To determine the function of immunoglobulin (Ig)α immunoreceptor tyrosine–based activation motif (ITAM) phosphorylation, we generated mice in which Igα ITAM tyrosines were replaced by phenylalanines (IgαFF/FF). IgαFF/FFmice had a specific reduction of B1 and marginal zone B cells, whereas B2 cell development appeared to be normal, except that λ1 light chain usage was increased. The mutants responded less efficiently to T cell–dependent antigens, whereas T cell–independent responses were unaffected. Upon B cell receptor ligation, the cells exhibited heightened calcium flux, weaker Lyn and Syk tyrosine phosphorylation, and phosphorylation of Igα non-ITAM tyrosines. Strikingly, when the Igα ITAM mutation was combined with a truncation of Igβ, B cell development was completely blocked at the pro-B cell stage, indicating a crucial role of ITAM phosphorylation in B cell development.

The purpose of the experiments described here was to test whether membrane-imperneant 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 ,ug of gentamicin per ml, but not when cultured with 0 to 5 ,ug 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.

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.

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.