Common pathways for receptor-mediated ingestion of Escherichia coli and LDL cholesterol by Entamoeba histolytica regulated in part by transmembrane kinase 39.

The single-celled parasite, Entamoeba histolytica, is an enteric pathogen that ingests bacteria and host cells. Inhibition of phagocytosis renders the parasite avirulent. The ligand/receptor interactions that allow E. histolytica to phagocytose are not well understood. We hypothesised that E. histolytica trophozoites might accomplish ingestion through the utilisation of a scavenger receptor for cholesterol. Here we show that acetylated low density lipoprotein cholesterol was phagocytosed by amoebae via receptor mediated mechanisms. Acetylated low density lipoprotein cholesterol competitively inhibited by 31 ± 1.3% (P < 0.005) the ingestion of Escherichia coli, but not erythrocytes and Jurkat T lymphocytes, suggesting a partially redundant phagocytic pathway for E. coli and cholesterol. Inducible expression ofa signalling-dead dominant-negative version of E. histolytica transmembrane kinase 39 inhibited ingestion of E. coli by 55 ± 3% (P < 0.005) but not LDL particles. We concluded that ingestion of E. coli was regulated by TMK39 and partially shared the acetylated low density lipoprotein cholesterol uptake pathway.

Chlamydophila pneumoniae attachment and infection in low proteoglycan expressing human lymphoid Jurkat cells.

This study investigated the proteoglycan (PG)-dependent mechanism of Chlamydophila pneumoniae attachment to lymphocytic cells. Lymphoid Jurkat cells and epithelial HEp-2 cells were statically infected with C. pneumoniae (TW183). Transmission electron microscopy and assessment of inclusion-forming units indicated that the bacteria grew normally in Jurkat cells and were capable of producing secondary infection; however, they grew at a slower rate than in HEp-2 cells. RT-PCR analysis indicated that HEp-2 cells strongly expressed PG-core protein encoding genes, thereby sustaining glycosaminoglycans (GAGs), such as heparin, on the cellular surface. Similar gene expression levels were not observed in Jurkat cells, with the exception of glypican-1. Immunofluorescence analysis also supported strong heparin expression in HEp-2 cells and minimal expression in Jurkat cells, although heparan sulfate pretreatment significantly inhibited bacterial attachment to both cell types. Immunofluorescent co-staining with antibodies against chlamydial LPS and heparin did not identify bacterial and heparin co-localization on Jurkat cells. We also confirmed that when C. pneumoniae was statically infected to human CD4(+) peripheral blood lymphocytes known not expressing detectable level of heparin, the bacteria attached to and formed inclusion bodies in the cells. Thus, the attachment mechanism of C. pneumoniae to Jurkat cells with low PG expression is unique when compared with HEp-2 cells and potentially independent of GAGs such as heparin.

Porphyromonas gingivalis stimulates TACE production by T cells.

INTRODUCTION: Tumour necrosis factor-alpha converting enzyme (TACE), also known as ADAM17, is a membrane-bound metalloprotease and disintegrin. It is produced by a number of host cells and is known to shed and release cell-bound cytokines, particularly members of the tumour necrosis factor family. The aim of this study was to investigate the effect of Porphyromonas gingivalis on TACE production by a human T-cell line, to identify putative virulence factors involved in this process, and to investigate the effect of doxycycline. METHODS: P. gingivalis 6-day culture supernatants were used to challenge Jurkat T cells for 6 h. Secreted and cell-associated TACE levels were measured by enzyme-linked immunosorbent assay, whereas messenger RNA expression was investigated by quantitative real-time polymerase chain reaction. To investigate the involvement of cysteine proteases or proteinaceous components in general, P. gingivalis culture supernatants were treated with the specific chemical inhibitor TLCK or heat-inactivated, respectively. The effect of doxycycline on the regulation of TACE secretion by P. gingivalis was also investigated. RESULTS: P. gingivalis challenge resulted in a concentration-dependent enhancement of TACE messenger RNA expression and protein release by Jurkat cells. TLCK treatment or heat treatment of P. gingivalis culture supernatants decreased TACE release to control levels. Doxycycline inhibited TACE secretion dose dependently. CONCLUSION: The induction of TACE by T cells in response to P. gingivalis may in turn favour the shedding of host cell-bound cytokines into the local microenvironment, potentially amplifying the inflammatory response. In the present experimental system, P. gingivalis cysteine proteases are involved in TACE release by T cells.

Helicobacter pylori-induced interleukin-12 p40 expression.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen-presenting cells that promotes the development of T-helper lymphocyte 1 (Th1). Chronic gastritis induced by Helicobacter pylori is considered a Th1-mediated process. IL-12 levels in gastric biopsy samples of H. pylori-infected patients are higher than in those of uninfected individuals, but the cellular source of IL-12 remains elusive. IL-12 staining was detected in mucosal epithelial cells, lymphocytes, and macrophages in specimens of patients with H. pylori-positive gastritis. Therefore, we investigated IL-12 p40 mRNA induction by H. pylori in gastric epithelial cells and T cells. Although cag pathogenicity island (PAI)-positive H. pylori induced IL-12 p40 mRNA expression, an isogenic mutant of the cag PAI failed to induce it in both cell types. Supernatants from H. pylori cultures and H. pylori VacA induced IL-12 p40 mRNA expression in T cells but not in epithelial cells. The activation of the IL-12 p40 promoter by H. pylori was mediated through NF-kappaB. The transfection of IkappaB kinase and NF-kappaB-inducing kinase dominant-negative mutants inhibited H. pylori-induced IL-12 p40 activation. Inhibitors of NF-kappaB, phosphatidylinositol 3-kinase, p38 mitogen-activated protein kinase, and Hsp90 suppressed H. pylori- and VacA-induced IL-12 p40 mRNA expression. The results indicate that H. pylori induces IL-12 p40 expression by the activation of NF-kappaB, phosphatidylinositol 3-kinase, and p38 mitogen-activated protein kinase. Hsp90 is also a crucial regulator of H. pylori-induced IL-12 p40 expression. In addition to the cag PAI, VacA might be relevant in the induction of IL-12 expression and a Th1-polarized response only in T cells.

Monocyte chemoattractant protein 1 (MCP-1) released from Helicobacter pylori stimulated gastric epithelial cells induces cyclooxygenase 2 expression and activation in T cells.

BACKGROUND: and aims: To clarify the interaction between gastric epithelial and mucosal T cells, we examined the role of cytokines released from epithelial cells in response to Helicobacter pylori water extract protein (HPWEP) in regulating T cell cyclooxygenase 2 (COX-2) expression and activation. METHODS: Media from MKN-28 cells incubated with HPWEP for 48 hours were added to Jurkat T cells and human peripheral T cells. C-C and CXC chemokine concentrations in MKN-28 cell media, and COX-2 expression, interferon gamma (IFN-gamma), and interleukin (IL)-4 secretions in T cells were determined by western blot analysis and ELISA methods. Distributions of COX-2 positive T cells and monocyte chemoattractant protein 1 (MCP-1) in tissue specimens with H pylori associated gastritis were determined as single or double labelling by immunohistochemistry. RESULTS: MCP-1, IL-7, IL-8, and RANTES were detected in media from MKN-28 cells incubated with HPWEP. Media as a whole, and MCP-1 alone, stimulated COX-2 expression and peripheral T cell proliferation. Anti-MCP-1 antibody inhibited media stimulated COX-2 mRNA expression in Jurkat T cells. Media stimulated IFN-gamma but not IL-4 secretion from peripheral T cells, while MCP-1 stimulated IL-4 but not IFN-gamma secretion. Both stimulated cytokine release, and peripheral T cell proliferation was partially inhibited by NS-398, a specific COX-2 inhibitor. In mucosa with gastritis, COX-2 was expressed in T cells and MCP-1 was localised mainly in epithelial and mononuclear cells. MCP-1 levels and the intensity of COX-2 expression in tissue samples were closely related. CONCLUSIONS: Cytokines such as MCP-1, released from gastric epithelial cells in response to HPWEP, seem to modulate T cell immune responses, at least in part via COX-2 expression.

Induction of HIV-1 long terminal repeat-mediated transcription by Neisseria gonorrhoeae.

Gonorrhoea enhances the transmission of HIV through increased viral shedding and the increased probability of seroconversion among previously HIV-negative individuals. However, the mechanism(s) underlying these influences remain poorly understood. We demonstrated that exposure to Neisseria gonorrhoeae induces the nuclear factor kappa B-dependent transcription from the HIV-1 long terminal repeat in derivatives of the Jurkat CD4 T cell line. These data suggest that gonococcal infection directly impacts HIV-1 transmission through the localized stimulation of viral expression.

Molecular mediators of Porphyromonas gingivalis-induced T-cell apoptosis.

Porphyromonas gingivalis produces virulence factors which can modify the molecular and cellular components of the host immune response. In the present work we investigated the role of specific virulence factors from P. gingivalis in the induction of apoptosis in Jurkat T cells. P. gingivalis culture supernatants mimicked the effect of butyric acid on T-cell apoptosis and this effect was associated with an increase in histone H4 acetylation. A role for proteases was excluded in experiments which demonstrated that neither protease inhibitors nor use of P. gingivalis mutants defective in protease synthesis had any effect on the stimulation of T-cell apoptosis in this system.