. This suggests that mechanisms responsible for these two biological activities may be different.

Silica decreases phagocytosis and bactericidal activity of both macrophages and neutrophils in vitro.

Silica, or silicon dioxide, has been shown to be toxic for macrophages. This is probably because it damages phagolysosomal membranes, allowing lysosomal enzymes to disrupt the cell. Neutrophils also take up particles such as silica and in addition they contain lysosomes. The purpose of this study was to determine whether incubation in vitro with silica inhibits function not only of mouse macrophages, but also of mouse neutrophils. The data show that incubation with silica for 1-3 hr decreases viability of both macrophages and neutrophils. Silica decreases the ability of macrophages and neutrophils to phagocytose both erythrocytes and bacteria, and it inhibits the ability of both cells populations to kill the facultative intracellular bacterium Listeria monocytogenes. Thus, it appears that silica, at least in vitro, is harmful to neutrophils as well as to macrophages.

Genetically determined resistance to listeriosis is associated with increased accumulation of inflammatory neutrophils and macrophages which have enhanced listericidal activity.

The C57BL/6 and A/J inbred strains of mice differ markedly in their resistance to the facultative intracellular bacterium Listeria monocytogenes. One possible explanation for this genetically determined resistance is that phagocytes from Listeria-resistant strains of mice can kill L. monocytogenes more effectively than phagocytes from Listeria-susceptible strains of mice. We report here that inflammatory neutrophils and macrophages from Listeria-resistant mice (C57BL/6) exhibit a slight but significantly enhanced ability to kill L. monocytogenes in vitro as compared to inflammatory phagocytes from Listeria-susceptible mice (A/J). More importantly, however, Listeria-resistant mice recruited more inflammatory neutrophils and macrophages to the peritoneal cavity in response to i.p. injection of heat-killed Listeria than did Listeria-susceptible mice. These data suggest that genetically determined resistance to listeriosis is dependent on the enhanced inflammatory responsiveness of Listeria-resistant mice. Further support for this hypothesis was provided by experiments in which the passive transfer to A/J mice (C5-deficient) of plasma from C57BL/6 mice (C5-sufficient) enhanced the ability of the recipient A/J mice both to recruit inflammatory neutrophils to the peritoneal cavity in response to i.p. injection of heat-killed Listeria, and to clear L. monocytogenes from the spleen after a sublethal challenge of viable Listeria.