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Infection and Immunity, May 2004, p. 2590-2597, Vol. 72, No. 5
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.5.2590-2597.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Caspase-8 Activation Precedes Alterations of Mitochondrial Membrane Potential during Monocyte Apoptosis Induced by Phagocytosis and Killing of Staphylococcus aureus

Kazimierz Weglarczyk,1,2 Jaroslaw Baran,2 Marek Zembala,2 and Juliusz Pryjma1,2*

Department of Immunology, Faculty of Biotechnology,1 Department of Clinical Immunology, Institute of Pediatrics, Medical College, Jagiellonian University, Cracow, Poland2

Received 30 September 2003/ Returned for modification 18 November 2003/ Accepted 3 February 2004

Human peripheral blood monocytes become apoptotic following phagocytosis and killing of Staphylococcus aureus. Although this type of monocyte apoptosis is known to be initiated by Fas-Fas ligand (FasL) interactions, the downstream signaling pathway has not been determined. In this work the involvement of mitochondria and the kinetics of caspase-8 and caspase-3 activation after phagocytosis of S. aureus were studied. Caspase-8 activity was measured in cell lysates by using the fluorogenic substrate Ac-IETD-AFC. Active caspase-3 levels and mitochondrial membrane potential ({Delta}{psi}m) were measured in whole cells by flow cytometry using monoclonal antibodies reacting with activated caspase-3 and chloromethyl-X-rosamine, respectively. The results show that caspase-8 was activated shortly after phagocytosis of bacteria. Caspase-8 activation was followed by progressive disruption of {Delta}{psi}m, which is associated with the production of reactive oxygen intermediates. The irreversible caspase-8 inhibitor zIETD-FMK prevented the disruption of {Delta}{psi}m and the release of cytochrome c from S. aureus-exposed monocytes. Caspase-3 activation occurred following disruption of {Delta}{psi}m. These results strongly suggest that apoptosis of monocytes that have phagocytosed and killed S. aureus is driven by the Fas-FasL-initiated pathway, which is typical for type II cells.

* Corresponding author. Mailing address: Department of Immunology, Faculty of Biotechnology, Jagiellonian University, Gronostajowa Str. 7, 30-387 Kraków, Poland. Phone: 48-12-664-6127. Fax: 48-12-664-6904. E-mail: pryjma{at}mol.uj.edu.pl.

Editor: J. B. Bliska

Infection and Immunity, May 2004, p. 2590-2597, Vol. 72, No. 5
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.5.2590-2597.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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