Neutrophil extracellular traps (NETs) are extracellular structures composed of chromatin and granule proteins that bind and kill microorganisms. We show that upon stimulation, the nuclei of neutrophils lose their shape, and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate, allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death process is distinct from apoptosis and necrosis and depends on the generation of reactive oxygen species (ROS) by NADPH oxidase. Patients with chronic granulomatous disease carry mutations in NADPH oxidase and cannot activate this cell-death pathway or make NETs. This novel ROS-dependent death allows neutrophils to fulfill their antimicrobial function, even beyond their lifespan.

N eutrophil extracellular traps (NETs) are extracellular structures composed of chromatin and granule proteins that bind and kill microorganisms. We show that upon stimulation, the nuclei of neutrophils lose their shape, and the eu-and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate, allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death process is distinct from apoptosis and necrosis and depends on the generation of reactive oxygen species (ROS) by NADPH oxidase. Patients with chronic granulomatous disease carry mutations in NADPH oxidase and cannot activate this cell-death pathway or make NETs. This novel ROS-dependent death allows neutrophils to fulfi ll their antimicrobial function, even beyond their lifespan.

The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns.

A group of T cells recognizes glycolipids presented by molecules of the CD1 family. The CD1d-restricted natural killer T cells (NKT cells) are primarily considered to be self-reactive. By employing CD1d-binding and T cell assays, the following structural parameters for presentation by CD1d were defined for a number of mycobacterial and mammalian lipids: two acyl chains facilitated binding, and a polar head group was essential for T cell recognition. Of the mycobacterial lipids tested, only a phosphatidylinositol mannoside (PIM) fulfilled the requirements for CD1d binding and NKT cell stimulation. This PIM activated human and murine NKT cells via CD1d, thereby triggering antigen-specific IFN-␥ production and cell-mediated cytotoxicity, and PIM-loaded CD1d tetramers identified a subpopulation of murine and human NKT cells. This phospholipid, therefore, represents a mycobacterial antigen recognized by T cells in the context of CD1d. I n contrast to classical MHC molecules, the nonpolymorphic CD1 proteins present lipid antigens to T cells (1). These evolutionaryconserved antigen-presenting molecules are divided into group I (consisting of CD1a, CD1b, CD1c, and CD1e in humans) and group II (represented by CD1d in mice and humans) (2, 3). CD1 molecules are 43-to 49-kDa cell-surface glycoproteins homologous to MHC class I molecules with a limited allelic polymorphism (4). Compared with MHC class I, they possess a deeper and more hydrophobic antigen-binding groove. Human CD1a, CD1b, and CD1c present mammalian and mycobacterial lipids to CD4 and CD8 T cells (2, 5). CD1d-restricted T cells appear to be primarily self-reactive, and they have been implicated in the control of autoimmune diseases (6, 7). The marine sponge-derived lipid ␣-galactosylceramide (␣-GalCer), in the context of CD1d, is a potent stimulator of all V␣14-J␣281 T cell receptor (TCR)-expressing natural killer T cells (NKT cells) in mice and their cognates in humans expressing V␣24-J␣Q TCR. Therefore, although ␣-GalCer is an artificial ligand of unclear physiological relevance, this lipid is a useful tool to study CD1d-restricted NKT cells in mammals (8, 9). The NKT cell subset is considered to perform regulatory, rather than host-defense, functions with the following two self antigens identified so far: phosphatidylinositol (PI) and the tumor-associated disialoganglioside GD3 (10, 11). To our knowledge, no bacterial antigen has been identified, which is presented by CD1d.In analyzing the structural determinants of mycobacterial and mammalian lipids for binding to CD1d and recognition by T cells, we identified a PI mannoside (PIM), which induced IFN-␥ release and cytotoxicity in a CD1d-restricted manner, from the mycobacterial cell wall. Hence, this PIM is a bacterial antigen for human and murine NKT cells. Materials and MethodsChemicals. All reagents were purchased from Sigma, unless indicated otherwise. ␣-GalCer was kindly provided by Pharmaceutical Research Laboratories (Kirin Brewery, Gumna, Japan).Mice. All mice were bred and housed under specific pat...

The gastric pathogen Helicobacter pylori translocates the CagA protein into epithelial cells by a type IV secretion process. Translocated CagA is tyrosine phosphorylated (CagA P-Tyr ) on speci®c EPIYA sequence repeats by Src family tyrosine kinases. Phosphorylation of CagA induces the dephosphorylation of as yet unidenti®ed cellular proteins, rearrangements of the host cell actin cytoskeleton and cell scattering. We show here that CagA P-Tyr inhibits the catalytic activity of c-Src in vivo and in vitro. c-Src inactivation leads to tyrosine dephosphorylation of the actin binding protein cortactin. Concomitantly, cortactin is speci®cally redistributed to actin-rich cellular protrusions. c-Src inactivation and cortactin dephosphorylation are required for rearrangements of the actin cytoskeleton. Moreover, CagA P-Tyr -mediated c-Src inhibition downregulates further CagA phosphorylation through a negative feedback loop. This is the ®rst report of a bacterial virulence factor that inhibits signalling of a eukaryotic tyrosine kinase and on a role of c-Src inactivation in host cell cytoskeletal rearrangements.

The effect of the tricyclic antidepressant desipramine on the processing of lysosomal sphingomyelinase (EC 3.1.4.12) was investigated by pulse-chase studies on [35S]methionine labeled cultured human skin fibroblasts. Desipramine induced rapid intracellular degradation of mature acid sphingomyelinase when added to the cells in the micromolar range, concomitantly abolishing the enzyme activity. Pulse chase labeling revealed the disappearance of mature enzyme forms when fibroblasts were treated with 25 microM desipramine. Incubation of cells with 25 microM leupeptin, an inhibitor of thiol proteases, 24 h prior to desipramine intoxication prevented this drug-induced effect. From these results we conclude that desipramine and possibly also similarly acting tricyclic antidepressants induce proteolytic degradation of acid sphingomyelinase.

Lipids from Mycobacterium tuberculosis are presented through CD1 proteins to T lymphocytes in humans, but the accessory molecules required for antigen loading and presentation remain unidentified. Here we show that fibroblasts deficient in sphingolipid activator proteins (SAPs) transfected with CD1b failed to activate lipid-specific T cells. However, the T cell response was restored when fibroblasts were reconstituted with SAP-C but not other SAPs. Lipid antigen and SAP-C colocalized in lysosomal compartments, and liposome assays showed that SAP-C efficiently extracts antigen from membranes. Coprecipitation demonstrated direct molecular interaction between SAP-C and CD1b. We propose a model in which SAP-C exposes lipid antigens from intralysosomal membranes for loading onto CD1b. Thus, SAP-C represents a missing link in antigen presentation of lipids through CD1b to human T cells.

CD4+ T cell help is important for the generation of CD8+ T cell responses. We used depleting anti-CD4 mAb to analyze the role of CD4+ T cells for memory CD8+ T cell responses after secondary infection of mice with the intracellular bacterium Listeria monocytogenes, or after boost immunization by specific peptide or DNA vaccination. Surprisingly, anti-CD4 mAb treatment during secondary CD8+ T cell responses markedly enlarged the population size of antigen-specific CD8+ T cells. After boost immunization with peptide or DNA, this effect was particularly profound, and antigen-specific CD8+ T cell populations were enlarged at least 10-fold. In terms of cytokine production and cytotoxicity, the enlarged CD8+ T cell population consisted of functional effector T cells. In depletion and transfer experiments, the suppressive function could be ascribed to CD4+CD25+ T cells. Our results demonstrate that CD4+ T cells control the CD8+ T cell response in two directions. Initially, they promote the generation of a CD8+ T cell responses and later they restrain the strength of the CD8+ T cell memory response. Down-modulation of CD8+ T cell responses during infection could prevent harmful consequences after eradication of the pathogen.