An antimicrobial activity of cytolytic T cells mediated by granulysin

Science

Volumn 282, Issue 5386, 1998, Pages 121-125

  Stenger, Steffen ^a   Hanson, Dennis A ^a   Teitelbaum, Rachel ^a   Dewan, Puneet ^a   Niazi, Kayvan R ^a   Froelich, Christopher J ^a   Ganz, Tomas ^a   Thoma Uszynski, Sybille ^a   Melián, Agustín ^a   Bogdan, Christian ^a   Porcelli, Steven A ^a   Bloom, Barry R ^a   Krensky, Alan M ^a   Modlin, Robert L ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

GRANULYSIN; PERFORIN; TUBERCULOSTATIC AGENT; UNCLASSIFIED DRUG;

ANTIMICROBIAL ACTIVITY; ARTICLE; CORRELATION FUNCTION; CYTOTOXIC T LYMPHOCYTE; HOST RESISTANCE; IMMUNITY; MACROPHAGE; MYCOBACTERIUM TUBERCULOSIS; PRIORITY JOURNAL; PROTEIN ANALYSIS; TARGET CELL; TUBERCULOSIS CONTROL;

ANTIGENS, DIFFERENTIATION, T-LYMPHOCYTE; CELL LINE; CELL MEMBRANE; CELLS, CULTURED; CYTOPLASMIC GRANULES; CYTOTOXICITY, IMMUNOLOGIC; HUMANS; MACROPHAGES; MEMBRANE GLYCOPROTEINS; MICROSCOPY, CONFOCAL; MICROSCOPY, ELECTRON, SCANNING; MYCOBACTERIUM TUBERCULOSIS; PORE FORMING CYTOTOXIC PROTEINS; RECOMBINANT PROTEINS; T-LYMPHOCYTES, CYTOTOXIC;

EID: 0032475823     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5386.121     Document Type: Article

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44. + APCs are adherent and therefore allow removal of non-phagocytized mycobacteria, (ii) they can be infected with virulent M. tuberculosis with high efficiency (3.9 bacteria per infected cell, 85% of the cells infected), and (iii) they present antigen to CD1-restricted CTLs.
45. H+L (2.5 μg/ml) (Molecular Probes) for 1 hour at 4°C. Subsequently, incubation with 10% mouse serum in PBS for 10 min and two washes with PBS and 5% goat serum were performed to block nonspecific binding. Cells were then stained with either anti-granulysin DH2 (4 μg/ml) (D. Hanson and A. Krensky, unpublished data) or P3, an IgG1 control, followed by two additional wash steps with PBS and 5% goat serum. For confocal microscopy, cells were mounted on glass slides in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) as an antifading agent. Cells were then examined with a Leica TCS-NT confocal laser scanning microscope fitted with krypton and argon lasers. Cells were illuminated with 488 and 568 nm of light after filtering through an acoustic optical device. Images of cells decorated with fluorescein isothiocyanate (FITC) and Texas Red were recorded simultaneously through separate optical detectors with a 530-nm band-pass filter and a 590-nm long-pass filter, respectively. Pairs of images were superimposed for colocalization analysis (22).
46. 6 CTLs 1 week after the last restimulation with antigen, lysing the cells in SDS sample buffer, and separating proteins on 15% SDS gels. Proteins were then transferred to nitrocellulose membranes. To ensure equal loading, we initially determined the protein concentration spectrophotometrically and confirmed it by staining the nitrocellulose membranes with Ponceau red. Granulysin protein was detected with 519/GST rabbit serum (1/1000) (5) followed by horseradish peroxidase-conjugated secondary antibodies. Immunodetection was performed with enhanced chemiluminescence following the manufacturer's guidelines.
47. + CTLs by strontium is not due to a nonspecific or toxic effect.
48. Mycobacterium. tuberculosis (virulent strain H37Rv) was grown in suspension with gentle, constant rotation in roller bottles containing Middlebrook 7H9 medium (Difco, Detroit, MI) supplemented with 0.05% Tween 80 (Sigma), 1% glycerol (ICN, Costa Mesa, CA), and 10% OADC (oleic acid, albumin, dextrose, catalase) (Becton-Dickinson). Portions from logarithmically growing cultures were frozen in PBS containing 10% glycerol. Comparison of microscope counts of mycobacteria and their growth on Middlebrook 7H11 agar plates revealed a viability of the bacteria above 90%. Adherent monolayers (25) were infected with live M. tuberculosis for 4 hours at a multiplicity of infection of 5:1. After extensive washing, macrophages were detached, and the efficiency of infection was determined by staining a portion with auramine-rhodamine acid-fast stain.
49. To determine the viability of intracellular mycobacteria, we lysed the macrophages with 0.1% saponin and plated fivefold dilutions of the lysates in duplicates on 7H11 agar plates. The number of colonies was counted after 3 weeks of incubation. The percentage of killing of intracellular M. tuberculosis mediated by CD8.TX (Fig. 1C) was calculated according to the following formula: [(CFUs in macrophages cultured with CD8.FP3 - CFUs in macrophages cultured with CD8.TX)/CFUs in macrophages cultured with CD8.FP3] × 100, where CD8.FP3 is an influenza peptide-specific CTL without lytic activity against mycobacteria-infected macrophages and was therefore included as a negative control. Killing of M. tuberculosis by granulysin and perforin (Fig. 3C) was calculated as follows: {[CFUs in macrophages cultured in diluent alone - CFUs in macrophages cultured in the presence of perforin or granulysin (or both)]/CFUs in macrophages cultured in diluent alone} × 100.
50. ++-treated or untreated CTLs were immobilized on poly-L-lysine-coated slides, fixed with 4% paraformaldehyde, and incubated with permeabilization/blocking solution (5% human serum, 5% goat serum, 0.1% Triton X-100, 0.01% saponin, and 1% nonfat dry milk). Cells were stained with a monoclonal mouse anti-human (DH2, 5 μg/ml) and detected with a FITC-conjugated goat anti-mouse.
51. 2-minimization. The principal protein used to control the activity of granulysin was a cloned fragment of HSP70, residues 549 to 646, previously used as a control for granulysin (5). It has a molecular weight of 10.8 kD, quite comparable to the size of the 9-kD form of granulysin. The HSP70(549-646) fragment was cloned into the same expression vector with the same cloning sites as granulysin, and both proteins were purified exactly the same way: nickel column under denaturing conditions, refolded, dialyzed, and finally purified by reversed-phase high-performance liquid chromatography. The endotoxin content of both preparations was <0.05 ng/ml. Granulysin, the HSP70(549-646) fragment, and all cell lines used in this study were of human origin. The HSP70(549-646) fragment was included in our initial screening of the antimicrobial activity of granulysin by radial diffusion assay and did not show any activity. We also used purified perforin as an additional control on the antimicrobial killing of several species of bacteria in vitro and found it to be without effect.
52. 490 spontaneous release × 100]}.
53. 6) was cultured in 7H9 media supplemented with OADC with or without the addition of purified granulysin (30 μg/ ml). After incubation at 37°C with gentle shaking for 80 hours, portions were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 4 hours. Bacteria were washed twice in PBS, placed on cover slips coated with poly-l-lysine or fibronectin-coated tissue culture inserts (BIOCOAT; Becton-Dickinson) for 45 min, and then processed for scanning electron microscopy. Specimens were viewed on a JEOL 6400 scanning electron microscope.
54. Supported by the AIDS Stipendium, Deutsches Krebsforschungszentrum, Heidelberg (S.S.), NIH (R.L.M., S.A.P., A.M.K., and B.R.B), Deutsche Forschungsgemeinschaft (C.B. and S.T.-U.), the Arthritis Foundation (S.A.P.), the American Cancer Society (S.A.P), the Howard Hughes Medical Institute (B.R.B.), the UNDP/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases (IMMLEP), and the Dermatology Research Foundation of California. A.M.K. is the Shelagh Galligan Professor of Pediatrics and a Burroughs Wellcome Scholar in Experimental Therapeutics. We thank M. Horwitz for allowing us to use his P3 laboratory, W. Ernst and M. Röllinghoff for critical reading of the manuscript, and R. A. Rogers, J. Lai, and J. Mervis for assistance with confocal microscopy. We thank the Genetics Institute for the gift of GM-CSF and Schering-Plough for the gift of IL-4.