Technical noteApplying caspase-1 inhibitors for inflammasome assays in human whole blood
Introduction
Caspases (cysteinyl aspartate proteases) represent the class of intracellular proteases evolved in multicellular organisms. They are essential mediators of cell death and inflammation (Siegel, 2006). Pro-apoptotic and pro-inflammatory caspases can be distinguished based on their substrate specificity and involvement in diverse signaling cascades. For instance, caspases-3, -6 and -7 play roles as effectors of apoptosis (Slee et al., 2001). Conversely, caspases-1, -4, -5, -11 and -12 regulate the proteolytic processing of the inflammatory cytokines precursors to their mature forms (Siegel, 2006).
Caspase-1, specifically is an important member of multiple adaptor complexes known as inflammasomes, which are assembled in response to microbial and/or damage-associated signals (Osuka et al., 2012). Activation of caspase-1 leads to proteolytic processing of immature pro-IL-1β and pro-IL-18 to their bioactive forms. Furthermore, activation of caspase-1 stimulates pyroptosis, a form of cell death characterized by rapid pore formation in the plasma membrane followed by osmotic cell lysis (Satoh et al., 2013).
Caspase-1 inhibitors are commonly used to investigate inflammasome activity (Halle et al., 2008, Dostert et al., 2009, Netea et al., 2009, Osuka et al., 2012). Often utilized, z-VAD-fmk, is a cell-permeable, irreversible pan-caspase inhibitor. Importantly, z-VAD-fmk also has affinity to caspases-3, -7 and -8, thereby stabilizing Beclin1 and promoting autophagy (Zhu et al., 2010). Also of use is ac-YVAD-cmk, a specific irreversible inhibitor of caspase-1, however it also exerts some activity against caspase-4 and caspase-5 (Santa Cruz Biotechnology I., 2013).
In order to demonstrate inflammasome/caspase-1 involvement in disease processes, caspase-1 inhibitors are utilized to block pre-IL-1β processing, however additional targets for these inhibitors are not always considered. In a simple model of whole blood culture stimulated with LPS, we demonstrate profound effects of z-VAD-fmk on other cytokines not processed by the inflammasome (IL-6, TNFα and IL-8), whereas ac-YVAD-cmk preferentially blocked IL-1β secretion. Therefore we strongly urge investigators to utilize the most specific caspase inhibitors available when trying to block caspase-1 mediated post-translational processing of IL-1 family of pro-cytokines.
Section snippets
Materials and methods
Briefly, we prepared whole blood cultures by diluting sodium heparinized blood obtained from healthy volunteers with RPMI media (1:1) and incubated in the absence or presence of 2 ng/ml lipopolysaccharide (LPS from Escherichia coli O111:B4) at 37 °C, 5% CO2 for only 3 h (in order to limit the contribution of de novo transcription of cytokines in this analysis). Culture supernatant was collected and assayed for IL-1β, TNFα, IL-6 and IL-8 by MSD (Mesco Scale Discovery) or ELISAs (R&D systems) as
Results and discussion
In whole blood, primary monocytes respond to LPS stimulation directly driving IL-1β secretion (Netea et al., 2009). In our culture model, LPS induces significant secretion of IL-1β in 3 h compared to unstimulated control cultures (Fig. 1), although peak levels typically occur approximately 6 h post stimulation (data not shown). Caspase inhibitors are reportedly effective in concentrations of 10–100 μM in different cell culture conditions. Initial evaluation of the dose required for z-VAD-fmk
References (10)
- et al.
Caspase-1 activates nuclear factor of the kappa-enhancer in B cells independently of its enzymatic activity
J. Biol. Chem.
(2004) - et al.
Differential requirement for the activation of the inflammasome for processing and release of IL-1beta in monocytes and macrophages
Blood
(2009) - et al.
Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis
J. Biol. Chem.
(2001) - et al.
Malarial hemozoin is a Nalp3 inflammasome activating danger signal
PLoS ONE
(2009) - et al.
The NALP3 inflammasome is involved in the innate immune response to amyloid-beta
Nat. Immunol.
(2008)