Listeriolysin O Is Strongly Immunogenic Independently of Its Cytotoxic Activity

The presentation of microbial protein antigens by Major Histocompatibility Complex (MHC) molecules is essential for the development of acquired immunity to infections. However, most biochemical studies of antigen processing and presentation deal with a few relatively inert non-microbial model antigens. The bacterial pore-forming toxin listeriolysin O (LLO) is paradoxical in that it is cytotoxic at nanomolar concentrations as well as being the source of dominant CD4 and CD8 T cell epitopes following infection with Listeria monocytogenes. Here, we examined the relationship of LLO toxicity to its antigenicity and immunogenicity. LLO offered to antigen presenting cells (APC) as a soluble protein, was presented to CD4 T cells at picomolar to femtomolar concentrations- doses 3000–7000-fold lower than free peptide. This presentation required a dose of LLO below the cytotoxic level. Mutations of two key tryptophan residues reduced LLO toxicity by 10–100-fold but had no effect on its presentation to CD4 T cells. Thus there was a clear dissociation between the cytotoxic properties of LLO and its very high antigenicity. Presentation of LLO to CD8 T cells was not as robust as that seen in CD4 T cells, but still occurred in the nanomolar range. APC rapidly bound and internalized LLO, then disrupted endosomal compartments within 4 hours of treatment, allowing endosomal contents to access the cytosol. LLO was also immunogenic after in vivo administration into mice. Our results demonstrate the strength of LLO as an immunogen to both CD4 and CD8 T cells

The mechanism of cell death in Listeria monocytogenes-infected murine macrophages is distinct from apoptosis.

Various pathogenic bacteria with the capacity to live within eukaryotic cells activate an apoptotic program in infected host cells. Induction of apoptosis by Listeria monocytogenes in murine dendritic cells and hepatocytes has been described. Here we address the questions of whether and how the pathogen kills macrophages, its most important habitat. Employing several complementary techniques aimed at discriminating between apoptosis and necrosis, we show that murine bone marrow-derived macrophages (BMM) undergo delayed necrosis but not apoptosis when infected with listeriolysin (Hly)-producing L. monocytogenes. This pathogen failed to elicit apoptotic morphology, DNA fragmentation, and surface annexin V binding of macrophages, in contrast to Shigella flexneri infection or gliotoxin treatment, which were used as positive controls. Furthermore, macrophages infected with L. monocytogenes released lower quantities of interleukin-1beta (IL-1beta) than did Shigella flexneri-infected ones, indicating diminished or even absent activation of IL-1-converting enzyme in macrophages harboring L. monocytogenes. We conclude that murine BMM die by necrosis after several hours of cytoplasmic replication of L. monocytogenes. The pathogen may benefit from this feature by the possibility of taking advantage of cells of "pseudo-healthy" appearance, thus avoiding rapid elimination by other phagocytes

Potentiation by granulocyte macrophage colony-stimulating factor of lipopolysaccharide toxicity in mice.

GM-CSF is known to prime leukocytes for inflammatory stimuli in vitro. The objective of this study was to investigate the role of GM-CSF in vivo in a systemic inflammatory reaction syndrome. The results demonstrate a potentiation of LPS toxicity by GM-CSF in a mortality model as well as in a septic liver failure model in mice. Pretreatment of animals with 50 micrograms/kg GM-CSF induced lethality within 24 h in mice challenged with a subtoxic dose of LPS while controls survived > 72 h. A monoclonal anti-GM-CSF antibody significantly protected against a lethal LPS dose. Serum GM-CSF was inducible by LPS and peaked at 2 h. GM-CSF pretreatment dramatically potentiated systemic TNF release and hepatotoxicity induced by a subtoxic dose of LPS in galactosamine-sensitized mice. Potentiation of LPS hepatotoxicity was possible until 30 min after LPS challenge. Polyclonal anti-GM-CSF IgG protected against septic liver failure in this model and attenuated serum TNF concentrations. In vitro an ex vivo experiments revealed that after GM-CSF pretreatment LPS-induced IL-1 release from bone marrow or spleen cells was also enhanced. These findings suggest that GM-CSF represents an endogenous enhancer of LPS-induced organ injury, possibly by potentiating the release of proinflammatory cytokines such as TNF and IL-1