Abstract
Apoptosis depends upon the activation of intracellular caspases which are classically induced by either an intrinsic (mitochondrial based) or extrinsic (cytokine) pathway. However, in the process of explaining how endotoxin activated monocytes are able to induce apoptosis of vascular smooth muscle cells when co-cultured, we uncovered a transcellular apoptosis inducing pathway that utilizes caspase-1 containing microvesicles. Endotoxin stimulated monocytes induce the cell death of VSMCs but this activity is found in 100,000 g pellets of cell free supernatants of these monocytes. This activity is not a direct effect of endotoxin, and is inhibited by the caspase-1 inhibitor YVADcmk but not by inhibitors of Fas-L, IL-1β and IL-18. Importantly, the apoptosis inducing activity co-purifies with 100 nm sized microvesicles as determined by TEM of the pellets. These microvesicles contain caspase-1 and caspase-1 encapsulation is required since disruption of microvesicular integrity destroys the apoptotic activity but not the caspase-1 enzymatic activity. Thus, monocytes are capable of delivering a cell death message which depends upon the release of microvesicles containing functional caspase-1. This transcellular apoptosis induction pathway describes a novel pathway for inflammation induced programmed cell death.
Highlights
Caspase-1 was first described as the IL-1 converting enzyme responsible for processing and activating proIL-1b to its active form [1,2,3]
Monocytes stimulated with LPS induced significant vascular smooth muscle cells (VSMCs) cell death, as compared to VSMC cocultured with control unstimulated monocytes (Figure 1A)
Intact, homogenized and heat-inactivated microvesicles were analyzed for caspase1 activity using both immunoblot and enzymatic assay systems. Both intact and homogenized microvesicles contained active caspase-1, whereas the caspase-1 activity was completely lost by heat-inactivation of microvesicles (Figure 5B). Executioner caspases such as caspases 3, 6 and 7 are recognized mediators of apoptotic cell death [4,31,32,33], whereas caspase-1 is the prime member of the inflammatory caspase family which functions to activate proIL-1b and proIL-18
Summary
Caspase-1 was first described as the IL-1 converting enzyme responsible for processing and activating proIL-1b to its active form [1,2,3]. It is recognized that caspase-1 regulation depends upon the assembly of a protein complex, termed the inflammasome This structure is centered upon the adapter molecule ASC and typically another member of the NOD-like receptor or RIG-I receptor family that is thought to provide an intracellular danger sensing function [6,7,8]. Either from pathogens (pathogen associated molecular patterns, PAMPs), from exogenous agents like silica, or endogenous signals such as ATP and uric acid, induces inflammasome assembly [6]. This assembly autoactivates caspase by a proximity mediated process with the resultant release from the cell of processed IL-1b and IL-18 [9,10,11,12]. It is generally recognized that with caspase-1 activation, is IL-1b and IL-18 processed and released by these danger sensing macrophages, but many of the inflammasome components themselves are released, notably caspase-1 and ASC [13]
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