DOI: 10.1126/science.285.5428.736. Cell Activation and Apoptosis by Bacterial Lipoproteins Through Toll-like Receptor-2

Cell Activation and Apoptosis by Bacterial Lipoproteins Through Toll-like Receptor-2

10.1126/science.285.5428.736

Crossref journal-article

American Association for the Advancement of Science (AAAS)

Science (221)

Abstract

Apoptosis is implicated in the generation and resolution of inflammation in response to bacterial pathogens. All bacterial pathogens produce lipoproteins (BLPs), which trigger the innate immune response. BLPs were found to induce apoptosis in THP-1 monocytic cells through human Toll-like receptor–2 (hTLR2). BLPs also initiated apoptosis in an epithelial cell line transfected with hTLR2. In addition, BLPs stimulated nuclear factor–κB, a transcriptional activator of multiple host defense genes, and activated the respiratory burst through hTLR2. Thus, hTLR2 is a molecular link between microbial products, apoptosis, and host defense mechanisms.

Bibliography

Aliprantis, A. O., Yang, R.-B., Mark, M. R., Suggett, S., Devaux, B., Radolf, J. D., Klimpel, G. R., Godowski, P., & Zychlinsky, A. (1999). Cell Activation and Apoptosis by Bacterial Lipoproteins Through Toll-like Receptor-2. Science, 285(5428), 736â739.

Authors 9

Antonios O. Aliprantis (first)
Ruey-Bing Yang (additional)
Melanie R. Mark (additional)
Shelly Suggett (additional)
Brigitte Devaux (additional)
Justin D. Radolf (additional)
Gary R. Klimpel (additional)
Paul Godowski (additional)
Arturo Zychlinsky (additional)

References 38 Referenced 1,156

10.1016/S0952-7915(97)80152-5
10.1128/mr.60.2.316-341.1996
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10.1046/j.1365-2567.1997.00365.x
Bessler W. G., et al., J. Immunol. 135, 1900 (1985). (10.4049/jimmunol.135.3.1900) / J. Immunol. by Bessler W. G. (1985)
10.1073/pnas.95.2.588
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10.1002/jlb.63.6.650
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10.1084/jem.188.11.2091
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Zhang H., Peterson J. W., Niesel D. W., Klimpel G. R., J. Immunol. 159, 4868 (1997). (10.4049/jimmunol.159.10.4868) / J. Immunol. by Zhang H. (1997)
R.-B. Yang M. R. Mark P. Godowski unpublished data.
Performed as in (9) with the following modifications. Cells were transfected in 15-cm dishes with pGL3-ELAM.tk and the Renilla luciferase reporter plasmid as an internal control and after 1 hour were seeded in an opaque 96-well plate. After 24 hours the indicated compounds were added in RPMI 1640 0.05% human serum albumin (HSA) (Grifols Miami FL). Luciferase activity was determined after 6 hours. sBLP was from Boehringer Mannheim Biochemica and Pam 3 Cys was from Novabiochem. MLP (12) contained <25 pg of LPS per milligram of protein. Unless indicated other reagents were from Sigma.
10.1016/S0171-2985(88)80036-6
Base treatment was done as in
10.1038/4007
. The products were resolved by reversed-phase high-performance liquid chromatography with a C4 column (Vydac) and a linear acetonitrile gradient. Fractions were analyzed by electrospray ionization mass spectrometry. A fraction was isolated that contained (i) a compound that has lost two of the three palmitate moieties (molecular weight = 1032.6 major peak) and (ii) a compound that has lost the entire S -[2 3-Bis(palmitoyloxy)-(2-RS)-propyl] moiety (molecular weight = 924.7 minor peak). The concentration was determined by ultraviolet absorption.
Radolf J. D., et al., J. Immunol. 154, 2866 (1995). (10.4049/jimmunol.154.6.2866) / J. Immunol. by Radolf J. D. (1995)
10.1111/j.1749-6632.1997.tb46249.x
mAb 2392 was generated by immunization of BALB/c mice with the purified extracellular domain of hTLR2 (9). The specificity of mAb 2392 for hTLR2 was confirmed by the following methods: (i) An enzyme-linked immunosorbent assay which demonstrated that mAb 2392 reacted with hTLR2-Fc but not hTLR4-Fc. (ii) A flow cytometric analysis with 293 cells expressing gD epitope–tagged versions of hTLR2 hTLR4 and hTLR6. Although each cell line was labeled with mAb 5B6 which recognizes the gD epitope only 293hTLR2 cells were labeled with mAb 2392. (iii) An immunoprecipitation (24) followed by protein immunoblotting with mAb 5B6 from the membrane extracts of 293 cells transiently transfected with gD epitope–tagged versions of hTLR2 hTLR1 hTLR4 and murine TLR2. mAb 2392 only precipitated hTLR2. However equivalent amounts of each receptor were obtained when mAb 5B6 was used for the immunoprecipitation. Supplemental data illustrating the specificity of mAb 2392 can be seen at www.sciencemag.org/feature/data/1040361.shl.
The test compounds were added to the 293 stable cell lines (9) in serum-free Dulbecco's modified Eagle's medium 0.05% HSA 5% fetal bovine serum (FBS). Cells were either photographed or stained with terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) by using the Apoptosis Detection System (Promega) and analyzed by flow cytometry. At least 5000 events were counted per sample. Results are presented as specific apoptosis (%) determined as [(% TUNEL-positive cells in experimental − % TUNEL-positive cells in control)/(100 − % TUNEL-positive cells in control] × 100. The % TUNEL-positive cells in controls was always <5%.
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Sellati T. J., et al., J. Immunol. 160, 5455 (1998). (10.4049/jimmunol.160.11.5455) / J. Immunol. by Sellati T. J. (1998)
Heumann D. et al. 148 3505 (1992). (10.4049/jimmunol.148.11.3505)
A. O. Aliprantis and A. Zychlinsky unpublished data.
Plasmids pKRN and phTLR2 (9) were prepared with the Endotoxin-free Plasmid Maxi kit (Qiagen). 293 cells were transfected with the Effectene Transfection reagent (Qiagen). Thirty hours after transfection an equal volume of diluent or diluent containing sBLP (200 ng/ml) was added. Thirty-six hours later the cells were stained with Annexin V–biotin (Pharmingen) and Streptavidin Tri-Color (Caltag) and analyzed by flow cytometry.
THP-1 cells (American Type Culture Collection) were maintained in RPMI 1640 10% FBS penicillin streptomycin and 2 mM l -glutamine. Six hours after seeding THP-1 cells in RPMI 1640 the indicated compounds diluted in RPMI 1640 0.05% HSA were added. Six hours later cell death was quantified by a lactate dehydrogenase release assay (Promega). Where indicated cells were first treated with 32 nM PMA for 6 hours or cotreated with cycloheximide (50 μg/ml). PMA and cylcoheximide diluents (dimethyl sulfoxide and ethanol) were not cytotoxic.
10.1126/science.285.5428.732
Ashkenazi A. Dixit V. M. 281 1305 (1998). (10.1126/science.281.5381.1305)
10.1016/S0074-7696(08)62419-5
10.1172/JCI119557
10.1002/jlb.61.4.375
10.1016/S0192-0561(97)00054-4
10.1073/pnas.94.20.10973
We thank D. Littman V. Dixit C. Scharff and J. Moss for critical reading of the manuscript M. Garabedian for discussions and T. Neubert for mass spectrometry analysis. pCDNA3 and pGFP were a gift of the Littman Lab. A.O.A. was supported by a grant from the Life and Health Insurance Fund. This work was supported by grants from the National Institute of Allergy and Infectious Diseases (AI 37720-04 to A.Z. and AI-38894 to J.D.R.).

Dates

Type	When
Created	23 years ago (July 27, 2002, 5:37 a.m.)
Deposited	1 year, 7 months ago (Jan. 13, 2024, 4:14 a.m.)
Indexed	2 days, 10 hours ago (Aug. 21, 2025, 12:56 p.m.)
Issued	26 years ago (July 30, 1999)
Published	26 years ago (July 30, 1999)
Published Print	26 years ago (July 30, 1999)

Funders 0

None

BibTeX

@article{Aliprantis_1999, title={Cell Activation and Apoptosis by Bacterial Lipoproteins Through Toll-like Receptor-2}, volume={285}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.285.5428.736}, DOI={10.1126/science.285.5428.736}, number={5428}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Aliprantis, Antonios O. and Yang, Ruey-Bing and Mark, Melanie R. and Suggett, Shelly and Devaux, Brigitte and Radolf, Justin D. and Klimpel, Gary R. and Godowski, Paul and Zychlinsky, Arturo}, year={1999}, month=jul, pages={736–739} }

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