ATP Treatment of Human Monocytes Promotes Caspase-1 Maturation and Externalization
1999; Elsevier BV; Volume: 274; Issue: 52 Linguagem: Inglês
10.1074/jbc.274.52.36944
ISSN1083-351X
AutoresRonald E. Laliberte, James F. Eggler, Christopher A. Gabel,
Tópico(s)Cellular transport and secretion
ResumoMechanisms that regulate conversion of prointerleukin-1β (pro-IL-1β) to its mature form by the cysteine protease caspase-1 are not well understood. In this study, we demonstrate that mature caspase-1 subunits are produced when human monocytes are treated with ATP and, like mature IL-1β, are released extracellularly. Characterization of the pharmacological sensitivity of this stimulus-coupled response revealed that some caspase-1 inhibitors allow pro-IL-1β secretion, whereas others do not. Two nonselective alkylating agents, N-ethylmaleimide and phenylarsine oxide, also blocked maturation and release of pro-IL-1β. Two inhibitors of anion transport, glyburide and ethacrynic acid, blocked maturation of both caspase-1 and pro-IL-1β and prevented release of the propolypeptides. Procaspase-3 was detected in monocyte extracts, but its proteolytic activation was not efficient in the presence of ATP. Maturation of procaspase-1 and release of the mature enzyme subunits therefore accompany stimulus-coupled human monocyte IL-1 post-translational processing. Agents that appear to selectively inhibit mature caspase-1 do not prevent ATP-treated cells from releasing their cytosolic components. On the other hand, anion transport inhibitors and alkylating agents arrest ATP-treated monocytes in a state where membrane latency is maintained. The data provided support the hypothesis that stimulus-coupled IL-1 post-translational processing involves a commitment to cell death. Mechanisms that regulate conversion of prointerleukin-1β (pro-IL-1β) to its mature form by the cysteine protease caspase-1 are not well understood. In this study, we demonstrate that mature caspase-1 subunits are produced when human monocytes are treated with ATP and, like mature IL-1β, are released extracellularly. Characterization of the pharmacological sensitivity of this stimulus-coupled response revealed that some caspase-1 inhibitors allow pro-IL-1β secretion, whereas others do not. Two nonselective alkylating agents, N-ethylmaleimide and phenylarsine oxide, also blocked maturation and release of pro-IL-1β. Two inhibitors of anion transport, glyburide and ethacrynic acid, blocked maturation of both caspase-1 and pro-IL-1β and prevented release of the propolypeptides. Procaspase-3 was detected in monocyte extracts, but its proteolytic activation was not efficient in the presence of ATP. Maturation of procaspase-1 and release of the mature enzyme subunits therefore accompany stimulus-coupled human monocyte IL-1 post-translational processing. Agents that appear to selectively inhibit mature caspase-1 do not prevent ATP-treated cells from releasing their cytosolic components. 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Although these peptides are potent inhibitors of purified caspase-1, much higher concentrations are required to prevent maturation of pro-IL-1β by intact cells due, in part, to the necessity that these agents must penetrate the plasma membrane to access the intracellular protease. For example, theK i of YVAD-CHO against caspase-1 is 0.76 nm, yet 5000-fold greater concentrations are required to inhibit IL-1β processing by human blood monocytes (4Thornberry N.A. Bull H.G. Calaycay J.R. Chapman K.T. Howard A.D. Kostura M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. Elliston K.O. Ayala J.M. Casano F.J. Chin J. Ding G.J.-F. Egger L.A. Gaffney E.P. Limjuco G. Palyha O.C. Raju S.M. Rolando A.M. Salley J.P. Yamin T.-T. Lee T.D. Shively J.E. MacCross M. Mumford R.A. Schmidt J.A. Tocci M.J. Nature. 1992; 356: 768-774Crossref PubMed Scopus (2267) Google Scholar). Evidence demonstrating that the various caspase inhibitors react exclusively with caspase-1 at these higher concentrations is lacking. The inhibitor acetyl-Tyr-Val-Lys(biotin)-Asp (acyloxy)methyl ketone displayed selectivity for mature caspase-1 subunits when assessed against an extract of THP-1 cells (38Thornberry N.A. Peterson E.P. Zhao J.J. Howard A.D. Griffin P.R. Chapman K.T. Biochemistry. 1994; 33: 3934-3940Crossref PubMed Scopus (253) Google Scholar), but similar studies employing intact cells have not been reported. In this study, ATP is employed as a stimulus to initiate IL-1β post-translational processing by LPS-activated human monocytes. This nucleotide triphosphate is shown to promote formation of mature caspase-1 subunits in addition to mature IL-1β and to elicit externalization of the mature products. In contrast, procaspase-3 is not efficiently processed by ATP-treated cells, suggesting that procaspase-1 and procaspase-3 require distinct signals for their maturation. The ATP-induced response is characterized with respect to its sensitivity to several distinct pharmacological agents. The results demonstrate that caspase-1 inhibitors do not act equivalently when evaluated in an intact cell system and provide insights into the novel cellular process employed by monocytes to generate and export the leaderless polypeptide IL-1β. Blood collected from normal volunteers in the presence of heparin was fractionated using lymphocyte separation medium obtained from ICN (Aurora, OH). The region of the resulting gradient containing banded mononuclear cells was harvested and diluted with 10 ml of maintenance medium (RPMI 1640 medium, 5% fetal bovine serum, 25 mm Hepes, pH 7.2, and 1% penicillin/streptomycin), and cells were collected by centrifugation. The resulting cell pellet was suspended in 10 ml of maintenance medium, and a cell count was performed. In an average metabolic experiment, 1 × 107 mononuclear cells were added to each well of 6-well multi-dishes in a total volume of 2 ml of maintenance medium. Alternatively, in experiments in which IL-1β production was measured by ELISA, 2 × 105 mononuclear cells were seeded into each well of a 96-well plate in a total volume of 0.1 ml. Monocytes were allowed to adhere for 2 h, after which the supernatants were discarded, and the attached cells were rinsed twice and then incubated in maintenance medium overnight at 37 °C in a 5% CO2environment. In the ELISA format, cultured monocytes in 96-well plates were activated with 10 ng/ml LPS (Escherichia coli serotype 055:B5; Sigma). Following a 2-h incubation, the activation medium was removed; cells were rinsed twice with 0.1 ml of chase medium (RPMI 1640 medium, 1% fetal bovine serum, 20 mm Hepes, and 5 mmNaHCO3, pH 6.9), 0.1 ml of chase medium containing a test agent was added, and the plate was incubated for 30 min. Each test agent concentration was evaluated in triplicate wells. ATP was then introduced (from a 100 mm stock solution, pH 7) to achieve a final concentration of 2 mm, and the plate was incubated at 37 °C for an additional 3 h. Media were harvested and clarified by centrifugation, and their IL-1β content was determined by ELISA (R&D Systems, Minneapolis, MN). In the metabolic format, cultured monocytes were incubated with 10 ng/ml LPS for 2 h and then labeled for 60 min in 1 ml of methionine-free RPMI 1640 medium containing 1% dialyzed fetal bovine serum, 25 mm Hepes, pH 7.2, and 83 μCi/ml [35S]methionine (1000 Ci/mmol; Amersham Pharmacia Biotech). The pulse medium was subsequently discarded; the radiolabeled cells were rinsed once with 2 ml of chase medium and then 1 ml of chase medium, with or without a test agent, was added to each well. Where indicated, ATP was added (from a 100 mm stock solution, pH 7) to achieve a final concentration of 2 mm. Radiolabeled monocytes were treated with ATP at 37 °C for various times, after which media were recovered and clarified by centrifugation; the resulting supernatants were harvested and adjusted to 1% in Triton X-100, 0.1 mm phenylmethylsulfonyl fluoride, 1 mm iodoacetic acid, 1 μg/ml pepstatin, and 1 μg/ml leupeptin by addition of concentrated stock solutions of these reagents. Adherent monocytes were solubilized by addition of 1 ml of extraction buffer composed of 25 mm Hepes, pH 7, 1% Triton X-100, 150 mm NaCl, 0.1 mm phenylmethylsulfonyl fluoride, 1 mm iodoacetic acid, 1 μg/ml pepstatin, 1 μg/ml leupeptin, and 1 mg/ml ovalbumin; 50 μl of this extraction buffer was also added to the pellets obtained after clarification of the media supernatants, and these samples were combined with their corresponding cell extracts. After a 30-min incubation on ice, both the media and cell extracts were clarified by centrifugation at 45,000 rpm for 30 min in a tabletop ultracentrifuge using a TLA 45 rotor (Beckman Instruments). Mononuclear cells (3 × 107 cells) in 10 ml of maintenance medium containing 10 ng/ml LPS were added to 10-cm dishes. These cultures were incubated for 2.5 h at 37 °C, after which non-adherent cells were discarded, and the adherent monocytes were washed three times with serum-free maintenance medium containing 10 ng/ml LPS. A test agent (in a total volume of 6.65 ml of maintenance medium containing 10 ng/ml LPS and 0.2% Me2SO) was introduced for 10 min; 5 mmATP was then added (from a 100 mm stock solution), and the cultures were incubated for an additional 90 min. Media were harvested, supplemented with protease inhibitors (1 μg/ml pepstatin, 1 μg/ml leupeptin, 0.1 mm phenylmethylsulfonyl fluoride, and 1 mm iodoacetic acid), and clarified by centrifugation to remove cell debris. Adherent monocytes were solubilized by addition of 3 ml of 10 mm Tris, pH 8, 1% Triton X-100, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 0.1 mm phenylmethylsulfonyl fluoride, and 1 mm iodoacetic acid and clarified by centrifugation. Media samples were concentrated with Centricon-30 concentrators (Amicon, Inc., Beverly, MA). For Western analysis, the media samples and cell lysates were treated with 30 μl of StrataClean resin (Stratagene, La Jolla, CA). The resin-bound protein samples were collected by centrifugation and washed once with 50 mmTris, pH 6.8; 30 μl of 2× SDS sample buffer was subsequently added to each sample, and the mixtures were boiled for 3 min. After centrifugation, the disaggregated protein samples were separated on 18% polyacrylamide gels (Novex, San Diego, CA). Proteins within the gels were transferred to nitrocellulose, and the blots were blocked by exposure to 5% nonfat milk in Tris-buffered saline containing 1% Tween (TBST). The blots were then incubated overnight at 4 °C with primary antibody in TBST containing 5% bovine serum albumin. After washing with TBST, these blots were incubated with a conjugate of goat anti-rabbit IgG and horseradish peroxidase (New England Biolabs Inc., Beverly, MA) at room temperature for 2 h. Blots were again washed with TBST, after which immune complexes were visualized using chemiluminescent reagents (New England Biolabs Inc.); images were captured with either x-ray film or a Lumi-Imager (Roche Molecular Biochemicals). The caspase-1 inhibitors YVAD-CHO and YVAD-CMK were obtained from Bachem (King of Prussia, PA); ZVAD-DCB was synthesized at Pfizer. Glyburide was purchased from BIOMOL Research Labs Inc. (Plymouth Meeting, PA), and ethacrynic acid was from Sigma. Rabbit anti-caspase-1 antibody and ELISA kits for measuring caspase-1 were obtained from Cistron Biotechnology, Inc. (Pine Brooks, NJ). Rabbit anti-caspase-3 antibody and A-431 nonstimulated cell samples (containing procaspase-3) were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Recombinant human mature caspase-3 was provided by D. Danley (Pfizer). Phenylarsine oxide (PAO) andN-ethylmaleimide (NEM) were obtained from Sigma and Pierce, respectively. The caspase-1 inhibitors YVAD-CHO, YVAD-FMK, and ZVAD-DCB were previously shown to inhibit mature IL-1β production in cell-based systems (4Thornberry N.A. Bull H.G. Calaycay J.R. Chapman K.T. Howard A.D. Kostura M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. Elliston K.O. Ayala J.M. Casano F.J. Chin J. Ding G.J.-F. Egger L.A. Gaffney E.P. Limjuco G. Palyha O.C. Raju S.M. Rolando A.M. Salley J.P. Yamin T.-T. Lee T.D. Shively J.E. MacCross M. Mumford R.A. Schmidt J.A. Tocci M.J. Nature. 1992; 356: 768-774Crossref PubMed Scopus (2267) Google Scholar, 15Nett-Fiordalisi M. Tomaselli K. Russell J.H. Chaplin D.D. J. Leukocyte Biol. 1995; 58: 717-724Crossref PubMed Scopus (33) Google Scholar, 23Miller B.E. Krasney P.A. Gauvin D.M. Holbrook K.B. Koonz D.J. Abruzzese R.V. Miller R.E. Pagani K.A. Dolle R.E. Ator M.A. Gilman S.C. J. Immunol. 1995; 154: 1331-1338PubMed Google Scholar). To assess their activity against ATP-induced post-translational processing, LPS-activated monocytes were treated with the nucleotide triphosphate in the presence of increasing inhibitor concentrations. All three caspase-1 inhibitors lowered production of ELISA-positive IL-1 in a dose-dependent manner (Fig. 1). Estimated IC50 values for YVAD-CHO, YVAD-CMK, and ZVAD-DCB were 4.3, 5.5, and 0.7 μm, respectively; these values are comparable to values reported for these compounds in other cell-based systems (4Thornberry N.A. Bull H.G. Calaycay J.R. Chapman K.T. Howard A.D. Kostura M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. Elliston K.O. Ayala J.M. Casano F.J. Chin J. Ding G.J.-F. Egger L.A. Gaffney E.P. Limjuco G. Palyha O.C. Raju S.M. Rolando A.M. Salley J.P. Yamin T.-T. Lee T.D. Shively J.E. MacCross M. Mumford R.A. Schmidt J.A. Tocci M.J. Nature. 1992; 356: 768-774Crossref PubMed Scopus (2267) Google Scholar, 15Nett-Fiordalisi M. Tomaselli K. Russell J.H. Chaplin D.D. J. Leukocyte Biol. 1995; 58: 717-724Crossref PubMed Scopus (33) Google Scholar, 23Miller B.E. Krasney P.A. Gauvin D.M. Holbrook K.B. Koonz D.J. Abruzzese R.V. Miller R.E. Pagani K.A. Dolle R.E. Ator M.A. Gilman S.C. J. Immunol. 1995; 154: 1331-1338PubMed Google Scholar,36Livingston D.J. J. Cell. Biochem. 1996; 64: 19-26Crossref Scopus (70) Google Scholar). For comparison, monocytes were treated with ATP in the presence of two inhibitors of ion transport, glyburide and ethacrynic acid (Fig. 1); these agents are known to block mature IL-1β production (39Laliberte R. Perregaux D. Svensson L. Pazoles C.J. Gabel C.A. J. Immunol. 1994; 153: 2168-2179PubMed Google Scholar, 40Hamon Y. Luciani M.-F. Becq F. Verrier B. Rubartelli A. Chimini G. Blood. 1997; 90: 2911-2915Crossref PubMed Google Scholar). Estimated IC50 values for glyburide and ethacrynic acid were 11 and 2.3 μm, respectively. It should be noted that the ELISA employed for these studies shows a preference for mature IL-1β relative to pro-IL-1β, but both forms are detected. The activity of the caspase antagonists was also evaluated in a metabolic assay format that allowed the fate of pro-IL-1β to be assessed. LPS-activated/[35S]methionine-labeled monocytes treated with ATP in the absence of an inhibitor released large quantities of 17-kDa mature IL-1β and smaller quantities of a 28-kDa species and of the 31-kDa precursor (Fig. 2 A); the 28-kDa species represents an alternate caspase-1 cleavage product (7Howard A.D. Kostura M.J. Thornberry N. Ding G.J.-F. Limjuco G. Weidner J. Salley J.P. Hogquist K.A. Chaplin D.D. Mumford R.A. Schmidt J.A. Tocci M.J. J. Immunol. 1991; 147: 2964-2969PubMed Google Scholar). Monocytes treated with ATP in the presence of YVAD-CHO generated less extracellular 17-kDa IL-1β (Fig. 2 A). Reduction in extracellular 17-kDa IL-1β was greater when the test agent was present at 60 μm rather than at 6 μm, but both concentrations significantly reduced extracellular mature cytokine levels (Fig. 2 A). Relative to the ATP-treated control cultures, quantities of extracellular 17-kDa IL-1β (determined by PhosphorImager analysis) were reduced by 66 and 87% at 6 and 60 μm YVAD-CHO, respectively. More important, the reduction in extracellular 17-kDa IL-1β caused by YVAD-CHO was accompanied by an increase in the quantity of extracellular pro-IL-1β (Fig. 2 A); this observation is consistent with previous findings indicating that YVAD-CHO blocks maturation of pro-IL-1β, but does not inhibit cytokine release (4Thornberry N.A. Bull H.G. Calaycay J.R. Chapman K.T. Howard A.D. Kostura M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. Elliston K.O. Ayala J.M. Casano F.J. Chin J. Ding G.J.-F. Egger L.A. Gaffney E.P. Limjuco G. Palyha O.C. Raju S.M. Rolando A.M. Salley J.P. Yamin T.-T. Lee T.D. Shively J.E. MacCross M. Mumford R.A. Schmidt J.A. Tocci M.J. Nature. 1992; 356: 768-774Crossref PubMed Scopus (2267) Google Scholar). After correcting for the loss of [35S]methionine that occurs when pro-IL-1β is converted to its mature species (2March C.J. Mosley B. Larsen A. Cerretti D.P. Braedt G. Price V. Gillis S. Henney C.S. Kronheim S.R. Grabstein K. Conlon P.J. Hopp T.P. Cosman D. Nature. 1985; 315: 641-647Crossref PubMed Scopus (1273) Google Scholar), the quantity of radioactivity associated with all extracellular IL-1β species released from the YVAD-CHO-treated cultures represented 69 and 61% at 6 and 60 μm, respecti
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