Monocyte derived microvesicles deliver a cell death message via encapsulated caspase-1

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-1beta 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.

Figure 1. VSMC apoptosis by monocytes is contact independent and inhibited by caspase-1 inhibitor.

Monocytes were stimulated with LPS (1 µg/ml) for 15 min in the presence or absence of YVADcmk. VSMCs were either co-cultured with these monocytes or subjected to conditioned medium from the stimulated monocytes. Apoptosis was measured by A) Annexin/PI and B) caspase-3 assays along with C) morphology. D) Monocytes were also stimulated with LPS (1 µg/ml) for indicated time and IL-18 and IL-1β release was measured from supernatants by ELISA. E) Supernatants were also analyzed for total and functional caspase-1 using ELISA and WEHD-assays. Cytokine and enzymatic assays depicted in graphs are based on average of n = 2 experiments.

Figure 2. Active caspase-1 release from stimulated monocytes occurs in microvesicles.

Monocytes were stimulated with LPS (1 µg/ml) for 15 min. Microvesicles were isolated from LPS stimulated monocytes supernatant by ultracentrifugation. Isolated microvesicles were analyzed by A) flow cytometry and B) transmission electron microscopy. C) Microvesicles were further analyzed for presence of catalytically active caspase-1 and inflammasome protein, ASC, by immunoblot. Activity of caspase-1 was also measured using WEHD-enzymatic assay. Immunoblots are representative of n = 3 experiments and caspase-1 enzymatic assay is average of n = 2 experiments.

Figure 3. Microvesicles released by LPS- stimulated monocytes induce VSMC apoptosis.

Monocytes were stimulated with LPS (1 µg/ml) for 15 min and microvesicles were isolated from supernatant by ultracentrifugation. VSMCs were then treated with microvesicles isolated from unstimulated, LPS, and LPS + YVAD treated monocytes. Cell death was measured by Annexin V/PI assays using flow cytometry. A) Representative data of apoptosis of VSMC by flow cytometry using Annexin V/PI assay. B) Average of quantitative analysis of the same (n = 2); C) VSMCs were pretreated with specific caspase inhibitors, YVAD (casp-1inhibitor), DEVD (casp-3 inhibitor) and ZVAD (pan-caspase inhibitor). MVs were isolated from monocytes stimulated with LPS for 15 min and subjected to the pretreated VSMCs for 18 h. Cell death was analyzed by Annexin V/PI assays (n = 2); D) Monocytes were again stimulated with LPS (1 µg/ml) for 15 min and both microvesicles (MV) and the non-vesicular fractions (non-MV) were isolated from supernatant of monocytes using ultracentrifugation. Both microvesicle (MV) and non-microvesicle (non-MV) fractions were subjected to VSMC and apoptosis was analyzed using Annexin V/PI assays.

Figure 4. Microvesicle encapsulated exogenous caspase-1 directly induces VSMC apoptosis.

A) Monocytes were either pretreated with YVAD-cmk or DMSO for 30 min. Cells were then rinsed with PBS and then stimulated with LPS (1 µg/ml) for 15 min. Microvesicles were isolated from the conditioned medium. Additionally, VSMCs were also pretreated with either saline, or with IL-1RA, IL-18 bp or sFAS for 30 min (* indicates VSMC pretreatments). VSMCs were then subjected to the isolated microvesicles from LPS stimulated monocytes. (▪) VSMC+ MV from control monocytes; () VSMC+ MV from LPS/YVAD monocytes; () VSMC treated with IL-1RA+ MV from LPS treated monocytes; (□) VSMC treated with IL-18 bp + MV from LPS treated monocytes; () VSMC treated with sFAS+ MV from LPS treated monocytes. Apoptosis of VSMC was measured using Annexin V/PI assay. Graph represents average of n = 2 experiments. B) HEK-293 cells were transfected with either wild type or mutant caspase-1 (0.2 µg) along with ASC (0.2 µg) using Lipofectamine 2000. Transfection was normalized using vector controls. Supernatants were collected from the transfected cells and subjected to VSMC. Apoptosis was measured using Annexin V/PI assays. THP-1 cells were lysed and cell lysates were either kept at 4°C or incubated at 30°C for 1 h. Cell lysates were then centrifuged at 15,000 X g for 20 min. Both lipid and non-lipid fractions were measured for caspase-1 activity using C) immunoblot and D) WEHD-enzymatic assay. E) Both fractions were also subjected to VSMC and cell death was analyzed by Annexin V/PI assays.

Figure 5. Vesiculation is necessary for exogenous caspase-1 mediated apoptosis of VSMC.

Monocytes were either stimulated with LPS (1 µg/ml) for 15 min, 30 min, 1 h and 2 h or left untreated prior to microvesicles isolation. Microvesicles were then either kept intact or disrupted by mild homogenization or subjected to heat inactivation of encapsulated caspase-1. A) Microvesicles isolated from different times of LPS stimulation were then subjected to VSMC for 18 h and induction of apoptosis of VSMC was analyzed by Annexin V/PI assay. B) Caspase-1 activity of each fraction was measured using WEHD enzymatic assay. Time (h) in the figure indicates time of LPS stimulation of monocytes prior to MV isolation.