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First published online 6 February 2007
doi: 10.1242/jcs.03377

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Caspase-1-dependent processing of pro-interleukin-1 is cytosolic and precedes cell death

Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester, M13 9PT, UK

View larger version (41K): [in this window] [in a new window]   Fig. 1. ATP-induced IL-1 release and cell death. (A) IL-1 immunoblot of murine macrophage cell lysates after LPS treatment (1 µg/ml), and stimulation with ATP (5 mM) for the indicated time in minutes (min). Shown are blots of lysates from experiments conducted in the absence (i) or presence (ii) of the caspase-1 inhibitor Ac-YVAD-fmk (50 µM). (B) IL-1 immunoblot of murine macrophage supernatant after the LPS and ATP treatments described in A. Blots reflect experiments conducted in the absence (i) or presence (ii) of the caspase-1 inhibitor Ac-YVAD-fmk (50 µM). (C) Effects of LPS (1 µg/ml) and ATP (5 mM, for the indicated time in minutes on cell death (LDH release, i) and IL-1 release (ii). The responses of macrophages isolated from wild-type (WT, C57BL/6J, black bars) and beige mice (white bars) were compared. (D) Effects of MDP (5 µg/ml, 2 hours) on IL-1 expression and release and cell death. Shown are immunoblots for IL-1 of macrophage lysate after incubation without MDP or LPS, with MDP, with LPS (1 µg/ml, 2 hours) or with MDP and LPS together (i); in addition, macrophages treated with LPS or LPS and MDP were incubated with ATP (5 mM, 10 minutes) and the supernatants were analyzed for IL-1 by immunoblot (ii). The effects of MDP on LPS and ATP-induced cell death (LDH release, black bars, left y-axis, iii) and IL-1 release and production as measured by ELISA (white bars, right y-axis, iii) are also shown. Data are presented as the mean ± s.d. of duplicate and triplicate wells from at least three independent cultures. Immunoblots are representative of three independent experiments.

View larger version (62K): [in this window] [in a new window]   Fig. 2. Distribution of cellular IL-1. (A) High-resolution, deconvolved image of a LPS-treated (1 µg/ml, 2 hours) murine macrophage immunostained for IL-1 (green) and EEA1 (red). Bar, 5 µm. (B) High-resolution, deconvolved image of a LPS-treated (1 µg/ml, 2 hours) murine macrophage immunostained for IL-1 (green) and cathepsin D (CD, red). Bar, 5 µm. Images are representative of three independent experiments.

View larger version (50K): [in this window] [in a new window]   Fig. 3. Sub-cellular localization of pro-IL-1. (A) Wide-field fluorescence images of LPS-treated (1 µg/ml, 2 hours) murine macrophages treated with digitonin for the indicated time seconds in (sec) after which they were immunostained for IL-1 (green) and cathepsin D (CD, red). Bar, 10 µm. (B) Immunoblot of the supernatants isolated from the experiment described in A for IL-1 (i) and cathepsin D (ii). The corresponding supernatants were also assayed for activity of the cytosolic LDH protein (iii). Images and blots are representative of three independent experiments.

View larger version (56K): [in this window] [in a new window]   Fig. 4. Sub-cellular localization of mature IL-1. (A) Wide-field fluorescence images of LPS-treated (1 µg/ml, 2 hours) murine macrophages in the absence or presence of ATP (5 mM, 5 minutes). Cells were immunostained for IL-1 (green) and cathepsin D (CD, red), before (non-permeabilized), and after digitonin treatment. Bar, 10 µm. (B) Immunoblot for IL-1 in supernatants isolated from the experiment described in A. C, control; T, supernatants isolated from macrophages permeabilized with Triton X-100. Images and blots are representative of three independent experiments.

View larger version (60K): [in this window] [in a new window]   Fig. 5. Expression of IL-1 proteins in HEK 293 cells. (A) HEK 293 cells were transfected with empty vector (C), and constructs encoding Flag-labeled pro-IL-1 (31), Flag-labeled 27-kDa IL-1 (27) and Flag-labeled mature, 17-kDa IL-1 (17). Cells were immunostained for IL-1 (green) and cathepsin D (CD, red). Bar, 10 µm. (B) IL-1 immunoblot of HEK 293 cell lysates transfected with empty vector (C), and constructs encoding Flag-labeled pro-IL-1 (31), Flag-labeled 27-kDa IL-1 (27) and - Flag-labeled mature, 17-kDa IL-1 (17). Images and blots are representative of three independent experiments.

View larger version (69K): [in this window] [in a new window]   Fig. 6. Blebbing macrophages retain their IL-1. (A) Wide-field fluorescence images showing IL-1 in LPS-treated (1 µg/ml, 2 hours) murine macrophages stimulated with ATP (5 mM) for the indicated times in minutes (min). Bar, 10 µm. (B) High-resolution, deconvolved images showing IL-1 in LPS-treated (1 µg/ml, 2 hours) murine macrophages stimulated with ATP (5 mM) for the indicated times. Bar, 5 µm. These images are representative of three independent experiments.

View larger version (83K): [in this window] [in a new window]   Fig. 7. Physical properties of IL-1 secretion. (A) Stills from a bright-field movie sequence of LPS-treated (1 µg/ml, 2 hours) murine macrophages stimulated with ATP (5 mM) in the absence (i) and presence (ii) of the caspase-1 inhibitor Ac-YVAD-fmk (50 µM). Time in minutes (min) after the addition of ATP is indicated. Arrows indicate blebbing cells. Bar, 10 µm. (B) The effects of ATP and Ac-YVAD-fmk in the above movies were quantified, with cells classified as dormant (white bars), alive but not blebbing (black bars), and alive and blebbing (gray bars). Data are the mean ± s.d. of cells counted from three independent experiments. (C) The effects of cholesterol depletion using -MCD (7.5 mM, 30 minutes) on cell death (LDH release, left y-axis) and IL-1 release (right y-axis) from LPS-treated (1 µg/ml, 2 hours) murine macrophages in the absence or presence of ATP (5 mM, 10 minutes). -MCD-treated cells, white bars; control cells, black bars. Data are the mean ± s.d. of duplicate wells from three independent cultures.

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