One representative experiment out of three is shown. Acute mycobacterial infections symbolize sites of inflammation that attract and induce DC maturation through the presence of maturation components. latent phase in the host. However, latent infections have the potential to reactivate and cause clinical tuberculosis (5, 6). The ability of to exist as a latent contamination of the host suggests that mycobacteria are able to suppress cellular immune responses. Although DCs are not the primary targets for contamination by mycobacteria, the specific function of DCs in the cellular immune response seems to be modulated by mycobacteria (7). Thus, knowledge about the conversation of DCs with mycobacteria and mycobacterial components is essential to fully understand and combat infections. The conversation of mycobacteria with macrophages has been extensively investigated and the mannose receptor (MR), CD11b, and CD11c have been exhibited to act as receptors on macrophages for mycobacteria (8, 9). Although these receptors also have been implicated in the conversation of mycobacteria with DCs (4, 7), little is known about the actual cell-surface receptors on DCs that are involved in DCCmycobacteria interactions. We have recently recognized the DC-specific C-type lectin DC-SIGN that plays a key role in the dissemination FR167344 free base of HIV-1 by DCs through HIV-1 gp120 binding (10). DC-SIGN has a high affinity for mannose-containing carbohydrates (11, 12) and we hypothesized that, based on its carbohydrate acknowledgement profile, DC-SIGN might function as a receptor for pathogens other than HIV-1. We have investigated the conversation of DC-SIGN with mycobacteria and demonstrate that DC-SIGN is an important receptor on DCs for viable mycobacteria, such as and bacillus Calmette-Gurin (BCG), and ManLAM, despite the presence of the other reported mycobacterial receptors. We demonstrate that mycobacteria specifically target DC-SIGN through ManLAM to impair DC maturation and to induce FR167344 free base production of the antiinflammatory cytokine IL-10. These conditions promote immunosuppression and may contribute to the survival of infections by shifting the precarious balance between immune activation and suppression to favor the removal of mycobacteria. Materials and Methods Antibodies. The following antibodies were used: anti-MR (Clone 19; BD Biosciences), CD11b (bear-1; reference 13), CD11c (SHCL3; reference 14), anti-DC-SIGN (AZN-D1, AZN-D2 , CSRD ), Light fixture-1 (H4A3; BD Biosiences), as well as the PE-conjugated antibodies Compact disc80, Compact disc86, HLA-DR (BD Biosciences), and Compact disc83 (Beckman Coulter). Cells. Immature DCs had been cultured from monocytes in the current presence of IL-4 and GM-CSF (500 and 800 U/ml, respectively; Schering-Plough; guide 16). K562 transfectants expressing wild-type DC-SIGN had been generated by transfection of K562 cells with 10 g pRc/CMV-DC-SIGN plasmid by electroporation as referred to previously (10). Mycobacteria. BCG (Pasteur), strains H37Rv and H37Ra, and were presents from A. Kolk (Royal Exotic Institute, Amsterdam, Netherlands). BCG was cultured in vitro using Middelbrook 7H9 broth supplemented with 0.05% Tween 80 and albumin-dextrose-catalase. The glycolipids AraLAM and ManLAM were extracted from J. Belisle, Colorado Condition College FR167344 free base or university, Fort Collins, CO, as well as the Country wide Institutes of Wellness, Bethesda, MD (agreement NO1 AI-75320). DCs had been contaminated with mycobacteria by coculturing them at Rabbit Polyclonal to RNF144B a proper multiplicity of infections (MOI) as indicated in the body legends. Internalization and Catch of mycobacteria by cells was evaluated using FITC-conjugated BCG. Bacteria (109/ml) had been tagged by incubation of 0.5 mg FITC per ml in phosphate-buffered saline (pH 7.4) in room temperatures for 1 h. The FITC-pulsed bacterias were washed 3 x to eliminate unbound FITC. Catch was dependant on calculating the percentage of cells that destined FITC-conjugated bacterias using movement cytometry (FACSCalibur?; Becton Dickinson). Phagocytosis was motivated utilizing a fluorescence-quenching technique as reported previously (17). In short, quenching of noninternalized membrane-bound FITC-conjugated BCG was attained by dealing with the cells with 0.05% trypan blue for 5 min. Fluorescent Bead Adhesion Assay. Carboxylate-modified TransFluorSpheres (488/645 nm, 1.0 m; Molecular Probes) had been coated using the glycolipid types of lipoarabinomannan (LAM). Streptavidin-coated beads (18) had been incubated with biotinylated F(ab)2 fragment goat antiCmouse IgG.