Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0404221 on July 7, 2004

Published online before print July 7, 2004
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(Journal of Leukocyte Biology. 2004;76:676-684.)
© 2004 by Society for Leukocyte Biology

Differing caspase-1 activation states in monocyte versus macrophage models of IL-1ß processing and release

J. Michelle Kahlenberg* and George R. Dubyak{dagger},1

* Department of Pathology and
{dagger} Department of Physiology and Biophysics, Case School of Medicine, Cleveland, Ohio

1 Correspondence: Department of Physiology and Biophysics, Case School of Medicine, 2109 Adelbert Road, Cleveland, OH, 44106. E-mail: george.dubyak{at}case.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The release of IL-1ß as an active, mature cytokine requires proteolytic processing by caspase-1, which is recruited to signaling complexes that facilitate its autocatalytic proteolysis and activation. Caspase-1 processing has been characterized in human monocyte and murine macrophage model systems, and comparative analyses indicate significant mechanistic differences in caspase-1 activation by these cell types. In this study, we used an in vitro processing assay to compare caspase-1 activation in THP-1 human monocytes vs. Bac1.2F5 murine macrophages. These in vitro caspase-1 and IL-1ß processing reactions indicated a higher rate of constitutive caspase-1 activation in lysates from THP-1 vs. Bac1 cells. Transfer of small amounts of THP-1 lysate to Bac1 lysate rapidly increased in vitro procaspase-1 and proIL-1ß processing in the latter preparation. The transferable activation factor(s) was heat-labile, ≥10 kDa, and unaffected by immunodepletion of procaspase-1 from the THP-1 lysate. This transactivating effect of THP-1 lysate on processing in Bac1 lysates could be mimicked by addition of purified recombinant human caspase-1. The constitutive caspase-1 and IL-1ß processing reactions in THP-1 lysates were insensitive to pharmacological blockade by the tyrphostin, AG126, and the phospholipase A2 inhibitor bromoenol lactone (BEL); contrarily, the same processing reactions were inhibited in lysates from Bac1 cells pretreated with either AG126 or BEL. These observations indicate significant biochemical differences in the assembly and regulation of caspase-1 signaling complexes within human monocyte and murine macrophage models of inflammatory activation. These differences need to be considered when comparing or pharmacologically manipulating IL-1ß processing and release in various model systems.

Key Words: AG126 • bromoenol lactone • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Strict regulation of the cytokine IL-1ß is crucial to prevent aberrant activation of pathways that can lead to chronic inflammation, septic shock, or death [1 ]. The study of IL-1ß maturation and release has been characterized mainly in either human monocytes or murine macrophages as model systems. IL-1ß accumulates as a 33 kDa pro-cytokine in the cytoplasm of monocytes and macrophages, and its activation depends on cleavage to an active 17 kDa (mIL-1ß) form by the enzyme caspase-1 [2 , 3 ]. In response to bacterial infection, LPS, or extracellular ATP, these cells can activate caspase-1 and subsequently process and release IL-1ß [4 5 6 ]. Caspase-1 is synthesized as a 45 kDa zymogen that is made active by cleavage of its C terminus into p10 and p20 subunits to form a heterotetramer (two p10 and two p20 subunits) [7 ]. Many in vitro and overexpression studies have suggested that the activation of procaspase-1 depends on the oligomerization of two or more procaspase-1 molecules via caspase association recruitment domain (CARD) interactions between various adaptor proteins that can bind to the CARD domain of procaspase-1 [8 9 10 11 12 13 14 ].

Macrophages are the prime physiologic source of released IL-1ß [1 ]. In these cells, LPS stimulates pro-IL-1ß production, but little secretion of mIL-1ß occurs in the absence of a secondary K+ release stimulus that triggers caspase-1 activation [15 16 17 18 19 20 ]. Such K+ release stimuli include activation of native ionotropic P2X7 receptors by extracellular ATP or exposure to pore-forming toxins and proteins derived from microbial (e.g., Staphylococcal {alpha} toxin) or mammalian (e.g., neutrophil cathelicidin) sources. Conversely, human monocytes, and THP-1 monocytes in particular, release significant amounts of processed IL-1ß in response to LPS alone [18 19 20 21 22 23 ], although the rate of release is increased further by secondary K+ release stimuli. Interestingly, as freshly isolated human blood monocytes are aged overnight on plastic (becoming more macrophage-like), they become less capable of releasing mature IL-1ß in response to LPS stimulation alone [16 ] and the rate of IL-1ß release becomes increasingly dependent on secondary stimulation by K+ release from the cell [20 , 24 ]. This condition indicates that during differentiation of monocytes to macrophages, a progression tends toward more tightly regulated caspase-1 activity, such that macrophages require a secondary K+-releasing stimulus for robust activation of caspase-1.

Many recent biochemical studies of the signal transduction machinery that underlies caspase-1 processing and IL-1ß maturation have used THP-1 human monocytic leukemia cells as a model system. These biochemical studies have used highly concentrated cell-free lysates from THP-1 cells for the in vitro identification, dissection, and manipulation of the adaptor proteins and factors that regulate caspase-1 activation [9 , 13 , 14 ]. However, similar biochemical studies have not been performed by using cell-free preparations from murine macrophages (freshly isolated or cell lines), the other major model system that has contributed to current understanding of the caspase-1/IL-1ß processing cascade. Here, we utilize an in vitro processing assay to examine and directly compare the biochemical activation states of two common model systems, THP-1 human monocytes and Bac1 murine macrophages, and thereby characterize biochemical and pharmacological parameters that may underlie cell-specific differences in the activation of caspase-1 and the subsequent processing of IL-1ß.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and antibodies
Purified recombinant human caspase-1 (highly active tetrameric complexes of p20 and p10 subunits) was purchased from Calbiochem (San Diego, CA). Cells were variously treated with the following reagents: AG126 (Calbiochem), bromoenol lactone (BioMol, Plymouth Meeting, PA), and Escherichia coli LPS serotype 01101:B4 (List Biologicals, Campbell, CA). Human and murine IL-1ß enzyme-linked immunosorbent assay (ELISA) antibodies (M-421B-E, M-420B-B, PM-425B, and MM-425B-B) were from Pierce Endogen (Rockford, IL). Anti IL-1ß used for Western blots (3ZD) was provided by the Biological Resources Branch of the National Cancer Institute–Frederick Cancer Research and Development Center (Bethesda, MD). Other antibodies were obtained from Santa Cruz: rabbit polyclonal anti-human caspase1 p10, rabbit polyclonal anti-mouse caspase-1 p10, all HRP-conjugated secondary antibodies, and anti-phosphotyrosine PY99.

Cell culture
Bac1.2F5 murine macrophages were cultured as described previously [25 ] in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO) supplemented with 25% L-cell conditioned media, 15% calf-serum (Hyclone, Logan, UT) and 1% Pen-Strep (100 U/ml penicillin and 100 µg/ml streptomycin; Gibco, Grand Island, NY) in the presence of 10% CO2. For experiments, cells were split 1:3 onto 150 cm2 culture dishes two to three days beforehand. THP-1 human monocytes were cultured in RPMI 1640 (Sigma) with 10% calf-serum and 1% Pen-Strep with 5% CO2 and were maintained at a cell density of 106/ml.

IL-1ß release assay
Bac1 or THP-1 cells (1 x 106) were plated into six-well plates. Cells were then treated with 500 ng/ml LPS for 16 h. The media were then collected, spun to remove any cell debris, and then used in a sandwich ELISA assay as described previously [26 ]. Briefly, 1–50 µl of media was added to a BSA-blocked ELISA plate that had been coated overnight with 1 µg/ml anti-murine IL-1ß. Biotin-conjugated IL-1ß antibody was then added, and the plates were incubated at room temperature for 2 h. The plate was then washed and incubated with HRP-conjugated streptavidin (Pierce Endogen) for 30 min and developed by using tetramethyl benzidine as substrate. The absorbance measurements were read at 450 nm with a Molecular Devices SoftMax Pro plate reader (Sunnyvale, CA) and were compared with IL-1ß standards.

In vitro processing assays
These assays were performed as described previously [26 ]. Briefly, for both THP-1- and Bac1-based assays, 1 x 108 cells were treated with 500 ng/ml LPS for 4 h. The cells were washed 1x in phosphate buffered saline (PBS) and resuspended in 1 ml buffer W (20 mM HEPES, pH 7.5; 10 mM KCl; 1.5 mM MgCl2; 1.0 mM EGTA; 1.0 mM EDTA) supplemented with 2 mM DTT, 2 µg/ml leupeptin, 100 µg/ml PMSF, and 2.5 µg/ml aprotinin. The cells were then pelleted, and all but ~50 µL of the buffer was removed. The cells were then allowed to swell for 10 min on ice and were subsequently lysed by 15 passages through a 22G needle. Lysates were then spun at 15,000 x g for 15 min, and the supernatant was removed into a new tube and kept on ice. Protein concentrations were determined by using the Bradford assay (BIO-RAD, Hercules, CA), and protein levels were adjusted to 22 mg/ml by using buffer W. Lysates (10 µL) were aliquotted into 1.5 ml tubes and placed at 30°C for the indicated times to facilitate proteolytic processing of the native caspase-1 and proIL-1ß under defined in vitro conditions. Processing reactions were stopped by adding an equal volume of 4x SDS-PAGE buffer followed by heat denaturation. Lysates were run on 15% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF; Millipore, Bedford, NH). Western Blots were probed with the following antibody concentrations: anti-IL-1ß: 5 µg/ml, anti-caspase-1: 5 µg/ml.

For transactivation of Bac1 lysates by factors in the THP-1 lysates, 1.25 x 108 THP-1 cells were treated with 500 ng/ml LPS for 2 h. THP-1 cells were lysed as above, and the lysates were kept on ice. Bac1 cells were treated with 500 ng/ml LPS for 4 h and then lysed as above and kept on ice. THP-1 lysate (1.35 µg) was added to 220 µg Bac1 lysate at a ratio of 1:11 (1 µL THP-1 lysate to 10 µL of Bac1 lysate), and the final protein concentration was adjusted to 22 mg/ml. Lysates were then incubated at 30°C as indicated above. This protocol was varied in some experiments by boiling the THP-1 lysate for 5 min before supplementation of Bac1 lysates. To determine the possible requirement for phosphorylated factors, THP-1 lysates were incubated with washed alkaline phosphatase-conjugated agarose beads (Sigma) at 166 U/ml for 1 h at 37°C. Lysates were spun to remove beads, and then THP-1 lysates were added to prepared Bac1 lysates as above. To determine the possible role of transferred human procaspase-1 in the transactivation of Bac1 lysates by THP-1 lysates, non-LPS treated THP-1 cells were lysed in buffer W at 16 mg/ml and then rotated at 4°C for 3 h in the presence of 30 µl protein A bead 50/50 slurry (Santa Cruz) and 5 µl (1 µg) of anti caspase-1 p10 antibody (Santa Cruz). Sample (30 µl) was run to determine depletion. The beads were washed 3x in buffer W and eluted with 30 µl of 4x SDS sample buffer to determine levels of caspase-1 bound. To determine the role of small proteins and factors from THP-1 cytosol in the transactivation of caspase-1/IL-1ß processing in Bac1 lysates, 3' 108 cells were washed 3' in PBS, lysed in buffer W as above, and diluted to 5 mg/ml. The lysate was placed in a Centricon-10 concentrator (Millipore, Billerica, MA) and spun for 60 min at 3000 x g. Equal amounts (~3.2 µg/220 µg Bac1 lysate) of filtrate or retentate protein were then added to Bac1 lysates, and processing was monitored as above.

To examine the acceleration of processing reactions in Bac1 lysates by purified human caspase-1 alone, Bac1 lysates were made as above at a concentration of 22 mg/ml. Purified, recombinant human active caspase-1 [10 ng; 0.125 µl to 10 µl (220 µg) of Bac1 lysate] was added to the Bac1 lysate, and then the lysates were placed at 30°C for the indicated time points and processing was monitored as above.

To examine the effects of AG126 or bromoenol lactone (BEL) on the constitutive rates of caspase-1/IL-1ß processing in lysates from THP-1 or Bac1 cells, 1.5 x 108 cells per treatment were stimulated with 500 ng/ml LPS for 4 h at 37°C, and inhibitors (AG126 or BEL) were added during the last 20 min of LPS treatment. Cells were then washed once in serum-free RPMI and were resuspended in buffer W and lysed as above. In vitro processing reactions were initiated and assayed as described above. In some cases, AG126 or BEL was added directly to the cell-free lysates prepared from control cells before initiation of the in vitro processing reactions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LPS-activated THP-1 monocytes exhibit increased mIL-ß secretion relative to LPS-activated Bac1 macrophages
Monocytes have been shown to release mIL-1ß in response to LPS stimulation alone; whereas other inflammatory cell types, and macrophages in particular, exhibit more stringently controlled mIL-1ß production that requires a secondary K+ release stimulus for initiation of IL-1ß processing and release [19 , 21 22 23 ]. Fig. 1 illustrates the differential release of mature IL-1ß from THP-1 monocytes vs. Bac1 macrophages, a cell line with tightly controlled IL-1ß processing [19 , 26 ], in response to LPS activation alone. Both cell types were treated with LPS for 16 h, followed by ELISA to detect mIL-1ß released to the extracellular media. Although both cell types accumulated proIL-1ß in equivalent amounts (Fig. 1 insert), Bac1 macrophages released very little mIL-1ß with LPS stimulation, whereas THP-1 monocytes readily processed and released IL-1ß in response to LPS (Fig. 1) . These data suggest the possibility of cell-specific differences in the signaling pathways that regulate caspase-1 activation and IL-1ß secretion in these two inflammatory cell models.



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  		Figure 1. THP-1 monocytes but not Bac1 macrophages release large amounts of mIL-1ß in response to LPS stimulation. THP-1 or Bac-1 cells were split into six-well dishes at 1 x 106/well. The cells were treated for 16 h with 500 ng/ml LPS, and the media was collected and centrifuged to remove cell debris. mIL-1ß released into the media was then quantified by ELISA. Inset: To control for production of pro-IL-1ß, the cells were lysed in SDS-PAGE buffer, and the lysates were subjected to Western blot analysis for pro-IL-1ß.

 
Rates of in vitro caspase-1/ IL-1ß processing in the cell-free lysates from THP-1 monocytes vs. Bac1 macrophages differ substantially
Previous studies have demonstrated that when inflammatory cells, including THP-1 and Bac1, are lysed in a hypotonic buffer, a "spontaneous" or constitutive activation of casapse-1 occurs that can be additionally influenced by poorly defined activation states or factors present in the intact cells prior to lysis [7 , 13 , 26 ]. Because intact THP-1 human monocytes, in contrast to Bac1 murine macrophages, significantly process and release IL-1ß in response to LPS alone, we hypothesized the possibility of fundamental differences in the kinetics of constitutive caspase-1 activation by cell-free lysates derived from these two cell types, even when assayed under defined and identical in vitro conditions. To test this, both THP-1 and Bac1 cells were treated with LPS for 4 h, lysed, and then analyzed by the in vitro processing assay as described in Materials and Methods. As shown in Fig. 2 , both the Bac1 macrophage and the THP-1 monocyte lysates could process IL-1ß in vitro when placed at 30°C. However, at similar protein concentrations, the THP-1 lysates processed IL-1ß much more rapidly, with nearly all proIL-1ß processed within 10 min compared with Bac1 lysates in which proIL-1ß was not processed to completion, even by 60 min. Additionally, Fig. 2 demonstrates that THP-1 lysates contain some active caspase-1 p10 subunits (indicative of the presence of the highly active p20/p10 tetrameric forms of processed caspase-1) even when the lysates are kept at 4°C, a condition previously shown to prevent caspase-1 activation in vitro [13 ]. Bac1 lysates, however, show no evidence of accumulation of active caspase-1 subunits when kept at 4°C. This finding suggests that before lysis, significant amounts of the caspase-1 within THP-1 monocytes exist in a pre-activated or "primed" state, which is then reflected in an extraordinarily rapid and complete processing of both caspase-1 and IL-1ß when the cell-free lysates are subsequently incubated in vitro. However, Bac1 macrophage lysates, which do not accumulate active caspase-1 in response to LPS treatment, exhibit a much slower rate of processing that is limited by the spontaneous activation of caspase-1 in vitro (Fig. 2) .



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  		Figure 2. Rates of caspase-1 and IL-1ß processing differ in cell-free lysates from Bac1 macrophages vs. THP-1 monocytes. Bac1 and THP-1 cells were treated for 4 h with LPS and then lysed in a hypotonic buffer at 22 mg/ml according to Materials and Methods. The lysates were placed at 4 or 30°C for the indicated time points, and processing was monitored by Western blot by using antibodies for IL-1ß and antibodies specific for either mouse or human caspase-1.

 
Caspase-1/IL-1ß processing reactions in Bac1 lysates can be transactivated by supplementation with THP-1 lysate
Because the caspase-1 signaling machinery in THP-1 monocytes appears to be substantially activated before lysis, we tested whether an activating factor(s) from THP-1 lysates could be transferred to Bac1 lysates to accelerate the much slower processing reactions that characterize this latter model system. We treated 1 x 108 THP-1 monocytes with LPS for 2 h, lysed the cells as described in Materials and Methods, and kept the lysates on ice until use. Bac1 macrophages were treated with LPS for 4 h to up-regulate pro-IL-1ß and were also lysed via the same protocol. Aliquots (220 µg; 10 µl) of Bac1 lysates were then supplemented with lysis buffer (1 µl buffer to 10 µl lysate) or with 1.35 µg (in 1 µl) of THP-1 lysate protein and then incubated at 30°C for the indicated time points. The addition of THP-1 lysate markedly accelerated the processing of both Bac1 IL-1ß and caspase-1 (Fig. 3 ). This finding was true even when THP-1 cells that were not treated with LPS were the source of transferred lysate (data not shown). The caspase-1 processing was detected with an antibody that is specific for the mouse caspase-1 p10 subunit, so the small amount of human caspase-1 p10 transferred from THP-1 lysates is not detected. The reverse experiment was then performed to determine whether any inhibitory factors within the Bac1 lysates could be found that might slow IL-1ß processing in vitro. When 3 µg (1 µl) of Bac1 lysate protein was added to 220 µg (10 µl) THP-1 lysate, no difference in THP-1-mediated processing was seen, which suggests that caspase-1/IL-1ß processing reactions have no transferable inhibitor(s) of within the Bac1 lysates (Fig. 3) .



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  		Figure 3. THP-1 lysate components can accelerate caspase-1 and IL-1ß processing in Bac1 lysates. (A) LPS-treated Bac1 cells were lysed according to Materials and Methods, and 1 µl (1.35 µg) of protein from LPS-treated THP-1 lysate was added to 10 µl (220 µg) Bac1 lysate while on ice. The reverse was also done where LPS-treated THP-1 cells were lysed and 1 µl (3 µg) of protein of Bac1 lysates was added to 10 µl of THP-1 lysate. Lysates were then placed at 30°C, and reactions were stopped after the indicated time points with SDS sample buffer. Processing was monitored as in Fig. 2 .

 
Experiments were designed to further characterize the transferable factor(s) within the THP-1 lysates that facilitated transactivation of the caspase-1/IL-1ß processing reactions in the Bac1 lysates. Boiling the THP-1 lysate prior to mixing with the Bac1 lysate prevented transactivation of the processing reaction, which suggested that a heat labile component, most likely a protein, in the THP-1 lysate is responsible (Fig. 4A ). THP-1 lysates were also centrifuged through a Centricon-10 ultrafiltration matrix (which will remove components <10 kDa from the lysate), and either the filtrate or retentate fraction was added to Bac1 lysates prior to initiation of the in vitro processing reactions. Although the added retentate markedly accelerated the Bac-1 processing reactions (Fig. 4B) , the filtrate, which contained all cytosolic factors <10 kDa, did not (Fig. 4B) . This finding suggested that the transactivating factors are not small molecules or peptides but rather are proteins or other macromolecules >10 kDa. Notably, the Centricon-10 retentate contained the p10 subunit of human caspase-1 (Fig. 4C) , consistent with its high-affinity association within the 60 kDa heterotetrameric complexes (2xp20 subunits; 2xp10 subunits) that comprise fully processed, active caspase-1 [7 ].



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  		Figure 4. Transactivation of processing reactions in Bac1 lysates is dependent on THP-1 lysate factors >10 kDa that are inactivated by boiling but not alkaline phosphatase treatment. (A) To check for heat lability, THP-1 lysates were generated as in Fig. 3 . Bac1 lysates were treated with 1 µl buffer W (left), 1 µl of untreated THP-1 lysate (center), or THP-1 lysate that was boiled for 5 min (right). (B) 1 ml THP-1 lysate (5 µg/ml) was centrifuged through a Centricon-10 filter; 1 µl of either the retentate (>10 kDa) or the filtrate (<10 kDa) was then added to Bac1 lysates as in Fig. 3 . (C). A quantity of 30 µl of the filtrate (F) or the retentate (R) was subjected to Western analysis with anti caspase-1 p10 antibody to determine the location of the p10 fragment. (D) Before addition to Bac1 lysates, THP-1 lysates were incubated with washed alkaline phosphatase (AP) conjugated agarose beads at 37°C for 1 h. Lysates were spun to remove the beads and were then added to Bac1 lysates as above. (E) AP-treated lysates were subjected to Western analysis with anti phospho-tyrosine antibodies as an index of decreased phosphoprotein levels.

 
Given the ubiquitous role of protein phosphorylation in the acute regulation of enzyme activities and signaling cascades, we considered the possibility that hyperphosphorylation of a caspase-1 regulatory protein might contribute to the markedly different rates of caspase-1/IL-1ß processing of the THP-1 vs. Bac1 lysates. To test this theory, we treated THP-1 lysate with immobilized alkaline phosphatase for 1 h before adding it to Bac-1 lysate. This alkaline phosphatase treatment markedly reduced (but did not eliminate) overall phosphoprotein levels within the THP-1 lysates, as determined by anti-phosphotyrosine Western blot analysis (Fig. 4E) . However, this treatment did not affect the ability of transferred THP-1 lysate to transactivate and accelerate processing of the murine proIL-1ß within the Bac1 lysates (Fig. 4D) . This finding indicates that hyperphosphorylation of some regulatory protein is an unlikely reason for the enhanced activation state of caspase-1 within THP-1 monocytes or the lysates derived from these cells.

The presence of caspase-1 p10 subunits within the retentate fraction of the Centricon-10 filtered THP-1 lysates suggested that one transactivating factor might be the tetrameric form of active caspase-1 itself. Alternatively or additionally, human procaspase-1 precomplexed with various adaptor proteins within the THP-1 lysate might act as a "seeding" factor to accelerate the processing of murine procaspase-1 when added to the Bac1 lysates. To test these possibilities, we first immunodepleted procaspase-1 from the THP-1 lysates by using an antibody directed against epitopes in the p10 domain of human procaspase-1. We found that this antibody immunoprecipitated the pro form of caspase-1, but not the p10 fragment, most likely due to epitope changes after cleavage. Despite the very efficient removal of procaspase-1 from the THP-1 lysates (Fig. 5B ), the addition of these immunodepleted lysates to Bac1 lysates still accelerated processing of the murine caspase-1 and IL-1ß within the Bac1 lysates (Fig. 5A) . This finding suggested that the transactivating factor is not human procaspase-1 or adaptor proteins that might co-precipitate with the anti-caspase-1 immune complexes. That this anti-caspase-1 antibody did not immunoprecipitate the p10 subunit of caspase-1 suggested that highly active, p20/p10-based complexes of caspase-1 within the THP-1 lysates might be responsible for the acceleration of the Bac1 processing reactions. Western blot analysis indicated that 5 µg of THP-1 lysate contains ~10 ng of caspase-1 (data not shown). To determine whether addition of active caspase-1 alone (as the complex of p10 and p20 subunits) was sufficient to accelerate the processing reactions in Bac1 lysates, we added 10 ng (14.5 units) of recombinant human caspase-1 to 220 µg of Bac1 lysate prior to initiation of the in vitro processing assay. Fig. 5C shows that this amount of purified caspase-1 was sufficient to accelerate processing of not only the murine proIL-1ß but also the murine procaspase-1 within the Bac1 lysates.



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  		Figure 5. Active caspase-1 heterotetramers can induce accelerated processing in Bac1 cell-free lysates. (A) Before addition to Bac1 lysates, THP-1 lysates were depleted of procaspase-1. THP-1 lysates were then added to Bac1 lysates as in Fig. 3 . (B) Caspase-1-depleted THP-1 lysates or the beads from the immunoprecipitation were probed with a caspase-1 p10 antibody to demonstrate effectiveness of the immunodepletion. (C) Bac1 cells were treated with LPS for 4 h and then lysed according to Materials and Methods. Buffer W vehicle or 10 ng of purified active caspase-1 was then added to the Bac1 lysates, and processing was monitored as in previous experiments.

 
AG126 and bromoenol lactone have differential inhibitory effects on in vitro caspase-1/ IL-1ß processing in cell-free lysates from THP-1 monocytes vs. Bac1 macrophages
To determine why THP-1 monocytes have an increased ability for caspase-1 activity with or without LPS stimulation, we used two inhibitors previously shown to block caspase-1 activation. Both the tyrphostin AG126 and bromoenol lactone are effective inhibitors of IL-1ß release and caspase-1 activation [26 27 28 29 ]. Our previous work has shown that both AG126 and BEL block the P2X7-mediated acceleration of caspase-1 processing in intact Bac1 cells but do not directly inhibit caspase-1 in vitro [26 ]. Preincubation of Bac1 macrophages with these inhibitors before lysis blocked the basal rate of IL-1ß processing seen in the in vitro processing assay (Fig. 6A ). Because THP-1 lysates exhibit an increased state of caspase-1 activation as compared with Bac-1 macrophages, we tested whether these inhibitors might have similar effects on in vitro IL-1ß and caspase-1 processing by THP-1 lysates. Fig. 6B demonstrates that neither AG126 nor BEL slowed the rate of IL-1ß or caspase-1 processing in THP-1 lysates, which indicates that the respective targets of these inhibitors are not required for processing per se in THP-1 lysates but rather for the initiation of caspase-1 activation in more regulated cell types, such as macrophages. Thus, this underscores a distinct difference between the signaling mechanisms required for activation of caspase-1 in THP-1 monocytes vs. Bac1 macrophages. Interestingly, when THP-1 lysate is added to BEL-treated Bac1 cell lysate, it cannot induce transactivation of the mouse caspase-1 (data not shown). This finding further suggests that the signaling mechanisms in Bac1 cells are regulated in a different fashion than in the THP-1 cells such that these inhibitors can maintain caspase-1 in an inaccessible and inactive state in macrophages, but not in THP-1 monocytes.



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  		Figure 6. Constitutive caspase-1 activation in THP-1 lysates is insensitive to inhibition by AG126 or BEL. (A) Bac1 cells were treated with LPS for 4 h followed by incubation with 50 µM AG126 or 20 µM BEL for 20 min before lysis. Cells were lysed in buffer W according to the Materials and Methods. Cell lysates were then subjected to the in vitro processing assay as in previous experiments. (B) THP-1 cells were treated with LPS for 4 h and treated with 50 µM AG126 or 20 µM BEL for 20 min before lysis. Cells were then washed and lysed as described in the Materials and Methods. Processing was analyzed via Western blot with IL-1ß and anti-human capsase-1 p10 antibodies.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although the processing and release of IL-1ß have been studied extensively, the mechanism of caspase-1 activation remains unresolved. Much research has focused on the two steps required for initiation of IL-1ß processing. Primary inflammatory stimuli, such as LPS, up-regulate IL-1ß synthesis in monocytes and macrophages. In most cells, such treatment elicits only slow rates of IL-1ß processing and secretion, and most of the IL-1ß made accumulates as its p33 kDa pro form within the cytoplasm [1 ]. However, treatment of freshly isolated blood monocytes or THP-1 monocytes with LPS alone results in mIL-1ß release that can be increased further with secondary K+-releasing stimuli [18 , 19 , 21 22 23 ].

The processing of IL-1ß into its mature form is dependent on the autoactivation of procaspase-1 into its p20/p10 heterotetramer. Recently, a complex of proteins, termed the inflammasome, was characterized in THP-1 monocyte lysates that oligomerize and subsequently activate procaspase-1 [13 ]. This complex, consisting of caspase-1, caspase-5, NALP-1/CARD7/NAC/DEFCAP, and ASC/PYCARD, can be detected when THP-1 cell lysates prepared in hypotonic buffer are incubated at 30°C. This activation is blocked by incubation with an anti-ASC antibody, which suggests that in vitro activation of caspase-1 is also dependent on ASC [13 ]. Another inflammasome complex was also recently described in THP-1 lysates that include caspase-1, ASC, and NALP3, and Cardinal [14 ]. However, the intracellular signals that induce inflammasome formation remain to be determined.

Using an in vitro processing assay, we now show a key difference between THP-1 human monocytes and Bac1 murine macrophages in that THP-1 cells possess the ability for immediate caspase-1 activation upon hypotonic lysis but macrophages do not. This "preactivation" can be transferred to Bac-1 lysates by addition of THP-1 lysate protein corresponding to only 1/200th the amount of Bac1 lysate protein. However, Bac1 lysates cannot act as a "brake" for the THP-1 lysates, which suggests that the slow rate of caspase-1 processing in Bac1 macrophage lysates reflects a lack of activating factors rather than an increased level of inhibitors. We have shown that this transactivation of Bac1 processing by THP-1 lysate may be due in part to the transfer of active caspase-1. First, we demonstrated that removal of procaspase-1, but not the p10 fragment, from THP-1 lysates did not affect their ability to activate Bac1 lysates. Second, the addition of recombinant active caspase-1 to Bac1 lysates can replicate the acceleration seen with THP-1 lysate alone, which underscores the difference in caspase-1 activation between the two cell types. Additionally, this finding raises an interesting question regarding the role of active p20/p10 caspase-1 tetramers in further activation of procaspase-1. Previous studies have examined the ability of purified, active p20/p10 caspase-1 tetramers to cleave S35-labeled recombinant p45 procaspase-1. These studies suggested that the reaction is inefficient and that after a 60-min incubation at 30°C, 12 units of active caspase-1 (an amount similar to that used in our studies) generates only small amounts of intermediate fragments and no p10 fragments of S35-labeled caspase-1 [7 ]. This finding suggests that active caspase-1 alone is not sufficient for efficient caspase-1 activation in vitro. Why then do we see a greatly accelerated activation of murine caspase-1 activation in Bac1 lysates with the addition of human p20/p10 caspase-1 heterotetramers? Most current models of caspase-1 activation are based on the hypothesis that the activation of caspase-1 depends on CARD-CARD oligomerization of procaspase-1 within inflammasome-like complexes. However, active p10/p20 heterotetramers no longer contain the CARD domain, so it has generally been assumed that they are released from the complex after cleavage [7 ]. One possibility to explain why active human caspase-1 so efficiently activates mouse caspase-1 when added to Bac1 lysates may be that the active caspase-1 tetramer is recruited to the scaffolding complex wherein procaspase-1 is activated. This condition would provide an efficient feed-forward mechanism for very rapid activation of caspase-1. In fact, the only major study that has investigated caspase-1 assembly within the inflammasome, a defined molecular platform for caspase-1 activation, used antibodies specific only for the CARD domain of caspase-1 (which is not part of the active heterotetramers) [13 ]. Thus, the issue of whether the active subunits of processed caspase-1 remain in the complex has not been unequivocally determined.

Additionally, other factors within the THP-1 cell lysates may also contribute to the transactivation of the murine caspase-1 in the Bac1 lysates. It has been shown that mutations in NALP3 that are associated with Muckle-Wells syndrome result in an increase in the activation state of monocytes such that increased levels of mIL-1ß are produced on a constitutive basis [14 ]. Likewise, it may be possible that the signals that are necessary for inflammasome formation in THP-1 cells are constitutively produced at an increased level of activity, and this is why these lysates so readily activate caspase-1. Because THP-1 is a cell line derived from a myelogenous leukemia, it is possible that these cells contain mutations of gene products that allow for increased activation of inflammasome-like complexes. However, Bac1 macrophages, which were generated by SV40 transformation and immortalization of normal Balb-C murine macrophages, as well as related murine macrophage cell lines such as RAW264 or J774, exhibit significant caspase-1 activation only when exposed to secondary stimulation, such as extracellular ATP. Indeed, we have recently reported that cell-free lysates prepared from ATP-stimulated Bac1 macrophages (i.e., intact cells treated with extracellular ATP prior to lysis) exhibit greatly accelerated rates of in vitro IL-1ß and caspase-1 processing that are comparable with the rates observed in lysates from unstimulated THP-1 cells [26 ]. Interestingly, ATP can also increase the rate of IL-1ß processing and release in THP-1 cells, but these cells are usually differentiated with IFN{gamma} [19 ] or phorbol ester [22 ] to first up-regulate the P2X7R. This condition raises the issue of whether additional factors are modified during differentiation that also regulate the ability for P2X7R to activate caspase-1. The identity and nature of these signals that regulate caspase-1 activation states remain to be determined, but increased phosphorylation of relevant proteins in THP-1 lysates does not appear to play a role, as treatment of THP-1 lysates with alkaline phosphatase, which decreased the overall phosphoprotein levels in the lysates, did not affect the THP-1-induced acceleration of processing reactions in Bac1 lysates.

An interesting clue to the signals that are active in the THP-1 lysates may lie in their insensitivity to known inhibitors of caspase-1 activation in macrophages and monocyte-derived macrophages. Recently, we demonstrated that AG126 and BEL completely block ATP-induced in vitro caspase-1 activation in Bac1 macrophages [26 ]. AG126 is a member of the tyrphostin family of compounds, many of which inhibit tyrosine kinases. The target of AG126 remains unknown, but its identification may reveal the regulated signaling pathway for caspase-1 activation. Additionally, because bromoenol lactone can inhibit Ca++-independent phospholipase A2 (iPLA2), lipid second messengers released as a result of this enzyme’s activation may also play an important part in caspase-1 activation. Although Bac1 macrophages cannot activate caspase-1 in the presence of these inhibitors, neither AG126 nor BEL had any effect on the in vitro caspase-1 processing in THP-1 lysates. This finding suggests that the targets of these inhibitors lie upstream of caspase-1 activation signals induced either by lysis or ATP stimulation of intact Bac1 macrophages. However, THP-1 monocytes can constitutively bypass the requirement for activation of these signaling pathways, which results in readily activated caspase-1. Further, incubation of intact THP-1 monocytes with YVAD to inhibit the active caspase-1 within the cell before lysis does not impair their ability to rapidly activate caspase-1 after hypotonic lysis or to accelerate Bac1 processing of IL-1ß with THP-1 lysate transfer (data not shown). This finding also suggests that THP-1 monocytes have an increased propensity to assemble complexes required for caspase-1 activation and that this phenotype is not dependent on preactivated caspase-1 within the cell. These biochemical differences in caspase-1 activity between Bac1 macrophages and THP-1 monocytes parallel the differences reported for mIL-1ß release from fresh human monocytes vs. plastic surface-differentiated human monocytes, mouse macrophages, and other macrophage cell lines [16 , 17 , 30 ]. Thus, it appears that the regulation and activation of caspase-1 in these cell types follow distinct but converging paths. The increased regulation of caspase-1 seen in macrophages may be important for controlling inflammation in tissues, where most exposure to inflammatory stimuli will occur. Monocytes, however, seem to have constitutive activation signals, even without LPS exposure, that would allow them to quickly produce mIL-1ß in conditions such as overwhelming septicemia. Although both monocytes and macrophages are important to the inflammatory response, these results indicate that the possible roles of caspase-1 activation or IL-1ß processing and release in this response need to be interpreted within the context of the cell type being studied. Thus, the ability of particular pharmacological agents to effectively regulate caspase-1 or IL-1ß processing/release may be dependent on the stage of differentiation or activation of the target proinflammatory cell.


   ACKNOWLEDGEMENTS
 
We would like to thank Sylvia Kertesy for her excellent technical assistance. This work was supported by NIH grant GM36387. M. K. is partially supported by NIH grant T32GM07250.

Received April 6, 2004; revised May 27, 2005; accepted June 1, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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