Membrane depolarization is induced in tolerant B lymphocytes by stimulation with antigen |
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| Authors: | M Aldo-Benson |
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| Affiliation: | 1. Divisions of Pulmonary, Allergy, & Critical Care Medicine, Emory University, Atlanta, GA 30322, USA;2. Pulmonary & Critical Care Medicine, University of Rochester Medical Center, Rochester, NY 14642, USA;3. Division of Rheumatology, Emory University, Atlanta, GA 30322, USA;4. Lowance Center for Human Immunology in the Departments of Medicine and Pediatrics, Emory University, Atlanta, GA 30322, USA;5. Divisions of Hematology/Oncology/James P. Wilmot Cancer Institute, University of Rochester Medical Center, Rochester, NY 14642, USA;6. Division of Allergy, Immunology, and Rheumatology, University of Rochester Medical Center, Rochester, NY 14642, USA;7. Cell Signaling Technology, Inc., Danvers, MA 01923, USA;8. Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Emory University, Atlanta, GA 30322, USA;9. Department of Pathology & Laboratory Medicine, Emory University, Atlanta, GA 30322, USA;10. Department of Pathology, University of Rochester Medical Center, Rochester, NY 14642, USA;11. Oregon Health & Sciences University, Beaverton, OR 97006, USA;12. Department of Biostatistics and Bioinformatics, Emory University, Atlanta, GA 30322, USA;13. Division of Infectious Diseases, University of Rochester Medical Center & Rochester General Hospital, Rochester, NY 14621, USA;14. Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, AL 35294, USA;1. Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, TN 37232, USA;2. Program in Chemical and Physical Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA;3. Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232, USA;4. National Institute for Communicable Diseases of the National Health Laboratory Service, Johannesburg 2131, South Africa;5. Division of Structural Biology, Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC 27710, USA;6. Department of Surgery, Duke University School of Medicine, Durham, NC 27710, USA;7. Ragon Institute of Massachusetts General Hospital, Harvard and Massachusetts Institute of Technology, Cambridge, MA 02139, USA;8. Vaccine Research Center, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA;9. Vanderbilt Center for Translational and Clinical Immunology, Vanderbilt University Medical Center, Nashville, TN 37232, USA;10. National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA;11. Faculty of Health Sciences, University of the Witwatersrand, Johannesburg 2000, South Africa;12. Centre for the AIDS Programme of Research in South Africa (CAPRISA), University of KwaZulu-Natal, Durban 4041, South Africa;13. Vanderbilt Institute for Infection, Immunology and Inflammation, Vanderbilt University Medical Center, Nashville, TN 37232, USA;14. Department of Electrical Engineering and Computer Science, Vanderbilt University, Nashville, TN 37232, USA;15. Center for Structural Biology, Vanderbilt University, Nashville, TN 37232, USA;1. Department of Microbiology and Immunology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden;2. Department of Biology and Biological Engineering, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Chalmers University of Technology, Göteborg, Sweden;3. Department of Biochemistry and Biophysics, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Stockholm University, Solna, Sweden;4. Immunology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA;5. Vaccine Evaluation Center, BC Children’s Hospital Research Institute, University of British Columbia, Vancouver, BC, Canada;6. Department of Pathology and Immunology, Washington University, St. Louis, MO, USA;7. Laboratory of Viral Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA;8. Department of Immunology, University of Oslo, Oslo, Norway;9. Region Västra Götaland, Department of Clinical Immunology and Transfusion Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden |
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| Abstract: | Membrane depolarization is one of the earliest events in activation of cells by ligand receptor interaction. It is known that crosslinking of antigen-specific Ig receptors on B cells by antigen can induce membrane depolarization and subsequent Ia antigen expression on the cell surface. To determine whether a tolerance-inducing form of the antigen can also induce membrane depolarization after Ig receptor binding we used splenic B cells enriched for dinitrophenyl (DNP)-specific cells and determined relative membrane potential in these cells after binding of DNP-murine IgG2a (MGG) (tolerogen) or antigens (DNP-keyhole limpet hemocyanin (KLH) and DNP-Ficoll). Relative membrane potential was determined by loading the cells with the dye, 3.3-dipentyloxacarboxyanine (DiOC5(3)) after 2 hr incubation with ligand and determining relative fluorescence intensity on the fluorescence-activated cell sorter (FACS). Carriers alone did not depolarize these normal cell populations, but 100% of DNP-specific cells were depolarized by DNP-KLH and DNP-MGG while 85% were depolarized by DNP-Ficoll. To determine if tolerant B cells could be depolarized by antigen we induced tolerance in vitro or in vivo with DNP-MGG and measured the depolarization of DNP-specific B cells in response to antigens and tolerogen. DNP-specific B cells made tolerant by DNP-MGG underwent membrane depolarization when incubated with either DNP-KLH, DNP-MGG, or DNP-Ficoll but not with carriers alone. These data suggest that tolerogen induces membrane depolarization equally as well as antigen in normal cells. In addition, tolerant cells can be depolarized by Ig receptor crosslinking with either antigen or tolerogen. Thus, tolerance does not block the early membrane events induced by antigen in B cells. |
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