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M. receptors (NLRs) and elicit an inflammatory response characterized by caspase-1 activation (40, 42). The NLR protein Nalp1b has also been linked to caspase-1 activation and macrophage cytolysis mediated by anthrax lethal toxin (LT) (6, 36). However, it is unclear how LT activates the proinflammatory protein Nalp1b and how this results in caspase-1 activation in murine macrophages. LT is considered the primary virulence factor produced by the gram-positive organism spores. LT is usually a protein toxin consisting of two subunits, protective antigen (PA) and lethal factor (LF) (10). PA binds to specific cell surface receptors and mediates endocytosis of LF, a zinc protease. The proteolytic activity of LF is essential for the cytopathic and lethal effects observed in LT-treated mice (15, 20). The response of murine macrophages to LT exposure is usually mouse strain dependent. Murine macrophages are either susceptible or resistant to LT-mediated caspase-1 activation and cytolysis (29, 30). Genetic mapping experiments have identified a single gene, alleles from susceptible murine strains in the resistant C57BL/6 background renders the resulting macrophages susceptible to rapid LT killing (6). Nalp1b belongs to the NLR family of intracellular surveillance proteins, which are able to recognize pathogen-associated molecular patterns, including lipopolysaccharide (LPS) (25, 34, 40). In contrast to murine Nalp1b, the human NLR proteins NALP1 and NALP3 have been well characterized (25, 40). Stimulation of NALP1 or NALP3 results in the recruitment of downstream components and the formation of the inflammasome complex, which appears to be a critical event associated with caspase-1 activation (1, 24, 26). The NOD of NALP proteins is required for dimerization, and the LRR domain name has a microbe-sensing function (40). The pyrin domain name (PYD) and the caspase recruitment domain name (CARD) of Rabbit polyclonal to GLUT1 NALP1 and NALP3 are essential for the recruitment of ASC and caspase-1, respectively (24, 26). In contrast to human NALP1, the PYD is usually absent in murine Nalp1b, and the involvement of murine Asc in Nalp1b inflammasome activation is usually therefore questionable (6). NLR stimulation by specific ligands results in activation of proinflammatory caspase-1 and cell death (11, 12, 16). Activated caspase-1 then processes pro-interleukin-1 (pro-IL-1), IL-18, and IL-33 into their mature forms (22, 37). Consistent with a role for Nalp1b in LT susceptibility, caspase-1 is usually activated in susceptible LT-treated macrophages but not in resistant cells (31). Studies with caspase-1-deficient murine macrophages LDS 751 and caspase-1 inhibitors suggest that caspase-1 is essential for LT killing of susceptible murine macrophages (6, 31, 41). The mechanism by which microbial components, including LT, activate the inflammasome and the way in which this results in caspase-1 activation are poorly comprehended. In contrast to bacteria, which LDS 751 contain multiple virulence factors that simultaneously activate several NLRs, LT is usually a single virulence factor and appears to represent an ideal LDS 751 LDS 751 model system to study microbial inflammasome induction and caspase-1 activation. Our findings indicate that LT triggers the formation of an inflammasome complex made up of Nalp1b and caspase-1 in murine macrophages. In untreated macrophages, caspase-1 was a part of low-molecular-weight fractions and shifted toward high-molecular-weight fractions following LT treatment. Formation of the high-molecular-weight complex, presumably the inflammasome, coincides with caspase-1 activation and macrophage lysis. Caspase-1-associated proteins also included caspase-11 and the caspase-1 target -enolase in LT-treated macrophages. MATERIALS AND METHODS Cell lines and plasmids. The BALB/c-derived murine macrophage cell line J774A.1 and the human kidney fibroblast line 293T were obtained from the American Type Tissue Culture Collection (ATCC, Manassas, VA). Cells were cultured in Dulbecco’s altered Eagle’s medium (DMEM) (Mediatech, Herndon, VA) made up of 10% fetal bovine serum (HyClone, Logan, UT) supplemented with 100 U/ml penicillin and 100 g/ml streptomycin (Gibco, Grand Island, NY). The procaspase-1 expression vector was purchased from ATCC. The.