Abstract
Activation of caspase-1 through the inflammasome protein Nalp1b controls anthrax lethal toxin (LT)-induced necrosis in murine macrophages. In this study we analyzed physiological changes controlled by caspase-1 in LT-treated murine macrophages. The caspase-1 inhibitor Boc-D-cmk blocked caspase-1 activity and membrane impairment in LT-treated cells. To determine the relationship between caspase-1 activation and membrane integrity, we added Boc-D-cmk to J774A.1 macrophages at different time points following LT exposure. Remarkably, Boc-D-cmk rescued LT-treated macrophages, even when added at the peak of caspase-1 activation. Late addition of the caspase-1 inhibitor reversed the losses of plasma membrane integrity and metabolic activity in these cells. Similar results were obtained with the proteasome inhibitor MG132, one of the most potent inhibitors of LT toxicity. LT-treated macrophages displaying evidence of membrane impairment recovered upon the addition of MG132, mirroring the Boc-D-cmk response. Strikingly, late addition of proteasome inhibitors also abrogated caspase-1 activity in LT-treated macrophages. Proteasomal control of caspase-1 activity and membrane impairment, however, was restricted to LT-induced cytolysis, because proteasome inhibitors did not block caspase-1 activation and cell death triggered by lipopolysaccharide and nigericin. Our findings indicate that proteasome inhibitors do not target caspase-1 directly but instead control an upstream event in LT-treated macrophages leading to caspase-1 activation. Taken together, caspase-1-mediated necrosis appears to be tightly controlled and differentially regulated by proteasomes depending on the source of caspase-1 induction.
Highlights
When injected into mice [1, 4]
Caspase-1 Activity Controls Membrane Integrity in LTtreated Macrophages—Nalp1b was recently identified as the genetic factor responsible for induction of lethal toxin (LT)-induced necrosis in BALB/c macrophages [22]
Caspase-1 activation was monitored by analysis of the cleavage of IL-18, and cytotoxic effects were determined by TOPRO-3 dye uptake using flow cytometry
Summary
Cell Culture, and Materials—C57BL/6 and BALB/c mice were obtained from Jackson Laboratories (Bar Harbor, MN). Propidium Iodide Uptake Assays—Macrophages were seeded on 96-well plates and exposed to LT or to LPS and nigericin, in the presence or absence of Boc-D-cmk or MG132. Membrane Permeability by Flow Cytometry—J774A.1 macrophages were seeded on 24-well plates and exposed to LT in the presence or absence of 40 M Boc-D-cmk, z-VAD-fmk, or Ac-YVAD-cmk. RAW 264.7 macrophages were cultured in 96-well plates and treated with LT in the presence or absence of 10 M MG132 and 80 M Boc-D-cmk. Western Blotting—Macrophages were cultured in 24-well plates and treated with LT or with LPS and nigericin, in the presence or absence of 40 M Boc-D-cmk, z-VAD-cmk, AcYVAD-cmk, or 10 M MG132. The membranes were probed with primary anti-murine polyclonal antibodies against IL-18 (BioVision, Mountain View, CA), actin (Sigma-Aldrich), or ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA). The experiments were performed with an Olympus IX81 electronically motorized microscope (Center Valley, PA)
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