Spontaneous Human Monocyte Apoptosis Utilizes a Caspase-3-Dependent Pathway That Is Blocked by Endotoxin and Is Independent of Caspase-1 1 Ruairi J. Fahy, 2 Andrea I. Doseff, 2 and Mark D. Wewers 3 Apoptosis is an important mechanism for regulating the numbers of monocytes and macrophages. Caspases (cysteine-aspartate- specific proteases) are key molecules in apoptosis and require proteolytic removal of prodomains for activity. Caspase-1 and caspase-3 have both been connected to apoptosis in other model systems. The present study attempted to delineate what role these caspases play in spontaneous monocyte apoptosis. In serum-free conditions, monocytes showed a commitment to apoptosis as early as 4 h in culture, as evidenced by caspase-3-like activity. Apoptosis, as defined by oligonucleosomal DNA fragmentation, was prevented by a generalized caspase inhibitor, z-VAD-FMK, and the more specific caspase inhibitor, z-DEVD-FMK. The caspase activity was specifically attributable to caspase-3 by the identification of cleavage of procaspase-3 to active forms by immunoblots and by cleavage of the fluorogenic substrate DEVD-AFC. In contrast, a caspase-1 family inhibitor, YVAD-CMK, did not protect monocytes from apoptosis, and the fluorogenic substrate YVAD-AFC failed to show an increase in activity in apoptotic monocytes. When cultured with LPS (1 mg/ml), monocyte apoptosis was prevented, as was the activation of caspase-3. Unexpectedly, LPS did not change baseline caspase-1 activity. These findings link spontaneous monocyte apoptosis to the proteolytic activation of caspase-3. The Journal of Immunology, 1999, 163: 1755–1762. M onocytes play a major role in initiating, maintaining, and resolving host inflammatory responses by differ- entiating into macrophages and dendritic cells and by releasing cell-signaling molecules, including cytokines. In the ab- sence of inflammation, more monocyte precursors develop from the marrow than are needed to replace normal tissue macrophage numbers (1). However, during inflammatory responses, a dramatic up-regulation of monocyte survival and differentiation may be re- quired. Thus, the processes involved in regulating monocyte re- moval and survival are critical to population control. The impor- tance of monocyte development and differentiation to disease pathogenesis has recently been highlighted by research in animal models in the field of atherogenesis and osteopetrosis (2, 3). In the absence of an appropriate stimulus, monocytes spontane- ously undergo programmed cell death (4 –7). Recently, a family of cysteine-aspartate-specific proteases called caspases has been found to play a major role in programmed cell death. Within this family, a central role has been suggested for caspase-3 (7–11) and more controversially for caspase-1 (12–21). Caspase-1 is the prototypical caspase, which was originally identified as IL-1b- converting enzyme (ICE) 4 (22). Caspase-1 mediates processing of both pro-IL-1b and pro-IL-18 (23). Additionally, caspase-1 may induce apoptosis, as evidenced by its effect when transfected into fibroblasts and its importance in Fas-mediated apoptosis in caspase-1 knockout animals (12–18). However, since the discovery of additional ICE-related molecules, other caspases such as caspase-3/CPP32 have been more consistently linked to apoptosis (8). Generally, caspases exist in cells in an inactive precursor form and require cleavage to generate the active caspase (7). For exam- ple, activation of procaspase-3 is tightly regulated by an apoptosis- activating complex, requiring proteolytic removal of an amino- terminal prodomain to produce the active caspase (24 –27). Once activated, caspase-3 performs a number of executioner functions, including the activation of a latent cytosolic endonuclease, caspase-activated deoxyribonuclease (CAD). CAD normally exists intracellularly in an inactive form bound to I-CAD. Caspase-3 cleaves I-CAD, resulting in the release of CAD (28 –30). CAD cleaves DNA into oligonucleosomal fragments that are released into the cytosol. The presence of these cytosolic fragments are landmarks for apoptotic cell death (31, 32). Due to the importance of caspases in determining either pro- grammed cell death or cytokine activation, we sought to study which caspases are important in human monocyte death and sur- vival. Although previous investigators have shown caspase activity in monocytic cell lines, this is the first study to address the issue in peripheral human monocytes. Our results identify an important role for caspase-3 activity in spontaneous monocyte apoptosis, which is prevented by endotoxin. Interestingly, we fail to identify a role for caspase-1 activation in monocyte death and unexpectedly after endotoxin stimulation. Although LPS protects monocytes Department of Internal Medicine, Division of Pulmonary and Critical Care, and The Heart and Lung Institute, Ohio State University, Columbus, OH 43210 Received for publication November 25, 1998. Accepted for publication May 27, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work is supported by National Institutes of Health Grants HL40871 and HL53229. R.J.F. is a Glaxo-Wellcome Pulmonary Fellowship recipient for 1998. 2 R.J.F. and A.I.D. contributed equally to this study. 3 Address correspondence and reprint requests to Dr. Mark D. Wewers, Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Ohio State University, N-325 Means Hall, 1654 Upham Drive, Columbus OH 43210. E-mail address: wewers.2@osu.edu 4 Abbreviations used in this paper: ICE, IL-1b-converting enzyme; AFC, amino tri- fluoromethyl coumarin; CAD, caspase-activated deoxyribonuclease; PVDF, polyvi- nylidene difluoride; DEVD, Asp-Glu-Val-Asp; YVAD, Tyr-Val-Ala-Asp; CMK, chloromethyl ketone; FMK, fluoromethyl ketone; aomk, acyloxymethyl ketone. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00