Published online before print July 7, 2004
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,1
* Department of Pathology and
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
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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
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 
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.
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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.
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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.
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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.
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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
.
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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.
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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.
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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.
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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
[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.
Received April 6, 2004; revised May 27, 2005; accepted June 1, 2004.
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