Human dendritic cells process and present Listeria antigens for in vitro priming of autologous CD4+ T lymphocytes

The role of human dendritic cells (DC) in the immune response toward intracellularly growing Listeria was analyzed under in vitro conditions using several morphological and functional methods. DC incubated with Listeria innocua and L. monocytogenes, respectively, readily phagocytosed the bacteria. Listeria did not impair viability and immunogenic potential of human DC. Listerial antigens were found to be processed within the lysosomal compartment of DC and colocalized with major histocompatibility complex (MHC) class II molecules, as shown by fluorescence and transmission electron microscopy. DC challenged with apathogenic L. innocua were highly effective in priming autologous naïve T cells (mainly CD4+) in vitro. The T cells strongly proliferated in the presence of DC incubated with L. innocua, which could be significantly inhibited by anti-MHC II mAb. L. innocua-primed T cells were also successfully stimulated by DC harboring the pathogenic L. monocytogenes, either the wild-type strain EGD or the p60 reduced mutant strain RIII. From our results, we conclude that human DC infected with nonpathogenic intracellular bacteria are able to efficiently prime naïve T cells, which are then suitable for recognition of antigens derived from related virulent bacterial species. This in vitro human model provides an interesting tool for basic research in infectious immunology and possibly for a new immunotherap

Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes

BACKGROUND: Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. Expression and transcription have been partially characterised in vitro and four transcripts (NKG5, 519, 520, and 522) were identified. However, only a single protein product of 15 kDa was found, which is subsequently processed to an active 9 kDa protein. RESULTS: In this study we investigated generation of granulysin in lymphokine activated killer (LAK) cells and antigen (Listeria) specific T-cells. Semiquantitative RT-PCR revealed NKG5 to be the most prominent transcript. It was found to be up-regulated in a time-dependent manner in LAK cells and antigen specific T-cells and their subsets. Two isoforms of 519 mRNA were up-regulated under IL-2 and antigen stimulation. Moreover, two novel transcripts, without any known function, comprising solely parts of the 5 prime region of the primary transcript, were detected. A significant increase of granulysin expressing LAK cells as well as antigen specific T-cells was shown by fluorescence microscopy. On the subset level, increase in CD4+ granulysin expressing cells was found only under antigen stimulation.Immunoblotting showed the 15 kDa form of granulysin to be present in the first week of stimulation either with IL-2 or with bacterial antigen. Substantial processing to the 9 kDa form was detected during the first week in LAK cells and in the second week in antigen specific T-cells. CONCLUSION: This first comprehensive study of granulysin gene regulation in primary cultured human lymphocytes shows that the regulation of granulysin synthesis in response to IL-2 or bacterial antigen stimulation occurs at several levels: RNA expression, extensive alternative splicing and posttranslational processing

Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes

BACKGROUND: Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. Expression and transcription have been partially characterised in vitro and four transcripts (NKG5, 519, 520, and 522) were identified. However, only a single protein product of 15 kDa was found, which is subsequently processed to an active 9 kDa protein. RESULTS: In this study we investigated generation of granulysin in lymphokine activated killer (LAK) cells and antigen (Listeria) specific T-cells. Semiquantitative RT-PCR revealed NKG5 to be the most prominent transcript. It was found to be up-regulated in a time-dependent manner in LAK cells and antigen specific T-cells and their subsets. Two isoforms of 519 mRNA were up-regulated under IL-2 and antigen stimulation. Moreover, two novel transcripts, without any known function, comprising solely parts of the 5 prime region of the primary transcript, were detected. A significant increase of granulysin expressing LAK cells as well as antigen specific T-cells was shown by fluorescence microscopy. On the subset level, increase in CD4+ granulysin expressing cells was found only under antigen stimulation.Immunoblotting showed the 15 kDa form of granulysin to be present in the first week of stimulation either with IL-2 or with bacterial antigen. Substantial processing to the 9 kDa form was detected during the first week in LAK cells and in the second week in antigen specific T-cells. CONCLUSION: This first comprehensive study of granulysin gene regulation in primary cultured human lymphocytes shows that the regulation of granulysin synthesis in response to IL-2 or bacterial antigen stimulation occurs at several levels: RNA expression, extensive alternative splicing and posttranslational processing

Cholesterol in Negatively Charged Lipid Bilayers Modulates the Effect of the Antimicrobial Protein Granulysin

The release of granulysin, a 9-kDa cationic protein, from lysosomal granules of cytotoxic T lymphocytes and natural killer cells plays an important role in host defense against microbial pathogens. Granulysin is endocytosed by the infected target cell via lipid rafts and kills subsequently intracellular bacteria. The mechanism by which granulysin binds to eukaryotic and prokaryotic cells but lyses only the latter is not well understood. We have studied the effect of granulysin on large unilamellar vesicles (LUVs) and supported bilayers with prokaryotic and eukaryotic lipid mixtures or model membranes with various lipid compositions and charges. Binding of granulysin to bilayers with negative charges, as typically found in bacteria and lipid rafts of eukaryotic cells, was shown by immunoblotting. Fluorescence release assays using LUV revealed an increase in permeability of prokaryotic, negatively charged and lipid raft-like bilayers devoid of cholesterol. Changes in permeability of these bilayers could be correlated to defects of various sizes penetrating supported bilayers as shown by atomic force microscopy. Based on these results, we conclude that granulysin causes defects in negatively charged cholesterol-free membranes, a membrane composition typically found in bacteria. In contrast, granulysin is able to bind to lipid rafts in eukaryotic cell membranes, where it is taken up by the endocytotic pathway, leaving the cell intac

Chlamydophila pneumoniae derived from inclusions late in the infectious cycle induce aponecrosis in human aortic endothelial cells

BACKGROUND: Atherosclerosis is still the leading cause of death in the western world. Besides known risk factors studies demonstrating Chlamydophila pneumoniae (C. pneumoniae) to be implicated in the progression of the disease, little is known about C. pneumoniae infection dynamics. We investigated whether C. pneumoniae induce cell death of human aortic endothelial cells, a cell type involved in the initiation of atherosclerosis, and whether chlamydial spots derive from inclusions. RESULTS: Lactate dehydrogenase release revealed host cell death to be dependent on the amounts of Chlamydia used for infection. The morphology of lysed human aortic endothelial cells showed DNA strand breaks simultaneously with cell membrane damage exclusively in cells carrying Chlamydia as spots. Further ultrastructural analysis revealed additional organelle dilation, leading to the definition as aponecrotic cell death of endothelial cells. Exclusive staining of the metabolic active pathogens by chlamydial heat shock protein 60 labelling and ceramide incorporation demonstrated that the bacteria responsible for the induction of aponecrosis had resided in former inclusions. Furthermore, a strong pro-inflammatory molecule, high mobility group box protein 1, was shown to be released from aponecrotic host cells. CONCLUSION: From the data it can be concluded that aponecrosis inducing C. pneumoniae stem from inclusions, since metabolically active bacterial spots are strongly associated with aponecrosis late in the infectious cycle in vascular endothelial cells and metabolic activity was exclusively located inside of inclusions in intact cells. Vice versa initial spot-like infection with metabolically inert bacteria does not have an effect on cell death induction. Hence, C. pneumoniae infection can contribute to atherosclerosis by initial endothelial damage

Perforin enhances the granulysin-induced lysis of Listeria innocua in human dendritic cells

Background: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells play an essential role in the host defence against intracellular pathogens such as Listeria, and Mycobacteria. The key mediator of bacteria-directed cytotoxicity is granulysin, a 9 kDa protein stored in cytolytic granules together with perforin and granzymes. Granulysin binds to cell membranes and is subsequently taken up via a lipid raft-associated mechanism. In dendritic cells (DC) granulysin is further transferred via early endosomes to L. innocua-containing phagosomes were bacteriolysis is induced. In the present study we analysed the role of perforin in granulysin-induced intracellular bacteriolysis in DC. Results: We found granulysin-induced lysis of intracellular Listeria significantly increased when perforin was simultaneously present. In pulse-chase experiments enhanced bacteriolysis was observed when perforin was added up to 25 minutes after loading the cells with granulysin demonstrating no ultimate need for simultaneous uptake of granulysin and perforin. The perforin concentration sufficient to enhance granulysin-induced intracellular bacteriolysis did not cause permanent membrane pores in Listeria-challenged DC as shown by dye exclusion test and LDH release. This was in contrast to non challenged DC that were more susceptible to perforin lysis. For Listeria-challenged DC, there was clear evidence for an Ca2+ influx in response to sublytic perforin demonstrating a short-lived change in the plasma membrane permeability. Perforin treatment did not affect granulysin binding, initial uptake or intracellular trafficking to early endosomes. However, enhanced colocalization of granulysin with listerial DNA in presence of perforin was found by confocal laser scanning microscopy. Conclusion: The results provide evidence that perforin increases granulysin-mediated killing of intracellular Listeria by enhanced phagosome-endosome fusion triggered by a transient Ca2+ flux

Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes-0

<p><b>Copyright information:</b></p><p>Taken from "Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes"</p><p>http://www.biomedcentral.com/1471-2172/8/9</p><p>BMC Immunology 2007;8():9-9.</p><p>Published online 27 Jun 2007</p><p>PMCID:PMC1914365.</p><p></p>d in LAK or antigen specific T-cells. Black boxes indicate coding and gray boxes noncoding exon parts. Exons are marked with roman numbers. Dotted lines correspond to intron regions. Arabic numbers 1 to 7 stay for primers numbered in the same way as in Table 1

Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes-8

<p><b>Copyright information:</b></p><p>Taken from "Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes"</p><p>http://www.biomedcentral.com/1471-2172/8/9</p><p>BMC Immunology 2007;8():9-9.</p><p>Published online 27 Jun 2007</p><p>PMCID:PMC1914365.</p><p></p>les were taken for RT-PCR analysis. Representative gel image shows transcripts present in LAK cells over the stimulation period (A). The same transcripts were found in different subpopulations (not shown). Graph (B) represents results of semiquantitative analysis of the transcripts normalized to actin (gel image, lines A) in LAK cells from three different donors. Each symbol represents one donor and bars correspond to the mean value. Graph (C) shows the same analysis in different subpopulations where one representative donor is shown

Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes-7

<p><b>Copyright information:</b></p><p>Taken from "Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes"</p><p>http://www.biomedcentral.com/1471-2172/8/9</p><p>BMC Immunology 2007;8():9-9.</p><p>Published online 27 Jun 2007</p><p>PMCID:PMC1914365.</p><p></p>d in LAK or antigen specific T-cells. Black boxes indicate coding and gray boxes noncoding exon parts. Exons are marked with roman numbers. Dotted lines correspond to intron regions. Arabic numbers 1 to 7 stay for primers numbered in the same way as in Table 1

Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes-6

<p><b>Copyright information:</b></p><p>Taken from "Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes"</p><p>http://www.biomedcentral.com/1471-2172/8/9</p><p>BMC Immunology 2007;8():9-9.</p><p>Published online 27 Jun 2007</p><p>PMCID:PMC1914365.</p><p></p>f cells and separation of proteins by SDS-PAGE. Proteins were blotted and detected using anti-granulysin Ab (1:1000). The blot was stripped and probed for the β-actin